DNA single-strand breaks and cytotoxicity induced by chromate(VI), cadmium(II), and mercury(II) in hydrogen peroxide-resistant cell lines.

The induction of cytotoxicity and DNA single-strand breaks by chromium(VI), cadmium(II), and mercury(II) were compared in H2O2-resistant Chinese hamster ovary (CHO(R)) cells and parental (CHO(P)) cells. Using a colony-forming assay, CHO(R) cells were found to be significantly more resistant than CHO(P) cells to the cytotoxicity caused by CdCl2 and HgCl2, but not to that caused by Na2CrO4. However, the DNA single-strand breaks produced by each of these metals were significantly lower in the CHO(R) cells. With respect to chromium reduction, the level of chromium(V) in CHO(R) cells was decreased. The role of intracellular active oxygen in the heavy metal-induced DNA damage and cytotoxicity is discussed.


Introduction
Toxic and carcinogenic heavy-metal compounds such as chromium(VI), cadmium(II), and mercury(II) have been shown to produce DNA single-strand breaks in cultured mammalian cells (1)(2)(3)(4)(5)(6). Previous studies have demonstrated that these metal compounds may cause adverse biologic effects, possibly through free radical species such as active oxygen, because active oxygen scavengers prevented metals-induced cellular injuries such as DNA breaks (4)(5)(6). With respect to chromium(VI), although active oxygen species have been reported to be generated during reduction of chromium(VI) to chromium(V) in vitro (3,(7)(8)(9)(10)(11), only one study examined the involvement of active oxygen species in chromium(VI)induced genotoxicity and cytotoxicity in intact cells (6). In addition, the relationship between active oxygen and cytotoxicity of mercury(II) and of cadmium(II) is not clear. In the present study, using hydrogen peroxide-resistant cell lines, we investigated the DNA breaks and cytotoxicity produced by these heavy metals to elucidate the role of active oxygen in the mechanism of their toxic effects (12).

Materials and Methods
Hydrogen peroxide-resistant Chinese hamster ovary (CHO ) cells were derived from the parental (CHO ) cells by adding progressively higher concentrations (6.25 to 100 pM) of H202 in stepwise fashion to the culture medium (12). Logarithmically growing cells were treated with H202 for 1 hr or with Na2CrO4, CdCl2, and HgCl2 for 2 hr in salts-glucose medium [50 mM Hepes (pH 7.2) containing 10 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 5 mM glucose]. DNA single-strand breaks and cytotoxicity were estimated by alkaline elution assay and colony forming assay, respectively (13)(14)(15). To detect chromium(V) in cells, the harvested cells were mixed with 400 pM Na2CrO4, and then placed in an electronic spin resonance (ESR) tube. ESR measurements were carried out during the incubation at room temperature (12). Table 1 shows that the concentration of H202 that reduced colony formation by 50% (ID50) was 5-fold less in CHOR cells than in CHOP cells. CHOR were also cross-resistant to CdCl2 and HgCl2, but not to Na2CrO4. Table 2 shows the induction of DNA single-strand breaks produced by H202 or the metal compared in CHOR and CHOp cells. The formation of DNA breaks induced by H202 was almost completely inhibited in CHOR cells. Although the cytotoxicity was not effected, the formation of DNA breakage produced by Na2CrO4 was significantly reduced by about 50% in CHOR cells as compared to that in CHOP cells. n CHOR were also cross-resistant to DNA breaks caused by CdCl2 and HgCl2.

Results
It is unlikely that the observed metalresistance in CHOR cells was due to alterations of metal uptake, because there was no difference in the uptake of the metals in these cell lines (12). Since chromium(VI) enters cells and is then reduced to chromium(III), the formation of chromium(III) and other oxidation states, in particular chromium(V), is believed to play a role in some of the biological effects of  0.510 ± 0.082 0.164 ± 0.096c aCells were treated with H202 for 1 hr or with the metals for 2 hr and then DNA breaks were measured as described (13,14). bEach value is the mean ± SD. cp<0.01 (Student's t-test). chromium(VI) (1)(2)(3). In fact, relatively long-lived chromium(V) species formed during reduction of chromium(VI) have been shown to induce DNA breaks by the generation of active oxygen radicals in vitro (7)(8)(9)(10). For this reason, we also examined the formation of chromium(V) in both cell lines. The results showed the appearance of the chromium(V) ESR signal with a maxi-mum peaks at g= 1.978 during the incubation of cells with Na2CrO4 and it was found that the level of this chromium(V) was about 2-fold lower in the resistant cells (12).

Discussion
Previous studies have reported that the induction by cadmium(II) and mercury(II) of damages such as DNA breaks is mediated by the formation of active oxygen species in cultured cells (4)(5)(6). The present results indicate that both the cytotoxicity and the DNA breakage induced by these two metals may be associated with active oxygen species inside cells, because CHOR cells were cross-resistant to the damage caused by CdCl2 and HgCl2. However, in the case of chromium(VI), CHOR were found to be resistant only to DNA breaks, but not to the cytotoxicity caused by Na2CrO4, indicating that active oxygen species inside cells may partly associated with chromium(VI)-induced DNA strand breaks. Since the levels of chromium(V) in CHOR cells were lower than those in CHOP cells, chromium(V) may also participate in the induction of DNA breaks. The relationship between active oxygen species and chromium(V) formation in cells is under investigation.

Conclusion
We examined the induction of cytotoxicity and DNA breaks by chromate(VI), cadmium(II), and mercury(II) in hydrogen peroxide-resistant cells. Cytotoxicity was not effected; however, a cross-resistance was found against DNA breaks caused by sodium chromate(VI). Cross-resistance was observed with respect not only to DNA breaks but also to the cytotoxicity produced by cadmium(II) and mercury(II).
Hydrogen peroxide-resistant cells may be useful for the examination of involvement of active oxygen species induced by toxic metal compounds.