Certain transition metals, including nickel, chromium, cadmium, and copper, are carcinogenic to humans and/or animals. They induce cancer of the respiratory tract in persons exposed to metal-containing aerosols and may also increase the incidence of childhood malignancies in progeny of the exposed fathers. However, mechanisms of the carcinogenic activity of these metals remain obscure. In recent years, we have been testing a hypothesis that the mechanisms would involve metal-mediated structural and/or oxidative damage to chromatin and some enzymatic proteins. In 2003/2004 we continued testing the above hypothesis. Our investigations of transition metals' interactions with chromatin components were focused on Ni(II)-assisted truncation and other modifications of histones H2A and H2B. As we have found before in test-tube and cell culture experiments, Ni(II) mediates hydrolytic cleavage of the C-terminal tail of histone H2A at its E-S peptide bond. In vivo, such cleavage has the potential of causing chromatin remodeling and gene expression derangement. To test this presumption, we obtained constructs of the truncated and full-length histones H2A, transfected them to cells, found their integration into chromosomes, and are actually looking for differences in gene expression using the microarray technique. Moreover, extensive modification of histone H2B, a close partner of H2A in chromatin, was also observed. Using various chromatografic, mass-spectrometric, and amino acid-sequencing techniques, we have identified three types of the H2B modifications in Ni(II)-exposed cells: (1)transient increase in ubiquitination, (2) decrease in acetylation, and (3) truncation at both termini. The mechanisms of these modifications and their biological significance are being investigated. Following our search for toxic metals' effects on enzymatic proteins, we continued investigations of possible role of the tumor suppressor protein Fhit, a diadenosine triphosphate phosphohydrolase, in the mechanisms of metal-induced carcinogenesis. Since Fhit is a pro-apoptotic protein, its suppression by nickel may contribute to the survival of cells mutated by nickel and thus assist in carcinogenesis. As we found earlier, Fhit' s enzymatic activity was inhibited by Cu(II), Ni(II) and other toxic metals. Also, Fhit expression was markedly lower in cells cultured with and/or tranformed by nickel carcinogens to malignant phenotype, and in nickel-induced skeletal muscle tumors in mice as compared with normal cells and tissues. In FY2004, using the immunostaining techniqe, we evaluated the results of a 2-year bioassay in rats aimed at determination of FHIT gene expression in target tissues of nickel carcinogenesis, kidney and muscle, during the entire course of carcinogenesis. The results indicate that the expression of Fhit protein becomes strongly down-regulated with the progress of malignant transformation and becomes virtually absent in nickel-induced muscle tumors. Another investigation has been focused on the MTH1 proteins, the oxidized purine nucleoside triphosphate pyrophosphohydrolases (8-oxo-dGTPases), a class of mammalian enzymes preventing incorporation of promutagenic oxidized purine nucleotides into DNA or RNA. To date, we have observed that the enzymatic activity of MTH1 varies greatly among different tumor types and does not depend on cell proliferation rate. These findings may help in using the MTH1 protein as a marker of certain tumors. Our studies also included the chemistry of Ni(II) interactions with MTH1 substrate and product, 8-oxo-dGTP and 8-oxo-dGMP. In FY2004, we continued to investigate metals' effects on yet another enzyme, HIF-1alpha prolyl hydroxylase (PH). PH is activated by Fe(II) and may therefore be sensitive to interference by other metals, such as the carcinogenic metals Ni(II) and Co(II) (and possibly others). This enzyme is crucial for up-regulation of the hypoxia-inducible factor HIF-1alpha that mediates cellular response to hypoxia. Such up-regulation by Ni(II) and Co(II), underlying the hypoxia-mimicking effect by these metals, was observed before. Our recent investigations in cultured cells have shown that this effect is indeed elicited through inhibition of PH by these metals. However, contrary to predictions, the major mechanistic reason for this inhibition appears to be depletion of ascorbic acid, a co-activator of PH (needed to reduce iron). Ni(II), Co(II) and Cr(VI) were found to greatly deplete cells of ascorbate, most likely through catalysis of its oxidation and hydrolysis. Most importantly, this depletion and HIF-1alpha activation could be prevented with ascorbic acid supplementation. This finding may help to develop prevention strategies against human cancer in metal industries. Studies on the biological implications of this finding are being continued. In addition to the above, our research provides an experimental basis for collaborative studies on oxidative DNA damage and enzyme inhibition by transition metals and other chemical carcinogens and toxicants, led by other PIs, Drs. L.M. Anderson, M.P. Waalkes, J.M. Phang, and L.K. Keefer.
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