UDP-Glycosyltransferase 3A Metabolism of Polycyclic Aromatic Hydrocarbons: Potential Importance in Aerodigestive Tract Tissues

Abstract

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.

Benzo[a]pyrene and Caenorhabditis elegans: defining the genotoxic potential in an organism lacking the classical CYP1A1 pathway

“…There are at least 72 genes that code for UGT-like proteins in C. elegans (Lindblom and Dodd 2006 ), 16 of which were shown to be up-regulated by BaP exposure. In humans, UGTs have been identified to play a role in the BaP detoxification process, which implies that nematode UGTs may have a similar function (Dellinger et al 2006 ; Zhang et al 2013a ; Vergara et al 2020 ).…”

Altered Metabolism of Polycyclic Aromatic Hydrocarbons by UDP-Glycosyltransferase 3A2 Missense Variants

“…Given that wt UGT3A2 activity is known to be significantly lower against complex PAHs, single point activity assays were used instead of kinetic analysis to determine the effects of UGT3A2 variants on glycosylation activity against B(a)P-7,8-diol. 11 For UDP-glucose, the rate for D140N, A344T, and S435Y variants decreased by 87% to 97% when compared to wt UGT3A2 (Figure S2, panel A). Similar results were observed when using UDP-xylose as the cosubstrate, with the rate for D140N, A344T, and S435Y variants decreased by 50% and 83% when compared to wt UGT3A2 (Figure S2, panel B).…”

“…Whole cell homogenates and microsomal fractions were prepared from UGT3A2-overexpressing cell lines through differential centrifugation, utilizing methods described previously. 11 Western blot analysis was performed using 20 μg of total microsomal protein using a 10% SDS-polyacrylamide gel and subsequent transfer to an Invitrolon PVDF membrane. The membrane was blocked with a 5% solution of ChromatoPur bovine albumin in TBST and probed with goat polyclonal UGT3A2 antibody (1:1000 dilution) followed by a donkey antigoat secondary antibody (1:2500 dilution).…”

“…Glycosylation assays using microsomes from the UGT3A2 missense variants overexpressed in the HEK293 cell line were performed with alternative cosubstrates as described previously for wt UGT3A2. 11 Briefly, pooled cell microsomes (total protein 0.26 to 54 μg) were incubated with alamethicin (50 μg/mg total protein) for 15 min on ice. Glycosylation reactions for the simple PAHs [1-OH-pyrene, 1-OH-B(a)P, 3-OH-B(a)P, 7-OH-B(a)P, and 9-OH-B(a)P] were performed in a final reaction volume of 25 μL at 37 °C containing 50 mM Tris-HCl (pH 7.4), 10 mM MgCl 2 , and 4 mM UDP-glucose or UDP-xylose.…”

“…UGT3A2 was shown to exhibit high affinity against PAHs, as compared to other UGTs. 11 Therefore, it is important to investigate the role genetic variations may have in altering the detoxification of PAHs from the human body. The goal of the present study was to assess the functional effect of UGT3A2 missense variants in the metabolism of PAHs and determine the utilization of the alternative cosubstrates (UDP-glucose and UDP-xylose) by wild-type (wt) vs UGT3A2 variants.…”