POLYMERASE CHAIN REACTION-BASED DELETION SCREENING OF BLEOMYCIN-INDUCED 6-THIOGUANINE-RESISTANT MUTANTS IN CHINESE-HAMSTER OVARY CELLS - THE EFFECTS OF AN INHIBITOR AND A MIMIC OF SUPEROXIDE-DISMUTASE

Bleomycin-induced 6-thioguanine-resistant mutants pretreated with or w ithout TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inhi bitor, or TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), an SOD mimic, were analyzed by polymerase chain reaction (PCR)-based dele tion screening in a Chinese hamster ovary (CHO) clone K1-BH4 and its d erivative AS52 cells. As we proposed earlier, TRIEN would decrease and TEMPOL would increase the intracellular level of hydroxyl radical lea ding to a higher and lower recovery of deletion mutants. We found that the proportion of the deletion mutants induced by bleomycin at the hy poxanthine-guanine phosphoribosyltransferase (hprt) locus in K1-BH4 ce lls was 45.5% (25/55). The proportion of deletion HPRT- mutants induce d by bleomycin pretreated with TRIEN was 31.0% (9/29) and with TEMPOL was 50.0% (14/28). The proportion of deletion mutants induced by bleom ycin on the xanthine-guanine phosphoribosyltransferase (gpt) gene in A S52 cells was 61.0% (36/59). The proportion of deletion GPT- mutants i nduced by bleomycin pretreated with TRIEN was 56.8% (21/37) and with T EMPOL was 61.4% (27/44). The trend of the change of the proportion of bleomycin-induced deletion mutants as affected by TRIEN and by TEMPOL provides molecular evidence for the involvement of reactive oxygen spe cies (ROS) in bleomycin mutagenesis in mammalian cells, in which delet ion is a major type of induced mutation.