animal clinical chemistry
TRANSCRIPT
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Animal Clinical Che
A Primer for Toxicologist
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Animal Clinical ChA Primer for Toxicologist
Edited by
G.O.EVANS
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This edition published in the Taylor & Francis e-
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British Library Cataloguing in Publication Data A catalogue
from the British Library
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Contents
Preface
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Abbreviations
Appendix I SI Units and Conversion Tables
Appendix II General References for Animal Clinical Che
Index
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Preface
In 1975 clinical chemists from several pharmaceutic
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Contributors
D T DAVIES
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1
General Introductio
C O EVANS
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For food additives, often with low biological activity, the
demonstrate a no observable adverse effect level (NOAEto identify separately any changes in data which may b
response to repeated overdosage. This is in contrast to the si
important to both demonstrate potential toxic response an
effect level (NOEL): here the difficulties are in distingu
pharmacological response(s), desired pharmacological actio
apparent toxic effect(s) (James, 1993). It remains debatab
should be based on demonstrating the absence of toxic stoxicity (Heywood, 1981). Toxicology studies are generally
adverse effects of a xenobiotic by identifying its effects
metabolic functions, and furthermore to determine if these e
f h d d i i l i i
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When the toxicity of chemical mixtures is under exam
data becomes more difficult: a mixture of two or more chemqualitative or quantitative response relative to that predicte
exposure to the mixture constituents. For example, the co-
hepatotoxic compounds, carbon tetrachloride and 1,2
expected to show an additive effect on plasma alanine amin
the resultant dual exposure reduces the degree of liver injur
to the effects produced by 1,2-dichlorobenzene alone (Mum
pretreatment of rats with retinol potentiates the hepatoassociated plasma ALT with carbon tetrachloride (ElSisi et a
Polypharmacy may complicate toxic changes, and a
associated with inactive ingredients in drug formulations (
hi l d f d d li k dl ff i
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Table 1.1Definitions of test spec
sensitivitySensitivityof an assay is the fraction of those with a specific diseas
predicts.
Specificityis the fraction of those without the disease that the assay
Where
TP=True positive, number of affected individuals correctly
FP=False positive, number of non-affected individuals mi
FN=False negative, number of affected individuals miscla
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The total variance for a single measurement may be expre
variance + biological variance + methodological (or study) these three components. In later chapters, we discuss some
approach the statistical analysis of the data.
Few if any of the common laboratory tests are intrinsica
most tests requiring some decision as to where to select the
and abnormality. Effective interpretation involves conside
probability values, it requires knowledge and experience
Clinical pathology measurements should always be interpother study data obtained from histopathology, experimenta
simple example is plasma or urine osmolality where water
mass should be collectively considered for the correct interp
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instrumentation and commercially available high-quality re
has been reduced, so that intra-batch coefficient of variatioachievable for most of the common tests. It is usually a lit
and manual tests. Thus as a general rule, analytical variance
the biological variance in the total variance sum. Wherev
changes during studies should be avoided.
The number of tests (or parameters) determined in a stud
size and the analytical methodology available, although
require small sample volumes for most of the common teststest). It is sometimes suggested that the choice of biochemi
been governed by investigators more familiar with human
has led to the inclusion of tests which are inappropriate for
S h f il i h l f h
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of sample collection and separation procedures, and w
collection tube systems are currently designed for collectinthose usually obtained with smaller laboratory animals.
disinfecting or autoclaving biological waste materials, an
analysers prior to selective maintenance procedures should b
1.5Summary
Biochemical measurements can help in:
Identifying target organ toxicity. Confirmation of other observations particularly changes fo
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EVANS, G.O. (1994) Removal of blood from laboratory mammals28, 1789.
FLETCHER, A.P. (1978) Drug safety tests and subsequent clinicalRoyal Society of Medicine,71,6936.
GLOCKLIN, V.C. (1983) The role of data organisation in the eval
drug application.Drug Information Journal,13951.
GOLIGHTLY, L.K., SMOLINSKE, S.S., BENNETT, M.L., SUTHB.H. (1988) Pharmaceutical excipients. Adverse effects associa
drug products.Medical Toxicology,3,20940.
GRANDJEAN, P., BROWN, S.S., REAVEY, P. & YOUNG, D.S.chemical exposure. The proceedings of the Arnold O.Beckman/Environmental Toxicology. Clinical Chemistry,40,1359476.
GRINER, P.F., MAYEWSKI, R.J., MUSHLIN, A.I. & GREENLA
interpretation of diagnostic tests and procedures Annals of Inte
General introduction 9
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2
Study Design and Regul
Requirements
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animals per group are also usually defined. For sub-acute ro
females per group are appropriate with double this number rodent studies (usually with dogs), four animals of each
sufficient for most regulatory studies. There are no clear g
animals that should be bled at each time-point but, conventi
be bled while 20 rodents (10 males and 10 females) per gro
Similarly, the frequency of blood sampling is left entirely to
or study director. As a minimum, blood samples should be c
For mice, where limitation of blood volume makes interimsamples can only reliably be collected at necropsy. Multi
jugular or cephalic veins is clearly not a problem in the lar
rat, several acceptable sites of interim blood sampling are de
l d h i d h l l b i fl
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livephase of a sub-chronic study, when pathological evalua
exploit the strengths of clinical pathology and provide add
of value. A blood-sampling regimen, similar to that descradopted provided that the sampling does not compromise the
This view is further supported by Davies (1992) who de
blood samples can provide valuable information on the
pathology. In the experiment described, blood samples take
study had normal plasma alkaline phosphatase (ALP) an
(AST) activities but there was a slight increase in alanactivity; morphologically the liver was normal. Early blood
1 and 2 exhibited elevated ALP and ALT activities and the
showed more than a 20-fold increase compared to the pre-
l i d f l k b d i i d
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Table 2.1Pre-clinical blood chem
regulatory documents
Glucose* Inorganic
Urea (or urea nitrogen)* Urate*
Creatinine* Cholester
Aspartate aminotransferase* Lactate dAlanine aminotransferase* Total pro
Total bilirubin* Albumin*
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change in rodents. Similarly, while gamma glutamyl tran
liver toxicity in the dog, an increase is rarely detected in
hepatotoxic compound. Other tests suggested by regulatoryassociations have also proved to be controversial.
Urate (uric acid) measurement is commonly used in hum
and treatment of gout, rheumatoid arthritis and other con
metabolism. Urate is the end point of purine metabolism in h
species are able to further metabolize urate to allantoin res
values which have little diagnostic value in generadehydrogenase activity is highly variable and lacks specifi
organ toxicity in animal species (Evans, 1991). The analyt
performance for plasma ornithine decarboxylase (Carakosta
i hi b l f (C k l 1986)
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suspected, several urinary enzymes can be measured
considered (see Chapter 7, Nephrotoxicity).
The debate about which tests should form the core proclinical toxicity and safety studies has exercised several
Recent noteworthy recommendations are discussed below. A
groups have been prominent in publishing recommenda
testing. In 1992, Stonard published the recommendations
Toxicology Group which formulated draft OECD Guideline
chemicals. Subsequently, the IHCPT Committee developefrom an earlier version prepared jointly by the Ameri
Chemistry and the American Association of Veterinary Clin
al.,1992). Both documents emphasize that they define mini
b i d f ll h i b
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2.4Good Laboratory Practice (G
Following the identification of serious flaws in toxicologFood and Drug Administration (FDA) of the United S
developed a code of practice designed, with periodic
agencies, to ensure the scientific validity of all studies subm
Table 2.2Recommendations for c
chemistry testsOECD Shadow Toxicology Group IHCPT
Non-fasting glucose Glucose
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Good Laboratory Practice (GLP) Regulation (FDA, 1
guidelines (OECD, 1982) were subsequently promulgated
countries.As for all other aspects of non-clinical toxicity and sa
pathology analyses must be shown to be scientifically
standards. These GLP regulations cover aspects of personn
training, general laboratory facilities, calibration and
performance of reagents and therefore assays, and character
variables. Great emphasis is placed on adequate documentaraw data and detailed description of any amendments mad
Appropriate statistical methods should be used to analyse
and Weil, 1989) and it must be stressed that concurrent con
h hi i l f f i i h
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(GEET-Italy); International Society for Animal Clinical Bio
Canada, Sweden); Japanese Pharmaceutical Manufacturers
References
ALDER, S., JANTON, C. & ZBINDEN, G. (1981) Preclinical safe
Federal Institute of Technology and University of Zurich.
CARAKOSTAS, M.C. (1988) What is serum ornithine decarboxyl
26067.CARAKOSTAS, M.C., GOSSETT, K.A., CHURCH, G.E. & CLE
Evaluating toxin-induced hepatic injury in rats by laboratory re
Veterinary Pathology,23,2649.
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3
Preanalytical and Analytical
J ROBINSON & G O EVANS
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Section 3.1Preanalytical Variab
J.ROBINSON& G.O.EVANSSeveral preanalytical variables must be considered, and th
influences, gender, age, environmental conditions, chro
biorhythms), nutrition, fluid balance and stress (Table 3.1.1
used for sample collection, separation and storage are also im
3.1.1Species, Strain, Age and Ge
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Fluid balance
Chronobiochemistry
Sampling procedures
Time of sampling
Sample volume
Site of sampling
Agents used for anaesthesia or euthanasia
Frequency of sampling
Anticoagulant
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Table 3.1.2Mean percentage cha
rats fasted overnight compared to controls at 4 and 13 weeks of age
Robinson, 1975)
Mean percentage c
4 weeks
Parameters Male Fema
Body mass 11
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intake with consequential perturbations of fluid balance also
Simple observations of water intake together with urinary
examining this preanalytical variable.Treatment prior to sequential blood sampling in toxicolo
in respect to food and water intake. The biochemical
alterations of nutritionary and fluid balance can be assoc
absorption or uptake of the test compound, changes in meta
mechanisms, modification of renal clearance or changes i
proteins with the test compound. Toxicity may affect both nexaggerate differences between dosed or treatment groups.
3 1 4 Chronobiochemical Effec
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reported differences in sample quality associated with indi
shows some of the common sites used for blood collection fr
Anaesthetics should be chosen on the basis of causing minimal interference effects on the analyte, and minimizing
Table 3.1.3Species and common
collection
Species Sites of collection
Mouse heart, vena cava, tail vein
Rat heart, vena cava, aorta, tail vein, retro-orbital plexu
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3.1.6AnticoagulantsLithium heparinate or heparin are suitable anticoagulants fo
Inappropriate use of anticoagulants, and incorrect proporti
volumes may also cause errors. Samples collected with se
citrate used for haematological investigations are not suitab
enzyme measurements; the inappropriate use of potassium
low calcium values due to chelation and high potassium check with local laboratories the correct anticoagulant requiWhen collecting blood, it is important to separate the
possible; this reduces the effects of glycolysis which result i
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3.1.8Urine CollectionsIn general urine collections should be made for fixed t
overnight) using well-designed metabolism cages. For so
enzymes the timing of collection period may produce dif
devices for separating faeces and urines in metabolism ca
reduction of faecal contamination of the urine. Bacterial
highly alkaline pH values, may occur if analysis is delayedbiochemical values. Some investigations require the use ofmay be collected over ice. Catheterization and random samp
in some studies.
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DAVY, C.W., TRENNERY, P.N., EDMUNDS, J.G., ALTMAN, J
Local myotoxicity of ketamine hydrochloride in the marmoset.
DROZDOWICZ, C.K., BOWMAN, T.A., WEBB, M.L. & LANGtransport on murine plasma corticosterone concentration and blAmerican Journal of Veterinary Research,51,18416.
EVANS, G.O. (1985) Lactate dehydrogenase activity in platelets a
31,1656.(1987) Post-prandial changes in canine plasma creatinine.Journal
31115.(1994) Removal of blood from laboratory mammals and birds.Lab
FALK, H.B., SCHROER, R.A., NOVAK, J.J. & HEFT, S.M. (198various serum chemistry parameters from common lab animals
FOUTS, J.R. (1976) Overview of the field: environmental factors a
in animals Federation Proceedings 35 11625
Preanalytical and analytical variables
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LINDENA, J., BUTTNER, D. & TRAUTSCHOLD, I. (1984) Biol
experimental components of variance in a long-term study of pl
of Clinical Chemistry and Clinical Biochemistry, 22,97104.LOEB, W.F. & QUIMBY, F.W. (1989) The Clinical Chemistry of
Pergamon Press.
MAEJIMA, K. & NAGASE, S. (1991) Effect of starvation and ref
of hematological and clinico-biochemical values, and water intaAnimals,40,38993.
MATSUZAWA, T., NOMURA, M. & UNNO, T. (1993) Clinical plaboratory animals.Journal of Veterinary Medical Science,55,
MATSUZAWA, T. & SAKAZUME, M. (1994) Effect of fasting ochemistry values in the rat and dog. Comparative Haematology
MCGOWAN, M.W., ARTISS, J.D. & ZAK, B. (1984) Description
from elevated serum solids Analytical Biochemistry 142 239
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SCHEVING, L.E., SCHEVING, L.A., FEUERS, R.J., TSAI, T.H.
Chronobiology as it relates to Toxicology, Pharmacology and C
Toxicology and Pharmacology,17,20918.SCHWARTZ, E., TORNABEN, J.A. & BOXILL, G.C. (1973) The
haematology, clinical chemistry and pathology in the albino ratPharmacology,25,51524.
SONNTAG, O. (1986) Haemolysis as an interference factor in clinClinical Chemistry and Clinical Biochemistry,24,12739.
STONARD, M.D., SAMUELS, D.M. & LOCK, E.A. (1984) The pinduced by different diets in female rats, and the effect on renal
Toxicology,22,13946.STREET, A.E., CHESTERMAN, H., SMITH, G.K.A. & QUINTO
diet on blood urea levels in the beagle.Journal of Pharmacy an
SUBER R L & KODELL R L (1985) The effect of three phlebot
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Section 3.2Analytical Variabl
J.ROBINSON& G.O.EVANSIn this section, we discuss some of the analytical variables
when performing studies and interpreting data. The go
Association of Societies of Pathology (1979) is that anal
equal to or less than one-half of the average within subanalytical CV
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reference materials may be obtained from a commercial so
an External Quality Assessment Scheme (EQAS).
Examples of methodological performance are indicated inwhere method A reflects an accurate and precise method, B
method, C a precise but inaccurate method, and method D
method.
Reproducibility, precision or perhaps more correctly
usually expressed as the degree of agreement between re
sample, and as a coefficient of variation (CV) where the s
measure of the dispersion of a group of values around
percentage of the mean, Values for the CV can be det
analytical run and between-batch or day.
l h i hi b h d b b
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Most laboratories participate in an External Quality A
where samples are distributed on a regular basis to particip
are compared to overall or group mean values as an indicato
3.2.2Other Examples of Analytical V
Sample EvaporationEven with the sophistication of todays analysers, sample
analytical procedures and this will affect results (Burtis et a1993). This evaporation problem is particularly important in
samples taken from laboratory animals.
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HormonesSee Chapter 8.
3.2.3Changing Analytical Meth
Although assay systems can be modified for use with a
always easily achieved. The problem for many manufact
market is too small to justify the expenditure required to
species-specific. Problems associated with lipid measureme
analysts to be vigilant when new reagent formulations are inRecognizing the limitations of certain methods in terms
it must be remembered that the analytical limitations will ap
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Section 3.3Analytical Interfere
G.O.EVANSWhen examining data from toxicological studies, differen
clinical chemistry data which cannot be explained simply
toxicity, histopathological findings, clinical observatio
preanalytical variables. In these cases it may be wor
differences are due to the effect(s) of the test compound
assay(s) in question, and this general consideration of metab
following section where only the test compound may be men
Xenobiotics may interfere with analytical methods in sev
may have a negative or positive effect on the result: these
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species. There is evidence that some of these serum separa
interact with hydrophobic drugs in human serum (Landt et a
Some other effects include inappropriate anticoagulantassociated with sequestrenated blood, iron chelators such
and effects due to intramuscular injections (see Chap
Myotoxicity).
3.3.1.2Plasma Protein Binding
Xenobiotics can bind to the various plasma protein fractionsbinding occurs can have a marked effect on the pharm
(Goldstein et al.,1974; Lindup and LE Orme, 1981). The
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3.3.3Testing for Analytical Interfe
Various guidelines have been proposed for testing for
chemistry measurements and they suggest the necessity fo
(Powers et al., 1986; Kallner and Tryding, 1989; Kroll an
several limitations to performing such studies.In vitrostudi
to clinical practice, e.g. high levels of ascorbic acid interfere
are rarely seen in vivo. More importantly xenobiotics are o
range of metabolites, and consideration then has to be gabundance of the metabolite and its availability for testing.
other drugs can complicate interpretation of effects due to in
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urine must be considered in deciding if the effects are du
assay, and additional problems occur when more than on
Following in vivo studies, testing the test compound andtechniques can be useful. Early recognition of potential in
chemistry measurements during the preclinical or developm
however, can assist and prevent misleading interpretations
development results.
References
APPEL, W., HUBBUCH, A. & KOLLER, P.U. (1991)In-vitro-Pro
h k b i i i i l b i i
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KROLL, M.H. & ELIN, R.J. (1994) Interference with clinical laboChemistry,40,19962005.
KROLL, M.H., NEALON, L., VOGEL, M.A. & ELIN, R.J. (1985negatively with the Jaffe reaction for creatinine. Clinical Chemi
LANDT, M., SMITH, C.H. & HORTIN, G.L. (1993) Evaluation o
tubes: effects of three types of polymeric separators on therapeu
Clinical Chemistry,39,171217.LETELLIER, G. & DESJARLAIS, F. (1985a) Analytical interferen
chemistry: 1. Study of twenty drugs on seven different instrume34551.
(1985b) Analytical interference of drugs in clinical chemistry: II. Tcephalosporins with the determination of serum creatinine conc
Clinical Biochemistry,18,3526.
LINDUP W E & LE ORME M C L (1981) Plasma protein bind
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4
Statistical Approach
A DICKENS & J ROBINSON
S i i l h 41
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In the final part of the chapter, different statistical techn
applied to different types of toxicological data are summariz
4.2Descriptive Statistics
4.2.1Diagrams
It is said that a picture is worth a thousand words. Certai
which words cannot convey can be very forcefully presente
the use of statistical diagrams is an important area (particu
computer packages) and there has been something of a resur
Statistical approaches 41
A i l li i l h i t 42
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Animal clinical chemistry 42
St ti ti l h 43
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1 Separate each observation into a stem and a leaf; in generadigits as needed, but each leaf should contain a single dig
value of 148, the stem would be 14 and the leaf 8.
Statistical approaches 43
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different from the others on the same treatment, though n
this should be so. Such an observation
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Making a graphical display is the first step towards und
calculations which give the location of the distribution and
observations is the second step.
Figure 4 5 Illustration of ne
Statistical approaches 45
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Since the number of observations, n=10, is even, the m
centre observations.
The median takes no account of the precise magnitude of mtherefore usually less efficient than the mean, because it w
the mean can be misleading. If we consider the previous ex
nature of the distribution the mean of 226.2 U/L is not rewhole, and the median of 177.5 U/L might be a more use
important weakness of the mean as a measure of centre,
influence of a few extreme values. Similarly, a mean should
Animal clinical chemistry 46
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The calculation of the quartiles leads to another e
distribution, the boxplot orbox and whisker plot. In a boxp
1 The ends of the box are at the quartiles, so that the length orange.
Statistical approaches 47
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There are several modifications of the above procedure b
same in all cases, the centre, spread and overall range
immediately apparent.The most commonly used measure of variability of a
deviation. This is a measure of spread about the mean, and
mean is employed as a measure of centre. The varianceof n
The standard deviation sis the square root of the variance s2
The idea behind the variance and the standard deviation
f ll Th d i ti di l th d f th
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4.3Some Basic Considerations in Experim
The design and analysis of experiments is an extensive subjhave been entirely devoted (Davies, 1954; Cochran and C
design are of course inseparable from those of analysis and
unless a suitable design is employed, it may be very difficulvalid conclusions from the resulting data.
Statistical experimental design was founded in the early at an agricultural research station at Rothamsted, Eng
comparing the yield from several varieties of wheat was pe
Statistical approaches 49
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4.3.3Blocking
If the individual units are homogeneous, respond in a c
treatments, then the experimental error variance will be sma
will have high precision and it will be relatively easy to d
individual treatment population means. However, Fisher
research, the individual plots in a field were anything but
with respect to fertility, drainage etc. Furthermore, it was
between units was desirable, in order to give the experimwould produce results applicable to the real world. Thus
apparently irreconcilable requirementsto design high-p
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3 The power (the probability of correctly detecting the differ4 The design itself (how well it controls experimental error)
5 The number of replicates tested (the number of units per trPrecise details of how to decide upon the appropriate size o
a variety of books, and the reader is referred to Desu (
stressing that an over-precise experiment is just as much
precise one. An experiment which can detect differences b
means, which are too small to be of any practical (as oppose
wasteful of valuable resources that could otherwise have beeDetermining sample size is a compromise between
expected variability of the outcome measure and effect sizeusually known or specified but the latter two are not Whe
Statistical approaches 51
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this is rather wasteful of data. Methods for ordinal data can
where one is uncertain of the underlying distribution, for ana
Although various guidelines (US Food and Drug Admistatistical tests be used to evaluate the data generated from t
no current standard statistical procedures being used to anal
(Waner, 1992). There are some published (Gad and
recommended approaches which summarize the method
approach for continuous data is shown in Figure 4.7. The m
be found in most standard statistical texts (Gad and Weil
1988; Campbell, 1989).The practice of statistics involves many numerical calcul
some very complex. As you learn how to perform these ca
l f i i i l l i f i k b i
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Suggested Reading
Basic StatisticsARMITAGE, P. & BERRY, G. (1988) Statistical Methods in Medi
Blackwell Scientific Publications.
CLARKE, G.M. & COOKE, D. (1992)Basic Course in Statistics,
GAD, S. & WEIL, C.S. (1987) Statistics and Experimental Design
Telford Press.
Intermediate StatisticsSNEDECOR G W & COCHRAN W G (1980) Statistical Metho
pp
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5
General Enzymolog
G O EVANS
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Table 5.1Plasma enzymes and th
half-lives in three species
Range of estimates for enz
Enzyme Dog R
Aspartate aminotransferase 3.3 to 4.4 2.
Alanine aminotransferase 2.5 to 60.9 4.
Creatine kinase 0.6 to 16.2 0.
Lactate dehydrogenase 1.6
y gy
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regeneration may also be accompanied by changes in pla
leakage from the cell, plasma enzyme activities may fall bu
on the rates of clearance from the circulation. Often theclearances (or half-lives) of plasma enzymes are found
methodologies used to establish these values. Table 5.1 s
elimination half-lives for enzymes, and it is particularly
variables in acute or time-course studies where enzyme chan
Urinary enzymes may originate from:
1 proteins of low molecular weight passing into the glomeru2 the renal tubular cells either by desquamation or injury
3 desquamation or injury of the epithelial cells of the urogen
4 secretion by the glands of the urogenital tract
y
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Table 5.2Enzymes, their abbrevi
Enzyme Commission (EC) numbe
Abbreviation Recommended name
ALT(GPT) alanine aminotransferase
AAP alanine aminopeptidase
ALP alkaline phosphatase
AMY amylase
AST(GOT) aspartate aminotransferase
CHE h li t
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5.1.2Aspartate Aminotransferase (
AST (also known as glutamic oxaloacetate transaminase, GO
As for ALT, this enzyme is widely distributed in the tissue
hepatic and renal tissues. It is commonly used in conjunct
site of tissue damage. Some common texts emphasize its us
plasma levels of this enzyme do change following th
hepatotoxins. Mitochondrial and cytosolic forms of AST
mitochondrial to cytosolic form is generally greater than fo
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5.1.4Aminopeptidases and Arylami
These enzyme assays have been linked here as they have s
diagnostic enzymology.Alanine aminopeptidase (AAP) and leucine arylamidase
at the N-terminal amino acid and some amino acid amidhydrolyse leucyl- and alanyl-4-nitroanilide substrates. They
also membrane bound, and they have been used in studie
nephrotoxicity. These two enzymes should not be conf
aminopeptidase (LAP): this enzyme is an aminopeptidase
acid residues of proteins, in particular those with an N-termi
-napthylamide is commonly used as substrate.
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appear to alter following renal injury but urinary GGT
monitoring renal tubular damage.In hepatic studies, plasma GGT can be used as an indica
where plasma GGT levels are normally very low, often less
as a marker of enzyme induction and of the presence of hep
in laboratory animals compared with data from human studi
al.,1992; see Chapter 6).
5.1.10Lactate Dehydrogenase (LD
This catalyses the reversible oxidation of lactate to pyruvate
It is distributed widely in the tissues and the tissue isoenzy
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5.1.14Other Enzymes
Arginase, malate dehydrogenase (MDH), isocitrate dehyd
phosphatase dehydrogenase (G6PDH) and alcohol dehydr
the many enzymes which have been measured in studies of h
Glutathione transferases (GST), essential in many detox
recently not been used widely due to the low levels in plas
and inhibition by bilirubin and bile acids in the assay. The
now available have been used in a few laboratories, but nmay widen the use of this enzyme assay.
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absence of data which show that these reagents are totall
differing species used in toxicology. Variations between spsubstrates have been shown for cholinesterase (Myers, 19
enzymes, e.g. angiotensin converting enzyme (Evans, 1989
the majority of methods employ 4-nitrophenylphosphate a
two main alternative buffersdiethanolamine and 2-amin
interspecies differences (Masson and Holmgren, 1992).
For isoenzymes, the majority of laboratories currently us
separation methods. Selective inhibition of isoenzymes w
increasingly, but there are problems associated with proisoenzyme concentrations in animal samples.
Preanalytical factors discussed in an earlier chapter also
i Si f li i l l f
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measurements in different species described here, it can be
one species cannot be used for any other species.In summary, several enzymes should be chosen wi
specificity for the detection of major organ toxicity. The ch
reflect their different tissue and intracellular locations. In m
times should be used with some reference to enzyme produ
the toxic insult. The use of such approaches is exemplified
nephrotoxicity, hepatotoxicity and cardiotoxicity. Whethe
simplicity or suitability for use with automated laborato
enzymes measured in regulatory studies has not increased dtwo decades.
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DO, T.-X., BOISNARD, P., GIRAULT, A., PLANCHENAULT, P
(1992) Etude exprimentale des variations diurnes et nocturnes
enzymatiques rnales de rats Sprague-Dawley. Science et techn
17,20711.DOOLEY, J.F. (1979) The role of clinical chemistry in chemical a
of laboratory animals. Clinical Chemistry,25,3457.
DORNER, J.L., HOFFMAN, W.E. & LONG, G.B. (1974) Corticoisoenzyme of alkaline phosphatase in the dog.American Journa
14578.ECKERSALL, P.D. (1986) Steroid induced alkaline phosphatase in
Veterinary Medicine,42,2539.EVANS, G.O. (1985) Lactate dehydrogenase activity in platelets a
31,165.
(1989a) More on orthinine decarboxylase Clinical Chemistry 35
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MASSON, P. & HOLMGREN, J. (1992) Comparative study of alk
animal samples using methods based on AMP and DEA buffersScandinavian Journal of Clinical and Laboratory Investigation
MILNE, E.M. & DOXEY, D.L. (1987) Lactate dehydrogenase andand sera of clinically normal dogs.Research in Veterinary Scie
Moss, D.W. (1982) Alkaline phosphatase isoenzymes. Clinical Ch
MYERS, D.K. (1953) Studies on cholinesterase: 9. Species variatiothe pseudocholinesterases.Biochemical Journal, 55,6779.
NAKAMURA, M., ITOH, T., MIYATA, K., HIGASHIYAMA, NNISHIYAMA, S. (1983) Difference in urinary N-acetyl--D-gl
male and female beagle dogs.Renal Physiology (Basel), 6,130NEPTUN, D.A., SMITH, C.N. & IRONS, R.D. (1985) Effect of sa
method on variations in baseline pathology parameters in FischFundamental and Applied Toxicology 5 11805
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6
Assessment of Hepatoto
D D WOODMAN
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Liver damage can conveniently be divided into two main
where direct hepatocyte damage or destruction occurs, awhere the normal flow of bile is reduced without nece
destruction. Within these two groups, however, many differe
identified, depending on the specificity of the original dama
In considering what tests are appropriate for assessing l
important to appreciate the wide variety of processes with w
is also vital to remember that functional deficit and damag
occur quite independently, especially in the early stages of to
6.2Functions of the Liver
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6.3.1Bilirubin
Bilirubin is the breakdown product of haem, the porphy
molecule. It is highly insoluble and toxic and it is partibecause the liver is responsible both for its conjugation with
more soluble, and for its excretion. It is most common to plasma, but the conjugated and unconjugated forms, for
indirect bilirubin respectively, can be measured separately
useful.In man, an increase in plasma bilirubin manifests its
concentrations above approximately 70 mol/L. Cholestasisignificantly in excess of 340 mol/L. The increase in
separated plasma becomes obvious at about 30 mol/L i
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6.3.2Bile Acids
Unlike bilirubin, bile acids are not waste products, but a
cholesterol and secreted into the intestine to act as lipid emubile acids has historically presented great problems, but the
and the availability of enzymatic methods have allowed measurement to become a tenable proposition. In most
practice, plasma bilirubin or alkaline phosphatase has been
confirming obstructive jaundice. Once clinical jaundicsensitivity of bile acids is of limited use, serving only to con
In the sphere of toxicology, the sensitivity of bile apositive advantage. Due to difficulties with bilirubin
measurements in laboratory animals bile acid measuremen
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6.3.3Dye Excretion Tests
The excretory capacity of the liver can be studied by
exogenous dyestuffs such as bromosulphthalein (BSP, sulp
green (ICG) or rose bengal.
Despite the fact that bromosulphthalein clearance (BSP
liver function in 1925 (Rosenthal and White, 1925) it rema
tests available if properly conducted. Other dyes have al
remains the most widely used. When administered intravento albumin (Baker and Bradley, 1966) and to a lesser exten
is removed from the circulation almost exclusively by the h
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of enzyme loss from a relatively small number of cells.
damage, cytoplasmic enzymes may leak from cells whosebeen increased by changes in cellular function. At this sta
subcellular organelles will be unable to escape and will
plasma. If the damage process continues and becomes more
mitochondrial and microsomal enzymes will be released and
plasma changes will be among the first measurable alteratio
This phased release can also act as an indicator of the tim
damage. Plasma half-lives of intracellular enzymes are shor
days at most, so a rapid rise following damage will quickdamage releases more enzymes. This makes plasma enzym
useful in acute studies but severely limits their use in ch
i d d b i i i l d i i h l d d i
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their use has been limited by methodological difficulties, a
(Carakostas et al.,1986).
Cholestasis
Alkaline phosphatase (ALP) has long been the standard en
despite its shortcomings. While ALP has been applied to mo
the rat and cat liver, the high intestinal component in rat
primate plasma activities do not make it an ideal choice
greatly improve the predictive specificity, but the required e
as a routine procedure (Kominami et al., 1984). Alternatused, but none has shown wide advantages over ALP. 5N
used as a more specific liver variant of ALP but wi
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stimulates the liver to synthesize and secrete acute-phase
damage which is minor or in its early stages may paradoxwhile reducing overall synthetic capacity, initially main
concentrations.The common pattern seen following significant hepatoc
in albumin accompanied by a relative increase in ga
synthesized by the B cells of the lymphoid system), often wi
total protein. These changes, however, produce an obvious e
ratio and on the plasma protein electrophoretogram. The
frequently used in the routine evaluation of liver functionalbumin, the calculation of albumin/globulin ratio and the p
pattern.
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Greater interest is being shown in drug-metabolizing en
and decrease. These are not plasma measurements, howeverliver tissue. A variety of demethylating enzymes can be me
can glucuronidating enzymes such as uridine diphosphate gmixed function oxidase system cytochrome P450 (Hinto
measurements are not easy because of the use of tissue rat
direct measurements of liver metabolic capacity and m
pharmaceutical development.
6.3.8Drug-induced Hepatotoxic
The majority of drugs are taken orally and following absorp
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aArithmetic means (and SD) and statistical significances of differen
test are shown. *p
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is dose dependent and rapid (Breen et al.,1975). Despite thi
levels is generally less than that seen with drugs inducing ne
6.3.10Cholestasis
A second major area of drug-induced toxicity is cholestasis.
shown capable of causing cholestasis but in animals
unpredictable. Oral administration of naphthylisothiocyan
produces a highly predictable and dose-dependent cholesta
Cholestasis occurs 1524 h after a single dose of ANIT i(Capizzo and Roberts, 1971) and damage to the cells lining
by electron microscopy 3 h after dosing (Schaffner et al
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Table 6.2aEnzyme activities in r
ANIT administrationa
AP ALT GD
Treatment group Activities, IU/L a
Control 587.3(86.8) 31.0(3.2)
ANIT, 30 mg/kg 576.2(106.0) 35.1(3.2)***
ANIT, 60 mg/kg 986.6(249.6)*** 234.5(129.9)*** 24
ANIT, 120 mg/kg 1285.8(193.8)*** 439.9(129.3)*** 39
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pyridoxal phosphate results in no loss in enzyme activity. Th
seen originally does not reflect any damage to the liver osynthesize the enzyme, but an action directly on the enzym
interferes with the binding of pyridoxal phosphate, aminotransferases, to the enzyme protein. Stripped of its co
has no catalytic activity and is not measured by normal e
treatment with excess pyridoxal phosphate restores the activ
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can provide a highly sensitive means of detecting even min
analytical techniques and the introduction of new indices wmay modify this choice from time to time. Examples of su
the thymol turbidity test and the resurgence of interest introduction of RIA and enzyme assays.
The importance of accurately assessing liver damage
constant re-examination and improvement of the laborator
needs to be done in particular on the peripheral metaboli
enzymes and on the metabolism and excretion of specific b
These areas offer some of the most promising possibilitiesof currently available tests and further clarifying the l
damage.
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DAVISON, S.C. & WILLS, D. (1974) Studies on the lipid compos
reticulum after induction with phenobarbitone and 20-methylchJournal,140,4618.
DE SCHEPPER, J. & VAN DER STOCK, J. (1971) Influence of sexcretion at increased free plasma haemoglobin levels in whole
normothermic perfused dog kidneys.Experientia,27,12645.
(1972) Increased urinary bilirubin excretion after elevated free plasVariations in the calculated renal clearance of bilirubin in whol
de Physiologie et de Biochimie,80,27991.DHAMI, M.S.I., DRANGOVA, R., FARKAS, R., BALAZS, T. &
aminotransferase activity of serum and various tissues in the ratClinical Chemistry, 25,12636.
DOOLEY, J.F., TURNQUIST, L.J. & RACICH, L. (1979) Kinetic
dehydrogenase activity with a centrifugal analyser Clinical Ch
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KORSRUD, G.O., GRICE, H.C. & MCLAUGHLAN, J.M. (1972)
enzymes in detecting carbon-tetrachloride-induced liver damagePharmacology,22,47483.
LEONARD, T.B., NEPTUN, D.A. & POPP, J.A. (1984) Serum gaspecific indicator of bile duct lesions in the rat liver.American J
9.
LEWIS, J.H. (1984) Hepatic toxicity of nonsteroidal anti-inflamma3,12838.
LOMBARDI, B. (1966) Considerations on the pathogenesis of fattInvestigation,15,120.
MARTIN, J.F., MIKULECKY, M., BLASCHKE, T.F., WAGGONBERK, P.D. (1975) Differences between the indocyanine green
men and women. Proceedings of the Society for Experimental B
17
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TUCKER, R.A. (1982) Drugs and liver disease: a tabular compilat
histopathological changes that can occur in the liver.Drug Intel
16,56980.
WHITBY, L.G., PERCY-ROBB, I.W. & SMITH, A.F. (1984)LecChemistry. Liver Disease,pp. 16991. Oxford: Blackwell.
WOODMAN, D.D. (1981) Plasma enzymes in drug toxicity. In Go
Toxicity Testing Methods,pp. 14556. London: Taylor & Franc(1988) Assessment of hepatic function and damage in animal speci
Toxicology,8,24954.WOODMAN, D.D. & MAILE, P.A. (1981) Bile acids as an index
Chemistry,27,8468.ZIMMERMAN, H. (1974) Hepatic injury caused by therapeutic ag
Liver,pp. 225302. New York: Marcel Dekker.
(1978) Hepatotoxicity: The Adverse Effects of Drugs and Other Ch
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7
Assessment of Nephroto
M D STONARD
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7.2Laboratory Investigations
Several of the current screening methods for nephrotoxicity
those used in clinical practice. However, it cannot be assumenzyme which has found extensive application in human cli
have the same diagnostic effectiveness in animal species. N
of certain blood plasma and urinary analytes in combinatio
nephrotoxicity (Berndt, 1976; Diezi and Biollaz, 1979; Pipe
et al.,1988; Stonard, 1990).
When the kidneys are unable to excrete some normal brate, the retention of these materials leads to an increase in t
creatinine and urea. However, urine rather than blood, is th
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detected conveniently by test strips using glucose oxidase
be affected by several interferents, including ascorbate. enhanced excretion because of elevated blood levels or be d
tubules, where glucose is reabsorbed.Changes of hydrogen ion concentration (pH) may reflec
or they may simply reflect dietary protein composition. The
cationic and anionic composition and will be influenced by
the excretion of individual ions, including hydrogen ion
metabolism produce an excess of acid substances, which a
system in the kidney. Urine samples from dog and man
ammonium ions, due to processes of ionic changes that oc
nephron and also reflecting the nature and composition of t
i
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Casts may form in the tubular lumen, and appear to cons
can trap exfoliated cells and debris. Casts may be hyamucoprotein is known to be the Tamm-Horsfall protein, w
region of the nephron and is thought to be involved in selectCrystals may also be detected microscopically in urine s
largely pH-dependent, e.g. phosphates or urates, and
corresponds to regions of the nephron where the tubular
changes in concentration. Crystals may also reflect
administered substance or metabolite whose solubility char
exceeded.
Reduced fluid intake or excessive fluid loss by vomitus
output. Although measurements of urine cations, e.g. sodiu
id f h id f l d f i hi i
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et al., 1981; Viau et al.,1986). However, the selection of
availability of suitably purified proteins and specific antiseraPrimary tubular disorders may be distinguished from glo
of high- and low-molecular-weight proteins in the urineelectrophoretic support media and buffers can provide a
composition of urinary proteins (Boesken et al.,1973; Allch
et al.,1987).
7.4Functional Assessment
The kidneys possess the capacity to filter out, via the g
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plasma or serum urea). Glomerular function, plasma cre
extent, all show variation with age (Corman et al.,1985; GoThe anionic compound,p-aminohippuric acid (PAH) ca
renal plasma flow, since it is filtered by the glomeruli and (Tune et al.,1969). PAH clearance can be used to estimat
the combined processes of glomerular filtration and tub
entering the kidney by the arterial supply almost completely
In the dog, approximately 8090% of PAH in arterial bloo
the venous blood supply leaving the kidneys. The proxim
primary site for the secretion of PAH. However, PAH clear
accurate estimate of renal plasma flow, as part of the rena
non-glomerular pathways.
A d i f A l b d l i
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Table 7.1Localization of renal tu
the rat
Enyzme EC No. Localization
Alanine aminopeptidase
(AAP)
EC 3.4.11.1
(cytosol)
EC 3.4.11.2
(microsomes)
PST>PCT
Alkaline phosphatase (ALP) EC 3.1.3.1 PCT>PST>distal tub
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dilution prior to analysis will suffice. This pretreatmen
essential for most urinary enzymes if erroneous results arremove interfering endogenous substances should be estab
The majority of enzymes show some instability in a hostilshould be assayed as rapidly as possible after any pretreatm
involved in the elimination of drugs, it may be necess
incubation of the enzyme with the drug at concentrations ex
order to demonstrate any potential interference. However
possibility that enzyme activity may be affected by wate
identity may not have been established.
Several experimental studies have established the role of
early markers of renal injury (Hofmeister et al.,1986) but a
h h l di i h h i
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measurements can be more informative than that of total en
1984).
References
ALDEN, C.L. (1986) A review of unique male rat hydrocarbon nepPathology,14,10911.
ALLCHIN, J.P. & EVANS, G.O. (1986a) A simple rapid method f
proteins by agarose electrophoresis and nigrosine staining.Labo
(1986b) A comparison of three methods for determining the concenComparative Biochemistry Physiology,85A,7713.
ALLCHIN, J.P., EVANS, G.O. & PARSONS, C.E. (1987) Pitfalls
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DIEZI, J. & BIOLLAZ, J. (1979) Renal function tests in experimenPharmacology and Therapeutics,5,13545.
ELLIS, B.G. & PRICE, R.G. (1975) Urinary enzyme excretion dur
induced in rats with ethyleneimine. Chemical Biological InteraELLIS, B.G., PRICE, R.G. & TOPHAM, J.C. (1973) The effect of
chloride on kidney function and some urinary enzymes in the do
Interactions,7,10113.EVANS, G.O. (1986) The use of an enzymatic kit to measure plasm
three other species. Comparative Biochemistry and Physiology,
(1987) Post-prandial changes in canine plasma creatinine.Journal
31115.
EVANS, G.O. & PARSONS, C.E. (1986) Potential errors in the mmale rat urine using test strips.Laboratory Animals,20,2731.
FENT K MAYER E & ZBINDEN G (1988) Nephrotoxicity sc
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renal protein fraction containing alpha 2globulin. Toxicology
18292.
LOVETT, D.H., RYAN, J.L., KASHGARIAN, M. & STERZEL, R
in glomerular cells of the rat.American Journal of Pathology,1MAGIL, A.B., MAVICHAK, V., WONG, N.L.M. et al.(1986) Lo
biochemical observations in Cisplatin induced hypomagnesaem
MATTENHEIMER, H. (1977) Enzymes in renal diseases.Annals 7,42232.
NEUHAUS, O.W. & FLORY, W. (1978) Age-dependent changes proteins by the rat.Nephron,22,5706.
PARIAT, C.L.,INGRAND, P., CAMBAR, J., DE LEMOS, E., PIR
(1990) Seasonal effects on the daily variations of gentamicin-inof Toxicology,64,2059.
PESCE A J (1974) Methods used for the analysis of proteins in th
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STROO, W.E. & HOOK, J.B. (1977) Enzymes of renal origin in u
nephrotoxicity. Toxicology and Applied Pharmacology,39,423
TUNE, B.M., BURG, M.B. & PATLAK, C.S. (1969) Characteristi
renal tubules.American Journal of Physiology,
217,
105763.VIAU, C., BERNARD, A. & LAUWERYS, R. (1986) Determinati
urine and in serum, 1. Development of an immunoassay based o
Journal of Applied Toxicology, 6,1859.WERNER, M. & GABRIELSON, D. (1977) Ultrafiltration for imp
Clinical Chemistry,23,7004.WERNER, M., MARUHN, D. & ATOBA, M. (1969) Use of gel fi
enzymes.Journal of Chromatography,40,25463.
Woo, J., FLOYD, M., CANNON, D.C. & KAHAN, B. (1978) Radalbumin. Clinical Chemistry,24,14647.
WRIGHT P J LEATHWOOD P D & PLUMMER D T (1972)
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8Assessment of Endocrine T
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3 interference with the release mechanism for the hormone4 alteration of capacity of carrier proteins
5 alteration of hormone catabolism, e.g. via hepatic or renal
6 interaction with a secondary messenger system, e.g. cyclic
and several of these are exemplified by cases of thyroid toxi
Various methods can be used to detect toxic effects on
include:
1 measurement of endocrine organ mass (absolute and relati
2 histological examination of endocrine organ3 measurement of circulating hormones
4 immunocytochemical examination of endocrine organ
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Table 8.1.1Trophic and releasing
their abbreviations
Hormone (abbreviation)
Adenohypophysis:
Adrenocorticotrophic hormone (ACTH)
Thyrotrophin or thyroid stimulating hormone (TSH)
Follitrophin or follicle stimulating hormone (FSH)Luteotrophin or luteinizing hormone (LH)
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The effects of stress and wide intra-animal variations ofte
of hormone assays in laboratory animals. Effects of procedures on prolactin, thyrotrophin, follitrophin, luteotr
T4 measurements have been described in rats (Wuttke an1972; Dohler et al.,1977; Gartner et al.,1980), rabbits (T
(Garnier et al.,1990), and monkeys (Torii et al.,1993). E
including corticosterone are given in Sections 8.2 and 8.3.
Some hormones show cyclical rhythms and the timing o
factor (Kreiger, 1979). Apart from the obvious changes in
particularly in females, periodic variations of other hormo
Circadian variations for TSH, T3 and T4 have been describeand for testosterone in dogs (Fukuda, 1990). LH, FSH, pr
b h f ll l h h i l h k
Assessment of endocrine toxicity
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MISTRY, A. & VOOGT, J.L. (1989) Role of serotonin in nocturna
in the pregnant rat.Endocrinology,125,287580.
RIBELIN, W.E. (1984) Effects of drugs and chemicals upon the strFundamental and Applied Toxicology,4,10519.
SARGENT, R.N. (1985) Determination of corticosterone in rat plaAnalytical Toxicology, 9,201.
TORII, R., KITAGAWA, N., NIGI, H. & OHSAWA, N. (1993) Efat 30-minute intervals during 24-hour serum testosterone, LH an
Japanese monkeys, (Macaca fuscata). Experimental Animals,4
TOTH, L.A. & JANUARY, B. (1990) Physiological stabilisation o
Laboratory Animal Science,40,3847.
WOODMAN, D.D. (1988) The use of clinical biochemistry for asstoxicology. In Keller, P. and Bogin, E. (eds), The Use of Clinica
Toxicologically Relevant Animal Models and Standardisation a
Animal clinical chemistry 102
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adenosine triphosphate. Thyroid hormones are also known
and messenger RNA (mRNA) synthesis while stimulatingATPase) of the cell membrane.
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normal cuboidal epithelial cells linked at their apical (inner
These cells show microvilli and pseudopodia at the surfaccolloid while their basal surfaces are closely apposed
morphology of the follicles vary according to the functiunstimulated glands show a flattened epithelium and dense
shows tall, columnar epithelium often with the follicular cel
together with a watery colloid in the follicular lumen. The
the highly stimulated gland consists largely of highly active
forming small follicles with small colloid spaces. Interspers
thyroid C (parafollicular) cells. A more detailed description
in Greaves (1990).
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released into the circulation is T4, which is usually consider
Tri-iodotyrosine (T3) is about fourfold more potent than thythe T4secreted undergoes 5-deiodination to the active T3in
ring deiodination of T4 can also occur with the formationreverse T3(rT3).
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0.04% of total unbound). It is the unbound form of each ho
active.The thyroid stimulating hormone (TSH), released from t
most aspects of thyroxine synthesis and secretion. TSiodination of thyroglobulin, endocytosis of thyroglobulin a
colloid with release of thyroid hormones. The rate of release
finely controlled by the amount of thyrotropin releasing ho
hypothalamus and by the circulating levels of T4 and T3.
there is a decrease in the circulating levels of thyroid hor
thyroid function is increased; if, on the other hand, circul
increase, TRH secretion is suppressed and eventually the thand regresses. This dynamic feedback mechanism func
i d bl i l i l l f h id h
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8.2.5.1Interference with Iodide Tra
The iodide-concentrating mechanism, which resides wit
membrane, can concentrate iodide to levels approximately
plasma. Thyroid stimulating hormone enhances the trainorganic ions such as thiocyanate, perchlorate, dithiocarbadichlorodiphenyl trichloroethane (DDT) inhibit iodide trap
Netter, 1974; Kuzan and Prahlad, 1975; Bowman and Rand,
8.2.5.2Interference with Iodide Oxi
The oxidation of the iodide (I) to iodine (I2) catalysed
inhibited by thyrotoxic agents such as thiouracil thiourea
b h b bi d i ifi i i li
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by phenobarbitone, produces a significant increase in liv
proliferation of hepatic endoplasmic reticulum. Induction ishepatic cytochrome P450 and a large number of oxida
oxygenases. The chlorinated hydrocarbon insecticides (Dexhibit induction characteristics similar to phenobar-bitone.
The effect of phenobarbitone-like inducers on thyroid
complex. Animals treated with phenobarbitone show increa
T4combined with enhanced biliary excretion of the hormon
1971). In rats, these changes result from an increased ra
compensated by the release of TSH and enhanced secretion
The second class of inducers, typified by the polycycholanthrene (3MC), do not cause an increase in liver size
h h 448 ( l 19 8) d f h 3
b d lb i Thi h l ffi i f h i d
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bound to albumin. This has a low affinity for thyroxine and,
T4in the rat is about 12 to 16 hours. In addition, the ratio orate of the liver in the rat is 2.5 times higher than in man (L
observations indicate a much higher turnover of the thyroserum TSH is 25 or more times higher in the rat compa
1979). This indicates a much higher activity in the rat t
primates, a conclusion supported by the histomorphology o
often appears to be hypertrophic and hyperplastic, even in h
This is supported by the observation that rats require a
rate per kilogram body weight than do humans to maintain
(Dohler et al., 1979). Several compounds have an indirecbinding. These compounds include phenytoin which reduces
l i i b h d d li l hi h
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1 Th d h it i ti it i i (i th id
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1 The compound has goitrogenic activity in vivo(i.e. thyroidhyperplasia).
2 There are clinical chemistry indications of changes in thyr
(i.e. reduced thyroid hormone and increased TSH plasma3 There is specific evidence that the agent either reduces thy
inhibits iodine uptake) or increases thyroid hormone clearexcretion).
4 A progression of lesions in studies of various duration, sho
and hyperplasia, nodular hyperplasia, and neoplasia (ben
tumours) can be demonstrated.
As for other carcinogens, it is important to quantify the rthreshold dose levels for the various toxicological end
ith a greater than 80% red ction in rat plasma th ro ine
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with a greater-than-80% reduction in rat plasma thyroxine
TSH being observed after five daily doses of PTU (Daviehave been noted in response to direct-acting goitrogens
particularly susceptible to these compounds. Following ethylene thiourea to rats and mice, thyroxine but not T3w
90-day duration of the study. Plasma TSH values, in contra
at 7, 28 and 90 days in the mouse but, in the rat, following
to control values by day 90, despite the continued suppress
(Elcombe, personal communication). However, in the beagl
PTU at the compounds maximally tolerated oral dose fo
plasma T3and T4values until at least day 14. A slight increaapparent after a further 3 days and the maximum va
i l d bl h b d d
1 U t l t 20 t
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1 Use at least 20 rats per sex per group.2 Collect blood samples at necropsy rather than multiple inte
3 Confine the time for collecting the blood to a 1-hour perio
associated with circadian rhythms.4 Samples should be collected at regular intervals after the s
observe plasma/serum changes prior to onset of homeost5 Store the samples at 20 or 70C and analyse all samples
the end of the study.
8.2.8.2Plasma Hormone Clearance Compound-induced changes in hepatic microsomal enzym
8 2 9 Conclusions
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8.2.9Conclusions
There is now a wealth of literature devoted to understand
thyroid-pituitary axis and to describe in detail how the func
be disrupted by various xenobiotics. The rat thyroid glansensitive to adverse effects on these compounds andoncogenicity studies, results in the development of thyroid f
time, ionizing radiation is the only acknowledged human t
well established in experimental systems also. Although hustimuli as do animals, with the development of cellular h
sometimes nodular lesions, even in its moderate to severe
aetiological factor for human thyroid cancer.
Because of marked species differences in thyroid gla
BOWMAN W C & RAND M J (1980) Textbook of Pharmacolo
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BOWMAN, W.C. & RAND, M.J. (1980) Textbook of Pharmacolo
BROWN, C.G. (1987) Application of thyroid cell culture to the stu
C.K. and Steele, C.E. (eds),In Vitro Methods in Toxicology,p.
University Press.
BROWN, C.G., HARLAND, R.F., MAJOR, I.R. & ATTERWILLdoses of novel histamine (H2) antagonist on the rat thyroid glan
Toxicology,25,78794.CAPEN, C.C. (1983) Chemical injury of thyroid: pathologic and m
Toxicology Forum:1983 Annual Winter Meeting, pp. 26073. Company.
CAPEN, C. & MARTIN, S. (1989) The effects of xenobiotics on th
thyroid follicular and C cells. Toxicologic Pathology,17,2669CAVALIERI, R.R. & PITT-RIVERS, R. (1981) The effects of dru
metabolism of thyroid hormones Pharmacological Review 33
HILL R N ERDREICH L S PAYNTER O E ROBERTS P A
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HILL, R.N., ERDREICH, L.S., PAYNTER, O.E., ROBERTS, P.A
WILKINSON, C.F. (1989) Thyroid follicular cell carcinogenesToxicology,12,62997.
HUFF, J.E., EUSTIS, S.L. & HASEMAN, J.K. (1989) Occurrence
induced benign neoplasms in long-term carcinogenicity studies122.
KANEKO, J.J. (1980) Thyroid function. In Kaneko, J.J. (ed.), ClinAnimals,3rd Edn, pp. 491512. New York: Academic Press.
KUZAN, F.B. & PRAHLAD, K.V. (1975) The effect of 1, 2, 3, 4, 8a-hexahydroxyendo,exo-5,8-dimethionaphthalene (aldrin) and
carbomate (Nabam) on the chick. Poultry Science,54,105464
LATROPOULOS, M.J. (1993/94) Endocrine considerations in toxiToxicologic Pathology,45,391410.
LUEPRASITSAKUL W FANG S L ALEX S & BRAVERMA
PENDERGRAST W J MILMORE B K & MACUS S C (1961
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PENDERGRAST, W.J., MILMORE, B.K. & MACUS, S.C. (1961
thyrotoxicosis in the United States. Their relationship with endeDisease,13,2238.
POTTER, C.L., SIPES, I.G. & RUSSEL, D.H. (1983) Hypothyrox
in responses to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin administrPharmacology,69,8995.
READER, S.J. (1987) Assessment of the biopotency of antithyroidcells.Biochemistry and Pharmacology,36,18258.
REFETOFF, S., ROBIN, N.I. & FANG, U.S. (1970) Parameters ofselected vertebrate species: a study of PBI, serum T4, free T4an
binding to serum protein.Endocrinology,86,793805.
RON, E., KLEINERMAN, R.A., BOICE, J.D., LIVOLSI, V.A., FLJ.F. (1987) A population-based case-control study of thyroid caCancer Institute 79 112
(1982c) Desensitisation of rat thyroid to the growth-stimulating act
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(1982c) Desensitisation of rat thyroid to the growth stimulating act
goitrogen administration.Acta Endocrinology,101,5629.
Section 8.3Other Endocrine Org
G.O.EVANSTests for assessing the functionality and injury to several o
than the thyroid glands will be discussed in this section. It
general frequency of toxic findings in these organs (see Sec
role of reproductive and teratological studies in the dreproductive systems.
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important for the biological activity in the target tissues.
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important for the biological activity in the target tissues.
plasma proteins including transcortin (or cortico-steroid albumin; these proteins have differing affinities and binding
Transcortin is synthesized in the liver and it appears to be
and Doe, 1966; Westphal, 1971), but its functions are not w
of hepatic toxicity or excessive renal loss of protein, some
caused by changes in plasma binding proteins.
The mineralocorticoids influence electrolyte transport by
and excretion of sodium, chloride, hydrogen and potassium
pressure homeostasis. The primary mineralocorticoid is al
dependent upon the renin-angiotensin system, plasmaconcentrations. Major changes due to adrenal toxicity, th
h i l l b l i h i h
A circadian rhythm has been demonstrated for plasma a
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A circadian rhythm has been demonstrated for plasma a
Sanchez et al., 1976) and in the rabbit (Vernay et al., 19influence levels of plasma aldosterone (Kotchen et al.,
measurements are of little value as less than 1% of secr
unchanged via the kidneys.
Problems in collecting suitable basal blood samples from
using in-dwelling catheters, as other blood-sampling method
epinephrine, norepinephrine and catecholamines (Kvetnans
1978).
8.3.2Gonads
Table 8 3 1 Major functions of pi
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Table 8.3.1Major functions of pi
Hormone Function
Adenohypophysis
ACTH Stimulates production of glucocorticosteroids by adren
FSH Stimulates ovarian follicle growth and spermatogenesi
GH Accelerates tissue growth.
LH Induces follicular maturation, ovulation, formation of c
secretion.Stimulates androgen secretion in males.
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such as alteration of ovarian microsomal mono-oxygena
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yg
Wilson and Leigh, 1992; Ratcliffe et al.,1993). Chemicalspituitary-ovarian axis can inhibit release of gonadotrophin
effect on ovarian steroid synthesis. Changes in oestrogenic
alterations of immune functions (Luster et al.,1985).
Ovulatory cycles are highly variable in the different spec
patterns of the plasma reproductive hormones together with
cause problems when interpreting results from these assay
8.1). Further complications include reproductive senescence
rats develop irregularities in their oestrous cycle at about ten
changes in plasma hormone levels induced by test compounAlthough some studies have shown correlation between
l i l l i d h l b i di
measure inactive fragments of PTH in addition to the in
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measure the amino- or N-terminal region of the polypeptidassays for the carboxy- or C-peptide fragment, which is bi
calcitonin are both measurable by radioimmunoassay (Selb
PTH values vary with age (Kalu et al.,1983; Kalu and H
status (Talmage et al,1975)
Parathyroid toxicity can sometimes be detected by measu
usually measured as total calcium. As approximately 30 t
bound to albumin with some binding to the globulin fract
total calcium levels should always allow for variatio
measurements of ionized or unbound plasma calcium fractioToxic injury to the parathyroids following acute o
bi i l b h id f i b
(1989) The calcium regulating hormones parathyroid hormone, cal
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McDonald, L.E. (ed.), Veterinary Endocrinology,4th Edn, pp. 9
Febiger.
CAPEN, C.C. & MARTIN, S.L. (1989) Mechanisms that lead to d
animals. Toxicologic Pathology,17,23449.CAPEN, C.C. & ROSOL, T.J. (1989) Recent advances in the struc
parathyroid gland in animals and the effect of xenobiotics. Toxi
CHENG, C.Y., MUSTO, N.A., GUNSALUS, G.L., FRICK, J. & B
two forms of androgen binding proteins in human testes.Journa
563140.
COLBY, H.D. (1987a) The adrenal cortex. In Hedge, G.A., Colby,
Clinical Endocrine Physiology,pp. 12759. Philadelphia: W.B(1987b) The adrenal medulla. In Hedge, G.A., Colby, H.D. & GooEndocrine Physiology pp 297315 Philadelphia: W B Saunde
HAQQI, T.M. & ADHAMI, U.M. (1982) Testicular damage and c
i d d b l i l b l h l d f h l i lb
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patterns induced by multiple sub-lethal doses of apholate in alb
199205.
HILFENHAUS, M. (1977) Urinary excretion of corticosterone as a
function in rats.Naunuyn Schmiedebergs Archives of PharmacoITOH, R. & OZASA, H.K. (1985) Changes in serum lactate dehyd
observed after cadmium administration. Toxicology Letters,28,
JOHNSON, A.N. (1989) Comparative aspects of contraceptive ster
dogs. Toxicologic Pathology,17,38995.JOHNSTON, S.D. & MATHER, E.C. (1978) Canine plasma cortis
by radioimmunoassay: clinical absence of diurnal variation and
and dexamethasone suppression tests.American Journal of VetJONES, M.K., WEISENBURGER, W.P., SIPES, I.G., RUSSELL,
alterations in prolactin corticosterone and thyroid hormone lev
MOORE, N.P., CREASY, D.M., GRAY, T.J.B. & TIMBRELL, J.
fil f d i i i f ll ifi i h
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profiles after administration of cell-specific toxicants to the rat.
43542.
ORTH, D.N., PETERSON, M.E. & DRUCKER, W.D. (1988) Plas
propiomelanocortin peptides and cortisol in normal dogs and dodiurnal rhythm and response to various stimuli.Endocrinology,
PLANT, T.M. (1981) Time courses of concentrations of circulatingtestosterone and cortisol in adult male rhesus monkeys (Macaca
light-dark cycle.Biology and Reproduction, 25,24452.POPPER, C.W., CHIEUH, C.C. & KOPIN, J.J. (1978) Plasma cate
unanaesthetized rats during sleep, wakefulness, immobilization
of Pharmacology and Experimental Therapeutics, 202,1448.RATCLIFFE, J.M., MCELHATTON, P.R. & SULLIVAN, F.M. (
Ballantyne B Marrs T & Turner P (eds) General and Appli
TIMBRELL, J.A., DRAPER, R. & WATERFIELD, C.J. (1994) Bi
f ld l l T i l d E i l N
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uses for some old molecules. Toxicology and Ecotoxicology Ne
VERNAY, M., MARTY, J. & MOALTI, J. (1984) Absorption of e
in the hind-gut of the rabbit. Circadian rhythm of hind-gut elect
British Journal of Nutrition, 52,41928.WESTPHAL, U. (1971) Steroid Protein Interactions,pp. 164350
WHEAT, T.E., HINTZ, M., GOLDBERG, E. & MARGOLIASH, specific multiple forms of lactate dehydrogenase and of cytochr
the mouse.Differentiation, 9,3741.WILSON, C.A. & LEIGH, A.J. (1992) Endocrine toxicology of the
Atterwill, C.K. and Flack, J. (eds),Endocrine Toxicology,pp. 3University Press.
WONG, C.-C., DOHLER, K.-D., GEAELINGS, H. & VON ZUR M
of age strain and season on circadian periodicity of pituitary go
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9Assessment of Gastrointestin
and Pancreatic Toxic
treatment groups and controls) as an indicator of gastrodi f f d d f d ddi i h f h li
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studies of foods and food additives, much of the literatumalignant and vascular disease states, with relativemeasurements.
It is generally thought that rodents and rabbits do not vprimates and cats do exhibit this reflex: when stimulated, fabsorption, toxicity and several biochemical measurements. gavage tube is correctly sited in the stomach, generally avoparticularly in rodents where regurgitation into the oesopharecognized that exposure by inhalation may result in second
While aqueous vehicles such as water or saline may bematerials may be administered with other vehicles such as off i f i ifi i i
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9.2Carbohydrate and Lipid Metab
Body glucose is mainly confined to the extracellular fluid wamounts in the liver and erythrocytes. Glucose enters nutritional sources or by hepatic glycogenolysis. Although constant in laboratory animals, it varies more than in man, maintaining glucose homeostasis removing approximately the portal circulation, and storing this as glycogen. In addi
glycerol, pyruvate and alanineresulting from tissue meglucose by hepatic gluconeogenesis. Thus, it is predictabl
been markedly altered. Blood collection procedures may l l h th i l i bj t t t i l
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plasma glucose where the animal is subject to stress inclu1980).
9.2.2Urine Glucose
Test strips (or dipsticks) commonly used for urinary glglucose oxidase, and these yield semi-quantitative results; thby urinary ascorbate (see Chapter 3). Semi-quantitative mealkaline copper sulphate reagent, can be used to measure
detect reducing substances other than glucose, e.g. fructose.to quantitative methods particularly for the detection of rena
h in the mouse to 5 h in the dog. Urinary amylase is genel i i d b t it ff j di
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amylase is increased, but it offers no major diagnosmeasurements because it is also affected by renal injury and
9.3.2Lipase
Pancreatic lipase is secreted in its active form and this actand bile salts. Other lipasesphospholipase a, phospholihydrolaseare also secreted by the pancreas. Followingassays, plasma lipase measurements are being used in
conjunction with plasma amylase can help diagnosis as pancreas (Banerjee et al.,1989). Some drugs, such as dexa
Table 9.1 Examples of pancreatic
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Table 9.1Examples of pancreatichormones
Hormone and location FunctionGastric antrum and duodenum
Gastrin (from G cells) Stimulates gastric H+secremucosa
Duodenum and jejunum
Secretin Stimulates pancreatic secrCholecystokinin (CCK) Stimulates secretion of pan
(Koop et al., 1982; Larsson et al., 1986), and the peptidsample collection procedures with protease inhibitors
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sample collection procedures with protease inhibitors.
9.5Other Tests of Gastrointestinal and Panc
Pancreatic function tests include the use of synthetic peaminobenzoic acid (BT-PABA) tests, fluoroscein dilauratabsorption, and stimulation of pancreatic enzyme sepancreozymin (CCK-PZ) or the Lundh test meal. As the ex
toxicity from oxygen free radicals, other measurements to radicals may be used in mechanistic studies (Braganza, 1
References
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ARGLEBE, C., BREMER, K. & CHILLA, R. (1978) Hyperamylasexperimental sialoadenosis in the rat.Archives of Oral Biology,
AUNGST, B. & SHEN, D.D. (1986) In Rozman, K. & Hanninen, OToxicology,pp. 2955. Amstersdam: Elsevier Science.
BANERJEE, A.K., PATEL, K.J. & GRAINGER, S.L. (1989) Drugcritical review.Medical Toxicology and Adverse Drug Experien
BERTHET, J. (1963). Pancreatic hormones: glucagon . In von EuleComparative Endocrinology,pp. 41028. London: Academic P
BOULAY, J.P., LIPOWITZ, A.J., KLAUSNER, J.S., ELLEFSON
(1986) Evaluation of a fluorimetric method for the quantitative dog.American Journal of Veterinary Research,47,12935.BOYD E J S RINDERKNECHT H & WORMSLEY K G (198
OMAYE, S.T. (1985) Effects of diet on toxicity testing. FederationPARENT, J. (1982) Effects of dexamethasone on pancreatic tissue
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PARENT, J. (1982) Effects of dexamethasone on pancreatic tissuelipase activities in dogs.Journal of the Veterinary Medical Asso
RAJASINGHAM, R., BELL, J.L. & BARON, D.N. (1971) A com
of mammalian alpha amylase.Enzyme,12,1806.SMITH, P.L. (1986) Gastrointestinal physiology. In Rozman, K. anGastrointestinal Toxicology.Amsterdam: Elsevier Science.
SRINIVAS, M., GHOSH, K., SHOME, D.K., VIRDI, J.S., KUMAK.C. (1986) Glycosylated hemoglobin (HbA1) in normal rhesus
Journal of Medical Primatology,15,3615.SZABO, S., SPILL, W.F. & RAINSFORD, K.D. (1989) Non-stero
induced gastropathy. Mechanism and management.Medical ToExperience,4,7794.
TANI S ISHIKAWA A YAMAZAKI H & KUDO Y (1987)
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10Assessment of Cardiotoxic
Myotoxicity
Table 10.1Plasma enzymes and th lf li (h) i t i ( ft
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half-lives (h) in two species (after
1986)
Enzyme Dog
AST 3.3 to 4.4
CK (total) 0.6 to 16.2
CK-MB 1.3 to 8.1
LDH (total) 1.6
LDHH4 3 3
isoenzymes are numbered according to decreasing anodic m
separation: LDH1 has four H subunits LDH5 has four M su
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separation: LDH1has four H subunits, LDH5has four M suLDH4 are hybrid combinationscontaining HHHM, HHM
(Markert and Whitt, 1975). The widespread tissue distribu
various species (Garbus et al.,1967; Ka