EPR Spectroscopic Study of the Interaction of E. coliCells with Ascorbic Acid and Sodium Nitrite

Ascorbic acid, an effective modulator and regulator of cell metabolism, was shown to induce the production of nitric oxide inE. colicells. This process was detected by EPR spectroscopy as the generation of a spectral signal typical of nitrosyl–iron–sulfur centers (Fe–S–NO) under anaerobic conditions. Incubation of E. colicells in the presence of ascorbic acid under aerobic conditions was shown to be accompanied by sodium nitrite formation. It is suggested that ascorbic acid is capable of supporting the system of energy supply to cells in hypoxia caused by reduced oxygen content or treatment with sodium nitrite.     

Non-Enzymatic Reduction of Nitrite by Means of Ascorbic Acid in Citrus and Other Higher Plant Tissues

It has been proved that the nitrite reduction in the leaves and other plant tissues of citrus and other green plants is partly or mainly a non-enzymatic chemical process, and a heat-stable factor present in these tissues is responsible for this reduction. It is suggested that ascorbic acid plays a major role in this chemical reaction since the reduction is inhibited by ascorbic acid oxidase. A significant association was also found between the ascorbic acid content and the nitrite reduction capacity of citrus leaves. Evidence has been presented that this non-enzymatic chemical reduction of nitrite occurs also in vivo as undetached citrus leaves on branches placed in NaNO₂ solution have shown diminution of their ascorbic acid content along with the absorption of nitrite. Stronger accumulation of nitrite in these leaf tissues was observed under dark conditions, apparently due to the inhibition of the biosynthesis of the ascorbic acid.     

Anti-microbial action against verotoxigenic Escherichia coli O157:H7 of nitric oxide derived from sodium nitrite

The levels of verotoxin-1 and verotoxin-2 released by verotoxigenic Escherichia coli O157:H7 treated in vitro with sodium nitrite, sodium chloride and several antibiotics were evaluated. Of the three strains of E. coli O157:H7 used in this study, two strains produced both verotoxin-1 and verotoxin-2, and one strain produced only verotoxin-2. Treatment of E. coli O157:H7 with sodium nitrite (6000 mg/l, minimum inhibitory concentration) did not increase the levels of verotoxin-1 and verotoxin-2 compared with a treatment by sodium chloride or antibiotics. When the electron paramagnetic resonance spectrum of sodium nitrite-treated bacterial cells was examined at 77 K to clarify the mechanism for the anti-bacterial activity of nitric oxide derived from sodium nitrite, electron paramagnetic resonance signals with g-values of 2.035 and 2.010 were observed. These were identified as being derived from iron-nitric oxide complexes. It appears that the dinitrosyl iron complexes in the E. coli O157:H7 cells were generated from the reaction of iron-sulfur proteins (enzymes) with nitric oxide formed by the reduction of sodium nitrite. The amount of ATP was decreased by the presence of sodium nitrite in the cell suspension. These findings indicate that nitric oxide derived from sodium nitrite penetrated the cells and inactivated enzymes related to the respiratory chain.     

Intragastric generation of antimicrobial nitrogen oxides from saliva--physiological and therapeutic considerations

Salivary nitrite is suggested to enhance the antimicrobial properties of gastric juice by conversion to nitric oxide (NO) and other reactive nitrogen intermediates in the stomach. Intubated patients exhibit extremely low gastric levels of NO, because they do not swallow their saliva. The present investigation was designed to examine the antibacterial effects of human saliva and gastric juice. Furthermore, we studied a new mode of NO delivery, involving formation from acidified nitrite, which could prevent bacterial growth in the gastric juice of intubated patients in intensive care units. The growth of Escherichia coli ATCC 25922 and the formation of NO and nitroso/nitrosyl species were determined after incubation of gastric juice with saliva from healthy volunteers that was rich (nitrate ingestion) or poor (overnight fasting) in nitrite. In a stomach model containing gastric juice from intubated patients, we inserted a catheter with a silicone retention cuff filled with ascorbic acid and nitrite and determined the resulting antibacterial effects on E. coli and Candida albicans. Saliva enhanced the bactericidal effect of gastric juice, especially saliva rich in nitrite. Formation of NO and nitroso/nitrosyl species by nitrite-rich saliva was 10-fold greater than that by saliva poor in nitrite. In our stomach model, E. coli and C. albicans were killed after exposure to ascorbic acid and nitrite. In conclusion, saliva rich in nitrite enhances the bactericidal effects of gastric juice, possibly through the generation of reactive nitrogen intermediates, including NO. Acidified nitrite inside a gas-permeable retention cuff may be useful for restoring gastric NO levels and host defense in critically ill patients.     

Reduction of nitrite to nitric oxide by enteric bacteria

Seven bacteria representing seven genera of enteric bacteria, in addition to Escherichia coli, were shown to reduce nitrite to NO under anaerobic conditions when the cells were grown as nitrate respirers. NO production was inhibited by nitrate and azide and was self limiting, just as was found to be the case previously with E. coli and its nitrate reductase. Maximum initial rates of NO production were observed at pH 5.5-6.     

The effect of incubation time and temperature on growth of Escherichia coli on gradient plates containing sodium chloride and sodium nitrite

Two-dimensional gradient plates are a convenient way of screening antimicrobial effects of preservative factors acting in combination across a broad range of physical and chemical conditions. We report the effects of sodium chloride, sodium nitrite and incubation temperature on the growth of Escherichia coli by staining, laser densitometry and computer graphics. Staining not only more easily distinguished the growth area but also gave an indication of the viability of cells present. 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride was the more useful of the two stains used. Inhibitory concentrations of sodium chloride decreased with reduced incubation temperature. The response of E. coli to combinations of salt and nitrite on gradient plates was very similar to its response in liquid medium.     

The effect of incubation time and temperature on growth of Escherichia coli on gradient plates containing sodium chloride and sodium nitrite

Two-dimensional gradient plates are a convenient way of screening antimicrobial effects of preservative factors acting in combination across a broad range of physical and chemical conditions. We report the effects of sodium chloride, sodium nitrite and incubation temperature on the growth of Escherichia coli by staining, laser densitometry and computer graphics. Staining not only more easily distinguished the growth area but also gave an indication of the viability of cells present. 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride was the more useful of the two stains used. Inhibitory concentrations of sodium chloride decreased with reduced incubation temperature. The response of E. coli to combinations of salt and nitrite on gradient plates was very similar to its response in liquid medium.     

Adaptation of E. coli cell method for micro-scale nitrate measurement with the Griess reaction in culture media

The E. coli cell method for nitrate measurement consists of two-steps: nitrate reduction by the E. coli cell usually under anaerobic conditions and subsequently nitrite measurement with the Griess reaction. It was found that the E. coli DSM 498k wildtype cell can reduce nitrate to nitrite under aerobic conditions. Therefore, the E. coli method for nitrate measurement was adapted to be performed under aerobic conditions in a microtiter plate. The adapted method is simpler than the original E. coli method and other nitrate methods such as those with inorganic reductants and with purified enzymes. Furthermore, it was found that for the Griess reaction the pH values of samples after addition of the Griess reagent A should be lower than 1.8 for a stable absorbance at 540 nm to be reached. It is important to add the two Griess reagents separately and to read the absorbance twice consecutively in a microtiter plate. The adapted E. coli method was successfully applied to measure the traces of nitrate in MRS and other medium components by measuring the standard curve of a dilution of each individual medium component. It was found that many organic medium components contain traces of nitrate, while none of them contain detectable nitrite. Among these, the extract of meat and yeast extract contain relatively high amounts of nitrate: 217 mg N/kg and 99 mg N/kg respectively. MRS broth contains nitrate from 0.3 to 0.6 mg N/l depending on the batch numbers of the product. The adapted E. coli can also be used for nitrate measurement in other matrices.     

Reversal of P-glycoprotein expressed in Escherichia coli leaky mutant by ascorbic acid

It has been reported that functional expression of the multidrug resistance protein P-glycoprotein (P-gp) in E. coli is useful for screening P-gp substrates and inhibitors. In the present study, we have constructed by nitrosoguanidine and UV mutagenesis 28 leaky mutants of E. coli UT5600. These mutants are significantly susceptible to the toxic effect of known P-gp substrates and lipophilic cancer drugs. Mouse mdr1 was functionally expressed in the most permeable E. coli mutant (UTP17). Expression of P-gp in this mutant confers cross-resistance to mitomycin C, tegafur, daunorubicin, rhodamine 6G, tetraphenylphosphonium bromide and ciprofloxacin. To examine the reversal of P-gp expressed in this heterologous system, UTP17 cells expressing mouse mdr1 or lac permease as negative control were treated with various concentrations of mitomycin C with or without ascorbic acid. We found that ascorbic acid abrogated P-gp mediated multidrug resistance, suggesting that ascorbic acid might be used in combination with anticancer drugs to reduce emergence of multidrug resistance. We also demonstrated that tomato lectin antagonized the inhibitory action of ascorbic acid. This study provide a heterologous system for mdr1 expression in E. coli leaky mutant that can be used as a system for the screening of P-gp inducers and inhibitors, since it is quick and simple.     

Acid-mediated mutagenicity of tobacco snuff: its possible mechanism

Polar solvent extracts of tobacco snuff under acidic conditions were mutagenic in Salmonella typhimurium. Using the Griess reagent test, nitrite ranging from approximately 1.8 to 5.4 mg/g of snuff was found in the polar fraction of extracts. After acid treatment, nitroso compounds in the amount corresponding to the nitrite concentration were detected. The mutagenic potency of the acid-treated extracts was consistent with the content of nitroso compounds generated. Formation of nitroso compounds and the mutagenic activity under acidic conditions was inhibited by ascorbic acid. The results indicate that a nitrosation process was involved in snuff extracts during acid treatment. Studies related to the source of nitrite in tobacco snuff demonstrated that snuff contained bacteria which were able to reduce nitrate to nitrite and that the amount of nitrite in snuff extracts could be further increased by incubation of the extracts with the bacteria. Since snuff contains a considerable amount of nitrate, it seems that reduction of nitrate in snuff to nitrite by bacteria, and nitrosation of certain constituents in snuff by nitrite under acidic conditions to form mutagenic nitroso compounds are possible mechanisms responsible for the acid-mediated mutagenicity of snuff extracts.     

The bactericidal effect and chemical reactions of acidified nitrite under conditions simulating the stomach

AIMS: To examine the hypothesis of non-immune defence mechanisms based on nitrite. METHODS AND RESULTS: The acidified media (nutrient broth or citrate-phosphate buffer) under aerobic conditions with additions of physiological levels of nitrite, L-ascorbic acid, iodide and thiocyanate were used to simulate gastric juice. The bactericidal effects of acidified nitrite on Escherichia coli and lactobacilli were investigated using bacterial plate counts. Conversion of acidified nitrite to nitric oxide, nitrogen dioxide and nitrate was also studied. Nitrite significantly increased the bactericidal effects on E. coli and lactobacilli. The bactericidal effects were enhanced by thiocyanate but not by L-ascorbic acid and iodide. L-Ascorbic acid and thiocyanate, but not iodide, enhanced the decomposition of acidified nitrite in nutrient broth. Acidified nitrite was converted to both nitric oxide and nitrate, but a portion of the acidified nitrite in nutrient broth may have been converted to other unidentified nitrogen compounds. Nitrogen dioxide was not detected in any of the samples. CONCLUSION: The bactericidal effects of nitrite appeared to be primarily related to nitrous acid, and possibly to other unidentified nitrogenous metabolites, but not to nitric oxide and nitrogen dioxide. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential role of nitrite as an antimicrobial substance in the stomach may be of some importance in the ecology of the gastrointestinal tract and in host physiology.     

Effects of pH, Nitrite, and Ascorbic Acid on Nonenzymatic Nitric Oxide Generation and Bacterial Growth in Urine

Nitrite may be generated by bacteria in urine during urinary tract infections. Acidification of nitrite results in the formation of nitric oxide (NO) and other reactive nitrogen oxides, which are toxic to a variety of microorganisms. We have studied NO formation and bacterial growth in mildly acidified human urine containing nitrite and the reducing agent vitamin C. Urine collected from healthy subjects was incubated in closed syringes at different pH values with varying amounts of nitrite and/or ascorbic acid added. NO generation was measured in headspace gas using a chemiluminescence technique. A similar setup was also used to study the growth of three strains of bacteria in urine. Mildly acidified nitrite-containing urine generated large amounts of NO and this production was greatly potentiated by ascorbic acid. The growth of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus saprophyticus was markedly reduced by the addition of nitrite to acidified urine. This inhibition was enhanced by ascorbic acid. In conclusion, we show that the growth of three common urinary pathogens is markedly inhibited in mildly acidified urine when nitrite is present. The bacteriostatic effect of acidified nitrite is likely related to the release of NO and other toxic reactive nitrogen intermediates. These results may help to explain the well-known beneficial effects of urinary acidification with, e.g., vitamin C in treatment and prevention of urinary tract infection.     

Anaerobic transfer of antibiotic resistance from Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic pathogen that often initiates infections from a reservoir in the intestinal tract, may donate or acquire antibiotic resistance in an anaerobic environment. Only by including nitrate and nitrite in media could antibiotic-resistant and -sensitive strains of P. aeruginosa be cultured in a glove box isolator. These anaerobically grown cells remained sensitive to lytic phage isolated from sewage. After incubation with a phage lysate derived from P. aeruginosa 1822, anaerobic transfer of antibiotic resistance to recipients P. aeruginosa PS8EtBr and PS8EtBrR occurred at frequencies of 6.2 x 10(-9) and 5.0 x 10(-8) cells per plaque-forming unit, respectively. In experiments performed outside the isolator, transfer frequencies to PS8EtBr and PS8EtBrR were higher, 1.3 x 10(-7) and 6.5 x 10(-8) cells per plaque-forming unit, respectively. When P. aeruginosa 1822 was incubated aerobically with Escherichia coli B in medium containing nitrate and nitrite, the maximum concentration of carbenicillin-resistant E. coli B reached 25% of the total E. coli B population. This percentage declined to 0.01% of the total E. coli B population when anaerobically grown P. aeruginosa 1822 and E. coli B were combined and incubated in the glove box isolator. The highest concentration of the recipient population converted to antibiotic resistance occurred after 24 h of aerobic incubation, when an initially high donor/recipient ratio (>15) of cells was mixed. These data indicate that transfer of antibiotic resistance either by transduction between Pseudomonas spp. or by conjugation between Pseudomonas sp. and E. coli occurs under strict anaerobic conditions, although at lower frequencies than under aerobic conditions.     

The Use of Ascorbate as an Oxidation Inhibitor in Prebiotic Amino Acid Synthesis: A Cautionary Note

It is generally thought that the terrestrial atmosphere at the time of the origin of life was CO₂-rich and that organic compounds such as amino acids would not have been efficiently formed abiotically under such conditions. It has been pointed out, however, that the previously reported low yields of amino acids may have been partially due to oxidation by nitrite/nitrate during acid hydrolysis. Specifically, the yield of amino acids was found to have increased significantly (by a factor of several hundred) after acid hydrolysis with ascorbic acid as an oxidation inhibitor. However, it has not been shown that CO₂ was the carbon source for the formation of the amino acids detected after acid hydrolysis with ascorbic acid. We therefore reinvestigated the prebiotic synthesis of amino acids in a CO₂-rich atmosphere using an isotope labeling experiment. Herein, we report that ascorbic acid does not behave as an appropriate oxidation inhibitor, because it contributes amino acid contaminants as a consequence of its reactions with the nitrogen containing species and formic acid produced during the spark discharge experiment. Thus, amino acids are not efficiently formed from a CO₂-rich atmosphere under the conditions studied.     

Nitric Oxide in Plants: The Roles of Ascorbate and Hemoglobin

Ascorbic acid and hemoglobins have been linked to nitric oxide metabolism in plants. It has been hypothesized that ascorbic acid directly reduces plant hemoglobin in support of NO scavenging, producing nitrate and monodehydroascorbate. In this scenario, monodehydroascorbate reductase uses NADH to reduce monodehydroascorbate back to ascorbate to sustain the cycle. To test this hypothesis, rates of rice nonsymbiotic hemoglobin reduction by ascorbate were measured directly, in the presence and absence of purified rice monodehydroascorbate reductase and NADH. Solution NO scavenging was also measured methodically in the presence and absence of rice nonsymbiotic hemoglobin and monodehydroascorbate reductase, under hypoxic and normoxic conditions, in an effort to gauge the likelihood of these proteins affecting NO metabolism in plant tissues. Our results indicate that ascorbic acid slowly reduces rice nonsymbiotic hemoglobin at a rate identical to myoglobin reduction. The product of the reaction is monodehydroascorbate, which can be efficiently reduced back to ascorbate in the presence of monodehydroascorbate reductase and NADH. However, our NO scavenging results suggest that the direct reduction of plant hemoglobin by ascorbic acid is unlikely to serve as a significant factor in NO metabolism, even in the presence of monodehydroascorbate reductase. Finally, the possibility that the direct reaction of nitrite/nitrous acid and ascorbic acid produces NO was measured at various pH values mimicking hypoxic plant cells. Our results suggest that this reaction is a likely source of NO as the plant cell pH drops below 7, and as nitrite concentrations rise to mM levels during hypoxia.     

Studies on the Activation Mechanism of the Myrosinase by L-Ascorbic Acid

The desmutagenic activity of various food components on C-nitro mutagens formed by the nitrite/sorbic acid reaction was assayed and several vegetable juices were found to be effective for eliminating the mutagenicity of the nitrite/sorbic acid system. Especially, the desmutagenic activity of pumpkin juice was investigated, and ascorbic acid, cysteine and other reducing compounds were found to be responsible for desmutagenic actions on 1,4-dinitro-2-methyl pyrrole, the main mutagen formed by the reaction of sorbic acid with sodium nitrite, by reduction of the conjugated C-nitro group to a C-amino group.     

Purification of Vibrio fischeri nitrite reductase and its characterization as a hexaheme c-type cytochrome

Dissimilatory nitrite reductase was isolated from anaerobically nitrate-grown Vibrio fischeri cells and purified to electrophoretic homogeneity. The enzyme catalyzes the six-electron reduction of nitrite to ammonia. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under either nonreducing or reducing conditions, the purified nitrite reductase migrated as a single protein band of Mr 57,000. Gel filtration chromatography revealed a native molecular weight of 58,000, indicating the enzyme as isolated to be present in the monomeric form. Purified nitrite reductase exhibited typical c-type cytochrome absorption spectra with the reduced alpha-band at 552.5 nm. Heme content analysis using the purified preparation indicated the enzyme to contain 5.5 heme c groups per molecule. Iron analysis showed the presence of 5.62 g iron atoms per mole of enzyme and no nonheme irons were detected. These results clearly indicate that, similar to the dissimilatory nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes, and Escherichia coli, the V. fischeri nitrite reductase is a hexaheme c-type cytochrome. Amino acid composition of V. fischeri also revealed close similarities to those of the other three hexaheme nitrite reductases previously studied. Based on this information, it is concluded that the four ammonia-forming, dissimilatory nitrite reductases isolated to date represent a homologous group of proteins with the distinct property of being hexaheme c-type cytochromes.     

The role of bacterial metabolism in transformation of non-mutagenic compounds into mutagens. I. Participation of Escherichia coli nitroreductase in creation of mutagens from non-mutagenic new pesticides]

Investigations concerned Escherichia coli nitroreductase in creation of mutagens from non-mutagenic pesticides-derivatives of urea. Three new compounds were studied: N-phenyl-N'-methylurea (IPO 4328), N-methyl,N-(2-hydroxyethyl)-N'phenylurea (IPO 2363), N-(2-hydroxyethyl), N-methyl-N'-(3,4 dichloroethyl) urea, and diurone-3-(3,4 dichlorophenyl)-1,1 dimethylurea. These compounds were incubated in anaerobic conditions with cells of E. coli K-12 (KF) strain and nitrate or nitrite. Using Ames test, mutagenicity of resulting metabolites was investigated. It was found that during incubation of herbicide IPO 4328 with cells of E. coli K-12 (KF) and nitrate, mutagenic product for strain of S. typhimurium TA 1537 is created. Very weak mutagenic metabolite for the same strain was appearing during incubation of herbicide IPO 2363 with cells of E. coli K-12 (KF) in presence of nitrite. Incubation of investigated compounds with E. coli K-12 (KF) cells alone did not result in appearance of mutagenic substances. Thus, role of Escherichia coli in creation of mutagenic compounds from non-mutagenic derivatives of urea consisted of nitrite from nitrate production with participation of nitroreductase, which afterwards in absence of bacteria or action of their enzymes reacted with investigated pesticides.     

Desmutagenic actions of ascorbic acid and cysteine on a new pyrole mutagen formed by the reaction between food additives; Sorbic acid and sodium nitrite

Mutagenicity of 1,4-dinitro-2-methyl pyrrole, a new mutagen isolated from the reaction mixture of sorbic acid and sodium nitrite, was found to be destroyed by treatment with ascorbic acid or cysteine. Chemical studies revealed that the loss of mutagenicity was due to reduction from the C-nitro to C-amino group.