ascorbic-acid and phenidone

Inhibition of soybean lipoxygenase (L-1) and potato 5-lipoxygenase (5-PLO) by the pyrazoline derivatives phenidone and BW755C only occurs after oxidation of these compounds by the peroxidase-like activity of the lipoxygenases. There is a clear relationship between this oxidation and the irreversible inactivation of L-1. The final product of phenidone oxidation by L-1, 4,5-didehydrophenidone, is not responsible of this inactivation, but the species derived from a one-electron oxidation of phenidone plays a key role in L-1 inactivation. In the absence of O2, inactivation of 1 mol of L-1 occurs after the oxidation of 34 mol of phenidone and the covalent binding of 0.8 mol of phenidone-derived metabolite(s) to L-1. In the presence of O2, inactivation of 1 mol of L-1 occurs already after oxidation of 11 mol of phenidone and only involves the covalent binding of 0.4 mol of phenidone-derived metabolite(s) to L-1. A mechanism is proposed for L-1 inactivation by phenidone, which involves the irreversible binding of a phenidone metabolite to the protein and the oxidation of an L-1 amino acid residue (in the presence of O2).

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Arachidonate 5-Lipoxygenase; Ascorbic Acid; Catalase; Electron Spin Resonance Spectroscopy; Enzyme Activation; Ferric Compounds; Glycine max; Humans; Leukocytes; Lipoxygenase; Lipoxygenase Inhibitors; Oxidation-Reduction; Oxygen; Pyrazoles; Solanum tuberosum; Sulfhydryl Compounds; Superoxide Dismutase

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

A bioassay technique was developed to analyze the effect of vasoactive substance(s) released from endothelial cells. Canine femoral arteries with or without endothelium were perfused with physiological salt solution at 37 degrees C. The perfusate was bioassayed with a ring of coronary artery without endothelium. A substance(s) released by the endothelial cells under basal conditions caused relaxation of unstimulated coronary arteries or relaxation of those contracted with prostaglandin F2 alpha. The release of the relaxing substance(s) was augmented by acetylcholine. The relaxation induced by acetylcholine was biphasic: an initial rapid phase followed by a partial recovery and a slowly developing prolonged relaxation; the half-life of the substance(s) causing the initial phase averaged 6.3 s. Norepinephrine, epinephrine, and ascorbic acid, given downstream of the femoral artery, reversibly prevented the second phase but only attenuated the initial relaxation. These observations indicate that an endothelium-derived relaxing substance(s) is released into the lumen of the femoral artery under basal conditions and during stimulation with acetylcholine. Catecholamines can inactivate the relaxing substance(s) but do not prevent either its production by endothelial cells or its action on vascular smooth muscle.

Topics: Acetylcholine; Animals; Ascorbic Acid; Coronary Vessels; Dogs; Endothelium; Epinephrine; Female; Femoral Artery; Male; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide; Norepinephrine; Pyrazoles; Stimulation, Chemical; Vasodilator Agents

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

Phenidone-ascorbic acid development in electronmicroscopic autoradiography, using Ilford L4 as photographic emulsion and microdol-x as reference developer. Grain yield and efficiency were studied on pale gold section of uniformly labeled tritium methacrylate. For determination of the resolution, a radioactive line source was prepared by cross-sectioning of an epon-embedded film of tritium labeled albumin. The spatial relationship between silver grains and silver bromide crystals was investigated by shadowing the emulsion with platinumcarbon before development. In shadowed autoradiographs both, silver grains and silver bromide crystal were visible. Phenidone was about twice as sensitive as microdol-x and had a half distance value (Salpeter et al., 1969) of 175 mm. Most of the silver grains of both developers were located within the perimeters of their parent silver bromide crystals. In the case of phenidone more than 80% of the excited crystals gave rise to just one silver deposit. These parameters, together with grain size and shape, and counting feasibility make phenidone a useful developer for quantitative EM-autoradiography.

Topics: Albumins; Ascorbic Acid; Autoradiography; Microscopy, Electron; Pyrazoles; Regression Analysis; Silver; Tritium