Copper Exposure Induced Chicken Hepatotoxicity: Involvement of Ferroptosis Mediated by Lipid Peroxidation, Ferritinophagy, and Inhibition of FSP1-CoQ10 and Nrf2/SLC7A11/GPX4 Axis.

Copper (Cu) is one of the most significant trace elements in the body, but it is also a widespread environmental toxicant health. Ferroptosis is a newly identified programmed cell death, which involves various heavy metal-induced organ toxicity. Nevertheless, the role of ferroptosis in Cu-induced hepatotoxicity remains poorly understood. In this study, we found that 330 mg/kg Cu could disrupt the liver structure and cause characteristic morphological changes in mitochondria associated with ferroptosis. Additionally, Cu treatment increased MDA (malondialdehyde) and LPO (lipid peroxide) production while reducing GSH (reduced glutathione) content and GCL (glutamate cysteine ligase) activity. However, it is noticeable that there were no appreciable differences in liver iron content and key indicators of iron metabolism. Meanwhile, our further investigation found that 330 mg/kg Cu-exposure changed multiple ferroptosis-related indicators in chicken livers, including inhibition of the expression of SLC7A11, GPX4, FSP1, and COQ10B, whereas enhances the levels of ACLS4, LPCAT3, and LOXHD1. Furthermore, the changes in the expression of NCOA4, TXNIP, and Nrf2/Keap1 signaling pathway-related genes and proteins also further confirmed 330 mg/kg Cu exposure-induced ferroptosis. In conclusion, our results indicated that ferroptosis may play essential roles in Cu overload-induced liver damage, which offered new insights into the pathogenesis of Cu-induced hepatotoxicity.

miR-181b-1-3p affects the proliferation and differentiation of chondrocytes in TD broilers through the WIF1/Wnt/ß-catenin pathway.

Thiram is a plant fungicide, its excessive use has exceeded the required environmental standards. It causes tibial dyschondroplasia (TD) in broilers which is a common metabolic disease that affects the growth plate of tibia bone. It has been studied that many microRNAs (miRNAs) are involved in the differentiation of chondrocytes however, their specific roles and mechanisms have not been fully investigated. The selected features of tibial chondrocytes of broilers were studied in this experiment which included the expression of miR-181b-1-3p and the genes related to WIF1/Wnt/ß-catenin pathway in chondrocytes through qRT-PCR, western blot and immunofluorescence. The correlation between miR-181b-1-3p and WIF1 was determined by dual luciferase reporter gene assay whereas, the role of miR-181b-1-3p and WIF1/Wnt/ß-catenin in chondrocyte differentiation was determined by mimics and inhibitor transfection experiments. Results revealed that thiram exposure resulted in decreased expression of miR-181b-1-3p and increased expression of WIF1 in chondrocytes. A negative correlation was also observed between miR-181b-1-3p and WIF1. After overexpression of miR-181b-1-3p, the expression of ACAN, ß-catenin and Col2a1 increased but the expression of GSK-3ß decreased. It was observed that inhibition of WIF1 increased the expression of ALP, ß-catenin, Col2a1 and ACAN but decreased the expression of GSK-3ß. It is concluded that miR-181b-1-3p can reverse the inhibitory effect of thiram on cartilage proliferation and differentiation by inhibiting WIF1 expression and activating Wnt/ß-catenin signaling pathway. This study provides a new molecular target for the early diagnosis and possible treatment of TD in broilers.

Curcumin alleviates AFB1-induced nephrotoxicity in ducks: regulating mitochondrial oxidative stress, ferritinophagy, and ferroptosis.

Aflatoxin B1 (AFB1), an extremely toxic mycotoxin that extensively contaminates feed and food worldwide, poses a major hazard to poultry and human health. Curcumin, a polyphenol derived from turmeric, has attracted great attention due to its wonderful antioxidant properties. Nevertheless, effects of curcumin on the kidneys of ducks exposed to AFB1 remain unclear. Additionally, the underlying mechanism between AFB1 and ferroptosis (based on excessive lipid peroxidation) has not been sufficiently elucidated. This study aimed to investigate the protective effects and potential mechanisms of curcumin against AFB1-induced nephrotoxicity in ducklings. The results indicated that curcumin alleviated AFB1-induced growth retardation and renal distorted structure in ducklings. Concurrently, curcumin inhibited AFB1-induced mitochondrial-mediated oxidative stress by reducing the expression levels of oxidative damage markers malondialdehyde (MDA) and 8-hydroxy-2 deoxyguanosine (8-OHdG) and improved the expression of mitochondria-related antioxidant enzymes and the Nrf2 pathway. Notably, curcumin attenuated iron accumulation in the kidney, inhibited ferritinophagy via the NCOA4 pathway, and balanced iron homeostasis, thereby alleviating AFB1-induced ferroptosis in the kidney. Collectively, our results suggest that curcumin alleviates AFB1-induced nephrotoxicity in ducks by inhibiting mitochondrial-mediated oxidative stress, ferritinophagy, and ferroptosis and provide new evidence for the mechanism of AFB1-induced nephrotoxicity in ducklings treated with curcumin.

Exposure to copper induces endoplasmic reticulum (ER) stress-mediated apoptosis in chicken (Gallus gallus) myocardium.

Copper (Cu), an omnipresent environmental pollutant, can cause potential harm to the public and ecosystems. In order to study the cardiotoxicity caused by Cu, molecular biology techniques were used to analyze the effect of Cu on ER stress-mediated cardiac apoptosis. In vivo investigation, 240 1-day-old chickens were fed with Cu (11, 110, 220, and 330 mg/kg) diet for 7 weeks. The consequence showed that high-Cu can induce ER stress and apoptosis in heart tissue. The vitro experiments, the Cu treatment for 24 h could provoke ultrastructural damage and upregulate the apoptosis rate. Meanwhile, GRP78, GRP94, eIF2α, ATF6, XBP1, CHOP, Bax, Bak1, Bcl2, Caspase-12 and Caspase-3 genes levels, and GRP78, GRP94 and Caspase-3 proteins levels were increased, which indicated that ER stress and apoptosis in cardiomyocytes. But the mRNA level of Bcl2 were decreased after Cu exposure. Conversely, Cu-induced ER stress-mediated apoptosis can be alleviated by treatment with 4-PBA. These findings generally showed that Cu exposure can contribute to ER stress-mediated apoptosis in chicken myocardium, which clarifies the important mechanism link between ER stress and apoptosis, and provides a new perspective for Cu toxicology.

Co-exposure to environmentally relevant concentrations of cadmium and polystyrene nanoplastics induced oxidative stress, ferroptosis and excessive mitophagy in mice kidney.

Nanoplastics (NPs) are defined as a group of emerging pollutants. However, the adverse effect of NPs and/or heavy metals on mammals is still largely unclear. Therefore, we performed a 35-day chronic toxicity experiment with mice to observe the impacts of exposure to Cadmium (Cd) and/or polystyrene nanoplastics (PSNPs). This study revealed that combined exposure to Cd and PSNPs added to the mice's growth toxicity and kidney damage. Moreover, Cd and PSNPs co-exposure obviously increased the MDA level and expressions of 4-HNE and 8-OHDG while decreasing the activity of antioxidase in kidneys via inhibiting the Nrf2 pathway and its downstream genes and proteins expression. More importantly, the results suggested for the first time that Cd and PSNPs co-exposure synergistically increased iron concentration in kidneys, and induced ferroptosis through regulating expression levels of SLC7A11, GPX4, PTGS2, HMGB1, FTH1 and FTL. Simultaneously, Cd and PSNPs co-exposure further increased the expression levels of Pink, Parkin, ATG5, Beclin1, and LC3 while significantly reducing the P62 expression level. In brief, this study found that combined exposure to Cd and PSNPs synergistically caused oxidative stress, ferroptosis and excessive mitophagy ultimately aggravating kidney damage in mice, which provided new insight into the combined toxic effect between heavy metals and PSNPs on mammals.

Toxicity of inorganic arsenic to animals and its treatment strategies.

In nature, arsenic is mostly found in the form of inorganic compounds. Inorganic arsenic compounds have a variety of uses and are currently used in the manufacture of pesticides, preservatives, pharmaceuticals, etc. While inorganic arsenic is widely used, arsenic pollution is increasing worldwide. Public hazards caused by arsenic contamination of drinking water and soil are becoming increasingly evident. Epidemiological and experimental studies have linked inorganic arsenic exposure to the development of many diseases, including cognitive impairment, cardiovascular failure, cancer, etc. Several mechanisms have been proposed to explain the effects caused by arsenic, such as oxidative damage, DNA methylation, and protein misfolding. Understanding the toxicology and potential molecular mechanisms of arsenic can help mitigate its harmful effects. Therefore, this paper reviews the multiple organ toxicity of inorganic arsenic in animals, focusing on the various toxicity mechanisms of arsenic-induced diseases in animals. In addition, we have summarized several drugs that can have therapeutic effects on arsenic poisoning in pursuit of reducing the harm of arsenic contamination from different pathways.

Curcumin attenuates AFB1-induced duck liver injury by inhibiting oxidative stress and lysosomal damage.

Aflatoxin B1 (AFB1), as the most toxic secondary metabolite produced by Aspergillus flavus, is a serious threat to human and animal health. Curcumin, a polyphenol from the plant turmeric, has demonstrated unique anti-damage properties in several studies. But, its ability to alleviate AFB1-induced liver damage in ducks and the underlying mechanisms are not completely elucidated. In this study, we investigated the intervention of curcumin on AFB1-induced hepatotoxicity in ducks. Research data showed that the combination of curcumin and AFB1 alleviated oxidative stress, reduced malondialdehyde (MDA) accumulation and relieved hepatotoxicity after 28 days of treatment, compared with AFB1. Also, curcumin upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream antioxidant enzymes (SOD, HO-1), which enhanced the antioxidant capacity of the liver. In addition, curcumin inhibited AFB1-induced lysosomal damage in the liver, with the character of reduced lysosomal membrane permeabilization, restored autophagic flux, and promoted lysosomal biogenesis, thereby enhancing the self-protective capacity of the liver. In conclusion, our results suggest that curcumin alleviates AFB1-induced duck hepatotoxicity by inhibiting oxidative stress and lysosomal damage.

MitomiR-504 alleviates the copper-induced mitochondria-mediated apoptosis by suppressing Bak1 expression in porcine jejunal epithelial cells.

Copper (Cu), an environmental heavy metal pollutant, has been widely researched in its toxicology. Recently, an increasing number of mitochondrial microRNAs (mitomiRs) have been shown to involve in the metabolic regulation. However, the underlying mechanisms of mitomiRs on regulating apoptosis under Cu exposure are still unclear. Here, we proved that Cu induced mitochondria-mediated apoptosis in porcine jejunal epithelial cells, concomitant with distinct reduction of mitomiR-504 in vivo and in vitro. The miR-504 mimic notably enhanced the mRNA and protein expressions of Bak1, Bax, Cleaved-caspase3 and Caspase-9, and significantly decreased the apoptosis rate and Bcl-2 mRNA and protein levels, indicating that overexpression of mitomiR-504 attenuated the Cu-induced mitochondria-mediated apoptosis. Besides, Bak1 was confirmed as a direct target of mitomiR-504 by the bioinformatics analysis and dual-luciferase reporter assay. Subsequently, transfection of siRNA targeting Bak1 significantly enhanced the alleviating effect of miR-504 mimic on the Cu-induced mitochondria-mediated apoptosis. Overall, these suggested that overexpression of mitomiR-504 alleviated the Cu-induced mitochondria-mediated apoptosis in jejunal epithelial cells by suppressing Bak1 expression. These findings are conducive to elucidating the mechanism of Cu-induced jejunal epithelial pathologies, providing a new research idea for the Cu toxicology.

N-acetyl-L-cysteine alleviates FUNDC1-mediated mitophagy by regulating mitochondrial dynamics in type 1 diabetic nephropathy canine.

Diabetic nephropathy (DN) is a major complication of type 1 diabetes mellitus, and hyperglycemia and hypertension are the main risk factors for the development of DN. N-Acetyl-Cysteine (NAC) has a variety of effects, interfering with the production and scavenging of free radicals and regulating the metabolic activity of tissue cells. However, the efficacy of NAC on DN treatment is unclear. Thus, this study investigated the protective mechanism of NAC combined with insulin on renal injury in dogs with DN. The forty dogs were selected and divided into control group, DM group, INS group, INS + NAC group and NAC group to establish the model for a trial period of 4 months. The results revealed that INS + NAC was effective in reducing and stabilizing blood glucose levels. Biochemical results showed that INS + NAC treatment significantly regulated the stability of UREA, CREA and fructosamine indicators. Meanwhile, histopathology staining showed significant glomerular wrinkling and fibrosis in the DM group, which could be reversed after INS + NAC treatment. In addition, INS + NAC could restore mitochondria homeostasis by upregulating the levels of mitochondrial fission (MFN1, MFN2 and OPA1) and inhibiting of mitochondrial fusion (DRP1, FIS1 and MFF) related indicators. Further studies revealed that INS + NAC regulated the expression levels of renal BNIP3, NIX and FUNDC1 in the DM group, thereby alleviating mitophagy. Collectively, these results suggested that NAC combined with insulin protects DN by regulating the mitochondrial dynamics and FUNDC1-mediated mitophagy.

N-acetylcysteine combined with insulin alleviates the oxidative damage of cerebrum via regulating redox homeostasis in type 1 diabetic mellitus canine.

Neurodegenerative diseases are one of the major complications of type 1 diabetes mellitus (T1DM). The effect of insulin monotherapy on controlling blood glucose and neurodegeneration associated with diabetes is unsatisfactory. It is revealed that oxidative stress is a key element in T1DM. Therefore, N-acetylcysteine (NAC) was used together with insulin to investigate the therapeutic effect on neuronal damage in T1DM in this study. A total of 40 beagles were randomly divided into 5 groups (control group, DM group, insulin monotherapy group, NAC combined with insulin group, and NAC monotherapy group) to explore the effects of NAC on alleviating the oxidative damage in cerebrum. Our results showed that the contents of H2O2, 8-OHdg and MDA were apparently increased in DM group, while DNA and lipid oxidative damage was alleviated by the treatment of NAC and insulin. Histopathology revealed the sparse of neurofibrils and vacuolar degeneration in DM group. Additionally, compared with the control group, the mRNA expression levels of HO-1, nqo1, GCLC and GSTM1 were significantly decreased in DM group, while the opposite trend could be shown under NAC combined with insulin treatment. Meanwhile, the tight junction proteins of ZO-1, occludin and Claudin-1 were up-regulated with the treatment of NAC combined with insulin. Additionally, NAC further alleviated oxidative damage by enhancing the activity of GSH, Trx and TrxR and reducing the activity of catalase, GSSG and Grx to maintain redox homeostasis. These results demonstrated that NAC combined with insulin exerted protective effects against T1DM-induced cerebral injury via maintaining cerebral redox homeostasis.

N-acetyl-L-cysteine ameliorates hepatocyte pyroptosis of dog type 1 diabetes mellitus via suppression of NLRP3/NF-κB pathway.

Type 1 diabetes mellitus (T1DM) is a chronic and represented by insulin-causing pancreatic ß-cell disruption and hyperglycemia. N-Acetyl-Cysteine (NAC) is regarded as facilitating endothelial cell function and angiogenesis and may have treatment effect in the case of diabetes. However, the impact of NAC on T1DM are unknown. Here we reported that inflammatory pathogenesis of canine type 1 diabetes liver disease and the therapeutic effect of NAC combined with insulin. For this purpose, the model was established by intravenous injection of streptozotocin (20 mg/kg). Forty adult dogs were used and divided into 5 groups: control group, DM group, insulin treatment group, NAC combined with insulin therapy, and NAC group, while study lasted for 16 weeks. Results showed that the level of liver function enzyme activity were apparently increased in DM group, while the NAC with insulin treatment remarkable decreased liver function enzyme levels. Histopathology revealed that obvious changes in liver structure of all DM group, as evidenced by hepatocyte disorder and cellular swelling. Liver structure was evaluated by Periodic Acid Schiff (PAS) and Masson staining, the tissues appeared glycogen deposition and collagen deposition, indicating that DM aggravated liver injury. Compared with control group, the protein and mRNA expression of NLRP3, Caspase-1, ASC, and GSDMD were significantly induced in the DM group, while INS and NAC combined with INS treatment reversed the above changes. The levels of NF-κB P65, p-NF-κB, and IFN γ were availably enhanced in the DM group, which decreased through insulin and NAC combined with insulin treatment. This study demonstrated that NAC combined with INS exerted protective effects against STZ-induced liver injury by inhibiting the NLRP3/NF-κB pathway. The findings indicated that NAC combined with INS may serve as a potential candidate therapy for the treatment of T1DM.

NAC alleviative ferroptosis in diabetic nephropathy via maintaining mitochondrial redox homeostasis through activating SIRT3-SOD2/Gpx4 pathway.

Diabetic nephropathy (DN) is known as a major microvascular complication in type 1 diabetes. The effect of insulin treatment alone on controlling blood glucose is unsatisfactory. N-acetylcysteine (NAC), a chemical agent with thiol group, is found to confer a protective effect in renal injury. However, whether NAC combined with insulin treatment can further enhance the therapeutic effect in DN remains unclear. Here, we firstly used large mammal beagle as DN model to explore the effect of NAC combined with insulin treatment on DN during 120 d. Our results showed that NAC further alleviated mitochondrial oxidative damage and ferroptosis by enhancing activity of mitochondria GSH and maintaining mitochondrial redox homeostasis in DN. Additionally, the upregulated acetylation level of SOD2 was further abrogated by NAC treatment. In MDCK cells, NAC reduced high glucose (HG)-caused ferroptosis via activating Gpx4 expression. Of note, inhibition of Gpx4 by FIN56 abolished the protective effects of NAC on HG-induced ferroptosis. More importantly, 3-TYP reversed the effect of NAC on the mitochondria ROS under HG treatment, as well as eliminated its following beneficial effects for ferroptosis against HG-stimulated cells. These results reveal that NAC attenuated ferroptosis in DN via maintaining mitochondrial redox homeostasis through activating SIRT3-SOD2-Gpx4 signaling pathway.

Curcumin antagonizes inflammation and autophagy induced by arsenic trioxide through immune protection in duck spleen.

Arsenic is a toxic heavy metal widely found in the natural environment and has adverse effects on the health of waterfowl and human. Curcumin (CUR), a natural pigment of the golden spice turmeric, exhibits excellent anti-tumor, anti-inflammatory and anti-oxidant activities. But the effects of CUR on duck spleen exposed to arsenic remain largely unknown. In this study, 75 ducks were divided randomly into Control, L-ATO, M-ATO, H-ATO and CUR + H-ATO groups to systematically analyze the underlying role of CUR. The results showed that arsenic trioxide (ATO) led to growth retardation of ducks, hyaline degeneration and sparse cell arrangement on their spleen. And in the ATO-exposed ducks, the levels of immunoglobulins (Ig; IgA, IgG, IgM) in the serum and the expression of autophagy-related genes (Atg5, P62, LC3I, LC3II, LC3II/I, Beclin-1) were significantly upregulated compared with the control ducks. Moreover, ATO also activated NF-κB signal pathway and upregulated the expression of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1ß, IL-2, IL-18). Meanwhile, application of CUR alleviated the ATO toxicity with the release of growth inhibition, and the reduced hyaline degeneration and distortion of the spleen capsule. CUR also suppressed ATO-induced NF-κB activation, pro-inflammatory cytokine addition and expression of autophagy-related genes. Overall, these results suggested that CUR might exert a protective effect against ATO-induced immunosuppression in ducks via anti-inflammation and autophagy restoring.

Residue of thiram in food, suppresses immune system stress signals and disturbs sphingolipid metabolism in chickens.

Thiram, a well-known sulfur containing organic compound is frequently and extensively used in agriculture because of high biological activity to control different pests. In certain cases, due to long persistence in the environment pesticides and other environmental contaminants induce undesirable toxic impacts to public health and environment. To ascertain the potential mechanisms of toxicity of thiram on different immune organs of broilers, a total of 100 one-day-old chicks were obtained and randomly divided into two groups including thiram group (50 mg/kg) and untreated control group. Thymus and spleen tissues were collected at the age of 14 days from the experimental birds. At necropsy level, thymus was congested, enlarged and hyperemic while spleen had no obvious lesions. The results on mechanisms (apoptosis and autophagy) of immunotoxicity showed significantly increased expression of bax, caspase3, cytc, ATG5, beclin1 and p62 in spleen of treated mice. Results indicated significantly decreased expression of m-TOR and bcl2 to activate apoptosis and autophagy. The expressions of bax, p53 and m-TOR were up-regulated in the thymus while the expressions of ATG5 and Beclin1 were down-regulated to mediate cell apoptosis and inhibit autophagy. The results on different metabolome investigation showed that the sphingolipid metabolism in the thymus of chicks exposed to thiram was disrupted resulting in up-regulation of metabolites related to cell membrane components such as SM, galactosylceramide and lactosylceramide. The results of our experimental research suggest that thiram can interfere with the sphingolipid metabolism in thymus and angiogenesis, inhibit the proliferation of vascular endothelial cells to induce potential toxic effects in chicken.

Toxicological mechanism of large amount of copper supplementation: Effects on endoplasmic reticulum stress and mitochondria-mediated apoptosis by Nrf2/HO-1 pathway-induced oxidative stress in the porcine myocardium.

Copper (Cu) is an essential micronutrient that is required by all living organisms. However, Cu can also be a potentially toxic metal if excessive dietary supplementation occurs. The current study aimed to investigate the mechanism of Cu toxicity in the cardiomyocytes of large mammal pigs. Here, we used pigs to explore Cu toxicity in the control group (10 mg/kg Cu) and treatment groups (125 mg/kg and 250 mg/kg Cu) for a period of 80 days. Consequently, we identified that large amount intake of Cu led to in oxidative damage, and activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)-mediated antioxidant pathway, indicating an imbalanced redox status in the myocardium. Furthermore, Cu exposure activated endoplasmic reticulum (ER) stress through upregulating levels of glucose-regulated protein 78 (GRP78), c-Jun N-terminal kinase (JNK), glucose-regulated protein 94 (GRP94), X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP). Additionally, mitochondrial fission and fusion homeostasis was disrupted and the copy number of mitochondrial DNA (mtDNA) was reduced under Cu exposure. Furthermore, Cu exposure could induce apoptosis, evidenced by the increased terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling (TUNEL)-positive staining, the upregulated expression levels of Cytoplasm-cytochrome C (Cytc), Bcl-2-associated X protein (Bax), and Cleaved-caspase3, and decreased expression level of B-cell lymphoma-2 (Bcl-2) and Mitochondrial-cytc. In summary, large amount of Cu could trigger Nrf2/HO-1 pathway-mediated oxidative stress, which promotes ER stress and mitochondrial damage pathways, causing apoptosis in cardiomyocytes.

Endoplasmic Reticulum Stress Contributes to Copper-Induced Pyroptosis via Regulating the IRE1α-XBP1 Pathway in Pig Jejunal Epithelial Cells.

Copper (Cu) is a common additive in food products, which poses a potential concern to animal and human health when it is in excess. Here, we investigated the relationship between endoplasmic reticulum (ER) stress and pyroptosis in Cu-induced toxicity of jejunum in vivo and in vitro. In in vivo experiments, excess intake of dietary Cu caused ER cavity expansion, elevated fluorescence signals of GRP78 and Caspase-1, and increased the mRNA and protein expression levels related to ER stress and pyroptosis in pig jejunal epithelium. Simultaneously, similar effects were observed in IPEC-J2 cells under excess Cu treatment. Importantly, 4-phenylbutyric acid (ER stress inhibitor) and MKC-3946 (IRE1α inhibitor) significantly inhibited the ER stress-triggered IRE1α-XBP1 pathway, which also alleviated the Cu-induced pyroptosis in IPEC-J2 cells. In general, these results suggested that ER stress participated in regulating Cu-induced pyroptosis in jejunal epithelial cells via the IRE1α-XBP1 pathway, which provided a novel view into the toxicology of Cu.

Arsenic trioxide-induced autophagy affected the antioxidant capacity and apoptosis rate of chicken hepatocytes.

Arsenic has recently received widespread attention due to its high toxicological effects on multiple animals; however, the mechanism underlying this toxicity is unclear. We investigated the damaging effects of arsenic trioxide (ATO) on hepatocytes and the effects of regulating autophagy on the hepatocyte damage induced by ATO exposure. First, we investigated the effects of ATO exposure (0, 0.6, 1.2, 2.4, and 4.8 µM) on the biochemical function and autophagy of chicken hepatocytes. The findings showed that as the concentration of ATO increased, the lactate dehydrogenase (LDH) concentration increased, more autophagosomes were observed via transmission electron microscopy (TEM), and the gene and protein expression levels of P62, LC3Ⅱ, and Beclin1 increased. Adding N-acetyl-l-cystine (NAC, 1 mM) attenuated autophagy and the hepatocyte damage induced by ATO. Then, we used rapamycin (Rapa) and 3-methylpurine (3-MA) to regulate the autophagy induced by exposure to 4.8 µM ATO and observed changes in the antioxidant capacity and apoptosis rate of chicken hepatocytes. Induction of autophagy reduced ATO-induced hepatocyte apoptosis but caused no significant effect on oxidative stress in chicken hepatocytes. Inhibition of autophagy exacerbated ATO-induced hepatocyte oxidative stress and apoptosis. These findings demonstrate that autophagy plays an important role in ATO-induced cell damage.

The potential risks of herbicide butachlor to immunotoxicity via induction of autophagy and apoptosis in the spleen.

Butachlor being an important member of chloroacetanilide herbicides, is frequently used in agriculture to control unwanted weeds. Exposure to butachlor can induce cancer, human lymphocyte aberration, and immunotoxic effects in animals. The current experimental trial was executed to determine the potential risks of herbicide butachlor to immunotoxicity and its mechanism of adverse effects on the spleen. For this purpose, mice were exposed to 8 mg/kg butachlor for 28 days, and the toxicity of butachlor on the spleen of mice was evaluated. We found that butachlor exposure led to an increase in serum ALB, GLU, TC, TG, and TP and changes in the morphological structure of the spleen of mice. More importantly, results showed that butachlor significantly increased the expression level of ATG-5, decreased the protein expression of LC3B and M-TOR, and significantly decreased the mRNA content of M-TOR and p62. Results revealed that the mRNA contents of APAF-1, CYTC, and CASP-9 related genes were significantly decreased after butachlor treatment. Subsequently, the mRNA levels of inflammatory cytokines (IL-1ß, TNF-α, IL-10) were reduced in the spleen of treated mice. This study suggested that butachlor induce spleen toxicity and activate the immune response of spleen tissue by targeting the CYTC/BCL2/M-TOR pathway and caspase cascading activation of spleen autophagy and apoptosis pathways which may ultimately lead to immune system disorders.

Curcumin alleviates arsenic-induced injury in duck skeletal muscle via regulating the PINK1/Parkin pathway and protecting mitochondrial function.

Arsenic is a well-known environmental pollutant due to its toxicity, which can do harm to animals and human. Curcumin is a polyphenolic compound derived from turmeric, commonly accepted to have antioxidant properties. However, whether curcumin can ameliorate the damage caused by arsenic trioxide (ATO) in duck skeletal muscle remains largely unknown. Therefore, the present study aims to investigate the potential molecular mechanism of curcumin against ATO-induced skeletal muscle injury. The results showed that treating with curcumin could attenuate body weight loss induced by ATO and reduced arsenic content accumulation in the skeletal muscle of duck. Curcumin was also able to alleviated the oxidative stress triggered by ATO, which was manifested by the increase of T-AOC and SOD, and MDA decrease. Moreover, we observed that curcumin could ease mitochondrial damage and vacuolate degeneration of nucleus. Our further investigation found that ATO disrupted normal mitochondrial fission/fusion (Drp1, OPA1, Mfn1/2) and restrained mitochondrial biogenesis (PGC-1α, Nrf1/2, TFAM), while curcumin could promote mitochondrial fusion and activated PGC-1α pathway. Furthermore, curcumin was found that it could not only reduce the mRNA and protein levels of mitophagy (PINK1, Parkin, LC3, p62) and pro-apoptotic genes (p53, Bax, Caspase-3, Cytc), but also increased the levels of anti-apoptotic genes (Bcl-2). In conclusion, curcumin was able to alleviate ATO-induced skeletal muscle damage by improving mitophagy and preserving mitochondrial function, which can serve as a novel strategy to take precautions against ATO toxicity.

Mitochondrial miR-1285 regulates copper-induced mitochondrial dysfunction and mitophagy by impairing IDH2 in pig jejunal epithelial cells.

Copper (Cu), a hazardous heavy metal, can lead to toxic effects on host physiology. Recently, specific mitochondria-localized miRNAs (mitomiRs) were shown to modulate mitochondrial function, but the underlying mechanisms remain undefined. Here, we identified mitomiR-1285 as an important molecule regulating mitochondrial dysfunction and mitophagy in jejunal epithelial cells under Cu exposure. Mitochondrial dysfunction and mitophagy were the important mechanisms of Cu-induced pathological damage in jejunal epithelial cells, which were accompanied by significant increase of mitomiR-1285 in vivo and in vitro. Knockdown of mitomiR-1285 significantly attenuated Cu-induced mitochondrial respiratory dysfunction, ATP deficiency, mitochondrial membrane potential reduction, mitochondrial reactive oxygen species accumulation, and mitophagy. Subsequently, bioinformatics analysis and luciferase reporter assay demonstrated that IDH2 was a direct target of mitomiR-1285. RNA interference of IDH2 dramatically reversed the effect that mitomiR-1285 knockdown relieved mitochondrial dysfunction and mitophagy induced by Cu, and the opposite effect was shown by overexpression of IDH2. Therefore, our results suggested that mitomiR-1285 aggravated Cu-induced mitochondrial dysfunction and mitophagy via suppressing IDH2 expression. These findings identified the important mechanistic connection between mitomiRs and mitochondrial metabolism under Cu exposure, providing a new insight into Cu toxicology.