Enhancement of superoxide production in vitro by the diphenyl ether herbicide nitrofen

Light-dependent reduction of p -nitro blue tetrazolium chloride (NBT) and 2,6-dichlorophenol-indophenol (DCPIP) was stimulated by nitrofen (2,4-dichlorophenyl p -nitrophenyl ether) in the presence or absence of the photosensitizer riboflavin. Enhancement of NBT reduction occurred at a concentration as low as 17 μ M nitrofen. Nitrofen had no effect on dark reduction of NBT by dithionite, ascorbate, or reduced phenazine methosulfate. NBT reduction proceeded in solution saturated with either air or nitrogen. Stimulation by nitrofen in the presence or absence of riboflavin also occurred independently of oxygen. Superoxide dismutase (superoxide:superoxide oxidoreductase; EC 1.15.1.1) inhibited light-dependent, aerobic reduction of NBT and DCPIP with and without riboflavin. The nitrogen-stimulated component was also eliminated. Thus, it appears nitrofen can effect reduction of NBT (and DCPIP) anaerobically by transferring electrons directly to NBT (and DCPIP) through an oxygen-independent mechanism or aerobically via superoxide radicals.