ascorbic-acid and 2-nitrosofluorene

2-Nitrosofluorene (NOF) and N-hydroxy-2-aminofluorene (N-HO-AF) are potent direct-acting mutagens, derived from metabolic activation of the carcinogen, N-acetyl-2-aminofluorene (AAF). To assess the ability of cellular glutathione (GSH) to detoxify these electrophilic derivatives, we examined the reaction of NOF and N-HO-AF with GSH in vitro. Two reaction products were isolated and identified as glutathionyl derivatives of 2-aminofluorene (AF) containing an N-S linkage. Amino acid analysis, infrared and NMR (500 MHz) spectroscopy, fast atom bombardment mass spectrometry and analysis of reaction characteristics and hydrolysis products established their structures as N-(glutathion-S-yl)-2-aminofluorene S-oxide (GS-AFI) and N-(glutathion-S-yl)-2-aminofluorene (GS-AFII). Ascorbic acid, which reduces NOF to N-HO-AF, was used to modify reaction yields. These results indicated that GS-AFI was derived from reaction with NOF and that GS-AFII could be formed from both NOF and N-HO-AF. A reaction scheme is proposed in which NOF reacts with GSH to form an intermediate addition product that can rearrange either to GS-AFI or be reduced to GS-AFII. The latter could also be formed by direct reaction with N-HO-AF.

Topics: Amino Acids; Ascorbic Acid; Chemical Phenomena; Chemistry; Drug Stability; Fluorenes; Glutathione; Hydroxylamines; Magnetic Resonance Spectroscopy; Mass Spectrometry; Nitroso Compounds; Time Factors

Horseradish peroxidase and H2O2 mediate N-hydroxy-N-acetyl-2-aminofluorene (N-OH-AAF) conversion into two more potent carcinogens, 2-nitrosofluorene and N-acetoxy-N-acetyl-2-aminofluorene. Optical studies of this system indicate that horseradish peroxidase is operating as a peroxidase with N-OH-AAF as the electron donor. Our studies confirm the earlier finding that 2-nitrosofluorene and N-acetoxy-N-acetyl-2-aminofluorene are the products of the type II enzyme-mediated oxidation of N-OH-AAF, but surprisingly, the results with the type VI enzyme indicate that more 2-nitrosofluorene was formed and, in addition, another product absorbing at 245 nm was formed. If ascorbate is present in the N-OH-AAF/horseradish peroxidase/H2O2 system, ascorbate is oxidized preferentially. Cyanide, a known inhibitor of the peroxidase, does not inhibit when N-OH-AAF is the electron donor. The reaction products are the same in the presence or absence of cyanide.

Topics: Acetoxyacetylaminofluorene; Ascorbic Acid; Chemical Phenomena; Chemistry; Cyanides; Fluorenes; Horseradish Peroxidase; Hydrogen Peroxide; Hydroxyacetylaminofluorene; In Vitro Techniques; Nitroso Compounds; Oxidation-Reduction; Peroxidases; Spectrophotometry, Ultraviolet