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

1998 ◽  
Vol 63 (6) ◽  
pp. 857-869 ◽  
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.

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

1995 ◽  
Vol 60 (12) ◽  
pp. 2189-2199 ◽  
Author(s):  
Marie Stiborová ◽  
Eva Frei ◽  
Heinz H. Schmeiser ◽  
Manfred Wiessler
Keyword(s):  
Dna Adducts ◽  
Microsomal Cytochrome ◽  
Aristolochic Acids ◽  
Nuclease P1 ◽  
Order Of Magnitude ◽  
Prostaglandin H ◽  
Cytochrome P 450

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.

Study on N-Demethylation of N,N-Dimethyl-4-aminoazobenzene and N-Nitrosamines by Prostaglandin H Synthase

1997 ◽  
Vol 62 (6) ◽  
pp. 971-980 ◽  
Author(s):  
Marie Stiborová ◽  
Eva Frei ◽  
Heinz H. Schmeiser
Keyword(s):  
Hydrogen Peroxide ◽  
Arachidonic Acid ◽  
Free Radicals ◽  
Seminal Vesicle ◽  
Oxidation Mechanism ◽  
Maximal Velocity ◽  
Prostaglandin H Synthase ◽  
Michaelis Constant ◽  
Prostaglandin H

The in vitro enzymatic metabolism of carcinogenic N,N-dimethyl-4-aminoazobenzene, N-nitroso-N-methylaniline and N-nitroso-N,N-dimethylamine was investigated using ram seminal vesicle microsomal prostaglandin H synthase. Both N-nitrosamines are not converted by the studied enzyme. Formaldehyde is produced by the prostaglandin H synthase catalyzed reaction from N,N-dimethyl-4-aminoazobenzene. Arachidonic acid and hydrogen peroxide serve as cofactors for the oxidation of N,N-dimethyl-4-aminoazobenzene. The apparent Michaelis constant and the maximal velocity values for N,N-dimethyl-4-aminoazobenzene as a substrate are 64 μmol/l and 51.2 nmol HCHO/min/mg protein, respectively. In addition to formaldehyde, N-methyl-4-aminoazobenzene and 4-aminoazobenzene, two unknown substances are the products of the N,N-dimethyl-4-aminoazobenzene oxidation. The oxidation of N,N-dimethyl-4-aminoazobenzene catalyzed by prostaglandin H synthase is inhibited by glutathione, ascorbate and NADH. The results suggest that prostaglandin H synthase metabolizes N,N-dimethyl-4-aminoazobenzene through a one-electron oxidation mechanism, giving rise to free radicals.

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

1985 ◽  
Vol 6 (9) ◽  
pp. 1379-1387 ◽  
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

In vitro inhibition of prostaglandin H synthase by compounds from the exocrine secretions of lace bugs

1989 ◽  
Vol 93 (2) ◽  
pp. 253-255 ◽  
Author(s):  
Russell A. Jurenka ◽  
John W. Neal ◽  
Ralph W. Howard ◽  
James E. Oliver ◽  
Gary J. Blomquist
Keyword(s):  
Prostaglandin H Synthase ◽  
Exocrine Secretions ◽  
Lace Bugs ◽  
In Vitro Inhibition ◽  
Prostaglandin H

In vitro prostaglandin H synthase- and monooxygenase-mediated binding of aflatoxin B1 to DNA in guinea-pig tissue microsomes

1990 ◽  
Vol 11 (11) ◽  
pp. 1915-1919 ◽  
Author(s):  
Ling Liu ◽  
Jonathan M. Daniels ◽  
Richard K. Stewart ◽  
Thomas E. Massey
Keyword(s):  
Guinea Pig ◽  
Aflatoxin B1 ◽  
Prostaglandin H Synthase ◽  
Prostaglandin H

Retinoid-induced differentiation regulates prostaglandin H synthase and cPLA2 expression in tracheal epithelium

1996 ◽  
Vol 270 (5) ◽  
pp. L854-L862 ◽  
Author(s):  
E. M. Hill ◽  
T. Bader ◽  
P. Nettesheim ◽  
T. E. Eling
Keyword(s):  
Airway Epithelium ◽  
Prostaglandin H Synthase ◽  
Induced Differentiation ◽  
Tracheal Epithelial Cells ◽  
Cytosolic Phospholipase ◽  
Low Levels ◽  
Differentiation Status ◽  
Prostaglandin H ◽  
Air Liquid Interface

Rat tracheal epithelial cells cultured in vitro at an air-liquid interface can differentiate into a mucociliary or squamous phenotype depending on the presence or absence of retinoic acid (RA). The airway epithelium is known to produce a number of eicosanoids. We propose that eicosanoid biosynthesis is dependent on the differentiation status of the epithelium. Therefore, prostaglandin production and the expression of cytosolic phospholipase A2 (cPLA2) and the prostaglandin H synthase (PGHS) isoforms were investigated during differentiation to these two phenotypes. The major eicosanoid produced by both phenotypes was prostaglandin E2 (PGE2). Proliferating undifferentiated cultures produced low levels of PGE2 regardless of retinoid status. Differentiated mucociliary cultures produced high levels of PGE2 (50 ng/10(6) cells), whereas differentiated squamous cultures produced low levels of PGE2 (< 5 ng/10(6) cells). Mucociliary cultures expressed high levels of cPLA2 and PGHS-2 isoform mRNA and protein. In contrast squamous cultures expressed very low levels of cPLA2 and PGHS-2 transcript and protein. The PGHS-1 isoform was expressed in squamous but not in mucociliary cultures. We investigated changes in expression of these enzymes during retinoid treatment of established squamous cultures. Treatment with RA resulted in a rapid (24 h) downregulation of PGHS-1 mRNA expression. However, the cPLA2 and PGHS-2 genes were expressed in squamous cultures only after 3 days of RA treatment coincident with redifferentiation of the culture to a mucociliary phenotype. These studies reveal that retinoid-induced differentiation of airway epithelium into either a mucociliary or squamous phenotype results in profound changes in the expression of cPLA2 and PGHS isozymes that regulate prostaglandin formation.

To the Mechanism of Horseradish Peroxidase-Mediated Degradation of a Recalcitrant Dye Remazol Brilliant Blue R

2001 ◽  
Vol 66 (4) ◽  
pp. 663-675 ◽  
Author(s):  
Markéta Mikšanová ◽  
Jiří Hudeček ◽  
Jan Páca ◽  
Marie Stiborová
Keyword(s):  
Molecular Weight ◽  
Horseradish Peroxidase ◽  
Oxidation Products ◽  
Brilliant Blue ◽  
Radical Mechanism ◽  
Remazol Brilliant Blue R ◽  
Radical Trapping ◽  
Optimum Ph ◽  
Mediated Oxidation

Thein vitroenzymatic metabolism of a recalcitrant dye Remazol Brilliant Blue R (RBBR) was investigated using horseradish peroxidase (HRP). At optimum pH (4.5), the apparent Michaelis constant (KM) value for the oxidation of RBBR catalyzed by HRP is 14.8 μmol l-1. HRP-mediated conversion of RBBR proceedsviaa conventional peroxidase reaction, by a sequential one-electron oxidation of two molecules of RBBR by the peroxidase Compounds I and II. The oxidation is inhibited by radical trapping agents (nicotinamide adenine dinucleotide reduced (NADH), ascorbate, glutathione). This confirms that the peroxidase-mediated oxidation of RBBR proceedsviaradical mechanism. Gel permeation profile of the RBBR oxidation products shows that the pattern of molecular weight distribution was shifted to the higher molecular weight region indicating formation of RBBR oligomers. In addition to HRP, the RBBR dye is also oxidized by another peroxidase, the mammalian lactoperoxidase.

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