Molybdenum and Cadmium co-induced the levels of autophagy-related genes via adenosine 5'-monophosphate-activated protein kinase/mammalian target of rapamycin signaling pathway in Shaoxing Duck (Anas platyrhyncha) kidney.

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.

Zhuang J ，Nie G ，Yang F ，Cao H ，Xing C ，Dai X ，Hu G ，Zhang C ... -  《POULTRY SCIENCE》

Molybdenum and cadmium co-exposure induces CaMKKβ/AMPK/mTOR pathway mediated-autophagy by subcellular calcium redistribution in duck renal tubular epithelial cells.

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 (IP3) activities and inositol 1,4,5-trisphosphate receptor (IP3R) 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/IP3/IP3R 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.

Cui T ，Wang X ，Hu J ，Lin T ，Hu Z ，Guo H ，Huang G ，Hu G ，Zhang C ... -  《-》

The activated ATM/AMPK/mTOR axis promotes autophagy in response to oxidative stress-mediated DNA damage co-induced by molybdenum and cadmium in duck testes.

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 H2O2 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.

Pu W ，Chu X ，Guo H ，Huang G ，Cui T ，Huang B ，Dai X ，Zhang C ... -  《-》

In vivo assessment of molybdenum and cadmium co-induced the mRNA levels of heat shock proteins, inflammatory cytokines and apoptosis in shaoxing duck (Anas platyrhyncha) testicles.

Cadmium (Cd) and high dietary intake of molybdenum (Mo) can lead to adverse reactions on animals, but the combined impacts of Mo and Cd on testicle are not clear. To investigate the co-induced toxic effects of Mo and Cd in duck testicles on the mRNA levels of heat shock proteins (HSPs), inflammatory cytokines, and apoptosis. A total of sixty 11-day-old male Shaoxing ducks (Anas platyrhyncha) were randomly divided into 6 groups and testicles were collected on day 120. The mRNA levels of HSPs (HSP60, HSP70, HSP90), inflammatory cytokines (TNF-α, NF-κB, COX-2), and apoptosis genes (Bcl-2, Bak-1, Caspase-3) were determined by real-time quantitative polymerase chain reaction (RT-qPCR), meanwhile the changes of ultrastructural were evaluated. The results showed HSPs mRNA levels were increased in high Mo and Cd groups, however, they were decreased in high dose Mo and Cd co-treated group. In all treatment groups, the mRNA levels of Bak-1 and Caspase-3 were upregulated, and Bcl-2 mRNA level was downregulated, especially in combination groups. The TNF-α, NF-κB, and COX-2 expression in co-exposure groups were higher than those in single groups. Furthermore, the ultrastructural changes showed nuclear deformation, mitochondria hyperplasia and cristaes rupture, and vacuolation in combination groups. Changes of all above factors indicated a possible synergistic relationship between the two elements, and the high expression of HSPs and inflammatory cytokines may play a role in the resistance of testicles toxicity induced by Mo or Cd or both.

Dai X ，Nie G ，Cao H ，Xing C ，Hu G ，Zhang C ... -  《POULTRY SCIENCE》

Cadmium and molybdenum co-exposure triggers autophagy via CYP450s/ROS pathway in duck renal tubular epithelial cells.

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 3CdSO4·8H2O (4.0 μM Cd), (NH4)6Mo7O24·4H2O (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 (H2O2) 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.

Zhang C ，Wang X ，Pi S ，Wei Z ，Wang C ，Yang F ，Li G ，Nie G ，Hu G ... -  《-》