Enhanced demethylation and denitrosation of N-nitrosodimethylamine by human liver microsomes from alcoholics.

The metabolism of N-nitrosodimethylamine (NDMA) was investigated in incubations with human liver microsomes from alcoholics and control patients who suffered from other diseases, but had a histological normal liver. All of the microsomal samples studied were able to metabolize NDMA at various concentrations to both formaldehyde and nitrite. Analysis of the liver microsomes from alcoholics revealed that both enzymatic activities--formaldehyde and nitrite formation--were enhanced several times as compared to the control patients. The results point to the fact that alcoholics metabolize NDMA at a higher rate probably due to the induction of one or more ethanol-inducible human liver cytochromes (cyt.) P450. The question if alcoholics therefore possess a higher risk for carcinogenic events is discussed.

Induction of nitrite/nitrate synthesis in murine macrophages by BCG infection, lymphokines, or interferon-gamma.

Macrophage synthesis of nitrite and nitrate after activation by BCG infection or by treatment in vitro with both T cell-derived (lymphokines (LK) or recombinant murine interferon-gamma (IFN-gamma] and bacterial (lipopolysaccharide (LPS) and heat-killed bacillus Calmette-Guerin (hk BCG] agents was studied by using macrophages from C3H/He and C3H/HeJ mice. Spleen and peritoneal macrophages isolated from BCG-infected donors that were producing nitrate continued to synthesize nitrite and nitrate in culture. LPS treatment in vitro (25 or 50 micrograms/ml) additionally increased this nitrite/nitrate synthesis. Thioglycolate-elicited macrophages from non-infected C3H/HeJ mice treated with LK also produced nitrite/nitrate, and concurrent LPS (0.1 to 50 micrograms/ml) treatment resulted in enhanced synthesis. Recombinant IFN-gamma also stimulated nitrite/nitrate synthesis by C3H/He and CeH/HeJ macrophages as did LPS (C3H/He only) and hk BCG. When given concurrently with either LPS or hk BCG, IFN-gamma enhanced C3H/He and C3H/HeJ macrophage nitrite/nitrate synthesis over that produced by macrophages treated with either LPS or hk BCG alone. Macrophages activated in vitro exhibited a 4 to 12 hr lag time before engaging in nitrite/nitrate synthesis, which then proceeded for 36 to 42 hr at linear rates. Daily medium renewal did not alter the synthesis kinetics but increased the total amount of nitrite/nitrate produced. Nitrate and nitrite were stable under the conditions of culture and when added did not influence additional macrophage synthesis. Taken together, these results indicate that T cell lymphokines and IFN-gamma are powerful modulators of macrophage nitrite/nitrate synthesis during BCG infection and in vitro, and nitrite/nitrate synthesis appears to be common property of both primed and fully activated macrophage populations.

The formation of nitrite and N-nitroso compounds in salivas in vitro and in vivo.

The considerable differences in salivary nitrite levels observed previously in subjects who had abstained overnight from dietary nitrate as completely as possible were again found in tests on ten separate days. A 25-fold variation was observed in the rate of production of nitrite in vitro during the incubation of salivas from different subjects with 16 mM-nitrate under standardized conditions. N-Nitroso compounds were detected as a group on 11/100 and 14/100 occasions after incubations in vitro without and with added nitrate, respectively. No significant changes in salivary nitrite level were found 1 hr after the volunteers had ingested water-borne nitrate at approximately the World Health Organization recommended limit for continuous use (50 mg NO-3/litre), but increases in nitrite concentration were consistently found after volunteers ingested nitrate in water at four times this concentration. Although the nitrite concentrations were markedly increased following the intake of nitrate at the higher level, the occurrence of N-nitroso compounds in the salivas of the volunteers was greater immediately prior to the ingestion of water-borne nitrate than 1 hr afterwards.

Metabolic nitrite formation from N-nitrosamines: evidence for a cytochrome P-450 dependent reaction.

Nitrite was formed on incubation of N-nitrosamines with a reconstituted monooxygenase system, consisting of cytochrome P-450 (P-450) and NADPH P-450 reductase from pig liver. Nitrite was not obtained when the nitrosamines were incubated with NADPH P-450 reductase alone or when molecular oxygen or NADPH were omitted. Interaction of nitrosamines with the reconstituted P-450 system or with hemoglobin under reducing conditions resulted in optical spectra identical with those obtained with nitrite. It is proposed that N-nitrosamines are denitrosated by electron transfer from the hemoprotein iron to the nitrosamine molecule.

[Bacterial flora and nitrite production in the stomach after gastroenterostomy. Experimental aspects of the pathogenesis of carcinoma in the operated stomach (author's transl)]. / Bakterielle Besiedlung und Nitritbildung im Magen nach Gastroenterostomie. Experimentelle Aspekte zur Pathogenese des Carcinoms im operierten Magen.

A gastroenterostomy without enteroanastomosis leads to a change in the bacterial flora of the stomach, where-by in particular the proportion of nitrite-decomposing bacteria, is enhanced. This results in an increase of nitrite concentration in the gastric fluid, which may possibly be accompanied by an augmented production of carcinogenic nitrosoamine. This latter aspect is considered with respect to the origin of carcinoma in the operated stomach. Since the reported changes will be largely prevented by a Roux-Y-gastroenterostomy, this should be taken into consideration for reconstruction of the alimentary tract after gastric surgery.

Metabolism of nitrophenols by bacteria isolated from parathion-amended flooded soil.

Two bacterial isolates from parathion-amended flooded soil, Pseudomonas sp. and Bacillus sp., were examined for their ability to decompose nitrophenols. Uniformly labelled 14C-p-nitrophenol was metabolized by both bacteria, 14CO2 and nitrite being end products. A substantial portion (23% for Pseudomonas sp. and 80% for Bacillus sp.) of radioactivity applied as p-nitrophenol was accounted for as 14CO2 at the end of a 72-h period; 8 to 16% remained in the water phase after solvent extraction. Pseudomonas sp. produced nitrite also from 2,4-dinitrophenol, but only after a lag, and not from o- and m-nitrophenols. Interestingly, m-nitrophenol, known for its resistance to biodegradation because of meta substitution, was decomposed by Bacillus sp., resulting in the formation of nitrite and phenol; o-nitrophenol and 2,4-dinitrophenol resisted degradation by this bacterium.

[Formation of nitrites from nitrates in the digestive tract]. / Formation des nitrites a partir des nitrates dans le tube digestif.

The authors performed in vitro incubations of gastrointestinal mucosae and contents in the rat, under anaerobiosis, in the presence of nitrates. Nitrate disappearance and nitrite appearance amounts were measured in the different incubation media. From these works it follows that a certain quantity of nitrates are actually reduced to nitrites specifically by the ileo-caecal microflora of rats and perhaps, to a lower degree, by the action of a nitrate-reductase which may occur in the digestive mucosae. Moreover, the authors carried out in situ perfusions into the small intestine of rats, by adding nitrates to the perfusion liquid at a concentration of 400 mg per litre. It was thus demonstrated that the nitrate-absorbing kinetics is very rapid but those nitrates do not gather in the blood; besides, the presence of nitrites in the perfusion medium indicates a possible nitrate reduction in the intestinal gut.

[Methemoglobinemias caused by ingestion of nitrites and nitrates]. / Les méthémoglobinémies induites par l'ingestion de nitrites et de nitrates.

Nitrites and nitrates are often responsible for methemoglobinemy. Infants, on account of their greater sensibility to oxidizing agents, are particularly liable to poison by nitrates and nitrites. Sodium nitrite can be responsible for poisoning after accidental ingestion or owing to an overdose when preparing salt provisions, or because it has been mistaken for another product. Then it is often a case of collective poisoning. Nitrates generally produce methenoglobinemy when they are changed into nitrites under the influence of a bacterial proliferation or of a reductase held in plants. It happens with spinach and carrot soup. Nitrates can pollute municipal water supply and chiefly well water. As regards therapy, cases of methemoglobinemy have been noted after an overdose of potassium nitrate and especially of bismuth subnitrate. The treatment of methemoglobinemy caused by nitrates and nitrites is not specific: suppression of the oxidizing agents, oxygenation, prescription of reducing agents.

Degradation of N-nitrosamines by intestinal bacteria.

A major proportion of bacterial types, common in the gastrointestinal tract of many animals and man, were active in degrading diphenylnitrosamine and dimethylnitrosamine, the former being degraded more rapidly than the latter. At low nitrosamine concentrations (is less than 0.05 micronmol/ml), approximately 55% of added diphenylnitrosamine, 30% of N-nitrosopyrrolidine, and 4% of dimethylnitrosamine were degraded. The route of nitrosamine metabolism by bacteria appears to be different from that proposed for breakdown by mammalian enzyme systems in that carbon dioxide and formate were not produced. In bacteria, the nitrosamines were converted to the parent amine and nitrite ion and, in addition, certain unidentified volatile metabolites were produced from dimethylnitrosamine by bacteria. The importance of bacteria in reducing the potential hazard to man of nitrosamines is discussed.

Method for in situ measurement of nitrification in a stream.

A method is described in which the oxidation of NH(3)-N to NO(2)-N and NO(3)-N in a stream was measured in situ by use of an equilibration chamber. The conversion was stoichiometric.

Ammonia or ammonium ion as substrate for oxidation by Nitrosomonas europaea cells and extracts.

The effect of pH on the K(m) values for ammonia was studied in its oxidation by Nitrosomonas cells and cell-free extracts. The K(m) values decreased markedly with increasing pH, suggesting (NH(3)) rather than (NH(4) (+)) as the actual substrate for oxidation.

Photoinactivation of ammonia oxidation in Nitrosomonas.

Photoinactivation of ammonia oxidation in cells of Nitrosomonas was shown to follow first-order kinetics with a rate constant proportional to incident light intensity. The action spectrum for photoinactivation consisted of a broad peak in the ultraviolet range, where both hydroxylamine and ammonia oxidation were affected, and a shoulder at approximately 410 nm where only ammonia oxidation was affected. In photoinactivated cells, hydroxylamine but not ammonia was oxidized to nitrite and hydroxylamine but not ammonia caused reduction of cytochromes in vivo. The amount per cell of the following constituents was not measurably altered by photoinactivation: cytochromes b, c, a, and P460; ubiquinone; phospholipid; free amino acids; hydroxylamine-dependent nitrite synthetase; nitrite reductase; p-phenylenediamine oxidase; and cytochrome c oxidase. Malonaldehyde or lipid peroxides were not detected in photoinactivated cells. Photoinactivation was prevented (i) under anaerobic conditions, (ii) in the presence of methanol, allylthiourea, thiosemicarbazide, hydroxylamine, ethylxanthate, or CO at concentrations wich caused 100% inhibition of ammonia oxidation, and (iii) at concentrations of ammonia or hydroxylamine which gave a rapid rate of nitrite production. Recovery of ammonia oxidation activity in 90% inactivated cells took place in 6 h, required an energy and/or nitrogen source, and was inhibited by 400 mug of chloramphenicol per ml.

Microbial decontamination of parathion and p-nitrophenol in aqueous media.

A mixed microbial culture was adapted to growth on parathion to determine the feasibility of using microorganisms to detoxify concentrated parathion in agricultural wastes. In a 600-ml chemostat, the culture was able to degrade 50 mg of parathion per liter per h. Para-nitrophenol, produced by enzymatic hydrolysis of parathion, caused delays in exponential growth which were directly proportional to its concentration. A pseudomonad, isolated from the mixed culture, exhibited optimal growth at 0.21 mM p-nitrophenol and grew in concentrations up to 3.5 mM. In metabolic studies using [(14)C]p-nitrophenol, the nitro group was removed in stoichiometric quantities as nitrite and hydroquinone was tentatively identified as a metabolite.

Comparative denitrification of selected microorganisms in a culture medium and in autoclaved soil.

The denitrifying behavior of selected soil bacteria was compared in a culture solution and in soil that was sterilized by autoclaving. The essential characteristics concerning nitrate reduction and the formation of nitrogenous gases did not change significantly for most bacteria in the two environments. Bacteria whose denitrification product was nitrous oxide evolved the same gas both in soil and in a liquid system, whereas other bacteria formed only nitrogen gas. The validity of laboratory observations in relation to field studies in the domain of denitrification is discussed and evaluated.

Enumeration of fecal Clostridium perfringens spores in egg yolk-free tryptose-sulfite-cycloserine agar.

The Shahidi-Ferguson perfringens, tryptose-sulfite-cycloserine (TSC), and egg yolk-free TSC agars have been tested for their suitability to enumerate fecal spores of Clostridium perfringens. When these spores comprised at least 20% of the total anaerobe spores, equally accurate counts were obtained in the three media. With lower ratios of C. perfringens spores, the most accurate counts were obtained in egg yolk-free TSC agar. The median C. perfringens spore count of 60 normal fecal specimens was log 3.4/g. A nonmotile, sulfite- and nitrate-reducing Clostridium, not identifiable with any known clostridial species, was isolated from 14 out of 60 fecal specimans. It was not differentiated from C. perfringens in the nitrite motility test, but could be distinguished by its inability to liquefy gelatin.