CELLULAR DNA-DAMAGE BY HYDROGEN-PEROXIDE IS ATTENUATED BY HYPOTONICITY

Chinese hamster fibroblasts (line V79) withstand well exposure for 30 min to hypotonic medium, corresponding to 25% physiological phosphate- buffered saline (PBS). Under these conditions, the cells become resist ant to two effects of H2O2: DNA damage and inhibition of cell clone fo rmation. The normal sensitivity to the DNA-damaging action of H2O2 is restored if, after exposure to hypotonic PBS, the cells are incubated in isotonic cell-culture medium. However, restoration of sensitivity i s not observed on incubation in isotonic PBS. The normal sensitivity t o H2O2 is also restored if one of the following reducing agents is add ed to hypotonic PBS: ascorbate, NADH and NADPH, in this order of decre asing efficiency. The recovery of sensitivity to H2O2 byascorbate is c ompletely inhibited by 1,10-phenanthroline, indicating that ascorbate is mediating the reduction of Fe(III). The decrease in the sensitivity to the DNA-damaging action of H2O2 is not a peculiarity of hypotonic PBS, since it appears to be caused by hypo-osmolarity in general: it i s also observed in culture medium of 25% the isotonic concentration, a nd in 0.07 M sucrose. One explanation for this phenomenon is that hypo tonic stress leads to a depletion of reducing species, in particular a scorbate. Under these conditions Fe(II) tends to be oxidized to Fe(III ) and the Fenton chemistry is mitigated. However, other possibilities are that hypotonicity brings about structural modifications in the chr omatin, rendering it less accessible to H2O2, or that it attenuates th e Ca2+-activation of endonuclease, induced by oxidative stress.