Nitrite nitrogen stress disrupts the intestine bacterial community by altering host-community interactions in shrimp.

Environmental stress can disrupt the intricate interactions between the host and intestine microbiota, thereby impacting the host health. In this study, we aimed to elucidate the dynamic changes in the bacterial community within shrimp intestines under nitrite nitrogen (nitrite-N) stress and investigate potential host-related factors influencing these changes. Our results revealed a significant reduction in community diversity within the intestine exposed to nitrite-N compared to control conditions. Furthermore, distinct differences in community structures were observed between these two groups at 72 h and 120 h post-stress induction. Nitrite-N stress also altered the abundances of some bacterial species in the intestine dramatically. It is noteworthy that, in comparison to the 72 h, intestine bacterial community structure of stressed shrimp exhibited a significantly higher degree of dispersion after 120 h of nitrite-N stress when compared to control shrimp, and the relative abundance of numerous bacterial species experienced a substantial decrease or even reached 0 %. Moreover, it led to a reduction in bacterial community interactions and decreased competitiveness within the intestine microbiota. Notably, the influence of bacterial community assemblies in the shrimp intestine shifted from a stochastic process to a deterministic one after 24 h and 72 h of nitrite-N stress, returning to a stochastic process at 120 h. We further observed a close association between this phenomenon and host's response to nitrite-N stress. Expression levels of differentially expressed genes in the intestinal tissue significantly impact the intestine bacterial diversity and abundance of species. In particular, the significant decline in bacterial diversity and abundances of quite a few species in intestine was attributed to the up-regulation of peritrophin-48-like. Overall, nitrite-N stress indeed disrupted the intestine microbiota and changed the host-microbiota interactions of shrimp. This study offered novel insights into environment-host-microbiota interactions and also provided practical guidance for promoting healthy shrimp cultivation practices.

Metabolomics revealed more deleterious toxicity induced by the combined exposure of ammonia and nitrite on Ruditapes philippinarum compared to single exposure.

NH3-N and NO2-N always co-exist in the aquatic environment, but there is not a clear opinion on their joint toxicities to the molluscs. Presently, clams Ruditapes philippinarum were challenged by environmental concentrations of NH3-N and NO2-N, singly or in combination, and analyzed by metabolomics approaches, enzyme assays and transmission electron microscope (TEM) observation. Results showed that some same KEGG pathways with different enriched-metabolites were detected in the three exposed groups within one day, and completely different profiles of metabolites were found in the rest of the exposure period. The combined exposure induced heavier and more lasting toxicities to the clams compared with their single exposure. ACP activity and the number of secondary lysosomes were significantly increased after the combined exposure. The present study shed light on the joint-toxicity mechanism of NH3-N and NO2-N, and provided fundamental data for the toxicity research on inorganic nitrogen.

mARC dependent NO synthesis activates CanA-Relish-AMPs signal pathway in Eriocheir sinensis during nitrite stress.

As a signal molecule, nitric oxide (NO) can induce the production of antimicrobial peptides (AMPs) in invertebrate innate immunity and is produced through NO synthase (NOS) oxidation or nitrite reduction. Although the role of NOS-derived NO has been extensively studied, studies on nitrite-dependent NO are relatively scarce. In this study, we identified a mitochondrial amidoxime reducing component (mARC), a kind of nitrite reductase, in Eriocheir sinensis. Under nitrite stress, the expression level of EsmARC in the intestine of E. sinensis increased, and the production of NO increased. Furthermore, EsmARC knockdown resulted in a remarkable decrease in NO concentration. These findings indicate that nitrite stress induces the expression of mARC, which promotes the production of NO in E. sinensis. In addition, the expression levels of AMPs in the intestine were upregulated under nitrite stress. Moreover, EsmARC knockdown resulted in the downregulated expression of AMPs. EsmARC plays a positive role in the synthesis of AMPs under nitrite stress. Calcineurin subunit A (CanA) is a serine/threonine protein phosphatase involved in the process by which NO regulates the expression of AMPs. EsCanA knockdown significantly inhibited the transcription of EsRelish and the expression of AMPs under nitrite stress, and EsRelish silencing resulted in the downregulated expression levels of AMPs under nitrite stress. These results indicate that nitrite stress activates the CanA-Relish-AMP pathway in E. sinensis. In summary, mARC-dependent NO synthesis activates the CanA-Relish-AMP signal pathway in E. sinensis during nitrite stress. This research provides novel insights into the relationship between nitrite stress and NO-dependent immune signal activation in crustaceans.

Duox mediated ROS production inhibited WSSV replication in Eriocheir sinensis under short-term nitrite stress.

Nitrite stress and white spot syndrome virus (WSSV) infection are major problems threatening the sustainable and healthy development of Eriocheir sinensis. Some studies have found that nitrite stress can lead to the production of reactive oxygen species (ROS), whereas synthetic ROS plays a vital role in the signaling pathway. However, whether nitrite stress influences the infection of crabs by WSSV remains unclear. NADPH oxidases, including NOX1-5 and Duox1-2, are important for ROS production. In the present study, a novel Duox gene (designated as EsDuox) was identified from E. sinensis. The studies found that nitrite stress could increase the expression of EsDuox during WSSV infection and decrease the transcription of the WSSV envelope protein VP28. Moreover, nitrite stress could increase the production of ROS, and the synthesis of ROS relied on EsDuox. These results indicated a potential "nitrite stress-Duox activation-ROS production" pathway that plays a negative role in WSSV infection in E. sinensis. Further studies found that nitrite stress and EsDuox could promote the expression of EsDorsal transcriptional factor and antimicrobial peptides (AMPs) during WSSV infection. Moreover, the synthesis of AMPs was positively regulated by EsDorsal in the process of WSSV infection under nitrite stress. Furthermore, EsDorsal played an inhibitory role in the replication of WSSV under nitrite stress. Our study reveals a new pathway for "nitrite stress-Duox activation-ROS production-Dorsal activation-AMP synthesis" that is involved in the defense against WSSV infection in E. sinensis during short-term nitrite stress.

scRNA-seq analysis reveals toxicity mechanisms in shrimp hemocytes subjected to nitrite stress.

In shrimp, hemocytes play an important role in detoxification and immune defense, and are where nitrite accumulates during exposure to this toxic environmental pollutant. However, the heterogeneity mechanisms of toxicity have not been reported under nitrite expose in shrimp. Here, we used single-cell RNA-seq to resolve 24,000 cells, which the responses of different cell populations of hemocytes under nitrite exposure in Penaeus vannamei. We identified 394 specific nitrite-responsive genes in 9 clusters of hemocytes, and found heterogeneity in the nitrite response of the three subpopulations of hemocytes (hyaline, semi-granular and granular cells). In hyaline, the response appeared modest, whereas nitrite-related dysregulation of metabolic processes in granular and semi-granular was pronounced. Ammonia nitrogen will rapidly accumulate in hemocytes of shrimp under nitrite stress. In semi-granular, excessive ammonia will interfere with oxidative phosphorylation and antioxidant system, thus inducing the production of reactive oxygen species. In granular, the abnormality of urea cycle caused by ammonia accumulation is the main toxic factor, which by inhibits arginase and arginine kinase. Collectively, our data provide a single-cell atlas for the dissection of shrimp hemocyte complexity, and reveal the toxicity mechanisms associated with nitrite exposure.

Performance of Daphnia simultaneously exposed to nitrite and predation risk: Reduced nitrite tolerance and aggravated predation-induced miniaturization.

Nitrogenous pollutants derived from human activities not only pose direct risk on aquatic organisms but may also indirectly endanger the stability of interspecific relations. To date, the effects of the nitrogen-containing pollutants on the induced defense remain unclear. Here, we aim to investigate the induced defense of an aquatic keystone species, Daphnia pulex, which responds to predation risk under nitrite pollution at environmentally relevant concentrations and simultaneously evaluate the effects of their induced defenses on nitrite tolerance. Results showed that increasing nitrite significantly reduced the survival time of D. pulex and posed severe reproductive toxicity, consequently reducing the offspring and broods. In the morphological defensive responses, early nitrite exposure interfered with the spine elongation, but the relative spine length induced by the predation risk was unaffected by the nitrite concentrations with exposure time prolonged, although high-dose nitrite inhibited the spine elongation and the increase of the body size. The integration of biomarker response index analyses further indicated that the reproductive capacity was more seriously impaired than the morphology and the survival. Moreover, the sensitivity analyses of growth and reproduction indicated that predation risk significantly reduced Daphnia's tolerance to nitrite. Conclusively, these findings highlight that long-term nitrite exposure exacerbates the predator-induced miniaturization of zooplanktons, and predation risk also reduces their tolerance to nitrite, which provides new insights into the performance changes of zooplanktons exposed to pollutants under predation risk and the vulnerability of predator-prey interspecific relationships in polluted environments.

Impact of nitrite exposure on oxidative stress and antioxidative-related genes responses in the gills of Procambarus clarkii.

Nitrite is the major environmental pollutant in the freshwater aquaculture environment, which has a negative impact on aquatic species growth. Currently, we know that the main way nitrite enters crustaceans is through their gills. In this study, a total of 96 h acute nitrite stress (60 mg/L) experiments were conducted, and the impact of the serum biochemical parameters, gill oxidase activity and oxidative-related gene expression of red swamp crayfish were evaluated. After exposure to nitrite for 0, 6, 12, 24, 48, and 96 h, hemolymph and gills samples were taken at each time point. In the serum, acute nitrite stress significantly increased glutamic-oxaloacetic transaminase (GOT) and alanine aminotransferase (ALT) activities after 6 h of exposure, decreased total protein (TP) and albumin (ALB) levels after 24 h and 48 h of exposure, respectively. In the gills, the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were enhanced to the maximum level at 12 h, 24 h and 24 h, respectively. The contents of malondialdehyde (MDA) and lipid peroxide (LPO) were increased significantly after 12 h and 24 h exposure, respectively. In addition, the expression levels of antioxidative-related genes, including hsp70, fer and mt, were significantly upregulated in the gills after 6 h of exposure. The results indicated that acute nitrite stress changed the serum physiological status, induced oxidative stress and caused damage to gill cells in P. clarkii.

Effect of sub-lethal ammonia and nitrite stress on autophagy and apoptosis in hepatopancreas of Pacific whiteleg shrimp Litopenaeusvannamei.

Oxidative stress caused by ammonia and nitrite, affect the health and growth of aquaculture animals, results in oxidative damages. However, the toxic mechanism and pathogenesis of ammonia and nitrite to aquatic invertebrates are not completely clear. The present study was conducted to investigate the effects of sub-lethal ammonia and nitrite on autophagy and apoptosis in hepatopancreas of Pacific whiteleg shrimp Litopenaeus vannamei. Shrimps were exposed to sub-lethal ammonia (20 mg/L) and nitrite (20 mg/L) for 72 h, respectively. Hepatopancreas was collected for investigating the autophagy and apoptosis under stress conditions. The results showed that ammonia stress could induce up-regulated of autophagy (ATG3, ATG4, ATG10 and ATG12) and apoptosis (Caspase3 and P53) genes transcription. Nitrite stress could also induce up-regulated of autophagy (ATG3, ATG4, ATG5 and ATG10) and apoptosis (Caspase3) genes transcription. The expression of the autophagy related genes increased at first and then decreased with increasing exposure time. The atrophy, lysis, vacuolation of cell and other tissue damages in hepatopancreas were observed after 72h exposure to ammonia and nitrite. The results indicated that ammonia and nitrite stress could induce autophagy and apoptosis, and results in oxidative damage.

Evaluation of Sublethal Toxicity of Nitrite to a Suite of Aquatic Organisms in Support of the Derivation of a Chronic Environmental Water Quality Benchmark.

Nitrite is a naturally-occurring inorganic compound that occurs in aquatic environments as an intermediary between nitrate and ammonia in the nitrogen cycle. It is a contaminant of potential concern resulting from anthropogenic activities in some cases. While the acute toxicity of nitrite has been characterized in previous studies, its sublethal toxicity is less understood. To determine the sublethal toxicity of nitrite on freshwater organisms, a suite of organisms was tested including: two salmonids (Oncorhynchus mykiss and O. kisutch), an alga (Pseudokirchneriella subcapitata), an aquatic macrophyte (Lemna minor), and three invertebrates (Ceriodaphnia dubia, Chironomus dilutus, and Neocloeon triangulifer). Test organisms were exposed to nitrite concentrations ranging between 0.02 and 1.28 mg/L nitrite (NO2-N). The toxicity tests were conducted according to procedures specified in standardized methods, allowing for the estimation of multiple endpoints for each test species. Species sensitivity distributions (SSDs) were generated using endpoints from the toxicity testing results, as well as data from previous studies, from which water chemistry approximated that used in the tests (i.e., < 5 mg/L chloride, an important toxicity-modifying factor for nitrite). The mayfly, N. triangulifer, was the most sensitive species, followed by the two salmonids (which represented the second and third most sensitive species), although they were not as sensitive to nitrite exposure as reported in previous studies. The fifth percentile hazard concentration (HC5) generated from the SSD could be used for derivation of regulatory benchmarks and threshold values for site-specific aquatic risk assessments.

Intestinal changes associated with nitrite exposure in Bufo gargarizans larvae: Histological damage, immune response, and microbiota dysbiosis.

Nitrite is a ubiquitous toxic compound in aquatic ecosystems and has negative effects on aquatic organisms. The intestine and the trillions of microbes that inhabit it, play an integral role in maintaining digestive and immune functions. However, the effects of nitrite on intestinal health and microflora have been poorly investigated. Therefore, the present study evaluated the response of intestinal histology, immunity, digestive enzyme activities and microbiota to nitrite exposure in Bufo gargarizans tadpoles. The results showed that nitrite caused damage to the intestine and impaired digestive performance. Significant changes in the transcriptional profiles of genes involved in oxidative stress (sod, gpx and hsp), inflammation, and immunity (socs3, il-27, il-1ß and il-17d) were observed in the NO2-N treatment groups. In addition, exposure to nitrite induced alterations of intestinal microbial diversity, structure and composition, suggesting that nitrite may lead to intestinal microbiota dysbiosis. It is noteworthy that probiotics (e.g., Bacteroidetes and Fusobacteria) were decreased after exposure to nitrite, whereas potentially opportunistic pathogens such as Proteobacteria and Enterobacteriaceae were elevated. Functional prediction and correlation analysis suggested that the above changes may interfere with metabolic function and trigger various diseases. Taken together, we concluded that nitrite exposure induced intestinal microbial dysbiosis, which may lead to immune dysfunction and metabolic disorder, and ultimately to histological damages in B. gargarizans. Further, this study will provide a scientific basis for further understanding the risk of nitrite pollution on the intestinal health of amphibians.

Nitrate and nitrite contamination in drinking water and cancer risk: A systematic review with meta-analysis.

BACKGROUND: Pollution of water sources, largely from wide-scale agricultural fertilizer use has resulted in nitrate and nitrite contamination of drinking water. The effects on human health of raised nitrate and nitrite levels in drinking water are currently unclear. OBJECTIVES: We conducted a systematic review of peer-reviewed literature on the association of nitrate and nitrite in drinking water with human health with a specific focus on cancer. METHODS: We searched eight databases from 1 January 1990 until 28 February 2021. Meta-analyses were conducted when studies had the same exposure metric and outcome. RESULTS: Of 9835 studies identified in the literature search, we found 111 studies reporting health outcomes, 60 of which reported cancer outcomes (38 case-control studies; 12 cohort studies; 10 other study designs). Most studies were set in the USA (24), Europe (20) and Taiwan (14), with only 3 studies from low and middle-income countries. Nitrate exposure in water (59 studies) was more commonly investigated than nitrite exposure (4 studies). Colorectal (15 studies) and gastric (13 studies) cancers were the most reported. In meta-analyses (4 studies) we identified a positive association of nitrate exposure with gastric cancer, OR = 1.91 (95%CI = 1.09-3.33) per 10 mg/L increment in nitrate ion. We found no association of nitrate exposure with colorectal cancer (10 studies; OR = 1.02 [95%CI = 0.96-1.08]) or cancers at any other site. CONCLUSIONS: We identified an association of nitrate in drinking water with gastric cancer but with no other cancer site. There is currently a paucity of robust studies from settings with high levels nitrate pollution in drinking water. Research into this area will be valuable to ascertain the true health burden of nitrate contamination of water and the need for public policies to protect human health.

Acute Nitrite Exposure Induces Dysfunction and Oxidative Damage in Grass Carp Isolated Hemocytes.

To evaluate the effects of nitrite on the oxidative damage of blood cells of Grass Carp Ctenopharyngodon idella, the isolated hemocytes were exposed to nitrite (0, 1, 10, or 100 mg/L) for up to 24 h. Hemoglobin (Hb) and methemoglobin (MetHb) concentrations, reactive oxygen species (ROS) and malondialdehyde (MDA) levels, mitochondrial membrane potential (∆Ψm), and antioxidant enzyme activity were assayed to assess hematological parameters and the antioxidant defense mechanism. Results showed a remarkable decrease in Hb concentration with increasing nitrite concentration after a 24-h exposure, while the MetHb concentration increased significantly in nitrite exposure groups. The levels of ROS, ∆Ψm, and MDA increased to varying degrees with increases in nitrite exposure concentration and time. The total antioxidant capacity, catalase (CAT) activity, glutathione peroxidase (GPx) activity, and glutathione content showed a trend of rising initially and then decreasing with prolonged exposure time. Superoxide dismutase (SOD) activity was higher in the 1-mg/L nitrite exposure group and lower in the 100-mg/L group than in the control. The relative messenger RNA expression ratios of cat, sod1, and gpx were up-regulated significantly in the 1- and 10-mg/L groups and then declined in the 100-mg/L group. Therefore, it can be concluded that nitrite exposure activates the antioxidant defense mechanism of Grass Carp hemocytes and that the balance of oxidant-antioxidant homeostasis will be undermined by higher nitrite doses or longer exposure periods.

Gut microbiota mediated the toxicity of high concentration of dietary nitrite in C57BL/6 mice.

Growing evidence indicates that exposure to high levels of nitrite for a prolonged time has adverse health effects. Although gut microbiota is responsible for the transformation of nitrite in the gut, the evidence concerning whether gut microbiota mediates the toxicity of nitrite is still lacking. The present study addressed the long-term effects of dietary nitrite on male C57BL/6 mice and employed fecal microbiota transplantation (FMT) to reveal whether gut microbiota mediated the effects of nitrite. Furthermore, the effect of azoxymethane (AOM) on gut microbiota was detected for mice drinking normal or nitrite-containing water. High nitrite had toxic effects on C57BL/6 mice. Meanwhile, high nitrite induced skin lesions in mice, accompanied with increased serum ALT, colon IL-6, TNF-α, and MDA levels, together with decreased serum Cr, colon sIgA, and T-AOC levels. After fecal microbiota was transplanted into the normal mice, the nitrite-regulated gut microbiota could also induce skin lesions, coupled with reduced serum Cr, and increased colon MDA. The high dose of nitrite caused the upregulations of Alistipes, Prevotella, and Ruminococcus, which could be transplanted into normal mice through FMT. Inversely, gut microbiota from normal mice reduced the effects of nitrite on serum ALT and Cr, together with colon sIgA and MDA. Gut microbiota from normal mice could also upregulate metabolic genes and downregulate stress genes in the nitrite-treated mice. It might due to the upregulation of Akkermansia and Parabacteroides caused by FMT from normal water-treated mice to nitrite-treated mice. In addition, AOM exhibited to be more toxic to the colon in the nitrite-treated mice in comparison with normal water-treated mice, and it might be due to the expression of Hspa1a and Hspa1b in the colon. Interestingly, gut microbiota was more influenced by AOM in the normal water-treated mice than the nitrite-treated mice. Overall, these data demonstrated that gut microbiota mediated the toxicity of a high concentration of dietary nitrite.

Transcriptomic analysis of gills in nitrite-tolerant and -sensitive families of Litopenaeus vannamei.

Nitrite stress is a major environmental factor that limits aquatic animal growth, reproduction and survival. Even so, some shrimps still can withstand somewhat high concentrations of nitrite environment. However, few studies have been conducted about the tolerance molecular mechanism of Litopenaeus vannamei in the high concentration nitrite. To identify the genes and pathways involved in the regulation of nitrite tolerance, we performed comparative transcriptomic analysis in the L. vannamei nitrite-tolerant (NT) and nitrite-sensitive (NS) families, and untreated shrimps were used as the control group. After 24 h of nitrite exposure (NaNO2, 112.5 mg/L), a total of 1521 and 868 differentially expressed genes (DEGs) were obtained from NT compared with NS and control group, respectively. Functional enrichment analysis revealed that most of these DEGs were involved in immune defense, energy metabolism processes and endoplasmic reticulum (ER) stress. During nitrite stress, energy metabolism in NT was significantly enhanced by activating the related genes expression of oxidative phosphorylation (OXPHOS) pathway and tricarboxylic acid (TCA) cycle. Meanwhile, some DEGs involved in innate immunity- related genes and pathways, and ER stress responses also were highly expressed in NT. Therefore, we speculate that accelerated energy metabolism, higher expression of immunity and ER related genes might be the important adaptive strategies for NT in relative to NS under nitrite stress. These results will provide new insights on the potential tolerant molecular mechanisms and the breeding of new varieties of nitrite tolerant L. vannamei.

Neurotoxicity induced by combined exposure of microcystin-LR and nitrite in male zebrafish (Danio rerio): Effects of oxidant-antioxidant system and neurotransmitter system.

With the intensification of water eutrophication around the world, cyanobacterial blooms have been becoming a common environmental pollution problem. The levels of microcystin-LR (MC-LR) and nitrite rise sharply during the cyanobacterial bloom period, which may have potential joint toxicity on aquatic organisms. In this study, adult male zebrafish were immersed into different joint solutions of MC-LR (0, 3, 30 µg/L) and nitrite (0, 2, 20 mg/L) for 30 days to explore the neurotoxic effects and underlying mechanisms. The results showed that single factor MC-LR or nitrite caused a concentration-dependent damage in brain ultrastructure and the effects of their joint exposure were much more intense. Downregulated expression of mbp and bdnf associated with myelination of nerve fibers further confirmed that MC-LR and nitrite could damage the structure and function of neuron. The decreases in dopamine content, acetylcholinesterase activity and related gene mRNA levels indicated that MC-LR and nitrite adversely affected the normal function of the dopaminergic and cholinergic systems in zebrafish brain. In addition, the significant increase in malondialdehyde content suggested the occurrence of oxidative stress caused by MC-LR, nitrite and their joint-exposure, which paralleled a significant decrease in antioxidant enzyme­manganese superoxide dismutase activity and its transcription level. In conclusion, MC-LR + Nitrite joint-exposure has synergistic neurotoxic effects on the structure and neurotransmitter systems of fish brain, and antioxidant capacity disruption caused by these two factors might be one of the underlying synergistic mechanisms. Therefore, there is a risk of being induced neurotoxicity in fish during sustained cyanobacterial bloom events.

Use of physiological activities to estimate the population growth of rotifer (Brachionus calyciflorus) under the stress of toxic Microcystis and nitrite.

Microcystis blooms disrupt aquatic systems and adversely affect zooplankton growth. Brachionus calyciflorus Pallas (rotifer) was introduced to different combinations of toxic Microcystis aeruginosa (0, 2 × 105, 2 × 106, and 2 × 107 cells mL-1) and nitrite (0, 2, 4, and 6 mg L-1) to evaluate their physiological activities and population growth under stress. Survival rate (S), population growth rate (r), grazing rate (G), antioxidant response, and metabolic and digestive enzyme activities were determined. Results revealed that G declined with the increasing nitrite doses and grazing time upon exposure to a certain Microcystis concentration. Toxic M. aeruginosa and nitrite inhibited the S, r, glutathione content, total antioxidant capacity level, and activities of alkaline phosphatase, xanthine oxidase, lactate dehydrogenase, and cellulase (p < 0.05) but increased the reactive oxygen species level, malondialdehyde content, and amylase activity (p < 0.05). The activities of superoxide dismutase, catalase, and pepsase were also increased in single low doses of nitrite solutions (p < 0.05). Therefore, the grazing intensity of rotifers affected B. calyciflorus physiological activities, which are useful in the estimation of its population growth in eutrophic water environments.

Whole body metabolism is improved by hemin added to high fat diet while counteracted by nitrite: a mouse model of processed meat consumption.

Nitrites and nitrates are traditional food additives used as curing agents in the food industry. They inhibit the growth of microorganisms and give a typical pink color to meat. Besides the positive effects of nitrite in foods, if present at high levels in the body, may induce hypoxia and contribute to the production of pro-carcinogenic secondary N-nitrosamines. This study investigated the whole-body metabolic effects of hemin and nitrite added to a high fat diet as red and processed red meat nutritional models. Mice were fed for 11 weeks with five different diets-(1) control diet (ND), (2) high fat diet (HFD) with 60% fat, (3) HFD with hemin (HFD + H, red meat model), (4) HFD with hemin and nitrite (HFD + HN, processed meat model), and (5) HFD with hemin, nitrite, and secondary amine (HFD + HNN, N-nitrosamine generating model)-and several metabolic parameters were determined and respiratory measurements were performed. Mice fed with the HFD + H or HFD + HNN diet had a lower epididymal white adipose tissue (eWAT) : body ratio and lower fasting glucose level than those fed the HFD alone. In addition, our results demonstrated a relief in hepatosteatosis grade among the HFD + H and HFD + HNN diet fed mice. Nitrite added to the HFD impaired the ability to use fat for energy, opposite to the effect of hemin. This study shows that nitrite in addition to pro-carcinogenesis and hypoxia can impact metabolic disease progression when added to meat.

Caenorhabditis elegans to Model the Capacity of Ascorbic Acid to Reduce Acute Nitrite Toxicity under Different Feed Conditions: Multivariate Analytics on Behavioral Imaging.

Nitrocompounds are present in the environment and human diet and form part of vegetables and processed meat products as additives. These compounds are related to negative impacts on human and animal health. The protective effect of ascorbic acid has been demonstrated by some biological systems as regards several nitrocompounds. This work focused on studying the possibility of modeling this effect on nitrite toxicity with the model Caenorhabditis elegans. The three factors studied in this work were ascorbic acid concentration, nitrite exposure concentration, and presence/absence of food. The protective effect was evaluated by scoring lethality and its impact on behavior by means of multivariate statistical methods and imaging analytics. The effects of nitrite and the influence of food availability were evidenced. Apart from increasing lethality, nitrite had disruption effects on movements. All the observed symptoms reduced when ascorbic acid was administered, and it diminished lethality in all cases. Ascorbic acid maintained nematodes' postural capacities. The results suggest that nitrites' nonspecific toxicity in C. elegans can be mitigated by ascorbic acid, as previously evidenced in other biological systems. Thus, our results reveal the ability of C. elegans to reproduce the known protective effect of ascorbic acid against nitrite.

S-Nitroso-N-acetylcysteine (NAC-SNO) vs. nitrite as an anti-clostridial additive for meat products.

Nitrite is added to meat products as a preservative and it acts as a bacteriostatic compound against Clostridium botulinum growth. Nitric-oxide (˙NO), myoglobin and S-nitroso-compounds seem to be the main molecules generated from nitrite in meat products, which by decomposition to ˙NO, form the main anti-clostridial factor. The growth of C. sporogenes from activated spores in the presence of 0.5-2.5 mM NAC-SNO was compared to nitrite, both at 37 °C for 5 days and at room temperature for 28 days. The present study demonstrates that NAC-SNO under the same conditions and concentrations, in meat products, acts as an anti-clostridial compound similar to nitrite. In contrast to nitrite which must be activated in meat by heating, NAC-SNO generates the anti-clostridial factor directly, without heating, as was evaluated in an unheated bacteriological medium. The toxic effect of NAC-SNO and nitrite in methaemoglobinaemia and generation of N-nitrosamines in vivo, in mice, were also determined. Mice were gavage fed milk containing 45 mg per kg per bw of nitrite or an equimolar equivalent of NAC-SNO in the presence of 50 mg per kg per bw of N-methylaniline. Nitrite generated methaemoglobinaemia and carcinogenic N-nitrosoamines (N-nitrosomethylaniline); however, NAC-SNO under the same conditions and concentrations generates much less methaemoglobin and no detectable N-nitrosoamines in the blood, in vivo.

Molecular characterization and expression dynamics of three key genes in the PI3K-AKT pathway reveal its involvement in the immunotoxicological responses of the giant river prawn Macrobrachium rosenbergii to acute ammonia and nitrite stress.

Ammonia nitrogen and nitrite are two common forms of environmental toxicants for aquatic organisms including crustaceans. The PI3K-AKT pathway is an important intracellular signaling pathway related to cellular stress response, but involvement of this pathway in the immunotoxicological response of decapod crustaceans to aquatic toxicants such as ammonia nitrogen and nitrite still remains enigmatic. In this study, based on transcriptome mining and molecular cloning techniques, three key genes (named as MrPI3K, MrAKT and MrFoxO) in the PI3K-AKT signaling pathway were identified from the giant river prawn Macrobrachium rosenbergii. Sequence homology and phylogenetic analysis revealed that all the three genes harbored signature sequences of corresponding protein families, and shared high levels of similarities with their respective homologs from other species. MrPI3K, MrAKT and MrFoxO all displayed ubiquitous tissue distribution profiles, but their expression levels varied to a great extend among different tissues and between sexes. Following exposure to nitrite (20 mg/L nitrite-N) or ammonia (25 mg/L total ammonia-N) stresses for 24 h and 48 h, the three genes all responded by altering their expression levels at different time points, but they didn't show uniform expression patterns following these stresses, indicating the diversified roles of these genes in different tissues and the complexity of this signaling pathway. Remarkably, MrPI3K and MrAKT were induced only in the hemocytes and intestine, respectively, indicating their specific roles in these organs. Our study demonstrated the potential utility of these genes as biomarkers of acute ammonia or nitrite toxicity in prawns, and also provided evidence that the PI3K-AKT pathway is involved in the immunotoxicological responses to nitrite and ammonia stress in M. rosenbergii.