Mechanisms of the Carcinogenic Chromium(VI)-induced DNA-Protein Cross-linking and Their Characterization in Cultured Intact Human Cells

Mattagajasingh, Subhendra N.; Misra, Hara P.

doi:10.1074/jbc.271.52.33550

Cited by 38 publications

(33 citation statements)

References 55 publications

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“…The decreased ability of the cell to repair DNA damage sustained during the replication process, could be the result of Cr(III) ion mediated inhibition of synthesome associated DNA polymerase δ and ε activity within the cell. In support of this suggestion, Cr(VI)-induced formation of binary Cr-DNA or DNA-DNA cross-links, and ternary Cr-DNA-protein complexes was shown to correlate with a metal ion induced increase in DNA damage (Mattagajasingh and Misra, 1996). Thus, the decrease in the activity and fidelity with which the DNA synthesome mediates DNA replication in the presence of Cr(III) ion, could be the result of the DNA synthesome forming DNA-protein-chromium complexes at these higher concentrations of Cr(III) ion.…”

Section: Discussionmentioning

confidence: 82%

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

Dai

Liu

Malkas

et al. 2009

Toxicology and Applied Pharmacology

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Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC 50 of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases α, δ and ε is 15, 45 and 125 μM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC 50 =88 μM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr (III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr (III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication correlates with the genotoxic and cytotoxic effects of this metal ion; and promotes cell killing via inhibition of the DNA polymerase activity mediating the DNA replication and repair processes utilized by human cells.

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Section: Discussionmentioning

confidence: 82%

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

Dai

Liu

Malkas

et al. 2009

Toxicology and Applied Pharmacology

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“…23,24 In addition, Cr(VI)/Cr(III) redox cycling produces ROS, which can contribute to the formation of DPCs by the free radical mechanism shown in Figure 2A. 25 …”

Section: Introductionmentioning

confidence: 99%

DNA–Protein Cross-Links: Formation, Structural Identities, and Biological Outcomes

2015

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CONSPECTUS Non-covalent DNA-protein interactions are at the heart of normal cell function. In eukaryotic cells, genomic DNA is wrapped around histone octamers to allow for chromosomal packaging in the nucleus. Binding of regulatory protein factors to DNA directs replication, controls transcription, and mediates cellular responses to DNA damage. Because of their fundamental significance in all cellular processes involving DNA, dynamic DNA-protein interactions are required for cell survival, and their disruption is likely to have serious biological consequences. DNA-protein cross-links (DPCs) form when cellular proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. DPCs progressively accumulate in the brain and heart tissues as a result of endogenous exposure to reactive oxygen species and lipid peroxidation products, as well as normal cellular metabolism. A range of structurally diverse DPCs are found following treatment with chemotherapeutic drugs, transition metal ions, and metabolically activated carcinogens. Because of their considerable size and their helix-distorting nature, DPCs interfere with the progression of replication and transcription machineries and hence hamper the faithful expression of genetic information, potentially contributing to mutagenesis and carcinogenesis. Mass spectrometry-based studies have identified hundreds of proteins that can become cross-linked to nuclear DNA in the presence of reactive oxygen species, carcinogen metabolites, and antitumor drugs. While many of these proteins including histones, transcription factors, and repair proteins are known DNA binding partners, other gene products with no documented affinity for DNA also participate in DPC formation. Furthermore, multiple sites within DNA can be targeted for cross-linking including the N7 of guanine, the C-5 methyl group of thymine, and the exocyclic amino groups of guanine, cytosine, and adenine. This structural complexity complicates structural and biological studies of DPC lesions. Two general strategies have been developed for creating DNA strands containing structurally defined, site-specific DPCs. Enzymatic methodologies that trap DNA modifying proteins on their DNA substrate are site specific and efficient, but do not allow for systematic studies of DPC lesion structure on their biological outcomes. Synthetic methodologies for DPC formation are based on solid phase synthesis of oligonucleotide strands containing protein-reactive unnatural DNA bases. The latter approach allows for a wider range of protein substrates to be conjugated to DNA and affords a greater flexibility for the attachment sites within DNA. In this Account, we outline the chemistry of DPC formation in cells, describe our recent efforts to identify the cross-linked proteins by mass spectrometry, and discuss various methodologies for preparing DNA strands containing structurally defined, site specific DPC lesions. Polymerase bypass experiments conducted with model DPCs indica...

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“…However, in earlier studies we have demonstrated that only about 19% of Cr(VI)-induced DPCs could be disrupted by EDTA-chelation and the remaining 81% require nuclease digestion (Mattagajasingh andMisra, 1996, 1999). Furthermore, we and others have also demonstrated that Cr(VI) treatment produces an 'oxidative stress' in cells and increases the cellular level of prooxidants (Mattagajasingh andMisra, 1995a, 1997;Travacio et al, 2001;Goulart et al, 2005;Kalahasthi et al, 2006;Fatima and Mahmood, 2007), and the majority of DPCs could be suppressed with antioxidant pretreatment (Mattagajasingh and Misra, 1996).…”

Section: Introductionmentioning

confidence: 94%

“…Cr(VI) is known to induce a variety of DNA lesions, including oxidation of DNA bases, single-and double-strand DNA breaks, and DNA-DNA and DNA-protein crosslinks (Wedrychowski et al, 1985;Mattagajasingh et al, 1996). Because Cr(VI) does not bind to DNA or proteins in cell-free systems (Fornace et al, 1981), but Cr(III) does (Cohen et al, 1990), some of the Cr(VI)-induced DNA-protein crosslinks (DPCs) could be disrupted by EDTA (Miller and Costa, 1989;Mattagajasingh andMisra, 1995b, 1996), a chelator of Cr(III) but not Cr(VI), and it is known that Cr(III) can induce DNA-amino acid crosslinks (Salnikov et al, 1992), it has been generally believed that a biologically reduced form of Cr(VI) such as Cr(III) physically mediates DNA-protein crosslinking.…”

Section: Introductionmentioning

confidence: 99%

Carcinogenic chromium(VI)‐induced protein oxidation and lipid peroxidation: implications in DNA–protein crosslinking

Mattagajasingh

Misra

2008

J of Applied Toxicology

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Hexavalent chromium [Cr(VI)] compounds are Group-I human carcinogens. Cr(VI)-induced DNA-protein crosslinks (DPCs) have been implicated in the mutagenic and carcinogenic effects of Cr(VI). Although multiple mechanisms have been suggested for Cr(VI)-induced DNA-protein crosslinking, the mechanism of formation of DNA-protein crosslinks is not well understood. In this study, we explored the hypothesis that Cr(VI)-induced DPCs could be formed via generation of protein carbonyls and malonaldehyde (MDA) through protein oxidation and lipid peroxidation, respectively. Treatment of human leukemic T-lymphocyte MOLT4 cells with potassium chromate induced the formation of protein carbonyls and DPCs within 2 h, but increased the level of MDA only after 4 h, in a dose-dependent manner. Chromate treatment of MOLT4 cell homogenates also resulted in increased formation of MDA and protein carbonyls in a dose-dependent manner. EPR spectrometry in combination with spin trapping techniques revealed that reaction of Cr(VI) with biological reductants such as NADPH, glutathione reductase or H(2)O(2) generates Cr(V) and (*)OH radicals. Pretreatment of cells with antioxidants such as alpha-tocopherol or Tiron inhibited chromate-induced increase in formation of protein carbonyls, MDA and DPCs, but pretreatment of cells with riboflavin or 3-aminotriazole, a catalase inhibitor, had the opposite effect. Our results, for the first time, demonstrate that Cr(VI) exposure increases the cellular level of protein carbonyls and that Cr(VI)-induced DPCs may be formed, at least in part, via generation of protein carbonyls.

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Mechanisms of the Carcinogenic Chromium(VI)-induced DNA-Protein Cross-linking and Their Characterization in Cultured Intact Human Cells

Abstract: DNA-protein complexes (DPCs

Cited by 38 publications

References 55 publications

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

DNA–Protein Cross-Links: Formation, Structural Identities, and Biological Outcomes

Carcinogenic chromium(VI)‐induced protein oxidation and lipid peroxidation: implications in DNA–protein crosslinking

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