ascorbic-acid and afimoxifene

High performance liquid chromatographic (HPLC) analysis showed that the prototype antioxidant ascorbate (vitamin C) inhibits the DNA adducts induced by synthetic estrogen diethylstilbestrol (DES) and the antiestrogen metabolite 4-hydroxytamoxifen (4-OHTam). Treatment of salmon testes DNA with 4-OHTam quinone or 4-OHTam in the presence of horseradish peroxidase and hydrogen peroxide (H(2)O(2)) generated the same DNA adduct profile. Vitamin C and N-acetylcysteine (NAC) inhibited the formation of 4-OHTam-dG adducts in a dose-dependent manner. To determine whether the same antioxidants also protect cellular DNA, HL-60 cells were used as cell culture model. Cells treated with 10 microM 4-OHTam in the presence of 1 microM H(2)O(2 )for 24 h gave 4-OHTam-dG adducts approximately 4 x 10(-7), n = 3. Treatment of the cells with 100 microM 4-OHTam, without H(2)O(2), produced the same level of adducts. Supplementation of the incubation media with vitamin C (2.5 mM) or NAC (5 mM) inhibited the formation of DNA adducts. Thus, antioxidants may protect susceptible cells from genotoxicity associated with 4-OHTam activation.

Topics: Acetylcysteine; Animals; Antioxidants; Ascorbic Acid; Chromatography, High Pressure Liquid; Diethylstilbestrol; DNA Adducts; Horseradish Peroxidase; Hydrogen Peroxide; Male; Salmon; Tamoxifen; Testis

The metabolism of tamoxifen was studied in female Sprague-Dawley rat and mouse liver slices and homogenates, and the three principal tamoxifen metabolites, 4-hydroxytamoxifen, N-desmethyl-tamoxifen and tamoxifen N-oxide, were identified by HPLC using authentic standards. It was not possible to identify any of the minor metabolites such as the epoxides using this technique. The N-oxide metabolite only appeared when NADPH was added to the system; this is because the production of tamoxifen N-oxide is primarily mediated by microsomal flavin monooxygenase (FMO) which is NADPH dependent. However, this metabolite did appear in incubations with mouse liver slices only, because they are rich in flavin monooxygenases (FMOs). It did not appear in female rat or mouse liver homogenates, because the NADPH present is destroyed during homogenisation, therefore it was necessary to add NADPH to the system to produce the N-oxide metabolite. The purpose of this study was to investigate the effect of inhibitors on the biotransformation of tamoxifen by female rat and mouse liver slices and homogenates. Female rat liver slices and homogenates were incubated with the following inhibitors (1 mM): cimetidine, ascorbate, sodium azide and reduced glutathione. Cimetidine, a general P-450 inhibitor, inhibited the production of the N-desmethyl metabolite by about 80%; this is in agreement with the action of the other inhibitors. Reduced glutathione, ascorbate and sodium azide are mainly peroxidase inhibitors, so therefore from these novel and interesting results it was possible to suggest that peroxidases play a role in the metabolism of tamoxifen. This observation was also strengthened when the production of the N-desmethyl metabolite increased when horseradish peroxidase was added to the incubate. The production of 4-hydroxytamoxifen was reduced and the N-oxide metabolite was completely inhibited in the presence of peroxidase inhibitors. When rat liver homogenates was incubated with superoxide dismutase (SOD) and catalase, it was observed that the N-desmethyl metabolite disappeared completely at 60 min and the N-oxide and 4-hydroxy metabolites were completely inhibited. However, this phenomenon was only observed when SOD and catalase were preincubated for 30 min with the rat liver homogenate at 37 degrees C; without preincubation the production of these metabolites was unaffected. Finally, the effect of long incubation periods (300 min) on the production of metabolites was examined.

Topics: Animals; Ascorbic Acid; Chromatography, High Pressure Liquid; Cimetidine; Cytochrome P-450 Enzyme Inhibitors; Enzyme Inhibitors; Female; Glutathione; In Vitro Techniques; Liver; Mice; NADP; Rats; Sodium Azide; Tamoxifen; Time Factors

Tamoxifen (TAM) is the antiestrogen most widely used in the chemotherapy and chemoprevention of breast cancer. It has been reported that TAM and its more active metabolite 4-hydroxytamoxifen (OHTAM) induce multiple cellular effects, including antioxidant actions. Here sarcoplasmic reticulum membranes (SR) were used as a simple model of oxidation to clarify the antioxidant action type and mechanisms of these anticancer drugs on lipid peroxidation induced by Fe2+/ascorbate and peroxyl radicals generated by the water-soluble 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) and by the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). Peroxidation was monitored by different assay systems, namely cis-parinaric acid (PnA) fluorescence quenching, production of thiobarbituric acid-reactive substances, polyunsaturated fatty acids (PUFA) degradation and oxygen consumption. TAM and OHTAM are efficient inhibitors of lipid peroxidation induced by Fe2+/ascorbate and strong intramembraneous scavengers of peroxyl radicals generated either in the water or lipid phases by AAPH and AMVN, respectively. However, these drugs are not typical chain-breaking antioxidant compounds as compared with vitamin E. Additionally, their antioxidant effectiveness enhances the protective capacity of vitamin E against lipid peroxidation induced by AMVN. OHTAM is a more powerful intramembraneous inhibitor of lipid peroxidation as compared with TAM; this effectiveness not correlating with alterations on membrane fluidity may be due to the presence of a hydrogen-donating HO-group in the OHTAM molecule and its preferential location in the outer bilayer regions where it can donate the hydrogen atom to quench free radicals capable of initiating the membrane oxidative degradation. The stronger OHTAM intramembraneous scavenger capacity over TAM also correlates with its higher partition in biomembranes. Therefore, the strong peroxyl radical scavenger activity of OHTAM in the hydrophobic membrane phase may putatively contribute to the mechanisms of cytostatic and chemopreventive action of its promoter TAM on development of breast cancer.

Topics: Amidines; Animals; Ascorbic Acid; Azo Compounds; Ferrous Compounds; Free Radical Scavengers; Hydrogen Peroxide; Intracellular Membranes; Lipid Peroxidation; Nitriles; Oxygen Consumption; Rabbits; Sarcoplasmic Reticulum; Tamoxifen; Time Factors