Role of paramagnetic chromium in chromium(VI)-induced damage in cultured mammalian cells.

Chromium(VI) compounds are known to be potent toxic and carcinogenic agents. Because chromium(VI) is easily taken up by cells and is subsequently reduced to chromium(III), the formation of paramagnetic chromium such as chromium(V) and chromium(III) is believed to play a role in the adverse biological effects of chromium(VI) compounds. The present report, uses electron spin resonance (ESR) spectroscopy; the importance of the role of paramagnetic chromium in chromium(VI)-induced damage in intact cultured cells is discussed, based upon our studies with antioxidants including vitamin E (alpha-tocopherol), B2 (riboflavin), C (ascorbic acid), and so on. These studies appear to confirm the participation of paramagnetic Cr such as chromium(V) and Chromium(III) in chromium(VI)-induced cellular damage.


Introduction
Chromium(VI) compounds are well-established human carcinogens based upon epidemiologic studies (1). They induce chromosomal aberrations, mutations, and transformation in cultured mammalian cells (2)(3)(4). Earlier studies have reported that chromium(VI) compounds produced a variety of DNA lesions such as DNA single-strand breaks, alkali-labile sites, and DNA-protein crosslinks, as well as selectively inhibiting the activity of enzymes such as glutathione reductase (2)(3)(4).
Chromium(VI) compounds have been shown to be more toxic and carcinogenic than chromium(III) compounds, because they readily enter cells by the sulfate transport system (4). However, after entering the cell, chromium(VI) is reduced to trivalent form, through chromium(V) and (IV) intermediates, by cellular reductants such as ascorbic acid, riboflavin, glutathione and flavoenzymes, including cytochrome P-45o reductase and glutathione reductase (2)(3)(4).
Thus, the levels of these biological reductants and paramagnetic chromium species in inside cells might be associated with the induction of chromium(VI)-induced damages. Therefore, electron spin resonance (ESR) studies have focused on the formation of paramagnetic chromium species such as chromium(V) and -(III) during reduction of chromium(VI) by biological reductants in vitro. The studies showed that this reduction process generates radical species such as active oxygen species, as well as glutathionyl radicals, with concomitant formation of chromium(V) species (4)(5)(6)(7)(8)(9)(10)(11)(12). In addition, previous studies reported that chromium(V) complex induced DNA breaks in vitro, and mutations in bacterial systems (7,9,13,14). Furthermore, recent in vitro studies have shown that biologically generated chromium(V) complexes react with hydrogen peroxide (H202), in a Fenton-type manner, to produce more hydroxyl radicals than a similar reaction with chromium(VI) (9,10,15). However, in intact cells, there have been very few studies that examined the role of paramagnetic chromium in the genotoxicity and toxicity caused by chromium(VI) compounds (16,17).
The present report discusses the relationship between paramagnetic chromium and chromium(VI)-induced damages in intact cultured cells using ESR spectrometry, based upon our study with the modification of chromium(V) and chromium(III) by reductants (18)(19)(20)(21)(22)(23)(24)(25)(26). The results indicate the important role of paramagnetic chromium in the genotoxicity and toxicity of chromium(VI) compounds in cells.

Formation of Chromium(V) and Chromium(lll) in Intact Cells
Incubation of cultured Chinese hamster V-79 cells with Na2CrO4 resulted in the appearance of the ESR signals of both chromium(V) and Cr(III) complex, as measured by ESR spectroscopy at a temperature of 153' K ( Figure 1). The signal of chromium(V) was observed with an anisotropy at g> = 2.016 and gA = 1.989, and the line width of the maximum absorption peak was 12 to 13G (22,23,26). On the other hand, a rather broad signal with a g value of 2.02 to 2.03 and a line width of 700 to 800G was also observed concomitantly with that of the chromium(V) signals (21)(22)(23)26). The formation of both chromium(V) and -(III) complex in cells was found to increase proportionally to the concentration of chromate (50 to 500 jiM) as well as to the time of exposure (30 min to 2 hr) to this metal (26). Since the line width of the chromium(III) hexaaqueous complex signal has been reported to be about 15OG (17), it is possible that the increased line width of chromium(III) in the cells may be related to the formation of chromium(III) complex with cellular components, leading to reduced mobility of this metal (26).
As shown in Figure 1, when the relative concentration of paramagnetic chromium species in cells was estimated by analyzing the areas of the ESR signals, the concentrations of chromium(III) were about 30 times greater than those of chromium(V), indicating that most of the chromium inside Environmental Health Perspectives the cells was in the form of chromium(III) (22,26). Following removal of extracellular chromate, the level of chromium(V) complex decreased quickly during the first hour but more slowly during the next hour, whereas the level of chromium(III) remained unchanged (26). Thus it appears that chromium(VI) may be subsequently reduced through chromium(V) to (III) complex in intact cells.
These studies showed that paramagnetic chromium species formed during reduction could be clearly detected in intact cultured cells, as measured by ESR spectroscopy.

Modification of the Levels of Paramagnetic Chromium and Chromium(VI)-induced Damage in V-79 Cells
Among biological reductants, an increase of the content of vitamins such as a-tocopherol, riboflavin, and ascorbic acid by the pretreatment of V-79 cells with tocopherol succinate, riboflavin, and ascorbic acid, respectively, resulted in alteration of the levels of paramagnetic chromium species, as estimated by ESR spectroscopy (18,(20)(21)(22)(23)27). As shown in Table 1, an increase of either a-tocopherol or ascorbic acid in cells reduced the level of chromium(V) complex, whereas an increase of riboflavin enhanced the level of this intermediate (20)(21)(22)(23). With respect to chromium(III), elevated ascorbic acid content was only found to increase chromium(III) content (20,22,23).
Under the same experimental conditions, the induction of DNA damage, enzyme inhibition and cytotoxicity by Na2CrO4, as well as cellular uptake of this metal were investigated, as summarized in Table 1. DNA single strand breaks and/or alkali-labile sites induced by chromate were decreased by increasing the levels of atocopherol or ascorbic acid, while increased riboflavin content enhanced the levels of both types of DNA damage (18,19,(21)(22)(23). Similarly, the elevation of a-tocopherol or ascorbic acid in cells restored the glutathione reductase activity suppressed by chromate, and an increase of riboflavin enhanced this enzyme inhibition (Table 1) (20)(21)(22)(23).
Recently, we also found that the cell-permeable metal chelator o-phenanthroline (OP) suppressed the formation of chromium(V) intermediates, as evaluated by ESR spectroscopy at room temperature, resulting in a decrease of chromate-induced DNA breaks and/or alkali-labile sites, as well as in a recovery of glutathione reductase inhibited by this metal in V-79 cells (28). In addition, hydrogen peroxide-resistant Chinese hamster ovary (CHO) cells were found to have less total chromium(V) and simultaneously fewer DNA strand breaks than those in the parental cells (29).
In all the cases mentioned above, cellular levels of chromium(V) were correlated with the levels of chromate-induced DNA single-strand breaks, alkali-labile sites, and the enzyme inhibition.
We further investigated the effects of increased levels of a-tocopherol or riboflavin on the induction of chromosomal aberrations and mutations at HGPRT locus by chromium(VI) in V-79 cells (24,25). The results showed that an increase of a-tocopherol suppressed the clastogenic and mutagenic action of chromate compounds (24), while the increased riboflavin resulted in an enhancement of both actions of this metal (25). These results suggest that chromium(V) may be associated with the clastogenic and mutagenic activity of chromate.
On the other hand, levels of DNA-protein cross-links produced by Na2CrO4 were unaffected by an increase of a-tocopherol or riboflavin in V-79 cells, but the levels were enhanced by an increase of ascorbic acid (Table 1) (19,22,23). The uptake of chromate was also unchanged by the elevation of a-tocopherol or riboflavin, but an increase of ascorbic acid caused an acceleration of metal uptake (Table 1) (20)(21)(22). Since we showed that most of the chromium inside the cells was in the form of chromium(III), and that ascorbic acid is capable of increasing the cellular level of chromium(III), these results suggest that the formation of chromium(III) in cells may be related to the level of DNA-protein cross-links and cellular uptake of this metal.
At the present time, the role of cellular chromium(III) in the clastogenicity, mutagenicity, and carcinogenicity of chromate(VI) is not well established. Although an elevation of ascorbic acid in cells caused an increase of chromium(III) and a decrease of chromium(V) levels, further study with ascorbic acid could lead to a better understanding of the role of chromium(III) in chromate(VI)-induced carcinogenicity.
With respect to cytotoxicity, as estimated by colony-forming assays, an increase of atocopherol in V-79 cells caused a marked reduction of cytotoxicity induced by Na2CrO4; the increased levels of riboflavin resulted in a decrease in the cell mortality caused by this metal, while the cytotoxicity of chromate was enhanced by the elevation of ascorbic acid (Table 1) (20)(21)(22)(23)(24). Thus, it is likely that the formation of paramagnetic chromium in cells may not be directly correlated with cell death.

Conclusion
The role of paramagnetic chromium in chromate-induced cellular damage in cul-  Environmental Health Perspectives tured mammalian cells was investigated utilizing ESR spectrometry. The results demonstrate that the formation of chromium(V) and -(III) can be clearly detected in cultured cells by ESR spectrometry and that the modification of cellular levels of these paramagnetic species by reductants such as vitamins influences the induction of the biological effects of chromium(VI) compounds. Based upon these results, intracellular chromium(V) appears to be the critical form that is responsible for DNA single-strand breaks, alkali-labile sites, chromosomal aberrations, and mutation, as well as for the inhibition of glutathione reductase. Intracellular chromium(III) may be the critical form responsible for the DNA-protein crosslinks induced by chromium(VI) compounds. In contrast, the formation of chromium(III) and chromium(V) may not be directly related to the metal-induced cytotoxicity.
Since ESR studies showed that chromium(VI) may be subsequently reduced to chromium(III) in cultured cells, it is necessary to elucidate the role of the ultimate cellular form, chromium(III), in the mutation and carcinogenicity of chromate(VI) compounds. However, a correlation between the level of chromium(V) intermediate and chromium(VI)-induced mutation was detected in cultured cells. Therefore, paramagnetic chromium, in particular chromium(V), may play an important role in the induction of carcinogenicity by chromium(VI) compounds.