Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
"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
10
¡
.E
'õ
oÊ,CD
EÈ-o-ØoEtrv
E
'õoo.ct)E