Drug metabolite toxicity assessed in human lymphocytes with a purified, reconstituted cytochrome P-450 system

Abstract

Evaluation of idiosyncratic drug reactions in predisposed individuals is limited by ethical concerns arising from rechallenge with the suspected offending agent. A previously developed in vitro method using human lymphocytes and a murine microsomal drug metabolizing system has been used to examine toxicity due to acetaminophen (APAP), sulfonamide antibiotics and aromatic anticonvulsants. An improved method is described in which toxic APAP metabolites are generated by a purified and reconstituted cytochrome P-450 system, minimizing the amount of exogenous detoxification enzymes in the assay. Toxicity is assessed by an objective, automated method based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to an insoluble purple formazan by the mitochondria of viable cells and correlates with that based on trypan blue exclusion. Toxicity required cytochrome P-450 and NADPH, and was inhibited by SKF 525A. Exogenous glutathione also decreased toxicity in a concentration-dependent manner. Lymphocytes from a glutathione synthetase-deficient patient exhibited markedly enhanced toxicity to APAP exceeding the 95% CL of 10 control subjects over a concentration range of 10 to 1000 micrograms/ml. The data are consistent with the generation of cytochrome P-450-dependent reactive metabolites which subsequently can be detoxified by glutathione. This method allows one to address specifically individual differences in detoxification pathways. The use of an automated assessment of cell viability may prove useful in preclinical screening of new compounds for their propensity to cause "idiosyncratic" drug reactions in a predisposed population.