Animal Clinical Chemistry

8/9/2019 Animal Clinical Chemistry

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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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All rights reserved. No part of this publication may be reproduced

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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

Animal clinical chemistry 2


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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

General introduction 3


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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



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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

Study design and regulatory requirement


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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

Preanalytical and analytical variables


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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



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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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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.




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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




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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




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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.




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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




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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




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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

General enzymology 55


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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

Assessment of hepatotoxicity 67


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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

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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

Assessment of endocrine toxicity


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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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107/231



108/231

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



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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



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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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115/231

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



116/231

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



117/231

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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119/231

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

Assessment of gastrointestinal toxicity and pancrea


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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

Assessment of cardiotoxicity and myotoxic


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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

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