Abstract 1698: Characterization of the activity and expression of UDP-Glucuronosyltransferase 2A1 variants and potential role in tobacco carcinogenesis

Abstract

Abstract UDP-glucuronosyltransferases (UGTs) play an important role in the metabolism and excretion of various endogenous and xenobiotic compounds including several carcinogens and chemotherapeutic agents. UGT2A1 is an extra-hepatic member of the UGT2A sub-family that was shown to be expressed in the lung in previous studies. The goal of the present study was to examine UGT2A1 expression in human tissues, assess its glucuronidation activity against exogenous substrates including lung carcinogens, and determine the potential functional role of UGT2A1 polymorphisms on UGT2A1 enzyme activity. Cell homogenates were prepared from HEK293 cells transfected with wild type or variant UGT2A1. Enzyme activity against carcinogens was determined through reverse-phase UPLC. Expression of UGT2A1 was determined by RT-PCR/sequencing, real time PCR (ABI), and western blot using a UGT2A1-specific antibody. In addition to the wild-type UGT2A1*1, two prevalent variants of UGT2A1 were investigated: a variant that causes a lysine to arginine amino acid change at codon 75 (UGT2A1*2), and a variant that causes a glycine to arginine amino acid change at codon 308 (UGT2A1*3). In addition, a newly-identified splice variant isoform lacking exon 3 (allele identified as UGT2A1*1a since it was not yet determined whether this variant was due to a genetic polymorphism or to altered splicing) was also investigated. As determined by RT-PCR and direct sequencing, UGT2A1 was expressed in lung, trachea, larynx, tonsil, mouth, and colon; no expression was observed in breast, whole brain, cerebral cortex or esophageal tissues. Reverse transcription-PCR demonstrated that tissues had variable levels of expression of UGT2A1 and the splice variant encoded by UGT2A1*1a; the UGT2A1*1a encoded variant was expressed in 5 of 6 individual lung specimens and a pooled larynx sample from 10 people. Homogenates of HEK293 cells over-expressing UGT2A1*1 or UGT2A1*2 both exhibited significant activity against 1-naphthol, with a Vmax/KM of 4.0 uL.min−1.ug homogenate protein−1 and 3.5 uL.min−1.ug homogenate protein−1 respectively. No activity was observed for cell homogenates over-expressing the UGT2A1*3 variant or the UGT2A1*1a isoform. A similar pattern of activity was observed for a variety of polycyclic aromatic hydrocarbons (PAHs) including 1-hydroxypyrene, dibenzo(a,l)pyrene-11,12-diol, benzo(a)pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 1-hydroxy (OH)-benzo(a)pyrene, 7-OH-benzo(a)pyrene, and 8-OH-benzo(a)pyrene. No activity was observed against substrates that form N-glucuronides such as NNAL, nicotine or N-OH-PhIP. UGT2A1 is expressed in various target organs for tobacco-induced carcinogenesis and exhibits activity against a variety of PAHs abundant in tobacco smoke. The UGT2A1308Arg variant could play an important role in tobacco-related cancer risk due to a lack of enzyme activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1698.