Polycyclic aromatic hydrocarbons (PAHs) are potent carcinogens and are a primary risk factor for the development of lung and other aerodigestive tract cancers in smokers. The detoxification of PAHs by glucuronidation is well-characterized for the UDPglycosyltransferase (UGT) 1A, 2A, and 2B subfamilies; however, the role of the UGT3A subfamily in PAH metabolism remains poorly understood. UGT3A enzymes are functionally distinct from other UGT subfamilies (which use UDP-glucuronic acid as a cosubstrate) due to their utilization of alternative cosubstrates (UDP-Nacetylglucosamine for UGT3A1, and UDP-glucose and UDP-xylose for UGT3A2). The goal of the present study was to characterize UGT3A glycosylation activity against PAHs and examine their expression in human aerodigestive tract tissues. In vitro metabolism assays using UGT3A2-overexpressing cell microsomes indicated that UGT3A2 exhibits glycosylation activity against all of the simple and complex PAHs tested. The V max /K m ratios for UGT3A2 activity with UDP-xylose versus UDP-glucose as the cosubstrate ranged from 0.65 to 4.4 for all PAHs tested, demonstrating that PAH glycosylation may be occurring at rates up to 4.4-fold higher with UDP-xylose than with UDP-glucose. Limited glycosylation activity was observed against PAHs with UGT3A1-overexpressing cell microsomes. While UGT3A2 exhibited low levels of hepatic expression, it was shown by western blot analysis to be widely expressed in aerodigestive tract tissues. Conversely, UGT3A1 exhibited the highest expression in liver with lower expression in aerodigestive tract tissues. These data suggest that UGT3A2 plays an important role in the detoxification of PAHs in aerodigestive tract tissues, and that there may be cosubstrate-dependent differences in the detoxification of PAHs by UGT3A2. SIGNIFICANCE STATEMENT UGT3A2 is highly active against PAHs with either UDP-glucose or UDP-xylose as a cosubstrate. UGT3A1 exhibited low levels of activity against PAHs. UGT3A1 is highly expressed in liver while UGT3A2 is well expressed in extrahepatic tissues. UGT3A2 may be an important detoxifier of PAHs in humans.

Women at high risk of developing breast cancer are prescribed selective estrogen response modulators, including raloxifene, as chemoprevention. Patients often seek complementary and alternative treatment modalities, including herbal products, to supplement prescribed medications. Milk thistle preparations, including silibinin and silymarin, are top-selling herbal products that may be consumed by women taking raloxifene, which undergoes extensive first-pass glucuronidation in the intestine. Key constituents in milk thistle, flavonolignans, were previously shown to be potent inhibitors of intestinal UDP-glucuronosyl transferases (UGTs), with IC 50 s £ 10 mM. Taken together, milk thistle preparations may perpetrate unwanted interactions with raloxifene. The objective of this work was to evaluate the inhibitory effects of individual milk thistle constituents on the intestinal glucuronidation of raloxifene using human intestinal microsomes and human embryonic kidney cell lysates overexpressing UGT1A1, UGT1A8, and UGT1A10, isoforms highly expressed in the intestine that are critical to raloxifene clearance. The flavonolignans silybin A and silybin B were potent inhibitors of both raloxifene 49-and 6-glucuronidation in all enzyme systems. The K i s (human intestinal microsomes, 27-66 mM; UGT1A1, 3.2-8.3 mM; UGT1A8, 19-73 mM; and UGT1A10, 65-120 mM) encompassed reported intestinal tissue concentrations (20-310 mM), prompting prediction of clinical interaction risk using a mechanistic static model. Silibinin and silymarin were predicted to increase raloxifene systemic exposure by 4-to 5-fold, indicating high interaction risk that merits further evaluation. This systematic investigation of the potential interaction between a widely used herbal product and chemopreventive agent underscores the importance of understanding natural product-drug interactions in the context of cancer prevention.

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The UDP-glycosyltransferase (UGT) family of enzymes are important in the metabolism of a variety of exogenous substances including polycyclic aromatic hydrocarbons (PAHs), a potent class of environmental carcinogens. As compared to the majority of UGT enzymes, which utilize UDP-glucuronic acid as a cosubstrate, UGT3A2 utilizes alternative cosubstrates (UDP-glucose and UDP-xylose). UGT3A2 is expressed in aerodigestive tract tissues and was highly active against multiple PAHs with both cosubstrates. The goal of the present study was to assess the functional effects of UGT3A2 missense variants (MAF ≥ 0.005) on PAH metabolism and the utilization of cosubstrates. The glycosylation activity (V max/K m) of all variants against simple PAHs using both cosubstrates was significantly (P < 0.05) decreased by 42–100% when compared to wild-type UGT3A2. When utilizing UDP-glucose, the variant isoforms exhibited up to a 362-fold decrease in V max/K m when compared to wild-type UGT3A2, with a 3.1- to 14-fold decrease for D140N, A344T, and S435Y, a 24- and 43-fold decrease for A436T and R445C, respectively, and a 147- and 362-fold decrease for Y474C and Y74N, respectively. When utilizing UDP-xylose, the variants exhibited up to a 4.0-fold decrease in V max/K m when compared to wild-type UGT3A2; Y74N did not exhibit activity, and Y474C did not reach saturation (K m > 4000 μM). Additionally, both wild-type and variant UGT3A2 exhibited a significant (P < 0.05) difference in their utilization of UDP-glucose vs UDP-xylose as cosubstrates using 1-OH-pyrene as substrate. These data suggest that UGT3A2 missense variants decrease the detoxification of PAHs, potentially resulting in altered individual risk for PAH-related cancers.