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.

In Crigler-Najjar type II patients and, recently, in CriglerNajjar type I patients treated with human hepatocyte cell therapy, phenobarbital has been used for reducing the serum bilirubin load. Its effect is attributed to induction of the enzyme required for hepatic bilirubin elimination, UDP-glucuronosyltransferase, UGT1A1. This study investigated the expression and inducibility of UGT1A1 in human donor livers and their corresponding primary hepatocyte cultures. Immunoblot analysis using a specific antibody directed against the amino terminal of the human UGT1A1 isoform showed that 5 hepatocyte donors exhibited a G50-fold difference in UGT1A1 level. UGT1A1 protein level correlated strongly with both liver microsomal bilirubin UGT activity and liver UGT1A1 mRNA level (r 2 ‫؍‬ .82 and .72, respectively). Of the 4 patients with the lowest UGT1A1 levels, 3 were homozygotes for the UGT1A1 promoter variant sequence associated with Gilbert's syndrome, and the fourth was a heterozygote. The 3 donors with the highest levels had a history of phenytoin exposure. Hepatocytes isolated from the phenytoin-exposed donors exhibited marked declines in UGT1A1 mRNA levels during culturing. Induction studies using hepatocytes treated for 48 hours with phenobarbital (2 mmol/L), oltipraz (50 mol/L), or 3-methylcholanthrene (2.5 mol/L) revealed UGT1A1-inducing effects of phenobarbital, oltipraz, and, in particular, 3-methylcholanthrene. Our data suggest that both genetic and environmental factors play an important role in the marked interindividual variability in UGT1A1 expression. An understanding of these mechanisms could lead to advances in the pharmacological therapy of life-threatening unconjugated hyperbilirubinemia. (HEPATOLOGY 1999;30:476-484.)Bilirubin is an endogenous waste product generated from the metabolism of heme. 1 At low concentrations (normal range Յ1 mg/dL), it is recognized as being beneficial as a result of its antioxidant properties. However, its accumulation in the serum to high concentrations (Ն20 mg/dL) is associated with serious toxicities, including neurological toxicity (kernicterus) and renal damage. 1 Therefore, the control of bilirubin levels in the body is critical.The major factor controlling the excretion of bilirubin is its rate of glucuronidation in liver. Glucuronidation of this substrate is catalyzed by a specific member of the UDPglucuronosyltransferase family, UGT1A1. 2-5 The level of bilirubin glucuronidation activity (and presumably UGT1A1) in liver is determined by genetic as well as environmental factors. Such factors include exposure to drugs 6-9 and xenobiotic compounds. 9-11 The most intensively studied class of bilirubin UGT inducers has been the barbiturates, which are used clinically for lowering serum unconjugated bilirubin levels in individuals with the type II form of Crigler-Najjar syndrome. 12 More recently, phenobarbital has been shown to be effective for lowering bilirubin in a child with the type I form of Crigler-Najjar who received human hepatocyte cell therapy. 13 The benef...

ABSTRACT:Gunn rats glucuronidate acetaminophen (APAP) at reduced rates and show increased susceptibility to APAP-induced hepatotoxicity. This defect is presumed to involve UDP-glucuronosyltransferase (UGT) 1A6, which is nonfunctional in Gunn rats, but it is currently unclear whether other 1A family members are also involved. In humans, two 1A isoforms are known to be active (1A6 and 1A9) but 1A6 form has a 25-fold lower apparent K m (2 mM). Rat liver microsomal APAP UGT activity is induced by in vivo treatment with ␤-naphthoflavone or oltipraz, an effect correlating with induction of 1A6 and 1A7. To address a possible role of 1A7 in APAP glucuronidation relative to other 1A forms, cDNAs encoding UGTs 1A1, 1A5, 1A6, 1A7, and 1A8 were expressed in human embryonic kidney cells and the contents of expressed enzyme in prepared membrane fractions determined by quantitative immunoblotting. At 2.5 mM APAP, 1A7 showed the highest specific activity (2.8 nmol/min/nmol 1A7 protein), followed by 1A6 (1.1 nmol/min/nmol), and 1A8 (0.27 nmol/min/nmol). 1A1 and 1A5 were essentially inactive. Kinetic comparisons indicated 1A7 had a similar apparent K m as 1A6 (4.7 versus 3.9 mM, respectively) but a 2.4-fold higher catalytic activity. These data suggest that in rats, 1A7 plays a major role in APAP glucuronidation and contributes to protection against APAP-induced hepatotoxicity. The involvement of other UGTs besides 1A6 is further underscored by the presence of significant residual APAP-glucuronidating activity by Gunn rat hepatocytes, indicating the activity of an unknown UGT2 family member. APAP1 (Tylenol) is a widely used over-the-counter analgesic/ antipyretic drug and model hepatotoxin. Although it is considered safe at normal doses, at higher doses it is associated with a predictable, dose-dependent centrilobular hepatotoxicity (Black, 1984) by a mechanism involving its metabolism to a toxic quinone imine (Cohen and Khairallah, 1997;Bessems and Vermeulen, 2001). APAP undergoes detoxification by competing phase 2 conjugation reactions, glucuronidation and sulfation, which convert APAP to nontoxic conjugates for elimination in bile or urine. The main type of APAP conjugation in most species, including rat and human, is glucuronidation. It has been proposed that individuals who metabolize APAP at slower rates are at higher risk of developing hepatotoxicity, based partly on the known greater susceptibility of animals that are deficient in the glucuronidation of phenols, such as cats (Feloidae) (Court and Greenblatt, 2000;Bessems and Vermeulen, 2001) and the Gunn rat Wells, 1988, 1989).A common assumption has been that the increased susceptibility of cats and Gunn rats to APAP toxicity is due to inactive UGT1A6, a major liver-expressed phenol UGT. The wild-type 1A6 2 enzyme from rat and human is effective in APAP catalysis (Bock et al., 1993), and in rats, 1A6 is induced by 3-methylcholanthrene (Munzel et al., 1994;Bock et al., 1999), which results in increased APAP glucuronidation (Gregus et al., 1990; Bock et al., 1993). In ca...

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The utility of oltipraz as a cancer chemopreventive agent is thought to depend on the induction of enzymes involved in phase 2 xenobiotic detoxification. Although studies of some enzymes induced by oltipraz implicate a novel transcriptional activating pathway involving Nrf2 and antioxidant-response elements (AREs), the mechanism of phenol UGT induction has remained unclear. Previous work showed that UGT1A6 is transcribed from two promoters, P1 and P2, that are both induced by oltipraz in rat liver. The effect also occurs in rat hepatocytes treated with oltipraz (concentrations Ͼ3 M). To investigate the mechanism, luciferase reporter plasmids under the control ofwere transfected into rat hepatocytes and tested for inducibility. P1, but not P2, showed responsiveness to oltipraz (2-to 5-fold increase) and 3-methylcholanthrene (10-to 30-fold increase). Because P1 contained no visible AREs, the role of a xenobiotic response element (XRE) centered between bases Ϫ134 and Ϫ129 was evaluated. Mutation of the XRE core reduced the effects of both oltipraz and 3-methylcholanthrene on the P1 reporter. The 1A6 XRE conferred oltipraz responsiveness on the simian virus 40 promoter of pGL3-Promoter. Comparative effects of oltipraz and 3-methylcholanthrene on transfected cytochrome P4501A1 reporters support the general but relatively weak XRE-stimulating activity of oltipraz. The involvement of the aryl hydrocarbon receptor (AHR) and aryl hydrocarbon nuclear translocator (ARNT) in mediating the effects of oltipraz on the XRE is supported by electrophoretic mobility supershift data and AHR/ARNT overexpression studies. These data raise questions about the contribution of AHR and other secondary induction pathways in the mechanism of oltipraz.