Hong Xie

Date of Award

5-2007

Level of Access Assigned by Author

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Advisor

John Pierce Wise, Sr.

Second Committee Member

Ah-Kau Ng

Third Committee Member

Stephen Pelsue

Abstract

Hexavalent chromium (Cr(VI)) is considered a particularly important and dangerous environmental chemical because it is carcinogenic to humans. Its water solubility has proven to be a key factor in the carcinogenicity of Cr(VI), with the insoluble compounds the more potent carcinogens. Because the potential for exposure to this carcinogen is high, it is imperative that we learn the mechanisms governing the carcinogenicity of Cr(VI). Lead chromate has been shown clastogenic in a concentration-dependent manner in human lung cells. Transmission electron microscopy showed that lead chromate particles were internalized into cells but intracellular dissolution is not occurred. Co-treating the cells with lead chromate and vitamin C eliminated both uptake of ionic chromium and the clastogenic activity, but had no effect on particle internalization. These data indicate that in human lung cells lead chromate clastogenesis is mediated by the extracellular dissolution of the particles. We modified the comet assay to detect DNA double strand breaks and found that lead chromate induced concentration-dependent increasing double strand breaks. Immunofluorescence assays showed that lead chromate induced concentration-dependent increase in phosphorylated H2A.X (gamma-H2AX) foci. In response to DNA damage, cells initiated S-phase arrest and increased levels of phosphorylated SMC1 expression. Lead chromate-induced DNA double strand breaks were repaired within 24 h. Ataxia telangiectasia mutated (ATM) protein and Mre11 expression was increased in response to the lesion. Mre11 co-localized with other DNA repair and signal factors such as ATM and gamma-H2A.X. The repair of lead chromate induced DNA double strand breaks in ATM-deficient or Mre 11-deficient cells was greatly reduced. These data indicate that Mre11 and ATM are important for lead chromate-induced DNA double strand break sensing and repair. A unique finding in this study was the neoplastic transformation of lead chromate. We use DNA double strand break repair-deficient cells to investigate the relationship between DNA double strand break repair and carcinogenecity. Mre11 or ATM gene deficiency impaired DNA double strand break repair which led to neoplastic transformation including cell contact inhibition and loss of anchorage independence. Therefore, our study indicates that DNA double strand breaks are a significant factor in particulate Cr(VI)-induced carcinogenesis.

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