Date on Master's Thesis/Doctoral Dissertation

1-2020

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Wise, Sr., John Pierce

Committee Co-Chair (if applicable)

Beverly, Levi

Committee Member

Beverly, Levi

Committee Member

Cai, Lu

Committee Member

States, J. Christopher

Committee Member

Zhang, Quenwei

Author's Keywords

hexavalent chromium; DNA repair; leatherback sea turtle; carcinogenesis; RAD51; E2F1

Abstract

Lung cancer is the leading cause of cancer death. Lung cancer is commonly associated with smoking, however, 1 in 5 women and 1 in 12 men who develop lung cancer are never-smokers. Environmental exposures, therefore, account for a significant portion of lung cancer cases. Hexavalent chromium [Cr(VI)] is a global environmental contaminant and known human lung carcinogen. Cr(VI) and other carcinogenic metals induce chromosome instability, an early event in lung cancer. Structural chromosome instability arises in part due to failed DNA repair. Particulate Cr(VI), the most potent form of Cr(VI), induces DNA double strand breaks and inhibits the high-fidelity DNA repair mechanism, homologous recombination. Specifically, the effector step of homologous recombination is affected shown by RAD51 failure. RAD51 failure is due to inhibited expression, inhibited localization to double strand breaks, or a combination of these two mechanisms. Little is known about the mechanisms of Cr(VI)-inhibited expression. However, Cr(VI) exposure results in downregulation of global expression, and it has been suggested epigenetic changes affect expression profiles after Cr(VI) exposure. Studies show changes in acetylation of the RAD51 promoter affect E2F1-mediated RAD51 transcription by altering the “histone code” as potential epigenetic mechanisms of inhibited expression. Studies also show changes in microRNAs are an additional epigenetic mechanism of Cr(VI)-altered expression, and this may provide an additional mechanism of inhibited RAD51 expression. The mechanisms of particulate Cr(VI)-induced RAD51 failure were investigated in a human lung cells, and key events were confirmed in a wildlife model, leatherback sea turtle (Dermochelys coriacea) cells. The leatherback model was included as a part of the One Environmental Health Approach to investigate particulate Cr(VI) carcinogenesis across species. This type of analysis is used to identify how two species with different environmental adaptations may have alternative responses to chemical exposures. Therefore, the hypothesis of this dissertation is: Prolonged exposure to particulate Cr(VI) inhibits RAD51 expression through E2F1-inhibited transcription and alteration of microRNA expression profiles, and these effects are paralleled in a leatherback sea turtle model. We found particulate Cr(VI) inhibits RAD51 and E2F1 nuclear and whole cell protein and mRNA levels in human lung cells. Therefore, we aimed to show E2F1 modulates the RAD51 response to particulate Cr(VI). We found E2F1 overexpression did not rescue particulate Cr(VI)-induced RAD51-failure after prolonged (120 h) exposure. However, when we knocked down E2F1 we found E2F1 knockdown does not inhibit RAD51 mRNA or protein expression but does reduce nuclear foci formation after acute (24 h) particulate Cr(VI) exposure when RAD51 is normally functional. These results suggest E2F1 may affect RAD51 localization to double strand breaks, but not expression after Cr(VI) exposure. As an alternative mechanism of inhibited RAD51 expression we next performed RNA sequencing (RNAseq) analysis to asses Cr(VI)-altered microRNA (miRNA) expression. This study showed Cr(VI) significantly affected global miRNA expression, a subset of which target homologous recombination genes and RAD51 expression directly. These data advance our understanding of how Cr interferes with a critical cellular pathway that contributes to carcinogenesis. We previously reported particulate Cr(VI) induces structural chromosome instability in leatherback lung cells similarly to data in human lung cells with some differences. In this dissertation we confirmed particulate Cr(VI) induces DNA double strand breaks in leatherback lung cells. In analysis of DNA repair we found lower levels RAD51 foci after prolonged particulate Cr(VI) exposure compared to acute exposure in leatherback lung cells. However, the sister chromatid exchange assay showed homologous recombination is functional after prolonged particulate Cr(VI) exposure. These results are dissimilar to results in human lung cells indicating there are significant differences in the mechanistic response to particulate Cr(VI) exposure between human and leatherback lung cells.

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