The generation of DNA single-strand breaks during the reduction of chromate by ascorbic acid and/or glutathione in vitro.

The potential role of iron and copper and the involvement of hydroxyl radicals in the DNA cleavage caused by chromate and glutathione (GSH) has been investigated. We have also studied the ability of chromate, on reaction with ascorbate as well as in mixed solutions of ascorbate and GSH, to cause DNA strand breaks. In both fully demetalated and conventional (i.e., metal contaminated) systems, chromate and GSH induced similar numbers of DNA strand breaks. This observation suggests that traces of iron or copper contaminating the reaction mixtures do not play a major role in the DNA cleavage caused by chromate and GSH. A series of hydroxyl radical scavengers exhibited a protective influence on the induction of DNA strand breaks. However, glucose and sucrose, both strong hydroxyl radical scavengers, showed no concentration-dependent inhibition of DNA cleavage. Competition kinetics studies yielded apparent rate constants that were not consistent with hydroxyl radicals being the species responsible for DNA strand breaks. Ascorbate in combination with chromate was also found to induce strand breaks in DNA; this damage could be attributed to reactive intermediates generated during the reduction. When mixed systems of ascorbate and GSH in the presence of chromate were investigated, there were clearly interactions between the two reductants. ImagesFigure 1.Figure 2.Figure 3.Figure 4.


Introduction
Despite considerable research effort during the last ten years, the mechanisms by which the known human carcinogen chromate can cause DNA impairment and mutations still remain obscure. Chromate is rapidly taken up by mammalian cells and readily reduced by cellular constituents. It is now generally accepted that the intracellular reduction of the metal compound is crucial for the induction of DNA lesions (1). On the basis of kinetic studies in vitro, it has been suggested that glutathione (GSH), ascorbic acid (AsA), and cysteine are the most important nonenzymatic cellular constituents for the reduction of chromate in the cytoplasm (2). However, the relative contribution of these various reductants to the generation of DNA lesions is far from clear. Furthermore, it has as yet not been possible to identify the ultimate carcinogen deriving from chromium(VI) upon reduction. systems in vitro (3) the significance of AsA as an intracellular reductant of chromate has, as yet, received comparatively little attention. Recently, Suzuki and Fukuda (4) reported that in the bronchoalveolar lining fluid and the lung parenchyma of rats AsA is more reactive than GSH in the reduction of chromate. In subsequent in vitro studies Suzuki (5) observed a synergistic effect of AsA and GSH on the rate of reduction of chromate. Later, Standeven and Wetterhahn (6) provided evidence suggesting that AsA may be the principal nonenzymatic reductant of chromate in rat liver and kidney. In view of these observations it is now necessary to consider the possibility that reduction by ascorbate mediates the generation of DNA lesions.
In the present article, we report some observations on the induction of DNA strand breaks by chromate and AsA. We also studied the generation of DNA cleavage in the presence of AsA, GSH, and chromate. Initial experiments in our laboratory revealed that AsA itself, in the absence of chromate, can cause DNA strand breaks. The effect is due to the generation of reactive oxygen species during the autoxidation of AsA (i.e., the oxidation of AsA in aqueous solution by dissolved dioxygen) (7). The reaction is catalyzed by traces of metal ions, namely copper(II) and iron(III). Ascorbate, in the absence of catalytic metals, is stable even in air-saturated solutions of neutral pH (8).
In the light of a controversy concerning the ability of chromate and GSH to induce DNA strand breaks in Fe-and Cu-free systems (9) it has also become necessary to reassess the role of traces of catalytic metal ions in the chromate/GSH reaction. While we have observed the induction of single strand breaks in PM2 DNA with chromate and GSH in both phosphate and HEPES buffer (10), Aiyar et al. (1]) using pBR322 DNA, have detected very little, if any, DNA cleavage in a Tris-buffered system. They removed contaminating iron from all solutions while we used all reagents without further purification. The results of subsequent studies carried out in our laboratory (12) in an attempt to clarify the situation suggested that Fe could be important in these reactions. Moreover, we observed a protective influence of a series of hydroxyl radical scavengers on the generation of strand breaks by chromate and GSH. Taken together, these observations led us to suggest hydroxyl radicals as the ultimate DNA-cleaving species. However, in an article primarily concerned with studying the effects of Cr binding on the cleavage by restriction endonucleases, the Wetterhahn group recently observed the induction of strand breaks by chromate and glutathione in a fully demetalated system (13). The ability of chromate and GSH to cause DNA cleavage seems to be a Environmental Health Perspectives distinct possibility, but more experimental work is needed before firm conclusions can be drawn. Here, we communicate our recent observations that might help to clarify some of the earlier, apparently contradictory, results.

Materials and Methods
All reagents used were of analytical grade. Glutathione, ascorbic acid, diethylendiaminopentaacetic acid (DTPA) and ethidium bromide were from Sigma (Poole, Dorset, UK); bromophenol blue and agarose were from Biorad (Hemel Hempstead, UK). Potassium chromate (K2CrO4) was purchased from BDH-Merck (Poole, UK) and PM2 DNA was obtained from Dr. Irene Witte, University of Oldenburg FRG, who isolated it as described by Espejo et al. (14). PM2 DNA (8 pg/ml), in a final volume of 20 pl, was treated with varying concentrations of K2CrO4, GSH, and/or AsA in phosphate buffer, 0.1 M, pH 7.0, for at least 3 hr at room temperature (close to 22°C). Where indicated ("demetalated system"), traces of Fe and Cu were removed from buffer solutions by stirring overnight with Chelex 100 resin. To remove metal traces from DNA, it was dialyzed (24 hr) against 1 mM DTPA (dissolved in demetalated phosphate buffer).
The successful removal of catalytic metals from buffer solutions was assessed by using the ascorbate method of Buettner (8). The method involves monitoring the autooxidation of ascorbate at 265 nm in the presence of test solution. After tr ment with Chelex resin, ascorbate was ble (i.e., loss of absorbance <0.5%) fc least 30 min, indicating that metal ion ( tamination was minimal. DNA was con ered free of metals when ascorbati concentrations up to 1 mM (in the abst of chromate) did not induce any str breaks.

Results and Discussion
Chromate and Glutathione Cause DNA Strand Breaks in Iron(III)an Copper(II)-fiee Systems In an attempt to probe the involvemen Fe in the generation of DNA strand br by chromate and GSH, we studied reaction in conventional (i.e., metal-co minated) and exhaustively demetala solutions. In both systems chromate GSH induced similar numbers of sin strand breaks in isolated PM2 D ( Figure 1). The level of cleavage incre; with rising concentrations of chromat phosphate buffer, 0.1 M, pH 7.0, cont ing 5 mM GSH.
These findings clearly indicate traces of Fe or Cu contaminating buffers or bound to DNA do not plh major role in the DNA cleavage causes chromate and GSH. Our results ar agreement with a recent report by Wetterhahn group that strand breaks formed in a demetalated system (13) observation that contradicts earlier reports by the same group (11,15).
The demonstration that there is no specific need for Fe or Cu in this reaction makes it necessary to reassess our earlier suggestion (12) of a thiyl radical/superoxide anion/hydrogen peroxide-mediated pathway leading to the generation of hydroxyl radicals as the ultimate DNAcleaving agent. This suggestion was based on observations of a concentration-dependent depression of the level of DNA cleavage by benzoate and formate upon addition to chromate and GSH. Consequently, we extended these studies and explored the influence of a wider range of hydroxyl radical scavengers.
The results confirm our earlier findings (12). We observed a concentration-dependent inhibition of DNA degradation with sodium formate and sodium benzoate.
Dimethyl sulfoxide (DMSO) and mannitol losed also showed a concentration-dependent inhibitory effect. To our surprise, glucose, a strong hydroxyl radical scavenger, exhibreat-ited no protective influence on the inducsta-tion of strand breaks by chromate/GSH at ir at concentrations of up to 10 mM. Sucrose con-showed effects very similar to those of gluisidcose (data not shown). Id The somewhat unexpected results with glucose and sucrose prompted us to probe further the involvement of hydroxyl radicals It of in the chromate/GSH system by using the eaks method of competition kinetics. This the method has been widely used in the deter-Fntamination of rate constants of radicals. It ated utilizes the fact that any hydroxyl radicals and (OH) generated in the present reaction glemixtures should react with DNA to form a NA strand break. Any other molecule S added ased that is capable of reacting with -OH should    Table 2, consistent values for kDNA are not obtained. Furthermore, far higher concentrations of scavengers are needed to inhibit the DNA cleavage induced by chromate and GSH than would be expected for a reaction involving free hydroxyl radicals. Consequently, the apparent values of kDNA exceed diffusion limit.
We conclude that simple Fenton chemistry does not explain the ability of chromate and GSH to induce single-strand breaks in DNA and that Fe or Cu is not necessary for this process. At this stage, we can only speculate which species are responsible for the DNA damage. Hypervalent Cr species known to be generated during reduction by GSH include several chromium(V) [Cr(V)] complexes. Both Lay and co-workers (18) and we (10) found evidence that Cr (V) complexes can cause DNA single-strand breaks. Thus, mechanisms involving the direct reaction of a high oxidation state complex with DNA, as suggested by Lay, are worth serious consideration. Furthermore, there is now evidence for some accumulation of Cr(IV) during the reaction of chromate with GSH (19), and chromium(IV) complexes are known to cause C-C bond scission (20). A further possibility includes the reaction of hypervalent Cr with molecular oxygen to form some reactive DNA cleaving species (P. O'Brien and A. Kortenkamp, this issue).
Another factor, which might help explain the conflicting results concerning controls 50 50 0 100 200 100 Figure 3. The influence of varying amounts of ascorbate on the DNA cleavage induced in the presence of constant amounts of chromate. Il: nicked, closed circular the ability of chromate and GSH to cause DNA cleavage, is the nature of the buffering agent. We showed earlier (10) that the buffering agent present in the incubation mixtures exhibits a strong influence on the level of strand breaks induced by chromate and GSH. When HEPES buffer was used instead of phosphate buffer, higher chromate concentrations were required to produce the same level of DNA strand breaks. The use of Tris seems to be problematic; it interferes with the reduction of chromate by AsA (21). We are exploring the effect of buffering agents in greater detail.

Induction ofDNA Stand Bks by Chromate in Combinaon with Ascorbate and Glutathione
Chromate and AsA, in the absence of traces of catalytic metal ions, caused DNA singlestrand breaks. Figure 2 shows the results of an experiment in phosphate-buffered solution containing 1 mM AsA. With rising concentrations of chromate, the level of strand breaks increased and reached a plateau as the ratio chromate:AsA approached 1. In the presence of 100 1M chromate, the number of strand breaks was still significantly higher than in the absence of chromate. These results indicate that chromate, upon reduction by AsA, has the potential to generate species that cleave DNA and lend further support to the idea that the reduction of chromate is a crucial step in the generation of DNA lesions. It remains to be seen whether this also applies to the induction of DNA-protein crosslinks. Suzuki has reported that the rate of reduction of chromate by AsA increases with rising concentrations of AsA in vitro (5). We conducted experiments using chromate and AsA at concentrations similar to those chosen by Suzuki and observed a decrease in the level of DNA cleavage with increasing concentrations of AsA ( Figure 3). These results could be indicative of an inverse relationship between the rate of reduction and the generation of DNA damage: the slower the reduction, the greater the damage. Alternatively, the effect could be due to an efficient scavenging of the cleaving species by an excess of AsA. The observation of a protective effect with increasing amounts of AsA clearly indicates that the DNA damage is caused by a species generated during the reduction of chromate by AsA rather than by the final product of the reaction. If the final product were responsible for the DNA impairment, rising amounts of AsA should have left the level of strand breaks unchanged.  We started exploring the DNA damaging effects of chromate in mixed solutions of AsA and GSH. Glutathione, when administered separately, appeared to be more effective than AsA in generating DNA strand breaks in the presence of identical amounts of chromate. As shown in Figure 4, samples containing chromate (100, 200, 500 pM), but with GSH (1 mM) instead of AsA, yielded slightly higher levels of strand breaks. The effect of We have demonstrated that the reduction of chromate by AsA leads to the generation of species that are able to cleave DNA. The final product of the reaction is not involved in the induction of these effects. The ultimately active species generated in such reaction mixtures are not yet known; however it is likely that they are different from the species present in solutions containing chromate and GSH.
The study of systems in mixed solutions of AsA and GSH has yielded results that are indicative of an inverse relationship between rate of reduction and generation of DNA damage. Another important factor is the influence of scavenging properties of AsA or GSH when present in excess. More work is needed to probe this suggestion further.
Although the levels of AsA and GSH in the human lung are not precisely known, available data for rats suggest that the cytoplasmic levels of both reductants are equimolar and fall in the millimolar range (4). Considerable amounts of Cr can accumulate inside cells after exposure to chromate. Sehlmeyer et al. (22) have shown that after the exposure of V79-cells to 10 jiM chromate the total intracellular, cytosolic, Cr concentrations can be in the millimolar range. Under similar conditions Cr levels in nuclei can be even higher than in the cytosol. The concentrations of chromate, AsA, and GSH used in our experiments are similar to levels that might be attained within cells; our observations are of relevance to the in vivo situation. However, it is too early for firm conclusions because the relative contribution of the extracellular reduction of chromate in lung tissue and the influence of intracellular compartmentalization is not known. Suzuki and Fukuda (4) have reported that no other effective reductant apart from AsA exists in the alveolar-lining fluid of rats. Thus, in the earlier periods of exposure to chromate, extracellular reduction may be the overall determining step. After depletion of extracellular AsA, intracellular reductions may become more important. However, more information is needed to delineate between these two routes of chromate reduction.