"t LI"L

EXPERIlvlENTAL STUDIES IN CHOLESTASIS

ftresis sr:bnitted to tJre Uníversity of Adelaíde for the degree of

Iloctor of Philosophy

ROGER DRË!'I

Departatent of Hr:ma¡ Physiology and Pha:macology

lftre UniversJ-ty of Ailelaide

Souttr Australia

byI

'<

{ra}¡UARY 1976

CONTENTS

DeclarationAcknowledgementsÀbstract of lllresisAbbreviations

GENERAL INTRODUCTION

(a) Introduction(b) BiTe SaTt Metabolism(c) MicrosomaT Mixed lwctìon Oxidase Sgstem(d) Schaffner-Popper Hg¡nthesìs for the Production

of IntrahePatic Cho-Z,estasjs(e) Drug-Induced ChoTestasjs(f) Broad Aims of the Thesis

(i)(ii)(iii)(vÍ)

736

707476

GENERAL METHODS

(a) Illeasuren:r-nt of BiTe FTow(b) PTasma Parameters(c) Estimatíon of Hepatic In Vitto Dtug MetaboTísm(d) MícrosomaT Cgtochrotæ P-450(e) MicrosomaT Cgtochtotte b

"(f) MicrosomaT NADPH Oxidasé@) HexobarbitaT STeePing Time(h) Glucose-6-PhosPhatase(i) Glucose-î-Phosphate Dehgdrogenase(j) Statistics'

CHAPTER ONE: CHOLESTAS]S INDUCED BY ALPHA-NAPHTHYLISOTHIOCYA}IATE

Introd.uctionMethodsResults

(a) The Effect of ANIT on BìLe FLow(b) PLasma Enzgme Changes(c) Effect of ANIT on Líver ÛIeìght and l[icrosomal

Protein YieTd(d) Cgtosol artd. Microsomal- Enzgme Changes(e)TheEffectofAN|TonMictosomaTMixedFunctìon

Oxìdase Activitg(Ð Effect of ANÍT on Hexobarbíta7 STeeping TimeG) Direct Intetaction of ANIT with Microsomes(h) ANIT Interaction with In vitro Incubation Mixtures(i) ANIT Inhibition of In Vitto NADPH Generation(j) Kinetícs of In Vitro Inhibítion bg ANIT

Discussion

2325

2527

33384042464654

CHAPTER TVIO: CHOLESTASIS TNDUCED BY BILE DUCT LTGATÏONPART A: PLASMA AND MICROSOMAL ENZYME CHANGES

77777879202027272722

2730

Introd.uctíon 60

Methods(a)(b)(c)(d)

(e)(t)G)

Surgical TechniquePlasma AnaTgsis.Assessment of Drug MetaboTìsmIn Vìvo PharmacoTogical Activitg of Dtugs DuringBDLBarbiturate Brain SensitivitgHexobarbitaJ- AssagThiopentaT Assag

61626262

63636364

707373

Results(a) Changes in PTasma Parameters after BDL(b) Effect of BDL on Liver tleight and MicrosomaT

Protein YieLd(c) Effect of BDL on Microsomal Glucose-6-Phosphatase

,:. Activitg(d) Effect of BDL on Hexobarbital STeepinq Time(e) Effect of BDL on In Vitro Drug MetaboTism(f) Effect of BDL on the PharmacoTogicaL Activitg of

Batbiturates and ZoxarcTamineG) Batbiturate Brain Sensitivitg

Discussion

IntroductionMethods

(a) Plasma Protein Binding of HexobarbítaL artd ThiopentaT(b) CLeatance of Hexobarbital bg rsoTated Petfused Livers(c) Hexobarbital Bfood Concentrations After

Intrapetitoneal or Intzavenous lnjectìon.Results

(a) Protein BÍndìng of HexobarbitaL and ThiopentalDuring BDL

(b) Clearance of Hexobarbital bg rsoTated PertusedLivers

(c) Pharmacokinetic AnaTgsís of HexobarbitaT BToodDísappearance after InttaperitoneaT otInttavenous Injection

(i) One Compartnent AnaTgsis(ii) nro Compartment Analgsis

Discussion

IntroductionMethods

(a) Acute Effects of Intravenous BìLe SaIt Injections(b) Subacwte Drug Pretreatment on the Responses

to Intravenous Bil-e SaTts in tJ¡e Rat(c) Subacute Combinations of Drugs and BiTe SaLÈs

in RaÈs(d) .Subacute Combinatìons of OraI LìthochoTìc Acid

and Antibiotics in Rabbits

65

65

798383

'cHAprEn rlrro: cHoLESTASTS TNDUcED By BILE DUcr LrcATroNPART B: HEXOBARBITAL PHARMACODYNAMÏCS

CHAPTER THREE: INTERACTION OF BILE SALTS IrIITH CHOLESTATIC DRUGS

90

9797

95

96

96

99707708115

L2L

724

725

725

126

Results(a) In Vitro MicrosomaT I'letabolism aftet Intravenous

Injection of TLC ot TCDC to RaËs(b) The Effects of Intravenous TLC ot TCDC on Rat

Bì7e FTow and Plasma Enzgmes and theit Modificationbg Subacute ChTorPromazine

(c) The Effect of Ergtltromgcin PretreaÞnent on BíIeEIow and Plasma Enzgme TeveLs after IntravenousTCDC in the Rat

(d) Interactìon between Dietarg LitltochoTic Acid andPatenteraT ChTorptomazine ot Ergtl'ttomgcin Estolatein Rats.

(e) Interaction betteen Orai LithochoTÍc Acid andAntìbiotics in Rabbits.

(f) Interaction between Oral LifJtochoTíc Acíd andInttaperitoneaT ChTorpromazìne in Rabbits

Discussion

Introduction. MethodsResults

(a) Production of Hgpertrophíc SER(b) Hg¡nactivitg of the Hgçtertrophic SER(c) Does HHSER cause ChoLestasis?(d) The Effect of HHSER pTus Litltocholic Acid(e) IIHSER ín Rabbits

Discussion

GENERAL DTSCUSSION

SUMMARY AND CONCLUSIONS

APPENDfX Chemicals and Drugs.Sources of

BIBLÏOGRAPHY

727

730

733

736

747

753156

161L62

762763768777777l.75

r77

190

192

193

CHAPTER FOUR: HYPOACTIVE HYPERTROPHIC SMOOTH ENDOPLASMTCRETICULUM (HHSER).

(Í)

DECLARATION

I declare ttrat this thesis contains no material which has

been accepted for the award of any other degreee or diploma in any

University, and to the best of my knowledge contains no material

previously pt¡blished by any other person, except where due reference

is made in the text. Material from ttrís thesis has or will form

part of the following publications.

Drew, R. and Priestly, B.G. CIin. Experim. Pharmacol. and Physiol.

2 , 44L-442 (1975) abstract

Drew, R. and Priestly, B.G. Biochem. Pharmacol. (L976) In press.

Drew, R. and. Priestly, B.G. Toxicol. App. Pharmacol. (L976) In press.

Results of this thesis have also been presenÈed to meetings

of tJle Australasian Society of ClinÍcal and Experimental Pharmacologists

in Sydney L972, Melbourne 1973 and Sydney L974, and to tt¡e

Australian Physiological and Pharmacological Society ourne 1974.

ROGER DREüI

(ii)

ACKNOl.ILEDGEIVIENTS

I would like to ttrank Dr" B.c. Príestly for supervising

my studies and experimental work and for his encour4gement, invaluable

advice and criticism throughout ttre project.

Dr. W.J. OrReilly of the School of Pharmaclz, South Australian

Institute of Technology for computing some of tlre pharmacokinetic data

ín chapter two.

Miss B. Arhns for her excellent technical assista¡ce and

!1s. A. Jaekel and Ms. P. Cassidy for typing the ttresis.

/ The experimental work descríbed herein was carríed out

during the years L972-L975 in ttre Department of Human Physiology

and Pharmacology, University of Adelaide. During this time ttre

auttror was the recipient of a Conuronwealttr Postgraduate Research

Scholarshíp.

(iii)

ABSTRACT OF THESIS

schaffner & Popper (1969) have suggested that intra-hepatic

cholestasis is the result of abnormal bile salt synthesis caused by

hypoactive hypertrophic smooth endoplasmic reticulum (HHSER) in the

hepatocyte.

The aims of the thesis v¡ere: (I) to investigate the function

of the SER in cr-naphthylisothiocyanate (ANIT) and bile duct ligation

(BDL)-induced chofestasist Q) Èo produce HHSER experimentally and

determine whether or not this condition could induce cholestasis;

(3) to evaluate the interaction between cholestatic bife salts and

potentially cholestatic drugs.

The activity of cytochrome P-450 dependent enzlzmes in the

SER \47as determined (a) in vitto in IO5.OOOxg microsomes and in

tO,OOOxg supernatants prepared from liver homogenates, and (b) in vivo

by the duration of hexobarbital sleeping time (HST). Bile flow'

plasma bilirubin, -alanine aminotransferase and -5r nucleotidase were

used to monitor the onset and duration of the cholestatic response.

ANIT: The onset of ANIT-induced, cholestasis preceded

decreases in SER specific activity. During Èhe pre-cholestatic phase

of ANIT toxicity, HST was prolonged but jn vitto drug metabolism in

lO5,OOOxg microsomal fractions r^/as unaffecÈed. Inhibition of substrate

metabolism in toroooxg supernatants from the same fivers was

attributed to residual ANIT or ANIT-metabolites in the in vitro

íncubations. ANIT \ttas shown to be a type I substrate and a potent

in vitro inhibitor of microsomal drug metal¡olism and NADPH generation.

pre-incubat.ion of microsomes with ANIT attered the kinetics of aniline

inhibition from competitive to non-competitive. It is concluded that

(iv)

prolongation of HST during the pre-cholestatic stage is the result of

a direct inhibitory interaction wiÈtr AI.IIT or AlilIT-metabolites.

BDL: It was not untit 48 hours after BDL that hlpoactive

SER was observed. As with ANIT, Ín vítto and :in rzjr¡o methods of

assessing SER activity did not agree during the early stages of

cholestasis but the discrepansy could not be accounted for in a

similar manner to ANIT. Changes in thiopental and zoxazolamine

induced loss of righting reflex, and ptrarmacokinetic studies, in vivo

and jn vitro, showed that the distribution of hexobarbital- was altered

by BDL, which may explain ttre early increase in HST wittrin 24 hours

of BDL.

BiTe salt/drug interactÍons: Various combinations of bile

salts and drugs were adminístered acutely and subacutely to rats and

rabbits. A non-specific interaction, compatible with the production

of intra-hepatic cholestasis, was obtained in rabbits between

lithocholic acid and antibiotics. Since some of the animals which

were cholestatic had normal SER, it was concluded that HHSER htas not

involved in the initiation of the response.

HHSER: IIHSER was experimentally produced. in rats by

simultaneous administration of phenobarbitone and cobalt chloride,

and was shown to be established for a period of at least 5 days.

During this time cholestasis was not detected, nor could it be

induced by adding oral lithocholic acid to the treatment regime.

Similar results were seen in rabbits.

Concl-usions: (t) HHSER is a result of cholestasis and is

not an initiating factor. (2) HHSER and bile salt/drug interactions

are not good toxicological models for investigating cholestasis.

(v)'

(3) Prolongation of HST. does not necessarily reflect impairment of

the activity of cytochrome P-450 dependent enzymes, and this jn vjrzo

test must be ínterpreted witlt caution.

AT

ALT

(vi)

ABBRTVIATIONS

aminotriazole (3-amino-L, 2, A'Etiazole)

alanine arnínotransferase (L-alaníne : 2-oxoglutarate aminotransferase

2.6.r.2.)alpha- naphttry I i so thie cyanate

bile duct tigationchlorpromazine

erytltromycin base

erythromycin estolateerythromycin stearate

times gravity; relative eentrifugal forcehlpoactive hypertrophic smooth endoplasmic reticulum

hexobarbital sleeping timeMichaelis-Menten constant for irùrilcítionMichaelis-Menten constant

spectral dissociation constantlittrochotic acidmicrosomal mixed function oxidase systemsigrnificance-level, probability index, probability of artevent due to chance alonepara-aminophenol

phenobarbitone

rough endoplasmic reticulumstandard error of the meansmootJt endoplasmic reticulumtaurochenodeoxycholic acid

taurolithocholic acid

Aù¡IT

BDL

æz

Ets

EE

ES

xg

HHSER

HST

Ki

Km

Ks

LC

MMTO

p

PAP

PB

RER

SEM

SER

TCDC

TLC

GENERAL II'{TRODUCTION

?¿\

(a) Introduction:

"Cholestasis may be defined as stagnation of bile formed by

the hepatocytes within the intrahepatic biliary passages, with

retention of all biliary substances in the blood." llltis definition

of cholestasis, first proposed by Popper (1968), emphasizes a

disÈurbance of bile flow and excludes other mechanísms leading to

hlperbilin:binaemia, such as altered transport and secretion oÍ

bí1in¡bin.

The clinical picture of cholestasis is one of jaundice'

pruritus(caused by deposition of retained bile salts in the skin),

.r increased serum alkaline phosphatase activity, raised serum

transaminase and cholesterol levels. In drug-induced cholestasis

these symptoms may be accompanied by hypersensitivity reactions

such as skin rash, fever, abdominal and back pain. Histological

evidence consists of ito"t pigrmentation of hepatocytes and Kupffer

cells d.ue to accumulation of bile pigrments, tl:ese cells tend to be

centrilobul-ar in location and may also have a vacuolated and

reticulated appearance termed "feathery degeneration" (GaIl and

Dabrogorski 1964). Inflammation of the portal tracts may also be

present. Visible evidence of stagnated bile in the form of bile

plugs in dilated bile canalícu1i may or may not be seen depending

upon the'gentleness'of the staining procedure and the stability of

the bile pigrments during hístologic processing.

There are two forms of liver disease which fit the above

definition of cholestasis (Popper and Schaffner 1970) and although

both have similar clinical and laboratory manifestations they

have different causes. Extrahepatic cholestasis is the result of

mechanical obstruction of the extrahepatic biliary tract, cholestasis

2

which cannot be attributed to a physical obstruction of bile flow is

termed intrahepatic cholestasis. Most instances of intrahepatic

cholestasis do not have an easily demonstrable explanation and much

effort has been expended searching for the mechanisms of intrahepatic

cholestasis.

As cholestasis refers specifically to the cessation of bile

flow an understanding of the biochemical mechanisms which promote and

regulate the secretion of bile is essential if cholestasis is to be

understood. The secretion of bile is a complex process consisting

of a nr¡nber of steps; these involve the uptake and/or synthesis of a

variety of sr¡bstances by the liver cell, the transport of substances

within the cell, and excretion of hraÈer and organic and inorganic

material into the biliary canaliculi. Bile flow Ís currently thought

ùo Ue promoted by ttrree basic mechanisms. The passive transport of

water from hepatocyte into canaticuli following an osmotic gradient

set up by the active secretion of bíle salts into the canaliculi,

t]¡e bile acid dependenL fraction of bile flow. The passive movement

of water following the active transport of inorganic ions, probably

linked to a sodium pump, through the canalicular membrane, the bile

acid índependent fraction of bite flow. The hormone secretin

facilitates bite flow lower down the biliary tree by stimulatíng

the active secretion of a bicarbonate rich solution into the lumen

of bile ducts and ductules.

The amounÈ of osmotic drive that can be provided for water

movement acïoss the canalicular membrane is dependent upon f-he number

of solute particles in the canalicular lumen. For many years it was

considered that the secretion of bile salts was the 'prime moverr of

bile, providing over 90% of the driving force for fluid flow (Sperber

3

1959). However, since'bile salts possess detergent properÈies the

osmotic gradient that can be provided by them depends upon the

nunloer of micelles that are formed. The characteristics of bile salt

micelles, notably their size and shape, are dependenÈ upon the type

and number of bile salt molecules present and the temperatut. "a which

the micelles are formed (Hofmann and Smal.I L967). At normal

physiological concentrations of bile salts, micelles are small

spherical aggregations, but as bile salt concentration increases,

large rod-like and then lamellar orientations or 'liquid crystalsl

develop. Íhe formation of 'liqr:id crystals' is associated with

a large increase in bile viscosity, this diminishes bile flow and

presents the possibility of precipitation of biliary sr:bstances in the

canaliculi. Monohydroxy bile salts have very poor micelle formíng

ability¡ they are not very water so}-:ble and when present in bile

salt micelles ín a.bnormal quantities exceeding the solubilising

capacity of oÈher bíie salts the monohyd.roxy salÈs are prone to form

'liquid crystals' relatively easily (Hofmann and Small L967). This

is the mechanism postulated for the experimental production of

cholestasis in animals by lithocholic acid, a monohydroxy bile

salt (Javitt and Emerman 1968, Schaffner and Javitt 1966). It

has been proposed that the abnormal production of monohydroxy bile

salts may be responsfüle for the initiation of íntrahepatic

cholestasis in man (Javitt 1969, Schaffner and Popper L969). Thus

the maintenance of a normal bile salt spectrum within the liver

appears to be necessary for the continuance of bile flow.

þ) BiTe SaIt Metabol-ism:

Íhe major biosynthetic pathways of hepatic bile acids

are shown in figure I. Mammalian bile acids are hydroxyl-substituted

4

H

HO-

Fig. I

CHOLESTEROL

OH

26

7e - hydroxylase

(nìicíosomal,

12 o< - hydroxylaso

(microsomal)

5p steto¡d reductase

3q - hydroxysleroid' dehydrogenase

(soluble)

'ox

Â5-3-katostcroid isomarase I

313 - hydroxyslsroid dshyd rog¿n¡s.l (microsomall

H"oH

side chain oxidation

several sleps

( mitochondrial)

HOI

OH

'oH HO" "oxCHOLIC ACID CHENODOXYCHOLIC ACID

Summary of the major biosynthetic pathways of hepatic bile

acids.

5.

derivatives of Sß-chelanoic acid. The fírst step in their synthesis

is microsomal 7a-hYdroxylation of cholesterol. Cholesterol is an

obligatory percursor of the bite acids and this inítia1 hydroxylation

is regarded as being the critícal step for possible feedback

inhibition of overall bile salt synthesis (Shefer et aI L97O' Mosbach

Lg72). Àfter 7o-hydroxylation, the As-double bond is shifted to the

A4 position and the 3cc-hydroxyJ- group, originally present on cholesterol,

is oxidised to a 3-keto grouP by microsomal enzymes to form 7cc-

hydroxycholest-4-en-3-one. The molecule may then undergo microsomal

l2cr-hydroxylation (to form 7cr, l2c-hydroxycholest-4-en-3-one and

ultimately cholic acid) or miss this reaction out to finally become

chenodeoxycholic acid. In either event the A4-double-bond is saturated

and the 3-keto group reduced by soluble enz)¡mes to form di-, or tri-l

hydroxylaÈed derivaÈives of Sß-cholestane. Ttre final step in the

for¡ration of the cholanoic acids, cholíc and chenodeoxycholic

involves removal of the termínal carbon atoms of the side chain

(C-25, C-26 and C-27) which leaves a carbonyl function aE C-24.

These oxidations are mediated by enzymes found predominantly in the

mitochondria and occur via several steps (Carey 1969, Mosbach L9T2) .

Under normal conditions bile acids are secreted into the bile as

conjugates of glycine or taurine; conjugates are the result of the

carboxyl group of the bile acid reacting with the primary amine

group of the amino acid to form a stable amide.

The main physiologic function of the bile acids is to aid

fat digestion and absorption from the intesÈine. As a result the

bile acids are efficiently reabsorbed ancl undergo extensive

enterohepatic recirculation. In the steady state, the net rate of

synthesis of bile salts equals their rate of loss from the body, this

occurs mainly by excretion in the faeces. The total bile acid pool

6

therefore remains fairly constant with hepatic bile acid synthesis

probably being regulated by a negative feed-back mechanism acÈivated

by bite saltsreturning to the liver from the gut.

BiIe acids syntltesized from cholesterol in the liver are

called pr5mary bile acids. However their steroid hydroxyl groups

undergo complex changes duríng enterohepatic cycling to form

secondary bile acids. Thus, the 7cc-dehydroxylation of cholic acid

by intestinal bacteria produces the secondary bile acid, deoxycholic

acid. Si:nilarly lithocholic acid is formed from chenodeoxycholic

acid. Deoxycholic acid undergoes enterohepatic circulation with the

prÍmary bile acids and comprises the third major bile acid of

marnmalian bile. On the other hand lithocholic acid is insoluble

and only trace amounts are normalty absorbed from the intestíne, most

òf ttre littrocholic acid formed ís excreted a'dsorbed to faeces.

Normally littte or no lithocholic acid is slmthesised

directly in the liver (He1strom and Sjovall 1961). However, Javitt

(1969) has postulated that hepatíc formation of lithocholic acid

(or its 3$-hydroxy analog) might occur if oxidation of the cholesterol

side chain preceded ring hydroxylation. It has been clai:ned that

once the side chain has been completely oxidised further ring

hydroxylation is ínhilcited (Berseus and Danielsson 1963). Such an

accumulation of monohydroxy bile salts within the liver might lead to

the production of intrahepatic cholestasis¡ a mechanism by which this

may occur has been proposed by Schaffner and Popper (1969).

(c) Microsomaf Mixed Functíon Oxidase Sgstem-'

The microsomal mixed function oxidase (MMFO) system is

an integral part of the synthesis of bile salts, and since the

7

Schaffner-Popper theory'implicates abnormal activity of this system

in ttre aetiology of cholestasis it is prudent Èo briefly describe

the components of the system so that it may be more easily understood

how ttreir function may be modified by cholestasis. Over the past few

years there have been many reviev¡s dealing with the hepatocyte MMFO

system (Gillette L963, 1966, Glaumann I97L, Estabrook L97L, Fouts L97L'

Mannering Ig7L, Remner L972, Wickramasinghe 1975). OnIy those aspects

which provide background information and are pertinent to the Schaffner-

Popper hypothesis, and to material presented in this thesis will be

discussed in this section.

Íhe MMFO systern" also call-ed the cytochrome P-450 dependent

or microsomal biotransformation system, is located exclusively in the

fraction of tissue homogenate which sedirnents at 105,000x9 after

differential centrifugation i.e. the microsomal fraction of the cell-

1lhe systern supports a number of oxidative reactions and meta-bolizes

a wide variety of exogenous substrates such as drugs, chemicals and

environmental pollutants, and endogenous substrates such as faÈty acids,

steroid hormones and bile acids, The system is essentiatly an electron

transfer complex embedded in the membrane of the SER; it has an

absolute requirement for NADPH and molecular oxygen and catalyses the

consumption of a molecule of oxygen for each molecule of drug or

substrate. One atom of the oxygen appears in the metabolised substrate

and the other in water, the overall reactíon may be represented as

follows:

R-H+O, I2e-+R-OH+H2O-

Although the precise mode of eLectron transfer has been the subject of

much controversy (see Mannering 1971) it is generally agreed that the

systen has two major components essential to its function; cytochrome

p-450 and a flavo-protein, NADPH-cytochrome c reductase.

Clztochrome P-450 is ttre terminal oxygenase of the system and is

regarded as the key component. NADPH-ryÈochrome c reductase is

responsible for the transfer of one electron from NADPH to a

cytochrome P-4so-substrate complex, the second electron may originate

from NADH and be transPorted Lo a cytochrome P-450-st¡bstrate-oxygen

complex via cytochrome bU (Eastabrook 1971). The rate limiting step in

q¡tochrome P-450 dependent biotransformations ís ttre rate of electron

transfere by the reductase, rather than the actual amount of

haemoprotein present.

Cytochromes ¿rre by definition haemoproteins (conjugated

proteins in which tJle prosthetic group ís haem) whose characteristic

/ function is electron transport by äeans of a reversible valency

change in their haern iron atom (ferrocytochromeãerricytochrome) -

C)rtochrome P-450 is widespread in nature and has been found in a

variety of plantsr insects and mammalian tissues (Wichramasinghe

1975); it gets its designation from the fact that in tfie reduced form

it binds with carbon monoxide to form a complex which has an absorbance

spectrum with a peak at 450nm. It is tttis property which enables

quantitative measurement of tl.e haemoprotein. Under physiological

conditions cytochrome P-450 alternates between the reduced and oxidized

forms. Onty the oxidized form is able to bind substrates for

biotransformation. C\ztochrome P-450 itself is strongly bound to membrane

phospholipids, an association that is required for full enzymatic activity.

When the association between haemoprotein and microsomal lipid is

disrupted cytochrome P-450 is converted to its inactive P-420 form'

A wide variety of agents are abie to achieve this dislocation which

frequently involves solubilizaÈíon of cytochrome P-420.

I

Not only does carbon monoxide bÍnding to cytochrome P-45O

produce a characteristic spectral change but sr:bstrates (mainly drugs)

binding to the haemoprotein also produce predictable difference

spectra patterns; these are loosely catalogued as tlpe I or type II

spectra and the drugs producing them as tlpe I or t!T)e II st¡-bstrates.

lYpe I and II spectra are approxj:oate mirror images of each other.

!!pe I compounds give a difference spectra whose maxj:num and minimum

absorption a-re in the wavelength range 385-390 nm and 4L8-427 rm

respectively, while the ùax and lmin of type II difference spectra

are 425-435 nm anil 390-405 nm. The spectral changes are brought

about by conformatior¡al changes in the haemoprotein molecule caused

by drugs binding to different hydrophobic areas of the molecular

complex. The majority of substraÈes ¿rre of the type I varíety, their

reaction site is on the protein moiety of cytochrome P-450. The type

II binding site is associated wittr tt¡e haeme portion of the cytochrome,

ttrus the rar.ge of t1þe II substrates is restricted to those molecules

that can combine with iron as a ligand e.g. amines. It is like1y that

few compounils combine exclusively with either the type I or type II

bincling site and it is probable that the resulting difference spectra

simply reflect preferential binding to one of the sites. Although

binding to cyÈochrome P-450 is an obligatory step prior to metabolísm

tJle production of a difference binding spectra does not guarantee

metabotism and alternatively the absence of a binding spectra does not

preclude meÈabolism.

Tt¡e MMFO system is inducible by its own substrates after

successive exposures. In vivo substrate administration causes a

progressive increase of liver weíght and SER. an increase of flavo-

proteì-n and cytochrome P-450 and subsequent increase of oxidative

activity. Hovrever not all substrates induce the biotransformatíon

10.

system to the same extenÈ. Most drugs and chemicals, for example

phenobarbital, DDT and related compgunds, increase the metabolism of a

nuch larger number of substrates than do the polycylic hydrocarbons

such as 3-methylcholanthrene or 3,A-benzpyrene. It is suggested that

polycylic hydrocarbons cause the synthesis of a modified cytochrome

P-450, called cytochrome P1-450 or P-448, which may have a defective

type I binding site (Remner L972)

Under the electron microscope the morphological appearance of

nuclei, mitochondria and RER of liver cells responding to inducing

agents d.o not differ from their appearance in normal cells. However

the SER is altered from small, hardly visible vesicles to a lattice

work of clearly defined interconnected tubules. These inductive

changes have commonly been regarded as toxic side effects of drugs

and environmental pollutants, however Renuner (L972) prefers to view

them as important adaptive measures which may be of pharmacological

significance. The analogy of induced SER looking "like an ingenuous

se\^¡er system" is very apt. The proliferation of SER membranes

enables the liver to quickly process Iípid soluble material to more

water soluble waste products which can then be excreted. UnforÈunately

some of the chemicals Çhat man has devised have metabolites which

are more toxic than the parent compound (e.9. CC14). Hence the

significance of SER proliferationrand inductíon of the MMFO systern

has to be re-evaluated in each sÍtuation.

(d) Schaffner-Popper Hgpothesis for the Production of Intrahepatic Chofestasis

On the assumption that cholestasis is a hepatocellular

alteration of the secretion of bile-salt-containing micelles, possiJcly

brought about by an increase in the amount of monohydroxy bile sa1ts,

Schaffner and Popper (1969) have put forward a theory to explain how

11

ttre intracellular concentration of monohydroxy bile salts may be

increased. Schaffner and Popper have proposed ttrat cholestasis is the

result of hypoactive hypertrophic smoottr endoplasmic reticulum (HHSER)

ín the hepatocyte.

iltre basic features of the theory are: non-specific he¡utic

injury + hypoactive hypertrophic SER + excess monohydroxy bile salts ->

cholestasis + hypoactive SER.

Hydroxylations of ttre steroid nucleus of cholesterol are the

initial steps in the slmthesis of bile salts (fig. 1). These reactions

are catalysed by enzlzmes which are part of the microsomal mixed

, function oxidase system located in tJ:e smooth endoplasmic reticulum.

It is postulated that impaired ring hydroxylation by hypoactive SER

wi'll force cholesterol to preferentiatly undergo mitochondríal side

ct¡ain oxidation, which in turn will preclude further ring hydroxylation.

If this occurs excess'-monohydroxy bile salts wilt be formed within the

Iiver. Monohydroxy bile salts have low agueous solubility and poor

mìcelIe forming ability, hence the fluidity and character of canalicular

bile wiII be altered and the cholestatic slmdrome initlated.

A basic premise of the Schaffner-Popper theory for tϡe

production of cholestasis is impaired 7c-hydroxylation of the

steroid nucleus of cholesterol. Because only 4% of an intravenous1L

dose -=c-littrocholic acid was converted to other bile acids prior

to bitiary excretion it is generally assr¡ned tlrat man is unable to

further metabolize littrocholic acid (Carey and Williams 1963).

However tJ e development of a micrornethod for the determination of bile

acids in needle biopsies of human liver (Greim et al L9-/3a) and its

adaption for measuring reaction products formed during the incubation

of bile acids with microsomes (Greim et aI 1973b) has enal¡Ied the

r.2.

Schaffner-Popper grouP Èo investigate the jn vitro metabolism of

taurolittrocholate by isolated human liver microsomes. These vùorkers

have shown that human liver microsomes are able Èo convert tithocholic

acid to hyodeoxycholic acid by cytochrome P-450 dependent 6a-hydroxylation

(Trulzsch et al 1972, Trulzsch et aI L974, Czygan et al J-974b). lltris

reaction has not yet been demonstrated to occur in vivo.

It has been found by electron microscopy that excess SER is

formed during cholestasis (Steiner et al L965, Javitt and Emerman

1968) which, in the case of alpha-naphthylisothiocyanate (ANIT)-

induced cholestasis (Plaa et al 1965) or cholestasis induced by bile

duct ligation (BDL) (McLuen and Fouts 196I, Schaffner et al L97Ll 'may have reduced ability to metabolise drugs. Schaffner and Popper

have suggested that HHSER, the initial lesion of cholestasis, can

result from any non-specific hepatocellular injury, from the

administration of steroids, various drugs or injurious chemicals.

Some of these agents are able to stimulate various components of the

mixed function oxidase system and at the same tj:ne interfere with its

normal function. Although the hypertrophic SER may be hypoactive in

respect to certain functions (e.g. those associated witJ. hydroxylation

reactions) other processes of Èhe SER may be enhanced by the increased

amount of membrane. llhus there is increased protein and cholesterol

synthesis in the SER during cholestasis (Lundborg and Hamberget L974,

Stakeberg eÈ al 1974). lltre increased amount of cholesterol may

aggravate the cholestasis by providing more precursor for the

formation of monohydroxy bile salts. Indeed, Schaffner and Popper

postulate ttrat biliary substances retaÍned within the ceII during

cholestasis aggravates cholestasis by decreasing the activity of the

SER, thus creating a vicious cycle which perpetuates the liver disease.

13.

Sínce proposinþ their ttreory for the production of

cholestasis the Popper and Schaffner group have accumulated a large

amount of data showing ttre detrimental effects that bile salts may have

on the MMFO system. Most of this information was obtained in ttre

rat after bile duct ligation (eOL). Using this model of experimental

cholestasis it was for:¡rd ttrat ttre SER became hypertrophied, microsomal

cytochrome P-450 content gradually decreased and arninoþyrine

demethylase and aniline hydroxylase acLivities decreased (Hutterer

et aI L97Oa, Schaffner et al L97L). In particular it was observed

tlrat the in vitro meta.bolism of a¡rinopyrine (a tlpe I sr:bstrate) by

microsomes isolated from ttre livers of BDL rats became depressed

r before the metabolism of aniline (a tYPe II sr:bstrate) . llhese effects

could be reproduced by adding bil-e acids to suspensions of control

microsomes, Thus bile salts, especially dihydroxy bile salts,

produced a typical t-1pe I binding spectra, and at low concentrations

competitively inhibitáa tfre in vitzo metabolism of aminopyrine but not

aniline (Hutterer et aI 1970b). At concentrations greater than l.0mM,

taurochenodeoxycholate destroyed the type I binding site and degraded

cytochrome P-450 (Hutterer et al 1970c). When taurocholate was added

to microsomes four times the concentration.that was used for the

dihydroxy bile salt was required to produce similar effects. Increased

levels of dihydroxy bile salt, chenodeoxycholate, have been found in

livers of BDL rats (Greim et aI L972a) and in obstructed human livers

(Greim et al L9l2b). Íkre fact that t]le in vitro effectsof dihydroxy

bile satts on the MMFO system could be reproduced by nonionic and

anioníc slmthetic detergents suggested that it was the detergent effect

of the dihydroxy bile salts which was producing hypoactive SER during

cholestasis (penf et al 1971). Alpha-naphthylisothiocyanate - induced

cholestasis has also been invesiigated by Schaffner et al (1973) but

discarded as a useful model of cholestasis on the grounds that the

14.

morphologic changes \,{ere different from BDL, the changes ocsurring in the

MMfp system were different from BDL, and the hepatic bile acid pattern

found in Al{IT-induced cholestasis was differenÈ to ttrat found in

norethandrolone-induced cholestasis and BDL (Czygan et al L974a).

It is interesting to note that ttre Schaffner-Popper grouP have not

reported tt¡e in vitro effects of monohydroxy bile salts on tJ:e MMFO

system¡ presumably this is because of the problem of soh:bilizing

ntonohydroxy bile salts in a form which could be added to microsomal

suspensions.

ft has been postulated that minor pathways of bile acid

metabolism may assume greater i:nportance during cholestasis and act asI'safety mecha¡risms against the toxic effects of accumulated hepatic

bile salts (Hutterer et al ]¡972). Ttrus rodents are able to 7cc-and

6$-hydroxylate lithocholic acið. in vivo (Emerman and Javitt L967).

In t]1e rat large increases in hepatic chenodeoxycholic acíd during BDL

are prevented by 7ß-epimerízation of chenodeoxycholic acid to form

ursodeoxycholic acid, followed by $ß-hydroxylation (cytochrome P-45O

dependent) to form ß-muricholic,acid (Greim et aI L972a, Greim et aI

1973b). While chenodeoxycholic acid is a strong detergent,

ursodeoxycholic acid and ß-muricholic acids have no detergent action

at the concentrations in which they are found in the liver. Sinilarly

accumulation of chenodeoxycholic acid in huma¡ cholestatic liver is

postulated to be prevented by l2a-hydroxylation to cholic acid

(Greim et aI L972c, Greim et aI 1973b).

(e) Drug-Induced ChoLestasis.'

Drug-induced hepatic injury has been described as the

"penalty for progress,' by Popper et al (1965) and there are nany

15.

reviews which deal with this problem, (Popper and Schaffner 1959,

Scherlock 1967, Klatskin L969, Berthelot L973, Perez et aI L972, Klatskin

L9?4). Based on Èheir mode of action, hepatotoxic chemicals can be

divided into two broad groups. Ttre so called hepatotoxic drugs are

ttrose which have a direct adverse effect on t}te liver, whose toxic

action is depenclent upon dose and length of exposure and whose effects

are easily and consistently reproducable in laboratory animals.

Ttre second group is made up of those compounds whose hepatotoxic

properties are thought to involve a hypersensitivity mechanism,

nost therapeutic drugs having liver injury as a possi-b1e side effect

are placed in this group. These sensitizing agents do not usually

produce adverse hepatic effects when fj¡st used, but rather on subsequent

administrations. Drug induced hepatitis and cholestasís are generally

regarded as being the result of hlpersensitivity. However, as pointed

out by Perez et al (L972) many d.rugs produce a spectrum of hepatic

reactions varying from pure cholestasis, cholestatic hepatitis

to a picture resembling viral hepatitis. Thus in some instances a

particular agent may produce pure cholestasis and in others

cholesÈatic hepatitis d,epending on ttre circumstances; or the

cholestatic response may be the initial phase of developing hepatitis.

lltre cholestatic potential of.some drugs mainly hormones such as lTcc-a1kyl

sr:bstituted steroid,s are able to provoke intrahepatic cholestasis by

direct toxicity; these are predictable and reproducal¡Ie in animals.

Unfortunately, because most cholestatic drug reactions may involve a

hypersensitivity mechanism, many drugs proven to be cholestatic in man,

do not elicit the same response in laboratory ani:nals. As a result there

are no relial¡Ie methocls for preclicting the cholestatic potenÈial of

drugs.

16.

(f) Broad Aims of the Thesis:

One of the aims of ttris thesis was to investigate the

possibility of using an in r¡irzo interaction between bile salts, and

cholestatic drugs as a toxicological screen for detecting the

ctrolestatic potential of drugs. fhis was part of an overall

investigation of the function of ttre SER in cholestasis

aimed at evaluating ttre possiJcle role of hypoactive SER in the

production of the condition. The problem htas apProached by studying

ttre time course of changes in the MMFO system in two models of

cholestasis, bile duct ligation (BDL) and alpha-naphthylisothíocyanate

(ANfT), and relating these to changes occurring in plasma parameters.

ftre Schaffner-Popper ttreory was tested by measuring changes in the

M!,ÍFO system and plasma parameters after producingr in experimental

animals rthe conditions postulated to be necessary for the manifestation

of cholestasis (i.e. HHSER and excess hepatic monohydro>ry bile salts).

lltre activity of tl:e MMFO system was assessed (a) in vivo,

by measuring the duration of hexobarbital-induced loss of righting

reflex (hexobarbital sleeping time' HST) and (b) in vitto, by

measuring the rate of drug oxidation by isolated hepatic microsomes.

During ttre course of these investigations it was found that in

cholestasis, jn viyo assessment of MMFO activity (i.e. HST) did not

always correlate with t]1e in vitto assessments, and a number of

investigations were therefore initiated to clarify this disparity.

Ttrroughout the experimental work hepatic excretary function

was assessed by monitoring plasma bilirubin levels; plasma alanine

aminotransferase (ALT) actívity was used as an indicator of hepatocellular

damage and. plasma 5'-nucleotidase activity as an index of bile

canalícular damage.

GENERAL IVITT|-|ODS

r7.

OnIy the methods which were used generally throughout the

ttresis are d.escribed in thís section. Mettrods pertaining to individual

experimental chapters are described under the methods section of those

chapters.. The source of drugs and chemicals used in the experimental

work are lisÈed in Appendix A. Male Wistar rats, nominally 200-3009,

were obtained from the University of Adelaide's central ani:nal

breeding house located at the Vtaite Agricultural Research Institute;

ñale DuE,ch rabbits were obtained from either ttre University of Adelaide

or the Institute of Medical and Veterinary Scíence.

(a) Measurement of BiLe FTow:

, Þts were anaethetised with sodium pentobarbiÈone (50m9/kg i.p.)

llt¡e conunon bile duct was exposed after abdominal midline incision

and cannulated with SP-10 polyvinyl cannular tr:bing. After a L5-2O

minute equilibrium period, during which Ehe body temperature was

maintained at 37oc by placing the rat on a warming table, bile flow

was measured gravimetrÍca1ly over lO-minute intervals. Results are

expressed. as mg bile flow per minute per kil-ogram body weight

(¡ng,/min/kg) .

(b) Pl-asma Patameters:

Total plasma bilirubin vras measured by the mett¡od of Nosslin

(1960.) Plasma aminotransferase (ALT), formerly known as glutamic-

pyruvate transaminase (SGOT) (L-alanine : 2-oxoglutarate aminotransferase

2.6.L.2.) was measured according to Reit¡nan and Frankel (1957) and plasma

5r-nucleotidase (5'-ribonucleotide phosphohydrolase 3.1.3.5.) was

measured, by the nickel inhibition meÈhod of Campbell (1962).

18.

(c) Estimation of Hepatic In-Vitro Drug'MetaboLism:

Rats htere killed by stunning and rapid exsanquinatíon.

lltre livers were perfused jn siÉu wittr ice-co1d saline via the porÈal

vein. After the livers were removed and weighed aLl subsequent

procedures were carried out at 4oc. fhe lívers !ì¡ere minced and

homogenised in 2Oml of 0.25M sucrose/O.05M Tris (pH 7.4) using a Potter-

Elvenhjem homogeniser and a motor-driven teflon pestle. llhe homogenate

was centrifuged f.or 20 minutes at 10r000xg, after which time the

supernatant was decanted and centrifuged for I hour at I05,000x9.

The microsomal pelleÈ was resuspended in cold 1.158 KCI (buffered with

O.OU',! phosphate buffer pH7.4) and recentrifuged for I hour at 105,000x9./The washed microsomes were resuspended with cold L15% KCl. Microsomal

protein was determined by the method of Lowry et aI (1951) and

related to the amount of liver originally homogenised.

inopyrine N-demethylase and aniline para-hydroxylase

activities were determined in incubation media as described by

Schenkman et al (1967), except that semicarbazide (4.1mM) was added

to trap formaldehyde produced during aminopyrine demethylatíon.

lltre media contained 5mM substrate and were íncubated with shakíng

for 15 minutes at 37oC. Either washed microsomes (3mg microsomal

proteinr/3ml incubate) or 10,000x9 supernatant (equivalent to

approximately 250mg wet weight l'iver/3 mI incr:bate) were used as a

source of enzyme. Formaldehyde was estimated by the method of Nash

(1953) and para-aminophenol, the metabolic product of aniline para-

hydroxylation, by a modified method of Imai et al (1966). Mercuric

chloride (500Ug) was ad,ded príor to colour clevelopment when l0r000xg

supernatant was used in order to prevent sulphydryl group interference

with the estj:nation of para-aminophenol (Chha-bra et al 1972) .

19.

Usíng this sys-tem the rate of metabolism of aminopyrine

and aniline by IOS,OOOxg isolated microsomes v¡as shown to be linear

with incubation tjmes up to 45 minutes (3mg microsomal protein/3 ml)

and with microsomal protein content up to approxi:nately 15 mg per

3 ml incr-rbaÈe (for 15 minute incr:bations). Símilarly when the 10,000x9

supernatant was used the reaction rates were linear up to I hour

incubation (250mg liver/3 mI) and up to the equivalent of 19 liver

per incr:bate (15 min incubations) .

Values of 13-16 mg microsomal protein per gram of liver for

control rats were consistently obtained during this study. Although

there is considerable variability in the literature on the yield of

i microsomal protein it should be pointed out that the above estÍmation

is approxlmately half Èhat guoted ín the literature. Íhere are a

number of factors that may be involved: (i) t}le strain of rats

available locally, (ii) the equipmenÈ for homogenisation and

centrifugation in tfris laboratory may result in an homogenate from

which a more d.ense peltet may be sedimented during the initial

IOrOOOxg spin, leading Èo the loss of microsomes. For example Lewis

and Tata (1973) have reported a eonsiderable degree of heterogeneity

in the centrifugal force at which various microsomal fractions may be

sedimented,. Àlthough the mierosornal yield is low, the microsomal

cytoch.rome P-450 content and specific activities of aminopyrine

N-demethylase and. aniline para-hydroxylase were coRsístent throughout

the experimenLal work and there is gtood agreement between these control

data and those quoted in the litera[ure (e.9. Denk L972, Schaffner et

al 1973, Czygan et al 1974, Wickramasinghe L975).

(d) MicrosomaT Cgtochrome P-450:

Cytochrome P-450 was determined from the carbon monoxide

20

difference spectrum of thionite-reduced microsomes assuming a molar

extinction coefficient of 91 cm-l m¡,t-l between 45Onm and 49Onm

(Omura and. Sato L964).

(e) IdicrosomaT Cgtochrome bU:

ClzÈochrome bU was determined from the difference spectrum

between NADPH reduced. and air saturated microsomes. Isolated 105r000x9

I4icrosomes were diluted to 2 mg proteinr/ml with O.IM phosphate buffer

(pH 7.4). 3.0m1 of suspension was added to each of 2 matched quartz

curvettes and. a baseline of equal light a-bsorbance run between

390-450nm on a Unicam SP1800 split beam spectrophotometer. 20Ul of

28 NADPH were added to the sample cell, 20UI of distilled water to the

reference cell and the spectrum between 390-450nm recorded after

thorough mixing. lttre cytochrome bU content was determined from ttre

difference spectrum betvveen 424nm and 409nm assuming a molar extinction

difference of 185 "*-l *-1. Resul-ts are expressed as nmols cytochrome

bU per mg microsomal protein.

(f) Microsomal- NADPH Oxid.ase:

Isolated l05r000xg microsomes !ìrere diluted to 5 mg protein,/ml

with I.I58 KCt (buffered witJ: 0.0J-lt4 phosphate buffer, pH7.4). In a

silica curvette 2.0 ml 0.IM phosphate buffer, pH7.4, was mixed with

I.0 mI of the microsomal suspension. 20UI NADPH (11.2m9rln1 in

distilled water) were added and mixed thoroughly and the change in

optical density at 340nm recorded with time. NADPH oxidase activity

was calculated as nmols NADPH oxidised per minute per mg microsomal

protein at room temperature¡assuming a molar extinction coefficient-'t -'lof 6.20 cm * mM ' for NADPH at 34Onm.

2r.

G) Hexobarbital Steepìng tìme (IIST).

Hexobarbital induced loss of righting reflex was measured

according to the technique described by Fouts (1971). Hexobarbital

(I25mg/kg) was administered intraperitoneally, and the rats placed

on their backs on pads of cotton wool on a heating table so that

ttreir body temperature was maintained at 37oC. The end point was

taken as the time at which rats could right themselves three times

in succession. Ttre time between the loss and spontaneous regaining

of the righting reflex was regarded as being tåe duration of

pharrnacological activity of hexobarbital.

, Either, sodium hexobarbital or hexobarbitone acid was used.

Ìùhen the latter was used a solution for injection (25mg,/m1) was

prepared by mixing the required amount of hexobarbitone in

approxi:nateLy 2/3 final volume of saline and gradually adding I N NaOH

until the hexobarbitorie dissolved. Ttre solutíon was back titrated

with a few drops of IN HCI until the hexobarbitone just came out of

solution, at this point 4-8 drops of IN NaOH were added to maintain a

clear solution and the fínal volume made up with saline. It hras found

that there was no difference in HST when either sodium hexobarbital

(dissolved in saline) or the above solution of hexobarbitone was

used.

(h) GTucose-6-Phosphatase:

Gruco s e -6 -pho spha tas e ( D- gluco se- 6 -pho sphate phosphohydrorase

3.1.3.9.) was estimated in 105,000xg isolated microsomes by the method

of Harper (1965).

(i) Glucose-6-Phosphate Ðehgdrogenase:

Rat livers were perfused jr¡ sjtu with íce cold saline,

22.

a portion h/as removed, blotted and weighed. llt¡is was homogenised

(Potter-Elvenhjem homogeniser) for exactly 2 minutes in EDTA 0 "66nll/

saline (O.O4rnl/mg tíssue) at 4oC ¿¡fl .the homogenate centrifuged. for

15 minutes at 20r0OOxg (ooC). [he time between removal of the liver

and beginning of centrifugation was not greater than 5 minutes.

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate :NADP

oxidoreductase 1.1.1.49) activity was estimated in the clear supernatant

according to Lohr and Waller (1965).

( j ) Statistics..

. fte effect of experimental treatment was evaluated by applying

tJ:e unpaired Student t-test ( two tailed) to values obtained from treated

,and control groups of anj:nals. A significance level of p

CHAPTER ONE

CHOLTSTASIS INDUCED BY ALPHA.NAPHTI1YLISOTHIOCYANATE GNIT)

ZJ.

]NTRODUCTTON

Alpha-naphthylisothiocyanate (ANIT) hepatotoxicity has been

extensively studied as a model of intrahepatic cholestasis. ANrr is

very ripid soluble and readily absorbed from the gastrointestinal

tract and distributed throughout the body, it rapidly decreases bile flow

and produces marked hyperbilirubinemia.

N=C =S

ALPHA-NAPHTHYLISOTHIOCYANATE (ANTT)

Most of the earlier investigations were concerned with

histological characterisation of the hepatic lesions produced by

ANIT, which in some respects resemble the l-esions seen in human

biliary cirrhosis. There is a marked reversible proliferation of

bire ducts and.infl-anatÍon of the portal_ tracts, but no detectable

l-iver cell injury und.er the light microscope (Moran and Ungar 1964 r

Goldfarb et aI 1962, Mclean and Rees 1958). Although it is generally

agreed that the biliary cirrhosis is free from biliary obstructionf

some workers thought that ANIT was causing jaundice by selectively

plugging smal-l- intrahepatic bile ductules (Goldfarb et at L962,

Desmet et al 1968) and that there was no, or only slight transient

disturbance of hepatic functíon (Mcl.ean and Rees 195,8, Griffiths

et al 1961). In contrast Steiner et al (1963) examined bile

canal-iculi and hepatocytes by electron microscopy and interpreted

the morphologic changes as disturbances in the excretory function

of hepatocytes. The alterations to cytoplasmic organelles were

regarded as being a nonspecific response to cellular injury (Steiner

and Baglio 1963).

24.

Plaa (1969) has rerziewed ,some of the literature dealing with

the possible biochemical mechanisms of action of ANIT and has also

concluded that the acute toxicity of ANIT is prìmarily due to altered

hepaÈocyte function. This was mainl]¡ based on the fact that ANIT

produces hyperbilirubinemía, BSP retention and affects the hepatic

uptake, storage and excretion of an exogenous bilirubin load long

before ttre cessation of bile flow (Becker a¡d. Plaa 1965, Clodi and

Stenfenelli 1967, Roberts and Plaa L967). Hepatic drug metabolism

is also affected by ANfT prior to a decrease in bile flow. lltrere is

a prolongation of hexobarbital and pentobarbital hlpnosis soon after

oral administration of ANIT, which corresponds wiÈh decreased in

tvitto drug metabolising activity by the Iiver 9,000x9 supernatant

fraction (Plaa et al 1965, Derr et aL ]-967, Buxton et al 1973).

ANIT is metabol.ised by liver microsomes (Roberts 1973) and

there is an increasing arnorrnt of evidence to suggest that metabolism

to a more toxic product may be responsiJcle for part of its hepatotoxic

action. Animals that have been pretreated wiÈh drugs which stimulate

microsomal metabolism show potenEiation of ANTT incluced cholestasis

whiLe inhibitors of microsomal enz)¡ne activity decrease the response

(Rol¡erts and Plaa L965, L966a). Protection against the cholestatic

effects can be afforded by hypothermia and inhil¡itors of protein

slmthesis (Roberts and Plaa Ig66b,Indacohea-Redmoncl et aI 1973). It

has been suggestecl that differentr. or alte::native, metabolic pathways

for the metabolism of ANIT may be responsible for the species variation

in ANIT induced hepatotoxicity (Cappizo and Roberts 1971a). Rats, mice

and guinea pi.gs show significant hyperbíIirubinemia' BSP retention and'

bile stasis while the hamster, rabbit ancl dog develop relatively nild

(if any) hepatic dysfunction (Indacohea-Redmond and Plaa L97L, Phillips

and. Steiner 1964).

25.

ANIT hepatoto>iicity was chosen in this investigation because

it presented a convenient model of chemically induced cholestasis in which

the onset and early sÈages of the condition coutd, be investigated. Íhe

investigation was initiated with three main aims. Firstly to establish

which biochemical parameters changed with the onset of cholestasis,

and coutd be used as a reliable index for the induction of cholestasis

in later toxicity experiments. Secondly, to investigate the relationship

between the early decrease in activity of the microsomal biotransformation

system and the later cessation of bile flow; i.e. does ANlT-induced

cholestasis fit the Schaffner-Popper ttreory of cholestasis? thirdly,

to Ínvestigate possible biochemical mechanisms for the effects of ANIT.

MHITODS

' MaIe albino Wistar rats (250-300g) were used throughout

the investigation and allowed food and water ad libitum. ANIT v¡as

dissolved in peanut oit "r,a administered by oral gavage (2O}mg/kg) ¡

control animals received the appropriate dose of peanut oil (lml/kS) '

At various times after ANIT bile flow was measured by

cannulation of the common bile duct and hexobarbital sleeping tíme

and jn vitro mixed function oxidase activity determined. A blood

sample was taken from the abdominal aorta for plasma estimation of

bilin:bin, AIT and 5'-nucleotidase.

RESULTS

(a) The Effect of ANIT on Bife FTow:

lfhe onset and time course of cholestasis measured by bile

duct cannulation after 2OOmg/kg ANIT (table I.1) was similar to that

reported by Indacochea-Redmond and Plaa (197I), who used an indirect

fluorescein method (PIaa and Becker 1965) to establish cholestasis

26.

Tab1e 1.I. The effect of ANIT on bile flow measured by cannulationof the connon bile duct.

TimeafÈer ANIT Control

BiIe flow (mg/mín/kg)

ÀI{IT t

2 hrs12 hrs

18 hrs23 hrs

3. days

7 days

86.8

86.8

77.O

77.O

67.7

91- r

t 1.52! L.52! 3.44! 3.44r I.94r 1.76

78. 0

72-7

28.5

0.04

32.L

92-I

t 3.60t 12.30t r8.5

t 5.34! 6.L2

(s)

(s)

(A',) *

(3)

(5) *(4)

89.8

83.7

37.0

o

47.3

101.1

(s)

(s)

(4)

(4)

(4)

(4)

*

a. 5/5 ANIT treated raÈs were also shown to be totally cholestaticaE 23 hrs by the indirect fluorescein method of Plaa and. Becker(1e6s).

Significantly different from control values (p

27-

after 300 mg/kg ANIT. During the first 12 hours after ANIT there \¡/as

little change in bile flow, but between 12 and. 24 hours a rapid drop

occurred, such that there was complete cholestasis aE 24 hours.

Zero bile flow 23 hours after ANIT vras confirmed by the indirect

fluorescein method of PIaa and Becker (1965), whereby 5/5 ANTT treated

rats were shown to be totally cholestatic. Ihree days after ANIT bile

flow was 47e. of respective controls and had returned to normal by 7

days. lltre data indicates that the period of complete cholestasis

starts approximately 20 hours after the administration of ANIT

and lasts for approximately another 24 hours.

(b) PLasma Enzgme Changes:

Sígnificant increases in plasma ALT activity occurred almost

immediately (2 hours) after ANIT (fig. 1.I) and prior to the onset of

cholestasis. fn contrast, plasma bilin:bin and 5'-nucleotidase did

not show a significant rise until cholestasis had been established

(24 hours after ANIT). Maximum increases of all the plasma parameters

were reached after two days and coincided r,r'ith the period of complete

bile stasis. After this time plasma levels gradually returned toward

control values with the return of bile flow. fhe haemolysis observed

in chronic ANIT experiments (Goldfarb et al 1962) vüas not seen in this

acute study and therefore did not contribute towards the increase

in plasma bilin:bin levels

(c) Effect of ANIT on Liver Weight and MicrosomaT Protein Yield:

f\¡o and three days after the administration of ANIT there

rrùas a small but significant increase in liver weight per 1009 body

weight, but no alteration in microsomal yield at any of the sampling

times (tabte 1.2). These results agree with Schaffner et aI (1973)

/) c)

't0

)fB0:q)

Ioo:o(1,

840z6

0 *-ts'NUcLEorrDAsE

*

*

*

*

ALT* *

*

I *¡I--I--I--r-- ¡----_ J-_--- r :---rrl-ú

* BILIRUBIN

/ ¡---"'I'--*

--.r.----. t--- -

I

t

c20E6t\t

o-- 0

0

200

0

150 :?

100 JJ

50tr

-oo\E''

E

¡¡

':t\t

EØñÀ

*I

6

i*4

2

234Days after ANIT

567

Fig. l. t . Changes ín plasma levels of bilin¡bin, ALT and 5'-

Nucleotidase after ANIT. Each point is the mean

(t s.e.u.) of at least 4 rats. The sofid line represents

ANIT treated animal-s and the dashed line placebo contro1s.

* Significantly different from control values (p

29.

Table I.2 ftre effect of ÀNIT on liver weight and microsomal proteinyield.

LIVER WEIGHT ( g/LOOg body weight)

Time afterAIIIT Control ANIT

2 hrs12 hrs

24 hrs

2 days3 days7 days1]- days

4. 8r

4.78

4.55

4.24

4.2L

4-23

4.28

t 0.I4t 0.18t 0.70r 0.09! o.241 0.11+ 0.06

4.25

5.22

4. 33

4.59

5 .14

4.62

4"36

! o.2L! o.24r 0.16t 0.06t 0.25! o.22t 0"34

(8)

(8)

(8)

(4) *(4) *

(4)

(4)

(8)

(8)

(8)

(4)

(3)

(4)

(3)

MICROSOMAL YIELD (mg proteinr/g liver)

T.ùne afterANIT Control AI{TT

2 hrs

12 hrs

24 hrs

2 days

3 days

7 days

ll days

14. 0

13.5

I3 .9

L6.66

L4.22

l3.62

12.7 4

1.40 (4)

o.8e (4)

L.O2 (4)

o.2s @)0.68 (3)

L.26 (4)

0.85 (.3)

r3 .8

t3-0

13.5

15. 36

13.3r

10.51

11.99

+

+

+

+

+

+

+

+

+

+

+

+

+

+

L.29

1.10

L.O7

o.47

0.53

o -26

0.73

(4)

(A',)

(4)

(4)

(4)

(4)

(4)

* Significantly different from controls (p

30.

and Denk (L972) who found that liver weight per 1009 body weight and

microsomal protein and phospholipid content' expressed as per gram

Iiver, were not altered after ANIT. However, hlpertrophic smooth

endoplasmic reticulum can be seen in electron micrographs during

AIiIIT toxicity (Schaffner et al L973, Czygan et aI 1974ù. Denk (L972)

claimed that hypertrophy of the SER was produced because the microsomal

yie1d, hrhen expressed as mg protein per whole liver per 1009 body weíght'

was significantty higher than controls. Animals tend to lose weight

during the first 3 days after ANIT (fig. L.2), which depending on the

severity of weight loss, explains why the latter e:

?l

+

+10

5

5

VEH ICLE

4

af ter AN lT

0

oct)

tú

oog)r!

rH

ooLoo-

AN IT

- 10

0 2

Days

I6

Fig. I.2. Percentage change of initial body weight in rats that

received a single oral dose of ANIT (2OOmg/kg) or vehicle(peanut oil 1 nl/kg). Each point is the mean (1 S.E-M.)

of at least 7 animals.

32.

Table 1.3. C)rtosol and microsomal enzlzme changes after AI{IT.

Ti¡ne (n) G6P-DH G6P'ase I{ADPH oxidase

Control

2 hrs

12 hrs

24 }rxs

3 days

(6)

(4)

(4)

(4)

(4)

64.2

66 .5

55.2

62.O

63 .5

6.9

7.O

14.2

6.5

6.5

14.0

16.5

18. i_

18.7

1-2-o

+

+

+

+

+

+

+

+

+

+

0.4

0.7 v

o.2 ,,

0.3 *

0.3 *

7.9 t O.7

7..2 ! t.L

8.3 t 1.1

7.9 10.8

5.9 t 0.3

G6P-DH:

G6P'ase:

glucose-6-phosphate dehydrogenase : Wroblewski units per mg

liver protein. (l Wroblewskí unit = 0.483 I.U.)

glucose-6-phosphatase : 1-tmols phosphate liberated per hour

per mg rnicroSomal protein.

nmols NADPH oxidised per min per mg microsomal protein.ÀIADPH oxidase:

v

*

Significantly different from controls

Significantly different frorn controls

(p

33.

(tirj. L.2) and j-t r.= rråt.d that the glycogen pellet after 105,0OOxg

centrifugation was very much smaller in ANIT treated animals than

in controls. fhe initial increase in glucose-6-phosphatase activity

night therefore be explained on the basis of stimulation due to starvation.

The decrease seen at 3 days occurs at the same time as the maximal

decrease in other enz)rmes located in the SER (table I.5) and probably

reflects destruction of the SER membranes by retained bite constituents

in tTre hepatocytes during the period of ANlT-induced cholestasis.

(e) The Effect of ANIT on lLicrosomal Mixed lunction Oxidase Activitg:

Irhe effects of ANIT on the microsomal mixed function oxidase

activity in preliminary experiments were different from those expected

from Èhe work of PIaa et aI (1965) and Buxton et al (1973) - lllris was

fòund to be due to the different "microsomal" preparations usedi

PIaa et aI (1965) and Buxton et aI (1973) used a 9,0O0xgt supernatant

fraction whereas fOSr-OOOxg isolated microsomes vtere used in the present

study. For the experiments reported in this section aliguots of Èhe

lO,OOOxg supernatant fraction were taken during tJle preparation of

tO5,OQOxg microsomes, thereby providíng activities for the two

different "microsomal" preparatíons from the same liver.

As was found by Plaa et aI (1965) and Buxton et al (1973) the

Ín vitro drug metabolizing activity of the 10r000x9 supernatant was

significantly decreased as early as 2 hours after ANIT administration

(table I.4). During the first 24 hours aniline hydroxylase was

inhibiteil (29-50% of controls) to a greater extent than was

aminopyrine demethylase (55-72% of controls), but this difference

was not as marked after 48 hours. Activities gradually returned to

control values by I1 days.

34.

Table 1.4. The effect of ANIT on the in vitro metabolism of aniline

and aminopyrine by l0,OOOxg liver supernatant.

ANILINE IIYDROXYLASE (rrmols PAP formed,/min/g liver)

TimeafteT ANIT Control AI{IT t

2 h.rs

12 hrs24 }:.rs

48 hrs

72 }rts7 days1l days

13 .20

13 .90

L2.94

L4.2L

Is .63

14.75

13"83

0.59

L.77

o.97

1.10

t.521" 36

0.90

(8) *(4) *(4) *(4) *(4) *(4) *(8)

49.8

39.4

29.8

32-9

28.4

60.9

92.9

+

+

+

+

+

+

+

(8)

(4)

(4)

(4)

(4)

(4)

(8)

6.57 r 0.505.47 t 0.503.86 1 0.124.68 ! O.424.45 ! O.378.99 t 0.6112.86 I 1.06

AII{INOPYRINE DEMETHYLASE (nmols HCOH formed/mín/g liver)

Timeafter ANIT Control ANIT *

2 hrs12 hrs24 }:rs

48 hrs

72 }rrs

7 days1I days

100.8

LLg.2

108.4

130. 5

145 .0

L27 .9

135 .7

6.3

l-6.4

10.4

L2.4

2L.7

8.8

L4"5

(8) *(4) v(4) *(4) *(4) *(4) v(8)

72.5

54.4

59.2

40.0

30.0

65.2

77.3

+

+

+

+

+

+

+

(8)

(4)

(4)

(4)

(4)

@)

(8)

73.I ! 5.764.3 ! 6.264.2 ! 2-652.2 ! 7.I43.5 r 8.683 .1 t 9.6IO4.9 ! 8.7

Y Significantly different.from controls (p

35.

In êontrast, the 1O5,000x9 microsomal aniline hydroxylase and

aminoplnrine demethylase activities were not changed during the first

24 hours of ANIT inÈoxication (table 1.5). It was not until 48 hours

after ANIT adninistraÈion, i.e. 24 hours after cholestasis had been

established that sigrnificant decreases in these enzlzmes were observed.

Ttris also differed from the decrease in activity of the 10,000x9

supernatant fraction in that aminopyrine and aniline metabolism were

depressed to a¡ equal extent. Ttre mixed function oxidase activity

reached a minimum 3 days after ANIT and then gradually retu::ned to

control values by 1l days with the return of bile flow. Changes in

nicrosomal cytochrome P-450 content (tab1e 1.6) closely followed the

¡ changes in in vitto microsomal enzyme activity.

Íhe changes in microsomal mixed function oxidase activity

observed in this study were different to the changes reported by

Schaffner et aI (1973), who re-reported the data of Denk (1972). Íhese

workers found. a decrease in the microsomal metabolism of aniline but

not aminopyrine 3 hours after ANIT (100m9/kg p.o.); after 48 hours the

ìn vitto metabolism of both substrates was decreased, but aniline

metabolism r,lras depressed nore than aminopyrine meta-bolism. Ttris

discrepancy between results may be explained by the lower concentrations

of aniline (0.06mM) and aminopyrine (0.7mM) used by Denk (1972) in his

in vitto assay mixtures compared to the 5mM substrate concentration

used in the present investigation.

llhe difference in in vitro drug metabolism in the f.írsE 24

hours between 10,000xg and I05,000x9 fractions from the livers of AI{IT

treatetf rats suggested that an inhibitor of -i.n vitro metabolism \^¡as

being removed during the preparation of 105,000x9 microsomes. Capizzo

and Roberts (1970) found that in rats treated 4 hours previously with

ób.

Tab1e I. 5. The effect of ANIT on jn vitro I05,000xg microsomalmetabolism of aniline and aminopyrine

ANILINE HYDROXYLASE (nmols PAP formed,/mg microsomal protein/min)

Time afterANIT Controls ANTT t

2 hrs12 hrs

24 t:-rs

48 hrs

72 }:rs7 days

, Il days

o.74

0.90

o.74

1.01

0.81

0.98

o.7 4

1 0.041t 0.05t 0.03r 0.07r 0.04r 0.075! o.o2

0.03

0.05

o.o2

0.055

o.o24

0.05

0.36

(8)

(8)

(4)

(4) *(4) *(8) *(8)

87.7

88. I97.8

56.4

31.9

70.4

48.2

(8)

(8)

(4)

(4)

(4)

(8)

(8)

0.65

0.80

o.72

0.57

o.26

0.69

0.65

+

+

+

+

+

+

+

AI\'lrNoPYRrNE DEMETHYLASE (nmoIs HCOH formed/mg microsomal protein/min)

Time afterANTT eontrols ANIT s

2 hrs

12 hrs

24 }lrs48 hrs

72 };'rs

7 days11 days

8.67

6.85

7.56

6.75

5 .57

7 .16

6.O2

t 0.50r 0.18! o.32t 0.53I 0.31! o.42! o.2L

7.92

6-42

7.26

3.89

1.69

5 .36

5.65

0.30

0. 39

0.18

0.3r0 .15

o.26

0.43

(8)

(8)

(4)

(4) *(4) *(4) *(4)

91.3

93.7

96 .0

57.6

30.3

7L.9

93 .8

(8)

(8)

(4)

+

+

+

+

+

+

+

(4)

(4)

(4)

(4)

* Significantly different from controls (pcO.OI)

Figures are mean + S.E.M. and. the nuñber of rats in each group isshown in parentheses".

37.

Table 1.6. fhe effect'of ANIT on 105,000x9 mícrosomal cytochromeP-45O content.

CYTOCHROME P-450 (nmols/mg microsomal Protein)

Tíme afterANIT Control AIIfT *

2 hrs

12 hrs

24 hrs48 hrs

72 }:xs

7 clays11 days

1.09

1.05

1.14

1. 18

1. 10

1. 02

I. 14

r 0.05r 0.04t 0.04I 0.06t 0.06t 0.06t 0.03

L.O2

0.98

1. 11

0.81

o.37

o.77

1.11

0.04

0.03

0.08

o.04

o.01

0 .05

0.04

(8)

(8)

(4)

(4) *(4) *(7) *(8)

93.6

92.8

97.4

68.6

33 .7

75.5

97.3

(8)

(8)

(4)

(4)

(4)

(8)

(8)

+

+

+

+

+

+

!

* Significantly different from controls (p

38.

ANIT-ruc, 638 of the total liver radioactivity was in the lO,OOOxg

supernatant fraction and that 508 of ttris was in the unwashed microsomes.

Since at least half of the radioactivity in the 10,000x9 supernatant

would be removed during the preparation of 105,000xg microsomes it is

possible that ANIT, or metabolites of ANIT, could act as inhibitors in

the lO,OOOxg preparation but be reduced to less than inhibitory

concentrations in the washed I05,000xg microsomal preparation.

In separate experiments' attempts were made to dialyse the

inhibitors out of the l0,000xg supernatants. fhese experiments h¡ere

not conclusive due to loss of activity in controls, however the

results suggested that the inhíbitor might be partially dialysable,

although Roberts (1973) suggests that the protein binding of ANIT

is irreversible

(f) Effect of ANIT on Hexobarbital STeeping Time:

Hexobarbital sleeping time was significantly prolonged

as early as 2 hours after ANIT, maximum elevation occurred after

48-72 hours and then gradually returned to control values by l0 days

(table I.7) .

The early increases in -hexobarbitat sleeping time could be

interpreted as indicating an early decrease of microsomal mixed

function oxidase activity. As seen in the preceding section, in vitro

105,000xg microsomal activity was noÈ significantly lowered until

cholestasis had been establ-ished, however in vitro drug metabolism

by the IOTOOOxg supernatant was decreased soon after ANIT administration.

Hence the inítial increases ín hexobarbital narcosis correlate with

changes in lOrOOOxg in vitro metabolism and represent in vivo competitíve

inhibition of hexobarbital metabolism rather than a decrease in specifíc

microsomal activity per se. Competitíve inhibition of this nature

39.

Table I.7. Changes in'hexobarbital sleeping tj:ne after ANIT

adninistration.

Hexobarbital sleeping time (min.)

Timeafter ANIT Control ANIT

2 hrs12 hrs

24 l:-rs

48 hrs

72 hrs7 days10 days

20.8

20-8

20.8

34.1

34.2

32.6

28.5

! 2.L! 2.L! 2.L! 2.6t 1.8+ro

! L.2

45.7

39"6

54. 3

62.6

87. 3

56.4

27.2

3.5

2.9

7.4

5.3

6.7

5.8

2.8

(5) *(6) *(5) *(7) t'(8) *(7) *(7)

(6)

(6)

(6)

(7)

(s)

(7)

(7)

+

+

+

+

+

+

+

* Significantly different from control value5 (p

40.

might be effected by AIIIT, or it.s metabolites. During the recovery

phase of the cholestasis there was good correlation between hexobarbital

sleeping time and microsomal nixed function oxidase activity.

(Ð Direct Interaction af ANIT with trIictosomes:

Íhe results of in vitro d-::ug netabolism by 10,OOOxg

supernatant fractions from the livers of ANIT treated rats suggested

tl¡at these preparations are likely to contain sr:bstantial amounts of

Ali¡IT' or ANlT-metabolites, which could act as competitive inhjlcitors

of metabolism. A series of experi:nents were therefore designed to

clarify the nature of the interaction between ANIT and the míxed

r funct-ion oxidase system.

The type of interaction between ANIT and microsomal

cytochrome P-450 was determined as foll.ows" Microsomes \¡rere suspended

in 0.L![ phosphate buffer pH7.4, to a concentration of 2.5mg protein/mI.

Cltochrome P-450 content was 1.15 nmols,/mg protein. Ttre sample and

reference curvettes of a split-beam spectrophotometer (Unicam SP1S00)

each had 3.0 mI of microsomal suspension. and a baseline of equal light

absorbance \Ias recorded. between 370 and 490nm. Microlitre amounts of

a 300 mM solution of ANIT in absolute ethanol were added to the sample

cell such thaÈ the final concentrations of ANIT were 0.05, 0.15, 0,25

and 0.5 mM. Equa1 volumes of absolute ethanol were added to the

reference cel1. The baseline was subtracted from the change in light

absorbance caused by the addition of ANIT to the sample curvette, and

the resul-tant difference spectra plotted.

!{hen ANIT was add,ed to the microsomal suspension a typical

type I difference spectrum was obtained (fi-g. 1.3). The peak lvas at

386rrm, the isobestic point at 402nm and the trough aE 422nm. When the

data was rerJrawn as a double reciprocal plot of change in absorbance,

ltl.

+ 0.01

0

-5

c

422

410 450Wavelength ( n m)

386

ouÊcl

-c¡

otn¡t

I102

d

370 490

t0 15 20

Ir-;rtr

AIôttt

IÀloçoo

300

200

100

50

Ks = 0'14 mM

Spectral- changes caused by consecutive additions of ANITto a suspension of rat liver microsomes.Final concentrations of ANIT were (a) 0.05rnM; (b) 0.l-Srnlvl;(c) 0.25rnM; (d) 0.5mM.Microsomal protein concentration was 2.Smg/rnl andcytochrome P-450 content 1.15 nmols/mg protein. Adouble reciprocal pJ-ot of changes in absorbance at422 nm relative lo 4O2 nm against ANIT concentrationyields a spectral dissociation constant (Ks) of 0.14mM.

T -t-l[mM ANIrJ

Fig. 1.3

-!-

42.

from 402-422rm, against- substrate concentration, the concentration

of ANIT required for half maxi¡nal spectral change (the spectral

dissociation constant; Ks) was 0.14mM.

(h) ANIT Interaction with In Vìtro Incubation Mixtures:

Plaa et al (1965) have shown ttrat ANIT can inhibit drug

metabolism when added directly to jn vitro incr:bation mixtures

containing 10,000x9 supernatant. To ensure that the inhibition of

in vitro metabolism by the 10,000x9 supernatant obtained from the

Iivers of ANIT treated rats was not dependent upon a particular

incr:bation systen, direct inhiloition of drug biotransformation by

ANIT was determined in three different incubation mixtures. fhe

incr:bation systems used were an isocitric dehydrogenase/isocitric

acid NADPH generating system coupled with either 10,000x9 supernatant

or 105¡000x9 microsomesi and an incr.:bation system which utilized

endogenous glucose-6Jpnosphatase in the l0,OOOxg supernatant and

exogenous glucose-6-phosphate to generate NADPH. Ttrese mixtures

are described in detail in table I.8. ANIT (twice recrystalized from

hot ethanol) was dissolved in absolute ethanol so that 0.1m1 gave the

requíred final concentration in the incubation vessels. Incu.bation

mixtures containing 0.lml ethanol but no ANIT served as controls.

lhe results are presented in fig. L.4. ANIT produced ttre

same degreee of inhilcition of in vitro drug netabolism in all of Èhe

three incubation systems, indicating that. in vitro inhibition by ANIT

is indepen

43.

Tab1e I.8. Insubation -media used for estimating in vitro drugmetabolism.

Incubation medium A B c

Tris buffer (pH 7.4)lisC12

NADP

Substrate: Aminopyrine (type 1)Aniline (type 11)

fsoeitric acidI Isocitric dehydrogenasea

L0.5,000x9 microsomal proteinI0,000x9 supernatant

Glucose-6-phosphate

50

5

0.33

50

5

0 .33

5 5

I 8trMapprox. 60 US

6mg

50

5

0. 33

m¡4

mlf

mM

nM5

equivalent to 250 mg liver

4.2 ml'{

Final volume 3.O 3.0 3.0 ml

lftre amount of isocitric dehydrogenase was such Ehat 0.1-0.2 UmolsNADPH vrere generated per min of incubation at 37oC.

a

44

60o

20

AN ILIN E

40

0 -_o--

AMINOPYRINE

40

20

0 o

-6 -5f0 10

o flo

o

o

10- 4 10-3 ß-2 f 0-t

60

o

=4t

octrrl'

ou(¡,

o.

Fig. r.4.

Molar concentration of ANIT

lnhibition of in vitro drug metabolism by addition ofdiffering concentrations of ANrr to various incubationmixtures. Open circles represent metabol_ism by lO5,OOOxgmicrosomes and open squares metaborism by toroooxg supernatantusing an isocitric dehydrogenase/isocitric acid NADpHgenerating system, media A & B of table I.g. Solid squaresrepresent metabolism by 10,000xg supernatant, utilizingendogenous glucose-6-phosphate dehydrogenase and addedglucose-6-phosphate to generate NADPH, medium C of tabler.8- Each point represents the average of 2-3 determinationsfor each ANIT concentration.

45.

was more sensitive to iñhibition than that of aniline, type 11

(10-b¡l ANIT caused a 3OE inhi-bition of aminopyrine demethylation but

only O-IOt inhibition of aniline hydroxylation) which would be expected

since ANIT is a type 1 substrate. However this finding contrasts the

in vitro drug metabolism by I0,000x9 supernatant fractions from livers

of rats treated with ANIT (ta¡te 1.4). In this case aniline metabolism

was inhibited to a greater extent than aminopyrine, which míght indicate

the involvement of an ANlT-metabolite, rather than ANIT ítself, in the

inhibition of drug metabolism by those preparations obtained from ANIT

treated rats.

It is interesting to note that the concentration of ANIT

(0.1n1'{) required for half maxjmal inhilcítion of aminopyrine demethylation

(fig. 1.4) is approximately the same as the spectral dissociation

constant (Ks = 0.14mM) of ANIT. Tl¡ese results differ from those of

Plaa et aI (1965) who. estimated that 100t inhilcition of in vìtro

hexobarbital oxidatíon could be obtained with 1O-stnl ANIT and that

the concentration of ANIT required to depress the metabolism of

hexobarbital (a type I substrate) to 50å of control values was about

Bx10-61¡. Íhis greater sensitivity of in vitro drug metabolism to

inhibition by ÃNIT may be explained by the different incubation

conditions used by Plaa et al. fhese workers used 9'000x9

supernatant, eguivalent to 0.59 liver and incubated in the presence

of ANIT for I hour. Hence there are líkely to be substantial

amounts of ANIT metabolites formed, whose Presence or production

may be more detrimental to cytochrome P-450 dependent reactions than

unchanqed ANIT (see section (i) below).

46.

(i) ANIT fnhibition of In Vitro NADPH Generation:

Besides binding competively with cytochrome p-450, direcÈ

inhibition of jn vitro drug metabolism by AI{IT could be accomplished

by liniting the production of NADPH during incr:bation.

The effect of AI.IIT on NADPH generation by isocitric

dehydrogenase/isocitrate and glucose-6-phosphate dehydrogenase/glucose-

6-phosphate systems is shown in fig. I.5. 1lt¡e mixtures v/ere A and C

of table 1.8. and ttre prod.uction of NADPH \iras followed by the change

in absorbance at 340nm. Both methods of generating NADPH were

ínhibited by ANIT. In separate experiments the addition of up to

/ lOymols of NADPH to incubation mixtures containing 2x1O-4¡¡ eUlt

failed to prevent any of the inhibitory effects of /\t{IT on clrug

metabolism; indicating that the inhibition of NADPH production is

probably not the major mode by which ANIT directly inhibits jn vitro

drug metabolism, hor.ir it cannot be discounted as being a contributing

mechanism.

(i) Kinetics of In Vítro Inhibition bg ANIT:

Preliminary studies of the kinetics of in vitro inhiJcition

of the microsomal mixed function oxidase system by ANIT revealed that

the amount of solvent used for AItrIT addition had a marked effect on

metabolism. ANIT was first added to incubation mixtures in 0.lnl

(1.7lmMol) absolute ethanol, but t-his quantity of ethanol produced

a substantial inhibition of aniline hydroxlzlation and aminopyrine

demethylation that appeared to be interfering with inhibitory kinetics

of ANIT. The amount of solvent was reduced to 51tl (0.085mMof) of

absofute ethanol which had no effect on microsornal aminopyrine

demethylation and produced either non-competitive or competitíve

L.7

75

.9Ë€50F

;o)rüs ôËia¿o(JLoo-

0-6

10-4

10

Molar concentrat ion ANIT

-DÉ10

Fig. I. 5 . In vitro ANIT inhibition of NADPH formation by isocitricdehydrogenase,/isocitrate (circles) and by glucose-6-phosphatedehydrogenase/glucose-6-phosphate (diamonds) . These systemswere rnedia A 6( C of table 1.8 but minus the 105,000xg andIO,OOOxg liver fractions. ANIT was added in 0.1 mIabsolute EtOH and results are expressed as the percentageinhibition of NADPH generation (at room temperature)relative to control incu-bations which received only 0.I mlEtOH. Points represent the average of two separatedeterminations for each ANIT concentration.

48.

inhibition of aniline hjrdroxylation (see fig. L.7). Competitive

inhibítion of aniline metal¡olism has been previously reported

(nu¡in et aI 1970).

In vitro inhibition of microsomal aniline and aminopyrine

metabolism by ANIT was investigated in the presence of varying amounts

of ANIT and ttre Michealis constant for inhibition (Ki) determined

(fig. 1.6). Ítre Ki of inhibition for both anitine hydroxylation and

aminopyrine demethylation was 0.lmM ANIT, this agrees with the previous,

but cruder, estimate of 0.1m14 ANIT (p45, - fig. 1.4) and is also very

similar to the spectral dissociation constant for ANIT (Ks = 0.14nM,

fis I.3).

The type of inhibition was further elucidated by Line!ìreaver-

Burke kinetic plots. ANIT inhibited the jn vÍtro hydroxylation of

aniline (fig. I.7) in a mixed-competitive manner but non-competitively

inhibited the in vitr6 demethylation of aminopyrine (fig. 1.8). Ttris

finding was surprising since ANIT is a type I substrate and as such would

be expected to competitively inhiJcit the netabolism of aminopyrine (type I)

rather than only aniline (type 1l). It also clisagrees with Czygan et al

(L974a) who found competitive inhibition of aminopyrine metabolism by

ANIT. It is not unusual that ANIT was able to conrpetitively inhibit

type 1I metabolísm, SKF 525-A (type 1) conpetitively inhibits the

in vitro meta-bolism of both aminopyrine and aniline (Schenkman et aI

L972). Prior incubation of metabo-l-ising systems with SKF 525-A before

the addition of aminopyrine and aniline changes the kinetics of

inhibition from competitive to non-competitive, this is attrilcuted to

the formation of a metabolite of SKF 525-4.

By analogy with sKF 525-A it was reasoned that the non-

competitive inhibition of aminopyr:ine meta-bolism by ANIT may be due to

Ki=0'1 mM

-10

0.3 3'0

0.2 2.0

0.1 1.0

0-5

49.

Aminopyr ine

Aniline

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