I. Brouet

Nitrotyrosine as a new marker for endogenous nitrosation and nitration of proteins

3-Nitrotyrosine (NTYR) in tissue or blood proteins was evaluated as a possible exposure marker for exogenous and endogenous nitrosating or nitrating agents. A sensitive and selective method for analysing NTYR by gas chromatography with a thermal energy analyser (GC-TEA) was developed. Using this method, a number of kinetic studies were carried out. It was found that free and protein-bound tyrosine residues easily react with nitrating/nitrosating agents to yield NTYR. NTYR formation in vivo showed a dose-dependent increase in NTYR in both plasma proteins and haemoglobin obtained from rats 24 hr after ip injection of various doses (0.5-2.5 mumol/rat) of tetranitromethane. Major urinary metabolites of NTYR, given orally to rats, were isolated and identified as 3-nitro-4-hydroxyphenylacetic acid (NHPA) and 3-nitro-4-hydroxyphenyllactic acid (NHPL). About 44% and 5% of the oral dose of NTYR (100 micrograms/rat) was excreted as NHPA and NHPL, respectively. Eleven 24-hr human urine samples were analysed for NHPA by GC-TEA after ethyl acetate extraction and HPLC purification: quantities ranging from 0 to 7.9 micrograms/24 hr, mean +/- SD 2.8 +/- 2.3 (n = 11) were detected (detection limit 0.2 micrograms/litre). NTYR in proteins or its metabolites in urine can be readily analysed by GC-TEA as a new/additional marker for endogenous nitrosation and nitration.

Formation of direct-acting genotoxic substances in nitrosated smoked fish and meat products: Identification of simple phenolic precursors and phenyldiazonium ions as reactive products☆

Epidemiological studies have associated the consumption of smoked fish and meat products with an increased risk of stomach cancer. Therefore, the reaction of such smoked foods with nitrite under acidic conditions was investigated and was shown to produce potent direct-acting genotoxic substances as detected by the SOS Chromotest. Similar genotoxic activity was observed in nitrosated samples of wood-smoke condensates. Simple phenolic compounds such as phenol, 3-methoxycatechol, catechol and vanillin were identified as the precursors of the genotoxic substances. These phenolic compounds also exhibited direct-acting genotoxicity after nitrosation. The major genotoxic substances formed after nitrosation of phenol were isolated and identified as 4- and 2-hydroxyphenyldiazonium ions. Nitrosation of various wood-smoke condensates was found to generate the same type of diazonium compounds, which in part account for the genotoxicity of nitrosated smoked foods.

Immunohistochemical localization of an inducible form of nitric oxide synthase in various organs of rats treated with Propionibacterium acnes and lipopolysaccharide

Immunohistochemical localization of an endotoxin‐inducible form of nitric oxide synthase was examined using rabbit polyclonal antibody against the enzyme purified from rat liver. In rats treated with Propionibacterium acnes and lipopolysaccharide, immunostaining was observed in macrophages, occasional lymphocytes, neutrophils and eosinophils in red pulp of spleen, Kupffer cells, endothelial cells and hepatocytes in liver, alveolar macrophages in lung, macrophages and endothelial cells in adrenal glands, and histiocytes, eosinophils, mast cells and endothelial cells in colon. Immunoreactivity was also evident in the following tissues: histiocytes and endothelial cells in kidney; histiocytes and neutrophils in esophagus; macrophages and eosinophils in duodenum; macrophages, some lymphocytes and mast cells in ileum; histiocytes in thymus; and endothelial cells in heart and aorta. Immunoreactivity was not detected in these organs from untreated rats. Positively staining cells in these rat organs appeared within 2.5 h after lipopolysaccharide administration; their number dramatically increased within the next 2.5 h, remained at high levels for a further 19 h, and then decreased over the following 24 h. The number of positive cells appearing correlated well with the nitric oxide synthase activity biochemically determined in the same organs.

Polyclonal antibody against an inducible form of nitric oxide synthase purified from the liver of rats treated with Propionibacterium acnes and lipopolysaccharide

A polyclonal antibody was raised in the rabbit against an inducible form of nitric oxide (NO) synthase (EC 1.14.23) purified from the liver of rats with acute liver necrosis induced by i.v. administration of Propionibacterium acnes and lipopolysaccharide. The antibody immunoprecipitated NO synthase activities in the soluble extract of the liver from treated rats. Western blot analysis showed that the cytosols of the liver, lung and spleen from the treated rats but not from non-treated rats, and that of murine macrophages cultured in the presence of lipopolysaccharide and interferon-gamma, contained immunoreactive protein with a molecular weight of 125 kDa. The antibody, however, does not cross-react with a 150 kDa constitutive form of NO synthase present in the brain of rats, indicating that the inducible and constitutive enzymes are immunologically distinguishable.

Oxidative Destruction of Hydrazines Produces N-Nitrosamines and Other Mutagenic Species

As part of the joint International Agency for Research on Cancer-National Cancer Institute program for the evaluation and development of methods for the degradation of chemical carcinogens, four oxidative techniques for the degradation of hydrazines were investigated. The oxidizing agents used were as follows: sodium hypochlorite, calcium hypochlorite, potassium iodate, and potassium permanganate in sulfuric acid. In each case, at least 99% of the hydrazine initially present was destroyed; however, the potential usefulness of these methods was compromised by the formation (in some reaction mixtures) of carcinogenic N-nitroso compounds and/or unknown mutagenic species. Oxidative degradation of hydrazines is recommended only for the decontamination of glassware and for the treatment of spills, for which reductive degradation methods are not suitable.

A method for the efficient degradation of melphalan into nonmutagenic products

Abstract A new chemical method has been developed for the destruction of an antineoplastic agent melphalane in laboratory waste, based on its oxidation by potassium permanganate in sodium hydroxide medium. To monitor the efficiency of destruction, analytical methods based on UV spectrophotometry, differential pulse voltammetry on a glassy carbon rotating disk electrode, and HPLC coupled with UV spectrophotometry have been developed. It has been demonstrated that the proposed method of destruction is highly efficient (more than 99.8% destruction) and the corresponding degradation products were found to be nonmutagenic to Salmonella typhimurium strains TA98, TA100, and TA1535, in both the presence and the absence of a rat liver S9 activation system.