nitrophenols and 7-hydroxycoumarin

Cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) are the most important enzymes for metabolic clearance. Characterization of phase I and phase II metabolism of a given drug in cellular models is therefore important for an adequate interpretation of the role of drug metabolism in toxicity. We investigated phase I (CYP) and phase II (UGT and SULT) metabolism of three drugs related to drug-induced liver injury (DILI), namely acetaminophen (APAP), diclofenac (DF) and tolcapone (TC), in cryopreserved primary human hepatocytes from 5 donors in suspension and monolayer. The general phase II substrate 7-hydroxycoumarin (7-HC) was included for comparison. Our results show that the decrease in CYP, UGT and SULT activity after plating is substrate dependent. As a consequence the phase I/phase II metabolism ratio is significantly affected, with a shift in monolayer towards phase I metabolism for TC and towards phase II metabolism for APAP and DF. Inter-donor variability in drug metabolism is significant, especially in sulfation of 7-HC or APAP. As CYP, UGT and SULT metabolism may lead to bioactivation and/or detoxification of drugs, a changed ratio in phase I/phase II metabolism may have important consequences for metabolism-related toxicity.

Topics: Acetaminophen; Aged; Benzophenones; Cells, Cultured; Cryopreservation; Cytochrome P-450 Enzyme System; Diclofenac; Female; Glucuronosyltransferase; Hepatocytes; Humans; Male; Middle Aged; Nitrophenols; Sulfotransferases; Tolcapone; Umbelliferones

UDP-glucuronosyltransferase (UGT) is an enzyme that catalyses a major phase II reaction in drug metabolism. Glucuronidation occurs mainly in the liver, but UGTs are also expressed in extrahepatic tissues, where they play an important role in local metabolism. UGT1A isoforms catalyse the glucuronidation of several drugs, neurotransmitters and neurosteroids that exert pharmacological and physiological effects on the brain. The aim of the current study was to determine UGT1A mRNA expression levels and glucuronidation activities in different regions of the rat brain (namely the cerebellum, frontal cortex, parietal cortex, piriform cortex, hippocampus, medulla oblongata, olfactory bulb, striatum and thalamus). It was found that all UGT1A isoforms were expressed in all the nine regions, but their expression levels differed between the regions. The difference between the regions of the brain where the mRNA levels were highest and those where they were lowest ranged between 2.1- to 7.8-fold. Glucuronidation activities were measured using the UGT substrates such as mycophenolic acid, p-nitrophenol and umbelliferone. Glucuronidation activity was detected in all nine regions of the brain. Activity levels differed between the regions, and were highest in the cerebellum, medulla oblongata and olfactory bulb. Differences in glucuronidation activity between regions with the highest rates and those with the lowest rates ranged from 5.3- to 10.1-fold. These results will contribute to our current understanding of the physiological and pharmacokinetic roles of drug-metabolizing enzymes in the brain. Copyright © 2016 John Wiley & Sons, Ltd.

Topics: Animals; Brain; Glucuronides; Glucuronosyltransferase; Male; Microsomes, Liver; Mycophenolic Acid; Nitrophenols; Rats, Sprague-Dawley; RNA, Messenger; Umbelliferones

Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.

Topics: Amino Acid Substitution; Bacterial Proteins; Biocatalysis; Carbon; Chlorzoxazone; Coumarins; Cytochrome P-450 Enzyme System; Enzyme Stability; Escherichia coli; Heme; Humans; Indigo Carmine; Indoles; Kinetics; Molecular Dynamics Simulation; NADPH-Ferrihemoprotein Reductase; Nitrophenols; Oxazines; Oxidation-Reduction; Phenacetin; Protein Engineering; Recombinant Proteins; Transformation, Genetic; Umbelliferones

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

1: The metabolism by HepG2 cell from two sources (M1, M2) of 12 substrates is reported: ethoxyresorufin, ethoxycoumarin, testosterone, tolbutamide, chlorzoxazone, dextromethorphan, phenacetin, midazolam, acetaminophen, hydroxycoumarin, p-nitrophenol and 1-chloro-2,4-dinitrobenzene (CDNB), and a pharmaceutical compound, EMD68843. 2: Activities varied markedly. Some were present in M1 (CYP1A, CYP2C9, CYP2E1) but absent in M2. M1 had a more complete set of Phase I enzymes than M2. CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A activities were present at levels similar to human hepatocytes. Phase II metabolism differed between M1 and M2. M1 conjugated hydroxycoumarin and p-nitrophenol to glucuronides only, whereas M2 produced sulfates. Glutathione conjugation of CDNB metabolism was 10-fold higher in M1 than in M2, but was still much lower than in human hepatocytes. CYP2E, CYP2C, CYP2B6 and CYP3A (but not CYP1A, glucuronyl S-transferase or S-transferase) were inducible in M1. Metabolites of EMD68843, produced by induced (but not uninduced) M1 were the same as those produced in human hepatocytes. 3: In conclusion, HepG2 cells have both Phase I and II enzymes, which activities and at what levels depend on the source and culture conditions. Therefore, HepG2 cells routinely used in in vitro assays should be characterized for their drug-metabolizing capabilities before any results can be fully interpreted.

Topics: Aryl Hydrocarbon Hydroxylases; Benzofurans; Carcinoma, Hepatocellular; Cell Culture Techniques; Cell Line, Tumor; Coumarins; Dinitrochlorobenzene; Enzyme Activation; Humans; Indoles; Kinetics; Liver Neoplasms; Nitrophenols; Oxazines; Piperazines; Substrate Specificity; Umbelliferones; Vilazodone Hydrochloride

Rates of glucuronidation and sulfation of 7-hydroxycoumarin were studied in perfused livers from normal chow-fed rats, or in livers from rats that had been fed liquid control or ethanol-containing diets. During infusion of 100 microM 7-hydroxycoumarin, rates of glucuronidation were similar in livers from chow-fed or control diet rats, but were 34% less in livers from ethanol-fed rats. These rates of glucuronidation in perfused livers could not be explained by changes of UDP-glucuronyltransferase activity, which was highest in hepatic microsomes from ethanol-treated rats and lowest in microsomes from chow-fed rats. The low rates of glucuronidation in livers from ethanol-treated rats were correlated with low hepatic concentrations of UDP-glucuronic acid, which were less than 70% of the levels measured in the other treatment groups. However, the diminished UDP-glucuronic acid levels could not be explained by alterations in adenine nucleotides, NAD+/NADH ratios, glycogen, UDP-glucose, or activity of UDP-glucose dehydrogenase. Rates of sulfation declined during prolonged 7-hydroxycoumarin infusion in livers from ethanol-treated rats, but not in livers from rats that had received the control diet. Similarly, hepatic concentrations of adenosine-3'-phosphate 5'-sulfatophosphate (PAPS) also decreased with time only in livers from ethanol-treated rats. Thus, chronic ethanol feeding impairs glucuronidation and sulfation in perfused livers as a result of diminished availability of the required cofactors for these conjugation pathways.

Topics: Adenosine Triphosphate; Alcoholism; Animals; Arylsulfotransferase; Female; Gluconeogenesis; Glucuronosyltransferase; Lactates; Liver; Nitrophenols; Phosphoadenosine Phosphosulfate; Pyruvates; Pyruvic Acid; Rats; Sulfurtransferases; Umbelliferones

The rates of metabolism of 7-ethoxycoumarin and subsequent conjugation of 7-hydroxycoumarin were studied in perfused livers from hypophysectomized or sham-operated control rats. Rates of 7-ethoxycoumarin O-deethylation were higher both in perfused livers and microsomes from hypophysectomized compared to sham-operated controls. However, conjugation of 7-hydroxycoumarin formed from 7-ethoxycoumarin was markedly decreased by hypophysectomy. p-Nitrophenol conjugation was also impaired in perfused livers from hypophysectomized rats. During infusion of 59-65 microM of p-nitrophenol into hypophysectomized livers, rates of glucuronidation were diminished by 45% (6.83 +/- 0.49 to 3.78 +/- 0.31 mumol/g/h) and rates of sulfation were decreased by 50% (1.25 +/- 0.12 to 0.62 +/- 0.07 mumol/g/h). Phenol sulfotransferase activity was decreased 57% by hypophysectomy, which is the likely explanation for the decrease in sulfate conjugation. However, hypophysectomy did not affect glucuronyltransferase activity. Perfused livers from hypophysectomized rats released glucose, pyruvate and lactate at lower rates than livers from sham-operated rats. Furthermore, infusion of glucose into perfused livers from hypophysectomized rats, but not sham-operated controls, increased the rate of conjugation. The results demonstrate that hypophysectomy decreased rates of conjugation in perfused livers, and that the decreased glucuronidation rates are probably the result of diminished carbohydrate reserves.

Topics: 7-Alkoxycoumarin O-Dealkylase; Animals; Arylsulfotransferase; Coumarins; Glucose; Glucuronidase; Glucuronosyltransferase; Hypophysectomy; Lactates; Liver; Male; Microsomes, Liver; Mixed Function Oxygenases; Nitrophenols; Oxidoreductases; Oxygenases; Perfusion; Pyruvates; Rats; Rats, Inbred Strains; Sulfurtransferases; Umbelliferones