This study is the first to demonstrate that UGT2A1 is expressed in a variety of tissues that are target sites for tobacco carcinogenesis and that UGT2A1 exhibits glucuronidation activity against members of the PAH class of carcinogens. This study confirmed previous studies demonstrating that UGT2A1 is expressed in trachea [9
] and for the first time demonstrated expression in other aerodigestive tract tissues including larynx and tonsil. Other PAH-metabolizing UGTs known to be well-expressed in multiple aerodigestive tract tissues include UGTs 1A7 and 1A10 [13
], but the relative level of expression of UGT2A1 versus UGTs 1A7 and 1A10 has not yet been determined. The present study also confirmed previous studies indicating relatively high UGT2A1 expression in lung [9
]. While UGT1A6 was expressed at a higher level in lung in a previous study [9
], this enzyme exhibits limited activity against simple B(a)P metabolites such as 7-OH B(a)P, and no reported activity against more complex activated B(a)P metabolites, such as B(a)P-7,8-diol [25
]. Of the other UGTs that are active against PAHs, UGT1A10 is expressed in lung but at relatively low levels [13
]. Together, these data suggest that UGT2A1 may be important in detoxifying PAHs in multiple aerodigestive tract tissues as well as in lung. UGT2A1 was also found to be expressed in colon, where dietary PAH exposure is a known risk factor for colorectal cancer [29
]. No glucuronidation activity was observed for UGT2A1 against carcinogens other than PAHs, including tobacco-specific nitrosamines or heterocyclic amines. Tobacco-specific nitrosamines and heterocyclic amines are glucuronidated at electrophilic nitrogen moieties, which suggests that UGT2A1 is not an efficient enzyme for N
Functional polymorphisms have been identified in many UGT genes, and several polymorphisms have been shown to significantly alter enzyme activity and impact cancer risk. Three variant protein isoforms exist for UGT1A7 that exhibit differences in enzyme activity against B(a)P metabolites and other substrates as compared to wild-type UGT1A7 [31
]. Low-activity UGT1A7 alleles have been linked to increased risk for hepatic and colorectal cancer [32
], and also have been linked to an increased risk for orolaryngeal cancer in smokers [16
]. A SNP at codon 139 in the UGT1A10 gene has been linked to altered orolaryngeal cancer risk in African Americans [17
]. The UGT2B17 whole-gene deletion polymorphism (UGT2B17*2) has been found to be associated with a gender-specific increased risk for lung adenocarcinoma, with this association likely due to UGT2B17’s glucuronidation activity towards NNAL [14
Results from the present study demonstrate that polymorphic variants in UGT2A1 exhibit decreased glucuronidation activity against PAHs. The UGT2A175Arg308Gly
variant exhibited a ~25% decrease in activity as compared to wild-type UGT2A1 against a variety of PAHs; this modest change in activity is unlikely to have major physiological relevance. -Conversely, the UGT2A175Lys308Arg
variant did not exhibit glucuronidation activity against any substrate tested. The glycine to arginine amino acid change at codon 308 is a non-conservative amino acid change in the C-terminus of the UGT protein, which is the UDPGA-binding region of the enzyme [36
]. Analysis of a crystal structure of the UDPGA-binding domain of UGT2B7 suggests that a glycine residue in UGT2B7, at codon 310, is critical to protein folding to create a UDPGA-binding pocket [37
]. This codon 310 glycine residue is conserved between all UGT2B members and the UGT1A common region, and corresponds to the glycine residue in UGT2A1 codon 308 upon amino acid sequence alignment (). Although the complete crystal structure of UGT2A1 is unknown, there is a high likelihood that UGT2A1 and UGT2B7 have similar UDPGA-binding regions due to high (70%) amino acid homology between these two enzymes [8
]. A recently completed homology model of UGT1A1 also predicts the Gly308 residue in UGT1A1 to be critical for UDPGA binding [38
]. Therefore, the polymorphic non-conservative glycine to arginine change in this highly conserved UGT region could inhibit UDPGA-binding by altering protein folding, thus significantly disrupting UGT activity.
Conservation of codon 308 glycine residue in UGT2A1
The UGT2A1*2 SNP at codon 75 was reported by HapMap to have a low allelic prevalence of 1.1% in Han Chinese individuals and was not observed in other racial groups [15
]. The data presented here suggest that this SNP may have a greater allelic prevalence in an Asian population (~8%) and may also be found in a significant proportion of Caucasians and African Americans (~4% allelic prevalence for both groups). Differences observed between our study and that reported in HapMap may be due to low subject numbers; a larger genotyping study may be warranted to determine the true allelic frequency of the codon 75 SNP in different racial groups. Another non-synonymous coding SNP in UGT2A1, a valine to isoleucine change at codon 391 (rs4148304), is reported to only be expressed in Caucasians, and at a low allelic frequency of 0.8% [15
]. This SNP was not analyzed in our study due to its low allelic frequency, but it may warrant further investigation into its functional effects particularly if the allelic frequency of this SNP is determined to be higher than what is currently published by HapMap.
UGT2A1 and UGT2A2 transcripts are comprised of individual first exons spliced to common exons 2–6 [5
]. The UGT2A1*2 SNP is located in exon 1, making the functional effects of this SNP unique to UGT2A1. The codon 308 SNP encoded by the UGT2A1*3 allele lies within the common region shared by UGT2A1 and 2A2. One study to date has characterized UGT2A2, reporting UGT2A2 to be expressed in the nasal mucosa and to have substrate specificity against simple phenols and estrogen metabolites [7
]. Additional studies analyzing UGT2A2, including a more widespread screen for expression in tobacco target organs and an activity screen against tobacco carcinogens, are necessary to determine UGT2A2’s potential role in tobacco carcinogen metabolism and possible codon 308 SNP-induced effects on UGT2A2 activity.
Together, the in vitro data presented in this study suggest that UGT2A1 may play an important role in PAH metabolism in multiple target organs and that prevalent SNPs within UGT2A1 alter its glucuronidation activity against these substrates. Large case-control studies will be required to examine the potential linkage between these SNPs and cancer risk.