Halothane-induced liver injury in outbred guinea pigs: role of trifluoroacetylated protein adducts in animal susceptibility.

Halothane causes a mild form of liver injury in guinea pigs that appears to model the hepatotoxicity seen in approximately 20% of patients treated with this drug. In previous studies, it was concluded that the increased susceptibility of some outbred guinea pigs to halothane-induced liver injury is not caused by their inherent ability to metabolize halothane to form toxic levels of trifluoroacetylated protein adducts in the liver. In this study, we reevaluated the role of trifluoroacetylated protein adducts in halothane-induced liver injury in guinea pigs. Male outbred Hartley guinea pigs were treated with halothane intraperitoneally. On the basis of serum alanine aminotransferase levels and liver histology, treated animals were designated as being susceptible, mildly susceptible, or resistant to halothane. Immunoblot studies with the use of anti-trifluoroacetylated antibodies showed that susceptible guinea pigs for the most part had higher levels of trifluoroacetylated protein adducts in the liver 48 h after treatment with halothane than did less susceptible animals. In support of this finding, the level of trifluoroacetylated protein adducts detected immunochemically in the sera of treated guinea pigs correlated with sera levels of alanine aminotransferase activity. In addition, the levels of cytochrome P450 2A-related protein but not those of other cytochrome P450 isoforms, measured by immunoblot analysis with isoform-specific antibodies, correlated with the amount of trifluoroacetylated protein adducts detected in the livers of guinea pigs 8 h after halothane administration. The results of this study indicate that the susceptibility of outbred guinea pigs to halothane-induced liver injury is related to an enhanced ability to metabolize halothane in the liver to form relatively high levels of trifluoroacetylated protein adducts. They also suggest that cytochrome P450 2A-related protein might have a major role in catalyzing the formation of trifluoroacetylated protein adducts in the liver of susceptible guinea pigs. Similar mechanisms may be important in humans.

Selective protein covalent binding and target organ toxicity.

Protein covalent binding by xenobiotic metabolites has long been associated with target organ toxicity but mechanistic involvement of such binding has not been widely demonstrated. Modern biochemical, molecular, and immunochemical approaches have facilitated identification of specific protein targets of xenobiotic covalent binding. Such studies have revealed that protein covalent binding is not random, but rather selective with respect to the proteins targeted. Selective binding to specific cellular target proteins may better correlate with toxicity than total protein covalent binding. Current research is directed at characterizing and identifying the targeted proteins and clarifying the effect of such binding on their structure, function, and potential roles in target organ toxicity. The approaches employed to detect and identify the tartgeted proteins are described. Metabolites of acetaminophen, halothane, and 2,5-hexanedione form covalently bound adducts to recently identified protein targets. The selective binding may influence homeostatic or other cellular responses which in turn contribute to drug toxicity, hypersensitivity, or autoimmunity.

UDP-glucose:glycoprotein glucosyltransferase associates with endoplasmic reticulum chaperones and its activity is decreased in vivo by the inhalation anesthetic halothane.

Halothane causes an idiosyncratic hepatitis that is thought to result, in part, from immune reactions against one or more lumenal endoplasmic reticulum (ER) proteins that have been covalently modified by the trifluoroacetyl chloride metabolite of halothane. In this study, we have identified a 170 kDa protein target of halothane in the liver of rats. The 170 kDa protein was first detected when proteins in lysates of hepatocytes from halothane-treated rats were immunoprecipitated with antisera against several resident ER proteins. This 170 kDa protein was found to be associated with other protein targets of halothane, including protein disulfide isomerase, a protein disulfide isomerase isoform, a 59 kDa carboxylesterase, and 78 kDa glucose-regulated protein. Immunoblotting with antiserum directed against the trifluoroacetylated hapten indicated that the 170 kDa protein was trifluoroacetylated. Based upon its subcellular localization, molecular mass, N-terminal amino acid sequence, and antigenicity, the trifluoroacetylated 170 kDa protein was identified as UDP-glucose:glycoprotein glucosyltransferase (UGGT), a lumenal ER protein that is thought to have a role in the folding of N-linked glycoproteins. Moreover, treatment of rats with halothane caused a 44% decrease in the activity of liver microsomal UGGT, and at least 36% of the change in the activity of the enzyme could be due to a decrease in the level of the protein. The results suggest that the function of UGGT in folding of N-linked glycoproteins may be affected by other resident ER proteins or xenobiotics such as halothane.

Processing of endoplasmic reticulum luminal antigens associated with halothane hepatitis in rat hepatocytes.

In this study we have investigated the mechanism of the processing of trifluoroacetylated liver microsomal protein antigens associated with halothane hepatitis to learn how the immune system might come in contact with these proteins to form antibodies directed against them. Rats were treated with halothane and parenchymal (PC) and non-parenchymal cells (NPC) were isolated 16 hours later. Immunoblotting of the cell lysates with antisera directed against the trifluoroacetyl hapten showed the presence of high levels of trifluoroacetylated proteins in parenchymal cells, whereas none of these proteins were detected in endothelial or Kupffer cells that were isolated by centrifugal elutriation. The half-lives of 100-, 82-, 80-, 63-, 59-, 58-, and 57-kd trifluoroacetylated and native carrier proteins of the trifluoroacetyl hapten in cultures of rat primary parenchymal cells were approximately 1 day. The turnovers of all of these trifluoroacetylated proteins, except for that of the trifluoroacetylated 100-kd protein, were inhibited by treatment of the cells with ammonium chloride, leupeptin, 4-(2-aminoethyl)-benzenesulfonyl fluoride, or 3-methyl-adenine (3-MA). These results indicate that, in liver, the major source of the formation of trifluoroacetylated antigens associated with halothane hepatitis is the parenchymal cells, It appears that most of the trifluoroacetylated antigens and possibly the native carrier protein of the trifluoroacetyl haptens are transferred from the endoplasmic reticulum (ER) to an acidic compartment of PCs, where they are enzymatically degraded. The processing of the trifluoroacetylated proteins by this pathway may be a protective mechanism that prevents these covalently altered proteins from inducing an antibody response in most patients who are administered halothane.

Identification of novel differentially expressed hepatic genes in cholesterol-fed rabbits by a non-targeted gene approach.

Several key genes involved in cholesterol metabolism are known to be directly regulated by cholesterol. The possible indirect effect, however, of increased levels of cellular cholesterol on gene expression and its possible role in cholesterol metabolism and atherosclerosis has not been thoroughly explored. In order to determine the overall effect of cholesterol on gene expression, we isolated differentially expressed genes from a PCR-based subtraction library prepared from the liver of chow-fed and cholesterol-fed rabbits. A total of nine upregulated and four down-regulated cDNA fragments were isolated. As determined by Northern blot analysis, the expression of the isolated cDNAs began to change as early as the first week on the cholesterol-rich diet or as late as 4 weeks, which corresponded with hepatic cholesterol accumulation. Three of the cDNAs were identified by DNA sequence homology, whereas the remaining cDNAs had no significant homology match. CYP1A1, a cytochrome P450 isoenzyme, was found to be down-regulated in hepatocytes by cholesterol feeding. Osteopontin and Mac-2, which are produced by macrophages, were found to be up-regulated in Kupffer cells by cholesterol feeding. Overall these results demonstrate the usefulness of the subtraction library approach for identifying new candidate genes for exploring the pathogenesis of atherosclerosis.

Biochemical and morphological alterations in xylazine-induced pulmonary edema.

Sprague-Dawley rats were given 42 mg/kg xylazine intramuscularly, and lungs were lavaged with phosphate-buffered saline 3, 6, and 12 hr later. Total protein, lactate dehydrogenase (LDH), xanthine oxidase (XO), tumor necrosis factor (TNF), and interleukin 1 (IL-1) were measured in bronchoalveolar lavage fluid (BALF). Protein concentration, LDH, XO, and TNF levels were increased (p < 0.05) in the BALF from xylazine-treated rats as compared to controls. IL-1 level was unchanged at 3 and 6 hr and was reduced (p < 0.05) at 12 hr. Another group of rats was given 42 mg/kg xylazine intramuscularly, and lungs were fixed 0.5 and 12 hr later. Histologically, severe pulmonary edema (PE) involving the alveoli and perivascular stroma was observed. Fibrin, increased numbers of eosinophils, and macrophages with foamy cytoplasm were present in the alveoli of all treated animals. Ultrastructurally, endothelial damage, characterized by thinning, detachment from basement membranes, or bleb formation, was observed. The lesions were similar in both xylazine groups, differing mainly in severity with the 12-hr group having more severe lesions than the 0.5-hr group. To determine whether endothelial injury is caused by direct toxicity of xylazine, bovine pulmonary artery endothelial cells (BPAECs) were incubated with xylazine (0.3, 3, and 30 micrograms) for 0.5 or 3 hr. Xylazine did not have any effects on BPAECs, as indicated by phase-contrast microscopy and dye-exclusion viability assay. These results indicate that xylazine-induced PE is due to increased permeability resulting from endothelial injury, which is not caused by direct effect of xylazine on pulmonary endothelium. While oxygen radicals and TNF are possibly involved, IL-1 does not appear to play a role in xylazine-induced PE.

Effects of hepatic P-450 enzyme inhibitors and inducers on the duration of xylazine + ketamine anesthesia in broiler chickens and mice.

We previously showed that pretreatment with inhibitors of hepatic P-450 microsomal enzymes prolonged xylazine-ketamine anesthesia, which sometimes led to mortality in rats. In this study we determined if similar effects were produced in broiler chickens and mice. A combination of 5 mg xylazine/kg and 15 mg ketamine/kg im was given to broiler chickens 5-6 w of age, and a combination of 10 mg xylazine/kg and 200 mg ketamine/kg ip was given to mice. The loss of righting reflex was used to measure duration of anesthesia. Pretreatment with 100 mg chloramphenicol/kg im, 150 mg cimetidine/kg im, 25 mg SKF-525A/kg im or 40 mg ketoconazole/kg po significantly increased the duration of xylazine + ketamine anesthesia in the chickens. Pretreatment with 40 mg phenobarbital/kg im bid for 3 d, 25 mg 3-methylcholanthrene/kg im sid for 3 d, or 50 mg rifampin/kg im bid for 2 d failed to alter the duration of xylazine + ketamine anesthesia in the broilers. None of the inhibitors tested altered the duration of anesthesia in mice. Some chickens pretreated with inhibitors (cimetidine, ketoconazole, SKF-525A) or inducers (phenobarbital, 3-MC, rifampin) died. This study suggests that hepatic metabolism of xylazine may be similar in the rat and broiler chicken and that the pulmonary metabolism of xylazine and ketamine may be different in chickens and rats.

Xylazine-induced pulmonary edema in rats.

Inhibitors of cytochrome P450, such as SK&F 525-A, prolong the duration of xylazine-ketamine anesthesia and cause pulmonary edema (PE) and death in rats. To determine the cause of PE, Sprague-Dawley rats were given a single dose of xylazine (21 mg/kg, im) alone or in combination with ketamine (45 mg/kg, im) and/or SK&F 525-A (50 mg/kg, ip) and percentage lung to body weight (%LW/BW) ratios (as an indicator of PE) were compared. The results indicated that xylazine caused PE which was independent of ketamine and was enhanced by SK&F 525-A. Subsequently, it was determined that 42 mg/kg xylazine, im, is an optimal edemagenic dose. Xylazine (42 mg/kg, im) increased the %LW/BW ratio as compared to control. Pleural effusion (PLE) of various amounts was observed in 75% of the animals. The pleural fluid to serum protein ratio for xylazine was similar to that obtained for alpha-naphthylthiourea (5 mg/kg, ip). Extensive serous PLE and alveolar edema with hemorrhage were found at necropsy in xylazine-treated rats. Pretreatment with yohimbine (4.2 mg/kg), prazosin (20 mg/kg), tolazoline (20 mg/kg), yohimbine (4.2 mg/kg) plus prazosin (20 mg/kg), atropine (20 mg/kg), dimethyl sulfoxide (DMSO) (7.8 g/kg), allopurinol (50 mg/kg), superoxide dismutase (20,000 U/kg), catalase (20,000 U/kg), BW755C (50 mg/kg), ibuprofen (50 mg/kg), cystathionine (100 mg/kg) plus taurine (100 mg/kg) did not affect the %LW/BW ratio. PLE was increased by yohimbine, yohimbine plus prazosin, and allopurinol, reduced by DMSO, and not changed in other groups. The results indicate that xylazine caused increased-permeability PE characterized by rapid onset, cellular damage and protein-rich pleural fluid. PE may not be mediated by adverse cardiovascular effects of xylazine and oxygen radicals are possibly involved in its etiology.

Effects of cimetidine and ranitidine on basal gastric pH, free and total acid content in pigs.

The effects of intramuscular administration of multiple doses of cimetidine and ranitidine on basal gastric pH, free and total acid content from young adult pigs were studied. Cimetidine (4.5 mg kg-1, four times a day, intramuscularly, for three days) significantly (P less than 0.05) raised the basal gastric pH above 3.5 with a simultaneous reduction in free acid content at two, three and 26 hours after the administration of the eighth dose. Ranitidine (0.75 mg kg-1, four times a day, intramuscularly, for three days) significantly (P less than 0.05) raised the basal gastric pH above 3.5 with a concomitant reduction in free acid content at three and 38 hours after the administration of the eight dose. Neither cimetidine nor ranitidine had any significant effects on total acid content. These results confirm that the pig is a basal acid secretor and that the pharmacodynamics and pharmacokinetics of cimetidine and ranitidine in pigs might be different from those in humans.

Effects of chloramphenicol, cimetidine and phenobarbital on and tolerance to xylazine-ketamine anesthesia in dogs.

The effects of chloramphenicol, cimetidine and phenobarbital on the xylazine-ketamine anesthesia and the development of tolerance to xylazine-ketamine and xylazine were studied in dogs. The duration of absence of pedal reflex, duration of return of consciousness, and duration for return of ambulation were determined. Pretreatment with chloramphenicol (33 mg/kg, iv, 15 min) and cimetidine (5 mg/kg, iv, 24 h) did not influence any of the above parameters significantly. Phenobarbital pretreatment (15 mg/kg, iv, 96 h) significantly reduced the duration of anesthesia. Although not significant, there is a trend toward the development of tolerance to repeated administration of xylazine (1.1 mg/kg, iv) and ketamine (10 mg/kg, iv) combination and xylazine (1/1 mg/kg, iv) alone once daily at 3 d intervals for 9 d. These results indicate that hepatic cytochrome P-450 drug metabolizing enzymes, inhibited by chloramphenicol and cimetidine, might not be involved in the metabolism of xylazine and/or ketamine, phenobarbital inducible hepatic cytochrome P-450 enzymes might play a role in metabolic disposition of xylazine and/or ketamine, and repeated administration of xylazine alone or in combination with ketamine might lead to development of tolerance in dogs.

Effects of streptozotocin-induced diabetes on xylazine-ketamine anesthesia.

The effects of streptozotocin-induced diabetes and insulin on the duration of xylazine-ketamine anesthesia were studied in rats. The duration of anesthesia was significantly reduced in diabetic group as compared to control group. In contrast, the duration of anesthesia in insulin-treated non-diabetic group was not significantly different from control group. The plasma glucose level in control group was increased significantly during and upon recovery from anesthesia as compared to pre-anesthetic levels. The plasma glucose level in diabetic group was not changed during anesthesia; however, it was reduced significantly upon recovery. The plasma glucose level was significantly reduced in insulin-treated group as compared to pre-anesthetic levels. The reduction in duration of anesthesia in diabetic rats is possibly due to enhanced metabolism xylazine and/or ketamine and is independent of plasma glucose level.

Effects of some hepatic microsomal enzyme inducers and inhibitors on xylazine-ketamine anesthesia.

Various inducers and inhibitors of hepatic microsomal enzymes were studied for their effects on xylazine-ketamine anesthesia. Pretreatment of Sprague-Dawley rats with chloramphenicol (100 mg/kg, ip), cimetidine (100 mg/kg, ip), ketoconazole (40 mg/kg, po), and SKF 525-A (25 mg/kg, ip) significantly (p less than 0.05) increased the duration of anesthesia in rats injected with ketamine (45 mg/kg, im) and xylazine (21 mg/kg, im). Pretreatment with phenobarbital (40 mg/kg, ip, once daily for 4 days) did not affect the duration of anesthesia significantly. The increase in duration of anesthesia in animals pretreated with SKF 525-A and ketoconazole was accompanied by secondary respiratory distress about 6 hr following recovery from anesthesia, often leading to death within 24 hr. Lesions consisting of extensive serous pleural effusion, alveolar edema rich in macrophages and extensive pulmonary hilar edema with hemorrhage were found at necropsy. These results indicate that inhibition of hepatic microsomal enzymes by commonly used therapeutic agents during xylazine-ketamine anesthesia to prolong the anesthetic effect, or as a result of concurrent pharmacotherapy, could have deleterious effects.

Effects of misoprostol and omeprazole on basal gastric pH and free acid content in horses.

The basal gastric pH and free acid contents from five young adult healthy horses were determined at one hour intervals for eight hours. The basal gastric pH and free acid contents varied from 1.63 +/- 0.06 to 1.97 +/- 0.11 and 26.42 +/- 4.14 to 17.92 +/- 5.28 mmol litre-1, respectively. Misoprostol, a methylester analogue of prostaglandin (5 micrograms kg-1, orally) produced a time-dependent increase in the basal gastric pH to above 3.5 (P less than 0.05) at three, four and five hours after administration with a concomitant reduction of 80 to 90 per cent in the basal gastric free acid contents throughout the eight hour period monitored. Omeprazole, a benzimidazole derivative (0.5 mg kg-1, intravenously) increased the basal gastric pH to above 3.5 at two and three hours after administration with a concomitant reduction of 65 to 90 per cent in the basal gastric free acid contents for seven of the eight hour periods monitored. These results confirm that the horse is a basal acid secretor, and both misoprostol and omeprazole are effective inhibitors of the basal gastric acid secretion, thus establishing that both prostaglandins and H+/K+-ATPase play an important role in controlling parietal cell function of the equine gastric mucosa.

Effects of cimetidine and ranitidine on basal gastric pH, free and total acid contents in horses.

The basal gastric pH, free and total acid contents from five adult horses were determined at two-hour intervals for six- to eight-hour periods. The basal gastric pH, free and total acid contents varied from 2.14 +/- 0.08 to 2.41 +/- 0.14, 28.63 +/- 8.27 to 17.89 +/- 2.86 mmol litre-1 and 41.38 +/- 9.72 to 37.38 +/- 3.70 mmol litre-1, respectively. Cimetidine (8.8 mg kg-1 orally) and ranitidine (2.2 mg kg-1 orally) increased the basal gastric pH to above 3.6 (P less than 0.05) with a concomitant reduction of 75 per cent and 75 to 100 per cent in the basal gastric free acid content, respectively, for an eight-hour period. Cimetidine (4.4 mg kg-1, intramuscularly) and ranitidine (1.4 mg kg-1, intramuscularly) increased the basal gastric pH to above 3.6 with a concomitant reduction of 54 to 93 per cent and 69 to 100 per cent in the basal gastric free acid content, respectively, for an eight-hour period. This study shows that the horse is a basal acid secretor, and that cimetidine and ranitidine, two widely used histaminergic-H2 type antagonists in human clinical practice are effective in horses with ranitidine being approximately four times more potent than cimetidine.

Failure of naloxone to attenuate fasting induced hyperphagia in broiler chicks.

Subcutaneous administration of naloxone at 1 to 10 mg/kg produced a dose-related decrease in feed intake of broiler chicks. Food deprivation for 3, 6, 12, and 24 hours produced a significant increase in feed intake compared to non-food deprived birds. Subcutaneous administration of naloxone at 1 to 10 mg/kg failed to attenuate hyperphagia of broiler chicks, deprived of food for 12 hrs. These data suggest that opiate receptors are involved in the regulation of spontaneous feeding behavior in broiler chicks. However, in contrast to other mammals and pigeons, a mechanism, other than endorphinergic system, not sensitive to naloxone blockade, might be involved in food deprivation induced hyperphagia in broiler chicks.