Glucuronosyltransferase Enzymes

Pharmacogenomics of human UDP- glucuronosyltransferase enzymes

C Guillemette1 ABSTRACT UDP-glucuronosyltransferase (UGT) enzymes comprise a superfamily of key 1Canada Research Chair in Pharmacogenomics proteins that catalyze the glucuronidation reaction on a wide range of Oncology and Molecular Endocrinology Research structurally diverse endogenous and exogenous chemicals. Glucuronidation Center, Laval University Medical Center (CHUL) is one of the major phase II drug-metabolizing reactions that contributes to and Faculty of Pharmacy, Laval University, Quebec, Canada drug biotransformation. This biochemical process is also involved in the protection against environmental toxicants, carcinogens, dietary toxins and Correspondence: participates in the homeostasis of numerous endogenous molecules, Dr C Guillemette, Canada Research Chair in including bilirubin, steroid hormones and biliary acids. Over the years, Pharmacogenomics, Oncology and significant progress was made in the field of glucuronidation, especially with Molecular Research Center, Laval University Medical Center and Faculty of Pharmacy, regard to the identification of human UGTs, study of their tissue distribution Laval University, Quebec, Canada G1V 4G2. and substrate specificities. More recently, the degree of allelic diversity has Tel: +1 418 654 2296 also been revealed for several human UGT genes. Some polymorphic UGTs Fax: +1 418 654 2761 have demonstrated a significant pharmacological impact in addition to being E-mail: [email protected] relevant to drug-induced adverse reactions and cancer susceptibility. This review focuses on human UGTs, the description of the nature of polymorphic variations and their functional impact. The pharmacogenomic implication of polymorphic UGTs is presented, more specifically the role of UGT polymorphisms in modifying cancer risk and their impact on individual risk to drug-induced toxicities. The Pharmacogenomics Journal (2003) 3, 136–158. doi:10.1038/sj.tpj.6500171

Keywords: UDP-glucuronosyltransferase; glucuronidation; genetic polymorphism; SNP; drug metabolism; cancer susceptibility

INTRODUCTION A number of detoxifying enzymes participate in host defense against endogenous toxins and xenobiotic chemicals. Among these, the UDP-glucuronosyltransferase (UGTs; EC 2.4.1.17) enzymes are found in many diverse living organisms, from bacteria to humans. UGTs are membrane-bound conjugating enzymes that catalyze the transfer of the glucuronic acid group of uridine diphosphoglucuro- nic acid (UDPGlcA) to the functional group (eg hydroxyl, carboxyl, amino, sulfur) of a specific substrate (Figure 1). Glucuronidation increases the polarity of the target compounds and facilitates their excretion in bile or urine. This biochemical system has been primarily studied for its role in the detoxification of exogenous compounds, mostly drugs. Indeed, virtually all classes of drugs are substrates for UGTs and this pathway has been estimated to account for approximately 35% of all drugs metabolized by phase II drug- metabolizing enzymes (DMEs).1 At least two additional biological functions are attributed to UGTs: (i) the contribution of UGTs is determinant in the mechanism of protection against some toxic dietary components, tobacco Received: 6 November 2002 2–9 Revised: 1 March 2003 smoke carcinogens and various environmental pollutants and (ii) they Accepted: 10 March 2003 represent key elements in the homeostasis of a number of endogenous Pharmacogenomics of human UGT enzymes C Guillemette 137

insertions or deletions, or single-nucleotide polymorphisms (SNPs). Polymorphisms are observed in various regions of a gene, including the regulatory and coding sequences, but also in the introns and the 50 and 30 untranslated regions. Depending on the nature of the variation and its location within a given gene, the phenotypic consequences may be barely noticeable, or they may alter the function or expression of the protein. In the case of UGTs, such genetic alterations would potentially modify the glucuronidation capacity of the individual carrying that polymorphism. The phenomenon is even more relevant considering that an increasing number of studies have brought to light examples of interindividual variations in the glucuronidating capacity towards various substances.7,16,20–60 Based on the diverse biological functions of UGTs, a significant alteration in a metabolic pathway involving these enzymes has the potential to modify significantly the pharmacokinetics of a given drug, carcinogen or endogenous molecule. Knowledge of the genetic mechanisms underlying variability in glucuronidation capacity are beginning to emerge but, to date, only a few clinically relevant genetic polymorphisms in UGTs have been described. Recent work has demonstrated that genetic variations affect glucuronidation rates and influence the risk of an individual to develop drug-induced toxicity.35,37,61–64 The role of genetic factors in determining variable rates of glucuronidation of hormones, tobacco Figure 1 The glucuronidation reaction. The UGTs belong to a smoke carcinogens, environmental pollutants and dietary multigene family of important enzymes involved in the inactivation and the elimination of a wide range of nucleophilic substances. components is poorly understood. Recent findings suggest UGTs conjugate both endogenous and exogenous molecules. In that metabolic alterations may actually determine the addition to these compounds, toxins and polycyclic and simple degree of exposure of an individual to toxic or carcinogenic phenols are among the most critical substrates of transferases, substances over a long time period and may contribute to although virtually all classes of drugs are substrates for UGT modify one’s susceptibility to diseases such as cancer.65–73 enzymes. UGT enzymes are localized to the internal membrane of This review is focused on recent work in the area of the 13 the endoplasmic reticulum. This conjugation reaction involves the pharmacogenomics of human UGTs. The following topics transfer of the ubiquitous cosubstrate uridine diphospho-glucuronic will be covered: (i) overview of human UGT enzymes; (ii) acid (UDPGlcUA) to hydrophobic molecules (also called aglycones), leading to the formation of b-d-glucopyranosiduronic acids or description of polymorphic human UGT enzymes; (iii) glucuronide derivatives (-G). The resulting glucuronides contain the pharmacogenomic phenomena involving polymorphic d-glucopyranuronosyl radical linked to -OR, -SR, -NR0R00 or -CR. UGT enzymes in drug response and (iv) pharmacogenomic Following the glucuronidation reaction, O-glucuronide ethers are phenomena involving polymorphic UGT enzymes in cancer. formed from substrates that contain functional groups such as aliphatic alcohols and phenols, while O-glucuronide esters are formed from substrates that contain functional groups like A BRIEF OVERVIEW OF THE GLUCURONIDATION carboxylic acids. These are the commonest glucuronides. Glucur- PATHWAY AND HUMAN UGTS onidation of primary, secondary aromatic and aliphatic amines and of tertiary amines leads to the formation of N-glucuronides. In The glucuronidation reaction, catalyzed by membrane- addition, the presence of a sulfhydryl functional group creates S- bound UGTs, involves the transfer of the ubiquitous glucuronides, whereas carbonyl groups yield C-glucuronides, cosubstrate UDPGlcA to hydrophobic molecules (also called although very few examples of these derivatives have been aglycones). This reaction leads to the formation of b-d- described. glucopyranosiduronic acids or glucuronide derivatives (-G) (Figure 1). The glucuronidation reaction confers polarity to xenobiotics, drugs and endogenous compounds, which are molecules, including bilirubin, steroid and thyroid hor- consequently more easily excreted from the body.13 The mones, fatty acids as well as biliary acids.10–14 carboxyl group from the glucuronic acid, which is ionized at In recent years, breakthroughs in the pharmacogenomic physiological pH, promotes excretion by increasing the field support the concept that genetic factors related to aqueous solubility of the glucuronide, otherwise highly genes encoding DMEs, transporters and drug targets play a hydrophobic. The glucuronide is recognized by the biliary significant role in the clinical response to therapeutic and renal organic anion transport systems, which enable drugs.1,15–19 These genetic factors include common genetic secretion into urine and bile. Compared to other conjugat- alterations or polymorphisms (41% frequency), small ing enzymes such as acetyltransferases (NAT), glutathione

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S-transferases (GST) and sulfotransferases (ST), UGTs have UGT2A and UGT2B (for detailed information see Mackenzie the most profound effect in both detoxification and et al81). In contrast to the UGT2B subfamily, which promotion of excretion, via urine and bile. Furthermore, comprises several independent genes,82–84 the entire UGT1 since the addition of glucuronic acid to compounds changes family is derived from a single gene locus (UGT1) spanning their structure, glucuronidation modifies the biological about 210 kb on chromosome 2 (2q37), which is composed activity of the parent foreign compound. Generally, the of 17 exons.85–87 To synthesize the final protein, only one of biological function of the aglycone is abolished by glucur- 13 different exon-1 sequences on the locus is associated with onidation. In few instances (glucuronides of morphine and four downstream exons, common to all UGT1A isoforms. Of retinoic acids), glucuronidation will maintain or increase the 13 exon-1 sequences, nine code for functional proteins their biological function.74,75 (UGT1A1, UGT1A3-1A10) and four correspond to pseudo- The knowledge about the substrate specificity of UGT genes (p) (UGT1A2p, UGT1A11p, UGT1A12p and UG- enzymes and their tissue expression and regulation has T1A13p).85–87 UGT proteins are composed of 527–530 progressed significantly in recent years. Although the liver is amino-acid residues, for a molecular weight of 50–57 kDa. recognized as the major site of glucuronidation, it is now The exon-1 sequence of UGTs codes for the substrate- clear that numerous organs also have the ability to make a binding domain (N-terminal half of the protein), while the significant contribution to the overall glucuronidation four common exons code for the cosubstrate-binding capacity of the organism. Similar to other DMEs, UGTs are domain (C-terminal half of the protein). The presence of localized in all ports of entry of chemicals, including the different possible substrate-binding domains confers the epithelial surfaces of the nasal mucosa, the gut, skin, white large substrate specificity and selectivity of UGT1A proteins. blood cells and lung (reviewed in Tukey and Strassburg76). The regulatory sequences flanking each of the exon-1 UGTs are also expressed in a wide variety of tissues, regions is thought to dictate the individual expression including brain, prostate, uterus, breast, placenta and profile of the UGT1A isoforms.76,87 Five of the nine UGT1A kidney, to name only a few.11,76–80 isoforms were isolated from human liver, while the three The molecular genetics of human UGTs is now more remaining isoforms were observed strictly in extrahepatic clearly understood, with evidence for the existence of tissues and demonstrated a tissue-specific expression pat- distinct subfamilies, which comprise more than 26 genes tern, mainly in the gastrointestinal tract.68,88–95 or cDNAs (Figure 2). Eighteen of them correspond to To date, the structures of several UGT2B genes (composed functional proteins and are encoded by two gene families, of six exons) and cDNAs have been published. Several UGT1 and UGT2 that are, based on their sequence UGT2Bs were isolated from liver as well from extrahepatic similarities, further divided into three subfamilies: UGT1A, tissues.76,81–84,96–101 In addition, numerous homologous pseudogenes have been found,83 all of which are clustered with UGT2B genes encoding functional proteins on chromosome 4 (4q13). Similar to the UGT1 family, members of the UGT2B subfamily share a high degree of similarity in the C-terminal portion of the protein and the highest degree of divergence in sequences encoded by exons 1. Two members of the UGT2A subfamily have also been characterized and are localized to chromosome 4q13. They share approximately 70% of identity with the UGT2Bs. The biological function of UGT2As was proposed to be the termination of the odorant signals, although it is not limited to this function since they are expressed in the liver, small intestine, brain and fetal lung.102–104 The wide range of substrates for human UGTs includes compounds of diverse chemical structures. Several studies have described the overlapping substrate specificities of UGT proteins.76,105 On the other hand, even in the case where the homology between UGTs of the same subfamily reaches 95%, the substrate specificity of individual transferases seems to remain distinctive. More importantly, even if several UGTs share a high degree of overlapping substrate specificity, UGTs often present distinct tissue-specific expression and even cell-type-specific expression within a 88–90,106 Figure 2 Phylogenetic tree for the different UGT isoenzymes. The tissue. As an example, using a specific anti-UGT2B17 dendrogram shows both UGT families that share less than 50% of antibody, the expression of this androsterone-conjugating homology. Percentage values represent the homology between UGT was found exclusively in basal cells of the epithelium of two groups or single isoenzymes at the amino-acid level. Pseudo- the prostate.106 Therefore, in situations where a specific genes were not analyzed for homology. UGT is inactive or less abundant, functional characteristics

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of other UGTs including the specificity of an enzyme and its lower level in approximately 0–3% of the Asian population, efficacy, tissue and cell-type-specific expression will not 2–13% of the Caucasian population and up to 16–19% of suffice to compensate for the deficient enzyme. Africans (Table 2). On the other hand, the UGT1A1*33 (TA5) To date, a number of polymorphisms have been described and UGT1A1*34 (TA8) polymorphisms found initially in the for both UGT1 and UGT2B genes. This area of research has African-American population are also observed at much become very active and reports of polymorphic UGT genes lower frequencies in Caucasians.67,118 Although interindivi- are constantly emerging. In the following sections, human dual variations in UGT1A1 expression are thought to be UGTs will be reviewed one by one for the presence of largely because of the UGT1A1*28 polymorphism, at least naturally occurring polymorphic forms of the protein, their one additional common polymorphic variant has been prevalence in human populations, their functional impact reported in the literature. A missense mutation caused the and their physiological, clinical and/or pathophysiological replacement of an arginine by a glycine at position 71 of the implications. corresponding protein (UGT1A1*6 Gly71Arg). This mutation is predominant in Japanese and does not seem to be linked 119–121 to the A(TA)7TAA polymorphic site. UGT1A1 Common Polymorphisms and Rare Mutations Associated Polymorphisms of the UGT1A1 Gene as Risk Factors for with Congenital Hyperbilirubinemia Syndromes Drug-Induced Toxicity UGT1A1 is the major UGT1 gene product that catalyzes the Patients affected by Gilbert’s syndrome display lower glucuronidation of bilirubin, a breakdown product of heme, glucuronidation rates for a number of therapeutic drugs which needs to be removed from the body. Historically, including acetaminophen, tolbutamide and lorazepam.20– genetic defects in the UGT1 gene complex have been found 22,34–37 Since Gilbert’s syndrome is associated with the to have a profound impact on the health of affected UGT1A1*28 (TA7 polymorphism), it is more likely that individuals, especially those modifying UGT1A1 activity. patients with this allele will present an altered drug Most genetic alterations in the UGT1 gene complex that clearance compared to patients with the wild-type genotype. have been described to date correspond to rare mutations However, a correlation between the presence of variant associated with two severe familial forms of the unconju- UGT1A1 alleles and altered rates of glucuronidation has not gated hyperbilirubinemia syndromes (Crigler–Najjar type I been systematically demonstrated. and II disorders). Breakthroughs in the molecular genetics of Recently, a strong correlation was observed between these syndromes have been made with the identification of UGT1A1 low (UGT1A1*28) and high (UGT1A1*1) promoter B60 rare mutations in the UGT1A1 gene, identified as point activity alleles and the corresponding rates of glucuronida- mutations, deletions and insertions (Table 1). Only a few of tion of the active metabolite of irinotecan, SN-38.122 these were found in the general population at a sufficiently Irinotecan {CTP-11 or 7-ethyl-10-[4-(1-piperidino)-1-piper- high frequency (41%) to be classified as polymorphisms. idino] carbonyloxy camptothecin} is an anticancer drug Among these polymorphisms, there is a dinucleotide repeat clinically used in combination with 5-fluorouracil and in the atypical TATA-box region of the UGT1A1 promoter. leucovorin as the first-line treatment of metastatic colorectal Variations in the number of A(TA)nTAA repeats lead to four cancer. CPT-11 is biotransformed by carboxylesterases into a variant alleles (n ¼ 5 UGT1A1*33; n ¼ 6 UGT1A1*1; n ¼ 7 pharmacologically active metabolite, SN-38 (7-ethyl-10- UGT1A1*28; n ¼ 8 UGT1A1*34) (Figure 3). Functional hydroxycamptothecin),123 which is responsible for severe studies revealed that increasing the number of repeats in toxicity. SN-38 undergoes significant glucuronidation to the promoter region leads to a decrease in the rate of form the corresponding inactive glucuronide (10-O-glucur- transcription initiation of the UGT1A1 gene.66,107–109 The onyl-SN-38 (SN-38-G))124 and UGT1A1 isoenzyme was ‘wild-type’ allele (UGT1A1*1) contains six TA repeats, suggested to be the predominant human UGT involved in whereas the most common variant allele (UGT1A1*28) the formation of SN-38-G.35 Individuals with Gilbert’s contains seven TA repeats and is associated with the mild syndrome are at greater risk to experience irinotecan- form of the inherited unconjugated hyperbilirubinemia induced toxicity.37 Therefore, inherited differences in syndrome (Gilbert’s syndrome).109,110 Gilbert’s syndrome is irinotecan glucuronidating capacity, such as those caused also characterized by the absence of liver disease and by by UGT1A1 promoter polymorphisms, may have an im- episodes of mild intermittent jaundice and is found in portant influence on the pharmacokinetics, the pharmaco- approximately 6–12% of the population.109–113 Studies logic effects and toxicity of this drug. This hypothesis is showed that mutations in the UGT1A1 gene, especially supported by the observation of an inverse relation between homozygosity for the UGT1A1*28 variant, are responsible SN-38 glucuronidation and the severity of diarrheas occur- for this syndrome. Moreover, this genetic variation of the ring in patients treated with irinotecan.122,124 In a prospec- UGT1A1 gene is a contributory factor in prolonged neonatal tive study comprising irinotecan-treated cancer patients, the jaundice.114–117 group of Iyer et al61 observed that UGT1A1 promoter activity A significant difference in the prevalence of the variant influences the extent of SN-38-G formation and is associated UGT1A1 promoter alleles has been observed. A detailed with the incidence of adverse reactions, mostly neutropenia analysis of data available in the literature revealed that and diarrhea. A retrospective clinical study of Japanese overall, the UGT1A1 isoform seems to be expressed at a cancer patients revealed an association between genetic

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Table 1 Genetic alterations of the UGT1A1 gene

Alleles Named allelesa Nucleotide changes Exon Protein changes Disease References

1 *1 Wild type A(TA)6TAA —— —91 2 *2 879 del 13 2 Truncated CN1 170 3 *3 1124 C-T 4 S375F CN1 171 4 *4 1069 C-T 3 Q357X CN1 172 5 *5 991 C-T 2 Q331 del 44 CN1 171 6 *6 211G-A 1 G71R Gilbert 173,174 7 *7 1456 T-G 5 Y486D CN2 173 8 *8 625 C-T 1 R209W CN2 175 9 *9 992 A-G 2 Q331R CN2 176 10 *10 1021 C-T 3 R341X CN1 177 11 *11 923 G-A 2 G308E CN1 178,179 12 *12 524 T-A 1 L175Q CN2 180 13 *13 508 del 3 1 F170del CN1 181 14 *14 826 G-C 1 G276R CN1 180 15 *15 529 T-C 1 C177R CN2 180 16 *16 1070 A-G 3 Q357R CN1 179 17 *17 1143 C-G 4 S381R CN1 179 18 *18 1201 G-C 4 A401P CN1 179 19 *19 1005 G-A 3 W335X CN1 179 20 *20 1102 G-A 4 A368T CN1 179 21 *21 1223 ins G 4 Frameshift CN2 179 22 *22 875 C-T 2 A291V CN1 179 23 *23 1282 A-G 4 K426E CN1 179 24 *24 1309 A-T 5 K437X CN1 179 25 *25 840 C-A 1 C280X CN1 182 26 *26 973 del G 2 Frameshift CN2 180 27 *27 686 C-A 1 P229Q Gilbert 183 28 *28 A(TA)7TAA Promotor Reduced Gilbert 109 29 *29 1099 C-G 4 R367G Gilbert 183 30 *30 44 T-G 1 L15R CN2 184 31 *31 1609 CC-GT 4 P387R CN1 185 32 *32 1006 C-T 3 R336W CN1 185 33 *33 A(TA)5TAA Promotor Increased — 66,107 34 *34 A(TA)8TAA Promotor Reduced — 107,186, 66 35 991 C-T 2 Q331X CN2 187 36 1201G-C/1308A-T 4/5 A401P/K437X CN1 179 37 872C-T/1282A-G 2/4 A291V/K426E CN1 179 38 826G-T 1 G276R CN1 180 39 120–121delCT 1 Frameshift CN1 185 - 40 1391A C/N (TA)7TAA 5 E464A CN2 188 41 881 T-C 2 I294T CN2 186 42 698 A-C 1 L233A — 62 43 115 C-G 1 H39D CN1 189 44 222C-A 1 Y74X CN1 189 45 517delC 1 Frameshift CN1 189 46 722–723delAG 1 Frameshift CN1 189 47 671T-G/722–723delAG 1 V224G/frameshift CN2 189 48 1046delA 3 Frameshift CN1 189 49 1223delA/N 4 Frameshift/N CN1 189 50 1130A-G/N 4 H377R/N CN2 189 51 1133G-T 4 G377V CN2 189 52 1451G-A 5 W484X CN1 189 53 1452G-A 5 W484X CN1 189 54 1490T-A/N 5 L497X/N CN1 189 55 Splice acceptor site Intron 1 CN1 190 56 Splice donor site Intron 3 CN1 190 57 145C-T 1 Q49X CN1 190 58 625C-T 1 R209W CN2 191 59 1186delG 4 Frameshift CN2 191 60 À3263T-A Promoter — Gilbert 192 61 Splice donor site G-A Intron 3 CN1 193 62 247T-C 1 L83F Gilbert 194 63 877T-A/878-890del 2 Frameshift/Del CN1 193 del : deletion; ins: insertion. In the case of rare hyperbilirubinemia, as in CN type I, the UGT1A1*28 variant promoter does not seem to play a significant role, while in the case of CN type II, the presence of the *28 allele was shown to affect the clinical characteristics of the disease.184,195 In addition to being associated with elevated bilirubin plasma levels, the UGT1A1*28 allele has been linked to various diseases including glucose-6-phosphate dehydrogenase (G6PD) deficiency, neonatal jaundice, hereditary spherocytosis, cholelithiasis in children with sickle cell anemia and b-thalassemia.196–201 aMackenzie et al81.

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66,67 66 alterations in the promoter region of UGT1A1*28 (A(TA)7- women. Based on functional studies, UGT1A1 promoter TAA) and in the coding region of the UGT1A1 gene alleles were divided into two classes; low transcriptional 229 (UGT1A1*27 (Pro Gln)) and the occurrence of severe activity alleles (A(TA)7TAA or A(TA)8TAA) and high transcrip- 64 irinotecan-induced toxicity. The SNP present in the tional activity alleles (A(TA)5TAA or A(TA)6TAA). It was UGT1A1*6 (Gly71Arg) allele was also investigated in this postulated that breast cancer cases have a higher frequency study and, although this allele is highly prevalent in the of low transcriptional activity alleles (high-risk genotypes) Asian population,120,125 the presence of this polymorphism compared to controls. An analysis of 200 African-American in cancer patients was not associated with irinotecan- women with invasive breast cancer and 200 matched induced toxicity.64 This is consistent with the recent controls revealed that the low-activity alleles (UGT1A1*28 observation showing that the impact of the UGT1A1*6 (A(TA)7TAA) and UGT1A1*34 (A(TA)8TAA)) were positively allele on in vitro rates of SN-38 glucuronidation is modest associated with breast cancer.66 The pattern of results further compared to that observed for UGT1A1*27.62 suggested a stronger association between UGT1A1 genotype The hepatic UGT1A9 and the extrahepatic UGT1A7 and estrogen receptor-negative breast cancer in premeno- enzymes were recently shown to be involved in the in vitro pausal women. On the other hand, no significant interac- metabolism of SN-38.62 Thus, polymorphisms in UGT1A7 tions were revealed with the use of oral contraceptives or and UGT1A9 along with the promoter polymorphism of hormone replacement therapy. These findings are consistent UGT1A1 and functional SNPs present in its coding region, with a role for estrogen-metabolizing UGTs in modulating potentially represent significant risk factors for severe the action of endogenous hormones and therefore in toxicity and could serve to predict the incidence of adverse affecting breast cancer risk in the African-American popula- events in patients undergoing irinotecan-based chemother- tion. This relation was further studied among 455 Caucasian apy. women with breast cancer and 603 women without breast cancer. In this nested case–control cohort within the Nurses’ Role of UGT1A1 Variants in Estrogen-Related Cancer Health Study, no significant risk was associated with the low The development of several cancers is modulated by steroid transcriptional activity allele UGT1A1*28.67 Stratification by hormones, which are important physiological substrates of age at menarche, menopausal status and by clinical and UGT enzymes. Alterations in the hormonal environment as pathological characteristics also failed to reveal any associa- a consequence of a change in metabolism may determine an tion. individual’s exposure over time and contribute to modify Since decreased transcription of the UGT1A1 gene has cancer risk. Several enzymes involved in the biotransforma- been found to be associated with increased plasma levels of tion of adrenal steroid precursors, progesterone, androgens other substrates metabolized by the UGT1A1 protein, such and estrogens have been studied as candidate genes, which as bilirubin, the relation between the A(TA)7TAA allele and could potentially modify the risk of developing cancer. circulating levels of estrogens was evaluated. In postmeno- Inherited variations in these genes are suggested to be pausal women not using hormone replacement therapy, associated with an increased risk of developing steroid- estrone and estradiol levels tended to vary as a function of related cancers including breast, ovarian, endometrial and UGT1A1 genotypes.67 The higher plasma concentrations of prostate cancers. The strategy for finding these low-pene- estradiol and estrone found in women with the low-activity trance susceptibility alleles has been the candidate gene UGT allele support a role of the glucuronidation pathway in approach, in which polymorphic variants of genes that estrogen metabolism and its contribution in the mainte- possibly influence cancer risk are studied by conventional nance of an adequate estrogenic environment in target cells. epidemiological studies using case–control cohorts. However, the elevation in the plasma levels of estrogenic The UGT1 and UGT2 loci harbor putative susceptibility hormones seems to be insufficient to alter one’s risk to genes that have not been explored in detail. Recent develop breast cancer. One can speculate that more studies revealed that specific genetic alterations in UGT important modifications in glucuronidation activity are genes could contribute to modify the risk factor of develop- needed to produce a significant alteration in breast cancer ing cancer.66–72 susceptibility. In fact, this situation might have been The first series of studies were case–control studies of breast achieved in African-Americans in whom the presence of cancer patients, in which the role of UGT1A1 as a candidate the UGT1A1*34 allele (TA8) leads to a 50% decrease in the gene was examined (Table 3). The hypothesis tested was that transcription of the UGT1A1 gene compared to 30% for the 66 constitutive alterations in UGTs involved in the inactivation TA7 allele. In addition to other UGTs that potentially of estradiol and catechol reactive metabolites could modify compensate for a deficient UGT1A1, other metabolic path- estrogen exposure and consequently estrogen-related cancer ways such as sulfation could also potentially compensate for risk. UGT1A1 was investigated as a first candidate gene, since the reduced glucuronide conjugation of estrogens. this UGT is expressed in human breast parenchyma and is In addition, it has been suggested that catechol metabo- involved in the formation of estradiol-glucuronide.66,126 Two lites of estradiol contribute to the development of estrogen- genetic epidemiological studies were designed to investi- induced cancers by acting as endogenous tumor initia- gate the association between genetic variability in the tors.127 Alterations in glucuronidation rates could modify UGT1A1 promoter region and the risk of breast cancer: one cancer risk by affecting the balance between estradiol and involving African-American subjects and the other, Caucasian C2, C4 and C16 catechol estrogens in the target cells. The

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a UGT1 genea

Commons Multiple Exons 1 Exons

c b 1A12p 1A11p 1A8 1A10 1A13p 1A9 1A7 1A6 1A5 1A4 1A3 1A2p 1A1 2534

Chromosome 2q37 T-106C 277 Cys Tyr Gly71Arg 181 158 173 Thr Ala Ala Gly Ala Gly Ser 7 Ala His225 Tyr A(TA)nTAA n= 5, 6,6 7 or 8 repeats 248 Trp 208 Arg Leu Ile

Glu139Asp Val249Leu

129 Asn Lys/ Gly259Arg Arg131 Lys

Gly115 Ser

b UGT2B genesd,e

UGT2B7 UGT2B4 f UGT2B15

1 2 3 4 5 6 12 34 5 6 12 3 45 6

Chromosome 4q13

His268 Tyr Asp458Glu Leu86 Ser

Asp85 Tyr

Figure 3 Common polymorphic variations in the UGT1 (a) and UGT2B (b) genes. (a) The structure of the UGT1 gene presented here is based on the Genbank accession number AF297093.87 (b) UGT1A5 alleles correspond to: UGT1A5*1 Ala158His225Leu248Val249Gly259; UGT1A5*2 Gly158His225Leu248Val249Gly259; UGT1A5*3 Ala158Tyr225Leu248Val249Gly259; UGT1A5*4 Ala158His225Ile248Leu249Arg259; UGT1A5*5 Gly158His225Ile248Leu249Arg259; UGT1A5*6 Ala158Tyr225Ile248Leu249Arg259; UGT1A5*7 Gly158Tyr225Ile248Leu249Arg259. (c) UGT1A7 alleles correspond to: UGT1A7*1 Gly115Asn129Arg131Glu139Trp208; UGT1A7*2 Gly115Lys129Lys131Glu139Trp208; UGT1A7*3 Gly115Lys129Lys131- Glu139Arg208; UGT1A7*4 Gly115Asn129Arg131Glu139Arg208; UGT1A7*5 Ser115Asn129Arg131Glu139Arg208; UGT1A7*6 Gly115Asn129Arg131- Glu139Trp208; UGT1A7*7 Gly115Lys129Lys131Asp139Trp208; UGT1A7*8 Gly115Lys129Lys131Asp139Arg208 and UGT1A7*9 Ser115Lys129Lys131Glu139Tr208. (d) The relative positions of the UGT2B4, UGT2B7 and UGT2B15 genes on chromosome 4q13 are based on the data reported by Riedy et al.101 (e) Polymorphic expression of two truncated UGT2B28 variants (types II and III) has been reported.99 UGT2B28 type II differs from type I by a deletion of 308 bp in the cofactor-binding domain, whereas UGT2B28 type III lacks 351 bp in the putative substrate-binding domain. (f) An additional cDNA clone isolated from human liver corresponds to the UGT2B4 Phe109Leu, Phe396Leu allele but appears to be very rare as it was not found in two independent population studies.

increased formation of catechol estrogens mediated by the relation between UGT1A1 polymorphisms and breast den- P450 enzymes combined with an insufficient conjugation of sity was investigated.128 Premenopausal women homozy- the estradiol and estrogen metabolites via glucuronidation gous for the UGT1A1*28 allele had significantly lower breast might alter the risk of estrogen-related cancers. Studies on density compared to those with the *1/*1 genotype (À43.1% the glucuronidation of estrogenic hormones and the con- difference; P ¼ 0.04). In contrast, postmenopausal women tribution of UGTs in altering the risk of hormone-dependent with the UGT1A1*28/*28 genotype had greater breast cancer need to be pursued. The role of the UGT1A1*28 allele density compared to those with the *1/*1 genotype in susceptibility to endometrial cancer is currently under (+32.0% difference; P ¼ 0.05), which was even greater evaluation. among current users of postmenopausal hormone replacement therapy (+56.8% difference; P ¼ 0.03).128 These UGT1A1 Polymorphisms and Variation in Breast Density results suggest that the UGT1A1 genotype is a predictor of A recent observation supports the role of UGT1A1 in breast density within groups of different menopausal status modulating the exposure of breast cells to hormones. The and would predict that interindividual differences in

The Pharmacogenomics Journal Pharmacogenomics of human UGT enzymes C Guillemette 143

Table 2 Polymorphic variations of the UGT1A1 gene

UGT1A1 Allele frequency a Genotype frequency (%)b References

Promoter n *1 *28 *33 *34 *1/*1 *1/*28 *28/*28

Polymorphism A(TA)6TA A(TA)7TA A(TA)5TA A(TA)8TA 6/6 6/7 7/7

Functional change ‘Wild type’ B30% reduced B20% increased B50% reduced expression expression expression

Population characteristicsc Caucasian (Scottish) 77 0.640 0.360 — — 40 48 12 110 Caucasian (European) 71 0.613 0.387 0.000 0.000 34 55 11 107 Caucasian (American) 202 0.698 0.295 0.005 0.002 51 37 11 135 Caucasian 88 0.715 0.285 0.000 0.000 75 23 2 202 Caucasian (American) 603 0.680 0.320 o0.001 o0.001 17 73 10 67 Caucasian 71 0.662 0.324 0.007 0.007 45 39 13 204 Caucasian (European) 70 0.693 0.307 — — 46 47 7 49 Inuit (Canadian) 88 0.585 0.415 — — 34 49 17 203 Asian (American) 47 0.840 0.160 0.000 0.000 70 28 2 107 Asian 30 0.867 0.133 0.000 0.000 73 27 0 135 Asian 90 0.855 0.145 0.000 0.000 74 23 3 202 Asian (Japanese) 27 0.981 0.019 — — 96 4 0 192 African (American) 200 0.520 0.380 0.080 0.020 28 36 17 66 African (American) 101 0.470 0.426 0.035 0.069 26 37 19 107 African (Sub-Saharan) 76 0.450 0.427 0.080 0.043 18 41 17 202 African (American) 54 0.519 0.407 0.065 0.009 24 46 17 204 Italian 98 0.640 0.360 — — 44 40 16 205 Amerindian 81 0.850 0.150 0.000 0.000 74 22 4 202 Parakana Indian 32 0.672 0.328 0.000 0.000 38 59 3 204 Hispanic (Admixed) 44 0.610 0.380 0.010 0.000 41 41 16 202

UGT1A1 Allele frequency Gentotype frequency (%) References

n *6 Heterozygous Homozygous (*1/*6) (*6/*6)

Polymorphism Gly71Arg

Population characteristicsc Caucasian (German) 50 0.000 0 0 120 Asian (Japanese) 101 0.130 23 2 120 Asian (Japanese) 27 0.170 26 4 192 Asian (Korean) 50 0.230 38 4 120 Asian (Chinese) 50 0.230 34 6 120 a The ‘—’ mean the data were not available in the original manuscript. b Due to their low frequencies, *33 and *34 allele-containing genotypes are not represented in the table, thus the sum of the percentages may not be 100. c When available, specific information on the ethinicity of subjects was added (parentheses). Some of the values presented in this table correspond to the sum of the reported frequencies from diverse ethnic subgroups included in the population cited. estrogen glucuronidation influence breast density. Since the Caucasian women studied in relation to breast density mammographic density is one of the strongest predictors of were a subset. breast cancer risk, these results would also suggest that Yet, it remains to be demonstrated if alterations in the risk of developing breast cancer is higher in premeno- UGT1A1 glucuronidation rates or expression levels result in pausal women with the UGT1A1*28 allele and lower in increased estrogen bioavailability within target cells. Since postmenopausal women with the UGT1A1*28 allele. high levels of estrogen-glucuronides have been observed in However, as described previously, the UGT1A1 genotype breast cyst fluid and the expression of UGTs confirmed in did not alter the risk of breast cancer in the large nested estrogen target cells, formation of glucuronides within the case–control study from the Nurses’ Health Study, of which breast, ovary and uterine tissues is expected.66,73

www.nature.com/tpj 144 h hraoeoisJournal Pharmacogenomics The Table 3 Association studies on (a) steroid-related cancer susceptibility and (b) orolaryngeal and colorectal cancers and HCC susceptibility

Population Characteristics (n) Hypothesis Genotype OR (95%Cl)a Exposure assessment References

(a) Steroid-related cancer susceptibility involving UGT1A1 and UGT2B15 Candidate gene: UGT1A1 breast cancer African-Americans Cases (200) A low glucuronidating 5/5, 5/6, 6/6 No association was 66 Recruited in North Women with a first activity would lead to Reference revealed in subgroups Carolina (USA) diagnosis of increased estrogen levels defined by use of histologically and to an increased 6/7, 6/8, 7/7, exogenous hormones.

confirmed, invasive susceptibility to estrogen- 7/8, 5/7, 5/8 enzymes UGT human of Pharmacogenomics breast cancer. dependent cancers. Overall: Controls (200) Women with the 1.30 (0-80-1.90)b Women frequency low-activity promoter Premenopause: matched to cases by genotypes (6/7, 6/8, 1.80 (1.00-3.10)b race and by age 7/7, 7/8, 5,7 or 5/8) Postmenopause: (5 years groups). compared to women 1.00 (0.50-1.70)b with the high-activity promoter genotypes

(5/5, 5/6, 6/6). Guillemette C Caucasians (99.3%) Recruited in the USA Cases (455) A low glucuronidating *1/*1 No association was 67 Nested case–control Women with pathologically activity would lead to Reference revealed in subgroups population within the confirmed breast cancer. increased estrogen levels defined by age at menarche, prospective Nurse’s Controls (609) and to an increased *1/*28 menopausal status, clinical Health Study cohort Women matched to susceptibility to estrogen- 0.93 (0.71–1.21)c,d and pathological (NHS). cases on the basis of dependent cancers. *28/*28 characteristicse. Mixed year of birth, menopausal Women with the 0.85 (0.54–1.33)c,d regression modelsf showed status and postmenopausal low-activity allele *28 *28 carriers an association between hormone use, as well as compared to women 0.91 (0.71–1.18)c,d genotype and circulating time of day, month, and homozygous for the estradiol and estrone levels. fasting status at blood draw. high-activity allele *1.

Candidate gene: UGT2B15 Prostate cancer Caucasians (91%) Cases (64) The presence of the Tyr85 (*2) *1/*1 None 72 African-Americans (9%) Patients with pathologically allele is associated with 3.00 (1.30–6.50) Recruited from the confirmed prostate cancer. increased rates of androgen Arkansas area (USA) Controls (64) glucuronidation, resulting Matched by age in lower intraprostatic (within 5 years), race androgen levels, possibly and county of residence. leading to a reduction in the risk of developing prostate cancer. UGT2B15 Tyr85 (*2)=high-activity alleleg UGT2B15 Asp85 (*1)=low-activity alleleg Table 3 (continued)

Population Characteristics (n) Hypothesis Genotype OR (95%Cl)a Exposure assessment References

Caucasians Cases (190) Low-activity *1/*1 None 71 Recruited in Patients with histologically allele linked Reference Vienna, Austria verified, previously to cancer. untreated prostate *1/*2 cancer. 1.25 (0.73–2.15) Controls (190) *2/*2 Men with lower urinary 1.20 (0.65–2.22) tract symptoms because of benign prostatic hyperplasia, matched by age.

(b) Orolaryngeal and colorectal cancers and HCC susceptibility involving UGT1A7hi Orolaryngeal cancer Caucasians (64%) Cases (192) The UGT1A7 is involved in Caucasians The risk for orolaryngeal 68 African-Americans (36%) Subjects diagnosed detoxification of tobacco- *1/*1 cancer was 8.5- to Recruited from institutions with primary squamous related carcinogens. Reference 10-fold higher for in New York City, cell carcinoma of the The subjects with the subjects with predicted USA (59%) oral cavity or of the larynx. UGT1A7 low-activity *3/*3, *3/*4, *4/*4* low-activity UGTIA7 and from the Temple Controls (381) genotypes are expected 2.80 (1.10–7.60)j genotypes compared University Hospital in Volunteers screened at to be low glucuronidators African-Americans to subjects with predicted

Philadelphia, USA (41%) participating hospitals and and thus, will be subjected *1/*1 high-activity genotypes Guillemette enzymes C UGT human of Pharmacogenomics that were consulting for to prolonged exposures 1.0 within each smoking conditions not related to or to greater concentrations Reference group. exposure to tobacco smoke. of tobacco-related They have not been carcinogens than *3/*3, *3/*4, *4/*4* previously diagnosed subjects with a high 6.20 (1.20–31.00)j with any type of glucuronidating capacity. cancer. They were Low-activity genotypes matched in a two-to-one were compared to ratio with patients for high-activity genotypes age (within 5 years), (reference). race and sex.k Hepatocellular Carcinoma Caucasians Cases (59) Low-detoxification-activity *1/*1 None 69 Recruited from Hannover Patients with HCC diagnosed allele linked to cancer. 9.73 (3.17–29.83) Medical School, Germany by ultrasonography and *1/*3 or *2/*4 abdominal computed 8.46 (2.69–26.57) tomography scans’. *2/*3

www.nature.com/tpj (Mean age=64) 7.61 (2.08–27.84) *3/*3 145 146 h hraoeoisJournal Pharmacogenomics The Table 3 (continued)

Population Characteristics (n) Hypothesis Genotype OR (95%Cl)a Exposure assessment References

Controls (70) NS Unmatched subjects were selected in order to represent the risk factors of the HCC group in the absence of cancer.m,n (Mean age = 45) Colorectal Cancer Caucasians Cases (78) UGT1A7*2, *3 and *1/*1 None 70 enzymes UGT human of Pharmacogenomics Recruited from Hannover Patients with sporadic *4 alleles were 0.39 (0.17–0.92)o Medical School, Germany. colorectal cancer (CRC) described as low-activity *1/*3 All of Northern German and diagnosed after alleles compared to 2.26 (1.09–4.68)o Caucasoid descent. histological evaluation of *1 allele. *2/*3 endoscopic surgical specimens. 2.39 (1.15–4.99)o (Mean age = 63 years) *3/*3 Controls (210) NS Unmatched healthy blood donorso. Guillemette C (Mean age = 48 years)

a OR=odds ratio; CI=confidence interval. b Unconditional logistic regression adjusted for age. cData represent the relative risk (RR). A conditional logistic regression adjusted for the following matching variables was applied: age, menopausal status, postmenopausal hormone use, date at blood draw, time at blood draw and fasting status at blood draw. d Similar adjusted RR were observed with a conditional logistic regression adjusted for the matching variables and age at menarche, parity, age at first birth, BMI at age 18, weight gain since age 18, benign breast disease, first-degree family history of breast cancer, and duration of postmenopausal hormone use. eUnconditional logistic regression adjusted for the matching variables, and age at menarche, parity, age at first birth, BMI at age 18, weight gain since age 18, age at menopause, benign breast disease, first- degree family history of breast cancer, and duration of postmenopausal hormone use. fMatched for all variables mentioned above (d ) and for laboratory batch. gBased on in vitro data.98 h Genotypes were stratified according to predicted activities based on allele-encoded isozyme glucuronidating activities toward benzo[>a]pyrene metabolites (Guillemette et al.65,66 ). iNS=not significant. jUnconditional logistic regression was performed and adjusted for age, sex, race, region of recruitment, smoking and alcohol consumption. kAllelic distribution followed Hardy–Weinberg equilibrium. lThey were also positive for HBV surface antigen (21%), HCV RNA (26%) and diagnosed for alcoholic cirrhosis (15%). m They were also positive for HBV surface antigen (20%), HCV RNA (33%) and diagnosed for alcoholic cirrhosis (13%). n A deviation from the Hardy–Weinberg equilibrium was noted in this "control" group. oLogistical regression analysis adjusted for sex. pA deviation from the Hardy–Weinberg equilibrium was noted. Allelic distribution was significantly different from the control group in Vogel et al.,69 although patients were recruited from the same geographical area. Pharmacogenomics of human UGT enzymes C Guillemette 147

UGT1A6 hormone replacement therapy, did not reveal any signifi- Common UGT1A6 Polymorphisms cant increase in the risk of developing adenoma in subjects UGT1A6 is a phenol-metabolizing UGT, primarily involved carrying variant UGT1A6 alleles. However, the UGT1A6 in the glucuronidation of simple phenols and planar genotype was shown to modulate colon adenoma risk in arylamines5,93 that mediates the conjugation of many aspirin users (OR ¼ 0.53 (95% CI ¼ 0.33–0.86)) and nonas- currently prescribed drugs including antidepressants, neu- pirin NSAID users (OR ¼ 0.47 (95% CI ¼ 0.24–0.92)) when roleptics and b-adrenoreceptor blockers.93,129–134 The compared to aspirin and nonaspirin NSAID users carrying UGT1A6 gene has been found to be polymorphic with at the UGT1A6 wild-type genotype, respectively. These results least four alleles characterized by three SNPs in the coding suggest that the UGT genotype modulates the efficacy of sequence. The unmodified ‘wild-type’ allele is referred to as chemopreventive drugs, such as NSAID. UGT1A6*1, while the allele characterized by the presence of 181 both missense polymorphisms at codons 181 (Thr Ala) UGT1A7 184 130 and 184 (Arg Ser) is referred as UGT1A6*2. Although Common UGT1A7 Polymorphisms these two mutations are usually linked, both mutations UGT1A7 metabolizes a vast panel of clinically and toxico- have been observed separately. A single mutation at codon logically important compounds. The genetic diversity of the 184 is designated UGT1A6*3 and a single mutation at codon UGT1A7 gene was revealed after resequencing the entire 130,135 181 is designated UGT1A6*4 (Table 4). Functional gene.65 Four forms of the UGT1A7 gene product were 130 studies by Ciotti et al revealed that the protein encoded initially found and more recently, five additional forms by the UGT1A6*2 allele has lower activity towards several were discovered. There are now nine alleles of the UGT1A7 phenolic compounds (27–75% of the rate of the wild-type gene, which vary at the following positions: Gly115Ser, enzyme). Also, 3-O-methyl-dopa and methyl salicylate were Asn129Lys, Arg131Lys, Glu139Asp and Trp208Arg (UGT1A7*1– conjugated at 41–74% of the wild-type rate, whereas a series UGT1A7*9)65,137 (Figure 3). The three nonsynonymous SNPs of b-blockers were metabolized at 28–69% of the normal at codons 129/131 and 208, two of which are tightly linked level. The clinical implications of these variant alleles still (129 and 131), create the variant allele UGT1A7*3, com- 130 remain to be determined. The data reported by Ciotti et al monly observed in the population (B17% of *3/*3). The has suggested that the UGT1A6*2 low-activity allele is UGT1A7*2 allele is composed of the linked Asn129Lys/ frequent in the population (frequency of 0.173). The impact Arg131Lys polymorphisms, whereas the UGT1A7*4 allele of single substitutions at codons 181 (*4) and 184 (*3) was carries only the Trp208Arg mutation. The additional genetic not assessed in vitro. The allelic distribution as a function of alterations at codons 115 and 139 were found to be less ethnicity was further assessed in a second study conducted common compared to the UGT1A7*3 allele within the 135 by Lampe et al. They reported higher frequencies of the general population (Table 4). UGT1A6*2 and UGT1A6*3 alleles compared to previous Since UGT1A7 is an important extrahepatic UGT that studies, with distributions being slightly different between inactivates a variety of precarcinogens, including hydro- Caucasian and Asian populations (Table 4). In a population xybenzo[a]pyrene metabolites, activities for variant composed of healthy Caucasian subjects (n ¼ 103), frequen- UGT1A7 recombinant enzymes (*1–*4) were initially de- cies of 0.010 and 0.024 were observed for the alleles scribed for benzo(a)pyrene (BaP) metabolites. Functional UGT1A6*3 and UGT1A6*4 and the genotype frequencies assays were subsequently performed for UGT1A7 variant were found to be in Hardy–Weinberg equilibrium (Table 4). alleles *1–*9 for a variety of substrates.65,137 Regardless of the The combined distribution of polymorphisms in the substrate tested, the UGT1A7 enzyme velocity was shown to TATA-box region of the UGT1A1 promoter and polymorph- be altered in the presence of polymorphisms at codons 115 135 isms in UGT1A6 was assessed in the study of Lampe et al. and 208, regardless of the presence of the mutations A significant linkage disequilibrium between genotypes for affecting codons 129/131 or 139. UGT1A1*28 and UGT1A6*2 was observed in both Caucasian and Asian populations. Homozygous variants were observed Role of UGT1A7 Variants in the Risk of Chemical-induced in 8% of the cases and 43% of individuals had at least one Cancers (Orolaryngeal and Lung Cancers) variant allele for both UGT1A1*28 and UGT1A6*2. There- UGT1A7 represents one of several UGTs that were shown to fore, for compounds metabolized by these two hepatic UGT conjugate chemical carcinogens. UGT1A7 has been impli- enzymes, neither of them would theoretically be able to cated in the glucuronidation of carcinogenic metabolites of compensate for the deficiency of the other enzyme since tobacco-specific nitrosamines138,139 and BaP.65,140 The ex- low-activity UGT1A6 and UGT1A1 alleles are closely linked. pression pattern of UGT1A7 is also consistent with a role in the protection against these chemicals. UGT1A7 is expressed Modulation of Colon Adenoma Risk by UGT1A6 Genotypes in extrahepatic tissues with high levels in the stomach and The group of Bigler et al136 have explored the role of the esophagus,68,138 as well as in other tissues of the UGT1A6 polymorphic variations in relation to the risk of aerodigestive tract including the tongue, tonsil, floor of developing colon adenoma. In their population-based study, the mouth and larynx.68,141 474 adenoma cases and 563 control subjects were genotyped Owing to the expression pattern of UGT1A7 and its for the variations at codons 181 and 184 of UGT1A6. contribution in carcinogen detoxification, the potential role Regression analyses, adjusted for age, sex, smoking and of UGT1A7 in the risk of developing tobacco-related cancers,

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more specifically orolaryngeal cancer, was recently ex- UGT1A7 *1/*2 and *2/*3 genotypes were at higher risk of plored.68 In a population-based case–control study, 125 HCC, OR ¼ 8.46 (95% CI ¼ 2.69–26.57) and OR ¼ 7.61 (95% Caucasian-American and 69 African-American subjects CI ¼ 2.08–27.84), respectively (Table 3). Based on the diagnosed with orolaryngeal cancer and 250 Caucasian- biological function of the UGT1A7 alleles and taking into American and 138 African-American control subjects were account the significant association between the UGT1A7*1/ genotyped for the presence of the UGT1A7*1–*4 alleles. In *2 and *2/*3 genotypes and HCC, one would have expected control subjects, the frequencies of the UGT1A7 alleles were the *3/*3 genotype to be associated with an increased risk of similar to those previously reported for Caucasians and HCC. However, no association with this genotype was followed Hardy–Weinberg equilibrium.65 In this study, the revealed in this study. Similarly, the UGT1A7*3/*3 allele authors demonstrated racial differences, with the UGT1A7*2 was not associated with colorectal cancer in the study allele occurring less frequently and the UGT1A7*3 allele conducted by Strassburg et al,70 although heterozygous occurring at a higher frequency in Caucasians compared to genotypes containing the UGT1A7*3 allele (*1/*3 and *2/ African-Americans. Since the isoenzymes encoded by *3) were associated with an approximately two-fold in- UGT1A7*3 and *4 are significantly less active against BaP- creased risk of developing colorectal cancer (Table 3). These hydroxylated metabolites compared to the enzymes en- findings were based on a case–control study composed of coded by the *1 and *2 alleles,65 genotypes of UGT1A7 were 210 Caucasian controls and 78 cases with colorectal cancer stratified into three categories based on their predicted recruited in the same area.70 Imbalance of the UGT1A7 phenotypes to assess the potential role of UGT1A7 alleles in genotypes was noted in the control group and allele the risk of developing cancer. UGT1A7*3 and *4 alleles, both frequencies were significantly different compared to the containing the Arg208 polymorphism, represented low- study of Vogel et al,69 although subjects of both studies were activity alleles while UGT1A7*1 and *2 alleles were con- recruited in the same geographical region. The rationale of sidered to be high-activity alleles. Unconditional regression studying UGT1A7 in relation with colorectal cancer is analysis was used to account for potential confounding mostly based on the role of this enzyme in the metabolism factors, such as sex, age, race, region of recruitment, and detoxification of dietary heterocyclic amines. The smoking and alcohol consumption. A statistically observation that the *3/*3 genotype is not associated with significant increase in the risk for orolaryngeal cancer an increased risk of developing HCC and colorectal cancer is was observed among all subjects who had the predicted intriguing and remains unexplained. lowest-activity UGT1A7 genotypes compared to subjects A potential limitation in some of these studies includes who had the highest-activity genotypes (OR ¼ 3.7; the fact that confounding factors such as age, race and sex of 95% CI ¼ 1.7–7.6) (Table 3). In addition, the risk was subjects in addition to region or institution of case higher among African-American subjects compared to recruitment were often not used as matching criteria Caucasian subjects (OR ¼ 6.2, 95% CI ¼ 1.2–31 compared between cases and controls. For example, since patient age to OR ¼ 2.8, 95% CI ¼ 1.1–7.6). Detailed information is a definite risk factor for colorectal cancer, controls have to was available for environmental exposure, which enabled be matched to cases for this criterion. Also in many of the authors to assess the association between UGT1A7 these studies, additional confounding factors including genotype and the degree of exposure. Within smoking smoking status and dietary exposures were not examined categories (light or heavy smokers), the risk for orolaryngeal in terms of gene : environment interactions. Such interac- cancer for subjects with predicted low-activity UGT1A7 tions are important factors that could potentially bias genotypes was 8.5- to 10-fold higher compared to subjects the results of risk assessment analysis of genetic variants with the predicted high-activity genotypes. These results since variability in the inherent metabolic activity of revealed for the first time that genetic variations in the enzymes such as UGTs against environmental carcinogens UGT1A7 gene that reduce carcinogen-detoxifying activity is being tested in such studies. Failure to find that genotype increase the risk of developing a smoking-related orolar- frequencies among controls are in Hardy–Weinberg yngeal cancer. equilibrium draws attention to potential bias and caution in the interpretation of the results of the association Role of UGT1A7 Variants in the Risk of Hepatocellular study.142 Optimally, population-based studies should be and Colorectal Cancers performed with a particular attention to these confounding The relation between polymorphic UGT1A7 and hepatocel- factors. lular cancer (HCC) risk was investigated by Vogel et al.69 Recently, a case–control study was conducted to assess the There is a strong epidemiological link between hepatitis B relation between characteristics of meat consumption, HCA virus (HBV) and hepatitis C virus (HCV) infections, liver exposure, the UGT1A7 genotype and colon cancer. A total of cirrhosis and hepatocarcinogenesis. Thus, the authors 400 subjects with colon cancer and 400 controls matched included 70 Caucasian subjects with HBV and HCV infec- for age, sex and race were recruited. In this population, the tions or alcoholic cirrhosis as the ‘control’ population and allele frequencies were similar to those obtained pre- 59 subjects with HCC. Allele frequencies were significantly viously.65,68 No main effect of the UGT1A7 genotype was different from previous studies in Caucasians and a devia- observed on colon cancer risk. On the other hand, the tion from the Hardy–Weinberg equilibrium was also evident association between dietary HCA exposure and colon cancer in the control group. In this study, subjects with the was increased in individuals with the low-activity UGT1A7

The Pharmacogenomics Journal Pharmacogenomics of human UGT enzymes C Guillemette 149

genotypes.143 These data suggest that the relation between if this polymorphism led to variable rates of glucuronida- dietary sources of HCA and colon cancer may be modulated tion, a positive association would have been expected. Patel by the UGT1A7 detoxification pathway. These results also et al24 once suggested the potential role of UGT2B7 point to HCA exposure as an important etiologic factor in polymorphisms in explaining interindividual differences in colon cancer. the biotransformation of oxazepam. However, the study of Coffman et al74 revealed that oxazepam does not appear to be a good substrate for both forms of the UGT2B7 enzyme, UGT2B7 which present similar catalytic efficiencies towards the Common UGT2B7 Polymorphisms molecule. Court et al153 recently confirmed this finding by UGT2B7 is one of the most important hepatic UGTs that showing that UGT2B15 is the main UGT involved in metabolizes a vast set of clinically, physiologically and oxazepam glucuronidation. Although UGT2B7 is the major toxicologically important compounds. The UGT2B7 protein UGT involved in the glucuronidation of epirubicin, the is also found in the brain, kidney, pancreas, mammary reported polymorphism at codon 268 in the UGT2B7 gland, lung and gastrointestinal tract, and several additional protein did not alter the formation rate of epirubicin tissues.80,134,138,144–146 A cytosine to thymine polymorphism glucuronide.154 at base pair 802, leading to a histidine (H)268 to tyrosine (Y) Barbier et al,150 reported a significant difference in the amino-acid substitution (UGT2B7*2), has been identi- ability of UGT2B7 alleles to conjugate zidovudine. The H fied.147,148 Data from large genotyping studies suggest and Y alleles had similar K values, but a 5.6-fold higher that approximately one-third of the Caucasian population m V was observed for the H allele compared to the Y allele. expresses the variant UGT2B7*2/*2 genotype (Table 5).46,149 max This result suggests a substrate-specific impact of this amino- The prevalence of the UGT2B7*2 allele appears to be lower acid variation. However, the clinical relevance of this in Asian individuals with only 5% of the Japanese popula- polymorphism for the metabolism of zidovudine remains tion being homozygous for the UGT2B7*2/*2 genotype to be demonstrated.150 It is clear that UGT2B7 is of major (Table 5).46 significance for the glucuronidation of a number of The in vitro glucuronidation activities of the most frequent clinically important drugs and further studies are needed allele (UGT2B7*1) and of the UGT2B7*2 variant allele have to uncover genetic variants of this gene and address their been thoroughly investigated by different groups.74,147,150 clinical effects. Coffman et al74 compared both isoenzymes in the same experimental conditions using HEK 293 cell lines that stably Polymorphic Expression of the UGT2B7 Gene in Breast express the *1 and *2 alleles. Reactivity towards androgenic Carcinogenesis steroids, xenobiotics, including morphinan derivatives and In addition to being involved in the metabolism of a variety menthol, propranolol and oxazepam was studied.74 of xenobiotic compounds, UGT2B7 is active on endobiotics Although the amino-acid change at codon 268 is a such as steroid hormones including androgens, estrogens, nonconservative substitution, the *2 allele (Tyr268) was pregnanes, biliary acids and retinoic acids.146–148,151,155 found to present similar catalytic properties compared to Recently, the expression of UGT2B7 was demonstrated in the *1 allele (His268). normal human breast parenchyma and in invasive and in situ breast cancers.73 A previous study reported that the No Significant Clinical Impact of the UGT2B7 Codon 268 glucuronidation activity was lower in breast cancer speci- Polymorphism mens compared to normal tissues.156 Using a specific anti- Therapeutic drugs that are glucuronidated by UGT2B7 UGT2B7 antibody, Gestl et al73 demonstrated that UGT2B7 include morphine (at position 3-OH and 6-OH), zidovudine, is expressed in the epithelium lining the mammary gland epirubicin and carboxylic acid-containing compounds such ductal system with variable intensities between individuals. as NSAIDS, clofibric acid and valproic acid.134,146,150,151 A variation in the expression of UGT2B7 among epithelial Functional studies suggested that the polymorphic varia- cells of the same tissue was also observed. In cancer cells, the tions in the coding region of the UGT2B7 gene do not result staining within invasive lesions was virtually absent while in in variable rates of glucuronide formation and/or altered situ lesions presented a high level of UGT2B7 expression. In affinity of the enzyme. Thus, this polymorphism cannot the same study, the levels of 4-hydroxyestrone glucuroni- account for the observed variability in biotransformation of dating activity were also assessed in normal tissues from the therapeutic drugs, which are substrates of UGT2B7 in mammoplastic surgeries and in normal and cancerous humans. tissues from neoplastic specimens. The glucuronidation rate A recent study by Holthe et al49 failed to show that the of estrogen was significantly lower in neoplastic tissues UGT2B7 H268Y polymorphism is associated with a variation compared to normal tissues, which is consistent with in the plasma ratios of morphine-3-G/morphine-6-G, mor- UGT2B7 expression data. The authors suggested an anti- phine-3-G/morphine or morphine-6-G/morphine among neoplastic function of the UGT2B7 within the mammary cancer patients undergoing analgesic therapy with mor- gland based on its role in the conjugation of estrogens and phine, thus confirming the results of an other group.46 retinoic acids. Interestingly, because of the increased in situ UGT2B7 is a major UGT responsible for the 3- and 6- expression of UGT2B7 in neoplastic foci, a preinvasive stage glucuronidation of morphine in humans;74,151,152 therefore, of carcinogenesis, a defense mechanism involving

www.nature.com/tpj 150 h hraoeoisJournal Pharmacogenomics The Table 4 Polymorphic variations of the UGT1A6, UGT1A7 and UGT1A8 genes

Allele frequency a Genotype References frequency (%)a

UGT1A6 n *1 *2 *3 *4 *1/*1 *1/*2 *2/*3 *1/*3 *1/*4 *2/*4

Amino-acid change T181 R184 A181 S184 T181 S184 A181 R184

Functional change ‘Wild type’ Decreased Not tested Not tested activity hraoeoiso ua G enzymes UGT human of Pharmacogenomics Population characteristics Caucasian (American) 202 0.673 0.307 0.020 — — — — — — — 135 Caucasian (American)b 103 0.669 0.296 0.010 0.024 45 37 10 2 4 1 g Asian (American) 31 0.758 0.226 0.016 — — — — — — — 135 Mixed population 98 0.821 0.173 0.005 — 65 34 1 — — — 130

UGT1A7 n *1 *2 *3 *4 *5–*9 Highc Intermediated Lowe

Amino-acid change N129 R131 W208 K129 K131 W208 K129 K131 R208 N129 R131 R208 Guillemette C

Functional change ‘Wild type’ Similar Decreased Decreased Variable activity activity activity activity

Population characteristics Caucasian (American)b 144 0.358 0.264 0.361 0.017 — 15 68 17 66 Caucasian (American) 245 0.420 0.240 0.320 0.010 — — — — 68 Caucasian (French Canadian)c 150 0.340 0.340 0.230 0.010 0.100 10 73 5 g African (American) 136 0.380 0.390 0.230 0.010 — — — — 68 Japanese 108 0.592 0.153 0.255 0.000 — 36 18 7 63

UGT1A8 n *1 *2 *3 *1/*1 *1/*2 *1/*3 *2/*2

Amino-acid changes A173 C277 G173 C277 A173 Y277

Functional change ‘Wild type’ Similar Decreased activity activity

Population characteristics Caucasian (French Canadian) 82 0.760 0.230 0.010 45 37 2 11 g Caucasian (American) 69 0.551 0.145 0.022 39 17 3 3 162

a The ‘—’ mean the data was not available in the original manuscript. b Recruited from the Boston area (USA). cHigh-activity genotype: UGT1A7 *1/*1. d Intermediate-activity genotypes: UGT1A7 *1/*2, *1/*3, *1/*4, *2/*2, *2/*3, *2/*4. eLow-activity genotypes: UGT1A7 *3/*3, *3/*4, *4/*4. fFor the genotype frequency, the 12% difference represents all the other genotypes (*1/*6, *1/*7, *1/*8, *2/*6, *2/*7, *2/*8, *2/*9, *2/*10, *3/*6, *3/*7, *4/*7, *5/*5 and *5/*9). Alleles are defined in Figure 2. gGuillemette et al, unpublished data. Pharmacogenomics of human UGT enzymes C Guillemette 151

the overexpression of UGT2B7 in these cells was also tissues such as the prostate, differences in the activity of this suggested. The study of Gestl et al73 also demonstrated the androgen-inactivating enzyme could result in a modified cell-type-specific expression of the UGT2B7 gene product degree of exposure of prostate cells to DHT. and the possible regulation of expression in normal and Two independent epidemiological studies have deter- cancerous tissues. A previous study by Barbier et al106 mined the potential role of the UGT2B15 gene in relation reported such a cell-type-specific expression of UGT2B to prostate cancer risk (Table 5).71,72 The first study described enzymes in the human prostatic tissue. The role of UGT2B7 the prevalence of variant UGT2B15 alleles in a case–control polymorphisms in relation to breast cancer risk has not been population of 64 patients with pathologically confirmed reported. prostate cancer and 64 controls recruited in the state of Arkansas.72 Controls were matched for age, race and county UGT2B15 of residence. The UGT2B15*1 allele occurred at a signifi- Common UGT2B15 Polymorphisms cantly high frequency (40.6%) in prostate cancer cases 2 UGT2B15 is involved in the inactivation of steroid hor- compared to controls (18.8%) (w ¼ 7.34; P ¼ 0.007). The risk mones, mainly androgens, and also displays a glucuronidat- of developing cancer in patients homozygous for the wild- 85 ing activity toward several classes of xenobiotic substrates, type D (*1) low-activity allele was three-fold higher including simple phenolic compounds, 7-hydroxylated [OR ¼ 3.0; IC 95% ¼ 1.3–6.5]. However, no trend was coumarins, flavonoids, anthraquinones and other drugs observed with the heterozygous UGT2B15 genotype. In a 71 and their hydroxylated metabolites.96,97,153,157 AG-T second study, Gsur et al genotyped 380 Caucasian men substitution at codon 85 results in an amino-acid change from Vienna (Austria), 190 cases with prostate cancer and (Asp85Tyr) within the putative substrate recognition site of 190 controls with lower urinary tract symptoms because of UGT2B15. A number of population genotyping studies were benign prostatic hyperplasia. The authors did not observe 85 completed to characterize the distribution of UGT2B15*1 any association between the D Y polymorphism and and *2 alleles among individuals of distinct ethnic back- prostate cancer. Based on these two studies, it remains ground (Table 5). UGT2B15*1 is less frequent in Caucasians unclear if UGT2B15 plays a significant role in altering compared to other populations (Asian, Hispanic and intraprostatic DHT levels and if this leads to a modified risk African). Overall, homozygous subjects for the variant *2 of prostate cancer. allele were found in 19–32% of the population. The functional impact of UGT2B15 polymorphisms on enzyme ADDITIONAL COMMON ALLELIC VARIANTS OF THE UGT1 activity was studied in vitro using intact cells (in situ assays) AND UGT2B GENES AND THEIR FUNCTIONAL IMPACT IN and microsomal preparations. These analyses revealed that VITRO the UGT2B15*2 allele presents similar Km values and UGT1A8 approximately two-fold different Vmax values for androgens Unlike most UGTs, UGT1A8 is not found in the liver and is (dihydrotestosterone (DHT)) and androstane-3alpha,17beta- predominantly expressed in different tissues of the intest- diol (3a-Diol)) in in situ cell assays.96 In a recent investiga- inal tract.102 UGT1A8 demonstrates a high reactivity tion, the catalytic properties of UGT2B15 variants were towards numerous endogenous and exogenous substrates assessed with cellular fractions of the same HEK 293 cells. including dietary and environmental carcinogens and The conjugating activity of the UGT2B15*1 allele for S- drugs.2,90,160,161 A recent study has shown that at least three oxazepam was five-fold higher than that of UGT2B15*2.153 alleles of the UGT1A8 gene exist: UGT1A8*1, the most As suggested by the authors, these differences could reflect frequent allele; UGT1A8*2, with a mutation at codon 173 differential stabilities of UGT2B15 proteins under the (Gly173Ala) and UGT1A8*3, bearing an amino-acid change experimental conditions used for microsome preparation at codon 277 (Cys277Tyr).162 These variants were discovered as opposed to enzymatic assays in intact cells.96,153 Never- after resequencing exon-1 from UGT1A8 in a population of theless, the role of UGT2B15 in the interindividual varia- 69 individuals. The observed frequencies were 0.55 (*1), 0.15 bility in S-oxazepam glucuronidation remains to be (*2) and 0.02 (*3). The altered function of variant UGT1A8 determined in vivo. Of interest, an additional polymorphic proteins was assessed by transfection of the corresponding site was also reported at codon 86 adjacent to the 85 cDNAs in HEK 293 cells to examine the catalytic activity on polymorphism (Figure 3). The function of this polymorph- substrates such as hydroxybenzo[a]pyrene metabolites and a ism has not been studied yet.158,159 number of additional simple phenols. Similar catalytic properties were observed for the UGT1A8*1 and *2 alleles, Potential Role of the UGT2B15 Gene in the Risk of whereas the tyrosine at codon 277 in UGT1A8*3 led to an Developing Prostate Cancer inactive protein. At this time, the clinical impact of a Among the androgen-metabolizing UGT2B enzymes, polymorphic UGT1A8 allele such as the relatively rare namely UGT2B28, UGT2B15 and UGT2B17, UGT2B15 is UGT1A8*3 remains to be elucidated. the most efficient UGT2B enzyme involved in the metabolism of the main metabolite of DHT, namely 3a-Diol.144 UGT2B4 Variations in androgen levels have been associated with the UGT2B4 plays a role in the metabolism of bile acids and risk of developing prostate cancer. Since UGT2B15 plays an detoxification of many phenols.98,144,148,155,163–169 UGT2B4 important role in the metabolism of androgens in target is expressed in the liver and in a wide range of extrahepatic

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Table 5 Polymorphic variations of the UGT2B4, UGT2B7 and UGT2B15 genes

Allele frequency Genotype (%)a References

UGT2B4 n *1 *2 *1/*1 *1/*2 *2/*2

Amino-acid change D458 E458

Functional change ‘Wild type’ Similar activity

Population characteristics Caucasian (French Canadian) 26 0.620 0.380 30 62 8 98 Caucasian (American) 273 0.815 0.185 — — — 101 Caucasian (American) 202 0.750 0.250 58 34 8 149 African (American) 188 0.832 0.168 — — — 101 Hispanic (American) 187 0.719 0.281 — — — 101 Asian (American) 32 1.000 0.000 100 0 0 149 Chinese (American) 78 1.000 0.000 — — — 101 Japanese (American) 77 1.000 0.000 — — — 101

UGT2B7 n *1 *2 *1/*1 *1/*2 *2/*2

Amino-acid change H268 Y268

Functional change ‘Wild type’ Similar or decreased activity

Population characteristics Caucasian (American) 202 0.460 0.540 22 49 29 149 Caucasian 91 0.510 0.490 28 47 25 46 Asian (American) 32 0.730 0.270 56 34 10 149 Asian (Japanese) 84 0.730 0.270 51 44 5 46

UGT2B15 n *1 *2 *1/*1 *1/*2 *2/*2

Amino-acid change D85 Y85

Functional change ‘Wild type’ Increased activityb

Population characteristics Caucasian (French Canadian) 27 0.480 0.520 19 59 22 96 Caucasian (American) 273 0.448 0.552 — — — 101 Caucasian (American) 202 0.450 0.550 22 46 32 149 African (American) 188 0.614 0.386 — — — 101 Hispanic (American) 187 0.630 0.370 — — — 101 Asian (American) 32 0.640 0.360 47 34 19 149 Chinese (American) 78 0.583 0.417 — — — 101 Japanese (American) 77 0.513 0.487 100 0 0 101 aThe ‘—’ mean the data was not available in the original manuscript. b(two-fold) for androgens (in situ assays)

tissues.98,144 One common polymorphism has been found 458 is unlikely to alter rates of glucuronidation. This in the UGT2B4 gene and the resulting variant allele is assumption is based on in vitro data, which suggest no designated as UGT2B4 Asp458Glu (UGT2B4*2).98 The single functional impact of the variation at position 458 on gene amino-acid change from aspartate to glutamate at codon product function when assessed with bile acids, phenol 458 is located in the putative cosubstrate binding domain. derivatives and catecholestrogens as substrates.98 An addi- The UGT2B4*2 allele is found at a frequency varying tional cDNA clone isolated from human liver corresponds to between 0.17 and 0.38 in the Caucasian, African and the UGT2B4 Phe109Leu, Phe396Leu allele165 and appears to Hispanic populations, while it is absent from the Asian be very rare as it was not found in 272 individuals that were population.98,101,149 The minor amino-acid change at codon genotyped in two independent studies.98,149

The Pharmacogenomics Journal Pharmacogenomics of human UGT enzymes C Guillemette 153

UGT2B28 phenylimidazo[4, 5-b]pyridine by human microsomal UDP-glucuro- UGT2B28 conjugates endogenous substrates including ster- nosyltransferases: identification of specific UGT1A family isoforms oids and bile acids, and exogenous molecules such as 4- involved. Carcinogenesis 1999; 20: 1107–1114. 99 3 Bartsch H, Petruzzelli S, De Flora S, Hietanen E, Camus AM, methylumbelliferone. UGT2B28 demonstrated much low- Castegnaro M et al. Carcinogen metabolism in human lung tissues er intrinsic clearances (Vmax/Km) towards multiple steroid and the effect of tobacco smoking: results from a case–control molecules compared to UGT2B15 and UGT2B17. Differen- multicenter study on lung cancer patients. Environ Health Perspect tial tissue expression profiles of two truncated UGT2B28 1992; 98: 119–124. 4 Jin CJ, Miners JO, Burchell B, Mackenzie PI. The glucuronidation of variants (types II and III) probably resulting from exon hydroxylated metabolites of benzo[a]pyrene and 2-acetylaminofluor- skipping, has been reported.99 UGT2B28 type II differs from ene by cDNA-expressed human UDP-glucuronosyltransferases. Carci- type I by a deletion of 308 bp (103 amino acids) in the nogenesis 1993; 14: 2637–2639. cofactor-binding domain, whereas UGT2B28 type III lacks 5 Orzechowski A, Schrenk D, Bock-Hennig BS, Bock KW. Glucuronida- tion of carcinogenic arylamines and their N-hydroxy derivatives by rat 351 bp (117 amino acids) in the putative substrate-binding and human phenol UDP-glucuronosyltransferase of the UGT1 gene domain. The expression of active UGT2B28 type I protein complex. Carcinogenesis 1994; 15: 1549–1553. was limited to the breast and liver, as well as to the prostatic 6 Bock KW, Schirmer G. Species differences of glucuronidation and cell line LNCaP. The biological function of the shorter sulfation in relation to hepatocarcinogenesis. Arch Toxicol Suppl 1987; 10: 125–135. UGTB28 isoforms has yet to be elucidated. 7 Stillwell WG, Turesky RJ, Sinha R, Tannenbaum SR. N-oxidative metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in humans: excretion of the N2-glucuronide conjugate of 2- CONCLUDING REMARKS hydroxyamino-MeIQx in urine. Cancer Res 1999; 59: 5154–5159. In summary, a high degree of interindividual variability in 8 Vienneau DS, DeBoni U, Wells PG. Potential genoprotective role for the glucuronidation rates of numerous compounds has been UDP-glucuronosyltransferases in chemical carcinogenesis: initiation of described. While genetic polymorphism is a possible cause micronuclei by benzo(a)pyrene and benzo(e)pyrene in UDP-glucuronosyltransferase-deficient cultured rat skin fibroblasts. Cancer Res for such phenotypic variations, alterations in glucuronida- 1995; 55: 1045–1051. tion activity may be related to age, gender, disease, diet or 9 Kim PM, Wells PG. Genoprotection by UDP-glucuronosyltransferases in environmental influences. Additional studies are needed to peroxidase-dependent, reactive oxygen species-mediated micronu- elucidate and characterize polymorphic UGTs to promote cleus initiation by the carcinogens 4-(methylnitrosamino)-1-(3-pyri- dyl)-1-butanone and benzo[a]pyrene. Cancer Res 1996; 56: 1526– the understanding of interindividual variations in the 1532. glucuronidation metabolic pathway, their pharmacological 10 Belanger A, Hum DW, Beaulieu M, Levesque E, Guillemette C, and toxicological importance, and their role in cancer Tchernof A et al. Characterization and regulation of UDP-glucurono- susceptibility. Much research in this area is needed although syltransferases in steroid target tissues. J Steroid Biochem Mol Biol 1998; 65: 301–310. promising leads have emerged, especially regarding the role 11 Hum DW, Belanger A, Levesque E, Barbier O, Beaulieu M, Albert C et of UGT genetic polymorphisms in cancer etiology and on al. Characterization of UDP-glucuronosyltransferases active on steroid their possible implication in modulating the degree of hormones. J Steroid Biochem Mol Biol 1999; 69: 413–423. exposure to several carcinogenic compounds. Additional 12 Liu J, Liu Y, Barter RA, Klaassen CD. Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: a dose–response well-designed case–control population-based studies are study. J Pharmacol Exp Ther 1995; 273: 977–985. needed to generalize these results. Special attention would 13 Dutton GJ. Glucuronidation of Drugs and other Compounds. 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