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 reaction with styrene epoxide though the products were too unstable to isolate. Thus, it was clear that BPs can effectively scavenge reactive metabolites, but the free carboxylic acids were significantly more effective at this than the dimethyl ester derivatives. This is not reflected in the anti-mutagenic activities of the compounds. Also, the ubiquitous nature of carboxylic acid groups in the cellular environment makes it unlikely that this reaction with activated epoxides would be unique to BPs. For these reasons we concluded that it is unlikely that chemical scavenging of reactive metabolites is the sole or even major mechanism of the inhibition of BPs.
Another possible mechanism of action of the BPs is that they inhibit the formation of the DNA modifying metabolites of the mutagens. We investigated this by performing in vitro experiments in which mutagens were co-incubated in the human liver S9 fraction in the presence and absence of BPs. The results indicated BPs were inhibitors of the metabolism of benzo[a]pyrene and 2-amino-1-methyl-6-phenylimido[4,5-b]pyridine by liver enzymes. The order of inhibitory effectiveness was bilirubin > protoporphyrin > biliverdin. Molecular modelling studies which examined the docking of the various BPs into the active sites of published crystal structures of the enzymes known to be responsible for the metabolism of the mutagens, suggested BPs could bind to the active sites of CYP1A1, 1A2, 1B1 and 3A4.
In summary, we conducted a series of experiments to evaluate the likely mechanisms of the inhibitory effects of BPs on known environmental mutagens. There are several theories postulated to explain the anti-mutagenic effects of BPs including the physical -stacking driven aggregation of BPs with the polyaromatic mutagens, the chemical scavenging of BPs towards reactive metabolites, and the inhibition of BPs of the P450 mediated activation of the mutagens. We have systematically tested each of these and found that the latter appears to be the most likely mechanism to explain the effects reported. In broader terms, this research will aid in understanding how BPs inhibit mutagenesis and thus may lead to the development of synthetic compounds that could decrease the risk to humans exposed to these environmental mutagens.