Gut Supplement, 1991, S34-S39

Drug metabolism and hepatotoxicity

J M Tredger, M Davis

Drug metabolism in the 1960sAt the time the Liver Unit was established in1966, interest in the study of drug metabolismwas growing, stimulated largely by the phar-maceutical industry, and the legislative require-ments imposed upon it. It represented aremarkable change from the situation a few yearspreviously when'. . . the study ofthe metabolismof foreign compounds was not a popular one;biochemists considered it rather a waste of timeand effort, both of which could be betterexpended on looking at naturally occurring subs-tances. Pharmacologists also had little interest inthe subject and regarded it as unrelated to theirimmediate problems. Toxicology was in a primi-tive state of development and still largely con-cerned with cataloguing and describing theeffects of various poisons'.' By the 1960s, how-ever, centres in Britain, Europe, and the UnitedStates had begun to characterise the drugmetabolising enzymes and their responses torepeated drug ingestion. In 1966, novel discov-eries emphasised: (i) the existence of more thanone form of cytochrome P-450,2 the basic func-tion3 and characterisation45 of which had beendefined only in the previous three years; (ii) theclinical implications ofenzyme induction to drugtherapy, as illustrated by three case reports, onedescribing fatal haemorrhage after withdrawal ofthe enzyme inducing sedative chloral hydrate in apatient on anticoagulants,' and two showingrelief of paediatric jaundice with phenobar-bitone; 8 (iii) the hepatotoxicity ofenvironmentaltoxins, after the outbreak of Epping jaundiceresulting from contamination of flour with 4,4-diamino-diphenylmethane.9 The limited under-standing of the mechanisms of hepatotoxic drugreactions at that time can be gauged from a 1969review by Schaffner and Raisfeld."' An abun-dance of important interactions between chemi-cals and endogenous biochemical pathways hadbeen documented, however, as exemplified inthe review ofcarbon tetrachloride hepatotoxicityby Recknagel" which continues to complementcontemporary texts. 12

In 1967 Conney published his almost timelessreview of the agents responsible for enzymeinduction in which he predicted that 'the geneticmake-up ofthe individuals ofa population withina given species may be important in determiningthe occurrence or magnitude of enzyme induc-tion'.'3 Support for these prophesies was someyears away, but the monitoring ofenzyme induc-tion in man and its clinical features and benefitswas the starting point for the King's Liver UnitDrug Metabolism Group in 1966. Studies on theclinical value of phenobarbitone in the treatmentofunconjugated hyperbilirubinaemia dominatedthe late 1960s 1116 and prompted the search foralternative inducing therapies with fewer hyp-notic side effects."' In 1965 Aarts" had suggestedthat urinary glucaric acid excretion might reflect

hepatic drug metabolising enzyme activity. Overthe next five years interest at King's centredaround screening for enzyme induction bymeasuring the urinary excretion of this com-pound.'9 The test was validated by showing anassociation with hepatic cytochrome P-450 con-tent,20 and was used to show the enzyme inducingproperties ofdrugs2' and environmental contami-nants such as DDT.22 The observation thaturinary D-glucaric acid excretion rose during thecourse of normal pregnancy indicated thatchanges in hepatic microsomal enzyme activitycould be associated with endogenous compoundsand physiological states.23 Other studies showed acorrelation between urinary D-glucaric acidexcretion and low circulating levels of calciumand folic acid in epileptic patients.2425 Thisprompted the suggestion that the enzyme induc-ing properties of anticonvulsant drugs could leadto increased consumption and eventual defi-ciency of vitamin D and folic acid.

The 1970s: the concepts ofenzyme multiplicityand covalent bindingThere was worldwide activity in cataloguing themetabolism and effects of many hundreds ofdrugs and chemicals throughout the 1970s, notleast in relation to the molecular features ofenzyme induction and inhibition. A growingawareness emerged of the multiplicity of bothphase I and phase II metabolising enzymes, withdifferent forms showing a specific yet overlap-ping capacity to catalyse particular drug transfor-mations, as well as a selective responsiveness toboth enzyme inducers and inhibitors.Perhaps the most significant advances of the

1970s in relation to drug induced hepatotoxicitywere the experimental observations by Gillette,Brodie and colleagues at the National Institutesof Health, USA, of the role played by reactivedrug metabolites,2" their covalent binding tocellular macromolecules27 and the hepatoprotec-tive role of glutathione.2" In retrospect, it isperhaps remarkable that 20 years had elapsedsince the Millers had first observed a similarphenomenon in aminoazo dye induced carcino-genesis.29 Their comment in 1966 after 20 yearswork that'. . . we are still not in a position todefine the molecular mechanism by which achemical induces cancer'3" had a prophetic paral-lel in arguments that raged for many years on therelevance of covalent binding of reactivemetabolites to chemical and drug induced liverdamage.The Liver Unit's interests in this area focused

initially on paracetamol hepatotoxicity.3' Suicidaloverdose with paracetamol was becomingincreasingly common in Great Britain,32 and thegrowing number of referrals of such patients tothe Liver Unit allowed the metabolic basis forhepatotoxicity from the drug to be studied in

Institute of LiverStudies, King's CollegeSchool of Medicine andDentistry, LondonJ M Tredger

Gastroenterology Unit,Royal United Hospital,BathM DavisCorrespondence to:Dr J M Tredger,Institute of Liver Studies,King's College School ofMedicine and Dentistry,Bessemer Road,London SE5 9PJ.

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DrugMetabolism and Hepatotoxicity

Paracetamol glutathione

PARACETAMOL

| | Paracetamol sulphate

2 electron1 election

f%viAe#-^xdation Uxwdation

P-450 IIEP-450 IA2

N-Acetylbenzoquinoneimine Parac

Free radical Glulathione

Intermediates deletatione

Covalent

Lipid bindingPeroxidation

Macrophageaccufmlation

Impaired calcium homeostasisand cellular function

Parenchymal / non-parenchymal cell damage

Cell death

HEPATIC NECROSIS

,etamol Glutathione

Figure 1: Current concepts in the mechanisms ofparacetamolinduced hepatotoxicity.

man. Early investigations showed that liver dam-age arises after ingestion ofhigh doses ofthe drugwhen the balance in metabolism switches fromthe detoxication pathways of glucuronide andsulphate conjugation to cytochrome P-450-dependent activation steps.33 This leads to anincreased production of an unstable metabolitewith the ability to bind irreversibly to liver cellproteins. Normally this metabolite is detoxifiedby conjugation with glutathione, but stores ofthis nucleophile become depleted leaving thereactive metabolite free to exert its toxic effects.By showing that the same mechanisms applied inman' (Fig 1), the value of studies in animalmodels for developing treatment regimens wasemphasised and stimulated the study of potentialnew antidotes. This led to the clinical use ofmethionine,35 cysteamine and dimercaprol,' ini-tiating an interest in the treatment ofparacetamolinduced fulminant hepatic failure discussed else-where in this volume.

Idiosyncratic drug hepatotoxicityIn the mid-1970s, no role had yet emerged forreactive metabolites in idiosyncratic drug tox-icity (hepatotoxicity produced unpredictably bytherapeutic doses of drugs in only a minority ofsusceptible individuals). Zimmerman, in hisauthoritative and still invaluable text of 1978commented that 'relatively few experimentalapproaches have thrown any light on the hepaticinjury caused by host idiosyncrasy.37 Within twoyears, however, important observations on indi-vidual variability in the rate of metabolism of

drugs and in the responses to their potentiallytoxic metabolites was to transform this area.

It was well known that the capacity of anindividual to acetylate drugs is genetically deter-mined,38 with the population divided into rapidand slow acetylators. Research from St Mary'sHospital Medical School, London showed that asimilar polymorphism applied to a pathway ofoxidative metabolism, the hydroxylation ofdebrisoquine.39 This provided a potential fordetermining susceptibility to liver damage fromsome therapeutic agents as a result of differencesin the rates at which individuals metabolised adrug through 'safe' or potentially hepatotoxicpathways. It was some years, however, beforewell characterised examples emerged. Thus, thehalf life of the antianginal drug, perhexilinemaleate, is much prolonged in poor hydroxyla-tors of debrisoquine& and this seems to beassociated with drug accumulation and micro-vesicular phospholipidosis.4' Different consid-erations apply to liver damage from theantituberculous drug isoniazid. Slow acetylatorsof the drug metabolise more through oxidationwhich can produce a reactive hydrazine deriva-tive, and there is a higher incidence of liverdamage amongst individuals with the slowacetylator phenotype.42 Genetic variations in oxi-dative metabolism are also likely to be important,as are responses to environmental influences. Forexample, concomitant exposure to rifampicininduces the metabolism of hydrazine andincreases the risk of hepatotoxicity.43Another factor determining susceptibility to

some types of hepatotoxic drug reaction is thecapacity to mount an immune response againstdrug altered liver components. A series of inves-tigations carried out in the Liver Unit identifiedan antibody directed against a hepatocyte surfaceantigen altered by a halothane metabolite.' Thealtered antigenic determinant probably resultsfrom oxidative halothane metabolism45 whichgenerates trifluroacetylated proteins"" (Fig 2).It is likely that all individuals exposed to the druggenerate altered hepatocyte membrane determi-nants,49 but only a small minority mount animmunological reaction against them.50 The factthat many patients with severe halothane hepa-titis have circulating antibodies directed againstother organs in the body5' strongly suggests anunderlying, genetically determined defect inimmune regulation.52 In contrast, some patientswith hepatitis from the drug have no evidence ofimmune involvement, and liver damage in thesecases is probably the result ofoverproduction of ahepatotoxic derivative produced by reductivehalothane metabolism. Preferential stimulationof this pathway in experimental animals pro-duces dose related hepatotoxicity53 (Fig 2).

Subsequently, the methods validated withhalothane have been used to show a possibleimmune basis for drug hepatotoxicity froma-methyldopa54 tienilic acid55 and ethanol.56 Inaddition to expressing an antibody reacting withtienilic acid derived neoantigens, more than 50%of patients with tienilic acid hepatitis produce anantibody (a-LKM2) directed against a cyto-chrome P-450 II isoenzyme (P-450 8; P-450MP). Both this and the analogous LKM1 anti-body (a-P-450 IID) have been associated with

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Tredger, Davis

Figure 2: Mechanisms underlying predictable and immune mediated hepatotoxicity fromhalothane.

autoimmune chronic active hepatitis57 and arediscussed in greater detail elsewhere in thisvolume.These and many other studies from groups

throughout the world have given support to HansPopper's thesis that improved understanding ofunderlying mechanisms could turn idiosyncraticdrug reactions into predictable ones.58

The 1980s: linking in vitro events with in vivoconsequencesThe profile of multiple cytochrome P-450 iso-enzymes present in different individuals is deter-mined primarily by genetic factors, but can bemodified by environmental manipulations, par-ticularly exposure to substances which induce orinhibit these enzymes.59 The implications of thisheterogeneity to drug metabolism and hepato-toxicity were to dominate most ofthe 1980s. Theidentification of multiple enzymic forms wasachieved initially with chemicals (specificinducers and inhibitors), then by purificationand isolation, and subsequently with specificantibodies which competitively inhibited sub-strate transformations. At the start ofthe decade,a role for gene probing of drug metabolisingenzymes was still some way off, with no mentionofit as an investigative tool made by Lu and Westin their 1979 review of the multiplicity of cyto-chromes P-450.6 Accumulating knowledge,however, showed that association could be madebetween the metabolism of a specific drug anddefined P-450 isoenzymes. This made it increas-ingly possible to predict which enzyme inducing

drugs might influence that compound's metabol-ism and which agents could competitively inhibitits clearance. So arose the concept that P-450doctors might emerge as a future subspeciality.6'Experimental studies in the Liver Unit weredirected towards characterising the enzymeinducing properties of rifampicin.62 Clinicallysignificant effects were demonstrated on cor-ticosteroid requirements in asthmatics63 and ontheophylline pharmacokinetics.TM Rifampicinalso induced the metabolism of testosterone andits hydroxylation at multiple sites on the mole-cule provided a powerful method for investigat-ing enzyme induction.65 6 The potential forurinalysis ofendogenous steroid metabolites as ameans of fingerprinting cytochrome P-450 iso-enzyme profiles in individuals, however, wasovertaken by the upsurge in molecular biologytechniques. These not only added a furtherdimension to detection and characterisation, butalso provided a long awaited basis for logicalclassification and nomenclature of a host ofP-450s previously existing under numerousaliases.

In the late 1970s and early 1980s enormousinformation accrued on the potential mecha-nisms by which drugs caused and antidotesprevented hepatic damage. A large number ofstudies were carried out in vitro and used singleend points such as covalent binding, depletion ofintracellular glutathione or the impairment ofcellular and biochemical events. The dangers ofinterpreting such in vitro observations in isola-tion soon became clear, however, stressing thevalue to toxicology of in vivo investigations forseveral reasons. First, the interplay betweendifferent enzymes acting on the same substratewas difficult to assess except in vivo, becausedrugs are not metabolised by a single isoenzyme.Secondly, competing detoxication pathwayscould determine a drug's toxic potential. Inaddition, non-parenchymal cells were increas-ingly being shown to be important in mediatingand promulgating hepatotoxic events,67 and theemerging complexity and polymorphism ofcyto-protective mechanisms could also influence sus-ceptibility to tissue damage. Experimentalstudies in the Liver Unit confirmed that chronicingestion of ethanol potentiated paracetamolhepatotoxicity, whereas acute administrationprovided a protective effect.68 These findingscould not be explained solely by induction orinhibition of the enzymes producing the toxicmetabolite, however, as had previously beenconsidered to be the case,69 and complementaryroles for effects on glutathione depletion andintermediary metabolism were proposed.70 Morerecent evidence from convincingly large series ofpatients suggests that chronic alcohol consump-tion and anticonvulsant therapy adversely affectoutcome after paracetamol overdose (Bray et al,in preparation).

From 1986 ... to the futureAdvances over the past five years have deendominated by the characterisation of geneticfactors regulating the control of drug metabolis-ing enzymes, together with an increased under-standing of the roles of glutathione and calcium

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Figure 3: Pharmacological influences on the response tocyclosporine immunosuppresswn.

homeostasis in mediating and moderating tissuedamage. Gene probing has provided a molecularbasis for phenotypic expression of extensive andpoor debrisoquine hydroxylation, where func-tionally defective gene products rather thancomplete gene deletions appear responsible.7'Parallel studies are strengthening links betweenthe functional activities of structurally relatedisoenzymes across species; the extent of struc-tural homology is providing clues as to the mostappropriate animal models for drug metabolismin some cases. Experimental work is also begin-ning to clarify the role ofendogenous compoundsin regulating drug metabolism. For example,glucocorticoids have been shown to enhance thetranscriptional activation of both cytochromeP-450 IA and glutathione S-transferase genesafter enzyme induction by polycyclic aromatichydrocarbons.72 The glutathione transferase iso-enzymes are important in a transport andmetabolic capacity and their polymorphism maycontribute towards variability in the intracellulartransport ofdrugs and the activity ofdetoxicationpathways with glutathione.73 Also emerging hasbeen the importance of subcellular glutathionepools and their relevance to the regulation andmaintenance oforganelle function - for example,the mitochondria.'4 There is increasing interestin the role of interorgan glutathione homeostasisin determining toxic events outside the liver. Forexample, the kidney and lung extract glutathionefrom the plasma pool, which is apparentlyreplenished only by the liver. Glutathione alsomaintains thiol/disulphide status, which in turnis crucial to the maintenance of subcellularintegrity by calcium, so controlling the activity ofplasma membrane ATPases, mitochondrialdehydrogenases and the activation of phospho-lipases and endonucleases. Disturbance of gluta-thione and calcium homeostasis is seen

increasingly as a unifying mechanism in toxinrelated cell injury of different types.75Recent studies in the Liver Unit have shown

that S-adenosylmethionine is beneficial and pos-sibly complementary to N-acetylcysteine,

another glutathione precursor, in preventingexperimentally induced paracetamol hepatotox-icity (Bray, Tredger, and Williams, in prepara-tion). The benefit of late treatment withN-acetylcysteine (after the 16 h limit previouslysuggested) for reducing mortality and preventingworsening encephalopathy in paracetamolinduced fulminant hepatic failure has also beenconvincingly shown.76 Another late treatmentstudied experimentally was calcium channelblockade with diltiazem. This reduced para-cetamol hepatotoxicity and stimulated regenera-tive activity when given to mice late after dosingwith paracetamol when conventional antidoteshad ceased to be maximally effective.77

LIVER TRANSPLANTATIONThe increasing use of liver transplantation atKing's has dominated research activity in thedrug metabolism group over the last five years.Selection of patients for transplantationdemanded improved methods for evaluatingprognosis and rekindled an interest in evaluatingliver function. Stimulated by Rudi Preisig'ssabbatical visit in 1987, studies were made of thevalue of the cytochrome P-450 I-based caffeineclearance test but it was found susceptible tointerference by intensive drug therapy in fulmi-nant hepatic failure and after liver transplanta-tion.78 Measurements of coagulation factors Vand VIII showed no such interference and theirvalue in predicting prognosis in paracetamol-induced fulminant hepatic failure79 and acutealcoholic hepatitis (Pereira, Langley, Bird et al,in preparation has been recently demonstrated.The widespread use of cyclosporine after liver

transplantation has stimulated Liver Unitresearch on drug monitoring and pharmacokine-tics. Immunosuppression with cyclosporin iscomplicated in liver transplant recipients by therole played by the graft in influencing absorption(through bile production) as well as metabolismand the biliary excretion of metabolites.Y' Themajority ofimmunosuppressive activity is associ-ated with the parent drug and management hasbeen improved by specific monitoring of cyclo-sporin itself. The value of radioimmunoassaywith a specific monoclonal antibody was vali-dated in liver graft recipients, showing a closeagreement of cyclosporine results with thosemeasured by liquid chromatography.8' In latercollaborative studies, the influence of biliaryT-tube status and graft function on cyclosporinebioavailability and clearance was clearlyobserved.82 Recent technical developments incyclosporine assay procedures have also beenappraised, but there is a growing need foralternative methods to routine drug monitoringin evaluating the efficacy of immunosuppressivetherapy, as Figure 3 illustrates. One optionpresently under consideration involves thescreening of activation markers on peripheralblood lymphocytes (Gonde, Cohen, Tredgeret al, in preparation).

It is clear that the future for research by thedrug metabolism group in the Institute of LiverStudies will extend beyond the perspective ofmetabolism and hepatotoxicity. Continuedresearch in particular areas of clinical relevance,

Therapeutic dose

Poor absorption Poor graft functionEnzyme induction Enzyme inhibitionRapid metabolism Slow metabolism

LOW DRUG THERAPEUTIC HIGH DRUGLEVELS DRUG LEVELS LEVELS

Reduce dosagIncreased dosageReduced dosageui Enzyme inhibitionEnzyme induction Graft dysfunction

Improved graht function * Biliary T-tube clamping

Increased time after transplant

GENETIC PREDISPOSITIONIN IMMUNE RESPONSE

UNDER ADEQUATE h OVERIMMUNOSUPPRESSION IMMUNOSUPPRESSION IMMUNOSUPPRESSION

Side effectsGraft rejection Graft retention

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S38 Tredger, Davis

however, such as paracetamol hepatotoxicity,seems assured. Here, and for many hepatotoxicdrugs, characterisation of qualitative aspects ofthe covalent binding process is of obvious rele-vance. Obtaining evidence for the intracellularsites involved, the enzymes and cellular func-tions affected and the possibility of reversingsuch interactions is of high priority. Thisapproach may be relevant to those drugs whichare toxic directly through reactive metabolitesand those invoking immune responses. For thelatter, one emphasis in the immediate futuremust be to identify how drug related antigens aretranslocated to the cell surface, complementinginvestigations into the role of endogenous mole-cules such as cytochrome P-450 in autoimmunedisease. Drug metabolists and immunologiststogether must then unravel the mechanismsunderlying idiosyncrasy in immune mediateddrug reactions. This may not only refine preclini-cal screening for drug toxicity, but will also leadto new treatment regimens to improve the safetyof existing drugs.

The authors wish to thank all previous and current colleagues andthe collaborators and sponsors whose support made our workpossible.

1 Neuberger A, Smith RL. Richard Tecwyn Williams: Theman, his work, his impact. Drug Metab Rev 1983; 14: 559-607.

2 Sladek NE, Mannering GJ. Evidence for a new cytochromeP-450 hemoprotein in hepatic microsomes frommethylcholanthrene treated rats. Biochem Biophys Res Com-mun 1966; 24: 668-74.

3 EstabrookRW, Cooper DY, Rosenthal 0. The light reversiblecarbon monoxide inhibition of the steroid C21-hydroxylasesystem of the adrenal cortex. Biochem Z 1963; 338: 741-55.

4 Omura T, Sato R. The carbon monoxide binding pigment ofliver microsomes. I. Evidence for its hemoprotein nature. JBiol Chem 1964; 239: 2370-8.

5 Omura T, Sato R. The carbon monoxide binding pigment ofliver microsomes. II. Solubilization, purification and prop-erties.J Biol Chem 1964; 239: 2379-85.

6 Cucinell SA, Odessky L, Weiss M, Dayton PG. The effect ofchloral hydrate on bishydroxycoumarin metabolism.JAMA1966; 197: 366-8.

7 Yaffe SJ, Levy G, Matsuzawa T, Baliah T. Enhancement ofglucuronide conjugating capacity in a hyperbilirubinaemicinfant due to apparent enzyme induction by phenobar-bitone. NEnglJMed 1966; 275: 1461-6.

8 Crigler JF, Gold N. Sodium phenobarbitone-induced diseasein serum bilirubin in an infant with congenital nonhaemoly-tic jaundice and kernicterus. J Clin Invest 1966; 45: 998-9.

9 Kopelman H, Scheuer PJ, Williams R. The liver lesion of theEpping jaundice. QJ Med 1966; 35: 553-64.

10 Schaffner F, Raisfeld IH. Drugs and the liver: a review ofmetabolism and adverse reactions. Adv Intern Med 1969; 15:221-51.

11 Recknagel RO. Carbon tetrachloride hepatotoxicity. Phar-macol Rev 1967; 19: 145-208.

12 Williams AT, Burk RF. Carbon tetrachloride hepatotoxicity:an example of free-radical-mediated injury. Semnin Liv Dis1990; 10: 279-84.

13 Conney AH. Pharmacological implications of microsomalenzyme induction. Pharmacol Rev 1967; 19: 317-66.

14 ThompsonRPH, Williams R. Treatment ofchronic intrahepa-tic cholestasis with phenobarbitone. Lancet 1967; ii: 646-8.

15 Thompson RPH, Strathers GM, Pilcher CWT, Robinson J,Williams R. Treatment of unconjugated jaundice withdicophane. Lancet 1969; ii: 4-7.

16 Thompson RPH, Eddleston ALWF, Williams R. Low plasmabilirubin in epileptics on phenobarbitone. Lancet 1969; i:21-3.

17 Hunter J, Thompson RPH, RakeMO, Williams R. Controlledtrial of phethnobarbital, a non-hypnotic barbiturate inunconjugated hyperbilirubinaemia. BMJ 1971; ii: 497-9.

18 Aarts EM. Evidence for the function of D-glucaric acid as anindicator for drug-induced enhanced metabolism throughthe glucuronic acid pathway in man. Biochem Pharmacol1965; 14: 3592-3.

19 Williams R, Hunter J and Maxwell JD. Measurement ofhepatic enzyme induction in man: use of B-glucaric acidexcretion test. In: Paumgartner G, Presig R, eds The liver:quantitative aspects of structuzre and function. Basel: Karger1973.

20 Hunter JP, Maxwell JD, Stewart DA, Williams R. UrinaryD-glucaric acid excretion and total liver content of cyto-chrome P-450 in guinea pigs: relationship during enzymeinduction and following inhibition of protein synthesis.Bioche,nPhantnacol 1973; 22: 743-7.

21 Hunter J, Maxwell JD, Carrella M, Stewart DA, WVilliams R.

Urinary D-glucaric acid excretion as a test for hepaticenzyme induction in man. Lancet 1971; i: 572-5.

22 Hunter J, Maxwell JD, Stewart DA, Williams R, Robinson J,Richardson A. Increased hepatic microsomal enzymeactivity from occupational exposures to certain organ chlo-rine pesticides. Nature 1972; 237: 399-401.

23 Davis M, Simmons CJ, Dordoni B, Maxwell JD, Williams R.Induction of hepatic enzymes during normal human preg-nancy.J Obstet Gynaecol Br Commonw 1973; 80: 690-4.

24 Hunter J, Maxwell JD, Stewart DA, Parsons V, Williams R.Altered calcium metabolism in epileptic children on anticon-vulsants. BMJ 1971; iv: 202-4.

25 Maxwell JD, Hunter J, Stewart DA, Ardeman S, Williams R.Folate deficiency after anticonvulsant drugs: an effect ofhepatic enzyme induction? BMJ 1972; i: 297-9.

26 Mitchell JR, Jollow DJ, Potter WZ, Davis DC, Gillette JR,Brodie BB. Acetaminophen-induced hepatic necrosis. I.Role of drug metabolism. J Pharmacol Exp Ther 1973; 187:185-94.

27 Jollow DJ, Mitchell JR, Potter WZ, Davis DC, Gillette JR,Brodie BB. Acetaminophen-induced hepatic mecrosis. II.Roleofcovalentbindingin vivo.JPharmacolExp Ther 1973;187: 195-202.

28 Mitchell JR, Jollow DJ, Potter WZ, Gillette JR, Brodie BB.Acetaminophen-induced hepatic necrosis. IV. Protectiverole ofglutathione.JPharmacolExp Ther 1973; 187: 211-7.

29 Miller JA, Miller EC. The presence and signifiance of boundaminoazodyes in the livers of rats fed p-dimethy-laminoazobenzene. CancerRes 1947; 7: 468-80.

30 Miller EC, Miller JA. Mechanisms ofchemical carcinogenesis.Nature of proximate carcinogens and interactions withmacromolecules. Pharmacol Rev 1966; 18: 805-38.

31 Toghill PJ, Williams R, Stephens JD, Carroll JD. Acutehepatic necrosis following overdosage with paracetamol.Gastroenterology 1969; 56: 773-8.

32 Clark R, Thompson RPH, Borirakchanyavat V, Widdop B,Davidson AR, Goulding R, Williams R. Hepatic damageand death from overdose of paracetamol. Lancet 1973; i: 66-70.

33 Davis M, Simmons CJ, Harrison NG, Williams R. Para-cetamol overdose in man: relationship between pattern ofurinary metabolites and severity of liver damage. Q J Med1976; 45: 81-91.

34 Williams R, Davis M Therapeutic, clinical and experimentalaspects of paracetamol overdose and hepatotoxicity includ-ing management of acute liver failure. In: Wickstron E,Dybing E, eds. Acta Pharmnacologica et Toxicologica. Pro-ceedings of the 7th International Congress of the. EuropeanAssociation of Poison Control Centres, Oslo, June 8-11,1976. Copenhagen: Munksgaard 1977: 282-310.

35 Crome P, Volans GN, Vale JA, Widdop B, Gouding R,Williams R. The use of methionine for acute paracetamolpoisoning.7IntMedRes 1976; 4: suppl 4: 112-7.

36 Hughes RD, Gazzard BG, Hanid MA, et al. Controlled trial ofcysteamine and dimercaprol after paracetamol overdose.BMJ 1977; ii: 1395.

37 Zimmerman HJ. Drug Hepatotoxicity: the adverse effects ofdrugsand other chemicals on the liver. New York: Appleton-CenturyCrofts 1978.

38 Weber WW. Commentary: The molecular basis of hereditaryacetylation polymorphism. Drug Metab Dispos 1986; 14:377-81.

39 Evans DA, Mahgoub A, Sloan TP, Idle JR, Smith RL. Afamily and population study of the genetic polymorphism ofdebrisoquine oxidation in a white British population. J MedGenet 1980; 17: 102-5.

40 Cooper RG, Evans DA, Price AH. Studies on the metabolismof perhexiline in man. EurJ Clin Pharmacol 1987; 32: 569-76.

41 Morgan MY, Reshef R, Shah RR, Oates NS, Smith RL,Sherlock S. Impaired oxidation of debrisoquine in patientswith perhexiline liver injury. Gut 1984; 25: 1057464.

42 Sarma GR, Immanuel C, Kailasam S, Narayana ASL, Ven-katesan P. Rifampicin-induced release of hydrazine fromisoniazid. Am Rev Resp Dis 1986; 133: 1072-5.

43 DavisM, Williams R. Textbook ofadverse drug reactions 1991 (inpress).

44 Vergani D, Mieli-Vergani G, Alberti A, et al. Demonstration ofa circulating antibody reacting with the surface membrane ofhalothane-altered hepatocytes in patients with fuiminanthepatitis following halothane anaesthesia. N Engl J Med1980; 303: 66-71.

45 Neuberger J, Mieli-Vergani G, Tredger JM, Davis M. Will-iams R. Oxidative metabolism of halothane in the produc-tion of altered hepatocyte membrane antigens in acutehalothane induced hepatic necrosis. Gut 1981; 22: 669-73.

46 Kenna JG, Neuberger J, Williams R. Identification by immu-noblotting of three halothane-induced liver microsomalpolypeptide antigens recognized by antibodies in sera frompatients with halothane-associated hepatitis. J PharmacolExp Ther 1987; 424: 733-40.

47 Pohl L. Drug-induced allergic hepatitis. Semin LiverDis 1990;10: 305-15.

48 Bird G, Williams R. Detection of antibodies to a halothanemetabolite hapten in sera from patients with halothane-associated hepatitis.J Hepatol 1989; 9: 366-73.

49 Kenna JG, Neuberger JM, Williams R. Identification inhuman liver of halothane induced neoantigens recognised byantibodies in sera from patients with halothane hepatitis.Hepatology 1988; 8: 1635-41.

50 Kenna JG, Neuberger J, Williams R. An enzyme-linkedimmunosorbent assay for detection of antibodies againsthalothane-altered hepatocyte antigens. J Immunol Meth1984; 75:3-14.

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53 Brown BR, Sipes IG. Biotransformation and hepatotoxicity ofhalothane. Biochem Pharmnacol 1977; 26: 2091-4.

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55 Neuberger J, Williams R. Immune-mediated mechanisms intienilic acid associated hepatotoxicity. Gut 1989; 30: 515-9.

56 Crossley IR, Neuberger J, Davis M, Eddleston ALWF,Williams R. Ethanol metabolism in the generation of newantigenic determinants on liver cells. Gut 1986; 27: 186-7.

57 Manns M. Autoantibodies and antigens in liver diseases -updated. J Hepatol 1989; 9: 272-80.

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