Demonstration of Halothane-induced Hepatic Lipid Peroxidation in Rats by Quantification of Flourine²-Isoprostanes

Male Sprague-Dawley rats (weighing 150-200 g) were purchased from Harlan Sprague-Dawley (Indianapolis, IN). The rats were housed in a facility approved by the American Association for Accreditation of Laboratory Animal Care with alternating 12 h/12 h light/dark cycles. Experimental protocols were reviewed and approved by the Vanderbilt University Animal Care Committee in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences. Pretreatment with phenobarbital (Mallinckrodt, Paris, KY) consisted of administering 75 mg/kg intraperitoneally on the first day followed by adding 1 mg/ml phenobarbital to drinking water for the next 4 days. Pretreatment with isoniazid (Aldrich Chemical, Milwaukee, WI) consisted of adding it to the drinking water (1 mg/ml) for 10 days. Both drugs were withdrawn and rats were fasted 16 h before anesthetic exposure.

Rats were placed in an 84-1 methyl methacrylate polymer chamber preequilibrated with warmed and humidified gas flowing at 6 l/min. Halothane (Fluothane donated by Wyeth-Ayerst, Princeton, NJ) was delivered using a Fluotec 3 vaporizer (Ohmeda, West Yorkshire, England) precalibrated using gas chromatography. Enflurane (Ethrane, donated by Anaquest, Murray Hill, NJ) was delivered using an Enfluratec vaporizer (Ohio Medical Products, Madison, WI). Desflurane (Suprane, Ohmeda) was delivered using a Tec6 vaporizer (Ohmeda). The oxygen-nitrogen mixture was controlled using two rotameters and oxygen concentration in the chamber was continuously monitored using a precalibrated Ohmeda 5120 oxygen monitor. The chamber halothane concentration was periodically confirmed using gas chromatography. Enflurane and desflurane concentrations were monitored using a Capnomac Airway Gas Monitor (Datex Instrumentarium, Helsinki, Finland). The body temperature of selected rats was monitored with a rectal probe and chamber temperature was adjusted to maintain normothermia.

Determination of the Effect of Halothane and Hypoxia on Flourine²-isoprostane Generation. All animals were pretreated with phenobarbital and divided into four experimental groups, 0% halothane/21% Oxygen², 0% halothane/14% Oxygen², 1% halothane/21% Oxygen², and 1% halothane/14% Oxygen². In each group, ten rats were exposed to the assigned treatment for 2 h. Five rats were then killed within minutes after removal from the anesthetic chamber to assess lipid peroxidation. Rats were killed under pentobarbital anesthesia (60 mg/kg) and blood was withdrawn from the dorsal aorta. The liver was removed and frozen in liquid nitrogen before storage at -70 degrees C. The remaining five animals in each group were fed and killed at 24 h to assess liver injury.

Determination of the Effect of Pretreatment on Flourine²-isoprostane Generation. Rats (15 per group) were divided into three pretreatment groups: no pretreatment, isoniazid pretreatment, or phenobarbital pretreatment. Ten animals from each group were exposed to 1% halothane/14% Oxygen²for 2 h. Five rats were killed immediately afterward and five were killed at 24 h as earlier. Five animals in each group were exposed to 0% halothane/14% Oxygen²for 2 h and killed immediately afterward to assess lipid peroxidation.

Determination of the Effect of Halothane, Enflurane, and Desflurane on Flourine²-isoprostane Generation. Three groups of ten rats were exposed to anesthetic concentrations of halothane (1.3%), enflurane (1.5%), or desflurane (5.2%) at 21% Oxygen²for 2 h. Five rats were killed immediately afterward and 5 were killed at 24 h as earlier. Five rats unexposed to anesthetic were used as controls for each experiment.

Flourine²-isoprostane concentrations were measured in freshly obtained plasma (1-3 ml) after purification and derivatization by gas chromatography/negative ion chemical ionization-mass spectrometry using selected ion monitoring as described previously. [17]This is a stable isotope dilution assay using [sup 2 Hydrogen⁴]-Prostaglandin Flourine sub 2 alpha as the internal standard. Phospholipid-esterified Flourine²-isoprostanes in liver were quantified after extraction of lipids by the method of Folch [18]and base hydrolysis as described previously. [13]Plasma alanine aminotransferase activities were measured using a commercial spectrophotometric kinetic assay kit (Sigma diagnostic procedures no. 59-10, St. Louis, MO).

Data were analyzed by t test or analysis of variance with Fisher's (protected) least significant difference test using Statview software (Abacus Concepts, Berkeley, CA). When variances were found to be heterogeneous, log transformation of the data was performed first. Differences between group means were considered significant at P < 0.05. Data are presented as mean+/-SEM.

The effect of hypoxia in the classic halothane-hypoxia model in which all animals were pretreated with phenobarbital was determined. Exposure of phenobarbital-pretreated rats to 1% halothane at atmospheric oxygen concentrations for 2 h increased plasma Flourine²-isoprostanes concentrations fivefold (Figure 1(A)). Exposure to 1% halothane under hypoxic conditions further increased plasma Flourine²-isoprostanes tenfold. However, hypoxia alone did not increase plasma Flourine²-isoprostanes at the end of 2 h. Hepatic Flourine²-isoprostanes increased similarly (Figure 1(B)). Half of the rats exposed to the four anesthetic conditions were killed at 24 h to assess liver damage by measurement of plasma ALT activity. Liver injury was mild in this experiment. Only the group exposed to halothane and hypoxia showed a significant ALT elevation (77+/-15 IU/l) when compared with no halothane/normoxemia (34+/-4 IU/l), halothane/normoxia (49 +/-3 IU/l), or hypoxia alone (25+/-2 IU/l).

The effect of pretreatment with phenobarbital or isoniazid on Flourine²-isoprostane production and liver injury induced by halothane and hypoxia or hypoxia alone was determined. As illustrated in Figure 2, a 2-h exposure to halothane under hypoxic conditions caused an increase in Flourine²-isoprostanes in plasma (A) and liver (B) in the absence of pretreatment with isoniazid or phenobarbital. Pretreatment with isoniazid did not enhance the production of plasma or liver Flourine sub 2 -isoprostanes caused by halothane and hypoxia. Phenobarbital, however, potentiated the effect of halothane and hypoxia on Flourine²-isoprostane concentrations in plasma and liver (9- and 20-fold, respectively, vs. hypoxia alone). Liver injury was assessed in the animals killed 24 h after anesthesia. Only rats pretreated with phenobarbital had elevated plasma ALT activity, 235+/-85 IU/l. Alanine aminotransferase activity was 41+/-4 and 44+/-3 IU/l for non-pretreated and isoniazid-pretreated rats exposed to halothane and hypoxia.

To determine whether Flourine²-isoprostane production was specific to metabolism of halothane, we determined the effects of halothane, enflurane, and desflurane on Flourine²-isoprostane generation at anesthetic concentrations of each anesthetic under normoxic conditions. As depicted in Figure 3(A), plasma Flourine²-isoprostane concentrations were elevated only in rats exposed to halothane (2.5-fold). Liver Flourine²-isoprostane concentrations were elevated fivefold in these animals. Although animals exposed to desflurane had statistically significant elevations of liver Flourine²-isoprostanes (60% increase), this increase was small and unaccompanied by an increase in plasma Flourine²-isoprostanes. None of the animals assessed for liver injury at 24 h in these experiments had ALT activities different from controls (data not shown).

Flourine²-isoprostane formation is a specific result of free radical-initiated lipid peroxidation. [19]Consequently, Flourine²-isoprostane quantification has been valuable in establishing the role of lipid peroxidation in a variety of free radical-induced hepatic and renal injury models in which existing techniques of measuring lipid in vivo peroxidation proved inadequate. [14,20-22]Plasma and tissue Flourine²-isoprostane elevations in these models are not simply a nonspecific consequence of tissue necrosis. Tissue injury not induced by free radicals (e.g., acetaminophen- or thioacetamide-induced liver necrosis) does not substantially increase Flourine²-isoprostanes. [14]Thus, Flourine²-isoprostane quantification confirms that halothane exposure causes lipid peroxidation in vivo as previously demonstrated with measurement of exhaled ethane. [11]Moreover, Flourine²-isoprostane quantification provides important new information. Documentation of increased hepatic Fluorine²-isoprostane-containing lipids establishes that lipid peroxidation occurs in the organ injured by halothane. In addition, for the first time, halothane-induced lipid peroxidation in vivo has been demonstrated even in the absence of drug pretreatment and/or hypoxia. Thus, quantification of the increase in lipid peroxidation caused by hypoxia and phenobarbital pretreatment was possible.

These results correlate well with data regarding the reductive metabolism of halothane in Sprague-Dawley rats. [5]Gourlay et al. measured reductive metabolites of halothane, 1-chloro-2,-2,-2,-triflouro ethane and 1-chloro-2,-2,-difluoro ethylene, during a 2-h exposure to 1% halothane. The quantity of these metabolites reflects the potential for halothane to initiate lipid peroxidation. They demonstrated that both 1-chloro-2,-2,-2,-triflouro ethane and 1-chloro-2,-2,-difluoro, ethylene were exhaled by phenobarbital-pretreated rats exposed to 1% halothane and 21% Oxygen². Exposure of rats to halothane at 14% Oxygen²approximately doubled the amount of these metabolites. This correlates well with the increase in halothane-induced Fluorine²-isoprostane concentrations in phenobarbital-pretreated rats caused by 14% Oxygen²in our study (Figure 1). Gourlay et al. also reported that formation of reductive metabolites caused by exposure of rats to 1% halothane and 14% Oxygen²was increased twofold to threefold by phenobarbital pretreatment. This is similar to the effect of phenobarbital pretreatment on Fluorine²-isoprostane concentrations that we observed under these conditions (Figure 2).

The relevance of these observations to human halothane hepatotoxicity is uncertain. Reductive metabolism of halothane is a minor, but established, metabolic pathway for halothane in humans. Specific reductive metabolites of halothane, 1-chloro-2,-2,-2,-triflouro ethane and 1-chloro-2,-2,-difluoro ethylene, have been measured in patients undergoing halothane anesthesia. [23]Thus, it is logical to postulate that some lipid peroxidation occurs in patients given halothane. Measurement of Fluorine²-isoprostanes in patients exposed to halothane should allow determination of whether this occurs. Nevertheless, detection of lipid peroxidation does not mean that injury detrimental to the individual will invariably result. Our data demonstrate that halothane administration to Sprague-Dawley rats can induce a degree of lipid peroxidation that does not cause sufficient liver necrosis to increase plasma ALT activity. However, the data also suggest that under conditions that increase reductive metabolism and lipid peroxidation in rats (i.e., hypoxia and phenobarbital pretreatment), liver injury becomes apparent. The Fluorine²-isoprostane concentrations observed in the halothane/hypoxia model are similar to those observed after administering low doses of CCl⁴(0.01-0.1 ml/kg) causing only modest and variable amounts of injury. [24]Mild hepatic biochemical abnormalities are noted in some patients, even after a single exposure to halothane [2,3]and it is possible that they are caused by reductive halothane metabolism. This would be more likely in patients in whom reductive metabolism of halothane is enhanced or antioxidant defenses are compromised.

Much evidence, including a history of exposure to halothane in most patients, suggests that severe halothane hepatotoxicity in humans is immunologically mediated. [25]A variety of microsomal neoantigens containing a triflouroacetyl group derived from oxidative metabolism of halothane and which react with antibodies from patients with halothane hepatotoxicity have been identified. [26,27]Interestingly, the immunogenicity of membranes can be increased by alteration of the phase structure of phospholipids [28]leading to speculation that halothane-induced peroxidation of endoplasmic reticulum lipids may alter the phase of hepatocyte membranes sufficiently to enhance the immune response to halothane-induced neoantigens. This could occur after a halothane exposure that was insufficient to cause cellular necrosis and provide an indirect mechanism for halothane-induced lipid peroxidation to contribute to immunologically mediated halothane hepatotoxicity.

Isoniazid pretreatment has been shown to increase oxidative, but not reductive, metabolism of halothane in older male Fischer 344 rats. [29]Halothane causes liver injury in these animals in the absence of hypoxia but reductive metabolism of halothane is not believed to play a role in this model of halothane-induced liver injury. We found that isoniazid pretreatment did not increase halothane-induced lipid peroxidation (Fluorine²-isoprostanes) or potentiate liver injury in Sprague-Dawley rats (Figure 2). We used the same isoniazid pretreatment protocol that we previously found to have increased CCl⁴-induced Fluorine²-isoprostane production sevenfold in Sprague-Dawley rats. [14]In that study, CCl⁴-induced Fluorine²-isoprostane generation was also inhibited by the P4502E1 inhibitor 4-methylpyrazole. Isoniazid, like ethanol, induces cytochrome P4502E1 [30]and this enzyme is largely responsible for reductive metabolism of CCl⁴. [31]However, the lack of an effect of isoniazid pretreatment on halothane-induced Fluorine²-isoprostane generation suggests preliminarily that cytochrome P4502E1 is not involved in reductive metabolism of halothane in Sprague-Dawley rats in vivo.

Phenobarbital pretreatment potentiated the effect of halothane on Fluorine²-isoprostane production. This is probably due to selective induction of cytochrome P450 enzymes, some of which are responsible for reductive metabolism of halothane. Interestingly, phenobarbital pretreatment alone caused an approximately twofold increase in baseline liver Fluorine²-isoprostane concentrations, probably by increasing endoplasmic reticulum mass in hepatocytes. Phenobarbital pretreatment raises the yield of microsomal lipid and protein isolated from rat livers 1.5-fold. [32]Thus, increased lipid content in livers from phenobarbital-pretreated animals would be expected to result in increased baseline hepatic Fluorine²-isoprostane concentrations.

In summary, lipid peroxidation caused specifically by halothane was demonstrated in Sprague-Dawley rats and was shown to be increased by hypoxia and phenobarbital pretreatment but not isoniazid pretreatment. Furthermore, Fluorine²-isoprostane quantification provides an important new tool for the evaluation of new and established halogenated anesthetics for the potential to cause lipid peroxidation in vivo.