Competing pathways in metabolism-mediated cytotoxicity in vitro

The metabolism of most xenobiotics to reactive metabolites is accompanied by competition with inactivation pathways, and the balance of these activation/inactivation pathways is critical in determining the precise circumstances in which metabolism-mediated cytotoxicity may be detected in vitro. Discussion of the importance of this balance must also take into account the nature of the question posed at the start of an in vitro study, and the choice of metabolizing system used. In quantitative terms, liver microsomal cytochrome P-450-mediated monooxygenase activity and conjugation with glutathione (GSH) are the major routes of activation and inactivation, respectively. Results of recent studies on the cytotoxicity of cyclophosphamide, bromobenzene and paracetamol measured in a liver homogenate fraction-cultured cell co-incubation have confirmed the importance of activation/inactivation balance and stability of the reactive metabolite as determinants of metabolism-mediated cytotoxicity in vitro. Competition between metabolism by P-450-mediated enzyme activity and conjugation with GSH may be used as the basis of a screen for detection of reactive metabolites by measurement of GSH depletion or conjugation with GSH. The use of cells in culture for studies of metabolism-mediated cytotoxicity is confounded by the time-dependent alterations in various enzyme activities that occur and so distort the balance of activation/inactivation. This, in turn, reduces the reliability of such systems as predictors of the events likely to occur in vivo; the lack of a distribution component in the in vitro system further distorts this in vivo/in vitro comparison. Rational development of in vitro systems for detection of reactive metabolites can only occur with an understanding of the role of competing pathways in metabolism.