Chantal Guillemette

Pharmacogenomics of human UDP-glucuronosyltransferase enzymes

ABSTRACTUDP-glucuronosyltransferase (UGT) enzymes comprise a superfamily of key proteins that catalyze the glucuronidation reaction on a wide range of structurally diverse endogenous and exogenous chemicals. Glucuronidation is one of the major phase II drug-metabolizing reactions that contributes to drug biotransformation. This biochemical process is also involved in the protection against environmental toxicants, carcinogens, dietary toxins and participates in the homeostasis of numerous endogenous molecules, including bilirubin, steroid hormones and biliary acids. Over the years, significant progress was made in the field of glucuronidation, especially with regard to the identification of human UGTs, study of their tissue distribution and substrate specificities. More recently, the degree of allelic diversity has also been revealed for several human UGT genes. Some polymorphic UGTs have demonstrated a significant pharmacological impact in addition to being 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.

Structural heterogeneity at the UDP-glucuronosyltransferase 1 locus: functional consequences of three novel missense mutations in the human UGT1A7 gene.

One of the most important mechanisms involved in host defense against xenobiotic chemicals and endogenous toxins is the glucuronidation catalysed by UDP-glucuronosyltransferase enzymes (UGT). The role of genetic factors in determining variable rates of glucuronidation is not well understood, but phenotypic evidence in support of such variation has been reported. In the present study, six single nucleotide polymorphisms were discovered in the first exon of the UGT1A7 gene, which codes for the putative substrate-binding domain, revealing a high structural heterogeneity at the UGT1 gene locus. The new UGT1A7 proteins differ in their primary structure at amino acid positions 129, 131 and 208, creating four distinct UGT1A7 allelic variants in the human population: UGT1A7*1 (N129 R131 W208), *2 (K129 K131 W208), *3 (K129 K131 R208), and *4 (N129 R131 R208). In functional studies, HEK cells stably transfected to express the four allelic UGT1A7 variants exhibited significant differences in catalytic activity towards 3-, 7-, and 9-hydroxy-benzo(a)pyrene. UGT1A7*3 exhibited a 5.8-fold lower relative Vmax compared to wild-type *1, whereas *2 and *4 had a 2.6- and 2.8-fold lower relative Vmax than *1, respectively, suggesting that these mutations confer slow glucuronidation phenotype. Kinetic characterization suggested that these differences were primarily attributable to altered Vmax. Additionally, it suggested that each amino acid substitutions can independently affect the UGT1A7 catalytic activity, and that their effects are additive. The expression pattern of UGT1A7 studied herein and its catalytic activity profile suggest a possible role of UGT1A7 in the detoxification and elimination of carcinogenic products in lung. A population study demonstrated that a considerable proportion of the population (15.3%) was found homozygous for the low activity allele containing all three missense mutations, UGT1A7*3. These findings suggest that further studies are needed to investigate the impact of the low UGT1A7 conjugator genotype on individual susceptibility to chemical-induced diseases and responses to therapeutic drugs.

Direct haplotyping of kilobase-size DNA using carbon nanotube probes

We have implemented a method for multiplexed detection of polymorphic sites and direct determination of haplotypes in 10-kilobase-size DNA fragments using single-walled carbon nanotube (SWNT) atomic force microscopy (AFM) probes. Labeled oligonucleotides are hybridized specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. We demonstrated this concept by detecting streptavidin and IRD800 labels at two different sequences in M13mp18. Our approach also permits haplotype determination from simple visual inspection of AFM images of individual DNA molecules, which we have done on UGT1A7, a gene under study as a cancer risk factor. The haplotypes of individuals heterozygous at two critical loci, which together influence cancer risk, can be easily and directly distinguished from AFM images. The application of this technique to haplotyping in population-based genetic disease studies and other genomic screening problems is discussed.

The Impact of UGT1A8, UGT1A9, and UGT2B7 Genetic Polymorphisms on the Pharmacokinetic Profile of Mycophenolic Acid After a Single Oral Dose in Healthy Volunteers

We studied whether polymorphisms in the UGT1A8, UGT1A9, and UGT2B7 genes, the enzymes producing the phenolic (MPAG) and acyl (AcMPAG) glucuronides of mycophenolic acid (MPA), could contribute to the interindividual variation observed in mycophenolate mofetil (MMF) pharmacokinetics (PKs). This study enrolled 17 healthy volunteers with no polymorphisms (controls) and 17 carriers of UGT1A9 —275/–2152 selected among 305 individuals genetically screened for UDP‐glucuronosyltransferase (UGT) polymorphisms. Additional investigative groups included carriers of UGT1A8*2 (A173G) (n=9), UGT1A8*3 (C277Y) (n=4), and UGT1A9*3 (M33T) (n=5). Genetic analysis also included UGT2B7 to detect UGT2B7*2 (His268Tyr) and the promoter haplotype −1248A>G, −1241T>C, −1054T>C, −842G>A, −268A>G, −102T>C. Kinetics were measured in plasma and urine after a single 1.5 g oral dose of MMF, by high‐performance liquid chromatography coupled with tandem mass spectrometry, over 12 h after drug intake. Compared to controls, MPA exposure was significantly lower for UGT1A9 −275/−2152 carriers, with no significant changes in MPAG. The estimates of enterohepatic (re)cycling (area under the concentration–time curve (AUC6–12 h/AUC0–12 h)) were significantly lower for MPA, MPAG, and AcMPAG in UGT1A9 −275/−2152 subjects. Compared with controls, UGT1A9*3 carriers had higher MPA and AcMPAG exposure, whereas homozygosity for the UGT1A8*2 allele and heterozygosity for UGT1A8*3 allele had no impact on MPA PKs. Compared with UGT2B7*1/*1 individuals (n=10), UGT2B7*2/*2 subjects (n=17) presented significantly higher free MPA Cmax values and elevated free and total MPA. Results indicate that after a single oral dose of MMF in healthy volunteers, specific UGT genotypes significantly alter MPA PKs and this clearly warrants additional studies with complete and detailed genetic profiling of UGT1A8, UGT1A9, and UGT2B7 genes.

A novel functional polymorphism in the uridine diphosphate–glucuronosyltransferase 2B7 promoter with significant impact on promoter activity

To clarify the molecular determinants of the metabolic variability of morphine, we searched for genetic polymorphisms in the gene for uridine diphosphate–glucuronosyltransferase 2B7 (UGT2B7) and evaluated their functional impact in vitro and in patients with cancer receiving long‐term morphine therapy. Genetic analysis revealed the existence of 8 single‐nucleotide polymorphisms (SNPs), 6 of which are tightly linked and are at positions −1248, −1241, −1054, −842, −268, and −102 relative to the hepatic start site. In contrast, an SNP at position −66 occurs independently, whereas a novel variation at position −79 appears to be in linkage disequilibrium with the codon 268 SNP (UGT2B7*2). At least 4 haplotypes were observed in white subjects included in the initial SNP screening. On functional in vitro characterization, promoter‐reporter gene constructs with the −79 variation displayed 2.5‐ to 7‐fold less activity compared with the wild‐type construct in Caco‐2 colon cells and HepG2 hepatoma cells, respectively (P = .015 and P < .001, respectively). To investigate a possible effect of the −79 variation in vivo, serum morphine and morphine glucuronide concentrations were measured by liquid chromatography–mass spectrometry in patients with cancer who received long‐term oral morphine therapy, and subjects were then genotyped for the −79 polymorphism. Among 175 patients with normal hepatic and renal function, 6 were heterozygous for the −79 variation, and the morphine‐6‐glucuronide (M6G)/morphine and morphine‐3‐glucuronide (M3G)/morphine ratios versus those in the 169 noncarriers were 5.9 ± 3.5 versus 7.1 ± 7.0 for M6G/morphine (P = .96) and 31.2 ± 17.1 versus 42.9 ± 31.2 for M3G/morphine (P = .53), respectively. Further studies in larger samples are needed to make conclusions about the possible clinical relevance of the −79 polymorphism in the UGT2B7 gene.

Glucuronidation of the Antiretroviral Drug Efavirenz by UGT2B7 and an in Vitro Investigation of Drug-Drug Interaction with Zidovudine

The non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) is directly conjugated by the UDP-glucuronosyltransferase (UGT) pathway to form EFV-N-glucuronide (EFV-G), but the enzyme(s) involved has not yet been identified. The glucuronidation of EFV was screened with UGT1A and UGT2B enzymes expressed in a heterologous system, and UGT2B7 was shown to be the only reactive enzyme. The apparent Km value of UGT2B7 (21 μM) is similar to the value observed for human liver microsomes (24 μM), whereas the variant allozyme UGT2B7*2 (Tyr268) displayed similar kinetic parameters. Because 3′-azido-3′-deoxythymidine (AZT), one of the most current nucleotide reverse transcriptase inhibitors prescribed in combination with EFV, is also conjugated by UGT2B7, the potential metabolic interaction between EFV and AZT has been studied using human liver microsomes. Glucuronidation of both drugs was inhibited by one another, in a concentration-dependent manner. At Km values (25 and 1000 μM for EFV and AZT, respectively), EFV inhibited AZT glucuronidation by 47%, whereas AZT inhibited EFV glucuronidation by 23%. With a Ki value of 17 μM for AZT-glucuronide formation, EFV appears to be one of the most selective and potent competitive inhibitor of AZT glucuronidation in vitro. Moreover, assuming that concentrations of EFV achieved in plasma (Cmax = 12.9 μM) are in a range similar to its Ki value, it was estimated that EFV could produce a theoretical 43% inhibition of AZT glucuronidation in vivo. We conclude that UGT2B7 has a major role in EFV glucuronidation and that EFV could potentially interfere with the hepatic glucuronidation of AZT.

UGT genomic diversity: beyond gene duplication

The human uridine diphospho (UDP)-glucuronosyltransferase (UGT) superfamily comprises enzymes responsible for a major biotransformation phase II pathway: the glucuronidation process. The UGT enzymes are located in the endoplasmic reticulum of almost all tissues, where they catalyze the inactivation of several endogenous and exogenous molecules, including bilirubin, sex steroids, numerous prescribed drugs, and environmental toxins. This metabolic pathway is particularly variable. The influence of inheritable polymorphisms in human UGT-encoding genes has been extensively documented and was shown to be responsible for a fraction of the observed phenotypic variability. Other key genomic processes are likely underlying this diversity; these include copy-number variations, epigenetic factors, and newly discovered splicing mechanisms. This review will discuss novel molecular aspects that may be determinant to UGT phenotypes.

Characterization of UDP-glucuronosyltransferases active on steroid hormones ☆

In recent years, the enzymes which are involved in the formation of DHT in steroid target tissues have been well investigated, however, enzymes responsible for the catabolism and elimination of steroids in these tissues, in particular the uridine diphospho-glucuronosyltransferase (UGT) family of enzymes, have received much less attention. We have recently demonstrated that human and monkey are unique in having high plasma levels of C19 steroid glucuronides. These circulating conjugates have been proposed to reflect the peripheral conversion of adrenal and gonadal C19 steroids to potent androgens, especially DHT. In humans, the presence of steroid UGT activities is found in the liver and several extrahepatic tissues including the prostate, mammary gland and ovary. In addition, UGT activities were observed in breast and prostate tumor cell lines such as MCF-7 and LNCaP, respectively. In agreement with the presence of steroid conjugating enzymes in extrahepatic tissues, UGT cDNA clones, which encode steroid conjugating proteins, have been isolated from libraries constructed from human and monkey prostate mRNA. The presence of UGT transcripts and proteins in extrahepatic tissues in both species, as determined by Northern blot, ribonuclease protection, specific RT-PCR, in situ hybridization, Western blot and immunocytochemistry analysis, indicate the relevance of steroid glucuronidation in tissues other than the liver. Knowing that both the human prostate and the human prostate cancer LNCaP cell line express steroid metabolizing proteins, including UGT enzymes, regulation of UGT mRNA and protein levels, as well as promoter activity was studied in these cells. The results demonstrate a differential regulation between the two highly related isoforms UGT2B15 and UGT2B17, where only the expression of UGT2B17 was affected following treatments of LNCaP cells with androgens, growth factors or cytokines. Steroid conjugation by UGT enzymes is potentially involved in hormone inactivation in steroid target tissues, thus modifications in UGT expression levels may influence hormonal responses.

Characterization and regulation of UDP-glucuronosyltransferases in steroid target tissues

Conjugation of compounds by glucuronidation is a pathway found in all vertebrates studied to date. Although, it is widely recognized that the liver is a major site of glucuronidation, it is now clear that extrahepatic tissues are also involved in the conjugation of compounds to which these tissues are exposed. High levels of androsterone glucuronide and androstane-3alpha,17beta-diol glucuronide found in the human prostate, breast cyst fluid and ovary follicular fluid suggest that glucuronidation of 5alpha-reduced C19 steroids occurs in these tissues. Recently, we have reported the tissue distribution of UGT2B15, which can conjugate steroids in several human extrahepatic steroid target tissues including the skin, breast and prostate. We have also isolated a new UGT2B cDNA encoding UGT2B17, that conjugates ADT which is the major 5alpha-reduced C19 steroid glucuronide in the circulation of humans. UGT2B17 is also widely distributed in several human steroid target tissues. This gene was mapped to human chromosome 4q13 and has an exon/intron structure similar to that of rat UGT2B1 and UGT2B2. Both UGT2B15 and UGT2B17, which are able to catalyze the glucuronidation of DHT, are expressed in LNCaP cells. Interestingly, glucuronidation of steroids is markedly regulated by several factors including androgens and growth factors. Treatment of LNCaP cells with dihydrotestosterone (DHT) and epidermal growth factor (EGF) caused a decrease of DHT glucuronidation and UGT2B mRNA levels. RNase protection assays showed a specific decrease of UGT2B17 transcript in LNCaP cells treated with DHT and EGF however, the level of UGT2B15 mRNA was not affected. As well, Western blot analysis demonstrated a diminution of UGT2B17 protein level in response to DHT and EGF. These results demonstrate a differential regulation of different isoforms of steroid conjugating UGTs present in human prostate LNCaP cells. In addition, UGT2B17 was shown to be more labile than UGT2B15 indicating that regulation of UGT2B17 expression would lead to a more rapid change in the level of glucuronidated steroids. Expression of exogenous UGT2B17 in LNCaP cells by gene transfer led to a significant decrease in the androgen response. This result indicates the ability of UGT enzymes to regulate the androgen response by conjugating androgens which abolishes their interaction with their receptor and facilitates their clearance from the cell. The glucuronidation of steroids by UGT enzymes is an important mechanism by which the levels of steroids is regulated in steroid target tissues.

Metabolic inactivation of estrogens in breast tissue by UDP-glucuronosyltransferase enzymes: an overview

The breast tissue is the site of major metabolic conversions of estradiol (E2) mediated by specific cytochromes P450 hydroxylations and methylation by catechol-O-methytransferase. In addition to E2 itself, recent findings highlight the significance of 4-hydroxylated estrogen metabolites as chemical mediators and their link to breast cancer development and progression, whereas, in opposition, 2-methoxylated estrogens appear to be protective. Recent data also indicate that breast tissue possesses enzymatic machinery to inactivate and eliminate E2 and its oxidized and methoxylated metabolites through conjugation catalyzed by UDP-glucuronosyltransferases (UGTs), which involves the covalent addition of glucuronic acid. In opposition to other metabolic pathways of estrogen, the UGT-mediated process leads to the formation of glucuronides that are devoid of biologic activity and are readily excreted from the tissue into the circulation. This review addresses the most recent findings on the identification of UGT enzymes that are responsible for the glucuronidation of E2 and its metabolites, and evidence regarding their potential role in breast cancer.