Thiocyanate Cannot Inhibit the Formation of Reactive Nitrogen Species in the Human Oral Cavity in the Presence of High Concentrations of Nitrite: Detection of Reactive Nitrogen Species with 4,5-Diaminofluorescein

Abstract

In the human oral cavity, nitrite is reduced to nitric oxide (NO) by certain bacteria. 4,5-Diaminofluorescein (DAF-2) was transformed to a fluorescent component triazolfluorescein (DAF-2T) in a bacterial fraction of saliva in the presence of nitrite. No detectable consumption of DAF-2 and formation of DAF-2T were observed in bacterial fraction in the absence of nitrite. The nitrite-dependent transformation of DAF-2 to DAF-2T was inhibited by catalase, SCN(-), and CN(-) suggesting the participation of peroxidases in saliva in the transformation. The formation of DAF-2T, which was observed by the addition of an NO generating reagent (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR 3) to bacterial fraction, was also inhibited by catalase, SCN(-), and CN(-). The degree of the inhibition by SCN(-) decreased as the concentration of nitrite or NOR 3 was increased. Superoxide dismutase (SOD) enhanced nitrite- and NOR 3-induced fluorescence increase in the bacterial fraction, and the degree of the enhancement decreased as the concentrations of nitrite and NOR 3 were increased. In whole saliva filtrate, the inhibitory effects of SCN(-) on the fluorescence increase decreased as the concentration of nitrite was increased, but the enhancement by SOD was not significantly affected by the increase in the concentration of nitrite. As salivary bacteria produce O(2)(-), H(2)O(2), and NO and as peroxidase/H(2)O(2)/nitrite systems in saliva produce NO(2), the effects of SCN(-) are discussed taking SCN(-)-dependent inhibition of NO(2) formation by peroxidases in saliva into consideration and the effects of SOD are discussed taking O(2)(-)-dependent consumption of NO into consideration. It is concluded that when the rate of the formation of NO is high, SCN(-) is not effective enough to inhibit the formation of N(2)O(3) in the oral cavity.