Determination of point mutational spectra of benzo[a]pyrene-diol epoxide in human cells

Abstract

The primary goal of our research consists of developing means sufficiently sensitive to allow assessment of human exposure to environmental carcinogens. We describe here a new approach for analyzing point mutational spectra and a test for its validity and precision using cultured human cells exposed to high doses of environmental carcinogens. The approach in its present form includes a) treatment of independent large cultures of human cells with a carcinogen, b) selection of mutant cells en masse by 6-thioguanine resistance, c) amplification of sequences of interest directly from 6TGR cells using high-fidelity polymerase chain reaction, and d) separation of mutant sequences from nonmutant sequences using denaturing gradient gel electrophoresis. We report use of this protocol to observe induced mutational spectra in exon 3 of the hprt gene in cultured human cells by benzo[a]pyrene-diol epoxide (BPDE), an active form of the widely distributed environmental carcinogen benzo[a]pyrene. BPDE induced predominantly G to T transversions within this target sequence. The variation of the frequency of the mutations among independent cultures is consistent with the interpretation that each of them corresponds to a hotspot.