Long-term Cu exposure alters CYP450s activity and induces jejunum injury and apoptosis in broilers.

Copper (Cu) is an essential trace element that plays a crucial role in numerous physiopathological processes related to human and animal health. In the poultry industry, Cu is used to promote growth as a feed supplement, but excessive use can lead to toxicity on animals. Cytochrome P450 enzymes (CYP450s) are a superfamily of proteins that require heme as a cofactor and are essential for the metabolism of xenobiotic compounds. The purpose of this study was to explore the influence of exposure to Cu on CYP450s activity and apoptosis in the jejunum of broilers. Hence, we first simulated the Cu exposure model by feeding chickens diets containing different amounts of Cu. In the present study, histopathological observations have revealed morphological damage to the jejunum. The expression levels of genes and proteins of intestinal barrier markers were prominently downregulated. While the mRNA expression level of the gene associated with CYP450s was significantly increased. Additionally, apoptosis-related genes and proteins (Bak1, Bax, Caspase-9, Caspase-3, and CytC) were also significantly augmented by excessive Cu, while simultaneously decreasing the expression of Bcl-2. It can be concluded that long-term Cu exposure affects CYP450s activity, disrupts intestinal barrier function, and causes apoptosis in broilers that ultimately leads to jejunum damage.

Co-exposure of arsenic and polystyrene-nanoplastics induced kidney injury by disrupting mitochondrial homeostasis and mtROS-mediated ferritinophagy and ferroptosis.

Arsenic (As) and polystyrene nanoplastics (PSNPs) co-exposure induced biotoxicity and ecological risks have attracted wide attention. However, the combined effects of As and PSNPs on the kidney and their underlying mechanisms of toxicities remain to be explored. Here, we investigated the effects of As and PSNPs co-exposure on structure and function in mice kidney, and further explored the possible mechanisms. In this study, we identified that co-exposure to As and PSNPs exhibited conspicuous renal structural damage and pathological changes, accompanied by renal tissue fibrosis (increased protein expression of Collagen I and α-SMA and deposition of collagen fibers), whereas alone exposure to As or PSNPs does not exhibit nephrotoxicity. Subsequently, our results further showed that combined action of As and PSNPs induced mitochondrial oxidative damage and impaired mitochondrial dynamic balance. Furthermore, co-treatment with As and PSNPs activated NCOA4-mediated ferritinophagy and ferroptosis in mice kidney and TCMK-1 cells, which was confirmed by the changes in the expression of ferritinophagy and ferroptosis related indicators (NCOA4, LC3, ATG5, ATG7, FTH1, FTL, GPX4, SLC7A11, FSP1, ACSL4 and PTGS2). Meaningfully, pretreatment with the mtROS-targeted scavenger Mito-TEMPO significantly attenuated As and PSNPs co-exposure induced mitochondrial damage, ferritinophagy and ferroptosis. In conclusion, these findings demonstrated that mtROS-dependent ferritinophagy and ferroptosis are important factors in As and PSNPs co-exposure induced kidney injury and fibrosis. This study provides a new insight into the study of combined toxicity of nanoplastics and heavy metal pollutants.

Arsenic and polystyrene-nano plastics co-exposure induced testicular toxicity: Triggers oxidative stress and promotes apoptosis and inflammation in mice.

Co-existing of polystyrene-nano plastics (PSNPs) and arsenic (As) in the environment caused a horrendous risk to human health. However, the potential mechanism of PSNPs and As combination induced testicular toxicity in mammals has not been elucidated. Therefore, we first explore the testicular toxicity and the potential mechanism in male Kunming mice exposed to As or/and PSNPs. Results revealed that compared to the As or PSNPs group, the combined group showed more significant testicular toxicity. Specifically, As and PSNPs combination induced irregular spermatozoa array and blood-testis barrier disruption. Simultaneously, As and PSNPs co-exposure also exacerbated oxidative stress, including increasing the MDA content, and down-regulating expression of Nrf-2, HO-1, SOD-1, and Trx. PSNPs and As combination also triggered testicular apoptosis, containing changes in apoptotic factors (P53, Bax, Bcl-2, Cytc, Caspase-8, Caspase-9, and Caspase-3). Furthermore, co-exposed to As and PSNPs aggravated inflammatory damage characterized by targeted phosphorylation of NF-κB and degradation of I-κB. In summary, our results strongly confirmed As + PSNPs co-exposure induced the synergistic toxicity of testis through excessive oxidative stress, apoptosis, and inflammation, which could offer a new sight into the mechanism of environmental pollutants co-exposure induced male reproductive toxicity.

Effect of arsenic and copper in kidney of mice: Crosstalk between Nrf2/ Keap1 pathway in apoptosis and pyroptosis.

Arsenic (As) and copper (Cu) are two common contaminants in the environment. When organisms are exposed to As or/ and Cu in large quantities or for sustained periods, oxidative stress is induced, adversely affecting kidney function. However, the molecular mechanisms involved in As or/ and Cu-induced nephrotoxicity remain elusive. In this experiment, wild-type C57BL/6 and Nrf2-knockout mice (n = 24 each) were exposed to arsenic trioxide and copper chloride alone or in combination. Our research findings indicate that exposure to As or/ and Cu can activate the Nrf2 antioxidant pathway by upregulating the levels of Nrf2, HO-1, CAT, and downregulating the level of Keap1, thereby reducing As or/ and Cu-induced oxidative stress. Meanwhile, exposure induced kidney cell pyroptosis and apoptosis by promoting the expression of NLRP3 inflammasomes and Caspase-3, which peaked in mice co-treated with As and Cu. Subsequently, we investigated its role in As or/ and Cu-induced kidney injury by knocking out Nrf2. Our results show that after knocking out Nrf2, the expression of antioxidant factors CAT and HO-1 significantly decreased. Based on the low antioxidant capacity after Nrf2 knockout, the levels of NLRP3 inflammasome, GSDMD, and Caspase1 were significantly upregulated after exposure to As and Cu, indicating more severe cellular pyroptosis. In addition, the level of Caspase3-mediated apoptosis was also more severe. Taken together, there is crosstalk between Nrf2-mediated antioxidant capacity and apoptosis/ pyroptosis induced by exposure to As or/ and Cu. Depletion of Nrf2 alters its antioxidant capacity, ultimately leading to more severe apoptosis, pyroptosis, and nephrotoxicity.

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.

Arsenic causes mitochondrial biogenesis obstacles by inhibiting the AMPK/PGC-1α signaling pathway and also induces apoptosis and dysregulated mitophagy in the duck liver.

Arsenic is a dangerous metalloid-material which is known to cause liver injury in many animals and humans. However, little is known about the underlying mechanism of arsenic-induced hepatotoxicity in poultry. This study was executed to systematically investigate the potential role of mitochondrial biogenesis, mitophagy and apoptosis in duck hepatotoxicity caused by arsenic. Results showed that the body weight and liver coefficient of duck had distinct changed after arsenic-exposure, and the arsenic content in serum and liver also increased significantly in a dose-dependent manner. Meanwhile, histopathological examination and metabolomics results showed that arsenic-exposure caused severe steatosis and metabolism disorder in liver tissues. Furthermore, arsenic-exposure significantly inhibited AMPK/PGC-1α-mediated mitochondrial biogenesis, determined by the ultrastructure observation and down-regulation of p-AMPKα/AMPKα, PGC-1α, NRF1, NRF2, TFAM, TFB1M, TFB2M and COX-Ⅳ expression levels. Besides, arsenic-treatment obviously increased the levels of mitophagy (PINK1, Parkin, LC3, P62) and pro-apoptotic (Caspase-3, Caspase-9, Cleaved Caspase-3, Cytc, Bax, P53) indexes, and simultaneously resulted in reductions in anti-apoptosis index (Bcl-2). Overall, our findings provided evidences that arsenic-induced duck hepatotoxicity may be caused by a combination of impaired mitochondrial biosynthesis, mitophagy, and mitochondrial-dependent apoptosis. To our knowledge, this is the first report to systematically investigate the potential mechanism of arsenic-induced hepatotoxicity in poultry.

Arsenic and antimony co-induced nephrotoxicity via autophagy and pyroptosis through ROS-mediated pathway in vivo and in vitro.

Arsenic (As) and antimony (Sb) are commonly accumulated environmental pollutants that often coexist in nature and cause serious widespread biological toxicity. To investigate the nephrotoxicity induced by As and Sb in detail, we explored the mechanism by which As and Sb cotreatment induced autophagy and pyroptosis in vivo and in vitro. In this study, mice were treated with 4 mg/kg arsenic trioxide (ATO) or/and 15 mg/kg antimony trichloride (SbCl3) by intragastric intubation for 60 days. TCMK-1 cells were treated with ATO (12.5 µM), SbCl3 (25 µM) or a combination of As and Sb for 24 h. The results of the in vivo experiment demonstrated that As or/and Sb exposure could induce histopathological changes in the kidneys, and increase the levels of biochemical indicators of nephrotoxicity. In addition, As and Sb can co-induce oxidative stress, which further activate autophagy and pyroptosis. In an in vitro experiment, As and/or Sb coexposure increased ROS generation and decreased MMP. Moreover, the results of related molecular experiments further confirmed that As and Sb coactivated autophagy and pyroptosis. In conclusion, our results indicated that As and Sb co-exposure could cause autophagy and pyroptosis via the ROS pathway, and these two metals might have a synergistic effect on nephrotoxicity.

Protective effects of curcumin on ATO-induced nephrotoxicity in ducks in relation to suppressed autophagy, apoptosis and dyslipidemia by regulating oxidative stress.

Arsenic trioxide (ATO) has been known as common environmental pollution, and is deemed to a threat to global public health. Curcumin (Cur) is a phytoconstituent, which has been demonstrated to have antioxidant effects. In the current experiment, we investigated the efficacy of Cur against ATO-induced kidney injury and explored the potential molecular mechanisms that have not yet been fully elucidated in ducks. The results showed that treatment with Cur attenuated ATO-induced body weight loss, reduced the content of ATO in the kidney, and improved ATO-induced kidney pathological damage. Cur also remarkably alleviated the ascent of ATO-induced MDA level and activated the Nrf2 pathway. Using the TEM, we found Cur relieved mitochondrial swelling, autolysosomes generating and nuclear damage. Simultaneously, Cur was found that it not only significantly reduced autophagy-related mRNA and protein levels (mTOR, LC3-Ⅰ, LC3-Ⅱ, Atg-5, Beclin1, Pink1 and Parkin) and but also decreased apoptosis-related mRNA and protein expression levels (cleaved caspase-3, Cytc, p53 and Bax). Furthermore, through nontargeted metabolomics analysis, we observed that lipid metabolism balance was disordered by ATO exposure, while Cur administration alleviated the disturbance of lipid metabolism. These results showed ATO could induce autophagy and apoptosis by overproducing ROS in the kidney of ducks, and Cur might relieve excessive autophagy, apoptosis and disturbance of lipid metabolism by regulating oxidative stress. Collectively, our findings explicate the potential therapeutic value of Cur as a new strategy to a variety of disorders caused by ATO exposure.

Arsenic (III) and/or Antimony (III) induced disruption of calcium homeostasis and endoplasmic reticulum stress resulting in apoptosis in mice heart.

Arsenic (As) and antimony (Sb) are known as an environmental contaminant with cardiotoxicity properties. The endoplasmic reticulum (ER) is the largest calcium reservoir in the cell, and its calcium homeostasis disorder plays a vital role in endoplasmic reticulum stress (ERS) and apoptosis. The objective of this study was to investigate whether As and Sb induced apoptosis via endoplasmic reticulum stress (ERS) linked to calcium homeostasis disturbance. In this study, thirty-two adult mice were gavage-fed daily with As2O3 (4 mg/kg), SbCl3 (15 mg/kg) and co-treat with SbCl3 (15 mg/kg) and As2O3 (4 mg/kg) daily for 60 days. It was observed that As or/and Sb caused histopathological lesions and ER expansion of the heart. Meanwhile, the gene expression of ER Ca2+ release channels (RyR2 and IP3R) and calmodulin-dependent protein kinase II (CaMKII) increased while the levels of mRNA and protein of ER Ca2+ uptake channel (SERCA2) downregulated significantly compared to the controls. Then, As or/and Sb induced ERS and triggered the ER apoptotic pathway by activating unfolded protein response (UPR)-associated genes ((PERK, ATF6, IRE1, XBP1, JNK, GRP78), and apoptosis-related genes (Caspase12, Caspase3, p53, CHOP). Above indicators in As + Sb group became more severe than that of As group and Sb group. Overall, our results proved that the cardiotoxicity caused by As or/and Sb might be concerning disturbing calcium homeostasis, which induced apoptosis through the ERS pathway.

Long-term exposure to copper induces autophagy and apoptosis through oxidative stress in rat kidneys.

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 CuCl2 (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.

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.

Multi-omics reveals the protective effects of curcumin against AFB1-induced oxidative stress and inflammatory damage in duckling intestines.

Aflatoxin B1 (AFB1) is the most prevalent and toxic class of aflatoxins, which is considered a significant risk factor for food safety. Curcumin, a phytoconstituent with anti-inflammatory and antioxidant properties, has potential therapeutic value for intestinal inflammatory diseases. In this study, the duckling model susceptible to AFB1 was selected for toxicity testing, aiming to explore the effect of curcumin on AFB1 enterotoxicity and its possible mechanism of action. The results showed that curcumin promoted the growth and development of ducklings and mitigated the changes in morphology and permeability serological index (DAO and D-LA) after AFB1 exposure. Curcumin also mitigated AFB1-induced oxidative stress by activating the Nrf2 pathway, and ameliorated intestinal inflammation by inhibiting the NF-κB/IκB signaling pathway and boosting intestinal autophagy. In terms of gut flora and their metabolites, we found that curcumin supplementation significantly increased the intestinal flora's abundance index and diversity index compared to the AFB1 group, mitigating the decline in the abundance of Actinobacteria and the rise in that of harmful bacteria Clostridia. Furthermore, untargeted metabolomic analysis revealed that the protective effect of curcumin on the intestine was mainly through the regulation of AFB1-induced disorders of lipid metabolism, involving linoleic acid metabolism, α-linolenic acid metabolism, and glycerolipid metabolism. Overall, the enteroprotective effects of curcumin may be of significant value in the future for treating chronic AFB1 poisoning and also provide new therapeutic ideas for other mycotoxicosis.

Arsenic induced neurotoxicity in the brain of ducks: The potential involvement of the gut-brain axis.

BACKGROUND: Arsenic is a widely distributed ecotoxic pollutant that has been found to cause neurotoxicity in a variety of species. Gut-brain axis is a two-way information network between the gut microbiome and the brain, which is closely related to organismal health. However, the role of the gut-brain axis in arsenic-induced neurotoxicity remains largely unknown. METHODS: In order to explore whether there is a relationship between brain and gut microbiota of meat ducks, we performed molecular biological detection including RT-qPCR and Western blot, as well as morphological detection including, HE staining and immunohistochemistry. Meanwhile, intestinal contents were analyzed using 16 S ribosomal RNA gene sequencing and analysis RESULTS: In this study, we investigated whether arsenic trioxide (ATO) can activate the gut microbiome-brain axis to induce intestinal and brain injury. The results showed that ATO-exposure disrupted the diversity balance of intestinal microbiota and integrity and injured the intestinal structure. ATO-exposure also reduced the number of glycogen and goblet cells in the duodenum. In addition, exposure to ATO caused intestinal inflammatory injury by activating NF-κB signaling pathway and promoting the expression of its target genes. Meanwhile, the tight junction-related proteins (ZO-1, occludin) of gut and brain were reduced by ATO exposure. Furthermore, results also revealed that ATO-exposure induced brain injury, including neuronal cell vacuolization and reduced numbers of neuronal cells in the cortex and hippocampus. Remarkably, ATO-exposure also disrupted neurotransmitter levels. Additionally, our further molecular mechanism study revealed that ATO-exposure increased the expression of autophagy and apoptosis related mRNA and proteins levels in the brain tissues. CONCLUSION: Altogether, these findings provide a new insight into that ATO-exposure induced intestinal injury and aggravated neurotoxicity via the gut-brain axis.

Curcumin attenuates aflatoxin B1-induced ileum injury in ducks by inhibiting NLRP3 inflammasome and regulating TLR4/NF-κB signaling pathway.

Aflatoxin B1 (AFB1) is a widespread toxic contamination in feed for animals. The primary active component of turmeric, curcumin (Cur), is an antioxidant and an anti-inflammatory. However, it is yet unknown how AFB1 affects the intestinal epithelial barrier and whether Cur acts as a protective mechanism when exposed to AFB1. Here, we explored the mechanism of AFB1-induced intestinal injury from intestinal epithelial barrier, inflammation, pyroptosis, and intestinal flora, and evaluated the protective role of Cur. We found that AFB1 caused weight loss and intestinal morphological damage that is mainly characterized by shortened intestinal villi, deepened crypts, and damaged intestinal epithelium. Exposure to AFB1 decreased the expression of Claudin-1, MUC2, ZO-1, and Occludin and increased the expression of pyroptosis-related factors (NLRP3, GSDMD, Caspase-1, IL-1ß, and IL-18) and inflammation-related factors (TLR4, NF-κB, IκB, IFN-γ, and TNF-α). Furthermore, ileal gut microbiota was altered, and simultaneously, the Lactobacillus abundance was decreased. The gut microbiota interacts with a wide range of physiologic functions and disease development in the host through its metabolites, and disturbances in gut microbial metabolism can cause functional impairment of the ileum. Meanwhile, Cur can ameliorate histological ileum injuries and intestinal flora disturbance caused by AFB1. We found that Cur reversed the effects of AFB1 through modulating both NLRP3 inflammasome and the TLR4/NF-κB signaling pathway. In conclusion, AFB1 can induce inflammatory damage and pyroptosis in duck ileum, while Cur has obviously protective effects on all the above damages.

Diagnosis and surgical management of testicular seminoma in captive giant panda (Ailuropoda melanoleuca).

A captive adult male giant panda (Guangzhou Chimelong Safari Park, CHINA) presented with azoospermia and enlarged left testicle. A tentative diagnosis of testicular neoplasia was made, and confirmed as testicular seminoma cases by testicular ultrasound, computed tomography (CT), testicular biopsy, and tumor marker examination findings. Based on the diagnostic results, the treatment of choice was surgical resection of the testicular tumor under general anesthesia. And the histopathological findings of the excised neoplasm were consistent with those of testicular seminoma. In addition, no tumor recurrence was observed after surgery, which indicates that our surgical and post-operative treatments were effective. The surgical treatment adopted in this case report is safe for patients and provides the best solution for the diagnosis and treatment of giant panda testicular seminoma. To our knowledge this is the first detailed report of surgical resection of testicular seminoma in the giant panda.

Arsenic Trioxide Triggers Mitochondrial Dysfunction, Oxidative Stress, and Apoptosis via Nrf 2/Caspase 3 Signaling Pathway in Heart of Ducks.

Arsenic is a common environmental pollutant and poses a serious threat to human and animal health. In this study, we used the ducks to mimic arsenic trioxide (ATO) exposure and investigated the mechanism of cardiac toxicity. The results indicated that ATO inhibited the body and organ growth of ducks, led to an increase in LDH content, and caused obvious deformity, ischemia infarction. It is found that ATO exacerbated the swell of mitochondrial and the contraction of cell nuclei in the heart of ducks through transmission electron microscopy (TEM). ATO also induced an increase in MDA content; inhibited the activation of the Nrf 2 pathway; downregulated the expression of mRNA and protein of Nrf 2, HO-1, and SOD-1; and upregulated the expression of mRNA and protein of Keap 1. At the same time, ATO induced apoptosis which not only upregulated the expression levels of mRNA and proteins (Caspase 3, Cyt-C, P53, Bax) but also decreased the mRNA and protein expression level of Bcl-2. These results indicated that ATO can lead to oxidative stress and apoptosis in the heart of ducks. In general, our research shows that ATO triggers mitochondrial dysfunction, oxidative stress, and apoptosis via Nrf 2/Caspase 3 signaling pathway in the heart of ducks.

Long-term oral tribasic copper chloride exposure impedes cognitive function and disrupts mitochondrial metabolism by inhibiting mitophagy in rats.

Copper (Cu) is an essential micronutrient element that commonly acted as a feed additive and antimicrobial in agricultural production. Tribasic copper chloride (TBCC) is a relatively new dietary Cu source, and its exposure directly or indirectly affects the safety of animals and ecological environment, thus posing a potential risk to human health. Cu overexposure would produce toxic reactive oxygen species (ROS) that may have toxic effects on the host, but the mechanism of neurotoxicity remains unclear. Herein, to explore the effects of long-term TBCC-induced neurotoxicity, 150 male Sprague-Dawley rats were randomly allocated and treated with different doses of TBCC, and the cortical and hippocampus tissues were harvested at 0, 6, and 12 weeks after treatment. Morris Water Maze (MWM) test showed that excessive intake of TBCC could induce cognitive dysfunction in rats. Moreover, after treatment with 160 mg/kg Cu (276 mg/kg TBCC) for 12 weeks, pathological changes were observed in the cortex and hippocampus, and the number of Nissl bodies decreased significantly in the hippocampus. Additionally, mitochondrial structure was significantly altered and neuronal mitochondrial fusion/fission equilibrium was disrupted in 80 mg/kg and 160 mg/kg Cu groups at 12 weeks. With an increase in TBCC dose and treatment time, the number of mitophagosomes and the expression of mitophagy-related genes were significantly decreased after initially increasing. Furthermore, metformin (Met) and 3-methyladenine (3-MA) were used to regulate the level of mitophagy to further explore the mechanism of Cu-induced nerve cell injury in vitro., and it found that mitophagy activator (Met) would increase mitochondrial fission, while mitophagy inhibitors (3-MA) would aggravate mitochondrial metabolic disorders by promoting mitochondrial fusion and inhibiting mitochondrial division. These results indicate that long-term oral TBCC could impede cognitive function and disrupts mitochondrial metabolism by inhibiting mitophagy, providing an insightful perspective on the neurotoxicity of dietary TBCC.

Cuproptosis is involved in copper-induced hepatotoxicity in chickens.

Copper (Cu) is an essential trace element, but it is also a ubiquitous environmental pollutant that threatens public health. Cuproptosis is a recently discovered cell death mode that unlike other programmed cell death, characterized by proteotoxic stress due to lipoylated protein aggregation and iron-sulfur cluster protein loss. Chickens as a high-trophic-level non-mammalian vertebrate that easily absorb and accumulate copper from the environment and food, but it is unclear whether the underlying molecular mechanisms that cause their hepatotoxicity under natural copper stress are related to cuproptosis. Therefore, we established animal models of chickens with different concentrations of copper exposure to dissect the role and mechanism of cuproptosis in chicken hepatotoxicity under natural copper stress. Our histopathological and biochemical results demonstrated that the liver structure with copper-treated exhibited dose-dependent damage. Meanwhile, copper treatment also dramatically increased serum and liver copper content and activated the expression of the membrane-associated copper transporter ATP7B. Furthermore, we found that Cu-exposure significantly increased the MDA content, and reduced the levels of T-AOC and SOD in serum and liver. Additionally, we found that the mRNA and protein levels of FDX1 were significantly upregulated in the 220 and 330 mg/kg Cu-treated groups. In our further studies, we found that copper did not alter protein levels of DLAT and DLST in chicken liver, but significantly increased Lipoylated-DLAT levels and oligomerization of Lipoylated-DLAT in the 330 mg/kg Cu-treatment group. Overall, we identified that FDX1-mediated protein lipoylation and proteotoxic stress indeed participate in copper-induced hepatotoxicity in chickens. Our results present novel insight into the pathogenesis of copper-induced hepatotoxicity in chickens and provide data to support filling in the role of cuproptosis in birds.

Effect of Copper Exposure on the Cholesterol Metabolism in Broiler Liver.

Copper (Cu) is a kind of widely used dietary supplement in poultry production, and a common environmental pollutant at the same time. Excess Cu exposure has been reported to accumulate in the liver and induce cytotoxicity, but the effect of Cu toxicity on hepatic cholesterol metabolism is still uncertain. Herein, we aimed to reveal the effect of excess Cu on the liver and primary hepatocytes of broilers at various concentrations. We found that 110 mg/kg Cu supplement remarkably increased blood cholesterol levels by detecting serum TC, LDL-C, and HDL-C in the broilers, while there was no significant difference in 220 and 330 mg/kg Cu supplements. In addition, high Cu exposure resulted in severe hepatic steatosis and hepatic cord derangement in the broilers. Oil red O staining of primary hepatocytes showed that Cu treatment caused intracellular neutral lipid accumulation. However, the hepatic TC content indicated a downward trend in both liver tissues and hepatocytes after Cu exposure. Furthermore, the expression of cholesterol metabolism-related indicators (SREBP2, HMGCR, LDLR, and CYP7A1) was notably decreased in the Cu-treated groups. While the expression of the key enzyme of cholesterol esterification (ACAT2) did not change significantly. Taken together, our findings preliminarily revealed excess Cu-induced hepatic cholesterol metabolism dysfunction, providing a deeper understanding of the molecular mechanisms of Cu-induced hepatotoxicity.

Mitochondrial miR-12294-5p regulated copper-induced mitochondrial oxidative stress and mitochondrial quality control imbalance by targeted inhibition of CISD1 in chicken livers.

Copper (Cu) is hazardous metal contaminant, which induced hepatotoxicity is closely related to mitochondrial disorder, but exact regulatory mechanism has not yet been revealed. Mitochondrial microRNAs (mitomiRs) are a novel and critical regulator of mitochondrial function and mitochondrial homeostasis. Hence, this study revealed the impact of Cu-exposure on mitomiR expression profiles in chicken livers, and further identified mitomiR-12294-5p and its target gene CISD1 as core regulators involved in Cu-induced hepatotoxicity. Additionally, our results showed that Cu-exposure induced mitochondrial oxidative damage, and mitochondrial quality control imbalance mediated by mitochondrial dynamics disturbances, mitochondrial biogenesis inhibition and abnormal mitophagy flux in chicken livers and primary chicken embryo hepatocytes (CEHs). Meaningfully, we discovered that inhibition of the expression of mitomiR-12294-5p effectively alleviated Cu-induced mitochondrial oxidative stress and mitochondrial quality control imbalance, while the up-regulation of mitomiR-12294-5p expression exacerbated Cu-induced mitochondrial damage. Simultaneously, the above Cu-induced mitochondrial damage can be effectively rescued by the overexpression of CISD1, while knockdown of CISD1 dramatically reverses the mitigating effect that inhibition of mitomiR-12294-5p expression on Cu-induced mitochondrial oxidative stress and mitochondrial quality control imbalance. Overall, these results suggested that mitomiR-12294-5p/CISD1 axis mediated mitochondrial damage is a novel molecular mechanism involved in regulating Cu-induced hepatotoxicity in chickens.