NADPH-cytochrome P450 reductase-mediated denitration reaction of 2,4,6-trinitrotoluene to yield nitrite in mammals

• Published in: Free radical biology & medicine 2016-02, Vol.91, p.178-187
• Main Authors: Shinkai, Yasuhiro, Nishihara, Yuya, Amamiya, Masahiro, Wakayama, Toshihiko, Li, Song, Kikuchi, Tomohiro, Nakai, Yumi, Shimojo, Nobuhiro, Kumagai, Yoshito
• Format: Article
• Language: English
• Subjects: 2,4,6-trinitrotoluene; Animals; Denitration; Enzyme purification; Hep G2 Cells; Humans; Inactivation, Metabolic; Male; Metabolism; Microsomes, Liver - enzymology; NADPH-cytochrome P450 reductase; NADPH-Ferrihemoprotein Reductase - chemistry; NADPH-Ferrihemoprotein Reductase - physiology; Nitrites - chemistry; Nitrites - metabolism; Rats, Wistar; Reactive oxygen species; Substrate Specificity; Superoxides - chemistry; Superoxides - metabolism; Trinitrotoluene - chemistry; Trinitrotoluene - metabolism; Xenobiotic
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2015.09.011

While the biodegradation of 2,4,6-trinitrotoluene (TNT) via the release of nitrite is well established, mechanistic details of the reaction in mammals are unknown. To address this issue, we attempted to identify the enzyme from rat liver responsible for the production of nitrite from TNT. A NADPH-cytochrome P450 reductase (P450R) was isolated and identified from rat liver microsomes as the enzyme responsible for not only the release of nitrite from TNT but also formation of superoxide and 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) under aerobic conditions. In this context, reactive oxygen species generated during P450R-catalyzed TNT reduction were found to be, at least in part, a mediator for the production of 4-HADNT from TNT via formation of 4-nitroso-2,6-dinitrotoluene. P450R did not catalyze the formation of the hydride-Meisenheimer complex (H--TNT) that is thought to be an intermediate for nitrite release from TNT. Furthermore, in a time-course experiment, 4-HADNT formation reached a plateau level and then declined during the reaction between TNT and P450R with NADPH, while the release of nitrite was subjected to a lag period. Notably, the produced 4-HADNT can react with the parent compound TNT to produce nitrite and dimerized products via formation of a Janovsky complex. Our results demonstrate for the first time that P450R-mediated release of nitrite from TNT results from the process of chemical interaction of TNT and its 4-electron reduction metabolite 4-HADNT. [Display omitted] •We examined the denitration reaction of 2,4,6-trinitrotoluene (TNT) in mammals.•P450R isolated from rat liver was identified as a catalyst for denitration of TNT.•TNT is metabolized to 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) by P450R.•Chemical interaction of TNT and 4-HADNT is involved in release of nitrite form TNT.•A novel mechanism of denitration reaction of TNT by P450R was proposed.