Comparison of Metabolic Stability and Metabolite Identification of 55 ECVAM/ICCVAM Validation Compounds between Human and Rat Liver Homogenates and Microsomes - a preliminary Analysis

Abstract

In vitro methods to produce metabolic information have increasingly been applied in toxicity risk assessment. In the current contract project of JRC/ECVAM In Vitro-Toxicology Unit, 55 organic chemicals, mostly drugs and pesticides, most belonging to ECVAM/ICCVAM validation compounds, expected to be analyzable by LC-MS technique, were subjected to a feasibility study. A simple experimental setup consisted of one concentration of a chemical (25 µM), enzyme preparation (human or rat liver homogenate or microsomes), a set of cofactors (NADPH, UDPGA, PAPS, GSH), 4 time points (0, 15, 30, 60 min, including cofactor-less tubes). Metabolites produced were analyzed and tentatively identified by the LC-MS techniques. Most of the chemicals were metabolized and metabolites were tentatively identified by the TOF-MS analysis. For some chemicals, about 10 or even more metabolites were detectable (e.g. thioridazine, verapamil, amitriptyline). Altogether 11 out of 55 did not display any metabolites under the experimental conditions of this study. Regarding the metabolites formed, there were mostly quantitative differences, but about 20 substances displayed also species-dependent qualitative differences, i.e. a major metabolite was formed in one species, but not in the other. For most chemicals, differences between microsomes and homogenates were relatively modest at least in the initial analysis. The results demonstrate that LC-MS approach is feasible and rather efficient in providing useful metabolic data from a simple experimental setup. More complex analyses, e.g. quantitative assessment of differences between species or biological preparations, or in vitro - in vivo extrapolations, require more complex approaches and a collection of appropriate, preferably curated, data bases of in vivo characteristics of the studied chemicals.