Mutagenic activity of ambient oxygen and mitomycin C in Fanconi's anaemia cells

Cellular evidence suggests that Fanconi's anaemia (FA) might be a condition of increased oxygen sensitivity. In order to test this hypothesis, a common shuttle vector assay with the plasmid pZ189 was utilized. We transfected intact, circular plasmid into FA and control lymphoblast and fibroblast host cells maintained at 5 and 20% O2 (v/v). In parallel 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% oxygen were significantly higher in plasmids passaged through FA lymphoblasts or FA fibroblasts in comparison with passage through the corresponding control cells. Lowering the oxygen concentration during the 48 h transfection period to 5% resulted in a significant decrease of mutation frequencies in plasmids passaged through FA cells. Sequence analysis of plasmids recovered from FA lymphoblasts revealed a mutation hot spot (22% of point mutations with G:C to A:T base substitutions) at base 117 of the supF tRNA gene. This hot spot was present only at 20% oxygen. 59% of the base changes at the hot spot and 39% of the changes elsewhere in the supF gene were C to T transitions (the corresponding figures 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% oxygen, FA cells displayed 4 times as many G:C to T:A transversions than FA cells kept at 5% O2. In MMC treated cells the decrease in plasmid survival 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 whose breakpoint involves an indirect repeat that corresponds to a heptamer signal sequence commonly seen at recombination sites. Together our data provide compelling evidence that the genetic defect in FA causes oxygen sensitivity and recombinational types of DNA lesions following exposure to MMC.