MUTAGENIC ACTIVITY OF AMBIENT OXYGEN AND MITOMYCIN-C IN FANCONIS-ANEMIA CELLS

Cellular evidence suggests that Fanconi's anaemia (FA) might be a cond ition of increased oxygen sensitivity, In order to test this hypothesi s, a common shuttle vector assay with the plasmid pZ189 was utilized, We transfected intact, circular plasmid into FA and control lymphoblas t and fibroblast host cells maintained at 5 and 20% O-2 (v/v), In para llel experiments, host cells were exposed to different concentrations of mitomycin C (MMC), a cross-linking agent towards which FA cells are known to be hypersensitive, Baseline mutation frequencies at 20% oxyg en were significantly higher in plasmids passaged through FA lymphobla sts or FA fibroblasts in comparison with passage through the correspon ding control cells, Lowering the oxygen concentration during the 48 h transfection period to 5% resulted in a significant decrease of mutati on frequencies in plasmids passaged through FA cells, Sequence analysi s of plasmids recovered from FA lymphoblasts revealed a mutation hot s pot (22% of point mutations with G:C to A:T base substitutions) at bas e 117 of the supF tRNA gene, This hot spot was present only at 20% oxy gen, 59% of the base changes at the hot spot and 39% of the changes el sewhere in the supF gene were C to T transitions (the corresponding fi gures are 0 and 27% at 5% oxygen), the most common type of base change induced by oxygen, The mutation spectrum observed suggests a role for 8-hydroxydeoxyguanosine in G:C to A:T base substitutions: at 20% oxyg en, FA cells displayed 4 times as many G:C to T:A transversions than F A cells kept at 5% O-2, In MMC treated cells the decrease in plasmid s urvival is dose dependent and more pronounced in FA than control cells , Mutation analysis shows similar rates of deletions for both control and FA cells, However, FA cells generate a specific type of deletion w hose breakpoint involves an indirect repeat that corresponds to a hept amer signal sequence commonly seen at recombination sites, Together ou r data provide compelling evidence that the genetic defect in FA cause s oxygen sensitivity and recombinational types of DNA lesions followin g exposure to MMC.