Genotoxic Assays for Measuring P450 Activation of Chemical Mutagens

Abstract

This review discusses using yeast as a model organism for studying the biological effects of P450-mediated metabolism of xenobiotics. We discuss the challenges of testing the safety of thousands of chemicals currently introduced into the market place, the limitations of the animal systems, the advantages of model organisms, and the humanization of the yeast cells by expressing human cytochrome P450 (CYP) genes. We discuss strategies in utilizing multiple genetic endpoints in screening chemicals and yeast strains that facilitate phenotyping CYP polymorphisms. In particular, we discuss yeast mutants that facilitate xenobiotic import and retention and particular DNA repair mutants that can facilitate in measuring genotoxic endpoints and elucidating genotoxic mechanisms. New directions in toxicogenetics suggest that particular DNA damaging agents may interact with chromatin and perturb gene silencing, which may also generate genetic instabilities. By introducing human CYP genes into yeast strains, new strategies can be explored for high-throughput testing of xenobiotics and identifying potent DNA damaging agents.Genotoxicity and Mutagenicity -Mechanisms and Test Methods 2 the Ames assays are carcinogenic, while others that test positive in the Ames assays are not carcinogens [8,9]. Many chemicals are not genotoxic per se but require metabolic bioactivation [10]. The bioactivated compound is generally a highly reactive intermediate in a pathway which renders hydrophobic compounds more hydrophilic to facilitate excretion. While bioactivation does occur in specific animal models, toxicity outcomes differ depending on the species [11]. Thus, there is a need for metabolic competent cell-based assays that can measure multiple genotoxic endpoints.Bioactivation occurs by phase I and phase II enzymes; phase I enzymes generally hydroxylate compounds so that phase II enzymes can conjugate larger molecules, facilitating the export and excretion of the modified compound. Phase I enzymes include cytochrome P450 monooxygenases (CYPs), which compose a superfamily of over 50 genes, and catalyze the formation of highly reactive electrophiles and epoxides, as intermediates in xenobiotic metabolism [12,13]. Up to 80% of all bioactivations require CYPs [14]. For catalytic efficiency, the CYP proteins must be reduced by oxidoreductases, which are colocalized with CYPs in the endoplasmic reticulum (ER [15]).Saccharomyces cerevisiae (budding yeast) is an excellent eukaryotic model organism for studying the genotoxicity of xenobiotics, including pharmaceuticals, pesticides, insecticides, and suspected carcinogens. Similar to bacterial organisms, yeast strains are easy to culture, grow rapidly, and can be manipulated genetically, rendering it possible to perform high-throughput analysis [16]. Many yeast genes are similar to human genes, and approximately 30% of can be functionally replaced by the human orthologue [17,18]. DNA repair pathways and genes are also similar [17,16]. Mitochondrial genotoxicity can also be measured [19]. Thus, identi...