The role ofglutathione and cytochrome P-450 in the production of DNA damage by chromium(VI) was examined In chicken embryo hepatocytes by the alkaline elution technique. Cellular levels of glutathione and cytochrome P-450 were altered by treating the hepatocytes with N-acetyl-Lcysteine, buthionine sulfoximine, isopentanol, or 8-naphthoflavone. A dramatic increase in chromium(VI)-induced DNA strand breaks was observed after increasing glutathione levels in the cells. Chromium(VI)-induced DNA strand breaks were even more numerous when the level of cytochrome P-450 was also increased. Upon depletion of glutathione levels and induction ofcytochrome P-450 or cytochrome P 448, little or no DNA strand breaks or DNA interstrand cross-links were observed after chromium(VI) treatment. Chromium(VI)-induced DNA-protein cross-links generally decreased after either increases or decreases in cellular levels of glutathione or cytochrome P-450 or P-448. These results suggest that glutathione enhances chromium(VI)-induced DNA damage through metabolic activation of chromium(VI). The possible production of reactive chromium species upon metabolism by glutathione and cytochrome P-450 or P448 and their involvement in DNA damage is discussed.Chromium(VI) compounds are recognized as carcinogens in humans and animals (1) and as mutagens in bacterial and mammalian cell systems (2). Chromium(VI) causes DNA strand breaks and cross-links in vivo and in cultured cells (3)(4)(5)(6). It has been proposed that cellular metabolism of chromium(VI) compounds is necessary for DNA damage, since chromium(VI) does not react with isolated DNA under physiological conditions (7). The DNA-damaging ability of other carcinogenic agents has been shown to be modulated by metabolic pathways such as the cytochrome P450 system, sulfotransferases, and glutathione (GSH) (8). Changes in cellular levels of cytochromes P-450 alter the levels of DNA damage produced by carbon tetrachloride (9), benzo-[a]pyrene (10), and dimethylnitrosamine (11) in rodents.In vitro studies have identified several cellular components that are capable of metabolizing chromium(VI) (12-14). Rat liver microsomes contain a NADPH-dependent chromium(VI) reductase activity (15). Induction of cytochrome(s) P-450 by phenobarbital enhances the chromium(VI) reductase activity ofliver microsomes; however, induction of cytochrome(s) P-448 by 3-methylcholanthrene has no effect (13). GSH also reduced chromium(VI) in vitro under physiological conditions (12,14). The reaction of GSH with chromium(VI) has been proposed to occur by a two-step mechanism that involves the rapid formation of a chromium(VI) thioester followed by the reduction of the chromium(VI) thioester to chromium(III) (12).The present report describes the effect of altering the intracellular concentrations of cytochrome P450 and P-448 and GSH on chromium(VI)-induced DNA damage in primary cultured chicken embryo hepatocytes. Chromium(VI) treatment is known to produce DNA damage in chicken embryo hepatocytes (5, 6). The hepatocytes ar...

The repair of DNA damage caused by chromate was examined in chick embryo hepatocytes. Treatment of chick embryo hepatocytes with 5 microM sodium chromate for 2 h caused the formation of DNA strand breaks, DNA interstrand cross-links, and DNA-protein cross-links. The maximal level of strand breaks and DNA interstrand cross-links was observed immediately after the 2 h chromate treatment. After removal of the chromate, strand breaks and DNA interstrand cross-links were completely repaired by 3 h and 12 h, respectively. In contrast, DNA-protein cross-links continued to form reaching a maximal level 3 h after chromate removal. Although the level of DNA-protein cross-links decreased at later times, a significant level persisted 40 h after chromate removal. The effect of these persistent DNA-protein cross-links on gene expression was examined by measuring induction of porphyrin accumulation by propylisopropylacetamide and deferoxamine methanesulphonate, a process known to depend on mRNA synthesis. Induction of porphyrin accumulation was decreased immediately following the 2 h chromate treatment. Only partial recovery of induction was observed even 40 h after chromate removal. The effect of chromate treatment on cellular glutathione levels was monitored. No change in cellular glutathione was observed after a 2 h treatment with 5 microM sodium chromate; however, a three-fold increase was observed 12 h after removal of chromate.

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