Physical and chemical interactions between bile pigments and polyaromatic mutagens

Abstract

A major cause of cancer in humans is exposure to mutagenic compounds. This raises the question of how humans can be protected from these environmental mutagens. Bile pigments (BPs) such as biliverdin, unconjugated bilirubin and protoporphyrin and their derivatives have recently been found to act as antioxidants and inhibit the mutagenic effects of several known environmental mutagens including 2-aminofluorene, benzo [α]pyrene, and 2-amino-1-methyl-6-phenylimido [4,5-b]pyridine. Despite these promising results, very little is known about the mechanisms by which this inhibition is achieved. Understanding these mechanisms would be useful for future drug development. Therefore, this PhD thesis aims to explore physical and chemical interactions between BPs and mutagens. Effects of BPs on the bioavailability and metabolism of mutagens were also examined in vitro using the colorectal adenocarcinoma (Caco-2 cell) monolayer model and the human liver S9 fraction.The physical interactions between mutagens and BPs were examined using three different methods:NMR, UV and effects of bioavailability. The results of the comparison of the NMR spectra of mutagens in the absence and presence of BPs showed very little changes in the chemical shifts of the protons and the changes that did occur were the result of acid/base interactions between the BPs and mutagens. The UV spectrum of each mutagen was measured in the presence and absence of varying concentrations of BPs, and there were no changes to the UV spectra of any of the compounds. Strong physical interactions or aggregation of compounds can also affect their absorption across cell monolayers and so the apparent permeability of mutagens across Caco-2 cell monolayers in the presence and absence of BPs were measured. The results indicated that BPs increased the permeability of the mutagens slightly and effected how much of the compounds remained in tight association with the monolayer but the effects were small. These experiments provided evidence to suggest that physical interactions and aggregations are unlikely to be a major contributing mechanism of the inhibitory effects of BPs on environmental mutagens.Chemical reactions between BPs and the DNA modifying metabolites of mutagens (epoxides) were studied using styrene epoxide as a model for the reactive metabolites. Styrene epoxide is commercially available, stable and less toxic than the reactive metabolites of the mutagens.Competitive reactions were performed in which BPs and their derivatives were placed in solution with guanine and allowed to react with styrene epoxide. These reactions showed that BPs and their dimethyl esters are more reactive to the epoxide than guanine. Bile pigments primarily react through their carboxylic acid groups with the mono-and di-styrene epoxide esters being the major products isolated form the reactions. The pyrrole rings in bilirubin also showed some evidence of iii reaction with styrene epoxide though the products were too unstable to isolate. Thus, it was clear that BPs can effecti...