Molecular mechanism of selenium against lead-induced apoptosis in chicken brainstem relating to heat shock protein, selenoproteins, and inflammatory cytokines.

Extensive application of lead (Pb) brought about environmental pollution and toxic reactions of organisms. Selenium (Se) has the effect of antagonizing Pb poisoning in humans and animals. However, it is still unclear how Pb causes brainstem toxicity. In the present study, we wanted to investigate whether Se can alleviate Pb toxicity in chicken brainstems by reducing apoptosis. One hundred and eighty chickens were randomly divided into four groups, namely the control group, the Se group, the Pb group, and the Se/Pb group. Morphological examination, ultrastructural observation, relative mRNA expressions of genes on heat shock proteins (HSPs); selenoproteins; inflammatory cytokines; and apoptosis-related factors were investigated. The results showed that Pb exposure led to tissue damage and apoptosis in chicken brainstems. Furthermore, an atypical expression of HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90); selenoprotein family glutathione peroxidase (GPx) 1, GPx2, GPx3, and GPx4), thioredoxin reductases (Txnrd) (Txnrd1, Txnrd2, and Txnrd3), dio selenoprotein famliy (diodothyronine deiodinases (Dio)1, Dio2, and Dio3), as well as other selenoproteins (selenoprotein (Sel)T, SelK, SelS, SelH, SelM, SelU, SelI, SelO, Selpb, selenoprotein n1 (Sepn1), Sepp1, Sepx1, Sepw1, 15-kDa selenoprotein (Sep15), and selenophosphate synthetases 2 (SPS2)); inflammatory cytokines (Interleukin 2 (IL-2), IL-4, IL-6, IL-12ß, IL-17, and Interferon-γ (IFN-γ)); and apoptosis-related genes (B-cell lymphoma-2 (Bcl-2), tumor protein 53 (p53), Bcl-2 Associated X (Bax), Cytochrome c (Cyt c), and Caspase-3) were identified. An inflammatory reaction and apoptosis were induced in chicken brainstems after exposure to Pb. Se alleviated the abnormal expression of HSPs, selenoproteins, inflammatory cytokines, and apoptosis in brainstem tissues of chickens treated with Pb. The results indicated that HSPs, selenoproteins, inflammatory, and apoptosis were involved in Se-resisted Pb poisoning. Overall, Se had resistance effect against Pb poisoning, and can be act as an antidote for Pb poisoning in animals.

EGCG alleviated Mn exposure-caused carp kidney damage via trpm2-NLRP3-TNF-α-JNK pathway: Oxidative stress, inflammation, and tight junction dysfunction.

Manganese (Mn), an essential trace metal element in organisms. However, with extensive use of Mn in industry and agriculture, Mn becomes a heavy metal pollutant in water. (-)-epigallocatechin gallate (EGCG), an tea polyphenols, can alleviate metal toxicity. Kidney is an important detoxifying organ, but toxic mechanism of Mn to kidneys is unclear, which needs further research. Carp is an Asian important economical species for fisheries and a biological model for studying environmental toxicology. Thus, we established excess Mn and EGCG-supplemented carp model to explore molecular mechanism of EGCG alleviating Mn-caused carp kidney damage. In this experiment, we set a control group (the Con group), a Mn treatment group (the Mn group, 90 mg/L Mn), a EGCG supplement group (the EG group, 75 mg/kg EGCG), and a combined group (the Mn + EG group, 90 mg/L Mn and 75 mg/kg EGCG). Transcriptome, qRT-PCR, kit, and morphology method results indicated that excess Mn caused oxidative stress, inflammatory damage, and tight junction dysfunction in carp kidneys. Excess Mn-triggered oxidative stress caused tight junction dysfunction via trpm2-NLRP3-TNF-α-JNK pathway and inflammation. EGCG reversed the harm of Mn to fish through the above mechanism. The findings of this study provided the evidence of EGCG-alleviated Mn poisoning and offered new ideas for reducing heavy metal environmental pollution risk.

Cadmium exposure caused cardiotoxicity in common carps (Cyprinus carpio L.): miR-9-5p, oxidative stress, energetic impairment, mitochondrial division/fusion imbalance, inflammation, and autophagy.

Cadmium (Cd), a toxic heavy metal pollutant, is a threat to human and eatable fish health. Common carps are widely cultivated and eaten by humans. However, there are no reports about Cd-damaged common carp hearts. Our experiment attempted to investigate the cardiotoxicity of Cd to common carps by establishing a common carp Cd exposure model. Our results showed that Cd injured hearts. Moreover, Cd treatment induced autophagy via miR-9-5p/Sirt1/mTOR/ULK1 pathway. Cd exposure caused oxidant/antioxidant imbalance and oxidative stress; and led to energetic impairment. Energetic impairment partook in oxidative stress-mediated autophagy through AMPK/mTOR/ULK1 pathway. Furthermore, Cd caused mitochondrial division/fusion imbalance and resulted in inflammatory injury via NF-κB-COX-2-PTGEs and NF-κB-COX-2-TNF-α pathways. Oxidative stress mediated mitochondrial division/fusion imbalance, further induced inflammation and autophagy via OPA1/NF-κB-COX-2-TNF-α-Beclin1 and OPA1/NF-κB-COX-2-TNF-α/P62 pathways under Cd treatment. Taken together, miR-9-5p, oxidative stress, energetic impairment, mitochondrial division/fusion imbalance, inflammation, and autophagy participated in the mechanism of Cd-cardiotoxicity to common carps. Our study revealed harmful effect of Cd on hearts, and provided new information for researches of environmental pollutant toxicity.

Nano-selenium protects grass carp hepatocytes against 4-tert-butylphenol-induced mitochondrial apoptosis and necroptosis via suppressing ROS-PARP1 axis.

4-tert-butylphenol (4-tBP) is a monomer widely used in the synthesis of industrial chemicals, and posed a high risk to aquatic animals. Our study focused on toxic phenotype and mechanism of detoxification in grass carp hepatocytes (L8824) after 4-tBP-treatment. In this experiment, L8824 displayed hallmark phenotypes of apoptosis and necroptosis after 4-tBP exposure, as evidenced by changes in cell morphology, increased rates of apoptosis and necrosis, the loss of MMP, the accumulation of ROS, and changes in associated factors (PARP1, JNK, Bid, Bcl-2, Bax, AIFM1, CytC, Caspase 9, APAF1, Caspase 3, TNF-α, TNFR1, RIPK1, RIPK3, and MLKL). Furthermore, we found that 4-tBP-induced apoptosis and necroptosis were reversed by pretreating with N-Acetylcysteine (a ROS scavenger) and 3-Aminobenzamide (a PARP1 inhibitor), indicating that 4-tBP induced the onset of mitochondrial apoptosis and necroptosis in L8824 via activating ROS-PARP1 axis. Nano-selenium (Nano-Se) is a novel form of Se with a noteworthy antioxidant capacity. Here, Nano-Se was found to have preventive, therapeutic, and resistance effects on 4-tBP-induced L8824 apoptosis and necroptosis. Nano-Se co-treatment with 4-tBP was an optimal way to alleviate 4-tBP-induced apoptosis and necroptosis. We demonstrated for the first time that Nano-Se protected L8824 against 4-tBP-induced mitochondrial apoptosis and necroptosis through ROS-PARP1 pathway. This study will provide a new theoretical basis for 4-tBP toxicology researches and aquatic animal protection.

Chlorpyrifos induced autophagy and mitophagy in common carp livers through AMPK pathway activated by energy metabolism disorder.

Water pollution caused by widely used agricultural pesticide chlorpyrifos (CPF) has aroused extensive public concern. While previous studies have reported on toxic effect of CPF on aquatic animal, little is known about its effect on common carp (Cyprinus carpio L.) livers. In this experiment, we exposed common carp to CPF (11.6 µg/L) for 15, 30, and 45 days to establish a poisoning model. Histological observation, biochemical assay, quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and integrated biomarker response (IBR) were applied to assess the hepatotoxicity of CPF in common carp. Our results displayed that CPF exposure damaged histostructural integrity and induced liver injury in common carp. Furthermore, we found that CPF-induced liver injury may be associated with mitochondrial dysfunction and autophagy, as evidenced by swollen mitochondria, broken mitochondrial ridges, and increased the number of autophagosomes. Moreover, CPF exposure decreased the activities of ATPase (Na+/K+-ATPase, Ca2+-ATPase, Mg2+-ATPase, and Ca2+Mg2+-ATPase), altered glucose metabolism-related genes (GCK, PCK2, PHKB, GYS2, PGM1, and DLAT), and activated energy-sensing AMPK, indicating that CPF caused energy metabolism disorder. The activation of AMPK further induced mitophagy via AMPK/Drp1 pathway, and induced autophagy via AMPK/mTOR pathway. Additionally, we found that CPF induced oxidative stress (abnormal levels of SOD, GSH, MDA, and H2O2) in common carp livers, which further contributed to the induction of mitophagy and autophagy. Subsequently, we confirmed a time-dependent hepatotoxicity caused by CPF in common carp via IBR assessment. Our findings presented a new insight into molecular mechanism of CPF induced-hepatotoxicity in common carp, and provided a theoretical basis for evaluating CPF toxicity to aquatic organisms.

Melatonin alleviates lead-induced intestinal epithelial cell pyroptosis in the common carps (Cyprinus carpio) via miR-17-5p/TXNIP axis.

Lead (Pb) has been concerned as one of the most severe hazardous contaminants, because it can cause pyroptosis in multiple tissues of mammals and birds. Melatonin (Mel) has attracted much interest for its role in governing intestinal injury via microRNAs (miRNAs). To explore the effect of Mel on Pb exposure-induced intestinal epithelial cell pyroptosis in common carps by regulating miR-17-5p/TXNIP axis, the Pb exposure and Pb-Mel treated models were constructed in vivo. The results elucidated that the suppressed expression of miR-17-5p and intensified level of TXNIP were primarily detected in Pb-exposed gut tissues, and both abolished with Mel addition, along with downregulated Pb-mediated elevated expression of NLRP3, CASP1, IL1ß and GSDMD. Additionally, the targeting relationship between miR-17-5p and TXNIP were demonstrated by dual-luciferase reporter assay, and on this basis, miR-17-5p NC, mimic and inhibitor cell models were established. Thereby, Thereby, the expression of TXNIP in the miR-17-5p mimic groups was significant lower in the Pb-exposure but still elevated than the Control group, and the expression of NLRP3 and NLRP3-dependent pyrotposis-related genes performed consistent alterations. Noticeably, the expression of TXNIP suppressed with Mel addition even in the miR-17-5p inhibitor cell model, resulting in the inactivation of NLRP3 inflammasome-dependent pyroptosis. Overall, we draw the conclusion as Mel attenuates Pb-induced intestinal epithelial cell pyroptosis via miR-17-5p/TXNIP axis. The present study provides a novel perspective for toxicological mechanism of Pb, and new insights for the detoxification mechanism of Mel.

HSP27-HSP40-HSP70-HSP90 pathway participated in molecular mechanism of selenium alleviating lead-caused oxidative damage and proteotoxicity in chicken Bursa of Fabricius.

Lead (Pb), a toxic environmental pollutant, is hazardous to the health of humans and birds. Bursa of Fabricius (BF) is a unique organ of birds. Toxic substances can attack BF and induce proteotoxicity. Increased heat shock proteins (HSPs) can induce oxidative damage. Selenium (Se) can alleviate harmful substance-caused oxidative damage. This study aimed to investigate whether Pb can cause oxidative damage and proteotoxicity, as well as Se reverse Pb-caused chicken BF toxicity. A model of chickens treated with Se and Pb alone and in combination was established. BFs were collected on days 30, 60, and 90. H&E and qRT-PCR were performed to observe the microstructure and to detect HSP27, HSP40, HSP60, HSP70, and HSP90 mRNA levels, respectively, in BFs. Multivariate correlation analysis and principal component analysis were conducted to explore the correlation among the five HSPs. In our results, Pb caused BF damage and up-regulated the five HSPs at three time points, causing oxidative damage and proteotoxicity via HSP27-HSP40-HSP70-HSP90 pathway. Furthermore, Pb caused time-dependent stress on HSP27, HSP40, HSP60, and HSP70. In addition, Se relieved Pb-caused damage and up-regulation of HSPs. Taken together, we concluded that Se alleviated Pb-caused oxidative injury and proteotoxicity in chicken BFs via the HSP27-HSP40-HSP70-HSP90 pathway.

Complex molecular mechanism of ammonia-induced apoptosis in chicken peripheral blood lymphocytes: miR-27b-3p, heat shock proteins, immunosuppression, death receptor pathway, and mitochondrial pathway.

Ammonia gas, a toxic environmental pollutant, is a vital component of PM2.5 aerosols, and can decrease human and animal immunity. Peripheral blood lymphocytes (PBLs) are main immune cells. Nevertheless, poisoning mechanism of PBLs under ammonia exposure remains unclear. Here, we established an ammonia poisoning model of chicken PBLs to explore poisoning mechanism of ammonia-caused apoptosis in chicken PBLs. Cell viability and apoptosis rate were detected using CCK8 assay and flow cytometry, respectively. Mitochondrial membrane potential (MMP) was observed using fluorescent staining. In addition, qRT-PCR was performed to measure mRNA levels of apoptosis-related genes (tumor necrosis factor-α (TNF-α), tumor necrosis factor receptor 1 (TNFR1), TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), Caspase-8, BH3-interacting domain death agonist (Bid), Bcl-2-associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak), B-cell lymphoma-2 (Bcl-2), Cytochrome-c (Cytc), apoptotic protease activating factor-1 (APAF1), Caspase-9, and Caspase-3), immune-related genes (interferon-γ (IFN-γ), interleukin-2 (IL-2), IL-4, IL-6, IL-1ß, IL-10, transforming growth factor-ß1 (TGF-ß1), IL-17, IL-21, and IL-22), heat shock protein (HSP) genes (HSP25, HSP40, HSP60, HSP70, HSP90, and HSP110), as well as miR-27b-3p. Western blot was used to determine protein levels of apoptosis-related factors (TNF-α, Caspase-8, Bcl-2, Caspase-9, and Caspase-3), as well as HSPs (HSP40, HSP60, HSP70, and HSP90). The results indicated that TRADD, FADD, and APAF1 were target genes of miR-27b-3p, as well as miR-27b-3p participated in molecular mechanism of apoptosis through targeting TNF-α/TNFR1/Caspase-8 death receptor pathway-triggered Bid/Cytc/Caspase-9 mitochondrial pathway in ammonia-treated chicken PBLs. In addition, our findings demonstrated that excess ammonia led to immunosuppression via Th1/Th2 imbalance and Treg/Th17 imbalance. Simultaneously, ammonia stress activated HSPs. In summary, for the first time, our data demonstrated that HSPs-triggered immunosuppression led to apoptosis under ammonia exposure. Our findings provided a new insight into molecular mechanism of ammonia poisoning and an important reference for environmental risk assessment related to ammonia.

4-tert-butylphenol triggers common carp hepatocytes ferroptosis via oxidative stress, iron overload, SLC7A11/GSH/GPX4 axis, and ATF4/HSPA5/GPX4 axis.

4-tert-butylphenol (4-tBP) is a toxic environmental pollutant with moderate bioaccumulation, environmental persistence, and long-term toxicity. Its toxicity to aquatic organisms has become an issue of concern. However, the molecular mechanism of 4-tBP toxicity to aquatic organisms remained unclear. Liver is a target organ for environmental pollutants. Here, we established 4-tBP-exposed toxicity model in vivo and primary hepatocyte model in vitro in common carp (Cyprinus carpio L.). We found increased hepatic-somatic index (HSI) and abnormal serum biochemical indexes (ALT, AST, and LDH) after 4-tBP exposure, indicating liver damage. We further revealed that 4-tBP damaged the structural integrity of the livers with typical features of ferroptosis. Based on toxicogenomics analysis, we found ferroptosis is likely to be involved in the mechanism of 4-tBP-induced liver damage. Moreover, our in vivo and in vitro experiment provided evidences that 4-tBP-exposure led to excess oxidative stress, iron overload, decreased MMP, and abnormal expression of ferroptosis-related factors. Interestingly, ferrostatin-1 (Fer-1, a ferroptosis inhibitor) pretreatment alleviated above changes. In summary, we demonstrated that 4-tBP triggered hepatocytes ferroptosis via oxidative stress, iron overload, SLC7A11/GSH/GPX4 axis, and ATF4/HSPA5/GPX4 axis. For the first time, we discovered that Fer-1 can ameliorate the toxicity of 4-tBP, which needs more investigations. Our results provided a scientific basis of molecular mechanism of 4-tBP-induced fish poisoning.

Energy metabolism disorder mediated ammonia gas-induced autophagy via AMPK/mTOR/ULK1-Beclin1 pathway in chicken livers.

Ammonia gas is a well-known environmental pollution gas, threatening human health. Ammonia gas is also one of the most harmful gases to livestock and poultry for many years. Many studies have demonstrated toxic effect of ammonia gas on animal health, such as eyes, respiratory system, and digestive system. However, the effect of ammonia gas toxicity on chicken livers and underlying molecular mechanism remains unclear. In this study, we selected chicken liver as research object and duplicated successfully ammonia gas poisoning model of chickens. 1-day-old Ross-308 broilers were randomly divided into the control group (the low ammonia gas group), and two treatment groups (the middle ammonia gas group and the high ammonia gas group) (3 replicates per group and 12 chickens per replicate). Ammonia gas concentration in the low ammonia gas group was ≤5 mg/m3 during day 1-42. Ammonia gas concentration in the middle group was set as 10 ± 0.5 mg/m3 during day 1-21, and 15 ± 0.5 mg/m3 during day 22-42). Ammonia gas concentration in the high ammonia gas group was set as 20 ± 0.5 mg/m3 during day 1-21, and 45 ± 0.5 mg/m3 during day 22-42. The ultrastructure of chicken livers was observed. The activities of four ATPases (Na+K+-ATPase, Mg++-ATPase, Ca++-ATPase, and Ca++Mg++-ATPase), the expression of twelve energy metabolism-related genes (HK1, HK2, PK, PFK, PDHX, CS, LDHA, LDHB, SDHA, SDHB, avUCP, and AMPK), as well as the expression of ten autophagy-related genes (PI3K, LC3I, LC3II, Beclin1, SQSTM1, mTOR, ULK1, ATG5, ATG12, and ATG13) were measured to explore the effect of ammonia gas on energy metabolism and autophagy in chicken livers. Our results showed that excess ammonia gas induced mitochondrial and autophagic damage in chicken liver tissue cells. Meanwhile, ATPases activities were inhibited and the expression of energy metabolism-related genes changed during ammonia gas treatment, meaning that excess ammonia gas caused energy metabolism disorder. Furthermore, ammonia gas exposure altered the expression of autophagy-related genes, suggesting that ammonia gas treatment caused autophagy in chicken livers. Moreover, ammonia gas-induced AMPK compensatory up-regulation activated autophagy process through inhibiting mTOR and promoting ULK1. In addition. there were dose-dependent and time-dependent effects on all detected indexes in ammonia gas-caused chicken liver cell damage. Taken together, AMPK/mTOR/ULK1-Beclin1 pathway participated in energy metabolism disorder-mediated autophagic injury caused by ammonia gas exposure in chicken livers.

Heat shock proteins took part in oxidative stress-mediated inflammatory injury via NF-κB pathway in excess manganese-treated chicken livers.

Manganese (Mn) is an essential metal in humans and animals. However, excess Mn entered environment due to the wide application of Mn in industry and agriculture, and became an environmental pollutant. Exposure to high doses of Mn is toxic to humans and animals (including chickens). Liver is a target organ of Mn poisoning. Nevertheless, there were few studies on whether Mn poisoning damages chicken livers and poisoning mechanism of Mn in chicken livers. Herein, the aim of this study was to explore if oxidative stress, heat shock proteins (HSPs), and inflammatory response were involved in the mechanism of Mn poisoning-caused damage in chicken livers. A chicken Mn poisoning model was established. One hundred and eighty chickens were randomly divided into one control group (containing 127.88 mg Mn kg-1) and three Mn-treated groups (containing 600, 900, and 1800 mg Mn kg-1, respectively). Histomorphological structure was observed via microstructure and ultrastructure. Spectrophotometry was used to detect total antioxidant capacity (T-AOC) and inducible nitric oxide synthase (iNOS) activity, as well as nitric oxide (NO) content. And qRT-PCR was performed to measure mRNA expression of inflammatory genes (nuclear factor kappa B (NF-κB), tumor necrosis factor α (TNF-α), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and iNOS) and heat shock protein (HSP) genes (HSP27, HSP40, HSP60, HSP70, and HSP90). Multivariate correlation analysis, principal component analysis, and cluster analysis were used to demonstrate the reliability of mechanism of Mn poisoning in our experiment. The results indicated that excess Mn led to inflammatory injury at three contents and three time points. Meanwhile, we found that NO content, iNOS activity, and NF-κB, TNF-α, COX-2, PGE2, and iNOS mRNA expression increased after Mn treatment, meaning that exposure to Mn induced inflammatory response via NF-κB pathway in chicken livers. Moreover, excess Mn decreased T-AOC activity, indicating that Mn exposure caused oxidative stress. Furthermore, mRNA expression of above five HSP genes was up-regulated during Mn exposure. Oxidative stress triggered the increase of HSPs and the increase of HSPs mediated inflammatory response induced by Mn. In addition, there were time- and dose-dependent effects on Mn-caused chicken liver inflammatory injury. Taken together, HSPs participated in oxidative stress-mediated inflammatory damage caused by excess Mn in chicken livers via NF-κB pathway. For the first time, we found that oxidative stress can trigger HSP70 and HSPs can trigger poisoning-caused inflammatory damage, which needs to be further explored. This study provided a new insight into environmental pollutants and a reference for further study on molecular mechanisms of poisoning.

miR-187-5p/apaf-1 axis was involved in oxidative stress-mediated apoptosis caused by ammonia via mitochondrial pathway in chicken livers.

Ammonia (NH3), a toxic gas, is an important cause of atmospheric haze and one of the main pollutants in air environment of poultry houses, threatening the health of human beings and poultry. However, little is known about the effect of NH3 on liver apoptotic damage. This study aimed to investigate the mechanism of oxidative stress-mediated apoptosis caused by NH3 in chicken livers and whether miR-187-5p/apaf-1 axis was involved in this mechanism. Here we duplicated NH3 poisoning model of chickens for fattening to study the ultrastructure of chicken livers, apoptosis rate, oxidative stress indexes, miR-187-5p, and apoptosis-related genes. Obvious apoptotic characteristics of liver tissues exposed to excess NH3 were observed, and the apoptosis rate increased. Excess NH3 decreased the activities of catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px), and increased the content of malondialdehyde (MDA), suggesting that oxidative stress occurred. miR-187-5p decreased, and apoptotic protease activating factor-1 (apaf-1) increased, indicating that excess NH3 dysregulated miR-187-5p/apaf-1 axis. The expression of tumor protein p53 (p53), Bcl-2 associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak), Cytochrome-c (Cyt-c), Caspase-9, Caspase-8, and Caspase-3 was promoted, and the expression of B-cell lymphoma-2 (Bcl-2) was inhibited, resulting in apoptosis. Moreover, oxidative stress indexes, miR-187-5p, and apoptosis-related genes changed in dose- and time-dependent manner. Altogether, miR-187-5p/apaf-1 axis participated in oxidative stress-mediated apoptosis caused by NH3 via mitochondrial pathway in the livers of chickens for fattening. This study may provide new ideas to study the mechanism of liver apoptotic damage induced by NH3 exposure.

Ammonia-triggered apoptosis via immune function and metabolic process in the thymuses of chickens by proteomics analysis.

Ammonia (NH3), an environmental pollutant with a pungent odor, is not only an important volatile in fertilizer production and ranching, but also main basic component of haze. In present study, we found that ultrastructural changes and 3167 differentially expressed proteins (DEPs) using proteomics analysis in the thymuses of chickens exposed to NH3 on day 42. Obtained DEPs were enriched using GO and KEGG; and 66 DEPs took part in immune function, metabolic process, and apoptosis in the thymuses of chickens treated with NH3. 9 genes of DEPs were validated using qRT-PCR, and mRNA expression of 2 immune-related genes (CTSG and NFATC2), 3 metabolic process-related genes (APOA1, GOT1, and GOLGA3), and 4 apoptosis-related genes (PIK3CD, CTSS, CAMP, and NSD2) were consistent with DEPs in chicken thymuses. Our results indicated that excess NH3 led to immunosuppression, metabolic disorder, and apoptosis in chicken thymuses. Present study gives a novel insight into the mechanism of NH3 toxicity and demonstrated that immune response, metabolism process, and apoptosis were important in the mechanism of NH3 toxicity of chicken exposure to high concentration of NH3.

Oxidative stress and mitochondrial dysfunction involved in ammonia-induced nephrocyte necroptosis in chickens.

Ammonia (NH3), an environmental pollutant, poses a serious threat to human and avian health. Although previous studies have showed that NH3 caused kidney injury, the molecular mechanisms of nephrotoxicity induced by NH3 remain unclear. To explore the mechanisms of NH3 nephrotoxicity, a total of 36 broiler chicks at one day of age were exposed to NH3. After 42 days of exposure, blood samples were collected to determine creatinine and uric acid; and kidney samples were weighted and then collected to detect ultrastructural changes, oxidative stress parameters, ATPases, necroptosis- and mitochondrial dynamics-related genes. The results showed that chickens exposed to NH3 showed lower relative kidney weight and an increase concentration in serum creatinine and uric acid. NH3 exposure caused nephrocyte necrosis and increased the expression of necroptosis-related genes (TNF-α, RIPK1, RIPK3, MLKL, and JNK). Besides, the activities of antioxidant systems (SOD, CAT, GSH-Px, and T-AOC) were reduced, whereas the concentrations of H2O2 and MDA were elevated. Lower activities of ATPases were obtained in NH3 treatment groups. Furthermore, the mitochondrial fission-related genes drp1 and mff were activated, and mitochondrial fusion-related genes opa1, mfn1 and mfn2 were suppressed after NH3 exposure. Based on the above results, we conclude that NH3 caused-oxidative stress and mitochondrial dysfunction mediated nephrocyte necroptosis in chickens. This study may provide new insight into NH3 nephrotoxicity.

The effect of ammonia exposure on energy metabolism and mitochondrial dynamic proteins in chicken thymus: Through oxidative stress, apoptosis, and autophagy.

Ammonia (NH3) gas is an atmospheric pollutant, produced from different sources. In poultry houses NH3 is produced from the biological process of liter, manure, and protein composition. It has been well documented that NH3 adversely effects the health of chickens. However, the underlying mechanism of NH3 toxicity on chicken thymus is still unknown. Thymus is an important immune organ, which play a critical role in eliciting protective immune responses to ensure healing process and elimination of harmful stimuli. The results showed that NH3 exposure reduced antioxidant activities and induced oxidative stress in thymus tissues. Histological observation showed normal morphology of chicken thymus in control group. In contrast, increased number of nuclear debris, vacuoles, and cristae break were seen in NH3 affected chickens. Ultrastructural analysis indicated mitochondrial breakdown, disappearance, vacuoles, and chromatin condensation in NH3 treated groups. The mRNA and protein expression of apoptosis related genes were significantly enhanced in the chicken thymus of NH3 affected chickens compared to control group. Moreover, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay results suggested that NH3 exposure increased positive stained nuclei in the chicken thymus. Meanwhile, NH3 exposure reduced the number of CD8+ T-lymphocytes, decreased the adenosine triphosphate (ATPase) activities. The mRNA and protein expression of autophagy, energy metabolism, and mitochondrial dynamics proteins were altered by NH3 exposure. In summary, these results showed that NH3 induced oxidative stress, apoptosis and autophagic cell death (ACD), which could be the possible causes of immune damage and structural impairment in chicken thymus.

Ammonia inhalation impaired immune function and mitochondrial integrity in the broilers bursa of fabricius: Implication of oxidative stress and apoptosis.

Ammonia (NH3) is considered as environmental pollutant and toxic agent for animals and humans including poultry. Previous reports demonstrated that NH3 suppressed broilers immunity. However, the harmful effects of NH3 on broilers bursa of fabricius (BF) is still unknown. Functionally, apoptosis is very important for many physiological processes including homeostasis of lymphocyte population. Therefore, the present study was aimed to investigate the underlying mechanisms of NH3 toxicity in the broilers BF. Histological observation showed lymphocyte accumulation, cavities and increased interstitial cells in BF. Ultrastructural observation indicated mitochondrial vacuoles, deformation and disappearance of mitochondrial membranes. Oxidative stress markers (CAT, MDA, H2O2, GGT, GSH-Px and GSH) showed that NH3-induced oxidative stress in BF. Meanwhile, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed increased apoptotic cells. In addition, the mRNA and protein expression of dynamin-related protein 1 (Drp1), mitochondrial fission factor (Mff), mitofusin 1 and 2 (Mfn1 and Mfn2), optic atrophy 1 (Opa1) indicated imbalance between mitochondrial inner and outer membrane and results in mitochondrial dysfunction in broilers BF. The mRNA and protein expression of apoptosis-related genes including Caspase-3, Caspase-9, Caspase-8, Cytochrome-C (Cyt-C), p53, B-cell lymphoma 2 (Bcl-2) and Bcl-2 associated X protein (Bax) were significantly altered in broilers BF. Conclusively, these results displayed that excessive NH3 causes BF damage and mitochondrial dysfunction through oxidative stress and apoptosis in BF and could affect immune function of BF. These findings provide possible therapeutic targets to prevent NH3 induced toxicity in the BF of broilers.

Impaired immune function and structural integrity in the gills of common carp (Cyprinus carpio L.) caused by chlorpyrifos exposure: Through oxidative stress and apoptosis.

As one of the mucosal lymphatic tissues, the gill is an important immune organ in fish. Water environmental pollutants enter fish body through the gill. Therefore, the gill is the initial site where pollutants produce toxic effects in water. Chlorpyrifos (CPF), a broad-spectrum organophosphate insecticide, is widely used for agricultural pests and causes river pollution. In the present study, we investigated histopathological effect, oxidative stress indexes (SOD, GSH, T-AOC, and MDA), and apoptosis-related genes (P53, PUMA, Bax, Bcl-2, Apaf-1, Caspase-9, and Caspase-3) in the gills of common carp exposed to CPF. The results indicated that CPF exposure decreased SOD, T-AOC, and GSH; increased MDA; decreased Bcl-2 mRNA expression; and increased P53, PUMA, Bax, Apaf-1, Caspase-9, and Caspase-3 mRNA expressions in common carp gills. Our results proved that CPF exposure caused oxidative stress and apoptosis in common carp gills; CPF exposure destroyed the structural integrity and affected the immune function through oxidative stress and apoptosis in common carp gills. These will provide evidence for the toxic effects of water environmental pollutants on immune function and structural integrity in fish gills.

Dietary selenium supplementation alleviates immune toxicity in the hearts of chickens with lead-added drinking water.

Lead (Pb) is an environmental pollutant and can damage organisms. Selenium (Se) can alleviate Pb poisoning. The present study aimed to investigate the alleviative effect of Se on Pb-induced immune toxicity in chicken hearts. One-hundred-and-eighty Hy-line male chickens were randomly divided into four groups at 7 days of age. The control group was offered a standard commercial diet (SD) and drinking water (DW); the Se group was offered SD supplemented with sodium selenite (SeSD) and DW; the Pb + Se group was offered SeSD and DW supplemented with lead acetate (PbDW); and the Pb group was offered SD and PbDW. Relative mRNA expression of inducible nitric oxide synthase (iNOS), interleukins (IL-2, IL-4, IL-6, IL-12ß, IL-17 and IFN-γ), and heat shock proteins (HSP27, HSP40, HSP60, HSP70, and HSP90) were determined by means of quantitative real-time PCR. Relative protein expression of iNOS, HSP60, HSP70, and HSP90 was assessed, as well as nitric oxide (NO) content and iNOS activity in heart tissue. The results indicated a down-regulation of interleukin (IL)-2 and IFN-γ and an up-regulation of NO, iNOS, interleukins (IL-4, IL-6, IL-12ß, IL-17), and heat shock proteins (HSP27, HSP40, HSP60, HSP70, and HSP90) in Pb-damaged hearts. Se alleviated all of the above Pb-induced changes. There were time-dependent effects on NO content, iNOS activity, and mRNA levels of iNOS, IL-2, IL-4, IL-6, IL-17, HSP27, HSP40, HSP60, HSP70, and HSP90 after Pb treatment in the chicken hearts. Se alleviated Pb-induced immune toxicity in the chicken hearts.

Identification of signal pathways for immunotoxicity in the spleen of common carp exposed to chlorpyrifos.

Chlorpyrifos (CPF) is an environmental pollutant due to its high toxicity to aquatic animals. Because CPF was detected in aquatic environments in many countries, it has been widely concerned by researchers. Although the immunotoxicity of CPF to fish had been reported, the immunotoxicity mechanism is still not clear. Recently, transcriptome analysis has become a major method to study the toxic mechanism of pollutants in environmental toxicology. However, the immunotoxicity identification of CPF on fish had not been reported by transcriptome analysis. In the present study, we examined the effects of CPF on organismal system in the spleen of common carp by transcriptome analysis. We have successfully constructed a database of transcriptome analysis of carp spleens under exposure to CPF and found 773 differentially expressed genes (DEGs) (including 498 up-regulated DEGs and 275 down-regulated DEGs) and 4 branches (containing 33 known KEGG pathways). Some genes associated with the 4 pathways (Complement and coagulation cascades, PPAR signaling pathway, Fat digestion and absorption, and Collecting duct acid secretion) contained in organismal system were validated by quantitative real-time PCR and showed significant improvement compared with the control group. Our results indicated that exposure to CPF caused a change in the signal pathways of organismal system in carp spleens. The present study provides new insights into the immunotoxicity mechanism and risk assessment of CPF, as well as references for comparative medicine.

The co-expression of circRNA and mRNA in the thymuses of chickens exposed to ammonia.

Ammonia (NH3) is one of major air pollutants in intensive poultry houses, affecting chicken health. Circular RNA (circRNA) is a novel type of RNA that can regulate gene expression and be associated with various biological activities. However, the changes of circRNA caused by excess NH3 in chickens have not been investigated. We found differentially expressed genes and morphological changes in the thymuses of chickens exposed to NH3 on day 42. We used a combination of RNA deep sequencing, qRT-PCR, and bioinformatic analysis to explore regulatory mechanism of circRNA and mRNA. Transcriptional profiling results showed that 5 circRNA genes and 100 mRNA genes were significantly dyregulated by high NH3. The results from GO items showed that immune response and the regulation of cytokine production were involved in the mechanisms of chickens exposed to NH3. Co-expression analysis found that circRNA-mRNA network was correlated with oxidative stress and inflammation. NH3 exposure decreased mRNA expression of antioxidant-related genes (GPx and GST4) and increased the mRNA expression of inflammation-related genes (IL-1ß, IL-6, IL-8, and iNOS) in chicken thymuses. Histopathologic analysis demonstrated that NH3 caused inflammatory injury in chicken thymuses. In conclusion, the co-expression of circRNA and mRNA took part in chicken thymus inflammatory injury caused by NH3. Our study further enriches the mechanism of NH3 toxicity on chickens, which may be valuable for human and animal health protection.