An investigation of the formation of cytotoxic, protein-reactive and stable metabolites from carbamazepine in vitro

doi:10.1016/0006-2952(92)90696-G

Biochemical Pharmacology

Volume 43, Issue 8, 15 April 1992, Pages 1675-1682

https://doi.org/10.1016/0006-2952(92)90696-G Get rights and content

Abstract

The formation of chemically reactive metabolites from carbamazepine (CBZ) in the presence of mouse and human liver microsomes has been investigated using cytotoxicity and irreversible binding of radiolabelled compoud as quantitative end-points. For comparison, the formation of the stable CBZ-10,11-epoxide (CBZ-10,11-E) has been measured. The formation of the cytotoxic, protein-reactive and stable metabolites of CBZ was increased by induction of the cytochrome P450 enzymes by phenobarbitone and reduced by co-incubation in vitro with ketoconazole (10–250 μM), suggesting that the formation of these metabolites is cytochrome P450 dependent. All human livers tested (N = 6) bioactivated CBZ to a protein-reactive metabolite, the mean covalent binding increasing from 0.08 ± 0.01% (without NADPH) to 0.27 ± 0.09% (with NADPH; P ⩽ 0.05). The formation of the chemically reactive metabolites was reduced by a subphysiological concentration of reduced glutathione (GSH) (500 μM), while ascorbic acid (100 μM) had no effect. Neither compound affected the formation of CBZ-10,11-E. Microsomal epoxide hydrolase (mEH), but not cytosolic epoxide hydrolase, caused a concentration-dependent inhibition of cytotoxicity reaching a maximum of 60% at 100 U od mEH. Covalent binding was also reduced by 60% by 100 U mEH. The separated T- and B-lymphocytes showed no difference in sensitivity when incubated with CBZ and mouse microsomes. The study demonstrates that the balance between activation of CBZ by the cytochrome P450 enzymes to a chemically reactive arene oxide metabolite and its detoxification by mEH and GSH may contribute to individual susceptibility to CBZ idiosynratic toxicity.

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Testing hepatotoxicity is a crucial step in the development and toxicological assessment of drugs and chemicals. Bio-activation can lead to the formation of metabolites which may present toxicity for the organism. Classical cytotoxic tests are not always appropriate and are often insufficient, particularly when non metabolically-competent cells are used as the model system, leading to false-positive or false-negative results. We tested over 24 h the effects of eight reference compounds on two different cell models: primary cultures of rat hepatocytes and FAO hepatoma cells that lack metabolic properties. We performed inter-assay validation between three classical cytotoxicity assays and real-time cell impedance data. We then complemented these experiments with high-content screening (HCS) to determine the cell function disorders responsible for the observed effects. Among the different assays used, the neutral red test seemed to be well suited to our two cell models, coupled with real-time cellular impedance which proved useful in the detection of bio-activation. Indeed, impedance monitoring showed a high sensitivity with interesting curve profiles yet seemed unsuitable for evaluation of viability on primary culture. Finally, HCS in the evaluation of hepatotoxicity is likely to become an essential tool for use in parallel to a classical cytotoxic assay in the assessment of drugs and environmental chemicals.
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An investigation of the formation of cytotoxic, protein-reactive and stable metabolites from carbamazepine in vitro

Abstract

Blood

J Biol Chem

Biochem Pharmacol

J Biol Chem

J Biol Chem

Biochem Pharmacol

Pharmacol Ther

Biochem Pharmacol

Metabolic basis of adverse drug reactions

J Roy Coll Physicians Lond

Adverse drug reactions and drug metabolism

Adv Drug Reaction Bull

Activation mechanisms to chemical toxicity

Arch Toxicol

Metabolism of carbamazepine

Drug Metab Dispos

Carbamazepine metabolism in man. Induction and pharmacogenetic aspects

Clin Pharmacokinet

Metabolism of carbamazepine and its epoxide metabolite in human and rat liver in vitro

Drug Metab Dispos

Drugs 5 years later: carbamazepine

Ann Intern Med

Hepatotoxic reactions associated with carbamazepine therapy

Epilepsia

Structural requirements for bioactivation of anticonvulsants to cytotoxic metabolites in vitro

Br J Clin Pharmacol

Anticonvulsant hypersensitivity syndrome: in vitro risk assessment

J Clin Invest

Carbamazepine hypersensitivity: assessment of clinical and in vitro chemical cross-reactivity with phenytoin and oxcarbazepine

Br J Clin Pharmacol

Genetic predisposition to drug hepatotoxicity: role in hepatitis caused by amineptine, a tricyclic antidepressant

Hepatology