ATLA::Alternatives to Laboratory Animals

Volume 29, Number 6

A genetically engineered cell-based system for detecting metabolism-mediated toxicity.

ATLA 29, 703-716, November/December 2001

Sarah Bull,^1,2 Ingrid Langezaal,¹ Richard Clothier³ and Sandra Coecke¹

¹ECVAM, Institute for Health & Consumer Protection, European Commission Joint Research Centre, 21020 Ispra (VA), Italy; ²Department of Veterinary Pharmacology, Pharmacy and Toxicology, University of Utrecht, Yalelaan 16, 3508 TD Utrecht, The Netherlands; ³School of Biomedical Sciences, University of Nottingham Faculty of Medicine and Health Sciences, Queen's Medical Centre, Nottingham NG7 2UH, UK

SUMMARY

Xenobiotics undergong bioactivation by CYP450 enzymes form reactive metabolites that may exert direct metabolism-mediated toxicity. An in vitro model was developed to study the direct toxic effects that follow the metabolic activation of chemicals. The model uses monolayer cultures of genetically engineered NIH-3T3 or V79 cells that express individual human or rat CYP450 isoforms, respectively. Following exposure to 1,3-dichloropropanol or cyclophosphamide, basal cytotoxicity endpoints, including neutral red uptake and Alamar Blue^TM reduction were used to assess changes in cell number and functional viability resulting from the formation of metabolites. Cell lines that express cytochrome P450 enzymes metabolised the test compounds, leading to increased toxicity compared with that observed in the control cell line. The use of specific inhibitors confirmed that the formation of reactive metabolites was CYP450-isoform dependent. These results indicate that a panel of genetically engineered cell lines expressing various cytochrome P450 enzyme isoforms can be used to reveal measurable metabolising capabilities, and could become a useful tool for the detection and possible determination of CYP450 isoforms in human liver metabolism-mediated toxicity.

Keywords: metabolism-mediated toxicity, genetically engineered cell lines, human CYP450, in vitro