Certain transition metals, including nickel, chromium, cadmium, and copper, are carcinogenic to humans and/or animals. Their effects include cancer in the progeny of fathers exposed to welding fumes and other metal dusts. However, mechanisms of the carcinogenic activity of these metals remain obscure. In recent years, we have been testing a hypothesis that one such mechanism would involve metal-mediated conformational and/or oxidative damage to DNA and nuclear proteins. In 1999/2000 we continued testing that hypothesis. Our investigations on transition metals' interactions with protamines were aimed at the conformational effect of Ni(II) on protamine HP2 N-terminal 15-mer peptide motif RTHGQ-SHYRR-RHCSR- [HP2(1-15)]. A 2D-NMR study revealed a strong long-range structuring effect of Ni(II) coordinated by the first three amino acids on HP2(1-15) folding in solution. The resulting folding of the peptide molecule explains the site-specificity of Ni(II)-mediated oxidative damage to this peptide, directed exclusively toward the His3 and Y8 residues, and the enhancement by Ni(II) of HP2(1-15) binding to DNA, observed by us before. The same modes of binding and structural effects are expected also for Cu(II), a known reproductive toxicant.P/> Another investigation focused on the MTH1 proteins, the oxidized purine nucleoside triphosphate pyrophosphohydrolases (formerly 8-oxo-dGTPases), a class of mammalian enzymes preventing incorporation of promutagenic oxidized purine nucleotides, such as 8-oxo-dGTP, 8-oxo-dATP, 2-OH-dATP, 2-OH-ATP, 8-oxo-GTP, and 8-oxo-ATP, into DNA or RNA. Using plasmid constructs carrying different upstream regions of murine MTH1 gene transfected into cultured cells, we found that Ni(II), but not Cd(II), at low concentrations could upregulate MTH1 gene expression, most likely through a direct interaction with the (CA)n, SINE, and (AT)n repeats. We postulate that such repeat sequences may be novel candidates for metal-responsive elements. In our next study, anti-MTH1 polyclonal antibody was used to test the effects of Ni(II) and Cd(II) on tissue and cellular expression and distribution of the MTH1 proteins in rats. The immunohistochemical staining revealed changing patterns of cytoplasmic and nuclear MTH1 expression in different cell populations, depending on the toxicity of the given metal in the liver, kidney, and testis. Also, the results showed for the first time variable levels of MTH1 expression in cell nuclei and its presence in the chromatoid bodies of spermatocytes. Using our in vivo assay for 8-oxo-dGTPase activity, developed previously, we have also shown that in spite of the apparent shifts of MTH1 expression among intracellular compartments, observed histochemically, the global enzymatic activity of MTH1 in a given cell population is quite stable and does not depend on cell cycle and proliferation rate. Our search for metal binding sites in enzymatic proteins led us to the """"""""fragile histidine triad,"""""""" -KVHVHVL-, motif of the tumor suppressor protein Fhit. We presumed that the carcinogenic metals might disrupt Fhit function through a deletion of the FHIT gene from its """"""""fragile site"""""""" (e.g., by metal-generated reactive oxygen species) and/or inhibition of the enzymatic activity of Fhit protein through binding to its Cys and His sites. Indeed, our in vitro study confirmed the inhibitory potential of several transition metals: Cu(II)>Ni(II)>Zn(II)>>Cd(II)>Cr(III)>>Co(II), and loss or a substantial decrease of Fhit expression in Ni(II)-induced murine sarcomas. The investigations are in progress. In addition, our research provides an experimental basis for collaborative studies on oxidative damage by other chemical carcinogens (see projects ZO1 BC 05352 and ZO1 BC 05488).
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