Br. J. Exp. Path. (I989) 70, 533-541

The pathology of halothane hepatotoxicity in a

guinea-pig model: a comparison withhuman halothane hepatitis

Christine A. Lunam, Pauline de la M. Hall* and Michael J. CousinstDepartment of Anatomy and Histology, School of Medicine, Flinders University of South Australia,

Bedford Park, SA 5042, *Department of Pathology and tDepartment of Anaesthesia and Intensive Care,Flinders Medical Centre, Bedford Park, SA 5042

Received for publication 30 November I988Accepted for publication I June I989

Summary. The pathology of halothane hepatotoxicity is described in detail in a guinea-pigmodel. Twenty-two of 40 guinea-pigs developed liver damage after exposure to I% halothanein 21% 02 for 4 h. The other i 8 animals showed no evidence of hepatic injury. Two distinctpatterns of damage were identified: mild damage, in which livers had focal areas of necrosis,and severe damage, where necrosis was confluent around the terminal hepatic venules, oftenextending to the portal tracts. Serum alanine aminotransferase activity was significantlyelevated in guinea-pigs with severe liver damage. Hepatocytes in the damaged areas showeddegenerative changes ranging from vacuolization to ballooning degeneration and necrosis.Inflammatory cells, predominantly lymphocytes, were often present in the areas of necrosis.The pathology of mild and severe liver injury in the guinea-pig closely resembles the spectrumof injury observed in non-fatal halothane hepatitis in man.

Keywords: halothane hepatotoxicity, light microscopy, pathology, guinea-pigs, humans

Halothane hepatitis is a recognized compli-cation of halothane anaesthesia. This clini-cal entity is particularly sinister in that itsoccurrence is unpredictable and does notappear to be influenced by either theduration of anaesthesia or type of surgicalprocedure (Benjamin et al. I985). Estimatesof the incidence of halothane hepatitis rangefrom i per 8ooo patients (Ranek 1978) toI per 14685 (Slater et al. I964) with amortality rate varying from I5% to greaterthan 50% (Touloukian & Kaplowitz I98I).Although the mechanisms of halothanehepatotoxicity are by no means clear, evi-

dence is accumulating that susceptibility tohalothane hepatitis is genetically determinedboth in humans (Otsuka et al. I985; Farrellet al. I985) and in experimental animals(Gourlay et al. I98I; Lunam et al. I986; Lindet al. I987).A variety of rat models have been deve-

loped in an attempt to investigate mecha-nisms and identify risk factors associatedwith halothane hepatitis. In each case, pre-treatment with at least one of a number ofagents is essential for lesion development(Sipes & Brown 1976; McLain et al. 1979;Wood et al. I980). As these agents them-

Correspondence: Dr C.A. Lunam, Department ofAnatomy and Histology, School of Medicine, FlindersUniversity of South Australia, Bedford Park, SA 5042 Australia.

533

selves elicit morphological changes (LunamI984) the histopathology associated withhalothane-induced liver damage in rats isdifficult to interpret. Furthermore, becausemany aspects of the lesion in the rat modelsare not comparable to those associated withhuman halothane hepatitis, doubt arises asto whether the pathogenesis of halothane-induced hepatotoxicity in rats is similar tothat in man. For example, in contrast tohumans, liver damage occurs in ioo% ofratswhich are given appropriate preconditioningtreatment. In addition, the different patternsof liver damage associated with halothanehepatitis in humans, that is, mild, focalnecrosis and fatal, massive necrosis, havenot been described in rat models ofhalothanehepatotoxicity.Hughes and Lang (I 9 72) reported guinea-

pigs developed liver necrosis following repeathalothane exposures administered in I00%oxygen. In contrast to the enzyme-inducedrat models, they demonstrated that guinea-pigs were susceptible to halothane-inducedliver injury in the absence of conditioningfactors. Because of the obvious advantages ofan animal model that allows the study of theeffects of halothane per se, we developed aguinea-pig model for halothane hepatotoxi-city and found that the expression of liverdamage is associated with both the meta-bolism of halothane (Lunam et al. I 98 5) anda heritable susceptibility (Lunam et al.I986). Here we describe in detail the liverpathology, at the light-microscope level, ofhalothane hepatotoxicity in the guinea-pigmodel and compare its patterns and severitywith that of human halothane hepatitis.

Methods

Male IMVS (Institute of Medical and Veterin-ary Science, Adelaide)-coloured guinea-pigs,5 months old, were bedded on straw for 2weeks prior to halothane anaesthesia. Foodand tap water containing i mg/ml vitamin Cwere supplied ad libitum.

Forty guinea-pigs received a mixture of I%halothane (ICI Chemicals, Australia) and

2I% 02-78% N2 for 4 h in a I50-1 Perspex(acrylic plastic) chamber with temperaturecontrol. The anaesthetic procedure andcontinuous monitoring of chamber concen-trations of halothane, carbon dioxide andoxygen were performed as described else-where (Lunam et al. I 98 5). During recoveryfrom anaesthesia, the animals were wrappedin aluminium foil and placed on a heatedelectric blanket to minimize loss ofbody heat.All animals were killed by cervical disloca-tion 48 h after halothane, the time ofmaximal liver injury (Lunam et al. I985).One millilitre blood samples were drawn bycardiac puncture 48 h post-halothane andserum alanine aminotransferase (ALT) acti-vity measured according to the procedure ofHenry et al. (I960). Ten unanaesthetizedcontrol guinea-pigs were placed in a Perspexchamber with temperature control andreceived a mixture of2I% 02 and 79% N2 for4 h.

Immediately after cervical dislocation,slices of liver were excised, fixed in I0%buffered formalin, dehydrated in acetone andembedded in resin for histological assess-ment. Sections (2 gm-thick) were treatedwith concentrated sodium hydroxide solu-tion to remove the resin and stained withhaematoxylin and eosin. Each section wasviewed at XI 2 5 magnification using a LeitzDialux microscope. A minimum of eightportal tract areas, five terminal hepaticvenules (THV) with adjacent hepatocytesand several 'indeterminate' areas whereneither THV nor portal tract areas wereobserved, were examined from each liver.The presence of fat droplets was confirmed bypost-fixation with potassium dichromate andosmium tetroxide, as described by Hall et al.(I982).

Liver damage was classified according tothe following criteria: no damage, normalpolygonal hepatocytes with rounded nuclei;mild damage, focal non-zonal distribution ofdamaged hepatocytes showing either hydro-pic degeneration and7or necrosis; moderatedamage, perivenular necrosis restricted towithin four cells from the THV; severe

C.A. Lunam et al.534

Pathology of halothane hepatotoxicitydamage, perivenular necrosis extending in astellate distribution to the portal tracts, thatis, the classic zone 3 necrosis first describedby Rappaport (I 958).

Differences in ALT values between gradesof liver damage were assessed using theKruskal-Wallis one-way analysis of vari-ance. Intergroup differences were analysedby Mann-Whitney U-tests using Bonfer-roni's correction for multiple comparisons. AP value of o os or less was accepted assignificant.

Results

Livers of non-anaesthetized control guinea-pigs had normal acinar architecture withanastomosing cords and plates of liver cellsarranged around hepatic venules (Fig. i).Polygonal-shaped hepatocytes with welldefined cell borders had one or more roundednuclei containing nucleoli. A few isolatedhepatocytes in some livers had pyknotic

nuclei. Fat droplets were not visible in hepa-tocytes of control animals.

Administration of halothane in normoxia(21% 02) resulted in hepatic necrosis in 22of 40 guinea-pigs. Liver damage was eithermild or severe. Focal necrosis in the mildlydamaged livers was non-zonal and wasfound with equal frequency in the periportal(zone i), perivenular (zone 3) and 'indeter-minate' regions of the liver acinus (Fig. 2).Hepatocytes in the damaged areas showeddegenerative changes ranging from vacuo-lization to ballooning degeneration andnecrosis. Necrotic cells had shrunken acido-philic cytoplasm and pyknotic nuclei thatoften displayed karyorrhexis and karyolysis.Variable numbers of inflammatory cells, pre-dominantly lymphocytes, were seen in areasof focal necrosis.

Livers with severe damage had classiczone 3 necrosis (Fig. 4) with a stellateconfiguration often extending to the portaltracts. An inflammatory cell infiltrate, con-

Fig. i. Section of liver from a non-anaesthetized guinea-pig. The hepatic architecture is normal: thecentrally situated terminal hepatic venule (THV) is surrounded by normal hepatocytes. H & E x I 30. Bar,250 Pm.

535

C.A. Lunam et al.

Fig. 2. Guinea-pig liver showing mild damage 48 h after halothane anaesthesia. A focal area of necrosis(arrowheads) is seen in the mid region ofa liver acinus. Hepatocytes adjacent to the THV (asterisk) appearnormal. H & E x I30. Bar, 250 Pm.

Fig. 3. Guinea-pig liver 48 h after halothane. There is no evidence of cell necrosis although most of thehepatocytes surrounding a THV (asterisk) show prominent fatty change. H & E x 280. Bar, I25 pm.

536

Pathology of halothane hepatotoxicity

0~ ~ ~Fig. 4. Guinea-pig liver showing severe liver damage after halothane. An area of confluent cell necrosissurrounds a THV (asterisk). Outlines of the necrotic cells with pyknotic nuclei are visible. A mild infiltrateof lymphocytes with occasional neutrophils is scattered within and at the periphery of the necrotic area(arrowheads). The surviving liver cells show prominent fatty change. H & E x 130. Bar, 250 tm.

sisting predominantly of lymphocytes withoccasional neutrophil polymorphs, was seenonly in the areas of damage. Hepatocytesadjacent to the necrotic zone contained largefat droplets. None of the livers showedmoderate damage; in all livers with perive-nular necrosis the damage extended wellbeyond four cells from the THV.

Eighteen of the guinea-pigs exposed tohalothane had livers with no evidence ofeither scattered foci of damage or zone 3necrosis. Sinusoidal ectasia was observed insome livers and macro or micro-vesicular fatdroplets were present in many liver cells (Figs3 and 5). Fat droplets in some livers weredistributed randomly within the acini whilein other livers they were confined to theperivenular regions.Serum ALT activities associated with each

grade of damage were significantly differentwhen compared with one another,P< o * ooI (Table i). Guinea-pigs with severe

damage had significantly higher serum ALTscompared to ALTs associated with all otherhistology grades. Mild liver injury was asso-ciated with a significantly higher mean ALTactivity compared to that of guinea-pigswithout evidence of liver damage afterhalothane (P<o os). Guinea-pigs with non-damaged livers after halothane had ALTactivities similar to control animals.

Discussion

The individual variation in sensitivity todevelopment of halothane hepatitis and thepattern and severity ofdamage in the guinea-pig model was similar to our previous findings(Lunam et al. I 98 5, I 986), as well as those ofLind et al. (I987). After administration ofhalothane, guinea-pigs developed eithersevere, mild, or no detectable liver injury.This spectrum of liver injury in guinea-pigsincorporates several of the histological distri-

537..8.:A-..X

C.A. Lunam et al.

Fig. 5. Guinea-pig liver 48 h after halothane. Hepatocytes around the THV (asterisk) show numerous fatdroplets after post-fixation with potassium dichromate and osmium tetroxide. x I30. Bar, 250 im.

Table i. Liver morphology and ALT activity afterhalothane anaesthesia

ALT*tHistology grade (IU/I)

Nodamage 82± 8(i8)Mild damage 217+ 37 (I3)Severe damage 719 I04 (9)Controls 65 ± 8 (io)

* P< oooi among histology grades.t P< 005 severe damage compared to mild

and no damage; mild damage compared to nodamage.ALT values are means ± s.e.m.Criteria for assessment of liver damage is given

under Methods. Numbers of animals are shown inparentheses.

butions of liver damage found in humanhalothane hepatitis. For example, confluentnecrosis of liver cells around the THV (zone 3necrosis) is found in liver biopsies from somepatients with non-fatal halothane hepatitisand also in the livers, at autopsy, of severalfatal cases (Brody & Sweet I963; Tornetta &Tamaki I963; Blackburn et al. I964; Tou-

loukian & Kaplowitz I98I; Benjamin et al.i985). In addition, the non-zonally distri-buted foci of hepatocyte necrosis closelyresembles the multifocal spotty necrosisobserved in some patients with halothanehepatitis (Tygstrup I963; Belfrage et a).I966; Klatskin & Kimberg I969; Klion et a].I969; Peters et al. I969; Uzunalimoglu et al.1970; Benjamin et al. I985).

It has been reported that mild liver dys-function, associated with mild liver damagein humans, occurs in 20% of patients afterhalothane anaesthesia (Thompson & Friday19 78). However, the actual incidence ofmildhalothane hepatitis in man is not knownsince liver biopsies are not conducted on themajority of patients with mild elevations ofliver enzymes following anaesthesia. Itshould be noted that the incidence of liverdamage in the guinea-pigs is considerablyhigher, greater than 50%, than that reportedin man. This relatively high incidence inguinea-pigs may be the result of inbreedingof a heritable susceptibility to halothanehepatotoxicity. Although considered anoutbred strain, the guinea-pig colony hasbeen closed for more than 30 years, so that a

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Pathology of halothane hepatotoxicity

trait for susceptibility to halothane hepato-toxicity may have been unknowinglyselected for. In support of this proposal, wewere able to significantly alter the percent-age of guinea-pigs susceptible to halothanehepatotoxicity by selected breeding studies(Lunam et al. I986).The incidence and spectrum of liver

damage in guinea-pigs after halothane ismarkedly different to that reported in rats.For example, only 23% of exposed guinea-pigs had livers with confluent zone 3necrosis, whereas I00% of rats developedthis pattern of injury (Cousins et al. 1979). Inaddition, the extent of the damage wasgreater than in most rat models (McLain et al.1979; Ross et al. I979) apart from theFischer 344 rat model (Cousins et al. 1979).In contrast to the severe lesion, some livershad several non-zonal foci of necrosis. Thisdistribution of hepatocyte necrosis is notobserved in phenobarbitone-hypoxic rats.The extent of liver damage in the guinea-

pig model is less severe than that observed infatal halothane hepatitis in man. At autopsy,none of the guinea-pigs had developed sub-massive or massive necrosis of the typeobserved on rare occasions in humans(Peters et al. I969; Benjamin et al. I985).Even in the most severely damaged livers ofthe guinea-pigs, necrosis remained zonal,involving all of zone 3 (Fig. 4). However,considering the small numbers of animalsexposed to halothane in this study, and themarked differences in response between indi-vidual guinea-pigs, it is perhaps not surpris-ing that none of the animals developed fatalmassive necrosis. Repeated halothane anaes-thetics at variable time intervals also failed toproduce submassive or massive necrosis inany guinea-pigs (Lunam et al. I986). This isin contrast to the situation in humans, inwhich the risk of developing fatal massivenecrosis is significantly increased after mul-tiple halothane anaesthetics (Touloukian &Kaplowitz I 98 I).Two mechanisms of halothane hepatitis

have been postulated in humans, namely adirect toxicity and a hypersensitivity reac-

tion. The onset of halothane hepatitis inpatients after repeated anaesthetics suggestsa hypersensitivity reaction (reviewed by Far-rell I988; Cousins et al. I989). In addition,in some patients with severe centrilobular orsub-massive liver damage after halothane,serum antibodies have been detected thatreact with halothane-altered hepatocytes(Vergani et al. I980). In contrast, patientswith mild liver damage after halothaneexposure do not develop serum antibodies(Davis et al. I980). It is difficult to explainthis discrepancy on the basis ofdirect toxicityresulting in mild damage and hypersensiti-vity resulting in severe damage. It remains tobe determined if immune responses initiateor even contribute to halothane-inducedliver injury.

In the guinea-pig, the delayed onset ofliver injury 48 h after halothane, and theappearance oflymphocytes at this time raisesthe possibility that an immune response mayat least be partially responsible for hepaticnecrosis (for review of a possible hypersensi-tivity response in guinea-pigs, see Farrell(I988)). However, zone 3 necrosis and thepresence of fat droplets are histological fea-tures consistent with direct toxicity ascaused by the classic hepatotoxins, carbontetrachloride and chloroform (KlatskinI968; Carney & Van Dyke 1972), and thussuggest a direct toxic effect by halothane.The individual variation in response of

guinea-pigs to the development of halothanehepatotoxicity is in keeping with the unpre-dictable occurrence and different patterns ofdamage found in halothane-induced liverdamage in humans. Moreover, we havedemonstrated that susceptibility of guinea-pigs to halothane hepatotoxicity is mostlikely a heritable characteristic (Lunam et al.I 98 6) as has been suggested to be the case inhumans (Hoft et al. I 98 I; Farrell et al. I 98 5;Otsuka et al. I985). We conclude thereforethat the guinea-pig model developed in ourlaboratory is the most appropriate animalmodel developed to date for the study of thepathogenesis of the non-fatal form of humanhalothane hepatitis.

539

540 C.A. Lunam et al.

Acknowledgements

This work was supported in part by grantsfrom the National Health and MedicalResearch Council of Australia and theFlinders University of South Australia. Wealso thank Dr John Plummer for his criticismof this manuscript.

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