Phenol UDP-glucuronosyltransferase Research Articles

Allelic variations in the plaice UGT1B1 gene

We have shown that there are large internanimal variations in hepatic phenol UDP-glucuronosyltransferase (UGT) activity in the plaice which may affect their capacity to detoxify metabolites of carcinogenic PAHS such as benzo(a)pyrene with obvious implications for their health and survival. By molecular studies we have only been able to identify one copy of the UGT1B1 gene coding for the major phenol-UGT in the diploid plaice genome and found that it is highly polymorphic about this locus. Sequencing of multiple UGT1B1 cDNA clones derived from RT-PCR revealed the existence of clustered SNPs, however, from their positions we contend that this is not sufficient to explain the observed phenotypic variability. We discovered a heterozygous null genotype derived from a dinucleotide deletion (and also found preliminary evidence for a corresponding phenotype) which has a much greater implication for survival not only in polluted environments but for embryonic survival if the gene product has an essential endogenous role. We propose that the observed interanimal variations are attributable to a combination of factors including genotypic variations and differential expression.

Presence of phenol UDP-glucuronosyltransferase in bovine alveolar macrophages and bronchial epithelial cells

Pulmonary organ and cells are the primary target of atmospheric pollutants. Phenol UDP-glucuronosyltransferase (UGT) was found in bovine alveolar macrophage cells by immunohistochemical staining and also was observed in bronchial epithelial cells of the lung. A high level of activity of UGT, which is one of the phase II drug-metabolizing enzymes, toward 1-naphthol was observed in the microsomes of both cell types. By Western blotting analysis, a 54-kDa band was detected in alveolar macrophage cells and in bovine lung using polyclonal antibodies against a purified rat UGT, which catalyze the glucuronidation of various phenolic xenobiotics such as 1-naphthol and have the same molecular mass (54 kDa). Reverse transcriptase-polymerase chain reaction (RT-PCR) amplified the common cDNA region in UGT1A subfamily isoforms, indicating that UGT1A subfamily isoform was expressed in alveolar macrophages and in bronchial epithelial cells of the lung. These results suggest that phenol UGT act as a primary barrier against various phenolic chemicals in the lung.

Tissue-specific regulation of canine intestinal and hepatic phenol and morphine UDP-glucuronosyltransferases by β-naphthoflavone in comparison with humans

UDP-glucuronosyltransferases (UGTs) are regulated in a species- and tissue-dependent manner by endogenous and environmental factors. The present study was undertaken to further our knowledge about regulation of UGTs in dogs, a species widely used in preclinical safety evaluation. β-Naphthoflavone (BNF) was selected as a known aryl hydrocarbon receptor agonist and antioxidant-type inducer. The latter group of inducers is intensively investigated as dietary chemoprotectants against colon cancer. Dog UGTs were investigated in comparison with related human UGTs by examples, (i) expression of dog UGT1A6, the first sequenced dog phenol UGT, and (ii) morphine UGT activities, responsible for intestinal and hepatic first-pass metabolism of morphine. The following results were obtained: (i) dog UGT1A6 was found to be constitutively expressed in liver and marginally increased by BNF treatment. Expression was low in small intestine but ca. 6-fold higher in colon than for example in jejunum. Conjugation of 4-methylumbelliferone, one of the substrates of dog UGT1A6, was also enhanced 7-fold in colonic compared to jejunal microsomes. (ii) Compared to the corresponding human tissues, canine 3-O- and 6-O-morphine UGT activities were found to be >10-fold higher in dog liver and ca. 10-fold lower in small intestinal microsomes. Small intestinal morphine and 4-hydroxybiphenyl UGT activities appeared to be moderately (2- to 3-fold) induced by oral treatment with BNF. (iii) In contrast to dogs, morphine UGT activities were found to be similar in homogenates from human enterocytes and liver. The results suggest marked differences in tissue-specific regulation of canine vs. human hepatic and intestinal phenol or morphine UGTs.

Glucuronidation of acetaminophen catalyzed by multiple rat phenol UDP-glucuronosyltransferases.

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 beta-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.

CDNA cloning and expression of a bovine phenol UDP-glucuronosyltransferase, BovUGT1A6

A full-length cDNA encoding a phenol UDP-glucuronosyltransferase was isolated by plaque hybridization, RT-PCR and 5′-RACE from a cDNA library prepared from the bovine liver. The deduced amino acid sequence (529 amino acid residues) has A signal sequence (23 amino acid residues) at the amino terminus and a transmembrane-anchoring domain (17 amino acid residues) at the carboxyl terminus. The encoded protein has a potential asparagine-linked glycosylation site (Asn291). The cloned cDNA was named bovUGT1A6 on the basis of the amino acid similarity. BovUGT1A6 cloned in the pAAH5 expression vector was transformed into Saccharomyces cerevisiea AH22 cells to obtain an active 54-kDa bovUGT1A6 enzyme. The expressed enzyme represented UDP-glucuronosyltransferase activities toward 1-naphthol and 4-methylumbelliferone, comfirming that the isolated cDNA is an isoform of bovine phenol UDP-glucuronosyltransferase. Microsomal UDP-glucuronosyltransferase activity toward 1-naphthol in the bovine kidney cortex was found to be higher than that in the liver and other organs, and mRNA of bovUGT1A6 was more strongly detected in the kidney on Northern blotting analysis. These results suggest that the bovine kidney, which strongly expresses bovUGT1A6, is a significant organ for xenobiotics glucuronidation.

Conjugation of 1-naphthol in primary cell cultures of rat ovarian cells

The present study concerns conjugation of 1-naphthol in primary cultures of rat ovarian cells. Two phase II enzymes catalyzing conjugation, i.e. phenol sulfotransferase (P-SULT) and phenol UDP-glucuronosyltransferase (P-UGT), were measured using 1-naphthol as substrate. The rates of conjugation by the different cell types of the rat ovary were the same at low concentrations and short incubation times. However, after 20 h of incubation the rate of conjugation in cells isolated from ovaries enriched in corpora lutea (CL) exceeded the rate in cells isolated from ovaries enriched in preovulatory follicles. In addition, when the granulosa cells were removed from the preovulatory follicles, the rate of conjugation was 1.7-fold higher, i.e. in the theca/stroma cells. When the cells were incubated with 1-[ 14C]naphthol and conjugates were subsequently separated by thin-layer chromatography, naphthyl glucuronide was the only conjugate observed. Pentachlorophenol (PCP), a commonly used inhibitor of P-SULT, inhibited 1-naphthol conjugation 50% in cell cultures, as well as in microsomal preparations. α-Naphthoflavone (ANF) and ellipticine (ELP), both cytochrome P450 (CYP) inhibitors, affected the conjugation of 1-naphthol in different ways; ANF did not affect P-UGT activity in microsomal preparations, but inhibited 1-naphthol conjugation in cell cultures by as much as 90%. On the other hand, ELP inhibited the conjugation of 1-naphthol up to 99% in the cell cultures, but only 75% in microsomal fractions. Testosterone (TST) and estradiol inhibited this activity ≈50% in both of these experimental systems. Clomiphene citrate (CLF), a drug used to induce ovulation and demonstrating both estrogenic and antiestrogenic effects, did not influence the conjugation of 1-naphthol significantly in the cell cultures. The present findings demonstrate that P-UGT is by far the major enzyme conjugating 1-naphthol in the rat ovary and that commonly used inhibitors of P-SULT and CYPs also inhibit P-UGT activity, either directly or via other mechanisms.

Hypophysectomy and/or peroxisome proliferators strongly influence the levels of phase II xenobiotic metabolizing enzymes in rat testis

The objectives of the present work were to determine the influence of hypophysectomy and/or peroxisome proliferators (PP) on certain xenobiotic-metabolizing enzyme activities, i.e. glutathione transferases (GST), glutathione peroxidase (GPX), phenol sulphotransferases (pSULT), phenol UDP-glucuronosyl transferases (pUGT), catalase, NADP(H) quinone oxidoreductase (QR) and epoxide hydrolases (EH) in the rat testes. Adult male rats, hypophysectomized and their sham-operated controls, were treated for 10 days with clofibrate (0.5%), perfluorooctanoic acid (0.05%, PFOA), acetylsalicylic acid (1%, ASA) and di(2-ethylhexyl)phthalate (2%, DEHP) in their diet. The results show that, in addition to both body and testis weight, hypophysectomy caused dramatic changes in most of the xenobiotic-metabolizing enzyme activities, which have been measured here. The most pronounced effects were seen in cytosolic QR (2.2-fold increase), pUGT (95% reduction), pSULT (75% reduction), mitochondrial catalase (75% reduction), microsomal EH (70% reduction) and microsomal GST (55% reduction). Treatment with PP, i.e. perfluorooctanoic acid (PFOA), clofibrate, acetyl salicylic acid (ASA) and di(2-ethylhexyl)phthalate (DEHP) showed varied effects on the xenobiotic-metabolizing enzyme activities, the highest effects (10–60% reduction) were seen in sham-operated animals. These effects were not so pronounced or were not seen in hypophysectomized rats except for the case of PFOA treatment, which caused increases of enzyme activities. The highest increases were seen with microsomal GST (70%), GPX (75%) and cytosolic EH (75%). It is concluded from these experiments that the regulation of several xenobiotic-metabolizing enzymes in the rat testis is affected by the pituitary and/or pituitary hormones and that different peroxisome proliferators have variable effects on the levels of these xenobiotic-metabolizing enzymes. The general trend of reduction in enzyme activities implies that the testis is less protected under conditions that can perturb hormonal status.

Small Intestinal UDP-Glucuronosyltransferase sheUGT1A07: Partial Purification and cDNA Cloning from Sheep Small Intestine

A phenol UDPglucuronosyltransferase (UGT) was partially purified, and the cDNA encoding the isoform was cloned and sequenced from sheep small intestine. The purified preparation containing a one major band (57 kDa) and one minor band (50 kDa) revealed high activities toward xenobiotics such as 1-naphthol (1-NA), 4-nitrophenol, and 4-methylumbelliferone. The preparation, however, had only little activity toward 4-hydroxybiphenyl and no activity toward bilirubin, suggesting that the preparation contains UGT1 isoforms. The NH2-terminal amino acid sequence of the major band was determined to be Gly-Lys-Leu-Leu-Val-Val-Pro-Met-Asp-Gly-Ser. A full-length UGT cDNA was obtained by reverse transcription–polymerase chain reaction with the degenerated 5′-primer from the NH2-terminal amino acid sequence of the purified major one and rapid amplification of cDNA ends from sheep small intestine. The cloned cDNA named sheUGT1A07 by amino acid similarity has a NH2-terminus sequence identical to that of the purified major one. Another phenol UGT cDNA named sheUGT1A6 was also cloned from sheep liver. sheUGT1A6 was expressed mainly in the liver, whereas sheUGT1A07 mRNA was expressed almost only in the alimentary organs, suggesting that sheUGT1A6 plays a role as a general drug metabolizing UGT isoform in the liver and sheUGT1A07 plays important role in the xenobiotics glucuronidation in the sheep small intestine.

Inhibitory effects of uridine diphosphate on udp-glucuronosyltransferase

Inhibitory effects of uridine diphosphate on the enzymatic activity of UDP-glucuronosyltransferase (UGT) were investigated. Pyrimidine nucleotides such as UDP, UTP and cytidine diphosphate reduced the activity of rat purified UGT (phenol UGT) to about 10%, 48% and 46% of the control, respectively, at the same concentration as a donor substrate, UDP-glucuronic acid. Purine nucleotides, uridine monophosphate, glucuronic acid and some UDP-sugars were only slightly inhibitory toward the transferase. Similar effects were observed in the expressed UGT (UGT1A6; corresponding to phenol UGT) in yeast cells and rat liver microsomal membrane-binding UGT, indicating that uracil and diphosphate residues are essential for the UDP inhibition. Interestingly, 2′-deoxy UDP was found to be a less effective inhibitor (about 50% inhibition) than UDP on the purified, the expressed (UGT1A6 and UGT2B1) and microsomal membrane-binding UGTs. These results indicate that not only uracil and diphosphate residues but also 2′-hydroxyl residue of UDP ribose participates in the interactions between UDP and UDP-glucuronosyltransferase.

Xenobiotic biotransformation enzyme gene expression in early larval stages of plaice

cDNA probes were utilised to determine the pattern of expression of genes coding for pollutant biotransformation enzymes and systems for detoxication of peroxidation products in eggs and the early stages of plaice larval development. The presence of mRNAs for metallothionein and glutathione S-transferase in eggs indicates that the potential for oxidative defence may be present in fertilised eggs. Genes for the enzymes non-specific carboxylesterase and phenol UDP-glucuronosyl transferase, which have roles in the metabolism of endobiotic substances as well as pollutants, showed a peak in expression at hatch. CYP1A was expressed at hatch and the mRNA levels increased by about four-fold by 20 d post-hatch indicating the potential for metabolic activation of toxic polyaromatic compounds at the earliest free-living stages.

Characterization of the UDP-glucuronosyltransferase isoenzyme expressed in rat ovary and its regulation by gonadotropins.

Earlier studies have demonstrated that phenol UDP-glucuronosyltransferase (UGT) activity is up-regulated by pregnant mare's serum gonadotropin (PMSG) in rat ovary, but not liver. This phenomenon was investigated in more detail in the present study. Ovaries and livers of immature rats, rats synchronized with respect to their preovulatory and corpus luteal phases by treatment with PMSG, and mature rats hyperstimulated with PMSG were compared. Under all of these conditions, only one immunoreactive band of UGT, shown to be phenol UGT, was detected in the rat ovary. The effects of oestradiol, progesterone and/or human chorionic gonadotropin (hCG) on the level of phenol UGT in immature rat ovary were also examined. Partial up-regulation was caused by progesterone or oestradiol, together with hCG, whereas progesterone or oestradiol alone had no up-regulating effect. Follicle-stimulating hormone also seemed to be required for the up-regulation in ovaries enriched in corpus luteum. The present findings demonstrate that progesterone is involved in the regulation of phenol UGT in rat ovary by gonadotropins. Regulation by both progesterone and oestradiol was dependent on induction of ovulation and steroidogenesis by luteinizing hormone.

Exposure of atlantic salmon parr (Salmo salar) to a combination of resin acids and a water soluble fraction of diesel fuel oil: A model to investigate the chemical causes of Pigmented Salmon Syndrome

AbstractPigmented Salmon Syndrome is a pollutant‐induced hemolytic anemia and hyperbilirubinemia. As part of an investigation of this condition, S2 Atlantic salmon parr (Salmo salar) were exposed to a diesel fuel oil, water soluble fraction (WSF) in combination with a mixture of three resin acids (isopimaric, dehydroabietic, and abietic acids) in a continuous‐flow freshwater system. The total nominal concentrations of resin acids in the exposure tanks were 10, 50, and 100 μg/L; the diesel WSF was generated in situ and provided a mean hydrocarbon concentration of 2.0 ±0.1 mg/L (n = 12) during the 9‐d exposure period. Exposure to the diesel WSF alone depressed liver bilirubin UDP‐glucuronosyl transferase (UDPGT) activity and induced phenol UDPGT activity. Exposure to the diesel WSF in the absence or presence of resin acids induced liver cytochrome P4501A and increased the concentrations in the plasma of the enzymes lactate dehydrogenase, alkaline phosphatase, and glutamic oxaloacetic transaminase. The combined exposure to diesel WSF with either 50 or 100 μg/L total resin acid caused significant elevations in the concentrations of bilirubin in the plasma and many of these fish had yellow pigmentation on the ventral surface and around the gill arches. The results demonstrate that exposure to combinations of two groups of contaminants can result in the manifestation of toxic effects not apparent from exposure to either of these chemicals in isolation.

High induction of phenol UDP-glucuronosyltransferase in the kidney medulla of β-naphthoflavone-treated rats

Phenol UDP-glucuronosyltransferase activity was highly induced in the microsomes of the kidney medulla of rats by β-naphthoflavone treatment. In the medulla, phenol UDP-glucuronosyltransferase and its mRNA were greatly increased in both immunoblotting and Northern blot analyses following β-naphthoflavone treatment of the rats. In untreated rat kidneys, phenol UDP-glucuronosyltransferase was detected by immunohistochemical analysis only in proximal convolution tubular cells located in the cortex. After β-naphthoflavone treatment of the rats, UDP-glucuronosyltransferase appeared in the epithelial cells in the straight portion of the distal tubules located in the medulla. In conclusion, the medullary distal tubular cells have high latent glucuronidation activity and are thought to play an important role in drug excretion.

A critical amino acid residue, asp446, in UDP-glucuronosyltransferase.

An amino acid residue, Asp446, was found to be essential for the enzymic activity of UDP-glucuronosyltransferase (UGT). We obtained a rat phenol UGT (UGT1*06) cDNA (named Ysh) from male rat liver by reverse-transcription (RT)-PCR using pfu polymerase. A mutant Ysh having two different bases, A1337G and G1384A (named Ysh A1337GC1384A), that result in two amino acid substitutions, D446G and V462M, was obtained by RT-PCR using Taq polymerase. Ysh was expressed functionally in microsomes of Saccharomyces cerevisiae strain AH22. However, the expressed protein from YshA1337GG1384A had no transferase activity. Two other mutant cDNAs with YshA1337G having one changed base, A1337G, resulting in one amino acid substitution, D446G, and YshG1384A having a changed base, G1384A, resulting in an amino acid substitution, V462M, were constructed and expressed in the yeast. The expressed protein from YshG1384A (named YshV462M) exhibited enzymic activity, but the one from YshA1337G (named YshD446G) did not show any activity at all. Asp446 was conserved in all UGTs and UDP-galactose:ceramide galactosyltransferases reported, suggesting that Asp446 plays a critical role in each enzyme.

Effects of peroxisome proliferators and/or hypothyroidism on xenobiotic-metabolizing enzymes in rat testis

The objectives of the present work were to study the effects of certain peroxisome proliferators on xenobiotic-metabolizing enzyme activities in the testes of normal and hypothyroid rats, i.e. phenol sulfotransferases (pST), phenol UDP-glucuronosyl transferases (pUDPGT), glutathione transferases (GST), catalase, epoxide hydrolase (EH), glutathione peroxidase (GPX) and NAD(P)H quinone oxidoreductase (QR). Adult male rats (normal and hypothyroid) were treated for 10 days with clofibrate (0.5%), perfluorooctanoic acid (0.05%, PFOA), acetylsalisylic acid (1%, ASA) and di(2-ethylhexyl)phthalate (2%, DEHP) in their diet. The results show that treatment of normal rats with peroxisome proliferators dramatically affects the activities of xenobiotic-metabolizing enzymes (40–60% reduction). The highest effects are seen in catalase activity (50–60% with PFOA and ASA), pUDPGT (55% with PFOA), pST (55% with PFOA) and QR (50% with DEHP). These effects are not seen or are weaker after induction of hypothyroidism. Taken together, it is concluded that different classes of peroxisome proliferators have different effects on rat testicular xenobiotic-metabolizing enzymes.

Genetic defects at the UGT1 locus associated with Crigler-Najjar type I disease, including a prenatal diagnosis

Characterization of the UGT1 gene complex locus encoding both multiple bilirubin and phenol UDP-glucuronosyltransferases (transferases) has been critical in identifying mutations in the bilirubin isoforms. This study utilizes this information to identify the bases of deficient bilirubin UDP-glucuronosyltransferase activity encoded by the UGT1A gene for the major bilirubin isozyme, HUG-Br1, in 3 Crigler-Najjar type I individuals and the genotype of an at-risk unborn sibling of one patient. A homozygous and heterozygous two-base mutation (CCC to CGT) created the HUG-Br1P387R mutant of the major bilirubin transferase in 2 different Crigler-Najjar type I patients, B.G. and G.D., respectively. Both parents of B.G. and his unborn sibling, J.G., were determined to be carriers of the P387R mutation. G.D. also contains the CAA to TAA nonsense mutation (G1n357st). Y.A. has a homozygous CT deletion in codons 40/41. The HUG-Br1P387R mutant protein was totally inactive at the major pH optimum (6.4), but retained 26% normal activity at the minor pH optimum (7.6), which was 5.4% of the combined activities measured at the two pH values.

Genetic defects at the UGT1 locus associated with Crigler‐Najjar type I disease, including a prenatal diagnosis

Characterization of the UGT1 gene complex locus encoding both multiple bilirubin and phenol UDP-glucuronosyltransferases (transferases) has been critical in identifying mutations in the bilirubin isoforms. This study utilizes this information to identify the bases of deficient bilirubin UDP-glucuronosyltransferase activity encoded by the UGT1A gene for the major bilirubin isozyme, HUG-Br1, in 3 Crigler-Najjar type I individuals and the genotype of an at-risk unborn sibling of one patient. A homozygous and heterozygous two-base mutation (CCC to CGT) created the HUG-Br1P387R mutant of the major bilirubin transferase in 2 different Crigler-Najjar type I patients, B.G. and G.D., respectively. Both parents of B.G. and his unborn sibling, J.G., were determined to be carriers of the P387R mutation. G.D. also contains the CAA to TAA nonsense mutation (G1n357st). Y.A. has a homozygous CT deletion in codons 40/41. The HUG-Br1P387R mutant protein was totally inactive at the major pH optimum (6.4), but retained 26% normal activity at the minor pH optimum (7.6), which was 5.4% of the combined activities measured at the two pH values. Am. J. Med. Genet. 68:173–178, 1997 © 1997 Wiley-Liss, Inc.

Glucuronidation of entacapone, nitecapone, tolcapone, and some other nitrocatechols by rat liver microsomes.

Nitrocatechol COMT inhibitors are a new class of bioactive compounds, for which glucuronidation is the most important metabolic pathway. The objective was to characterize the enzyme kinetics of nitrocatechol glucuronidation to improve the understanding and predicting of the pharmacokinetic behavior of this class of compounds. The glucuronidation kinetics of seven nitrocatechols and 4-nitrophenol, the reference substrate for phenol UDP-glucuronosyltransferase activity, was measured in liver microsomes from creosote-treated rats and determined by non-linear fitting of the experimental data to the Michaelis-Menten equation. A new method that combined densitometric and radioactivity measurement of the glucuronides separated by HPTLC was developed for the quantification. Apparent K(m) values for the nitrocatechols varied greatly depending on substitution pattern being comparable with 4-nitrophenol (0.11 mM) only in the case of 4-nitrocatechol (0.19 mM). Simple nitrocatechols showed two-fold Vmax values compared with 4-nitrophenol (68.6 nmol min-1 mg-1), while all disubstituted catechols exhibited much lower glucuronidation rate. Vmax/K(m) values were about 10 times higher for monosubstituted catechols compared to disubstituted ones. The kinetic parameters for COMT inhibitors were in the following order: K(m) nitecapone > > entacapone > tolcapone; Vmax nitecapone > entacapone > tolcapone; Vmax/K(m) tolcapone > nitecapone > entacapone. Nitrocatechols can in principle be good substrates of UGTs. However, substituents may have a remarkable effect on the enzyme kinetic parameters. The different behaviour of nitecapone compared to the other COMT inhibitors may be due to its hydrophilic 5-substituent. The longer elimination half-life of tolcapone in vivo compared to entacapone could not be explained by glucuronidation kinetics in vitro.

Formation of mono- and diglucuronides and other glycosides of benzo( a)pyrene-3,6-quinol by V79 cell-expressed human phenol UDP-glucuronosyltransferases of the UGT1 gene complex

Glucuronidation of quinols of polycyclic aromatic hydrocarbons (PAHs) represents an important detoxication pathway preventing toxic quinone/quinol redox cycles. Therefore, mono- and diglucuronide formation of benzo( a)pyrene-3,6-quinol was investigated and compared to that of structurally related 3,6-dihydroxychrysene and simple phenols (1-naphthol and 4-methylumbelliferone) using V79 cell-expressed human UGT1.6 (= P1) and human UGT1.7 (= P4). Properties of human UGT1.6 were compared to those of the rat ortholog. Cofactors related to UDP-glucuronic acid such as UDP-galacturonic acid and UDP-glucose were also studied. It was found that rat and human UGT1.6 and human UGT1.7 catalyse monoglucuronide formation of planar PAH quinols. Diglucuronide formation was only detectable with human UGT1.7. The UGT isozymes studied also formed galacturonides and, although only to a minor extent, glucosides. Rat UGT1.6 (but not the human ortholog) catalysed digalacturonide formation of benzo( a)pyrene-3,6-quinol; the in vivo significance of galacturonide formation remains to be established. The results suggest that planar PAH phenols and quinols are conjugated more efficiently by human UGT1.7 than by UGT1.6, which preferentially conjugates simple planar phenols.

Isolation of cDNAs for mouse phenol and bilirubin UDP-glucuronosyltransferases and mapping of the mouse gene for phenol UDP-glucuronosyltransferase (Ugtla1) to chromosome 1 by restriction fragment length variations.

The mouse gene for phenol UDP-glucuronosyltransferase (UDPGT; Ugtla1) was mapped at 42 cM on chromosome 1, a position identical to that of the gene for bilirubin UDPGT (Ugtla1), from linkage analysis of a three-point cross test with Idh-1, En-1, and Ugtla1 as marker genes. The cDNAs for mouse phenol and bilirubin UDPGTs, isolated after amplification by PCR, shared an identical 3'-half region. Our results strongly suggest that mouse bilirubin and phenol UDPGTs are expressed from a single gene and involve alternative splicing events. We also detected duplication of the gene for phenol UDPGT in all mouse strains examined with the exception of MOL-MIT and SUB-SHH.