Search

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 resistant to two effects of H2O2: DNA damage and inhibition of cell clone formation. 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 is not observed on incubation in isotonic PBS. The normal sensitivity to H2O2 is also restored if one of the following reducing agents is added to hypotonic PBS: ascorbate, NADH and NADPH, in this order of decreasing efficiency. The recovery of sensitivity to H2O2 by ascorbate is completely 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 is also observed in culture medium of 25% the isotonic concentration, and in 0.07 M sucrose. One explanation for this phenomenon is that hypotonic stress leads to a depletion of reducing species, in particular ascorbate. 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 chromatin, rendering it less accessible to H2O2, or that it attenuates the Ca(2+)-activation of endonuclease, induced by oxidative stress.

We have previously derived 2 V79 clones resistant to menadione (Md1 cells) and cadmium (Cd1 cells), respectively. They both were shown to be cross-resistant to hydrogen peroxide. There was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Md1 presented an increase in catalase and glutathione peroxidase activities whereas Cd1 cells exhibited an increase in metallothionein and glutathione contents. The susceptibility of the DNA of these cells to the damaging effect of H2O2 was tested using the DNA precipitation assay. Both Md1 and Cd1 DNAs were more resistant to the peroxide action. In the case of Md1 cells it seems clear that the extra resistance is provided by the increase in the two H2O2 scavenger enzymes, catalase and glutathione peroxidase. In the case of Cd1 cells the activities of these enzymes as well as of superoxide dismutases (Cu/Zn and Mn) are unaltered as compared to the parental cells. The facts that parental cells exposed to 100 microM Zn2+ in the medium exhibit an increase in metallothionein but not in glutathione and that these cells become more resistant to the DNA-damaging effect of H2O2 suggest that this protein might play a protective role in vivo against the OH radical attack on DNA.

Menadione (Md)-resistant variants of V79 Chinese hamster cells were derived by culturing cells in progressively higher concentrations of this drug. Along with Md resistance these cells acquired cross-resistance to H2O2, which was retained when cells were cultured back in the absence of Md for 18 cell passages. This indicates that some stable alteration is responsible for the modification and may suggest that the toxicity of Md is mediated by oxygen activation. In agreement with the latter a 2.8-fold increase in catalase activity and a 1.5-fold increase in glutathione content were observed in Md-resistant cells as compared to parental cells, whereas superoxide dismutase and glutathione peroxidase remained unaltered. The use of inhibitors of Fenton reaction, inhibitors of enzymatic and nonenzymatic lipid peroxidation and OH radical scavengers, indicated that both DNA damage and cytotoxic effects of H2O2 and Md are mediated by OH radical, without intervention of lipid peroxides. However whereas the catalysis by iron was required for toxicity of extracellular H2O2, it was not involved in Md-induced toxicity. Possible explanations for this difference have been considered, one of them assuming that an excess of the semiquinone form of Md in the cell might replace iron II as a H2O2 reductant, producing OH radical by an organic Fenton reaction.