Role of the Uridine Diphosphate Glucuronosyltransferase 2a Family in Tobacco Carcinogen Metabolism

Open Access
- Author:
- Bushey, Ryan Taylor
- Graduate Program:
- Pharmacology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 18, 2012
- Committee Members:
- Philip Lazarus, Dissertation Advisor/Co-Advisor
Shantu G Amin, Committee Member
John Ellis, Committee Member
Melvin Lee Billingsley, Committee Member
Thomas E Spratt, Committee Member - Keywords:
- UDP-glucuronosyltransferase
tobacco carcinogen metabolism
single nucleotide polymorphism
alternate splicing
UGT regulation - Abstract:
- Tobacco use is considered the most preventable cause of death in the world today, with tobacco-related cancers causing millions of deaths annually. Environmental and genetic factors are known to impact cancer susceptibility, and using environmental exposure and/or genetic information to identify a subset of individuals at high-risk for developing tobacco-related cancers has the potential to save many lives. Inter-individual differences in enzymes that activate and metabolize carcinogens are thought to influence cancer risk. UDP-glucuronosyltransferases (UGTs) are phase II detoxifying enzymes that play a critical role in the metabolism of endogenous and exogenous compounds, including multiple classes of tobacco carcinogens. The effects of coding and non-coding SNPs on UGT activity have been analyzed for many UGT isoforms, and multiple UGT variants have been determined to be significantly associated with cancer risk. The entire UGT2A family has been neglected in prior research studies, with UGT2A tissue expression and enzyme activities relatively unknown. With recent reports suggesting UGT2A1 expression in the lung and trachea and UGT2A1 glucuronidation activity against simple polycyclic aromatic hydrocarbon (PAH) substrates, the overall hypothesis of this research project was that UGT2A1 detoxifies PAH carcinogens in target organs for tobacco carcinogenesis. Due to the sequence homology between all UGT2A enzymes, and with little information reported on UGT2A2 or UGT2A3, we also hypothesized that UGT2A2 and UGT2A3 enzymes are involved in extra-hepatic tobacco carcinogen metabolism. An initial set of experiments was completed to characterize the role of UGT2A1 in tobacco carcinogen metabolism. Quantitative real-time PCR showed highest relative UGT2A1 expression in the lung, followed by trachea > tonsil > larynx > colon. Significant UGT2A1 glucuronidation activity was observed against a variety of PAHs, including the proximate carcinogens benzo(a)pyrene(B(a)P)-7,8-diol, dibenzo(a,l)pyrene-11,12-diol, and 5-methylchrysene-1,2-diol. No UGT2A1 glucuronidation activity was observed against additional classes of tobacco carcinogens, including tobacco specific nitrosamines or heterocyclic amines. In vitro experiments suggested that UGT2A1 over-expression in a HEK293 cell system prevents B(a)P-mediated cytotoxicity and covalent binding. These data suggested that UGT2A1 is an important detoxification enzyme in the metabolism of PAHs within aerodigestive and respiratory tract tissues. The next set of experiments focused on characterizing two prevalent UGT2A1 non-synonymous coding SNPs, the UGT2A175Lys and UGT2A1308Arg variants. The UGT2A175Arg variant exhibited a significant (p<0.05) ~25% decrease in glucuronidation activity (Vmax/KM) against all PAH substrates examined compared to wild-type UGT2A175Lys activity, while no detectable glucuronidation activity was observed for the UGT2A1308Arg variant against all substrates examined. Results from a lung cancer case-control study showed the inactive UGT2A1308Arg variant to be significantly associated with lung non-small cell carcinoma (p=0.04) and lung squamous cell carcinoma risk (p=0.02). A significant decrease (p<0.001) in wild-type UGT2A1 activity against multiple PAH substrates was observed following UGT2A1308Arg co-expression with wild-type UGT2A1 at approximately a 1:1 ratio. Co-immunoprecipitation experiments showed dimerization between wild-type UGT2A1 and UGT2A1308Arg. The decrease in wild-type UGT2A1 activity following UGT2A1308Arg co-expression is a novel regulatory mechanism for UGT2A1 and may have implications on cancer risk. In the initial cloning of UGT2A1 from human lung RNA a novel UGT2A1 exon 3 deletion splice variant was identified (termed UGT2A1Δexon3), and subsequent studies were completed to characterize the expression and function UGT2A1Δexon3 and the corresponding UGT2A1_i2 protein. Through qualitative PCR, UGT2A1Δexon3 was shown to be expressed in various tissues including lung, trachea, larynx, tonsil, and colon. The ratio of UGT2A1Δexon3:wild-type UGT2A1 expression was highest in colon (0.79 ± 0.08) and lung (0.42 ± 0.12). An antibody specific to UGT2A1 revealed the ratio of UGT2A1_i2:UGT2A1_i1 protein expression in lung and colon homogenates to be 0.5-0.9. UGT2A1_i2 exhibited no glucuronidation activity against a variety of substrates, including PAHs such as 1-hydroxy-pyrene and B(a)P-7,8-diol. An inducible in vitro system was created to determine the effect of UGT2A1_i2 co-expression on wild-type UGT2A1_i1 activity. Increasing UGT2A1_i2 levels resulted in a significant (p<0.01) decrease in UGT2A1_i1 activity (Vmax) against 1-hydroxy-pyrene, 3-OH-B(a)P and B(a)P-7,8-diol. Co-IP experiments suggested the formation of UGT2A1_i1 and UGT2A1_i2 hetero-oligomeric complexes and UGT2A1_i1 homo-oligomeric complexes. These data suggested that a novel exon 3 deletion UGT2A1 splice variant specifically regulates UGT2A1-mediated glucuronidation activity via protein-protein interactions, and that expression of this variant could impact the local detoxification of carcinogens. The final set of experiments described in this dissertation focused on determining the functions of UGT2A2 and UGT2A3 in the local metabolism of tobacco carcinogens. UGT2A2 was determined to be expressed in the trachea and larynx, and a novel splice variant of UGT2A2 lacking exon 3 (UGT2A2Δexon3) was identified. UGT2A3 was determined to be expressed a variety of extra-hepatic tissues, with UGT2A3 well expressed in the colon, lung, tonsil, trachea, and larynx. Cell homogenates prepared from HEK293 cells over-expressing UGT2A2Δexon3 had no detectable glucuronidation activity against all substrates examined. Cell homogenates prepared from HEK293 cells over-expressing UGT2A2 and UGT2A3 showed activity against simple PAHs. Both UGT2A2 and UGT2A3 were determined to have no detectable activity against complex PAH proximate carcinogens, tobacco specific nitrosamines, or heterocyclic amines. Data presented here suggested UGT2A2 and UGT2A3 are both expressed in various aerodigestive and respiratory tract tissues; however, these enzymes lack enzyme activity against PAH proximate carcinogens. This dissertation has laid the groundwork for understanding the physiological role of UGT2A enzymes in the local detoxification of PAH tobacco carcinogens. Results presented in this dissertation suggest that UGT2A1 is a major metabolizer of PAH carcinogens in the lung and other target tissues for tobacco carcinogenesis. UGT2A1 coding SNPs and a novel UGT2A1Δexon3 splice variant were characterized for the first time in this study. Results presented here suggest that the UGT2A1308Arg variant is associated with increased lung cancer risk, and we propose that inter-individual variability in UGT2A1Δexon3 expression may also impact cancer risk. Although additional work is needed to confirm these findings, results presented in this dissertation suggest that UGT2A1 variants negatively regulate wild-type UGT2A1 activity and may play a role in tobacco-related cancer susceptibility.