Determination of Mutational Spectra Induced by Environmental Toxicants in Complex Human Cell Populations

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment and have potent mutagenic and carcinogenic activities. Studies of mutations induced by these compounds in human cells can help acquire an understanding of their mutagenic pathways. In this chapter, independent cultures of a human cell line expressing cytochrome P450 CYP1A1 (cell line MCL-5) were treated with benzo(a)pyrene (BaP) or dibenzo(a,l)pyrene (DBP), and mutants at the hypoxanthine phosphoribosyltransferase (HPRT) locus were selected en masse by 6-thioguanine resistance (6TG^R). The kinds and positions of the mutations occurring in the third exon of the HPRT gene were analyzed in the mixed HPRT^R mutant cell populations using a combination of polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Mutant bands were excised from the gel, amplified using PCR, and sequenced to determine the kinds and positions, or spectrum of mutations.