Metabolism of ¹⁴C‐Labelled 5‐Nitro‐1,2,4‐Triazol‐3‐One by Rat Liver Microsomes Evidence for the Participation of Cytochrome P‐450

Abstract

In the present study, we synthesized '"C-labelled 5-nitro-l,2,4-triazol-3-one (NTO) and investigated its hepatic metabolism by dexamethasone-induced murine hepatic microsomes. Under the nitrogen atmosphere, 5-amino-l,2,4-triazol-3-one was the only detected metabolite of NTO. The microsomal nitroreductase activity was dependent on NADPH, totally inhibited by carbon monoxide and partially inhibited by oxygen. In aerobic conditions, beside a low amount of amine, the major metabolite formed is the 5-hydroxy-triazolone, urazole. This compound resulted from the oxidative denitrification of NTO, which produced equivalent amount of nitrite. This reaction, like the nitroreductaae activity, was dependent on NADPH and totally inhibited by carbon monoxide. Both nitroreduction and oxidative denitrification were inhibited by imidazole-related inhibitors : miconazole and methimazole, and to a less extent by N-octylamine. The microsomal denitrification was induced by the treatment of rats with dexamethasone and phenobarbital. The microsomal reductare activity is present in untreated rat microsomes, and recovered with various inducers. The results of this study indicate the role played by cytochrome P-450 in the metabolism of NTO, supported by its transformation with reconstituted cytochrome P-450 systems.Keywords: nitrotriazolone; cytochrome P-450; nitroreductase; denitrification; dexamethasone.The explosive 5-nitro-1,2,4-triazol-3-one (NTO) 1 is thermally stable and impact-insensitive [ 11. Its high performance and stability [2, 31 made it of considerable interest to both the defense and civilian sectors. NTO is produced in many countries in multi-ton amounts for large scale formulation and evaluation. Consequently, it is a potential source of pollution in soils and waters around facilities where it is made or used. Several examples of such ecological and health damage caused by explosives in and around military facilities have been reported [4-61. The impact of NTO on the environment has not yet been investigated, and any potential risk can only be accurately predicted when knowledge of its metabolism in mammalian tissues is available. Toxicological studies on NTO indicate that the molecule is not toxic to mice or rats when given orally (LD,,, > 5g/ kg), and is a mild irritant when applied to rabbit skin [7]. However, these investigations did not assess the carcinogenic properties that a molecule having the structure of NTO may possess, based on findings for other nitro-aromatic or nitro-heterocyclic explosives [6,8, 91. Thus, the metabolic intermediates produced during the nitroreduction of nitro compounds cause local damage in the liver [lo]. These intermediates are responsible for the cytotoxicity, neurotoxicity and chemosensitization of 2-nitroimidazole derivatives [II, 121, and for the mutagenicity, genotoxicity and carcinogenicity of nitro aromatic [8, 101 and nitro aliphatic compounds [ 131. Intermediates, including hydroxylamines, ring-opening products and activated oxygen species, are probably responsible for the bi...

Microbial remediation of NTO in aqueous industrial wastes

“…13 101.0645, found: 101.0465. These characteristics are identical to those obtained for authentic 2 prepared by Pd/C-catalyzed hydrogenation [15].…”

“…The isolated strain B. licheniformis operated in the presence of high concentrations of compounds (15 g l 31 NTO, and 11.5 g l 31 ATO). We previously reported the nitroreduction of NTO by rat liver microsomes [15]. Contrary to the microbial reaction, the microsomal nitroreduction catalyzed by cytochrome P-450 enzymes is drastically inhibited by oxygen, and the formed amine is not further metabolized.…”

“…14 C-labelled NTO and 14 C-labelled ATO were prepared as described earlier [15]. Biomass from 3-day-old cultures was recovered by centrifugation and washed twice with distilled water.…”

Nitroreductases and Azoreductases

“…Nitroreductase activity was found in mammalian liver and adrenal cortex, and channel cat®sh (Ictalurus punctatus) (Washburn and Di Giulio 1988), as well as in different bacteria, such as Bacillus licheniformis isolated from industrial waste containing high concentrations of 5-nitro-1,2,4-triazol-3-one (Le Campion et al 1999).…”

In Vitro Endocrine Disruption Screening of 3-nitro-1,2,4-triazol-5-one (NTO)

“…If testicular toxicity of NTO is a priority concern, the in vitro assays could be used to screen metabolites of NTO. Thus far, urazole has been identified as metabolite in isolated rat hepatocytes (Le Campion et al, 1997;Le Campion et al, 1998). Urazole is commercially available and it is recommended that it be screened with the EDC in vitro assays.…”