Abstract
We have developed a new cytotoxicity assay to study cellular detoxification capacity. Human lymphocytes are exposed in vitro to reactive acetaminophen (APAP) metabolites generated by purified and reconstituted rat cytochrome P-450 and NADPH-dependent cytochrome P-450 reductase. Cells (2 × 105) are incubated with APAP, 12 pmol P-450, 0.015 units of reductase, and an NADPH generating system in a microtitre plate well (250 μl total volume) for 2 hours at 37°C, washed, resuspended in 5 μg/ml albumin in HEPES and incubated for a further 16 hours at 37°C before assessment of cell death. Viability is based on the ability of live cells to reduce the tetrazolium salt MTT to a purple formazan which is measured with a multiwell scanning spectrophototometer. APAP toxicity, expressed as the percentage dead cells, is dependent on the presence of P-450 and NADPH, and can be inhibited by 25 μM SKF 525A. To validate this assay, lymphocytes from a patient with glutathtone synthetase (GSH-S) deficiency and 10 control subjects were challenged in vitro. Those of the GSH-S deficient patient exhibited toxicity to APAP which exceeded the 95% confidence limits of the control subjects over a concentration range of 10 - 1000 ug/ml : 7.0% toxicity at 10 ug/ml (control 95% confidence limit 0 - 0.4 %), 24.6% toxicity (3 9 - 9.5%) at 100 μg/ml and 43.1% toxicity (23.0 - 32.1%) at 1000 μg/ml. The data indicate a markedly enhanced susceptibility to reactive APAP metabolites presumably due to reduced GSH synthesis and thus detoxification capacity in GSH-S deficient cells. This new assay is free of detoxification enzymes such as microsomal epoxide hydrolase, requires a relatively small number of cells, and utilizes an objective, automated estimate of cell viability. It may prove useful for screening the cytotoxte potential of drugs implicated in idiosyncratic adverse reactions, and for determining the biochemical basis of susceptibility to drug toxicity.
Article PDF
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Leeder, J., Cannon, M., Nakhnoda, A. et al. DRUG METABOLITE TOXICITY ASSESSED IN HUMAN LYMPHOCYTES WITH A PURIFIED, RECONSTITUTED CYTOCHROME P-450 SYSTEM. Pediatr Res 21 (Suppl 4), 237 (1987). https://doi.org/10.1203/00006450-198704010-00422
Issue Date:
DOI: https://doi.org/10.1203/00006450-198704010-00422