Covalent DNA Adduct Formation Mediated by Cytochrome P450

In this chapter, the experimental details are described for the utilization of cytochrome P450-mediated reactions to examine the potential of chemicals (drugs) to be activated to reactive species, leading to the formation of covalent deoxyribonucleic acid (DNA) adducts. Methods for the isolation of subcellular fractions containing activating enzymes (microsomes or cytosols containing cytochromes P450 or other activating enzymes such as reductases xanthine oxidase, DT-diaphorase, and aldehyde oxidase, respectively), those for the activation of test drugs by these enzymatic systems (incubations) and the DNA isolation procedure are shown. In addition, the suitable techniques to detect and quantify covalent DNA adducts such as two enhancement procedures of ³² P-postlabeling assay (nuclease P1 version and extraction of adducts into n -butanol) and utilization of radioactive-labeled test compounds are described in detail in the chapter.