ascorbic-acid and benzidine

Benzidine (Bz), a human bladder carcinogen, was strongly mutagenic to Salmonella TA102 tester strain in the Ames Salmonella microsome/mutagenicity assay in the presence of rat liver S9 mix. Various non-mutagenic plant polyphenols were included in the assay to test their inhibitory effects on the Bz-induced mutations. Coumestrol, ellagic acid (EA), (-)-epicatechin (EC), (-)-epichatechingallate (ECG), gallic acid (GA), (-)-gallocatechin (GC), plumbagin, propyl gallate (PG), taxifolin, and 2,2',4'-trihydroxychalcone were found to have a strong inhibitory effect on Bz-induced mutations. (-)-Epigallo-catechingallate (EGCG), fisetin, (-)-gallocatechingallate (GCG), and piceatannol were moderately inhibitory to the mutations; whereas, (-)-catechin, (-)-catechingallate (CG), and reseveratrol were weakly inhibitory to the mutations. (-)-Epigallocatechin (EGC) and 7,3',4'-trihydroxy isoflavon were not inhibitory to the Bz-induced mutations. Isoliquirtigenin, quercetin dihydrate, and rhein were found to be mutagenic in tester strain TA102. Benzidine mediated lipid peroxidation was conducted employing the thiobarbituric acid reactive substances (TBARS) assay using linoleic acid as a substrate. In the presence of rat liver S9 mix, Bz could cause lipid peroxidation as an outcome of production of oxygen free radicals. Incorporation of the above mentioned non-mutagenic plant polyphenols significantly inhibited benzidine mediated lipid peroxidation in a time dependent manner. These polyphenols also effectively reduced the iron mediated lipid peroxidation. Thus, it is concluded that the inhibition of oxidative mutagenicity of Bz by plant polyphenols could be due to an inhibitory effect of plant polyphenols on the bioactivating enzymes such as cytochrome P-450 and peroxidase and the chelation of iron present in the cytochrome P-450 in the S9 mix. Thus, these plant polyphenols play a significant inhibitory role on Bz-induced mutagenicity.

Topics: Animals; Antimutagenic Agents; Ascorbic Acid; Benzidines; Dose-Response Relationship, Drug; Flavonoids; In Vitro Techniques; Lipid Peroxidation; Molecular Weight; Mutagenicity Tests; Mutagens; Phenols; Plants; Polyphenols; Rats; Salmonella; Structure-Activity Relationship; Thiobarbituric Acid Reactive Substances; Urinary Bladder Neoplasms

Human thyroid peroxidase (hTPO) catalyzes a one-electron oxidation of benzidine derivatives by hydrogen peroxide through classical Chance mechanism. The complete reduction of peroxidase oxidation products by ascorbic acid with the regeneration of primary aminobiphenyls was observed only in the case of 3,3',5,5'-tetramethylbenzidine (TMB). The kinetic characteristics (k(cat) and K(m)) of benzidine (BD), 3,3'-dimethylbenzidine (o-tolidine), 3,3'-dimethoxybenzidine (o-dianisidine), and TMB oxidation at 25 degrees C in 0.05 M phosphate-citrate buffer, pH 5.5, catalyzed by hTPO and horseradish peroxidase (HPR) were determined. The effective K(m) values for aminobiphenyls oxidation by both peroxidases raise with the increase of number of methyl and methoxy substituents in the benzidine molecule. Efficiency of aminobiphenyls oxidation catalyzed by either hTPO or HRP increases with the number of substituents in 3, 3', 5, and 5' positions of the benzidine molecule, which is in accordance with redox potential values for the substrates studied. The efficiency of HRP in the oxidation of benzidine derivatives expressed as k(cat)/K(m) was about two orders of magnitude higher as compared with hTPO. Straight correlation between the carcinogenicity of aminobiphenyls and genotoxicity of their peroxidation products was shown by the electrophoresis detecting the formation of covalent DNA cross-linking.

Topics: Aminobiphenyl Compounds; Ascorbic Acid; Benzidines; Dianisidine; DNA; DNA Damage; Humans; Hydrogen-Ion Concentration; Iodide Peroxidase; Kinetics; Oxidation-Reduction

In the absence of its substrate hydrogen peroxide, peroxidase exhibits perturbations in its Fe(3+)-heme center, when incubated with ascorbic acid. The electron paramagnetic pattern sprang towards a higher g-value side, denoting a sharpening of the rhombic axial symmetry around the heme-center. The interpretation is that the ascorbate dependent peroxidase action starts with the formation of an Fe(3+)-ascorbate charge transfer complex intermediate.

Topics: Ascorbic Acid; Benzidines; Electron Spin Resonance Spectroscopy; Guaiacol; Heme; Horseradish Peroxidase; Iron; Protein Conformation

Metabolism of benzidine was assessed with rabbit renal inner medullary slices. 3-(Glutathion-S-yl)-benzidine was identified as a product of metabolism. This thioether conjugate was shown to be identical to synthetic conjugate by chromatographically assisted hydrodynamic voltammetric and enzymatic techniques. A good correlation between PGE2 synthesis and conjugate formation was observed with a variety of incubation conditions including tissue weight, arachidonic acid concentration and incubation time. With 0-0.01 mM idomethacin, an inhibitor of the fatty acid cyclo-oxygenase component of prostaglandin H synthase (PHS), a linear relationship between conjugate formation and prostaglandin E2 synthesis was observed. In contrast, the peroxidase cosubstrates propylthiouracil, phenidone, ascorbate and methimazole inhibited arachidonic acid stimulation of conjugate formation but not prostaglandin E2 synthesis. These cosubstrates may be functioning as competitive inhibitors of benzidine co-oxidation. The results are consistent with peroxidatic metabolism of benzidine in intact tissue by a PHS-mediated process. 3-(Glutathion-S-yl)-benzidine may be a useful marker for studying peroxidatic metabolism in intact tissue and in investigating selective inhibition of this process.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Ascorbic Acid; Benzidines; Dinoprostone; In Vitro Techniques; Indomethacin; Kidney Medulla; Kinetics; Methimazole; Peroxides; Propylthiouracil; Prostaglandin-Endoperoxide Synthases; Prostaglandins E; Pyrazoles; Rabbits

Carcinogens which cause cancers in tissues distal to their entry are thought to require metabolic activation before covalent binding to macromolecules. The hydroperoxidase component of prostaglandin H synthase (PHS) activates certain carcinogens and a model describing this process is presented. The procarcinogen benzidine was used to identify sites at which microsomal PHS-catalyzed binding might be inhibited by pharmacologic agents. Activation of benzidine was determined by assessing free radical cation formation and covalent binding to protein. Reduction of benzidine diimine to diamine was also assessed. This study provides the first demonstration of inhibition of PHS-activated benzidine binding by propylthiouracil, methimazole, MK447, vitamin C and phenidone. The agents tested identified the following sites at which PHS-catalyzed binding of benzidine can be prevented: 1) inhibition of generation of the peroxide cosubstrate for benzidine oxidation; 2) inhibition of prostaglandin hydroperoxidase; 3) reduction of oxidized intermediate(s) to the parent compound; and 4) conjugation of the activated intermediate(s). This study provides a basis for further investigations of the pharmacologic intervention of chemical carcinogenesis.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Ascorbic Acid; Benzidines; Binding Sites; Butylated Hydroxytoluene; Carcinogens; Dose-Response Relationship, Drug; Drug Interactions; Indomethacin; Male; Methimazole; Microsomes; Models, Chemical; Peroxidases; Propylthiouracil; Prostaglandin Endoperoxides; Prostaglandin-Endoperoxide Synthases; Prostaglandins H; Protein Binding; Pyrazoles; Rabbits; Stimulation, Chemical