expression of recombinant human cytochrome p450...
TRANSCRIPT
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EXPRESSION OF RECOMBINANT HUMAN CYTOCHROME P450 IN AMES TEST
SThUNS FOR MUTAGENICITY TESTING
A Thesis
Presented to - the Faculty of Graduate Studies
of
The University of Guelph
by
TRACEY HENRY
In partial fir&ent of the requirements
for the degree
Master of Science
June 1997
Q T. Henry, 1997
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EXPRESSION OF RECOMBINANT HUMAN CYTOCHROME P4501A1 IN AMES TEST STRAINS FOR MUTAGENICITY TESTING
Tracey L. Henry University of Guelph, 1997
Dr. P- David Josephy Advisor
Heterologous expression of mammalian bioactivation e-es in bacteria can obviate the
requirement for the addition of exogenous enzymes in bacterial genotoxicity testing. Ames tester
strains bearing a plasmid which canies the cDNA of human P450 IAl were constmcted. It was
determineci that both NADPH-cytochrome P450 reductase and plasmid pKM101, which carries
the genes for error-prone DNA repair (mucAB), were likely required for the activation of P4SO
1Al-dependent mutagens. Subsequently, tester strains bearing plasmids coding for a P450
1 A1 :NADPH-cytochrome P450 reductase ftsion protein and the MucAB proteins were
constmcted. Strain testing indicated that fùnctional MucAB proteins were produced. However,
fùnctional P450 IAl was not detected in any of the strains. b u n o b l o t analysis indicated that
the level of P4SO 1Al expression was low in strains bearing both ptasmids. The presence of the
plamid bearing the mucAB genes may have afkted the level of P4SO 1Al expression.
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For Non& Nana, and Pcrpa
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Acknowledgments
There are many people who have made significant contributions to my thesis work
My advisor, David losephy, whose advice, support, encouragement, and humour were
invaluable. Dr. A Meiiors and Dr. C. Whitfield, my advisory cornmittee members, who
provided many usefül suggestions.
Thanks to Lian DeBruin for many helpfùl discussions, and to Heather Lord and
James Oak for keeping excellent Iab notes.
1 also wish to express my gratitude to my f d y in Niagara Faiis, who have been a
constant source of support and encouragement.
And W y , thanks to my sisters and brothers at C.U.PE. 3913 , the Central Student
Association, and the Graduate Student Association who cuntributed to my informai
education in labour studies, sociology, and poiitics.
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List of Figures:
Figure I :
Figure 2:
Figure 3 :
Figure 4:
Figure 5:
Figure 6:
Figure 7:
Figure 8:
Figure 9:
Figure 10:
Figure 1 1 :
Figure 12:
Figure 13:
Figure 14:
Figure 15:
Figure 16:
Figure 17:
Active site of cytochrome P450, showing the reactive ùon-0x0 species
Proposed cataiytic cycle of cytochrome P450
Possible mechanism for hydroxylation of aromatic rings
Schematic representaîion of a polycistronic gene wnstruct
Structures of various polycyclic aromatic hydrocarbons
Mechanism for the hydroxyIation of acetanilide, illustrating the NiH shifk
Activation of benzo[a]pyrene
Activation of aflatoxin B , Fe+* vs. Fe+'-CO dinerence spechim of membranes isolateci fkom DHSa pP450 1A2
hmunoblot malysis of P4SO 1Al expression in S. iphimwim
Construction of TA1535 pP45O 1 Al-reductase pGW249
Construction of YG7 1û4 pGW249 pP450 1Al and YG7 104 pGW249 pP45O IAl-reductase
Mutagen spot test results
Maps of plasrnids pP450 1 Aland pP450 1Al-reductase and agarose gel electrophoresis of Bm36Ldigested plasmid DNA
Mutagenicity of BP-7,8diol+ S9 in YGïlM pGW249, YG pGW pP4SO 1A1, and YG pGW pP45O 1Al -reductase
hunoblot analysis of P45O 1A1 and P45O 1 Al-teductase expression in S. &phimurhm membranes
Unmunoblot analysis of P45O 1A1 and P450 1Al-reductase expression in E. coli and S. îyphimun'm membranes
.. II
Page
6
7
8
20
27
29
31
3 3
54
56
60
63
65
69
72
74
76
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List of Tables:
Page
Table 1 :
Table 2:
Table 3 :
Table 4:
Table 5:
Mammaiian P450s expressed in E. coli. 12
Bacterial strains and plasmids 41
Isolation of bacterial membrane proteins 53
Spot tests with MucAB-dependent mutagens 66
Spot tests with P4501AI-dependent mutagens 70
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List of Abbreviations:
2-AA
AF-2
6-ALA
ampR
m p S
B[alP
DCA
BP 7,8-di01
BP 7,s-diol-9,lû-epoxide
BP 7,8-epoxide
CmR
DMSO
EDTA
EROD
FAT3
FMN
GAPDH
HMG-COA
HPLC
kmR
2-aminoanthracene
firry @amide
6 -aminolmlinic acid
ampicillin-resistant
ampicillin-sensitive
benzo[dpyrene
bicinchoninic acid
7,s-diydroxy-7,8-dihydrobenzo[a]pyrene
7,8-dihydroxy-9,lO-epoxy-7,8,9,10- teîrahydrobenzo[a]pyrene
7,8-epoxy-7,8-dihydrobenzo[a] pyrene
chloramphenicol-raistant
diethyl sulfoxide
disodium ethylenediamine tetraacetate
7-ethoxyresoru6n O-deethylase
fiavin adenine dinucleotide
flavin mononucleotide
glyceradehyde-3-phosphate dehydrogenase
P-hydroxy- P -methylglutaryl-coenyme A
high-performance iiquid chromatography
kanamycin-resistant
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kb
kDa
LPS
PTG
MeIQ
MHC
MNNG
NADPH
NAT
4-NQO
2-NF
OD,
PAGE
PAH
PhIP
PMSF
RFLP
S-9
SDS
2,4,5-TMA
tep
TpP- 1
kilobase
kilodalton
Epopolysaccharide
isopropyl- Q a-thiogaiactoside
2-amino-3,5-dimethylunid~~[4,5-~quinoline
major histocompatibility cornplex
N-methyl-N'-nitro-N-nitrosoguanidine
nicotinamide adenine diiucleotide phosphate
N-acetyltransferase
4-nitroquinoline- 1 -oxide
2-nitrosofluorene
optical density measured at 650 nm
polyacrylamide gel electrophoresis
polycyclic aromatic hydrocarbon
2-amino- 1-rnethyl-6-phenylimidazo-[4,5-bl pyridine
phenylmethylsulfonyl fluoride
restriction fiagrnent length polymorphism
hepatic 9 000 x g supernatant
sodium dodecyl sulfate
2,4,5-trimet hylaniline
tetracycline-resistant
3-amino-l,4-diimethyl-SH-pyrido[4,3-b]indole
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.................................................... Acknowledgements i C C
LiaofFigures ........................................................ u ... LiaofTables ..................................................... ui
.................................................... List of Abreviations iv
htroduction ................................................... 3 1.0 Perspective .............................................. 3 1.1 CytochromeP450 ......................................... 4 1.2 P450 analfical techniques .................................. 4
............ 1.2.1 Fe+'-CO vs Fe+* optical diifference spectroscopy 4 1.2.2. Enzyme assays ..................................... 5 1.2.3. Cytochrome c reductase activity ........................ 5
............................... 1.3. Cytochrome P450 mechanism 6 ..................................... 1.4. Cytochrome P450 1A1 9
.............................. I .5 . Bacterial expression ofP450s 10 1.6. Electron transport to P450: NADPH cytochrome P450 reductase .... 15
...................... 1 .7 . Recombinant cytochrome P450 reductase 18 1.8. Expression of recombinant human enzymes in SaIrnonella whimurim 22 1 .9 . P450 expression in kchatomyces cerevisiae .................. 24
............................ 1.10. Polycyciic aromatic hydrocarbons 26 1.11. Polycyclic arornatic hydrocarbon metaboiism ................... 28 1.12. AflatoxhB, ............................................ 32 1.13. Mutagenicity testhg ..................................... 35 1.14. SOSDNArepair ........................................ 37
.......................................... 2 . Materials and Methods 40 .............................................. 2.1 Chemids 40
................................................. 2.2 Media 40 2.3 Bacterial strains and plasrnids ............................... 41
. . . . . . . . . . 2.4.1. Reisolation and storage of bacterial strains 42 ..................... 2.4.2. Bacterial growth conditions 42
2.4.3. Plasmid mini-prep ............................ 42 ................. 2.4.4 . Electrotransfonnation of bacteria 43
2.4.5. Bacterid Conjugation . . . . . . . . . . . . . . . . . . . . . . . . 43 2.4.5.1. Liquid incubation . . . . . . . . . . . . . . . . . . . . . . . 43 2.4.5.2. Plate incubation ........................ 44
. .. . . . . . . . 2.4.6. Phenotypic confirmation of S t v p h i ~ u m strains 44 .................... 2.4.6.1. Histidine requirement 44
. . . . . . . 2.4.6.2. va mutation (deep-rough phenotype) 45 2.4.6.3. AuvrB mutation ....................... 45
.................. 2.4.6.4. S pontaneous mutations 45
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1. Introduction
1 .O Perspective
The Ames test is a bacteriai (&dmoneella typhimurïum) mutageniCity assay which
is used widely to screen potentialiy mutagenic and carcinogenic chemicals. Many
compounds require metabolic activation by mammalian enzymes to exert theu
carcinogenic effects- Since bacteria do not carry out the 'bioactivation' process, an
exogenous source of activating enzymes is fiequently required to observe chemical
mutagenicity in bacteriai assays. Mammalian liver homogenate is cornmonly used as the
source of activation enzymes in bacteriai mutagenicity testing. Heterologous expression
of enzymes of bioactivation in Salmonella can obviate this requirement. In addition to
the obvious advantage of reducing the use of animais in rnutagenicity experiments,
heterologous expression of bioactivation enzymes provides i n c r d speciticity and
sensitivity in bacterial mutagenicity tests.
Cytochrome P450 monooxygenases are among the most sipifkant
biotransforrnation enzymes. Metaboiic activation of a vast number of chemical
carcinogens proceeds through P450-catalysed oxidations. The focus of the present
study is the development of Ames tester strain bacteria which express recombinant
human P450s and c m detect P450-dependent mutagens, in the absence of rniunmalian
Iiver homogenate.
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1.1 Cytochrome P450
Cytochrome P450s are a super-My of enzymes which metaboiize a diverse
range of endogenous and xenobiotic wmpounds. Multiple isoforms of cytochrome P450
are found in mammals, insects, plants, yeast, and bacteria (Groves and Han, 1995).
P450s have been identitied in many rnarnmalian tissues, including liver, kidney, h g ,
adrenal cortex, and intestine (Groves and Han, 1995). Eukaryotic P450s are membrane
proteins, with the exception of the fungal P450 55A1 (Shoun et al., 1989). The number
of substrates of cytochrome P450s is weii into the thousands, due to multiple isoforms
and the ability of many isoforms to metaboiize a broad range of substrates. Reactions
cataiysed by cytochrome P450s include hydroxylation, epoxidation, dealkylation,
suIfoxidation, and dehdogenation (Ortiz de Montellano, 1995). P450s oxidiie nonpolar
xenobiotics, producing compounds of increased polarity which are more eady excreted
or detoxifïed. However, some P450 metabolites are far more reactive than their
precursors and can pose a threat to the integrity of suscepn%le nucleophiles, such as
DNA
1.2 P450 analyticd techniques
1.2.1 Fe+2-C0 vs Fe'? optical düference spectroscopy
Many ferrous heme proteins, includiig cytochrome P450, fonn a characteristic
carbon monoxide complex in the presence of CO gas and a reducing agent. This ferrous-
CO complex can be detected using optical difference spectroscopy, which records the
difference in absorbame between CO-bound and CO-fiee P450 samples. P450 was first
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identified, and so named, because of its characteristic absorption maximum at 450 nm
(Omura and Sato, 1964). Since the iron must be in a reduced state to b i d CO, a
reducing agent is added to the samples prior to the addition of CO gas. Fe+2-C0 vs Fe+2
dîerence spectroscopy is routinely used to determine P450 holoenzyme content in
microsomes (artificiai vesicles produced though hornogenation of membranous
structures, especidiy endoplasrnic reticulum) and bacterial membrane preparations.
1.2.2. E-e assays
The catalytic activity of a P450 sample is measured using hctional markers
which are P450 substrates, A vast number of P4SO isozyme-specific enzyme activity
assays have been developed (Correia, 1995). 7-Ethoxyresorufin Odeethylase (EROD) is
a fûnctional marker substrate cornmonly used to measure P450 1Al catalytic activity.
The product fomed in a P450-catalysed deethylation of the substrate has a fluorescence
ernission maximum at 590 m. Measurement of fluorescence intensity at this wavelengtb
provides a simple measurement of P4SO 1Al activity. ûther common P450 assays may
use colourimetric or HPLC analysis of the products (Josephy, 1997).
1.2.3. Cytochrome c reductase activity
NADPH cytochrome P450-reductase ('450-reductase) is an essential component
of P450-catalyseci xenobiotic metaboüsm in mammalian species (see 1.6.; Strobel et ai.,
1995). P450-reductase activity is comrnonly assayed by measuring its associated
cytochrome c reductase activity. This indirect measurement is much simpler than direct
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detemination of P450-reductase activity, which requires anaerobic conditions and rapid
reaction techniques (Guengerich, 1989). Redudon of cytochrome c can be meaiured by
recordmg the change in absorbante at 550 nm, upon the addition of NADPH to a sample
wntaining cytochrome c and P450-reductase (Guengerich, 1989). Cytochrome c
reductase activity is an accurate and cunvenieat method for assayhg P450-reductase
activity.
1.3. Cytochrome P450 mechanism
The general mechanism of the P450 catalytic cycle is weii estabiished (Groves
and Han, 1995). The common features of the P450 active site are an iron protoporphyrin
IX (heme), with an axial cysteine and a coordination site for oxygen (figure 1). The
Fig. 1. Active site of cytochrome P450, showing the reactive iron-0x0 species.
cataiytic cycle of cytochrome P450 is outiined by the following paradigm: (1) substrate
binding; (2) reduction of femc heme to the ferrous state; (3) binding of molecular oxygen
to give the Fe+*(OJ complex; (4) transfer of a second electron to produce a peroxo-
iron(m) complex; (5) protonation of the complex and cleavage of the 0-0 bond to give
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water and a highly reactive iron-0x0 species; (6) trarisfer of the oxygen atom fkom the
iron-oxo complex to the subsbate; and (7) release ofthe product (figure 2).
Fig. 2. Proposed catalytic cycle of cytochrome P450.
The widely accepted mechanism of cytochrome P45O-catalysed hydroxylation of
aikane hydrocarbons entails the transfér of a hydrogen atom fiom the substrate to the
0x0-iron complex to produce a meta1 bound hydroxyl radical and an akyl radical (Ortiz
de Monteilano, 1995). Subsequent transfer of the metal bound hydroxyl radical to the
alhl radical intermediate produces the hydroxylated product. An dternative mechanism,
proposed by Newcomb et al., suggests that the hydroxylation reaction must be a
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concerted step and that the radical species generated is a transition state, as opposed to
an intermediate (1995). Further investigation is required to determine wnclusiveIy the
detaüs of this complicateâ mechanism.
The hydroxylation of an aromatic substrate by P450 commonly pro& through
a mechanism distinct fiom that of alkane hydroxylation. P450-catalysed insertion of a
hydroxyl group into an aromatic ring entails epoxidation, foiiowed by openhg of the
epoxide ~ g , and migration of the hydride to the adjacent carbo'n (the 'WEI-shifY'; see
1.1 1). Subsequent tautomerisation of the ketone produces the hydroxylated product (see
figure 6). Formation of the epoxide can occur via concertai formation of the two
carbon-oxygen bonds of the epoxide or addition of the oxygen to one carbon of the x
w
Figure 3. Possible mechanism for hydroxylation of aromatic rings with electron donating substituents, without formation of an epoxide.
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bond, foilowed by ring closure of the epoxide (Ortiz de Montellano, 1995).
Alternatively, aromatic substrates with strong electrondonating groups may circumvent
the formation of the epoxide through the formation ofa single carbon-
oxygen bond, foiiowed by electron transfér fiom the substrate to the P450 heme (figure
3; losephy, 1996). The ewidence for the proposed mechanisms of hydroxylation of
aromatic TC bonds is ambiguous, and fbrther studies are rquired to establish W y the
pathway(s) of polycyclic aromatic hydrocarbon oxidation.
1.4. Cytochrome P4501Al
Cytochrome P450 1Al (P4SO 1Al) is an extrahepatic enyme which has
generated considerable interest because of its potential role in smoking-induced lung
cancer. P4501Al is strongly induced by cigarette smoke in Iung, placenta, and
lymphocytes (Fujino et ai., 1982). The constitutive level of P4SO 1Al expression in
lymphocytes has been associated with Iung cancer risk in smokers (Kouri et ai., 1982).
Cytochrome P450 IAl metaboiizes many potentidy carcinogenic substrates,
including benzo[a]pyrene and other PAHs. Benzo[a]pyrene is metaùolized by P450 1Al
to numerous hydroxy, dihydrodiol, and quinone products (Hail et al., 1989). in addition
to polycyclic aromatic hydrocarbons, P450 1Al has been shown to activate a variety of
aromatic amines, includig MeIQ Trp-P- 1, and PhIP (Shimada et al., 1996). Other
procarcinogens, such as aflatoxin B, and Znitropyrene, may also be activated by P450
1 Al (Shirnada et ai., 1996).
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P4501Al is a polymorphic gene in humans. A Japanese study has show a
correlation between a restriction hgment Iength polymorphism (RFLP) in human P450
1Al and incidence of lung cancer in Asian populations (Kawajiri et al., 1990). Digestion
of lymphocyte DNA with MspI reveals a clear DNA polymorphism. Genetic fiequencies
of the predominant homozygous (0.49), heterozygous (0.40), and rare homozygous
alleles (0.1 1) were determinecl in a hedthy population. The fiequency of the rare dele
was 3-fold higher in lung cancer patients. In a subsequent study, Hayashi et al.
demonstrated a genetic linkage between the MqI polymorphism and an amino acid
replacement (Ile-Val) in the heme-bindiig region of human P45O 1Al (1991). In a study
of foundry workers who were exposed to PAHs, individuais with both the rare
homozygous MqI aiiele and the isoleucineto-valine amino acid replacement were shown
to have an increased level of leukocyte DNA adducts (Hernminki et al., 1997). The
increased fiequency of the M.1 polymorphism in lung cancer patients, and the
association of the polymorphism with an increased level of DNA adducts, suggest that
P450 1Al genotypes may contribute to lung cancer risk.
1 S. Bacterial expression of P4SOs
Heterologous expression of cytochrome P450s in bacterial systems has been the
focus of extensive research. interest in recombinant expression of cytochrome P450s
stems fiom the potential of such systems to provide substantial quantities of catalytically-
active protein. Such systems are particularly usefiil in the study of those proteins, which
cannot be easily puritied, or obtained in sdcient quantities, fiom natural sources.
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M c a t i o n of mammalian P45Os h m natural sources is confounded by contamination
with closely related P450 isoforms. Studies of human P450s are fiirther limiteci by lack
of avaiiability of human tissue samples. Bacterial expression systems circumvent these
probIems and provide additional advantages, including ease of manipulation and wst-
effectiveness.
Initial attempts to express tùli-length, unaltered, mammalian cytochrome P450s,
using standard vectors, were wisuccessfiil (Barnes et al., 1991). Several modifications of
the P450 cDNA sequence were required to achieve high-level protein expression.
Various modifications of the nucIeotide sequence enwdiig the N-terminal region of the
polypeptide enhanceci the expression of mammalian P450s in E. coli. These
modifications included truncation of the hydrophobic segment (Larson et al., 1991; Li et
al., 1991); replacement of the second codon with GCT, which occurs fiequently in this
position in E. coli open read'ig fiames; introduction of silent mutations which increase
the IeveIs of A and T in mRNA transcripts, to resemble more closely the composition of
this region in E. coli mRNAs; and introduction of silent mutations which minimize
mRNA-secondary structure formation (Barnes et al., 1991). The N-terminal region was
targeted for alterations because it fùnctions as a signal-anchor sequence, directing
insertion of the protein into the endoplasmic reticulum and inner mitochondrial
membrane, as opposed to playing a direct role in catalysis (Wachenfeldt and Johnson,
i 995).
Barnes et ai. were successfùl in expressing bovine 17a-hydroxylase (P450 17A)
in E. coli, with the N-temiinal modifications describeci above (1991). Rabbit P450 2E1
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Table 1
II - -
4A4 17 A terminus 1 Nishimoto et d, 1993 11
Il 4A1 1 tnincation A-1 Aia. replacement of the m d codon with GCT. min-fol4 modi6cation of bases in the 5' temiinus to
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Rat
2B 1
7
ceduce & formation ofsecondeiy sauchire in the &A; 1 7 ~ terminus, replacement of the N-temiinus with that used for reçombinant bovine P450 17A ; 2C primer, inaoduction of a consenstis sequence for P450 2C directly following ik d e d P450 17A t&al sequence; tnincation, ternoval of a portion of the N- terminus; AT, optimized the AT content of the 5' terminal codons.
- -
Ala
tmncation
John et al., 1994
Li and Chiang, 199 1
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(Larson et al., 1991) and rat 7a-hydroxylase (P450 7; Li and Chian& 1991) were
expressed in E. coli with a truncated N-terminal sequence. Subsequently, many other
mammalian P450s have been expressed in E coli using the previously describeci
approaches (see Table 1; Guengerich et al., 1996).
Recombinant, N-terminal-modied P450s are predominantly located in the
bacterial inner membrane (Pemecky et ai., 1995; Guengerich et al., 1996). This result
was unexpected, since the hydmphobic N-terminal region fùnctions as a membrane
anchor. Pemecky et al. (1995) found that the N-terminal region of modiied P450 2E1
(A 3-29) had a large number of uncharged, hydrophobic amino acids, which could
facilitate the translocation of proteins into the inner membrane. Replacement of the first
17 N-terminal amino acids of P4SO 2E1 (63-29) with the 6rst 19 of the soluble bacteriai
P45OBM (BM-3:2El) resulted in 800A of the expressed protein being locaiized in the
cytosol. These results suggest that the N-terminal region of modiied P450 2E 1 is
primarily responsible for the membrane insertion of the protein. (However, the existence
of the 20% of BM-32E1 which remains membrane bound indicates that peptides other
than those in the N-terminal region are also involved in membrane localization of P45O
2E 1 .)
The N-terminal region appears to be of greater importance to membrane bindiig
in P450 2B4 than in P450 2E1. The N-teminai modied P450 2B4 (A2-29) is
predominantly cytosolic, unlike the &Il-length protein, which is membrane-bound.
Substitution of the P450,,, N-termina1 sequence in P450 284 (A2-29; BM-3 :2B4)
resulted in only a modest decrease in the proportion of the protein which was membrane-
-
bound. These results further suggest that the N-terminal region is primarily responsible
for insertion of recombinant P450s into the membrane-
The activities of purüïed, reconstituted recombinant mammalian P450s, relative to
their native forms, Vary with the isoform. Purifieci recombinant human P450 2E1 activity
is comparable to that of P45O 2E1 purified fiom human tissues, with turnover numbers
for chlorzoxazone 6-hydroxylation of 5.6 min" and 5.5 min", respectively (Giiiam et aL,
1994; Peter et al., 1990). In contrast, the N-demethylase activity of purifieci recombinant
rabbit P450 2B4 with benzphetamine was found to be ody 41 % that of purified rabbit
P450 2B4 (Pernecky et al., 1995). The reported advities of purified recombinant
human P450 3A4 and 3AS are also substantidiy lower than of the analogous native
proteins (Gillam et ai., 1995; Wrighton et al., 1990). It has been proposed that the N-
terminal modifications may intiuence activity through interaction with NADPH-
cytochrome P450 reductase (Pernecky et al., 1995). However, preIiminary expetiments
in this area have been inconclusive. Diftérences in activities of native and recombinant
P450s rnay also be attributable to the diiculties in optimiPng the reconstitution
conditions for the purified proteins (ûuengerich et al., 1996).
Typically, the activities of recombinant human P450s in intact ceUs or isolated
bacterial membranes have been signiscantly lower than those obsewed in reconstituted
systems (Guengetich et al., 1996). Human P450 1Al or 1A2 activity in isolated
membranes was less than 10% that of the correspondhg purified recombinant enzyme
(Guo et al., 1994; Sandhu et al., 1994). Similady, purified reconstituted P450 2E1
activity was five times greater than that observed in intact membranes (Gillam et al.,
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1994). The diminished P450 activity in membrane preparations may be due to
inaccessibiiity of the NADPH-P450 reductase (see 1.6. and 1.7.; Guengerich et al., 1996)
or the presence of an inhibitor in the membrane preparation (Sandhu et al., 1994). The
level of P45O catalytic activity in intact cells is reIevant to the present investigation
because we are developing a system which relies on activity of the recombinant P45Os in
whole bacterial celis.
Blake et al. (1996) reported very high levels of human P450 3A4 activity, in both
membranes and intact E. coli ceus, when human NADPH-cytochrome P450 reductase
was coexpressed with the P450. intact ceils and isolated membranes cataiysed
testosterone 6 9 -hydroxylation with turnover rates of 17.3 min'' and 25.5 min'',
respectively. These rates exceed the turnover rate of 6 min-' reported for P450 3A4
activity in human Liver microsomes (Yarnazaki et QI., 1996). Hydroxylase activity was
not detectabte in cells or membranes which lacked either P450 3A4 or P450-NADPH
reductase (Blake et al., 1996). These results highlight the ftnctionai importance of
ensuring facile electron transport to the P450 protein.
1.6. Electron transport to cytochrome P450: NADPK cytochrome P450 reductase
Cytochrome P450-catalysed oxidations require electron transfer tiom reduced
pyridme nucleotides to the P450. Electron transfer to microsomal cytochrome P450s
fiom NADPH is mediated by NADPH-cytochrome P450 reductase (P450 reductase).
The reductase is anchored to the endoplasmic reticulum by a single transmembrane helut,
with the bulk of the protein exposed to the cytosol (Black and Coon, 1982). P450
-
reductase is widely distributed in rnammalian species and tissues in association with
cytochrome P450 (Wachenfeldt and Johnson, 1995). UnWre cytochrome P450, only one
form of cytochrome P450 reductase has been identifiai (Wachenfèldt and Johnson,
1995).
Purifieci P450 reductase contains equimolar amounts of the fiavins FMN and FAD
(Dignam and StrobeL 1977), which mediate electron transfier from the two-electron
donor, NADPH, to the singie-electron acceptor, cytochrome P450. Studies of an FMN-
depleted reductase, which was able to oxidize NADPH, but unable to reduce cytochrome
P450, suggested that electrons flow fiom NADPH to reductase-bound FAD (Vennilion
and Coon, 1978). Furthemore, the FMN-depleted reductase was unable to produce an
air-stable semiquinone, as observeci in the hoIoenzyme (Strobel et aï-, 1995). The
formation of the semiquinone is required for direct electron transfer to the cytochrome
P450 heme iron. Thuq the hypothesired pathway for the flow of electrons is:
NADPH ---D FAD - FMN - acceptor (Vermilion and Coon, 1978). Subsequent "P-NMR shidies demonstrated that the air-
stable semiquinone detected in the reductase is forrned exclusively on FMN
(Narayanasami et ai., 1992). Kunban and Strobel(1986) produced an FADaepleted
reductase which could not accept electrons fiom NADPH, fiirther c o n f i d g the
pathway of electron tlow in NADPH-cytochrome P450 reductase proposed by Verrnilion
and Coon,
Bacterial and rnitochondrial systems rely on a flavoproteinhron-sulfur protein
system to transfer electrons from reduced pyridine nucleotides to cytochrome P450
16
-
(Josephy, 1997). Mitochondrial cytochrome P450 enzymes are supported by
adrenodoxin reductase, an FAD-contauiùig flavoprotein, and adrenodoxin, an iron-
sulphur protein (Lambeth et al,, 1982). The anaIogous e1ectron transfer system for the
soluble bacterial cytochrome P450, consists of putidaredoxin reductase and
putidaredoxin (Lambeth et al., 1982).
Soluble flavoproteins have been shown to support electron transport to
recombinant eukaryotic cytochrome P450s, expresseci in bacterial strains which do not
contain endogenous P450s (Jenkins and Watennan, 1994; Yamazaki et al., 1995). The
flavoproteins were first identified after recombinant cytochrome P450 17A hydroxylase
activity was observed in E . d i , in the absence of an exogenous source of NADPH-
cytochrome P450 reductase (Barnes et al., 1991). Flavodoxin and NADPH-flavodoxh
reductase were identified as the E, d i proteins supporting the activity of recombinant
bovine P440 17A hydroxylase (Jenkins and Waterman, 1994). E. coli flavodoxin binds
P450 17A diectiy, with an apparent K, of -0.2 W. Furthemore, amino acid sequence
aiignment revealed signiticant sequence similady between the E. coli flavoproteins and
homologous domains in rat microsoma1 P450 reductase. These results suggested that the
flavoproteins were responsible for the cytochrome P450 activities, in reconstituted,
recombinant E. coli systems However, puritied flavodoxin and NADPH-Eavodoxin
reductase were show to be ten-foId less efficient than purified, recombinant rat P450
reductase in supporting P450 17A hydroxylase activity (Jenkins and Waterman, 1994).
-
The si@cantly lower enzyme activity of P45O 17A hydroxylase when supported by
endogenous bacteid flavoenzymes suggests that the in vivo catalytic activity of
recombinant P4SOs wuld be improved ifP450 reductase were also present in the
bacterial cell.
1 -7. Recombinant cytochrome P450 reductase
Coexpression of recombinant NADPH-cytochrome P450 reductase and
cytochrome P450 is a significant step towards the development of systems that can
perform cytochrome P450-dependent oxidations in Live bacteria. Several approaches are
possible, including: (i) coexpression of NADPH-P450 reductase on separate plasmids;
(Ü) expression of cytochrorne P450 and NADPH-P45O reductase fiom a single plasmid,
in a poly-cistronic manner; and (üi) generation of a cytochrome P450:NADPR-P450
fiision protein (Guengench et al., 1996). The &st approach is undesirable, due to the
fiequent instability of systems containing more than one plasmid. However, the two
latter possibilities, which entail expression of both proteins f?om a singfe plasmid, are the
focus of current research in bacterial P450 expression systems (Guengench ef al., 1996).
A single polypeptide which is fùnctionaiiy anaiogous to the microsornai
monooxygenase systern has been identified in Bacillus megatetium (Nahri and Fulco,
1986). Cytochrome P450 102 is a catalytically seif-sufficient protein containing heme,
FAD, and FMN, in a 1: 1: 1 ratio. Upon trypsinolysis, the protein is cleaved into two
domains: a substrate-binding hemoprotein, and a flavoprotein domain capable of
NADPH-dependent cytochrome c reduction. Similar artifid fiision proteins have been
-
deveIoped for recombinant P45O expression systems in yeast (Murakami el al., 1987)
and E. coli (Guengerich et al., 1996). Reductase ftsion proteins of several isoforms of
human P450 have been constmcted, incluâiig P450 1Al (Chun et al., 1996), P450 3A4
(Shet et al., 1993), P450 1A2, and P450 3AS (Chun and Guengerich, unpublished
results).
As mentioned above, a human cytochrome P45O 1A1 :NADPH-cytochrome P450
reductase (P4501Al- reductase) fùsion protein has been constructed and expressed in E.
coli (Chun et ai., 1996). The compIete cDNA sequence coding for human cytochrome
P450 1Al was firsed to the sequence codig for the soluble portion of rat NADPH-
cytochrome P450 reductase, and sub-cloned into the expression vector, pCW. The
plasmid was transformed into E. coli strain DMa, and expression of the fùsion protein
was optunized through isopropyl-P-D-thiogalactoside (IPTG) induction, and incubation
with 6 -aminolevulinic acid (&%LA), a heme precursor. The human P450 1 Al -reductase
was isolateci fiom bacterial membranes and characterized. The molecular weight of the
fiision protein was 129 Ha, as expected. The NADPH-cytochrome c reductase activity
(see 1.2.3 .) of the purified fùsion protein (20 pmoVmidmg protein) was only about one-
half of the expected activity of puriiied, endogenous rat NADPH-P450 reductase
(Yasukochi and Masters, 1976). This may be due to a tighter interaction between the
reductase and the P450, which does not favour electron transfer to exogenous elemon
acceptors such as cytochrome c. Altematively, the reductase moiety could be inherently
less stable than the fk reductase. The V,, and K, values for cytochrome P450-
dependent 7-ethoxyresorufin O-deet hylation and B[a]P 3-hydroxylation were 0.65 and
-
1.04 nmol product/min~nmol enzyme and 0.34 and 19 p ~ , respectively. These rates are
comparable to those achieved in previous studies of purifieci recombinant human P450
1Al activity in reconstituted systems, with an exogenous source ofNADPH-cytochrome
P450 reductase (Guo et al., 1994). However, the rates of catalytic activity achieved with
either the recombinant human cytochrome P450 1Al with an exogenous source of
reductase, or the P4501Al:rat reductase fbsion protein are considerably lower than those
reported for purifieci rat liver cytochrome P450 IAl (e.g B[a]P 3-hydroxylation rates of
10-25 mol product/rnin~nmol enzyme; Chun et d., 1996 ).
An alternative approach to the construction of ftsion protehs, for the production
of catalytidy active, recombinant cytochme P450 systems in bacteria, is the
developrnent of polycistronic expression vectors. Polycistronic gene organization was
employed to develop a prokaqotic systern which expressed various eukaryotic proteins,
includig cyclophiün, FKSO6-bindig protein, and a domain ofan MHC class La
molecule at detectable levels (Ito and Kurosawa, 1992). The expression of the various
eukaryotic proteins was achieved through the construction of a vector that placed the
eukaryotic genes downstream fiom a weli- expressed gene and incorporateci a ribosome-
bindig site (RBS; see figure 4; Ito and Kurosawa, 1992).
- - -- - --
Figure 4. Schematic representation of a polycistronic gene constnict. GST; glutathione tramferase. GST, a well-expressed gene, is placed upstream fiom the foreign gene.
-
Polycistronic gene organization has been usefiil in the construction ofvectors
which coexpress human cytochrome P450s and P450-reductase in E. coli (Dong and
Porter, 1996; Blake et al., 1996). An expression vector bearing the cDNAs for human
cytochrome P450 2E1 and rat NADPH-P450 reductase, arranged in tandem, was
constructed and transfonned into E. coli (Dong and Porter, 1996). Immunoblot analysis
confirmed the production of two distinct polypeptides, with the expected molecular
weights. The reported rates of aniline andpnitrophenol hydroxylation, by solubiiized
membranes fiom the transformed E. coli cells, were 1.8 and 2.0 nmol/min~nmol P450,
respectively. However, P450 activity was not demonstrated in intact E. coli.
As mentioned above, a polycistronic-cytochrome P450 monooxygenase system
with very high levels of catalytic activity in E. coli has been reported by Blake et ai.
(1 996). A vector bearing the cDNA for human cytochrome P450 3A4 and human
cytochrome P450 reductase, organized in tandem, was constructed. The bacterial PelB
signal sequence was translationally fised to the P450 reductase. The PelB leader
sequence was isolated from the phytopathogenic enterobacterium Envinia chrysanthemi,
and diicts the transport of the reductase into the inner bacterial membrane (Anderson et
al., 1994). The cytochrome P450 3A4 yield obtained was relatively high: 200 nmoM
culture vs 0.8 nrnoVl culture in the P450 2E1 polycistronic construct (Dong and Porter,
1996). Rates of 6 -hydroxylation of testosterone and oxidation of nifedipine, by whole
cells, were 15.2 and 17.3 nrnol,min/nmol P450, respectively. These turnover rates are
higher than those previously reported for both reconstituted recombinant cytochrome
P450 3A4 membranes (Yamazaki et al., 1995) and human liver microsomes (Yamazaki et
-
al,, 1996). The levels of cataiytic activity reported by Blake et aL (1996) far swpass the
activities reported in previous studies of recombinant cytochrome P450-bacterial
expression systems. This success may be due to one or more of the foiiowing: (i)
increased human cytochrome P450 activity when coupled to human P450 reductase, as
opposed to rat reductase; (ii) incorporation of the bacterial PelB signal sequence into the
recombinant reductase protein; and (iii more sensitive methods for detection of oxidized
products.
1.8. Expression of recombinant human enzymes in SdmonelIa typhimurium
Systems expressing recombinant -es in S. typhmurium have proven usefiil
in the development of improved bacterial mutagenicity assays (see 1.13). A system was
developed in S. &phimurium to investigate the mutagenic activation of aromatic amines
by human enzymes (Grant et al., 1992). Subsequently, .unes tester strains which
activate aromatic amines in the absence of an exogenous source of bioactivation enzymes
were developed (Josephy et al, 1995; Josephy el ai., 1997). In these studies, plasrnid
vectors developed for E. coli systems were used for expression in S. typhimurium.
Activation of aromatic amines requires both N-hydroxylation and N-acetylation,
catalyzed by P450 1A2 and NAT, respectively :
-
Ames tester strains bearing plasmids coding for either human NAT 1 or NAT 2, yielded
fûnctional enzymes which supported mutagenic activation of aromatic amines in
mutagenicity assays (Grant et al., 1992). An exogenous source of PGO 1A2
(mammalian liver homogenate or S9) was provided to catalyze the initiai hydroxylation
of aromatic amines to N-hydroxyarylamines. Subquent N-aceîylation, catalyzed by
NAT, gives rise to N-acetoxy esters. Spontaneous heterolysis of the highly reactive N-
acetoxy esters produces the ultimate DNA reactive species, presumably a nitrenium ion
(RNW). Both strains expressing recombinant human NAT activated aromatic amines to
mutagenic species in Ames assays. The NAT-overexpressing strains demonstrated
greatly enhanceci sensitivity to arylamhes, compared to the parental strain which was
NAT. These strains provide tools for studying the role of human NAT enzymes in the
metaboiic activation of aromatic amines to genotoxic products.
The NAT over-expressing Ames tester strains were further developed by Josephy
et al. (1995)- The strains were transformed with a plasmid encoding human P450 lA.2
@J450 1A2) and were show to be sensitive to aromatic amine mutagenicity in the
absence of an exogenous source of activating enzymes. in modiied Ames tests,
mutagenicity of 2-aminofluorene was p a t e r in DJ450 1 A.2, in the absence of S9, than in
either of the standard tester strains with S9 activation. The expressed P450 protein
demonstrated the spectroscopie, immunological, electrophoretic, and catalytic properties
of the human protein. However, the level of expression of the recombinant P450 1A2 in
S. typhimurium was only one-quarter of that observeci in E. coli. The activity of
proteases in Ames tester strains may account for the decreased level of expression
-
obsewed in S. fjphimurium (Guengerich et al., 1996), although many other factors may
be contributhg to this ciifference.
There are severai advantages to the engineered expression of bioactivation
enzymes in Ames tester strains: (i) reduction in the requirement for mammalian tissue
preparations; (i) generation of reactive metabolites inside the bacterial ce& as opposed
to their production outside the ceU; Ci) the ability to study enzymes f?om specific tissues
or species, in the absence of competing isoforms; and (iv) the ability to study chimeric or
mutant enzymes (Josephy et al., 1995). The goal of the current research project is to
engineer the expression of human P4SO 1Al in an Ames tester background and to detect
activation of P4SO 1Al-dependent mutagens in the absence of an exogenous source of
bioactivation enzymes.
1.9. P450 expression in Saccharomyces cerevisiae
Expression of recombinant human P450s has been studied extensively in
eukaryotic Saçcharomyces cerevisiae. Yeast and bacterial systems are similar in that they
both offer simple, cost-effective production and purification of large quantities of
prote&. An additional advantage of the yeast system is the presence of endoplasmic
reticulum and mitochondria. However, expression levels in recombinant yeast celIs Vary
considerably (Bellamine et al., 1994) and the presence of endogenous P450s can
complicate the development of heterologous expression systems.
Human P450 1Al has been successfiiIIy expressed in S. cerevisiae, with P450
1Al-speci6c activity detected in isolated microsomal fiactions (Eugster et al., 1990).
-
The observed P450 1Al activity was supported by a heterologous electron donor,
presumably yeast oxidoreductase. In a subsequent study, human P450 1Al activity in S.
cerevisiae was increased sixteen-fold when the P450 1Al was coexpressed with human
NADPH-P450 reductase (Eugster et al., 1992). EROD activity (see 1.2.2.) in whole
ceils expressing the reductase and P450 1Al was 45.5 pmoI productlOD, units/min. A
concomitant decrease in the amount of immunoreactive P450 1 Al was observed in the
coexpression system, suggesting that the increased activity was a result of more efficient
coupling between the human oxidoreductase and the human P450 1Al.
in further studies, which paraliel approaches to bacterial P450 expression
systems, human P450 1 Al-human NADPH-P450 reductase fbsion proteins have been
constructed and expressed in S. cerevisiae (Wittekindt et al., 1995). The cDNA
encoding the entire human P450 reductase sequence was fiised to the coding region of
human P450 1Al and expressed under the control of the constitutive S. cerevisiae
GAPDU promoter. In a second constmct, the amino tenninus of the reductase was
replaced by the membrane-anchor domain of yeast HMG-Cok CO-difference
spectroscopy indicated that the holoenzyme content of microsumes containing the fusion
proteins was approximately 0.1 nrnoVmg, which is comparable to bvels observed in a
strain containing only P450 1Al. Western blot analysis of whole ceUs indicated the
presence of intact fûsion constmcts. However, immunoblots of microsomes revealed
degradation of the fiision proteins. Whole ceU EROD activities ofthe fiil-length
reductase fusion and the amino terminal-m&ed reductase fiision proteins were found
to be 15% and 8%, respectively, of those obsewed in ceils expressing only P450 lA1.
-
The turnover number for EROD activity in microsrimes isolated fiom oxidoreductase-
deficient yeast ceils expressing the hi-length reductase fusion protein was 0.3 pmoles
product/pmoIe enzymdmin. These results suggest that, while the level of fùsion protein
production in this system may be adequate, the stabïity of the protein and the abüity of
the fùsed enzymes to metaboüze their substrates are limiteci.
In addition to heterologous expression of proteins in yeast, there has been some
interest in the development of mutation assay systems in S. cerevisiae. Forward
mutation to qcloheximide resistance, L-canavanine resistance, and DL-a-arninoadipic
acid resistance in a wild-type S. cerevisiae strain was used to test for sensitivity to
polycyclic and heterocyclic compounds (Mitchel and Gilbert, 1991). While mutations
were detected, the doses required were up to 100-fold higher than those detected by the
Ames assay (see 1.13). Furthemore, ce11 counting was impeded by heavy background
growth and ceU clumping. Mutagenicity assays have yet to be developed in S. cerevisiae
which are comparable to the Ames test in sensitivity and simplicity.
1.10. Polycyclic aromatic hydrocarbons
PoIycyclic aromatic hydrocarbons (PAH) have long been impiicated in certain
types of cancer. Over 200 years ago, it was observeci that chimney sweeps had a high
incidence of scrotai cancer, a disease which is very rare among the general population- in
the early 1900q it was demonstrated experimentally that cancer could be induced in
rodents which were exposed to coai tar. Further studies focused on identifying one or
more individual chernicals that were the cancer- causing agents in coal-tar. in 1933,
-
more than 150 years after the first description of an occupational cancer, a polycyclic
aromatic hydrocatbon (PAH), benzo[a]pyrene @[aJP), was identified as an active CO ai-
tar carchogen.
The simplest PAHs are naphthalene and anthracene, which are composed of two
or three six-membered rings, respectively (Figure 5). More complex PAHs include
pyrene (four fiised rings) and various substituted pyrenes and anthracenes. Uniike
benzene, the carbon-carbon bond lengths in anthracene and other PAHs are not
equivalent. This can be accounted for by considering the resonance structures for a given
PAH.
naphthalene anthracene pyrene benzo[a]pyrene
Fig. 5. Stmctures of various polycyclic aromatic hydrocarbons.
Analysis of bond length data reveals the type of carbon-carbon bond, at a given
position, which contributes most prominently to a resonance hybrid. For.example, the
length of the 1,2 carbon bond of naphthalene is 1.36q cornpared with 1 . M in benzene.
Since two of the three resonance structures of naphthaiene shows an olefinic bond in this
position, it is shorter than a typical aromatic carbon-carbon bond. The 1,2; 43; 7,s;
9,10; and 11,12 bonds of B[a]P are activated relative to benzene, and contribute to the
biological reactivity of P AHs.
-
1.1 1. PolycycIic aromatic hydrocarbon metabohm
Metabolic activation of benzo[a]pyrene proceeds through an arene oxide
intermediate. The first evidence which suggested that an arene oxide intermediate is
fonned during enzymatic hydroxylations was reported in 1967 (Guroff et al.). They
unexpectedly observed the migration, to an adjacent position on an aromatic ring, of the
group displaced by hydroxide during the P450-catalyzed hydroxylation of acetadide
(Figure 6). The migration of the hydrogen atom at the position of hydroxylation was
coined the 'WEI-sW because it was discovered in the laboratories of the National
Institute of Health, Bethesda, MD. This observation indicated that an arene oxide
intermediate may be formed, since an electron-deficient carbon can aise from the
opening of an epoxide. Subsequent migration of an adjacent group, to the nascent
carbenium ion results in the observed shift. Further studies, which trapped radiolabeiied
naphthdene 1,Zoxide as a metabolite of naphthalene, provided direct evidence that
epoxide intermediatas are formed during the metabolism of aromatic hydrocarbons
(Jerina and Daly, 1974).
-
NHA= NHAc I
NHAc I
NHAc
I
migration of hydrogen with its clcctroa pair
Fig. 6. Mechanisrn for the hydroqdation of acetaniiide, illustrating the NIH shift .
Epoxides are highly reactive, due to their strahed three-membered ring structure.
The major reaction pathways of arene oxides include spontaneous isomerization to
phenols and quinones, hydrolysis (catalyzed by epoxide hydrolase), and conversion to
glutathione conjugates (cataiyzed by cytosolic glutathione S-transferase). Depending
upon which ring position is initially oxidized, and which metaboliking enzymes are
present, one or more of these pathways may occur.
Isomenzation to phenols and quinones is a major pathway for transformation of
arene oxides produced during primary metabolism of arene oxides. B[a]P 2,3-, 4,s-, 7,8-
-
and 9,IO-oxides can potentialiy isomerize to their respective phenols. 3-Hydroxy-B[a]P
and 9-hydroxy-B[a]P were reported as two major B[a]P metaboiites forrned in the
presence of rat iiver microsornes (Waterfd and Sims, 1972). 1- and 7- hydroxy-B[aJP
have also been identifid as B[a]P metaboiites (Thakker et al,, 1985). The formation of
phenols fiom epoxides is energetically favourable, since it restores aromaticity to the
molecule. This is confinned by the fact that isomerization proceeds in the absence of
fiinctional enzymes (Waterfdi and Sims, 1972 ). Phenols may be fùrther oxidized to
quinones by rat iiver microsornai enzymes. Indeed, 1,6-, 3,6-, and 6,12-B[a]P-
quinones have been identified as products of BP metaboiism (Thakker et al., 1985).
Metaboiism of arene oxides to dihydrodiols is a crucial step in the bioactivation of
PAHs. In the presence of rat liver homogenates, 7,8-dihydro-7,8-diiydroxy B[a]P (BP
7,8-diol) and 9,lO-dihydro-9,lO-diiydroxy B[a]P are formed (Waterfd and Sims, 1972).
The hydrolysis of arene oxides to diiydrodiols is cataiyzed by microsomal epoxide
hydrolase (Josephy, 1997). The 7,8-, and 9, 10-diiydrodiol products are not directly
reactive. However, these species possess an olennic bond in the terminal ring, which can
be fiirther metabolized to the direct-acting dihydrodiol-epoxide (Figure 7).
-
Figure 7. Activation of benzo[a]pyrene.
In 1973, Sirns et ai. identified 7,8-dihydro-7,&diydroxybenzo[a] pyrene 9,lO-
oxide (BP 7,8-diol-9, IO-epoxide) as the major B[a]P metabolite which reacts directly
with DNA Their experiments compared DNA hydrolysates from hamster embryo cells
that had been incubated with [%+BP 7,8-diol-9,lO-epoxide and with [''Cl-
benzo[a]pyrene. The adducts formed from cellular metabolism of B[a]P were show to
be identical to the BP 7,8-diol-9,lO-epoxide-derived adducts. The high chernicd and
-
biologid reactivity of this molecule is attriiuted to the presence of an qoxide group on
the saturated angular benzo ring in the bay region of the molecule ( M e r et al., 1985).
Bay region epoxides are very reactive molecules which are not scavenged effectively by
detoxication enzymes. Many studies have indicated that these molecules are very poor
substrates for epoxide hydrolase (Thakker et d., 1985 and refk therein), The generation
of reactive electrophilic molecules, in the absence of effective biologid scavengers,
poses a threat to the integrity of susceptible nucleophiies, such as DNA
1.12. MatoxinB,
Matoxin B, is a fiingal toxin produced by certain strains of Asper.lIz1sf7ms
and related molds. Aspergiilusflavus is a ubiquitous mold which grows on a variety of
agricultural products, when adequate moisture and heat are avaiiable. Under such
conditions, foodstuffs can become contaminated with toxins and pose a serious health
risk to both animals and humans. Matoxin B, is acutely hepatotoxic and carcinogenic.
In South Asian and Atncan countries, where food supplies have been heavily
contaminated with afiatoxin B,, Liver cancer is one of the most fiequently observed
malignancies (Hayes and Campbell, 1986). Epidemiological studies have revealed a
significant increase in respiratory cancers in workers who are occupationaiiy exposed to
dusts fiom afiatoxin B,-contaminateci products (Hayes et al., 1984). Further evidence
has suggested that lung tissue, in addition to liver, is also exposed to aflatoxin B, as a
result of ingestion (Harrison and Garner, 1991).
-
Afiatoxin B, is metaûolicaiiy activated to afiatoxin B,-8,9-epoxide, the ultimate
DNA binding species (Miller, 1978). Two stereoisomers of the epoxide, an e& and
exo-epoxide, have been identifieci as products of the microsomal bioactivation of
aflatoxin B, (see figure 8; Raney et ai., 1992a). The ero-epoxide was shown to be 500-
fold more mutagenic than the enrio-isomer, in S. tvphimurium reversion assays (Iyer et
al., 1994). Mechanistic studies revealed that, upon intercalation of the epoxides into
DNA, only the exo-epoxide is correctiy positioned for a backside S,2 attack on the N'
position of guanine (Iyer et al., 1994). W e both isomers are formai upon
bioactivation of datoxin B,, the emepoxide is the principal DNA-binding metabolite.
I
Fig. 8. Activation of aflatoxin B,.
Aflatoxin B, is a substrate for several of the cytochrome P450 isozymes. P450
1Al and P450 LA2 were s h o w to activate aflatoxin BI, in S. ~rphimzirium mutagenicity
-
assays (Shimada et al., 1996). In a r e ~ o ~ t u t e d system, E. c d membranes fiom a main
expressing human P450 3A4 preferentially activated aflatoxin B, to the em-epoxide, with
no endo-epoxide detected by HPLC analysis (Ueng et al,, 1995). P450 3A4 is the
predominant P450 isoform identified in human iiver tissues, and is primarily responsibb
for afiatoxin B, metabolism in human livw microsornes (Guengerich, 1995). In rabbit
lung tissue, P450 2B4 and P450 4 8 1 are the principle isoforms wfiich catalyze aflatoxin
B1 bioactivation (Massey, 1996). These isofonns are preferentidy expresseci, and have
higher catalytic activity than other P450s in rabbit lung @aniels et ai., 1990).
Ailatoxin B, metabolism is complex: the endo- and exo-epoxides comprise only a
smaü fiaction of the total afiatoxin B, metabolites. Hydroxyiation and O-dealkylation,
catdyzed by various P450 isofonns, both occur during the initiai metabolism of afiatoxin
B, (Massey, 1996). The 3a-hydroxylation product, aflatoxin Q,, is the predominant
afiatoxin B, metabolite produced by P450 3A4-cataiyzed metabolism (Ueng et al., 1995).
P450 1A2 preferentiaiiy metabolizes afiatoxh B, to afiatoxin FMl, via hydroxylation in
the 9a position (see Figure 8; Ueng et ai., 1995). Hydroxyiation at position 8 and 0-
dealkylation can also occur, for a total of four initial aflatoxin B, metabolites, in addition
to the two epoxides. Since the non-epoxide metabolites are not DNA-binding species,
hydroxylation and O-dealkylation are considered deactivating pathways.
The primary detoxication pathway for afiatoxin B, is glutathione S-transferase
(GST)-catalyzed conjugation of aflatoxin B,-8,9-epoxide (Massey, 1996). GST
conjugates of both the endo- and exo- epoxide are formed (Raney et al., 1992b). GSTs
are a superfamily of enzymes, which vary considerably in their substrate specificity and
-
distribution. Two classes of GSTs, Alpha and Mu, have demonstrated high activity
toward activated datoxh B, in vitro (Stewart et al., 1996).
1-13. Mutagenicity testing
The Ames test is a mutagenicity assay developed in the laboratory of Dr. Bruce
N. Ames (Ames et al., 1973). Due to its simpIicity and sensitivity, the Ames test is
currently one of the most widely used mutagenicity assays. The Ames test detects
mutagenicity through the reversion of histidiie-auxotrophic mutants to the prototrophic
phenotype. The S. typhi-um tester strauis developed by Ames and coiieagues each
bear a mutation in one of the genes of the histidine biosynthesis operon. Exposure of
these strains to mutagenic compounds results in reversion of the non-fùnctionai allele to
the wild-type. This phenotypic change is easily detectable by plating on minimal agar
with trace amounts of histidine (to support the first few ceU divisions and allow
expression of the reversion mutation). The number of colonies which revert to the His'
phenotype in the presence of a mutagen is a measure of its potency. As a routine control,
the number of colonies which revert to His' spontaneously is also deterrnined, in each
experiment.
The Ames tester strains bear severai mutations which make them highiy sensitive
to chernical mutagens. Galactose (go0 and deep rough (va) mutations were introduced
into the
S. typhimurium tester strains to disrupt the polysaccharide side-chah of the
iipopolysaccharide (LPS) which coats the bacterial surface (Ames et al., 1973). These
-
mutations increase the cell's permeabity to chemicals. A deletion in the g d operon
eliminates galactose synthesis fiom the ce4 resulîing in the disruption of O-antigen
biosynthesis. The rfa mutation inhibits synthesis of polysaccharides distal to the
ketodeoxyoctanoate core. A deletion through the m B region of the bacteriai
chromosome, which eliminates the DNA nucleotide excision repair system, was also
introduced. In the absence of excision repair, pyrirnidie dimers and other buQ pre-
mutagenic adducts are not repaired by the UvrABC system, resulting in a higher
fiequency of mutagenesis.
Some details of the mutational specificity of a mutagen can be elucidated using
the Ames assay. Two of the Ames tester strains (TA1 537 and TA1 53 8) were derived by
treatment with the fiameshift mutagen ICR364-OH (Ames et al., 1972). As a result,
TA1538 contains a h e s h i f t mutation in the hisD gene, which codes for histidinol
dehydrogenase. In an Ames assay, reversion of this strain to the his' phenotype indicates
that a particular chemical is a fi-ameshift mutagen. Similady, TA1537 is a "fhnesW1
strain with a mutation in the hisC gene, which codes for histidiiol-phosphate
aminotransferase. TA1535 was derived fiom a spontaneous mutant of LT-2, the wild-
type strain. This straùi has a singie nucleotide substitution in the gene coding for ATP
phosphoribosyltransferase. Mutagens give characteristic patterns of responses when
tested in difEerent Ames strains. For example, Znitrosofluorene (2-NF) is mutagenic in
the fiameshifi strains TA1537 and TA1538, but not in TA1535, indicating that 2-NF is a
fiameshift mutagen.
-
Many mutagens are only detected in the Ames assay in the presence of an
exogenous source of bioactivation enzymes, Incubation of the test compound and the
bacteria with hepatic post-mitochondriai supernatant (S9) is a standard method in the
Ames assay. S9 is rich in cytochrome P450s, the most sigdicant enzymes ofxenobiotic
bioactivation. The addition of S9 in the Ames assay approximates the in vivo conditions
under which procarcinogenic compounds are metabolized to DNA-readve species.
1.14. SOS DNA repair
The sensitivity and scope of the Arnes test are signincantiy increased by
the incorporation of pKM10 1, an R-factor derivative carrying the genes for SOS DNA
repair, to the original Ames tester strains (McCann et al., 1975). SOS DNA repair is a
highly mutagenic process which allows DNA replication to proceed past non-instructive
DNA lesions. TAI OO and TA98 are pKMl0l-bearing anaiogs of TA1 535 and TA1 538,
respectively. The pKM10l-bearing strains are more sensitive to a number of mutagens,
including 4-nitroquinoline- 1-oxide (4-NQO), N-methyl-N'-nitro-N-nitrosoguanid'u~e
(MNNG), atlatoxin B,, benzo[a]pyrene, and niridazole. Indeed, severai mutagens
previously undetectable in the Ames test, such as fùryffiramide (AF-2), mitomycin C,
and I-phenyl-I-(3,4-xylyt)-2-propynyl-N-cyclohexy1carbamate, are mutagenic in TAI00
and TA98. Plasmid pKM1Ol enhances mutagenesis, while providing resistance to kiliing
by UV and chemicals. The increase in susceptibility to mutagenesis is observed only in
RecA" and LexA* strains (Waker, 1977).
-
Walker et al. sub-cloned the region of plasmid pKMlOl required for mutation
enhancement. Two plasmid-encoded protebu, MucA and Mu&, are responsible for
pKMl0 1 -mediated mutagenesis (Perry and Walker, 1982). The muc region of pKMlO 1
(about 1.6 kb) codes for the 16 kDa MucA and 45 kDa Mu& proteins. The mucAB
genes are organized in an operon which is regulated by IexR (Eiiedge and Walker, 1983).
Furthemore, the m u M operon is homoIogous to the u d C operon (Perry et al.,
1985), which encodes chrornosomal genes required for mutagenic DNA repais in E. coli
(Shïnagawa et al., 1983) and S. typhimwium (Smith et al., 1990; Thomas et al., 1990).
The E. coli umuDC operon codes for a 16 kDa and a 45 kDa protein, and the m u d B
genes can restore induced mutability to E. coli umuDC mutants (Walker and Dobson,
1979).
Le* RecA, UmuD, and UmuC are essential elements of the SOS regulon,
which facilitates DNA replication past a chemical- or UV-induced lesion (Walker, 1995).
The SOS response is highly mutagenic, since it o£ten results in the insertion of an
incorrect nucleotide opposite a noncoding or miscoding lesion. LexA fiinctions as a
repressor of umuD, umuC (EIledge and Waiker, 1983)- and recA (Walker, 1984). in
response to DNA damage, RecA binds to regions of single-stranded DNA in the presence
of a nucleoside triphosphate to fonn a nucleoprotein filament (Walker, 1995). in this
tripartite cornplex, RecA is activateci, this fonn is referred to as RecA*. LexA molecules
d i s e to RecA*, where they undergo autodigestion, which inactivates LexA as a
repressor (Slilaty et al., 1986). Upon derepression, umuD and umuC are transcribed.
UmuD, which shares significant homology with the carboxy-terminai domain of LexA
-
(Perry et al., 1985), also undergoes RecA*-mediated cleavage to produce an activated
poIypeptide, UmuD' (Woodgate and Sedgewick 1992). UmuD' exists as homodimer
and associates with one molecuie of UmuC to produce an active trimer. The exact
mechanism of translesion synthesis has not been elucidated, although studies have
concluded that DNA polymerase III, UmuD', WmuC, and RecA are required for the
process (Rajagopalan et al., 1992).
S. typhimrrrnmr LT2 and its derivatives carry a 60-MDa phsrnid (the cryptic
plasmid) which codes for d, a m&C-like operon (Nohrni et al., 1 99 1). LT2 and
TA1538 contain both the mu- and d operons. However, S. typhimrium
demonstrates a weak mutagenic response to UV and many chernicals in the absence of
pKMlO 1 (McCann et al., 1975). Nohmi et al. demonstrated that the samRB operon,
when subcIoned ont0 a high-copy number vector, could restore signiricant levels of W
mutability to UmuD and UmuC deficient E: coli strains. Poor expression of the
umuDC, and samAZ3 operons, when they are in the single-copy state, the absence of a
factor which is requued for operon fùnction, a d o r the presence of an inhibitor of the
operon, are fàctors which may contribute to the suppression of the operons in S.
&phimurium (Nohmi et al., 199 1).
In summary, the MucAB proteins significantly enhance the scope and sensitivity
of the Arnes test. Many P4SO-dependent mutagens require the presence of pKMlOl for
theu detection in the Ames test. The present study focuses on the development of Ames
tester strain bacteria which can activate and detect P45O-dependent mutagens in the
absence of exogenous activation enzymes.
-
2. Materials and Methods
2.1 Chemicals
The foUowing chernids were purchased Eiom Sigma Cheinical Co. (St. Louis,
MO): alkaiine phosphatase-conjugated anti-rabbit immunogiobin, datoxin B,, 2-AA, 2-
Al?, benzo[a]pyrene, MNNG, & A L 4 ampicillin, kanamycin, and IFTG. MeIQ and
Trp-P-1 were obtained 6om Chemsyn Science Labotatories (Lenexa, KA). BP 7,s-di01
was supplied by Midwest Research Institute (Kansas City, MO). Lysozyme and
tetracycline were purchased 6om Boehger-Mannheim Ltd. (Laval, Que.). Protein
molecular weight markers and Bsu36I were obtained f3om New England Biolabs Ltd.
(Mississauga, Ontario). Chloramphenicol was purchased fiom Aldrich Chernid Co.
(Miiwaukee, WI). 2,4,S-TMA was supplied by Pfaltz and Bauer Research Chemicals
(Waterbury, CT). An aroclor-induced rat liver S-9 preparation was obtained 6om
Molecular Toxicology, hc. (Annapolis, MD). Rabbit anti-human P450 1Al antibody
was provided by Dr. F. Guengerich. AU other chernicals were reagent grade or better.
2.2 Media
Nutrient broth and agar were prepared using Oxoid Nutrient Broth No. 2 (Oxoid
Canada Ltd., Nepean, Ont.) and supplemented with ampiciUin (100 pg/mL), tetracycline
(8 pg/mL), kanamycin (50 pg/mL), chloramphenicoI(15 pg/mL), IPTG (1.0 mM), or 8-
ALA (0.3 mM), as required. Minimal giucose medium containing Vogel-Borner saIts,
glucose, histidine, and biotin was prepared as described (Maron and Ames, 1983).
40
-
2.3 Bacterial sirains and plasmids
Table 2.
-
2.4. General Procedures
2.4. f . Reisolation and storage of bacterial strains
Bacteriai colonies were isolated by streaking out fieshiy thawed permanent
bacteriai culture ont0 a nutrient agar plate containhg the appropriate antibiotics, and
incubating ovemight at 37°C. A single bacteriai colony was picked 6om the plate and
inoculated into nutrient broth (75 mL) containing the proper antibiotics, and grown
oveniight at 37T, with gentle shakuig. Spectral grade DMSO (0.9 ni') was added to
k h l y grown overnight culture (10 mL) and aliquots (1 mL) were transferred to
cryovials. The cryoviais were placed on a bed of crushed dry ice and then transferred to
a -70°C 6eezer.
2.4.2. Bacterial growth conditions
Bacteriai cultures were grown in screw-top Erlenmeyer flasks (250 mL)
containing nutrient broth (75 d) and the appropriate antibiotics. Flasks were inoculated
with thawed stock bacterial culture (O. 1 d) and incubated overnight, at 37"C, with
gentle shaking. CuItures were grown until the desired OD,, was reached.
2.4.3. Plasmid mini-prep
Small-sale preparations of plasmid DNA were obtained by an alkalie lysis
method, as described (Sambrook et al., 1989). The optional phenol:chioroform
extraction step was perforrned only when the DNA preparation was to be anaiysed by
-
restriction enzyme analysis. Isolated DNA was dissolved in Tris-EDTA bufEèr (50 fi),
pH 8.0 and stored at -20°C.
2.4.4. Electrotransforrnation of bacteria
Ovemight bacterial culture (75 mL) was washed twice with cold s t d e water,
then with 1W glycerol. Ceiis were resuspended in the residual 10% glycerol. CeU
suspension (48 PL) and plasmid DNA (600 ng) were mixed in a 1 mm-gap
electroporation cuvette and placed on ice for one minute. The chiiled cuvette was then
transferred to a BTX Transporator Plus electroporator (San Diego, CA) and pulsed once
at 1.4 kV. Nutrient broth (1 mi,, 20-fold dilution) was immediately added to the cuvette.
Cells were then transferred to a sterile tube and incubated for one hou, at 37°C.
Aiiquots of the diluted electroporated celis (10 CiL, 100 pL, and 500 &) were plated
onto selective agar plates and incubated ovemight at 37°C. Weli-isolateci single colonies
were streaked ont0 selective media and incubated ovemight. Single colonies were
chosen fiom these plates and used to prepare bacteriai stocks.
2.4.5. Bacteriai Conjugation
2.4.5.1. Liquid incubation
As indicated in section 3.2.1 ., the liquid incubation method described by McCann
et ai. (1975) was followed. Briefly, aliquots of ftlly grown cultures of the donor strain
(0.1 mL) and the recipient strain (1.0 mC) were diluted into 10 mL of nutrient broth and
-
incubated at 37°C for 20 hours. Aliquots of the incubation mixture (10 w, 100 & 500 pL) were plated ont0 selective medium.
2.4.5.2- Plate incubation
The bacterial conjugation method described by Kunter and Gaünski (1995) was
performed as indicated. Donor and recipient strains were grown ovemight at 37"C7 with
gentle shaking. Both strains were subatltured and grown untii the OD,, reading of the
donor strain and the recipient strain reached 0.5 and 1.0, respectively. Both the donor
and recipient strain (0.5 mL ofeach) cultures were microcentrifuged and resuspended in
nutrient broth (O. I mL). The conjugation mixture was pipetted ont0 a Milfipore filter
disk (type HA, 45 CiM) that had been placed on the surface of a nutrient agar plate. Mer
incubation ovemight at 37"C, the flter disk was removed from the plate and the ceils
were resuspended in 1 mL of phosphate buffered saline. Dilutions of the ceiis (IO1-, 102-,
1 03 , 1 O*, and 1 Os-fold) were plated ont0 selective media
2.4.6. Phenotypic confinnation of S. fv~him~iunr strains
The foliowing methods for the phenotypic confirmation of Ames tester strains
were described by Maron and Arnes (1983).
2.4.6.1. Histidine requirement (Hi$ awrotrophic Ames tester strains)
Histidine (0.1 mL of O. 1 n/î) and biotin (0.1 mL of 0.5 mM) were applied to a
minimal glucose plate. Samples were streaked across the plate using a Cotton swab, and
plates were incubated ovemight at 3î°C. Control plates containkg biotin but no
-
histidine were prepared. The His' phenotype was confimeci by the inability of the ceUs to
grow in the absence of histidine.
2.4.6.2. Ma mutation (deep-COU@ phenotype)
Bacteriai culture (0.1 mL) was ovedaid onto nutrient agar plates. A BBL füter
disk (Becton-Dickinson, Cockeysville, MD) was placed in the middle of the plate and
crystal violet solution (10 pi., of 10 mg/mL) was pipetted ont0 the disk. Plates were
incubated overnight at 37°C. The presence of the éfa mutation, which makes the ceUs
permeable to tortins such as crystai violet, was confirmecl by a 14 mm zone of inhibition
of growth around the disk.
2.4.6.3. AuvrB mutation
Bacterial cultures were streaked onto nutrient agar plates using a Cotton swab.
Half of the plate was irradiated, with ultra-violet light (plates were held at 20 cm fiom a
30 Watt UV hght source), for 10 seconds. Plates were incubated ovemight at 37°C.
The presence of the A uvrB mutation was wn6rmed by growth oniy on the non-
irradiated side of the plate.
2.4.6.4. Spontaneous mutations
Bacteriai culture (0.1 rnL) was overlaid ont0 a minimai glucose plate. Plates
were incubated at 37"C, for 2-3 days, as required for colonies to grow to an approximate
diarneter of 3-5 mm.
-
2.4.6.5. Antibiotic resistance
Bacteriai culture (0.1 mi,) was overlaid onto a nutrient agar plate. A BBL Glter
disk was placed in the middle of the plate, and ampiciJiin (1 mg), kanamycin (0.5 mg), or
tetracycline (0.8 mg; in a 10 pL volume) was pipetted onto the disk. Plates were
incubated ovemight at 37°C. Antibiotic resistance was demonstrated by growth to the
edge of the disk (ampR) or with a s 10 mm zone of inhibition (kmR).
2.4.7. Restriction enzyme analysis of plasmid DNA
Plasmid DNA was heated at 70°C for 15 minutes. Wpon cooling, Bsu36I (30
units) was added to the plasmid DNA, and incubated at 37"C, for one hour. RNase A
(0.5 mg) was added to the digested DNA, and the entire sarnple was loaded ont0 a 1%
agarose gel. Bands were visualized by stainllig with ethidium bromide and fluorescence.
2.4.8. Isolation of bacterial membranes
The foiiowing was adaptai fiom sr method for the isolation of bacterial
membranes f?om E. coli (Sandhu et al., 1994). Bacterial cultures were supplemented
with thiamine (1 mM), antibiotics, trace elements, 64LA (0.3 m M as indicated), and
iPTG (1 mM, as required), and grown for 24-36 hours, at 3 7 OC or 3 0 OC as noted, with
vigorous shaking. Cultures were chilled ( -20°C) for 1 hour, and ail subsequent steps
were performed at 4°C. Cells were centrifùged at 4000 x g, resuspended in Tris-acetate
buffer containhg lysozyme (2 mg/mL), and gently shaken for 30 minutes. The resulting
spheroplasts were centrifiged at 4000 x g , resuspended in potassium phosphate buffer
-
containing DTT (O. 1 mM), and fiozen at -70°C. Protease inhibitors, leupeptin (2.0 pM),
PMSF (1 .O mM), and aprotinin (0.04 UImL) were added upon thawing. Thawed
spheroplasts were lysed with three 15 second bursts (at 60% power) fiom a Microson
(HL-SX2005) sonicator (Mandel ScientSc Company Ltd.). The lysate was centrifiiged
for 10 minutes at 10 000 x g. The resuiting ceii debris was coiiected as a pellet and
discarded. The supernatant was centrüùged at 180 000 x g for 35 minutes. Membrane
pellets were resuspended in Tris-acetate buffer (1-2 mL).
2.4.9. Pre-adsorption of antisera to remove bacterial antibodies
The removai of antibacteriai antibodies from antisera was perfomed as descnied
by Gmber and ZingaIes (1995). Bacterial culture (150 mL) was grown overnight, at
37"C, with vigorous shaking. Half the culture was autoclaveci at 12 1 OC, for 1 hour.
Formaldehyde was added to the remainder (0.5% &vol) and the culture was incubated
for 2 hours at 37°C. The treated bacterial suspensions were mixed, washed twice with
phosphate buffered saline, aiiquoted into ten centrifirge tubes, centrifùged, and stored at - 20 OC. The bacterid pellets were resuspended in semm (1.5 mL, of 150 dilution) and
incubated at roorn temperature under rniid agitation. After centrifkgation, the serum was
transferred to another bacteriai pellet and the absorption cycle was repeated. Foiiowing
eight serial absorption cycles, the serum was filter-sterilized (0.22 pM MilIipore
membrane, Bedford, MA) and stored at -20 O C .
-
2.4. IO. Western ImmunobIotting
Western immunoblotting as described by Sarnbrook et ai. (1989) was used to
analyse bacterial sarnples for P4501Al expression. B r i e , bacteriai membrane proteins
were separateci on a 10% sodium dodecyl sulphate-polyacrylaniide gel, ushg a Bio-Rad
Mini-Protean II system and electrophoreticaiiy transferred to a nitroceliulose membrane.
The nitroceliulose membrane was incubated in blocking solution (5% skirn m . powder)
for 8 hours, and then transferred to the primary antibody solution (1: 1000 dilution of pre-
adsorbed rabbit anti-human antiserum), Mer a 2 hour incubation with the primary
antiserum, the nitroceliulose membrane was washed three times with phosphate-buffered
saline, foliowed by a final wash in phosphate-fie, azide-free buffet (50 m M Tris-CI, 150
m M NaCl). The membrane was then incubated with alkaline phosphatase-conjugated
anti-rabbit immunoglobulin (1:5000 dilution), for 1 hour. The blot was developed by the
addition of nitro blue tetrazoiium (0.1 rnL) and 5-bromo-4-chloro-3-indolyl phosphate
(O. 1 mL).
2.4.11. MutageniCity assays
Mutagenicity assays were based on an adaptation of the method of Muon and
Ames (1 983). Cultures of the appropriate S. îypkimurium strains were grown overnight
at 37°C with gentle shaking, untit an OD,, reading of 1.0 was reached. The required
amount of test çompound and bacteria (O. 1 mi,,) were incubated at 37°C for 30 minutes
with gentle shaking. At the end of the incubation period, molten top agar (2 mL
containing 50 p M histidine, 50 PM biotin) was added to each tube, and the contents
-
were overlaid ont0 minimal glucose plates. Mer incubation at 37°C for 2-3 days, the
number of revertant colonies on each plate was counted.
2.4.12. Mutagen spot tests
Cultures of the appropriate S. ryphrtturium strains were grown overnight at 37°C
with gentle shaking, until an OD,, of 1.0 was reached. Bacterial culture (0.1 mL) in
molten top agar (2 mL containhg 50 pM histidine and 50 pM biotin) was overlaid ont0
mininal glucose plates. A sample of the test compound O M S 0 solution, 10 FL volume)
was pipetted directly ont0 the plate. Mer incubation at 37°C for 2-3 days, the size of
the zone of inhibition and the distribution of revertant colonies was determined.
2.4.13. Preparation of benzo[a]pyrene-7,8-dihydrodiol stock solution
A 5 mg sample of benzo[a]pyrene-7,s-dihydrodiol was dissolvecl 95% ethanol(5
mL). The approximately 1 mg/mL solution was diluted 1000-fold and the absorbance at
368 nm (A-) was measured using a Hewlett-Packard diode array spectrophotometer
(Corvallis, OR). The concentration of the solution was calculated using the Ber-
Lambert equation: A=~cl where A is the absorbance measured at a given wavelength, E
is the compound's molar extinction coefficient, c is the concentration of the sample, and Z
is the Iength of the Iight path through the sample. The molar extinction coefficient used
for the calculation of benzo[a]pyrene-7,8-dihydrodiol stock solution concentration was
E3(B = 3.69 x 104 L mol" cm" (NCI Chernical Carcinogen Reference Standard Repository,
data sheet NCI No. L01I3).
-
2.4.14. Fe'2/Fe+2-C0 diifference spectroscopy
The presence of P4SO holoprotein was determined as describeci by Omura and
Sato (1964). An aliquot of thawed bacterial membranes (2 mg total protein) was
incubated on ice for 30-60 minutes, after the addition of Na$,O, (5-10 mg). The
membranes were divideci into two cuvettes, sample and reference, and placed in an SLM-
Aminco DW-2C spectrophotometer (Rochester, NY). Approxhately 20 CO bubbles
were delivered into the sample cuvette, and the difference spectmm fiom 370 nm to 520
nm was measured, A baseiiie difference spectmm was recorded prior to the addition of
CO.
-
3 - Results and Discussion
3.1.1. Strain construction
The Ames test strain TA1538 (hisDSO52) was chosen as the background strain
for the expression of P45O lAl, in Our îïrst series of experirnents. in standard S9-
mediated Ames tests, TA1538 was more sensitive to benzo[a]pyrene mutagenicity than
was TA1 535 (hisG46; McCann et al., 1975). S9 was required to observe the mutagenic
effects of benzo[a]pyrene in ail Ames tester strains. The expression of P45O lA1 in
TA1538 was our first strategy for mutagenic activation of PAHs, in the absence of an
exogenous source of enzymes.
A plasmid containhg the fuii-length cDNA of the human P450 1Al (pP450 1A1)
was obtained fiom the laboratory of Dr. F. Peter Guengerich. Since plasmid pP4501A1
had been isolated fiom E. coli, it was moved hto an S. rjphimurium strain which lacks
restriction enzyme functions but can stil methylate DNA (LB5000; Buiias and Ryu,
1983; McGowan-Jordan and Josephy, 1990). This step was required to cucumvent the
S. typhimurium restriction enzymes present in TA1538, which may have cleaved plasmid
DNA which was isolated directly fiom E coli. Plasmid pP4501AI was isolated 6om
LB5000 and used to transform TA1538 by efectroporation. Transformants were
detected by growth on selective medium (100 pg/mL ampicillin). Typical transformation
yields were 300-500 colonies per pg of plasmid DNA
Six isolates of TA1538/pP4501Al were selected for strain testing (see 2.4.6.).
The testing conhned the presence of the plasrnid (ampicillin resistance) and the Ames
5 1
-
tester strain phenotype (histidine requirernent, UV-sensitivity, crystal-violet sensitivity).
The spontaneous reversion rate was 14 to 17 colonies per plate, which is typical of
TA1 538.
3.1 -2. Fe+21Fe+2-C0 difference spectroscopy
After the construction of the TA1538fpP450 1 AI, bacterial membranes were
harvested and the presence of P4SO 1Al holoprotein was assayed using Fe+2/~e+2-C0
diEerence spectroscopy. A trial run of the membrane isolation technique described by
Sandhu et al (1994) was performed and foiiowed at each step, using a standard Pierce
bicinchoninic acid ( K A ) assay kit (Rockfiord, IL; see table 3). Previous studies have
detected the presence of PdSO, using Fe'2/Fe+2-C0 ciifference spectroscopy, with sample
concentrations of I mglml (Josephy er al., 1995). Results of the BCA assay indicated
that the technique recovered a suffiCient amount of protein. DHS ar pP45O 1A2 was
chosen as a positive control for the spectral analysis because this strain was shown to
express P450 1A2 in previous studies (Sandhu et al., 1994; see figure 9).
-
Table 3
~ -
Description of the santple 1 Major steps in the isohtion of 1 I bactend membranes !
Bacteriai cultures [ Grow bacteria overnight 1 Centrifiige and resuspend ceUs (4 OOQg)
Supematant (nutrienî broth)
Centrifuge and resuspend spheroplasts (4 O W )
S onicate sp heroplasts and centrifuge (10 OOOg)
Centrifuged œii debris (resuspended)
Centfige 1 OOOOg supernatant (180 OOOg)
180 ûûûg supernatant
Bacterid membranes 2.0 1 2 assay kit Bolded d using the Pierce Licinchoninic acid (BCI
lines represent samples wbich were retained and used in subsequent steps.
-
- -
Figure 9. Fe2-CO versus Fe+* dierence spectrurn of membranes prepared fiom DH5a p F 4 ~ ~ 1A.2 (basdine corrected). The sp&m indiutes a level of expression of approximately 10 m o l P4501 liter of culture.
-
P4SO 1Al holoenzyme was not detected in bacterial membranes isolated h m
TA1 538/pP45O 1A1 using Fe+2/Fe+2-C0 diierence spectroscopy. Initially, membranes
were isolated fiom cultures grown as describecl in section 2.4.8., at 37°C with a 12 hour
period oflPTG induction. Subsequent trials focused on attempting to in- the level
of P4SOlAl production through m o d i i g bacterial growth conditions. 6-ALA, a heme
precursor which has been shown to increase leveis of heterologous P450 production, was
added to bacterial cultures (ûiüam et al., 1995a). The length of IPTG induction time
was hcreased to 24 hours and the growth temperature was lowered to 30°C, to match
the conditions used for expression of pP4SO 1Al -encoded proteins in E. coli (Guo et al.,
1994). In aii trials, the P450 peak was not observed in the Fe'*/W2-CO diierence
spectrum of the samples.
3.1.3. Immunoblot analysis of TA1 538/pP450 1A1
Since the presence of the recombinant protein had not been spectrally detected,
immunoblot (Western blot) analysis was perfonned to conlïrm that the recombinant
P45O 1 A1 protein was being produced in the S. typhiimriurn strain. Rabbit anti-human
P450IA1 antiserum was obtained t