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The induction by ambient NO3- and NO2- of the NO3- and NO2- uptake and reduction systems in roots of 8-d-old intact barley (Hordeum vulgare L.) seedlings was studied. Seedlings were induced with concentrations of NaNO3 or NaNO2 ranging from 0.25 to 1000 [mu]M. Uptake was determined by measuring the depletion of either NO3- or NO2- from uptake solutions. Enzyme activities were assayed in vitro using cell-free extracts. Uptake and reduction systems for both NO3- and NO2- were induced by either ion. The Km values for NO3- and NO2- uptake induced by NO2- were similar to those for uptake induced by NO3-. Induction of both the uptake and reduction systems was detected well before any NO3- or NO2- was found in the roots. At lower substrate concentrations of both NO3- and NO2- (5-10 [mu]M), the durations of the lag periods preceding induction were similar. Induction of uptake, as a function of concentration, proceeded linearly and similarly for both ions up to about 10 [mu]M. Then, while induction by NO3- continued to increase more slowly, induction by NO2- sharply decreased between 10 and 1000 [mu]M, apparently due to NO2- toxicity. In contrast, induction of NO3- reductase (NR) and NO2- reductase (NiR) by NO2- did not decrease above 10 [mu]M but rather continued to increase up to a substrate concentration of 1000 [mu]M. NO3- was a more effective inducer of NR than was NO2-; however, both ions equally induced NiR. Cycloheximide inhibited the induction of both uptake systems as well as NR and NiR activities whether induced by NO3- or NO2-. The results indicate that in situ NO3- and NO2- induce both uptake and reduction systems, and the accumulation of the substrates per se is not obligatory.
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Isobutyl nitrite is used to a limited extent as an intermediate in the syntheses of aliphatic nitrites. It is also an ingredient of various incenses or room odorizers and is used as a euphoric. The chemical has also been used as a jet propellant and in the preparation of fuels. Isobutyl nitrite was nominated by the Consumer Product Safety Commission to the NTP for toxicology and carcinogenicity studies because of its possible contribution to the high incidence of Kaposi's sarcoma among male homosexual acquired immune deficiency syndrome patients and because of the lack of available data on the potential carcinogenicity of isobutyl nitrite. Male and female F344/N rats and B6C3F1 mice were exposed to isobutyl nitrite (purity of 93% or greater) by inhalation for 16 days, 13 weeks, or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, cultured Chinese hamster ovary cells, Drosophila melanogaster, and mouse peripheral blood. 16-DAY STUDY IN RATS: Groups of five male and five female F344/N rats were exposed to 0, 100, 200, 400, 600, or 800 ppm (approximately 420, 840, 1,700, 2,500, or 3,300 mg/m(3)) isobutyl nitrite by inhalation for 6 hours per day, 5 days per week for a total of 12 exposures during a 16-day period. All males and females exposed to 600 or 800 ppm and one 400 ppm female died on the first day of the study. Final mean body weights and mean body weight gains of 400 ppm males and females were significantly lower than those of the controls. Clinical findings observed in 400 ppm males and females included ocular discharge, lethargy, hunched posture, and rough coats. Absolute and relative lung weights of all exposed groups of males and of 200 and 400 ppm females were less than those of the controls. Chemical-related hyperplasia of the bronchial epithelium was observed in 200 and 400 ppm males and females and hyperplasia of the nasal turbinate epithelium was observed in rats exposed to 400 ppm or less. Hemosiderin pigmentation was observed in the spleen of 200 and 400 ppm males and females and bone marrow hematopoietic hyperplasia was observed in rats exposed to 400 ppm or less. 16-DAY STUDY IN MICE: Groups of five male and five female B6C3F1 mice were exposed to 0, 100, 200, 400, 600, or 800 ppm (approximately 420, 840, 1,700, 2,500, or 3,300 mg/m(3)) isobutyl nitrite by inhalation for 6 hours per day, 5 days per week for a total of 12 exposures during a 16-day period. Three males and four females exposed to 800 ppm died before the end of the study. Final mean body weights and mean body weight gains of 600 and 800 ppm males and females were significantly lower than those of the controls. Mice exposed to 400 ppm or greater were lethargic and exhibited hunched posture and rough coats. Absolute and relative lung weights of 600 and 800 ppm males and the relative lung weight of 600 ppm females were significantly greater than those of the controls. Chemical-related hyperplasia of the bronchiolar epithelium was observed in all exposed groups of males and females. Lymphocytic atrophy of the spleen and thymus was observed in males and females exposed to 400 ppm or greater. 13-WEEK STUDY IN RATS: Groups of 10 male and 10 female F344/N rats were exposed to 0, 10, 25, 75, 150, or 300 ppm (approximately 42, 105, 315, 630, or 1,260 mg/m(3)) isobutyl nitrite by inhalation for 6 hours per day, 5 days per week for 13 weeks. All rats survived to the end of the study. Final mean body weights and mean body weight gains of 300 ppm males and females were significantly lower than those of the controls, as was the mean body weight gain of 150 ppm females. Clinical findings observed during the study included ruffled fur in 300 ppm males and females, hypoactivity in 300 ppm males, and hyperactivity in 150 and 300 ppm females. A very mild chemical-related methemoglobinemia and anemia occurred in male and female rats in the 75, 150, and 300 ppm groups. Hematopoietic hyperplasia occurred in the bone marrow of all exposed groups of males and females and was considered to be a secondary response to the anemia and methed methemoglobinemia. There was minimal hemosiderin pigment accumulation in the spleens of males and females exposed to 75 ppm or greater, mild to moderate epithelial cell hyperplasia of the nasal mucosa was observed in 300 ppm males and females, and minimal hyperplasia occurred in 150 ppm males and females. Hyperplasia of the bronchial epithelium was observed in 300 ppm males and females. 13-WEEK STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were exposed to 0, 10, 25, 75, 150, or 300 ppm (approximately 42, 105, 315, 630, or 1,260 mg/m(3)) isobutyl nitrite by inhalation for 6 hours per day, 5 days per week for 13 weeks. There were no chemical-related deaths. Final mean body weights and mean body weight gains of 150 and 300 ppm females were significantly less than those of the controls. Final mean body weights and mean body weight gains of exposed groups of males were similar to those of the controls. There were no chemical-related clinical findings. A very mild chemical-related methemoglobinemia occurred in male and female mice in the 150 and 300 ppm groups. A very mild anemia occurred in the 300 ppm groups. In the lung, increased incidences of mild to moderate hyperplasia of the bronchiolar epithelium occurred in males and females exposed to 300 ppm. Minimal hyperplasia occurred in males exposed to 75 ppm or greater and in females exposed to 150 ppm. Minimal epithelial cell hyperplasia of the nasal mucosa was observed in 300 ppm males. Increased hematopoiesis of the spleen, secondary to the hematotoxicity, occurred in males exposed to 75 ppm or greater and in females exposed to 150 or 300 ppm. Increased hemosiderosis of the spleen occurred in males exposed to 300 ppm and in females exposed to 75 ppm or greater. 2-YEAR STUDY IN RATS: Based on the low final mean body weights, anemia, and the mild to moderate nasal mucosal lesions and the hyperplastic bronchial lesions observed in 300 ppm males and females, isobutyl nitrite exposure concentrations selected for the 2-year inhalation study in rats were 37.5, 75, and 150 ppm. Groups of 56 male and 56 female rats were exposed to 0, 37.5, 75, or 150 ppm (equivalent to 0, 158, 315, or 630 mg/m(3)) isobutyl nitrite by inhalation for 6 hours per day, 5 days per week, for 103 weeks. Ten male and 10 female rats from each group were evaluated at 15 months for clinical pathology and histopathology. Survival, Body Weights, Clinical Findings, Hematology, and Clinical Chemistry: Survival rates of exposed groups of rats were greater than those of the controls, and the survival rates of 75 and 150 ppm males were significantly greater than that of the control. Mean body weights of 150 ppm males and females were 3% to 11% lower than those of the controls throughout the course of the study. There were no clinical findings considered to be related to isobutyl nitrite exposure. A very mild methemoglobinemia and anemia occurred in male and female rats exposed to 75 or 150 ppm. Pathology Findings: Incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) occurred with significant positive trends in exposed males and females, and the incidences of these neoplasms in 75 ppm males and in 150 ppm males and females were significantly greater than those in the controls. The incidence of alveolar/bronchiolar carcinoma was significantly greater in 150 ppm male rats than that in the controls. The incidences of alveolar epithelial hyperplasia were also increased in 75 and 150 ppm males and in all exposed groups of females. The incidences of mononuclear cell leukemia in exposed groups of males and females were significantly less than those in the controls. 2-YEAR STUDY IN MICE: Based on the low final mean body weight of 300 ppm females and the mild to moderate bronchiolar hyperplasia observed in 300 ppm males and females, isobutyl nitrite exposure concentrations selected for the 2-year inhalation study in mice were 37.5, 75, and 150 ppm. Groups of 60 male and 60 female mice were exposed to 0, 37.5, 75, or 150 ppm (equivalent to 0, 158, 315, or 630 mg/m(3)) isobutyl nitrite by inhalation for 6 hours per day, 5 days per week, for 103 weeks. As many as 10 male and 10 female mice from each group were evaluated at 15 months for clinical pathology and histopathology. Survival, Body Weights, Clinical Findings, and Hematology and Clinical Chemistry: Survival rates of exposed groups of males were similar to those of the controls. Survival rates of exposed groups of females were greater than those of the controls, and the survival rate in 37.5 ppm females was significantly greater than that of the controls. Mean body weights of exposed groups of males and of 37.5 and 75 ppm females were similar to those of the controls throughout the study. Mean body weights of 150 ppm females were lower than those of the controls from week 20 until the end of the study. There were no biologically significant clinical findings noted in the 2-year study in mice. A very mild methemoglobinemia and anemia occurred in male and female mice exposed to 75 or 150 ppm. Pathology Findings: Incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) occurred with significant positive trends in exposed males and females, and the incidences of these neoplasms were significantly greater than those in the controls in 75 ppm males and in 150 ppm males and females. Incidences of alveolar epithelial hyperplasia were significantly increased in 75 and 150 ppm male and female mice. Thyroid gland follicular cell adenoma occurred with a significant positive trend in male mice; the incidences of thyroid gland follicular cell hyperplasia were increased in all exposed groups of males, and the incidences in males exposed to 37.5 or 150 ppm were significantly greater than those in the controls. Incidences of serous exudate and olfactory epithelium atrophy in the nose of 150 ppm females were significantly greater than those in the controls. Incidences of minimal to mild hemosiderin pigment in the spleen of 75 and 150 ppm male mice were significantly greater than those in the controls. GENETIC TOXICOLOGY: Isobutyl nitrite was found to be mutagenic in vitro and in vivo. It induced base-pair substitution mutations in Salmonella typhimurim strains TA100 and TA1535 and sister chromatid exchanges and chromosomal aberrations in cultured Chinese hamster ovary cells. Positive responses in the S. typhimurium tests required S9 activation, but isobutyl nitrite induced chromosomal effects in cultured Chinese hamster ovary cells with and without S9. In vivo, no induction of sex-linked recessive lethal mutations was noted in the germ cells of male Drosophila melanogaster exposed to isobutyl nitrite via feeding or injection. However, significant increases in micronucleated normochromatic erythrocytes were observed in the peripheral blood of male and female mice treated with isobutyl nitrite for 90 days by inhalation. CONCLUSIONS: Under the conditions of these 2-year inhalation studies, there was clear evidence of carcinogenic activity of isobutyl nitrite in male and female F344/N rats based on the increased incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined). There was some evidence of carcinogenic activity of isobutyl nitrite in male and female B6C3F1 mice based on the increased incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) in males and females. The increased incidence of thyroid gland follicular cell adenoma in male mice may have been related to isobutyl nitrite exposure. Exposure of rats and mice to isobutyl nitrite by inhalation for 2 years resulted in increased incidences of alveolar epithelial hyperplasia (male and female rats and mice), thyroid gland follicular cell hyperplasia and splenic hemosiderin pigmentation (male mice), and serous exudate and atrophy of the olfactory epithelium of the nose (female mice). Exposure of rats to isobutyl nitrite by inhalation for 2 years resulted in decreased incidences of mononuclear cell leukemia in males and females.
Two polytopic membrane proteins, NarK and NarU, are assumed to transport nitrite out of the Escherichia coli cytoplasm, but how nitrate enters enteric bacteria is unknown. We report the construction and use of four isogenic strains that lack nitrate reductase Z and the periplasmic nitrate reductase, but express all combinations of narK and narU. The active site of the only functional nitrate reductase, nitrate reductase A, is located in the cytoplasm, so nitrate reduction by these four strains is totally dependent upon a mechanism for importing nitrate. These strains were exploited to determine the roles of NarK and NarU in both nitrate and nitrite transport. Single mutants that lack either NarK or NarU were competent for nitrate-dependent anaerobic growth on a non-fermentable carbon source, glycerol. They transported and reduced nitrate almost as rapidly as the parental strain. In contrast, the narK-narU double mutant was defective in nitrate-dependent growth unless nitrate transport was facilitated by the nitrate ionophore, reduced benzyl viologen (BV). It was also unable to catalyse nitrate reduction in the presence of physiological electron donors. Synthesis of active nitrate reductase A and the cytoplasmic, NADH-dependent nitrite reductase were unaffected by the narK and narU mutations. The rate of nitrite reduction catalysed by the cytoplasmic, NADH-dependent nitrite reductase by the double mutant was almost as rapid as that of the NarK+-NarU+ strain, indicating that there is a mechanism for nitrite uptake by E. coli that is in-dependent of either NarK or NarU. The nir operon encodes a soluble, cytoplasmic nitrite reductase that catalyses NADH-dependent reduction of nitrite to ammonia. One additional component that contributes to nitrite uptake was shown to be NirC, the hydrophobic product of the third gene of the nir operon, which is predicted to be a polytopic membrane protein with six membrane-spanning helices. Deletion of both NarK and NirC decreased nitrite uptake and reduction to a basal rate that was fully restored by a single chromosomal copy of either narK or nirC. A multicopy plasmid encoding NarU complemented a narK mutation for nitrite excretion, but not for nitrite uptake. We conclude that, in contrast to NirC, which transports only nitrite, NarK and NarU provide alternative mechanisms for both nitrate and nitrite transport. However, NarU might selectively promote nitrite ex-cretion, not nitrite uptake.
The purpose of the current study was to ascertain whether ethyl nitrite could be detected in vitro from the reaction of ethanol with peroxynitrite, as well as after administration of ethanol to mice. Ethyl nitrite analyte was determined by using gas chromatography--mass spectrometry with headspace analysis with the use of a solid-phase microextraction device. Peroxynitrite was allowed to react with ethanol under a variety of conditions in vitro. Ethyl nitrite was generated when peroxynitrite was allowed to react with ethanol. Male, inbred short-sleep mice were injected intraperitoneally with either ethanol [5.2 g/kg; 15.0% (weight/volume) ethanol in saline] or a 50:50 mixture of deuterium-labeled ethanol (D5-ethanol) and ethanol. Blood samples, as well as whole brain and liver sections, were obtained from mice 30 min later for determination of ethanol, D5-ethanol, ethyl nitrite, and deuterium-labeled ethyl nitrite (D5-ethyl nitrite). Time courses for the appearance of ethyl nitrite in blood samples, as well as in whole brain and liver sections, obtained from mice were carried out. After ethanol administration, ethyl nitrite was detected and quantitated in mouse blood, brain, and liver. A small fraction of ethyl nitrite was present. When a 50:50 mixture of ethanol and D5-ethanol was given to animals, both ethyl nitrite and D5-ethyl nitrite were found in blood and brain in approximately the same ratio as that of ethanol and D5-ethanol. The level of D5-ethyl nitrite in liver was more than twice that of ethyl nitrite, indicating a possible isotope effect in the metabolism of ethyl nitrite. Ethyl nitrite is a new metabolite of ethanol in vivo. The mechanism of ethyl nitrite formation is most likely the reaction of ethanol with peroxynitrite generated in vivo from nitric oxide.
OBJECTIVES: To measure cerebrospinal fluid and plasma nitrite and nitrate concentrations as indicators of nitric oxide production in adults after severe closed-head injury. To determine if there is an association between cerebrospinal fluid and plasma nitrite and nitrate concentrations, and cerebral blood flow, arterio-jugular oxygen content difference, injury severity, and outcome after severe closed-head injury. DESIGN: A prospective, clinical study. SETTING: Multidisciplinary intensive care unit. PATIENTS: Fifteen comatose (Glasgow Coma Scale score of < or = 7) adult patients with severe closed-head injury were studied during the prospective, randomized evaluation of the effect of moderate hypothermia (32 degrees C for 24 hrs) on neurologic outcome after closed-head injury. Seven patients were in the hypothermic group and eight patients were in the normothermic treatment group. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patients were examined sequentially, every 12 hrs for 2 days. Intraventricular cerebrospinal fluid was assayed for nitrite and nitrate concentrations. Cerebral blood flow was measured by the 133xenon intravenous method. Simultaneous blood samples were obtained for measurements of arterio-jugular oxygen content difference and plasma nitrite and nitrate concentrations. Cerebral metabolic rate for oxygen was calculated. Cerebrospinal fluid nitrite and nitrate concentrations were highest at 30 to 42 hrs vs. 6 to 18, 18 to 30, and 42 to 54 hrs (26.4 +/- 3.3 vs. 17.3 +/- 2.1, 20.0 +/- 2.2, and 18.8 +/- 2.4 microM, respectively, p < .05). There was no difference over time in plasma nitrite and nitrate concentrations. Cerebral blood flow was increased and arterio-jugular oxygen content difference was reduced at 18 to 30, 30 to 42, and 42 to 54 hrs vs. 6 to 18 hrs (p < .05). At 30 to 42 hrs, cerebrospinal fluid nitrite and nitrate concentrations were 80% higher in patients who died vs. survivors (36.4 +/- 3.2 vs. 20.2 +/- 3.6, p < .05). Using a generalized, multivariate, linear regression model, both plasma nitrite and nitrate concentrations and injury Severity Score independently predicted cerebrospinal fluid nitrite and nitrate concentrations (p < .00001 and p = .0053, respectively). Cerebral blood flow and arterio-jugular oxygen content difference were not associated with cerebrospinal fluid or plasma nitrite and nitrate concentrations using this model. Cerebrospinal fluid nitrite and nitrate concentrations were increased over time in hypothermic vs. normothermic patients. But, where this difference occurred could not be determined by multiple comparisons (p = .03). The hypothermic patients had lower admission Glasgow Coma Scale scores than normothermic patients (p = .04) and tended to have higher injury Severity Scores (p = .09). CONCLUSIONS: Increases in cerebrospinal fluid nitrite and nitrate concentrations peaked at 30 to 42 hrs after severe closed-head injury. This increase in cerebrospinal fluid nitrite and nitrate concentrations was greater in nonsurvivors. Also, cerebrospinal fluid and plasma nitrite and nitrate concentrations were associated with injury Severity Score, suggesting that increased nitric oxide production in the brain is associated with injury severity and death. Hypothermia did not prevent the increase in cerebrospinal fluid nitrite and nitrate concentrations. Further study is required to determine the source of this increase in cerebrospinal fluid nitrite and nitrate concentrations and to further define the relationship to outcome and the effect of hypothermia on this process.
Nitrite reduction to nitric oxide (NO) may be potentiated by a nitrite reductase activity of deoxyHb and contribute to systemic hypoxic vasodilation. The effect of nitrite on the pulmonary circulation has not been well characterized. We explored the effect of nitrite on hypoxic pulmonary vasoconstriction (HPV) and the role of the red blood cell (RBC) in nitrite reduction and nitrite-mediated vasodilation. As to method, isolated rat lungs were perfused with buffer, or buffer with RBCs, and subjected to repeated hypoxic challenges, with or without nitrite. As a result, in buffer-perfused lungs, HPV was reduced at nitrite concentrations of 7 muM and above. Nitrite inhibition of HPV was prevented by excess free Hb and RBCs, suggesting that vasodilation was mediated by free NO. Nitrite-inhibition of HPV was not potentiated by mild acidosis (pH = 7.2) or xanthine oxidase activity. RBCs at 15% but not 1% hematocrit prevented inhibition of HPV by nitrite (maximum nitrite concentration of approximately 35 muM) independent of perfusate Po(2). Degradation of nitrite was accelerated by hypoxia in the presence of RBCs but not during buffer perfusion. In conclusion, low micromolar concentrations of nitrite inhibit HPV in buffer-perfused lungs and when RBC concentration is subphysiological. This effect is lost when RBC concentration approaches physiological levels, despite enhanced nitrite degradation in the presence of RBCs. These data suggest that, although deoxyHb may generate NO from nitrite, insufficient NO escapes the RBC to cause vasodilation in the pulmonary circulation under the dynamic conditions of blood flow through the lungs and that RBCs are net scavengers of NO.
UNLABELLED: Sodium nitrite is used as a color fixative and preservative in meats and fish. It is also used in manufacturing diazo dyes, nitroso compounds, and other organic compounds; in dyeing and printing textile fabrics and bleaching fibers; in photography; as a laboratory reagent and a corrosion inhibitor; in metal coatings for phosphatizing and detinning; and in the manufacture of rubber chemicals. Sodium nitrite also has been used in human and veterinary medicine as a vasodilator, a bronchial dilator, an intestinal relaxant, and an antidote for cyanide poisoning. Sodium nitrite was nominated by the FDA for toxicity and carcinogenesis studies based on its widespread use in foods. Male and female F344/N rats and B6C3F1 mice were exposed to sodium nitrite (99% pure) in drinking water for 14 weeks or 2 years. Genetic toxicology studies were conducted in Salmonella typhimurium, rat and mouse bone marrow, and mouse peripheral blood. 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were exposed to 0, 375, 750, 1500, 3,000, or 5000 ppm sodium nitrite (equivalent to average daily doses of approximately 30, 55, 115, 200, or 310 mg sodium nitrite/kg body weight to males and 40, 80, 130, 225, or 345 mg/kg to females) in drinking water for 14 weeks. Clinical pathology study groups of 15 male and 15 female rats were exposed to the same concentrations for 70 or 71 days. One female exposed to 3000 ppm died before the end of the study. Body weights of males exposed to 3000 or 5000 ppm and females exposed to 5000 ppm were significantly less than those of the controls. Water consumption by 5000 ppm males and 3000 and 5000 ppm females was less than that by the controls at weeks 2 and 14. Clinical findings related to sodium nitrite exposure included brown discoloration in the eyes and cyanosis of the mouth, tongue, ears, and feet of males exposed to 3000 or 5000 ppm and of females exposed to 1500 ppm or greater. Reticulocyte counts were increased in males and females exposed to 3000 or 5000 ppm. The erythron was decreased on day 19 but increased by week 14 in males and females exposed to 5000 ppm. Methemoglobin concentrations were elevated in almost all exposed groups throughout the 14 week study; a no-observed-adverse-effect level was not achieved. The relative kidney and spleen weights of males and females exposed to 3000 or 5000 ppm were significantly greater than those of the controls. Sperm motility in 1500 and 5000 ppm males was significantly decreased. Increased erythropoietic activity in the bone marrow of exposed males and females was observed. The incidences of squamous cell hyperplasia of the forestomach in 5000 ppm males and females were significantly increased. 14-WEEK STUDY IN MICE: Groups of 10 male and 10 female B6C3F1 mice were exposed to 0, 375, 750, 1500, 3000, or 5000 ppm sodium nitrite (equivalent to average daily doses of approximately 90, 190, 345, 750, or 990 mg/kg to males and 120, 240, 445, 840, or 1230 mg/kg to females) in drinking water for 14 weeks. Body weights of males exposed to 5000 ppm were significantly less than those of the controls. Water consumption by males exposed to 1500 ppm or greater was slightly less than that by the controls at week 13. Relative spleen weights of 3000 and 5000 ppm males and absolute and relative heart, kidney, liver, and spleen weights of females exposed to 3000 or 5000 ppm were greater than those of the control groups. Sperm motility was decreased in 5000 ppm males, and the estrous cycles of 1500 and 5000 ppm females were significantly longer than in the controls. There were increased incidences of squamous cell hyperplasia of the forestomach in 5000 ppm males and females, extramedullary hematopoiesis of the spleen in 3000 and 5000 ppm males and 1500 ppm or greater females, and degeneration of the testis in 3000 and 5000 ppm males. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were exposed to 0, 750, 1500, or 3000 ppm sodium nitrite (equivalent to average daily doses of approximately 35, 70, or 130 mg/kg to males and 40, 8d 40, 80, or 150 mg/kg to females) in drinking water for 2 years. For toxicokinetic studies of plasma nitrite and blood methemoglobin, 10 male and 10 female special study rats were exposed to the same concentrations for 12 months. Survival of exposed groups was similar to that of the controls. Mean body weights of males and females exposed to 3000 ppm were less than those of the controls throughout the study. Water consumption by males and females exposed to 3000 ppm was less than that by the controls throughout the study, and that by the other exposed groups was generally less after week 14. The incidences of hyperplasia of the forestomach epithelium in males and females exposed to 3000 ppm were significantly greater than those in the control groups. The incidence of fibroadenoma of the mam mary gland was significantly increased in females exposed to 1500 ppm, and the incidences of multiple fibroadenoma were increased in 750 ppm and 1500 ppm females; however, these neoplasms occur with a high background incidence, and no increase was seen in the 3000 ppm group. The incidences of mononuclear cell leukemia were significantly decreased in males and females exposed to 1500 or 3000 ppm. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female B6C3F1 mice were exposed to 0, 750, 1500, or 3000 ppm sodium nitrite (equivalent to average daily doses of approximately 60, 120, or 220 mg/kg to males and 45, 90, or 165 mg/kg to females) in drinking water for 2 years. Survival of exposed groups was similar to that of the controls; mean body weights of 3000 ppm females were less than those of the controls throughout the study. Exposed groups generally consumed less water than the control groups. The incidences of squamous cell papilloma or carcinoma (combined) in the forestomach of female mice occurred with a positive trend. The incidence of hyperplasia of the glandular stomach epithelium was significantly greater in 3000 ppm males than in the controls. GENETIC TOXICOLOGY: Sodium nitrite was mutagenic in Salmonella typhimurium strain TA100, with and without Aroclor 1254-induced hamster and rat liver S9 enzymes; no mutagenicity was observed in strain TA98. Results of acute bone marrow micronucleus tests with sodium nitrite in male rats and mice by intraperitoneal injection were negative. In addition, a peripheral blood micronucleus assay conducted with mice from the 14-week study gave negative results. CONCLUSIONS: Under the conditions of this 2-year drinking water study, there was no evidence of carcinogenic activity of sodium nitrite in male or female F344/N rats exposed to 750, 1500, or 3000 ppm. There was no evidence of carcinogenic activity of sodium nitrite in male B6C3F1 mice exposed to 750, 1500, or 3000 ppm. There was equivocal evidence of carcinogenic activity of sodium nitrite in female B6C3F1 mice based on the positive trend in the incidences of squamous cell papilloma or carcinoma (combined) of the forestomach. Exposure to sodium nitrite in drinking water resulted in increased incidences of epithelial hyperplasia in the forestomach of male and female rats and in the glandular stomach of male mice. Decreased incidences of mononuclear cell leukemia occurred in male and female rats.
Chloroplasts were prepared from peas (Pisum sativum) in glucose-phosphate medium. In the presence of dl-glyceraldehyde, they catalyzed nitrite-dependent O(2) evolution (mean of 13 preparations, 17.5 mumole per mg chlorophyll per hour, sd 3.64). The optimum concentration of nitrite was 0.5 mm; 0.12 mm nitrite supported V(max)/2. The reaction was accompanied by the consumption of nitrite; 55 to 80% of the nitrite-N consumed was recovered as ammonia. In short experiments (less than 10 minutes) the O(2) to nitrite ratio approached 1.5, but thereafter decreased. There was no nitrite-dependent O(2) evolution with chloroplasts from plants grown without added nitrate but such chloroplasts could assimilate ammonia at about the usual rate. The results are consistent with the reduction of nitrite to ammonia involving nitrate-induced nitrite reductase and a reductant generated by the chloroplast electron transport chain.In the presence of ADP, pyrophosphate, and MgCl(2) the O(2) to nitrite ratio was typically 0.5 to 0.6 and the recovery of nitrite-N as ammonia about 60%. Under these conditions, alpha-ketoglutarate increased the O(2) to nitrite ratio (0.9-1.35) and the recovery of nitrite-N as ammonia decreased to 27%. These data and the results of nitrite plus ammonia addition experiments (with and without alpha-ketoglutarate) are attributed to incorporation of nitrite-N into glutamate via the chloroplast enzymes nitrite reductase, glutamine synthetase, and glutamate synthetase.
Biological nitrogen removal via the nitrite pathway in wastewater treatment is very important in saving the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulation were carried out in a biofilm airlift reactor with autotrophic nitrifying biofilm. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.5 to 3.5 kg N/m3 x d. Nitrite accumulation was stably achieved by the selective inhibition of nitrite oxidizers with free ammonia and dissolved oxygen limitation. Stable 100% conversion to nitrite could also be achieved even under the absence of free ammonia inhibition on nitrite oxidizers. Batch ammonium oxidation and nitrite oxidation with nitrite accumulating nitrifying biofilm showed that nitrite oxidation was completely inhibited when free ammonia is higher than 0.2 mg N/L. However, nitrite oxidation activity was recovered as soon as the free ammonia concentration was below the threshold level when dissolved oxygen concentration was not the limiting factor. Fluorescence in situ hybridization analysis of cryosectioned nitrite accumulating nitrifying biofilm showed that the beta-subclass of Proteobacteria, where ammonia oxidizers belong, was distributed outside the biofilm whereas the alpha-subclass of Proteobacteria, where nitrite oxidizers belong, was found mainly in the inner part of the biofilm. It is likely that dissolved oxygen deficiency or limitation in the inner part of the nitrifying biofilm, where nitrite oxidizers exist, is responsible for the complete shut down of the nitrite oxidizers activity under the absence of free ammonia inhibition.
Ferredoxin-nitrite reductase (EC 1.7.7.1.) from spinach has been purified to homogeneity with a specific activity of 110 units/mg of protein. The enzyme, Mr = 61,000 has 3 iron atoms (of which one is in siroheme) and 2 labile sulfides, i.e. 1 (Fe2-S2) per molecule, with absorption maxima at 276, 386 (Soret), 573 (alpha), and 690 nm, with an E386 of 3.97 X 10(4) M-1-cm-1, and A276/A386 absorptivity ratio of 1.8. Anaerobic addition of dithionite results in the loss of the 690 nm peak and the splitting of the 573 nm absorption band into two broad peaks at 545 and 585 nm. Reduction by dithionite is enhanced by cyanide (Fig. 7) and requires about 3 electron eq per mol of enzyme. With nitrite or hydroxylamine (substrates of the enzyme), cyanide (a competitive inhibitor with respect to nitrite), or sulfite, the 690 nm absorption band of substrate-free enzyme disappears and the absorbance in the Soret and alpha region are altered. The high spin EPR signals disappear (J. M. Vega, H. Kamin, N. R. Orme-Johnson, and W. H. Orme-Johnson, unpublished observations). Titration permits calculation of 1 mol of nitrite bound/mol of enzyme with a Kdiss of 3.2 X 10(-6) M. Dithionite-reduced enzyme also forms complexes with added nitrite, hydroxylamine, or cyanide, characterized by marked alterations in the 573 (alpha) absorption band. THus, substrates or competitive inhibitors can be bound to the oxidized or reduced enzyme forms. CO inhibits nitrite reductase and forms a complex with reduced enzyme (epsilonmax at 395, 543, and 585 nm). Formation or dissociation of the spectrophotometrically detectable CO complex correlates with inhibition or inhibition-reversal of nitrite reduction catalysis. During steady state turnover with dithionite and nitrite, the enzyme forms a complex with added nitrite with absorption difference maxima at 445, 538, and 580 nm with respect to reduced enzyme. When nearly all substrate is depleted the spectrum of a new species appears, indicating that nitrite reductase may form complexes with nitrogen compounds of more than one oxidation state. Nitrite is stoichiometrically reduced to ammonia without detectable free nitrogen compounds of intermediate reduction state. p-Chloromercuribenzoate (pCMB) inhibits nitrite reductase activity and nitrite partially protects against this inhibition. Titration of native enzyme with the mercurial shows that 6 mol of pCMB can be bound/mol or nitrite reductase. The Soret absorption band of the native nitrite reductase is altered and partially bleached in the pCMB-treated enzyme, and the 573 (alpha) band disappears.
CONTEXT: Delayed cerebral vasospasm causes permanent neurological deficits or death in at least 15% of patients following otherwise successful treatment for ruptured intracranial aneurysm. Decreased bioavailability of nitric oxide has been associated with the development of cerebral vasospasm. OBJECTIVE: To determine whether infusions of nitrite will prevent delayed cerebral vasospasm. DESIGN, SETTING, AND SUBJECTS: A total of 14 anesthetized cynomolgus monkeys had an autologous blood clot placed around the right middle cerebral artery. Cerebral arteriography was performed before clot placement and on days 7 and 14 to assess vasospasm. The study was conducted from August 2003 to February 2004. INTERVENTIONS: A 90-mg sodium nitrite intravenous solution infused over 24 hours plus a 45-mg sodium nitrite bolus daily (n = 3); a 180-mg sodium nitrite intravenous solution infused over 24 hours (n = 3); or a control saline solution infusion (n = 8). Each was infused continuously for 14 days. MAIN OUTCOME MEASURES: Nitrite, S-nitrosothiol, and methemoglobin levels in blood and cerebrospinal fluid and degree of arteriographic vasospasm. RESULTS: In control monkeys, mean (SD) cerebrospinal fluid nitrite levels decreased from 3.1 (1.5) micromol/L to 0.4 (0.1) micromol/L at day 7 and to 0.4 (0.4) micromol/L at day 14 (P = .03). All 8 control monkeys developed significant vasospasm of the right middle cerebral artery, which was complicated by stroke and death in 1 animal. Sodium nitrite infusions increased the nitrite and methemoglobin levels (<2.1% of total hemoglobin) in the blood and cerebrospinal fluid without evoking systemic hypotension. Nitrite infusion prevented development of vasospasm (no animals developed significant vasospasm; mean [SD] reduction in right middle cerebral artery area on day 7 after subarachnoid hemorrhage of 8% [9%] in nitrite-treated monkeys vs 47% [5%] in saline-treated controls; P<.001). There was a negative correlation between the concentration of nitrite in cerebrospinal fluid and the degree of cerebral vasospasm (P<.001). Pharmacological effects of nitrite infusion were also associated with the formation of S-nitrosothiol in cerebrospinal fluid. There was no clinical or pathological evidence of nitrite toxicity. CONCLUSION: Subacute sodium nitrite infusions prevented delayed cerebral vasospasm in a primate model of subarachnoid hemorrhage.