Hexavalent chromium (Cr-6) is a firmly established human carcinogen with widespread exposure in metal- processing and other industries. There are also significant concerns about potential health effects of environmental exposure due to the presence of Cr-6 at numerous Superfund sites and its ambient and drinking water contamination. Cr-6 is a pro-carcinogen that is reduced inside the cell yielding stable Cr-3 and causing the formation of abundant Cr-DNA adducts. Cr-DNA adducts are mutagenic and genotoxic in human cells although they are weakly duplex-distorting and do not block DNA replication in vitro. We have found that genotoxicity of Cr-DNA adducts in human or mouse cells required the presence of mismatch repair (MMR) proteins. Cells lacking MMR activity were highly resistant to the induction of cell death and chromosomal damage by Cr-6. Chronic exposure to Cr-6 can therefore lead to the selection of resistant cells with inactive MMR, which provides an explanation for the very high frequency of microsatellite instability (a hallmark of inactive MMR) among lung cancers in chromate workers. Our recent findings also showed that MMR enhances Cr-6 genotoxicity via a rapid production of DNA double-strand breaks (DSB) in G2 cells immediately after replication of Cr-damaged DNA. The induction of DSB required a sequential recruitment of MSH6- and MSH3-containing complexes, indicating the unprecedented cooperation of both MMR branches in processing of Cr-DNA adducts. This application is designed to address three central questions related to the formation and the biological importance of MMR-induced DSB. These include: (1) mechanisms of DSB generation and the role of individual MMR proteins, (2) significance of error-prone and error-free pathways in repair of MMR-induced DSB, and (3) crosstalk between DSB processing and checkpoint signaling. The completion of the proposed work should identify the key processes involved in the formation and repair of highly toxic DNA lesions responsible for chromosomal breakage and other adverse genetic effects resulting from human exposure to Cr-6. Elucidation of molecular mechanisms involved in the unique processing of Cr-damaged DNA is also expected to uncover novel functions of MMR, a key genome maintenance system and a major factor in the clinical efficacy of platinum-based and some other DNA alkylating chemotherapeutic drugs.

Public Health Relevance

Hexavalent chromium is a potent human carcinogen with widespread environmental and occupational exposures. This toxic metal causes DNA breaks that remain in cells for a long period of time leading to adverse genetic effects even at very low chromium doses. This proposal is focused on the determination of the molecular causes of DNA breaks in chromium-exposed human cells and the characterization of the unique cellular systems required for their repair. The completion of the proposed work can lead to the identification of biochemical factors that increase or decrease individual susceptibility to genetic damage by carcinogenic chromium.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Special Emphasis Panel (ZRG1-DKUS-B (03))
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Reinlib, Leslie J
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Brown University
Schools of Medicine
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Luczak, Michal W; Zhitkovich, Anatoly (2018) Monoubiquitinated ?-H2AX: Abundant product and specific biomarker for non-apoptotic DNA double-strand breaks. Toxicol Appl Pharmacol 355:238-246
Krawic, Casey; Zhitkovich, Anatoly (2018) Toxicological Antagonism among Welding Fume Metals: Inactivation of Soluble Cr(VI) by Iron. Chem Res Toxicol 31:1172-1184
Krawic, Casey; Luczak, Michal W; Zhitkovich, Anatoly (2017) Variation in Extracellular Detoxification Is a Link to Different Carcinogenicity among Chromates in Rodent and Human Lungs. Chem Res Toxicol 30:1720-1729
Luczak, Michal W; Zhitkovich, Anatoly (2017) Nickel-induced HIF-1? promotes growth arrest and senescence in normal human cells but lacks toxic effects in transformed cells. Toxicol Appl Pharmacol 331:94-100
Luczak, Michal W; Green, Samantha E; Zhitkovich, Anatoly (2016) Different ATM Signaling in Response to Chromium(VI) Metabolism via Ascorbate and Nonascorbate Reduction: Implications for in Vitro Models and Toxicogenomics. Environ Health Perspect 124:61-6
Sawant, Akshada; Kothandapani, Anbarasi; Zhitkovich, Anatoly et al. (2015) Role of mismatch repair proteins in the processing of cisplatin interstrand cross-links. DNA Repair (Amst) 35:126-36
DeLoughery, Zachary; Luczak, Michal W; Ortega-Atienza, Sara et al. (2015) DNA double-strand breaks by Cr(VI) are targeted to euchromatin and cause ATR-dependent phosphorylation of histone H2AX and its ubiquitination. Toxicol Sci 143:54-63
DeLoughery, Zachary; Luczak, Michal W; Zhitkovich, Anatoly (2014) Monitoring Cr intermediates and reactive oxygen species with fluorescent probes during chromate reduction. Chem Res Toxicol 27:843-51
Luczak, Michal W; Zhitkovich, Anatoly (2013) Role of direct reactivity with metals in chemoprotection by N-acetylcysteine against chromium(VI), cadmium(II), and cobalt(II). Free Radic Biol Med 65:262-269
Green, Samantha E; Luczak, Michal W; Morse, Jessica L et al. (2013) Uptake, p53 pathway activation, and cytotoxic responses for Co(II) and Ni(II) in human lung cells: implications for carcinogenicity. Toxicol Sci 136:467-77

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