The chemical characteristics of different sodium iron ethylenediaminetetraacetate sources and their relative bioavailabilities for broilers fed with a conventional corn-soybean meal diet.

BACKGROUND: Our previous studies demonstrated that divalent organic iron (Fe) proteinate sources with higher complexation or chelation strengths as expressed by the greater quotient of formation (Qf) values displayed higher Fe bioavailabilities for broilers. Sodium iron ethylenediaminetetraacetate (NaFeEDTA) is a trivalent organic Fe source with the strongest chelating ligand EDTA. However, the bioavailability of Fe when administered as NaFeEDTA in broilers and other agricultural animals remains untested. Herein, the chemical characteristics of 12 NaFeEDTA products were determined. Of these, one feed grade NaFeEDTA (Qf = 2.07 × 108), one food grade NaFeEDTA (Qf = 3.31 × 108), and one Fe proteinate with an extremely strong chelation strength (Fe-Prot ES, Qf value = 8,590) were selected. Their bioavailabilities relative to Fe sulfate (FeSO4·7H2O) for broilers fed with a conventional corn-soybean meal diet were evaluated during d 1 to 21 by investigating the effects of the above Fe sources and added Fe levels on the growth performance, hematological indices, Fe contents, activities and gene expressions of Fe-containing enzymes in various tissues of broilers. RESULTS: NaFeEDTA sources varied greatly in their chemical characteristics. Plasma Fe concentration (PI), transferrin saturation (TS), liver Fe content, succinate dehydrogenase (SDH) activities in liver, heart, and kidney, catalase (CAT) activity in liver, and SDH mRNA expressions in liver and kidney increased linearly (P < 0.05) with increasing levels of Fe supplementation. However, differences among Fe sources were detected (P < 0.05) only for PI, liver Fe content, CAT activity in liver, SDH activities in heart and kidney, and SDH mRNA expressions in liver and kidney. Based on slope ratios from multiple linear regressions of the above indices on daily dietary analyzed Fe intake, the average bioavailabilities of Fe-Prot ES, feed grade NaFeEDTA, and food grade NaFeEDTA relative to the inorganic FeSO4·7H2O (100%) for broilers were 139%, 155%, and 166%, respectively. CONCLUSIONS: The bioavailabilities of organic Fe sources relative to FeSO4·7H2O were closely related to their Qf values, and NaFeEDTA sources with higher Qf values showed higher Fe bioavailabilities for broilers fed with a conventional corn-soybean meal diet.

Polystyrene microplastics with different sizes induce the apoptosis and necroptosis in liver through the PTEN/PI3K/AKT/autophagy axis.

The production of plastics worldwide has been instrumental in the progress of modern society, while the increasing accumulation of plastics castoff in oceans, soils and anywhere else has become a major pressure source on environmental sustainability and animal health. Meanwhile, from a biological perspective, our understanding of the toxicological fingerprints of plastics, especially microplastics (MPs), is still poor. Here, we reported a phenomenon of hepatotoxicity dominated by MPs in the form of polystyrene (PS), was observed in mice model systems and cellular assays. Apoptosis and necroptosis related to the size of particles were seen upon PS-MPs introduction, as revealed by transmission electron microscopy, fluorescence microscopy, flow cytometry, and quantitative analysis of signaling pathways in vivo and vitro. Collectively, the current study demonstrated that the levels of liver cell injury caused by PS-MPs were negatively correlated with the particle diameters. Small-sized particles (1-10 µm) induced cell death primarily as necroptosis whereas the large-sized particles (50-100 µm) mainly induced apoptosis, which was directly accomplished by PTEN/PI3K/AKT signaling axis and its targeted autophagy flux. More interestingly, inhibition of autophagy not only alleviated PS-MPs-triggered cell death, but also changed the form of death injury to a certain extent. This uncovered crosstalk relationship opens up a new avenue for investigating the biological and toxicological effects of MPs, and may provide important insights for preventing and limiting of health hazards from MPs.

TBBPA induces inflammation, apoptosis, and necrosis of skeletal muscle in mice through the ROS/Nrf2/TNF-α signaling pathway.

Tetrabromobisphenol A (TBBPA) is present in large quantities in the environment due to its widespread use. And TBBPA is capable of accumulating in animals, entering the ecological chain and causing widespread damage to organisms. TBBPA is capable of causing the onset of oxidative stress, which induces tissue damage and cell death, which in turn affects the physiological function of tissues. Skeletal muscle is a critical tissue for maintaining growth, movement, and health in the body. However, the mechanism of TBBPA-induced skeletal muscle injury remains unclear. In this study, we constructed mouse skeletal muscle models (10, 20, and 40 mg/kg TBBPA) and mouse myoblasts (C2C12) cell models (2,4, and 8 µg/L TBBPA) at different concentrations. The results of this experiment showed that under TBBPA treatment, the levels of reactive oxygen species (ROS) and Malondialdehyde (MDA) in mouse skeletal and C2C12 cells were increased significantly, but the activities of some antioxidant enzymes decreased. TBBPA can inhibit Nuclear factor E2-related factor 2 (Nrf2) entry into the nucleus, thus affecting the expression of the Nrf2 downstream factors. With the increase of TBBPA concentration, the expression levels of inflammatory factors were significantly increased, while the anti-apoptotic factors were significantly decreased. The expression of pro-apoptotic factors increased in a dose-dependent manner. Programmed necrosis-related factors were also significantly elevated. Our results suggest that TBBPA induces oxidative stress and inflammation, apoptosis, and necrosis in the skeletal muscle of mice by regulating Nrf2/ROS/TNF-α signaling pathway.

Profiling Substrate Specificity of the SUMO Protease Ulp1 by the YESS-PSSC System to Advance the Conserved Mechanism for Substrate Cleavage.

SUMO modification is a vital post-translational regulation process in eukaryotes, in which the SUMO protease is responsible for the maturation of the SUMO precursor and the deconjugation of the SUMO protein from modified proteins by accurately cleaving behind the C-terminal Gly-Gly motif. To promote the understanding of the high specificity of the SUMO protease against the SUMO protein as well as to clarify whether the conserved Gly-Gly motif is strictly required for the processing of the SUMO precursor, we systematically profiled the specificity of the S. cerevisiae SUMO protease (Ulp1) on Smt3 at the P2-P1↓P1' (Gly-Gly↓Ala) position using the YESS-PSSC system. Our results demonstrated that Ulp1 was able to cleave Gly-Gly↓ motif-mutated substrates, indicating that the diglycine motif is not strictly required for Ulp1 cleavage. A structural-modeling analysis indicated that it is the special tapered active pocket of Ulp1 conferred the selectivity of small residues at the P1-P2 position of Smt3, such as Gly, Ala, Ser and Cys, and only which can smoothly deliver the scissile bond into the active site for cleavage. Meanwhile, the P1' position Ala of Smt3 was found to play a vital role in maintaining Ulp1's precise cleavage after the Gly-Gly motif and replacing Ala with Gly in this position could expand Ulp1 inclusivity against the P1 and P2 position residues of Smt3. All in all, our studies advanced the traditional knowledge of the SUMO protein, which may provide potential directions for the drug discovery of abnormal SUMOylation-related diseases.

Polystyrene microplastics induce apoptosis and necroptosis in swine testis cells via ROS/MAPK/HIF1α pathway.

Microplastics (MPs) generally refer to the plastic fragments or particles smaller than 5 mm in diameter, which are closely concerned due to their widespread presence in the environment. Recent studies have shown that MPs have a serious threat on the reproductive health of organisms. Pigs are often selected as the model animals because of their high similarity to human tissues and organs. However, there are no reports on the effects and mechanisms of MPs exposure on swine germ cells. In the present study, we established swine testis (ST) cell models exposed to 250, 500, and 1000 µg/ml polystyrene microplastics (PS-MPs, 1-10 µm), respectively. The findings revealed that PS-MPs reduced cell viability dose-dependently. Acridine orange/ethidium bromide staining and flow cytometry results indicated the occurrence of apoptosis and necrosis in ST cells under PS-MPs exposure, and the expression changes of relevant marker genes (B-cell lymphoma-2, Bcl-2 Associated X, Caspase-3, Caspase-9, Receptor-interacting protein kinase 1, Receptor-interacting protein kinase 3, Mixed lineage kinase domain-like, and Caspase-8) were clarified via quantitative real-time PCR and western blot. Further mechanistic studies found that PS-MPs treatment induced excessive intracellular reactive oxygen species (ROS) production, which promoted the phosphorylation of mitogen-activated protein kinase (MAPK) pathway-related genes (P38, c-Jun N-terminal kinase, extracellular regulated protein kinases) and activated the downstream gene hypoxia-inducible factor (HIF1α). In conclusion, our study suggests that PS-MPs treatment causes apoptosis and necroptosis in ST cells via ROS/MAPK/HIF1α signaling pathway.

Cadmium induces apoptosis by miR-9-5p targeting PTEN and regulates the PI3K/AKT pathway in the piglet adrenal gland.

Cadmium (Cd) is an environmental pollutant that can cause endocrine organ damage. To explore the effect of subacute CdCl2 exposure on piglet adrenal gland tissue and its mechanism based on the establishment of this model, bioinformatics, TUNEL assay, western blot (WB), and qRT-PCR methods were used to detect related indicators. The results showed that after Cd exposure, antioxidant enzymes decreased, heat shock protein increased, and miR-9-5p-gene of phosphatase and tensin homolog (PTEN) upregulates the phosphatidylinositol-3-kinase (PI3K/AKT) pathway. After this pathway was activated, the expression of the apoptosis-related factors cysteinyl aspartate-specific proteinase 3 and 9 (caspase 3 and 9), B-cell lymphoma-2-associated X (BAX) was increased sharply, and the expression of B-cell lymphoma-2 (BCL2) was significantly decreased. The changes in these indicators indicate that Cd exposure induces apoptosis and causes tissue damage in the adrenal gland of piglets. This study aims to reveal the toxic effects of CdCl2 in animals and will provide new ideas for the toxicology of Cd.

A new discovery of polystyrene microplastics toxicity: The injury difference on bladder epithelium of mice is correlated with the size of exposed particles.

Microplastics (MPs), as widespread hazardous substances in the environment, can cause potential adverse effects on biological health. However, reports on the toxic effects of different diameters MPs on urinary system are limited. Here, we investigated the types and mechanisms of damage to mice bladder epithelial cells treated with diameter (1-10 µm and 50-100 µm) polystyrene microplastics (PS-MPs). The results showed that exposure to PS-MPs of both diameters resulted in necroptosis and inflammation to bladder epithelium. However, 1-10 µm PS-MPs posed more severe necroptosis and 50-100 µm PS-MPs led to a higher degree of inflammatory injury at the same exposure concentration. Mechanistically, PS-MPs were found to induce necroptosis as well as p-NFκB-mediated inflammation by triggering oxidative stress and excessive release of reactive oxygen species (ROS). Furthermore, N-Acetyl-l-cysteine (NAC) attenuated the toxic effects of PS-MPs on bladder epithelial cells. In conclusion, our study demonstrated for the first time that PS-MPs caused necroptosis and inflammation in mice bladders tissues, and the difference of injury correlates with the size of PS-MPs particles.

Exposure to imidacloprid induce oxidative stress, mitochondrial dysfunction, inflammation, apoptosis and mitophagy via NF-kappaB/JNK pathway in grass carp hepatocytes.

Imidacloprid (IMI) is a neonicotinoid compound widely used in agriculture production, causing surface water pollution and threatening non-target organisms. The aim of this study was to analyze the effects of IMI on grass carp (Ctenopharyngodon idellus) liver cell (L8824) injury. The L8824 cells were exposed to different doses of IMI (65 mg/L, 130 mg/L and 260 mg/L) for 24 h. Our results demonstrated that exposure IMI significantly suppressed the activity of anti-oxidant enzymes (SOD, CAT and T-AOC) and accumulated oxidase (MDA) levels, and promoting reactive oxygen species (ROS) generation in L8824 cells. Additionally, mitochondrial membrane potential (ΔΨ m), mitochondria-derived ROS and ATP content and the MitoTracker Green indicated that IMI aggravated mitochondrial dysfunction, thereby inducing inflammation and enhancing pro-inflammatory genes (NF-kappaB, TNFα, IL-1ß and IL-6) expressions. However, the addition of 2 mM N-acetyl-l-cysteine (NAC) can reverse these adverse effects of high-dose IMI- induced. Hence, ROS is the main factor of IMI-induced mitochondrial dysfunction and inflammation. We further found that exposure to IMI induced apoptosis, which is characterized by promoting release of cytochrome c (Cyt-C), and increasing the expression of Bcl-2-Associated X (BAX), cysteinyl aspartate specific proteinases (Caspase 9 and 3), decreasing Bcl-2 level. Immunofluorescent staining, qRT-PCR and Western Blot results indicated that IMI exposure also activated mitophagy, which was demonstrated by the expression of mitophagy-related genes (BNIP3, LC3B and P62). Conversely, scavenging JNK by SP600125(10 µM) alleviated the expression of mitochondrial apoptosis and mitophagy-related gene induced by high-dose IMI. Therefore, these results of study demonstrated that IMI-induced oxidative stress to regulate mitochondrial dysfunction, thus causing inflammation, mitochondrial apoptosis and mitophagy in grass carp hepatocytes through NF-kappaB/JNK pathway.

Quercetin antagonizes imidacloprid-induced mitochondrial apoptosis through PTEN/PI3K/AKT in grass carp hepatocytes.

Imidacloprid (IMI) is widely used in agriculture, and is toxic to non-target aquatic species. Quercetin (Que) is a flavonoid abundant in fruits and vegetables that exhibits anti-oxidant activity. In the present study, we treated grass carp hepatocytes (L8824) with 0.1 µM Que and/or 1 mM IMI for 24 h to explore the effect of Que on IMI-induced mitochondrial apoptosis. We found that IMI exposure enhanced reactive oxygen species (ROS) generation, inhibiting the activities of SOD, CAT and T-AOC, exacerbating the accumulation of MDA, aggravating the expression of mitochondrial apoptosis pathway (Cyt-C, BAX, Caspase9 and Caspase3) related genes and decreased the expression of anti-apoptosis gene B-cell lymphoma-2 (Bcl-2). In addition, Que and IMI co-treatment significantly restored the activity of anti-oxidant enzymes, downregulated ROS level and apoptosis rate, thereby alleviating the depletion of mitochondrial membrane potential (ΔΨm) and the expression of cytochrome c (Cyt-C), Bcl-2-associated X (BAX), and cysteinyl aspartate specific proteinases (Caspase9 and 3), increasing the Bcl-2 level. Furthermore, we elucidated that Que could inhibit the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), thus activating phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway to attenuate IMI-induced apoptosis. Molecular docking provides assertive evidence for the interaction between Que ligand and PTEN receptor. Consequently, these results indicate that Que effectively antagonizes IMI-induced mitochondrial apoptosis in grass carp hepatocytes via regulating the PTEN/PI3K/AKT pathway.

Subacute cadmium exposure promotes M1 macrophage polarization through oxidative stress-evoked inflammatory response and induces porcine adrenal fibrosis.

Cadmium (Cd) is a widely distributed environmental pollutant with immunotoxicity and endocrine toxicity. M1/M2 macrophages participate in the immune response and exert an essential influence on fibrosis. Nevertheless, whether Cd can induce porcineadrenal fibrosis by affecting the polarization of M1/M2 macrophages and its potential regulatory mechanism have not been explored. We added 20 mg/kg CdCl2 to the pig diet for 40 days to investigate the fibrogenic effect of subacute Cd exposure on the adrenal gland. The results indicated that the ACTH and CORT in serum were decreased by 15.26 % and 21.99 %, respectively. The contents of adrenal mineral elements Cd, Cr, Mn were increased up to 34, 1.93, 1.42 folds and Co, Zn, Sn were reduced by 21.57 %, 20.52 %, 15.75 %. Concurrently, the pro-oxidative indicators (LPO, MDA and H2O2) were increased by 1.85, 2.20, 2.77 folds and 3.60, 11.15, 4.11 folds upregulated mRNA levels of TLR4, NF-κB, NLRP3 were observed. Subsequently, the expression of M1 macrophages polarization markers (IL-6, iNOS, TNF-α, CCL2 and CXCL9) were raised by 2.03, 2.30, 2.35, 1.58, 1.56 folds, while M2 macrophages (IL-4, CCL24, Arg1, IL-10, MRC1) showed a 62.34 %, 31.88 %, 50.26 %, 74.00 %, 69.34 % downregulation. The expression levels of AMPK subunits and genes related to glycolysis, oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) were also markedly increased. Additionally, the expression level of TGF-ß1, Smad2/3 and downstream pro-fibrotic markers was obviously upregulated. Taken together, we conclude that Cd activates the oxidative stress-mediated TLR4/NF-κB/NLRP3 inflammatory signal transduction, leading to porcine adrenal fibrosis by promoting macrophage polarization toward M1.

Selenomethionine alleviates LPS-induced JNK/NLRP3 inflammasome-dependent necroptosis by modulating miR-15a and oxidative stress in chicken lungs.

Selenium (Se) was involved in many physiological processes in humans and animals. microRNAs (miRNAs) also played important roles in lung diseases. However, the regulatory mechanism of miRNA in chicken lungs and the mechanism of lipopolysaccharide (LPS)-induced pneumonia remained unclear. To further study these mechanisms, we established a supplement of selenomethionine (SeMet) and/or LPS-treated chicken model and a cell model of LPS and/or high and low expression of miR-15a in chicken hepatocellular carcinoma (LMH) cells. We detected the expression of some selenoproteins, p-c-Jun N-terminal kinase (JNK), nod-like receptor protein 3 (NLRP3), caspase1, receptor-interacting serine-threonine kinase 1 (RIPK1), receptor-interacting serine-threonine kinase 3 (RIPK3), mixed lineage kinase domain-like pseudokinase (MLKL), miR-15a, and oxidative stress kits. Additionally, we observed the morphology of lungs by H.E. staining in vitro. The results indicated that necroptosis occurred in LPS-treated chicken and LMH cells. Moreover, LPS stimulation inhibited miR-15a, and increased the expression of JNK, NLRP3, caspase1, RIPK1, RIPK3, and MLKL. We also found that LPS treatment not only increased the content of H2O2 and MDA in the lungs but also increased the activities of iNOS and CAT and the content of GSH decreased. Conclusion: SeMet could reduce the oxidative damage and activate NLRP3 inflammasome reaction by stimulating miR-15a/JNK, thus reduced the pulmonary necroptosis induced by LPS.

Cadmium exposure induces TNF-α-mediated necroptosis via FPR2/TGF-ß/NF-κB pathway in swine myocardium.

Cadmium (Cd) is one common environmental pollutant with systemic toxicity. Lipoxin A4 (LXA4) can regulate transforming growth factor-ß (TGF-ß) pathway and alleviate tissue injury via binding to formyl peptide receptor 2 (FPR2). The activation of nuclear factor-κB (NF-κB) pathway can promote the occurence of necroptosis. However, whether Cd exposure induces necroptosis in swine myocardium and the role of FPR2/TGF-ß/NF-κB pathway in this process are unclear. Hence, we established Cd-exposed swine myocardial injury model by feeding a CdCl2 added diet (20 mg Cd/kg diet). Hematoxylin-eosin (H&E) staining was used to observe the morphological changes, and inductively coupled plasma mass spectrometry (ICP-MS) was performed to detect the levels of ion elements in myocardium. We further detected LXA4 and its receptor FPR2, TGF-ß, Nrf2, NF-κB pathway and necroptosis related-genes expressions by RT-PCR and western blot. The results showed that Cd exposure induced necrotic cell death and ion homeostasis imbalance in swine myocardium. Moreover, Cd exposure increased the LXA4 content, inhibited the FPR2 expression, activated TGF-ß pathway and suppressed Nrf2 pathway, activating the NF-κB pathway. In addition, Cd exposure increased the expressions of necroptosis related-genes TNF-α, TNFR1, RIP1, RIP3 and MLKL. It indicated Cd exposure induced necroptosis via FPR2/TGF-ß/NF-κB pathway, revealing the potential mechanism of Cd-induced cardiotoxicity in swine myocardium.

MAPK/iNOS pathway is involved in swine kidney necrosis caused by cadmium exposure.

Cadmium (Cd) pollution in the environment could cause toxic damage to animals and humans. MAPK pathways could regulate their downstream inflammatory factors, and plays a crucial role in necrosis. Since the swine kidney tissue is an important accumulation site of Cd and target organ of its toxic damage, but the damage form of Cd to swine kidney and the role of MAPK pathways in it are still not clear, we selected six week old weaned piglets as the research object, and fed a diet supplemented CdCl2 (20 mg/kg) to establish the model of liver injury induced by Cd. The expressions and phosphorylation of MAPK pathways (ERK, JNK, p38), expression levels of inflammatory factors (TNF-α, NF-κB, iNOS, COX-2 and PTGE) and necrosis related genes (MLKL, RIPK1, RIPK3 and FADD) and heat shock proteins (HSPs) were detected by RT-PCR and Western blot. H.E. staining was used to determine the damage of kidney caused by Cd exposure. The results showed that Cd exposure could activate p38 and JNK pathway phosphorylation, rather than ERK 1/2, up regulated the expressions of inflammatory factors, finally induced programmed necrosis (increasing the expressions of MLKL, RIPK1, RIPK3 and FADD) in swine kidney. Our study elucidated the mechanism of Cd-damage to swine kidney and the relationship among MAPK pathways, inflammatory factors and programmed necrosis in swine.

Cadmium exposure induces mitochondrial pathway apoptosis in swine myocardium through xenobiotic receptors-mediated CYP450s activation.

Cadmium (Cd) pollution has become an important public and environmental health issue. Xenobiotic receptors (XRs, aryl hydrocarbon receptor, AHR; constitutive androstane receptor, CAR; pregnane X receptor, PXR) modulate downstream cytochrome P450 enzymes (CYP450s) expression to metabolize xenobiotics and environmental contaminants. However, the underlying mechanisms of cardiotoxicity induced by Cd(II) in swine and the roles of XRs and CYP450s remain poorly understood. In this study, the cardiotoxicity of Cd(II) was investigated by establishing a Cd(II)-exposed swine model (CdCl2, 20 mg Cd/Kg diet). Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay and transmission electron microscope were used to observe the apoptosis. Antioxidant capacity was evaluated by free radicals contents and antioxidant enzymes activities. RT-PCR and western blot were used to measure the expression of XRs, CYP450s and apoptosis-related genes. Our results revealed that Cd(II) exposure activated the XRs and increased the CYP450s expression, contributing to the production of reactive oxygen species (ROS). Cd(II) exposure restrained the antioxidant capacity, causing oxidative stress. Moreover, mitogen-activated protein kinase (MAPK) pathway including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38) was activated, triggering the mitochondrial apoptotic pathway. In brief, we concluded that Cd(II) caused mitochondrial pathway apoptosis in swine myocardium via the oxidative stress-MAPK pathway, and XRs-mediated CYP450s expression might participate in this process through promoting the ROS.

Resveratrol relieves chlorothalonil-induced apoptosis and necroptosis through miR-15a/Bcl2-A20 axis in fish kidney cells.

Chlorothalonil (CT) is a commonly used fungicide and its excessive application seriously threatens aquatic life and human health. Resveratrol (RSV) is a natural polyphenol and can be used as a therapeutic and preventive agent for the treatment of various diseases. To explore the toxic mechanism of CT exposure on fish kidney cell, as well as the alleviation effect of RSV, we established CT poisoning and/or RSV treatment fish kidney cell models. Ctenopharyngodon idellus kidney (CIK) cell line was treated with CT (5 µg/L) and/or RSV (10 µM) for 48 h. The results showed that CT exposure activated cytochromeP450s (CYPs) including CYP1A1, CYP1B1 and CYP1C, caused malondialdehyde (MDA) accumulation, inhibited glutathione (GSH) levels and glutathione peroxidase (GPX) activities, increased the expression of miR-15a and downregulated BCL2 and TNFα-induced protein 3 (TNFAIP3, A20), triggered mitochondrial pathway mediated apoptosis and receptor interacting serine/threonine kinase (RIP)-dependent necroptosis in CIK cells. However, cell death under CT exposure could be relieved by RSV treatment through inhibiting the expression of CYP1 family genes and restoring miR-15a/BCL2-A20 axis disorders. Overall, we conclude that RSV could relieve CT-induced apoptosis and necroptosis through miR-15a/Bcl2-A20 axis in CIK cells. These results enrich the toxicological mechanisms of the CT and confirm that RSV can be used as a potential antidote for CT poisoning.

Cadmium-induced oxidative stress promotes apoptosis and necrosis through the regulation of the miR-216a-PI3K/AKT axis in common carp lymphocytes and antagonized by selenium.

Cadmium (Cd) is a primary environmental pollutant which causes the immune dysfunction of aquatic animals. MicroRNAs (miRNAs) play a key role in programmed necrosis and apoptosis of immune organs. Selenium (Se), known as an important element, can antagonize Cd toxicity in birds, but the impact of Se on common carps (Cyprinus carpio) has not been reported. To investigate the Cd-induced immunotoxicity mechanism mediated by miR-216a in splenic lymphocytes of common carp and antagonized by Se, we extracted lymphocytes from the spleen and divided them into control group, Se group (10-6 mol/L of Na2SeO3), Se + Cd group and Cd group (4 × 10-5 mol/L of CdCl2). After 6 h of incubation, AO/EB staining, Flow cytometry, qPCR and Western blot were performed. The results showed that Cd exposure caused the apoptosis (BAX, Bcl-2, Caspase 3, Caspase 9) and programmed necrosis (RIP, RIP3, MLKL) in lymphocytes, increased the expression of CYP enzymes, glycometabolism-related enzymes and production of ROS, while irritated the oxidative stress (MDA, SOD, CAT and GSH-PX), upregulated the expression of miR-216a which attenuated the levels of PI3K. However, those variations were apparently mitigated in the Se + Cd group. In short, we have proven that Cd activates oxidative stress and miR-216a-PI3K/AKT axis disorder, thus promoting apoptosis and necrosis in lymphocytes. Moreover, Se can antagonize Cd-triggered apoptosis and necrosis in lymphocytes.

Environmental contaminant ammonia triggers epithelial-to-mesenchymal transition-mediated jejunal fibrosis with the disassembly of epithelial cell-cell contacts in chicken.

Ammonia (NH3) is an environmental contaminant that is causing increasing problems with human and animal health due to the development of poultry industry. There are limited studies on the effect of NH3 inhalation toxicity on the intestinal tract of animals, and underlying molecular mechanisms remain unclear. In the present study, we established a chicken model of NH3 aspiration-induced injury for 42 days and observed histopathological changes of the jejunum. Tandem mass tag-based quantitative proteomic analysis was applied to investigate changes in the protein profile in the jejunum tissue of chickens that were exposed to NH3. Overall, 48 significantly differentially expressed proteins (DEPs) were identified. GO and KEGG analyses revealed that most DEPs were closely related to epithelial-to-mesenchymal transition (EMT), cell-cell junctions, and fibrosis-related factors. Regarding fibrosis, type I collagen and fibronectin were significantly increased. With respect to EMT, epithelial marker proteins (such as E-cadherin and keratin) were repressed, while mesenchymal marker proteins (such as vimentin) were activated. Loss of epithelial cell-cell junctions (such as tight junctions, adherens junctions and desmosomes) were observed. Additionally, overexpression of transforming growth factor-beta (TGF-ß) may play a key role in the EMT process and fibrosis. Taken together, these findings suggested that NH3 triggered the EMT and disassembly of epithelial cell-cell contacts, resulting in jejunal fibrosis that was mediated by TGF-ß in chickens. The results of our study will contribute to provide a technical reference regarding the research methods of intestinal toxicity of NH3 and have largely regulatory implications for ecological risk assessment of human health.

DEHP induces immunosuppression through disturbing inflammatory factors and CYPs system homeostasis in common carp neutrophils.

Di-(2-ethylhexyl) phthalate (DEHP), a common pollutant in the water environment, has been reported to be associated with immune functions, especially aquatic organisms. However, whether DEHP exposure causes neutrophils toxicity in common carp is still unclear. To investigate the toxic effect of DEHP on immune functions, common carp neutrophils were exposed to DEHP (40 µmol/L and 200 µmol/L) for 2 h. The common carp neutrophils exposed to DEHP showed a decrease in neutrophil phagocytosis rate compared with control group. DEHP exposure induced a significant decrease in mRNA expression levels of inflammatory cytokines-related genes (Interleukin-6, Interleukin-8, transforming growth factor, tumor necrosis factor (TNF)-α, TNF-R1, TNF-T1, Interferon (IFN)-2a, IFN-g2b, IFN-g1) in common carp neutrophils, while the expression levels of IL-1ß and IL-10 were increased compared with control group (P < 0.05). Furthermore, the detection of cytochrome P450 enzyme related genes showed that the mRNA expression levels of CYP (cytochrome P450 proteins)-1A, CYP-1B1, CYP-C1, CYP-2K were significantly decreased, and the mRNA expression level of CYP-3A was significantly reduced (P < 0.05). The results indicated that DEHP could affect the phagocytic ability of neutrophils by regulating the expression of inflammatory cytokines and disrupting cytochrome P450 homeostasis, which caused the immunosuppression in common carp.

Ammonia regulates chicken tracheal cell necroptosis via the LncRNA-107053293/MiR-148a-3p/FAF1 axis.

Ammonia (NH3) is a known harmful gas that causes injury to the respiratory system. Ammonia also exists in haze, forming secondary organic aerosols. However, the specific damage caused by NH3 in chicken trachea has not been determined. The regulatory mechanism of ceRNA and its multiple roles have been proposed in many pathomechanisms; therefore, we investigated the functional role of ceRNA in chicken trachea after NH3 inhalation. Broiler chicken trachea exposed to NH3 was selected as the research object. The pathological ultrastructure was observed by transmission electron microscopy. Transcriptome analyses were applied and referenced, and lncRNA-107053293 and miR-148a-3p and FAF1 were selected. A dual-luciferase reporter assay verified the target relationship. Real-time quantitative PCR (RT-PCR) and western blotting were performed to examine the expression levels of necroptosis genes, such as RIPK1, RIPK3, MLKL, caspase 8, and FADD. Our results indicated that lncRNA-107053293 regulated necroptosis by acting as a competing endogenous RNA of miR-148a-3p. FAF1, as a gene target of miR-148a-3p, also affects necroptosis.

Avermectin inhibits neutrophil extracellular traps release by activating PTEN demethylation to negatively regulate the PI3K-ERK pathway and reducing respiratory burst in carp.

Excessive residual avermectin (AVM) in the environment can have toxic effects on non-target organisms. AVM can exert immunotoxicity by inducing genomic demethylation, but its effect on neutrophil extracellular traps (NETs) release in carp is unclear. In this study, carp neutrophils were pretreated with 5 µg/L AVM or 4 µM DNA demethylation inhibitor (aurintricarboxylic acid, ATA), alone or in combination, and then treated with 4 µM phorbol 12-myristate 13-acetate (PMA) to stimulate NETs release. The results showed that exposure of carp neutrophils to AVM significantly suppressed NETs release and MPO expression, increased ROS production, and dramatically reduced PMA-induced cellular respiratory burst. In addition, AVM could bind to the MBD2 molecule, markedly upregulate MBD2 expression to cause demethylation, and clearly activate PTEN expression, thereby inhibiting the expression of PI3K, AKT, Raf, MEK, and ERK. However, these effects were alleviated by ATA. In conclusion, our study showed that AVM could inhibit NETs release in carp by inducing demethylation of PTEN to negatively regulate NETs synthesis pathways and reducing respiratory burst level. Our findings clarify the mechanism of AVM immunotoxicity to fish and are of great significance for efforts to protect the ecological environment and human health.