Flavonoid metabolism: Induction of UDP-glucuronosyltransferase and its potential role in inactivation of carcinogens

Flavonoids, polyphenolic compounds, are abundant in the diet. Animal studies in vivo and in vitro data demonstrated that flavonoids could play a protective role in cancer through inhibition of key enzymes, such as CYP1A1/1A2 and SULT1A1 both involved in the initiation of cancer, and inhibition of pathways involved in cell proliferation. The beneficial effects of flavonoids in humans could be limited by poor absorption and extensive presystemic metabolism. This dissertation describes the pathways involved in metabolism of one such flavonoid, chrysin (5,7-dihydroxyflavone), by cultured intestinal Caco-2 cells and hepatic Hep G2 cells. In addition, we examined the inducing effect of chrysin on UDP-glucuronosyltransferases (UGTs) in Caco-2 cells. The general hypothesis of this dissertation is that dietary flavonoids are extensively metabolized in the human intestine and act as inducers of UGTs, increasing the metabolic clearance of colon carcinogens.; At concentrations that may be achieved from the diet, chrysin was extensively metabolized in both Caco-2 and Hep G2 cells to two conjugated metabolites, M1 and M2, with no CYP-mediated oxidation. M1 was identified as a glucuronide conjugate, and M2 as a sulfate conjugate by liquid chromatography/mass spectrometry (LC/MS) and other spectroscopic and biochemical techniques. Pretreatment of cells with 3-methylcholantherene (3-MC) did not result in oxidation of chrysin but rather increased glucuronidation. M1 was catalyzed by UGT1A6 with a K m of 12 muM. M2 was catalyzed by M- and P-form phenolsulfotransferases (SULT1A3 and SULT1A1) with very low Km values of 3.1 and 0.05 muM, respectively. Also, M1 and M2 were the only metabolites formed from chrysin with fresh rat hepatocytes. These observations suggest that both sulfation and glucuronidation are critical determinants of the oral bioavailability in humans.; Exposure of Caco-2 cells to 50 muM chrysin resulted in a 3.8-fold increase in chrysin glucuronidation in the intact cells (p < 0.0001) with a 38% decrease in sulfation (p < 0.01). In cell homogenates prepared from chrysin treated cells glucuronidation was increased by 14-fold. The induction was slow to develop with maximum induction after 3--4 days of pretreatment. Immunoblot analysis of Caco-2 cell microsomes with a UGT1A subfamily-selective antibody showed a markedly increased band at about 59 kDa, consistent with induction of one or more UGT1A isoforms.; Using isoform specific antibodies we demonstrated that pretreatment of Caco-2 cells with chrysin resulted in a potent induction of UGT1A1. Chrysin pretreatment did not affect the expression of UGT1A6 or UGT2B7. Northern blot analysis showed markedly increased expression of UGT1A1 mRNA after chrysin treatment. In addition, we showed that UGT1A1 glucuronidated a potent colon carcinogen 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP). The induction of UGT1A1 by chrysin in Caco-2 cells resulted in a 10-fold increase in the glucuronidation of N-hydroxy-PhIP.; Based on these results, we conclude that dietary flavonoid-mediated induction of intestinal UGT1A1 may be important for the glucuronidation and detoxification of colon carcinogens and other toxic chemicals as well as therapeutic drugs.