Richard C. Lind

The Role of Oxidative Biotransformation of Halothane in the Guinea Pig Model of Halothane-associated Hepatotoxicity

The role of the oxidative pathway of halothane biotransformation in mediating the hepatotoxicity of halothane in the guinea pig was examined by utilizing the deuterated form of halothane (d-halothane), which is resistant to oxidative metabolism. Male outbred Hartley guinea pigs were exposed to either 1% v/v halothane or d-halothane, FIO2 = 0.21, for 4 h. Significant reductions in both oxidative and overall halothane biotransformation were observed with the use of d-halothane as indicated by decreased plasma levels of trifluoroacetic acid and bromide ion, respectively, immediately following exposure. Plasma fluoride ion, indicative of the reductive metabolism of halothane, was significantly increased with the use of d-halothane. These changes in metabolism were accompanied by a reduced hepatotoxic response as indicated by significantly decreased plasma ALT levels 24-96 h following exposure and a significantly lesser incidence of centrilobular necrosis. Thus, the oxidative biotransformation of halothane is implicated as a mechanism of injury in guinea pigs.

Factors affecting the formation of chlorotrifluoroethane and chlorodifluoroethylene from halothane

Since CF3CH2Cl and CF2CHCl are probably the products of reactive intermediates formed during the reductive metabolism of halothane (CF3CHClBr), factors affecting their in vitro and in vivo formation were investigated. In vitro studies with rat hepatic microsomes showed that CF3CH2Cl and CF2CHCl are produced by cytochrome P-450 mediated reductive pathways which were inhibited by the presence of CO. Under conditions of exposure known to promote halothane hepatotoxicity in phenobarbital treated rats (1 per cent halothane, 14 per cent oxygen), the hepatic and blood concentrations of the volatile metabolites were enhanced. Central venous levels of the volatile metabolites were much higher than the concentration in peripheral vessels. The CF3CH2Cl/CF2CHCl ratio in blood was approximately three, whereas the ratio in vitro was almost unity. Liver levels of the two volatile metabolites greatly exceeded the blood levels, but interestingly they were present in equivalent concentrations. The differences in the ratio of CF3CH2Cl to CF2CHCl may be explained by the fact that CF2CHCl is further degraded under oxidative conditions, whereas CF3CH2Cl appears relatively stable. Measurement of these metabolic products in patients undergoing halothane anesthesia may permit rapid detection of an unusually high level of halothane biotransformation along its hepatotoxic pathway.

The Involvement of Endotoxin in Halothane-associated Liver Injury

Since endotoxin, lipopolysaccharides (LPS), have been implicated as a causative factor in the development of hepatic necrosis in rats exposed to hepatotoxic levels of several chemical agents, the role of LPS in the halothane–hypoxia (HH) model of hepatic damage in male Sprague–Dawley rats was investigated. When injected intravenously immediately after halothane anesthesia, a subnecrotic dose of LPS (0.5 mg/kg; Escherichia coli 026:B6) was found to markedly potentiate HH-induced hepatic necrosis. Pretreatment of the animals with the antiendotoxin agent, lactulose, prior to exposure to halothane reduced the hepatic damage normally seen from HH. A possible mechanism of LPS-induced potentiation was indicated by changes in hepatic calcium levels at 24 h after treatment. Endogenous LPS may play a role in HH-induced hepatic necrosis, and the mechanism of LPS-induced potentiation may be due to an LPS-related membrane dysfunction.

Covalent Binding of Oxidative Biotransformation Intermediates Is Associated with Halothane Hepatotoxicity in Guinea Pigs

In vivo covalent binding of halothane biotransformation-reactive intermediates to hepatic protein and lipid was examined in association with the subsequent development of hepatic necrosis in the guinea pig. Oxidative halothane biotransformation was inhibited by the use of deuterated halothane, whereas reductive metabolism was enhanced by low inspired oxygen concentrations. Male outbred Hartley guinea pigs (n = 8) were exposed to either 1% (v/v) halothane or deuterated halothane--with a fractional inspired O2 concentration (FIO2) of 0.40 or 0.10--for 4 h. Livers removed from half of the animals immediately after anesthesia were evaluated for organic fluorine bound to protein and lipid. The remaining animals were evaluated for a hepatotoxic response up to 96 h after exposure. Only guinea pigs that received 1% halothane at an FIO2 of 0.40 had centrilobular necrosis develop with significantly increased plasma alanine aminotransferase activities. All other treatment conditions significantly reduced oxidative halothane biotransformation, as indicated by decreased plasma trifluoroacetic acid concentrations. These reductions were associated with a significant decrease in organic fluorine bound to hepatic proteins. An FIO2 of 0.10 during halothane anesthesia significantly enhanced reductive biotransformation, as indicated by plasma fluoride ion concentrations. This was associated with a significant increase in organic fluoride bound to hepatic lipids. Centrilobular necrosis did not develop under these conditions. Thus, covalent binding to subcellular proteins by the trifluoroacetyl acid chloride intermediate generated by oxidative halothane biotransformation is implicated as a mechanism of centrilobular necrosis in guinea pigs. Binding to lipids by reductive pathway generated free radicals does not appear to be involved in production of the lesion.

Age and Gender Influence Halothane-Associated Hepatotoxicity in Strain 13 Guinea Pigs

The factors of age and gender, which have been linked to development of fulminant halothane hepatitis in humans, were evaluated in a guinea pig model of acute halothane-associated hepatotoxicity. Since nitrous oxide is commonly coadministered with halothane and has been shown to exacerbate halothane-associated liver injury in rats; this combination of anesthetics was also evaluated in guinea pigs. Male and female strain 13 guinea pigs (300-1000 g) were exposed to 1% v/v halothane and 39% O2 for 4 h with a balance of either 60% N2 or 60% N2O. Both animal age, as determined by body weight, and gender proved to be factors in the model with older (approximately 6.2 +/- 1.0 month) guinea pigs of both sexes, demonstrating significantly greater elevations in plasma ALT and a greater incidence of centilobular necrosis versus younger (approximately 3.1 +/- 0.6 month) animals. Older females showed a greater hepatotoxic response than older males. There were no significant differences in halothane plasma metabolite levels between older and younger animals of either gender. The addition of nitrous oxide affected neither plasma concentrations of halothane metabolites nor the degree of resultant hepatic injury. Older (approximately 5-6 month) male guinea pigs, from a strain (inbred Hartley) previously shown to be resistant to the halothane lesion, did not develop centrilobular necrosis following halothane exposure even though they generated plasma metabolite concentrations equivalent to those generated by strain 13 animals. The lack of differences in the biotransformation of halothane between groups indicates that other intrinsic factors must be involved in the observed variations in susceptibility to hepatic injury.

Halothane hepatotoxicity in guinea pigs.

A recently reported animal model of halothane-associated hepatotoxicity in males of a colored strain of guinea pig was further characterized as to possible sex and strain specificity in outbred albino Amana, inbred albino Hartley, inbred colored strain 2, and inbred colored strain 13 guinea pigs. Exposure to 1 % halothane for 4 hr in 21 % O2 proved to be hepatotoxic in both sexes. Forty-eight hours after halothane exposure fatty vacuolization of hepatocytes was present in all animals. Histologically identifiable hepatic necrosis occurred in 60% of the guinea pigs exposed, along with concomitant increases in SGPT. Approximately one half of these responding animals had extensive centrilobular necrosis, which was still present 96 hr after halothane exposure. Females of the inbred strain 2 and males and females of strain 13 were the most susceptible to halothane-induced hepatic necrosis whereas the inbred Hartley strain was almost totally refractory to necrosis. Outbred Amana and male inbred strain 2 animals exhibited an intermediate hepatotoxic response. Comparison of the halothane-associated hepatic lesion with that induced by anoxidischemic mechanisms, (exposure to low (8%) oxygen during 1.7% enflurane anesthesia) showed obvious differences in the morphology of the hepatic necrosis and the apparent time course of lesion development. This guinea pig model of halothane-associated hepatotoxicity appears to be superior to previous animal models in that no pretreatment of the guinea pigs is required, both sexes are affected, and the resulting hepatic lesion is more persistent.

A model for fatal halothane hepatitis in the guinea pig

BackgroundIn the guinea pig, depleting hepatic glutathione before inhaling subanesthetic 0.1% halothane increases covalent binding of halothane biotransformation intermediates to hepatic protein and potentiates resultant liver injury. Because inhalation of a higher concentration of halothane is known to produce greater levels of covalent binding than with subanesthetic halothane, this study was undertaken with 0.25–1.0% halothane concentrations to further examine glutathione depletion as an etiology for halothane hepatitis. MethodsMale Hartley guinea pigs were injected intraperitoneally with either vehicle control solution (Veh) or 1.6 g/kg buthionine sulfoximine (BSO), to decrease hepatic glutathione by >80%, 24 h before a 4-h exposure to 0.25%, 0.5%, or 1.0% (v/v) halothane with 40% O2. Some BSO-pretreated animals also received 2.0 g/kg glutathione monoethyl ester (GEE), intraperitoneally, 2 h before inhaling halothane to replenish hepatic glutathione. ResultsGlutathione-depleted animals developed significantly worse hepatic injury with each halothane concentration. One-third to one-half of BSO + halothane-treated animals developed fatal submassive to massive hepatic necrosis. Covalent binding of halothane intermediates to hepatic protein increased by 45% in BSO + 1.0% halothane-treated guinea pigs. Administration of GEE to BSO-pretreated animals before 1.0% halothane decreased binding to protein and blunted development of liver necrosis. Following Veh + 1.0% halothane, hepatic glutathione was found to be decreased by 60%. ConclusionsGlutathione would appear to help protect hepatocytes to some degree from covalent binding by reactive halothane biotransformation intermediates. These studies present the first animal model to produce fatal halothane-induced hepatic necrosis.

Comparison of the requirements for hepatic injury with halothane and enflurane in rats.

A rat model of cnflurane-associated hepatotoxicity was compared with the halothane-hypoxia (HH) model (adult male rats, phenobarbital induction, 1% halothane, 14% O2, for 2 hr). The enflurane-hypoxia heating (EHH) model involved exposing phenobarbital-pretreated male adult rats to 1.5–1.8% enflurane at 10% O2 for 2 hr with external heating to help maintain body temperature. Exposure to either anesthetic without temperature support led to a decrease in body temperature of 7–9°C, while heating the animals during anesthesia resulted in only a 0.5–2°C decrease. Reducing the oxygen tension to 10% O2 combined with heating the animals during exposure produced significant decreases in the oxidative metabolism of both halothane and enflurane as compared to exposures of 14% O2. The same conditions also caused a significant increase in the reductive metabolism of halothane, indicating that a severe hepatic hypoxia or anoxia occurs during anesthesia at 10% O2 with external heating. The time course of lesion development in the HH model paralleled results obtained with an oral dose of CCl4: gradual progression of necrosis up to 24 hr. EHH resulted in a classic hypoxic/anoxic injury with elevated serum glutamate pyruvate transaminase values and a watery vacuolization of centrilobular hepatocytes immediately after exposure. The HH model required phenobarbital pretreatment of the rats for expression of hepatic injury; EHH did not. Heating of the animals during anesthesia exposure was necessary for enflurane-induced hepatotoxicity but had little effect on the HH model. Exposure to 5% O2 without anesthetic mimicked EHH in both requirements for and type of hepatic injury. Thus the HH model appears to act via a bioactiva- tion/chemotoxic mechanism, whereas models of anesthetic- induced hepatotoxicity that require very low oxygen tensions and heating of the animals during exposure result from a severe hypoxia/anoxia of the liver.

Hepatoprotection by dimethyl sulfoxide. II. Characterization of optimal dose and the latest time of administration for effective protection against chloroform and bromobenzene induced injury.

Dimethyl sulfoxide (DMSO) has previously been shown to attenuate chloroform (CHCl3) and bromobenzene (BB) induced hepatotoxicity in the rat when a dose of 2.0 ml/kg is given 24 hr after the toxicants. However, the optimal dose of DMSO and the latest time at which DMSO can be administered and still provide effective protection have not been determined. In order to determine the latest time at which DMSO can interrupt the development of necrosis, male Sprague Dawley rats received either 0.75 ml/kg CHCl3 or 0.5 ml/kg BB, 20% in corn oil, p.o., followed by single dose of 2 ml/kg DMSO, 50% in saline, i.p., at 24, 26, 28 or 30 hr later. Positive control groups received either CHCl3 or BB and then 4.0 ml/kg saline, i.p., 24 hr later. All of the animals were then killed 48 hr after toxicant dosing. The extent of liver injury present when DMSO was administered was examined by killing animals at 24, 26, 28 or 30 hr after toxicant dosing. The optimal dose of DMSO for providing protection was estimated by administering either 0, 1.0, 2.0, 3.0 or 4.0 ml/kg DMSO at 24 hr after toxicant dosing and then killing the animals at 48 hr. Delaying DMSO administration to times later than 24 hr after toxicant dosing led to a loss of protection as indicated by both plasma ALT activity and the light microscopic appearance of liver tissue. The distinctive liver lesions present at 24 hr after CHCl3 or BB dosing rapidly expanded from being limited around central veins to bridging between centrilobular areas in only a few hours. This was accompanied by large increases in plasma ALT. With both toxicants, doses of DMSO greater than 2 ml/kg did not enhance its protective action while the lower dose of 1 ml/kg DMSO was not as effective. The loss of DMSOs antidotal action when given at times later than 24 hr after the toxicants indicates irreversible changes were underway as the centrilobular lesions progressed from being limited to more bridging in nature. Hopefully, further elucidation of the mechanism(s) by which DMSO interrupts the rapid progression of injury will both help to understand the steps involved in lesion development and provide insights into therapeutic interventions for drug and chemical induced hepatitis.

Subanesthetic halothane is hepatotoxic in the guinea pig.

Subanesthetic concentrations of halothane were examined for their hepatotoxic potential in the guinea pig. Outbred male, Hartley guinea pigs (600–700 g) were exposed to either 1.0%, 0.25%, or 0.10% (vol/ vol) halothane, 40% O2, for 4 h. Plasma isocitrate dehydrogenase (ICDH) activity was compared to plasma alanine aminotransferase (ALT) for sensitivity as an indicator of hepatic injury. As previously seen, exposure to the anesthetic concentration of 1.0% halothane produced limited to confluent centrilobular necrosis in 50% (4/8) of the guinea pigs. The Subanesthetic concentrations of 0.25% and 0.1% halothane were also hepatotoxic. After exposure to 0.25%, confluent centrilobular necrosis developed in 2 of 8 animals, whereas 0.10% halothane produced limited centrilobular necrosis in 3 of 8. Plasma ICDH activity was a more sensitive indicator of halothane-induced hepatic injury than ALT. Mean plasma ALT activity increased significantly after 1.0% halothane exposure only. However, ICDH activity was significantly increased after exposure to all three concentrations of halothane. Comparison of peak plasma enzyme activities demonstrated significantly larger increases in ICDH than in ALT when centrilobular necrosis was present. Use of Subanesthetic concentrations of halothane should help overcome the many transient effects that high concentrations of halothane have on whole liver and hepatocyte functions. By being able to isolate and titrate the bioactivation of halothane, the mechanisms through which halothane biotrans-formation produces acute hepatotoxicity should be more easily elucidated.