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Aflatoxin B1 (AFB1) is a stable mycotoxin that contaminates animal feed on a large scale and causes severe damage to intestinal cells, induces inflammation and stimulates autophagy. Transient receptor potential mucolipin subfamily 1 (TRPML1) is a regulatory factor of autophagy, but the underlying mechanisms of TRPML1-mediated autophagy in AFB1 intestine toxicity remain elucidated. In the present study, AFB1 (0, 5, 10 μg/mL) was shown to reduce cell viability, increase reactive oxygen species (ROS) accumulation and apoptosis rate. Additionally, AFB1 caused structural damage to mitochondria and lysosomes and increased autophagosomes numbers. Furthermore, AFB1 promoted Ca 2+ release by activating the TRPML1 channel, stimulated the expression of autophagy-related proteins, and induced autophagic flux blockade. Moreover, pharmacological inhibition of autophagosome formation by 3-methyladenine attenuated AFB1-induced apoptosis by downregulating the levels of TRPML1 and ROS, whereas blockade of autophagosome-lysosomal fusion by chloroquine alleviated AFB1-induced apoptosis by upregulating TRPML1 expression and exacerbating ROS accumulation. Intriguingly, blocking AFB1-induced autophagic flux generated ROS- and TRPML1-dependent cell death, as shown by the decreased apoptosis in the presence the free radical scavenger N-Acetyl-L-cysteine and the TRPML1 inhibitor ML-SI1. Overall, these results showed that AFB1 promoted apoptosis of IPEC-J2 cells by disrupting autophagic flux through activation of the ROS/TRPML1 pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Excessive molybdenum (Mo) and cadmium (Cd) have toxic effects on animals. However, hepatotoxicity co-induced by Mo and Cd in ducks is still unclear. To evaluate the effects of Cd and Mo co-exposure on autophagy by nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant defense and endoplasmic reticulum stress (ERS) in duck livers, 40 healthy 7-day-old ducks were randomly assigned to 4 groups and fed diets containing different doses of Mo and/or Cd for 16 weeks, respectively. The results verified that Mo and/or Cd induced oxidative stress via decreasing glutathione peroxidase (GSH-Px), catalase (CAT), and total-superoxide dismutase (T-SOD) activities and increasing hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) concentrations; inhibited Nrf2 axis by downregulating the pathway-related genes and proteins expression levels, and activated ERS through upregulating the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2a (eIF2a), inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) pathway-related genes and proteins expression levels, which triggered autophagy via increasing autophagosomes, light chain 3 (LC3) puncta, LC3A, LC3B, autophagy-related gene 5 (Atg5), Bcl-2-interacting protein (Beclin-1) mRNA levels and Beclin-1, microtubule-associated protein light chain 3 II/I (LC3II/LC3I) protein levels, decreasing Dynein, p62, mammalian target of rapamycin (mTOR) mRNA levels and p62 protein level. Additionally, the changes in Mo and Cd group were the most obvious. Briefly, our study reveals that autophagy induced by Mo and/or Cd may be associated with the activation of crosstalk between Nrf2-mediated antioxidant defense response and ERS in duck livers. Mo and Cd may aggravate toxic damage to the liver., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cadmium (Cd) is a toxic heavy metal that accumulates in the brain and causes a series of histopathological changes. Selenium (Se) exerts a crucial function in protecting damage caused by toxic heavy metals, but its potential mechanism is rarely studied. The main purpose of this study is to explore the protective effects of Se on Cd-induced oxidative stress and autophagy in rabbit cerebrum. Forty rabbits were randomly divided into four groups and treated as follows: Control group, Cd (1 mg/kg⋅BW) group, Se (0.5 mg/kg⋅BW) group and Cd (1 mg/kg⋅BW)+Se (0.5 mg/kg⋅BW) group, with 30 days feeding management. Our results suggested that Se treatment significantly suppressed the Cd-induced degenerative changes including cell necrosis, vacuolization, and atrophic neurons. In addition, Se decreased the contents of MDA and H 2 O 2 and increased the activities of CAT, SOD, GST, GSH and GSH-Px, alleviating the imbalance of the redox system induced by Cd. Furthermore, Cd caused the up-regulation of the mRNA levels of autophagy-related genes (ATG3, ATG5, ATG7, ATG12 and p62), AMPK (Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag2, Prkag3) and Nrf2 (Nrf2, HO-1 and NQO1) signaling pathway, and the expression levels of LC3II/LC3I, p-AMPK/AMPK, Beclin-1, Nrf2 and HO-1 proteins, which were alleviated by Se, indicated that Se inhibited Cd-induced autophagy and Nrf2 signaling pathway activation. In conclusion, our study found that Se antagonized Cd-induced oxidative stress and autophagy in the brain by generating crosstalk between AMPK and Nrf2 signaling pathway., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Increasing evidence indicates autophagy and apoptosis are involved in the toxicity mechanism of heavy metals. Our previous studies showed that cadmium (Cd) could induce autophagy and apoptosis in duck kidneys in vivo, nevertheless, the interaction between them has yet to be elucidated. Herein, the cells were either treated with 3CdSO 4 ·8H 2 O (0, 1.25, 2.5, 5.0 μM Cd) or/and 3-methyladenine (3-MA) (2.5 μM) for 12 h and the indictors related autophagy and apoptosis were detected to assess the correlation between autophagy and apoptosis induced by Cd in duck renal tubular epithelial cells. The results demonstrated that Cd exposure notably elevated intracellular and extracellular Cd contents, the number of autophagosomes and LC3 puncta, up-regulated LC3A, LC3B, Beclin-1, Atg5 mRNA levels, and Beclin-1 and LC3II/LC3I protein levels, down-regulated mTOR, p62 and Dynein mRNA levels and p62 protein level. Additionally, autophagy inhibitor 3-MA decreased Beclin-1, LC3II/LC3I protein levels and increased p62 protein level. Moreover, co-treatment with Cd and 3-MA could notably elevate Caspase-3, Cyt C, Bax, and Bak-1 mRNA levels, Caspase-3 and cleaved Caspase-3 protein levels, and cell apoptotic rate as well as cell damage, decreased mitochondrial membrane potential (MMP), Bcl-2 mRNA level and the ratio of Bcl-2 to Bax compared to treatment with Cd alone. Overall, these results indicate Cd exposure can induce autophagy in duck renal tubular epithelial cells, and inhibition of autophagy might aggravate Cd-induced apoptosis through mitochondria-mediated pathway., (Copyright © 2020 Elsevier Inc. All rights reserved.)

To investigate Molybdenum (Mo) and Cadmium (Cd) co-induced the levels of autophagy-related genes via AMPK/mTOR signaling pathway in Shaoxing Duck (Anas platyrhyncha) kidney, 60 healthy 11-day-old ducks were randomly divided into 6 groups, which were treated with Mo or/and Cd at different doses on the basal diet for 120 d. Kidney samples were collected on day 120 to determine the mRNA expression levels of adenosine 5'-monophosphate (AMP)-activated protein kinase α1 (AMPKα1), mammalian target of rapamycin (mTOR), Beclin-1, autophagy-related gene-5 (Atg5), microtubule-associated protein light chain A (LC3A), microtubule-associated protein light chain B (LC3B), sequestosome-1, and Dynein by real-time quantitative polymerase chain reaction. Meanwhile, ultrastructural changes of the kidney were observed. The results indicated that the mTOR and P62 mRNA expression levels were significantly downregulated, but the Atg5 and Beclin-1 mRNA levels were remarkably upregulated in all treated groups compared to control group, and their changes were greater in joint groups. Additionally, compared to control group, the Dynein mRNA expression level was apparently downregulated in co-treated groups, the LC3B, LC3A, and AMPKα1 expression levels were dramatically upregulated in single treated groups and they were not obviously different in co-treated groups. Ultrastructural changes showed that Mo and Cd could markedly increase the number of autophagosomes. Taken together, it suggested that dietary Mo and Cd might induce autophagy via AMPK/mTOR signaling pathway in duck kidney, and it showed a possible synergistic relationship between the 2 elements., (© 2019 Poultry Science Association Inc.)

Cadmium (Cd) and excess molybdenum (Mo) are multiorgan toxic, but the detrimental impacts of Cd and/or Mo on poultry have not been fully clarified. Thence, a 16‐week sub‐chronic toxic experiment was executed with ducks to assess the toxicity of Cd and/or Mo. Our data substantiated that Cd and Mo coexposure evidently reduced GSH‐Px, GSH, T‐SOD, and CAT activities and elevated H2O2 and MDA concentrations in myocardium. What is more, the study suggested that Cd and Mo united exposure synergistically elevated Fe2+ content in myocardium and activated AMPK/mTOR axis, then induced ferroptosis by obviously upregulating ACSL4, PTGS2, and TFRC expression levels and downregulating SLC7A11, GPX4, FPN1, FTL1, and FTH1 expression levels. Additionally, Cd and Mo coexposure further caused excessive ferritinophagy by observably increasing autophagosomes, the colocalization of endogenous FTH1 and LC3, ATG5, ATG7, LC3II/LC3I, NCOA4, and FTH1 expression levels. In brief, this study for the first time substantiated that Cd and Mo united exposure synergistically induced ferroptosis and excess ferritinophagy by AMPK/mTOR axis, finally augmenting myocardium injure in ducks, which will offer an additional view on united toxicity between two heavy metals on poultry. [ABSTRACT FROM AUTHOR]

Excessive amounts of molybdenum (Mo) and cadmium (Cd) are toxicant, but their combined immunotoxicity are not clearly understood. To estimate united impacts of Mo and Cd on pyroptosis and autophagy by PI3K/AKT axis in duck spleens, Mo or/and Cd subchronic toxicity models of ducks were established by feeding diets with different dosages of Mo or/and Cd. Data show that Mo or/and Cd cause oxidative stress by increasing MDA concentration, and decreasing T‐AOC, CAT, GSH‐Px and T‐SOD activities, restrain PI3K/AKT axis by decreasing PI3K, AKT, p‐AKT expression levels, which evokes pyroptosis and autophagy by elevating IL‐1β, IL‐18 concentrations and NLRP3, Caspase‐1, ASC, GSDME, GSDMA, NEK7, IL‐1β, IL‐18 expression levels, promoting autophagosomes, LC3 puncta, Atg5, LC3A, LC3B, LC3II/LC3I and Beclin‐1 expression levels, and reducing expression levels of P62 and Dynein. Furthermore, the variations of abovementioned indexes are most pronounced in co‐treated group. Overall, results reveal that Mo or/and Cd may evoke pyroptosis and autophagy by PI3K/AKT axis in duck spleens. The association of Mo and Cd exacerbates the changes. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is detrimental to animals, but nephrotoxic effects of Cd on duck have not been fully elucidated. To evaluate the impacts of Cd on Ca homeostasis and autophagy via PLC‐IP3‐IP3R pathway, primary duck renal tubular epithelial cells were exposed to 2.5 μM and 5.0 μM Cd, and combination of 5.0 μM Cd and 10.0 μM 2‐APB or 0.125 μM U‐73122 for 12 h (U‐73122 pretreated for 1 h). These results evidenced that Cd induced [Ca2+]c overload mainly came from intracellular Ca store. Cd caused [Ca2+]mit and [Ca2+]c overload with [Ca2+]ER decrease, elevated Ca homeostasis related factors (GRP78, GRP94, CRT, CaN, CaMKII, and CaMKKβ) expression, PLC and IP3 activities and IP3R expression, but subcellular Ca2+ redistribution was reversed by 2‐APB. PLC inhibitor U‐73122 dramatically relieved the changes of the above indicators induced by Cd. Additionally, U‐73122 obviously reduced the number of autophagosomes and LC3 accumulation spots, Atg5, LC3A, LC3B mRNA levels and LC3II/LC3I, Beclin‐1 protein levels induced by Cd, and markedly elevated p62 mRNA and protein levels. Overall, the results verified that Cd induced [Ca2+]c overload mainly originated from ER Ca2+ release mediated by PLC‐IP3‐IP3R pathway, then triggered autophagy in duck renal tubular epithelial cells. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is an environmental pollutant that has an enormous influence on agricultural production, but selenium (Se) can alleviate its toxicity. The present study aimed to illustrate the effects of Se on Cd‐induced heart injury. All 40 rabbits were randomly divided into four groups: control group, Se [0.5 mg kg−1·body weight (BW)] group, Cd (1 mg kg−1·BW) group, and Se + Cd group. After 30 days of feeding, morphological changes, the levels of oxidative stress and myocardial enzyme, the content of cardiac troponin T, programmed cell death (pyroptosis, autophagy and apoptosis), and PI3K/AKT/PTEN transduction capacity were observed. The results showed that Cd destroyed the physiological balance of trace elements and caused myocardial damage, increased the cardiac oxidative damage and led to programmed cell death. Coadministration of Se prominently ameliorated histological lesions and improved cardiac function of hearts in Cd‐induced rabbits. Furthermore, Se exerted detoxification and oxidation resistance, maintained trace element homeostasis, and alleviated the changes of mRNA and protein levels of pyroptosis‐, autophagy‐ and apoptosis‐controlling factors and PI3K/AKT/PTEN signal molecules caused by Cd. In conclusion, Se might protect against Cd‐induced pyroptosis, autophagy and apoptosis by interfering with PI3K/AKT/PTEN signaling in heart. [ABSTRACT FROM AUTHOR]

Fatty liver haemorrhagic syndrome (FLHS) is characterized by hepatic rupture and haemorrhage leading to sudden death in laying hens. Resveratrol (Res) is a natural polyphenol with antioxidant and anti-inflammatory effects that can ameliorate chronic liver disease. The aim of this study was to investigate the improved effect of Res on the altered expression of autophagy and apoptosis-related genes in laying hens with FLHS. A total of 144 healthy 150-day-old laying hens were randomly divided into four groups: control group (standard diet), HELP group (high-energy-low-protein (HELP) diet), HELP + Res group (HELP diet with 400 mg/kg Res) and Res group (standard diet with 400 mg/kg Res). Histopathological lesions of the liver and the mRNA levels of Beclin-1, Atg5, Atg7, p62, Bcl-2, Bax and Caspase-3 on days 40, 80, and 120 were measured. The results showed that lipid accumulation and hepatocyte damage in the HELP group were more serious than those in the HELP + Res group. The mRNA levels of Beclin-1, Atg5, Atg7, and Bcl-2 in the HELP and HELP + Res groups were strikingly declined (P < 0.01) compared to the control group, and their mRNA levels were markedly higher in HELP group than those in the HELP + Res group (P < 0.05). Additionally, the mRNA levels of p62, Bax and Caspase-3 were significantly increased in the HELP and HELP + Res groups (P < 0.01 or P < 0.05), but their mRNA levels in the HELP group were higher than those in the HELP + Res group (P < 0.05). Collectively, FLHS could induce severe lipid accumulation, abnormal mRNA levels of liver autophagy and apoptosis-related genes. Res as a dietary supplement could attenuate these abnormal changes. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) and excess molybdenum (Mo) have multiple organ toxicity, and testis is one of their important target organs, but the reproductive toxicity of Mo and Cd combined treatment is still unclear. To explore the effects of Mo and Cd co-exposure on DNA damage and autophagy from the insight of ATM/AMPK/mTOR axis in duck testes, we randomly assigned 40 healthy 8-day-old ducks to control, Mo (100 mg/kg Mo), Cd (4 mg/kg Cd), and Mo + Cd groups for 16 weeks. Results found that Mo and/or Cd exposure caused trace elements imbalance, oxidative stress with a decrease in the activities of GSH-Px, CAT, T-SOD and GSH content, an increase in the concentrations of H 2 O 2 and MDA and pathological damage. Additionally, Mo and/or Cd markedly raised DNA damage-related factors expression levels and 8-OHdG content, caused G1/S arrest followed by decreasing CDK2 and Cyclin E protein levels and increasing CDK1 and Cyclin B protein levels, and activated ATM/AMPK/mTOR axis by enhancing p-ATM/ATM, p-AMPK/AMPK and reducing p-mTOR/mTOR protein levels, eventually triggered autophagy by elevating LC3A, LC3B, Atg5, Beclin-1 mRNA levels and LC3II/LC3I, Beclin-1 protein levels and reducing P62, Dynein, mTOR mRNA levels and P62 protein level. Moreover, these changes were most apparent in the combined group. Altogether, the results reveal that autophagy caused by Mo and/or Cd may be associated with activating the DNA damage-mediated ATM/AMPK/mTOR axis in duck testes, and Mo and Cd co-exposure exacerbates these changes. [Display omitted] • Mo and/or Cd caused trace elements imbalance and oxidative stress to duck testes. • Mo and/or Cd triggered DNA damage-mediated autophagy through ATM/AMPK/mTOR axis. • Mo and Cd co-treatment aggravated these changes in duck testes. [ABSTRACT FROM AUTHOR]

Excess molybdenum (Mo) and cadmium (Cd) are widespread environmental and industrial metal pollutants. To evaluate the combined effects of Mo and Cd on calcium homeostasis and autophagy in duck kidneys. 160 healthy 7-day-old ducks (Anas platyrhyncha) were randomized into 4 groups and given to a basic diet, adding various doses of Mo or/and Cd for 16 weeks. On the 4th, 8th, 12th and 16th weeks, kidney tissues were collected. The study exhibited that Mo or/and Cd caused histological abnormality, reduced the activities of Ca2+ ATPase, Mg2+ ATPase, Na+-K+ ATPase and Ca2+-Mg2+ ATPase, K and Mg contents, and increased Na and Ca contents, upregulated CaMKKβ, CaMKIIɑ, CaN, IP 3 R, GRP78, GRP94, CRT mRNA levels and CaMKIIɑ, CaN, IP 3 R protein levels. Moreover, exposure to Mo or/and Cd notably promoted the amount of autophagosomes and LC3II immunofluorescence, upregulated AMPKα1, ATG5, Beclin-1, LC3A, LC3B mRNA levels and Beclin-1, LC3II/LC3I protein levels, downregulated mTOR, Dynein, P62 mRNA levels and P62 protein level. The changes of above indicators in combined group were more obvious. Overall, the results suggest that Mo and Cd co-exposure may can synergistically induce nephrotoxicity via causing calcium homeostasis disorder and autophagy in ducks. [Display omitted] • The co-toxicity of Mo and Cd was assessed in duck kidneys. • Mo and Cd could synergistically induce Ca2+ homeostasis imbalance. • Mo and Cd could synergistically induce Ca2+ homeostasis imbalance and autophagy. [ABSTRACT FROM AUTHOR]

Pyroptosis and autophagy are two different biological processes that determine cell fates. Our previous studies revealed that pyroptosis and autophagy were involved in cytotoxicity co-induced by molybdenum (Mo) and cadmium (Cd) in duck renal tubular epithelial cells, but crosstalk between them is unclear. Hence, the cells were treated with 500.0 μM Mo, 4.0 μM Cd, 10.0 μM Z-YVAD-fluoromethylketone (YVAD), 2.5 μM 3-methyladenine (3-MA) and 10.0 μM chloroquine (CQ) alone or in combination for 12 h (CQ for the last 4 h). Under Mo and Cd co-stress, data evidenced that YVAD addition decreased the number of autophagosomes, LC3 puncta, and AMPKα-1, Atg5, Beclin-1, LC3A, LC3B mRNA levels and LC3-II/LC3-I, Beclin-1 protein levels, and increased p62 expression levels. Besides, both 3-MA and CQ addition increased NLRP3, Caspase-1, NEK7, ASC, GSDMA, GSDME, IL-1β, IL-18 mRNA levels, NLRP3, Caspase-1 p20, ASC, GSDMD protein and ROS levels, and NO, LDH, IL-1β, IL-18 releases. Collectively, our results revealed that pyroptosis and autophagy co-induced by Mo and Cd were interrelated in duck renal tubular epithelial cells, and inhibiting pyroptosis might attenuate Mo and Cd co-induced autophagy, but inhibiting autophagy might promote Mo and Cd co-induced pyroptosis. [Display omitted] • The co-toxicity of Mo and Cd was assessed in duck renal tubular epithelial cells. • Crosstalk between pyroptosis and autophagy co-induced by Mo and Cd was explored. • Autophagy inhibitor both 3-MA and CQ might promote pyroptosis co-induced by Mo and Cd. • Pyroptosis inhibitor YVAD might attenuate autophagy co-induced by Mo and Cd. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) and excessive molybdenum (Mo) are detrimental to animals, but the combined nephrotoxic impacts of Cd and Mo on duck are still unclear. To evaluate the combined impacts of Cd and Mo on autophagy via Cytochrome P450s (CYP450s)/reactive oxygen species (ROS) pathway, duck renal tubular epithelial cells were treated with 3CdSO 4 ·8H 2 O (4.0 μM Cd), (NH 4) 6 Mo 7 O 24 ·4H 2 O (500.0 μM Mo), butylated hydroxy anisole (BHA) (100.0 μM) and combination of Cd and Mo or Cd, Mo and BHA for 12 h, and combined cytotoxicity was investigated. The results indicated that Mo or/and Cd induced CYP1A1, CYP1B1, CYP2C9, CYP3A8 and CYP4B1 mRNA levels, decreased superoxide dismutase (SOD), catalase (CAT) activities and glutathione peroxidase (GSH-Px) content, and increased malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2) contents. Besides, Mo or/and Cd elevated the number of autophagosome and microtubule-associated protein light chain 3 (LC3) puncta, upregulated mRNA levels of Beclin-1, LC3A, LC3B, Atg5 and adenosine 5′-monophosphate (AMP)-activated protein kinase α1 (AMPKα-1), inhibited Dynein, p62 and mammalian target of rapamycin (mTOR) mRNA levels, increased Beclin-1 and LC3II/LC3I protein levels. Moreover, the changes of these factors in Mo and Cd co-treated groups were more apparent. Additionally, BHA could efficiently alleviate the changes of above these indicators co-induced by Mo and Cd. Overall, these results manifest Cd and Mo co-exposure may synergistically trigger autophagy via CYP450s/ROS pathway in duck renal tubular epithelial cells. Unlabelled Image • The combined effects of Cd and Mo on autophagy by CYP450s/ROS pathway were assessed. • Mo or/and Cd induced autophagy via CYP450s/ROS pathway. • Mo and Cd exhibited synergic interactions. • BHA inhibited autophagy co-induced by Mo and Cd via CYP450s/ROS pathway. [ABSTRACT FROM AUTHOR]

Excessive molybdenum (Mo) has adverse effects on animals. To elucidate the effects of autophagy on Mo-induced nephrotoxicity, the duck renal tubular epithelial cells were cultured in medium in absence and presence of (NH 4) 6 Mo 7 O 24.4H 2 O (0, 480, 720, 960 μM Mo), 3-Methyladenine (3-MA) (2.5 μM), and the combination of Mo and 3-MA for 12 h. After 12 h exposure, the MDC staining, morphologic observation, LC3 puncta, cell viability, autophagy-related genes mRNA and proteins levels, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) level, antioxidant indices, mitochondrial membrane potential (MMP), mitochondrial mass, mitochondrial respiratory control ratio (RCR) and oxidative phosphorylation rate (OPR) were determined. The results showed that excessive Mo exposure significantly elevated the number of autophagosome and LC3 puncta, upregulated Beclin-1, Atg5, LC3A and LC3B mRNA levels, and LC3II/LC3I and Beclin-1 protein levels, decreased mTOR, p62 and Dynein mRNA levels and p62 protein level. Besides, co-treatment with Mo and 3-MA dramatically increased LDH release, ROS level, hydrogen peroxide (H 2 O 2) and malondialdehyde (MDA) contents as well as cell dam age, reduced cell viability, the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), MMP, mitochondrial mass, mitochondrial RCR and OPR compared to treatment with Mo alone. Taken together, these results suggest that excessive Mo exposure can induce autophagy in duck renal tubular epithelial cells, inhibition of autophagy aggravates Mo-induced mitochondrial dysfunction by regulating oxidative stress. • Toxicity of excessive Mo was assessed in duck renal tubular epithelial cells. • Excessive Mo could induce autophagy. • Inhibition of autophagy exacerbated oxidative stress induced by excessive Mo. • Inhibition of autophagy aggravated excessive Mo-induced mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Excessive molybdenum (Mo) and cadmium (Cd) are toxic environmental pollutants. Our previous research confirmed excessive Mo and Cd co-induced calcium homeostasis disorder and autophagy in duck kidneys, but how calcium ion (Ca2+) regulates autophagy is unclear. The results revealed that the Mo- and/or Cd-induced cytosolic Ca2+ concentration ([Ca2+] c) increase mainly came from intracellular calcium stores. Mo and/or Cd caused mitochondrial Ca2+ content ([Ca2+] mit) and [Ca2+] c increase with endoplasmic reticulum (ER) Ca2+ content ([Ca2+] ER) decrease and upregulated calcium homeostasis-related factor expression levels, but 2-Aminoethoxydiphenyl borate (2-APB) reversed subcellular Ca2+ redistribution. Increased Phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP 3) activities and inositol 1,4,5-trisphosphate receptor (IP 3 R) expression level were observed in Mo- and/or Cd-treated cells, which was reversed by the PLC inhibitor U-73122. 2-APB and 1,2-Bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) addition mitigated [Ca2+] c and autophagy (variations in microtubule-associated protein light chain 3 (LC3), LC3B-II/LC3B-I, autophagy related 5 (ATG5), sequestosome-1(P62), programmed cell death-1 (Beclin-1) and Dynein expression levels, LC3 puncta, autophagosomes and acid vesicle organelles) under Mo and/or Cd treatment, respectively, while thapsigargin (TG) had the opposite impacts. Additionally, the calmodulin-dependent protein kinase kinase β (CaMKKβ) inhibitor STO-609 reversed the increased CaMKKβ, adenosine 5′-monophosphate-activated protein kinase (AMPK), Beclin-1, and LC3B-II/LC3B-I protein expression levels and reduced mammalian target of rapamycin (mTOR) and P62 protein expression levels in Mo- and/or Cd-exposed cells. Collectively, the results confirmed that [Ca2+] c overload resulted from PLC/IP 3 /IP 3 R pathway-mediated ER Ca2+ release, and then activated autophagy by the CaMKKβ/AMPK/mTOR pathway in Mo- and/or Cd-treated duck renal tubular epithelial cells. Excessive molybdenum (Mo) and/or cadmium (Cd) destroy intracellular calcium homeostasis, promote endoplasmic reticulum (ER) calcium ion (Ca2+) release and cytosolic Ca2+ concentration ([Ca2+] c) overload in duck renal tubular epithelial cells, triggering autophagy through calmodulin-dependent protein kinase kinase β (CaMKKβ)/ adenosine 5′-monophosphate-activated protein kinase (AMPK)/ mammalian target of rapamycin (mTOR) pathway. [Display omitted] • The co-toxicity of Mo and Cd was assessed in duck renal tubular epithelial cells. • Mo and/or Cd caused subcellular calcium redistribution. • [Ca2+] c overload induced by Mo and/or Cd came from ER Ca2+ release. • Mo and/or Cd induced ER Ca2+ release by PLC/IP 3 /IP 3 R pathway. • Mo and/or Cd induced autophagy by CaMKKβ/AMPK/mTOR pathway. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the crosstalk between oxidative stress and autophagy through the ROS/HO-1/NQO1 pathway caused by copper (Cu). Duck renal tubular epithelial cells were treated in Cu sulfate (CuSO 4) (0, 100 and 200 μM) for 12 h, and in the combination of CuSO 4 (200 μM) and reactive oxygen species (ROS) scavenger (butyl hydroxyanisole, BHA, 100 μM), or HO-1 inhibitor (zinc protoporphyrin, ZnPP, 10 μM) for 12 h. Results revealed that Cu could significantly elevate the levels of intracellular ROS, superoxide dismutase, hydrogen peroxide, malondialdehyde, glutathione, simultaneously reduce catalase and glutathione peroxidase levels, and upregulate HO-1, SOD-1, CAT, NQO1, GCLM mRNA levels and HO-1, SOD-1 protein levels. Additionally, Cu could observably increase the number of autophagosomes, acidic vesicle organelles (AVOs) and LC3 puncta; upregulate mRNA levels of mTOR, Beclin-1, ATG7, ATG5, ATG3, LC3II and protein levels of Beclin-1, LC3II/LC3I, downregulate LC3I mRNA level. Both treatments with BHA and ZnPP could significantly alleviate the changes of antioxidant indexes levels and ROS accumulation, reduce the increase of the number of autophagosomes, AVOs and LC3 puncta, and mitigate the above changed oxidative stress and autophagy related mRNA and protein levels induced by Cu. In summary, our findings indicated that excessive Cu could induce oxidative stress and autophagy by activating the ROS/HO-1/NQO1 pathway, and inhibition of HO-1 might attenuate Cu-induced oxidative stress and autophagy in duck renal tubular epithelial cells. Unlabelled Image • Copper (Cu) induced oxidative stress in duck renal tubular epithelial cells. • Autophagy occurred in duck renal tubular epithelial cells induced by Cu. • High levels of heme oxygenase-1 (HO-1) aggravated Cu-induced oxidative stress. • Inhibition of HO-1 might attenuate Cu-induced autophagy. • Cu caused oxidative stress and autophagy through ROS/HO-1/NQO1 pathway. [ABSTRACT FROM AUTHOR]