Advisor(s)

Paul Vouros

Contributor(s)

Penny J. Beuning, David A. Forsyth, Roger W. Giese

Date of Award

2011

Date Accepted

5-2011

Degree Grantor

Northeastern University

Degree Level

Ph.D.

Degree Name

Doctor of Philosophy

Department or Academic Unit

College of Science. Department of Chemistry and Chemical Biology.

Keywords

Chemistry, analytical chemistry, biochemistry, 4-aminobiphenyl, chemopreventive agents, DNA adducts, LC-MS, mass spectrometry, sulforaphane

Subject Categories

Liquid chromatography, Mass spectrometry, Cancer - Chemoprevention, Bladder - Cancer

Disciplines

Medicinal-Pharmaceutical Chemistry

Abstract

This dissertation demonstrates the significance of liquid chromatography-mass spectrometry (LC-MS) methods to the development of targeted early intervention chemopreventive strategies against bladder cancer. Here, LC-MS techniques facilitate a quantitative analysis of DNA adducts of the human bladder carcinogen 4-aminobiphenyl (4-ABP). These DNA adducts are a measure of exposure and risk, and chemopreventive agents can mitigate this risk by inhibition of adduct formation or promotion of adduct repair. Accordingly, DNA adducts are effective biomarkers of the efficacy of chemopreventive agents.

Chapter 1 provides an overview of the metabolism of 4-ABP and methods for quantifying its genetic damage. Chapter 2 reviews the application of LC-MS in the quantitative analysis of DNA adducts for the specific evaluation of chemopreventive agents. Many of the findings in this dissertation contribute to this new and exciting field.

The development of a validated LC-MS/MS method for quantification of 4-ABP-induced genotoxicity was crucial for this research and is described in chapter 3. This method overcomes analytical challenges that are often encountered in the quantitative analysis of DNA adducts from human samples, including limited sample availability and the need to reach detection limits approaching the part-per-billion threshold. By operating at nano-flow rates and incorporating a capillary analytical column in addition to an automated sample enrichment step, we have developed a sensitive HPLC-MS/MS method appropriate for quantifying dG-C8-4-ABP. The Limit of Detection (LOD) of 5 adducts in 10^9 nucleotides (20 attomol dG-C8-4-ABP and 1.25 ug DNA nucleosides on column) and low sample requirement of 5 ug DNA per sample makes this method an improvement from the current methods. Subsequent chapters describe our efforts to expand this research by considering DNA adducts not only as biomarkers of exposure but as significant to the discovery of chemopreventive agents.

In Chapter 4, we investigate the widespread chemopreventive strategy of activating the cytoprotective protein nuclear factor (erythroid-derived 2)-like 2 (Nrf2) as a potential mechanism for 4-ABP DNA adduct inhibition in bladder cells and tissues. We show that Nrf2 inhibits 4-ABP DNA adduct formation locally in human bladder RT-4 cells. However, in vivo research comparing 4-ABP DNA adduct levels in Nrf2+/+ and Nrf2-/- C57BL/6 mice revealed that in the process of detoxifying 4-ABP and its metabolites from the liver, Nrf2 increased the bioavailability of 4-ABP in the bladder. Subsequent experiments showed that UDP-glucuronosyltransferase (UGT) was at least partly responsible for the detoxification and transport of 4-ABP from the liver to the bladder: Nrf2 up-regulated UGT, promoted conjugation of 4-ABP with glucuronic acid in the liver, and increased urinary excretion of the conjugate. We concluded that Nrf2 activation that doesn't result in up-regulation of liver UGT should be considered and investigated further as a chemopreventive strategy against 4-ABP induced bladder carcinogenesis.

In Chapter 5, two cancer chemopreventive agents Sulforaphane (SF) and 5,6-Dihydrocyclopenta[c][1,2]-dithiole-3(4H)-thione (CPDT) were evaluated for their inhibition of 4-ABP DNA adducts. SF and CPDT, both derived from compounds found in cruciferous vegetables, are well known chemopreventive agents that induce Nrf2 and have been shown to be specific for bladder tissue. We found that SF and CPDT both activate Nrf2 and the Nrf2 cytoprotective signaling pathway in human bladder carcinoma RT-4 cells and C57BL/6 mouse bladder tissue. Both agents inhibit 4-ABP DNA adduct formation in bladder cells and tissues and require Nrf2 to exert substantial cytoprotective effects. Furthermore, neither agent induced UGT liver enzymes that are highly significant in the transport of 4-ABP to the bladder. These results suggest that SF and CPDT are promising chemopreventive agents against 4-ABP-induced bladder carcinogenesis and provide mechanistic insight into their cytoprotective effects.

Future directions of this research are considered in Chapter 6. This includes quantification of the isomers of 4-ABP DNA adducts, a comprehensive human smoke-quit study involving the quantitative monitoring of 4-ABP DNA adducts in urothelial cells over an extended period of time, an analysis of additional metabolites of 4-ABP, and the evaluation of structural analogs of SF and CPDT.

Document Type

Dissertation

Rights Holder

Kristen L. Randall


View Full Text

Click button above to open, or right-click to save.

Share

COinS