Date of Award

2014

Document Type

Open Access Dissertation

Department

Biological Sciences

First Advisor

Bert Ely

Abstract

Xenobiotic Metabolizing Pathway works to detoxify the cell from numerous carcinogenic, mutagenic, and toxic hydrophobic compounds. As a member of the phase I enzymes (the first phase in the Xenobiotic Metabolizing Pathway) the Cytochrome P-450 Family 1 Sub-family B Protein 1 (Cyp1B1) works to attach an oxygen molecule to its hydrophobic substrate. In performing this reaction, Cyp1B1 often increases the reactivity of the xenobiotic compound. If these reactive Cyp1B1 products migrate into the nucleus and they can cause damage by reacting with DNA. However, the Glutathione S-Transferase Theta 1 (GSTT1) and Glutathione S-Transferase Mu 1 (GSTM1), members of the phase II xenobiotic metabolizing pathway, are able to inactive these reactive Cyp1B1 products through the addition of a glutathione molecule. Previous studies have shown that four single nucleotide polymorphisms, which lead to amino acid substitutions, in the cyp1B1 gene and gene deletions in gstt1 and gstm1 genes lead to differences in cancer susceptibility. However, our analysis genotyped 473 European Americans and 177 African Americans at each locus and found no correlation between genotype and any of 13 tumor characteristics and breast cancer risk factors that impact breast cancer progression or development. Due to high substrate overlap between xenobiotic metabolizing enzymes, we hypothesize the cell can compensate for differences in protein levels and enzymatic rates can by increased expression of highly related enzymes.

We have also investigated the role of four cyp1B1 polymorphisms on protein stability using endogenous Cyp1B1 variant proteins in human cell lines. We determined that an Asparagine to Serine amino acid substitution at amino acid position 453 decreases stability by 20% compared to our ancestral control. This result was modest when compared to previously published data, which used African Green Monkey cells with overexpressed Cyp1B1 proteins. Furthermore, we showed that Arginine to Glycine substitutions at amino acid position 48 in conjunction with Alanine to Serine substitution at amino acid position 119 increased stability by 50%. We hypothesize that these differences in protein stability have little effect on the production of carcinogenic compounds and thus cancer development and progression.

Rights

© 2014, Thomas Daniel Crowder

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