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Mutagenic activity and mutational specificity of antiprotozoal drugs with and without nitrite treatment.

We examined the mutagenic activities of six antiprotozoal drugs (three diaminopyrimidine compounds [pyrimethamine, diaveridine, and trimethoprim] and three 8-aminoquinoline derivatives [primaquine, pentaquine, and pamaquine]) in Escherichia coli WP2uvrA/pKM101 and Salmonella typhimurium TA100 and TA98 with and without nitrite treatment. The diaminopyrimidine compounds showed no mutagenic activity under any condition in any strain. The 8-aminoquinoline derivatives after nitrite treatment at 5-20 mM for 5 min at pH 3, on the contrary, showed clear mutagenicity in TA100 and WP2uvrA/pKM101 in the presence and absence of S9 mix. We concluded that 8-aminoquinoline derivatives became mutagenic following nitrite treatment. In the Lac(+) reversion assay with E. coli WP3101P-WP3106P, these nitrite-treated compounds induced G:C --> A:T transitions and G:C --> T:A transversions in the absence of S9 mix. On the other hand, A:T --> T:A transversions were induced only in the presence of S9 mix, suggesting a different kind of products may be responsible for the mutagenicity.

Aminoquinolines↗

Some effects of adrenaline on anion transport and nitrite-induced methaemoglobin formation in the rainbow trout (Salmo gairdneri Richardson).

The effects of adrenaline on branchial anion transport and nitrite-induced methaemoglobinaemia have been investigated in rainbow trout. Nitrite uptake and efflux results suggest that adrenaline effects a net anion efflux principally by stimulation of the unidirectional branchial anion efflux. In oxygenated whole blood nitrite-induced methaemogloblin was significantly reduced in the presence of adrenaline. The physiological and environmental consequences of nitrite-induced stress are discussed.

Animals↗

Elevated TNF-alpha and inducible nitric oxide production by alveolar macrophages after exposure to a nitrite inhalant.

Abuse of nitrite inhalants, widespread among male homosexuals, has been identified by epidemiological studies as an independent risk factor for AIDS and for Kaposi's sarcoma. Subchronic exposure of mice to inhaled isobutyl nitrite was previously found to impair the tumoricidal activity of peritoneal macrophages. Because inhalants would be expected to have the greatest effects on cells in the lung, alveolar macrophages from exposed mice were examined in this study. Mice were exposed to 900 ppm isobutyl nitrite in an inhalation chamber for 45 min/day for 14 days. Following this treatment, the lungs of exposed mice had large increases in cellularity, both in the alveolar septa and within the alveoli. Bronchoalveolar lavages also contained increased numbers of cells. Alveolar macrophages collected from treated mice had increased tumoricidal activity compared with controls and produced higher levels of inducible nitric oxide and tumor necrosis factor-alpha (TNF-alpha). The frequency of alveolar cells secreting TNF-alpha was increased ninefold in mice exposed to the inhalant. Cell influx into the lung, as indicated by the presence of red blood cells in lung lavages, was evident after only a single 45-min exposure to inhaled isobutyl nitrite at doses as low as 300 ppm.

Aerosols↗

Toxicity of nitrite to three species of freshwater invertebrates.

Nitrite is a compound with a high toxicity to aquatic animals. Several anthropogenic pollution sources are increasing the concentrations of this component of the nitrogen cycle. Despite this toxicity, there is little available literature on its effects on freshwater invertebrates. Laboratory bioassays were performed to obtain data on the lethal effects of nitrite to three species of freshwater invertebrates: the planarian Polycelis felina and the amphipods Echinogammarus echinosetosus and Eulimnogammarus toletanus. The LC(50), LC(10), and LC(0.01) values (mg/L NO(2)--N) at 24, 48, 72, and 96 h were calculated for each species. E. toletanus and E. echinosetosus were the most sensitive species, with 96 h LC(50) values of 2.09 and 2.59 mg/L NO(2)--N, respectively. In contrast, the planarian P. felina showed a higher tolerance to nitrite, with a 96 h LC(50) value of 60.0 mg/L NO(2)--N. The obtained results were compared with the reported nitrite data for other freshwater invertebrates. This study may contribute to a more appropriate assessment of the ecological risk of this compound in freshwater ecosystems.

Amphipoda↗

Synthesis of peroxynitrite in a two-phase system using isoamyl nitrite and hydrogen peroxide.

A new method for the preparation of high concentrations of peroxynitrite (up to 1 M) is described. The synthesis uses a two-phase system and involves a displacement reaction by the hydroperoxide anion (in the aqueous phase) on isoamyl nitrite (in the organic phase). The product peroxynitrite remains in the aqueous phase, whereas isoamyl alcohol forms a new organic phase along with the unreacted isoamyl nitrite. The aqueous phase contains some 0.15 M isoamyl alcohol and the unreacted hydrogen peroxide, but no isoamyl nitrite. Removal of isoamyl alcohol or traces of isoamyl nitrite is accomplished by washing the aqueous phase with dichloromethane, chloroform, or hexane. A near total removal of hydrogen peroxide is then achieved by passing the solutions through a short column of manganese dioxide. The peroxynitrite in these postprocessed solutions has broad absorption spectrum with a maximum around 302 nm, follows a characteristic first-order decomposition at pH 7.2 and 25 degrees C (k = 0.34 +/- 0.1 s-1), and reacts with organic compounds to give either nitrated or one-electron transfer products. When stored frozen at -20 degrees C, these peroxynitrite solutions decompose at a rate of about 1.7 % per day and should be used within 2-4 weeks. For short-term storage of about 1 week or less, these solutions can be stored at refrigerator temperatures (approximately 5 degrees C) where peroxynitrite has a half-life of about 7 days.

Amyl Nitrite↗

Nitrite reductase from Desulfovibrio desulfuricans (ATCC 27774)--a heterooligomer heme protein with sulfite reductase activity.

The membrane bound cytochrome c nitrite reductase from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) was found to have a high specific activity in the reduction of sulfite, producing stoichiometric amounts of sulfide. The K(m) for sulfite in the MV+.:sulfite oxidoreductase assay is 0.75 mM, and the specific activity 2.06 mumolH2/min/mg. Visible and EPR spectroscopies studies indicate that the enzyme high-spin heme reacts with sulfite in the oxidised state, and that sulfide partially reduces the enzyme. The redoxcycled enzyme, using H2/Hydrogenase/MV+. as a reductant, is identical to the resting enzyme. This is the first time that a c-type nitrite reductase has been shown to reduce sulfite. These findings, besides revealing a new function for the nitrite reductase, raise a major question regarding the sulfur metabolism in the sulfate reducing bacteria as well as the cellular localization of the enzymatic activities involved in the dissimilatory reduction of sulfate. The purified nitrite reductase is a heterooligomer, containing two types of subunits of 62 kDa (+/- 5 kDa) and 18.8 kDa (+/- 1 kDa), and forms a complex or aggregate with a molecular mass of approximately 750 kDa.

Chromatography, Gel↗

Reduction of organic nitrites to nitric oxide catalyzed by xanthine oxidase: possible role in metabolism of nitrovasodilators.

Xanthine oxidase (XO) was shown to catalyze the reduction of isoamyl and isobutyl nitrites to nitric oxide (NO) in the presence of xanthine under anaerobic conditions. NO was produced at a stoichiometric ratio of 2:1 versus urate generation, steady-state analysis of which showed Michaelis-Menten kinetics with xanthine as varied substrate and substrate inhibition with varied organic nitrite. Under the conditions of NO generation from isoamyl nitrite, XO was progressively inactivated by a mechanism involving conversion of Mo=S to Mo=O, yielding "desulfo" enzyme. It is proposed that XO is involved in the metabolism of organic nitrites to NO in vivo and that the observed inactivation serves to explain the phenomenon of tolerance.

Animals↗

Formation of nitrotyrosine by methylene blue photosensitized oxidation of tyrosine in the presence of nitrite.

Methylene blue photosensitized oxidation of tyrosine in the presence of nitrite produces 3-nitrotyrosine, with maximum yield at pH 6. The formation of 3-nitrotyrosine requires oxygen and increases using deuterium oxide as solvent, suggesting the involvement of singlet oxygen in the reaction. The detection of dityrosine as an additional reaction product suggests that the first step in the interaction of tyrosine with singlet oxygen generates tyrosyl radicals which can dimerize to form dityrosine or react with a nitrite-derived species to produce 3-nitrotyrosine. Although the chemical identity of the nitrating species has not been established, the possible generation of nitrogen dioxide (*NO(2)) by indirect oxidation of nitrite by intermediately produced tyrosyl radical, via electron transfer, is proposed. One important implication of the results of this study is that the oxidation of tyrosine by singlet oxygen in the presence of nitrite may represent an alternative or additional pathway of 3-nitrotyrosine formation of potential importance in oxidative injures such as during inflammatory processes.

Animals↗

Role of glutathione in macrophage activation: effect of cellular glutathione depletion on nitrite production and leishmanicidal activity.

We have examined the effects of two agents depleting the intracellular pool of glutathione (GSH) on macrophage activation induced by IFN-gamma + LPS, as measured by nitrite production and leishmanicidal activity. Diethylmaleate (DEM), which depletes intracellular GSH by conjugation via a reaction catalyzed by the GSH-S-transferase, strongly inhibited nitrite secretion and leishmanicidal activity when added before or at the time of addition of IFN-gamma + LPS; this inhibition was progressively lost when addition of DEM was delayed up to 10 hr. A close correlation was observed between levels of intracellular soluble GSH during activation and nitrite secretion. Inhibition was partially reversed by the addition of glutathione ethyl ester (GSH-Et). Buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, also inhibited macrophage activation, although to a lesser extent than DEM despite a more pronounced soluble GSH depletion. This inhibition was completely reversed by the addition of GSH-Et. DEM and BSO did not alter cell viability or PMA-triggered O2- production by activated macrophages, suggesting that the inhibitory effects observed on nitrite secretion and leishmanicidal activity were not related to a general impairment of macrophage function. DEM and BSO treatment reduced iNOS specific activity and iNOS protein in cytosolic extracts. DEM also decreased iNOS mRNA expression while BSO had no effect. Although commonly used as a GSH-depleting agent, DEM may have additional effects because it can also act as a sulhydryl reagent; BSO, on the other hand, which depletes GSH by enzymatic inhibition, has no effect on protein-bound GSH. Our results suggest that both soluble and protein-bound GSH may be important for the induction of NO synthase in IFN-gamma + LPS-activated macrophages.

Amino Acid Oxidoreductases↗

Effect of lead acetate on nitrite production by murine brain endothelial cell cultures.

One of the mechanisms by which lead may cause a perturbation in the nervous system is the alteration of endothelial cell function. This study investigated the effect of lead acetate on constitutive and cytokine-induced production of nitrite, a marker of nitric oxide, in brain microvascular endothelial cells. Nitric oxide synthase may be a target for lead and changes in its function can result in a cascade of physiological effects seen in vivo. Concentrations of 10, 100, and 1000 nM lead acetate, in the presence or absence of 100 ng lipopolysaccharide/ml, 400 U interferon-gamma/ml and 100 U tumor necrosis factor-alpha/ml, were added to confluent cultures of brain microvascular endothelial cells. Concentrations of lead acetate as low as 10 nM decreased constitutive levels of nitrite by 50% without inhibiting the inducible levels. Addition of 1 microM lead acetate had no effect on [3H]L-leucine incorporation, lactate dehydrogenase release, or cellular morphology, indicating that the effect was selective. Increasing the concentration of extracellular calcium to 2 mM abolished the inhibitory effect of lead acetate on the constitutive production of nitrite. These studies suggest that low concentrations of lead are capable of inhibiting nitrite produced by the calcium-dependent constitutive form of nitric oxide synthase while the calcium-independent, inducible form of nitric oxide synthase is not affected. These data provide another testable hypothesis for the as yet undetermined mechanisms of lead neurotoxicity.

Amino Acid Oxidoreductases↗

Microbial assimilation of alkyl nitro compounds and formation of nitrite.

66 representative strains of bacteria, yeasts and fungi were tested for their ability to grow in a semidefined medium containing 0.5% nitroethane as a nitrogen source. About half of them were found capable of growing in the medium. Hansenula beijerinckii, Candida utilis, and Penicillium chrysogenum were most active in assimilating nitroethane. 2-Nitropropane inhibited growth of most of the microorganisms tested in a medium containing 0.2% peptone and 0.2% glycerol. Hansenula mrakii was found to grow rapidly in the nitroethane-peptone medium after a lag phase. Nitrite was accumulated in the culture fluid after the phase of logarithmic multiplication, and increased with increase of the growth, followed by a decline after the maximum growth. The alkyl nitro compounds were oxidatively denitrified to form nitrite by the crude enzyme from Hansenula mrakii. Nitroethane was generally a poor substrate, but was the best inducer to produce the nitro compounds oxidizing enzyme. 2-Nitro-propane and nitroethane were enzymatically oxidized to nitrite, and acetone and acetaldehyde, respectively, which were isolated as 2,4-dinitrophenylhydrazones and identified. Nitrite formed was found to be reduced into ammonia by the intact cells and also the crude enzyme.

Acetaldehyde↗

Effect of nitrites and nitrates on irrigation and secretion of gastric mucosa in rat.

An aspect of the vasomotor effect of nitrites and nitrates was studied by investigating the influence of these substances on the mucosal blood flow and hence on gastric secretion in the rat. Firstly, point measurements were obtained using an aminopyrine clearance technique after intravenous nitrite and nitrate treatment. Then with an appreciably different technique (14C-aniline clearance on perfused stomach) the kinetics of the phenomenon were followed after, firstly, intravenous administration of the two agents and secondly their intragastric administration. It is interesting to note that nitrites and nitrates are active to differing degrees and present opposing effects: nitrites decrease whereas nitrates globally enhance the factors under consideration (gastric mucosal blood flow, gastric juice volume, acory effect and cuase no modification on intragastric administration. The flow rate-gastric parameter correlations are significant in all cases and show modifications of the aqueous compartments of the body.

Aminopyrine↗

Effects of nitrite exposure on blood respiratory properties, acid-base and electrolyte regulation in the carp (Cyprinus carpio).

Adult carp were subjected to 1 mM environmental nitrite for 48 h and nitrite uptake and changes in blood respiratory properties, extracellular electrolyte composition and acid-base status were examined. A constant influx of nitrite caused an accumulation of NO2- in plasma to 5.4 mM in 48 h. The fraction of methaemoglobin rose with plasma [NO2-] to 83%, and the arterial oxygen content decreased to extremely low values. Arterial PO2 increased as a compensation to this O2-shortage, whereas the O2 saturation of the functional (unoxidized) haemoglobin decreased, revealing a reduction in its O2 affinity. Blood haematocrit decreased as a result of red cell shrinkage, which caused very high red cell haemoglobin (Hb) concentrations. The erythrocytic nucleoside triphosphate (NTP) concentration showed a parallel increase whereby NTP/Hb, as well as the relative contributions of ATP and GTP to NTP, remained unchanged. Plasma [Cl-] declined by 15 mM in 48 h, offsetting the plasma [NO2-] increase, minor changes in plasma [HCO3-] and a considerable increase in plasma [lactate]. Arterial pH and [HCO3-] rose slightly during the first 24 h of nitrite exposure, but returned to control values at 48 h. The rise in plasma [lactate] was not reflected in an extracellular metabolic acidosis. Plasma [K+] increased by 94% in 48 h, revealing an uncompensated extracellular hyperkalemia, whereas plasma [Na+] decreased, and plasma [Ca++] was unchanged. Plasma osmolality remained essentially constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗

Model experiments on nitrite and nitrate in simulated primeval conditions.

In experiments on the prebiotic formation of nitric oxides, anoxic mixtures of N2 and water vapour were sparked in contact with phosphate buffer solutions at various pH values. Nitrite was found in the aqueous phase, and nitrate grew from it, presumably by reaction with H2O2. In acid solutions, these anions were reduced and destroyed by Fe2+, and the same was true of nitrite in solutions kept at a pH value similar to that of the contemporary ocean (8.2) with HEPES buffer. Nitrate was not destroyed in short-term experiments, but as in sparking nitrate is formed only vianitrite, neither anion could accumulate. In further sparking experiments with alkaline sulphide, both nitrite and nitrate were reduced entirely. It is concluded that it is unlikely that the primeval ocean contained appreciable concentrations of nitrite or nitrate either at the reducing or at the redox-neutral stage.

Anaerobiosis↗

Accumulation of nitrite and sulfite in yeast cells and synergistic depletion of the intracellular ATP content.

When nitrite or sulfite are applied to yeast cells below pH 5.0, an enormous intracellular accumulation occurs. It is assumed that nitrite and sulfite penetrate the cell membrane in their undissociated forms as nitrous acid (pK = 3.3) or sulfurous acid (pK = 1.8), respectively. Due to the neutral intracellular pH they are trapped inside the cell in their anionic forms, which are impermeable to the cell membrane. It has previously been shown that sulfite causes a rapid depletion of the ATP content of yeast cells [Schimz, K.L. and Holzer, H. (1979) resp. Hinze et al. as above]. Similarly, millimolar concentrations of nitrite decrease the ATP level to less than 10% of the initial value. Nitrite and sulfite in combination deplete the ATP content of yeast cells much stronger than expected for the sum of the separate effects of these compounds ("synergistic effect").

Adenosine Triphosphate↗

N-nitrosamines and residual nitrite in cured meats from the Dutch market.

A total of 140 samples of 16 kinds of cured meats were analyzed for contents of residual nitrite and N-nitrosamines. Nitrite was determined by reaction with sulfanilamide/naphthylethylenediamine and colorimetric measurement. N-nitrosamines were isolated from the samples by vacuum distillation and determined by gas-chromatography with chemiluminescence detection (GC-TEA). In six samples no nitrite was detectable (less than 1 mg NaNO2/kg), the remaining samples contained 1-140 mg NaNO2/kg, median value 6.8 mg/kg. In 46 samples (33%) no N-nitrosamines were detected, i.e. less than 0.1-0.5 microgram/kg of the individual nitrosamines, depending upon their structure. N-nitrosodimethylamine (NDMA) was the nitrosamine present most frequently, in 75 samples, contents were 0.1-0.9 microgram/kg, mean 0.3 microgram/kg. Other N-nitrosamines found were: N-nitrosopiperidine (NPIP), 10 times, 0.3-25 micrograms/kg; N-nitrosodiethylamine (NDEA), three times, 0.2-0.9 microgram/kg; N-nitrosopyrrolidine (NPYR), three times, 1.3-4.2 micrograms/kg; N-nitrosomorpholine, once, 0.7 microgram/kg and N-nitrosothiazolidine (NTHZ), 36 times, 0.5-91 micrograms/kg, mean 5.7 micrograms/kg. NTHZ was found most often and with the highest contents in smoked products. Frying of bacon and cured, smoked pork bellies led to substantially increased levels of NPYR in both products, and for the pork bellies also of NTHZ. In five samples of cured, smoked pork bellies after frying NTHZ-contents of 3.6-490 micrograms/kg (mean 179) were found. No correlation between residual nitrite levels and N-nitrosamine contents could be established. Investigations during the nineteen seventies gave much higher levels for NDMA, NDEA, NPIP and NPYR in Dutch cured meats than now found; at that time NTHZ was not measured.

Animals↗

Determination of nitrite at low level without prior extraction.

A method has been devised for the determination of nitrite at low level that is directly applicable to food or other dried matrices without prior extraction. Nitric oxide released from nitrite through the action of acetic acid is determined using a chemiluminescence analyser. The limit of detection is approximately 0.02 microgram, the coefficients of variation being 5.7 and 8.2% using 0.1 and 0.05 microgram of sodium nitrite, respectively. The chemiluminescence analyser response is diminished when water in excess of 0.5 ml is present in the assay system unless hydrogen bromide in acetic acid is used instead of acetic acid alone. The application of the method to the direct determination of nitrite in freeze-dried cod fish has indicated a content of 0.25 mg NaNO2 per kg, equivalent to 0.050 mg per kg of the original undried material.

Animals↗

Nitrite from inflammatory cells--a cancer risk factor in ulcerative colitis?

Elevated levels of luminal nitrite and a lowered luminal pH were found in 77 percent of patients with acute ulcerative colitis. No luminal nitrite was found in healthy control subjects. Nitrites are a secretory product of activated macrophages and neutrophils of the lamina propria, whereas the lowered luminal pH is due to diminished bicarbonate formation by impaired colonocytes. A hypothesis is put forward that nitrites, lowered pH, and bacterial amines are conducive to formation of carcinogenic n-nitroso compounds, which reflect a cancer risk in patients with ulcerative colitis dependent on the type and extent of inflammatory cell activation as well as metabolic impairment of colonic epithelial cells.

Animals↗