Detoxification of Polycyclic Aromatic Hydrocarbons (PAHs) by UDP‐glycosyltransferase 3A2 (UGT3A2) Variants using Alternative Sugars

Abstract

The UDP‐glycosyltransferase (UGT) family of enzymes detoxify endogenous and exogenous substances by conjugating lipophilic chemicals with a UDP‐sugar, thereby making them more hydrophilic, water‐soluble and ultimately more readily excreted from the body. Examples of such chemicals are steroids, drugs, and a variety of carcinogens including polycyclic aromatic hydrocarbons (PAHs), a class of procarcinogenic chemicals produced by incomplete combustion such as during tobacco smoking. The UGT1A/2A/2B subfamily of enzymes are known to detoxify PAHs; however, the UGT3A subfamily has been understudied in the detoxification of tobacco carcinogens, although they have been shown to detoxify 1‐hydroxypyrene (1‐HP), a PAH metabolite. Additionally, the UGT3A subfamily also uses different sugars than the UGT1A and UGT2 enzymes, which use UDP‐glucuronic acid. The objective of the present study is to characterize the UGT3A2‐mediated detoxification of PAHs using the alternative sugars UDP‐glucose and UDP‐xylose and to assess the potential functional role of UGT3A variants in tobacco‐related cancer susceptibility. Microsomes prepared from UGT3A2‐overexpressing HEK293 cells exhibited significant glycosylation activity against a variety of PAHs including 1‐HP, 1‐hydroxybenzo(a)pyrene (1‐OH‐B(a)P), 3‐OH‐B(a)P, 7‐OH‐B(a)P, 8‐OH‐B(a)P, 9‐OH‐B( a)P, benzo(a)pyrene‐7,8‐diol, and dibenzo(a,l)pyrene‐11,12‐diol, using both UDP‐glucose and UDP‐xylose as the co‐substrate. Using UDP‐glucose, UGT3A2 exhibited Km ‘s ranging from 3.3 – 11.8 μM for 1‐HP, 1‐OH‐B(a)P, 3‐OH‐B(a)P, 7‐OH‐B(a)P, and 9‐OH‐B(a)P. The Km's were comparable when using UDP‐xylose, with Km's ranging from 1.2 – 9.6 μM for the same substrates. The Vmax/Km ratio of UDP‐xylose/UDP‐glucose ranged from 1.0 – 2.1, demonstrating that PAH glycosylation may be occurring at rates up to two‐fold higher with UDP‐xylose as the co‐substrate. To assess the potential functional role of co‐substrate selectivity for UGT3A2 genetic variants (MAF ≥ 0.5%), site‐directed mutagenesis was performed on the wild‐type UGT3A2 plasmid to create seven variants that were also overexpressed in HEK293 cells. Microsomes from UGT3A2D140N overexpressing cells exhibited up to a two‐fold higher level of glycosylation activity with UDP‐xylose than UDP‐glucose. All variants had decreased glycosylation activity (Vmax/Km) than microsomes from wild‐type UGT3A2‐overexpressing cells. These data suggest there may be differences in co‐substrate‐dependent detoxification of PAHs by UGT3A2 and UGT3A2 variants may play a role in lung and other tobacco‐related cancers.Support or Funding InformationThis study is supported by the National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health to P Lazarus (Grant R01‐ES025460), a NIEHS Research Supplement to Promote Diversity in Health‐Related Research to AG Vergara (Grant R01‐ES025460‐02S1), and a grant from the Health Sciences and Services Authority of Spokane, WA (Grant WSU002292).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.