The objective of this continuation application is to investigate the interaction of particulate and soluble chromium compounds with normal human small airway epithelial cells (HSAE) and fibroblasts (HLF) which are representative target cells for chromium toxicity and carcinogenesis. These compounds are human respiratory toxins and/or carcinogens which directly damage DNA and after DNA replication, and cause gene-inactivating mutations and neoplastic transformation at exposure levels which also induce some apoptotic cell death. We have discovered chromium- induced DNA-DNA interstrand crosslinks (Cr-DDC) as a major lesion in chromium-treated HSAE and HLF cells which had previously escaped detection because of its unique biochemical characteristics. Cr-DDC cause base-specific arrest of DNA polymerases, are sensitive to redox potential, are likely responsible for S phase cell cycle blockade and probably contribute to apoptotic cell death. Thus, the objective of this research is to investigate the formation, modulation, repair, and biological and molecular consequences of Cr-DDC. Virtually nothing is known about Cr-DDC, thus one goal is to investigate the biochemical and structural parameters of its formation in the presence of several physiologically relevant reductants of Cr(VI). The hypotheses to be tested are: (i) that Cr-DDC formation in HSAE and HLF cells results in base-specific DNA polymerase arrests and that these can be modulated by altering the reductive metabolism of Cr(VI), (ii) that formation and repair of genomic and gene-specific Cr-DDC correlates with DNA replication status, (iii) that repair of Cr-DDC is deficient in certain complementation groups of human Fanconi's Anemia (FA) cells leading to increased sensitivity of those cells to chromium-induced growth arrest and apoptosis, (iv) that Cr-DDC block replication and induce deletion recombination of transfected shuttle vectors, and (v) that Cr-DDC in reconstituted chromatin will interfere with the processivity of an intact DNA polymerase replication complex. This research will help elucidate molecular mechanisms of chromium toxicity and carcinogenesis and will have practical value in contributing to the evaluation of risk to humans in contact.
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