hymecromone and 4-nitrophenol

4-Azido-2-hydroxybenzoic acid (4-AzHBA), a novel photoactive benzoic acid derivative, has been synthesized and used as a photoprobe to identify the phenol binding site of UDP-glucuronosyltransferases (UGTs). Analysis of recombinant His-tag UGTs from the 1A family for their ability to glucuronidate p-nitrophenol (pNP) and 4-methylumbelliferone (4-MU) revealed that UGT1A10 shows high activity toward phenols and phenol derivatives. Purified UGT1A10 was photolabeled with 4-AzHBA, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry. A single modified peak corresponding to amino acid residues 89-98 (EFMVFHAQWK) of UGT1A10 was identified. The attachment site of the 4-AzHBA probe was localized to the quadruplet Phe(90)-Met(91)-Val(92)-Phe(93) using ESI LC-MS/MS. Sequence alignment revealed that the Phe(90) and Phe(93) are conserved in UGT1A7-10. Site-directed mutagenesis of these two amino acids was then followed by kinetic analysis of the mutants with two phenolic substrates, pNP and 4-MU, containing one and two planar rings, respectively. Using the combination of photoaffinity labeling, enzymatic digestion, MALDI-TOF and LC-MS mass spectrometry, and site-directed mutagenesis, we have determined for the first time that Phe(90) and Phe(93) are directly involved in the catalytic activity of UGT1A10 toward 4-MU and pNP.

Topics: Amino Acid Sequence; Azides; Binding Sites; Chromatography, Liquid; Glucuronosyltransferase; Humans; Hymecromone; Kinetics; Mass Spectrometry; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitrophenols; Phenol; Phenylalanine; Photoaffinity Labels; Recombinant Proteins; Salicylates; Sequence Alignment; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Amination of 4-nitrophenol, umbelliferone and 4-methylumbelliferone gave the corresponding oxyamines 1-3. These oxyamines react with aldehydes and ketones to form oximes. In the case of aliphatic aldehydes and electron-poor aromatic aldehydes, the oximes undergo base-catalyzed fragmentation in aqueous buffer in the presence of bovine serum albumin to give the parent phenols, which is the acyclic analog of Kemp's elimination reaction of 5-nitrobenzisoxazole 28. The process can be used as a spectrophotometric assay for formaldehyde under aqueous neutral conditions.

Topics: Aldehydes; Amination; Chromatography, Thin Layer; Chromogenic Compounds; Ethers; Fluorescent Dyes; Formaldehyde; Hymecromone; Ketones; Kinetics; Lipoprotein Lipase; Magnetic Resonance Spectroscopy; Molecular Structure; Nitrophenols; Oximes; Pseudomonas; Serum Albumin, Bovine; Spectrophotometry; Umbelliferones

UDP-glucuronosyltransferases (UGTs) are important enzymes involved in glucuronidation of various exogenous and endogenous compounds. Studies were undertaken on the variability of three UGT enzyme activities in human livers. Enzyme activities were associated with genetic polymorphisms in UGT1A1 (UGT1A1*28) and UGT1A6 (UGT1A6*2). UGT1A1*28 is associated with Gilbert's syndrome, a deficiency in glucuronidation of bilirubin leading to mild hyperbilirubinemia, whereas UGT1A6*2 may result in low glucuronidation rates of several drugs.. Enzyme activities and genetic polymorphisms were assessed in 39 human liver samples, and polymorphisms were also assessed in blood of 253 healthy controls.. Associations were found between UGT enzyme activities of bilirubin (B) and 4-nitrophenol (NP; r=0.47, P=0.0024), B and 4-methylumbelliferone (MUB; r=0.54, P=0.0003), and NP and MUB (r=0.89, P<0.0001). In addition to the association between B-UGT enzyme activity and UGT1A1*28 (r=0.45, P=0.0034) as reported earlier, an association between B-UGT and UGT1A6*2 (r=0.43, P=0.007) was found. In 253 Dutch Caucasian controls, co-occurrence of UGT1A1*28 and UGT1A6*2 was found (r=0.9, P<0.0001).. Most patients with Gilbert's syndrome, in addition to their reduced B-UGT enzyme activity, may have abnormalities in the glucuronidation of aspirin or coumarin- and dopamine-derivatives, due to this combination of UGT1A1*28 and UGT1A6*2 genotypes.

Topics: Adult; Bilirubin; Case-Control Studies; Female; Genotype; Gilbert Disease; Glucuronosyltransferase; Humans; Hymecromone; Male; Nitrophenols; Polymorphism, Genetic

1. To elucidate the determining factors for elimination pathways of sulfate and glucuronide metabolites of xenobiotics, a single-pass perfusion of 4-methylumbelliferone (4MU) or p-nitrophenol (pNP) was performed with an isolated rat liver preparation. 2. Without bovine serum albumin in the perfusion system, clearance calculated based on the unbound concentration in the liver clearly showed that the net efflux clearances (CLeff) of sulfates from the sinusoidal membrane were much higher than those of glucuronides and that the biliary excretion clearances (CLb) of glucuronides were approximately two times larger than those of sulfates. 3. The ratios of CLeff to CLb were much higher for sulfates than those for glucuronides. The bile-oriented elimination of glucuronides or sinusoidal efflux-oriented elimination of sulfates was observed even using the perfusate including 3% bovine serum albumin, but the sinusoidal efflux of sulfates was extensively enhanced by bovine serum albumin in the perfusate. The mechanisms behind this stimulatory effect remain to be elucidated. 4. For both compounds, CLb of glucuronide was comparable with CLb of sulfate, meaning that CLb is not responsible for the biliary excretion of glucuronides at extensively higher rate than sulfates. 5. Higher concentration of glucuronides in the liver, partly caused by much lower CLeff of glucuronides than that of sulfates, is likely responsible for the bile-oriented excretion of glucuronides. The extensive sinusoidal efflux of sulfates, leading to the urine-oriented excretion, is attributed to the substantially higher CLeff than CLb. 6. In conclusion, the sinusoidal efflux is an important factor for determining elimination pathways of both sulfates and glucuronides, although further studies are needed to clarify the mechanisms of the sinusoidal efflux.

Topics: Acetaminophen; Animals; Bile; Glucuronides; Hymecromone; In Vitro Techniques; Liver; Male; Nitrophenols; Protein Binding; Rats; Rats, Wistar; Serum Albumin, Bovine; Sulfates; Xenobiotics

1. The hepatic and renal handling of glucuronides and sulphates of three phenolic compounds, 4-methylumbelliferone (4-MU), p-nitrophenol (pNP) and acetaminophen (APAP), were evaluated pharmacokinetically by in vivo constant infusion experiments in rat. It was shown that the urinary excretion rate at steady-state was larger than the biliary excretion rate for both glucuronides and sulfates, and sulfates, in particular, were extensively excreted into the urine. 2. For each glucuronide, however, biliary excretion clearances (CL(b)) calculated based on the total concentration and unbound concentration in the liver were much larger than the corresponding renal excretion clearances (CL(r)). Even in the case of sulfates, there was not any large difference between CL(r) and CL(b) based on the total and unbound concentration in tissues, which could not explain their extensive urinary excretion. From these results, these excretion clearances were recognized not to reflect necessarily the actual excretion rate obtained. 3. On the other hand, the tissue-to-plasma concentration ratio (K(p)) of both glucuronides and sulfates for every phenolic compound was much higher in the kidney than that in the liver. The results suggested that one of the most important determinants for the preferential excretion of these conjugates into the bile or urine is the extent of disposition of each compound to the liver or kidney. 4. In addition, K(p) of both glucuronides and sulfates in the liver, where these conjugates are mainly formed, was small. The K(p) of sulfates was quite low, suggesting that sulfates generated in the liver were subject to extensive sinusoidal efflux.

Topics: Acetaminophen; Animals; Glucuronides; Hymecromone; Infusions, Intravenous; Kidney; Kinetics; Liver; Male; Nitrophenols; Pharmaceutical Preparations; Rats; Rats, Wistar; Sulfates

A UDP-glucuronyltransferase isoform glucuronizes phenolic xenobiotics such as 4-nitrophenol, and an isoform glucuronizing 4-hydroxybiphenyl has also been found in rat liver. We purified a UDP-glucuronyltransferase isoform glucuronizing 4-hydroxybiphenyl from bovine liver microsomes by solubilization with 0.7% sodium cholate followed by three column chromatographic separations using DEAE-Toyopearl 650S, UDP-hexanolamine Sepharose 4B, and hydroxyapatite. The purified bovine liver 4-hydroxybiphenyl UDP-glucuronyltransferase (named Bovine 4HBGT) had glucuronidation activities toward 4-hydroxybiphenyl and 4-methylumbelliferone but had little activity toward 4-nitrophenol and 1-naphthol. The apparent molecular mass of Bovine 4HBGT was 54,000 Da on SDS-PAGE, and this was decreased to 50,000 Da by digestion with endo-beta-N-acetylglucosaminidase H. These data suggest that Bovine 4HBGT consists of a 50,000 Da polypeptide and a high mannose type oligosaccharide chain(s) of about 4,000 Da. The NH2-terminal sequence of GT-3 was GKVLVWPVDFSXWINI. These properties of Bovine 4HBGT were very similar to those of rat UDP-glucuronyltransferase glucuronizing xenobiotics. However, the NH2-terminal sequence of Bovine 4HBGT had higher homology with that of rat liver 4-hydroxybiphenyl UDP-glucuronyltransferase than with that of rat liver 4-nitrophenol UDP-glucuronyltransferase.

Topics: Amino Acid Sequence; Animals; Biphenyl Compounds; Catalysis; Cattle; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Glucuronosyltransferase; Hymecromone; Male; Microsomes, Liver; Molecular Sequence Data; Naphthols; Nitrophenols; Sequence Homology, Amino Acid; Substrate Specificity

Crigler-Najjar syndrome, type I (CN-I) is a potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from a recessively inherited deficiency of hepatic UDP-glucuronosyl-transferase (UGT) activity toward bilirubin (B-UGT). Two forms of B-UGT exist in human liver. mRNAs for these two forms and that for another isoform with activity toward simple phenols (P-UGT) have unique 5' regions, but their 3' regions are identical. The three mRNA species are derived from a single locus; the unique 5' regions are encoded by single unique exons and the identical 3' regions consist of four consecutive exons that are shared by all three isoforms. In this paper, we determined genetic lesions in two CN-I patients with deficiency of hepatic B-UGT and P-UGT activities. In one patient, there was a C----T substitution in exon 4 (common region) predicting the substitution of a serine residue with a phenylalanine residue; this mutation was present in the identical region of B-UGT and P-UGT mRNAs. In the other patient, a C----T substitution in exon 2 (common region) of the B-UGT/P-UGT locus resulted in a premature stop codon. This exon (132 nt) was absent in heptic B-UGT and P-UGT mRNAs of this patient due to exon skipping during pre-mRNA processing. Sequence abnormality of three distinct mRNA species explains the abnormality of multiple UGT isoforms in these patients. Presence of identical abnormalities in the common regions of the three mRNAs is consistent with the finding that the common 3' regions of the two B-UGT mRNAs and the P-UGT mRNA are encoded by four shared exons.

Topics: Androsterone; Base Sequence; Bilirubin; Blotting, Northern; Chromosome Mapping; Crigler-Najjar Syndrome; Electrophoresis, Agar Gel; Estradiol; Glucuronosyltransferase; Humans; Hymecromone; Isoenzymes; Liver; Molecular Sequence Data; Nitrophenols; Polymerase Chain Reaction; RNA, Messenger

Studies on the induction of non-oxygenative detoxication enzymes in mice by anticarcinogenic thionosulfur compounds have been extended to include hepatic and pulmonary UDP-glucuronosyltransferases. Dietary administration of disulfiram and of bisethylxanthogen to female CD-1 mice enhanced microsomal glucuronidation of 4-methylumbelliferone, a characteristic GT1 substrate, and of 4-hydroxybiphenyl, a GT2 substrate. Latency of the activity toward 4-methylumbelliferone was not affected appreciably. Disulfiram also enhanced glucuronidation of 4-nitrophenol. Diethyldithiocarbamate was ineffective under the conditions used. These thionosulfur compounds caused no significant change in beta-glucuronidase activity measured in homogenates of 7 organs.

Topics: Animals; Biphenyl Compounds; Diet; Disulfides; Disulfiram; Ditiocarb; Female; Glucuronates; Glucuronidase; Glucuronosyltransferase; Hymecromone; Lung; Mice; Microsomes; Microsomes, Liver; Nitrophenols; Thiones

In order to further establish optimal and reproducible conditions for the use of primary hepatocyte cultures in studies of drug metabolism, the effect of culture age on the basal and induced activities of ethoxycoumarin O-deethylase (ECDE), UDP-glucuronyltransferase (GT) [methylumbelliferone (MU) and p-nitrophenol (pNP) as substrates] and sulfotransferase (MU) were measured. In contrast to the monooxygenase activity conjugating activities were maintained for 2-3 weeks in culture, although especially sulfate conjugation showed a transient decline during the first days, and GT activity increased later on during culture. Low induction of ECDE with both phenobarbital (PB) and 3-methylcholanthrene (MC) was seen during the first day in culture, and maximum induction was obtained when inducer was added on the second or third day. The MC inducible GT (pNP) exhibited a similar behaviour indicating that the coordinated induction of the MC inducible activities is preserved in culture. The results show that primary cultures of hepatocytes can be used to study conjugating enzymes and their regulation. However, each functional parameter that is to be investigated in hepatocyte cultures should first be studied as a function of culture age to establish the optimum time.

Topics: 7-Alkoxycoumarin O-Dealkylase; Animals; Cells, Cultured; Cytochrome P-450 Enzyme System; Enzyme Induction; Glucuronosyltransferase; Hymecromone; Liver; Male; Nitrophenols; Oxygenases; Rats; Rats, Inbred Strains; Sulfurtransferases; Time Factors