Date on Master's Thesis/Doctoral Dissertation

5-2025

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 Member

Hein, David W.

Committee Member

Kouokam, J. Calvin

Committee Member

Cai, Lu

Committee Member

Wise, Sandra S.

Committee Member

Liu, Jim

Author's Keywords

Hexavalent chromium; homologous recombination repair; BCDX2 complex; CX3 complex

Abstract

Lung cancer remains the leading cause of cancer death, often linked to smoking. However, smoking alone does not account for all cases. Exposure to environmental metals like hexavalent chromium [Cr(VI)] also contributes to lung cancer. Cr(VI) causes lung cancer by inducing chromosome instability (CIN), an early hallmark of its carcinogenic mechanism. While it has been established that Cr(VI) induces chromosome instability, the precise mechanisms underlying the chromosome instability are poorly understood. This dissertation investigates the mechanisms of carcinogenesis associated with zinc chromate, a particularly potent particulate form of hexavalent chromium. Studies show Cr(VI) induces DNA double-strand breaks and impairs the high-fidelity DNA repair mechanism, homologous recombination (HR). Cr(VI) specifically targets RAD51, a critical protein in the HR pathway, preventing its loading onto the DNA strand. The mechanism underlying this disruption remains unclear. RAD51 is regulated by RPA and two complexes, namely BCDX2 (RAD51B, RAD51C, RAD51D, XRCC2) and CX3 (RAD51C, XRCC3). How Cr(VI) affects these mediator protein interactions remains poorly understood and largely unexplored. In Aim 1, we focused on how Cr(VI) affects key proteins in HR repair. We found acute and prolonged Cr(VI) exposure increased RPA foci formation, indicating loss of RPA is not responsible for the loss of RAD51 on the filament. Similarly, RAD51B, RAD51C, and XRCC2 foci formation increased following acute exposure to Cr(VI) but decreased with prolonged exposure. In contrast, XRCC3 remained unchanged after acute exposure and showed minimal response, ultimately failing to function after prolonged exposure. Notably, acute exposure significantly impacted RAD51D, leading to a reduction in RAD51D foci formation, protein levels, and mRNA expression. Together, these data suggest that RAD51D plays an initial role in the loss of RAD51 filament formation and the subsequent failure of the HR repair response. The One Environmental Health approach emphasizes the interconnectedness of humans, wildlife, and ecosystems, all of which share a common environment and are exposed to toxicants like Cr(VI). Aim 2 characterizes the effects of prolonged Cr(VI) exposure on alligator cells to better understand the mechanisms of Cr(VI) carcinogenesis. Long-lived alligators are often exposed to environmental contaminants long-term, yet they show low cancer rates. We measured the effects of Cr(VI) to induce DNA double-strand breaks, HR repair, and chromosome damage in alligator lung cells. Cr(VI) induced DNA double-strand breaks in alligator cells, but HR repair remained functional. These data suggest that alligator cells are resistant to Cr(VI)-induced HR repair failure. These findings highlight important differences between how human and alligator cells respond to Cr(VI) exposure. To further investigate the underlying mechanisms of Cr(VI)-induced DNA repair and Cr(VI) tolerance in alligators, we conducted a transcriptomic analysis of alligator cells. In Aim 3, transcriptome analysis showed distinct patterns of gene expression changes across different exposure levels. We found acute and prolonged Cr(VI) exposure induced differentially expressed genes in alligator cells, leading to both upregulation and downregulation of gene expression. Notably, Cr(VI) induced upregulation of DNA repair genes. Particularly, RAD51, RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 were significantly upregulated in alligators compared to humans. These findings suggest that alligator cells may possess more efficient repair systems. Overall, this dissertation shows Cr(VI) suppresses RAD51 and disrupts HR by specifically targeting RAD51D within the BCDX2 complex. These findings suggest that RAD51D may be key in Cr(VI)-reduced RAD51 function and HR repair inhibition. We showed alligator lung cells are resistant to Cr(VI)-inhibited HR repair. Our studies translate Cr(VI)-induced DNA double-strand breaks and HR repair to alligator lung cells.

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