James R. Gillette

Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite.

This laboratory has previously postulated that bromobenzene-induced hepatic necrosis results from the formation of a reactive metabolite that arylates vital cellular macromolecules. Accordingly, the severity of liver necrosis has been compared with the formation of metabolites of bromobenzene and with covalent binding of metabolites in vivo and in vitro after various pretreatment regimens that alter hepatotoxicity. These data provide direct kinetic evidence that 3,4-bromobenzene oxide is the reactive hepatotoxic metabolite. The studies also demonstrate that the hepatotoxic metabolite is preferentially conjugated (detoxified) with glutathione, thereby depleting glutathione from the liver. Liver necrosis and arylation of cellular macromolecules occur only when glutathione is no longer available. Thus, a dose threshold exists for bromobenzene-induced hepatic necrosis.

Biochemistry of Drug Oxidation and Reduction by Enzymes in Hepatic Endoplasmic Reticulum

Publisher Summary This chapter discusses the biochemistry of drug oxidation and reduction by enzymes in hepatic endoplasmic reticulum. In studying the action of drugs in the body, it is important not only to determine the mechanisms through which drugs elicit their pharniacological effect, but, also to study the factors which control their concentration at active sites. The conccntration of some drugs are controlled mainly by their relative rates of absorption and excretion. Most drugs, however, are converted to other substances before they are excreted into air, bile, and urine, and thus their rates of metabolism may be a major factor in limiting their action. Indeed, the pharmacological effects of highly bound drugs, such as chlorpromazine, phenylbutazone, and thiopental, would last for virtually a lifetime if they were not converted to inactive metabolites which were readily excreted.

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: Role of cytochrome P-450

Abstract In order to define the site of bioactivation of CCl4, CHCl3 and CBrCl3 in the NADPH cytochrome c reductase-cytochrome P-450 coupled systems of liver microsomes, the 14C-labeled hepatotoxins were incubated in vitro with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O2 (8:2) and addition of a cytochrome c reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome c reductase activity was unchanged, the covalent binding of CCl4, CHCl3, and CBrCl3 was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N2 enhanced the binding of CCl4, inhibited the binding of CHCl3 and did not influence the binding of CBrCl3. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome c reductase and that CCl4 bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl3 activation proceeds by cytochrome P-450 dependent oxidative pathways.

Studies on the mechanism of the lung toxicity of paraquat: Comparison of tissue distribution and some biochemical parameters in rats and rabbits

Abstract After iv injection of [14C]paraquat (20 mg/kg) tissue localization was preferential in lungs of rats as well as rabbits although the latter did not show any histopathologic or biochemical signs of lung damage. No preferential subcellular localization of [14C]paraquat was found in lungs of either species, but all subcellular levels decreased more rapidly in the rabbit than in the rat. [14C]Paraquat was not covalently bound to tissue macromolecules. In vitro measurements of lipid peroxidation, H2O2 formation and lung lysosomal stability failed to account adequately for the lung damage in the rat.

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.

Studies on the destruction of liver microsomal cytochrome P-450 by carbon tetrachloride administration

Carbon tetrachloride impairs the oxidative enzymes in liver microsomes by decreasing the amount of cytochrome P-450. The destruction is probably not mediated by an increase in lipid peroxidation, since ethanol which promotes lipid peroxidation does not decrease cytochrome P-450 or the metabolism of ethylmorphine. The decrease in cytochrome P-450 is not due to destruction of liver cells or to altered permeability of endoplasmic reticulum because NADPH cytochrome c reductase was not detected in plasma and was not decreased in liver microsomes. The solvent effect of CCl4 probably does not account for the decrease in cytochrome P-450 because CHCl3 and CH2Cl2 did not decrease the cytochrome. Our results are in accord with the view that CCl4 acts through an active metabolite, but they do not necessarily imply that the active metabolite is formed by a cytochrome P-450 enzyme.

Induced Synthesis of Liver Microsomal Enzymes Which Metabolize Foreign Compounds

The administration of 3,4-benzpyrene to rats markedly increases the activities of certain liver microsomal enzymes which metabolize foreign compounds. Evidence based on studies of enzyme induction is presented which suggests the presence in liver microsomes of several enzymes which can catalyze the same type of reaction.

Prevention of carbon tetrachloride-induced necrosis by inhibitors of drug metabolism—Further studies on their mechanism of action

Abstract Liver necrosis caused by CCl 4 is known to be decreased by the administration of SKF 525-A (2-diethylaminoethyl 2,2-diphenylvalerate), Sch 5705 (ethyl 2-diethyl-aminoethyl-2-phenyl-2-ethyl malonate), Sch 5706 [ethyl N -(2-diethylaminoethyl) 2-phenyl-2-ethyl malonate], Sch 5712 (ethyl 2-diethylaminoethyl 2-ethyl-2-butyl malonate), CFT 1201 [2-diethylaminoethyl 2-phenyl (2-propene) 4-penten-1-oate], Lilly 18947 (2,4-dichloro-6-phenyl phenoxyethyl diethylamine) and DPEA (2,4-dichloro-6-phenyl phenoxyethylamine). Although these substances are known to inhibit cytochrome P-450 dependent drug-metabolizing enzymes in liver microsomes, they apparently do not evoke their protective effects by slowing the elimination of CCl 4 . In fact, SKF 525-A, but none of the other inhibitors, partially prevents the impairing effects of CCl 4 on cytochrome P-450 in liver. Although SKF 525-A markedly decreased the liver necrosis and the rise in serum isocitrate dehydrogenase (ICD) caused by CCl 4 , it only partially prevented the CCl 4 -induced decrease in body temperature.

Isolation from rat brain of a metabolic product, desmethylimipramine, that mediates the antidepressant activity of imipramine (Tofranil)

Nach Verabreichung von Imipramin (Tofranil) wurde aus dem Gehirn von Ratten Desmethylimipramin, das N-Monomethylderivat, isoliert. Dieser Metabolit beziehungsweise dessen Anreicherungin vivo scheint für die antidepressive Wirkung von Imipramin verantwortlich zu sein.

Species and sex differences in electron transport systems in liver microsomes and their relationship to ethylmorphine demethylation.

Abstract Species differences in the metabolism of ethylmorphine parallel differences in NADPH cytochrome P-450 reductase more closely than they do differences in NADPH-cytochrome c reductase, cytochrome P-450 content or the type I spectral change caused by ethylmorphine.