Cytochrome P-450 and Peroxidase Oxidize Detoxication Products of Carcinogenic Aristolochic Acids (Aristolactams) to Reactive Metabolites Binding to DNA in vitro

We report the analysis of DNA adducts formed from aristolactams I and II, which are the final metabolites derived from carcinogenic aristolochic acids in vivo, after their oxidation by microsomal cytochrome P-450 and horseradish peroxidase in vitro. DNA adducts were detected and quantified using the nuclease P1-enhanced variation of the 32P-postlabeling assay. Quantitative analysis revelead that the extent of modification of DNA by aristolactams activated by peroxidase was more than one order of magnitude higher than for activation by microsomal cytochrome P-450. Peroxidase catalyzes the formation of active oxygen in the presence of NADH, H2O2 and aristolactams. Aristolactams are also oxidized by mammalian peroxidase prostaglandin H synthase. The possible role of aristolactams in carcinogenesis induced by aristolochic acid is discussed.

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The role of cytochrome P-450 in cholesterol biogenesis and catabolism

Biochemical Journal ◽

10.1042/bj1280237 ◽

1972 ◽

Vol 128 (2) ◽

pp. 237-242 ◽

Cited By ~ 30

Author(s):

Sandra D. Atkin ◽

Eileen D. Palmer ◽

P. D. English ◽

B. Morgan ◽

M. A. Cawthorne ◽

...

Keyword(s):

Cholesterol Metabolism ◽

Carbon Disulphide ◽

Normal Serum ◽

Liver Cholesterol ◽

Drug Metabolizing Enzyme ◽

Metabolizing Enzyme ◽

Cytochrome P 450

1. Adjuvant-induced arthritis in rats is accompanied by a loss of activity of the drug-metabolizing enzyme system and a decrease in hepatic cytochrome P-450. 2. Arthritic rats have normal serum and liver cholesterol concentrations. 3. The rate of biogenesis of cholesterol in vivo and in vitro from either [14C]acetate or [14C]mevalonate in arthritic rats was the same as or greater than that found in control rats. 4. Treatment of rats with carbon disulphide (1ml/kg) resulted in a loss of drug-metabolizing-enzyme activity and increased cholesterol biogenesis. 5. The activity of cholesterol 7α-hydroxylase in adjuvant-induced arthritic rats did not differ significantly from that in control rats. 6. Rats fed with cholestyramine had an elevated hepatic cholesterol 7α-hydroxylase activity, but neither the concentration of cytochrome P-450 nor the activity of the drug-hydroxylating enzyme, aminopyrine demethylase, was affected. 7. The relationships between drug hydroxylation and cholesterol metabolism are discussed.

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Metabolic activation of acetylenic substituents to derivatives in the rat causing the loss of hepatic cytochrome P-450 and haem

Biochemical Journal ◽

10.1042/bj1740853 ◽

1978 ◽

Vol 174 (3) ◽

pp. 853-861 ◽

Cited By ~ 51

Author(s):

Ian N. H. White

Keyword(s):

Covalent Binding ◽

Metabolic Activation ◽

Microsomal Protein ◽

Significant Loss ◽

Microsomal Cytochrome ◽

Rf Values ◽

Green Pigments ◽

Cytochrome P 450

1. A number of acetylenic-substituted steroidal and non-steroidal compounds, including 2,2-dipropargylacetamide, pregna-2,4-dien-20-yno[2,3-d]isoxazol-17-ol (Danazol) and acetylene gas, when administered to rats in vivo brought about a decrease in the concentrations of hepatic microsomal cytochrome P-450 and haem. Abnormal haem-breakdown products, ‘green pigments’, and porphyrins accumulated in the livers of these animals. 2. For loss of microsomal cytochrome P-450 to occur in vitro, metabolic activation of the acetylenic substituent was necessary. The enzyme system responsible required NADPH and air, and was induced by pretreatment of rats with phenobarbitone; these are characteristics typical of the microsomal mixed-function oxidases. 3. When rats were dosed with 17α-ethynyl-17β-hydroxyandrost-4-en-3-one (ethynyltestosterone, 1mmol/kg) the pattern of green pigments extracted from the liver 4h after dosing and separated by t.l.c. was quite different from that in rats given 17β-hydroxy-17α-vinylandrost-4-en-3-one (vinyltestosterone), suggesting that reduction of the unsaturated triple bond to a double bond is not normally part of the metabolic activation pathway of the acetylenic substituent. 4. The green pigments extracted from the livers of rats 4h after the administration of the acetylenic-substituted compounds (1mmol/kg) when separated by silica-gel t.l.c. had variable RF values. The number and distribution of green pigments was characteristic for each compound examined. There was little correlation between the total loss of hepatic microsomal haem and the apparent intensity of the green pigments seen on the thin-layer chromatograms. 5. After incubation of [14C]acetylene in vitro with microsomal preparations from phenobarbitone-pretreated rats and a NADPH-generating system, no significant covalent binding to microsomal protein was detected over a 30min incubation period, although under similar conditions there was a significant loss of cytochrome P-450.

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DNA adducts formed by ring-oxidation of the carcinogen 2-naphthylamine with prostaglandin H synthase in vitro and in the dog urothelium in vivo

Carcinogenesis ◽

10.1093/carcin/6.9.1379 ◽

1985 ◽

Vol 6 (9) ◽

pp. 1379-1387 ◽

Cited By ~ 46

Author(s):

Y. Yamazoe ◽

D.W. Miller ◽

C.C. Weis ◽

K.L. Dooley ◽

T.V. Zenser ◽

...

Keyword(s):

Dna Adducts ◽

Prostaglandin H Synthase ◽

Ring Oxidation ◽

Prostaglandin H

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32P-Postlabelling Analysis of DNA Adducts with 1-(Phenylazo)-2-naphthol (Sudan I, Solvent Yellow 14) Formed in vivo in Fisher 344 Rats

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19991335 ◽

1999 ◽

Vol 64 (8) ◽

pp. 1335-1347 ◽

Cited By ~ 11

Author(s):

Marie Stiborová ◽

Heinz H. Schmeiser ◽

Andrea Breuer ◽

Eva Frei

Keyword(s):

Cytochrome P450 ◽

Urinary Bladder ◽

Oral Administration ◽

Dna Adducts ◽

Initiation Phase ◽

Negative Results ◽

Nuclease P1 ◽

Sudan I

We report the analysis of DNA adducts with 1-(phenylazo)-2-naphthol in the liver and urinary bladder of Fisher 344 rats treated orally with this dye. DNA adducts were detected and quantitated using the nuclease P1-enhanced version of the 32P-postlabelling assay. Two variations of multidirectional chromatographic systems were used to resolve either bulky and/or smaller (polar) 32P-labelled adducts by TLC. In the present study, a double oral administration of the dye (500 mg/kg) for one day yielded negative results in 32P-postlabelling assay of liver DNA (24 h after dosing). However, three DNA adducts in the urinary bladder were detected under the same conditions of treatment. Chromatography experiments indicated that the two principal DNA adducts detected in the urinary bladder of Fisher 344 rats were the same as those detected in DNA modified by 1-(phenylazo)-2-naphthol and its metabolite 1-(phenylazo)naphthalene-2,6-diol after their activation with peroxidase in vitro. The results presented here strongly suggest that peroxidase itself or in a combination with cytochrome P450 participates in the initiation phase of 1-(phenylazo)-2-naphthol carcinogenesis in the urinary bladder.

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The Effect of pH on Peroxidase-Mediated Oxidation of and DNA Adduct Formation by Ellipticine

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc20061169 ◽

2006 ◽

Vol 71 (8) ◽

pp. 1169-1185 ◽

Cited By ~ 12

Author(s):

Jitka Poljaková ◽

Martin Dračínský ◽

Eva Frei ◽

Jiří Hudeček ◽

Marie Stiborová

Keyword(s):

Horseradish Peroxidase ◽

Dna Adducts ◽

Cytochromes P450 ◽

Antineoplastic Agent ◽

Ph Optimum ◽

Effect Of Ph ◽

Order Of Magnitude ◽

Enzymatic Activation

In order to understand the mechanism of enzymatic activation of an antineoplastic agent ellipticine, we investigated the effect of pH on the efficiency of three model peroxidases (bovine lactoperoxidase, human myeloperoxidase and horseradish peroxidase) in oxidation of ellipticine and in formation ellipticine-DNA adducts. The formation of the major ellipticine metabolite, ellipticine dimer, in which two ellipticine residues are connected through nitrogenN6in the pyrrole ring of one of the ellipticine moieties and carbon C9 of the other ellipticine, and formation of four ellipticine-DNA adducts were analyzed. All three peroxidases oxidize ellipticine to dimer and form ellipticine-DNA adducts, but lactoperoxidase and myeloperoxidase were less efficient in these processes than horseradish peroxidase. More than one order of magnitude higher rates of formation of dimer and amounts of the DNA adducts were found upon horseradish peroxidase than in reactions with lactoperoxidase or myeloperoxidase. An acid pH optimum was found for the formation of ellipticine dimer (pH 6.4), while the highest binding of ellipticine activated by peroxidases to DNA was detectable at pH 8.4. Likewise, the highest binding of 5-(hydroxymethyl)ellipticine, a metabolite of ellipticine generated by cytochrome P450, to DNA was found at pH 8.4. The results presented here are a contribution to the explanation of the reaction mechanism of formation of the major deoxyguanosine adduct in DNA generated from ellipticinein vivoandin vitroby its activation with cytochromes P450 and peroxidases.

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Cycle Network Model of Prostaglandin H Synthase-1

Pharmaceuticals ◽

10.3390/ph13100265 ◽

2020 ◽

Vol 13 (10) ◽

pp. 265

Author(s):

Alexey Goltsov ◽

Maciej Swat ◽

Kirill Peskov ◽

Yuri Kosinsky

Keyword(s):

Enzyme Activation ◽

Prostaglandin H Synthase ◽

Experimental Conditions ◽

Peroxidase Enzyme ◽

Inflammatory Drugs ◽

Prostaglandin H ◽

Regulatory Properties

The kinetic model of Prostaglandin H Synthase-1 (PGHS-1) was developed to investigate its complex network kinetics and non-steroidal anti-inflammatory drugs (NSAIDs) efficacy in different in vitro and in vivo conditions. To correctly describe the complex mechanism of PGHS-1 catalysis, we developed a microscopic approach to modelling of intricate network dynamics of 35 intraenzyme reactions among 24 intermediate states of the enzyme. The developed model quantitatively describes interconnection between cyclooxygenase and peroxidase enzyme activities; substrate (arachidonic acid, AA) and reducing cosubstrate competitive consumption; enzyme self-inactivation; autocatalytic role of AA; enzyme activation threshold; and synthesis of intermediate prostaglandin G2 (PGG2) and final prostaglandin H2 (PGH2) products under wide experimental conditions. In the paper, we provide a detailed description of the enzyme catalytic cycle, model calibration based on a series of in vitro kinetic data, and model validation using experimental data on the regulatory properties of PGHS-1. The validated model of PGHS-1 with a unified set of kinetic parameters is applicable for in silico screening and prediction of the inhibition effects of NSAIDs and their combination on the balance of pro-thrombotic (thromboxane) and anti-thrombotic (prostacyclin) prostaglandin biosynthesis in platelets and endothelial cells expressing PGHS-1.

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Interaction of monocrotophos and its novel thion analogues with microsomal cytochrome P-450: in vivo and in vitro studies in rat

Toxicology ◽

10.1016/0300-483x(92)90160-g ◽

1992 ◽

Vol 76 (2) ◽

pp. 133-139 ◽

Cited By ~ 5

Author(s):

M.K.J. Siddiqui ◽

M. Mahboob ◽

M. Mustafa

Keyword(s):

In Vitro Studies ◽

Microsomal Cytochrome ◽

Cytochrome P 450

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To the Mechanism of 2-Nitroanisole Carcinogenicity: in vitro Metabolism of 2-Nitroanisole Mediated by Peroxidasesand Xanthine Oxidase

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19980857 ◽

1998 ◽

Vol 63 (6) ◽

pp. 857-869 ◽

Cited By ~ 8

Author(s):

Marie Stiborová ◽

Heinz H. Schmeiser ◽

Eva Frei

Keyword(s):

Horseradish Peroxidase ◽

Xanthine Oxidase ◽

Dna Adducts ◽

Active Oxygen Species ◽

Radical Mechanism ◽

Maximal Velocity ◽

Prostaglandin H Synthase ◽

Nuclease P1 ◽

Prostaglandin H

The in vitro enzymatic metabolism of carcinogenic 2-nitroanisole was investigated using peroxidases (horseradish peroxidase and prostaglandin H synthase) and xanthine oxidase catalyzing oxidative and reductive reactions, respectively. The oxidation of 2-nitroanisole catalyzed by horseradish peroxidase exhibits the Michaelis-Menten kinetics. The Michaelis constant (Km) and the maximal velocity (Vmax) values for this substrate were determined at pH 5.0, 7.0, 7.6 and 8.0. At optimal pH (7.6), the Km and Vmax values are 0.219 μmol/l and 34.45 pmol/min per nmol peroxidase, respectively. The oxidation of 2-nitroanisole is inhibited by radical trapping agents (NADH, ascorbate, glutathione and nitrosobenzene). This indicates that the peroxidase-mediated oxidation of 2-nitroanisole proceeds via a radical mechanism. Active oxygen species are formed during the horseradish peroxidase-catalyzed reactions in the presence of NADH, hydrogen peroxide and 2-nitroanisole. 2-Nitroanisole is also oxidized by mammalian prostaglandin H synthase. Using the nuclease P1-enhanced variation of the 32P-postlabelling assay, the formation of DNA adducts was detected in DNA treated with 2-nitroanisole and xanthine oxidase. No DNA binding was detected after oxidation of 2-nitroanisole with horseradish peroxidase and prostaglandin H synthase. The results presented (the formation of DNA adducts after 2-nitroanisole activation by xanthine oxidase and that of radicals and/or superoxide radicals during the reactions with peroxidases) strongly suggest the participation of 2-nitroanisole both in the initiation and in the promotion phases of carcinogenesis.

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