Effects of vitamins on chromium(VI)-induced damage.

The effects of vitamin E and vitamin B2 on DNA damage and cellular reduction of chromium(VI) were investigated using Chinese hamster V-79 cells. Pretreatment with alpha-tocopherol succinate (vitamin E) resulted in a decrease of DNA single-strand breaks produced by Na2CrO4, while similar treatment with riboflavin (vitamin B2) enhanced levels of DNA breaks. In contrast, levels of DNA-protein crosslinks induced by Na2CrO4 were unaffected by these vitamins. Electron spin resonance (ESR) studies showed that incubation of cells with Na2CrO4 resulted in the formation of both chromium(V) and chromium(III) complexes, and cellular pretreatment with vitamin E reduced the level of the chromium(V) complex, whereas pretreatment with vitamin B2 enhanced the level of this intermediate. However, the levels of chromium(III) were unchanged by these vitamins. The uptake of chromate was not affected by vitamin E or vitamin B2, nor were the levels of glutathione or glutathione reductase activity, which are both capable of reducing chromate. ESR studies demonstrated that a chromium(V) species was formed by the reaction of Na2CrO4 with vitamin B2 and that vitamin B2 enhanced the formation of hydroxyl radicals during the reaction of Na2CrO4 and hydrogen peroxide. Treatment cells with Na2CrO4 resulted in a decrease of glutathione reductase activity, and pretreatment with vitamin E restored the enzyme activity suppressed by this metal. However, pretreatment with vitamin B2 enhanced the inhibition of this enzyme by Na2CrO4. Using a colony-forming assay, pretreatment with vitamin E dramatically decreased the cytotoxicity of Na2CrO4, while pretreatment with vitamin B2 was found to result in only a decrease of cell lethality of this metal.(ABSTRACT TRUNCATED AT 250 WORDS)

As illustrated in Figure 1, it is speculated that free radical species such as hydroxy radicals and glutathionyl radicals are generated during the reduction of chromium(VI) in cells (18,21,25). However, in spite of these evidences, the effects of cellular vitamins on chromate-

DNA Damage
The alkaline elution technique for analysis of DNA lesions was performed as described (9). To quantify the extent of DNA single-strand breaks and DNA protein crosslinks, the strand scission factor and crosslink factor were calculated from the alkaline elution patterns (34)(35)(36).

ESR Spectroscopy
The formation of paramagnetic chromium in cells was determined by electron spin resonance (ESR) analysis (36,37). Briefly, 40 x 106 cells were placed into an ESR tube and were rapidly frozen in liquid nitrogen. ESR measurements were made at temperatures of 153 K using a JES-FE3X spectrometer with 100 KHz modulation, 8 mW microwave power, and 4.0 G modulation amplitude.
Glutathione Reductase and Glutathione, Flavin Derivatives, and Cytotoxicity Glutathione reductase was measured according to the method of Staal et al. (39) as detailed in Sugiyama et al. (36). The total glutathione (oxidized and reduced) was determined as described by Tietze (40) with minor modifications (34).
Flavin derivatives were extracted from cells and were measured by means of high pressure liquid chromatography (HPLC) following the procedure detailed in Sugiyama et al. (37).

Results and Discussion
In the present study, cells were pretreated with vitamins for 24 hr before treatment with chromate because if the cells are treated with vitamins in the presence of chromate, it is difficult to tell whether the action of the vitamins occurs in extracellular or intracellular systems. Thus, first of all, the effect of 24-hr vitamin treatment on cell growth was examined. As shown in Table 1, treatment with vitamin E at 50 pM resulted in an inhibition of cell growth, whereas similar treatment with vitamin B2 did not change cell growth even at 200 pM.
The concentration at which cells were able to grow at rates similar to those of the controls was less than 25 pM for vitamin E and less than 200 pM for vitamin B2. Therefore, these concentrations were used throughout the subsequent experiments (34).
Effects of Vitamins on DNA Damage and Cellular Reduction of Chromium(VI) Table 2 shows the effects of vitamins on Na2CrO4induced DNA damages in V-79 cells. Pretreatment with nontoxic levels of vitamin E resulted in a significant decrease of DNA single-strand breaks produced by chromate. In contrast, similar pretreatment with vitamin B2 enhanced the number of breaks by about four times above that seen with chromate alone. As shown in Table 2, these effects were not due to changes in the cellular uptake of chromate.
In contrast to DNA breaks, there was no change from initial levels of Na2CrO4-induced DNA-protein crosslinks in cells pretreated with vitamin E or vitamin B2 (Table  2). Although the formation of DNA-protein crosslinks has been shown to require time (7,41), the level of  (34) and Sugiyama et al. (35). bCells were pretreated for 24 hr with vitamins and then treated for 2 hr with Na2CrO4. Following treatment, cellular DNA was analyzed by alkaline elution. cCellular uptake of chromate was measured by radioisotope 51Cr analysis as described (36). dEach value is the mean ± SE for at least four determinations. SSF, strand scission factor; CLF, crosslink factor. *p<0.01. tp < 0.001 compared to un-pretreated chromate-treated values. crosslinks 4 hr after chromate treatment was not influenced by these vitamins (data not shown). Thus, cellular pretreatment with vitamin E or vitamin B2 has a specific effect on the formation of DNA breaks but not that of protein crosslinks induced by chromate.
Since chromium(VI) easily passes through the cell membrane and is then reduced to chromium(III) (Fig.  1), the formation of the intermediate oxidation states such as chromium(V) and (IV) may play a role in the induction of DNA damage. Thus, the effects of vitamin E and vitamin B2 on the production of paramagnetic chromium in cells were investigated directly by ESR spectrometry. Figure 2 shows the ESR spectra of both chromium(V) (sharp spectra) and chromium(III) (broad spectra) in V-79 cells treated with Na2CrO4. The formation of a chromium(V) signal was confirmed with an anisotropy at g = 2.016 and g = 1.989 and the line width of the maximum absorption peak was 12 to 13 G (36,37). On the other hand, the ESR signal due to chromium(III) complex was characterized by a g value of about 2.03 and a line width of 700 to 800 G (37). These levels of chromium(V) and (III) increased in a concentrationdependent manner (50-500 ,uM).
As shown in Table 3, when cells were pretreated with vitamins, the ESR signal intensity of chromium(V) (g = 1.989) was significantly decreased by vitamin E, while similar treatment with vitamin B2 resulted in an approximately 2-fold increase of chromium(V) compared to un-pretreated Na2CrO4-treated cells. These results were correlated with the effects of vitamin E and vitamin B2 on chromate-induced DNA breaks (Table 2). untreated 50MM 6000 FIGURE 2. ESR spectra of chromium(V) and chromium(III) complex at 153 K. Cells were treated for 2 hr with various concentrations of Na2CrO4. Followingtreatment, an ESR signal was obtained from the cells as described (37). Several recent studies using ESR spectroscopy have reported that the reactive chromium(VI) is relatively long lived (16,17,23) and that it causes DNA breaks in vitro (21,42,43), indicating that the production of DNA breaks might be closely related to the level of this reactive intermediate. Therefore, these results suggest that the protective effect of vitamin E and the enhancing effect of vitamin B2 on chromate-induced DNA breaks may be due to modification of the formation of chromium(V) in cells.
Recently, isolated chromium(V) intermediates have been shown to potentially induce mutation in bacterial cell systems (43). Our results show that cellular levels of chromium(V) were reduced by vitamin E. In addition, under similar conditions, vitamin E was found to suppress the clastogenic and mutagenic action of chromate compounds (unpublished observation). Collectively, these results suggest that chromium(V) might be the critical form that is responsible for the genotoxic and clastogenic, as well as the mutagenic, activity of chromate.
On the other hand, the formation of chromium(III) was not affected by pretreatment with vitamin E or vitamin B2 (Table 3). Several in vitro studies have shown that only chromium(III) can form a ternary complex with DNA and protein (3,5,10), and present results show that DNA-protein crosslinks induced by chromate were not influenced by vitamins (Table 2). Therefore, these results indicate that cellular levels of chromium(III) should play a role in the induction of DNA-protein crosslinks. However, it is not clear why these vitamins have an ability to change the chromium(V) but not the chromium(III) complex in cells. With respect to DNA damage, DNA breaks induced by chromate have been reported to be associated with cellular levels of glutathione and the activity of cytochrome P-450 reductase, whereas protein crosslinks were not dependent upon these factors (12). Furthermore, our previous studies have shown that in three different cell lines of human, mouse, and hamster origin, the order of sensitivity to DNA-protein crosslinks was not consistent with the sensitivity to formation of DNA breaks by chromate (8), suggesting that chromium-induced DNA-protein crosslinks may be formed by a different mechanism than that for single-strand breaks. Therefore, it is possible that the formation of chromium(III) complexes including DNA-protein crosslinks might be controlled by a different cellular components than for chromium(V).
Since ascorbate (vitamin C) is capable of reducing chromium(VI) directly to chromium(III) (37), we are investigating the effect of pretreatment with this vitamin. The results show that cellular levels of ascorbate were increased, resulting in a decrease of chromium(V) and an increase of chromium(III) in V-79 cells (unpublished observation). Thus, further study with vitamin C could lead to a better understanding of the role of intracellular paramagnetic chromium on DNA damage induced by chromate.

Mechanism of Action of Vitamins
To examine whether vitamin E and vitamin B2 had an effect on chromate-reducing flavoenzymes, glutathione reductase was examined following treatment with vitamins. However, as shown in Table 4, no alteration of glutathione reductase activity was observed in V-79 cells treated with vitamin E or vitamin B2 (36,37). Among cellular components, glutathione has been shown to be one of major reductants of chromate (16)(17)(18)(19), but cellular treatment with these vitamins did not affect the content of glutathione (Table 4) (34). Since riboflavin is a precursor molecule for FAD and FMN, and all the chromate-reducing enzymes have been shown to be flavoenzymes, it might be possible that the increase of chromium(V) by vitamin B2 is related to other chromate-reducing flavoenzymes activated by FAD and FMN. However, the treatment with Vitamin B2 did not influence the content of FAD and FMN in V-79 cells ( Table 5) (37). All of this suggests that the effects of vitamins on the formation of chromium(V) might not be due to the modification of glutathione and chromatereducing flavoenzyme activity.
The antioxidant effect of vitamin E is well documented in the literature, and this effect may be due in part to efficient radical scavenging (26,27,30). Thus, the protective mechanism of vitamin E in preventing DNA breaks produced by chromate might be due to its scavenging of paramagnetic chromium(V) during reduction of chromium(VI) in cells. In fact, cellular treatment with vitamin E resulted in a 10-fold increase of ct-tocopherol  as determined by HPLC analysis (unpublished observation). Since chromium(VI) has been shown to be metabolized to chromium(V) with simultaneous formation of active oxygen (21,25) and glutathione radicals (18), it is difficult to exclude the possibility of the scavenging effects of vitamin E against these radical species.
In the case of vitamin B2, as indicated in Table 5, cellular pretreatment with this vitamin resulted in a marked increase of riboflavin content. Thus, we further examined the direct interaction of vitamin B2 and chromate in vitro using ESR spectrometry. As shown in Figure 3, neither vitamin B2 nor Na2CrO4 alone could produce an ESR signal. However, a new signal with g-value of 1.977 was detected during the reaction of chromate with vitamin B2 (38), indicating that chromate reacts with vitamin B2 to form chromium(V) species. These in vitro studies suggest that the enhancement of chromium(V) formation by vitamin B2 might be due to the increase in cellular riboflavin.
With respect to DNA breaks, chromium(VI) reacts with hydrogen peroxide to form chromium(V), leading to the generation of hydroxyl radicals, which caused DNA breaks in vitro (21). As shown in Figure 4, The ESR spin trapping study demonstrated that the formation of DMPO spin adduct with intensity 1:2:2:1 represents the adduct of the hydroxyl radical during the reaction of Na2CrO4 and hydrogen peroxide (38). When vitamin B2 was added to this reaction mixture, a signifl- cant increase in DMPO-OH adduct was detected, indicating the enhancement of hydroxyl radical formation. During the reaction of chromate and hydrogen peroxide, a tetraperoxochromate(V) was also formed and addition of vitamin B2 resulted in an increase of this chromium(V) species (data not shown) (38). The hydroxyl radical has been shown to be particularly active, reacting with and breaking DNA (44). Thus, these results suggest that one possible mechanism of enhanced chromate-induced breakage by vitamin B2 might involve an increase of chromium(V)-related hydroxyl radical formation. Further studies are necessary to elucidate how hydrogen peroxide as well as hydroxyl radicals would become available for chromate-induced DNA breaks to occur in vivo.

Effects of Vitamins on Chromate Inhibition of Glutathione Reductase
Previous studies have shown that chromate compounds selectively inhibit the activity of glutathione reductase not only in erythrocytes (13,14), but also in the liver (15), and that this inhibition was prevented by antioxidants such as vitamin C (13) and N-acetylcysteine (15). Thus, the effects of vitamin E and vitamin B2 on chromate inhibition of glutathione reductase was examined in V-79 cells treated with Na2CrO4. As shown in Figure 5, the treatment of cells with chromate decreased glutathione reductase activity in a concentrationdependent fashion (5-15 ,uM), whereas pretreatment with vitamin E resulted in a recovery of this enzyme activity suppressed by chromate (36). On the other hand, similar pretreatment with vitamin B2 enhanced this inhibition (37). The mechanism of chromate inhibition of this enzyme remains obscure. However, other studies have shown that the enzyme inhibition was accompanied by reduction of chromium(VI) to chromium(III), and trivalent chromium could not inhibit this enzyme in vitro (13). The present results show that vitamins affected cellular levels of chromium(V) species but not those of chromium(III). These results strongly suggest that enzyme inhibition might be closely related to the cellular formation of chromium(V) during reduction of chromium(VI).
Effects of Vitamins on Chromate Cytotoxicity Figure 6 shows the effects of vitamin E and vitamin B2 on chromate-induced cytotoxicity using the colonyforming assay. Pretreatment with vitamin E resulted in a marked reduction of the cytotoxicity induced by Na2CrO4. On the other hand, pretreatment with vitamin B2 had no effect on the cytotoxicity of sublethal concentrations (5-7.5 ,uM), but there was a significant decrease of cytotoxicity induced by a lethal concentration (15 rM) of chromate (37). This result was unexpected, because similar pretreatment with vitamin B2 enhanced the formation of DNA breaks as well as the inhibition of enzyme activity induced by chromate. A recent study has shown that antioxidant enzymes such as catalase and superoxide dismutase are effective in reducing the formation of chromium(VI)-induced DNA breaks, but these antioxidants have no effect on the cytotoxicity caused by this metal (45 Na2CrO4 (pM) FIGURE 6. Effects of vitamin E and vitamin B2 on Na2CrO4-induced cytotoxicity. Cells were pretreated for 24 hr with vitamin E ( 0 ), vitamin B2 ( A ), or DMSO alone ( 0 ) and then treated for 2 hr with Na2CrO4. Following treatment, appropriate numbers of cells were plated and allowed to form colonies. Modified from Sugiyama et al. (36,37).
DNA damage induced by chromate may contribute to the cytotoxicity but apparently is not the only lesion associated with cell death induced by this metal.

Conclusion
The effects of vitamin E and vitamin B2 on chromateinduced damage as well as on the formation of chromium(V) and (III) were studied using V-79 cells. The results indicate that a) the level of chromium(V) in cells treated with chromate shows a strong correlation with the induction of DNA breaks and enzyme inhibition. Therefore, chromium(V) might be one critical ultimate form which is responsible for the toxic action of chromate; b) DNA-protein crosslinks are not as dependent upon chromium(V) formation as are DNA strand breaks, but may be associated with chromium(III) formation; c) chromium(VI)-induced cytotoxicity is not directly related to the induction of DNA damage by this metal, indicating that DNA damage might not be the only lesion required for cell death; d) in particular, vitamin E protected cells from all of the chromate-induced damages tested. Therefore, vitamin E might be a useful antitoxic agent for chromium compounds; and e) these vitamins are capable of altering the biological effects of chromate, indicating the importance of the action of vitamins on the chromate-induced toxicity.
Since the importance of vitamins in both human nutrition and cancer prevention has been well docu-mented and since not all vitamins have similar effects on chromate-induced damage, studies are necessary to elucidate whether other vitamins have an effect on chromate-induced damage.
This work was supported in part by Fukuoka Cancer Society and Chiyoda Mutual Life Foundation.