Characterization of UDP-glucuronosyltransferase 2A1 (UGT2A1) variants and their potential role in tobacco carcinogenesis.

OBJECTIVE: To examine UGT2A1 expression in human tissues, determine its glucuronidation activity against tobacco carcinogens, and assess the potential functional role of UGT2A1 missense single nucleotide polymorphisms on UGT2A1 enzyme activity. METHODS: Reverse transcription polymerase chain reaction and real time polymerase chain reaction were used to assess UGT2A1 gene expression in various human tissues. A glucuronidation assay measured by reverse phase ultra-performance liquid chromatography was used to determine UGT2A1 activity. RESULTS: UGT2A1 was expressed in aerodigestive tract tissues including trachea, larynx, tonsil, lung, and colon; no expression was observed in breast, whole brain, pancreas, prostate, kidney, liver, or esophagus. UGT2A1 exhibited highest expression in the lung, followed by trachea >tonsil >larynx >colon >olfactory tissue. Cell homogenates prepared from wildtype UGT2A1(75Lys308Gly) overexpressing HEK293 cells showed significant glucuronidation activity against a variety of polycyclic aromatic hydrocarbons including, 1-hydroxy-benzo(a)pyrene, benzo(a)pyrene-7,8-diol, and 5-methylchrysene-1,2-diol. No activity was observed in UGT2A1 overexpressing cell homogenate against substrates that form N-glucuronides, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), nicotine, or N-OH-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (N-OH PhIP). A significant (P<0.05) decrease (approximately 25%) in glucuronidation activity (Vmax/KM) was observed against all polycyclic aromatic hydrocarbons substrates for the UGT2A1(75Lys308Gly) variant compared with homogenates from wildtype UGT2A1(75Lys308Gly); no activity was observed for cell homogenates overexpressing the UGT2A1 variant for all substrates tested. CONCLUSION: These data suggest that UGT2A1 is an important detoxification enzyme in the metabolism of polycyclic aromatic hydrocarbons within target tissues for tobacco carcinogens and functional polymorphisms in UGT2A1 may play a role in tobacco-related cancer risk.

Potential role of UGT pharmacogenetics in cancer treatment and prevention: focus on tamoxifen.

Tamoxifen (TAM) is a selective estrogen receptor modulator that is widely used in the prevention and treatment of estrogen receptor-positive (ER(+)) breast cancer. Its use has significantly contributed to a decline in breast cancer mortality, since breast cancer patients treated with TAM for 5 years exhibit a 30-50% reduction in both the rate of disease recurrence after 10 years of patient follow-up and occurrence of contralateral breast cancer. However, in patients treated with TAM there is substantial interindividual variability in the development of resistance to TAM therapy, and in the incidence of TAM-induced adverse events, including deep vein thrombosis, hot flashes, and the development of endometrial cancer. This article will focus on the UDP glucuronosyltransferases, a family of metabolizing enzymes that are responsible for the deactivation and clearance of TAM and TAM metabolites, and how interindividual differences in these enzymes may play a role in patient response to TAM.

Functional significance of UDP-glucuronosyltransferase variants in the metabolism of active tamoxifen metabolites.

Tamoxifen (TAM) is a selective estrogen receptor modulator widely used in the prevention and treatment of breast cancer. A major mode of metabolism of the major active metabolites of TAM, 4-OH-TAM and endoxifen, is by glucuronidation via the UDP-glucuronosyltransferase (UGT) family of enzymes. To examine whether polymorphisms in the UGT enzymes responsible for the glucuronidation of active TAM metabolites play an important role in interindividual differences in TAM metabolism, cell lines overexpressing wild-type or variant UGTs were examined for their activities against TAM metabolites in vitro. For variants of active extrahepatic UGTs, the UGT1A8(173Ala/277Tyr) variant exhibited no detectable glucuronidation activity against the trans isomers of either 4-OH-TAM or endoxifen. Little or no difference in TAM glucuronidating activity was observed for the UGT1A8(173Gly/277Cys) or UGT1A10(139Lys) variants compared with their wild-type counterparts. For active hepatic UGTs, the UGT2B7(268Tyr) variant exhibited significant (P < 0.01) 2- and 5-fold decreases in activity against the trans isomers of 4-OH-TAM and endoxifen, respectively, compared with wild-type UGT2B7(268His). In studies of 111 human liver microsomal specimens, the rate of O-glucuronidation against trans-4-OH-TAM and trans-endoxifen was 28% (P < 0.001) and 27% (P = 0.002) lower, respectively, in individuals homozygous for the UGT2B7 Tyr(268)Tyr genotype compared with subjects with the UGT2B7 His(268)His genotype, with a significant (P < 0.01) trend of decreasing activity against both substrates with increasing numbers of the UGT2B7(268His) allele. These results suggest that functional polymorphisms in TAM-metabolizing UGTs, including UGT2B7 and potentially UGT1A8, may be important in interindividual variability in TAM metabolism and response to TAM therapy.

Glucuronidation of nicotine and cotinine by UGT2B10: loss of function by the UGT2B10 Codon 67 (Asp>Tyr) polymorphism.

Nicotine, the major addicting agent in tobacco and tobacco smoke, undergoes a complex metabolic pathway, with approximately 22% of nicotine urinary metabolites in the form of phase II N-glucuronidated compounds. Recent studies have shown that UGT2B10 is a major enzyme involved in the N-glucuronidation of several tobacco-specific nitrosamines. In the present study, microsomes of UGT2B10-overexpressing HEK293 cells exhibited high N-glucuronidation activity against both nicotine and cotinine with apparent KM's that were 37- and 3-fold lower than that observed for microsomes of UGT1A4-overexpressing cells against nicotine and cotinine, respectively. The KM of microsomes from wild-type (WT) UGT2B10-overexpressing cells for nicotine and cotinine was similar to that observed for human liver microsomes (HLM) against both substrates. The level of glucuronidated nicotine or cotinine in 112 HLM samples was correlated with UGT2B10 genotype; the levels of nicotine- and cotinine-glucuronide were 21% to 30% lower in specimens from subjects with the UGT2B10 (*1/*2) genotype compared with specimens from subjects with the WT UGT2B10 (*1/*1) genotype; a 5- and 16-fold lower level of nicotine- and cotinine-glucuronide formation, respectively, was observed in HLM from subjects with the UGT2B10 (*2/*2) genotype. In contrast to the relatively high activity observed for cells overexpressing WT UGT2B10 in vitro, little or no glucuronidation was observed for microsomes from cells overexpressing the UGT2B10*2 variant against either nicotine or cotinine. These data suggest that UGT2B10 is the major hepatic enzyme involved in nicotine/cotinine glucuronidation and that the UGT2B10*2 variant significantly reduces nicotine- and cotinine-N-glucuronidation formation and plays an important role in nicotine metabolism and elimination.

Glucuronidation of active tamoxifen metabolites by the human UDP glucuronosyltransferases.

Tamoxifen (TAM) is an antiestrogen that has been widely used in the treatment and prevention of breast cancer in women. One of the major mechanisms of metabolism and elimination of TAM and its major active metabolites 4-hydroxytamoxifen (4-OH-TAM) and 4-OH-N-desmethyl-TAM (endoxifen; 4-hydroxy-N-desmethyl-tamoxifen) is via glucuronidation. Although limited studies have been performed characterizing the glucuronidation of 4-OH-TAM, no studies have been performed on endoxifen. In the present study, characterization of the glucuronidating activities of human UDP glucuronosyltransferases (UGTs) against isomers of 4-OH-TAM and endoxifen was performed. Using homogenates of individual UGT-overexpressing cell lines, UGTs 2B7 approximately 1A8 > UGT1A10 exhibited the highest overall O-glucuronidating activity against trans-4-OH-TAM as determined by Vmax/K(M), with the hepatic enzyme UGT2B7 exhibiting the highest binding affinity and lowest K(M) (3.7 microM). As determined by Vmax/K(M), UGT1A10 exhibited the highest overall O-glucuronidating activity against cis-4-OH-TAM, 10-fold higher than the next-most active UGTs 1A1 and 2B7, but with UGT1A7 exhibiting the lowest K(M). Although both N- and O-glucuronidation occurred for 4-OH-TAM in human liver microsomes, only O-glucuronidating activity was observed for endoxifen; no endoxifen-N-glucuronidation was observed for any UGT tested. UGTs 1A10 approximately 1A8 > UGT2B7 exhibited the highest overall glucuronidating activities as determined by Vmax/K(M) for trans-endoxifen, with the extrahepatic enzyme UGT1A10 exhibiting the highest binding affinity and lowest K(M) (39.9 microM). Similar to that observed for cis-4-OH-TAM, UGT1A10 also exhibited the highest activity for cis-endoxifen. These data suggest that several UGTs, including UGTs 1A10, 2B7, and 1A8 play an important role in the metabolism of 4-OH-TAM and endoxifen.

Glucuronidation of PhIP and N-OH-PhIP by UDP-glucuronosyltransferase 1A10.

The UDP-glucuronosyltransferase (UGT) 1A10 is an extra-hepatic enzyme that plays an important role in the glucuronidation of a variety of endogenous and exogenous substances and is expressed throughout the aerodigestive and digestive tracts. Two classes of carcinogens that target the colon, heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons, are known to be detoxified by the UGT family of enzymes. Recently, our laboratory demonstrated that UGT1A10 has considerably more activity against polycyclic aromatic hydrocarbons in vitro than any other UGT family member. In this study, we focused on the glucuronidation of the HCA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and its bioactivated metabolite, N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP). We demonstrated that UGT1A10 exhibited a significantly higher glucuronidation rate against PhIP and N-OH-PhIP than any other UGT family member in vitro using whole-cell homogenates of HEK293 cells over-expressing individual UGTs. Kinetic analysis revealed a 9- and 22-fold higher level of activity for UGT1A10 homogenates as compared with the next most active UGT, UGT1A1, against N-OH-PhIP as determined by maximum rate/apparent Michaelis constant (V(max)/K(M)) at the N3 and N2 positions, respectively. The polymorphic UGT1A10(139Lys) variant exhibited a 2- to 16-fold decrease in glucuronidation activity against PhIP and N-OH-PhIP, as compared with the wild-type UGT1A10(139Glu) isoform. These data suggest that UGT1A10 is the most active UGT against PhIP and N-OH-PhIP and that UGT1A10 may play an important role in susceptibility to HCA-induced colon cancer.