nitrophenols and ethoxyresorufin

The hepatic cytochrome p450 enzymes 1 A, 2A19 and 2E1 is very important for the elimination of skatole from the body of pigs. Impaired skatole metabolism, results in skatole accumulation, which give rise to off flavor of the meat. Several metabolites of skatole has been identified, however the role of these metabolites in the inhibition of the skatole metabolizing enzymes are not documented. Using microsomes from pigs and fish, we determined the ability of several skatole metabolites to inhibit CYP1 A, CYP2A19 and CYP2E1 dependent activity. Our results show that 2-aminoacetophenone is an inhibitor of porcine CYP2A19 and CYP2E1 activity, but not the piscine orthologues. In conclusion, there is species specific differences in the inhibition of CYP1 A and CYP2A19 dependent metabolism of probe substrates. This is relevant to the evaluation of different model systems and to the reduction of off flavor of meat.

Topics: Acetophenones; Animals; Coumarins; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP2E1 Inhibitors; Cytochrome P-450 Enzyme System; Fishes; Liver; Male; Microsomes, Liver; Nitrophenols; Oxazines; Red Meat; Seafood; Skatole; Species Specificity; Swine

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

The cytochrome P450 enzymes (P450s) that mediate mammalian xenobiotic metabolism are highly versatile monooxygenases, which show wide and overlapping substrate ranges but generally poor catalytic rates. Re-engineering of these P450s may enable the development of useful biocatalysts for industrial applications. In the current study, restriction enzyme-mediated DNA family shuffling was used to create a library from human CYP1A1 and CYP1A2. Among sequenced clones (four randomly selected and eight functional clones), 5.9 +/- 2.3 crossovers and 1.5 +/- 1.5 spontaneous mutations (mean +/- S.D.) were detected per mutant. A high level of structural integrity as well as diverse functionality were found, with 53% of clones expressed at significant levels (>50 nM P450 hemoprotein) and 23% of clones showing activity on one or more of the following compounds: luciferin 6'-chloroethyl ether (luciferin-CEE), luciferin 6'-methyl ether (luciferin-ME), 6'-deoxyluciferin (luciferin-H), the ethylene glycol ester of luciferin 6'-methyl ether, 7-ethoxyresorufin, and p-nitrophenol (PNP). Different activity profiles were seen with higher specific activity on individual compounds (e.g., clone 22; 9 times the CYP1A1 specific activity toward luciferin-CEE), novel activities (e.g., clone 35; activity toward luciferin-H and PNP), and broadening of substrate range observed in particular clones (e.g., clone 9; activity toward both selective substrates luciferin-ME and luciferin-CEE as well as toward luciferin-H and PNP). In summary, forms were found with distinct and novel activity profiles, despite the relatively small number of mutants examined. In addition, the whole-cell metabolic assays described here provide simple, high-throughput methods useful for screening larger libraries.

Topics: Carbon Monoxide; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; DNA Mutational Analysis; DNA Restriction Enzymes; DNA Shuffling; Firefly Luciferin; Gene Library; Genotype; Humans; Luminescent Agents; Mutation; Nitrophenols; Oxazines; Phenotype; Spectrophotometry; Substrate Specificity

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

The inhibitory effect of the isoflavonoids genistein and equol on cytochrome P450 activities has been investigated. Genistein and equol inhibited the high capacity component of p-nitrophenol (CYP2E1 substrate) metabolism in liver microsomes from acetone-induced mice with IC50 values of approximately 10 mM and 560 microM, respectively (cf. diethyldithiocarbamate, IC50, 69 microM). Using human CYP2E1 from a specific expression system (which overcame multienzyme involvement in the rodent system), non-competitive inhibition was also seen with both isoflavonoids. Genistein and equol also inhibited the high capacity component of ethoxyresorufin (CYP1A substrate) metabolism in liver microsomes from beta-naphthoflavone-induced mice with IC50 values of 5.6 mM and 1.7 mM, respectively (cf. alpha-naphthoflavone, IC50 0.8 microM). Using human CYPIA2 from a specific expression system, noncompetitive inhibition was seen with both isoflavonoids. CYP1A1 inhibition offers a possible explanation for the chemopreventative effect of genistein against, for example, dimethylbenz[a]anthracene genotoxicity reported in animals but the IC50 values negate the relevance of this specific chemopreventative action at the levels likely to be achieved from the human diet.

Topics: Animals; Cell Line, Transformed; Chromans; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP1A2 Inhibitors; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP2E1 Inhibitors; Equol; Genistein; Humans; Isoflavones; Male; Mice; Mice, Inbred BALB C; Microsomes, Liver; Monoamine Oxidase Inhibitors; Nitrophenols; Oxazines; Rats; Rats, Wistar; Substrate Specificity

The dealkylations of 7-ethoxy- and 7-pentoxyresorufin,p-nitrophenol hydroxylation, and regio- and stereoselective hydroxylation of testosterone were measured to study the stability and inducibility of cytochrome P450 activities in cultured human hepatocytes. The results showed that human hepatocytes in primary culture retain the ability to increase specific cytochrome P450 activities upon incubation with inducers. 3-Methylcholanthrene produced a strong increase (6- to 21-fold over control) in 7-ethoxyresorufin O-deethylase activity and a small enhancement (1.5- to 2.5-fold) of the p-nitrophenol hydroxylation rate. Incubation of cells with phenobarbital resulted in moderate increases in 7-pentoxyresorufin O-depentylation (1.5- to 2-fold) and in testosterone hydroxylation at 16 alpha (1.5- to 4.5-fold) and 16 beta (1.3- to 4-fold) positions. Ethanol specifically increased p-nitrophenol hydroxylase activity (1.5- to 3.5-fold) and reduced 15 beta- and 6 beta-hydroxylations of testosterone. Treatment of hepatocytes with dexamethasone produced an increase of almost all the activities studied, with 6 beta- (2- to 3-fold) and 16 beta-hydroxytestosterone (1.4- to 2.4-fold) formation showing the greatest enhancement. Clofibric acid exposure resulted in 1.5- to 3-fold increases in 7-pentoxyresorufin O-depentylase and in testosterone 6 beta- and 2 beta-hydroxylase activities. Isosafrol selectively increased 7-ethoxyresorufin O-deethylase activity (2- to 3-fold), and it moderately reduced the other activities studied.

Topics: Adult; Aged; Cells, Cultured; Clofibric Acid; Cytochrome P-450 Enzyme System; Dexamethasone; Enzyme Induction; Ethanol; Female; Humans; Isoenzymes; Liver; Male; Methylcholanthrene; Middle Aged; Nitrophenols; Oxazines; Oxidation-Reduction; Phenobarbital; Safrole; Substrate Specificity; Testosterone

The ability of ethanol to induce the deethylation of ethoxyresorufin and hydroxylation of p-nitrophenol in rat lung and liver was investigated. The administration of 10% ethanol in the drinking water for 7 d increased hepatic p-nitrophenol hydroxylation twofold whereas ethanol ip (3 ml/kg/d for 7 d) decreased the activity. Both ethanol treatments decreased the activity in the lung. Ethoxyresorufin deethylation was induced in the liver by both ethanol treatments, and in the lung the bolus dosing ip increased the activity four-fold. The results indicate that the lung microsomes have p-nitrophenol hydroxylase and ethoxyresorufin deethylase activities, but only the latter was increased by ethanol treatment.

Topics: Administration, Oral; Animals; Ethanol; Hydroxylation; Injections, Intraperitoneal; Lung; Male; Microsomes, Liver; Nitrophenols; Oxazines; Rats; Rats, Inbred Strains

Rainbow trout kidney was subfractionated by differential centrifugation to obtain preparations suitable for the study of xenobiotic metabolizing enzymes and to ascertain the distribution of these activities in the cell. The cytochrome P-450-dependent monooxygenase, NADPH-cytochrome c reductase, and UDP glucuronosyltransferase, which are enzymes important in the biotransformation of xenobiotics, were enriched in the microsomal fraction. Another xenobiotic-metabolizing enzyme, epoxide hydrolase, was enriched in the mitochondrial and microsomal fractions almost to the same extent. Cytochrome P-450-dependent monooxygenase and UDP glucuronosyltransferase activities were characterized in the trout kidney microsomes. The cytochrome P-450 deethylation of 7-ethoxycoumarin and 7-ethoxyresorufin as well as the glucuronidation of p-nitrophenol in the kidney were found to proceed at rates comparable to those occurring in the liver. The difference spectrum of the complex between carbon monoxide and reduced trout kidney microsomes showed a peak at 448.5 nm. Addition of 7-ethoxycoumarin to kidney microsomes produced an absorbance change in difference spectrum similar to the substrate binding spectrum found in rainbow trout liver and rat liver microsomes.

Topics: Animals; Cell Fractionation; Coumarins; Cytochrome P-450 Enzyme System; Glucuronosyltransferase; Kidney; Microsomes; Microsomes, Liver; Mitochondria; NADPH-Ferrihemoprotein Reductase; Nitrophenols; Oxazines; Salmonidae; Trout