Effects of antioxidants on streptonigrin-induced DNA damage and clastogenesis in CHO cells.

The effect of several free radicals scavengers on DNA damage and clastogenesis induced by streptonigrin (SN) in CHO cells was investigated. The addition of the antioxidant enzymes superoxide dismutase and/or catalase on CHO cell cultures did not prevent the induction of DNA and chromosome damage by SN. In fact, when superoxide dismutase was added to the culture medium an increase on the frequency of SN-induced chromosome aberrations was observed. Moreover, the addition of the hydroxyl radicals scavenger mannitol caused a significant increase in DNA and chromosome damage induced by SN. On the contrary, when all the antioxidants mentioned above were added-alone or in different combinations-encapsulated into liposomes, a significant decrease in the yield of SN-induced chromosome aberrations and DNA damage was observed. These findings indicate that free radicals are involved in the production of DNA and chromosome damage by SN and that this damage can be partially inhibited through the incorporation of antioxidants by the cells.

Enhancement of chromosome aberrations by the combination of DNA substitution with halogenated deoxyuridine and streptonigrin treatments.

We treated CHO cells with streptonigrin (SN) alone, in combination with BrdUrd or IdUrd substitution, and with or without the addition of caffeine. The cells assessed for chromosome damage by SN were in the G2 period and the magnitude of the damage was expressed as monosubstituted chromatid breaks, bisubstituted chromatid breaks and boundary regions breaks (boundary regions indicate the point of exchange of mono- and bisubstituted chromatids). We found that the combination of BrdUrd or IdUrd substitution with SN treatments produced a remarkable increase in the frequency of breaks over the frequencies observed with the halogenated compound only. The effect was more evident with IdUrd than with BrdUrd, and more dramatic in bisubstituted than in monosubstituted chromatids. The frequency of boundary breaks in cells treated with BrdUrd plus SN was similar to the frequency of breaks in monosubstituted chromatids treated similarly. Conversely, the damage in boundary regions was almost similar to that in bisubstituted chromatids in cells challenged with IdUrd plus SN. The addition of caffeine to BrdUrd-substituted chromosomes gave rise to a marked enhancement of breakages with a gradient of chromatid damage that was: bisubstituted > monosubstituted > boundary regions. A further increase of chromatin breaks maintaining the gradient indicated above was obtained when the cells were treated with BrdUrd plus SN plus caffeine. We propose that BrdUrd and IdUrd substitution alone or in combination with caffeine treatments and with SN in its capacity to bind DNA, give rise to different chromatin structures capable of modulating the DNA damage induced along the chromatin fibril by the active oxygen species liberated by SN-DNA complexes.

The kinetics of chromosome and DNA damage by streptonigrin in CHO cells.

CHO cells were pulse-treated for 20 min with increasing doses of streptonigrin (SN). First division cells analyzed 18 h after the end of treatment showed a combination of chromosome and chromatid aberrations with a typical dose-response curve. The frequency of SCEs in second division cells (24 h harvesting time) also exhibited a positive correlation with the SN dose. A high incidence of chromatid aberrations was detected in second and third division metaphases. This indicates that SN has a persistent clastogenic action that lasts for at least three cell cycles after the end of treatment. The kinetics of DNA damage and repair was studied by the alkaline unwinding and single cell gel methods. It was found that a pulse treatment with SN elicited a triphasic response characterized by repair-damage-repair. It is proposed that SN forms stable complexes with the DNA. These complexes by a continuous cycling redox process would produce active oxygen species inducing direct damage to DNA.