Subrahmanyam, Vangala Venkata Satya (1986) Hydrogen peroxide/peroxidase-mediated co-oxidation (a potential pathway for the activation of carcinogenic and toxic xenobiotics in the cell). Doctoral (PhD) thesis, Memorial University of Newfoundland.
PDF (Migrated (PDF/A Conversion) from original format: (application/pdf))
- Accepted Version
Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission.
Products of the metabolism of arylamine carcinogens ¹⁴C-benzidine, N-¹⁴C-methylaminoazobenzene and ¹⁴C-aminofluorene and ¹⁴C-phenol bind irreversibly to nuclear DNA in intact polymorphonuclear leukocytes activated by the tumor promoter phorbol myristate acetate. Little binding occurred under anaerobic conditions indicating that the binding was dependent upon oxygen. Sulfhydryl inhibitors p-chloromercuribenzoate and N-ethylmaleimide inhibited oxygen uptake as well as decreased the binding of arylamine carcinogens and phenol to DNA in leukocytes activated by tumor promoter. Phenolic antioxidants butylated hydroxyanisole or nordihydroguaiaretic acid also had a similar inhibitory effect. Azide increased both the oxygen uptake and binding, presumably as a result of intracellular catalase inhibition. However, higher concentrations of azide and cyanide inhibited binding without affecting the oxygen uptake indicating that the binding is catalysed by myeloperoxidase. Granules isolated from the activated leukocytes catalysed a cyanide-sensitive binding of benzidine to calf-thymus DNA in the presence of hydrogen peroxide. -- Products of the metabolism of ¹⁴C-phenol also bind irreversibly to calf-thymus DNA in the presence of horseradish peroxidase and hydrogen peroxide. Binding also occurred to the homopolyribonucleotides polyriboadenylic acid, polyriboguanylic acid, polyribocytidylic acid and polyribouridylic acid, suggesting that the binding is relatively non-specific with respect to nucleotide bases. -- DNA, binding of ¹⁴C-phenol oxidized with horseradish peroxidase/hydrogen peroxide was prevented by glutathione and N-acetylcysteine and the mechanism was shown to involve reduction of the activated phenol intermediates and the formation of conjugates with glutathione and N-acetylcysteine. Ascorbate prevented binding by reduction of the activated phenol intermediates. -- Phenol oxidation by horseradish peroxidase/hydrogen peroxide initially results in p,p'-biphenol and o,o'-biphenol formation and subsequently results in polymer formation. o,o'-Biphenol is the major product formed but is rapidly oxidized to polymer, particularly in the presence of phenol. Phenol oxidation with bone marrow homogenate and hydrogen peroxide also results in 0,0'-biphenol as the major product. p,p'-Biphenol is very rapidly oxidized to p,p'-biphenoquinone which can also be involved in polymer formation. -- Enzymic or acid-catalyzed hydrolysis of DNA releases the bound products. o,o'-Biphenol, but not p,p'-biphenol binds to DNA following peroxidase-catalysed oxidation. Enzymic hydrolysis of the DNA, to which the products of the oxidation of o,o'-biphenol had bound, resulted in the release of products derived from the biphenol. -- Peroxidase also catalysed the formation of active oxygen species in the presence of NADH or GSH and traces of hydrogen peroxide and arylamine or phenolic substrates. Some oxygen activation occurred with some arylamines even in the absence of NADH or GSH. Oxygen consumption was proportional to the NADH oxidized or GSSG formed. Approximately 0.80 and 0.40 moles of oxygen were consumed per mol of NADH or GSH oxidized respectively. The requirement for trace amounts of hydrogen peroxide and arylamine or phenolic substrate suggests that the redox cycling resulted in hydrogen peroxide formation. It is proposed that initially formed phenoxv radicals or arylamine cation radicals oxidize NADH or GSH to radicals which react with oxygen to form superoxide radicals and hydrogen peroxide. -- Non-carcinogenic arylamines mesidine, aniline and 1-naphthylamine were poor at initiating redox cycling in the presence of NADH or GSH, whereas carcinogenic 2-naphthylamine, 4-aminobiphenyl, methylaminoazobenzene, N,N'-dimethyl p-toluidine or 2-aminofluorene were highly effective in initiating redox cycling in this system with resultant hydrogen peroxide formation. -- Only phenol and o,o'-biphenol were effective among phenolic substrates in redox cycling with resultant hydrogen peroxide formation. p,p'-Biphenol, hydroquinone and catechol were ineffective in activating oxygen. p,p'-Biphenol formed a glutathione conjugate when oxidized with horseradish peroxidase and hydrogen peroxide in the presence of glutathione. The major conjugate formed was identified as 3-S-(glutathion-yl)-p,p'-biphenol using FAB-Mass spectroscopy and NMR spectroscopy.
|Item Type:||Thesis (Doctoral (PhD))|
|Additional Information:||Bibliography: leaves -240.|
|Department(s):||Science, Faculty of > Biochemistry|
|Library of Congress Subject Heading:||Free radical reactions; Peroxidase; Carcinogenesis|
Actions (login required)