Formation Of Nitroso Compounds Research Articles

Kinetic study on the reaction of sodium nitrite with neurotransmitters secreted in the stomach

Nitroso-compounds are potentially mutagenic and carcinogenic compounds due to their ability to alkylate DNA bases. One of the most common sources of human exposure to nitroso-compounds is their formation in the acidic environment of the stomach by the reaction between electron-rich molecules present in the lumen and sodium nitrite ingested in the diet. To date, the formation of nitroso-compounds by the reaction of nitrite with food components has been investigated in depth, but little attention has been paid to substances secreted in the stomach, such as dopamine or serotonin, whose reaction products with nitrite have proven mutagenic properties. In this article, we present a kinetic study with UV–visible spectroscopy of the nitrosation reactions of both molecules, as well as of L-tyrosine, the amino-acid precursor of dopamine. We determined the kinetic parameters and reaction mechanisms for the reactions, studying the influence of the reactants concentration, pH, temperature, and ionic strength on the reaction rate. In all cases, the favoured reaction product was a stable nitroso-compound. Serotonin, the molecule whose product was the most mutagenic, underwent two consecutive nitrosation reactions. These findings suggest that additional biological research is needed to understand how this reaction alters the function of these neurotransmitters as well as the potentially toxic effects they may have once nitrosated.

Interaction of Persulfate and Hydroxylamine Produces Highly Reactive Species in Aqueous Systems

Activation of persulfate is a hot topic in environmental remediation via generating reactive species such as SO4•– and 1O2. This study investigated the activation of peroxydisulfate (PDS) by HA with respect to the formation mechanisms of reactive species and their roles in contaminant transformation. SO4•–, HO•, and reactive nitrogen species (RNS, e.g., •NO, •NO2, and NH2O•) were proved to be predominant reactive species in the HA/PDS system by electron paramagnetic resonance analysis and radical scavenging tests. The reaction stoichiometric ratio of HA to PDS was about 0.63, involving two major steps: HA initially reacted with PDS to generate SO4•– and NH2O•, and NH2O• further reacted with PDS to form SO4•– and HNO. Ultimately, N2O was the major product of HA via the dimerization of HNO. •NO was formed via the reactions of HNO with O2 and HO•, and •NO2 could be formed via the reaction of SO4•– and NO2–. In the HA/PDS system, SO4•– and HO• primarily contributed to the degradation kinetic of carbamazepine, while RNS also played important roles in the transformation pathways of carbamazepine, resulting in the formation of nitroso compounds. As pH increased from 2 to 7, the removal rate of carbamazepine was highest at pH 3–4 (66–69% at 60 min), which is mainly due to NH2OH showing higher activation efficiency for PDS but faster consumption for SO4•– and HO• compared to NH3OH+. This study broadens the understanding of peroxide activation by HA and demonstrates the important roles of formed RNS in contaminant transformation.

New Insights into the Chemical Reactivity of Dry-Cured Fermented Sausages: Focus on Nitrosation, Nitrosylation and Oxidation.

Nitrite and nitrate are added to cured meat for their bacteriological, technological and sensorial properties. However, they are suspected to be involved in the formation of nitroso compounds (NOCs), such as potentially mutagenic nitrosamines, nitrosylheme and nitrosothiols. Controlling the sanitary and sensorial qualities of cured meat products by reducing these additives requires elucidating the mechanisms involved in the formation of NOCs. To this end, we studied the dose-response relationship of added sodium nitrite and/or sodium nitrate (0/0, 80/80, 0/200, and 120/120 ppm) on the formation of NOCs in dry cured fermented sausages. The results showed a basal heme iron nitrosylation in the absence of NaNO2/NaNO3 due to starter cultures. This reaction was promoted by the addition of NaNO2/NaNO3 in the other conditions. Reducing the dose to 80/80 ppm still limits lipid oxidation without the formation of non-volatile nitrosamines. Conversely, the addition of NO2/NO3 slightly increases protein oxidation through higher carbonyl content. The use of 80/80 ppm could be a means of reducing these additives in dry-cured fermented meat products.

Chemical reactivity of nitrite and ascorbate in a cured and cooked meat model implication in nitrosation, nitrosylation and oxidation

Nitrite, added to cured meat for its bacteriological and technological properties, is implicated in the formation of nitroso compounds (NOCs), such as nitrosylheme, nitrosamines and nitrosothiols, suspected to have a potential impact on human health. The mechanisms involved in NOC formation are studied in regard with the dose–response relationship of added nitrite and its interaction with ascorbate on NOC formation in a cured and cooked meat model. The impact of a second cooking stage on nitrosation was evaluated. The addition of nitrite in the cured and cooked model promoted heme iron nitrosylation and S-nitrosation but not N-nitrosation. Nitrite reduced lipid oxidation without an additional ascorbate effect. The second cooking sharply increased the nitrosamine content while the presence of ascorbate considerably lowered their levels and protected nitrosothiols from degradation. This study gives new insights on the chemical reactivity of NOCs in a cured meat model.

Reactions of polyphenols in pomegranate peel with nitrite under simulated stomach conditions.

Punicalagin and ellagic acid are the major polyphenols present in pomegranate peels. The contents of α‐punicalagin, β‐punicalagin, and ellagic acid in the pomegranate peels were approximately 75, 72, and 20 µM, respectively. The reactions of polyphenols in pomegranate peels with sodium nitrite under simulated stomach conditions were studied. The reactions decreased the polyphenolic contents of the pomegranate peels and accompanied the formation of nitroso compounds. The oxidation rates followed the order ellagic acid ＜α‐punicalagin ≈ β‐punicalagin. The results suggested that the reactions can occur in the stomach after a meal, while the pH changes from 2 to 4.5.

Inulin-fortification of a processed meat product attenuates formation of nitroso compounds in the gut of healthy rats

Intake of red and processed meat has been suspected to increase colorectal cancer risk potentially via endogenous formation of carcinogenic N-nitroso compounds or increased lipid and protein oxidation. Here we investigated the effect of inulin fortification of a pork sausage on these parameters. For four weeks, healthy Sprague-Dawley rats (n = 30) were fed one of three diets: inulin-fortified pork sausage, control pork sausage or a standard chow diet. Fecal content of apparent total N-nitroso compounds (ATNC), nitrosothiols and nitrosyl iron compounds (FeNO) were analyzed in addition to liver metabolism and oxidation products formed in liver, plasma and diets. Intriguingly, inulin fortification reduced fecal ATNC (p = 0.03) and FeNO (p = 0.04) concentrations. The study revealed that inulin fortification of processed meat could be a strategy to reduce nitroso compounds formed endogenously after consumption.

Red Meat-Derived Nitroso Compounds, Lipid Peroxidation Products and Colorectal Cancer.

About 20 years ago, the research group of Sheila Anne Bingham in Cambridge, UK, showed for the first time that volunteers consuming large amounts of red meat excrete high amounts of nitroso compounds via feces. In the meantime, it has been demonstrated that heme leads to the enhanced formation of nitroso compounds in the gastrointestinal tract and that the main nitroso compounds formed in the gastrointestinal tract are S-nitrosothiols and the nitrosyl heme. Moreover, it has been postulated that these endogenously formed nitroso compounds may alkylate guanine at the O6-position, resulting in the formation of the promutagenic DNA lesions O6-methylguanine and O6-carboxymethylguanine, which, if not repaired (in time), could lead to gene mutations and, subsequently to the development of colorectal cancer. Alternatively, it has been postulated that heme iron could contribute to colorectal carcinogenesis by inducing lipid peroxidation. In the present review, the evidence supporting the above-mentioned hypotheses will be presented.

Reactions of polyphenols in masticated apple fruit with nitrite under stomach simulating conditions: Formation of nitroso compounds and thiocyanate conjugates

By the ingestion of fresh apple fruit, it is masticated squeezing apple juice into the oral cavity and the juice is mixed with saliva. The mixture of saliva and apple juice is swallowed into the stomach where the pH is around 2. This paper deals with the reactions of polyphenols in the juice obtained by mastication of apple fruit with salivary nitrite under acidic conditions. The concentrations of catechins, procyanidins, and chlorogenic acid in the apple juice were approximately 55, 55, and 170μM, respectively, and the polyphenols were oxidized by salivary nitrite under conditions of the stomach. Rates of the oxidation increased in order chlorogenic acid<catechins<procyanidins. The oxidation of catechins and procyanidins resulted in the formation of the nitroso compounds, and the oxidation of chlorogenic acid resulted in the formation of the thiocyanate conjugate. The production of dinitroso compounds is proposed to be due to the addition of nitric oxide (NO) to radicals of catechins and procyanidins. As the mechanism of thiocyanate conjugate formation, reaction of o-quinone of chlorogenic acid with a salivary component thiocyanate is proposed, and the formation of the thiocyanate conjugate is discussed from the point of detoxification of chlorogenic acid quinone.

Effects Of Polyphenols On The Endogenous Formation Of Nitroso Compounds In Simulated Gastric Conditions

There is considerable epidemiological evidence supporting a positive association between red and processed meat intake and risk of colorectal cancer. There are several potential mechanism for this positive association, in particular the formation of carcinogenic N-nitroso compounds. Previously, we could show a close relationship between red meat and ATNC (apparent total nitroso compounds) formation. Polyphenols can reduce the formation of N-nitroso compounds in vitro. In this study, we have investigated the protective effect of different polyphenols in a simulated gastric system. The in vitro effect of quercetin, rutin, (-)-epicatechin, rosmarinic acid, ascorbic acid and several plant extracts containing these compounds were tested in an acidic gastric juice environment (bovine serum albumin, haemin and haemoglobin were dissolved in 7mM HCl, pH2.2, 1 mM NaNO2 were added to start the reaction). Different classes nitroso compounds were determined using chemical denitrosation with chemiluminescence detection. This enabled us not only to measure ATNC but also nitrosothiols and iron-nitrosyl (nitrosyl haem) complexes separately to have more information of the mechanism. The highest concentration of ATNC were observed after 20 mins of reaction time, while nitrite concentrations gradually decreased to almost 0 µmol/L. The formation of nitroso compounds were investigated in a simplified stomach environment to simulate high nitrate and high red meat intake with and without the addition of polyphenols. Our results did not show a consistent effect of phenolic compounds on the formation of total nitroso compounds, nitrosothiols or iron-nitrosyl complexes.

Traditional Indian spices and their health significance.

India has been recognized all over the world for spices and medicinal plants. Both exhibit a wide range of physiological and pharmacological properties. Current biomedical efforts are focused on their scientific merits, to provide science-based evidence for the traditional uses and to develop either functional foods or nutraceuticals. The Indian traditional medical systems use turmeric for wound healing, rheumatic disorders, gastrointestinal symptoms, deworming, rhinitis and as a cosmetic. Studies in India have explored its anti-inflammatory, cholekinetic and anti-oxidant potentials with the recent investigations focusing on its preventive effect on precarcinogenic, anti-inflammatory and anti atherosclerotic effects in biological systems both under in vitro and in vivo conditions in animals and humans. Both turmeric and curcumin were found to increase detoxifying enzymes, prevent DNA damage, improve DNA repair, decrease mutations and tumour formation and exhibit antioxidative potential in animals. Limited clinical studies suggest that turmeric can significantly impact excretion of mutagens in urine in smokers and regress precancerous palatal lesions. It reduces DNA adducts and micronuclei in oral epithelial cells. It prevents formation of nitroso compounds both in vivo and in vitro. It delays induced cataract in diabetes and reduces hyperlipidemia in obese rats. Recently several molecular targets have been identified for therapeutic / preventive effects of turmeric. Fenugreek seeds, a rich source of soluble fiber used in Indian cuisine reduces blood glucose and lipids and can be used as a food adjuvant in diabetes. Similarly garlic, onions, and ginger have been found to modulate favourably the process of carcinogenesis.

Dietary meat, endogenous nitrosation and colorectal cancer

Colorectal cancer is the third most common cancer in developed countries such as the U.K., but incidence rates around the world vary approx. 20-fold. Diet is thought to be a key factor determining risk: red and processed meat, but not white meat or fish, are associated with an increased risk of colorectal cancer. The endogenous formation of N-nitroso compounds is a possible explanation because red and processed meat, but not white meat or fish, cause a dose-dependent increase in faecal ATNCs (apparent total N-nitroso compounds) and the formation of nitroso-compound-specific DNA adducts in humans. Red meat is particularly rich in haem which has been found to promote the endogenous formation of ATNC. Nitrosyl haem and nitroso thiols have been identified as major constituents of both faecal and ileal ATNC with a significant increase in the formation of these compounds following a diet rich in red meat. In vitro incubations show that, under simulated gastric conditions, nitroso thiols are the main species of nitroso compound formed, suggesting that acid-catalysed thionitrosation is the initial step in the endogenous formation of nitroso compounds. Nitrosyl haem and other nitroso compounds can then form under the alkaline and reductive conditions of the small and large bowel.

Red meat and colon cancer: Heme proteins and nitrite in the gut. A commentary on “Diet-induced endogenous formation of nitroso compounds in the GI tract”

Red meat and colon cancer: Heme proteins and nitrite in the gut. A commentary on “Diet-induced endogenous formation of nitroso compounds in the GI tract”

Diet-induced endogenous formation of nitroso compounds in the GI tract

Red or processed meat, but not white meat or fish, is associated with colorectal cancer. The endogenous formation of nitroso compounds is a possible explanation, as red or processed meat—but not white meat or fish—causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC) and the formation of nitroso-compound-specific DNA adducts. Red meat is particularly rich in heme and heme has also been found to promote the formation of ATNC. To investigate the underlying mechanism of ATNC formation, fecal and ileal samples of volunteers fed a high red meat or a vegetarian diet were analyzed for nitrosyl iron, nitrosothiols, and heme. To simulate the processes in the stomach, food homogenates and hemoglobin were incubated under simulated gastric conditions. Nitrosyl iron and nitrosothiols were significantly ( p < 0.0001) increased in ileal and fecal samples after a high red meat diet compared with a vegetarian diet; significantly more nitrosyl iron than nitrosothiols was detectable in ileal ( p < 0.0001) and fecal ( p < 0.001) samples. The strong correlation between fecal nitrosyl iron and heme (0.776; p < 0.0001) suggested that nitrosyl heme is the main source of nitrosyl iron, and ESR confirmed the presence of nitrosyl heme in fecal samples after a high red meat diet. Under simulated gastric conditions, mainly nitrosothiols were formed, suggesting that acid-catalyzed thionitrosation is the initial step in the endogenous formation of nitroso compounds. Nitrosyl heme and other nitroso compounds can then form under the alkaline and reductive conditions of the small and large bowel.

Formation of Nitroso Compounds in the Reaction of Nitrite and Hemoglobin.

Nitrite is being studied as a therapeutic agent for several pathologies, largely due to the demonstration that infusions of nitrite into the human forearm results in increased blood flow. Thus, nitrite may serve as a storage pool for the endothelium derived vasodilator, nitric oxide (NO). Nitrite is known to react with deoxyhemoglobin to form NO, which would need to diffuse from the red blood cells to the smooth muscle cells in order to activate its target enzyme soluble guanylyl cyclase. However, hemoglobin scavenges NO extremely quickly so that NO formed in the red blood cell cannot get out. The export of nitrite-derived NO activity is likely to require the formation of either a diffusible intermediate or alternative species for the transport of NO. One of the suggested intermediates is S-nitrosohemoglobin, small amounts of which have been reported to have formed during nitrite infusion studies. Other investigators recently reported that S-nitrosohemoglobin is directly formed in the reaction of nitrite and hemoglobin via the formation of a quasi-stable Fe(III)NO-hemoglobin intermediate. We have used absorption spectroscopy, electron paramagnetic resonance spectroscopy, and several chemiluminescence-based assays to the nitrite/hemoglobin reaction, focusing on the formation of S-nitrosohemoglobin and other nitroso species such as S-nitrosoglutathione (GSNO). We have also developed a sensitive chemiluminescence-based assay to explore Fe(III)NO-Hb formation. We applied these techniques to a variety of conditions, varying oxygen saturation and reactant concentrations in cell-free and red cell encapsulated hemoglobin. Based on our results, we conclude (contrary to several other recent reports) that little or no nitroso species are formed in the reaction of nitrite and hemoglobin at neutral pH (7.4) and a quasi-stable Fe(III)NO-Hb intermediate is also not formed. We find that, under thoroughly deoxygenated conditions, one nitrite molecule results in the formation of one Fe(II)NO-Hb and one MetHb species, with some nitrite bound MetHb being formed subsequently. Importantly, we find that at pH 6.5, significant nitrosation does occur. These data suggest that much or all of the nitroso species measured in nitrite therapeutics may be formed in metabolically active muscle or other tissues of mild acidity.

The effect of haem in red and processed meat on the endogenous formation of N-nitroso compounds in the upper gastrointestinal tract

Red and processed meat (PM) consumption increases the risk of large bowel cancer and it has been demonstrated that haem in red meat (RM) stimulates the endogenous production of N-nitroso compounds (NOCs) within the human intestine. To investigate whether N-nitrosation occurs in the upper gastrointestinal tract, 27 ileostomists were fed diets containing no meat, or 240 g RM or 240 g PM in a randomly assigned crossover intervention design carried out in a volunteer suite. Endogenous NOC were assessed as apparent total N-nitroso compounds (ATNC) in the ileostomy output. ATNC concentration in the diets was 22 microg ATNC/kg (RM) and 37 microg ATNC/kg (PM), and 9 microg ATNC/kg in the no meat diet. Levels significantly increased to 1175 microg ATNC/kg SEM = 226 microg ATNC/kg) following the RM (P=0.001) and 1832 microg ATNC/kg (SEM=294 microg ATNC/kg) following PM (P<0.001) compared to the no meat diet (283 microg ATNC/kg, SEM=74 microg ATNC/kg). ATNC concentrations in the ileal output were equivalent to those measured in faeces in similarly designed feeding studies. Supplementation with either 1 g ascorbic acid or 400 IU alpha-tocopherol had no effect on the concentration of ATNC detected in the ileal output. In in vitro experiments, N-nitrosomorpholine (NMor) was formed in the presence of nitrosated haemoglobin, at pH 6.8 but not in the absence of nitrosated haemoglobin. These findings demonstrate that haem may facilitate the formation of NOC in the absence of colonic flora in the upper human gastrointestinal tract.

Endogenous versus exogenous exposure to N -nitroso compounds and gastric cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC-EURGAST) study

The risk of gastric cancer (GC) associated with dietary intake of nitrosodimethylamine (NDMA) and endogenous formation of nitroso compounds (NOCs) was investigated in the European Prospective Investigation into Cancer and Nutrition (EPIC). The study included 521,457 individuals and 314 incident cases of GC that had occurred after 6.6 average years of follow-up. An index of endogenous NOC (ENOC) formation was estimated using data of the iron content from meat intake and faecal apparent total NOC formation according to previous published studies. Antibodies to Helicobacter pylori and vitamin C levels were measured in a sub-sample of cases and matched controls included in a nested case-control within the cohort. Exposure to NDMA was < 1 microg on average compared with 93 mug on average from ENOC. There was no association between NDMA intake and GC risk (HR, 1.00; 95% CI, 0.7-1.43). ENOC was significantly associated with non-cardia cancer risk (HR, 1.42; 95% CI, 1.14-1.78 for an increase of 40 microg/day) but not with cardia cancer (HR, 0.96; 95% CI, 0.69-1.33). Although the number of not infected cases is low, our data suggest a possible interaction between ENOC and H.pylori infection (P for interaction = 0.09). Moreover, we observed an interaction between plasma vitamin C and ENOC (P < 0.02). ENOC formation may account for our previously reported association between red and processed meat consumption and gastric cancer risk.

Effect of Additives Containing a Heavy Atom on the Yield of Photooxidation Products of Arylazides

The effect of additives containing a heavy atom on the yield of nitroso and nitro compounds, the photooxidation products of arylazides, was studied. The addition of ethyl bromide to the reaction mixture increases the total yield of photooxidation products and the contribution of nitroso compounds. The assumption was made that the intersystem crossing (ISC) of nitrene from the singlet to the triplet state is the rate-determining step of the reaction, and the ISC of the singlet adduct of nitrene with oxygen is one of the steps of the reaction path leading to the formation of nitroso compounds.

Acid-mediated mutagenicity of tobacco snuff: its possible mechanism

Polar solvent extracts of tobacco snuff under acidic conditions were mutagenic in Salmonella typhimirium. Using the Griess reagent test, nitrite ranging from ∼ 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 (1) reduction of nitrate in snuff to nitrite by bacteria, and (2) 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.

Formation of DNA-damaging nitroso compounds by interaction of drugs with nitrite. A preliminary screening for detecting potentially hazardous drugs.

Fifty-seven theoretically nitrosatable widely used drugs that are commonly administered orally have been screened to determine the formation of nitroso compounds by drug-nitrite interaction and to evaluate the genotoxicity of their nitrosation products against Chinese hamster ovary (CHO) cells, measured as DNA-damaging potency by the alkaline elution technique. The drug (0.1 mmol) was reacted with NaNO2 (0.4 mmol) at pH 3-3.5 for 1 h. Nitroso compounds were present in varying yield in the nitrosation mixture of 47 drugs. Twenty-two drugs formed direct-acting nitroso compounds capable of producing DNA fragmentation, i.e., a statistically significant (p less than 0.01) increase in the elution rate of CHO cell DNA. On a molar basis, their DNA-damaging potency varied over a 570-fold range, with 12 exhibiting greater potency than that of N-nitroso-N-methylurea.

Formation of nitroso compounds and mutagens from cinnarizine, ethambutol, piromidic acid, pyridinol carbamate and tiaramide by drug/nitrite interaction

The formation of nitroso compounds and mutagens from 5 nitrogen-containing drugs (cinnarizine, ethambutol, piromidic acid, pyridinol carbamate and tiaramide) by drug/nitrite interaction was examined. On the reaction of 50 mM drug and 500 mM nitrite at pH 3.0–3.4 and 37°C for 4 h, the considerable formation of nitroso compounds was observed for ethambutol, cinnarizine and pyridinol carbamate. The reaction product of pyridinol carbamate and nitrite was remarkably mutagenic to Salmonella typhimurium TA100 in the absence of S-9 mix. Under conditions presumed to resemble those in the stomach after ingestion of a therapeutic dose of the drug, the formation of nitroso compounds was observed for ethambutol and significant mutagenicity was detected in the reaction product of pyridinol carbamate.