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Title page for ETD etd-04102010-225656


Type of Document Dissertation
Author Loecken, Elisabeth Mary
URN etd-04102010-225656
Title DNA-Protein Cross-Links Induced by Bis-Electrophiles
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
F. Peter Guengerich, Ph.D. Committee Chair
Carmelo Rizzo, Ph.D. Committee Member
Daniel C. Liebler, Ph.D. Committee Member
David L. Hachey, Ph.D. Committee Member
Richard N. Armstrong, Ph.D. Committee Member
Keywords
  • carcinogens
  • bis-electrophiles
  • DNA damage
  • DNA-protein cross-links
Date of Defense 2010-03-18
Availability unrestricted
Abstract
Diepoxybutane is a mutagenic and carcinogenic oxidation product of the important industrial chemical and environmental contaminant butadiene. The mutagenic potential of diepoxybutane is thought to be due in part to its bifunctional electrophilic character. One mechanism by which bis-electrophiles can exert their toxic effects is through the induction of genotoxic and mutagenic DNA-protein or –peptide cross-links. This mechanism has been shown in systems overexpressing the DNA repair protein O6-alkylguanine DNA-alkyltransferase (AGT) or glutathione transferase and involves reactions with nucleophilic cysteine residues. The hypothesis that DNA-protein crosslink formation is a more general mechanism for genotoxicity by bis-electrophiles was investigated by screening nuclear proteins for reactivity with model monofunctional electrophiles. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was identified as a candidate due to the nucleophilicity of two cysteine residues (Cys152 and Cys246) in reaction screens with model electrophiles (Dennehy, M. K. et al. (2006) Chem. Res. Toxicol. 19, 20-29). Incubation of GAPDH with bis-electrophiles resulted in inhibition of its catalytic activity but only at high concentrations of diepoxybutane. In vitro assays indicated DNA-GAPDH crosslink formation in the presence of diepoxybutane, and bis-electrophile reactivity at Cys246 was confirmed using mass spectral analysis. In contrast to AGT, overexpression of human GAPDH in Escherichia coli did not enhance mutagenesis by diepoxybutane. The candidate proteins histones H2b and H3 were identified in screens using human liver nuclei and the bis-electrophile 1,2-dibromoethane. Incubation of these proteins with diepoxybutane resulted in DNA-protein cross-links and produced protein adducts, and DNA-histone H2b cross-links were identified (immunochemically) in E. coli cells expressing histone H2b. However, heterologous expression of histone H2b in E. coli failed to enhance bis-electrophile-induced mutagenesis, although histone H2b bound DNA with even higher affinity than AGT. The extent of DNA cross-linking of isolated histone H2b was similar to that of AGT, suggesting that differences in post-cross-linking events explain the difference in mutagenesis. In a related experiment, reactive diepoxybutane-glutathione conjugates believed to contribute to enhanced mutagenesis observed in bacterial cells overexpressing glutathione transferases were investigated. Mass spectral analysis of incubations containing purified glutathione transferase, glutathione, and diepoxybutane yielded a glutathione conjugate that retained the epoxide. Diepoxybutane also produced glutathione-DNA cross-links upon incubation.

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