QUANTITATIVE AND MOLECULAR ANALYSES OF GENETIC RISK - A STUDY WITH IONIZING-RADIATION

Mammalian cells in culture have been used to study the genetic effects of physical and chemical agents. We have used Chinese hamster ovary ( CHO) cells, clone K1-BH4, to quantify mutations at the X-linked, large (35 kb) hypoxanthine-guanine phosphoribosyltransferase (hprt) locus ( the CHO/HPRT assay) induced by environmental agents. By transfecting a n hprt-deletion mutant CHO cell line with the plasmid vector pSV2gpt, we isolated a transformant, AS52. AS52 cells carry a single functional copy of an autosomal, small (456 bp) xanthine-guanine phosphoribosylt ransferase (gpt) gene (the bacterial equivalent of the mammalian hprt gene; AS52/GPT assay). We found that ionizing radiations such as X-ray s and neutrons and oxidative genotoxic chemicals such as Adriamycin, b leomycin, hydrogen peroxide, and potassium superoxide are much more mu tagenic to the gpt gene in AS52 cells than to the hprt locus in KI-BH4 cells. The hypermutability of the gpt gene probably results from a hi gher recovery of multilocus deletion mutants in AS52 cells than in K1- BH4 cells, rather than a higher yield of induced mutants. These result s demonstrate that the use of the hprt locus alone could lead to an un derestimate of the genetic risk of these agents. Analyses of the mutat ion spectrum using polymerase chain reaction-based deletion screening and DNA sequencing procedure showed that a high proportion of HPRT and GPT mutants induced by X-rays carry deletion mutations. ThuS, both th e mutant frequency and mutation spectrum need to be considered in asse ssing the genetic risk of ionizing radiation and oxidative genotoxic c hemicals.