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With the development of Type 1 diabetes mellitus (T1DM), various complications can be caused. Hyperglycemia affects the microenvironment of cardiomyocytes, changes endoplasmic reticulum homeostasis, triggers unfolding protein response and eventually promotes myocardial apoptosis. However, insulin therapy alone cannot effectively combat the complications caused by T1DM. Forty adult beagles were randomly divided into five groups: control group, diabetes mellitus group, insulin group, insulin combined with NAC group, and NAC group. 24-hour blood glucose, 120-day blood glucose, 120-day body weight, and serum FMN content were observed, furthermore, hematoxylin-eosin staining, Periodic acid Schiff reagent staining, and Sirius red staining of the myocardium were evaluated. The protein expressions of GRP78, ATF6, IRE1, PERK, JNK, CHOP, caspase 3, Bcl2, and Bax were detected. Results of the pathological section of myocardial tissue indicated that insulin combined with NAC therapy could improve myocardial pathological injury and glycogen deposition. Additionally, insulin combined with NAC therapy down-regulates the expression of GRP78, ATF6, IRE1, PERK, JNK, CHOP, caspase3, and Bax. These findings suggest that NAC has a phylactic effect on myocardial injury in beagles with T1DM, and the mechanism may be related to the improvement of endoplasmic reticulum stress-induced apoptosis. • A canine model of T1DM was established by the combination of alloxan and streptozotocin. • NAC combined with insulin therapy maintained glucose stability better than insulin therapy alone. • NAC can reduce myocardial injury by regulating ERs and apoptosis in diabetes myocardium. • NAC can be used as an adjuvant drug for the treatment of T1DM to alleviate the complications of T1DM. [ABSTRACT FROM AUTHOR]

Abstract The purpose of this study was to investigate the effects of copper (Cu) on hepatocyte pyroptosis and the relationship between pyroptosis and apoptosis in the mechanisms of Cu toxicity. Primary chicken hepatocytes were cultured in different concentrations of Cu sulfate (CuSO 4) (0, 10, 50, and 100 μM), N-acetylcysteine (NAC) (1 mM), and Z-YVAD-fluoromethylketone (Z-YVAD-FMK) (10 μM) for 24 h, and the combination of Cu and NAC or Z-YVAD-FMK for 24 h. Cellular morphology and function, cell viability, mitochondria membrane potential (MMP), apoptosis rate, mRNA expression of pyroptosis-related and apoptosis-related genes, and Caspase-1, Caspase-3 proteins expression were determined. These results indicated that Cu markedly induced the mRNA expression of pyroptosis-related genes (Caspase-1, IL-1β, IL-18, and NLRP3) and Caspase-1 protein expression. Furthermore, contents of Caspase-1, IL-1β, and IL-18 in the supernatant fluid of culture hepatocytes were significantly increased in hepatocytes. NAC relieved excess Cu-caused the changes of above genes and proteins. Additionally, Z-YVAD-FMK, caspase-1 inhibitor, which attenuated Cu-induced the increased lactic dehydrogenase (LDH), aspartate amino transferase (AST), alanine aminotransferase (ALT) activities. Furthermore, treatment with Cu and Z-YVAD-FMK could down-regulate the mRNA levels of Caspase-3, Bak1, Bax, and CytC and Caspase-3 protein expression, up-regulate the mRNA expression of Bcl2, increase the MMP and reduce cell apoptosis compared to treatment with Cu in hepatocytes. Collectively, these finding evidenced that excess Cu induced pyroptosis by generating ROS in hepatocytes, and the inhibition of Caspase-1-dependent pyroptosis might attenuate Cu-induced apoptosis. Highlights • Cu has hepatotoxicity that causes Caspase-1-dependent pyroptosis in hepatocytes. • Cu induces Caspase-1-dependent pyroptosis by generating excessive ROS in hepatocytes. • Inhibition of Caspase-1-dependent pyroptosis attenuates Cu-induced apoptosis in hepatocytes. [ABSTRACT FROM AUTHOR]

Cu is a metallic element that widely spread over in the environment, which have raised wide concerns about the potential toxic effects and public health threat. The objective of this study aimed to investigate the impression of copper (Cu)-triggered toxicity on mitochondrial dynamic, oxidative stress, and unfolded protein response (UPRmt) in fundic gland of pigs. Weaned pigs were randomly distributed into three groups, fed with different Cu of 10 mg/kg (control group), 125 mg/kg (group I), and 250 mg/kg (group Ⅱ). The trial persisted for 80 days and the fundic gland tissues were collected for further researches. Moreover, the markers participated to mitochondrial dynamic, UPRmt，and oxidative stress in fundic gland were determined. Results revealed that vacuolar degeneration were observed in the treated groups contrast with control group, and the Cu level was boosted with the increasing intake of Cu. Besides that, the levels of CAT, TRX, H 2 O 2 , and G 6 PDH were reduced in group Ⅰ and group Ⅱ, the mRNA levels of NRF2 , HO-1 , SOD-1 , CAT , SOD-2 , GSR , GPX1 , GPX4 , and TRX in the treated groups were promoted contrast to control group. Furthermore, the protein expression of KEAP1 was dramatically decreased, and the protein expression of NRF2 , TRX and HO-1 were markedly enhanced in group Ⅰ and Ⅱ at 80 days. Moreover, the mRNA and protein expression levels of MFN1 , MFN2 , and OPA1 down-regulated and protein level of DRP1 was increased with the adding levels of Cu. Nevertheless, the UPRmt-related mRNA levels of CLPP , HTRA-2 , CHOP , HSP10 , and HSP60 were enhanced dramatically in Cu treatment group compared with control group. In general, our current study demonstrated that excessive absorption of Cu in fundic gland were related with stimulating UPRmt, oxidative stress, and the NRF2 interceded antioxidant defense. These results could afford an updated evidence on molecular theory of Cu-invited toxicity. [Display omitted] • Copper exposure induced fundic gland toxicity. • Copper triggers the NRF2-mediated antioxidant defense. • Copper induced mitochondrial damaged via inhibition of UPRmt. [ABSTRACT FROM AUTHOR]

Copper (Cu), one of the heavy metals, is far beyond the carrying capacity of the environment with Cu mining, industrial wastewater discharging and the use of Cu-containing pesticides. Intaking excess Cu can cause toxic effects on liver, kidney, heart, but few studies report Cu toxicity on brain tissue. It is noteworthy that most toxicity tests are based on rodent models, but large mammals chosen as animal models has no reported. To explore the relationship of the Cu toxicity and mitochondria-mediated apoptosis on hypothalamus in pigs, the content of Cu, histomorphology, mitochondrial related indicators, apoptosis, and AMPK-mTOR signaling pathway were detected. Results showed that Cu could accumulate in hypothalamus and lead to mitochondrial dysfunction, evidenced by the decrease of ATP production, activities of respiratory chain complex I-IV, and mitochondrial respiratory function in Cu-treated groups. Additionally, the genes and proteins expression of Bax, Caspase-3, Cytc in treatment group were higher than control group. Furthermore, the protein level of p-AMPK was enhanced significantly and p-mTOR was declined, which manifested that AMPK-mTOR signaling pathway was activated in Cu-treated groups. In conclusion, this study illuminated that the accumulation of Cu could cause mitochondrial dysfunction, induce mitochondria-mediated apoptosis and activate AMPK-mTOR pathway in hypothalamus. [Display omitted] • Excess Cu accumulation could cause mitochondrial dysfunction in hypothalamus. • Cu could induce mitochondrial-mediated apoptosis in hypothalamus. • AMPK-mTOR pathway could be involved in Cu-induced mitochondrial dysfunction and apoptosis in hypothalamus. [ABSTRACT FROM AUTHOR]

Copper poses huge environmental and public health concerns due to its widespread and persistent use in the past several decades. Although it is well established that at higher levels copper causes nephrotoxicity, the exact mechanisms of its toxicity is not fully understood. Therefore, this experimental study for the first time investigates the potential molecular mechanisms including transcriptomics, metabolomics, serum biochemical, histopathological, cell apoptosis and autophagy in copper-induced renal toxicity in pigs. A total of 14 piglets were randomly assigned to two group (7 piglets per group) and treated with a standard diet (11 mg CuSO 4 per kg of feed) and a high copper diet (250 mg CuSO 4 per kg of feed). The results of serum biochemical tests and renal histopathology suggested that 250 mg/kg CuSO 4 in the diet significantly increased serum creatinine (CREA) and induced renal tubular epithelial cell swelling. Results on transcriptomics and metabolomics showed alteration in 804 genes and 53 metabolites in kidneys of treated pigs, respectively. Combined analysis of transcriptomics and metabolomics indicated that different genes and metabolism pathways in kidneys of treated pigs were involved in glycerophospholipids metabolism and glycosphingolipid metabolism. Furthermore, copper induced mitochondrial apoptosis characterized by increased bax, bak, caspase 3, caspase 8 and caspase 9 expressions while decreased bcl-xl and bcl2/bax expression. Exposure to copper decreased the autophagic flux in terms of increased number of autophagosomes, beclin1 and LC3b/LC3a expression and p62 accumulation. These results indicated that the imbalance of glycosphingolipid metabolism, the impairment of autophagy and increase mitochondrial apoptosis play an important role in copper induced renal damage and are useful mechanisms to understand the mechanisms of copper nephrotoxicity. [Display omitted] • Transcriptomics and metabolomics investigated nephrotoxicity induced by copper in pig. • Compound lipids metabolism was disrupted. • Copper induced mitochondrial apoptosis and decreased the autophagic flux. [ABSTRACT FROM AUTHOR]

Among different synthetic compounds copper (Cu) is persistently and frequently used as growth promoter, antibacterial, antifungal and antiparasitic agent and has become common environmental pollutant. Therefore, this study explores the cardio-toxic effects of control group (10 mg/kg bw Cu) and treatment group (125 and 250 mg/kg bw Cu), and it association with process of autophagy and metabolomics in myocardium of pigs kept in three different experimental treatments for a period of 80 days. The results of serum biochemical parameters showed a significantly increase in creatinine kinase (CK), creatine kinase-MB (CK-MB), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and aspartate aminotransferase (AST) in pigs exposed to 125 mg/kg bw and 250 mg/kg bw Cu. Meanwhile, the severe structural abnormalities in cardiomyocytes were found when exposed to 250 mg/kg Cu at day 80. In addition, the mRNA and proteins (Beclin1, ATG5 and LC3II) expression levels were significantly increased and p62 was significantly decreased in cardiomyocytes exposed to 250 mg/kg Cu at day 80 of the trial. Further, UPLC-QTOF/MS technique showed that 7 metabolites were up-regulated and 37 metabolites were down-regulated in cardiomyocytes after 250 mg/kg Cu treatment, with a principal impact on the metabolic pathways including glycerophospholipid metabolism, one carbon pool by folate, fatty acid elongation and fatty acid degradation, which were related to autophagy. Overall, our study identified the autophagy processes and metabolites in metabolic pathways in Cu-induced myocardium injury, which provided useful evidence of myocardium toxicity caused by Cu exposure via metabolomics and multiple bioanalytic methods. [Display omitted] • Autophagy caused by Cu in myocardia was evaluated by metabolomics. • A total of 44 differential metabolites were detected in Cu-induced autophagy. • Metabolisms differences played a key role in Cu-induced autophagy in myocardia. • Excess intake of Cu could induced autophagy in myocardia. • Cu can pose an ecological risk to pig. [ABSTRACT FROM AUTHOR]

To explore the effects of copper (Cu) on energy metabolism and AMPK-mTOR pathway-mediated autophagy in kidney, a total of 240 one-day-old broiler chickens were randomized into four equal groups and fed on the diets with different levels of Cu (11, 110, 220, and 330 mg/kg) for 49 d. Results showed that excess Cu could induce vacuolar degeneration and increase the number of autophagosomes in kidney, and the adenosine triphosphate (ATP) level and mRNA levels of energy metabolism-related genes were decreased with the increasing dietary Cu level. Moreover, immunohistochemistry and immunofluorescence showed that the positive expressions of Beclin1 and LC3-II were mainly located in cytoplasm of renal tubular epithelial cells and increased significantly with the increasing levels of Cu. The mRNA levels of Beclin1, Atg5, LC3-I, LC3-II, Dynein and the protein levels of Beclin1, Atg5, LC3-II/LC3-I and p-AMPKα1/AMPKα1 were markedly elevated in treated groups compared with control group (11 mg/kg Cu). However, the mRNA and protein levels of p62 and p-mTOR/mTOR were significantly decreased with the increasing levels of Cu. These results suggest that impaired energy metabolism induced by Cu may lead to autophagy via AMPK-mTOR pathway in kidney of broiler chickens. Image 1 • Excessive intake of copper could induce energy metabolism disorder in kidney. • Excessive intake of copper could induce autophagy in kidney. • Excessive intake of copper could activate AMPK-mTOR pathway in kidney. • Impaired energy metabolism may contribute to copper-induced autophagy through AMPK-mTOR signaling pathway. [ABSTRACT FROM AUTHOR]

Copper (Cu) is a necessary trace mineral due to its biological activity. Excessive Cu can induce inflammatory response in humans and animals, but the underlying mechanism is still unknown. Here, 240 broilers were used to study the effects of excessive Cu on oxidative stress and NF-κB-mediated inflammatory responses in immune organs. Chickens were fed with diet containing different concentrations of Cu (11, 110, 220, and 330 mg of Cu/kg dry matter). The experiment lasted for 49 days. Spleen, thymus, and bursa of Fabricius (BF) on day 49 were collected for histopathological observation and assessment of oxidative stress status. Additionally, the mRNA and protein levels of NF-κB and inflammatory cytokines were also analyzed. The results indicated that excess Cu could increase the number and area of splenic corpuscle as well as the ratio of cortex and medulla in thymus and BF. Furthermore, excessive Cu intake could decrease activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); but increase contents of malondialdehyde (MDA), TNF-α, IL-1, IL-1β; up-regulate mRNA levels of TNF-α, IFN-γ, IL-1, IL-1β, IL-2, iNOS, COX-2, NF-κB and protein levels of TNF-α, IFN-γ, NF-κB, p–NF–κB in immune organs. In conclusion, excessive Cu could cause pathologic changes and induce oxidative stress with triggered NF-κB pathway, and might further regulate the inflammatory response in immune organs of chicken. Image 1 • Excessive dietary Cu could induce oxidative stress and inflammatory responses in immune organs of chickens. • Excessive dietary Cu might regulate the inflammatory response in immune organs of chicken via NF-κB pathway. • Excessive dietary Cu could cause pathologic changes in immune organs of chicken. [ABSTRACT FROM AUTHOR]

Copper (Cu) is an essential trace element for most organisms. However, excessive Cu can be highly toxic. The purpose of this study was to elucidate the mechanism underlying Cu toxicity in the kidneys of rats after treatment with CuCl 2 (15 [control], 30, 60, or 120 mg/kg in the diet) for 180 days. Histological and ultrastructural changes, antioxidant enzyme activity, and the mRNA and protein levels of apoptosis and autophagy-related genes were measured. The results showed that Cu exposure led to significant accumulation of copper in kidneys and disorganized kidney morphology. The activities of total anti-oxidation capacity (T-AOC) and superoxide dismutase (SOD) in the kidneys decreased significantly, while the malondialdehyde (MDA) content increased. Furthermore, excessive Cu markedly upregulated the expression of autophagy and apoptosis-related genes (LC3A , LC3B , ATG-5 , Beclin-1 , Caspase3 , CytC , P53 , Bax), but downregulated the expression of P62, mTOR and BCL-2. Moreover, the LC3B/LC3A, ATG-5, Beclin-1, P53, Caspase3 proteins were up-regulated while P62 was down-regulated in the kidney tissues of the treatment groups. Overall, these findings provide strong evidence that excess Cu can trigger autophagy and apoptosis via the mitochondrial pathway by inducing oxidative stress in rat kidneys. Image 1 • Excessive intake of Cu has nephrotoxicity that causes damage in kidneys. • Excessive intake of Cu could induce oxidative stress in kidneys. • Excessive intake of Cu could induce autophagy in kidneys. • Excessive intake of Cu could induce apoptosis in kidneys. [ABSTRACT FROM AUTHOR]

The purpose of this research was to discuss the effects of copper (Cu)-induced toxicity on oxidative stress and autophagy in hypothalamus of broilers. In this study, 240 one-day-old broilers were randomly divided into 4 groups and the contents of dietary Cu in 4 groups were 11 mg/kg (control group), 110 mg/kg (group I), 220 mg/kg (group II), and 330 mg/kg (group III). The experiment lasted for 49 days and the hypothalamus tissues were collected for histological observation and detection of Cu content. Additionally, the indicators related to oxidative stress in hypothalamus were determined. Moreover, the mRNA expression levels of autophagy-related genes and the protein expression levels of Beclin1, LC3-II/LC3-I, and p62 in hypothalamus were measured. Results showed that the treated groups were observed vacuolar degeneration in hypothalamus compared to control group, and the Cu content in hypothalamus was increased with the increase of dietary Cu. Furthermore, the activities of SOD, CAT, T-AOC were increased in group I and group II and then decreased in group III, and the content of MDA and the mRNA levels of Nrf2, HO-1, SOD-1, CAT, GCLC, GCLM, and GST in treated groups were elevated compared to control group. Moreover, the mRNA expression levels of Beclin1, Atg5, LC3-I, LC3-II and the protein expression levels of Beclin1 and LC3-II/LC3-I up-regulated significantly with the increasing levels of Cu. However, the mRNA expression levels of p62 and mTOR and the protein expression level of p62 down-regulated remarkably. Taken together, our present study evidenced that excessive intake of Cu could induce oxidative stress and autophagy in hypothalamus of broilers. • Excessive intake of Cu has neurotoxicity that causes damage in hypothalamus. • Excessive intake of Cu could induce oxidative stress in hypothalamus. • Excessive intake of Cu could induce autophagy in hypothalamus. [ABSTRACT FROM AUTHOR]