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Cytochrome P-450 of the common mussel, Mytilus edulis L. : partial purification and characterization
A thesis submitted to the University of Surrey for the degree of Doctor of Philosophy
by
Miles Andrew Kirchin B.Sc.
November, 1988 Department of Biochemistry,
University of Surrey,
Guildford, Surrey,
GU2 5XH, England
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Summary
Studies were carried out on microsomes of the digestive gland of the
common mussel, Mytilus edulis L. Cytochrome P-450 specific content, and
the specific contents or activities of other mixed-function oxidase (MFO)
components, and the oxidative activities benzo[a]pyrene hydroxylase (BPH)
and NADPH-independent 7-ethoxycoumarin O-deethylase (ECOD), all varied
seasonally. To varying extents, correlations were seen between changes in
these parameters, and changes in the mussel reproductive cycle and/or the
seasonal variation in water temperature. The existence of P-450 isoenzymes
was indicated by ^synchrony in the seasonal changes in BPH and NADPH-
independent ECOD activities relative to the changes in P-450 specific
content, and by seasonal changes in cytochrome P-450 Amax and the
microsomal protein profile on SDS PAGE. Indications of P-450 isoenzymes
were also obtained from purification studies, and from kinetic studies of
NADPH-independent ECOD activity (multiphasic kinetics seen with 7-
ethoxycoumarin concentration).The purification scheme essentially comprised
sodium cholate solubilization, (NH^)2 S0 ^-protein fractionation and
affinity and ion-exchange column chromatographic steps. Two cytochrome P-
450 peaks were obtained on DEAE-sephacel ion-exchange chromatography (KC1
elution), the overall purification for the major peak being x 2 0 , with a
yield of 5%. The final detergent-free P-450 preparation was largely in the
low-spin state, and had a monomeric molecular weight of 53.0 Kd.
Ligand-binding experiments were performed on partially-purified
cytochrome P-450 preparations. Type II spectra were obtained with N-
substituted imidazole compounds, metyrapone and pyridine, but with
compounds giving type I spectra with mammalian cytochromes P-450
(i)
(testosterone, 7-ethoxycoumarin, a-naphthoflavone and SKF 525-A), reverse
type I spectra were seen. In vitro MFO metabolic activity toward possible
xenobiotic and endogenous substrates, was limited, and surprisingly,
largely NADPH-independent. NADPH-independent ECOD activity was susceptable
to modulators of mammalian MFO activity, and indicated to be cytochrome P-
450-mediated. Mussel microsomal fraction and microsomal-extract were shown
to inhibit rat MFO activities and hexobarbital binding to rat cytochrome P-
450. These and other results are discussed in terms of a possible
"endogenous blocking" of the substrate-binding site of the mussel P-450,
and in terms of possible mechanisms of cytochrome P-450 catalytic action in
addition to monooxygenation, such as peroxidation.
Mussel cytochrome P-450 specific content was relatively unaffected by
a variety of mammalian model P-450 inducers, with the exception of a small
elevation with exposure to 3-methylcholanthrene (3MC). In contrast, NADPH-
cytochrome c (P-450) reductase activity was slightly more responsive. An
increase in NADPH-independent ECOD activity with 3MC-exposure was seen at
one time of the year, but not at other times.
(ii)
For my parents,
for all their help and encouragement.
(iii)
Acknowledgements
There are a number of people I would like to thank for their help and
support during the time taken to produce this thesis. In particular, I
wish to thank my supervisors, Dr. David R. Livingstone of P.M.L. (formerly
I.M.E.R.), Plymouth and Dr. Alan Wiseman of the Department of Biochemistry,
University of Surrey. Their guidance, enthusiasm and knowledge has been
invaluable. I am grateful also to my colleagues and to many of the staff
of both institutions. In particular, I would like to thank Mr. David M.
Lowe for his help with the stereological assessment of mussel gametogenic
stage, Jane Addy for her help with much of the statistical analysis, and
Suzanne Callow (nee Farrar) for her help during the early exposure
experiments, and for her considerable help during times of mussel
collection and dissection. The 3-methylcholanthrene and sodium
phenobarbital water exposure experiments were carried out jointly with Dr.
D. R. Livingstone.
For the preparation of the manuscript, I am indebted to Dr. Robert
Willows for his help in preparing many of the figures, and to Angie Smith,
Linda Jones and Dr. Howard Bottrell for the time and effort they put in to
the typing and final presentation of the thesis.
Lastly, I would like to thank Laura Canesi for giving me the final
incentive when it was most needed.
This research was carried out during the tenure of a studentship from
the Natural Environment Research Council.
(iv)
List of main abbreviations
NADH /3-nicotinamide adenine dinucleotide (reduced form)
NADPH /3-nicotinamide adenine dinucleotide phosphate (reduced form)
BPH benzo[a]pyrene hydroxylase activity
ECOD 7-ethoxycoumarin O-deethylase activity
EROD 7-ethoxyresorufin O-deethylase activity
3MC 3-methylcholanthrene
PB phenobarbital
SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis
PEG polyethylene glycol
(NH^^SO^ ammonium sulphate
DTT dithiothreitol
EDTA ethylenediaminetetraacetic acid
Tris Tris (hydroxymethyl)aminomethane
HC1 hydrochloric acid
DMF dimethylformamide
DMSO dimethylsulphoxide
Amax wavelength maximum
V__„ maximum rate of reactionI I l a A
apparent concentration of substrate giving half Vmax
maximum absorbance change
apparent Kg concentration of substrate giving half AA j
(v)
Note on nomenclature
The discovery of multiple forms of cytochrome P-450 has led to
considerable confusion in the literature concerning cytochrome P-450
nomenclature. The term "cytochrome P-450" is now recognised as a
collective name for a family of enzymes with Soret peaks in their reduced
carbon monoxide-difference spectra at or around 450 nm. This includes
cytochrome P-448 forms of the enzyme. The P-450 Amax of M. edulis shows
considerable seasonal variation. However, for convenience, it is referred
to as "cytochrome P-450" throughout the thesis.
(vi)
Contents
Section
1. General introduction1.1 Cytochrome P-450 and the mixed-function
oxidase system
1.1.1 General
1.1.2 The cytochrome P-450 catalytic cycle
1.1.3 Substrate interaction with cytochrome P-450:spin-state and substrate/inhibitor - induced binding spectra
1.1.4 Multiplicity and inducibility of mammaliancytochromes P-450
1.2 Mixed-function oxidase systems of aquaticvertebrate and invertebrate species
1.3 The common mussel Mytilus edulis - general
1.4 Cytochrome P-450 and the MFO system in M.edulis and other molluscs
1. 5 Aims of the proj ect
2 Seasonal variation of the mussel digestivegland mixed-function oxidase system and related activities
2.1 Introduction
2.2 Materials and methods
2.2.1 Collection of M. edulis from Whitsand Bay,Cornwall, and preparation of tissue pools
2.2.2 Chemicals
2.2.3 Preparation of digestive gland microsomalfraction
2.2.4 Enzyme and protein assays
2.2.4.1 Cytochrome P-450 and '416' nm peak content
(vii)
Page
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2
2
5
13
19
27
31
34
42
45
46
49
49
50
51
52
53
Section
2.2.4.2
2 .2 .4.32 .2 .4.4
2 .2 .4.5
2.2.4.6
2.2.4.7
2 .2 .4.8
2.2.5
2 .2.6
2.2.7
2 .2 .7.1
2 .2 .7.2
2.3
2.3.1
2.3.2
2.3.3
2.4
2.4.1
2.4.2
2.4.3
2.5
Total haemoprotein content
Cytochrome content
NADPH- and NADH-dependent cytochrome c reductase activities
NADH-dependent ferricyanide reductase activity
NADPH-independent 7-ethoxycoumarin 0- deethylase activity
Benzo[a]pyrene hydroxylase activity
Lowry determination of total microsomal protein
Sodium dodecyl sulphate polyacrylamide gel electrophoresis of microsomal proteins
Reproductive stage analysis
Statistical methods
Tests for sex differences
Tests for seasonal correlations
Results
Seasonal variations in components of the mussel MFO system and changes in reproductive condition
Seasonal variation in NADPH-independent 7- ethoxycoumarin O-deethylase activity and benzo[a]pyrene hydroxylase activity
SDS gel electrophoretic comparison of microsomal proteins
Discussion
Seasonal changes in cytochromes P-450 and b^ and reductase activities
Seasonal changes in BPH and NADPH-independent ECOD activities and haemoproteins
Summary
Appendix
Page
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55
56
56
57
58
60
60
63
66
67
67
69
69
93
99
103
103
110
118
120
(viii)
Section
2.5.1
2.5.2
3
3.1
3.2
3.2.1
3.2.2
3.2.3
3.2.4
3.2.5
3.2.5.1
3.2.5.1.1
3.2.5.1.2
3.2.5.2
3.2.5.2.1
3.2.5.2.2
3.2.6
3.2.6.1
3.2.6 .2
3.2.6 .3
3.2.7
3.2.8
3.2.8.1
Statistical formulae used for analysis of data
Global tests of correlation
Partial purification and properties of digestive gland microsomal cytochrome P-450 and other MFO components
Introduction
Materials and methods
Chemicals and materials
Enzyme assays and SDS PAGE electrophoresis
Preparation of digestive gland microsomal fraction
Solubilization of digestive gland microsomal cytochrome P-450 and NADPH-cytochrome c (P-450) reductase activity
Fractionation of solubilized microsomal cytochrome P-450 and NADPH-cytochrome c (P-450) reductase activity
Fractionation with ammonium sulphate
Optimization of conditions
(NH^^SO^-fractionation as used in purification
Fractionation with polyethylene glycol (PEG)
Optimization of conditions
PEG fractionation as used in purification
Affinity chromatography on 8 -aminooctyl sepharose 4B
Preparation of 8 -aminooctyl sepharose 4B
Column pouring and equilibration
Chromatography on 8 -aminooctyl sepharose 4B
Ultrafiltration and dialysis of pooled fractions from 8 -aminooctyl sepharose 4B chromatography
Ion-exchange chromatography on DEAE-sephacel
Preparation of DEAE-sephacel columns
(ix)
Page
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126
129
129
129
130
131
133
133
134
134
135
136
136
136
136
137
138
139
140
140
Section
3.2.8.2
3.2.9
3.2.10
3.2.10.1
3.2.10.2
3.2.11
3.2.11.1
3.2.11.2
3.3
3.3.1
3.3.2
3.3.3
3.3.4
3.3.5
3.3.6
3.3.7
3.4
Chromatography on DEAE-sephacel
Ultrafiltration and dialysis of pooled samples from DEAE-sephacel chromatography
Removal of emulgen 911 on hydroxylapatite
Preparation of hydroxylapatite columns
Chromatography on hydroxylapatite
Further purification of NADPH-cytochrome c (P-450) and NADH-cytochrome c reductase activities on 2',5'- ADP sepharose 4B
Preparation of 2',5'- ADP sepharose 4B columns
Chromatography on 2',5'- ADP sepharose 4B
Results
Solubilization of digestive gland microsomal cytochrome P-450 and NADPH-cytochrome c (P-450) reductase activity
Ammonium sulphate and PEG fractionation of solubilized digestive gland microsomal cytochrome P-450 and NADPH-cytochrome c (P-450) reductase activity
8 -aminooctyl sepharose 4B affinity chromatography of cytochrome P-450 and other MFO components
A purification scheme, involving (NH^^SO^ fractionation, for digestive gland microsomal cytochrome P-450
Spectral properties, molecular weight and purity of the partially purified cytochrome P-450
Partial purification of digestive gland microsomal cytochrome P-450 following fractionation by PEG
The partial purification of digestive gland microsomal NADPH-cytochrome c (P-450) and NADH-cytochrome c reductase activities
Discussion
Page
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142
142
142
143
143
143
144
145
145
153
159
164
173
176
180
185
(x)
Section
3.4.1 Solubilization and protein fractionation ofdigestive gland microsomes
3.4.2 Purification and properties of digestive glandmicrosomal cytochrome P-450
3.4.3 Purification of NADPH-cytochrome c (P-450)and NADH-cytochrome c reductase activities
3.5 Appendix
4 Ligand-interaction with mussel digestive glandmicrosomal cytochrome P-450
4.1 Introduction
4.2 Materials and methods
4.2.1 Chemicals
4.2.2 Preparation of solubilized and (NH^^SO^ -fractionated cytochrome P-450 samples
Assays for cytochrome P-450 and protein
Ligand - induced binding spectra
Measurement of ligand-induced binding spectra
Determination of the apparent spectral dissociation constant Kg and theoretical maximum peak to trough absorbance change AAjjj
4.3 Results
4.4 Discussion
5 Studies on the metabolism of possible xenobioticand endogenous substrates by mussel digestive gland microsomal preparations
5.1 Introduction
5.2 Materials and methods
5.2.1 Chemicals and materials
5.2.2 Preparation of digestive gland microsomalfraction
5.2.3 Studies on NADPH-independent 7-ethoxycoumarinO-deethylase activity
4.2.3
4.2.4
4.2.4.1
4.2.4.2
Page
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187
196
198
200
201202202202
203
203
203
204
206
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230
232
232
232
233
(xi)
Section
5.2.4
5.2.5
5.2.6
5.2.7
5.2.8
5.2.9
5.2.10
5.2.11
5.3
5.3.1
5.3.2
5.3.3
5.3.4
5.4
5.4.1
5.4.2
5.4.3
5.4.4
NADPH-independent 7-ethoxyresorufin O-deethylase (EROD) activity
Testosterone hydroxylase activity
Arachidonic acid hydroxylase activity
Autoradiography of developed T.L.C. plates
Laurie acid hydroxylase activity
Benzphetamine N-demethylase activity
Other mammalian MFO activities assayed in mussel digestive gland microsomes
Inhibition of rat liver microsomal MFO activities by mussel digestive gland microsomes and ethyl acetate-extract of mussel microsomes
Results
Studies on digestive gland microsomal NADPH- independent ECOD activity
Studies on the ability of digestive gland microsomes to metabolise various xenobiotic substrates (N-demethylase, O-deethylase, hydroxylase activities)
Studies on the ability of digestive gland microsomes to metabolise the endogenous substrates, arachidonic acid, "lauric acid" and testosterone
Studies on the inhibition of rat hepatic microsomal MFO activities by digestive gland microsomes and ethyl acetate-extract of mussel microsomes
Discussion
NADPH-independent 7-ethoxycoumarin O-deethylase activity
Other xenobiotic NADPH-independent oxidative activities
NADPH-independent endogenous substrate oxidative activities
Possible endogenous inhibition of M. edulis oxidative metabolism
Page
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235
236
237
237
238
239
240
242
242
256
259
266
274
274
285
287
290
(xii)
Section Page
5.5 Appendix 292
6 Responses of digestive gland microsomal MFO 294components and NADPH-independent ECOD activity to xenobiotic exposure
6.1 Introduction 295
6 .2 Materials and methods 298
6.2.1 Chemicals 298
6.2.2 Static tank exposure experiments 298
6 .2.2.1 Collection and equilibration of M. edulis prior 298to exposure
6 .2.2.2 Exposure of M. edulis to xenobiotics in sea- 299water (without feeding)
6 .2.2.3 Exposure of M. edulis to xenobiotics by direct 301inj ection
6 .2.2.4 Three-week exposure of M. edulis to xenobiotics 303(with feeding)
6.2.3 Measurement and analysis of the effects of 304xenobiotic exposure on contents and activitiesof the mussel digestive gland microsomal MFO system
6 .3 Results 306
6.3.1 Exposure of M. edulis to xenobiotics in sea- 306water (without feeding)
6.3.2 Exposure of M. edulis to xenobiotics by direct 316injection
6.3.3 Three-week exposure of M. edulis to xenobiotics 324(with feeding)
6.4 Discussion 327
6.5 Appendix 337
7. General discussion 339
References 345
(xiii)
Section 1
General Introduction
1
1.1 Cytochrome P-450 and the mixed-function oxidase system
1.1.1 General
Cytochrome P-450 is the collective name for a distinct group of
haemoproteins (isoenzymes) showing absorption maxima at or around 450 run in
dithionite-reduced carbon monoxide-difference spectra. They have monomeric
molecular weights of approximately 45,000-55,000 and are the terminal
enzymic components of electron transport chains (termed mixed-function
oxidase (MFO) systems; see below) catalysing the oxidation of numerous,
structurally-diverse endogenous (e.g. steroids, sterols, fatty acids, bile
acids and vitamins) and exogenous or foreign (xenobiotic) (e.g. drugs,
pesticides, carcinogens and environmental pollutants) substrates (Ioannides
and Parke, 1987; Guengerich, 1988). The overall reaction catalysed by
cytochromes P-450 is :
SH + O2 + 2H+ ------»\£0H + H2O (where SH denotes the substrate)
The reaction is referred to as either a mono oxygenation or a mixed-
function oxidation, the former because only one atom of molecular oxygen is
introduced into the substrate, the other being reduced to water, and the
latter because both oxidative and oxygenative components are apparent in
the reaction. Cytochromes P-450 are, therefore, distinct from purely
oxidase enzymes which use oxygen solely as a hydrogen acceptor (e.g.
cytochrome c oxidase), and from dioxygenase enzymes which catalyse the
incorporation of both atoms of molecular oxygen into the substrate (e.g.
lipoxygenase). The requirement for an external source of reducing
equivalents indicates that cytochromes P-450 are "external" monooxygenases
as opposed to "internal" monooxygenases in which the substrate itself acts
as the reductant (e.g. lactate monooxygenase) (Ishimura et al., 1978).
Drugs and other xenobiotics are metabolised by two major processes,
namely, phase I metabolism or biotransformation, and phase II metabolism or
conjugation. The involvement of cytochromes P-450 in the metabolism of
xenobiotics constitutes the majority of phase I biotransformations. In
processes of detoxication, relatively lipophilic compounds are rendered
more polar to facilitate their subsequent phase II conjugations (with
endogenous compounds such as glucuronic acid, glutathione, glycine or
glutamine) and eventual elimination. Recently, the primary function of
phase I reactions has been regarded not to make the substrates more polar
per se, but to introduce a functional group such as -OH, -NH2 etc.
(functionalization phase) into the xenobiotic so preparing it for
subsequent conjugation in phase II (the real "water-soluble creating
phase") (Gibson and Skett, 1986). However, although it is essentially a
system for detoxication, phase I metabolism can also result in activation
of certain chemicals to reactive intermediates or ultimate carcinogens.
These are poor substrates for the various conjugases, and tend to react
non-enzymically with glutathione, or to form covalent-bound ligands with
proteins and DNA (Ioannides and Parke, 1987; Parke and Obreska - Parke,
1987; see below).
The discovery of a "carbon monoxide - binding pigment" was first
reported in 1958 in liver microsomes of rat (Klingenberg, 1958) and pig
(Garfinkel, 1958). However, it was not until 1962 that the haemoprotein
nature of the pigment was elucidated (Omura and Sato, 1962), and not until
the mid-1960s that direct evidence was presented for its involvement in
hydroxylation reactions (Estabrook et al., 1963; Cooper et al., 1965) (for
reviews of the discovery of cytochrome P-450, see Omura, 1978 and
Schenkman, 1982). Today, cytochrome P-450 is known to have a ubiquitous
distribution in nature, occurring in species ranging from bacteria
(Appleby, 1967), to higher plants (Madyastha et al. , 1976, 1977) to
mammals. In species of bacteria, cytochrome P-450 is found in soluble form
(Appleby, 1967; Katagiri et al., 1968), whereas in plant and mammalian
species it is membrane-bound in the endoplasmic reticulum (Klingenberg,
1958), mitochondria (Cheng and Harding, 1973; Jefcoate and Orme-Johnson,
1975) and the nuclear envelope (Khandwala and Kasper, 1973) of the cell.
Although in mammalian species, cytochromes P-450 are most abundent in the
liver, they also occur in extrahepatic tissues, including kidney, lung,
intestine, steroidogenic tissues, skin and white blood cells (Burke and
Orrenius, 1982). However, they are apparently not present in striated
muscle or erythrocytes (Guengerich, 1988).
As mentioned above, cytochromes P-450 are the terminal oxygenases of
mixed-function oxidase systems. The second major component of the MFO
system of the endoplasmic reticulum (obtained experimentally, following
tissue homogenization and centrifugation as small vesicles termed
microsomes) is an NADPH-linked flavoprotein termed NADPH-cytochrome P-450
reductase (monomeric molecular weight approximately 78,000). This enzyme
contains one mole each of flavin adenine dinucleotide (FAD) and flavin
mononucleotide (FMN) per mole of enzyme (Iyanagi and Mason, 1973), and
serves to provide cytochrome P-450 (at least in part; see later) with the
necessary reducing equivalents from NADPH for the monooxygenation reaction
(NADPH rather than NADH is the primary reduced coenzyme utilized by the MFO
system; see Section 1.1.2). The MFO system of mitochondria differs from
those of either the endoplasmic reticulum or the nuclear envelope in that a
4
third electron-transferring component, a non-haem iron-sulphur protein
termed adrenodoxin, is present in between the NADPH-linked flavoprotein and
the terminal cytochrome P-450 (Kimura and Suzuki, 1967). In this respect*
the MFO system of mitochondria resembles the soluble systems of bacteria,
which also contain an intermediate iron-sulpher protein termed
putidaredoxin (Tsai et al., 1971). The flavoprotein reductase of the
mitochondrial MFO system also differs from the NADPH-cytochrome P-450
reductase of microsomal systems in that only one mole of FAD is present per
mole of enzyme (Chu and Kimura, 1973).
Aspects of mammalian MFO systems, in particular the cytochrome P-450
component, are discussed more fully in the next sections.
1.1.2 The cytochrome P-450 catalytic cycle
In addition to the monooxygenation (hydroxylation) of substrates,
cytochromes P-450 are also known to catalyse dealkylations, deaminations,
desulphurations, dehydrogenations or dehalogenations (Lampe et al. , 1984),
the particular resulting reaction being dependent upon the chemical nature
of the substrate. However, in all of these cytochrome P-450-mediated
reactions, the first step is always an hydroxylation (Gillete, 1966).
Figure 1.1 shows the catalytic cycle for mammalian hepatic microsomal
cytochrome P-450 (adapted from the schemes of Schenkman and Gibson, 1981
and Ortiz de Montellano, 1986). The initial step 1, consists of the3 ireversible binding of substrate to the oxidized (Fe ) form of cytochrome
P-450 (discussed more fully in Section 1.1.3). This binding alters the
mid-point redox potential of the cytochrome (making it more positive), so
facilitating a one-electron reduction catalysed by NADPH-dependent
SH e“
NADPHSOH
SH*P-450[Fe2+] (SH#P-450[ Fe3 ]
NADH PD
e"
SH*P-450[ Fe2+]*Oo2 H
Figure 1.1The hepatic microsomal cytochrome P-450 catalytic cycle.
SH denotes substrate
P-450[Fe^+] and P-450 [Fe2+] denote cytochrome P-450 in the oxidized and reduced states respectively
Fp^ denotes NADPH-cytochrome P-450 reductase
Fpp denotes NADH-cytochrome reductase
b^ denotes cytochrome b^
SOH denotes hydroxylated product
(Adapted from Schenkman and Gibson, 1981 and Ortiz de Montellano, 1986)
6
cytochrome P-450 reductase (step 2 of the cycle). Step 3 involves the
binding of molecular oxygen to form a ternary oxygenated complex capable of
accepting a necessary second electron. This second electron is provided
(step 4) either from NADPH via NADPH-cytochrome P-450 reductase, or from
NADH via NADH-cytochrome b^ reductase and cytochrome b^ (another microsomal
electron transport system involved in fatty acid desaturation; Shimakata et
al., 1972). Step 5, the least understood part of the cycle, involves
cleavage of dioxygen. One oxygen atom is reduced to water, while the other
atom forms an activated oxygen-cytochrome P-450-substrate complex.
Finally, in step 6 , the activated oxygen atom is inserted into the
substrate to form the hydroxylated product. At this point, oxidized
cytochrome P-450 is regenerated. The nature of the active oxygen species
is unclear, but may involve a number of radical species (White and Coon,
1980).
The catalytic cycle requires the sequential input of two electrons.
As noted above, the first of these is supplied from NADPH via NADPH-
cytochrome P-450 reductase (Prough and Burke, 1975; Dignam and Strobel,
1977). The second electron, however, may be supplied either as the first,
or from NADH via NADH-cytochrome b^ reductase and cytochrome b^
(Hildebrandt and Estabrook, 1971; Sasame et al., 1974; Peterson and Prough,
1986). Using purified components in reconstituted MFO systems, different
isoenzymes of cytochrome P-450 have been shown to differ in their
requirement for a cytochrome b^-mediated input of reducing equivalents
(Imai, 1981). Thus, for example, cytochrome b^ has been shown to stimulate
the metabolism of aminopyrine, benzphetamine, testosterone and p-
nitroanisole by cytochrome P-450 RIM5 or RLM«ja of the untreated rat, but to
have no effect when these compounds are metabolised by cytochrome P-450
7
RLM2 or RLM3 (Jansson et al. t 1985). In addition to the particular
cytochrome P-450 isoenzyme, the MFO substrate-type is also a factor
determining the contribution of the cytochrome electron-transfer system.
For example, cytochrome has been shown to be obligatory for the
metabolism of methoxyflurane by rabbit liver cytochrome P-450 LM2 but not
for the metabolism of benzphetamine by this same isoenzyme (Canova - Davies
et al., 1985; Jansson and Schenkman, 1987). Evidence that the cytochrome
b^ system is required for the supply of the second electron for certain MFO
activities has come from both kinetic and immunochemical studies. For a
review of the reduction of cytochrome P-450 during catalysis, see Peterson
and Prough (1986).
The mechanism of cytochrome P-450-mediated monooxygenation would be
expected to produce a stoichiometric consumption of one mole of (NADPH +
H+) and one mole of oxygen for each mole of product formed. However, with
both microsomal and purified and reconstituted systems, this is often not
observed, and excessive amounts of oxygen and NADPH are consumed
concomitant with the production of hydrogen peroxide (Hildebrandt et al. ,
1975; Nordblom and Coon, 1977). It is possible that the extra oxygen and
NADPH are used in the formation of oxygenated cytochrome P-450 which then
acts as an oxidase, producing hydrogen peroxide, as shown by the equation
below :
cytochrome02 + NADPH + H+ ---------- ► H202 + NADP+
P-450
However, it is more likely the observed hydrogen peroxide is produced by
disproportionation of superoxide anion radical, itself being produced from
the auto-oxidation of oxygenated cytochrome P-450 and/or cytochrome P-450
8
reductase (Winston and Cederbaum, 1983).
2H+°2 + °2 ” * H2°2 + °2
This reaction may also be catalysed by superoxide dismutase (EC 1.15.1.1)
(Estabrook et al., 1984), although this is a cytosolic, not a microsomal,
enzyme (Steinman, 1982). An alternative fate for superoxide anion radical
is conversion to other radical species, such as the hydroxyl radical, or
involvement in the peroxidation of fatty acids to lipid peroxides
(Rahimtula and O'Brien, 1978; White and Coon 1980; Blake and Coon, 1981).
In addition to its involvement in the two-electron monooxygenase
pathway, cytochrome P-450 can also function in an oxygen/NADPH-linked one-
electron monooxygenative pathway (Cavalieri and Rogan, 1984) . The
mechanism is thought to involve initial abstraction of one electron from
the substrate to the oxycytochrome P-450 complex. This produces a reactive
substrate radical-cation and a nucleophilic oxygen species, which interact
to produce an oxygenated product. This mechanism has been shown to
function in the cytochrome P-450-catalysed oxygenation of sulphides to
sulphoxides, and sulphoxides to sulphones (Watanabe et al., 1980, 1982),
and in the oxidation of N-hydroxynorcocaine (Rauckman et al., 1982). In
the absence of NADPH and oxygen, cytochrome P-450 can act as a nonselective
peroxidase, utilizing for example, hydrogen peroxide, organic
hydroperoxides or peroxyacids as oxygen-donor substrates (Hrycay et al.,
1975; Nordblom et al. , 1976). . This is termed the "peroxide-shunt" (Coon
and Vaz, 1987) and operates in studies in which NADPH and oxygen are
replaced by, for example, cumene hydroperoxide (Kadlubar et al., 1973;
Rahimtula and O'Brien, 1974; Ellin and Orrenius, 1975) or sodium periodate
9
(Hrycay et al.,1975; Gustafsson et al., 1976). The reaction is represented
by the equation :
cytochromeROOH + SH ------------ ► ROH + SOH
P-450
(where ROOH represents the peroxy compound and SH the substrate).
The exact mechanism of the reaction is uncertain, and may involve either
homolytic or heterolytic cleavage of the peroxide bond, or both (Thompson
and Wand, 1985; Coon and Vaz, 1987). The heterolytic mechanism is that
generally seen with peroxidase enzymes (Coon and Vaz , 1987). The overall
mechanism for the peroxidase pathway has been considered to be essentially
similar to that of the NADPH/oxygen-linked one-electron oxygenation
described above (Cavalieri and Rogan, 1984). It is shown, together with
proposed schemes for cytochrome P-450-mediated monooxygenase and oxidase
activities, in Figure 1.2 (adapted from Estabrook et al., 1984 and Coon and
Vaz, 1987). Dissociation of the cytochrome P-450-hydroxylated product
complex regenerates the oxidized form of cytochrome P-450. The peroxidase
pathway is independent of electron transfer from NAD(P)H and does not
require molecular oxygen to produce a ternary peroxygenated-cytochrome P-
450 complex. Many of the reactions occurring by this pathway are similar
to those that occur in NADPH/oxygen-supported catalysis, for example,
alkanes are hydroxylated (Nordblom et al., 1976; Blake and Coon, 1981),
amines are N-dealkylated (Kadlubar et al., 1973; Griffin et al., 1980) and
olefins are epoxidated (McCarthy and White, 1983). However, that the
mechanism is intrinsically different from that of the MFO system is
indicated, for example, by the different patterns of benzo[a]pyrene
metabolites obtained in NADPH- and cumene hydroperoxide-supported
10
Figure 1.2
A scheme for the involvement of cytochrome P-450 in oxygenase, peroxidase
and oxidase pathways.
XOOH denotes Peroxides
SH denotes substrate
SOH denotes hydroxylated product
X denotes fatty acids or lipids
C>2 denotes superoxide radical
SOD denotes superoxide dismutase
CO denotes carbon monoxide
(Adapted from Estabrook et al., 1984 and Coon and Vaz, 1987)
11
oIT) ID
Oin
in
n:oC/D
O oin oinin
CM
CM
Oin
12
reactions. In the presence of NADPH, a preponderence of phenols and
dihydrodiols are formed, whereas, in the presence of cumene hydroperoxide,
a preponderence of quinones are formed (Capdevila et al., 1980).
1.1.3 Substrate interaction with cytochrome P-450 : spin-state and substrate/inhibitor-induced binding spectra
Cytochrome P-450 is classified as a b-type cytochrome, containing
protoporphyrin IX as its prosthetic group (Omura and Sato, 1962). The
unique properties of cytochrome P-450 are due, in part at least, to the
nature of its haem ligation (Gibson and Tamburini, 1984). The haem-iron of
cytochrome P-450 can exist in both hexa- and penta- coordinate forms (see
Figure 1.3). In both cases, the four equatorial coordination ligands are
pyrrole nitrogen atoms from the protoporphyrin IX, and one of the axial
ligands (the 5th ligand), a cysteinate residue from the apoprotein (Hahn et
al., 1982; Mansuy, 1983). The nature of the other axial ligand (the 6 th
ligand) in the hexa-coordinate form is unclear, but is likely to be a
hydroxyl group from an amino acid of the apoprotein (Dawson et al., 1982;
White and Coon 1982; Andersson and Dawson, 1984). In the hexa-coordinate3+state, the oxidized (ferric, Fe ) haem-iron exists in an octahedral
complex, and contains five electrons in its 3d-subshell (Schenkman et al.,
1981). Due to differences in their spatial arrangement, these electrons
are subject to differential electron repulsion by lone-pair electrons from
the coordination ligands. This differential repulsion is such that the
electrons occupying the dz 2 and dx2 _y2 orbitals (the eorbitals)
experience a greater repulsion (ligand effect) than electrons occupying the
dXy, dZy or dxz orbitals (the t2g orbitals). Consequently more energy is
required for an electron to occupy an eg orbital than a t£g orbital. The
Protein Protein
Haem
Cys
Protein
.3+Haem
Cys
(a) (b)
Figure 1.3 Haem iron coordination in cytochrome P-450
(a) Hexa-coordinated, low-spin with in-plane iron
(b) penta-coordinated, high-spin with out-of-plane iron
(taken from Gibson and Skett, 1986)
en orbitels
Low spinS=T
11 e!
11KHigh spin
Figure 1.4 d-orbital electron distribution in the low- and high-spin states of cytochrome P-450
AE^ = large e - t2g energy difference
AE2 *= reduced eg - t2g energy difference
(taken from Gibson and Skett, 1986)
14
net effect is a splitting in the energy levels of the ferric ion d-orbitals
as shown in Figure 1.4. The difference in energy (AE) between the e andOt2g orbitals is known as the crystal field splitting (or stabilization)
energy, and is a function of the strength of the ligand to ferric ion d-
orbital repulsion forces, and thus the ligand field strength.
In a strong ligand field, such as in the hexa-coordinate state, AE
exceeds the energetic instability resulting from electron-electron
repulsion, and the five 3d electrons occupy the 3 t2 g lower energy
orbitals. Consistent with the Pauli exclusion principle that no more than
two electrons can occupy a single orbital, and that these electrons must
have opposite spins, the single unpaired electron gives rise to a total
spin, S, of 1/2. This is termed low spin. In a weak ligand field, such as
in the penta-coordinate state, AE is reduced, such that it is energetically
more favourable for each electron to separat^ occupy each 3d-orbital. In
this case, all five electrons are unpaired, and the total spin is 5/2,
termed high spin. This is described in more detail in Schenkman et al.
(1981) and Gibson and Tamburini (1984) . The two spin states are
characterized optically in absolute spectra by absorption maxima in the
Soret region at 385-390 nm (high spin) and 416-418 nm (low spin) (Gibson et
al., 1980).
In the absence of substrate, cytochromes P-450 exist as an equilibrium
of high and low spin states (S.ligar, 1976; Cinti et al., 1979). However,
both substrates and inhibitors of cytochrome P-450, as well as changes in
temperature and ionic strength (Cinti et al., 1979), influence this
equilibrium. This is achieved through changes in the ligand field
strength, mediated by changes in axial ligand coordination. Perturbations
15
of the spin state equilibrium by substrates and inhibitors of cytochrome P-
450 can be observed by difference spectrophotometry (Schenkman et al.,
1967). The spectral changes which are generally classified as being type
I, type II or reverse type I are described below, and shown in Figure 1.5.
Type 1: The type I spectral change is characterized by an absorption peak
at 385-390 nm and a trough at 420 nm and corresponds to a low to high spin
transition (a decrease in the absorbance of low spin cytochrome concomitant
with an increase in the absorbance of high spin cytochrome; Schenkman et
al., 1967). The compounds evoking such a spectral change are generally
known substrates for cytochrome P-450 MFO systems, and include both
xenobiotic and endogenous compounds (Schenkman et al., 1981). It is likely
that the spin perturbation is due to a reduction in the ligand field
strength resulting from a change in the axial ligand coordination from a
hexa-coordinate state (with in-plane iron) to a penta-coordinate state
(with out-of-plane iron) (see Figure 1.3). This results from substrate
binding to the cytochrome P-450 apoprotein (probably the active site),
causing a change in protein conformation and hence the ligation of the haem
(iron) with the protein (Gibson and Skett, 1986).
Type II: The type II spectral change is characterized by an absorbance
minimum at 390-405 nm and a maximum at 425-435 nm. It is, therefore,
associated with a high to low spin transition (Schenkman et al., 1981).
The compounds capable of eliciting such a change are mainly nitrogenous
bases containing, unhindered, non-bonded electron pairs capable of ligating
directly to the cytochrome P-450 haem-iron as the sixth ligand (the
criterion used to determine type II interaction is the compounds ability to
displace carbon monoxide from reduced cytochrome P-450 - Remmer et al.,
1969). By ligating directly to the haem iron, the ligand field strength is
16
350
h iOz<co ocoCOCD<
WAVELENGTH (nm)
400 450 500
TYPE 1
REVERSE TYPE 1
TYPE 2
350 400 450 500WAVELENGTH (nm)
Figure 1.5
Types of spectral change resulting from compound interaction with
cytochrome P-450.
17
markedly increased such that the electrons occupy a low spin configuration.
Most type II compounds, therefore, tend not to be substrates for cytochrome
P-450, but rather are inhibitors of MFO activity.
Reverse type I : The reverse type I spectrum, (previously called a
modified-type II spectrum) which is characterized by a peak at about 420 nm
and a trough at 390 nm, appears to be the mirror image of the type I
spectral change. It, again, reflects a high to low spin transition. The
cause of this spectral change is uncertain. One suggestion has been that
it is due to the displacement of endogenous substrates from the type I
binding site by the added substrate (Schenkman et al., 1969, 1972, 1973;
Powis et al., 1977). However, the more recent suggestion is that it is due
to the direct interaction of hydroxyl groups with the haem iron (Yoshida
and Kumaoka, 1975).
The binding of substrate to the type I binding site, and the resulting
increase in the proportion of high-spin cytochrome, appears to facilitate
subsequent steps in the cytochrome P-450 catalytic cycle (Schenkman and
Gibson, 1981). The spin-state of cytochrome P-450 controls the redox
potential of the molecule, with the high-spin form having a less negative
potential (Sligar, 1976). Since, in the absence of substrate, (rat
hepatic) cytochrome P-450 has a redox potential approximately similar to
that of NADPH ("350 mv compared with "365 mv for NADPH - Schenkman and
Gibson, 1981), electron transfer from NADPH is not favoured. However,
following substrate binding, the increase in the proportion of high-spin
cytochrome effectively changes the redox potential to a more positive
value, allowing electrons to more readily flow to the cytochrome P-450
(Sligar et al., 1979). Thus, the substrate-induced change in spin-state
results in an accelleration of reduction rate of the cytochrome (Rein et
18
al., 1979). Further, it is likely that the change in coordination state to3+the penta-coordinate form (and thus the change in Fe positioning; see
Figure 1.3) facilitates the subsequent interaction of the necessary oxygen
molecule.
1.1.4 Multiplicity and inducibility of mammalian cytochromes P-450
The existence of more than one liver microsomal drug-metabolising
enzyme was first postulated more than 25 years ago (Conney et al., 1959).
Today, nearly 20 different forms of cytochrome P-450 have been purified and
characterized from rat liver alone (Ortiz de Montellano, 1986), and it
seems likely that similar numbers of cytochrome P-450 forms are also
present in other mammalian species (Guengerich, 1987). An attempt to
characterize cytochromes P-450 into different gene families has recently
been made for cytochromes P-450 from various species for which the full-
length amino acid sequences were known (Nebert and Gonzalez, 1987) (see
Table 1.1). There appears to be at least ten distinct gene families, of
which eight are present in mammalian species. The major features of these
gene families are as follows. (1) The cytochromes from any of the ten
families are <36% similar in gene sequence to cytochromes from any of the
other families. (2) Two families (II and XI) contain more than one
subfamily. Cytochrome P-450 genes considered to be in the same subfamily
have >68% similarity in gene sequence to other genes in that subfamily.
Genes considered to be in different subfamilies of the same family have
between 40 and 65% similarity. (3) The gene superfamily as a whole
represents an example of divergent evolution. Even among cytochromes with
very little overall similarity (<25%), enough localized similarity (>33%)
19
Table 1.1 The cytochrome P-450 gene superfamily
(Taken from Nebert and Gonzalez, 1987)
Family, subfamily, and gene designation S o m e of the existing n a m e s in the literatureP450I (polycyclic aromatic compound-induciblc)
O nly one subfamilyP 450IA1 Rat c, rabbit form 6, m o u s e P lt h u m a n P|P 4 5 0 I A 2 Rat d, rabbit form 4, m o u s e P 3, h u m a n P,
P450II (major)P450IIA subfamily
P450IIAI Rat aP450IIA2 H u m a n P450(l)
P450IIB subfamily (phenobarbital-inducible)P450IIB1 Rat b, rabbit form 2P450IIB2 Rat e
P450I1C subfamilyP450IICI Rabbit P BcIP450IIC2 Rabbit PBc2, KP450IIC3 Rabbit PBc3, form 3bP450IIC4 Rabbit PBc4, form 1-8P450I1C5 Rabbit form 1P4501IC6 Rat P B 1P450IIC7 Rat fP450IIC8 H u m a n form 1P450IIC9 H u m a n m pP450IICI0 Chicken P B 1 5P450 I I C I 1 Rat M -l, h
P4S0IID subfamilyP450IID1 Rat dbl, h u m a n dblP 450IID2 Rat db2
P4501IE subfamily (ethanol-inducible)P450IIE1 Rat j, rabbit form 3a, h u m a n j
P450III (steroid-inducible)O nly one subfamily
P450IIIA1 Rat pcnlP4$0IIIA2 Rat pcn2
P 4 5 0 I V (peroxisome proliferator-inducible)Only one subfamily
P 4 5 0 I V A 1 Rat L A wP 4 5 0 X I (mitochondrial proteins)
P 4 5 0 X I A subfamilyP 4 5 0 X I A 1 Bovine (and hum a n ) see
P 4 5 0 X I B subfamilyP 4 5 0 X 1 B 1 Bovine (and hu m a n ) 11/3
P 4 5 0 X V I I (steroid !7a-hydroxylase)O nly one subfamily
P 4 5 0 X V 1 I A I Bovine (and h u m a n ) 17aP 4 5 0 X 1 X
O nly one subfamilyP 4 5 0 X I X A 1 H u m a n a r o m
P 4 5 0 X X I (steroid 21-hydroxylase)O nly one subfamily
P 4 5 0 X X I A 1 Bovine (and mouse, hu m a n ) C 2 1 AP 4 5 0 X X I A 2 Bovine (and mouse, hu m a n ) C 2 1 B
P 450LI (plant P450)O n l y one subfamily
P 4 5 0 L I A 1 Yeast lanP 4 5 0 C I (prokaryote P450)
O nly one subfamilyP 4 5 0 C I A ! Pseudomonas putida c a m
20
is evident (for example, a 21-residue cysteinyl fragment associated with
the haem-binding pocket), to suggest that all the genes evolved from a
common ancestor. In this latter respect, it has been suggested that
cytochromes P-450 originally evolved from a bacterial enzyme primarily
concerned with the detoxication of oxygen (Wickramsinghe and Villee, 1975).
A unique feature of cytochrome P-450 enzymes are their indueibility by
foreign chemicals. The induction response, seen as an increase in
cytochrome P-450 specific content and/or particular monooxygenation
activities, is achieved mainly through transcriptional activation leading
to an increase in specific mRNA (Hardwick et al., 1983; Gonzalez et al.,
1985; Whitlock, 1986; Fonne and Meyer, 1987). However, it can also be
achieved through posttranscriptional regulation such as through mRNA
stabilization (Simmons et al., 1987) or through posttranslational protein
stabilization (Song et al.,1986; Watkins et al., 1986). In most cases,
xenobiotics induce a range of mammalian cytochrome P-450 isoenzymes (Ryan
et al., 1979; Imai et al., 1980; Funae and Imaoka, 1985). For example, in
rat liver microsomes, cytochrome P-450s c and d (collectively termed
cytochromes P-448) are both induced by polycyclic aromatic hydrocarbons of
which 3-methylcholanthrene (3MC) is an example; whereas phenobarbital (PB)
induces cytochrome P-450s b and e (which are 97% similar and termed
cytochromes P-450), as well as. various cytochromes from the cytochrome P-
450 lie subfamily (Nebert and Gonzalez, 1987). All of these particular
cytochromes P-450 are induced by the polychlorinated biphenyl mixture,
Aroclor 1254 (Ryan et al., 1982). However, conversely, certain xenobiotics
appear to induce only one major form of cytochrome P-450. For example,
clofibrate, a hypolipidaemic drug, primarily induces cytochrome P-452 (P-
450 LAw) (Tamburini et al., 1984; Hardwick et al., 1987), a cytochrome P-
21
450 efficient in the 12-hydroxylation of lauric acid, and characterized as
belonging to the cytochrome P-450 IV gene family (Nebert and Gonzalez,
1987).
In addition to full-length amino acid sequencing, a number of criteria
are used to distinguish cytochrome P-450 isoenzymes (Lu and West, 1980;
Astrom and Depierre, 1986).
(1) Molecular weight. Isoenzymes often have different monomeric molecular
weights and, therefore, show different mobilities on sodium dodecyl
sulphate polyacrylamide electrophoretic (SDS PAGE) gels.
(2) Spectral properties. As indicated above, absorption maxima for
reduced carbon monoxide-difference spectra may vary for different
isoenzymes. Also, the spin state (high or low) of purified cytochromes may
be different.
(3) Catalytic activity. Different isoenzymes often have different but
overlapping substrate specificities and/or exhibit positional and
stereoselectivity in the metabolism of substrates. For example, 7-
ethoxyresorufin is a specific substrate for cytochrome P-450c, the major
3MC-inducible cytochrome, whereas 7-pentoxyresorufin is a specific
substrate for cytochrome P-450b, the major PB-inducible cytochrome. Both
cytochromes, however, are capable of metabolising 7-ethoxycoumarin
(Rodrigues et al.,1987). Also, both cytochromes metabolise steroids such
as testosterone, cytochrome P-450c primarily at the 6)9 position and
cytochrome P-450b at the 16a and 16)9 positions (Astrom and Depierre, 1986;
Sonderfan et al., 1987).
(4) Immunological properties. Antibodies raised to one form of cytochrome
22
P-450 are highly specific for that form, and often show little or no cross-
reactivity with other forms. For example, clofibrate-induced cytochrome P-
452 exhibits a complete lack of immunocrossreactivity with cytochromes P-
450 induced by polycyclic hydrocarbons or phenobarbital (Tamburini et al. ,
1984). In contrast, PB-inducible cytochrome P-450s ^ and e are
immunologically identical (Ryan et al., 1982).
(5) Peptide mapping. Following limited proteolysis peptide fragments can
be separated by SDS PAGE. The resulting peptide map can be used to
differentiate between the primary structures of different cytochrome P-450
isoenzymes (Ryan et al., 1979, 1982).
Table 1.2 shows a comparison of the characteristics of different rat
cytochrome P-450 isoenzymes. The most extensively studied cytochromes P-
450 are the 3MC-inducible cytochromes P-448 and the PB-inducible
cytochromes P-450. These cytochromes constitute between 60 and 80% of the
total cytochrome content of treated animals, but often less than 5% of the
total in untreated animals (Ryan et al., 1982; Ioannides and Parke, 1987).
In contrast, cytochrome P-450s a and pen appear to be constitutively
expressed in untreated animals (Elshourbagy and Guzelian, 1980; Ryan et
al. , 1982; Nebert and Gonzalez, 1987). The different substrate
specificities of cytochromes P-448 and P-450 are due to fundamental
differences in the substrate binding sites (Lewis et al., 1986; Ioannides
and Parke, 1987). Thus, cytochromes P-448 accept large, relatively
lipophilic molecules, with small depths and large area/depth ratios,
whereas cytochromes P-450 accept smaller, more hydrophilic molecules, with
small area/depth ratios. For this reason, cytochromes P-448 metabolise
large planar aromatic compounds to reactive intermediates and are,
23
Table 1.2
A comparison of some of the characteristics of rat hepatic microsomal
cytochrome P-450 isoenzymes.
* Nomenclature as used by Nebert and Gonzalez, 1987. see references for original nomenclature.
f Metabolism by P-450d is considerably less than that with P-450c
| Metabolism by P-450e is considerably less than that with P-450b
References
1. Ryan et al., 19792. Ryan et al., 19823. Rodrigues et al., 19874. Guengerich et al., 19825. Goldstein et al., 19826. Koop et al., 19827. Tamburini et al., 19848. Elshourbagy and Guzelian, 19809. Graves et al., 1987
24
CytochromeP-450
isoenzyme*
Modelinducers
MonomericMolecularweight
reduced-CO
Amax(nm)
Characteristic I substrate/ activities
References
P-450a untreated/ Aroclor 1254/ Phenobarbital
48000 452 Testosterone 7a- hydroxylation
1.2
P-450b Phenobarbi tal 52000 450 7-pentoxyresorufin/ Benzphetamine Dealkylations Testorerone 16a- hydroxylation
1.2
3
P-450c 3-methylcholanthrene/ /9-naphthof lavone
56000 447 Ethoxyresorufin O-deethylation
Testosterone 6/3-/ Benzo[a]pyrene hydroxylations
1.2
4
P-450d Isosafrole/3-me thylcholanthrene
52000 447 EthoxyresorufinO-Deethylase^
25
P-450e Phenobarbital 52500 450.6 BenzphetamineN-demethylation^
2
P-450J Ethanol 51000 452 Aniline hydroxylation Alcohol oxidation
6
P-450LAU Clofibrate 51500 452 Laurie acid 12- hydroxylation
7
p-450pcn Pregnenalone 16a-carbonitrile/Dexamethasone
51000 450 Ethylmorphine N-demethylation
warfarin hydroxylation
89
25
therefore, associated with carcinogenicity and toxicity. Cytochromes P-
450, on the other hand, are associated with the metabolism of compounds to
more polar, biologically-inactive metabolites, and, hence, with conjugation
and detoxication. These aspects of cytochromes P-448 and P-450 are
discussed fully in Lewis et al., (1986), Ioannides and Parke, (1987), and
Parke and Obreska-Parke, (1987).
26
1.2 Mixed-function oxidase systems of aquatic vertebrate andinvertebrate species.
Table 1.3 compares cytochrome P-450 contents and NADPH- cytochrome c
(P-450) reductase activities (N.B. NADPH-cytochrome c reductase activity
is the catalytic activity used to assay for cytochrome P-450 reductase -
Masters and Okita, 1982) of aquatic vertebrate and invertebrate species
with those of mammalian, avian and other species. That there is wide
variation in both cytochrome P-450 microsomal specific content and
reductase activity, even between species of the same phylum, is apparent.
The MFO systems of aquatic, poikilothermic species are fundamentally
similar to those of mammalian species in a number of respects, vis. they
are membrane-bound in the endoplasmic reticulum of mainly the liver
(vertebrates) or hepatopancreas (invertebrates) (Stegeman, 1981) and have
similar physicochemical properties; the cytochrome b^ system may be
important in providing the second electron, at least in some species (Klotz
et al., 1986); and they catalyse essentially the same monooxygenation
reactions (Pohl et al., 1974; James et al., 1979; Noshiro and Omura,
1984). However, in at least two respects, the MFO systems of aquatic
species are distinct from those of mammalian species. Firstly, due to the
poikilothermic nature of fish, amphibia and marine invertebrates, the MFO
systems often function optimally at lower temperatures (Stegeman, 1979;
Andersson and koivusaari, 1986). Further, due to seasonal changes in
environmental water-temperature and/or endocrine factors, both components
and activities of aquatic MFO systems can show marked seasonal variations
(Chambers and Yarbrough, 1976; Stegeman and Kaplan, 1981; Walton et al.,
1983; Bihari et al., 1984; Miura, 1985). Secondly, although cytochrome P-
27
Table 1.3
Comparison of cytochrome P-450 contents and NADPH-cytochrome c (P-450)
reductase activities in various tissues of different species.
Note Contents and activities were determined at optimal temperatures for
each species - see particular reference for details.
References
1. Cheng and Schenkman, 1982;2. Orton and Parker, 19823. Guengerich, 19774. Souhaili-el Amri et al., 19865. Dalvi et al., 19876. Noshiro and Omura, 19847. Monod et al., 19878. Anderson et al., 19859. Maemura and Omura, 198310 Stegeman and Binder, 197911. James et al., 197912. James and Bend, 198013. Elmamlouk et al., 197414. Stegeman and Kaplan, 198115. Batel et al., 198616. Stanton et al., 197817. Wilkinson, 197918. Lee et al., 198119. Azari and Wiseman, 1982a20. Milligan et al., 1986
28
Species Tissues Cytochrome P-450 content nmoles
mg protein
NADPH-cyt.c.(P450)reductase activity
nmoles min ^-1mg protein
Reference
MammalsRat Liver 0.81 204-3982 1.2Rabbit Lung 0.27 114 3Human (male) Liver 0.31±0.09 99.8±14.4 4BirdsDuck Liver 0.18±002 5Turkey Liver 0.36±0.03 - 5AmphibiaRana catesbeiana Liver 0.43 74 6Rana nigromaculata Liver 0.77 116 6Xenopus laevis Liver 0.10 82 6FishRoach (Rutilus rutilus) Liver 0.36 30 7Nase (Condrostoma nasus) Liver 0.58 40 7Trout (Salmo gairdneri) Liver 0.19±0.05 15.2±1.7 8Goldfish (Carassius auratus) Liver 0.18±0.01 112±26 9Scup (Stenatomus versicolor) Liver 0.62±0.08 107±5 10Mullet (Mugil cephalus) Liver 0.47±0.31 51 - 55.3 11Stingray (Dasyatis sabina) Liver 0.50±0.18 49.3±20.1 11Skate (Raja erlnacea) Liver 0.32±0.08 60 12CrustaceaLobster (Panulirus argus) Hepatopancreas 0.91±0.39 4.3±2.7 11Lobster (Homarus americanus) Hepatopancreas 0.04±0.13 - 13Barnacle (Balanus ebumeus) Hepatopancreas 0.11±0.01 28.6±10 14Blue Crab (Callinectes sapidis) Hepatopancreas 0.18±0.08 5.2±4.8 11Spiny crab (Maja crispata) Hepatopancreas 0.47±0.06 12.25±2.99 15InsectsHouse fly (Musca domestica) Whole animal 0.1B±0.005 - 16Armyworm (Spodoptera eridanis) Midgut 1.37 - 17OtherSandworm (Nereis virens) Intestine 0.089±0.024 8.8±2.0 18Yeast (Saccharomyces cerevisiae) Whole organism 0.036 8.3 19Earthworm (Dendrobaena veneta) Intestine 0.164 - 20
29
450 isoenzymes are present in both fish (Klotz et al., 1983, 1986; Williams
and Buhler, 1983, 1984; Goksoyr, 1985) and crabs (Quattrochi and Lee,
1984), there are differences in their inducibility by xenobiotics. For
example, there is little or no indication of induction by phenobarbital or
by PB-type inducers, such as l,l-di(p-chlorophenyl)-2,2,2-trichloroethane
(DDT), in either fish, amphibia or crustacea (Addison et al., 1977; Elcombe
and Lech, 1979; Harri, 1980; Schwen and Mannering, 1982a; Batel et al.,
1988). In contrast, however, 3MC-type induction does occur in fish and
amphibia (Stegeman, 1981; Williams and Buhler, 1983, 1984; George and
Young, 1986; Stegeman and Kloepper-Sams, 1987) and is indicated for certain
crustacea (Batel et al., 1988). In particular, in fish, the major (though
not all) induced isoenzymes of different species appear similar in terms of
physicochemical properties, catalytic activity and immunocrossreactivity
(Stegeman et al., 1981; klotz et al., 1983; Williams and Buhler, 1983,
1984; Goksoyr, 1985; Stegeman and Kloepper-Sams, 1987). As in mammals,
induction in fish by 3MC-type xenobiotics appears to be by de novo
synthesis of cytochrome P-450 (Klienow et al., 1987). Aspects of aquatic
vertebrate and invertebrate MFO systems are discussed in more detail in
subsequent sections, in relation to observations made with the common
mussel, Mytilus edulis L.
30
1.3 The common mussel Mytilus edulis - general
The common mussel, Mytilus edulis L. , a bivalve mollusc, is a
sedentary filter-feeder of ubiquitous, global distribution. It attains a
relatively large size (upto approximately 10 cm in length), particularly in
warmer waters, and is frequently the dominant organism of intertidal and
sublittoral ecosystems of coastal and estuarine habitats (Yonge, 1976;
Krieger et al., 1981). Mussels live on a variety of substrata such as
rock, stones, shingle or sediment. The intertidal mussels, experience
marked diurnal and seasonal changes in, for example, water- and air-
temperature, salinity, oxygen availability and humidity. A photograph and
annotated diagram showing the major tissues and their organization in M.
edulis are given in Figure 1.6.
The major tissue with respect to the mussel MFO system is the
digestive gland (Livingstone and Farrar, 1984; also see next section),
lying centrally and dorsally near the mouth of the digestive system and the
cerebral ganglion. The digestive gland has also been, questionably, called
the hepatopancreas, but neither a hepatic nor a pancreatic function is
obviously evident (see below) (Viarengo et al., 1986). The tissue, as the
name suggests, is part of the molluscan digestive system. It contains
several cell types, and is rich in lysosomes involved in intracellular
digestion and sequestration of heavy metals and organic xenobiotics (Owen,
1972; Moore, 1985). However, very little is known of the biochemical or
physiological functions of the tissue other than digestion and storage.
The digestive gland, and other tissues such as the mantle, are
relatively rich in lipids (Gabbot, 1983), and the consequence of this and
31
mantleedge
digestivegland
posterioradductormuscle
shell
mantle
pedalganglion
i c w i i c i i l i n n u s t v i e
rectum intestine \
cerebrovisceral connective
recurrent limb C*W,Q pericardiumventricle
auricle
anus
posterior adductor muscle
visceral ganglion
pallial nerve
digestive gland
intestine
stomach
inner demibranch
outer demibranch
byssus
oesophagus
\ cerebral ganglion
m outh
anterior adductor muscle
caecum
Figure 1.6 The common mussel Mytilus edulis
32
the mode of feeding of M. edulis is that lipophilic xenobiotics from the
water-column are readily concentrated in these tissues (Geyer et al.,
1982). This aspect, together with other features, such as the size and
occurrance of M. edulis in large, accessable populations and its ability to
tolerate a wide range of environmental conditions (Krieger et al., 1981),
has lead to its use as a sentinel organism for monitoring environmental
pollution in such studies as "Earthwatch" (Jensen et al., 1975) and "Mussel
watch" (Goldberg et al., 1978), the concentration of xenobiotics in its
tissues being much greater than in the surrounding water-column.
33
1.4 Cytochrome P-450 and the MFO system in M. edulis and other molluscs
Knowledge of molluscan biochemistry, particularly intermediary
metabolism, is extensive and shows both basic similarities and fundamental
differences with the biochemistry of other invertebrate and vertebrate
groups (Hochachka et al., 1983; Livingstone, 1983; Livingstone et al.,
1983). In contrast, information on endogenous and xenobiotic oxidative
metabolism, particularly of cytochrome P-450 and the MFO system, is
considerably more limited.
A number of early studies failed to demonstrate any xenobiotic
metabolic activity in bivalve molluscs. The clams Mercenaria mercenaria
(Carlson, 1972) and Mya arenaria (Vandermeulen and Penrose, 1978), the
oyster, Ostrea edulis (Vandermeulen and Penrose, 1978) and the mussel M.
edulis (Lee et al., 1972; Payne, 1977; Vandermeulen and Penrose, 1978;
Payne and May, 1979) were indicated to lack either aryl hydrocarbon
hydroxylase activity or N- or 0- dealkylase activity. Thus, it was
suggested that bivalve molluscs lacked the enzyme mechanisms for
metabolising xenobiotics and that this was, in part, the reason for the
long-term persistence of, for example, hydrocarbons in their tissues (Lee
et al., 1972; Payne, 1977). In contrast to these observations, however,
were the results of a number of other early studies which did indicate the
presence of an MFO and other xenobiotic detoxication systems in bivalves.
The reported in vitro, activities included the epoxidation of aldrin to
dieldrin by the freshwater bivalves Anodonta sp. (Khan et al. , 1972), the
californian mussel, Mytilus califomianus (Gee et al., 1979; Krieger et
al., 1979) and M. edulis (Bayne et al., 1979; Moore et al., 1980), biphenyl
34
hydroxylation by M. edulis (Willis and Addison, 1974) p-nitroanisole 0-
demethylation (Trautman et al., 1979) and antipyrine hydroxylation (Krieger
et al., 1979) by M. califomianus, and benzo[a]pyrene hydroxylation by both
the oyster Crassostrea virginica (Anderson, 1978) and M. edulis (Stegeman,
1980, 1981; Mix et al., 1981; Payne et al., 1983). Further indication of
the existence of a bivalve MFO system came from the demonstration of
epoxide hydratase (EC 4.2.1 64) in the digestive gland of M. edulis (Bend
et al., 1977), mutagenicity studies (Parry et al., 1981; Dixon et al.,
1985), the in vivo transformation of some xenobiotics such as antipyrine
(Krieger et al., 1979), and the apparent inducibility of the MFO-
associated, cytochemically-measured, NADPH-neotetrazolium reductase
activity (another measure of cytochrome P-450 reductase) (Moore et al.,
1980). Today there is unequivocal evidence for the existence of bivalve
cytochrome P-450 and other components of an MFO system (see below) .
Cytochromes P-450 and b^, and NADH- and NADPH- dependent reductase
activities were first demonstrated in Mytilus sp. in Mytilus
galloprovincialis in 1982 (Ade et al., 1982) and in M. edulis in 1984
(Livingstone and Farrar, 1984). Tissue and subcellular characterization
studies of M. edulis demonstrated that, in common with other phyla, the
putative cytochrome P-450 monooxygenase system is primarily membrane-bound
in the endoplasmic reticulum (microsomes) and localized predominantly in
the digestive gland (Livingstone and Farrar, 1984). Cytochrome P-450 is
also present in the gills (Stegeman, 1985), whereas cytochrome b5, the
NADPH- and NADH- dependent reductase activities and benzo[a]pyrene
hydroxylase (BPH) activity have a wider tissue distribution, occurring also
in both the gills and mantle (reproductive) tissue (Livingstone and Farrar,
1984). Reductase and BPH activities, but not cytochromes P-450 or b^, have
been detected in blood cells (Livingstone and Farrar, 1984; Stegeman,
1985).
MFO components and BPH activity have been indicated to vary seasonally
in both M. edulis (digestive gland) (Livingstone, 1985, 1987; Stegeman,
1985) and M. galloprovincialis (whole tissues) (Suteau et al. , 1985;
Suteau, 1986). Variously, maximum specific contents, activities or
monooxygenase activities of the MFO system have been indicated to reach a
maximum over the summer or autumn, and a minimum over the colder, winter
months (Livingstone, 1985; Suteau et al., 1985), the seasonal changes
possibly being related to changes in water-temperature or mussel
reproductive condition (Livingstone, 1985; Stegeman, 1985; Suteau et al.,
1985). Sex differences in the MFO system have also been observed for M.
edulis and M. galloprovincialis. On occasions during the-year, BPH
activities were higher in female than male mussels, in digestive gland
microsomes of M. edulis (Livingstone, 1985) and whole body microsomes of M.
galloprovincialis (Suteau et al., 1985). Smaller sex differences have been
observed in the levels of MFO components, for example, cytochromes P-450
and b^ were higher in female than male mussels (Suteau et al., 1985;
Livingstone, 1987). A comparison of digestive gland microsomal BPH
activity, cytochromes P-450 and b^ contents and NADPH- cytochrome c
reductase activity in different species of mollusc is shown in Table 1.4.
The levels of the different parameters are considerably lower than those
seen in, for example, vertebrate species (e.g. compare values for
cytochrome P-450 content and NADPH- cytochrome c reductase activity with
corresponding values for vertebrates in Table 1.3). However, in relation
to their own metabolism, it has been argued that the in vitro activities
and contents are those to be expected i.e. the proportional difference in
Table 1.4
Digestive gland microsomal cytochrome P-450 and associated MFO components
and activities in mollusc species.
The data are for animals from the field, from both clean and polluted
sites.
Mean values are quoted and the ranges are different populations or times of
year.
* present but measured in arbitrary fluorescence units"If including ovotestis and based on a microsomal protein yield of 5 mg g
fresh weight - not measured.
References1. Livingstone and Farrar (1984)2. Livingstone (1985)3. Livingstone (1987)4. Stegeman (1985)5. Suteau (1986)6. Gilewicz et al., (1984)7. Koivusaari et al., (1980)8. Moore et al., (1987)9. Wilbrink (pers. comm)10. Schlenk and Buhler (1988)
37
Species cytochromeP-450pmoles mg ^
cytochrome
\ -ipmoles mg
NADPH-cyt.creductasenmoles min 1 -1mg
Benzo[a]pyrenehydroxylasepmoles min ^
-1mg
Reference
Mussel (Mytilus edulis) 47-134 26-76 10.3-15.4 3-25 1,2,3
Mussel (Mytilus edulis) 101 37 8.1 35 4
Mussel (Mytilus galloprovincialis) 47-52 205 11.8-21.8 24-28 5
Mussel (M. galloprovincialis) 3-88 75-400 - - 6
Mussel (Area zebra) 106 76 8.3 - 4
Mussel (Anodonta cygnea) 100 - - - 7
Clam (Macrocalista maculata) 79 81 4.0 - 4
Cockle (Cardium edule) 20-85 80-94 5.3-10.6 6-10 8
Periwinkle (Littorina littorea) 8-54 26-70 7.5-11.2 * 3
Snail (Lymnaea stagnalis) 40* - - - 9
Chiton (Cryptochiton stelleri) 110 - 5.5 26 10
38
enzyme activity between M. edulis and vertebrates, such as rat, is the same
for most enzyme activities measured, including, for example, key regulatory
enzymes of intermediary metabolism (Livingstone and Farrar, 1984).
A number of in vitro xenobiotic oxidative activities have been
detected in the digestive gland of Mytilus sp., vis. biphenyl hydroxylase
in M. edulis (Willis and Addison, 1974), N,N-dimethylaniline N-demethylase
in M. galloprovincialis (Ade et al., 1982) and M. edulis (Kurelec, 1985;
Livingstone et al. , 1988a); aminopyrine N-demethylase in M.
galloprovincialis (Gilewicz et al., 1984), and aldrin epoxidase, antipyrine
hydroxylase, p-nitroanisole O-demethylase and p-chloro-N-methylaniline N-
demethylase in M. califomianus (Krieger et al., 1979, 1981; Trautman et
al., 1979). In addition, 7-ethoxyresorufin O-deethylase activity has been
observed in M. edulis, but this was only occasionally (Stegeman, 1985). The
most studied putative monooxygenase activity however, has been digestive
gland microsomal BPH activity of M.edulis measured by radiometric and
fluorometric methods and by h.p.I.e. metabolite analysis (Livingstone 1984,
1985; Livingstone and Farrar 1984; Livingstone et al., 1985; Stegeman,
1985; Livingstone et al., 1988a; Moore et al., 1988). In contrast to the
absolute requirement for NADPH seen in mammals, surprisingly, an NADPH-
independent BPH activity has been demonstrated in digestive gland
microsomes of M. edulis (Livingstone, 1985; Stegeman, 1985; Moore et al.,
1988) and whole body microsomes of M. galloprovincialis (Suteau et al.,
1985; Suteau, 1986). N,N-Dimethylaniline N-demethylase activity
(formaldehyde formation) was also found to be NADPH-independent in M.
edulis digestive gland microsomes (Livingstone et al., 1988a). The in
vitro benzo[a]pyrene metabolite profiles of M. edulis also differ from
those produced by other species, in either the presence or absence of
39
NADPH, in that the major metabolites formed are quinones rather than
phenols or dihydrodiols (Stegeman, 1985; Livingstone et al., 1988a). This,
and other observations, has lead to suggestions that more than one process
may be involved in the metabolism of benzo[a]pyrene in vitro by digestive
gland microsomes of M. edulis (Stegeman, 1985; Livingstone, 1988; Moore et
al., 1988). Despite the observations on in vitro xenobiotic metabolism,
little is known of the in vivo function of the MFO system in M. edulis or
other molluscs (Livingstone, 1985, 1988). The sex and seasonal variations
have been suggested to indicate an endogenous function (Livingstone and
Farrar, 1984), but studies performed on this aspect are few, for example,
the demonstration in M. edulis tissue homogenates of arachidonic acid
metabolism to prostaglandins (Srivastava and Mustafa, 1985) and vitamin D^
metabolism to unknown polar metabolites (Lehtovaara and Koskinen, 1986).
A number of studies have been performed in which Mytilus sp. have been
exposed to xenobiotics. In M. galloprovincialis, both digestive gland
microsomal cytochrome P-450 and cytochrome b^ specific contents increased
in response to laboratory exposures to paraffin, anthracene, perylene and
3MC, and in response to field exposures to hydrocarbons (Gilewicz et al.,
1984). In digestive gland microsomes of M. edulis, the specific contents
of cytochromes P-450 and b^, and the specific activities of NADPH- and
NADH- dependent reductases, (but not fluorometric BPH activity), increased
in response to both short-term and long-term experimental mesocosm-
exposures to polycyclic aromatic hydrocarbons (Livingstone et al., 1985;
Livingstone, 1987, 1988). Following long-term recovery, the contents or
activities of the MFO components of M. edulis returned to control levels
(Livingstone et al., 1985, Livingstone, 1987). In addition to those
changes, a 2-3 nm blue shift in wavelength maximum of the carbon monoxide-
40
difference spectrum of digestive gland microsomal cytochrome P-450 was also
observed in hydrocarbon-exposed mussels, possibly suggesting the synthesis
of new forms of cytochrome P-450 (see above references). The observed
induction responses in M. edulis were seasonally-variable, with little or
no response to polycyclic aromatic hydrocarbons being seen when the mussels
were ripe with gametes (Livingstone, 1987). Hydrocarbon exposure
experiments have also been carried out on other molluscs with increases
generally being seen more often in MFO component levels (cytochromes P-450
and b^, NADPH-cytochrome c reductase activity) than in BPH activity, vis.
in the periwinkle, Littorina littorea (Livingstone et al. , 1985;
Livingstone, 1987, 1988), the cockle, Cerastoderma (Cardium) edule
(Livingstone and Farrar, 1985; Moore et al. , 1987), the whelk, Thais
haemastoma (Livingstone et al., 1986), the oyster Crassostrea virginica
(Anderson, 1978, 1985) and the chiton, Cryptochiton stelleri (Schlenk and
Buhler, 1988). Considerable variability in responses were observed,
however, and the "induction picture" is far from clear (see Section 6).
41
1.5 Alms of the project
The main objective of the study was to investigate the nature and
functioning of cytochrome P-450 and the MFO system of the digestive gland
microsomes of M. edulis, principally by purification of cytochrome P-450,
and a study of its properties in control and xenobiotic-exposed mussels.
The reasons for this were, essentially, three-fold:
(1) Comparative /phylogenetic An understanding of the molluscan MFO
system, for example its properties, activities and inducibility, would
be of interest from both a comparative and an evolutionary viewpoint.
(2) Biological effects monitoring It is possible that changes in MFO
components or activities, in response to organic pollutants, could be
used in pollution monitoring as a specific indicator of biological
impact for this group of chemicals. Such an application has been
proposed for mussels (Bayne et al., 1985, 1988; Livingstone, 1988),
and is, at present, being used in fish, in the form of cytochrome P-
450 specific content, antibodies for cytochrome P-450E (3MC-
inducible), BPH and 7-ethoxyresorufin O-deethylase activities
(Stegeman et al., 1988).
(3) Fate of xenobiotics An understanding of the in vivo function of the
molluscan MFO system would enable a better understanding of the fate
of xenobiotics taken up by molluscs. This would be important in two
respects. Firstly, it may help explain, in part, the incidence of
neoplasms in molluscs (Mix, 1986), and secondly, since molluscs are a
source of food to humans, it may be of benefit in assessing the
possible hazards for consumption.
The thesis is divided into six additional sections. The first
42
investigates and establishes the seasonal pattern of variation of the
digestive gland microsomal MFO system principally as a prelude for the
purification of cytochrome P-450. The results are compared with changes in
environmental water-temperature and mussel reproductive condition (measured
quantitatively by stereology) and discussed in relation to information
available for mussels and other marine species. Section 3 describes the
partial purification of digestive gland microsomal cytochrome P-450, by
ammonium sulphate or polyethylene glycol protein fractionation, followed by
various affinity and ion-exchange chromatographic methods. Cytochrome P-
450 being membrane-bound, initially requires solubilization from the
microsomes, and the optimization experiments for this and the other
purification steps are described. Limited properties of the final
partially-pure cytochrome P-450 preparation were determined, and these and
the purification procedures are compared with results for other species.
The possibility of the existence of cytochrome P-450 isoenzymes in M.
edulis is discussed. Section 4 describes substrate/inhibitor-ligand binding
spectra with partially-purified digestive gland microsomal cytochrome P-
450. The main objective of the study was to investigate the nature of the
compounds that might possibly be substrates for the mussel cytochrome P-450
enzyme. The results are discussed in relation to known substrates of
mammalian and other cytochromes P-450. Section 5 investigates the putative
MFO activities of mussel digestive gland microsomes. The ability of
microsomes to metabolize a range of xenobiotic and endogenous substrates
was examined. The effect of various cofactors and modulators of cytochrome
P-450 activity were investigated, and the results compared to those of
other organisms and of known mammalian cytochrome P-450 isoenzymes. The
results are also discussed in relation to possible catalytic mechanisms of
molluscan cytochrome P-450. Section 6 describes xenobiotic-exposure
43
experiments performed with M. edulis. Mussels were exposed, short-term and
long-term to a number of known inducers of the mammalian MFO system, to
examine their effect on digestive gland microsomal MFO components and
activities. The results are discussed in terms of the type of cytochrome
P-450 which may be present, and in terms of the potential for using the MFO
system in biological effects monitoring. The final section, section 7,
summarizes the major findings and conclusions of the research. The
evidence for cytochrome P-450 isoenzymes and other aspects is discussed
and, where possible, related to findings for other species.
44
Section 2
Seasonal variation of the mussel digestive gland mixed-function oxidase system and related activities.
45
2.1 Introduction
Seasonal variation in the common mussel, M. edulis, has been observed
for most major areas of metabolism (see Section 1). The mussel MFO system
is primarily localized in the digestive gland (hepatopancreas) (Livingstone
and Farrar, 1984), but relatively little information is available on its
seasonality in this or any other tissue.
One of the earliest indications of mussel MFO seasonality was the
observation of seasonal variation in the ability of total tissue microsomes
of M. edulis to activate chemical extracts from its tissues to mutagens
(Parry et al., 1981). Activation of these extracts was greater with summer
microsomes than winter microsomes. More recently, seasonal variation in
the activity of benzo[a]pyrene hydroxylase (BPH) in digestive gland
microsomes of M. edulis has been demonstrated (Livingstone, 1985). Maximal
activity was indicated for late summer/early autumn, followed by a decline
in late winter/early spring. It was suggested the decline might be
associated with the approach of spawning. Although a detailed study of
sex-related differences was not performed, in November, female mussels had
higher BPH activity than males. Similar results have been obtained for the
closely related species Mytilus galloprovincialis (Suteau et al., 1985).
BPH activity in whole body microsomes was highest during the late summer
(period of gametogenesis), compared to the winter and spawning period.
Seasonal variations in BPH activity, and also in whole body microsomal
cytochrome P-450 content, were observed to be correlated with seasonal
variations in mussel total lipids and the environmental water temperature.
Females were indicated to have a higher BPH activity than males at certain
times of the year. More recently seasonal differences have been observed
in the microsomal specific contents and activities of MFO components of the
digestive gland of both M. edulis and the gastropod Littorina littorea L.
(Livingstone, 1987). The patterns of change in the mussel, for both
cytochrome P-450 content and NADH- and NADPH-dependent cytochrome c
reductase activities, were indicated to be similar to that for BPH
activity, with minimum values around June, corresponding to the period of
spawning. Mussels were only sexed at one time of the year but the levels
of MFO components were indicated to be higher in females than males.
In contrast to molluscs, a number of studies have been carried out on
the seasonal variation of MFO systems in other poikilothermic aquatic
species. Examples include fish eg. Salmo gairdneri (Forlin et al., 1984),
Coregonus albula (Lindstrom-Seppa, 1985), Tautogolabrus adspersus (Walton
et al., 1983); crabs e.g. Maja crispata (Bihari et al., 1984), Carcinus
maenas (O'Hara et al., 1982); the barnacle Balanus ebumeus (Stegeman and
Kaplan, 1981) and frogs e.g. Rana temporaria (Harri, 1980) and Rana
catesbeiana (Miura et al., 1986).
The aim of the study presented in this section was to comprehensively
describe the seasonal variation of cytochrome P-450 and other components
and activities of the MFO system of the digestive gland microsomal fraction
of M. edulis. Samples were taken every six to eight weeks from a rocky-
shore population located in a relatively clean environment. Male and
female mussels were examined separately. Measurements were made on
cytochromes P-450 and b , NADPH- and NADH-dependent cytochrome c reductase
activities, NADH-dependent ferricyanide reductase activity, NADPH-
independent 7- ethoxycoumarin O-deethylase (ECOD) activity, BPH activity
and microsomal proteins (by SDS-gel electrophoresis). Water temperature
was recorded and the mussel reproductive stage quantified stereologically
47
by the methods described in Lowe et al. (1982). The results are discussed
in relation to known seasonal metabolic changes in M. edulis and in
relation to the findings for species from other phyla. The implications of
the seasonal variations in M. edulis for the purification of cytochrome P-
450 and the other MFO components are considered.
48
Materials and Methods
Collection of M. edulis from Whits and Bay, Cornwall, and preparation of tissue pools.
Mussels were obtained at low tide at six to eight week intervals, from
7th April 1986 to 30th March 1987, from a stable, open coast population at
Sharrow Point, Whitsand Bay, Cornwall (O.S. 393521). On each occasion,
approximately 150 mussels (4.5 - 5.5 cm in length) were collected and
removed to a system of ambient recirculating seawater at PML, Plymouth.
Here, the animals were kept without food for two to three days to clear the
gut contents prior to dissection (for details of seawater-system, see Bayne
and Thompson, 1970).
Individual mussels were sexed and the digestive glands dissected out
and damp-dried on filter-paper. After removal of the crystalline style,
the tissue was quickly frozen in liquid nitrogen as either 'male' or
'female'. Sexing was achieved by examining a smear of mantle
(reproductive) tissue under a light-microscope for the presence of sperm or
oocytes. Once the digestive glands of 48 male and 48 female mussels were
obtained, each tissue pool was removed from the liquid nitrogen and stored
at -70°C until required. The digestive glands from any remaining unsexed
mussels were also excised, frozen and stored at -70°C for possible later
complementary investigations after the main study was completed. On two
occasions, sexing of the mussels was not possible by light-microscopy
because of the stage of gametogenesis. On these occasions the digestive
glands from 48 mussels only were dissected out. These were termed
'indeterminate'. Immediately prior to preparation of the microsomal
2.2
2.2.1
49
fraction (Section 2.2.3), each tissue pool was divided into six replicate
samples of eight digestive glands each.
On each sampling occasion, the mantle tissues of 10 male and 10 female
mussels were also dissected out. These were used for subsequent
stereological quantitative analysis of the reproductive stage of the
mussels (Section 2.2.6). The freshly excised tissues were immersed in the
fixative, Baker's Formol Calcium (2.0% sodium acetate, 10% formalin, 2.5%
sodium chloride) and stored at 4°C. On the occasions the mussels could not
be sexed, 25 'indeterminate' mantle tissues were excised and fixed. Of
these 25 tissues, at least 10 of each sex would later be distinguishable
during the reproductive stage analysis.
2.2.2 Chemicals
Trizma base for Tris(hydroxymethyl)aminomethane - HCL (Tris-HCl)
buffers, ethylenediaminetetracetic acid (EDTA), dithiothreitol (DTT),
bovine serum albumin (BSA), {5-nicotinamide adenine dinucleotide, reduced
form (NADH), £-nicotinamide adenine dinucleotide phosphate, reduced form
(NADPH), cytochrome c (horse heart), 7-ethoxycoumarin, 7-hydroxycoumarin,
sodium dodecyl sulphate (SDS), tetramethylethylenediamine (TEMED),
acrylamide, N'N-methylene bis-acrylamide, glycine, coumassie blue G-250 and
standard proteins for SDS-gel electrophoresis were obtained from Sigma
Chemical Co. Ltd. (Poole, Dorset, UK). Glycerol was obtained from May &
Baker Ltd. (Dagenham, Essex, UK). Benzo[a]pyrene, bromophenol blue and 2-
mercaptoethanol were obtained from Aldrich Chemical Co. Ltd. (Gillingham,3 -1Dorset, UK). [G- H]-Benzo [a]pyrene (24 /iCi mmole ) was from Amersham
International PLC (Amersham, UK). Optiphase 'Safe' scintillant was from
LKB scintillation products (Loughborough, UK). Histosol and Paraplast wax
50
were from Shandon Southern products (London, UK). Potassium ferricyanide,
dimethylformamide (DMF), dimethylsulphoxide (DMSO), ammonium persulphate
and all other chemicals were from BDH Chemicals Ltd. (Poole, Dorset, UK)
and were of analar grade.
2.2.3 Preparation of digestive gland microsomal fraction
Mussel digestive gland microsomes were prepared at 4°C by a method
essentially similar to that of Livingstone and Farrar (1984). Each frozen
sample of eight digestive glands was first weighed and then homogenized in
a 1:4 w/v ratio (tissue weightibuffer volume) in 20 mM Tris-HCl pH 7.6
containing 0.5 M sucrose, 0.15 M potassium chloride, 1 mM EDTA and 1 mM
DTT. Gentle homogenization was performed using an electrically driven,
variable-speed Potter-Elvehjem teflon homogenizer. Sufficient passes were
made to remove any visible particulate tissue material. The homogenate was
centrifuged in an M.S.E. Hi-Spin 21 centrifuge at 500 x g for 30 mins. and
the resulting supernatant at 12,000 x g for 45 mins. These spins removed
cell debris and large cellular organelles such as mitochondria. The 12,000
x g supernatant was then centrifuged in an M.S.E. Prepspin 50
ultracentrifuge at 100,000 x g for 90 mins., using a 6 x 16.5 ml swing-out
rotor. The resulting supernatant (cytosolic fraction) was discarded and
the microsomal pellet 'washed' by overlaying with a small volume of 20 mM
Tris-HCl pH 7.6 containing 20% w/v glycerol, 1 mM EDTA and 1 mM DTT and
spinning at 12,000 x g for 30 mins. The microsomal pellet was then
resuspended in this buffer to a protein concentration of 4 - 8 mg ml”
using a hand-held Potter-Elvehjem teflon homogenizer.
The resuspended microsomal fraction was divided into three aliquots.
51
One aliquot was used immediately for the determinations of cytochrome P-
450, cytochrome and total haemoprotein content. The remaining aliquots
were stored overnight at -70°C. One of these aliquots was used for
determination of BPH activity and the other for determinations of NADH- and
NADPH-dependent cytochrome c reductase activities, NADH-ferricyanide
reductase activity, NADPH-independent ECOD activity and total protein
content.
Remaining microsomal sample was stored at -70°C and later used in an
SDS-gel electrophoretic, seasonal comparison of microsomal proteins.
All procedures of sample collection, preparation, storage and enzyme
analysis were rigorously standardized to minimise experimental error.
2.2.4 Enzyme and protein assays
Measurement of cytochromes and all enzyme activities, except BPH and
ECOD, were carried out at 25°C on a Pye Unicam SP8-200 dual beam UV/VIS
spectrophotometer, fitted with a thermostated four-cell holder and a 10
cell preheater in series with a Grant cooler/heater to maintain an
incubation and assay temperature of ± 1°C. The spectrophotometer was
fitted with automatic baseline correction and recorder dampening
facilities.
Microsomes and substrates were kept on ice at 4°C until required for
use. All reagents were made up in double-distilled water unless stated
otherwise. Enzyme activities and cytochrome contents were shown to be
linearly-dependent on sample concentration over a 5-fold variation of the
latter. All assays were carried out in duplicate.
52
2.2.4.1 Cytochrome P-450 and *416f nm peak content
Cytochrome P-450 and '416' nm peak content in digestive gland
microsomes were determined using a modification of the dithionite-reduced
carbon monoxide difference spectrum method of Omura and Sato (1964). An
aliquot of resuspended microsomes (normally 2 0 0 fil), diluted to 2 ml with
50 mM Tris-HCl pH 7.6, was first divided equally into two semi-micro
cuvettes - the reference and sample beam cuvettes - to give a cuvette
protein concentration of about 0.5 - 1.0 mg ml"^. Carbon monoxide (CO) was
then bubbled through the sample beam cuvette at a rate of about one bubble
sec"^ for 40 seconds. A baseline was recorded over the range 400 - 500 nm.
Both cuvettes were then removed and a few grains of fresh sodium dithionite
added to each. A difference spectrum was then recorded over the range 400
- 500 nm. Scans were repeated over 5 - 1 0 mins until maximum peak sizes
were observed. A typical difference spectrum is shown in Figure 2.1.
The bubbling of CO through the sample cuvette, before addition of
dithionite, helped eliminate any spectral interference resulting from an
interaction between CO and 'oxidised' sample (Livingstone and Farrar,
1984); any spectral aberrations were largely removed by automatic baseline
correction and recorder dampening. For each sample, scans were made
without the recorder dampening facility to enable an accurate determination
of the wavelength Amax values for both cytochrome P-450 and the '416' nm
component.
Cytochrome P-450 content was calculated from the difference in
absorbance between 450 and 490 nm using an extinction coefficient of 91 mM"
cm"^ (Estabrook and Werringloer, 1978). The content of the '416' nm
component was calculated in arbitrary absorbance units from the difference
53
0 . 0 0 5 A
4 0 0 4 2 5 4 5 0 4 7 5 5 00
WAVELENGTH (nm)
Figure 2.1A typical dithionite-reduced carbon monoxide difference spectrum
showing the cytochrome P-450 peak at approximately 450 nm, and the low wavelength peak at about 416 nm. 0.5 - 1 mg digestive gland microsomal fraction was used. For other details, see text.
0 . 0 0 5 A
I ------------ 1----------- :--14 0 0 4 2 5 4 5 0
WAVELENGTH (nm)
Figure 2.2A typical cytochrome b^ difference spectrum. 0.5 - 1 mg digestive
gland microsomal fraction was used. For other details, see text.
54
in absorbance between the Am_„ at about 416 nm and 490 nm.n ia X
2.2.4.2 Total haemoprotein content
An estimation of total haemoprotein content was obtained from the
absorbance at 417 nm of 50 /il microsomal sample in 1 ml of 100 mM Tris-HCl,
pH 7.6 relative to a buffer blank. Haemoprotein content was calculated in-1 -1terms of arbitrary absorbance units ml microsomal sample or mg
microsomal protein. Measuring the absorbance at 417 nm is a general method
for haemoprotein determination and is described by a number of authors, eg.
Cheng and Schenkman (1982).
2.2.4.3 Cytochrome b content
Cytochrome b^ content was determined from the difference in absorbance
of the ferrous form of the haemoprotein relative to the ferric form
according to the method of Estabrook and Werringloer (1978).
Reference and sample semi-micro cuvettes were set up with the
reference cuvette containing 500 /il of 100 mM Tris-HCl pH 7.6, 400 /il of
water and 1 0 0 /il of microsomal sample, and the sample cuvette containing
500 /tl of 100 mM Tris-HCl pH 7.6, 350 /il of water and 100 /il of microsomal
sample. A baseline was recorded between 400 - 450 nm using the baseline
correction facility on the spectrophotometer. 50 /il of NADH (in 1% NaHCO^,
final concentration: 600 /zM) was then added to the sample cuvette and the
difference spectrum recorded. The spectrum was rescanned until no further
difference in absorbance between 426 and 409 nm was observed. A typical
difference spectrum is shown in Figure 2.2. Cytochrome b^ content was1 1determined using an extinction coefficient of 185 mM cm (426 - 409 nm)
(Estabrook and Werringloer, 1978). A correction was applied for the
absorbance of NADH at 409 nm.
2.2.4.4 NADPH- and NADH-dependent cytochrome c reductase activities
NADPH- and NADH-dependent cytochrome c reductase activities were
determined spectrophotometrically by measuring the increase in absorbance
at 550 nm, in a reaction mixture containing in a final volume of 1.0 ml :
50 mM Tris-HCl pH 7.6, 1 mM potassium cyanide, 30 /iM cytochrome c and 0.26
mM NADPH or NADH. 25 /il of microsomal sample (0.1 - 0.25 mg protein) was
first added to the pre-incubated reaction mixture lacking NAD(P)H and a
baseline recorded giving the background rate of reaction between sample and
cytochrome c. This rate was relatively low and linear. NADPH or NADH (in
1% NaHCO^) was then added and the reaction proper started. The initial
reaction was linear for the first 1 - 2 mins. From this rate was
subtracted the background rate and the previously determined non-enzymatic
rate of reaction between NAD(P)H and cytochrome c in the absence of
microsomal sample. The activities of NAD(P)H cytochrome c reductase were
determined from this net rate of reaction using an extinction coefficient
of 19.6 mM"'*" cm"'*'. (Shimakata et al., 1972).
2.2.4.5 NADH-dependent ferricyanide reductase activity
NADH-dependent ferricyanide reductase activity was determined
spectrophotometrically by measuring the decrease in absorbance at 420 nm in
a reaction mixture containing in a final volume of 1.0 ml : 50 mM Tris-HCl
pH 7.6, 1 mM potassium cyanide, 0.2 mM potassium ferricyanide and 0.26 mM
NADH. The methodology was essentially the same as that described for the
56
determination of cytochrome c reductase activities. 25 /il (0.1 - 0.25 mg
protein) of microsomal sample was used and reaction started by the addition
of NADH (in 1% NaHCO^). The initial rate of reaction was linear for the
first 1 - 2 mins. The net rate of NADH-supported reduction of ferricyanide
was determined after subtraction of the background (sample + ferricyanide)
and non-enzymatic, chemical (NADH + ferricyanide) rates. The activity of
NADH-dependent ferricyanide reductase was calculated from the net rate
using an extinction coefficient of 1.02 mM"^ cm"^. (Ichikawa et al.,
1969).
2.2.4.6 NADPH-independent 7-ethoxycoumarin O-deethylase activity
NADPH-independent 7-ethoxycoumarin O-deethylase (ECOD) activity (see
Section 5) was determined fluorometrically at 37°C using a Perkin-Elmer
3000 fluorescence spectrometer linked to a Perkin-Elmer 56 recorder, by a
modification of the method of Ullrich and Weber (1972). 7-Ethoxycoumarin
is O-deethylated to 7-hydroxycoumarin:
7-ethoxycoumarin 7-hydroxycoumarin
The rate of deethylation was determined from the rate of production of
7-hydroxycoumarin. This was measured fluorometrically with excitation at
370 nm and emission at 450 nm. The ECOD method used for M. edulis differed
from that of Ullrich and Weber (1972) in that no NADPH was required for
measurable activity. The reaction was started by the addition of sample
57
and was termed the NADPH-independent ECOD activity. The addition of even
low concentrations of NADPH substantially inhibited the activity (the I^q
was about 2-14 /tM; see Section 5).
The microsomal sample (200 /il; normally 1 - 2 mg protein) was added to
a pre-equilibrated reaction mixture, containing in a final volume of 2.5
ml: 54 mM Tris-HCl, pH 7.6 and 0.1 mM 7-ethoxycoumarin in DMF. After an
initial lag of 1 - 2 mins, a progressive, linear increase in fluorescence
was observed for between 5 and 15 mins. The rate of increase was taken as
a measure of 7-hydroxycoumarin production. For each sample, a standard
fluorescence change was determined from the mean increase in fluorescence
from successive 0.1 nmole additions of 7-hydroxycoumarin to the above
incubation mixture minus 7-ethoxycoumarin (7-ethoxycoumarin itself had no
effect on fluorescence changes due to 7-hydroxycoumarin addition). This
procedure corrected for quenching by the sample. The lowest detectable_ ifluorescence change was about 0.06 units min , corresponding to a mussel
ECOD specific activity of about 0.1 - 0.2 pmoles min"^ mg"^ microsomal
protein.
2.2.4.7 Benzo[a]pyrene hydroxylase activity
Benzo[a]pyrene hydroxylase was assayed by the radiometric method of
Van Cantfort et al. (1977), using the modifications described by
Livingstone and Farrar (1984). The method measures the total polar
metabolites of benzo[a]pyrene (essentially phenols, dihydrodiols and
quinones).
The microsomal sample (200 /tl; 1 - 2 mg protein) was incubated in a
reaction mixture containing in a final volume of 1.0 ml : 50 mM Tris-HCl pH
58
7.6, 0.2 mM NADPH and 0.06 mM [G-%]-benzo[a]pyrene (« 300 /iCi jumole" ).
Although 0.2 mM NADPH inhibits total metabolism of benzo[ajpyrene by about
50% in this assay (Livingstone, 1985; Moore et al., 1987), it was included
to standardize conditions for the seasonal survey.
Reaction was initiated by the addition of benzo[a]pyrene substrate in
DMF. 50 fil aliquots of the assay mixture were removed after 1 and 15 mins.
of incubation and the reaction stopped by transfer to tubes containing 1 . 0
ml of 0.15 M potassium hydroxide in 85% (v/v) DMSO. The stopped reaction
mixtures were extracted twice with 7.0 ml of analar-grade hexane and the
aqueous phase counted after addition of 700 /xl of 0.35 M HC1. 10 ml of LKB
Optiphase 'Safe' scintillant was used per scintillation vial and counting
was performed on a Beckman LS230 liquid scintillation counter. Aliquots of 3H-benzo[ajpyrene substrate were also counted in 10 ml of scintillant to
determine its specific radioactivity. Counting efficiencies were
determined by use of spiked internal standards and were about 39%. Counts
at time 1 min. were kept to a minimum by routine purification of the H-
benzo[a]pyrene substrate to remove polar material. This was achieved by
the method of Depierre et al. (1975). The toluene solvent from 100 /zl of
stock H-benzo[a]pyrene was evaporated under nitrogen and the residue
dissolved in 3 ml of hexane. The solution was extracted three times with 2
ml of 0.25 M NaOH in 40% ethanol to remove polar material. After removal3of the aqueous phase, the hexane layer containing purified H-
benzo[a]pyrene was blown-off under nitrogen and the residue redissolved in
'cold' benzo[a]pyrene (1.5 mM) in DMF.
Benzo[a]pyrene hydroxylase activity was linear over 15 mins. The
method was sensitive to rates of less than 1 pmole min"^ (equivalent to a
minimum specific activity detectable of about 0.4 pmoles min"'*' mg"'*').
59
2.2.4.8 Lowry determination of total microsomal protein
Microsomal protein was determined by the method of Lowry et al.
(1951). 50 /il aliquots of microsomal sample (approximately 4 - 8 mg
protein ml"^) were diluted x 50, x 75 and x 100 with distilled water.
Duplicate 0.5 ml aliquots of each dilution were transferred to clean tubes.
A protein standard curve over the concentration range 0 - 200 /ig proteini _ iml was prepared, to 0.5 ml final volumes, from a 1 mg ml stock solution
of bovine serum albumin. To both samples and protein standards was added
2.5 ml of a 1:1 (v/v) mixture of 1% copper sulphate and 2% sodium/potassium
tartrate, diluted x 50 with 2% sodium carbonate in 0.1 M sodium hydroxide.
Tubes were vortexed and left to stand at room temperature for 10 mins.
before addition of 0.25 ml of Forlin-Ciocalteau reagent (diluted 1:1 with
distilled water). Tubes were again vortexed and left to stand at room
temperature for 30 mins. before the absorbance was read
spectrophotometrically at 750 nm.
Microsomal protein content was determined, using the protein standard
curve, as the mean value of the different readings.
2.2.5 Sodium dodecyl sulphate polyacrylamide gel electrophoresis of
microsomal proteins
The method of Laemmli (1970) was used for molecular weight
distribution analysis of microsomal proteins. Microsomal samples were
solubilized with sodium dodecyl sulphate (SDS) in the presence of a thiol
reagent, 2 -mercaptoethanol, and the denatured proteins separated on
polyacrylamide using a discontinuous buffer system.
60
Solutions used were prepared as follows:
Gel stock : 30% (w/v) acrylamide containing
0.8% (w/v) N,N'-methylene bis-
acrylamide
Buffer 1 (separating gel buffer): 1.5 M Tris-HCl pH 8 . 8 containing
0.4% (w/v) SDS
Buffer 2 (stacking gel buffer) : 0.5 H Tris-HCl pH 6 . 8 containing
0.4% (w/v) SDS
Buffer 3 (electrode buffer) : 0.025 M Tris-HCl pH 8.3 containing
0.192 M glycine and 0.1% (w/v) SDS
Buffer 4 (sample buffer) : 0.125 M Tris-HCl pH 6 . 8 containing
15% glycerol, 10% 2-mercaptoethanol,
4.6% SDS and 0.002% bromophenol blue
A vertical slab gel apparatus made in the workshop of PML, Plymouth
to specifications described by Hanies (1981) was used. The gel was
contained in a cuvette comprising two glass plates held 1.5 mm apart by
perspex strips along the vertical and lower horizontal sides of the plates.
The glass plates were first washed sequentially in 50% nitric acid, 0.02%
Decon and tap water before being rinsed with distilled water and acetone
and allowed to dry. The cuvette was then assembled, as described above,
and sealed with 2% (w/v) molten agar. This was then clamped in a vertical
position. The lower (separating) gel was prepared, to a final acrylamide
concentration of 10%, by mixing buffer 1 (10 ml), gel stock (13.3 ml) and
water (16.6 ml). Polymerization was initiated by addition of 20 fil of
tetramethylethylenediamine (TEMED) and 240 /il of freshly prepared ammonium
persulphate (from a 100 mg ml"^ stock solution). The solution was poured
61
into the cuvette to a height of 80 mm. A layer of distilled water was
introduced above the gel mixture to ensure a flat interface between the gel
and the subsequently added stacking gel. When polymerization was complete,
the water layer was removed and the upper (stacking) gel added to the
cuvette above the separating gel. The stacking gel consisted of a mixture
of buffer 2 (2.5 ml), gel stock (1.0 ml) and water (6.5 ml).
Polymerization was initiated by the addition of 20 /zl of TEMED and 60 /zl of
ammonium persulphate. A perspex comb was immediately introduced into the
stacking gel to enable formation of sample wells upon polymerization. The
comb was removed when polymerization was complete and a small amount of
electrode buffer (buffer 3) introduced into the sample wells to prevent
them collapsing. The perspex strip along the lower horizontal side of the
cuvette was then removed and the cuvette placed in the electrophoresis
tank. Electrode buffer was added to the upper and lower reservoirs of the
tank and any air bubbles trapped underneath the gel removed with a syringe.
Microsomal samples were diluted with sample buffer (buffer 4) to a
final concentration of 1.3 mg protein ml"'*'. Samples and protein standards
were placed in a boiling water-bath for 5 mins. After cooling, 100 /zg of
solubilized, denatured protein and 25 /zg of mixed protein standard was
applied to the stacking gel using a syringe. Protein standards (each at 1
mg ml’ ) over the molecular weight range 12,400-92,500 were used. These
were: cytochrome c (horse heart, M.W. 12,400); trypsin inhibitor (soybean,
M.W. 20,100); chymotrypsinogen A (bovine pancreas, M.W. 25,700); lactate
dehydrogenase (pig heart, M.W. 29,000); ovalbumin (M.W. 43,000); fumarase
(pig liver, M.W. 48,500); glutamate dehydrogenase (bovine liver, M.W.
53.000); catalase (bovine liver, M.W. 57,500); bovine serum albumin (M.W.
68.000) and phosphorylase a (rabbit muscle, M.W. 92,500).
62
A constant current of 20 mA was run across the system until the
bromophenol blue dye front from the sample buffer, had entered the
separating gel. This current had the effect of moving the proteins through
the stacking gel and concentrating them at the interface of the gels. The
constant current was then increased to 40 mA until the dye front had moved
to within 5 mm of the end of the gel. The current was turned off and the
cuvette removed from the tank. The glass plates were separated, the
stacking gel removed and the gel placed overnight in staining solution
consisting of propan-2-ol: acetic acid: water (5:2:13 v/v/v) containing
0.05% (w/v) coumassie blue G-250. Destaining of the gel background was
performed by immersing the gel in several changes of propan-2 -ol: acetic
acid: water (1:1:8 v/v/v) over a period of 48 hours. Destained gels were
photographed and stored in 3% glycerol at 4°C.
Apparent molecular weights of the microsomal proteins were determined
from their electrophoretic mobilities relative to the mobilities of the
standard proteins. A standard curve was constructed from a plot of log^Q
(protein molecular weight) against electrophoretic mobility. The
electrophoretic mobility of the protein bands was calculated as:
distance moved by band ------------------------- x 1 0 0distance moved by dye front
2.2.6 Reproductive stage analysis
The reproductive stage of the mussels was determined stereologically
by the method described by Lowe et al. (1982). The mantle tissue of
63
mussels is composed predominantly of three cell-types which in various
combinations, depending on the time of year, account for around 90% of the
total mantle cell composition. These cells are the adipogranular (ADG)
cells (concerned with the storage of lipids, protein and some glycogen),
the vesicular connective tissue (VCT) cells (concerned with storage of
large reserves of highly labile glycogen), and the reproductive germinal
cells, (Lowe and Pipe, 1986). The method employed determines the volume
fraction of each of these cell types in the tissue. This is expressed as a
percentage of the total volume analysed. Although the method is capable of
distinguishing ripe from developing gametes, in this study only the total
gamete fraction was determined at each time of the year.
Mantle tissue excised and fixed as described in Section 2.2.1, was
first dehydrated through sequential 1 hour immersions in an ascending
alcohol series (70% - absolute ethanol). The alcohol was then removed from
the tissue by immersion in two 1 hour changes of the clearing agent
'histosol'. The cleared tissue was finally embedded in paraplast wax. The
processing scheme described was performed overnight on a Shandon Southern
products, 2L processor MK II. Having prepared a wax block, 7 pm tissue
sections were cut using a Richeart rotary microtome. These sections were
flattened on a waterbath at 40°C and collected on acid-cleaned microscope
slides. After dewaxing in 'histosol', the sections were stained by the
Papanicolaou method (Culling, 1963) and mounted in colophonium resin.
Stained by this method, sperm appear blue-grey to blue and oocytes blue to
pink, depending on the degree of ripeness. ADG cells appear orange, while
the VCT cells remain unstained. Each of these cells is shown in Figure
2.3.
64
Male
ADG
Female
Figure 2.3
Light level micrographs of major mantle tissue cell types
RGC - reproductive germinal cells ADG - adipogranular cells VCT - vesicular connective tissue cells Photographs courtesy of Mr D.M. Lowe.
65
The proportion of each cell type in a particular tissue section was
determined under a light microscope using a weibel point-counting graticule
as a sampling matrix. This graticule has 42 specific end-points and a
count was made each time one of these end-points fell on a particular cell
type. Five different fields of view were taken for each tissue section and
the volume fraction of each cell type determined by:
Total number of counts per cell type ---------------------------------- x 1 0 0
210(Lowe, 1985).
The overall volume fraction for each cell type was expressed as the
mean ± SEM of 10 individual values obtained for each of the 10 male and 10
female mantles.
2.2.7 Statistical methods
Statistical tests were carried out on the data to determine (a) if sex
differences exist for MFO components or activities, both at individual
sampling times and over the one year sampling period; and (b) if
correlations exist between the seasonal profiles of the different MFO
components and activities, and whether these profiles correlate with
seasonal changes in either water temperature or reproductive condition.
Statistical tests (described in detail in Scheffe, 1959) were carried out
using established statistics packages on a GEC 4190 minicomputer.
66
2.2.7.1 Tests for sex differences
Seasonal sex differences were tested at the 5% level (P < 0.05) using
two-way analysis of variance. For most variables, both males and females
had an equal number of replicates and the 'TWOWAY' command in the
statistics package 'MINITAB' was used. For BPH activity, there was an
unequal number of replicates and the 'FIT' command in the 'GLIM' package
for unbalanced data was used. Both programs gave, for each variable,
values for the degrees of freedom and the sums of squares. From these
values, the F-ratio of each variable was calculated and compared with the
relevant theoretical values from F-distribution tables.
Sex differences at individual sample times were tested using Students
t-tests. Calculated values were again compared with the relevant
theoretical values given in t-distribution tables.
2.2.7.2 Tests for seasonal correlations
Correlation coefficients were calculated according to Pearsons formula
(see Appendix, Section 2.5) using the 'CORR' command in 'MINITAB'. If
there were significant sex differences in MFO contents or activities,
correlation coefficients were calculated separately for the individual
sexes. If there were no differences between the sexes, data for males and
females were combined before calculating correlation coefficients. Since
data from reproductive stage analyses were in terms of percentages,
correlations were determined after transformation by sin"'*' [%] .
A global test of correlation was also performed on the seasonal data
as a whole. A correlation matrix was established using the program 'CSPRO'
67
(a PML 'in house' program). The true set of coefficients were plotted on a
graph together with 50 random sets. If the real set was distinguishable
from the simulated random sets, then correlations were present within the
data. This test did not specify where the correlations were. The results
of this test are given in the appendix.
68
2.3 Results
2.3.1 Seasonal variations in components of the mussel MFO system andchanges in reproductive condition
Figures 2.4 to 2.8 show seasonal changes occurring in the specific
contents of cytochrome P-450 and cytochrome b^, and in the specific
activities of NADPH-cytochrome c reductase, NADH-cytochrome c reductase and
NADH-ferricyanide reductase, in mussel digestive gland microsomal fraction
over the one year period from 7-4-86 to 30-3-87. Although contents and
activities appeared on the whole to be slightly higher in females than
males, particularly over the autumn and winter months, there was no
statistical difference between the sexes for any parameter (P > 0.05),
either over the one year sampling period or on any of the individual
sampling occasions.
The seasonal profiles differed for the different parameters.
Significant (P < 0.05) peaks in cytochrome P-450 specific content were seen-1in late June and mid-November (50-60 pmoles mg microsomal protein) (Fig.
2.4). Content was lowest in mid-February (approximately 25 pmoles mg"^).
Cytochrome b^ specific content was relatively constant throughout the year
at about 20-30 pmoles mg"^, apart from in May when a significant peak of
about 40 pmoles mg"^ was seen (Fig. 2.5). A second slight peak in late
December/January of about 35 pmoles mg"^ was also evident. NADPH-dependent
cytochrome c reductase specific activity differed markedly from the NADH-
dependent activity. NADPH-cytochrome c reductase activity "oscillated"
between about 7 and 1 1 nmoles cytochrome c reduced min” mg"^ microsomal
protein, with the higher activities in late June, early October and late
69
CYTOCHROME b5
pM/mg
protei
n CYTOCHROME P-4
50 pM/
mg protein 70 -
60 -
40 -
30 -
20 -
10 -
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR1986/87
SEX ----- MALE FEMALEFigure 2.4. Seasonal variation in digestive gland microsomal
Cytochrome P-450 specific content.Values are means + 1 S.E.M. (n - 6) .
50-
40 -
30 -
20 -
10 -
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR1986/87
SEX ----- MALE FEMALEFigure 2.5. Seasonal variation in digestive gland microsomal
Cytochrome b5 specific content.Values are means + 1 S.E.M. (n - 6) .
70
Figure 2.6. Seasonal variation in digestive gland microsomal NADPH-dependent cytochrome c reductase activity.Values are means + 1 S.E.M. (n - 6).
Figure 2.7. Seasonal variation in digestive gland microsomal NADH-dependent cytochrome c reductase activity.Values are means + 1 S.E.M. (n - 6) .
Figure 2.8. Seasonal variation in digestive gland microsomal NADH-dependent ferricyanide reductase activity.Values are means ± 1 S.E.M. (n - 6) .
SEX ---- MALE FEMALE
71
NADH
ferricy.
red. n
M/min/mg protein
NADH
cyt.c
red.
nM/min/mg
protein
Q)4JOC.a.12 -
E3:cTJUC.u•p>*oXQ.a<2 0 -
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR19B6/B7
120 -
100 -
80 -
60 - — r
40 -
20 -
OCT NOV DEC JAN FEB MAR APR19B6/B7
BOO -700 -600 -500 -400 -300 -200 -
100 -
OCT DEC JAN FEB MAR APRJUN AUG1986/87
72
December/January (Fig. 2.6). The profile for NADH-dependent cytochrome c
reductase specific activity resembled that of NADH-ferricyanide reductase
specific activity, apart from the presence of a more severe trough in
activity in late August (Figs. 2.7 and 2.8). Both activities decreased
from early April to mid-February, before rising sharply in late
February/March to about the original levels. NADH-cytochrome c reductase-1 -1 -1activity decreased from about 90 nmoles min mg to about 50 nmoles min
mg"^, while NADH-ferricyanide reductase activity fell from about 700 nmoles- 1 - 1 - 1 - 1 min mg to about 400 nmoles min mg .
The degrees of correlation between the seasonal profiles of the MFO
components are given in Table 2.1. Since there were no significant
differences between the sexes for any component, the data for males and
females were pooled for the correlations. Table 2.1 indicates there was
little correlation between any of the profiles, except for the two NADH-
dependent reductase activities (r = 0.73). Despite the overall differences
between the seasonal profiles of the MFO components, some similarities were
evident. Firstly, the lowest specific contents and activities occurred in
February 1987, and secondly, prior to spawning between June and August (see
Fig. 2.12), the specific contents and activities of each component showed
slight but consistent decreases i.e. between June and August 1986.
Figures 2.9 and 2.10 show, respectively, seasonal variations in the
'416' nm low wavelength peak and total haemoprotein. There were no
significant differences between the sexes on any individual sampling
occasion, for either parameter. However, over the sampling period as a
whole, sex differences were evident for the '416' nm peak, with males
having higher levels. The seasonal profiles of the two parameters were
73
Table 2.1
Degrees of correlation between seasonal profiles of the specific
contents and activities of the digestive gland MFO components (pooled male
and female data). See Materials and Methods for statistical procedures.
Correlation CytochromeP-450
Cytochromeb5
NADPH- Cyt. c red.
NADH- Cyt. c red.
NADH-Ferric;red.
CytochromeP-450
0.11 0.15 0.39 0.25
Cytochromeb5
0.11 0.10 0.31 0.11
NADPH-Cyt. c reductase
0.15 0.10 0.16 0.20
NADH-Cyt. c reductase
0.39 0.31 0.16 0.73
NADH-Ferricy. reductase
0.25 0.11 0.20 0.73
74
Haemoprotein
units/mg pro
tein
'416'n
m pea
k units/mg protein
300 -
250 -
200 -
150 -
100 -
50 -
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR1986/87
SEX ---- MALE FEMALEFigure 2.9. Seasonal variation in digestive gland microsomal
'416Knm low wavelength peak.Estimation is in arbitrary units/mg microsomal protein. Values are means ± 1 S.E.M. (n - 6) .
.5
.0
5
.0
5
0
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR1986/87
SEX --- — MALE FEMALEFigure 2.10. Seasonal variation in digestive gland microsomal haemoprotein.
Estimation is in arbitrary units/mg microsomal protein.Values are means ± 1 S.E.M. (n - 6) .
75
very similar with peaks in late June and mid-November. The lowest levels
were during the winter, from December to February. Levels of both
parameters decreased sharply prior to spawning (June to August). Table 2.2
gives the degrees of correlation between the '416' nm peak and total
haemoprotein profiles and the profiles for the MFO component specific
contents and activities. Sexes were treated separately for the '416' nm
peak but data were pooled for total haemoprotein. For both parameters, a
relatively high degree of correlation was seen with cytochrome P-450
content (r = 0.82 for total haemoprotein, r — 0.88 and 0.73 for the '416'
nm peak, male and female, respectively). Correlations with the profiles
for other MFO components were relatively low, with the exception perhaps of
NADH-cytochrome c reductase activity. A high degree of correlation was
seen between the '416' nm peak and total haemoprotein (r = 0.91 for males,
r = 0.82 for females).
The seasonal variation in water temperature at Whitsand Bay, Cornwall
over the 12 month sampling period, is given in Figure 2.11. The highest
temperatures were recorded in the summer, between late June and early
October, declining through the winter. Table 2.3 gives the degrees of
correlation between the seasonal changes in water temperature and the
seasonal profiles of MFO and other microsomal components. The highest
correlations were seen with cytochrome P-450 content, 'total haemoprotein'
and the '416' nm peak. Low correlations were seen with cytochrome b^
content and the reductase activities.
Seasonal changes in the reproductive condition of M. edulis are shown
in Figure 2.12. The total gamete volume fraction was significantly higher
in males than in females over the sampling period as a whole (Fig. 2.12a).
76
Table 2.2
Degrees of correlation between the seasonal profiles of the digestive gland
microsomal '416' nm low wavelength peak (male and female) and total
haemoprotein (pooled male and female data) and the seasonal profiles of the
MFO components (pooled male and female data). See text for statistical
procedures.
Correlation CytochromeP-450
Cytochromeb5
NADPH- Cyt. c red.
NADH- Cyt. c red.
NADH-Ferricyred.
Male '416' nm peak
0 . 8 8 0.13 0.05 0.58 0.28
Female '416' nm peak
0.73 0.19 -0.08 0.61 0.51
Total 0.82 0.14 0.04 0.54 0.31haemoprotein
TEMP
ERAT
URE
2 0 -
cj 15O
10 -
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR1986/87
Figure 2.11. Seasonal variation in water temperature at Whitsand Bay. Cornwall, over the 12 month sampling period (7.4.86 to 30.3.87).
78
Table 2.3
Degrees of correlation between the seasonal changes in water temperature and the
seasonal profiles of the digestive gland MFO and other microsomal components
(pooled male and female data except for '416' nm peak). See text for
statistical procedures.
Correlation Cyto- Cyto- NADPH NADH NADH Total '416' nm '416' nm
chrome chrome Cyt-c Cyt-c Ferricy haemo- peak peak
P-450 b_ red. red. red. protein (male) (female)5
Water 0.70 0.29 0.05 0.14 0.09 0.60 0.72 0.65temperature
79
Figure 2.12a. Volume fraction of total gametes (ripe and developing) over the 12 month sampling period.Values are means + 1 S.E.M. (n - 10) .
Figure 2.12b. Volume fraction of adipogranular (ADG) cellsover the 12 month sampling period.Values are means ± 1 S.E.M. (n - 10) .
Figure 2.12c. Volume fraction of vesicular connective tissue(VCT) cells over the 12 month sampling period.Values are means ± 1 S.E.M. (n - 10) .
SEX ---- MALE FEMALE
80
PERCENTAGE V.C.T. CEL
LS PERCENTAGE A.D.G. CEL
LS PERCENTAGE TOT
AL GAMETES
X\J\J -
0 -i— '— i— 1— i— 1— i— '— i— '— i— •— i— '— i— 1— i— 1— i— '— i— 1— i— • rAPR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR
1986/87
40 -
30
20
10
MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR1986/87
50-
40 -
30 -
20 -
10 -
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR1986/87
81
Differences were not seen, however, when the total gamete volume fractions
were either at their lowest (approx. 10% of total) in late August following
spawning or at their highest (approx. 65-75% of total) in February/March
prior to spawning. No sex differences were seen in the volume fractions of
either adipogranular (ADG) cells (Fig. 2.12b), or vesicular connective
tissue (VCT) cells (Fig. 2.12c). The volume fractions of both ADG and VCT
cells increased from late June to maximum levels of about 24-32% and 40-
45%, respectively, in late August/September. Their decline through autumn
and winter was accompanied, in both sexes, by an increase in the volume
fraction of total gametes. Slight spawning possibly occurred in spring
(April/May 1986 and February/March 1987), although the major spawning
period was clearly in the summer between late June and August.
Table 2.4 gives the degrees of correlation of the seasonal changes in
the MFO and other microsomal components, and water temperature, with the
reproductive conditions of M. edulis in terms of seasonal changes in total
gamete volume fraction and ADG cell volume fraction. Sexes were treated
separately for correlations involving gamete volume fractions, but were
pooled for correlations involving the ADG cell volume fractions.
Correlations were not carried out with the VCT cell data which was
essentially similar to that of the ADG cells. Correlations with gamete
volume were low, being highest for cytochrome P-450 specific content and
total haemoprotein (Table 2.4a). There was relatively little correlation
between gamete volume fraction and water temperature, although it was
apparent that spawning occurred at higher water temperatures (Figs. 2.11
and 2.12). The correlations with ADG cell volume fraction were also low
(Table 2.4b). The highest correlations were seen with seasonal changes in
NADH-cytochrome c reductase activity and NADH-ferricyanide reductase
82
Table 2.4
Degrees of correlation between the seasonal changes In reproductive condition and the seasonal profiles of the
digestive gland MFO and other microsomal components (pooled male and female data except for '416' nm peaks)
and water temperature.
(a) Correlation with changes In gamete volume fraction (male and female).
(b) Correlation with changes In ADG cell volume fraction (pooled male and female except where Indicated
otherwise).
See text for statistical procedures
(a)
Correlation CytochromeP-450
Cytochrome
b5
NADPH Cyt. c reductase
NADH Cyt. c reductase
NADHFerricy.reductase
Totalhaemoprotein
'416 nm Water peak' temperature
Maletotalgamete
-0.45 0.13 -0.12 0.02 0.01 -0.49 -0.29 -0.35
Femaletotalgamete
-0.41 -0.18 0.01 0.14 0.05 -0.44 -0.16 -0.32
b)
Correlation CytochromeP-450
Cytochrome
b5
NADPH Cyt. c reductase
NADH Cyt. c reductase
NADHFerricy.reductase
Totalhaemoprotein
'416 nm Waterpeak' Temperature
male female
ADGCells 0.03 -0.17 -0.07 -0.53 -0.47 -0.08 -0.26 -0.34 0.33
83
activity, the correlations reflecting the similar declines in these
parameters between August and February.
Figure 2.13 shows the seasonal changes in the total tissue yields of
microsomal protein and the various MFO and microsomal components i.e.
microsomal enzyme activities and contents of cytochromes, haemoprotein and
'416' nm peak expressed in terms of per gram wet weight of digestive gland
tissue. Microsomal protein was significantly higher in females than in
males, both on a number of individual sampling occasions and over the
sampling period as a whole (Fig. 2.13a). As there were no significant
differences between sexes in the specific contents or activities of the
various MFO and microsomal parameters (with the exception of the '416' nm
peak; see above), not surprisingly the differences in microsomal protein
tissue yields resulted in there being significant sex differences in the
total contents or activities of all these parameters (Figs. 2.13, b-h). In
each case the total microsomal content or activity per gram wet weight was
higher in females than in males over the 12 month sampling period.
Microsomal protein content varied between approximately 5.5 and 11 mg1 -1 g wet weight in females, and 5.5 and 9.5 mg g wet weight in males.
Content in both sexes was lowest in November and highest in February. A
second protein peak was indicated in late June/July in females, but this
was less evident in males. The seasonal variation in microsomal protein
content resulted in different seasonal profiles for the MFO component total
contents and activities compared to the seasonal profiles for the MFO
component specific contents and activities (Figs. 2.4 to 2.10 compared to
Fig. 2.13). Cytochrome P-450, total haemoprotein and the '416' nm peak had
roughly similar profiles, reaching maxima in late June/July (Figs. 2.13 b,
84
Figure 2.13a. Seasonal variation in total tissue microsomal proteincontent per gram wet weight of digestive gland tissue.
Values are means ± 1 S.E.M. (n - 6) .
Figure 2.13b. Seasonal variation in total tissue microsomal cytochromeP-450 content per gram wet weight of digestive gland tissue.Values are means ± 1 S.E.M. (n - 6) .
Figure 2.13c. Seasonal variation in total tissue microsomal haemoproteincontent per gram wet weight of digestive gland tissue.Values are means ± 1 S.E.M. (n - 6) .
Figure 2.13d. Seasonal variation in total tissue microsomal *416' nm peak content per gram wet weight of digestive gland tissue.Values are means ± 1 S.E.M. (n - 6) .
SEX ---- MALE - FEMALE
85
<o oooo oooooo
snse;} B/wd OSfr-d 3W0UH001A0 anssT^ 6/sq;un -qjB >iv3d mu .gTfr.
<(OCDOC\J oinin oosnEEjq B/fiuj NI310dd ansBTq B/sqTun -qjB NI310dd0W3VH
86
19B6/B7
198B
/B7
Figure 2.13e. Seasonal variation in total tissue microsomal cytochromeb5 content per gram wet weight of digestive gland tissue.
Values are means ± 1 S.E.M. (n - 6) .
Figure 2.l3f. Seasonal variation in total tissue microsomal NADPH- cytochrome C reductase activity per gram wet weight of digestive gland tissue.Values are means + 1 S.E.M. (n - 6) .
Figure 2.13g. Seasonal variation in total tissue microsomal NADH- cytochrome C reductase activity per gram wet weight of digestive gland tissue.Values are means + 1 S.E.M. (n - 6) .
Figure 2.l3h. Seasonal variation in total tissue microsomal NADH- ferricyanide reductase activity per gram wet weight of digestive gland tissue.Values are means + 1 S.E.M. (n - 6) .
SEX ---- MALE FEMALE
87
Lf -r-in
-|---t - " T -o in ’ i
o— i--- 1— -i iJ
in oni O N in OJanssT^ B/utui/WU ' 03H 0-1A0 HdOVN
ooooo
snesf3 O/uxui/wu -03U ‘AOIHbGd HOVN
ooooo
0TTTo oo ino n
anBBT} fi/Wd Sd 3H0UHD01A0 anssi; 0/UTUi/wu ‘038 0-1A0 HOVN
88
1986/87
1986
/87
c and d, respectively). Total cytochrome P-450 content at this time was-1 -1 approximately 580 pmoles g wet weight in females and 460 pmoles g wet
weight in males. Total content fell to approximately 300 pmoles g” wet_ iweight in females and 225 pmoles g wet weight in males during the winter.
Cytochrome b^ total content varied in females between approximately-1 -1 315 pmoles g wet weight in late May and 125 pmoles g wet weight in mid-
November, and in males between approximately 240 and 125 pmoles g"^ wet
weight at the same times (Fig. 2.13e). A second distinct peak seen in
females in late December/January was not evident in males. The seasonal
profiles for NADPH-cytochrome c reductase, NADH-cytochrome c reductase and
NADH-ferricyanide reductase total microsomal activities were roughly
similar (Figs. 2.13 f, g and h, respectively). For each enzyme, a peak of
activity in late June/July was more evident in females than in males. The
lowest activities for each enzyme were in mid-November.
Table 2.5 gives, for males and females, the degrees of correlation
between the individual seasonal profiles given in Figure 2.13 (excluding
microsomal protein), and between these profiles and the seasonal changes in
water temperature and total gamete volume fraction. As observed for
correlations between specific contents (see Table 2.2), high coefficients
of correlation were seen between the seasonal profiles for total cytochrome
P-450, total haemoprotein and the '416' nm peak (r = 0.9 to 0.92 for males;
0.81 to 0.91 for females). The profiles for these parameters also showed
some correlation with the seasonal.changes in water temperature (r =
approx. 0.6). Other correlations were low with the exception of total
tissue microsomal NADH-cytochrome c reductase activity which showed some
correlation, in both males and females, with the tissue microsomal levels
of cytochrome P-450, total haemoprotein and the '416' nm peak (r = 0.6 to
Table 2.5
Degrees of correlation between the seasonal profiles of total gamete
fraction, water temperature and the total tissue microsomal contents and
activities of the MFO and other components (male and female data treated
separately). See text for statistical procedures.
90
Correlation
Cytochrome
Cytochrome
NADPH
NADH
NADH
'Total
'416
nm $
Water
P-450
bg Cyt. c
Cyt. c
Ferricy.
haemoprotein'
peak'
Gamete
Temperature
Reductase
Reductase
Reductase
ocm o CM CM rH CO in
vO CM o rH vO VO COO
o o o1 o 1 O O 01
VOH rH CM CM CO VO inH ■M* rH CO O o CO
Odi
o o o o1 o 01
O*o o o vO CO CM COo\ co CM VO Q* Ov O vOo o O o O o o
f*o CO rH in CO CM vO rHcr» CO CO vO Ov o VO
oo o O o O 01
o
H CO vO •M* r- 0> vO oCM r CO CO CO CM
o o o o o o o 01
o CM o r- in vO CM CMVO VO vO CO rH
oo o o o o o o
vOVO o rH o CM oCO CM CO CM rH oo O o o o o o
1
COin a* CM CM CO o rH CMCM CM CO CO •M* CMo d O o o o o
rHin VO O o o rHCM CO VO o\ o> rH VO
OO o O o o O1
o
c•H a)o 0) 0) 0) a 0) u6 B o w 0 U w p d0 O X (0 03 0 c0 i—i 0 pb o u tu P X P ffi •H P (0 b b c0
in x Q 0 q a q u P a X CD 0) u0 u in rtj p d p d b d 0 o CO p P 0)0 i O A £ >i *d £ >1 *d fc a) 'd H 6 vO <u a) c0 04P P U 0) o a) a> c0 rH a d? B 5 6
>1 0) (0 0)o o .c o £-»
91
Correlation
Cytochrome
Cytochrome
NADPH
NADH
NADH
’Total
'416
nm %
Water
P-450
b_ Cyt.c
Cyt.c
Ferricy.
haemoprotein'
peak'
Gamete
Temperature
5Reductase
Reductase
Reductase
CO CO in CM CMvO CO O rH o in vO coo d o O o o O 01
inrH 10 VO O Ov CM in CMrH CO vO rH CO co COo O o o o o o O1
rH Ov rH in CMrH CM r-* <N CO CO VOCO o o o O o o oo
rH CO rH in Ov CM inOV CO r* rH co CO ino o o o o o o d
rH CM in in Ov rH o corH O CM rH CM rH oo o o o O O O d
in o in in Oin cm VO rHo o o d o o o o
CM cm o in rH o\ vO «**m* o CM CO o
o o o 01 o o o o
o CM CM CO rH vO r-*CO rH COo o d o o o o o
o CM in rH rH rHin rH O* CO in COrH VOo o o O o o o o
c-rl d)0) 0) V • 0) 0)B B o W 0) tx 10 P 30 0 X (d u w u <d rH 0 pP o u x P X fd X •H P id p fi u idX2 in Q u Q p q U p X c 0) <L)u u in flj P 3 < o a 3 0 o * p P 0)0 i 0 pQ 2 03 P 3 X 0) *0 H B vO id 0) id 04p 04 P o 0) tx 03 x 01 id rH 0) 6 3 e>1 >1 X o 0) X 0) x id 0)o o X x: o Eh
92
0.75), and also with NADH-ferricyanide reductase activity in males only (r
= 0.74). There was little correlation of any parameter with the seasonal
changes in the volume fractions of total gametes.
Seasonal variations in the carbon monoxide difference spectra Amax
values for cytochrome P-450 and the '416' nm low wavelength peak are shown
in Fig. 2.14. Sex differences were not significant for either parameter,
but changes over time were. Cytochrome P-450 Amax values were relatively
high from February to June (449.5 to 450 nm), fell more or less steadily to
a seasonal minimum in late December (448 nm) and then rose sharply again in
January and early February (Fig. 2.14a). There was no correlation between
the seasonal changes in cytochrome P-450 Amax values and the seasonal
profile for cytochrome P-450 specific content (r = 0.14; values for males
and females were pooled for correlation).
The seasonal profile in the Amax.values of the '416' nm low wavelength
peak resembled that of cytochrome P-450 Amax, except that a distinct
minimum in late December was not evident (Fig. 2.14b). Again, there was no
correlation between the seasonal changes in the '416' nm peak Amax values
and the changes in its specific content (r = 0.007, pooled male and female
data).
2.3.2 Seasonal variation in NADPH-independent 7-ethoxy coumarin O-
deethylase activity and benzo[a]pyrene hydroxylase activity
Figure 2.15 shows the seasonal variation of NADPH-independent ECOD
activity over the one year sampling period. Although activity appeared
consistently higher in males than in females, the differences were not
93
452-
451
4500 in1CLUJ 4490 DC1o 448 H >- U447 -
— i— |— .— |— i— i— i— |— i— |— .— |— i — |— i— |— i— |— .— |— i— i— i— r
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR1986/87
SEX MALE FEMALEFigure 2.14a. Seasonal variation in the wavelength maximum
for digestive gland microsomal cytochrome P-450.Values are means ± 1 S.E.M. (n - 6) .
418-
417 -
£ 416
‘to 415
414 -APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR
1986/87
SEX ---- MALE FEMALEFigure 2.14b. Seasonal variation in the wavelength maximum
of the digestive gland microsomal '416’nm peak.Values are means ± 1 S.E.M. (n - 6) .
94
CD 3
u
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR19B6/B7
SEX ---- MALE FEMALEFigure 2.15. Seasonal variation in digestive gland microsomal NADPH-
independent 7-ethoxycoumarin O-deethylase (ECOD) activity.Values are means ± 1 S.E.M. (n - 6) .
BO -
c 60
40
20 -
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR1986/87
SEX MALE FEMALEFigure 2.16. Seasonal variation in theoretical 7-ethoxycoumarin (7-EC)
turnover by digestive gland microsomal cytochrome P—450.Values are means ± 1 S.E.M. (n - 6) .
95
significant. ECOD activity varied from being undetectable in late-1 -1June/July to a maximum activity of about 3 pmoles min mg microsomal
protein in mid-November. The maximum activity occurred as a distinct peak
over autumn and winter, activity rising sharply from late August/September,
and decreasing sharply to mid-February. The theoretical turnover of 7--1 -1ethoxycoumarin by cytochrome P-450, in pmoles min nmole P-450 is given
in Figure 2.16. Essentially the same profile was obtained, although the
decline through the winter from mid-November was less sharp. Again,
turnover appeared higher in males over the sampling period although the
differences were not statistically significant.
Figure 2.17 shows the seasonal variation in BPH activity. Although
data were available for fewer sampling occasions, higher activities in
males were seen in April 1986 and March 1987, but not over the sampling
period as a whole. High variability and a complicated seasonal picture were
seen but minimal activities for both sexes were evident in June/July and
possibly the following winter, with peaks of activity between. Specific-1 -1activities varied between about 2 and 11 pmoles min mg . A similar
seasonal profile was obtained for the theoretical turnover of
benzo[a]pyrene by cytochrome P-450, with again, higher values for males in
April 1986 and March 1987 (Fig. 2.18).
Table 2.6 gives the degrees of correlation of the seasonal changes in
NADPH-independent ECOD and BPH activities with those for the MFO
components, the difference spectra Amax of cytochrome P-450 and the '416'
nm peak, the volume fractions of male and female gametes and water
temperature. Pooled male and female data were used in all cases, except
for volume fraction which was analysed separately (see before). All BPH
96
30 -
CD
20
£ 10w
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR1986/87
SEX ---- MALE FEMALEFigure 2.17. Seasonal variation in digestive gland microsomal
benzo [a] pyrene hydroxylase (BPH) activity.Values are means ± 1 S.E.M. (n - 6 or less) .
400-
300
200 -i
p 100
APR MAY JUN JUL AUG SEP OCT NOV DEC JAN FEB MAR APR19B6/B7
SEX MALE FEMALEFigure 2.18. Seasonal variation in theoretical benzofa)pyrene (B [a]P)
turnover by digestive gland microsomal cytochrome P-450.Values are means + 1 S.E.M. (n - 6 or less) .
97
Table 2.6
Degrees of correlation between the seasonal profiles for NADPH-independent 7-
ethoxycoumarin O-deethylase and benzo[a]pyrene hydroxylase specific activities
and the seasonal profiles of the specific activities and contents of the
digestive gland MFO components and the difference spectra A of cytochrome P-max
450 and ’416' nm peak (pooled male and female data), total gamete volume
fraction and water temperature. Sea text for statistical procedures .
Correlation CytochromeP-450
Cytochrome
b5
NADPH- NADH- Cyt.c Cyt.c Reductase Reductase
NADH-Ferricy.Reductase
ECODactivity -0.39 -0.17 -0.10 -0.12 -0.09
BPHactivity -0.29 0.14 0.08 0.31 0.16
Correlation AmaxP-450
Amax'416' nm peak
MaleGamete
FemaleGamete
WaterTemperature
ECODactivity -0.66 -0.65 -0.43 -0.65 0.01
BPHactivity 0.10 0.06 0.03 -0.27 0.01
98
and most ECOD activity correlations were low. The only exception was a
relatively high correlation between ECOD activity and the difference
spectra Amax values for cytochrome P-450 and the '416' nm peak (r = -0.66
and -0.65 respectively). No correlation was seen between ECOD and the
seasonal changes in water temperature.
2.3.3 SDS-Gel electrophoretic comparison of microsomal proteins
An SDS-gel electrophoretic comparison of pooled male and female
digestive gland microsomal proteins from each seasonal sampling date, is
shown in the photograph Figure 2.19. Although the photograph is of limited
quality, protein bands with similar electrophoretic mobilities were present
at each time of the year. The major electrophoretic bands correspond to
proteins with molecular weights of approximately 94.5 Kd, 66.0 Kd, 53.0 Kd,
51.5 Kd, 41.5 Kd, 27.5 Kd and 22.5 Kd. Although each electrophoretic
profile had bands corresponding to very high molecular weight proteins
(above about 180.0 Kd), the major concentrations of bands corresponded to
proteins with molecular weights below 30.0 Kd and between 40.0 Kd and 95.0
Kd. Although 130 /xg of protein sample was applied to each sample well,
the profiles from 28-6-86, 26-8-86 and 6 -1 0 - 8 6 appear darker than profiles
from other sampling dates. This difference was not apparent on the actual
gel indicating the darker appearance was due to photographic artefact.
A diagrammatic representation of the gel is given in Figure 2.20. An
electrophoretic protein band of approximately 53.0 Kd, which most likely
corresponded to a cytochrome P- 450 (see Section 3), was present on each
sampling occasion. It was not possible to examine the seasonality of the
band by a direct comparison between seasonal samples (because of the
99
Figure 2.19
Comparison of digestive gland microsomal protein by SDS-gel electrophoresis
S Molecular weight standards
Seasonal samples 1 07-04-862 22-05-863 28-06-864 26-08-865 06-10-866 14-11-86 .7 30-12-868 12-02-879 30-03-87
Figure 2.20
Diagrammatic representation of the gel showing major protein bands and areas of interest.
100
M.Wt.Kd
f * ' ►
92. 5---
68.0--57. 5 ---53. 0 ---48.5 ——43. 0 ---
29. 0---25.7---20 . 1 ----
12.4____________________ ______________-— — — _____ ________ ________S 1 2 3 4 5 6 7 8 9
M.Wt.Kd
92.5
68.0-57.5-53.0-48.5-43.0-
■64.5
29.025.720.1 ■22.5
12.4S 21 3 54 6
101
photographic artefacts), however, some indication of seasonality was
obtained from a comparison of the 53.0 Kd band with other bands within the
same sample. The density of this band relative to other bands varied with
the time of year. During winter and spring it was relatively faint in
comparison to other bands, while during the early summer, it was
considerably more dense. On three occasions, 6-10-86, 14-11-86 and 30-12-
8 6 , an electrophoretic band at 51.5 Kd could be seen. The 53.0 Kd band was
more faint relative to surrounding bands when the 51.5 Kd band was evident.
Other bands, possibly showing seasonality, were evident at 64.5 Kd and 22.5
Kd.
102
2.4 Discussion
The metabolism of many intertidal molluscs and other marine
invertebrates is seasonally variable, showing regular and marked patterns
of change, often linked to annual cycles of food storage and utilization
and gametogenesis (Gabbot, 1983). Seasonal changes in the tissues of M.
edulis are numerous and include alterations in the steady-state
concentrations of metabolites, in rates of processes, and in the specific
activities and kinetic characteristics of enzymes of intermediary
metabolism (Livingstone and Clarke, 1983). Seasonal changes in MFO systems
and related activities have been observed in a variety of poikilothermic
organisms (see Introduction), including the mussel species M. edulis
(Livingstone, 1985, 1987) and M. galloprovincialis (Suteau et al., 1985).
The results of this study confirm a number of the observations of these
earlier studies and, in addition, provide a comprehensive picture of the
variability and levels of the MFO components and activities of the
digestive gland microsomal fraction of M. edulis in relation to season and
the reproductive cycle. The possible functional and mechanistic
interpretations of the observed changes, in relation to the limited
information available on xenobiotic and endogenous monooxygenase metabolism
in M. edulis, are discussed below.
2.4.1 Seasonal changes in cytochromes P-450 and b and reductase activities
The seasonal variation in cytochrome P-450 specific content, with
relatively high contents during the early summer and autumn followed by a
decline in mid-winter, was partially correlated with the seasonal variation
103
in water temperature (r = 0.70). A similar degree of correlation with
temperature (r = 0.68) was obtained for the cytochrome P-450 of whole body
microsomes of M. galloprovincialis from the Arachon Bay, France (Suteau et
al.t 1985), suggesting that, for at least part of the year, changes in
cytochrome P-450 content may be regulated by, or dependent on, changes in
water temperature. However, that the situation is not so simple, and that
other regulatory factors might be involved, is indicated by a more detailed
comparison of the seasonal profile. For example, a seasonal peak of
temperature in early October corresponded to a slight trough in cytochrome
P-450 content (see Figs. 2.4 and 2.11). More dramatically, correlations
between seasonal changes in water temperature and in NADPH- and NADH-
dependent cytochrome c reductase and NADH-ferricyanide reductase activities
and cytochrome b^ content were non-existent, suggesting any regulatory
effect of temperature on these MFO components is minimal. With respect to
enzyme activities, however, the data must be interpreted with some caution
as activities were assayed at 25°C, not at the ambient water or tissue
temperature, and the temperature-dependencies of the activities have not
been investigated. For example, it is well known that species of fish are
capable of metabolic acclimation to altered water temperature (Dewaide,
1970). Measured at constant temperature, MFO activities from cold-
acclimated fish can be higher than those from warm-acclimated fish
(Koivusaari, 1983). Similarly, in both fish (Stegeman, 1979) and mammals
(Matsuyama et al., 1986) the levels of NADPH-cytochrome c (P-450) reductase
were reduced at warmer temperatures.
The increase in cytochrome P-450 content in spring coincided with
an increase in water temperature, and peak levels of P-450 were reached
immediately prior to spawning in late June/July. By analogy with the MFO
systems of bacteria, vertebrates and other organisms (Sato, 1978; Snyder
104
and Remmer, 1981), an increase in cytochrome P-450 content would be
expected to result in enhanced or altered monooxygenation in vivo. A
similar consequence might be anticipated from increased cytochrome c (P-
450) reductase activity through its effect on flux-regulation in the MFO
system (Bjorkhem, 1982). The levels of both NADPH- and NADH-dependent
cytochrome c reductase activities were maximal during the pre-spawning
period. NADH-cytochrome c reductase activity was high throughout the
spring while NADPH-cytochrome c reductase activity fluctuated but showed a
peak with cytochrome P-450 content in June. These observations, together
with the very low BPH and NADPH-independent ECOD activities during this
period, (NADPH-independent ECOD activity, for example, was undetectable
when cytochrome P-450 content was maximal), suggest a function for the
digestive gland microsomal MFO system in the endogenous metabolism of the
mussel, possibly involved in metabolic changes associated with gamete
development and the build-up to spawning. Metabolic interactions between
tissues are known for M. edulis. For example, the transfer of carbohydrate
from the digestive gland to the reproductive mantle tissue occurs at
certain stages during gametogenesis (Gabbot, 1983). The MFO system is
known to have a role in the endogenous metabolism of steroids and fatty
acids in mammals (Kupfer, 1982; also see Section 1), and the same is
established or indicated for a number of poikilothermic invertebrate and
vertebrate phylogenetic groups, for example, the Crustacea Callinectes
sapidus (Singer and Lee, 1977), Balanus eburneus (Stegeman and Kaplan,
1981), Carcinus maenas (O'Hara et al., 1982) Maja crispata (Bihari et al.,
1984) and Panulirus argus (James and Shiverick, 1984) ; the fish
Tautogolabrus adspersus (Walton et al., 1983), Caregonus albula (Lindstrom-
Seppd, 1985) and the amphibia Rana temporaria (Harri, 1980) and Rana
catesbeiana (Muira, 1985). Little is known about the monooxygenation of
105
endogenous substrates in the digestive gland of M. edulis, although the
microsomal fraction is capable of both testosterone and arachidonic acid
metabolism (see Section 5). M. edulis contains cholesterol and a wide
range of other steroids and sterols (Goad, 1981; Khan and Goad, 1983) and
the biosynthesis of steroid hormones in the mantle tissue has been
demonstrated (De Longcamp et al., 1974). A role for the digestive gland
microsomal MFO system in steroid metabolism, associated with the pre
spawning period and subsequent spawning, is therefore a possibility. Other
information on endogenous metabolism in M. edulis is limited to the
demonstration of the hydroxylation of vitamin in whole tissue
homogenates (Lehtovaara and Koskinen, 1986) and the conversion of
arachidonic acid to prostaglandins in homogenates of several tissues
(Srivastava and Mustafa, 1984, 1985).
Cytochrome b^ and cytochrome b^ reductase are known to function in
the MFO dependent metabolism of endogenous compounds such as steroids and
fatty acids (Oshino, 1982; Katagiri et al., 1982; Nakojin and Shinoda,
1983) and this has been shown in the fish Stenatomus chrysops (Klotz et
al., 1986). Although there was no correlation between the seasonal
profiles for cytochromes P-450 and b^, it is interesting that cytochrome b^
content also increased in early spring with water temperature, peaking in
May. Similarly, it may be significant that digestive gland glucose-6 -
phosphate dehydrogenase (EC 1.1.1.49) activity has been observed to be
higher in early summer than in autumn or winter (Livingstone, 1981).
Glucose-6 -phosphate dehydrogenase catalyses the first step in the pentose
phosphate pathway, the oxidation of glucose - 6 -phosphate to 6 -
phosphoglucolactone. The primary functions of the pentose phosphate
pathway are the production of pentose phosphates for nucleotide synthesis
106
and the formation of NADPH for a variety of reductive processes and
important biosynthetic pathways, such as monooxygenation and lipid
synthesis. An increase in NADPH flux in the digestive gland in the early
summer might be required to supply the necessary reducing equivalents for
the observed increased cytochrome P-450 content.
Studies on several species of fish - for example, the trout Salmo
gairdneri - have demonstrated a decrease in cytochrome P-450 and MFO
activities such as BPH activity during or shortly after the spawning period
(Stegeman and Chevion, 1980; Koivusaari et al., 1982). In the present
study, the specific content of digestive gland microsomal cytochrome P-450
similarly decreased during the spawning period. Decreases were also
apparent for cytochrome b^ content and the specific activities of NADPH-
and NADH- cytochrome c reductase and NADH-ferricyanide reductase.
Following the completion of spawning in August, the specific contents and
activities of all the microsomal components began to increase. Similarly,
increases in activity occurred for BPH and NADPH-independent ECOD which,
prior to spawning, were, respectively, minimal or undetectable. Decreased
specific content of digestive gland cytochrome P-450 with spawning has been
observed before for a population of M. edulis from a Norwegian fjord
(Livingstone, 1987). The results, therefore, indicate that spawning or
reproductive state may be factors affecting or regulating the levels of MFO
components and activities in the digestive gland of M. edulis. The
reproductive cycle of the Whitsand mussel population in the study was
typical for Mytilus sp. from temporate waters (Lowe et al., 1982)
gametogenesis occurred during the autumn and winter, utilizing glycogen
reserves that had been synthesized and stored in the adipogranular and
vesicular connective tissue storage cells during the summer. The
utilization of glycogen reserves continued into spring concomitant with the
107
ripening of gametes. The level of total gametes remained high until the
main spawning occurred between June and late August.
The mechanism underlying a possible relationship between gamete
release and changes in the mussel digestive gland MFO system can only be
speculated at. However, by analogy with observations on fish, for example,
that oestradiol-17y3 lowers specific activity and cytochrome P-450 content
in S. gairdneri (Hansson and Gustafsson, 1981; Koivusaari et al., 1981;
Stegeman et al., 1982; Forlin et al., 1984), it is possible that hormonal
changes within the mussel at the time of spawning could have an effect on
the seasonality of the MFO system. Both oestradiol-17/2 and testosterone
are known to be present in the mantle of M. edulis (De Longcamp et al.,
1974). That the digestive gland might be a target organ for hormonal
impact has been indicated by studies on M. edulis in which injection of
oestradiol-17^9 and progesterone at physiological concentrations caused
membrane destabilization of intracellular lysosomes (Moore et al., 1978).
The digestive gland of M. edulis, as in bivalve and gastropod molluscs in
general, contains many large and highly developed lysosomes (Owen, 1972)
within which are present various hydrolytic enzymes including
aminopeptidases, arylsulphatases, hexoseaminidases and glucuronidases
(Moore and Clarke, 1982; Pipe and Moore, 1985, 1986). Although lysosomes
function in the intracellular digestion of ingested material (Owen, 1972),
they are also involved in the turnover and catabolism of intracellular
proteins (Segal, 1975; Amenta and Brocher, 1980; Moore, 1980). Increased
levels of sex hormones during the spawning period could result in the
destabilization of digestive cell lysosomes and the release of proteolytic
enzymes which function either in the specific catabolism and turnover of
cytochrome P-450 and other MFO components or in the general breakdown of
108
proteins and tissues. Equally possible, however, is that such hormones
could act directly on the synthesis of MFO components. The observed
declines in MFO components with spawning could, therefore, either be due to
a specific regulatory event, or simply be a consequence of the general
tissue breakdown, resorption and rearrangement that is known to accompany
spawning (Lowe et al., 1982).
Sex differences in MFO systems, reflecting both endogenous and
xenobiotic aspects of monooxygenation metabolism, are well known for
mammals (Sato, 1978) and poikilothermic vertebrates and invertebrates, for
example, S. gairdneri (Stegeman and Chevion, 1980; Koivusaari et al.,
1982), C. sapidus (Lee et al., 1977) and C. maenas (O'Hara et al., 1982).
On occasions during a year, higher specific activities and contents (up to
a factor of x 2) in females than in males of NADPH- and NADH-cytochrome c
reductases, BPH and cytochromes P-450 and b^ have been recorded in
digestive gland microsomes of M. edulis (Livingstone, 1985, 1987) and
indicated for whole body microsomes of M. galloprovincialis (Suteau et al.,
1985). In contrast, in this seasonal study of the digestive gland of M.
edulis no differences in specific activities or contents of the MFO system
were observed between the sexes at any time of the year, suggesting that
sexual differences are of short duration (less than six weeks - the
sampling interval of the study) and possibly linked to particular events in
the reproductive cycle. The general pattern of the reproductive cycle (as
described earlier) is fixed, but the timing and duration of the various
phases, and the occurrence of events such as multiple spawnings and gamete
resorption, will vary from year to year depending on the particular
climatic and other conditions. For example, water temperature, or more
exactly the rate of change of water temperature, is considered to be one of
the most influential factors affecting the timing of the reproductive cycle
(Sastry, 1975; Lubet et al., 1986). Similarly, nutritional status of the
mussel is important and the occurrence of starvation can delay the onset of
spawning (Newell et al., 1982; Gabbot, 1983).
In contrast to specific activities and contents, seasonal and
sexual differences in microsomal total protein content were seen, with the
result that the tissue contents and activities of the digestive gland MFO
system, expressed in terms of per gram wet weight, were higher in female
than male mussels for most of the year. The higher microsomal protein
content in females was in contrast to earlier studies on mussels from the
same population (Livingstone and Farrar, 1984; Livingstone, 1985). The
apparent greater "capacity" of the MFO system in female mussels is
presumably related to either general or reproductive aspects of their
metabolism and, interestingly, is consistent with other observations on the
biochemistry of M. edulis, viz. the following, expressed in terms or per
gram wet weight, are higher in female than male mussels for much of the
year - digestive gland glucose-6 -phosphate dehydrogenase activity and total
soluble protein (Livingstone, 1981), mantle hexokinase activity and total
soluble protein (Livingstone, 1981; Livingstone and Clarke, 1983) and many
glycolytic enzyme activities of both the mantle and posterior adductor
muscle (Churchill, 1987).
2.4.2 Seasonal changes in BPH and NADPH-independent ECOD activities and haemoproteins
NADPH-independent ECOD activity showed a marked and regular
change during the year. Activity increased sharply from July/August to a
peak in November and then declined over the following winter and early
spring to undetectable levels. A similar profile was obtained for the
110
theoretical turnover of 7-ethoxycoumarin by cytochrome P-450 but the
decline over the winter into spring was less steep. Similar observations
of maximal activities in autumn followed by a decline into late
winter/early spring have been seen for digestive gland microsomal BPH in
mussels from the same Whitsand population (Livingstone, 1985). Digestive
gland microsomal BPH activity was low around spawning in the seasonal study
of this section but unfortunately no data were obtained for the late
autumn/winter period.
In contrast to these results, a partly different seasonal BPH
activity profile was obtained for a population of M. galloprovincialis,
with a partial positive correlation with water temperature (r = 0 .6 ) being
demonstrated (Suteau et al., 1985). The different results are possibly due
to a number of factors. For example, in the M. galloprovincialis study,
the mussels were sub-littoral and not intertidal, were from a warmer
climate with a different and less synchronised reproductive cycle (see Lowe
et al., 1982) and whole body microsomes were used rather than digestive
gland microsomes. That a different seasonal cycle of biotransforming or
MFO activity might be anticipated for whole body compared with digestive
gland microsomes is possibly indicated by mutagenicity studies on M. edulis
(Parry et al., 1981) - levels of mussel chemical extracts active in
bacterial Salmonella typhinurium Ames tests were found to increase through
the year reaching a maximum in summer, and the ability of whole mussel
microsomes to further activate such tissue extracts was higher in summer
than in winter. However, it should be noted that the origin of the
mutagenic chemicals was unknown, i.e. whether they were produced in the
mussel or simply bioconcentrated from the seawater. Also, the activating
ability of microsomes was only tested at two times of the year. A
111
different seasonal cycle of biotransforming and mutagenic activating
ability for whole body compared with digestive gland microsomes could be
produced on the basis of the differential tissue distribution of the MFO
system (Livingstone and Farrar, 1984; Stegeman, 1985) but, presumably given
the apparent absence of cytochrome P-450 in most tissues, would also
require the existence of other mechanisms of bioactivation in the different
tissues. For example, NADPH- and NADH-cytochrome c reductase and NADH-
ferricyanide reductase activities are detectable in most tissues of M.
edulis (Livingstone and Farrar, 1984) and the involvement of cytochrome P-
450 reductase and other flavoprotein reductases in redox cycling, leading
to the generation of reactive oxygen species, mutagenicity and possibly
biotransformation of xenobiotics, is well established in mammals (Kappus
and Sies, 1981). The in vitro generation of superoxide anion radical,
hydrogen peroxide and hydroxl radical from both NADPH and NADH has been
demonstrated in digestive gland microsomes of M. edulis (Livingstone et
al., 1988a). Another activating system that could be involved, and which
has been indicated in both M. edulis and M . galloprovincialis, is the
flavoprotein FAD-containing monooxygenase system (Kurelec, 1985; Kurelec et
al., 1986).
The seasonal profile of digestive gland microsomal NADPH-
independent ECOD activity had distinct mimima and maxima. Activity was
lowest during spawning periods. As discussed previously for cytochrome P-
450, decreases in MFO activities with spawning have been observed for a
number of poikilothermic species such as T. adspersus (Walton et al.,
1983), S. gairdneri (Stegeman and Chevion, 1980; HAnninen et al., 1982;
Koivusaari et al. , 1982) and C. albula (Lindstrom-Seppd, 1985). The
previously observed spring decline in digestive gland BPH activity in M.
edulis was also suggested to be associated with the approach of spawning
112
(Livings tone, 1985).
Maximal NADPH-independent ECOD activity in November coincided
with a second seasonal peak in levels of cytochrome P-450. Of particular
significance, however, was the marked lack of overall seasonal correlation
between total cytochrome P-450 content and NADPH-independent ECOD activity,
reflected in changing seasonal values for the theoretical turnover of 7-
ethoxycoumarin. Assuming that the NADPH-independent ECOD activity is
catalysed by cytochrome P-450 (see Section 5), then presumably this is
indicative of the existence of more than one form or isoenzyme of
cytochrome P-450 in the digestive gland of M. edulis. That the increase in
NADPH-independent ECOD and BPH activities over the autumn and spring could
be due to the synthesis of a new or altered amounts of a particular
cytochrome P-450 is possibly supported by several observations. Firstly,
on three sampling occasions in autumn and early winter when NADPH-
independent ECOD activity was highest, viz. 6-10-86, 14-11-86 and 3-12-86,
a second electrophoretic band of approximately 51.5 Kd could be discerned
in the SDS-gel electrophoretic profiles of digestive gland microsomal
proteins. This band was in addition to the major 53.0 Kd band which was
present throughout the year and which from purification studies is thought
to be cytochrome P-450 (see Section 3). An apparent molecular weight of
approximately 51.5 Kd is consistent with cytochrome P-450 isoenzymes from
mammals (Lu and West, 1980; Nebert and Negishi, 1982), fish (Williams and
Buhler, 1983; Klotz et al., 1986) and several species of crab (Quattrochi
and Lee, 1984). It is possible that the supposed 51.5 Kd cytochrome P-450
isoenzyme was also present during the rest of the year but at levels too
low to be detected under the loading conditions employed in the
electrophoresis. Secondly, concomitant with the peak of NADPH-independent
113
ECOD activity was a reduction in the cytochrome P-450 difference spectra
wavelength maximum ( max) from about 449.5/450 nm (in spring and early
summer) to about 448 nm (in early winter), possibly indicative of an
altered cytochrome P-450 composition or the synthesis of a new isoenzyme.
A correlation coefficient of -0.66 was obtained between the seasonal
profiles for NADPH-independent ECOD specific activity and the difference
spectra Amax values.
Other, perhaps less likely, possibilities also exist to account for
the seasonal variation in BPH and NADPH-independent ECOD activities. For
example, if there is an endogenous supply of reducing equivalents for the
in vitro measured MFO activities (see Section 5) , then this could vary
seasonally. Similarly, a seasonal profile of MFO activity could result
from the presence of a seasonally-variable inhibitor of MFO activity. Such
an inhibitor, probably a component of the digestive juices, has been
reported for the crustacea Callinectes sapidus and Homarus americanus (Pohl
et al., 1974).
Assuming that the seasonal profile of NADPH-independent ECOD activity
is due to the appearance of a particular isoenzyme of cytochrome P-450, as
is possibly indicated by the 51.5 Kd protein band and the change in Amax,
then of interest is the possible regulation and function of such an enzyme.
The seasonal profile could be partly or wholly determined by water
temperature and/or the reproductive cycle. For example, water temperature
was at a maximum in early October when the 51.5 Kd protein band was first
detectable. Changes in water temperature have been shown to alter the
quantitative composition of cytochrome P-450 isoenzymes in certain aquatic
poikilotherms, such as the fish Lepomis machrochirus (Karr et al., 1985).
It is possible that following spawning when the requirement for
114
monooxygenase metabolism is presumed to be minimal (see earlier
discussion), that the subsequently increasing water temperature induces the
synthesis of one cytochrome P-450 isoenzyme relative to another. Although
somewhat intangible, the presence of the 53.0 Kd and 51.5 Kd protein bands
appear to an extent reciprocal (see Fig. 2.19). The 53.0 Kd band was much
more evident in June/July, around the spawning period, and less so later on
in the autumn and winter when the 51.5 Kd band appeared. If the bands are
representative of isoenzymes of cytochrome P-450, then the possibility
exists that the 53.0 Kd band is intimately linked to endogenous functions
and reproductive events, whereas the 51.5 Kd band, as evidenced by the
appearance of NADPH-independent ECOD activity has different catalytic
specifications, directed towards endogenous or xenobiotic metabolism. It
should be noted, however, that 7-ethoxycoumarin is a substrate for several
different isoenzymes of cytochrome P-450 (Ullrich and Weber, 1972; Rogiers
et al., 1986), and the appearance of NADPH-independent ECOD activity is not
necessarily indicative of an increased capacity for xenobiotic metabolism.
For example, the cytochrome P-450 species responsible for steroid
aromatization in human placental microsomes has ECOD activity (Meigs,
1987). A changing cytochrome P-450 isoenzyme composition in response to
temperature and/or hormonal status has been shown in crabs and postulated
to reflect changing requirements for the MFO system (Quattrochi and Lee,
1984).
An alternative to endogenous regulation for the seasonal
appearance of NADPH-independent ECOD activity (and BPH activity -
Livingstone, 1985) is chemical induction. Environmental xenobiotics such
as polynuclear aromatic hydrocarbons (PAH) are accumulated in many tissues
of molluscs but primarily in lipid-rich tissues such as the digestive gland
(Lee et al., 1972; Palmork and Solbakken, 1981; Widdows et al., 1983). PAH
115
and other xenobiotics have been shown to induce cytochrome P-450 and
cytochrome P-450-dependent monooxygenase activities in numerous vertebrate
and some marine invertebrate species (Bend et al., 1979; Lee and Singer,
1980; Stegeman, 1981; also see Section 1). Elevation of cytochrome P-450
content and other responses have been seen in various bivalve and gastropod
molluscs including both M. galloprovincialis (Gilewicz et al., 1984) and M.
edulis (Livingstone, 1985, 1987; Livingstone et al., 1985; also see Section
6 ) . An observed seasonal difference of BPH activity in several tissues of
the barnacle B. eburneus was suggested to possibly reflect seasonally-
variable environmental induction associated with a higher input of
petroleum hydrocarbons into the water from local boat-traffic (Stegeman and
Kaplan, 1981). Although there are no data available on seasonal seawater
and mussel tissue PAH concentrations at Whitsand Bay, Cornwall, higher
tissue levels of PAH in autumn and winter than in summer have been observed
in several populations of M. edulis from temporate to sub-tropical waters
from around the world (Fossato et al., 1979; Mix et al. , 1982). In
contrast, higher tissue PAH levels in the summer than the rest of the year
were seen in M. edulis and the periwinkle Littorina littorea
experimentally-exposed to diesel oil in a mesocosm system, but this was
argued to be due to a transient rather than a seasonal event (Livingstone,
1987). A factor that could lead to higher body burdens of organic
xenobiotics in autumn and winter is tissue lipid levels which in M. edulis
change during gametogenesis and are higher in autumn and winter than in
summer (Pieters et al., 1979; Gabbot, 1983). Many factors affect the rates
and amounts of organic xenobiotics taken up by molluscs but of importance
are the lipid solubility of the xenobiotic and lipid levels (Geyer et al.,
1982). Thus, for example, uptake of various PAH increased with increased
lipid levels in different tissues of M. edulis (Widdows et al., 1983). In
the case of M. galloprovincialis,seasonal correlations were observed
between whole body lipid content and whole body microsomal cytochrome P-450
content and BPH activity (Suteau et al., 1985). Possibly of particular
significance is that the difference spectra Amax of digestive gland
cytochrome P-450 of M. edulis decreased in the late summer/autumn. The
induction by, and metabolism of, PAH and PAH-like compounds in mammals and
fish are generally characteristically associated with cytochrome P-450
isoenzymes with low Amax values, for example, cytochrome P-448, cytochromes
P-450c and d and cytochrome P-450 LM^ (Parke, 1985; Lewis et al., 1986).
Decreases and increases in cytochrome P-450 Amax with, respectively,
experimental-exposure to and recovery from PAH, have been observed in M.
edulis (Livingstone et al., 1985; Livingstone, 1987). If chemical
induction is a factor in the seasonal profile of NADPH-independent ECOD
activity, then the low or undetectable activities in late spring/early
summer could have been due either to lower tissue levels of xenobiotics
such as PAH or, perhaps more likely, to the reproductive state of the
mussels. For example, in a seasonal study on the effects of diesel oil on
M. edulis, no response of the digestive gland MFO system was seen when the
mussels were ripe with gametes (Livingstone, 1987). Similarly, the
induction of aryl hydrocarbon hydroxylase activity in the fish T. adspersus
was suppressed (Walton et al., 1983), and the time-course for induction of
BPH activity in the fish Archosargus probatocephalus (James and Bend, 1980)
were both affected by season.
Previous studies on the digestive gland cytochrome P-450 of M. edulis
have identified a large peak, between about 415 and 418 nm, in the
cytochrome P-450 carbon monoxide-difference spectrum (Livingstone and
Farrar, 1984; Livingstone et al., 1985; Livingstone, 1987, 1988). The
117
identity of the peak is unknown but there is some evidence to suggest that
it may be wholly or partly due to cytochrome P-420, the denatured form of
cytochrome P-450. Alternatively it may be due to another haemoprotein such
as a peroxidase. The seasonal results support both of these possibilities.
High correlations were seen between the seasonal content profiles for
cytochrome P-450, the '416' nm peak and total haemoprotein (r - 0.73 to
0.91), with, in the case of the latter pair, two marked peaks being seen in
June/July and November. In addition, the Amax for the '416' nm peak also
varied seasonally showing, like cytochrome P-450, minimum values in autumn.
2.4.3. Summary
Seasonal variations occurred in most of the components and
activities of the digestive gland microsomal MFO system of M. edulis and,
to varying extents, associations with the reproductive cycle were seen.
Sex differences were limited to higher microsomal protein yields, with
resultant higher tissue levels of MFO components and activities, in female
than male mussels for most of the year. Marked seasonal changes in BPH and
NADPH-independent ECOD activities, in relation to cytochrome P-450 content,
were highly suggestive of the existence of isoenzymes of cytochrome P-450.
Possibly supportive of this suggestion were seasonal changes in the Amax
for cytochrome P-450 and the SDS PAGE microsomal protein profile. Two
possible isoenzymes of cytochrome P-450 have been tentatively suggested. A
major protein band of molecular weight 53.0 Kd, the same as partially
purified digestive gland cytochrome P-450 (see Section 3), was present
throughout the year, and a second band of 51.5 Kd molecular weight appeared
for part of the year approximately coincident with maximal NADPH-
independent ECOD activity. The possible catalytic functions (endogenous or
xenobiotic metabolism) and regulation (endogenous or chemical induction) of
118
the putative isoenzymes have been speculated at. A marked coincidence in
seasonal peaks occurred in the microsomal contents of the '416' nm peak and
total haemoprotein, and to a lesser extent of cytochrome P-450.
The seasonal variations have significance for the study of the
digestive gland MFO system. Purification of cytochrome P-450 might give
different results in terms of, for example, isoenzyme patterns at different
times of the year, and would be worst carried out during the period
approximately before spawning when microsomal specific contents are lowest.
Similarly, it would be difficult to study NADPH-independent ECOD activity
during spawning when the activities are minimal or undetectable.
119
2.5 Appendix
2.5.1. Statistical formulae used for analysis of data
The methods used for data analysis were standard statistical
techniques taken from Scheffe (1959) (see Section 2.2.7).
(i) Students t-test (one-way analysis of variance):
This test was used to determine if sex differences were significant on
individual sampling occasions (Section 2.2.7.1)
At time-point t:
- h
S
where 3^ and are the means of the data values for males and females
respectively, n^ and n^ are the numbers of male and female data values (in
this study, = n^ = 6 ), and S is the standard deviation of the sample.
(ii) Two-way analysis of variance:
This test was used to determine if sex differences were
significant over the 12 month sampling period as a whole (Section 2.2.7.1).
The test takes into account both sex and time effects. The formula is
comparatively complicated but is represented below (taken from Sheffe,
1959).
1 1+"m nf
120
Source SS d.f. MS J?(MS)
A main effects ss4 -JKZiy^.-y...)2 i
1 - 1 o2+JKo\
B main effects SSB -iK2(y-j--y---)2 j
J-l 02+IKo%
AB interactions •U .)2 (I-1)(J-1) as
Error ssc -sss(7i -fc-7i -.)2 IJ(K-1) o2
"Total" ss'tot' -zsz(y1Jk-y---Yijk J
IJK-1
where A are the main sex effects; B, the main time effects; SS are the sums
of squares; d.f. are the degrees of freedom; I is the number of sexes (2);
J is the number of time-points (in this study,9); K is the number of2replicates for each time/sex combination (in this study, 6 ) and a is the
variance.
(iii) Test of correlation:
The degree of correlation between the seasonal profiles of two
different parameters was determined using Pearson's formula (Section
2 .2 .7 .2).
[(xi-x) (y±-y)}
[(xi-x) 2 (y^-y)2]
121
where, at a certain time-point t, and y^ are individual data values of
each parameter, and x and y are the respective mean values of each
parameter.
2.5.2 Global tests of correlation
Global tests of correlation were performed using the program
'CSPRO' (Section 2.2.7.2). Figure 2.21a shows for males and females, the
correlations between the seasonal profiles of cytochrome P-450 and
cytochrome b^ specific contents, the seasonal profiles of NADPH- and NADH-
cytochrome c reductase and NADH-ferricyanide reductase specific activities,
the seasonal changes in microsomal protein and cytochrome P-450 content
g"^ wet weight, the changes in 'total haemoprotein' and the '416' nm peak
mg"^ protein, the changes in BPH and ECOD activity, the changes in the
volume fractions of total gamete and ADG cells, and the seasonal variations
in water temperature. For both sexes, approximately 25 of the 91
correlations were indistinguishable from a random, simulated set of
correlations generated by the program. Of the remainder, approximately 40
positive correlations of each sex were distinct from the simulated set.
Approximately 20 male and 16 female negative correlations were also
distinct from the simulated set. In each case, the most positive
correlations corresponded to those between the profiles for cytochrome P-
450, the '416' nm peak and total haemoprotein. The most negative
correlations corresponded to those between the seasonal changes in gamete
and ADG cell volume fractions.
Figure 2.21b shows for males and females, the correlations
between the seasonal profiles of total cytochrome P-450 and cytochrome b^
content g"^ wet weight, the profiles of total NADPH- and NADH-cytochrome c
122
8 z
w o
N0I1V13UU00
c/5
N0I1V13UU00
<
■6 0C•in CD
0(MCU0 0
ocCCoou.
in OccHIm2uz
N0liV13bU00 N0I1V-13HU00
Figure 2.21Global correlation profiles.The parameters included in each global test are given in the text.
Solid lines - random simulated correlations Dashed lines - true set of correlations
123
1reductase and NADH-ferricyanide reductase activity g wet weight, the
changes in total haemoprotein and the '416' nm peak g wet weight, the
seasonal changes in gamete volume fraction and the seasonal variation in
water temperature. Of the 36 correlations, almost all are positive
correlations significantly distinct from the simulated set. The most
negative correlation corresponded to that between changes in gamete volume
fraction and changes in the water temperature. Again, the more positive
correlations were between the seasonal profiles of cytochrome P-450, total
haemoprotein and the '416' nm peak.
The true set of correlations in Figure 2.21b were more distinct from
the simulated set than the true set in Figure 2.21a because fewer
correlations were made. Figure 2.21 indicates that although correlations
might appear low, they may still be distinct from a random, simulated set.
124
Section 3
Partial purification and properties of digestive gland
microsomal cytochrome P-450 and other MFO components
125
3.1 Introduction
To date there have been no reports of cytochrome P-450 purification
from molluscs. In contrast, forms of cytochrome P-450 have been purified
or partially purified from a number of other poikilothermic, marine
species, for example, from fish, Salmo gairdneri (Williams and Buhler,
1982; Arin? and Adali, 1983), Stenotomus chrysops (Klotz et al., 1983) and
Gadus morhua (Goks^yr, 1985) and from crustacea, Callinectes sapidus
(Conner and Singer, 1981), Libinia emarginata, C. sapidus, Menippe
mercenaria and Uca minax (Quattrochi and Lee, 1984) and Maja crispata
(Batel et al., 1986). The aim of this section was to purify cytochrome P-
450 from M. edulis to as near homogeneity as possible, and to compare such
aspects as yield, extent of purification and isoenzyme molecular weight
with values reported in the above studies, and in studies of other marine
invertebrates and animal and plant species. The final purification
procedure consisted of a number of stages, viz. solubilization of proteins
from the microsomal membrane, fractionation of cytochrome P-450 from other
proteins by precipitation techniques, affinity chromatography on 8 -
aminooctyl sepharose 4B, ion-exchange chromatography on DEAE-sephacel, and
finally adsorption chromatography on hydroxylapatite.
Solubilization of microsomal cytochrome P-450 with ionic or non-ionic
detergents is .prerequisite in any purification procedure (Imai, 1978).
Commonly used ionic detergents include the anionic sodium cholate (e.g.
Guengerich, 1977; West et al. , 1979; King et al., 1984) and sodium
deoxycholate (e.g. Comai and Gaylor, 1973) and the zwitterionic 3-[(3-
cholamidopropyl) -dimethylammonio] - 1-propanosulphonate (CHAPS) (e.g.
Williams and Buhler, 1982). Non-ionic detergents are generally based on
polyoxyethylenephenyl ethers (Guengerich, 1979) and include, for example,
126
Triton X-100 (e.g. Miki et al., 1980; Karanlampi et al., 1986). Each of
these detergents was investigated for its ability to solubilize mussel
digestive gland microsomal cytochrome P-450 and NADPH-cytochrome c (P-450)
reductase activity. The most effective detergent, in terms of cytochrome
P-450 yield, was further utilized in purification studies.
In certain cytochrome P-450-rich tissues of non-molluscan species,
such as fish and mammalian liver, a protein fractionation step is often
unnecessary in the purification procedure (e.g. Imai et al., 1980; Williams
and Buhler, 1982; Wang et al., 1983). Because cytochrome P-450 specific
and total contents are relatively low in M. edulis (Livingstone and Farrar,
1984; see Section 1), it was found necessary to include a protein
fractionation step, partly to remove much of the contaminating material
before the chromatographic steps and partly to concentrate the cytochrome
P-450. Conditions were optimized and are described for two protein
fractionation techniques involving polyethylene glycol (PEG) and ammonium
sulphate. The further purification of cytochrome P-450 using the
chromatographic methods referred to above is described following protein
fractionation with each of these agents.
Also described in this section, are the attempts to further purify,
after 8 -aminooctyl sepharose 4B chromatography, the NADPH- and NADH-
dependent cytochrome c reductase activities by affinity chromatography on
2',5'-ADP sepharose 4B, and the effects of some of the purification steps
on NADH-ferricyanide reductase activity.
The digestive gland is rich in lysosomes which, upon homogenization,
normally break and release their proteolytic enzymes (Livingstone and
127
Farrar, 1984). Attempts to improve the microsomal specific content of
cytochrome P-450 and other MFO components by inclusion of various reagents
in the homogenization buffer were not successful, viz.
phenylmethy1sulphonyl fluoride (100 /zM; trypsin-inhibitor), leupeptin (0.1
/zM; lysosomal protease (cathepsin D) inhibitor) and cortisol (1 mM;
lysosomal stabilizer) (data not shown).
128
3.2 Materials and Methods
3.2.1 Chemicals and Materials
Sodium cholate, sodium deoxycholate, glutathione (reduced), flavin
mononucleotide (FMN) and Adenosine 2'-monophosphate (2'-AMP) were obtained
from Sigma Chemical Co. Ltd. (Poole, Dorset, U.K.). Triton X-100,
polyethylene glycol (6000) and 'Aristar' ammonium sulphate (low in heavy
metals) were from BDH Chemicals Ltd, (Poole, Dorset, U.K.). CHAPS and 1,8-
diaminooctane were from Aldrich Chemical Co. Ltd. (Gillingham, Dorset,
U.K.). CNBr-activated sepharose 4B, DEAE-sephacel, 2',5'-ADP sepharose 4B
and columns for chromatography were obtained from Pharmacia (Hounslow,
Middlesex, UK). Biogel HT (hydroxylapatite) was from Bio-rad laboratories
(Watford, Herts, UK). Emulgen 911 was from Kao-atlas corporation (Tokyo,
Japan). PM-30 ultrafiltration membranes were from Amicon Ltd, (Stonehouse,
Gloucs., UK). All other chemicals were of analar grade from sources
described in Section 2.2.2.
3.2.2 Enzyme assays and SDS PAGE
Cytochrome P-450, NAD (P)H-cytochrome c reductase, NADH-ferricyanide
reductase and total protein were measured, as described in Section 2. SDS
PAGE was as described in Section 2 and molecular weight determination by
interpolation with known protein standards in a plot of % mobility (on SDS
PAGE) versus Log molecular weight (of proteins).
129
3.2.3 Preparation of digestive gland microsomal fraction
Collection of mussels from Whitsand Bay, Cornwall, and dissection and
freezing of digestive glands were as described in Section 2.1.1.
Microsomes were prepared at 4°C by a procedure similar to that described in
Section 2.2.3, but with modifications because of the nature of the study
and the scale of preparation. Digestive gland tissue (normally 30-40 g for
solubilization and protein fractionation experiments; 80-100 g for full-
scale purifications) was homogenized in a 1:4 ratio (tissue weight:buffer
volume) in 20 mM potassium phosphate, pH 7.6, containing 0.5 M sucrose,
0.15 M potassium chloride, 1 mM EDTA and 1 mM DTT. Gentle homogenization
was performed as described in Section 2.2.3. The homogenate was
centrifuged in an MSE Hi-spin 21 centrifuge at 500 x g for 30 mins and the
resulting supernatant at 12,000 x g for 45 mins. The 12,000 x g
supernatant was centrifuged at 100,000 x g for 90 mins, using an 8 x 35 ml
angle-head rotor in either an MSE Prepspin 50 ultracentrifuge or a Beckman
L5-65 ultracentrifuge. The resulting supernatant was discarded and the
microsomal pellet 'washed' by overlaying with a small volume of 100 mM
potassium phosphate pH 7.6, containing 20% (w/v) glycerol, 1 mM EDTA and 1
mM DTT (microsomal buffer) and spinning at 12,000 x g for 30 mins. The
microsomal pellet was resuspended in this buffer to a protein concentration
of about 8 mg ml~^ for solubilization experiments, and 10-15 mg ml"^ for
fractionation and full-scale purification studies, using a hand-held
Potter-Elvehjem teflon homogenizer. The microsomal suspensions were stored
at -70°C, in 15-20 ml aliquots until required.
130
3.2.4 Solubilization of digestive gland microsomal cytochrome P-450 and
NADPH-cytochrome c (P-450) reductase activity
Solubilization of membrane-bound proteins is achieved by disruption of
hydrophobic interactions between protein and membrane using detergents
possessing lipophilic domains which bind to proteins in lieu of the normal
membrane (Scopes, 1982). The ability to solubilize cytochrome P-450 and
NADPH-cytochrome c (P-450) reductase activity from digestive gland
microsomes was determined for sodium cholate, sodium deoxycholate, CHAPS
and Triton X-100 (see Figure 3.1). The microsomal fractions were prepared
as described in Section 3.2.2. A small aliquot was removed for accurate
determinations of protein, cytochrome P-450 content and NADPH-cytochrome c
(P-450) reductase activity and the remainder divided into eight aliquots of
equal volume (normally 15 ml). Detergent was added to each aliquot to
duplicate, final concentrations of 0.5, 1.0, 1.5 and 2.0% (w/v) for
cholate, deoxycholate and CHAPS, and 0.3, 0.6, 0.9 and 1.2% (v/v) for
Triton X-100. These concentrations gave detergent to protein ratios of
approximately 0 .6 -2 .5 mg detergent per mg protein for cholate, deoxycholate
and CHAPS, and 0.375-1.5 mg detergent per mg protein for Triton X-100.
Cholate, deoxycholate and CHAPS were added slowly as the solid, while
Triton X-100 was added drop-wise as a 10% (v/v) solution in microsomal
buffer (100 mM potassium phosphate, pH 7.6, containing 20% (w/v) glycerol,
1 mM EDTA and 1 mM DTT). In each case, glutathione (reduced form) was also
added to a final concentration of 0 .1% (w/v) to prevent any autooxidation
of sample (King et al., 1984). Solubilization was carried out for 1 hour
at 4°C under an atmosphere of nitrogen before centrifugation of the
solubilized microsomal fraction at 100,000 x g for 1 hour. Protein,
cytochrome P-450 content and NADPH-cytochrome c reductase activity were
determined in both supernatant and pellet (resuspended to 5 ml in
SODIUM CHOLATE
SODIUM DEOXYCHOLATE
CHAPS
TRITON X-100
Figure 3.1
Structures of detergents used in cytochrome P-450 solubilization experiments.
microsomal buffer). Recovery of cytochrome P-450 in the supernatant was
taken as a measure of the effectiveness of solubilization.
Sodium cholate proved to be the most effective detergent. Conditions
for its use were optimized further by repeating the above experiment over
the final concentration range 1 .0 -1 .6% (w/v) (a cholate to protein ratio
range of approximately 1.25-2.0 mg per mg protein), and by determination of
the optimum length of time for solubilization at the ideal cholate
concentration.
Fractionation and purification studies were performed following
solubilization of microsomal cytochrome P-450 with 1.3% (w/v) sodium
cholate (generally, at or around the ideal cholate to protein ratio) plus
0.1% (w/v) reduced glutathione for 1 hour at 4°C under nitrogen.
Solubilized cytochrome P-450 and reductase activities were collected in the
supernatant of a 1 hour centrifugation at 1 0 0 , 0 0 0 x g.
3.2.5 Fractionation of solubilized microsomal cytochrome P-450 and
NADPH-cytochrome c (P-450) reductase activity
3.2.5.1 Fractionation with ammonium sulphate
Precipitation (salting-out) of proteins following the addition of
ammonium sulphate ((NH^^SO^) occurs because salt ions upon hydration,
effectively "trap" water molecules. The trapping of water molecules, from
areas surrounding hydrophobic regions of the protein, leads to increased
hydrophobic attractions between proteins, resulting in their aggregation
and precipitation (Scopes, 1982). Fractionation of proteins is achieved by
133
changing the (NH^^SO^ concentration by increments and spinning to
precipitate aggregated proteins. The more hydrophobic proteins precipitate
first.
3.2.5.1.1 Optimization of conditions
Digestive gland microsomes were prepared and solubilized as described
in Sections 3.2.2 and 3.2.3, respectively. The supernatant from the
solubilization procedure was divided into six aliquots of equal volume and
(NH^^SO^ added to each to cover the range 15-70% saturation.
Centrifugation was performed at 100,000 x g for 20 mins and aliquots of
supernatant and pellet (resuspended in 5 ml microsomal buffer) taken for
determination of protein, cytochrome P-450 content and NADPH-cytochrome c
(P-450) reductase activity. For ease of solubili Trcitythe (NH^^SO^ was
ground to a fine powder by pestle and mortqr prior to addition. Because of
the slight acidifying effect of (NH^^SO^, ammonium hydroxide (NH^OH) was
added drop-wise from a 0.2 M stock to maintain the pH between pH 7.0 and pH
7.6 (departure from the pH 7.6 of the microsomal buffer toward pH 7.0 was
not deleterious to the cytochrome P-450). The amount of (NH^^SO^ required
was determined from tables in Green and Hughes (1955).
3.2.5.1.2 (NH/jgSO/-fractionation as used in purification
(NH^^SO^ was added to solubilized cytochrome P-450 and NADPH-
cytochrome c (P-450) reductase activity to 35% saturation, and the sample
centrifuged at 100,000 x g for 20 mins. To the supernatant, (NH^^SO^ was
further added to 65% saturation. Centrifugation at 25,000 x g for 20 mins
was sufficient to pellet the cytochrome P-450 and reductase activity. The
134
pellet was resuspended in a reduced volume (to approximately 1 0 - 1 2 mg
protein ml’ ) in 20 mM potassium phosphate, pH 7.0, containing 20% (w/v)
glycerol, 1 mM EDTA, 1 mM DTT and 0.3% cholate. This was dialysed
overnight against 2 x 30 volumes of 10 mM potassium phosphate, pH 7.0,
containing 20 % (w/v) glycerol, 1 mM EDTA, 1 mM DTT and 0.3% (w/v) cholate
to remove the (NH^^SO^. Insoluble material was removed by centrifugation
at 100,000 x g for 50 mins, and the supernatant containing fractionated
cytochrome P-450 and reductase activity applied to a column of 8 -aminooctyl
sepharose 4B (Section 3.2.6)
3.2.5.2 Fractionation with polyethylene glycol (PEG)
PEG is a polymerized organic solvent and causes precipitation of
proteins by a mechanism similar to that by which organic solvents
precipitate proteins (Scopes, 1982). Aggregation and precipitation in the
presence of PEG is likely due to electrostatic and dipolar Van der Waals
forces between proteins caused by a reduction in water activity, rather
than through an increase in hydrophobic attractions (organic solvents and
PEG would tend to increase the solubility of hydrophobic areas of
proteins). The solvating power of aqueous solutions for charged,
hydrophilic areas of protein is decreased as the concentration of PEG
increases. Larger proteins tend to aggregate sooner because they have a
greater chance of possessing charged areas on their surface that match up
with other proteins (although the composition and net charge of proteins
also have effects).
135
3.2.5.2.1 Optimization of conditions
Conditions for fractionation by PEG were optimized as described in
Section 3.2.5.1.1 for fractionation by (NH^^SO^. Only the fractionation
of cytochrome P-450 by PEG was assessed. PEG was added drop-wise from a
50% (w/v) stock to cover the range 3-18% (w/v). Centrifugation was
performed at 1 0 0 , 0 0 0 x g for 2 0 mins.
3.2.5.2.2 PEG fractionation as used in purificaton
PEG was added to solubilized cytochrome P-450 to a final concentration
of 4% (w/v) and the sample centrifuged at 100,000 x g for 20 mins. To the
supernatant, PEG was further added to a final concentration of 15% (w/v)
and the sample centrifuged at 35,000 x g for 20 mins. The supernatant was
discarded, and the pellet resuspended in a small volume of 15 mM potassium
phosphate, pH 7.0, containing 20% (w/v) glycerol, 1 mM EDTA, 1 mM DTT and
0.3% (w/v) cholate (to about 10 mg protein ml"^) and applied directly to a
8 -aminooctyl sepharose 4B affinity column.
3.2.6 Affinity chromatography on 8-aminooctyl sepharose 4B
3.2.6.1 Preparation of 8-aminooctyl sepharose 4B
Cyanogen bromide-activated sepharose 4B (normally 30 g; supplied as a_ 3freeze-dried powder) was swollen in an excess of 10 M HC1 (approximately
200 ml g"^ sepharose 4B) on a sintered-glass filter under vacuum-suction.
The washed gel (approximately 120 ml final volume) was coupled to 1,8-
diaminooctane (normally 30 g dissolved in 150 ml coupling buffer; 0.1 M
136
sodium hydrogen carbonate, pH 8.3, containing 0.5 M sodium chloride) by
gently stirring at room-temperature for 4-5 hours. Excess ligand was
washed away with coupling buffer on the sintered-glass filter. Any
remaining unreacted groups on the activated sepharose 4B matrix were
blocked by gently stirring for 1 hour in 0.1 M TRIS, pH 8.0. The fully
derivatised sepharose 4B was washed on the sintered filter under suction
with three cycles of 0.1 M sodium acetate, pH 4.0, containing 0.5 M sodium
chloride, followed by 0.1 M TRIS, pH 8.0, containing 0.5 M sodium chloride.
The product was finally washed in 100 mM potassium phosphate, pH 7.0,
containing 20% (w/v) glycerol, 1 mM EDTA, 1 mM DTT and 0.3% (w/v) sodium
cholate and, either used immediately for chromatography, or stored at 4°C
as an approximately 2 0% (v/v) suspension in the final wash buffer, also
containing 0 .0 2 % (w/v) sodium azide.
3.2.6.2 Column pouring and equilibration
Prior to the pouring of the column, the gel was washed on the sintered
filter under suction with 15 mM potassium phosphate, pH 7.0, containing 20%
(w/v) glycerol, 1 mM EDTA, 1 mM DTT and 0.3% (w/v) sodium cholate
(equilibration buffer). This step also removed sodium azide from 8 -
aminooctyl sepharose 4B previously stored at 4°C. A small volume
(approximately 5 ml) of equilibration buffer was poured into the bottom of
an assembled chromatography column and allowed to run through the outlet
tap to displace air trapped under the column support-bed. With the outflow
fully closed, 8 -aminooctyl sepharose 4B was carefully poured into the
column as a 2 0 % (v/v) suspension and allowed to settle under gravity for
24-36 hours at 4°C. In early experiments, typical bed-volumes were 25-30
ml in columns 1.5 cm in diameter and approximately 15 cm in length.
137
Improved chromatographic results were later achieved with bed-volumes of
approximately 50 ml in columns 2.6 cm in diameter and approximately 10 cm
in length. When settling of the gel was complete, at least four column
volumes of equilibration buffer were run through at the flow rate to be
used during chromatography (normally 24 ml h"^). The pH of the eluate was
monitored during equilibration to ensure a pH of 7.0.
3.2.6.3 Chromatography on 8-aminooctyl sepharose 4B
The solubilized and fractionated sample containing cytochrome P-450
and reductase activities (approximately 1 0 - 1 2 mg protein ml"^) was applied
carefully to the top of the equilibrated column and allowed to run onto the
gel by gravity. At least three column volumes of equilibration buffer were
run through the column to wash off unadsorbed protein. Cytochrome P-450
was then eluted as a relatively broad peak with equilibration buffer also
containing 0.2% (v/v) emulgen 911 (a non-ionic detergent). During column
washing and cytochrome P-450 elution, 8 ml fractions of eluate were
collected. In early studies on the efficiency and applicability of 8 -
aminooctyl sepharose 4B chromatography, each eluted fraction was assayed
for both cytochrome P-450 content and reductase activity. In later
experiments, elution of haemoprotein (absorbance at 417 nm) was used as an
indicator of cytochrome P-450 elution. Only fractions with a high 417 nm
absorbance were assayed for cytochrome P-450. To enable its further
purification on DEAE-sephacel, fractions containing cytochrome P-450 were
pooled and the total volume reduced by ultrafiltration (Section 3.2.7).
On occasions, the purifications of NADPH-cytochrome c(P-450) and NADH-
cytochrome c reductase activities were attempted. Following the complete
elution of cytochrome P-450, the column was washed with a further two
138
column volumes of cytochrome P-450 elution buffer. The cytochrome c
reductase activities were then eluted from the column with cytochrome P-450
elution buffer also containing 0.2% (w/v) sodium deoxycholate and 2 /zM
flavin mononucleotide (FMN). Fractions containing reductase activity were
also pooled.
3.2.7 Ultrafiltration and dialysis of pooled fractions from 8-aminooctyl sepharose 4B chromatography
The cytochrome P-450 pool from affinity chromatography was reduced in
volume to 10-15 ml by ultrafiltration, at 4°C, in Amicon Diaflow
ultrafiltration cells of either 100 or 500 ml capacity. Ultrafiltration
was performed under nitrogen at 30 psi using PM-30 ultrafiltration
membranes (30 kd cut-off). The pH of the concentrated sample was adjusted
to pH 7.6 by dialysis for 6 - 8 hours against 3 x 30 volumes of 0.01 M
potassium phosphate, pH 7.7, containing 20% (w/v) glycerol and 0.2% (v/v)
emulgen 911. This step also removed EDTA, DTT and cholate from the sample.
When attempts were made to further purify NAD(P)H-cytochrome c
reductase activity on 2',5'-ADP sepharose 4B (Section 3.2.11), the pooled
reductase sample from affinity chromatography was concentrated by
ultrafiltration as described above, and dialysed overnight against 2 x 30
volumes of 0.3 M potassium phosphate, pH 7.7, containing 20% (w/v)
glycerol, 1 mM EDTA, 1 mM DTT and 0.2% (v/v) emulgen 911.
139
3.2.8 Ion-exchange chromatography on DEAE-sephacel
3.2.8.1 Preparation of DEAE-sephacel columns
The required amount of medium was transferred to a sintered-glass
filter and washed under gravity with approximately 3 L of distilled water
to remove the storage solution (DEAE-sephacel was supplied as a suspension
in 20% ethanol). The chloride counter-ion was exchanged with phosphate and
the medium set to the correct pH by transferring to an excess volume of
0.4 M potassium phosphate, pH 7.6, and leaving overnight at 4°C. The
medium was then washed on the sintered filter with a further 500 ml of
0.4 M potassium phosphate, pH 7.6, followed by 1 L of 0.01 M potassium
phosphate, pH 7.6, containing 20% (w/v) glycerol and 0.2% (v/v) emulgen 911
(equilibration buffer). DEAE-sephacel was poured as a 20% (v/v) suspension
as described in Section 3.2.6 .2 for the pouring of 8 -aminooctyl sepharose
4B. Column bed-volumes were normally approximately 10 ml, in columns of
diameter 0.9 cm and approximate length 16 cm. Once the column was fully
packed, approximately two column volumes of equilibration buffer were run
through at the flow rate to be used during chromatography (normally 24 ml
h'1).
3.2.8 .2 Chromatography on DEAE-sephacel
The concentrated and dialysed cytochrome P-450-containing pool from
affinity chromatography was loaded carefully onto an equilibrated DEAE-
sephacel column by gravity. The column was washed with at least three
column volumes of equilibration buffer to remove unadsorbed protein, before
elution of cytochrome P-450 with increasing ionic strength in the form of a
linear potassium chloride (KC1) gradient. An increasing ionic strength
140
gradient was used (in preference to a step-wise increase in ionic strength)
in an attempt to detect cytochrome P-450 isoenzymes. The eluate from both
washing and elution stages was collected as 2 or 3 ml fractions and
measured spectrophotometrically at 417 nm for the presence of haemoprotein.
The KC1 gradient was established between either 0 and 0.4 M or 0 and 0.5 M
over a total volume of 400 ml using the apparatus outlined in Figure 3.2
(below) .
-►TO COLUMN
MAGNETIC STIRRERFigure 3.2
Apparatus for the generation of a linear KC1 gradient. Beaker A contained 200 ml DEAE-sephacel equilibration buffer. Beaker B contained 200 ml equilibration buffer containing 0.8 M or 1.0 M KC1. (From Scopes, 1982)
The approximate concentration of KC1 in any given fraction was
determined by interpolation between the KC1 concentration at the start of
the gradient (zero) and the concentration after 400 ml of gradient (0.4 M
or 0.5 M) . Fractions from individual peaks were pooled, assayed for
cytochrome P-450 content and protein, concentrated by ultrafiltration, and
dialysed before attempts were made to remove emulgen 911 by chromatography
on hydroxylapatite (Section 3.2.10).
141
3.2.9 Ultrafiltration and dialysis of pooled samples from DEAE-sephacel
chromatography
Pooled fractions from DEAE-sephacel chromatography were reduced in
volume to 4-5 ml by ultrafiltration over an Amicon PM-30 membrane as
described in Section 3.2.7. The concentrated pool containing the highest
content of cytochrome P-450 was then dialysed for 6 - 8 hours against 2 x 30
volumes of 0.02 M potassium phosphate, pH 7.6, containing 20% (w/v)
glycerol and 0.2% (v/v) emulgen 911 to remove KC1 from the sample before
application to hydroxylapatite.
3.2.10 Removal of emulgen 911 on hydroxylapatite
3.2.10.1 Preparation of hydroxylapatite columns
The required amount of hydroxylapatite was transferred to a 5-fold
excess volume of 0.02 M potassium phosphate, pH 7.6, containing 20% (w/v)
glycerol and 0.2% (v/v) emulgen 911 (column equilibration buffer) and
gently swirled into suspension. The gel was allowed to settle, leaving a
cloudy buffer volume above containing slowly sedimenting fine particles,
which were decanted. Fresh buffer was added and the procedure repeated
four times. The equilibrated hydroxylapatite was then poured, as described
previously, to give, after packing by gravity, a final bed-volume of
approximately 2 ml in a column 0.9 cm in diameter and approximately 3 cm in
length. A further 30-40 ml of equilibration buffer was run through the_ ncolumn at the flow rate (normally 8 ml h ) used during chromatography.
142
3.2.10.2 Chromatography on hydroxylapatite
The concentrated and dialysed cytochrome P-450 pool from DEAE-sephacel
was loaded onto the hydroxylapatite column by gravity. The column was
washed with 0.02 M potassium phosphate, pH 7.6, containing 20% (w/v)
glycerol (minus emulgen 911), and 1 ml fractions of eluate collected. Each
fraction was monitored at 417 nm for the presence of haemoprotein and at
280 nm for the presence of emulgen 911. Washing was continued until the
absorbance at 280 nm had decreased to zero. The cytochrome P-450 that
bound was eluted with 300 mM potassium phosphate, pH 7.6, containing 20%
(w/v) glycerol. These fractions were pooled and concentrated by
ultrafiltration using an Amicon, Centricon 10 (10 Kd cut-off)
microconcentrator before assessment of purity on SDS PAGE. The final
cytochrome P-450 sample obtained was stored in freeze-drying vials, under
nitrogen, at -70°C.
3.2.11 Further purification of NADPH-cytochrome c (P-450) and NADH-
cytochrome c reductase activities on 2 *,51-ADP sepharose 4B
3.2.11.1 Preparation of 2*,5f-ADP sepharose 4B columns
2',5'-ADP sepharose 4B (supplied pre-swollen) was transferred to 0.3 M
potassium phosphate, pH 7.6, containing 20% (w/v) glycerol, 1 mM EDTA, 1 mM
DTT and 0.2% (v/v) emulgen 911 (equilibration buffer). The column was
poured as described previously to give a final packed bed-volume of
approximately 20 ml (1.5 cm in diameter by approximately 12 cm in length).
A further 2-3 column volumes of equilibration buffer were run through the
column at the flow-rate used during chromatography (normally 30 ml h” ).
143
3.2.11.2 Chromatography on 21,5' -ADP sepharose 4B
The concentrated and dialysed reductase activity pool from 8 -
aminooctyl sepharose 4B chromatography was loaded onto the 2 ',5'-ADP
sepharose 4B column by gravity. The column was washed with 40-50 ml of
equilibration buffer and then the reductase activities eluted with
equilibration buffer also containing 5 mM 2'-AMP. 3 ml fractions of both
washing and elution stages were collected.
144
3.3 Results
3.3.1 Solubilization of digestive gland microsomal cytochrome P-450 andNADPH-cytochrome c (P-450) reductase activity
A comparison of the ability of sodium cholate and sodium deoxycholate
to solubilize cytochrome P-450 from microsomes is shown in Table 3.1.
Similar trends were observed for solubilization by both detergents,
although yield of solubilized cytochrome P-450 i.e. that present in the
supernatant, was higher with cholate at each concentration. Maximum yields
of solubilized cytochrome P-450 were obtained following treatment with
either detergent at a concentration of 1.5% (w/v) (equivalent to a
detergent to protein ratio of approximately 1.75 mg per mg protein). Only
partial solubilization was observed at a detergent concentration of 0.5%
(w/v) (approximately 0 . 6 mg detergent per mg protein), whereas at a
concentration of 2.0% (w/v) (approximately 2.3-2.4 mg detergent per mg
protein) a reduced recovery in the supernatant was indicative of
denaturation of the cytochrome P-450 by detergent. Following treatment
with either detergent, the total recovery of cytochrome P-450 i.e. that in
the supernatant plus pellet, decreased as the ratio of detergent to protein
increased, indicative, again, of denaturation. At a detergent to protein
ratio of about 1.75 mg per mg protein (1.5% w/v), approximately 92% of
total recovered cytochrome P-450 was fully solubilized by cholate, but only
approximately 81% by deoxycholate. Of the two detergents, cholate was the
better choice.
The cholate to protein ratio required to maximize the yield of
solubilized cytochrome P-450 was further optimized (Table 3.2). Also shown
145
Table 3.1
Solubilization of cytochrome P-450 from mussel digestive gland microsomes by (a) sodium cholate and (b) sodium deoxycholate
(a)
Sample, % cholate and ratio of cholate to to protein
Protein mg ml~^
cytochrome P-450 , pmoles mg
fold-purificationYield
%
unsolubilizedmicrosomes 8.6 54.3 1 1 0 0
0.5% supernatant 0.58 pellet
5.2 34.06 0.64 34.210.7 76.5 1.41 58.4
1 .0 % supernatant 1.16 pellet
6.3 48.8 0.90 63.76.5 41.2 0.76 19.1
1.5% supernatant 1.74 pellet
6 .6 52.6 0.97 71.45.1 16.2 0.30 5.9
2 .0 % supernatant 2.30 pellet 6.7 45.7 0.84 63.4
5.3 14.3 0.26 5.4
(b)
Sample, % deoxycholate ratio of deoxycholate to protein
Protein mg ml~^
cytochrome P-450 , pmoles mg
fold-purification
Yield%
unsolubilizedmicrosomes 8 .2 56.8 1 1 0 0
0.5% supernatant 0.61 pellet
4.9 28.1 0.49 28.613.3 64.9 1.14 61.8
1 .0 % supernatant 1 . 2 2 pellet 5.9 45.2 0.79 54.66.4 64.8 1.14 29.7
1.5% supernatant 1.83 pellet
6.4 47.2 0.83 62.35.6 36.9 0.65 14.8
2 .0 % supernatant 2.44 pellet
6.4 45.7 0.80 60.75.2 25.5 0.45 9.5
(Pellets were resuspended in 5 ml; 1 hour solubilization period; values are means of duplicate samples; for other details see text).
146
Table 3.2
Determination of the optimum ratio of cholate to protein for the solubilization of digestive gland
microsomal cytochrome P-450 and NADPH- cytochrome c reductase activity.
Sample. % cholate Protein Cytochrome P-450 NADPH-cytochrome c reductaseand ratio of cholate to protein
mg ml ^ content pmoles mg
fold-purification
Yield(*)
activitynmoles. -1 -1m m mg
fold-purification
Yield(*)
unsolubilizedmicrosomes 7.8 53.7 1 100 9.7 1 100
1.0 supernatant 5.8 48.9 0.91 65.0 9.3 0.96 68.41.28 pellet 6.4 32.9 0.61 16.8 5.5 0.57 15.6
1.2% supernatant 6.2 49.2 0.92 70.4 9.2 0.95 72.31.54 pellet 5.5 23.8 0.44 10.4 5.3 0.55 12.8
1.4% supernatant 6.0 51.7 0.96 71.2 9.3 0.96 71.31.79 pellet 5.7 17.9 0.33 8.1 4.7 0.48 11.7
1.6% supernatant 6.2 48.6 0.90 69.5 9.1 0.94 71.62.05 pellet 5.1 15.0 0.28 6.1 5.4 0.56 12.1
(other details as for Table 3.1)
147
in Table 3.2 are the effects of cholate on the solubilization of NADPH-
cytochrome c (P-450) reductase activity. The highest yields of solubilized
cytochrome P-450 (approximately 70-71%) were obtained at cholate to protein
ratios of 1.54 to 1.79 mg per mg protein. Although the total recovery at
these concentrations was only about 80%, the solubilized fraction
corresponded to about 87-89% of that recovered. A slightly higher fraction
of recovered cytochrome P-450 (approximately 92%) was solubilized at a
cholate to protein ratio of 2.05 mg per mg protein, but the total recovery
was lower. The results of Tables 3.1 and 3.2 indicate that irrespective of
the cholate to protein ratio, no more than 92% of total recovered
cytochrome P-450 is in the supernatant following centrifugation at 100,000
x g. (It is likely that a small amount of P-450 would be precipitated
under these centrifugation conditions). The solubilization of NADPH-
cytochrome c (P-450) reductase activity was roughly equivalent at each
cholate to protein ratio. Total recovery of activity was about 85%, of
which approximately 85% was solubilized.
The results obtained with sodium cholate suggested a ratio of
approximately 1.75 mg cholate per mg protein would be optimal for the
solubilization of cytochrome P-450, and suitable also for the
solubilization of NADPH-cytochrome c (P-450) reductase activity. A time-
course, performed from 0.5 h to 2 h, indicated the optimal length of time
for solubilization was 1 h (data not shown). No increase in yield was seen
at times above 1 h, and at 2 h the total recovery of P-450 was
significantly reduced, presumably because of denaturation.
Table 3.3 shows the effect of different concentrations of CHAPS on the
solubilization of cytochrome P-450 and NADPH- cytochrome c (P-450)
reductase activity. The pattern of solubilization was similar to that
148
Table 3.3
Determination of the optimum ratio of CHAPS to protein for the solubilization of digestive gland
microsomal cytochrome P-450 and NADPH-cytochrome c reductase activity.
Sample, % CHAPS Protein Cytochrome P-450 NADPH-cytochrome c reductaseand ratio of CHAPS to protein
.-1mg ml content pmoles mg
fold- purification
Yield(*)
activitynmoles. -1 -1min mg
fold-purification
Yield(*)
unsolubilizedmicrosomes 8.1 51.6 1 100 9.2 1 100
0.5 supernatant 4.8 34.2 0.66 36.6 6.5 0.70 40.60.62 pellet 11.2 65.2 1.26 56.2 10.9 1.18 54.8
1.0 supernatant 5.8 47.8 0.93 61.8 8.6 0.93 64.71.23 pellet 6.4 49.4 0.96 24.3 5.9 0.64 16.9
1.5% supernatant 6.3 47.3 0.92 66.4 8.8 0.96 71.91.85 pellet 5.7 29.4 0.57 12.9 3.3 0.36 8.4
2.0% supernatant 6.4 45.7 0.89 65.2 8.5 0.92 70.62.45 pellet 5.5 18.5 0.36 7.8 3.1 0.34 7.6
(other details as for Table 3.1)
149
observed for cholate (Table 3.1). The total recovery decreased with
increasing ratios of CHAPS to protein, and the maximum recovery of
solubilized cytochrome P-450 occurred at a CHAPS to protein ratio of
approximately 1.7-1.85 mg per mg protein. With the exception of the lower
cholate to protein ratios, the total recovery of cytochrome P-450 was
slightly higher following CHAPS treatment, although the fraction in the
supernatant was lower. The solubilization of NADPH-cytochrome c (P-450)
reductase activity by CHAPS was similar to that by cholate. A maximum
recovery of solubilized activity (approximately 72%), corresponding to
approximately 90% of the total recovered activity, was obtained with 1.85
mg CHAPS per mg protein.
The ability of different concentrations of Triton X-100 to solubilize
P-450 and NADPH-cytochrome c (P-450) reductase activity is shown in Table
3.4. Unfortunately the ratio of Triton to protein required to obtain
maximum solubilization of P-450 was not determined. However, at a Triton
to protein ratio of approximately 1.45 mg per mg protein, only 58.4% of
total P-450 (approximately 73% of the total recovered after solubilization)
was in the supernatant after centrifugation. Although the total recovery
was approximately the same, this was considerably less than that obtained
with cholate at approximately the same ratio of detergent to protein (Table
3.2). With this ratio, both the total recovery of reductase activity
(approximately 77%) and the yield of solubilized reductase activity
(approximately 78% of the total recovered activity) were less than that
obtained with cholate.
The yields of solubilized P-450 following treatment with cholate,
deoxycholate, CHAPS and Triton X-100 are shown in Figure 3.3. Cholate at a
150
Table 3.4
Solubilization of digestive gland microsomal cytochrome P-450 and NADPH-cytochrome c reductase
activity by Triton X-100 over a range of Triton : protein ratios.
Sample, $ triton Protein Cytochrome P-450 NADPH-cytochrome c reductaseand ratio of Triton to protein
.-1mg ml content pmoles mg
fold-purification
Yield(*)
activitynmoles. -1 -1min mg
fold- Yield purification ($)
unsolubilizedmicrosomes 8.3 57.2 1 100 9.5 1 100
0.3$ supernatant 3.9 27.1 0.47 21.5 5.4 0.57 25.80.36 pellet 13.5 75.1 1.31 71.2 12.0 1.26 68.5
0.6$ supernatant 5.1 36.4 0.64 37.8 6.9 0.73 42.80.72 pellet 9.7 74.9 1.31 51.0 10.8 1.14 44.4
0.9$ supernatant 5.8 44.6 0.78 52.5 7.5 0.79 53.41.08 pellet 7.7 57.9 1.01 31.3 8.0 0.84 26.1
1.2$ supernatant 6.0 48.1 0.84 58.4 8.1 0.85 60.21.45 pellet 7.1 43.6 0.76 21.7 5.5 0.58 16.5
(see text for other details)
151
CYTOCHROME
P-450
RECOVERED
(X)
BO
60
40
20
1.5 2.50.0 0.5 1.0 2.0DETERGENT : PROTEIN RATIO
Figure 3.3. Yield of solubilized digestive gland microsomal cytochrome P-450 following treatment with :cholate (-------- ): deoxycholate (--------):CHAPS (-------- ); Triton X-100 (--------);
152
ratio of about 1.75 mg per mg protein was clearly the most effective
detergent. The results presented in Figure 3.3 were obtained over a few
days from the same collection of mussels and give no indication of any
seasonal variation of solubilization. Although preliminary experiments,
comparing cholate, Triton and deoxycholate and performed on different
batches of mussels, all indicated cholate to be the more effective
detergent, yields from the solubilization stage of cytochrome P-450
purifications varied between about 60 and 80%. Generally, higher
recoveries were obtained when the seasonal microsomal specific content was
high.
3.3.2 Ammonium sulphate and PEG fractionation of solubilized digestive
gland microsomal cytochrome P-450 and NADPH-cytochrome c (P-450)
reductase activity
The precipitation of solubilized cytochrome P-450 and NADPH-cytochrome
c (P-450) reductase activity by (NH^^SO^ is shown in Table 3.5. At
concentrations of (NH^^SO^ up to about 40% saturation, most of the
cytochrome P-450 and reductase activity was retained in the supernatant.
At concentrations above 40% saturation, both the cytochrome P-450 and
reductase activity began to be precipitated, until at 70% (NH^^SO^ all the
recovered cytochrome P-450 and 97% of recovered reductase activity was
precipitated. Figure 3.4(a) shows the precipitation profile for cytochrome
P-450 by (NH^^SO^ at different concentrations. A similar profile was
obtained for NADPH-cytochrome c (P-450) reductase activity (data not
shown). Also shown is the decrease in the ratio of cytochrome P-450 in the
supernatant to cytochrome P-450 in the pellet with increasing (NH^^SO^.
153
The
precipitation
of solubilized
digestive
gland
microsomal
cytochrome
P-450
and
NADPH-cytochrome
c reductase
activity
by (NH.).SO,.
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154
SUPERNATANT
: PELLET
RATIO
SUPERNATANT
: PELLET
RATI
O
- 10025-
6020 -
6015-
4010 -
20
705040 600 10 20 30AMMONIUM SULPHATE SATURATION (X)
- 10015-
60
1060
40
20
180 12 153 6 9PERCENTAGE PEG (v/v)
Figure 3.4. The precipitation of solubilized digestive glandcytochrome P-450 (----- ). and the ratio of cytochromeP-450 in the supernatant to cytochrome P-450 in thepellet (------), at different concentrations of(a) ammonium sulphate and (b) polyethylene glycol (PEG).
155
CYTOCHROME P-450
IN PELLET
(X) CYTOCHROME
P-450 IN
PELLET
Despite the relatively low yields at (NH^^SO^ saturations greater
than 50%, a partial purification of both cytochrome P-450 and NADPH-
cytochrome c (P-450) reductase activity was obtained. Addition of
(NH^^SO^ to 30 and 40% saturation caused the precipitation of,
respectively, 19 and 25% of the total protein, but only 9 and 13.5% of the
total cytochrome P-450 (respectively, 10 and 16% of the total recovered),
and 5.5 and 11% of the total reductase activity (6 and 14% of the total
activity recovered). Addition of (NH^^SO^ to 60 and 70% saturation caused
the precipitation of, respectively, 45 and 50% of the total protein, but 57
and 64% of the total cytochrome P-450 (83 and 100% of that recovered), and
55.5 and 6 6% of the reductase activity (76.5 and 97% of that recovered).
As a step in the purification of either cytochrome P-450 or NADPH-
cytochrome c (P-450) reductase activity, an acceptable compromise was to
take the fraction precipitating between 35 and 65% (NH^^SO^ saturation.
The yield of protein in this fraction was indicated to be about 25%, while
the yields of both cytochrome P-450 and reductase activity were
approximately 50% of the original content or activity. The net
purification for a 35-65% fractionation step was thus indicated to be about
2-fold. Slightly higher yields might have been obtained using a larger
(NH^^SO^ fraction (e.g. 25-70% saturation), but with a concomitant
reduction in purification.
Table 3.6 and Figure 3.4(b) shows the precipitation of solubilized
cytochrome P-450 by PEG. Figure 3.4(b) also shows how the ratio of
cytochrome P-450 in the supernatant to cytochrome P-450 in the pellet
decreases with increasing concentrations of PEG. The pattern of
precipitation was essentially the same as that observed for (NH^^SO^,
although possibly less abrupt. At a concentration of 3% (v/v) PEG,
156
Table 3.6
The precipitation of solubilized digestive gland microsomal cytochrome P-450 by PEG.
Sample and concentration of PEG
Protein.-1 mg ml
Content pmoles mg
Foldpurification
Yield%
Total contenl ratio*
supernatant fromsolubilisation 6.15 43.89 1 100 -
step
supernatant3%
5.39 46.24 1.02 86.6414.70
pellet 2.72 17.96 0.41 6.03
supernatant6%
4.74 41.97 0.96 71.254.02
pellet 4.41 32.54 0.74 17.72
supernatant9*
4.37 33.04 0.75 52.421.60
pellet 5.57 47.70 1.09 32.81
supernatant12%
4.04 18.08 0.41 26.700.48
pellet 6.64 68.47 1.56 56.14
supernatant15%
3.40 6.43 0.15 8.100.11
pellet 8.13 72.45 1.65 72.74
supernatant18%
3.06 ND - -
pellet 9.14 67.63 1.54 76.34
Values are means of duplicate samples.* ratio of cytochrome P-450 in supernatant to that in pellet ND = not detectable
157
approximately 93% of the recovered cytochrome P-450 was retained in the
supernatant. Above 3% (v/v) PEG, increasing amounts of cytochrome P-450
were precipitated, until at a PEG concentration of 18% (v/v) all of the
recovered cytochrome P-450 (76.3% of the total) was in the pellet following
centrifugation.
The fraction precipitating between 4 and 15% (v/v) PEG, comprising
approximately 28% of the total protein, was selected as being suitable for
subsequent studies on cytochrome P-450 purification. The yield of total
cytochrome P-450 in this fraction was indicated to be approximately 63%,
giving an overall step-purification of about 2.3. Again, improved recovery
at the expense of purification would have been achieved with a larger PEG
fraction.
Comparison of Tables 3.5 and 3.6 suggests that of the two techniques,
cytochrome P-450 fractionation by PEG was the more preferable. Although a
4-15% PEG fraction was indicated to contain slightly more protein than a
35-65% (NH^^SO^ fraction, the yield and step-purification of cytochrome P-
450 were greater. Unfortunately, these results were obtained from mussels
collected at different times of the year, and it is possible the observed
differences between PEG fractionation and (NH^^SO^ fractionation reflected
seasonal differences between the samples. Yields of cytochrome P-450
following (NH^^SO^ fractionation were generally between 45 and 60%,
whereas those from PEG fractionation were between 55 and 70%. Again, the
highest yields tended to be when the microsomal specific contents were
high. Of the two techniques, protein fractionation by (NH^^SO^ is
considered the more harsh (Scopes, 1982). The fractionation of reductase
activity by PEG was not assessed.
158
3.3.3 8-aminooctyl sepharose 4B affinity chromatography of cytochrome
P-450 and other MFO components
The elution of solubilized and (NH^^SO^-fractionated cytochrome P-
450 and other components of the mussel MFO system from an 8 -aminooctyl
sepharose 4B affinity column was typically as shown in Figure 3.5.
Cytochrome P-450 co-eluted with NADH-ferricyanide reductase activity with
column equilibration buffer containing 0.2% (v/v) emulgen 911. NADPH- and
NADH-cytochrome c reductase activities remained bound to the column during
cytochrome P-450 elution, but were subsequently eluted in cytochrome P-450
elution buffer containing also 2% (w/v) deoxycholate and 2 /xM FMN. Table
3.7 gives the yields and purification factors of each of these components
after elution and pooling of, respectively, cytochrome P-450/NADH-
ferricyanide reductase activity-containing fractions, and NADPH-/NADH-
cytochrome c reductase activity-containing fractions. Although both the
yields and purifications of cytochrome P-450 after solubilization and
(NH4/ 2SO4 - fractionation were typical of values normally obtained, the
step-yield and purification values for affinity chromatography (54.6% and
4.76, respectively) in this particular experiment were particularly high.
Yields from 8 -aminooctyl sepharose 4B, following (NH4/ 2SO4 -fractionation,
were normally 40-50%, and the purification between 2- and 4-fold. It was
possible the high values obtained in this study were a consequence of the
seasonally high microsomal specific content (72.6 pmoles mg"^ protein). The
yields of reductase activities following affinity chromatography were less
than that for cytochrome P-450, although higher recoveries of each,
particularly of NADH-ferricyanide reductase activity, would have been
obtained, at the expense of purity, had a larger number of reductase-
containing fractions been pooled.
159
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160
161
Although a final cytochrome P-450 yield of 18.2% was obtained in this
study, the yield normally obtained after (NH^)2 ^0 ^-fractionation and
affinity chromatography was between 8 and 14%. Attempts to increase the
yield by omitting the protein fractionation step were largely unsuccessful
because the high protein and pigment content of the sample appeared either
to overload the capacity of the column, or to interfere with the binding of
cytochrome P-450, as very little of the latter was generally observed. In
addition, problems were encountered with maintaining flow rate (a column
1.5 cm in diameter was used) and with cleaning up the affinity column after
use. However, on an occasion when the microsomal specific content was
relatively high, a degree of binding was achieved. The results (given in
Table 3.8) indicate that no improvements on either yield or purification
were gained from the omission of the (NH^) 2 *0 ^-fractionation step.
Although values for total cytochrome P-450 recovery and purification (16.9
and 3.6, respectively) were similar to values obtained in studies
incorporating an (NH^^SO^-fractionation step, only 27% of the cytochrome
P-450 loaded onto the column was successfully recovered as a partially pure
fraction. Over 40% of the cytochrome P-450 failed to bind and eluted
directly during column-washing. A similar situation was apparent for NADH-
ferricyanide reductase activity, whereas the yields and purifications for
NADPH- and NADH-cytochrome c reductase activities were considerably lower.
In the absence of a protein fractionation step, the time required for
sample loading and elution was greatly increased. Although the time
involved may have been reduced and the yields perhaps improved by using a
column of greater diameter, the results of this study generally highlighted
the necessity for a protein fractionation step in the purification
procedure, particularly during times of the year when microsomal specific
content was low.
162
163
3.3.4 A purification scheme, involving (NH7|)g^O/, -fractionation, for
digestive gland microsomal cytochrome P-450
A number of cytochrome P-450 purifications were performed with
(NH^^SO^ as the protein fractionating agent. In early studies the
affinity chromatography step was performed on columns 1.5 cm in diameter.
In later studies a column of 2.6 cm diameter was used. Although similar
results for P-450 yield and purification were obtained for both columns,
the greater capacity of the wider column enabled the loading and
purification of a larger amount of P-450.
Table 3.9 shows a complete scheme for the partial purification of
cytochrome P-450, incorporating (NH^)2 ^0 ^-fractionation followed by
affinity chromatography on a column 2.6 cm in diameter. The haemoprotein
elution profile from the affinity column (Figure 3.6) was typical of
profiles normally obtained. Approximately 15% of the total haemoprotein
failed to bind to the column. Figure 3.7 shows cytochrome P-450 carbon
monoxide-difference spectra for crude microsomes, the 35-65% (NH^^SO^-
fractionated sample and the concentrated, post-affinity-column sample. The
spectra up to and including the protein fractionation step were essentially
similar, having a large '416' nm component relative to the cytochrome P-450
peak. Affinity chromatography, however, removed a large proportion of the
'416' nm component, as illustrated by the P-450/'416' nm peak ratio (Table
3.9; column 12).
The haemoprotein-containing fractions from the affinity
chromatography step were pooled, concentrated and dialysed as described in
Section 3.2.7, assayed for cytochrome P-450 and applied finally to a DEAE-
sephacel ion-exchange column. The elution of haemoprotein from this column
164
The partial
purification of
digestive
gland
microsomal cytochrome P-450
by sodium cholate
solubilization,
(NH^)2SO^-fractionation,
8-aminooctyl sepharose
4B affinity chromatography and DEAE-
sephacel ion-exchange chromatography.
(mussels collected
spring
1987)
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165
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166
tein
measured
in arbitrary
absorbance
units
at 41
7nm.
collected
in April.
1987.
Figure 3.7
Cytochrome P-450 carbon monoxide- difference spectra from stages of the
digestive gland microsomal purification procedure upto and including the 8 -
aminooctyl sepharose 4B affinity chromatography stage.
a. Crude microsomes(50 /il sample, 0.9 mg protein/cuvette)
b. 35-65% (NH^^SO^ fraction after dialysis (100 jul sample, 1 . 2 mg protein/cuvette)
c. Pooled fraction from 8 -aminooctyl Sepharose 4B after concentration and dialysis
(125 n1 sample, 0.34 mg protein/cuvette)
167
ABSO
RBAN
CE0.050
LUor<g 0.025OWm<
0.000 500475400 450 WAVELENGTH
425nm
0.030
0.015-
0.000 500475nm
450 WAVELENGTH
425400
0.010
LUOz<g 0.000-owm<
- 0.010 4 >5 500450 WAVELENGTH
425400nm
168
is shown in Figure 3.8. A fraction of haemoprotein failed to bind to the
column (peak 1) and washed directly off. After commencement of the KC1
gradient, two peaks eluted at KC1 concentrations of approximately 12 mM
(peak 2) and 42 mM (peak 3). Peak fractions were again pooled,
concentrated and assayed for cytochrome P-450. A total step-yield of over
64% was obtained for cytochrome P-450 from the ion-exchange step.
Approximately 85% of this eluted in peak 3, giving an overall step-
purification of almost 7-fold (almost 32-fold in total). Of the remaining
recovered cytochrome P-450, approximately 12% eluted in peak 2 and only
3.6% in peak 1 (unbound haemoprotein). Cytochrome P-450 carbon monoxide-
difference spectra on aliquots from each of these three peaks are shown in
Figure 3.9. Of interest is the distribution of the '416' nm component
between the peaks. Peak 1 consists almost entirely of the '416' nm
component with no discernable cytochrome P-450 (Fig. 3.9A). This is of
significance since it strongly suggests the '416' nm component is a
haemoprotein, and possibly denatured P-450. Peak 2 contains both
cytochrome P-450 and the '416' nm component, whereas peak 3 contains
entirely cytochrome P-450 (Figs 3.9B and C; see also P-450/'416' nm
peak ratios, Table 3.9, column 12). The presence of cytochrome P-450 in
peaks 2 and 3 is suggestive of the existence of at least two distinct
isoenzymes in M. edulis. The presence of both P-450 and the '416' nm
component in peak 2 possibly indicates similar ionic properties and,
therefore, is possibly further evidence for the '416' nm component being
denatured cytochrome P-450.
KC1 was removed from the cytochrome P-450-rich peak 3 by dialysis
(Section 3.2.9) and the sample applied to a small hydroxylapatite column
for removal of emulgen 911. The elution of haemoprotein from this column
169
MOLAR CONCENTRATION OF KCLoorl
innooino
inC\loooo
” (0
trinmxDZZoMHCJ<cctl_
HI ELU< inin • •a o crl •rtE EO 1 \£_ rH0) EEC 3.rtrH •V O O■P >O 1 •L. T3 Ea 0 0 CE torH0 c C_ID E3 5 •PrH O 0H O rH(0 a «P toE -PO • • ■rt0 £_ . . Co (0 rH 3£_ rH Eu O 01 >.•rt E L.E 1 0CT3 • 0 •PC O E «h(0 1 3 13rl o rH £_O) O 0>a) c> -p c •rt-rtc o■P 0) ■rt■a(0•H •P 0a) •o a £_cn (0 0 3•H £_ c. 0•O O) «*- 00H- ■ Eo CJX E Cc U ■rto 0•rt -0) ■P■P rH • o3 0 o £_rH u X a0) (0 in oJC • EQ) a to 0JC a) rH 0H 0 X
CDciV£.3OIli-
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170
Figure 3.9
Cytochrome P-450 carbon monoxide-difference spectra from the ion-exchange
and hydroxylapatite stages of the digestive gland microsomal purification
procedure.
a. Peak 1 from DEAE- sephacel after concentration (200 \i 1 sample, 0.48 mg protein/cuvette)
b. Peak 2 from DEAE- sephacel after concentration (100 fil sample, 0.83 mg protein/cuvette)
c. peak 3 from DEAE- sephacel after concentration (50 //I sample, 0.056 mg protein/cuvette)
d. Pooled fraction from hydroxylapatite(1000 fj, 1 sample, 0.044 mg protein/cuvette)
171
ABSO
RBAN
CE
ABSO
RBAN
CE0.020
0.005-
- 0.010500450 475400 425
0.004
- 0.001 -
-0.006 500475425 450400WAVELENGTH WAVELENGTH
0.015
0.005-
-0.005 500475450400 425WAVELENGTH
0.006
0.002-
- 0.002 500475400 425 450 WAVELENGTH nm
172
is shown in Figure 3.10. Surprisingly, only about 5% of the cytochrome P-
450 loaded onto the column was successfully recovered free of emulgen, the
remainder not binding. Of the successfully recovered cytochrome P-450, an
overall loss of purity of approximately 38% resulted from the procedure.
This was presumably due to denaturation of the P-450, although the P-450
carbon monoxide-difference spectrum showed little, if any, '416' nm
contamination (Fig. 3.9D). (It is possible the reduced purity, following
hydroxylapatite chromatography, was due to a loss of haem, leaving
spectrally-inactive apo-P-450 -see Discussion) . Although there is no
indication of stability of P-450 from this study, in a similar experiment
(described by Kirchin et al., 1988; see Appendix), a partially-pure post
ion-exchange cytochrome P-450 preparation (equivalent to peak 3 of this
study) was stable in storage, at 4°C, for several months). However, it
should be noted that emulgen 911 was still present in this preparation and
may have contributed to stability.
3.3.5 Spectral properties, molecular weight and purity of the partially-
purified cytochrome P-450
The of the carbon monoxide-difference spectrum of themax remulgen-free partially-purified cytochrome P-450 was indicated to be 449 nm
(Figure 3.9D). The absolute absorption spectrum showed a Soret peak at 417
nm (Figure 3.11). This spectrum is characteristic of a cytochrome P-450
largely in the low-spin state. A slight shoulder between 390 and 410 nm
possibly indicates the presence of a degree of high-spin P-450 (see Section
1). Unfortunately, there was too little sample to examine peaks in the
visible (535-580 nm) region i.e. the noise-to-signal ratio was too great.
SDS-polyacrylamide gel electrophoresis of samples from different
173
EMULGEN ABSORBANCE AT 2B0nmooocu
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ooin
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ECllimzDZOHhCJ<DCli.
UlUZTfr' IV 3ONV8HOS0V NI3±0Hd0W3VH
c # * c4-1 rl 4-10) . EJp d C TO0 d rl 4-1 0c. 0) c_a 1 TJ D0 c • 0 0E 0 •H £_ 0O □) O D 0(0 rl > 0 En D 0E c 0 rHiH O □ E 0to 4-1 >E «»-4J c 0O O a •rl E0 to 0 0□ rH L. 4J L. •c_ (0 «*- O E0 > L. C C4-1 O % m a 0 0E E O D D0 E E «H CUTOC C_ O 0 3 0 E -P(0 O 0) IU1 0rl JC •Dl•P OX 0 • +>a) ■O cn E -H> C • C C0 C h* ZO•P rH rl rl(0 0 E E r 0(D 4J \ 0Ol4H CU H4J C•H -P E 0 0T3 to 1 13a •cn 0 L.H- 0 1—I■H 4J OO rH O • 4-1 0>» > O C S3c X O 00 0 T3 14-1 L. 0 0 >. 5-»■P ■D 13 4J L LD >» 0 0 0i—1r: C L. L. £_0) E •P JPE D 5 4-1 4-10 O rH 0 n nC_ O rl L t.
Figure
3.10
. T H- U H- 0 0
174
0.035
LLIO2<cc 0 - 0 2 OCOCO<
0 . 0 1 0 500400 600300WAVELENGTH nm
Figure 3.11
Absolute spectrum of partially-pure (emulgen-free) cytochrome P-450 (oxidized form) from the digestive gland of M. edulis (1 0 0 /il sample, 0.044 mg protein/cuvette).
175
stages of the purification procedure shows diagrammatically the
purification of mussel digestive gland microsomal cytochrome P-450 (Figure
3.12). A single major band was observed for the emulgen-free partially-
purified P-450. Interpolation of the electrophoretic mobility of the
protein band presumed to be cytochrome P-450 against the electrophoretic
mobilities of the protein standards indicated a molecular weight for this
particular P-450 isoenzyme of approximately 53.0 Kd. Assuming the
molecular weight of the P-450 to be 53.0 Kd, and the extinction coefficient_ ifor the carbon monoxide difference spectrum of mussel P-450 to be 91 mM
cm"^, the theoretical specific content of a 100% pure sample is 18.87
nmoles mg~^ protein. The highest specific content obtained in this study
(1.85 nmoles mg~^ protein; peak 3 from DEAE-sephacel chromatography),
therefore, was 9.8% pure.
3.3.6 Partial purification of digestive gland microsomal cytochrome
P-450 following fractionation by PEG
The result of a typical attempt at cytochrome P-450 purification
following fractionation of solubilized microsomal protein with PEG is shown
in Table 3.10. Both the solubilization and protein fractionation steps
gave typical values for yield and purification (see Sections 3.3.1. and
3.3.2). The yield for the affinity chromatography stage, however, was only
8 .6%, considerably lower than values obtained in typical purifications
incorporating an (NH^^SO^ fractionation step (see Sections 3.3.3 and
3.3.4). The purification obtained after elution of the successfully-bound
cytochrome P-450 was, however, of the order normally obtained. The
reason(s) for the lack of binding of PEG-fractionated cytochrome P-450 to
the 8 -aminooctyl ligand is unknown. A similar lack of binding was observed
on a number of occasions suggesting it was not a seasonally-related effect.
176
Figure 3.12
Top: SDS - polyacrylamide gel electrophoresis of fractions from the
digestive gland microsomal cytochrome P-450 purification procedure.
A crude microsomes
B supernatant from solubilization
C 35-65% (NH^^SO^ fraction
D post-affinity column fraction
E post-ion exchange peak 3
F post-ion exchange peak 1
G post-ion exchange peak 2
*H Post-hydroxylapatite fraction
S Protein standards
(^concentrated x 5 by ultrafiltration prior
(0.18 mg protein)
(0 . 2 0 mg protein)
(0 . 2 0 mg protein)
(0.06 mg protein)
(0.04 mg protein)
(0.06 mg protein)
(0.04 mg protein
(0 . 0 1 mg protein)
to loading)
Bottom: The mobility of the protein standards. The molecular weight of the
partially pure cytochrome P-450 was determined by interpolation.
177
LOG MOL
ECULAR
WEIGHT
M.Wt.Kd
92.5
68.0•57.553.0
-43.0•40.0
-29.025.720.1
12.4
B D F G H
o
8 4.724
6
4
2
56.840
0 25 50 75 100PERCENTAGE MOBILITY
178
The partial
purification of
digestive
gland
microsomal cytochrome P-450
by sodium cholate
solubilization,
PEG-fractionation,
aminooctyl sepharose
4B chromatography and DEAE-sephacel
ion-exchange chromatography.
(mussels collected
autumn
1985)
cO0) tAE •PO o 1 <du in 'O uA H -H0 i 0 *Ho cu -H4-> P>1 3o 04
C CM (M r-H •H (M (M o*Id H 0) 0)4-> 0) 4J E O o\ CO0 ■H 0 (M (M os
b04
CO Os H
0)erH E O ^ >
Os CM vO o in OsCO in in CM CO<* Os o o o o
0o >1 rH i—i rH•H 4-> 'd 0) 0) 0)
•P P •H <u u E u E u(0 e rd c 0) rH 4-> P id O id O id0 m N o •H E o id 0 .C P A P AB 4-> “H w C O o u <H 04 <H 04 «H 040 id 6 •H 04 cu 0 P 04 P 0) 0) Vco C o •H 0) Ti id .C c »—i (0 rH (0 CM (00 c u .Q P d? P 1 O o Q) 0 1 1 1M d) 0 10 in O P o in U 0 w rH w rH wU 04 rH rH id 10 P c 04 rt O rt O rt
•H 0 O 1 p o >4 i 0 3 0 w 0 w6 CO 10 <H 04 O 04 u Q 04 Q cu Q
179
Despite the low recovery, cytochrome P-450-containing fractions
eluted from the affinity column were pooled, concentrated by
ultrafiltration, and applied to a DEAE-sephacel ion-exchange column. The
concentration of cytochrome P-450 in individual fractions was too low for
detection but the elution of total haemoprotein was determined (Figure
3.13). After commencement of the KC1 gradient, two major haemoprotein
peaks were detected, eluting at KC1 concentrations of 7.5 mM and 41 mM.
Possibly a third minor peak eluted at a KC1 concentration of 67.5 mM.
Although the identity of these peaks could not be ascertained, it is
possible they are further indication of the presence of isoenzymes
suggested from the purification procedure involving (NH^^SO^-fractionation
(see Section 3.3.4). The use of PEG as a protein fractionating agent was
largely abandoned in favour of (NH^^SO^. Improved yields may have been
achieved with affinity columns of larger diameter, but no attempts were
made to investigate this.
3.3.7 The partial purification of digestive gland microsomal NADPH-
cytochrome c (P-450) and NADH-cytochrome c reductase activities
Experiments were performed to improve the purification of NADPH-
and NADH-cytochrome c reductase activities (see Section 3.3.3). The
results of one such experiment, a continuation of that described in Section
3.3.3, are given in Table 3.11. The elution of the activities from an 8 -
aminooctyl sepharose 4B affinity column was previously shown in Figure 3.5.
The yields and purifications after cholate solubilization, (NH^^SO^-
fractionation and 8 -aminooctyl sepharose 4B chromatography were limited
i.e. respectively, for NADPH- and NADH-cytochrome c reductase activities,
yields of 11 and 5% and purifications of x 3.1 and x 1.3. These figures,
180
inMOLAR CONCENTRATION OF KCL
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181
The
partial
purification
of digestive
gland
microsomal
NADPH-cytochrome
c (P-450)
and
NADH-cytochrome
c reductase
activities by
sodium cholate
solubilization,
8-aminooctyl sepharose
4B and ADP-sepharose
4B affinity chromatography.
c0•HP
1 to CO in»a u 1 CO CO•H *HO <H H o rH TJ*
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4) to P *H Z *H 4) 0 u < uH 4) o tfl ^ ^ P H 1 *H (0 1 toOl *0 rH p O Oi P 8 p Ae P 0 O rH 'd to E M to Ol to Olto U *H JC O C U to 0 1 4) O 4)w U Q o n a <h (o Oi CO to cu to
182
however, are almost certainly underestimates and not representative simply
of activity loss through inactivation or non-binding to the affinity
column. The recovery of NADH-cytochrome c reductase activity in
preliminary solubilization experiments was always considerably lower than
for either the NADPH-dependent activity or the NADH-ferricyanide reductase
activity (data not shown) and was of the order of 25%. This was presumably
a reflection of this microsomal reductase activity being dependent on the
presence of not one, but at least two correctly associated components in
the endoplasmic reticulum (see Sato, 1978). In the case of NADPH-
cytochrome c reductase activity, it has subsequently been discovered that,
unusually, the enzyme activity in mussel digestive gland microsomes, unlike
the NADH-cytochrome c reductase activity, is markedly inhibited by
phosphate buffer (I50 ~ 3 mM, pH 7.6 -Livingstone and Garcia Martinez,
pers.comm.). The samples during the purification procedure would have
contained variable amounts of phosphate buffer.
The pooled reductase fraction from the 8 -aminooctyl sepharose 4B
column was concentrated and applied to an 2',5'-ADP sepharose 4B affinity
column. The elution from this column is shown in Figure 3.14.
Approximately 10% of each activity failed to bind to the column. The
yields and purifications for the step were good, however, vis, for NADPH-
and NADH- cytochrome c reductase activities respectively, yields of 47 and
71% and purifications of x 2.4 and x 3.6. Of interest was the improved
yield and purification of NADH- cytochrome c reductase activity over the
NADPH- dependent activity in this step compared with the previous
purification steps.
183
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184
3.4 Discussion
3.4.1 Solubilization and protein fractionation of digestive gland
microsomes
Studies have been performed on a number of species comparing different
ionic and non-ionic detergents for their ability to solubilize cytochrome
P-450 from microsomal membranes (Miura et al., 1980; Azari and Wiseman,
1982a; Monod et al., 1987). In most of the studies detergents were compared
at just one concentration. The study presented here compared four
detergents over a range of concentrations. Sodium cholate, at a ratio of
about 1.75 mg per mg microsomal protein for 1 h, was shown to be the most
effective detergent for solubilization of mussel digestive gland microsomal
cytochrome P-450.
Sodium cholate is extensively used for mammalian P-450 purification
(e.g. West et al., 1979; Tamburini et al., 1984) and has been used for the
solubilization of microsomal P-450 from species of Crustacea (Conner and
Singer, 1981; Quattrochi and Lee, 1984; Batel et al., 1986) and fish (Arin?
and Adali, 1983; Klotz et al., 1983). A ratio of 1.75 mg cholate per mg
protein is consistent with the above studies on Crustacea and fish, which
utilized cholate to protein ratios ranging from about 1.3 to 2.7 mg per mg
protein. (A ratio of 0.5 mg cholate per mg protein was used on scup, S.
chrysops hepatic microsomes, but in this study solubilization was achieved
using a mixture of cholate and emulgen 911 - Klotz et al., 1983). 1.75 mg
cholate mg"^ protein, however, is slightly lower than the 3 to 1 ratio
often used in mammalian studies (e.g. West et al., 1979; Ryan et al., 1982;
Abe and Watanabe, 1983).
185
Unfortunately, yields of P-450 following microsome solubilization have
not been reported for Crustacea or any other form of marine invertebrate.
In comparison to studies on fish, the 60-80% recovered in this study is
slightly lower than the 85% and 91% reported for S. gairdneri and S.
chrysops (respectively, Arin? and Adali, 1983; Klotz et al., 1983), but
considerably higher than values reported for the freshwater nase,
Chondrostoma nasus and roach Rutilus rutilus (40% and 29% respectively-
Monod et al., 1987). The cholate to protein ratio of this latter study was
relatively high (approximately 3.13 to 1), and therefore, the low yields
may have been due to denaturation.
Improved yields may have been achieved by the use of different
detergents, for example the non-ionic detergents Renex 690 or Tween 80, or
by a combination of detergents, as used for example, for P-450 purification
from S. chrysops (cholate and emulgen 911) (Klotz et al., 1983; see above),
the Atlantic cod, Gadus morhua (CHAPS and cholate) (Goksoyr, 1985) and from
yeast, Saccharomyces cerevisiae (100% yield following treatment with 1%
cholate and 0.1% Triton X-100 compared with a 36% yield following treatment
with 1% cholate alone) (Azari and Wiseman, 1982a).
The protein precipitation study indicated fractionation by PEG to be a
more efficient procedure than fractionation by (NH^^SO^. This is
presumably the same as that observed for other species, since the vast
majority of P-450 purifications, which incorporate a protein fractionation
step, utilize PEG fractionation. However, for the mussel digestive gland
P-450 purification, (NH^^SO^ was routinely preferred over PEG because of
186
problems encountered in the subsequent affinity chromatography step with
use of the latter. Omission of the protein fractionation step was,
similarly, found impractical. The choice of a suitable fraction was
determined by a compromise between yield and purification, with the former
being of greater importance due to the low specific contents and microsomal
yields of mussel digestive gland. Hence, 4-15% PEG fractions and 35-65%
(NH^^SO^ fractions were selected. Similarly, studies on several species
of crab have utilized relatively wide 0-16% PEG fractions (Quattrochi and
Lee, 1984). More narrow PEG fractions can be utilized in mammalian P-450
purifications, since, to an extent, yield can be sacrificed for
purification (e.g. 10-16%, West et al., 1979; Elshourbagy and Guzelian,
1980; 8-12% Koop et al., 1982; 9-15%, Ryan et al., 1982; 6-13%, Miki et
al., 1987). Cytochrome P-450 precipitation by 15% PEG and 65% (NH^^SO^,
in this study, is similar to that observed for a variety of species (see
above for PEG, and Azari and Wiseman, 1982a; King et al., 1984 for
(NH^^SO^) and presumably indicates basic similarities, in terms of
molecular weight and hydrophobicity, between the cytochrome P-450 from M.
edulis and those forms from other species.
3.4.2 Purification and properties of digestive gland microsomal cytochrome
P-450
The patterns of elution of components of the mussel digestive gland
microsomal MFO system from an 8 -aminooctyl sepharose 4B column were similar
to those observed for other species (e.g. Guengerich, 1977; King et al.,
1984). Although the behaviour of cytochrome b^ was not determined, by
analogy with the above studies it may have been elutable from the column
187
after the reductase activities by raising the concentration of
deoxycholate.
The reason for the general failure of PEG - fractionated P-450 to bind
to 8 -aminooctyl sepharose 4B affinity columns is not known. It may have
been related to other components or interactions retained in the
preparation after PEG fractionation, such as carotenoids or other pigments.
There are no reports of PEG - fractionated cytochrome P-450 from other
species showing an inability to bind to the extent observed in this study,
although PEG - precipitated P-450 from two species of Crustacea,
Callinectes sapidus and Maja crispata, showed only a comparatively limited
binding of, respectively, 12.7% and approximately 30%. N.B. the
solubilization step is also included in the value for C. sapidus (Conner
and Singer, .1981; Batel et al., 1986). The lack of binding of PEG -
fractionated cytochrome P-450 was observed for microsomal preparations from
mussels collected at different times of the year, so a seasonally-dependent
phenomenon is unlikely. However, since cytochrome P-450 fractionated by
(NH^^SO^ successfully bound to the column there is indication of physico
chemical similarities between cytochrome P-450 from M. edulis and
cytochrome P-450 from other species.
The binding and elution of P-450 (fractionated by (NH^^SO^ in this
study) compare favourably with results obtained from studies on other
species. The step-yield of 40-50% and the step-purification of 2-4 fold
were similar to those achieved for microsomal cytochrome P-450 preparations
from mammalian liver (Guengerich and Martin, 1980; Fisher et al., 1981;
Funae and Imaoka, 1985) and adrenal gland (Narasimhulu and Eddy,
1985).Interestingly, studies on cytochrome P-450 purification from fish
188
have either used tryptamine-sepharose 4B, for example, trout (Williams and
Buhler, 1982), or phenyl-sepharose 4B, for example, cod (Goksoyr, 1985),
rather than 8 -aminooctyl sepharose 4B. . Other studies on fish (Arin? and
Adali, 1983; Klotz et al., 1983) and crabs (Quattrochi and Lee, 1984) have
not included an affinity chromatographic step. Although the step-results
with tryptamine- and phenyl-sepharose 4B were no better than achieved in
this study with 8 -aminooctyl sepharose 4B, it is possible that either or
both of these affinity ligands might have improved the performance with
mussel cytochrome P-450. Similarly, if the functions of molluscan
cytochrome P-450 are particularly directed towards the metabolism of
endogenous substrates (see Sections 2 and 5), then the use of an endogenous
substrate as an affinity ligand might have potential. For example, the use
of lauric acid as an affinity ligand has been successful for the
purification of constitutive forms of rat liver cytochrome P-450 (Cheng and
Schenkman, 1982; Schenkman et al., 1982).
Application of the (NH^^SO^- fractionated post-affinity sample to a
DEAE - sephacel ion exchange column, followed by column washing and a KC1
(ionic) gradient, produced three major haemoprotein peaks eluting during
column washing (peak 1), and at KC1 concentrations of 12 mM (peak 2) and 42
mM (peak 3) . Peak 1 was devoid of cytochrome P-450 and contained only the
'416'nm component. That the '416'nm component might be partly or wholly
denatured P-450 is supported by SDS PAGE (Fig. 3.12, track F). Despite the
absence of measurable P-450 from the sample, a faint band of approximately
53.0 Kd (the same as that of cytochrome P-450; track H) can be seen.
Interestingly, the equivalent peak was absent from the purification
involving fractionation by PEG, possibly because of a differential effect
on the '416'nm component or because of further denaturation, involving haem
189
loss, to a spectrally-inactive apoprotein (Ingleman-Sundberg and Paul,
1986).
Haemoprotein peaks 2 and 3 both contained cytochrome P-450, suggesting
the existence of isoenzymes. Assuming the haemoprotein peaks eluting at
7.5 mM KC1 and 41 mM KC1 of the PEG-purification scheme were equivalent to
these peaks, it is interesting to compare the ratios in each experiment of
the total haemoprotein in the peak eluting at 41-42 mM KC1 with the total
haemoprotein in the peak eluting at 7.5-12 mM KC1. In the PEG -
purification scheme this ratio was 2 .1 :1 , whereas in the (NH^^SO^ -
purification scheme it was 3.4:1. It is possibly significant that the
mussels for the PEG-purification were collected during autumn, while those
for the particular (NH^^SO^ - purification were collected during April, at
or around the spawning period. These observations lend support to the
suggestion (see Section 2) of seasonal changes in the digestive gland
microsomal cytochrome P-450 isoenzyme composition of M. edulis, with one
major isoenzyme before and during the spawning period, and a second being
more apparent during the autumn. Such seasonal changes in a haemoprotein
elution profile, indicating changes in isoenzyme composition, have been
observed for the crab, Libinia emarginata (Quattrochi and Lee, 1984).
However, it should be noted for the mussel studies that the suggestions are
based on a number of assumptions which are open to question. Firstly, that
the microsomal P-450 isoenzyme composition is not differently affected by
the PEG- and (NH^^SO^ - purification schemes. Secondly, that the
haemoprotein peaks of the PEG- purification contain cytochrome P-450, at a
concentration too low to be determined, and, in the case of peak 2 , at a
ratio to the '416'nm component and other haemoproteins equivalent to that
of the (NH^^SO^ - purification. The latter is possibly likely considering
190
the high correlation between P-450, the '416'nm component and total
haemoprotein observed seasonally (see Section 2). The suggestions are not
supported by the SDS PAGE which showed molecular weight bands of 53.0 Kd
for both putative isoenzyme peaks i.e. not 53.0 and 51.5 Kd (see Section 2)
(Fig. 3.12, tracks E and G equivalent to ion-exchange elution peaks 3 and
2). However, this observation may be due to, in peak 2, amounts of
cytochrome P-450 too low to be detected by electrophoresis and the presence
of the '416'nm component which is indicated also to have a molecular weight
of 53.0 Kd (see before).
The presence of at least two cytochrome P-450 isoenzymes in the
digestive gland of M. edulis is not surprising. In addition to the
multiple forms present in mammals (see Section 1), at least five forms have
been reported for the scup, S. chrysops (Klotz et al., 1983), and four
forms each for the trout, S. gairdneri (Williams and Buhler, 1982) and cod,
G. morhua (Goksoyr, 1985). At least three isoenzymes have been shown to
exist in each of the crabs C. sapidus, L. emarginata, Uca minax and Menippe
mercenaria (Quattrochi and Lee, 1984). Interestingly, the crab cytochrome
P-450 isoenzymes were resolved on hydroxylapatite after an initial ion-
exchange chromatography step. In contrast, in a previous report on crab
cytochrome P-450 purification, only two isoenzymes were resolved in a
procedure incorporating an initial 8 -aminooctyl sepharose 4B affinity step,
(Conner and Lee, 1982). The use of ion-exchange chromatography, as a first
step in the purification of mussel cytochrome P-450, might, therefore, also
enable the detection of more isoenzyme forms. Only one form of cytochrome
P-450 was reported for the spiny crab, M. crispata (Batel et al., 1986),
although in this study it was not possible to prevent the denaturation of
P-450 to P-420 following affinity chromatography, and therefore more forms
191
may have existed.
There is virtually no information on cytochrome P-450 isoenzymes from
fish or crustacea eluting from DEAE-sepharose columns at KC1 concentrations
at or around 7.5-12 mM or 41-42 mM, although a haemoprotein peak from M.
crispata eluted "soon after starting" a linear 0-0.3 M KC1 gradient (Batel
et al., 1986). Forms of cytochrome P-450 from rabbit pulmonary microsomes
eluted from a DEAE-sephacel column with 35 mM phosphate (Guengerich, 1977).
Similarly, P-450d from rat liver microsomes eluted from a Whatman DE-52
sepharose column at an NaCl concentration of about 30 mM (Ryan et al. ,
1982).
Attempts to remove emulgen 911 from, and further purify, the
cytochrome P-450 comprising peak 3 of the (NH^^SO^ - purification were
largely unsuccessful because of the failure of over 90% of the P-450 to
bind to the hydroxylapatite column. Removal of emulgen 911 and other non
ionic detergents on hydroxylapatite is a standard method in the cytochrome
P-450 purification procedures of many species (e.g. Saito and Strobel,
1981; Klotz et al., 1983; King et al., 1984). At present, there is no
obvious explanation for the general lack of binding observed for partially-
pure mussel cytochrome P-450.
An absolute oxidised spectrum recorded on an aliquot of the emulgen-
free partially-pure sample from hydroxylapatite revealed an absorption
maximum at 417 nm, with possibly a slight shoulder between 390 and 410 nm.
Such a spectrum is indicative of a form of cytochrome P-450 largely in the
low-spin state, but containing also a slight high-spin presence. Low-spin
192
forms of cytochrome P-450 have been solubilized from a number of species,
for example, rat (Saito and Strobel, 1981; Tamburini et al., 1984), rabbit
(Bonfils et al., 1986), human (Wang et al., 1980), Scup (Klotz et al.,
1983), trout (Williams and Buhler, 1982), several species of crab
(Quattrochi and Lee, 1984) and yeast (Azari and Wiseman, 1982a). It is
possible the cytochrome P-450 of M.edulis was initially in a mixed-spin
state, but was converted to an essentially low-spin state upon treatment
with emulgen 911. Such a conversion of spin states by non-ionic detergents
has been reported previously (Fujita et al., 1973; Yoshida and Kumaoka,
1975). A cytochrome P-450 carbon monoxide-difference spectrum on the same
emulgen-free partially-pure fraction revealed a rather broad absorption
maximum at 449 nm. The broadness of the peak may be real or possibly a
consequence of the high sensitivity needed to measure the low amounts of
cytochrome P-450. The Amax of 449 nm is slightly lower than the wavelength
maxima of about 449.5-450 nm recorded during the seasonal survey at about
the same time of the year (see Section 2).
The purity of the digestive gland microsomal cytochrome P-450
preparation was calculated as 9.8% after DEAE-sephacel chromatography (peak
3), and 6.1% after hydroxylapatite removal of detergent (Section 3.3.4).
The reduced purity may be due to partial haem loss. (Final purified
cytochrome P-450 preparations from other species often contain 20-60%
apoenzyme - Ingelman-Sundberg and Paul, 1986). A comparison of this
purification with those for microsomal cytochrome P-450s from a number of
other species is presented in Table 3.12. Although the final purity
achieved for M. edulis was considerably less than that for yeast and
untreated mammalian species, the values compare favourably with those for
each of the poikilothermic species, with the exception of L. emarginata.
193
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194
The 1.8 nmoles mg“ protein final specific content reported for C. sapidus
(Conner and Singer, 1981) was for a preparation still containing non-ionic
detergent (Triton-NlOl). Interestingly, this value is almost identical to
the 1.85 nmoles mg for the mussel peak 3 from the DEAE-sephacel column
before removal of emulgen. The 9.0% purity for cytochrome P-450 from C.
sapidus (Conner and Singer, 1981) is perhaps artificially high, the
approximate value of 5.2% obtained by Quattrochi and Lee (1984) possibly
being more realistic. The purity for mussel P-450 compares well with the
9.0% for P-4501b from untreated trout (Arin? and Adali, 1983), but is
considerably less than the 69% purity for a cytochrome P-450 isoenzyme from
trout pretreated with /3-naphthoflavone (Williams and Buhler, 1984).
Significantly, the 19.6-fold purification for mussel, although much less
than that for yeast, is of the order obtained for rat cytochrome P-450
(Cheng and Schenkman, 1982). The considerably lower purity obtained with
mussel is thus, presumably, a reflection of much lower starting microsomal
specific content. Values for the mussel cytochrome P-450 isoenzyme
molecular weight and difference spectra Amax, are similar to those of
constitutive forms of cytochrome P-450 from other species. However, it is
not possible to say from this whether other properties and functions are
also similar.
Finally, in a number of marine vertebrate and invertebrate species,
multiple isoenzymes of cytochrome P-450 have been resolved by
hydroxylapatite chromatography after ion-exchange chromatography failed to
resolve more than one or two haemoprotein peaks (e.g. see Williams and
Buhler, 1982, 1984; Arin? and Adali, 1983; Quattrochi and Lee, 1984). This
possibility was not extensively investigated for mussel digestive gland due
to the limited amounts of P-450 remaining at this stage of purification.
195
3.4.3 Purification of NAD PH- cytochrome c (P-450) and NADH- cytochrome c
reductase activities
The purpose of the study was to develop a procedure for the
purification of NADPH- cytochrome c (P-450) reductase activity as part of
the cytochrome P-450 purification procedure. The procedure incorporated,
the use, after 8 -aminooctyl sepharose 4B chromatography, of affinity
chromatography on 2', 5'-ADP sepharose 4B. This affinity-ligand is specific
for NADP+-specific reductase and dehydrogenase enzymes, and has been used
successfully for the purification of NADPH - cytochrome P-450 reductase
from a variety of species, for example, rabbit (Guengerich, 1977; French
and Coon, 1979), rat (Guengerich and Martin, 1980; Shephard et al., 1983),
pig (Yasukochi et al., 1980), hamster (Ardies et al., 1987) and trout
(Williams et al. , 1983). Similar procedures for the simultaneous
purification of NADPH- dependent reductase activity and cytochrome P-450
have been reported from, for example, rabbit pulmonary microsomes,
(Guengerich, 1977), rat liver microsomes (Guengerich and Martin, 1980) and
the yeast, S. cerevisiae (King et al., 1984). Despite the mussel NADPH-
and NADH- cytochrome c reductase activities binding to and being eluted
from the 2' , 5' -ADP sepharose 4B column, the overall purification from
digestive gland microsomes compared badly with the results in the above
studies. The total purification for mussel digestive gland microsomal
NADPH-cytochrome c(P-450) reductase activity of 7.5-fold contrasts with,
for example, 420-fold for rabbit pulmonary microsomal cytochrome P-450
reductase (Guengerich, 1977) and 199-fold for the same enzyme from
phenobarbital - pretreated rats (Guengerich and Martin, 1980). Similarly,
the final yield for mussel digestive gland microsomal NADPH-cytochrome c
(P-450) reductase activity was less than 5%, compared with 47% and 63% for
196
rabbit and rat, respectively. Two main possibilities exist to explain the
relatively poor performance of the mussel purification compared to those of
the above species. Firstly, that the physico-chemical properties of the
mussel P-450 reductase are significantly different from the same in other
species. However, although differences have been reported between the
cytochrome P-450 reductase of rat and the poikilothermic trout, designed to
allow the MFO system of the latter to function at lower temperatures, the
purification of the trout enzyme by the same procedures was still 1230-fold
(Williams et al., 1983). Secondly, as mentioned in Section 3.3.7, the
purification and yield results for the mussel NADPH-cytochrome c reductase
activity are distorted because of its inhibition by phosphate buffer which
is present at varying concentrations throughout the purification procedure
(this observation in itself indicates different properties for the mussel
digestive gland microsomal P-450 reductase). Clearly, substantially more
work is required to design a purification procedure for the mussel
reductase and, presumably, preferably not one involving phosphate buffers.
197
3.5 Appendix
The partial purification of cytochrome jP-450 from the digestive gland of the common mussel Mytilus edulis
MILES A. K IRCH IN , ALAN WISEMAN and DAVID R. LIVINGSTONE*Department o f Biochemistry, University o f Surrey, Guildford, Surrey, GU2 5XH, U.K., and *N.E.R.C. Institute fo r Marine Environmental Research, Prospect Place, The Hoe, Plymouth, Devon, PL1 3DH, U.K.
Cytochrome P-450 in the common mussel Mytilus edulis is of great interest in relation to environmental xenobiotic metabolism and from a comparative viewpoint (Lee, 1981; Livingstone, 1985; Stegeman, 1985). The enzyme is located primarily in the endoplasmic reticulum of the digestive gland (hepatopancreas) and can be collected in the microsomal fraction (microsomes). We report here a procedure for the partial purification (8-fold) of this enzyme from digestive gland microsomes by solubilization with sodium cholate, fractionation by ammonium sulphate precipitation followed by affinity chromatography with 8-amino-/z-octyl Sepharose 4B (S4B) (Pharmacia) and ion-exchange chromatography with DEAE-Sephacel (DEAE-S) (Table 1).
Microsomes prepared by the method of Livingstone & Farrar (1984) were resuspended in lOOmM-phosphate buffer pH 7.6 containing 20% (w/v) glycerol, 1 riiM-EDTA and 1 mM-dithiothreitol (DTT), to a concentration of 12- 15 mg/ml of protein. Solubilization of these microsomes was carried out by the addition of sodium cholate (1.3% w/v) with glutathione (0.1% w/v), followed by stirring at 4°C under nitrogen for 1 h. This preparation was centrifuged at 100 000 g for 50min and the supernatant taken as a source of solubilized cytochrome P-450. The yield for this stage is in our experience normally greater than 50%. The value (45.5%) reported here is therefore rather low, which is possibly a reflection of seasonal variation in cytochrome
Abbreviation used: D TT , dithiothreitol.
P-450 and the mixed-function oxidase system in M . edulis (Livingstone, 1985).
Ammonium sulphate fractionation was carried out on the supernatant by collecting the prepipitate at 35-65% saturation at pH 7.0 according to the method of King et al. (1984). The precipitate was resuspended in a reduced volume of 0.015 M-phosphate buffer at pH 7.0 containing 20% (w/v) glycerol, 1 mM-EDTA, 1 mM-DTT and 0.3% (w/v) sodium cholate, and dialysed against 30 volumes of this buffer for 12 h. Insoluble material was removed by centrifugation (lOOOOOg, 50min) and the supernatant applied directly to an affinity column of 8-amino-/?-octyl Sepharose 4B, preequilibrated with 0.01 M-phosphate buffer pH 7.0 containing 20% (w/v) glycerol, 1 mM-EDTA, 1 mM-DTT and 0.3% (w/v) cholate. The equilibration buffer minus DTT (100 ml) was run through in a washing step, and cytochrome P-450 was then eluted as a relatively sharp peak with the wash buffer containing 0.2% (v/v) Emulgen 911 (a non-ionic detergent). The cytochrome P-450 recovery for this step is high (40-50%) and there is a purification for the step of more than 2-fold (range 2.5-3.5-fold). The affinity stage provides an effective means of separating the P-450 from the NADPH-dependent cytochrome c (P-450) reductase activity.
Eluted fractions containing cytochrome P-450 were then combined and the total volume reduced by ultrafiltration at 4°C (membrane, 30kDa cut-off). The pH of the resulting cytochrome P-450-rich fraction was then adjusted to pH 7.6 by dialysing for 4h against 0.01 M-phosphate buffer pH 7.7 containing 20% (w/v) glycerol and 0.2% (v/v) Emulgen 911. The dialysate was applied to a DEAE-Sephacel ion- exchange column, pre-equilibrated with 0.01 M-phosphate pH 7.6 containing 20% (w/v) glycerol and 0.2% (v/v) Emulgen 911. The column was washed (3 volumes) with this buffer and eluted with a KC1 gradient (0-500 mM). One major cytochrome P-450 peak eluted at 10-40mM-KCl and
Table 1. The partial purification o f cytochrome P-450from the digestive gland o f M. edulisCytochrome P-450 was quantified from the carbon monoxide difference spectrum of sodium dithionite reduced samples in lOOmM-potassium phosphate buffer pH 7.6 using an extinction coefficient of 91mM_Icm-1 (450-490nm) (Estabrook & Werringloer, 1978). Protein was measured by the method of Lowry et al. (1951). The ‘418’ peak was quantified from A A 418-490 nm).
Ratio
FractionSpecific content
(pmol • m g-1 protein)Total content
(nmol)Purification
factorYield(%)
P-450/'-peal
Crude microsomes 51.2 38.8 1.0 100 0.4M icrosomes solubilized
in 1.3% cholate 31.6 18.1 0.6 45.5 0.135-65% (N H 4)2S 0 4
precipitate 41.3 7.5 0.8 18.9 0.28-amino-n-octyl S4B
affinity step 108 3.0 2.1 7.5 1.1DEAE-Sephacel
ion-exchange step 417.0 2.4 8.2 5.9 00
198
t remains to be determined whether this is homogeneous or f it comprises more than one isoenzyme. The recovery for his step is between 80 and 90% and normally gives a purifi- ation of approximately 4-fold. Ion-exchange chromato- raphy provides the largest single purification step in the cheme presented in Table 1. Following this step, a large ontaminating haemoprotein peak absorbing at 416-418nm n the reduced carbon monoxide difference spectrum of the riginal microsomal preparation, and present to a varying xtent in all the subsequent preparations, is finally completely emoved (Table 1, column 5).
Overall purification factors of between 5 and 10 have been btained using this purification procedure although the final ields are generally less than 10%. The partially purified ytochrome P-450 chromatographic elutant is stable in
storage at 4°C and represents a significant improvement on crude microsomes for spectral binding studies using substrates and inhibitors of the enzyme. The purification procedure appears suitable for use with molluscan tissues of
low specific content and low total content of microsomalcytochrome P-450.
M. A. Kirchin is indebted to the Natural Environment Research Council for a C.A.S.E. Ph.D. award.
Estabrook, R. W. & Werringloer, J. (1978) M ethods Enzymol. 52C, 212-220
King. D. J., Azari, M. R. & Wiseman, A. (1984) Xenobiotica 14, 187— 206
Lee, R. F. (1981) Mar. B io l Lett. 2, 87-105 Livingstone, D . R. (1985) Mar. Pollut. Bull. 16, 158-164 Livingstone, D . R. & Farrar, S. V. (1984) Sci. Total Environ. 39, 209 -
235Lowry, O. H ., Rosebrough, N . J., Farr, A. L. & Randall, R. J. (1951)
J. Biol. Chem. 193, 265-275 Stegeman, J. J. (1985) M ar. Biol. (Berlin) 89, 21-30
Received 18 June 1987
(published in Biochem. Soc. Trans. 15 1100-1101)
199
Section 4
Ligand - interaction with mussel digestivegland microsomal cytochrome P-450
200
4.1 Introduction
Ligands binding to either the substrate - binding site (type I
binding) or directly to the haem moeity (type II binding) of cytochrome P-
450 cause shifts in the haem iron spin-equilibrium toward either high spin
(type I compounds) or low spin (reverse type I and type II compounds)
(Schenkman et al., 1981). The changes in spin state equilibrium upon
ligand addition (discussed more fully in Section 1) result in changes in
absorbance which can be observed by difference spectroscopy.
In addition to mammals, spectral changes due to ligand (substrate or
inhibitor) binding to cytochrome P-450 have been observed in aquatic
species such as fish (e.g. James et al., 1979) and Crustacea (e.g. Lee et
al., 1981). The aim of the study presented in this section was to examine
spectral changes with a wide range of ligands in an attempt to gain some
idea of potential substrates (xenobiotic and endogenous) and inhibitors of
the mussel digestive gland MFO system. For compounds showing spectral
interaction with mussel cytochrome P-450, spectral titrations were
performed, where possible, to determine AAmax, the maximal absorbance
change n m o l e P - 4 5 0 (this is a measure of the intensity of binding i.e.
the ability of the compound to perturb the haem spin-equilibrium) and Kg,
the spectral dissociation constant i.e. that concentration of ligand
causing half-maximal absorbance. The latter value is independent of
cytochrome P-450 concentration. Ligand binding studies were mainly carried
out on dialysed 35-65% (NH^^SO^-fractionated solubilized microsomes.
Occasionally the crude microsomal preparation was used. The results are
discussed in relation to findings for other species and to aspects of
possible cytochrome P-450 function in molluscs.
4.2 Materials and Methods
4.2.1 Chemicals
Miconazole and ketoconazole were obtained from Janssen Pharmaceuticals
(Wantage, Oxon, U.K.). a-Naphthoflavone, clotrimazole, imidazole,
arachidonic acid, lauric acid, testosterone, cholesterol, 7-ethoxycoumarin,
androstenedione, metyrapone, aminopyrine, p-aminophenol, phenacetin,
hexobarbital, benzylamine, nicotinamide, cortisol, )9-oestradiol and
phenobarbital were from Sigma Chemical Co. Ltd. (Poole, Dorset, U.K.).
Pyridine and benzo [a]pyrene were from Aldrich Chemical Co. Ltd.
(Gillingham, Dorset, U.K.). Biphenyl, aniline, toluene and n-octane were
obtained from BDH Ltd. (Poole, Dorset, U.K.). Benzphetamine was obtained
from Upjohn Chemical Co. (Kalamazoo, Michigan, U.S.A.). SKF 525-A was a
generous gift from Mr. S. O'Hara, Marine Biological Association, Plymouth,
U.K. All other chemicals and solvents were from sources already described
in other sections.
4.2.2 Preparation of solubilized and (NH/^SO/- fractionated microsomal
cytochrome P-450 samples
Digestive gland microsomal cytochrome P-450 for substrate binding
studies was prepared in 100 mM phosphate buffer pH 7.6, containing 20% w/v
glycerol, 1 mM EDTA and 1 mM DTT as described in Section 3.2.2. Cholate
solubilization and (NH^^SO^ - fractionation of cytochrome P-450 were
performed essentially as described in Sections 3.2.3 and 3.2.4. The final
35-65% (NH^^SO^ - pellet was dialysed overnight, at 4°C, against 2 x 30
202
volumes of 100 mM phosphate buffer pH 7.6 containing 20% (w/v) glycerol.
In addition to removing (NH^^SO^, this step also removed much of the small
molecular weight compounds, including sodium cholate, from the preparation.
The post-dialysis cytochrome P-450 preparation was either used immediatly,
or stored in small (approx. 1-2 ml) aliquots at -70°C until required.
4.2.3 Assays for cytochrome P-450 and protein
Cytochrome P-450 and protein were assayed as described in Sections
2.2.4.1 and 2.2.4.8 , respectively. Cytochrome P-450 content was determined
for each fresh sample and for each frozen sample following thawing.
4.2.4 Ligand-induced binding spectra
4.2.4.1 Measurement of ligand-induced binding spectra
Ligand binding studies were performed at 25°C on a kontron Unicon 860
dual-beam scanning spectrophotometer using the standard procedure described
by Schenkman et al. (1967). Solubilized and (NH^)2 ^0 ^-fractionated
digestive gland microsomal cytochrome P-450 (or occasionally crude
microsomes) was added to each of two 1 ml masked cuvettes (sample and
reference), to a final cuvette concentration of approximately 2 0 0 pmoles
ml"^ in 100 mM phosphate buffer, pH 7.6. The cuvettes were placed in the
spectrophotometer and, after temperature-equilibration, a corrected
baseline was recorded between 350 and 500 nm. Ligand was then added to the
203
sample cuvette and an equal volume of the solvent vehicle (either water or
DMF) added to the reference (DMF alone showed no spectral interaction with
mussel cytochrome P-450). The cuvette contents were gently stirred and the
difference spectrum recorded between 350 and 500 nm. For determination of
the apparent spectral dissociation constant of a particular compound,
ligand and solvent were added to their respective cuvettes as aliquots
(generally ranging from 1 to 5 /il) until further addition of ligand failed
to produce further changes in the spectrum.
Certain compounds such as benzo [a] pyrene and 7-ethoxycoumarin were
'optically-active' between 350 and 500 nm. For these compounds, the split
cell technique described by Azari and Wiseman (1982b) was used to eliminate
interference from this absorbance. Solubilized and (NH^^SO^- fractionated
cytochrome P-450 (1 ml) was added to one compartment of each cuvette (to
give approximately 200 pmoles ml”'*') and an equal volume of 100 mM phosphate
buffer, pH 7.6, to the other. After recording the baseline, difference
spectra were recorded following addition of ligand to the 'sample'
compartment of the sample cuvette and the 'buffer' compartment of the
reference cuvette, and the solvent vehicle to the 'sample' compartment of
the reference cuvette and 'buffer' compartment of the sample cuvette.
Determination of the apparent spectral dissociation constant was again
achieved by incremental addition of ligand and solvent as described above.
4.2.4.2 Determination of the apparent spectral dissociation constant (K„)
and theoretical maximum peak to trough absorbance change (AA ^^)
The apparent spectral dissociation constant (Kg; that concentration of
204
ligand causing half-maximal spectral change) and the theoretical maximum
peak to trough absorbance change (AA^^ were determined from a plot of
peak to trough absorbance difference (AA) against ligand concentration
([S]) after fitting the data to both the Michaelis-Menten model
and the Hill model
AA - ---------Ksn + [S]n
using the Plymouth Marine Laboratory computer programme 'LSFITS' (described
in Livingstone and Clarke, 1983). In the latter equation, the value of n
(Hill coefficient) represents a third unknown parameter (in addition to Kg
and AAmax) not present in the Michaelis-Menten model. As used in this
study, a value of n significantly greater than or less than 1 would
indicate more complex binding than defined by the Michaelis-Menten model.
However, for each of the compounds showing spectral interaction with mussel
cytochrome P-450, it was found that the 95% confidence interval for n
contained the value n = 1 , and, therefore, it demonstrated that the more
complex Hill model did not represent a significant improvement over the
Michaelis-Menten model for calculating kg and AAj . Direct fitting of the
data to a AA versus [S] plot, to calculate Kg and A A ^ ^ is statistically a
more rigorous and accurate procedure than an unweighted or weighted
Lineweaver - Burk plot of 1/AA versus 1/[S] (Livingstone and Clarke,
1983).
205
4.3 Results
Figures 4.1, 4.2 and 4.3 show, respectively, the binding to
solubilized and (NH^^SO^- fractionated mussel digestive gland cytochrome
P-450 of clotrimazole, metyrapone and pyridine. Each compound (known to
give type II binding spectra with mammalian cytochromes P-450) gave typical
type II binding spectra (see Section 1), with absorbance maxima at about
424-427 nm and minima at about 384-389 nm. The binding spectra produced by
clotrimazole (an //-substituted imidazole anti-fungal agent) was typical of
spectra produced by two related //-substituted imidazole compounds,
miconazole and ketoconazole (data not shown). The binding characteristics
for each of these compounds, and also for imidazole (also a mammalian"*" type
II ligand) are presented in Table 4.1
The apparent Kg values for each of the //-substituted imidazole
compounds was an order of magnitude lower than the values for metyrapone
and pyridine, indicating mussel digestive gland microsomal cytochrome P-450
to have a far stronger affinity for these compounds. The apparent kg for
the binding of imidazole could not be determined because saturation (less
than 0.005 absorbance units) was achieved after only a single substrate
addition. The background-noise, created by the turbidity of the cytochrome
P-450 preparation, prevented the use of a higher spectrophotometer
sensitivity or a greater initial dilution of imidazole stock.
With the exception of imidazole, and to an extent, also pyridine,
(nmole"’*' P-450) values for each of the compounds producing type II
spectra were approximately similar, indicating each to have a roughly
2061. The term mammalian is used to identify the list of type I, type II
and reverse type I compounds as given in Schenkman et al. (1981).
0.005-
- 0.010 390 4^5 460WAVELENGTH nm500350
,Cl
CLOTRIMAZOLE
0.024LUg 0.015-<CD CCO 0.011 * CD CD <
0.0054<
* * *'
" I " “ i 1 “ 1 ( . . . i * * * • i *— ■— r— ■— i— “ — r-5 10 15 20 25 30 35 40CLOTRIMAZOLE (yuM)
Figure 4.1Top: Type II difference spectrum given by 29 /xM clotrimazole with
solubilized and (NH^^SO^-fractionated mussel cytochrome P-450.
Cytochrome P-450 and protein concentrations were 215 pmoles ml 2.82 mg ml" , respectively.
-1 and
Bottom: Plot of peak to trough absorbance change against concentration of clotrimazole.
207
UJoz<£ 0.015OcoCO<
wnm
390 425WAVELENGTH
500
0=C
METYRAPONE
0015-
001 -
LU O z <COa;Ococo< 0 005 <
... js-..
100 200 300 400 500 600 700 800 900 METYRAPONE (jM)
Figure 4.2Top: Type II difference spectrum given by 750 /iM metyrapone with
solubilized and (NH^^SO^-fractionated mussel cytochrome P-450.
Cytochrome P-450 and protein concentrations were 206 pmoles ml” and 2.55 mg ml ” , respectively.
Bottom: Plot of peak to trough absorbance change against concentration of metyrapone
208
LDOZ<£ 0.007-OcoCD<
0.002. 460nm390 425
WAVELENGTH500350
PYRIDINE
I *0 0 0 6 -j
w 0 005-I Xz< 0 0044COO 0 0034tf\ t v< 0 002
0001
“ I....... t-- --- i.... — ,-------- r .-----r-100 200 300 400 500 600
PYRIDINE (juM)
Figure 4.3
Top: Type II difference spectrum given by 475 /zM pyridine with solubilized and (NH^^SO^-fractionated mussel cytochrome P-450.
Cytochrome P-450 and protein concentrations were 218 pmoles ml~^ and 2.74 mg ml"\ respectively.
Bottom: Plot of peak to trough absorbance change against concentration ofpyridine.
209
Table 4.1
The binding to solubilized and (NH^^SO^- fractionated mussel digestive
gland microsomal cytochrome P-450 of compounds known to give type II
spectral changes with mammalian cytochromes P-450.
Difference Spectra Ks ^max .1 95%Compound Maxima Minima (nmole confidence in
nm nm (/iM) P-450) for "n" (H
Clotrimazole 427 387 3.8 0.114 0.72 - 1.02
Miconazole 424 384 12.7 0.097 0.93 - 1.39
Ketoconazole 425 384 1 1 . 0 0.077 0.49 - 1.10
Imidazole 427 386 - <0.005 -
Metyrapone 424 389 132.7 0.096 0 . 6 8 - 1.08
Pyridine 427 389 216.3 0.041 0.88 - 1.31
210
similar ability to perturb the cytochrome P-450 spin-equilibrium toward the
low spin state (see Section 1). In addition to the compounds listed in
Table 4.1, the mammalian type II compounds aniline, nicotinamide,
benzylamine and p-aminophenol were also tested, but showed no detectable
binding.
Addition of a number of mammalian type I compounds to solubilized and
(NH^^SO^- fractionated mussel digestive gland microsomal cytochrome P-450
preparations failed to produce binding spectra, viz. for benzo[a]pyrene,
benzphetamine, biphenyl, aminopyrine, arachidonic acid, lauric acid,
cortisol, cholesterol, /?-oestradiol, hexobarbital, n-octane, safrole,
phenobarbital, and toluene. Spectral interactions were observed, however,
for the type I compounds testosterone, androstenedione, SKF 525-A, 7-
ethoxycoumarin and a-naphthoflavone but the spectra produced by each of
these compounds were not characteristic type I spectra. Rather, they
resembled the type II spectra described previously, but possibly may be
reverse type I in nature (see Discussion). Figures 4.4 to 4.7 show the
spectra produced by testosterone, 7-ethoxycoumarin, a-naphthoflavone and
SKF 525-A, respectively. The spectrum produced by androstenedione was
essentially similar, but for this compound, like imidazole, the AAmax was
too small for a spectral titration to be performed. Assuming the spectra
obtained are indicative of ligand-binding, the binding characteristics for
these compounds are presented in Table 4.2. Unlike the binding of type II
compounds, the absorbance maxima and particularly the minima for these
compounds occurred over a relatively wide range of wavelengths (maxima 422-
431 nm, minima 384-406 nm) . It is possible the wide range of minima values
may, in part, be explained by the relatively broad troughs making it
difficult for the peak detection facility of the spectrophotometer to
211
Illo2<“ 0.015Ocom<
0.005 500460425390 WAVELENGTH
350nm
TESTOSTERONE
0 -012-
001 -LUz 0-008- <£ 0 006- oS 0-004- <<] 0-002-f,
I
.X-"'X,.-
— I— ■— I— ■— I— ■— I— ■— I------1-------1200 400 600 800 1000 1200 1400TESTOSTERONE (uM)
Figure 4.4Top: Difference spectrum produced by 845 /iM testosterone with solubilized
and (NH^^SO^- fractionated mussel cytochrome P-450.
Cytochrome P-450 and protein concentrations were 205 pmoles ml"^ and2.48 mg ml" , respectively.
Bottom: Plot of peak to trough absorbance change against concentration oftestosterone.
212
Illoz<“ 0.00-o <n c o <
- 0.01 500425 460390WAVELENGTH
350nm
7-ETHOXYCOUMARIN
0-015-
0014LUo z <CO DCO COCO< 0 005-<
*xx
2000 4000 6000 80007-ETHOXYCOUMARIN ( iM)
Figure 4.5
Top: Difference spectrum produced by 6.54 mM 7-ethoxycoumarin with solubilized and (NH^^SO^- fractionated mussel cytochrome P-450.
_ iCytochrome P-450 and protein concentrations were 216 pmoles ml and2.52 mg ml" , respectively.
Bottom: Plot of peak to trough absorbance change against concentration of7-e thoxyc oumar in.
213
u.uu
LUOz<COCCoCOCO<
0.01<
- 0.01
oC-NAPHTHOFLAVONE
350 390 425 460WAVELENGTH nm
500
0014-LU 0012-oz 001-<CDCC 0008-oCOCD 0006-<<1 0004-
0-002-w0 J 1-------; t 1 l 1-------- 1------- 1 -t ----- i—5 10 15 20 25 30 35 40 45 50
c<-NAPHTHOFLAVONE (fJM)
Figure 4.6Top: Difference spectrum produced by 30 /zM a-naphthoflavone with
solubilized and (NH^^SO^- fractionated mussel cytochrome P-450.
Cytochrome P-450 and protein concentrations were 205 pmoles ml"^ and2.48 mg ml’ , respectively.
Bottom: Plot of peak to trough absorbance change against concentration ofa-naphthoflavone.
214
LUo2“ 0.04-o£0m<
0.02 460 500350 425390WAVELENGTH nm
CH-CHCH
c=o
CH2c h 2
3h c 2hc/ n c h 2ch3SKF 525-A
0 -012-
uj ° 01'01 0-0084 mo 0 006-(f)co< 0 004
0-002-
0
Figure 4.7
...X.-"
X.-''
X''
500 1000 1500 2000SKF 525-A tyjM)
Top: Difference spectrum produced by 990 /jM SKF 525-A with solubilized and (NH^^SO^- fractionated mussel cytochrome P-450
•1Cytochrome P-450 and protein concentrations were 207 pmoles ml and2.52 mg ml~ , respectively.
Bottom: Plot of peak to trough absorbance change against concentration ofSKF 525-A.
215
Table 4.2
The binding to solubilized and (NH^^SO^- fractionated mussel digestive
gland microsomal cytochrome P-450 of compounds known to give type I
spectral changes with mammalian cytochromes P-450.
CompoundDifference spectra Kg ^Amax 95%Maxima Minima (nmoleconfidence intervalnm nm (/zM) P-450) for "n" (Hill)
Testosterone 428
Androstenedione 422
7-Ethoxycoumarin 427
a-Naphthoflavone 431
SKF 525-A 428
384 203.6 0.070
404 - <0.005
392 1440 0.074
406 7.81 0.076
394 946.5 0.072
0.96 - 1.71
0.76
0.98
0.99
1.43
1.43
1.93
216
determine values accurately. With the exception of androstenedione,
(nmoleP-450) values were similar for each compound, indicating each
binds to solubilized and (NH^^SO^- fractionated mussel digestive gland
microsomal cytochrome P-450 with a similar intensity (and hence perturbs
the spin-equilibrium toward the low spin state to a similar extent).
The spectrum for a-naphthoflavone binding (Figure 4.6) showed an extra
peak at about 466 nm. Although an extra peak was not observed in binding
spectra produced by type II compounds, high wavelength shoulders were
observed in the spectra produced by both testosterone (Figure 4.4) and 7-
ethoxycoumarin (Figure 4.5) binding. This is seen more clearly in Figure
4.8, which shows the binding of testosterone to a cytochrome P-450
preparation different to that used for the spectral titration. Unlike the
peak observed with a-naphthoflavone (466 nm), the shoulders observed with
both testosterone and 7-ethoxycoumarin occurred at about 455-460 nm. The
spectrum produced by SKF 525-A also contained a shoulder, but this occurred
between about 410 and 425 nm, rather than at about 455-465 nm.
Although spectra of a reverse type I/type II shape were obtained after
addition of both type II and type I compounds, no such spectra were
observed after addition of mammalian reverse type I compounds, viz.
ethanol, n-butanol and phenacetin.
On occasions, attempts were made to obtain ligand binding spectra with
crude microsomes. Unfortunately, the low cytochrome P-450 specific content
(approx. 40-60 pmoles P-450 mg~^ protein) necessitated a relatively high
cuvette protein content, and consequently a high turbidity, in order to
217
0.030
LUo2<COCCoCOm<
0.020-
0.010 500460425390350WAVELENGTH nm
Figure 4.8
Difference spectrum produced by 850 /iM testosterone with solubilized and (NH^^SO^- fractionated mussel cytochrome P-450.
Cytochrome P-450 and protein concentrations were 220 pmoles ml"^ and 2.81 mg ml" , respectively.
218
have sufficient cytochrome P-450 in the cuvette. To avoid an inpractical
background noise level only a low spectrophotometer sensitivity could be
used and consequently, only very marked spectral interactions could be
detected. For most compounds, spectral interaction with cytochrome P-450
in digestive gland microsomes was not detectable. The binding of the N-
substituted imidazole compounds, however, could be observed. Figure 4.9
shows a typical type II spectrum obtained for clotrimazole and crude
digestive gland microsomes.
219
0.045
wOz<Jr 0.030-OcoCO<
0.015-1— 350 500390 460425WAVELENGTH nm
Figure 4.9
Type II difference spectrum produced by 15 /xM clotrimazole with mussel digestive gland microsomal cytochrome P-450.
Cytochrome P-450 and protein concentrations were 210 pmoles ml"^ and 4.31 mg ml" , respectively.
220
4.4 Discussion
Type II ligand-binding spectra, indicative of cytochrome P-450 spin
shifts toward a new low spin state, are believed to be caused by direct
ligation to the haem iron (ferric form) of compounds containing non-bonded
electron pairs (see Section 1). In this study, it is assumed the spectra
given by imidazole, //-substituted imidazoles, metyrapone and pyridine (all
mammalian type II compounds) are indicative of such binding, although
carbon monoxide displacement experiments (Schenkman et al., 1972) which
would have verified this were not performed. With the exception of
metyrapone binding to cytochrome P-450 of hepatopancreas microsomes of the
freshwater crayfish, Procambrus clarkii (Jewell, 1987), none of the type II
compounds shown to interact with mussel digestive gland microsomal
cytochrome P-450 have been examined in other aquatic species. Of the type
II compounds tested, only aniline has been examined in aquatic species (see
later). The apparent high affinity binding to mussel cytochrome P-450,
observed for the //-substituted imidazole compounds (Kg = 4-12 /uM) , although
an order of magnitude less, bears comparison with the high affinity binding
to rat microsomal cytochrome P-450, (Murray and Zaluzny, 1988). However,
caution must be taken with such direct comparisons of, for example, Kg
values, because, of necessity, preparations of solubilized and (NH^^SO^-
fractionated cytochrome P-450 were used in this study compared with, for
example, microsomal or purified preparations used in mammalian and other
studies.
Despite addition of a wide variety of mammalian type I ligands to
preparations of digestive gland microsomal cytochrome P-450, characteristic
221
type I binding spectra were not observed. This included biphenyl although
biphenyl hydroxylase activity has been detected in microsomes of M. edulis
(Willis and Addison, 1974). Benzo[a]pyrene hydroxylase activity is also
measurable in M. edulis (see Section I and elsewhere) but absence of this
binding is perhaps not surprising given that 'uninduced' mussels were used
i.e. type I benzo[ajpyrene spectra are only seen in rats containing the 3-
methylcholanthrene-induced cytochrome P-450 isoenzyme (Schenkman et al.,
1981) . The lack of binding of many of the compounds contrasts with studies
on various teleost and elasmobranch fish (James et al., 1979; Schwen and
Mannering, 1982b; Fabacher, 1982) and crustacea and polychaetes (James et
al., 1979; Conner and Singer, 1981; Lee et al., 1981; Jewell, 1987), in
which type I binding spectra were observed with lauric acid, benzphetamine,
SKF 525-A, aminopyrine, hexobarbital, phenobarbital and other compounds
However, a lack of type I binding, similar to that observed in this study
for the mussel, has been reported for human foetal and adult liver
microsomes (Pelkonen et al., 1973), insects (Kulkarni et al., 1974) and the
trout Salmo trutta lacustris (Ahokas et al., 1976). Type I binding spectra
are indicative of cytochrome P-450 low to high spin shifts and are thought
to be caused by substrate, and other ligands, binding to the type I
(active) site (see Section 1). Binding of substrate to the type I site is
a prerequisite for cytochrome P-450-mediated metabolism (Ortiz de
Montellano, 1986), although equally well, all compounds binding to the type
I site are not necessarily substrates of (metabolized by) cytochrome P-450
(Schenkman et al., 1981). The lack of type I binding to mussel cytochrome
P-450 might possibly be due to endogenous substrates, or some sort of
inhibitor, already present at the type I site, so preventing binding of the
added type I compounds. An inability of type I substrates to bind to
mussel cytochrome P-450 might partly be responsible for the difficulties
222
encountered in measuring certain xenobiotic MFO activities in digestive
gland microsomes (see Section 5). The presence of an endogenous ligand was
suggested as being a reason for the lack of type I binding in trout (Ahokas
et al., 1976). A number of possibilities exist for an endogenous ligand
preventing type I binding in mussels. If a function of the cytochrome P-
450 is endogenous metabolism, then substrates such as fatty acids or
steroids could be bound. Alternatively, the digestive gland is rich in
carotenoids and other pigments, and, following homogenization, one or more
of such molecules might bind to the type I site. Similarly, a ligand could
be derived from the so-called "digestive juices" of the tissue. In the
rock crab, Cancer borealis and the lobster, Homarus americanus, the
digestive juices have been suggested as possibly being responsible for the
negligable xenobiotic metabolism observed in hepatopancreas microsomal
fraction (Pohl et al., 1974), although this inhibitory effect could have
been due to a proteolytic rather than a ligand-binding action.
The apparent absence of type I binding to mussel cytochrome P-450
might have been a contributory factor toward the non-detection of type II
spectra with other substrates, such as aniline. Aniline hydroxylase
activity has, in fact, been detected in digestive gland microsomes of the
mussel, Mytilus galloprovincialis (Ade et al. , 1982). Although this
compound produces type II spectra with uninduced cytochrome P-450 from
mammals (Imai and Sato; 1967; Schenkman et al., 1967; Topham, 1970), it is
also a substrate for cytochrome P-450 and, therefore, has a type I
component, in addition to ligating directly to the haem moeity. The lack
of a type II spectrum with aniline could, therefore, be partly due to its
exclusion from the substrate-binding site. However, in this respect it
should be noted that a type II aniline spectrum was observed for trout,
despite the noted absence of type I binding (Ahokas et al., 1976). Type II
spectra for aniline have also been observed for microsomal fractions of the
crab, Sesarma cinerum (Lee et al., 1981), the crayfish, P. clarkii (Jewell,
1987) and various other fish species (James et al., 1979; Fabacher, 1982).
Another possibility that must be considered to explain the non
detection of difference spectra with many of the compounds is not lack of
binding, but rather a combination of perhaps a lowered affinity of the
ligands for digestive gland microsomal cytochrome P-450 with the
limitations imposed by the experimental working conditions. For example,
about 200 pmoles of mussel cytochrome P-450 were in each assay compared
with about 500 to 1500 pmoles used in many mammalian and non-mammalian
aquatic studies (Schenkman et al., 1967; Fabacher, 1982; Jewell, 1987).
Also, the maximal absorbance change nmolecytochrome P-450 of the
compounds that did give spectra with M. edulis microsomes were generally of
the order 0.04 to 0.11 compared with values of 0.008 to 0.03 for the above
references. Similarly low absorbance change nmole"^ P-450 for some
compounds with mussel cytochrome P-450 might make the detection of spectral
changes difficult, if not impossible. On the other hand, spectra were seen
for imidazole and androstenedione with AA n m o l e v a l u e s of <0.005.
Clearly, further studies are required, with higher concentrations of mussel
cytochrome P-450, to examine these possibilities.
The spectra obtained with testosterone, 7-ethoxycoumarin, a-
naphthoflavone and SKF 525-A were unexpected. Each are mammalian type I
compounds, and, as possible substrates (testosterone and 7 -ethoxycoumarin)
and substrate-analogue inhibitors (a-naphthoflavone and SKF 525-A) of
224
mussel MFO activity (see Section 5), would be expected to bind to mussel
digestive gland microsomal cytochrome P-450 to give type I spectra.
However, assuming the mussel cytochrome P-450 to be structurally similar to
the cytochromes P-450 of other species, the spectra obtained were
characteristic of either type II or reverse type I binding. The spectra
are unlikely to be indicative of type II binding, however, since none of
the compound structures contain accessable (nitrogenous) non-bonded
electron pairs to ligate directly to the haem iron. As discussed in
Section 1, causes of reverse type I spectral change are uncertain. They
have been suggested as being due to either endogenous ligand displacement
from the type I site (Schenkman et al., 1969, 1972, 1973) or to alcohol and
sometimes ketone group interaction with the haem iron (Yoshida and Kumaoka,
1975; Ebel et al., 1977; Andersson and Dawson, 1984). Direct interaction of
protein with cytochrome P-450 has also been observed (Schenkman et al.,
1981) . Although the range of wavelength maxima and minima obtained for the
mussel are slightly higher than the, respectively 420 nm and 390 nm
obtained for reverse type I binding to mammalian cytochromes P-450
(Schenkman et al. , 1981), reverse type I binding seems a possibility.
Reverse type I binding has been observed previously for steroids, for
example, for testosterone with rat kidney microsomes (Ellin et al., 1972)
and pregnenalone with human placental microsomes (Canick and Ryan, 1978;
N.B. in this study, androstenedione and various testosterone derivatives
produced type I spectra) . Causes of the spectral changes seen for
testosterone, SKF 525-A, 7-ethoxycoumarin and a-naphthoflavone with mussel
cytochrome P-450 can only be speculated at. If, as suggested above, the
absence of type I binding is due to the presence of an endogenous ligand at
the type I site, then the spectral changes might be due to ligand
displacement and, therefore, lend support to the endogenous ligand
displacement theory (Schenkman et al., 1972). However, equally noticeably,
each of the compounds possesses an alcoholic or ketone group and so direct
interaction with the haem is a possibility. Arguing against the latter
explanation, is possibly the observation that for certain substrates, for
example cyclohexanone binding to cytochrome (Andersson and Dawson,
1984), reverse type I spectra occur at high substrate concentrations, due
to alcohol or ketone interaction with the h4£m, whereas at low substrate
concentrations type I spectra are seen. With mussel cytochrome P-450,
reverse type I - like spectra were observed at all substrate
concentrations. A third explanation, of course, is that the structure of
molluscan cytochrome P-450 is sufficiently different from mammalian or
bacterial P-450s, such that binding of the substrate to the apoprotein
stabilizes hexacoordinate coordination of the haem iron, so shifting spin
equilibrium towards a low spin state.
Comparison of spectral dissociation constants for these type I
compounds with values for other species is of limited value, given that
they did not show type I spectra and the amount of data available are not
extensive. Also, as for the type II compounds, the mussel studies were not
on crude microsomes or purified cytochrome P-450. In addition, the
spectral shoulders, and in the case of a-naphthoflavone the extra
absorbance peak, generated in the spectra by these type I compounds are not
normally seen in substrate-binding spectra of other species. The reason
for their presence is unknown, although again, endogenous ligands, at or
around the active site, might be involved.
Whatever the reason for the observed spectral changes caused by the
226
cytochrome P-450 substrates testosterone and 7-ethoxycoumarin, the effect
is an apparent transition of the haem spin towards the low spin state. If
these compounds are substrates for the mussel cytochrome P-450 system , as
they appear to be (see Section 5), then the transitions are in contrast to
the generally accepted mechanism of cytochrome P-450 action, viz. binding
of the substrate to the active site induces a low to high spin shift and a
concomitant change in redox potential (to a less negative value) , to
facilitate electron-transfer from reduced NADPH- cytochrome P-450 reductase
to cytochrome P-450 (Sligar et al., 1979; Gibson and Tamburini, 1984; see
Section 1). Possibly the high to low spin transition observed here,
through generating a more negative, and hence unfavourable, redox
potential, is related to the lack of any requirement for NADPH of digestive
gland microsomal 7-ethoxycoumarin O-deethylase and testosterone hydroxylase
MFO activities (see Section 5). Presumably, in the intact animal such a
situation may not arise and, if a low to high spin transition is necessary
for metabolism, then substrate binding will facilitate this. If this is
the case, then the argument is further support for the idea of a bound
endogenous inhibitor or substrate at the active site of the mussel
cytochrome P-450.
All the difference spectra obtained for digestive gland microsomal
cytochrome P-450 were best described by a simple Michaelis-Menten model,
with no deviations towards a sigmoidal binding response (Hill model) or
more complex changes indicative of isoenzymes of cytochrome P-450. This
included 7-ethoxycoumarin, which from 7-ethoxycoumarin O-deethylase
activity kinetic studies is indicated to be metabolized by more than one
mussel cytochrome P-450 isoenzyme (see Section 5). Another approach to the
227
indication of the possible existence of isoenzymes of cytochrome P-450 is a
consideration of the molefcular geometry (shape and size) of the ligands
that bind to produce spectra (Lewis et al., 1986; Ioannides and Parke,
1987; Rodrigues et al., 1987). Application of computer-graphics techniques
to the different binding sites and structures of the phenobarbital- and 3-
methylcholanthrene - type groups of cytochrome P-450 isoenzymes revealed
that whereas the former interact with non-planar globular molecules, the
latter interact with flat, planar molecules. The former ligands2 2 (substrates/inhibitors) have low A/D ratios (area/(depth) of ligand)
(e.g. 1 . 1 for phenobarbitone) whereas the latter ligands have high ratios
(e.g. 12.0 for benzo[a]pyrene) (Ioannides and Parke, 1987). The A/D
values available for the ligands binding to the mussel cytochrome P-450
were all low, suggesting a structural similarity of the binding sites more
akin to that of the phenobarbital-type cytochrome P-450 than the 3-omethylcholanthrene-type, viz. 7-ethoxycoumarin (A/D , 3.7), imidazole
(3.6), SKF 525-A (2.4), metyrapone (2.0), ketoconazole (2.0), clotrimazole
(1.0) and miconazole (0.8) (Lewis et al., 1986; Rodrigues et al., 1987).
Against this, it must be noted that compounds of intermediate molecular
dimensions such as 7-ethoxycoumarin and aflatoxin, are substrates or
inhibitors of both families of cytochromes P-450 (Ioannides and Parke,
1987), and that benzo[a]pyrene is a substrate for mussel digestive gland
microsomal cytochrome P-450 (see Section 1).
228
Section 5
Studies on the metabolism of possible xenobiotic and endogenous substrates by mussel digestive gland
microsomal preparations.
229
5.1 Introduction
Despite reports of the absence of "aryl hydrocarbon hydroxylase" and
other MFO system-associated activities in bivalves (Lee et al. , 1972;
Payne, 1977; Vandermeulen and Penrose, 1978), such activities have been
detected in vitro, in M. edulis but at low levels compared with vertebrates
or certain invertebrates of different phyla, viz. benzo [a]pyrene
hydroxylase (BPH) activity (Stegeman, 1980, 1981, 1985; Mix et al., 1981;
Livingstone and Farrar, 1984; Livingstone, 1985); biphenyl hydroxylation to
4-hydroxybiphenyl (Willis and Addison, 1974) and aldrin epoxidation to
dieldrin (Bayne et al., 1979; Moore et al., 1980). In other bivalve
molluscs, including different species of mussel, xenobiotic metabolising
activities are also evident in digestive gland microsomes or homogenates,
vis. BPH activity in the oyster Crassostrea virginica (Anderson, 1978) and
M. galloprovincialis (Ade et al., 1982; Suteau et al., 1985; Viarengo et
al., 1986); aldrin epoxidase activity in Anodonta sp. (Khan et al., 1972)
and M. californianus (Krieger et al. , 1979, 1981) and both antipyrine
hydroxylase (Krieger et al. , 1979) and p-nitroanisole 0 -demethylase
activities (Trautman et al., 1979) in M. californianus. One of the aims of
the study in this section was to further investigate the xenobiotic
metabolising capability of the digestive gland microsomal fraction of M.
edulis, the following MFO activities being examined: O-deethylase activity
towards 7-ethoxycoumarin and 7-ethoxyresorufin; //-demethylase activity
towards benzphetamine, aminopyrine, N,N-dimethylaniline and ethylmorphine;
and hydroxylase activity towards aniline and biphenyl. Previous studies
have recorded slight biphenyl hydroxylase (see before) and possibly 7-
ethoxyresorufin (Stegeman, 1985) activities in M. edulis. There are no
230
reports to date however, of activity towards any of the other compounds
examined; for example, Payne and May (1979) failed to detect 7-
ethoxycoumarin O-deethylase activity.
As discussed in Section 2, a possible major function of cytochrome P-
450 in M. edulis may be in the metabolism of endogenous substrates.
Information on the monooxygenation of such substrates in molluscs is
limited. The conversion of arachidonic acid to prostaglandins and other
more polar metabolites has been demonstrated in various tissues, but not
including the digestive gland of M. edulis (Srivastava and Mustafa, 1984,
1985), and more recently Vitamin metabolism to polar metabolites has
been observed in whole tissue homogenates of M. edulis (Lehtovaara and
Koskinen, 1986). The second major aim of the study was to investigate the
capability, if any, of the digestive gland microsomal fraction to
metabolize endogenous substrates, vis. fatty acids (arachidonic acid and
lauric acid) and steroids (testosterone).
The results of the studies are discussed in relation to the
information available on xenobiotic and endogenous monooxygenase metabolism
in M. edulis and other species. The possibility, for certain substrates at
least, that more than one metabolic process may be involved in the observed
in vitro microsomal activity is considered, as is the possibility that an
endogenous inhibitor of cytochrome P-450-mediated activity is present
within the digestive gland microsomal preparations. With respect to the
latter, a series of experiments were performed on the inhibitory effect of
mussel digestive gland microsomal preparations and ethyl acetate-extracts
of the same on certain rat liver microsomal MFO activities.
231
5.2 Materials and Methods
5.2.1 Chemicals and Materials
[4-^C]-Testosterone, [1-^C]-arachidonic acid and [1-^C]-lauric acid
were obtained from Amersham International (Amersham, Bucks., U.K.). Laurie
acid, arachidonic acid, ethylmorphine, resorufin, cyclophosphamide and
ellipticine were from Sigma Chemical Co. (Poole, Dorset, U.K.). 7-
ethoxyresorufin was from Pierce Chemicals (Rockford, Illinois, U.S.A.).
Biphenyl, 2- and 4- hydroxybiphenyls, p-aminophenol, semicarbazide
hydrochloride, formaldehyde, acetylacetone, N,N-dimethylaniline and Merck
silica gel GF thin layer chromatography (T.L.C.) plates (20cm x 20cm x
0.25mm) were obtained from B.D.H. Ltd. (Poole, Dorset, U.K.). Industrex MX
x-ray film, LX24 developer and FX40 fixer were obtained from Kodak Ltd.
(Hemel Hempstead, Herts., U.K.). All other chemicals and materials were
obtained as stated elsewhere or were of analar grade from B.D.H. Ltd.
(Poole, Dorset, U.K.). Hepatic microsomes from untreated, clofibrate pre
treated or phenobarbital pre-treated rats were the generous gifts of Mr. P.
Kentish or Mr. M. Milton, Dept. Biochemistry, University of Surrey,
Guildford, U.K.
5.2.2 Preparation of digestive gland microsomal fraction
Mussels were collected from Whitsand Bay, Cornwall, and dissected as
described in Section 2.1.1. Digestive gland microsomal fractions were
prepared from 20-30 g pools of frozen (-70°C) tissue, essentially as
described in Section 2.2.3. The microsomal pellets were resuspended to a
final protein concentration of 5-10 mg ml"^ in 20 mM Tris-HCl, pH 7.6,
232
containing 20% (w/v) glycerol and 1 mM EDTA. These were frozen until
required at "70°C in aliquots of 2-3 ml. Once thawed, samples were used
immediatly and not refrozen.
5.2.3 Studies on NADPH-independent 7-ethoxycoumarin O-deethylase activity
7-Ethoxycoumarin O-deethylase (ECOD) activity in digestive gland
microsomes was observed in the absence of NADPH and was inhibited by the
addition of NADPH (see Section 2) . Studies were carried out on the
properties of the NADPH-independent ECOD activity using the assay
procedure described in Section 2.2.4.6 , except that a 7-ethoxycoumarin
concentration of 0.24 mM rather than 0.1 mM was used. This concentration
was established from preliminary substrate concentration optimization
experiments. A variety of compounds (see Section 5.3.1), including
classical inhibitors of cytochrome P-450, were examined over a range of
concentrations for stimulatory or inhibitory effects on NADPH-independent
ECOD activity. Blank determinations were performed using the appropriate
solvent vehicle for the particular compound (water, dimethyl formamide
(DMF) or acetone). NADPH-independent ECOD activity was determined, as the
mean of triplicate assays, after correcting for the blank controls.
Also described is a study of the tissue and sub-cellular distribution
of NADPH-independent ECOD activity in Mytilus galloprovincialis.
Microsomal, mitochondrial (12,000 x g pellet) and cytosolic (100,000 x g
supernatant) fractions were prepared, as described in Livingstone and
Farrar (1984), from digestive gland, gill, mantle, foot, posterior adductor
muscle and remainder tissues and assayed for NADPH-independent ECOD
activity. The mussels for this study were from a sub-tidal population in
233
the Arachon Bay, France. Activities were expressed as the mean ± SEM of
four samples of each fraction. The tissues of six mussels comprised each
sample.
5.2.4 NADPH-independent 7-ethoxyresorufin O-deethylase (EROD) activity
An attempt to measure digestive gland microsomal EROD activity was
performed on M. edulis collected during the autumn of 1986. The method
used, based on that of Burke et al. (1977), measures the 0-deethylation of
7-ethoxyresorufin to resorufin:
7-ethoxyresorufin 7-hydroxyresorufin
Product formation is measured fluorometrically with excitation at 560 nm
and emission at 586 nm. The fluorescence spectrophotometer and
experimental procedure were similar to those described for NADPH-
independent ECOD activity (Section 2.2.4.6 ). A 7-ethoxyresorufin
concentration of 0.2 mM was used (added as 10 /il of a 50 mM stock in
methanol), in a final reaction volume of 2.5 ml and reaction was started by
addition of microsomal sample (200 /il; 1-2 mg protein). EROD activity was
investigated in the presence and absence of 0.25 mM NADPH, and the activity
calculated by reference to the fluorescence produced by successive
additions of 5 /il (25 pmoles) of the product, resorufin (see ECOD activity
determination, Section 2.2.4.6 ).
234
5.2.5 Testosterone hydroxylase activity
Mussel digestive gland microsomes were assayed for testosterone
hydroxylase activity using an adaption of the method of Shiverick and Neims
(1979). An initial incubation mixture contained, in a volume of 4.9 ml
(assay concentrations in brackets): 3.5 ml of 100 mM potassium phosphate
buffer, pH 7.4 (70 mM); 0.2 ml of 3 mM testosterone substrate in ethanol
(containing 30 /tCi [4 - ^C]-testosterone) (0.12 mM); 0.2 ml of 125 mM
MgCl2 (5 mM) and 1 ml of microsomal fraction (5-10 mg protein ml"^). After
incubating for 5 mins, at 25°C, 0.1 ml of either water or NADPH (20 mM in
1% NaHCOg) (0.4 mM) was added. Incubation, at 25°C, was continued for a
further 25 mins before being stopped by addition of 5 ml of methylene
chloride. Stopped reaction mixtures were vortexed and centrifuged at 2,500
rpm for 5 mins, before removing the methylene chloride layers to clean
tubes. Following two further extractions with 5 ml of methylene chloride,
the combined extracts of each sample (containing testosterone and any
testosterone metabolites) were dried down under nitrogen and the residues
resuspended in 1 0 0 /il of diethyl ether for application to silica gel thin
layer chromatography (T.L.C.) plates. Normally, the extracts of eight
samples were applied to each plate. Loaded T.L.C. plates were developed
with methylene chloride/acetone (4:1 (v/v)) followed in the same dimension
by chloroform/ethyl acetate/ethanol (4:1:0.7 (v/v/v)). The radioactive
zones of developed plates were located by autoradiography (see Section
5.2.7), individually scraped into scintillation vials and counted directly
in 5 ml of Optiphase 'safe' scintillant on a Beckman LS 230 liquid
scintillation counter. Testosterone hydroxylation was expressed in terms
of per cent conversion to metabolites, making the estimation of recoveries
unnecessary.
235
5.2.6 Arachidonic acid hydroxylase activity
The assay for arachidonic acid hydroxylase activity utilized
incubation and extraction procedures based on those of Capdevila et al. ,
(1981). An initial reaction volume of 1.96 ml (assay concentrations in
brackets) contained: 1 ml of 100 mM Tris-HCl, pH 7.4 (50 mM); 0.1 ml of 3
mM stock arachidonic acid in ethanol (containing 30 /xCi [1 -^C]-
arachidonic acid (0.15 mM); 0.2 ml of 50 mM MgC^ (5 mM); 0.5 ml of
digestive gland microsomal fraction (5-10 mg protein ml“ ) and 0.16 ml of
distilled water. After a 5 min pre-incubation at 25°C, the reaction volume
was made up to 2 ml with 40 /xl of either distilled water or 20 mM NADPH (in
1% NaHCOg) (0.4 mM). Incubation was continued for 1 hour before being
stopped by removal to ice and the addition of 50 /xl of 1 M HC1.
Arachidonic acid and any metabolites were extracted into 6 ml of ethyl
acetate containing 0.01% butylated hydroxytoluene. This was achieved by a
twice repeated procedure involving addition to the stopped reaction
mixtures of 2 ml aliquots of ethyl acetate containing butylated
hydroxytoluene, mixing for 10 mins in sealed (sovril) tubes, centrifugation
for 10 mins at 2,500 rpm and removal of the upper (ethyl acetate) layers to
clean tubes. The extracted samples were evaporated to dryness under
nitrogen, reconstituted in 1 0 0 /x 1 of ethyl acetate and applied to silica
gel T.L.C. plates. Loaded plates were developed with the upper phase of
ethyl acetate/iso-octane/acetic acid/water (110:50:20:100 (v/v/v/v)).
Radioactive areas of developed plates were located by autoradiography
(Section 5.2.7), scraped into scintillation vials and counted, as described
in Section 5.2.5, in 5 ml of Optiphase 'safe' scintillant.
236
5.2.7 Autoradiography of developed T.L.C. plates
Autoradiography of developed T.L.C. plates was performed in a
photographic dark-room. One sheet of Industrex MX x-ray film was placed
over each T.L.C. plate and left in darkness for 3-4 days. This film was
then removed from the plate and developed for 4 mins in a 1:5 dilution of
LX24 developer. After rinsing in tap water for 30 secs, the developed film
was fixed for 2 mins in a 1:5 dilution of FX40 fixer. The fixed film was
again rinsed in tap water for a further 30 secs and allowed to dry. The
developed and fixed film showing radioactive zones was used to locate
radiolabelled substrate and metabolites on the T.L.C. plate.
5.2.8 Laurie acid hydroxylase activity
The assay for lauric acid hydroxylase activity was based on that of
Parker and Orton (1980). An initial 1.96 ml incubation mixture (assay
concentrations in brackets) contained: 1 ml of 100 mM Tris-HCl, pH 7.4, (50
mM); 0.4 ml of 1 mM lauric acid (freshly diluted from a 200 mM stock
solution in methanol with 10 mM Tris-HCl, pH 7.4) (0.2 mM) ; 0.1 pCl of [1 -
^C]-lauric acid (added in 10 pi methanol); 0.5 ml of digestive gland
microsomal fraction (5-10 mg protein ml"^) and 50 pi of distilled water.
After pre-incubation at 25°C for 5 mins, the reaction volume was made up to
2 ml with 40 pi of either distilled water or 20 mM NADPH (in 1% NaHCOg)
(0.4 mM). Following a further 15 min incubation at 25°C, reaction was
stopped by the addition of 0.2 ml of 3M HC1, and removal of the tubes to
ice. Lauric acid and any metabolites were extracted into 10 ml of diethyl
ether by transferring the stopped incubation mixtures (plus diethyl ether)
to 15 ml screw cap sovril tubes and rotating end-over-end for 10 mins.
237
After centrifugation at 2,500 rpm for 10 mins, the upper (ether) layers
were transferred to clean tubes and evaporated to dryness under nitrogen.
Any radiolabelled metabolites produced during the incubation were separated
from unmetabolised [1 - ^C]-lauric acid by T.L.C. The dried ether
extracts were reconstituted in 60 /il of methanol and applied to silica gel
T.L.C. plates. Loaded plates were developed in hexane/diethyl
ether/glacial acetic acid (70:28:1.5 (v/v/v)). Radioactive areas were
located and measured using a Berthold LB2842 automatic T.L.C. linear
analyser.
5.2.9 Benzphetamine demethyl as e activity
The N-demethylation of benzphetamine and concomitant production of
formaldehyde result from the breakdown of an unstable amino - carbinol
intermediate formed by a cytochrome P-450-mediated hydroxylation of the
benzphetamine amino-methyl group:
N— CH2OH3HC-<
r N'— H
The rate of benzphetamine N-demethylation is determined from the rate of
production of formaldehyde, measured using the method of Nash (1953).
Incubation mixtures were prepared containing, in an initial volume of 1.96
ml (assay concentrations in brackets): 1 ml of 0.3 M Tris-HCl, pH 7.4, (150
mM); 0.2 ml of 15 mM benzphetamine (pre-adjusted to about pH 7.0) (1.5 mM) ;
50 /il of 150 mM MgC^ (3.75 mM); 0.16 ml of 0.2% (w/v) semicarbazide - HC1
(also pre-adjusted to about pH 7.0); 0.5 ml of microsomal fraction (5-10 mg
protein ml‘ ) and 50 fil of distilled water. After a 5 min pre-incubation
at 25°C, the incubation volume was made up to 2 ml with 40 y.1 of either
distilled water or NADPH (25 mM in 1% NaHCO^) (0.5 mM). Incubation was
then continued at 25°C for periods up to 2 h, before stopping the reaction
with 1 ml of 15% (w/v) zinc sulphate followed by 1 ml of a 2:1 mixture of
saturated solutions of barium hydroxide and disodium tetraborate. After
centrifugation at 2,500 rpm for 10 mins to remove the protein, 2 ml
aliquots of supernatant were used in the Nash assay for formaldehyde. The
aliquots of supernatant were added to 2 ml of fresh Nash reagent (15% (w/v)
ammonium acetate and 0.2% (v/v) acetylacetone, taken to pH 6.0 with glacial
acetic acid) and incubated in darkness at 37°C for 45 mins. After cooling
for 5 mins, the absorbance was measured at 420nm on a Cecil CE 292
spectrophotometer. Formaldehyde standards over the range 0-200 nmoles
were prepared from a 5 mM stock solution in distilled water and were
included in incubation mixtures lacking benzphetamine and NADPH. These
were taken through the same procedure as 'test' samples. Benzphetamine N-
demethylase activity was determined as nmoles of product formed min” mg~^
microsomal protein. ' _
5.2.10 Other mammalian MFO activities assayed in mussel digestive gland microsomes
The metabolism of a number of other xenobiotic substrates of mammalian
cytochromes P-450 were investigated in mussel digestive gland microsomes.
239
Of the compounds undergoing demethylation (and therefore detectable using
the Nash assay for formaldehyde), aminopyrine, ethylmorphine and N,N-
dimethylaniline (in dimethylsulphoxide) were, in turn, substituted for
benzphetamine, at the same substrate concentration, in the assay for
benzphetamine N-demethylase activity (Section 5.2.9). Preliminary studies
were also carried out on compounds undergoing direct hydroxylation in
mammalian systems, vis. aniline and biphenyl hydroxylase activities
(hydroxylated to p-aminophenol and 2- and 4-hydroxybiphenyls, respectivley)
were investigated using the methods of Guarino et al. (1969) and Willis and
Addison, (1974), respectively (see references for details of assays).
5.2.11 Inhibition of rat liver microsomal MFO activities by mussel digestive gland microsomes and ethyl acetate-extracts of mussel microsomes
A series of experiments were carried out to investigate the
possibility that endogenous compounds within digestive gland microsomal
preparations inhibit in vitro mussel MFO activities. The experiments
examined the effects of mussel digestive gland microsomes and ethyl
acetate-extracts of the microsomes (presumably containing carotenoids and
other lipid-soluble pigments) on the rat hepatic microsomal metabolism of
7-ethoxycoumarin, lauric acid and benzphetamine. The assays for the
metabolism of these substrates were as described previously, but used
approximately 2 mg of rat liver microsomal protein (from untreated or
clofibrate pre-treated rats) in the absence and presence of a range of
concentrations of either mussel digestive gland microsomes or ethyl
acetate-extracts of the same. Incubations were performed at 30°C, rather
than 25°C, and reactions were started by the addition of NADPH. In the
240
case of benzphetamine N-demethylase activity, formaldehyde standards were
prepared over the range 0-200 /imoles. Ethyl acetate-extraction of pigments
and other lipid-soluble material from mussel digestive gland microsomes was
carried out as follows. Ethyl acetate (4 ml) was added to 10-20 mg of
digestive gland microsomal protein and rotated end-over-end for 1 - 2 h in 10
ml screw cap sovril tubes. After centrifugation at 2,500 rpm for 15 mins,
the ethyl acetate layer was removed and evaporated to dryness under
nitrogen. The dried extract was then reconstituted to its original volume
in microsomal buffer, and stored until required at 4°C.
241
5.3 RESULTS
5.3.1 Studies on digestive gland microsomal NADPH-independent ECOD
activity
The subcellular distribution and specific activities of NADPH-independent
ECOD activity in the digestive gland of M. galloprovincialis are given in
Table 5.1. The distribution of activity was similar for both sexes, with
approximately 90% in males and 80% in females being located in the
microsomal fraction and the remainder in the mitochondrial fraction. The
mitochondrially-located activity is probably due to microsomal
contamination (see Livingstone and Farrar, 1984). The total NADPH-
independent ECOD activity was greater in males than in females although the
specific activities in both fractions were similar for both sexes.
Subcellular microsomal and mitochondrial fractions prepared from gill,
mantle, foot, adductor and remainder tissues showed no detectable activity.
The major site of NADPH-independent ECOD activity, therefore, appears to be
the digestive gland microsomal fraction.
Figure 5.1 shows typical variation of NADPH-independent ECOD activity
with 7-ethoxycoumarin concentration. The substrate concentration versus
velocity plot indicates possible biphasic kinetics. At low concentrations
of 7-ethoxycoumarin (0 - 0.01 mM) , an apparent Michaelis constant (app. 1^)
of 35.0 ± 7.4 /aM and a Vmax of 13.6 ± 1.1 (means ± SEM) pmoles min"^ mg"^
protein were obtained. At higher concentrations (0.01 - 0.32 mM), the
apparent was 249.7 ± 107.0 /aM and the Vmax 32.6 ± 7.4 pmoles min"^ mg"^
protein. Similar biphasic kinetics were observed for different microsomal
preparations, although values for Vmax and, more particularly, apparent
242
Table 5.1
Specific activities and subcellular distribution of NADPH-independent 7-
ethoxycoumarin O-deethylase activity in digestive gland of M.
galloprovincialis.
Sex Parameter Microsomal
fraction
Mitochondrial
fraction
Cytosolic
fraction
Male Specific activityf 5.56 ± 0.54 0 . 6 8 ± 0 . 0 1 N.D.
Total activity! 116.8 ± 10.3 11.6 ± 1.5 0
Female Specific activity! 6.04 ± 0.73 0.92 ± 0.01 N.D.
Total activity! 64.8 ± 9.2 16.0 ± 1 . 8 0
f pmoles . -1 -1 m m mg protein; _ i! pmoles min , values are means ± SEM (n=4);
N.D.= not detectable;
No activity was detected in subcellular fractions of gills, mantle, foot,
posterior adductor muscle or the remainder tissues.
243
35- Vmax 32.64
25-
o 20-
15- Vmax 13.63
B 10-
HH
3500 50 100 150 200 250 300
7-ETHOXYCOUMARIN CONCENTRATION (pM)
Figure 5.1
Variation of digestive gland microsomal NADPH-independent ECOD activity of
M. edulis with 7-ethoxycoumarin concentration.
Microsomal protein = 1.9 mg
Cytochrome P-450 = 74 pmoles mg"^ protein
Values for apparent and Vmax calculated using Michaelis-Menten model
(see Section 4.2).
The two portions of the curve indicating apparent biphasic kinetics are
shown by the two lines.
13.63 x 32.64 xEquations of lines: (*) y = -------- and (A) y = --------
35.04 + x 249.7 + x
244
were variable, especially at higher 7-ethoxycoumarin concentrations (app.
values for the two portions of the curve ranged from 14-35 /zM and 250-
580 /zM; see Kirchin et al., 1988; Section 5.5). The use of 0.24 mM 7-
ethoxycoumarin in subsequent studies on digestive gland microsomal NADPH-
independent ECOD activity was a compromise between the two parts of the
substrate concentration versus velocity plot.
Normally, 1-2 mg of microsomal protein were used in the NADPH-
independent ECOD incubation mixtures. This was also a compromise between
the need for high microsomal (cytochrome P-450) levels for enzyme activity
and the necessity for low microsomal levels to minimise protein-quenching
of the 7-hydroxycoumarin (product) fluorescence. Figure 5.2 shows the
extent of quenching by digestive gland microsomal fraction. Using, for
example, 1.2 mg of microsomal protein, 7-hydroxycoumarin fluorescence was
reduced by about 65%. This amount of protein, however, was generally
sufficient (allowing for seasonality) for a reasonable, reproducible rate
of NADPH-independent ECOD activity to be measured.
The inhibitory effect of NADPH on microsomal NADPH-independent ECOD
activity is shown in Figure 5.3. Generally, complete inhibition of
activity was observed at final NADPH concentrations of between 75 and 100
/zM, whereas concentrations giving 50% inhibition varied with the microsomal
preparation between about 2 and 14 /zM. In the example shown, 50%
inhibition was obtained at an NADPH concentration of approximately 10 /zM.
In addition to NADPH, NADPH-independent ECOD activity was also inhibited by
NADH (Figure 5.4 (a)). Although an inhibition profile was not determined,
the extent of inhibition appeared similar to that observed with NADPH, vis.
88.2% inhibition at 40 /zM NADH and complete inhibition at 80 /zM. In
245
1000 -
800-
600-
=? 400 -
200 -
0.70.5 0.60.0 0.1 0.2 0.3 0.47-HYDROXYCOUMARIN CONC. (jjM)
Figure 5.2
The reduction of 7-hydroxycoumarin fluorescence by digestive gland
microsomal fraction of M. edulis.
Individual values were the mean of two determinations.
7-Ethoxycoumarin was omitted from the incubation mixture (final volume =
2.5 ml).Symbol Protein (mg) Fluorescence (%)
+ 0 1 0 0X 0.4 74* 0 . 8 ;/ 49o 1 . 2 /•' 35A 1 . 6 / 26
246
100 -
80-
z0P 60-MtnM1h 40 -
*
20 -
8040 600 20NADPH CONCENTRATION tyjM)
Figure 5.3
The inhibitory effect of NADPH on digestive gland microsomal NADPH-independent
ECOD activity of M. edulis.
Values determined are mean ± SEM (n = 3)
Microsomal protein = 1.61 mg_ 1Cytochrome P-450 = 54.4 pmoles mg
Values for apparent and Vmax calculated using Michaelis-Menten model (see
Section 4.2)
Equation of line : 107.3 xy -----------------
9.675 + x
247
Figure 5.4
Effects on digestive gland microsomal NADPH-independent ECOD activity of M.
edulis of 40 /iM NAD+, NADP+’ NADH and NADPH in the absence (a) and presence
(b) of 12 /iM cytochrome c.
Microsomal protein = 1.6 - 1.8 mg
Cytochrome P-450 = 54.4 - 57.2 pmoles mg~^
Control ECOD activity =1 1in absence of cytochrome c: 1.86 - 2.47 pmoles min mgi iin presence of cytochrome c: 1.3 - 1.73 pmoles min’ mg’
Values are mean ± range of duplicate assays except where indicated*
248
OF CONTROL
% OF
CONT
ROL
300
250
200
150
100
50
0 CONTROL NAD NADH NADP NADPH
300
250
200
150
* 100
50
0CONTROL NAD+ NADH NADP NADPH
LVNXM LVSXSl
249
+ +contrast, the oxidized coenzymes, NAD and NADP , at concentrations of 40
/zM, caused slight stimulation of NADPH-independent ECOD activity
(approximately 20%) (Figure 5.4 (a)). However, further stimulation with+ +higher concentrations of NAD or NADP was not observed (data not shown).
To test the hypothesis that the microsomal NADPH-independent ECOD
activity might utilize an endogenous source of reducing equivalents,
digestive gland microsomes were first incubated with cytochrome c before
being assayed for ECOD activity. Addition of oxidised cytochrome c to
microsomes results in a small but finite conversion to reduced cytochrome c
(observed at 550 nm) , and it was considered possible that this might
represent the removal of reducing equivalents from the microsomal material.
The pre-incubation with cytochrome c involved addition of 50 /tl of 0.6 mM
(12 /zM) cytochrome c to 200 /zl of microsomal sample (approximately 1.6 mg
protein). After 5 mins at 25°C, the 250 /zl total volume was then used in
the final 2.5 ml ECOD incubation mixture. The presence of 12 /zM cytochrome
c inhibited NADPH-independent ECOD activity by about 30% (see Figure 5.4
legend). The effects of 40 /zM concentrations of NAD+, NADP+, NADH and
NADPH on this activity are given in Figure 5.4 (b). Both NAD+ and NADP+
stimulated NADPH-independent ECOD activity in the presence of cytochrome c,
but whereas the stimulation by NADP+ was similar to that observed in the
absence of cytochrome c, the stimulation by NAD+ was considerably higher in
the presence of cytochrome c (see Figure 5.4). The reasons for the
stimulation by NAD+ and NADP+ are unknown. Different results were obtained
for NADH, but not for NADPH, in the presence of cytochrome c compared with
its absence (Figure 5.4). Whereas NADPH still markedly inhibited enzyme
activity, NADH produced a large stimulation. The latter is likely due to
reduction of the cytochrome c by microsomal NADH-cytochrome c reductase
250
activity, and the concomitant oxidation of NADH to NAD+. Given a longer
incubation period, the same result would probably have been obtained for
NADPH, through the lower NADPH-cytochrome c reductase specific activity,
vis. NADH-dependent cytochrome c reductase activity is about ten times
higher than the NADPH-dependent activity (see Section 2) : assuming-1 -1specific activities of, respectively, 80 and 8 nmoles min mg microsomal
protein in each incubation containing approximately 1.6 mg of protein, NADH
will be oxidized at a rate of 128 nmoles min"^ and NADPH at a rate of 12.8
nmoles min"^. The 100 nmoles of NADH present per incubation will therefore
be oxidized in less than a minute, whereas almost 8 minutes would be
required for total oxidation of the NADPH.
Various compounds were tested for their effects on the microsomal
NADPH-independent ECOD activity. The results for four of these, sodium
periodate, a-naphthoflavone, SKF 525-A and metyrapone, are shown in Figures
5.5 to 5.8. Sodium periodate and a-naphthoflavone both stimulated enzyme
activity with maximally-observed stimulations of, respectively 130% (400 /M
periodate) and 50% (100 |iM a-naphthoflavone) (Figures 5.5 and 5.6,
respectively). Above a concentration of 400 /jM, sodium periodate
stimulation of NADPH-independent ECOD activity was reduced (Figure 5.5).
SKF 525-A and metyrapone both inhibited ECOD activity (Figures 5.7 and 5.8,
respectively). In both cases, inhibition appeared roughly linear with
concentration over the ranges studied. Inhibition by SKF 525-A was far
greater than by metyrapone. Whereas 200 /jM SKF 525-A caused almost 80%
inhibition, only about 18% inhibition was observed with 400 fM metyrapone.
In this latter case however, an element of seasonality was apparent. The
experiments described here were performed on digestive gland microsomal
preparations from mussels collected in late autumn/early winter. In an
251
150-
125
10020 Hh*<_I1 75H
50
25
0 -•
0 . B0.60.40.20.0SODIUM PERIODATE (mM)
Figure 5.5Stimulation of digestive gland microsomal NADPH-independent ECOD activity
of M. edulis by sodium periodate.
Microsomal protein = 1.82 mg
Cytochrome P-450 *= 49.09 pmoles mg”^-1 -1Control NADPH-independent ECOD activity — 2.43 pmoles min mg
Values are mean ± SEM (n *= 3)
252
80-
60-
5 40-M
20
0 --
0.00 0.02 0.04 0.06 0.08 0.10o<-NAPHTHOFLAVONE (mM)
Figure 5.6
Stimulation of digestive gland microsomal NADPH-independent ECOD activity
of M. edulis by a-naphthoflavone.
Microsomal protein = 1.73 mg
Cytochrome P-450 = 51.4 pmoles mg~^i _ iControl NADPH-independent ECOD activity = 2.09 pmoles min mg
Values are mean ± range (n = 2)
253
100 -
80-
2Oh 60 f—HmHx2H 40
20
0 -
0.200.150.100.050.00SKF 525-A (mM)
Figure 5.7
Inhibition of digestive gland microsomal NADPH-independent ECOD activity of
M. edulis by SKF 525-A.
Microsomal protein = 1.92 mg
Cytochrome P-450 =52.3 pmoles mg"^_ i 1Control NADPH-independent ECOD activity =2.72 pmoles min mg
Values are mean ± SEM (n = 3)
254
40-
302OHh*MH 20-X2H
10
0 - -
0.40.30.20.0 0.1METYRAPONE (mM)
Figure 5.8
Inhibition of digestive gland microsomal NADPH-independent ECOD activity of
M. edulis by metyrapone.
Microsomal protein = 1.69 mg
Cytochrome P-450 = 56.41 pmoles mg"^1 -1Control NADPH-independent ECOD activity = 3.53 pmoles min’ mg
Values are mean ± SEM (n = 3).
255
earlier experiment performed on a microsomal preparation from mussels
collected during summer, a single 2 0 0 /iM metyrapone concentration caused
almost 35% inhibition of NADPH-independent ECOD activity. Unfortunately,
confirmatory or other studies on seasonality of NADPH-independent ECOD
stimulation/inhibition by these compounds were not performed.
Cyclophosphamide, ellipticine and nicotinamide at concentrations of up to
400 /iM had no effect on NADPH-independent ECOD activity. Carbon monoxide,
bubbled through microsomes at a rate of 1 bubble sec"^ for 40 secs,
resulted in inhibition of 48.3 ± 5.1% (mean ± SEM; n = 3). No NADPH-
independent ECOD activity was seen with microsomal fraction pre-boiled for
5 minutes.
5.3.2 Studies on the ability of digestive gland microsomes to metabolise
various xenobiotic substrates (.N-demethylase, O-deethylase,
hydroxylase activities).
The metabolic activity of mussel digestive gland microsomal fraction
towards a range of xenobiotic substrates was investigated. Compounds
undergoing N-demethylation by mammalian cytochromes P-450 were either very
slightly metabolised (benzphetamine, aminopyrine or N,N-dimethylaniline) or
not metabolised at all (ethylmorphine). Formaldehyde production in each
case was less than 0 . 1 nmoles min"^ mg"^ microsomal protein (equivalent to
a maximum spectrophotometric absorbance change of no more than 0 . 0 2 0 . D.
units for incubations of 30 mins at 25°C). No activity for any substrate
was observed using microsomes pre-boiled for 5 minutes. Given the
relatively low levels of microsomal cytochrome P-450, theoretical substrate-1 -1turnovers of 1-2 nmoles min nmole cytochrome P-450 were obtained. In
each case, activity was observed in the absence of added NADPH. The low
256
rates of formaldehyde production limited investigation of the effects of
modulators on the N-demethylase activities. A slight inhibition by 500 /im
NADPH (approximately 10-20%) was possibly indicated, but this was clearly
much less of an effect than for the NADPH-independent ECOD activity
(Section 5.3.1). Sodium periodate and SKF 525-A (at concentrations of,
respectively, 2 0 0 pm and 1 0 0 /jm) had no apparent effect on N-demethylase
activities. The benzphetamine N-demethylase activity was characterized
further and optimized for both substrate concentration and incubation time.
Maximal activity was obtained at a benzphetamine concentration of
approximately 2 mM. The time-course of the reaction is given in Figure
5.9. Total formaldehyde produced increased up to about 30 minutes before
plateauing and eventually decreasing after about 1 hour. The studies on
benzphetamine N-demethylase activity were performed on microsomal
preparations from mussels collected in early spring. The possibility of
seasonal variability of this or other N -demethylase activities was,
however, not examined.
Mussel digestive gland microsomes also catalysed the NADPH-independent
0-deethylation of 7-ethoxyresorufin (EROD). The rate, however, was
considerably lower than that observed for the 0 -deethylation of 7-
ethoxycoumarin. For example, when NADPH-independent ECOD activities were
high during the autumn, NADPH-independent EROD activities of 0.5 - 1.5
pmoles min"^ mg"^ microsomal protein were obtained (equivalent to
theoretical 7-ethoxyresorufin turnovers of about 15-30 pmoles min"^ nmole
cytochrome P-450).
Attempts to detect aniline and biphenyl hydroxylase activities were
unsuccessful. It should be noted however, that the sensitivities of the
257
0.025-
0.020
0.015H
* 0.010
o.ooo-:12010060 8020 400
INCUBATION TIME (MINS)
Figure 5.9Reaction time-course (formaldehyde production) of digestive gland
microsomal benzphetamine demethylase activity of M. edulis.
Microsomal protein = 3.76 mg
Cytochrome P-450 content = 38.13 pmoles mg"^.
258
assays are not particularly high (minimum specific activities detectable-1 -1are approximately 5-6 pmoles min mg protein.
5.3.3 Studies on the ability of digestive gland microsomes to metabolise the endogenous substrates arachidonic acid, "lauric acid" and testosterone
Autoradiographic results for the metabolism of arachidonic acid by
microsomes are shown in Figure 5.10. Incubations were carried out in the
presence of various modulators of mammalian cytochrome P-450 activity, vis.
NADPH and sodium periodate, and the cytochrome P-450 inhibitors metyrapone
and SKF 525-A. In each case, a number of radioactive bands were seen. Of
these, the major bands had Rf values of approximately 0.35 and 0.37
(scraped as area 3) and 0.42 (scraped as area 4) (see Figure ). The
slightly higher Rf values observed in tracks E and F were probably due to
"edge11 effects of the T.L.C. plate. Each of these radioactive bands was
more polar than the unmetabolised arachidonic acid (Rf approximately 0.67).
A less polar band, seen to run ahead of the unmetabolised arachidonic acid,
had an Rf value of approximately 0.88. Comparison of the radioactivity in
each of the scraped areas from each track (A to F) is given in Table 5.2.
It is difficult to draw many definite conclusions concerning the effects of
the cytochrome P-450 modulators because the extent of total metabolism in
each track was approximately similar, and any variation in the percentage
radioactivity of each scraped area slight. However, certain observations
can be made. Firstly, the metabolism appeared to be NADPH-independent, the
absence or presence of NADPH having little, if any, effect on the extent of
metabolism. Secondly, there was an indication of stimulation by sodium
periodate (the combined radioactivity in areas 3 and 4 was increased over
B
ID
Figure 5.10
T .L .C ./Autoradiograph showing the metabolism of arachidonic acid by
digestive gland microsomes of M. edulis under a variety of conditions._ i _ i(Protein = 9.33 mg ml ; Cytochrome P-450 = 37.27 pmoles mg )
N.B. Numbers up the sides show areas scraped and counted.
A -NADPH B +NADPH (0.4 mM)C +Sodium periodate (0.4 mM)D +Metyrapone (0.4 mM)E +Ethanol (20 /z1)F +SKF 525-A (0.2 mM applied in 20 /zl ethanol)
260
Table 5.2
The metabolism of arachidonic acid by digestive gland microsomes of M. edulis
Condition Counts: (% of Total)
Area of plate scraped
1 2 3 4 5 6 7
-NADPH 1.47 5.89 6.97 4.12 2.39 70.27 8 . 8 8
+NADPH 1.56 5.94 6.85 3.64 3.33 72.89 6.09
+Sodiumperiodate 1.46 4.90 7.62 4.81 2.37 71.54 7.31
+Metyrapone 1.30 3.31 4.77 2.48 1.56 77.58 8.99
+Ethanol 0.85 2 . 6 8 4.55 3.92 1.62 72.02 14.37
+ SKF 525-A (in Ethanol) 0.93 3.60 3.99 3.52 1.69 75.72 10.54
261
the -NADPH condition (track A). Thirdly, there was an indication of
inhibited metabolism in the presence of metyrapone or SKF 525-A. In the
presence of either of these cytochrome P-450 inhibitors, both the total
metabolism and the radioactivity in the major metabolite bands
(particularly in area 3) were reduced. However, it should be noted that
even in the presence of ethanol alone the radioactivity in area 3 was
reduced, although total metabolism was not. Interestingly, in both
incubations in which ethanol was present (tracks E and F) , the
radioactivity in the less polar band above the unmetabolised arachidonic
acid was increased, whereas, the radioactivity remaining at the origin was
decreased. Metabolite identification, using either arachidonic acid
metabolite standards, or by reference to metabolite profiles from mammalian
microsomal incubations, were not carried out. Attempts to demonstrate
lauric acid hydroxylation were unsuccessful. Scans for radioactivity on
the T.L.C. plate showed only one radioactive spot per track, corresponding
to unmetabolised lauric acid. The digestive gland samples used were from
mussels collected during late spring, 1987.
The metabolism of testosterone by mussel digestive gland and rat
hepatic (untreated animals) microsomes is shown in Figure 5.11. The
metabolite profile for mussel microsomes showed radioactive bands with Rf
values both similar to and different from bands obtained with rat
microsomes. The Rf values of major radioactive bands similar in mussel and
rat were approximately 0.53 and 0.57 (scraped as area 5), 0.64 and 0.66
(scraped as area 6 ) and 0.88, 0.95 and 0.99 (scraped as area 9). Of these,
the bands in areas 5 and 6 were more polar than the unmetabolised
testosterone (Rf approximately 0.83), while those in area 9 were less
polar. Table 5.3 shows the percentage radioactivity in each of the scraped
262
8
*¥A B C D E F G H
Figure 5.11
T .L.C./Autoradiograph showing the metabolism of testosterone by M. edulis
digestive gland and rat hepatic microsomes.
(Mussel: Protein = 10.61 mg ml’^; Cytochrome P-450 = 35.09 pmoles mg(Rat : Protein = 15.72 mg ml"^; Cytochrome P-450 = 372.54 pmoles mg )
N.B. Numbers up the sides show areas scraped and counted.
A Pre-boiled mussel microsomal fractionB Pre-boiled rat microsomal fractionC Rat microsomal fraction - NADPHD Rat microsomal fraction + NADPH (0.4 mM final)E and G Mussel microsomal fraction - NADPHF and H Mussel microsomal fraction 4- NADPH (0.4 mM final)
9
8
7
6
5
4
3
263
Table 5.3
The metabolism of testosterone by M. edulis digestive gland and rat hepatic
microsomes.
Condition Counts (% of total)
Area of plate scraped
1 2 3 4 5 6 7 8 9
Boiledmussel 0.33 0.08 0.04 0.18 0.36 0.25 0.49 89.40 8 .8 6*
Boiledrat 0.27 0.09 0.03 0 . 2 1 0.34 0.30 0.46 91.22 7.07*
Rat-NADPH 0.28 0.06 0.31 0.47 0.82 0.43 0 . 2 1 89.72 7.69
Rat+NADPH 0.57 0.27 1.42 9.54 7.32 6.77 4.52 51.46 18.15
Mussel-NADPH 0.23 1 . 6 6 0 . 1 2 0.26 1.03 0.06 0.43 87.24 8.42
Mussel+NADPH 0.23 1.58 0.13 0.51 1.24 0.76 0.58 85.83 9.15
Mussel-NADPH 0.34 1 . 2 2 0.16 0.65 1 . 1 2 0.42 0.47 90.05 5.56
Mussel+NADPH 0.53 1.17 0.06 0.36 1.09 0.61 0.53 89.31 6.35
These areas possibly contained contaminating radioactivity from area 8
(unmetabolised testosterone).
264
areas. It was apparent that, as observed for mussel metabolism of
arachidonic acid, any metabolism of testosterone by mussel microsomes was
partially or totally independent of NADPH. (Areas 6 , 7 and 9 were either
similar or slightly higher in radioactivity in the presence than the
absence of NADPH). The NADPH-independence of mussel microsomal metabolism
was in marked contrast to the requirement for NADPH of the rat microsomes
for metabolism of testosterone. The radioactivity present in each area,
particularly areas 5, 6 and 7 when boiled microsomes were used was
presumably a result of non-enzymic metabolism of testosterone, either in
storage or during the incubation. The higher levels of radioactivity in
the same areas following incubation with viable microsomes presumably
suggests the metabolites formed in each condition were similar. No attempt
to experimentally characterise the metabolites was carried out (see also
Discussion).
A major unique area of radioactivity was seen only following
incubation with mussel microsomes and had an Rf value of approximately 0.15
(scraped in area 2). The presence of this band was again independent of
the addition of NADPH. Because of its comparatively low Rf value, it is
possible the band may have been due to the binding of testosterone to some
extracted digestive gland pigment or other molecule (the samples applied to
the T.L.C. plate were coloured brown). However, the absence of such a spot
following testosterone incubation with boiled mussel microsomes argues
against this, and suggests it possibly represents a unique metabolite, not
formed by rat microsomal preparations. No attempts were made to further
characterise this putative metabolite.
265
5.3.4 Studies on the inhibition of rat hepatic microsomal MFO activities
by digestive gland microsomes and ethyl acetate-extract of mussel
microsomes
It was considered possible that an endogenous inhibitor of cytochrome
P-450-mediated MFO activity present (following homogenization) within the
mussel digestive gland microsomal fraction, might be responsible, at least
in part, for both the observed low levels of MFO activities and the NADPH-
independence of these activities (see Section 4 and Discussion) . To
investigate this, the effects of mussel digestive gland microsomes and/or
an ethyl acetate-extract of the microsomes on various MFO activities of rat
hepatic microsomes were examined. Figures 5.12, 5.13 and 5.14 show,
respectively, the inhibition of rat ECOD activity by ethyl acetate mussel
microsomal extract (50% inhibition with about 90 /j1 of extract) , the
inhibition of benzphetamine N-demethylase activity by mussel microsomes
(50% inhibition with about 130 /zl, 1.16 mg of mussel microsomal protein),
and the inhibition of lauric acid hydroxylase activity of hepatic
microsomes from clofibrate pre-treated and untreated rats by, respectively,
ethyl acetate-extract (50% inhibition with about 300 /il of extract) and
microsomes (50% inhibition with about 250 /il, 2.1 mg protein) of mussel.
Autoradiographs for the lauric acid hydroxylase activities are shown in
Figure 5.15. The ethyl acetate-extracts used were prepared to their
original microsomal concentration (see Section 5.2.11). The volumes of
mussel microsomes and ethyl acetate-extracts effecting 50% or more
inhibition of rat liver MFO activities were similar to those employed in
the mussel microsomal MFO activity assays.
In addition to these results, rat liver microsomal benzphetamine N-
266
100 -
80-
z0P 60-HmH1 Zh 40 -
K
H
20 -
20016040 80 1200
ETHYL ACETATE EXTRACT
Figure 5.12
Inhibition of rat hepatic microsomal ECOD activity by an ethyl acetate-
extract of digestive gland microsomes of M. edulis.
Hepatic microsomes from untreated rats : 1.44 mg protein;
287 pmoles cytochrome P-450 mg"^ protein1 _ qControl ECOD activity = 179.3 pmoles min mg protein
Each 1 ml aliquot of digestive gland ethyl acetate - extract was re
constituted from 1 ml microsomal fraction (Protein content *= 9.39 mg ml"^)
Values presented as Mean ± SEM (n «= 3)
267
100 -
80-
z0£ 60- HmM1zh 40 -
20 -
500400200 3001000
MUSSEL D.G. MICROSOMES
Figure 5.13Inhibition of rat hepatic microsomal benzphetamine N-demethylase activity
by digestive gland microsomes of M. edulis.
Hepatic microsomes from untreated rat : 1.74 mg protein;
317 pmoles cytochrome P-450 mg"^ protein.-1 -1Control benzphetamine N-demethylase activity =3.71 nmoles min mg protein
Mussel microsomal protein content = 8.93 mg ml”'*'
Values presented as Mean ± range of duplicate assays
268
Figure 5.14
The inhibition of rat hepatic microsomal lauric acid hydroxylase activity by
(A) ethyl acetate-extract and (B) microsomes of digestive gland of M. edulis.
(A) Microsomes from clofibrate pre-treated rats : 1.26 mg protein;
638 pmoles cytochrome P-452 mg’ protein.1 iControl lauric acid hydroxylase activity =9.73 nmoles min mg
Each 1 ml aliquot of digestive gland ethyl acetate-extract was
reconstituted from 1 ml of microsomal fraction (Protein content
= 8.42 mg ml’ )
(B) Microsomes from untreated rats : 1.52 mg protein;
579 pmoles cytochrome P-450 mg"^ protein._ i _ iControl lauric acid hydroxylase activity =2.36 nmoles min mg
Mussel microsomal protein content = 8.42 mg ml"^
Activities measured as total 11- and 12-hydroxylauric acids formed.
Only single determinations made.
269
N0I1I8IHNI %
N0I1IQIHNI %
75 -
60-
45-
30-
15-
0 -)
200 300100 5000 400ETHYL ACETATE EXTRACT (j-lL)
B75-
60-
30 -
15-
500300200 400100MUSSEL D.G. MICROSOMES (JJL)
270
0 10 25 50 100 150 250 500MUSSEL DIGESTIVE GLAND ETHYL ACETATE-EXTRACT (jjL)
B
4 - ’> i*' 4> *
0 10 20 30 50 100 150 250 500MUSSEL DIGESTIVE GLAND MICROSOMES (fiL)
Figure 5.15
T .L.C./Autoradiographs showing the inhibition of rat hepatic lauric acid
hydroxylase activity by (A) Ethyl acetate-extract and (B) microsomes of
digestive gland of M. edulis. Conditions as described in legend to Figure
5.14.
271
demethylase activity was also inhibited by boiled mussel digestive gland
microsomes (35% inhibition by 50 /il (0.45 mg protein) of microsomes) and
ethyl acetate-extract of mussel digestive gland microsomes (50% inhibition
by 100 /il extract; original mussel microsomal concentration of 8.93 mg
protein ml"^).
The effects of the ethyl acetate digestive gland microsomal extract on
the type I binding spectra of hexobarbital with rat hepatic microsomes was
also examined (Figure 5.16). Using microsomes from phenobarbital pre
treated rats, addition of 50 /il of ethyl acetate-extract (prepared from an_ -loriginal mussel microsomal protein concentration of 9.4 mg ml ),
obliterated the spectrum. The addition of ethyl acetate-extract had no
effect on rat hepatic microsomal cytochrome P-450 specific content, as
judged by the dithionite-reduced carbon monoxide difference spectrum (data
not shown).
272
0.005 A
r T T 13 5 0 4 0 0 4 5 0 5 0 0
WAVELENGTH ( nm)
B0 . 0 0 5 A
3 5 0 4 0 0 4 5 0 5 0 0
WAVELENGTH ( nm)
Figure 5.16
Inhibition of hexobarbital binding to rat hepatic microsomal cytochrome P-
450 by an ethyl acetate-extract of digestive gland microsomes of M. edulis
(cytochrome P-450 from phenobarbital pre-treated rats).
B
Binding spectrum in the absence of ethyl acetate-extract.
Final cuvette concentrations of cytochrome P-450, protein and hexobarbital were, respectively, 1.43 nmoles ml‘\ 0.92 mg ml"^ and 0.5 mM.
Spectrum in the presence of 50 /tl of ethyl acetate-extract.
Conditions as in (A) above plus 50 /il of extract from a 9.39 mg ml"^ microsomal preparation.
273
5.4 Discussion
Of the various types of MFO reactions catalysed by mammalian and other
cytochrome P-450 isoenzymes (see Section 1), the ability to 0-deethylate
(substrates: 7-ethoxycoumarin, 7-ethoxyresorufin), N-demethylate
(benzphetamine, aminopyrine, ethylmorphine, N,N-dimethylaniline) and
hydroxylate (aniline, biphenyl, arachidonic acid, lauric acid,
testosterone) was examined in digestive gland microsomes of M. edulis.
Activities towards most of these model substrates, in the samples examined,
were either undetectable or low relative to the assay procedures employed.
The only activity sufficiently high for more detailed characterization was
7-ethoxycoumarin O-deethylase (ECOD).
5.4.1 NADPH-independent 7-ethoxycoumarin O-deethylase activity
Although ECOD activity is well-characterized in mammals (e.g. Ullrich
and Weber, 1972; Boobis et al., 1981; Goksoyr et al., 1986; Rogiers et al.,
1986), birds (e.g. Riviere, 1980; Rivi&re et al., 1985; Miranda et al.,
1987); amphibia (e.g. Noshiro and Omura, 1984), fish (e.g. James et al.,
1977; Koivusaari et al., 1982; Andersson et al., 1985; Funari et al., 1987)
and, to a lesser extent, in crustacea (e.g. Singer et al., 1980; James,
1984), there are no reports to date on it in mussels or any other molluscan
species. The results for M. edulis indicate the ECOD activity to be
primarily localized in the microsomal fraction of the digestive gland.
This is consistent with previous findings for this species of the digestive
gland microsomal fraction being the principle location for both cytochrome
P-450 and another xenobiotic metabolic activity, benzo[a]pyrene hydroxylase
(BPH) (Livingstone and Farrar, 1984; Stegeman, 1985). The ECOD activity
274
detected in the mitochondrial fraction of the digestive gland was probably
due to microsomal contamination, as was indicated for BPH activity, by
glucose-6 -phosphatase marker studies, in another study on subcellular
fractions prepared in an identical manner (Livingstone and Farrar, 1984).
That a larger proportion of (contaminating) ECOD activity should be seen in
the mitochondrial fraction of female mussels is consistent with the
observed distribution of BPH activity (see Livingstone and Farrar, 1984).
Of particular interest was the NADPH-independence of the digestive
gland microsomal ECOD activity and the inhibitory effect of NADPH on this
activity. The NADPH-independence is in marked contrast to the absolute
requirement for NADPH of ECOD activity in vertebrates and Crustacea (see
previous references) , and is inconsistent with the fundamental
monooxygenase requirement of MFO systems for NADPH (Omura, 1978). The
inability to demonstrate ECOD activity in M. edulis (Payne and May, 1979),
and the chiton, Cryptochiton stelleri (Schlenk and Buhler, 1988) may
possibly have been due to the inhibitory effect of NADPH included in the
assays. NADPH-independence and inhibition by NADPH has also been seen for
BPH activity (total metabolism measured radiometrically) in both M. edulis
(Livingstone, 1985; Stegeman, 1985) and M. galloprovincialis (Suteau et
al., 1985). For example, NADPH-independent total benzo [ a] pyrene
metabolism in M. edulis digestive gland microsomes was inhibited 50-60% by
0.2 mM NADPH (Livingstone, 1985; Livingstone et al., 1988a). Speculative
suggestions put forward to account for the observations with molluscan BPH
activity have been that a microsomal endogenous supply of reducing
equivalents exists, that the added NADPH stimulates some competitive
pathway so causing inhibition of BPH activity, and that some cooxidation
via lipid peroxidation or prostaglandin synthase could occur (Stegeman,
1985). However, with respect to the latter suggestion, recent studies on
microsomal BPH activity in C. stelleri have indicated that any effect of
lipid peroxidation is minimal, vis. EDTA had no quantitative or qualitative
effect on in vitro benzo[a]pyrene metabolism (Schlenk and Buhler, 1988).
The NADPH-independence and inhibitory effect of NADPH on both BPH
(total metabolism) and ECOD activities of digestive gland microsomes of M.
edulis suggests some common underlying mechanism for the two, and possibly
other (see later), oxidative activities. One possible explanation could be
that the O-deethylation of 7-ethoxycoumarin and the hydroxylation of
benzo[a]pyrene are catalysed, at least in part, by a cytochrome P-450-
mediated one-electron oxidatic free-radical mechanism (see Cavalieri and
Rogan, 1984, 1985). Such a mechanism, as proposed in mammals for
metabolism of polycyclic aromatic hydrocarbons, such as benzo[a]pyrene,
represents an alternative pathway to two-electron oxidatic monooxygenation.
It involves initial abstraction of an electron from the substrate to the3+cytochrome P-450-(FeO) complex, followed by a reaction between the
resulting substrate radical-cation and the generated nucleophilic oxygen of2+the cytochrome P-450-(FeO) complex to leave an oxygenated product (see
Section 1). The effect of NADPH, and other reducing compounds, on such a
pathway would be to inhibit, by reducing the radical-cation back to the
parent compound (O'Brien, 1984). The inhibition of digestive gland
microsomal NADPH-independent ECOD activity by both NADPH and NADH, but not
by the oxidized coenzymes, or by NADH in the presence of cytochrome c
(removes electrons from NADH) provides support for such a proposal. The
stimulation of the NADPH-independent ECOD activity by NAD+ and NADP+ , could
possibly be due to an enhanced formation of the putative free-radical
intermediates.
276
That one-electron oxidation might be important in molluscs is possibly
indicated from metabolite and other studies on BPH activity. The three
major classes of benzo[a]pyrene metabolites are phenols, dihydrodiols and
quinones (e.g. Cavalieri et al., 1987). The major metabolites formed by
mammalian microsomal MFO systems in the presence of NADPH (i.e. two-
electron oxidatic monooxygenation) are phenols and dihydrodiols, with
minimal formation of quinones (Capdevila et al., 1980; Cavalieri and Rogan,
1985). Under peroxidatic conditions, for example with cumene hydroperoxide
as substrate instead of NADPH and O2 (which is presumed to stimulate
cytochrome P-450-mediated oxygenation via a one-electron oxidatic process -
Capdevila et al. , 1980; Wong et al. , 1986), the major metabolites of
benzo[a]pyrene are quinones, with substantially lower levels of phenols and
dihydrodiols evident. Significantly, of the total benzo[a]pyrene
metabolites produced by digestive gland microsomes of M. edulis, in the
absence (Livingstone et al., 1988a) or presence (Stegeman, 1985) of NADPH
(and in the absence of any added hydroperoxide), between 60 and 70% have
been found to be quinones, with the remainder consisting of about 30%
phenols and less than 2% dihydrodiols. Similarly, in digestive gland
microsomes of the chiton C. stelleri (Schlenk and Buhler, 1988), 40-80% of
metabolites formed were quinones. The quinones produced by M. edulis have
been identified as occuring at the 1,6-, 3, 6 - and, unusually, the 6,12-
positions of the benzo[a]pyrene molecule (Stegeman, 1985; Livingstone et
al., 1988a). This is consistent with initial metabolism at the 6 -carbon
(Stegeman, 1985), the position which is also the site of highest charge
density in the benzo[a]pyrene radical-cation (a high charge density in the
radical intermediate facilitates one-electron oxidation - Cavalieri and
Rogan, 1985). Further support for the free-radical mechanism of quinone
formation is that the putative 6 -hydroxybenzo[a]pyrene, which would be an
intermediate of quinone formation by an MFO two-electron ("ionic") process,
has never been isolated (Nagata et al., 1984; Cavalieri and Rogan, 1985).
The three quinones observed in M. edulis have been shown to be cytotoxic in
studies on rat liver homogenate (Lesko et al., 1975), and to have mutagenic
activity, and possibly therefore to be important in benzo[a]pyrene
carcinogenesis (Lorentzen et al., 1979; Chesis et al. , 1984). Their
possible formation in vivo could, therefore, contribute to the incidence of
neoplasms in M. edulis (Mix et al., 1981; Mix, 1983). The high quinone
formation in the digestive gland microsomal fraction of M. edulis and other
molluscs is in contrast to that generally observed for mammals (see
before), fish (e.g. Bend et al., 1979; Stegeman et al., 1981; Varanasi et
al., 1986) and crustacea (e.g. Singer et al., 1980; Bend et al., 1981;
Stegeman and Kaplan, 1981). It is possible, therefore, that a cytochrome
P-450-mediated peroxidation or one-electron oxidatic process is an
important feature of molluscs in general. Low levels of quinones were
observed for in vitro metabolism of benzo [a]pyrene by the clam Mercenaria
mercenaria, but in this case digestive gland homogenates rather than
microsomes were used (Anderson, 1985). Results for the oyster C. virginica
have been variable with both high (Anderson, 1978) and low (Anderson, 1985)
levels of quinones being recorded.
The inhibition of NADPH-independent digestive gland microsomal BPH
activity of M. edulis by NADPH is only observed for the radiometric assay
i.e. for total metabolism. Measured fluorometrically (phenol formation)
NADPH has either no effect on the NADPH-independent rate (fresh microsomes)
or stimulates it (previously frozen microsomes) (Livingstone, 1985; Moore
et al., 1988). Both the NADPH-independent and NADPH-dependent fluorometric
BPH activities are inhibited by SKF 525-A (Moore et al., 1988). The
278
radiometric BPH activity, in the presence of NADPH (no data are available
in the absence of NADPH), is inhibited by this and other cytochrome P-450
inhibitors, vis. carbon monoxide and a-naphthoflavone (Livingstone and
Farrar, 1984; Stegeman, 1985). These results, together with the finding
that 30% of benzo[a]pyrene metabolites formed are phenols (Stegeman, 1985),
suggest functions for both cytochrome P-450-mediated monooxygenation and
one-electron oxidation in the in vitro metabolism of benzo[a]pyrene by
digestive gland microsomes of M. edulis (Moore et al., 1988). That more
than one process might be involved in the oxygenation of benzo [a]pyrene and
other xenobiotics by molluscs is possibly supported by a recent study on
digestive gland microsomes of C. stelleri in which the proportion of
quinones produced in vitro in relation to phenols and dihydrodiols was
reduced following exposure of the chitons to /?-naphthoflavone for three
weeks (Schlenk and Buhler, 1988). Significantly cumene hydroperoxide-
dependent metabolism of benzo[a]pyrene to quinones in rats is not increased
by exposure of animals to 3-methylcholanthrene-type inducers (Wong et al.,
1986).
The results for the effects of modulators of cytochrome P-450 activity
on digestive gland microsomal NADPH-independent ECOD activity of M. edulis
possibly also support the suggestion of more than one cytochrome P-450-
mediated oxidatic catalytic process in M. edulis. Metyrapone, SKF 525-A
and carbon monoxide inhibited NADPH-independent ECOD activity whereas a-
naphthoflavone and sodium periodate stimulated it. The stimulatory effect
of a-naphthoflavone on NADPH-independent ECOD activity contrasts with its
inhibitory effect on the BPH activity (Livingstone and Farrar, 1984;
Stegeman, 1985). a-Naphthoflavone stimulates BPH activity catalysed by
control or phenobarbital-induced rat hepatic microsomal cytochrome P-450,
279
but inhibits it when catalysed by the 3-methylcholanthrene or
naphthoflavone-inducible cytochromes P-448 (Wiebel et al., 1971; Huang et
al., 1981), possibly suggesting, therefore, the existence of cytochrome P-
450 isoenzymes in M. edulis. The effect of a-naphthoflavone on microsomal
BPH activity, presumably reflecting cytochrome P-450 isoenzyme composition,
appears species-dependent. Inhibition of activity has been seen, for
example, in untreated trout, S. gairdneri (Ahokas et al., 1975; Statham et
al., 1978), scup, S. chrysops (Stegeman and Binder, 1979), carp, Cyprinus
carpio (Melancon et al., 1981), roach, Rutilus rutilus (Monod et al.,
1987), the crab C. sapidus (Singer et al., 1980) and the barnacle B.
eburneus (Stegeman and Kaplan, 1981), whereas either no effect or
stimulation of activity has been seen, for example, in untreated
sheepshead, Archosargus probatocephalus (Bend and James, 1978; James and
Bend, 1980), flounder, Paralichthys lethostigma (Little et al., 1984),
croaker, Micropogon undulatus (Stegeman et al., 1981), little skate, Raja
erinacea (Bend et al., 1979), guppy Poecilia reticulata (Funari et al.,
1987) and spiny lobster, Panulirus argus (James, 1984; James and Little,
1984). The inhibition of BPH activity in M. edulis by a-naphthoflavone
possibly indicates the major form responsible for this activity resembles
the 3-methylcholanthrene or ^-naphthoflavone-inducible cytochromes P-448 of
mammalian species. However, the inability to demonstrate a benzo[a]pyrene
binding spectrum with digestive gland microsomes (type I or otherwise; see
Section 4), may indicate fundamental differences between a P-448-type
cytochrome in M. edulis and the inducible cytochromes P-448 of mammalian
species, or that more than one cytochrome P-450 isoenzyme is involved in
the in vitro metabolism of benzo [a] pyrene. For example, the digestive
gland microsomal BPH activity of M. edulis was also inhibited by SKF 525-A
(see before), an inhibitor of mammalian P-450-type cytochromes (see later).
280
The cytochrome P-450 isoenzyme(s) responsible for microsomal BPH activity
in M. edulis could either be constitutive (and possibly seasonal), or the
result of environmental induction, as has been suggested for certain fish
species in which, for particular animals caught in the field, a-
naphthoflavone inhibited BPH activity rather than stimulated it, as is
normally observed (James and Bend, 1980). In contrast to digestive gland
microsomal BPH activity, the stimulation of NADPH-independent ECOD activity
by a-naphthoflavone possibly suggests that the major catalyst of this
reaction in M. edulis digestive gland microsomes is a P-450 -type
cytochrome, although again, it should be remembered that both
phenobarbital- and 3-methylcholanthrene-inducible cytochromes P-450 in
mammals are capable of catalysing the ECOD reaction (Ioannides and Parke,
1987).
The involvement of a P-450-type cytochrome in the catalysis of the
NADPH-independent ECOD activity is supported by the observed inhibitory
effects of metyrapone and SKF 525-A. Both compounds inhibit control and
phenobarbital-inducible cytochrome P-450 activity in mammalian species
(e.g. Goujon et al., 1972; see Lewis et al., 1986). Metyrapone has been
shown to inhibit ECOD activity in both fish and Crustacea, vis. the guppy,
P. reticulata (Funari et al., 1987), nase, Chondrostoma nasus (Monod et
al., 1987) and the spiny lobster, P. argus (James, 1984). Although there
are no data on the effects of metyrapone on mussel BPH activity, the
compound had no effect on microsomal BPH activity in trout (Elcombe and
Lech, 1979) and carp (Melancon et al, 1981), both species in which BPH
activity is inhibited by a-naphthoflavone (see above) . The seasonal
variation in metyrapone inhibition of M. edulis NADPH-independent ECOD
activity is possibly indicative of seasonal variation in digestive gland
281
microsomal cytochrome P-450 isoenzymes (see Section 2). Seasonal variation
in cytochrome P-450 isoenzyme composition has been indicated for various
crab species (Quattrochi and Lee, 1984). Further evidence for the
involvement of a P-450-type cytochrome in the mussel NADPH-independent ECOD
activity comes from the fact that 7-ethoxycoumarin, a-naphthoflavone,
metyrapone and SKF 525-A all show spectral interaction with the digestive
gland microsomal cytochrome P-450 (see Section 4), and that each compound
has a molecular dimension compatible with a P-450-type cytochrome rather
than with a P-448-type cytochrome (Lewis et al., 1986).
In all these speculations and suggestions as to the nature of M.
edulis cytochrome P-450 isoenzymes, however, it should be remembered that
the assumption is being made that much of what is known regarding the
fundamental properties of mammalian cytochrome P-450 isoenzymes can be
applied to other organisms, and this may, or may not, be true; for example,
some similar and some unique non-mammalian isoenzymes are indicated for
fish (Stegeman and Kloepper-Sams, 1987) and Crustacea (James, 1984).
\The existence of cytochrome P-450 isoenzymes in M. edulis is perhaps
also indicated by the biphasic kinetics observed for the dependence of
NADPH-independent ECOD activity on 7-ethoxycoumarin concentration. Similar
biphasic kinetics have been observed in mammals (e.g. Ullrich and Weber,
1972; Boobis et al., 1981) and birds (e.g. Riviere, 1980; Riviere et al.,
1985) and have been attributed to the presence of several forms of
cytochrome P-450 catalysing the ECOD reaction (Rogiers et al. , 1986).
Significantly, a-naphthoflavone and metyrapone have been shown to have
different effects on the two phases in mammals (Boobis et al., 1981) and,
as mentioned above, both phenobarbital-type and 3-methylcholanthrene-type
282
cytochromes P-450 catalyse the ECOD reaction. The biphasic kinetics in M.
edulis were observed in the Autumn, when cytochrome P-450 isoenzymes are
most apparent (see Section 2), and, therefore, could be due to metabolism
by different cytochrome P-450 isoenzymes. Equally however, the biphasic
kinetics could represent metabolism by two different processes
(monooxygenation and one-electron oxidation), dependent on the
concentration of 7-ethoxycoumarin.
The inhibition of NADPH-independent ECOD activity by carbon monoxide
indicates that a cytochrome P-450-mediated monooxygenase reaction is at
least partly involved, although some caution must be attached to such data
as the inhibitory effect could be exerted simply through the displacement
of oxygen (Stegeman, 1985) . That the inhibition was only partial is
consistent with the incomplete inhibition of microsomal BPH activity seen
in M. edulis (Livingstone and Farrar, 1984) and in other marine
invertebrates such as the ragworm Nereis virens (Lee and Singer, 1980) and
the barnacle, B. ebumeus (Stegeman and Kaplan, 1981). The incomplete
inhibition is possibly further indication that other mechanisms, possibly
peroxidative (Stegeman, 1985), are partly responsible for the BPH and ECOD
activities in M. edulis. Sodium periodate stimulated M. edulis NADPH-
independent ECOD activity. Similar stimulation is well-known in mammalian
species and has been observed in several marine species, including the
spiny lobster P. argus (James et al., 1977). The sodium periodate could
presumably stimulate activity either by supplying both electrons and the
oxygen atom for monooxygenation (two-electron oxidation - Bend et al.,
1981), or by stimulating one-electron oxidation in a manner similar to that
proposed for cumene hydroperoxide (Wong et al. , 1986). Cumene
hydroperoxide also stimulated ECOD activity in P. argus (James et al.,
1977; James, 1984). Other inhibitors of mammalian cytochrome P-450-
mediated activity (ellipticine, cyclophosphamide and nicotinamide) had no
effect on M. edulis NADPH-independent ECOD activity. The lack of any
effect with cyclophosphamide may have been because its mode of action is
thought to be through inhibition of NADPH-cytochrome P-450 reductase
(Marinello et al., 1981) which is possibly not required for the NADPH-
independent mussel ECOD activity. The same may be true for ellipticine
(Guenthner et al. , 1980), although, in this case, the compound is
relatively planar and is, therefore, a greater inhibitor of cytochrome P-
448-mediated activity than cytochrome P-450-mediated activity (Lewis et
al., 1986). The lack of inhibition with nicotinamide indicates that the
inhibition by NADPH is not related to its nicotinamide mot&ty.
Maximal specific activities determined for the NADPH-independent ECOD
activity in mussel digestive gland microsomes (0.24 mM 7-ethoxycoumarin)-1 -1were 16 pmoles min mg protein, compared with, for example, NADPH-
- 1 - 1dependent ECOD activities of 500 pmoles min mg in untreated rat (0.3 mM1 17-ethoxycoumarin - Guengerich et al., 1982), 4 to 640 pmoles min" mg in
various species of (untreated) teleost and elasmobranch fish (0.2 mM 7-
ethoxycoumarin - James et al., 1979; James and Bend, 1980) and 20 pmoles
min"^ mg"^ in the crab C. sapidus (0.2 mM 7-ethoxycoumarin - James et al.,
1979). Although the digestive gland microsomal specific activity in M.
edulis is low compared to fish and mammalian species (only the bluntnose
ray, Dasyatis sayi, and the flounder, P. lethostigma in the above
references had lower specific activities; respectively, 4 and 5 pmoles min"
mg’ ), the cytochrome P-450-mediated theoretical turnovers for 7-
ethoxycoumarin are similar in all the species; for example, 0.21 nmoles 7--1 -1ethoxycoumarin min nmole cytochrome P-450 in M. edulis compared to 0.22
284
- 1 - 1nmoles min nmole cytochrome P-450 in untreated rat (Guengerich et al.,
1982). The values for apparent 1^ for 7-ethoxycoumarin for the mussel
NADPH-independent ECOD activity were approximately 35 and 250 /xm for the
two parts of the substrate concentration versus velocity curve. These
values are considerably less than the Kg value of 1440 /xm for 7-
ethoxycoumarin determined from the substrate binding studies (see Section
4). However, it should be noted that the two studies were performed on
different biological samples, the binding studies employed solubilized and
ammonium sulphate- fractionated cytochrome P-450, and a reverse type I
rather than a type I spectrum was obtained for the binding of 7-
ethoxycoumarin.
5.4.2 Other xenobiotic NADPH-independent oxidative activities
Of other known xenobiotic substrates of mammalian cytochromes P-450
examined, slight digestive gland microsomal oxidative activity was seen
towards 7-ethoxyresorufin and benzphetamine. Like the activity towards 7-
ethoxycoumarin, the 0-deethylation of 7-ethoxyresorufin (EROD) was observed
in the absence of added NADPH and was inhibitable by NADPH, suggesting the
involvement of processes other than monooxygenation, such as peroxidation
and/or one-electron oxidation (see before). If monooxygenation is partly
or wholly responsible for the NADPH-independent EROD activity, then this is
possibly further evidence for the existence of cytochrome P-450 isoenzymes.
7-Ethoxyresorufin is a planar compound and is almost entirely specific for
P-448-type cytochromes (Lewis et al., 1986). As such, EROD activity is
generally seen in aquatic species containing constitutive P-448-type
cytochromes in which for example, a-naphthoflavone is inhibitory to BPH
285
activity. Such species include the trout, S. gairdneri (Ahokas et al.,
1975; Statham et al., 1978; Elcombe and Lech, 1979), scup, S. chrysops
(Stegeman et al., 1979), carp, C. carpio (Melancon et al., 1981), cod,
Gadus morhua (Hansen et al., 1983), the deep-sea rattail, Coryphaenoides
armatus (Stegeman et al., 1986) and the crab, C. sapidus (Singer et al.,
1980). Significantly, the digestive gland microsomal NADPH-independent
EROD activity was determined in mussels collected during Autumn when the
possibility of isoenzymes is more apparent (see Section 2). EROD activity
has occasionally been seen before in digestive gland microsomes of M.
edulis (Stegeman, 1985).
The mussel digestive gland microsomal N-demethylase activities (towards
benzphetamine, aminopyrine and N,N-dimethylaniline) , although NADPH-
independent, differed from the ECOD and EROD activities in that NADPH had
little or no inhibitory effect on the activities. For N,N-dimethylaniline
N-demethylase, this observation is in contrast to a recent report showing
50% inhibition of the activity by 50 pK NADPH (Livingstone et al., 1988a).
The observed NADPH-inhibition of N,N-dimethylaniline N-demethylase activity
(Livingstone et al., 1988a) may indicate a radical-mediated mechanism.
Similar mechanisms have been proposed for the N-demethylation of N,N-
dimethylaniline and aminopyrine by mammalian hepatic microsomal cytochromes
P-450 acting peroxidatively (Griffin, 1978; Griffin and Ting, 1978).
However, possibly inconsistent with this is the relatively limited NADPH
inhibition of the mussel activity, compared to that observed for NADPH-
independent ECOD activity, and the absence of any stimulatory effect of
sodium periodate. Also, a more recent report (O'Brien, 1984) has suggested
a cytochrome P-450-mediated two-electron oxidation is more likely than a
one-electron radical-mediated mechanism for the aminopyrine N-demethylase
286
activity observed by Griffin and Ting (1978).
Benzphetamine is mainly a substrate for P-450-type cytochromes in
mammals (Guengerich et al., 1982; Lewis et al., 1986). In aquatic species,
benzphetamine N-demethylase activity is seen in many species of fish and
crustacea (James et al., 1977, 1979), but particularly in species with
possibly constitutive P-450-type cytochromes in which, for example, a-
naphthoflavone is stimulatory (e.g. the spiny lobster P. argus - James,
1984). The presence of, albeit NADPH-independent, benzphetamine N-
demethylase activity in digestive gland microsomes, is, therefore, possibly
further support for the existence of a P-450-type cytochrome in M. edulis,
although it should be noted that SKF 525-A had no effect on this activity.
5.4.3 NADPH-independent endogenous substrate oxidative activities
Limited activities were seen towards testosterone and arachidonic
acid. Like the digestive gland microsomal xenobiotic oxidative activities,
these activities were independent of the presence of NADPH. The lack of
effect of known modulators of cytochrome P-450 on these activities could
have several bases, vis. the reactions are not cytochrome P-450-mediated,
the mammalian inhibitors are ineffective for the molluscan P-450(s)
responsible for the activities, or the putative endogenous inhibitors in
the mussel microsomes (see Sections 4 and 5.4.4) could have prevented an
effect. The absence of an inhibitory effect of NADPH possibly suggests the
absence of radical-cation-mediated mechanisms.
Metabolism of testosterone and other androgens is well-known in marine
invertebrates, for example, in P. argus (James and Shiverick, 1984). The
287
metabolites formed by the mussel digestive gland microsomes were not
identified, but some reference can be made to published Rf values in the
literature. The major metabolites scraped in the areas indicated are
likely to be as follows: area 4 is 7a- and 16a-hydroxytestosterone; area 5
is 6/?-hydroxytestosterone; areas 6 or 7 is 2/?-hydroxytestosterone, and area
9 is androstenedione. The Rf values for these metabolites are similar to
those for known testosterone metabolites from untreated rat (see Shiverick
and Neims, 1979; Cheng and Schenkman, 1983). Of these, the major
metabolite occuring in M. edulis, with an Rf value similar to a metabolite
of the rat, was in area 5, and possibly corresponded to 6yS-
hydroxytestosterone. Significantly, this is the major metabolite catalysed
by cytochrome P-450 RLMg °f t*16 untreated rat (Cheng and Schenkman, 1983).
It is possible the major cytochrome P-450 isoenzyme of M. edulis involved
in testosterone metabolism has a degree of similarity to this rat
isoenzyme. A major radioactive spot observed for M. edulis, but not seen
in metabolite profiles of mammalian species had an Rf value of 0.15. That
this is a metabolite unique to the mussel may suggest either novel
cytochrome P-450-mediated pathways of testosterone metabolism, or that
testosterone is metabolised in mussels, at least in part, via pathways that
differ from those of vertebrate systems. Significantly, vitamin Dg, a
known endogenous substrate of mammalian cytochromes P-450 (Dahlback and
Wikvall, 1987), has also been shown to be converted by whole body
homogenate of M. edulis to polar metabolites that differ from known
mammalian metabolites (Lehtovaara and Koskinen, 1986).
The metabolites produced from arachidonic acid by mussel digestive
gland microsomes were also not identified. Comparison with rat microsomal
arachidonic acid profiles must be made with caution (as no rat incubations
288
were carried out), but by analogy with published profiles it is likely the
metabolites correspond to mono-, di-, and tri-hydroxyeicosatetraenoic acids
(HETES), with the more non-polar mono-hydroxymetabolites running further up
the T.L.C. plate (Capdevila et al., 1981; Oliw et al., 1982). Metabolism
of arachidonic acid has been observed previously in both in vivo and in
vitro studies on M. edulis (Srivastava and Mustafa, 1984, 1985).
Identified metabolites included cyclooxygenase products (prostaglandins and
thromboxane B2 ). However, a large proportion of metabolites were more
polar than unmetabolised arachidonic acid, and remained unidentified. It
is possible the metabolites produced by the digestive gland microsomal
fraction in this study are similar to some or all of these metabolites,
although it should be noted that in the above studies, extracts of
adductor, mantle and gill only were investigated and again, it is possible
different pathways of metabolism may exist in different tissues.
The observed metabolism of testosterone and arachidonic acid, even in
the presence of a postulated inhibitor of cytochrome P-450 (see Section
5.4.4) supports the idea that the cytochrome P-450 of M. edulis is involved
in endogenous metabolism (see Section 2). However, it cannot be stated
that these compounds are natural endogenous substrates since, as noted
earlier, M. edulis contains, for example, cholesterol and a wide range of
other steroids and sterols (Goad, 1981; Khan and Goad, 1983). The
possibility of unique testosterone metabolites may also argue for natural
substrates other than testosterone.
289
5.4.4 Possible endogenous inhibition of H. edulis oxidative metabolism
The limited and NADPH-independent xenobiotic and endogenous oxidative
metabolic activities in M. edulis could be partly due to seasonal effects
or simply reflect the fact that these compounds are not good substrates for
the mussel cytochrome P-450. Another possibility, however, is the presence
of an endogenous inhibitor of cytochrome P-450-mediated activity. As
suggested in Section 4, the inability to obtain type I binding spectra may
be due to an endogenous inhibitor blocking the type I substrate - binding
site. The findings that an ethyl acetate-extract of mussel digestive gland
microsomal fraction prevented hexobarbital binding to phenobarbital-induced
rat microsomal cytochrome P-450, and that both mussel digestive gland
microsomes and ethyl acetate-extracts of the same, inhibited rat microsomal
MFO activities, possibly support this suggestion. The postulated
inhibitory, possibly lipid-soluble, substance may well be compartmentalized
in the intact digestive gland and only inhibitory following "release" by
homogenization (Lee, 1981). In preventing substrate-binding and the
concomitant shift in spin-state from low to high (to facilitate electron
transfer from NADPH cytochrome P-450 reductase), the addition of NADPH
would be expected to have little if any effect. The slight enzyme activity
observed could be due to a restricted binding of substrate sufficient for
endogenous NADPH to provide the necessary reducing equivalents for
reaction. The presence of an endogenous inhibitor is possibly supported by
observations of endogenous inhibition in other marine invertebrates. For
example, addition of "digestive juice" from either the rock crab Cancer
borealis or the lobster, Homarus americanus has been shown to inhibit
cytochrome P-450 activity (including ECOD and benzphetamine N-demethylase
activities) of the little skate R. erinacea (Pohl et al., 1974).
290
Similarly, digestive gland microsomal or cytosolic fractions of the spiny
lobster P. argus is inhibitory to MFO activity of sheepshead, A.
probatocephalus and stingray D. sabina hepatic microsomes (James et al.,
1977, 1979). More significantly, in M. edulis, a post-mitochondrial
supernatant of digestive gland inhibited mantle aldrin epoxidase activity
(Moore et al., 1980). In contrast to these results, however, is the
observation that digestive gland microsomes from M. edulis had no effect on
BPH activity in the dogfish, Squalus acanthias (Stegeman, 1985). Further,
any inhibitory substance within M. edulis has possibly a different
mechanism of action from that in P. argus since both type I and type II
binding spectra (with benzphetamine and aniline, respectively) have been
obtained with hepatopancreas microsomal fractions of the latter (James et
al., 1979).
291
5.5 Appendix
STUDIES ON THE MIXED FUNCTION OXYGENASE SYSTEM
OF THE MARINE BIVALVE MYTIUJS EDULIS1 2 1 2 Miles A. Kirchin * , Alan Wiseman and David R. Livingstone
^Dept. Biochemistry, University of Surrey Guildford, Surrey GU2 5XH U.K.
2 NERC Institute for Marine Environmental Research Prospect Place, The Hoe, Plymouth Devon PL1 3DH U.K.
The common mussel, M. edulis has a cytochrome P-450 monooxygenase or mixed
function oxidase (MFO) system primarily located in the endoplasmic
reticulum of the digestive gland. The system or its components are
indicated to be involved in the metabolism of xenobiotics such as
benzo[a]pyrene and are elevated in response to exposure to pollutants such
as diesel oil. Little, however, is really known about the nature of the
molluscan MFO system or its function in vivo.
Digestive gland microsomal MFO activity has been studied using assays for
7-ethoxycoumarin O-deethylase (ECOD), benzphetamine N-demethylase and
testosterone hydroxylase (TH) . In each case, as with benzo [ a] pyrene
hydroxylase (BPH) activity, activities were partially or totally NADPH-
independent. ECOD activity varied seasonally, up to 3.20 ± 0.48 (n=6 ) (±
SEM) pmoles min’ mg"^ protein in the Autumn, and the changes did not
parallel changes in total cytochrome P-450 content. Biphasic kinetics are
seen with respect to 7-ethoxycoumarin with apparent values for the two
portions of the substrate vs. velocity curve of 14-35 /xm and 250-580 /xM.
292
ECOD activity is stimulated by a-naphthoflavone (NF) (35% increase at 100
/zM NF) , in contrast to mussel BPH activity which is inhibited. A
T.L.C./autoradiographic assay for TH activity indicated metabolite spots
both similar to and different from those obtained with rat liver
microsomes. Digestive gland cytochrome P-450 has been partially purified
by solubilization of microsomes with sodium cholate, fractionation with
ammonium sulphate (35-65%) or polyethylene glycol (4-14%) and emulgen
elution from an 8 -aminooctyl sepharose 4B affinity column. Purification
factors of up to 5 were obtained but final yields were less than 10%.
However, the partially purified P-450 appears stable and represents a
significant improvement on crude microsomes for spectral studies. It is
concluded that both similarities and differences exist between the
molluscan and other animal group MFO systems and that a fundamental
understanding of its function(s) is necessary before application in
pollution studies will be possible.
(Published in Marine Environmental Research 1988 24 117-118).
293
Section 6
Responses of digestive gland microsomal MFO components and NADPH-independent
ECOD activity to xenobiotic exposure
294
6.1 Introduction
A major feature of MFO systems of vertebrate and some invertebrate
groups are their inducibility by drugs and other foreign chemicals. In
mammals, two major classes of inducing agents are the 3-methylcholanthrene
(3MC)-type inducers which induce P-448-type cytochromes, and the
phenobarbital-type inducers which induce P-450-type cytochromes (Kato,
1982; see Section 1). In fish, inductive responses are generally seen
toward 3MC-type inducers, but apparently not toward phenobarbital-type
inducers (Forlin, 1980; Schwen and Mannering, 1982a; Goksoyr et al. ,
1986b). In Crustacea, wide species variation is apparent, with some
species showing similar responses to those for fish (Batel et al., 1983,
1988; Bihari et al., 1984), and others showing fundamental differences from
both mammals and fish (Singer et al., 1980; James, 1984; James and Little,
1984).
An understanding of the molluscan MFO system, and its response to
xenobiotic exposure, is of interest not only from the comparative and
evolutionary viewpoint, but also in relation to processes of neoplastic
disease (Anderson, 1978, 1985; Anderson and Doos, 1983) and the potential
for its use in biological effects monitoring (Lee et al., 1980; Livingstone
et al., 1985; Moore et al., 1986; Livingstone, 1987; Payne et al., 1987).
To these ends, laboratory and field studies have demonstrated changes
(increases) in MFO component contents and/or activities in response to
xenobiotic exposure in a number of molluscan species, vis. M. edulis and
the periwinkle, Littorina littorea (Livingstone et al., 1985; Livingstone,
1987, 1988); the cockle, Cardium edule (Moore et al., 1987); the oyster
drill, Thais haemastoma (Livingstone et al., 1986) and the mussel, Mytilus
295
galloprovincialis (Gilewicz et al., 1984). Changes in digestive gland
benzo[a]pyrene hydroxylase (BPH) specific activity following xenobiotic
exposure have been demonstrated in the oyster, Crassostrea virginica
(Anderson, 1978, 1985) and more recently in the chiton, Cryptochiton
stelleri (Schlenk and Buhler, 1988). Further indications of a
responsiveness of the MFO system of M. edulis to xenobiotics has come from
studies on sister chromatid exchange (Dixon et al., 1985) and cytochemical
NADPH-neotetrazolium reductase activity, (Moore, 1979, 1985, 1988).
The aim of the study presented in this section was to further
investigate the effects of xenobiotic exposure on components of the M.
edulis MFO system. Two methods of approach were used to effect the
exposures, either administration of the xenobiotic into the seawater, or
direct injection of the xenobiotic into the mussels. Individual pure
compounds were used rather than mixtures such as diesel oil (e.g. see
Livingstone et al., 1985). Experiments were carried out at different times
of the year, to investigate the possibility of seasonal variability in
response. The effect, if any, of algal food upon the response to
xenobiotics was also investigated. Emphasis was placed on the MFO
components (cytochromes P-450 and b^, P-450 ^max, reductase activities)
because of the practical and conceptual difficulties with the MFO activity
measurements (see Section 5). However, in the major seawater exposures (3-
methylcholanthrene, phenobarbital and clofibrate), NADPH-independent 7-
ethoxycoumarin 0-deethylase (ECOD) activity was also measured. In these
studies, BPH activity (radiometric, in the presence of NADPH) was also
measured by D. R. Livingstone but no responses were seen and the data are
not given. With the exception of phenanthrene, which has been shown to
cause a stimulation of NADPH-neotetrazolium reductase activity in M. edulis
296
blood cells (Moore, 1979), the xenobiotics used were all chemicals known to
induce particular cytochrome P-450s in mammalian species, vis.
phenobarbital (Fonnd and Meyer, 1987), 3MC (Astrom et al., 1986),
clofibrate [ethyl 2-(4-chlorophenoxy)-2-methylpropanoate] (Gibson et al.,
1982), ^-naphthoflavone (Guengerich et al. , 1982), Aroclor 1254 (a
polychlorinated biphenyl mixture) (Ryan et al., 1979), prochloraz [N-
propyl-N-[2-(2,4,6 -trichlorophenoxy)ethyl]- imidazole-1-carboxamide]
(Riviere, 1983), pregnenalone 16a-carbonitrile (Elshourbagy and Guzelian,
1980), isosofrole [5-(1-propenyl)-1,3-benzodioxole] (Cook and Hodgson,
1985) and acetone (Patten et al., 1986). The solvent vehicles were also
investigated for effect, vis. dimethy1formamide, ethanol and corn oil. The
results are discussed in relation to the information available for other
molluscs and animal groups, and in relation to the problems involved in
carrying out molluscan laboratory xenobiotic exposure experiments.
297
6.2 Materials and Methods
6.2.1. Chemicals
/?-Naphthoflavone was obtained from Sigma Chemical Co. Ltd., (Poole,
Dorset, U.K.). Phenobarbital (sodium salt) was from B.D.H. Chemicals Ltd.,
(Poole, Dorset, U.K.) and ^C-phenobarbital was from Amersham International
pic (Amersham, Bucks, U.K.). 3-Methylcholanthrene, isosafrole and
phenanthrene were from Aldrich Chemical Co. Ltd., (Gillingham, Dorset,
U.K.). Pregnenalone 16a-carbonitrile was obtained from the Upjohn Company
(Kalamazoo, Michigan, U.S.A.). Aroclor 1254 was from Applied Science
products (Oud-Beijerland, Holland). Mazola pure corn oil was obtained from
Sainsburys supermarket chain (U.K.). 'Optisorb' liquid scintillant was
from Fisons pic (Loughborough, Leics., U.K.). Clofibrate (sodium salt) and
prochloraz were the generous gifts of, respectively, Dr. G. Frost (Robens
Institute, Guildford, Surrey, U.K.) and Mr. J. T. Borlakoglu (University of
Reading, Bucks., U.K.). All other chemicals were of analar grade from
sources described previously.
6.2.2 Static tank exposure experiments
6 .2.2.1 Collection and equilibration of M. edulis prior to exposure
Mussels (approximately 4.5 - 5.5 cm in length), collected from
Whitsand Bay, Cornwall (see Section 2.2.1), were placed initially in a
flow-through seawater system, at ambient field temperature, and, in all
experiments except one (see Section 6 .2.2.4), kept without food for 3 - 4
days. This 3-4 day period enabled both acclimation to tank conditions and
298
clearance of any gut contents prior to exposure. Following the acclimation
period, mussels were transferred to 20 or 50 litre plastic static-seawater
tanks (again, pre-equilibrated to the ambient temperature), equivalent to
each mussel occupying 400 ml of filtered (0.45 /im mesh size)-seawater.
Dosing with xenobiotics was begun after a further 24 hours in these tanks.
Filtered-seawater was changed daily for the duration of the experiments.
For each experiment, a daily light/dark cycle was maintained, approximating
the hours of day and night at the time of collection. Each tank was
vigourously aerated using a pump and air-stone.
6 .2.2.2 Exposure of M. edulis to xenobiotics in seawater (without feeding)
Exposure of M. edulis to xenobiotics in seawater was performed at two
times of the year, during February and June, 1986. For the February
experiment, mussels were exposed to phenobarbital (adjusted to pH 7.0) at-1 -1 -1 150 /ig L seawater day (assumed to be approximately 15 /ig g wet weight
-1 -1 -1 of mussel day ), 3MC at 75 /ig L seawater day (approximately 7.5 /ig g1 -1 1 1 wet weight day ) and clofibrate at 150 /ig L seawater day
(approximately 15 /ig g"^ wet weight day"^). Control exposures to
dimethylformamide (DMF) the solvent vehicle for 3MC and to seawater alone
were also performed. Dosing was by daily injection of 400 /il or 1 ml
volumes (depending on tank size) of stock solution into the seawater;
injection was into the air-bubble source to effect rapid dispersal of the
xenobiotic or solvent. Samples were taken on day 0, immediately prior to
exposure, and on days 1,3,7 and 14 of the exposure. For the 3MC and
phenobarbital exposures, five replicate samples, each containing the pooled
digestive glands of six mussels, were taken on each sampling day, whereas
for the clofibrate exposure and the DMF and untreated controls, only four
299
replicate samples of similarly six digestive glands each were taken. For
the June experiment, mussels were exposed under similar conditions to the-1 -1same water levels of 3MC (75 /ig L seawater day ) , to higher levels of
phenobarbital (adjusted to pH 7.0) (1500 /ig L"^ seawater day"'*’;
approximately 150 /ig g"^ wet weight day"^) and again to DMF (3-
methylcholanthrene control) . For each exposure and for the DMF and
untreated controls, six replicate samples of six digestive glands were
taken on each sampling day (taken on a shorter time-course of 0,1,3 and 7
days). Dissected digestive glands (see Section 2.2.1) were frozen at -70°C,
until required for preparation of microsomes and analysis (see Section
6.2.3).
For each exposure experiment, HPLC analysis of the tissue (digestive
gland and the remainder) levels of 3MC was performed. On each sampling
day, digestive gland and remainder tissues from three mussels were taken
and each divided into two replicate pools. Pools were soponified overnight
in 1 ml of 4 M NaOH, at 37°C. 4 ml of distilled water and 0.5 ml of
absolute ethanol were added, and 4 x 1 ml hexane extractions carried out.
The pooled hexane extracts were made up to 6 ml with hexane and then 250 /il
aliquots of the extract transferred to 500 /il acetonitrile for application
to an LKB isocratic reverse-phase HPLC system (Waters C^g standard bondapak
column, methanol solvent, 1 ml min"^ flow rate). 3-Methylcholanthrene
content was determined from the height of the eluting peak (measured as
absorbance at 292 nm). Efficiencies of extraction, determined from the use
of pyrene (10 /ig in 30 /il DMF added to homogenate before soponification) as
an internal standard (measured at 240 nm), were normally greater than 90%.
The tissue levels of phenobarbital on each sampling day, of the
300
February experiment only, were determined radioisotopically. A separate“I / 1but identical exposure to C-phenobarbital (0.08 /*Ci mmole ; 150 /ig
-1 -1 -1 sodium phenobarbital L seawater day ; added as 10 /il of a 15 /ig /il
stock) was performed simultaneously with the main exposure experiment. On
each sampling day, ten mussels were dissected and the digestive gland and
remainder tissues frozen, at -70°C, until required. For ^C-phenobarbital
analysis, each tissue fraction was homogenized and duplicate aliquots of
homogenate taken for oxidation using a Harvey Biological oxidizer 0X400.
The resulting ^C-carbon dioxide was bubbled through 10 ml of 'optisorb'
liquid scintillant and counted on a Packard 'Tricarb' liquid scintillation
counter. Recoveries of radioactivity were approximately 90%, based on the
recovery of standard -phenobarbital (5000 dpm), oxidized and counted in
an identical manner.
The reproductive stage of the mussels i.e. the percentage of total
gametes, at the time of exposure, was determined as described in Section
2 .2 .6 , for each exposure experiment.
6.2.2.3 Exposure of M. edulis to xenobiotics by direct injection
A series of three-day experiments were performed during January and
April, 1987, exposing M. edulis to a number of xenobiotics by direct
injection. The procedure for each exposure was essentially similar. Each
condition (control or exposed) consisted of 48 mussels occupying 20 L of
filtered seawater in aerated static-tanks. Following the initial 24 hour
acclimation to the static-tank environment, mussels from each condition
(treated sequentially) were individually injected with either xenobiotic or
the solvent vehicle of the xenobiotic. The method for injection was based
301
on that of De Zwaan et al. (1983), and utilized a small guage needle and
syringe. Small ( < 100 /i 1) volumes were injected to minimize stress and,
in the case of water-soluble compounds particularly, to limit tissue
changes in osmotic concentration. Injection was into the mantle cavity,
the needle being placed between the shell (valve) and the mantle edge. The
xenobiotic was dispensed in two equal portions into the two sides of the
mantle cavity. During this procedure, the mussel was kept open by a small
plastic pipette tip wedged between the shells. The mussels were then left
dry for six hours, with an elastic band placed around the two shells to
prevent any opening of the animal and possible discharge of the xenobiotic.
After the six hour period, the elastic bands were removed and the mussels
returned to the tanks. In an attempt to prevent overstressing and possible
spawning (observed on occasions following xenobiotic exposure) the mussels
were not treated on the second day of the experiments, but were similarly
injected on the third day. On the fourth day, the digestive gland of each
mussel was dissected out and frozen, at -70°C, until required for analysis.
The 48 mussels in each condition were analysed as six replicate samples of
eight pooled digestive glands each.
Two experiments were performed in January, 1987. In the first,
mussels were exposed to phenobarbital (adjusted to pH 7.0) (approximately-1 -1 -1 1 25 /ig g wet weight day ), clofibrate (80 /ig g wet weight day ) and
DMF (500 /ig g"^ wet weight day"^). Distilled water (25 /il) was also
injected into another group of mussels as a vehicular control. In the
second experiment, mussels were exposed to 3MC (20 /ig g’ wet weight day"
■) , the polychlorinated biphenyl mixture, Aroclor 1254 (50 /ig g"^ wet-1 1 1 weight day ) and the fungicide, prochloraz (50 /ig g wet weight day ).
Corn oil (25 /il), the solvent vehicle for each of these xenobiotics, was
302
injected into a fourth group of mussels as the vehicular control.
One experiment was performed in April, 1987. In this, mussels were
either left untreated or were injected with one of eight different
xenobiotics or vehicular solvents vis. phenobarbital (adjusted to pH 7.0)-1 -1 -1 -1 (35 /ig g wet weight day ), clofibrate (150 /ig g wet weight day ),
acetone (25 /il of "stock" analar grade), pregnenalone 16a-carbonitrile in-1 -1 1acetone (25 /ig g wet weight day ), phenanthrene in acetone (25 /ig g
-1 -1 -1wet weight day ), isosafrole in acetone (40 /ig g wet weight day ), corn
oil (25 /tl) or /9-naphthof lavone in corn oil (40 /ig g"^ wet weight day"^) .
6.2.2.4 Three week exposure of M. edulis to xenobiotics (with feeding)
A three-week exposure of M. edulis to phenobarbital, /8-naphthof lavone
and ethanol (the solvent vehicle for /?-naphthoflavone) was performed in
June, 1987. Each condition, plus an untreated control condition, comprised
48 mussels in 20 L of static, aerated, unfiltered seawater. Dosing was by
injection of 400 /il volumes of stock solutions into the seawater, and
involved addition of phenobarbital (at pH 7.0) to a concentration of 100 /ig-1 -1 -1 L seawater day and /8-naphthoflavone to a concentration of 25 /ig L
seawater day"^. 400 /il of ethanol was added each day to the /S-
naphthoflavone-control (ethanol) condition. During the experiment, the
mussels in each condition were fed a maintenance ration of the diatom
Phaeodactylum tricomutum. 2.5 L of the algal culture (approximate cell
density = 5 x 10^ cells ml"^) were added to each tank per day. After the
three week dosing period, the mussels were starved for three days to clear
the gut contents before being dissected. Dissected digestive glands were
again stored at -70°C until required for analysis.
In all the exposure experiments (in seawater and by injection), the
concentration of xenobiotics used were a compromise between those in the
literature effective for other species (see Discussion), those thought
likely to be effective for M. edulis, and the need to minimize possible
deleterious and interfering consequences of exposure, such as, for example,
spawning or lysosomal destabilization.
6.2.3 Measurement and analysis of the effects of xenobiotic exposure on contents and activities of the mussel digestive gland microsomal MFO system
The preparation of microsomes and the enzyme assays were similar for
all exposed and untreated conditions, in each exposure experiment.
Digestive gland microsomes were prepared as described in Section 2.2.3 to
final protein concentrations of 4-8 mg ml"^ (resuspended in 20 mM Tris-
HC1, pH 7.6, containing 20% (w/v) glycerol, 1 mM EDTA and 1 mM DTT). The
assays performed on the microsomes were all as described in Section 2.2.4.
For both the February, 1986 seawater exposures, and the January, 1987
direct injection exposures, measurements were made of the contents of
cytochrome P-450 (and Amax) and cytochrome b^, and of the activities of
NADPH-cytochrome c reductase, NADH-cytochrome c reductase and NADH-
ferricyanide reductase. In the June, 1986 seawater exposures, measurements
were made of the contents of cytochrome P-450 (and ^max) and cytochrome b^
but only of the activity of NADPH-cytochrome c reductase. In both 1986
seawater exposure experiments, measurements were also made of the NADPH-
independent ECOD activity. The low wavelength '416' nm peak was also
determined on occasions. For the April, 1987 direct injection exposures
304
and the June, 1987 seawater exposures, measurements were made only of
cytochrome P-450 content and of the activities of NADPH-cytochrome c
reductase and NADH-ferricyanide reductase.
For all experiments, two or more groups of values were compared by
means of students t-test as described in Section 2.2.7. One-way analysis
of variance over time, for several groups of values, were also performed.
305
6.3 Results
6.3.1 Exposure of M. edulis to xenobiotics in seawater (without feeding)
Changes over time of digestive gland microsomal MFO component contents
and activities, the cytochrome P-450 Amax, and NADPH-independent ECOD
activity as a result of February, 1986, seawater exposures to
phenobarbital, clofibrate, DMF and 3MC (in DMF), are shown in Figure
6.1(a). Consistent changes for each of the parameters were not evident.
Exposure to either phenobarbital or 3MC had little if any effect (relative
to untreated or DMF-treated controls, respectively) on the specific
contents of either cytochrome P-450 or cytochrome b^, apart from after 14
days exposure when cytochrome P-450 content was observed to be reduced in
phenobarbital-treated animals but elevated in 3MC-treated animals (P <
0.05). Cytochrome b«j specific content was indicated to be elevated by
exposure to 3MC but the change was not statistically significant (P > 0.1).
Cytochrome P-450 content was reduced following exposure to clofibrate (P <
0.05) (except on day 3). A significant (P < 0.05) increase in the Amax of
the cytochrome P-450 carbon monoxide-difference spectrum was apparent after
exposure to phenobarbital for one day only, but not subsequently.
Indications of a lowered cytochrome P-450 Amax following 3 to 14 days
exposure to 3MC were apparent, but not statistically significant (P > 0.1).
NADPH-cytochrome c reductase specific activity was elevated after
exposure to phenobarbital for one day (P < 0.05), and was indicated to be
higher subsequently, although this was not statistically significant (P >
0.1). The activity was also higher afer 14 days exposure to DMF, compared
to the untreated control (P < 0.05). NADH-cytochrome c reductase activity
306
Figure 6.1
Exposure of H. edulis to phenobarbital (PB), clofibrate (CL),
dimethylformaraide (DMF) and 3-methylcholanthrene (3MC) (in DMF) in
seawater. (February, 1986).
a) Changes in digestive gland microsomal MFO component contents and
activities and NADPH-independent ECOD activity
Values are means ± SEM (n = 6 )
b) Uptake of 3-methylcholanthrene into the digestive gland and remainder
tissues
Values are means ± range (n — 2)
See Materials and Methods for other details of exposures. (xenobiotic
water concentration etc).
-------- Control untreated-------- PB pre-treated-------- CL pre-treated-------- DMF pre-treated--------- 3MC/DMF pre-treated
★ P < 0.05 PB or CL vs untreated control, 3MC vs DMF control
♦ P < 0.01 Cl or DMF vs untreated control only
P < 0.001 PB vs untreated control, 3MC vs DMF control
307
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was elevated after 3 days exposure to 3MC (P < 0.05), and a general trend
of elevation was indicated. Phenobarbital, however, had little, if any,
effect on either this activity or NADH- ferricyanide reductase activity.
Both NADH-cytochrome c reductase and NADH-ferricyanide reductase activities
were reduced following exposure to clofibrate for either 3 or 7 days (P <
0.05). For NADPH-independent ECOD activity, elevations above control
activities were seen in phenobarbital-treated animals after 7 days exposure
and in 3MC-treated animals after each of 1,3 and 7 days exposure (P <
0.001). Higher activity , above the untreated control activity, was also
seen after 7 days exposure to either DMF or clofibrate (P < 0.01).
Figure 6.1(b) shows the uptake of 3MC into the digestive gland and
remainder tissues during the exposure period. The main site of uptake was
the digestive gland with maximum levels of, approximately, 155 fig g"^ wet
weight of tissue occurring after 7 days exposure. The radiometric tissue
analysis of phenobarbital content indicated that the compound was not
bioaccumulated and that tissue levels approximated those of the seawater;
further that maximum tissue concentrations were achieved within 3 hours of
exposure (data not shown).
The results of a similar, but only seven day exposure, to
phenobarbital, DMF and 3MC (in DMF), performed later in the year during
June, 1986, are shown in Figure 6.2. The changes over time of the
digestive gland microsomal MFO component contents and activities, the
cytochrome P-450 Amax, and the NADPH-independent ECOD activity are given in
Figure 6.2(a). Despite variations over time in the levels for control
mussels, slightly more consistent trends of change with xenobiotic exposure
were indicated than for the February experiment. Cytochrome P-450 specific
311
Figure 6.2
Exposure of M.edulis to phenobarbital (PB) , dime thy lformamide (DMF) and 3-
methylcholanthrene (3MC) (in DMF) in seawater. (June, 1986).
a) Changes in digestive gland microsomal MFO component contents and
activities and NADPH-independent ECOD activity.
Values are means ± SEM (n = 6 )
b) Uptake of 3-methylcholanthrene into the digestive gland and remainder
tissues
Values are means ± range (n = 2)
See Materials and Methods for other details of exposures (xenobiotic water
concentration etc)
—■ Control untreated--------- PB pre-treated--------— DMF pre-treated--------- 3MC/DMF pre-treated
P < 0.05 PB or DMF vs untreated control, 3MC vs DMF control
* P < 0.025 3MC vs DMF control
$ P < 0.01 DMF vs untreated control, 3MC vs DMF control
312
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content was indicated to be elevated in response to both phenobarbital and
3MC but this was only statistically significant for 3MC exposure after 7
days (P < 0.05). The cytochrome P-450 Amax values of mussels exposed to
3MC were decreased after 3 and 7 days exposure (P < 0.05 and 0.025
respectively), whereas no such trend was evident for phenobarbital-exposed
mussels. The decrease in cytochrome P-450 Amax with 3MC-exposure was
consistent with the same trend (indicated but not statistically
significant) observed in the February experiment (see Figure 6.1 (a)).
Elevations of cytochrome b«j specific content were seen only on day one,
following exposure to either phenobarbital or DMF (P < 0.05). The response
of NADPH-cytochrome c reductase activity to xenobiotic exposure was much
greater in the June experiment than in the February experiment. Increases
in this activity were seen after exposure to DMF for 3 days (P < 0.01), and
after exposure to 3MC for 3 (P < 0.01) or 7 (P < 0.025) days (compared to,
respectively, untreated and DMF-treated control activities). There were
also indications of a response to phenobarbital on days 1 and 3, but these
increases were not statistically significant (P > 0.1). In marked contrast
to the February experiment, there were no increases in NADPH-independent
ECOD activity in the June experiment with exposure to any of the
xenobiotics. Control NADPH-independent ECOD specific activities were lower
in the June than the February experiment (compare Figures 6.1(a) and
6 .2 (a)).
The tissue levels of 3MC during the June exposure experiment are given
in Figure 6.2(b). Although the major site of uptake was again the
digestive gland, the tissue content, after 7 days exposure, of about 27 /zg
g"^ wet weight, was considerably lower than at the corresponding time in
the February experiment.
315
The reproductive stages of the mussels used in the two experiments
were approximately similar at both times of the year, vis. in terms of %
total gamete (combined male and female data), 36.8 ± 9.1% (February) and
29.3 ± 6.4% (June) (means ± SEM; n — 10)
6.3.2 Exposure of M. edulis to xenobiotics by direct injection
The results of experiments in which mussels were exposed to a number
of xenobiotics by repeated (2 days out of 3) direct injection over a 3 day
period were largely inconclusive. The results of two such experiments
performed in January, 1987 are given in Figures 6.3 (phenobarbital,
clofibrate or DMF injection) and 6.4 (3MC, Aroclor 1254, prochloraz or corn
oil injection). Mussels were sampled before injection (day 0 control) and
after injection of the xenobiotic or vehicular control (corn oil or
distilled water) was complete. There were no significant elevations above
the day 0 value or vehicular controls for the contents of cytochrome P-450,
the 416 ran component or cytochrome b^, in either experiment (P > 0.1). A
decrease (P < 0.05) in cytochrome P-450 content relative to the day 0
value, but not to the distilled water control, was seen following
phenobarbital exposure (Figure 6.3). The '416' nm peak was elevated over
the distilled water control following phenobarbital exposure, but the
reverse was seen for cytochrome b^ specific content (P < 0.05). Cytochrome
b^ specific content was also reduced after Aroclor 1254 treatment (P <
0.05). No changes were observed in cytochrome P-450 Amax values for any
treatment. An increase in NADPH-cytochrome c reductase activity was seen
only after exposure to phenobarbital (P < 0.05). A decrease in this
activity was indicated with clofibrate exposure (values were lower (P <
316
Figure 6.3
Exposure of M. edulis to distilled water, phenobarbital (PB), clofibrate
(CL) and dimethylformamide (DMF) by direct injection.
Effects on digestive gland microsomal MFO component specific contents and
activities.
Experiment performed during January, 1987.
See Materials and Methods for other exposure details (doseage etc)
Values are means ± SEM (N = 6 )
★ P < 0.05 Exposed condition vs distilled water control
317
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Exposure of M. edulis to corn oil, 3-methylcholanthrene (3MC), Aroclor 1254
(AR) and prochloraz (PZ) by direct injection.
Effects on digestive gland microsomal MFO component specific contents and
activities.
Experiment performed during January, 1987.
See Materials and Methods for other exposure details (doseage etc)
Values are means ± SEM (n = 6 )
★ P < 0.05 Exposed condition vs corn oil control
319
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0.05) relative to the day 0 value but not the distilled water control).
Xenobiotics administered in distilled water (phenobarbital and clofibrate)
had no effect on either NADH-cytochrome c reductase or NADH-ferricyanide
reductase activities. Corn oil alone caused an elevation of NADH-
ferricyanide reductase activity above the day 0 control activity (P <
0.05), and for this reason, apparent elevations in response to 3MC, Aroclor
1254 and prochloraz were not statistically significant (P > 0.1). NADH-
cytochrome c reductase activity was reduced following prochloraz exposure
(P < 0.05).
The results of a similar direct injection exposure experiment
performed in April, 1987 are shown in Figure 6.5. Measurements were
restricted to cytochrome P-450 content and NADPH-cytochrome c and NADH-
ferricyanide reductase activities. In this experiment untreated control
mussels were taken after day 3 rather than day 0 and no distilled water
injections were made (the day 3 control was used as a control for the
clofibrate and phenobarbital exposures). With the exception of a slightly
reduced cytochrome P-450 content following isosafrole exposure (P < 0.05),
the only effects of the xenobiotics were on NADPH-cytochrome c reductase
activity. Elevations above the untreated control activity were seen
following exposure to phenobarbital, clofibrate, isosafrole and, again,
corn oil. The clofibrate response was in contrast to the reduced activity
seen after exposure in the January experiment (see Figure 6.3). The
activity following ^-naphthoflavone exposure was also significantly greater
than the untreated control (P < 0.05), but was not greater than its
vehicular (corn oil) control. Activity following exposure to pregnenalone
16a-carbonitrile was significantly lower than the activities of either the
untreated control or the vehicular control (acetone).
321
Figure 6.5
Exposure of M. edulis to phenobarbital (PB), clofibrate (CL), acetone,
pregnenalone 16a-carbonitrile (PCN), phenanthrene (PH), isosafrole (IS),
corn oil and /?-naphthoflavone (BNF) by direct injection.
Effects on digestive gland microsomal MFO component specific contents and
activities.
Experiment performed during April, 1987
See Materials and Methods for other exposure details (doseage etc)
Values are means ± SEM (n = 6 )
★ P < 0.05 Exposed condition vs vehicular control
322
20o>Eina>OEa.
10
BNF.C0RN-0ILPCN PH.ISACETONEPBCONTROL CL
14
12 -
1 0 -
o>EcEin<DO 6‘Ec
4-
BNF.CORN—OILPH.PCN.IS.ACETONEPB.CONTROL CL
aUJcc o
700
600-
500 -UJCC cno .5 4oo-
O 300-
200 -
100 -
CORN-OIL BNF.PH.PCNIS.ACETONEPB.CONTROL CL
323
6.3.3 Three week exposure of M. edulis to xenobiotics (with feeding)
A relatively long-term exposure of M. edulis to phenobarbital, ethanol
and /3-naphthoflavone (in ethanol), with algal feeding, failed to produce
any significant changes in either digestive gland microsomal cytochrome P-
450 specific content or NADPH-cytochrome c reductase and NADH-ferricyanide
reductase specific activities (P > 0.1) (Figure 6 .6 ).
324
Figure 6.6
Three-week exposure of M. edulis (with algal feeding) to phenobarbital
(PB), ethanol (ETOH) and /3-naphthoflavone (BNF).
Effects on digestive gland microsomal MFO component specific contents and
activities.
Experiment performed in June, 1987
See Materials and Methods for other exposure details (doseage etc)
Values are means ± SEM (n = 6 )
325
NADH-FERRICY.
RED.
0CONTROL PB. ETOH. BNF.
§5CONTROL ETOH. BNF.
E 300-
100-
CONTROL PB. ETOH. BNF,
326
6.4 Discussion
Increases in monooxygenase activities, resulting from the induction of
particular cytochrome P-450 isoenzymes, are not always accompanied by
detectable increases in cytochrome P-450 specific content (Jayyosi et al. ,
1987; Payne et al., 1987). However, increases in total cytochrome P-450
levels are generally common following 3MC exposure of fish, amphibians and
reptiles and, to a lesser extent, of certain Crustacea (Schwen and
Mannering, 1982a; Maemura and Omura, 1983; Batel et al. , 1988), and
following both 3MC and phenobarbital exposure of mammals (Schwen and
Mannering, 1982a; Yeowell et al., 1985). The results of the mussel
exposure experiments indicate that cytochrome P-450 content is unchanged
following short-term exposure to either of these xenobiotics by direct
injection, but is increased slightly to 3MC only with longer-term exposure
in seawater (14 days exposure in February or 7 days in June). The absence
of any cytochrome P-450 specific content response towards phenobarbital is
consistent with observations for fish, amphibians, reptiles and Crustacea
(Elcombe and Lech, 1979; Singer et al., 1980; Schwen and Mannering, 1982a;
Goksoyr et al. , 1986b; Batel et al. , 1988). From an evolutionary
viewpoint, such a lack of response might be expected if the phenobarbital-
inducible isoenzymes are a relatively recent development among higher
vertebrates (Nebert and Gonzalez, 1987). The response toward 3MC was low
compared with responses seen in species from other phyla. Although
possibly seasonally variable, the increase in cytochrome P-450 specific
content above the vehicular control ranged from 0 to 34% in this study.
This compares with increases in specific content of, for example,
approximately 2 0 0% in rats and almost 90% in guinea pig (Astrom et al.,
1986), 253% in the goldfish, Carassius auratus (Maemura and Omura, 1983),
327
130% in the frog, Xenopus laevis (Noshiro and Omura, 1984), and 100% in the
crab, Maja crispata (Batel et al., 1988). The low response to 3MC in M.
edulis could reflect limitations of the experimental design, such as the
static tank environment imposing stress on the mussels or the doseage and
dosing regimen being inappropriate. With respect to the latter, whole body
microsomal cytochrome P-450 specific content of M. galloprovincialis has
been observed to increase after 48 hours exposure to about 90 to 333 ppb
benzo [a]pyrene (seawater concentration) but not to 900 ppb or above
(Suteau, 1986). However, equally likely is that the response was
characteristic of the M. edulis MFO system.
Table 6 .1 shows the responses of digestive gland microsomal cytochrome
P-450 specific content and NADPH-cytochrome c (P-450) reductase specific
activity from the two seawater exposure experiments described here,
compared with other available data on the responses of the M. edulis and
other molluscan MFO systems to various xenobiotics, including polycyclic
aromatic hydrocarbons (PAH). As with the 3MC exposure experiments, great
variability has been seen in all the molluscan xenobiotic exposure studies
with, on occasions, responses not being seen. Compared to the responses
following exposure to diesel oil (mixed PAH) (up to 200%) (Livingstone et
al., 1985; Livingstone, 1987, 1988), the increases in cytochrome P-450
specific content with 3MC exposure in this study (up to 34%) were small.
The greater increase in cytochrome P-450 in the former may possibly reflect
the presence of better particular PAH inducers in the diesel oil mixture
than 3MC. For example, in the study of Livingstone (1988), marked
increases in M. edulis digestive gland microsomal cytochrome P-450 specific
content resulted from exposure to a diesel oil high in 2- and 3-ring PAH
but low in 4- and above ring PAH. The same exposure had no effect on
328
Table 6.1
Responses of molluscan microsomal cytochrome P-450, NADPH-cytochrome c
(P-450) reductase and benzo[a]pyrene hydroxylase (radiometric and
fluorometric activities) to experimental exposure to xenobiotics (table
modified from Livingstone et al., 1988b)
Data are for digestive gland microsomes except for C. virginica (digestive
gland homogenate) and M. galloprovincialis (Suteau et al.) (whole body
microsomes).
f see references for doseages and exposure periods which vary greatly
$ no statistical treatment of data
Abbreviations: 3,4,3',4'-tetrachlorobiphenyl (TCBP) (3MC-type inducer),
2,4,5,2',4',5'- hexachlorobiphenyl (HCBP) (phenobarbital-type inducer).
Polycyclic aromatic hydrocarbons (PAH).
- Not determined.
329
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330
haemastoma
crude
oil
(PAH)
61 -
- Livingstone
et al.
(1986)
flounder Platichys flesus hepatic microsomal cytochrome P-450 content,
cytochrome P-450E (3MC-type isoenzyme), BPH or EROD activities (Addison and
Edwards, 1988; Stegeman et al., 1988). However, it should also be noted
that in the diesel oil experiments, mussels were exposed for long periods
(weeks to months) in an experimental mesocosm facility closely resembling
the natural habitat (see Livingstone et al., 1985), rather than short-term
in the static tank environment of this study.
In studies in which M. edulis was exposed to PAH (Livingstone et al.,
1985; Livingstone 1987, 1988) the increases in digestive gland microsomal
cytochrome P-450 specific content were accompanied by a blue shift (~2 nm)
of the Amax of the carbon monoxide-difference spectrum of cytochrome P-450.
A similar observation was made in this study following 3MC exposure.
Interestingly, the decrease in cytochrome P-450 Affiax was apparently
seasonal in that a more significant shift was seen following exposure in
June than following exposure in February. Reasons for this are unclear,
although it could be a reflection of the isoenzyme compositions at
different times of the year. For example, if as postulated before (see
Section 2) at least two isoenzymes are more apparent over the autumn and
winter months, and only one is responsive to 3MC treatment, then a reduced
blue shift of the cytochrome P-450 Amax might be anticipated in February
compared to June. That a seasonal aspect was apparent in the response to
exposure was further indicated by the tissue levels of 3MC over the
exposure periods of the two experiments. Considerably higher levels were
observed in digestive glands from the February than the June experiment.
Reasons for this are, again, unclear, but may, for example, reflect
seasonal differences in the tissue lipid levels. PAH are most readily
accumulated in lipid-rich tissues (Widdows et al., 1983) and, as has been
331
noted previously, higher tissue concentrations of PAH in natural mussel
populations are normally observed over the autumn and winter than over the
summer periods (Fossato et al., 1979; Mix et al., 1982).
As discussed above, induction of particular activities are not
necessarily accompanied by detectable changes in the total contents or
specific activities of the MFO components. In this study, a marked
stimulation of NADPH-independent ECOD activity was seen following 3MC
exposure in February but not in June. It is possible this is a direct
reflection of the different tissue levels of 3MC having different inducing
capabilities. However, as the seasonal study showed (see Section 2), NADPH-
independent ECOD activity is low or negligible around June, and, therefore,
the lack of response to 3MC exposure at this time may reflect a refractory
period to induction. That responses were also seen toward clofibrate, DMF
and phenobarbital at certain times of the February experiment, may,
however, indicate that factors other than induction were responsible for
the stimulations seen, or, that given the right conditions, these
xenobiotics can also induce NADPH-independent ECOD activity.
In mammals, induction of MFO systems by 3MC and other PAH is mediated
through cytosolic receptors (Nebert et al., 1975; Astrom and DePierre,
1986; Whitlock, 1986). A similar mechanism has been proposed for induction
by these compounds of MFO systems in fish and other lower vertebrates
(Schwen and Mannering, 1982a). The induction seen in certain Crustacea
(Batel et al., 1988) and in molluscs (see Table 6.1), following exposure to
either 3MC or other PAH, may indicate that such a mechanism is also present
in these marine invertebrate species. However, that fundamental
differences may also exist in the MFO system induction mechanism of
332
molluscs is indicated by the responses of NADPH - cytochrome c (P-450)
reductase activity to diesel oil (Livingstone et al., 1985; Livingstone,
1987, 1988) or 3MC exposure (this study). 3-Methylcholanthrene is known
not to simultaneously induce NADPH - cytochrome P-450 reductase (with
cytochrome P-450) in mammals (Ioannides and Parke, 1987), nor to have any
effect on this activity in species of fish, frog or snake (Schwen and
Mannering, 1982a). Following 3MC treatment of M. edulis in the June
experiment of this study, and following exposure of both M. edulis and the
gastropod mollusc L. littorea to diesel oil, marked increases in digestive
gland microsomal NADPH-cytochrome c ( P-450) reductase activity were seen
(Table 6.1). A responsiveness of this activity to phenobarbital was seen
after day one of the February experiment, and after both the January and
April direct injection experiments. Phenobarbital is known to
simultaneously induce NADPH-cytochrome P-450 reductase and cytochrome P-450
in mammals (Ioannides and Parke, 1987). That no obvious response towards
phenobarbital is seen for cytochrome P-450 in this or any other aquatic
species further suggests possible intrinsic differences in the induction
mechanisms of xenobiotics in molluscs. A responsiveness of digestive gland
NADPH-cytochrome P-450 reductase to phenobarbital exposure in M. edulis is
also indicated from cytochemical studies on NADPH-neotetrazolium reductase
activity, which was increased following phenobarbital injection (Moore,
1979) (this activity is thought to measure some aspect, though not exactly
the same aspect as NADPH-cytochrome c reductase activity - Masters and
Okita, 1982). More recently, phenobarbital pre-exposure of M. edulis was
found to increase the frequency of sister chromatid exchange produced by
exposure to cyclophosphamide, a compound requiring metabolism by the MFO
system to exert its mutagenic effect (Dixon et al., 1985). It is possible
that such a response could have been elicited by increased flux through the
333
MFO system, as a result of elevated cytochrome P-450 reductase activity.
The marked responsiveness of digestive gland microsomal NADPH-cytochrome c
(P-450) reductase activity, particularly in gastropods, has led to its
suggested use as a biochemical indicator in biological effects measurement
(Livingstone, 1987).
In addition to phenobarbital, a response of digestive gland microsomal
NADPH-cytochrome c (P-450) reductase activity was seen following direct
injection of DMF, isosafrole and corn oil, and was indicated for 3MC,
Aroclor 1254 and prochloraz, suggesting a general responsiveness of this
activity to xenobiotics. In contrast, no response was observed for
cytochrome P-450 specific content or the levels or activities of any other
of the MFO components measured. Each of the compounds examined is a known
inducer of MFO systems in mammals and, in many cases, also lower
vertebrates such as fish (see Sections 1 and 6.1). There are a number of
possible explanations for the observed lack of responses. Firstly, the
xenobiotics, like phenobarbital, are not inducers of molluscan cytochromes
P-450. Secondly, the doseages and exposure times were inappropriate; for
example, as with the seawater exposure experiments, the choice of dos x e,
was somewhat arbitrary and no doseage-regimen experiments were carried out.
Thirdly, the experimental design of using injection may have been stressful
to the mussels, although this technique has been successfully used in other
biochemical studies on M. edulis, for example, anaerobic metabolism (De
Zwaan et al., 1983). Fourthly, no measurements were made of more specific
(and presumably more sensitive) MFO activities such as ECOD and BPH
activity. However, emphasis in the injection, and most of the xenobiotic
exposure experiments, was placed on measurement of MFO system components
rather than monooxygenase activities because of the difficulties
334
encountered in measuring such activities (see Section 5), their seasonal
variability and, most important, the significance of such measurements
given the phenomenon of their NADPH-independence.
In conclusion, a number of points can be made from the results of the
xenobiotic exposure experiments of this section.
(1) Despite variability in response, increases in digestive gland
microsomal cytochrome P-450 did occur with exposure to 3MC, which is
in agreement with other PAH studies on M. edulis (Livingstone et al.,
1985; Livingstone, 1987, 1988) and M. galloprovincialis (Gilewicz et
al., 1984; Suteau et al. , 1988). Also, the synthesis of new
isoenzymes was indicated.
(2) Qualitative and quantitative differences in the response of the MFO
system exist from other animal groups, for example, the low percentage
changes in cytochrome P-450 content and the nature of the cytochrome
P-450 reductase response to 3MC, phenobarbital and other xenobiotics.
(3) The results are in agreement with other molluscan studies (Table 6.1),
showing apparent induction but great variability in the occurrence of
such responses.
(4) Laboratory molluscan induction studies are greatly limited at present
by three factors, vis. (a) the absence of seasonally-stable, easily
measured, sensitive, cytochrome P-450-dependent monooxygenase
activities for routine use, (b) the conceptual difficulties associated
with the phenomenon of the NADPH-independence of the monooxygenase
activities, and (c) the lack of knowledge or "tricks" that could be
used to guarantee induction with "wild" mussels from the field; for
example, biological variability in cytochrome P-450 specific content
was low, and the increase following diesel oil exposure high, in the
335
experiments of Livingstone (1988) in which, in the mesocosm facility,
the mussels were kept at low temperature and without food i.e.
starvation and low temperature may have combined to minimize metabolic
activity (reduce biological variability) and maximize the subsequent
response to xenobiotic exposure.
336
6.5 Appendix
RESPONSES OF THE DIGESTIVE GLAND CYTOCHROME P-450 MONOOXYGENASE SYSTEM OF THE COMMON MUSSEL (Mytilus edulis) TO 3-METHYLCHOLANTHRENE
AND SODIUM PHENOBARBITAL
1 1 7David R. Livingstone , Suzanne V. Farrar , Cristina Fossi ,
Miles A. Kirchin^’ and Michael N. Moore^
"hsfERC Institute for Marine Environmental Research,Prospect Place, The Hoe, PLYMOUTH, Devon PL1 3DH, U.K.
2Dipartimento di Biologia Ambientale,Universita di Sienna, Via delle Cerchia 3,
53100 Sienna, Italy.
3Dept. Biochemistry, University of Surrey,GUILDFORD, Surrey, GU2 5XH, U.K.
Sodium phenobarbital (PB) and 3-methylcholanthrene (3MC) induce
different isoenzymes of cytochrome P-450 in mammals. Mussels were exposed
to these compounds in order to obtain comparative data and provide
information on the fundamental nature of the molluscan mixed function
oxidase (MFO) system. Experiments were carried out in February and June in
static tanks (controlled to field-ambient temperature) and the mussels
exposed to daily single doses of 3MC (dissolved in dimethylformamide) or PB
injected into the seawater giving water concentrations of, respectively, 75
ppb and 150 or 1500 ppb; exposure periods were from 1 to 7 days.
3MC was readily taken up into the digestive gland, largely in the
lysosomes, giving concentrations of from 2.8 to 155 pg g"^ wet weight
depending on the exposure-time. In contrast, tissue concentrations of PB
approximated those of the seawater. Microsomal MFO components in control
337
mussels were generally higher in February than June and different responses
were seen. In June, following 7 days exposure to 3MC, cytochrome P-450 and
NADPH-cytochrome c reductase activity were respectively, increased from
23.8 ± 2.0 to 31.8 ± 1.7 pmoles mg’ protein and from 8.75 ± 1.34 to 12.76
± 0.54 nmoles min” mg’ (± SEM, n - 6 ). P < 0.05), but no changes were
demonstratable in February. In contrast, NADPH-neotetrazolium reductase
activity was elevated by 3MC in February. PB made little impact but a
slight increase in NADPH-cytochrome c reductase activity was seen in June.
Benzo[a]pyrene hydroxylase activity (total metabolites) did not change but
an increase in 'NADPH-independent 7-ethoxycoumarin 0-deethylase' activity
occurred in February with 3MC exposure. It is concluded that the responses
of cytochrome P-450 and NADPH-cytochrome c reductase activity are similar
to those observed previously in experiments with diesel oil exposure.
(Published in Marine Environmental Research 1988 24 118-119.
338
Section 7
General discussion
339
Indications of the existence of isoenzymes of digestive gland
microsomal cytochrome P-450 have been obtained from several of the
investigations, the most tangible evidence perhaps being provided by the
purification studies (Section 3) . Two haemoprotein peaks were eluted
during ion-exchange chromatography, using a salt (KC1) gradient. The peaks
were obtained in purifications in which either PEG or (NH^^SO^ was used as
an initial protein fractionating agent, and in the latter case the two
peaks were both shown to contain cytochrome P-450. The (NH^^SO^-
experiments also demonstrated that the large peak at '416' nm (in the
dithionite-reduced carbon monoxide difference spectrum of cytochrome P-450)
was also a haemoprotein, possibly cytochrome P-420, the denatured form of
cytochrome P-450. The existence of cytochrome P-450 isoenzymes has also
been indicated from in vitro MFO activity studies, particularly on NADPH-
independent ECOD activity (Section 5). The dependence of this activity on
7-ethoxycoumarin concentration showed biphasic kinetics, which may be
indicative of the existence of, at least, two cytochrome P-450 isoenzymes
(Rogiers et al., 1986) (N.B. it may also be suggestive of two different
cytochrome P-450 mechanisms of action - see below). Further indication of
the existence of isoenzymes came from the stimulatory effect of a-
naphthoflavone on NADPH-independent ECOD activity, which was in contrast to
its inhibitory effect on mussel digestive gland microsomal BPH activity
(Livingstone, 1985). Significantly, in mammals, a-naphthoflavone has been
shown to inhibit BPH activity catalysed by 3MC-inducible cytochromes P-448,
but to stimulate activity catalysed by PB-inducible cytochromes P-450
(Wiebel et al., 1971; Huang et al., 1981). Finally, some supportive
evidence for cytochrome P-450 isoenzyme existence was given by the seasonal
variability in the SDS PAGE digestive gland microsomal protein profile
(Section 2). For most of the year, only one band corresponding to
340
cytochrome P-450 could be discerned, having a monomeric molecular weight of
53.0 Kd. However, over the autumn and early winter months, a second band
of molecular weight 51.5 Kd appeared. Interestingly, the emergence of the
second protein band coincided with a sharp increase in NADPH-independent
ECOD activity, and with a decrease in the wavelength maximum (carbon
monoxide-difference spectrum) for cytochrome P-450, suggesting a
relationship between the three observations. That two or more isoenzymes
could be present in M. edulis and molluscs in general, is consistent with
observations on other marine invertebrates. For example, species of crab
have been shown to contain two or more isoenzymes which are apparently
seasonally variable (Quattrochi and Lee, 1984).
Despite the evidence for the likely existence of multiple forms of
cytochrome P-450 in M. edulis, it is impossible, for several reasons, to
identify analogous mammalian isoenzymes. Firstly, the nature of the NADPH-
independence of the MFO activities make comparisons with other species
difficult. Secondly, the inability to demonstrate type I ligand-binding
limits insight into the type of compounds that may be substrates for the
mussel cytochrome P-450. Thirdly, the low yield of partially-purified P-
450 prevented comparisons being drawn by way of, for example, peptide
mapping or immunochemical studies, although, with a scale-up of procedures,
the potential for this now exists. Fourthly, other observations, not
consistent with properties of characterised mammalian cytochrome P-450
isoenzymes, were apparent. For example, the seasonal increase in NADPH-
independent ECOD activity coincided with a lowering of the cytochrome P-450
Amax (carbon monoxide-difference spectrum). In mammals, a lowering of the
P-450 Amax is elicited by synthesis of P-448-type cytochromes, and the
effect of a-naphthoflavone is to inhibit MFO activity mediated by mammalian
341
cytochromes P-448. In contrast, mussel NADPH-independent ECOD activity was
shown to be stimulated by a-naphthoflavone. (N.B. as mentioned previously,
7-ethoxycoumarin can be metabolised by both P-450 and P-448-type
cytochromes; Rodrigues et al., 1987).
Since M. edulis is phylogenetically a lower, more primative animal
than, for example, a mammal, it is perhaps to be expected that differences
as well as similarities should be seen in their cytochrome P-450s. Thus,
the cytochromes P-450 of H. edulis may have characteristics of, for
example, both P-450 and P-448-type cytochromes, as well as unique
characteristics of their own. In this respect, it is interesting to note
that, recently, some homology with a particular mammalian cytochrome P-450
isoenzyme has been reported; specifically, using hybridization of total
mRNA from mussel digestive gland to rat liver cDNA probes, transcript
sequences 60% or greater to rat P-450 IV Al (also called P-450LAw or P-
452), at the nucleotide level, were detected (Spry, pers. comm.). This P-
450 isoenzyme, which in rat is inducible by clofibrate, is catalytically
relatively specific for w and w-l hydroxylation of endogenous fatty acids
(Tamburini et al., 1984). That a cytochrome P-450 of M. edulis may be
structurally similar to this mammalian form, is presumably indicative of a
role in endogenous metabolism. Such a function has been suggested
previously (Livingstone and Farrar, 1984), and is consistent with
discussions from the seasonal study (Section 2) and the study of MFO
activities (Section 5).
The differences between mussel and mammalian MFO systems, are further
indicated by the formers apparent limited response to model xenobiotics,
including PB and 3MC (Section 6 ). Thus, induction of components and MFO
342
activities of the order seen in mammals and other vertebrate species, was
not seen in M. edulis. The exposure methodologies employed for the mussel
experiments may have contributed to the limited response, but, equally,
real differences likely exist in inducibility and possibly the type of
xenobiotics which elicit a response; for example, marked induction of MFO
components, including cytochrome P-450, have been seen for M. edulis in
response to mixtures of hydrocarbons in diesel oil (Livingstone, 1987,
1988).
The in vitro ECOD activity of digestive gland microsomes was
independent of the presence of NADPH and was inhibited by reducing agents
(NADPH and NADH). These results are consistent with a one-electron
peroxidatic mechanism (assuming an endogenous source of peroxide) for
mussel cytochrome P-450 catalysis (Section 5). Such, or a similar,
mechanism is also indicated, in part, for benzo[a]pyrene metabolism (to
quinones) in this and other molluscan species (Stegeman, 1985; Livingstone
et al. , 1988a; Moore et al., 1988), and is perhaps consistent with the
possibility of a more primitive cytochrome P-450 catalytic mechanism in
lower animals. Other in vitro mussel MFO activities were NADPH-independent
but not inhibited by NADPH, for example, testosterone hydroxylase, and
fluorometric (phenols) BPH activity (Livingstone, 1985; Moore et al.,
1988). Cytochrome P-450 isoenzymes may, therefore, exist which operate
different catalytic mechanisms, for example, monooxygenation and one-
electron oxidation, or, alternatively, the catalytic mechanism for one
isoenzyme might change with substrate concentration or other factors. The
latter scenario would be an alternative explanation for the biphasic
kinetics seen with 7-ethoxycoumarin (see before).
343
Another explanation for the NADPH-independence of ECOD and other MFO
activities could be that the substrate-binding site of the P-450 is blocked
or hindered by the presence of an endogenous ligand (Section 4). Mussel
digestive gland microsomes and ethyl acetate-extracts of these microsomes
were thus found to inhibit in rat, both MFO activity and hexobarbital
binding to cytochrome P-450. The prevention of the binding of substrates
to cytochrome P-450, and the inhibition of a shift in spin-state from low
to high, would inhibit electron transfer from NADPH via cytochrome P-450
reductase to cytochrome P-450. The presence of endogenous ligands would
also explain the inability of mussel cytochrome P-450 to elicit type I
spectral changes. Interestingly, reverse type I spectra were obtained with
the type I ligands testosterone, 7-ethoxycoumarin, SKF 525-A and a-
naphthoflavone. Whether these spectra were indicative of endogenous ligand
displacement from the type I site, or due to direct interaction with the
haem iron, is not known. However, that spectra were seen is consistent
with other observations (Section 5) of these compounds being substrates or
inhibitors for mussel cytochrome P-450.
Titration of 7-ethoxycoumarin with digestive gland microsomal
cytochrome P-450 produced binding spectra compatible with a simple
Michaelis-Menten model (Section 4). This was in contrast to the biphasic
NADPH-independent ECOD enzyme kinetic studies (Section 5). However, the
two results are not obviously inconsistent because different biological
samples were used for the two studies (biphasic kinetics were less obvious
or absent with some samples), and comparison is limited by the unusual
phenomena of NADPH-independence of enzyme activity and the reverse type I
spectra with the type I substrate.
344
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