Molecular mechanisms of the carcinogen chromium(VI)-induced DNA-protein crosslinking in cultured intact human cells

Chromium(VI)-induced DNA-protein crosslinking is implicated in chromium Carcinogenicity. However, the mechanism of chromate-induced DNA-protein crosslinking has not been established. Based on the current literature and our preliminary data, we hypothesized that Cr(VI)-induced DNA-protein crosslinks may, in part, be due to generation of active oxygen species following the intracellular reduction of Cr(V1) and that antioxidants may ameliorate the genotoxic and carcinogenic effects of Cr(VI). We proposed to test our hypothesis with the following specific aims: 1) To characterize the DNA-protein complexes induced by the carcinogen Cr(VI) in MOLT4 cells, 2) To investigate the effect of Cr(VI) on "oxidative-stress status" of the cell, 3) To distinguish between proteins directly cross-linked by Cr(III) and those that are cross-linked oxidatively during intracellular Cr(VI) reduction, and 4) To identify major proteins that are complexed to DNA by Cr(III) or via oxidative mechanisms, in MOLT4 cells treated with chromate. Together, the proposed studies should provide new information regarding the mechanism of chromium genotoxicity and carcinogenicity. The identification of specific protein(s) involved in DNA-protein crosslinks may also provide an useful tool to develop biomarkers for assessment of chromium risk.

The results of our studies indicate the following: (1) Characterization of chromate-induced DNA-protein complexes in MOLT4 cells: A selected group of non-histone proteins were found to crosslink to DNA upon chromate exposure. The subcellular localization of these proteins was found to be in the nuclear fraction, specifically in the nuclear matrix, suggesting the proximity of these proteins to DNA. Analysis of the stability of the crosslinks to disruptive chemicals and enzymes suggested that Cr(III) and sulfhydryl groups are partially involved in such complexes. However, resistance of some proteins to treatments such as EDTA, B-mercaptoethanol or thiourea, and their need for nuclease digestion to release them by fragmenting DNA indicated that those proteins may be covalently crosslinked to DNA via oxidative mechanisms.

(2) Effect of Cr(VI) on the "oxidative-stress status" of MOLT4 cells: Chromate treatment of cells was found to not only decrease the level of low molecular weight antioxidants and antioxidant enzymes, but also induce the formation of active oxygen species, such as hydrogen peroxide. A mechanism for this hydrogen peroxide-inducing effect of chromate was proposed. A close correlation was observed between the cellular levels of oxidants and DNA-protein crosslinking following chromate exposure. Pretreatment of cells with antioxidants also illustrated correlative changes in chromate-induced DNA-protein crosslinking and the cellular level of oxidants. Intracellular generation of reactive species of chromium, such as Cr(V), and generation of active oxygen species during the reaction of Cr(VI) and its reduced forms such as Cr(V) and Cr(III), with its biological reductants were studied by EPR spectrometric techniques. Furthermore, exposure of cells to chromate was also found to induce protein oxidation and lipid peroxidation that were effectively suppressed by antioxidant pretreatment of cells, suggesting a role of reactive oxygen species, protein carbonyls and malonaldehyde in inducing DNA-protein crosslinking.

(3) Detection of proteins crosslinked to DNA by direct participation of Cr(III) and via oxidative mechanisms upon chromate exposure of cells: Chromate-induced DNA-protein complexes that were formed by direct participation of Cr(III) were distinguished from those that were formed via oxidative mechanisms by using EDTA to specifically dissociate Cr(III) mediated crosslinks, and α-tocopherol succinate to suppress oxidatively crosslinked proteins, respectively. DNA-protein complexes induced by x-ray irradiation of cells and incubation of isolated nuclei with Cr(III) were used as positive controls for crosslinking of proteins to DNA by oxidative mechanisms and by Cr(III), respectively. A common group of identical non-histone proteins were found to complexed to DNA by Cr(VI), Cr(III), and x-ray, however a 51 kD basic protein appeared to be predominantly crosslinked to DNA by participation of Cr(III), while a 49 kD acidic protein appeared to be primarily crosslinked by oxidative mechanisms. Chromate-induced DNA-protein crosslinks isolated from α-tocopherol pretreated cells exhibited an increase in the fractionation of DNA by restriction enzymes as compared to that isolated from cells treated with chromate alone, further supporting the involvement of oxidative mechanisms in the process. Formaldehyde, on the other hand, primarily crosslinked histones to DNA, indicating that specificity in protein-DNA crosslinking is dependent on the chemical nature of the crosslinking agent.

(4) Identification of major proteins complexed to DNA upon chromate treatment: Attempts were made to identify the proteins that crosslink to DNA upon chromate exposure of cells by using antibodies to candidate proteins, and N-terminal sequencing followed by searching for their homology in GeneBanks. Actin, lectin, and aminoglycoside nucleotidyltransferase were identified as participants in chromate-induced DNA-protein crosslinking.

(5) DNA protein complexes as a biomarker of exposure to chromate: Finally, DNA-protein crosslinks were detected immunologically by developing an antiserum to chromate-induced DNA protein crosslinks. The antiserum predominantly reacted with nuclear proteins, and DNA-protein crosslinks induced by Cr(VI), Cr(III) and x-ray, but reacted poorly with formaldehyde-induced DNA-protein crosslinks, further suggesting specificity in DNA-protein crosslinks induced by different crosslinking agents. The antiserum did not react with DNA-protein crosslinks isolated from control cells.

Taken together, we conclude that DNA-protein crosslinks are caused by intracellularly generated Cr(III) as well as active oxygen species formed following exposure to Cr(VI). Hence, it appears that antioxidants may be useful in reducing the genotoxic and carcinogenic effect of chromium(VI) in human populations exposed to this toxicant. Participation of the three nuclear proteins, actin, lectin, and aminoglycoside nucleotidyltransferase, in chromate-induced DNA-protein crosslinks suggest that these proteins may be parts of chromatin structure. Since these proteins are crosslinked to DNA by chromate, they may be used as biomarkers of chromate exposure.