Importance of Udp-glucuronosyltransferase 1a10 (ugt1a10) in the Detoxification of Polycyclic Aromatic Hydrocarbons: Decreased Glucuronidative Activity of the Ugt1a10 Isoform

UDP-glucuronosyltransferase 1A10 (UGT1A10) is an extrahepatic enzyme expressed in aerodigestive tract tissues that exhibits significant glucuronidation activity against the important procarcinogenic benzo(a)pyrene (BaP) metabolite, BaP-trans-7,8-dihydrodiol (BPD), and the UGT1A10 codon 139 (Glu>Lys) polymorphism was previously implicated in risk for orolaryngeal cancer by Elahi et al. in their 2003 study. To better assess the potential role of UGT1A10 in risk for tobacco-related cancers, the glucuronidation activity of UGT1A10 was compared with that of other known UGT enzymes against selected polycyclic aromatic hydrocarbons, and the effects of the codon 139 polymorphism on UGT1A10 function were examined in vitro. UGT1A10 exhibited considerably more glucuronidation activity as determined by Vmax/Km against 3-hydroxy (OH)-BaP, 7-OH-BaP, 9-OH-BaP, and 1-OH-pyrene than any other UGT1A family member. Although a kinetic comparison using Vmax could not be performed against family 2B UGTs, UGT1A10 exhibited a 1.7to 254-fold lower Km than active family 2B UGTs against 3-OH-BaP, 7-OH-BaP, and 1-OH-pyrene. A significantly (p < 0.01) higher Vmax/Km was observed for homogenates from wild-type UGT1A10-overexpressing cells against all four BaP metabolites tested (3-OH-BaP, 7-OH-BaP, 9-OH-BaP, and BPD). A similarly significant (p < 0.05) increase in Vmax/Km was observed for homogenates from wild-type UGT1A10-overexpressing cells against 1-OH-pyrene. Significant differences in Km were observed for homogenates from wild-type UGT1A10-overexpressing cells against 1-OH-pyrene (p < 0.05) and 3-OH-BaP (p < 0.01). Reverse transcription-polymerase chain reaction of total lung RNA showed low levels of UGT1A10 expression in human lung tissue. Together, these studies implicate UGT1A10 as an important detoxifier of polycyclic aromatic hydrocarbons in humans and that the UGT1A10 codon 139 polymorphism may be an important determinant in risk for tobacco-related cancers. The UDP-glucuronosyltransferase (UGT) superfamily of enzymes catalyzes the glucuronidation of a variety of endogenous compounds such as bilirubin and steroid hormones, as well as xenobiotics such as drugs and environmental carcinogens (Tephly and Burchell, 1990; Owens and Ritter, 1995; Gueraud and Paris, 1998; Ren et al., 2000). Based on structural and sequence homology, UGTs are classified into several families and subfamilies (Jin et al., 1993). UGT family 2B members are derived from independent genes, whereas the entire UGT1A family is derived from a single gene locus in chromosome 2. This locus codes for nine functional proteins that differ only in their amino terminus as a result of alternate splicing of independent exon 1 regions to a shared carboxy terminus encoded by exons 2 through 5 (Beaulieu et al., 1997). Several family 1A UGTs have been implicated in the detoxification of tobacco carcinogen metabolites including the tobacco-specific nitrosamine, NNK (Ren et al., 2000; Wiener et al., 2004b), and polycyclic aromatic hydrocarbons (PAHs) like benzo(a)pyrene (BaP) (Ciotti et al., 1997; Levesque et al., 1997; Beaulieu et al., 1998; Bélanger et al., 1998; Carrier et al., 2000). Although most family 1A UGTs are expressed in the liver (Ciotti et al., 1997; Burchell and Hume, 1999; Levesque et al., 1999; Guillemette et al., 2000), several UGTs are extrahepatic (Tukey and Strassburg, 2000) and are expressed in several target tissues for tobacco-induced cancers, including tissues in the aerodigestive tract (Zheng et al., 2002). Polymorphisms have been previously identified for many of the UGT genes, and several recent studies have examined their potential role in tobacco carcinogenesis and in risk for tobacco-induced cancers. In studies examining UGT family 1A variants, the “TATA” box polymorphism in the promoter region of UGT1A1, commonly associated with Gilbert’s syndrome, is associated with reduced function in the UGT1A1 transcriptional promoter (Burchell and Hume, 1999) and is associated with decreased formation of the glucuronide conjugate of These studies were supported by U.S. Public Health Service Grants R01DE13158 (National Institute for Dental and Craniofacial Research) and P01CA68384 (National Cancer Institute) from the National Institutes of Health, Department of Health and Human Services to P.L. 1 Current affiliation: Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.009100. ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; PAH, polycyclic aromatic hydrocarbon; BaP, benzo(a)pyrene; BPD, BaP-trans-7,8-dihydrodiol; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; 3-OH-BaP, 3-hydroxybenzo(a)pyrene; 7-OH-BaP, 7-hydroxy-benzo(a)pyrene; 9-OH-BaP, 9-hydroxy-benzo(a)pyrene; UDPGA, UDP-glucuronic acid; 1-OH-pyrene, 1-hydroxypyrene; HEK, human embryonic kidney; PCR, polymerase chain reaction; RT, reverse transcription. 0090-9556/06/3406-943–949$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 6 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 9100/3113825 DMD 34:943–949, 2006 Printed in U.S.A. 943 at A PE T Jornals on A ril 6, 2017 dm d.aspurnals.org D ow nladed from the important procarcinogenic BaP metabolite, BaP-trans-7,8-dihydrodiol (BPD), in liver microsomes (Fang and Lazarus, 2004). UGT1A7-specific genetic variants are associated with reduced UGT1A7 metabolic function against BaP phenols (Guillemette et al., 2000) and are strongly linked to increased risk for orolaryngeal (Zheng et al., 2001), pancreatic (Ockenga et al., 2003), and lung (Araki et al., 2005) cancer. Recent studies have shown an association between liver microsomal O-glucuronide conjugate formation activity against NNAL, the major metabolite of NNK, and a Pro Thr polymorphism at codon 24 of the UGT1A4 gene (Wiener et al., 2004a). Among the family of 2B polymorphic variants, recent studies have shown an association between both the UGT2B7 codon 268 polymorphism and the UGT2B17 gene deletion polymorphism and the Oglucuronidation of NNAL in liver microsomes (Wiener et al., 2004a; Lazarus et al., 2005). UGT1A10 is an extrahepatic enzyme expressed in aerodigestive tract tissues (Zheng et al., 2002) that exhibits significant glucuronidation activity against BPD (Fang et al., 2002). Previous studies have shown that polymorphism in codon 139 of the UGT1A10 gene, resulting in a nonconservative Glu Lys amino acid change, was linked to altered risk for orolaryngeal cancer (Elahi et al., 2003). No such association was observed for the UGT1A10 codon 244 polymorphism, which results in a conservative amino acid change of Leu Ile. To better assess the potential role of UGT1A10 in risk for tobaccorelated cancers, the glucuronidation activity of UGT1A10 was compared with that of other known UGT enzymes against selected PAHs, UGT1A10 expression was examined in lung tissue, and the effects of the codon 139 polymorphism on UGT1A10 function were examined in vitro. Results are presented showing that UGT1A10 appears to be the most highly active UGT against a number of tobacco carcinogen metabolites, that UGT1A10 is expressed in lung, and that the UGT1A10-encoded variant exhibits reduced enzyme activity against all the substrates tested compared with the wild-type UGT1A10-encoded isoform in vitro. Materials and Methods Chemicals and Materials. 3-Hydroxy (OH)-BaP, 7-OH-BaP, 9-OH-BaP, and BPD were obtained from the National Cancer Institute Chemical Carcinogen Repository (Midwest Research Institute, Kansas City, MO). UDP-glucuronic acid (UDPGA), DL-2-lysophosphatidyl choline palmital C16:0, 1-hydroxypyrene (1-OH-pyrene), 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone were purchased from Sigma (St. Louis, MO). C-UDPGA (specific activity: 300 mCi/mmol) was obtained from PerkinElmer (Wellesley, MA). Dulbecco’s modified Eagle’s medium was obtained from Mediatech (Herndon, VA), and both fetal bovine serum and geneticin (G418) were purchased from Invitrogen (Carlsbad, CA). Taq DNA polymerase (HotMaster) was purchased from PerkinElmer Biosystems (Foster City, CA); Moloney murine leukemia virus reverse transcriptase and the pcDNA3.1/V5-His-TOPO mammalian expression vector were obtained from Invitrogen (Carlsbad, CA); the human UGT1A Western blot kit that includes the anti-UGT1A polyclonal antibody was purchased from BD Gentest (Woburn, MA), and the anti-actin monoclonal antibody (1:5000 dilution) was provided by Sigma. The human embryonic kidney (HEK) 293 cell line was purchased from The American Type Culture Collection (Manassas, VA), and the human oral squamous cell carcinoma MSK1483 cell line was provided by Peter Sacks (New York University, New York, NY). Cell lines stably overexpressing UGT1A4, UGT1A6, UGT1A8, UGT2B4, and UGT2B7 were described previously (Ren et al., 2000; Wiener et al., 2004b). Baculosomes (BD Gentest) were used for screening UGT1A1, UGT1A3, UGT1A7, UGT1A9, UGT2B15, and UGT2B17 glucuronidation activities. UGT1A10 Cloning and Reverse Transcription-Polymerase Chain Reaction of Lung Tissue. The amplification of UGT1A10 cDNA was performed after an initial reverse transcriptase reaction using 3 g of total RNA from the MSK1483 cell line (shown previously to express UGT1A10; P. Lazarus, unpublished data), 2.5 M oligo(dT) primer, and 200 units of reverse transcriptase in a 50-min incubation at 42°C. Polymerase chain reaction (PCR) amplification was subsequently performed using 2 l of the reverse transcriptase reaction in a 50l reaction volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM concentration each of deoxynucleotide triphosphates, 20 pmol of both sense (UGT1A10S1, 5 -TCCGCCTACTGTATCATAGCAG-3 , corresponding to nucleotides 61 to 40 relative to the UGT1A10 translation start site; GenBank accession no. BC020971) and antisense (UGT1A10AS1, 5 -TTTTACCTTATTTCCCACCC-3 , corresponding to nucleotides 6 to 25 relative to the UGT1A10 translation stop codon) primers, and 2.5 units of Pfx DNA polymerase. Incubations were performed in a GeneAmp 9700 thermocycler (Applied Biosystems, Foster City, CA) as follows: 1 cycle of 94°C for 2 min, 41 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min, followed by a final cycle of 7 min at 72°C. The PCR product (1679 bp) was purified after electrophoresis in 1.5% agarose using the QIAEX II gel extraction kit (QIAGEN, Valencia, CA) and subsequently subcloned into the pcDNA3.1/V5-His-TOPO mammalian expression vector using standard methodologies. Confirmation of insert orientation was performed by restriction enzyme digestion, and the UGT1A10 sequence was confirmed by dideoxy sequencing of the entire PCR-amplified UGT1A10 cDNA product (performed at the Molecular Biology Core Facility at Penn State University College of Medicine) using two vector primers (T7 and BGH; Integrated DNA Technologies, Coralville, IA) and one internal sense primer (UGT1A10S2; 5 CCTCTTTCCTATGTCCCCAATGA-3 , corresponding to nucleotides 556 to 578 relative to the UGT1A10 translation start site in the UGT1A10 cDNA by comparison with the UGT1A10 cDNA sequence described in GenBank accession no. BC020971). Reverse transcription (RT)-PCR from normal human lung tissue was performed as described above except that total RNA from histologically normal human lung tissue specimens, obtained from the Tissue Procurement Facility at the H. Lee Moffitt Cancer Center (Tampa, FL), was used as a template. The primers used to amplify exon 1 of UGT1A10 were described previously (Zheng et al., 2002). All the protocols involving the analysis of tissue specimens were approved by the Institutional Review Board at Penn State College of Medicine and in accordance with assurances filed with and approved by the U.S. Department of Health and Human Services. Site-Directed Mutagenesis, Generation of Cell Lines, and Cell Homogenate Preparation. The UGT1A10 variant was generated by PCR amplification of the pcDNA3.1/V5-His-TOPO vector containing the wild-type UGT1A10 sequence using site-directed mutagenesis primers specific for the polymorphic site. The primers used to generate this variant were UGT1A10m139F (5 -GTAGAATACTTAAAGAAGAGTTCTT TTGATGCAGTGTTTCTGG-3 ) and UGT1A10-m139R (5 -CCAGAAACACTGCATCAA AAGAACTCTTCTTTAAGTATTCTAC-3 ), corresponding to bases 400 to 442 relative to the UGT1A10 translation start site, with the polymorphic base in bold for both primers. PCR was performed using 10 units of Pfx polymerase, 1 Pfx buffer, 2 enhancer solution (Invitrogen), 100 to 500 ng of template, 1 mM MgSO4, 300 M deoxynucleoside-5 -triphosphate, and 0.6 to 2.4 M concentrations of each primer. The products were amplified in a Bio-Rad (Hercules, CA) MyCycler with an initial denaturation of 95°C for 2 min, followed by 25 cycles of 95°C for 30 s, 59 to 61°C for 30 s, and 68°C for 18 min. After amplification, 20 units of the DpnI restriction enzyme were added to each reaction and incubated for 1.5 h at 37°C to specifically digest the wild-type template DNA. The nondigested, PCR-amplified plasmid (which has incorporated the polymorphism) was then transformed into competent DH5 Escherichia coli; individual colonies were isolated; and subsequent plasmid DNA minipreps were screened for the codon 139 variant by digestion with the EarI restriction enzyme, which specifically recognizes the UGT1A10 but not the UGT1A10 variant. UGT1A10 sequences were confirmed by dideoxy DNA sequencing analysis using the same primers used to confirm the cloning of wild-type UGT1A10 as described above. HEK293 cell lines overexpressing wild-type or variant UGT1A10 were generated by stable transfection using the LipofectAMINE reagent (Invitrogen) procedure according to the manufacturer’s protocol. Briefly, pcDNA3.1/ V5-His-TOPO/UGT1A10 constructs were transfected into HEK293 cells grown in 5% CO2 to 80% confluence in Dulbecco’s modified Eagle’s medium supplemented with 4.5 mM glucose, 10 mM HEPES, 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin. At 24 h post-transfection, 944 DELLINGER ET AL. at A PE T Jornals on A ril 6, 2017 dm d.aspurnals.org D ow nladed from cells were passaged and subsequently grown in geneticin (700 g/ml medium) for the selection of geneticin-resistant cells, with selection medium changed every 3 to 4 days. Cell homogenates were prepared by resuspending pelleted cells in Tris-buffered saline (25 mM Tris base, 138 mM NaCl, 2.7 mM KCl, pH 7.4) and subjecting them to three rounds of freeze-thaw before gentle homogenization. Cell homogenates (5–30 mg/ml homogenate protein) were stored at 70°C in 100l aliquots. Total cell homogenate protein concentrations were determined using the BCA assay from Pierce Biotechnology (Rockford, IL) after protein extraction using standard protocols. Western Blot Analysis. Levels of UGT1A protein in UGT-overexpressing cell lines were measured by Western blot analysis using the anti-UGT1A antibody (1:5000 dilution as per the manufacturer’s instructions), whereas -actin protein levels were assayed using a 1:5000 dilution of the monoclonal anti-actin antibody. UGT1A protein was detected by chemiluminescence using the SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology). Secondary antibodies supplied with the Dura enhanced chemiluminescence kit (anti-rabbit and anti-mouse) were used at 1:3000. UGT1A protein levels were quantified against a known amount of human UGT1A protein (100 ng, supplied in the Western blot kit provided by BD Gentest) by densitometric analysis of X-ray film exposures (5-s to 2-min exposures) of Western blots using a GS-800 densitometer with Quantity One software (Bio-Rad). Quantification was made relative to the levels of -actin observed in each lane (also quantified by densitometric analysis of Western blots as described above). X-ray film bands were always below densitometer saturation levels as indicated by the densitometer software. Relative UGT1A protein levels are reported as the mean of three independent Western blot experiments, with Western blot analysis performed using the same UGT1A-containing cell homogenates used for activity assays. Glucuronidation Assays. The rate of glucuronidation by cell homogenates was determined essentially as described previously (Fang et al., 2002; Wiener et al., 2004a). Cell homogenate protein (0.10–3.0 mg) was incubated (100– 250 l final volume) in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, DL-2lysophosphatidyl choline palmital C16:0 (0.2 mg/mg protein), 4 mM UDPGA, and 0.025 to 5 mM aglycone at 37°C for 1 to 2 h (as indicated in the text). C-UDPGA (1 Ci/100 l reaction volume) was added to all the assays except those incubations with PAH as substrate. For glucuronidation rate determinations, aglycone concentrations, cell homogenate protein levels, and incubation times for individual assays were chosen to maximize levels of detection within a linear range of uptake and were similar to established protocols (Fang et al., 2002; Wiener et al., 2004a). For kinetic analysis, incubations were performed using 0.2 mg (for analysis of 1-OH-pyrene, 3-OH-BaP, 7-OH-BaP, and 9-OH-BaP) or 1 mg (for analysis of BPD) of UGT1A10-overexpressing cell protein homogenate, with equal amounts of protein homogenate assayed for the wild-type and variant UGT1A10-overexpressing cell lines in any given experiment. Aglycone concentrations ranged between 0.0025 and 1 mM, a range that encompassed the Km for all the metabolites tested. Reactions were terminated by the addition of an equal volume of 100% acetonitrile on ice. Glucuronidation assays were analyzed by high-performance liquid chromatography with appropriate controls as described previously (Ren et al., 2000; Fang et al., 2002; Wiener et al., 2004b). Experiments were always performed in triplicate as independent assays. Statistical Analysis. The Student’s t test (two-sided) was used for comparing rates and kinetic values of glucuronide formation for the UGT1A10 and UGT1A10 isoforms against the different substrates examined in this