salicylates and 3-3--5-5--tetramethylbenzidine

We have previously shown that the anticancer agent doxorubicin undergoes oxidation and inactivation when exposed to myeloperoxidase-containing human leukemia HL-60 cells, or to isolated myeloperoxidase, in the presence of hydrogen peroxide and nitrite. In the current study we report that commercial fetal bovine serum (FBS) alone oxidizes doxorubicin in the presence of hydrogen peroxide and that nitrite accelerates this oxidation. The efficacy of inactivation was dependent on the concentration of serum present; no reaction was observed when hydrogen peroxide or serum was omitted. Peroxidase activity assays, based on oxidation of 3,3',5,5'-tetramethylbenzidine, confirmed the presence of a peroxidase in the sera from several suppliers. The peroxidative activity was contained in the >10000 MW fraction. We also found that hemoglobin, a heme protein likely to be present in commercial FBS, is capable of oxidizing doxorubicin in the presence of hydrogen peroxide and that nitrite further stimulates the reaction. In contrast to intact doxorubicin, the serum + hydrogen peroxide + nitrite treated drug appeared to be nontoxic for PC3 human prostate cancer cells. Together, this study shows that (pseudo)peroxidases present in sera catalyze oxidation of doxorubicin by hydrogen peroxide and that this diminishes the tumoricidal activity of the anthracycline, at least in in vitro settings. Finally, this study also points out that addition of H2O2 to media containing FBS will stimulate peroxidase-type of reactions, which may affect cytotoxic properties of studied compounds.

Topics: Aniline Compounds; Anthracyclines; Antibiotics, Antineoplastic; Benzidines; Blood Proteins; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Doxorubicin; Doxylamine; Hemeproteins; Humans; Hydrogen Peroxide; Mass Spectrometry; Methemoglobin; Methimazole; Molecular Structure; Oxidation-Reduction; Peroxidases; Phthalic Acids; Salicylates; Sodium Nitrite

5-Aminosalicylic acid (5-ASA) inhibited by a mixed mechanism the peroxidase catalyzed oxidation of tetramethylbenzidine (TMB) in 0.015 M phosphate-citrate buffer (pH 6.4) supplemented with 5% DMSO and 5% DMF. Poly(salicylic acid 5-aminodisulfide) (poly(SAADS)) in 0.01 M phosphate buffer (pH 6.2-7.4) supplemented with 5% DMSO and 5% DMF effectively activated the peroxidase-catalyzed oxidation of TMB. The activation was quantitatively characterized by coefficients alpha (M-1) determined at different pH values: alpha increased linearly with increase in pH up to the maximal value of 2.44*105 M-1 at pH 7.0. The activating effect of poly(SAADS) on the peroxidase-catalyzed oxidation of TMB is explained by the activator properties of polyelectrolyte, with its anionic form interacting with peroxidase sites responsible for the acid-base catalysis.

Topics: Antioxidants; Benzidines; Catalysis; Disulfides; Enzyme Activation; Horseradish Peroxidase; Hydrogen-Ion Concentration; Kinetics; Mesalamine; Oxidation-Reduction; Polymers; Salicylates; Structure-Activity Relationship

Tetramethyl benzidine (TMB) as a substrate for horseradish peroxidase (HRP) histochemistry offers a great advantage over other substrates in its sensitivity but the reaction product is unstable in alcohol. Immersing TMB-reacted sections in methyl salicylate renders the reaction product stable enough so that exposure to ethanol for 10 min causes no detectable loss of HRP labelled neurons. A rapid thionin stain is introduced which, after stabilizing the TMB-based reaction product in methyl salicylate, causes no detectable loss of HRP labelled neurons. These procedures should prove valuable to users of the popular HRP techniques for studying neural connections.

Topics: Benzidines; Brain Mapping; Chemical Phenomena; Chemistry; Coloring Agents; Ethanol; Histocytochemistry; Horseradish Peroxidase; Microtomy; Peroxidases; Phenothiazines; Salicylates