Terrence J. Monks

Acetaminophen-induced hepatotoxicity

Abstract In large doses the commonly used analgesic acetaminophen produces a centrilobular hepatic necrosis in man and experimental animals. The toxicity is mediated by a reactive metabolite formed by a cytochrome P-450 mixed-function oxidase system in hepatic microsomes. Following therapeutic doses the reactive metabolite is efficiently detoxified by glutathione. Following large doses, however, the total hepatic glutathione concentration is decreased to approximately 20% of normal and the reactive metabolite covalently binds to protein. Changes in protein covalent binding caused by various treatments correlates with changes in the incidence and severity of the hepatic necrosis. The reactive metabolite is believed to be N-acetylimidoquinone and is apparently formed by a previously uncharacterized mechanism for cytochrome P-450.

Bromobenzene and p-bromophenol toxicity and covalent binding invivo

A hepatotoxic dose of bromobenzene (3 mmoles/kg) decreases hepatic glutathione concentration in rats by approximately 80% within 5 hr following ip injection. A major bromobenzene metabolite, p-bromophenol at a similar dose did not significantly alter hepatic glutathione levels compared to controls. Twenty four hr after administration, serum glutamate pyruvate transaminase (SGPT) levels were significantly increased by bromobenzene but not by p-bromophenol. After 14C-bromobenzene administration, a significant amount of covalently bound radiolabel was detected in liver, kidney and small intestine. A small amount of covalently bound radiolabel was also detected in the lung. After a similar dose of 14C-bromophenol, covalently bound radiolabel was found in liver (62% of the amount detected with 14C-bromobenzene) and smaller amounts were detected in kidney, small intestine and lung. These data are consistent with the view that the hepatotoxicity and glutathione depleting ability of bromobenzene are mediated mainly by bromobenzene-3,4-oxide rather than by chemically reactive metabolites of p-bromobenzene. Covalently bound radiolabel from 14C-bromobenzene, however, may be derived from both bromobenzene-3,4-oxide and the nontoxic reactive metabolites of p-bromophenol.

The role of ortho-bromophenol in the nephrotoxicity of bromobenzene in rats

ortho-Bromophenol (1.92 mmol/kg, ip) caused a 50% decrease in renal glutathione levels within 90 min. In contrast, hepatic glutathione levels remained 80% of control values 5 hr after ortho-bromophenol administration. Renal glutathione was far more susceptible to the initial rapid depleting effects of ortho-bromophenol than was hepatic glutathione, the dose-response curve for hepatic glutathione depletion being shifted to the right. ortho-Bromophenol at doses greater than 1.6 mmol/kg caused severe renal necrosis in noninduced rats, with consequent elevations in BUN levels. This dose was one-fifth as large as that required by bromobenzene to produce a similar necrosis in phenobarbital-treated rats (W. D. Reid, Exp. Mol. Pathol., 19, 197-214, 1973). Phenobarbital pretreatment and depletion of tissue glutathione with diethyl maleate caused significant increases in BUN levels over controls. Pretreatment with piperonyl butoxide decreased the incidence of elevated BUN levels following ortho-bromophenol administration. While liver microsomes converted ortho-bromophenol to covalently bound material, kidney microsomes did not. However, in vivo, ortho-bromophenol covalently bound to kidney protein of control rats four times greater than to liver protein. Phenobarbital pretreatment increased the in vivo covalent binding to kidney protein but not to liver protein. The degree of covalent binding to kidney protein correlated with BUN levels (r = 0.91, p less than 0.001). The nature of the nephrotoxic metabolite of ortho-bromophenol is not known, but an intermediate may be generated in the liver and transported to the kidney. These findings suggest that ortho-bromophenol may play a role in the nephrotoxicity observed following bromobenzene administration.

Stereoselective formation of bromobenzene glutathione conjugates.

Two bromobenzene-glutathione conjugates have been detected as both in vivo and in vitro metabolites of bromobenzene. Separation and purification by high pressure liquid chromatography (HPLC) and analysis by 13C and 1H-NMR spectroscopy indicated that the metabolites are trans-3-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol and trans-4-bromo-6-(glutathion-S-yl)-cyclohexa-2,4-dien-1-ol. The two conjugates are formed in unequal amounts; over a dose range of 25-500 mg/kg the ratio of the two conjugates excreted into bile in 6 h was 1.6 +/- 0.1 (mean +/- S.E.). Pretreatment of rats with either phenobarbital or 3-methyl-cholanthrene did not significantly alter the ratio of the two conjugates excreted into bile. When bromobenzene was incubated with rat liver microsomes and glutathione, the same two conjugates were formed in the presence but not in the absence of 100 000 x g supernatant. Furthermore, in the presence of 100 000 x g supernatant from control animals, microsomes from rats treated with phenobarbital formed both conjugates 6 times more rapidly than did microsomes from control rats, whereas microsomes from rats treated with 3-methylcholanthrene formed both conjugates less rapidly than did those from control rats. Thus, the data suggest that both conjugates are formed via bromobenzene 3,4-oxide and that their formation requires in liver cytosol.

Detection and half-life of bromobenzene-3,4-oxide in blood

Bromobenzene-3,4-oxide can be detected in venous blood of rats by trapping it as the corresponding 35[S]glutathione conjugates. More bromobenzene-3,4-oxide is detected in venous blood of rats treated with phenobarbital and diethyl maleate than in venous blood of rats treated with phenobarbital alone. The half-life of bromobenzene-3,4-oxide in venous blood was about 13.5 s. Bromobenzene-3,4-oxide may contribute to the extrahepatic covalent binding and presumably the toxicity observed after bromobenzene administration. The present technique may be used to determine in blood, the presence or absence of other reactive metabolites that form glutathione conjugates.

Activation and detoxification of bromobenzene in extrahepatic tissues

Bromobenzene causes hepatic and extrahepatic toxicity in rats. Toxicity is related to the presence of covalently bound material in these tissues. A major bromobenzene metabolite, p-bromophenol, has been shown to give rise to covalently bound material in liver, lung and kidney in vivo, but is not toxic. p-Bromophenol is formed from bromobenzene in liver, lung and kidney microsomes and is subsequently metabolized to 4-bromocatechol and covalently bound material. Bromobenzene-3,4-oxide generated in situ by liver microsomes, is detoxified by kidney, liver and lung cytosol. The results suggest that the kidney toxicity caused by bromobenzene is probably not mediated by either bromobenzene-3,4-oxide or the reactive metabolites of p-bromophenol. In contrast, bromobenzene-3, 4-oxide may play a role in the lung toxicity observed after bromobenzene administration. However, the covalently bound material found in extrahepatic tissues may be derived from both bromobenzene-3,4-oxide or the reactive metabolites of p-bromophenol, which may be formed directly by these tissues or transported there from the liver.

Free radical intermediates and liver cell necrosis

It is now well established that differences in the severity and incidence of tissue damage caused by foreign compounds frequently correlate with differences in the covalent binding of chemically reactive metabolites of the foreign compounds with various intracellular components (Snyder et al, 1982). Although such correlations suggest that the toxicities are caused by chemically reactive metabolites, they usually do not by themselves provide direct proof for the identity of the toxic metabolites nor the sequence of events that results in the manifestation of the tissue damage. Indeed, they simply imply that the covalently bound material and the toxic metabolites are derived from common intermediates (Gillette, 1974a,b).