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Human Cell-mediated Benzo(a)pyrene Cytotoxicity and Mutagenicity in Human Diploid Fibroblasts¹

Carcinogenesis Laboratory-Fee Hall, Departments of Microbiology and Biochemistry, Michigan State University, East Lansing, Michigan 48824

A human epithelial cell-mediated cytotoxicity and mutagenicity assay system for benzo(a)pyrene [B(a)P] has been developed with human fibroblasts as the target cells. Lethally X-irradiated human kidney carcinoma-derived epithelial cells, which had constant levels of B(a)P-metabolizing activity, were cocultivated with target human skin fibroblasts (XP12BE), which lack excision repair capability for B(a)P-DNA adducts. The optimal conditions determined for the cell-mediated cytotoxicity assay were a 48-hr exposure to B(a)P concentrations ranging from 0.1 to 1 µM at a metabolizing cell:target cell ratio of at least 1:1. Under these conditions, the frequency of mutations to 6-thioguanine resistance induced in the target XP12BE cells by B(a)P as well as the binding of tritium-labeled B(a)P to DNA was also shown to be concentration dependent. High-pressure liquid chromatography analysis of enzymatically degraded B(a)P-DNA adducts revealed two peaks: a major peak (82%) which cochromatographed with the guanosine adduct-standard synthesized from anti-isomeric-7,8-dihydrodiol-9,10-epoxide of B(a)P; and a minor peak (18%) which cochromatographed with the guanosine adduct-standard synthesized from syn-isomeric-7,8-diol-9,10-epoxide of B(a)P. Human liver carcinoma- and lung carcinoma-derived cell lines, capable of metabolizing B(a)P, proved equal to or better than the kidney carcinoma-derived cell line in producing cytotoxic B(a)P metabolites in the cell-mediated cytotoxicity assay with target XP12BE cells.

¹ Supported in part by Grant R805563 from the Environmental Protection Agency, by Department of Health, Education and Welfare Grant CA 21253 from the National Cancer Institute, and by Department of Health, Education and Welfare Post Doctoral Training Grant ES 07076 awarded by the National Institute of Environmental Health Sciences.

² To whom requests for reprints should be addressed, at Carcinogenesis Laboratory-Fee Hall, Michigan State University, East Lansing, Mich. 48824.

Received 12/12/79. Accepted 6/23/80.