A Universal Radiochemical High-Performance Liquid Chromatographic Assay for the Determination of UDP-Glucuronosyltransferase Activity

Ethell, Brian T.; Anderson, Gail D.; Beaumont, Kevin; Rance, David J.; Burchell, Brian

doi:10.1006/abio.1997.2443

Cited by 44 publications

(37 citation statements)

References 19 publications

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“…Therefore, UGT1As in the brain would be involved in mediating the pharmacological effects of several central-acting drugs as UGT substrates, such as acetaminophen (APAP) (Court et al, 2001), amitriptyline (Green et al, 1998), and valproic acid (Ethell et al, 1998). UGT1As also catalyze the glucuronidation of neurotransmitters and neurosteroids, such as serotonin (Krishnaswamy et al, 2003), dopamine (Itäaho et al, 2009), and estradiol (Court, 2005), as endogenous substrates in the brain.…”

Section: Introductionmentioning

confidence: 99%

Effects of Phenobarbital on Expression of UDP-Glucuronosyltransferase 1a6 and 1a7 in Rat Brain

Sakakibara

Katoh

Kondo

et al. 2015

Drug Metabolism and Disposition

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UDP-glucuronosyltransferase (UGT), a phase II drug-metabolizing enzyme, is expressed in the brain and can catalyze glucuronidation of endogenous and exogenous substrates in the brain. Thus, changes in UGT1A expression could affect homeostasis and drug efficacy. Phenobarbital (PB), a typical inducer of drug-metabolizing enzymes, has been reported to induce oxidative stress and epigenetic changes, which could alter UGT expression in the brain. Here, we aimed to clarify the effects of PB on Ugt1a6 and Ugt1a7 gene expression in rat brains. Sprague-Dawley rats were treated intraperitoneally with PB (80 mg/kg), once daily for 7 days. Ugt1a6 and Ugt1a7 mRNA expression levels were increased in the striatum and thalamus (Ugt1a6, 3.0-and 2.9-fold, respectively; Ugt1a7, 2.6-and 2.6-fold, respectively). Acetaminophen glucuronidation was also increased in the medulla oblongata and thalamus by 1.8-and 1.2-fold, respectively. The induction rates within different brain regions were correlated with Ugt1a6 and Ugt1a7 mRNA expression, and the degree of induction also correlated with that of NF-E2-related factor-2 mRNA. Measurement of oxidative stress markers suggested that PB induced oxidative stress in brain regions in which Ugt1a6 and Ugt1a7 mRNAs were increased. Moreover, histone modifications were altered by PB treatment, resulting in increased histone H3 lysine 4 trimethylation in the striatum and thalamus and decreased histone H3 lysine 9 trimethylation in the thalamus. These results suggested that oxidative stress and histone modifications may promote transcriptional activation of Ugt1a6 and Ugt1a7 genes. In summary, Ugt1a6 and Ugt1a7 mRNA levels were increased by PB treatment, which may alter pharmacokinetics in the brain.

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Section: Introductionmentioning

confidence: 99%

Effects of Phenobarbital on Expression of UDP-Glucuronosyltransferase 1a6 and 1a7 in Rat Brain

Sakakibara

Katoh

Kondo

et al. 2015

Drug Metabolism and Disposition

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“…The typing of human liver and kidney microsomes and the assays used as controls for the LC/MS-MS assays were performed by the method of Ethell et al (1998). Briefly, substrates were incubated with microsomes or recombinant UGTs in 100 mM Tris-maleate buffer containing 5 mM MgCl 2 , 2 mM UDPGA (0.1 Ci of [ 14 C]UDPGA) for 40 min in a volume of 100 l. After this time, reactions were terminated by the addition of 100 l of prechilled methanol.…”

Section: Chemical Synthesis Of Hmr1098-s-glucuronide Methyl Estermentioning

confidence: 99%

GLUCURONIDATION OF HMR1098 IN HUMAN MICROSOMES: EVIDENCE FOR THE INVOLVEMENT OF UGT1A1 IN THE FORMATION OFS-GLUCURONIDES

Ethell

Riedel²,

Englert³

et al. 2003

Drug Metab Dispos

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:HMR1098, a novel K ATP -blocking agent, is metabolized to form an S-glucuronide in rat and dog bile. Synthesis of the S-glucuronide metabolite was studied in human liver and kidney microsomes. Recombinant UPD-glucuronosyltransferases (UGTs) were screened for activity, and kinetic analysis was performed to identify the isoform or isoforms responsible for the formation of this novel S-glucuronide in humans. S-Glucuronidation is relatively rare, but from this study it appears that S-glucuronides are not generated exclusively by a single UGT isoform. From the panel of recombinant isoforms used, both UGT1A1 and UGT1A9 catalyzed the glucuronidation of HMR1098. The V max values in both instances were similar, but the K m for UGT1A1 was substantially lower than that measured for UGT1A9, 82 M compared with 233 M, respectively. Liver and kidney microsomes displayed similar K m values, but the V max in kidney was more than 20-fold less than in liver microsomes, which is suggestive of a significant role for the bilirubin UGT in catalysis of HMR1098, although other UGTs may play a secondary role.HMR1098 is a new K ATP -blocking agent being developed as a drug for prevention of sudden cardiac death by Aventis Pharma Deutschland GmbH. Metabolic studies in rats, dogs, and humans have shown that the main metabolite, M1, was characterized by 2D 1 NMR as HMR1098-S-␤-D-glucuronide (see this paper), and the glucuronide was linked via the thiourea group (Fig. 1). S-Glucuronides are rare metabolites, and the individual UDP-glucuronosyltransferases (UGTs) catalyzing their formation have never been identified.UGTs are a family of microsomal enzymes in humans catalyzing the glucuronidation of thousands of compounds by transferring glucuronic acid from UDP-glucuronic acid to the aglycone substrate (Burchell et al., 1997). The substrate specificity of individual UGTs has been partially characterized by study of cloned/expressed transferases. This work has determined that UGT subfamily 1 is responsible for glucuronidation of bilirubin, phenols, and drugs, whereas subfamily 2 enzymes catalyze glucuronidation of steroids, bile acids, and drugs (Burchell et al., 1997). Key probe substrates such as bilirubin for UGT1A1 are used to indicate the specific activity of an individual isoform (Burchell et al., 1995).The formation of thiol drug glucuronides is a relatively rare event, and few examples have been reported up to 1980 (see Dutton, 1980). The antithyroid drug 6-n-propyl-2-thiouracil forms a confirmed S-glucuronide in rats (Lindsay et al., 1977). A pyrithone (Omedine) metabolite, 2-mercaptopyridine-N-oxide, was detected as an S-glucuronide in rat, rabbit, and rhesus monkey urine (Mitoma et al., 1983). The thiocarbonate herbicide, SUTAN [bis(2-methylpropyl)carbamothioic acid S-ethyl ester], was significantly metabolized to an S-glucuronide found in rat urine (Peffer et al., 1991). A recent search of the literature failed to reveal any reference to formation of S-glucuro...

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“…The resulting supernatant was then transferred to an HPLC vial and 150 l of this volume directly injected onto gradient HPLC using solid scintillant radioactive detection as described previously (Ethell et al, 1998). Product formation was linear for 1-naphthol glucuronidation, with a V max value in the same range as substrates for UGT1A6 and UGT1A9, which were most rapidly glucuronidated (Ethell et al, 1998). Control V79 cell lines showed no detectable activity toward these substrates.…”

Section: Methodsmentioning

confidence: 99%

“…Briefly, the incubations contained 100 mM Tris/maleate buffer, pH 7.4, containing 5 mM MgCl 2 , typically 500 M substrate, 250 to 350 g of cellular sonicate, 2 mM UDPGA (0.1Ci [ 14 C] UDPGA/assay) in a total volume of 100 l. Incubations were run for 40 min and terminated by the addition of 100 l of methanol that had been prechilled at Ϫ20°C. The resulting supernatant was then transferred to an HPLC vial and 150 l of this volume directly injected onto gradient HPLC using solid scintillant radioactive detection as described previously (Ethell et al, 1998). Product formation was linear for 1-naphthol glucuronidation, with a V max value in the same range as substrates for UGT1A6 and UGT1A9, which were most rapidly glucuronidated (Ethell et al, 1998).…”

Section: Methodsmentioning

confidence: 99%

“…UGT assays for phenolic glucuronidation were performed as described previously (Ethell et al, 1998). Briefly, the incubations contained 100 mM Tris/maleate buffer, pH 7.4, containing 5 mM MgCl 2 , typically 500 M substrate, 250 to 350 g of cellular sonicate, 2 mM UDPGA (0.1Ci [ 14 C] UDPGA/assay) in a total volume of 100 l. Incubations were run for 40 min and terminated by the addition of 100 l of methanol that had been prechilled at Ϫ20°C.…”

Section: Methodsmentioning

confidence: 99%

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Quantitative Structure Activity Relationships for the Glucuronidation of Simple Phenols by Expressed Human UGT1A6 and UGT1A9

Ethell¹,

Ekins²,

Wang³

et al. 2002

Drug Metab Dispos

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:UGT1A6 and UGT1A9 have both been demonstrated to rapidly glucuronidate simple phenolic compounds. A series of simple phenols were selected and screened with both isoforms and then used as model substrates for the generation of V max and K m values. UGT1A6 showed a more restricted acceptance of phenolic substrates compared with UGT1A9. However, the affinity of UGT1A6 for these compounds exhibited higher K m values than UGT1A9, although rates of turnover were similar. Molecular surfaceweighted holistic invariant molecular descriptors were generated for each substrate and used to produce the first quantitative structure activity relationship models generated for expressed human UGTs. Models relating log of the K m value to the generated descriptors correlated well with the experimental data r 2 value of

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A Universal Radiochemical High-Performance Liquid Chromatographic Assay for the Determination of UDP-Glucuronosyltransferase Activity

Cited by 44 publications

References 19 publications

Effects of Phenobarbital on Expression of UDP-Glucuronosyltransferase 1a6 and 1a7 in Rat Brain

Effects of Phenobarbital on Expression of UDP-Glucuronosyltransferase 1a6 and 1a7 in Rat Brain

GLUCURONIDATION OF HMR1098 IN HUMAN MICROSOMES: EVIDENCE FOR THE INVOLVEMENT OF UGT1A1 IN THE FORMATION OFS-GLUCURONIDES

Quantitative Structure Activity Relationships for the Glucuronidation of Simple Phenols by Expressed Human UGT1A6 and UGT1A9

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