Molybdenum and Cadmium Co-induce Pyroptosis via Inhibiting Nrf2-Mediated Antioxidant Defense Response in the Brain of Ducks.

Excess molybdenum (Mo) and cadmium (Cd) are harmful to animals, but the neurotoxic mechanism co-induced by Mo and Cd is unclear. To estimate the effects of Mo and Cd co-exposure on pyroptosis by nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant defense response in duck brains, 40 healthy 7-day-old ducks were randomly assigned to 4 groups and fed diet supplemented with Mo or/and Cd for 16 weeks, respectively. Results showed that Mo or/and Cd markedly increased Mo and Cd contents; decreased iron (Fe), copper (Cu), zinc (Zn), and selenium (Se) contents, elevated malondialdehyde (MDA) content; and decreased total-antioxidant capacity (T-AOC), total-superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities accompanied by pathological damage in brain. Additionally, Mo or/and Cd inhibited Nrf2 pathway via decreasing Nrf2, CAT, SOD1, glutathione S-transferase (GST), hemeoxygenase-1 (HO-1), NAD (P) H:quinone oxidoreductase 1 (NQO1), glutamate-cysteine ligase catalytic subunit (GCLC), and modifier subunit (GCLM) mRNA levels and Nrf2 protein level, which induced pyroptosis through upregulating nucleotide oligomerization domain-like receptor protein-3 (NLRP3), apoptosis-associated speck-like protein (ASC), gasdermin A (GSDMA), gasdermin E (GSDME), interleukin-1ß (IL-1ß), interleukin-18 (IL-18), Caspase-1, NIMA-related kinase 7 (NEK7) mRNA levels and NLRP3, Caspase-1 p20, gasdermin D (GSDMD), ASC protein levels and IL-1ß, and IL-18 contents. Besides, the changes of these indicators were most apparent in the Mo and Cd co-treated group. Collectively, the results certificated that Mo and Cd might synergistically induce pyroptosis via inhibiting Nrf2-mediated antioxidant defense response in duck brains, whose mechanism is closely related to Mo and Cd accumulation.

Cadmium and molybdenum co-induce pyroptosis and apoptosis via the PTEN/PI3K/AKT axis in the livers of Shaoxing ducks (Anas platyrhynchos).

Cadmium (Cd) and excessive molybdenum (Mo) have adverse impacts on animals. However, the hepatotoxicity co-induced by Cd and Mo in ducks has not been fully elucidated. In order to explore the impacts of Cd and Mo co-exposure on pyroptosis and apoptosis by the PTEN/PI3K/AKT pathway in the livers of ducks, 40 healthy 7-day-old Shaoxing ducks (Anas platyrhynchos) were randomly assigned into 4 groups, and Cd or/and Mo were added to the basic diet per kilogram (kg): control group (0 mg Mo and 0 mg Cd), Mo group (100 mg Mo), Cd group (4 mg Cd), and Mo + Cd group (100 mg Mo and 4 mg Cd), with 16 weeks feed management. Results signified that Cd or/and Mo caused trace element imbalance, liver function and histomorphological abnormalities in the duck liver, and activated the PTEN/PI3K/AKT pathway through increasing PTEN mRNA and protein levels, reducing PI3K, AKT mRNA and p-AKT/AKT protein levels, which triggered pyroptosis and apoptosis via increasing Caspase-1, NLRP3, NEK7, ASC, GSDME, GSDMA, IL-1ß and IL-18 mRNA levels, Caspase-1 p20, NLRP3, ASC and GSDMD protein levels, and IL-1ß and IL-18 contents, and increasing Bak-1, Bax, Cyt C and Caspase-3 mRNA levels and cleaved Caspase-3/Caspase-3 protein level, and downregulating Bcl-2 mRNA level and the ratio of Bcl-2 to Bax, respectively. Overall, the results illustrate that pyroptosis and apoptosis induced by Cd or/and Mo may be associated with activating the PTEN/PI3K/AKT pathway in the livers of ducks. There may be a synergy between these two elements.

Involvement of calcium homeostasis and unfolded protein response in autophagy co-induced by molybdenum and cadmium in duck (Anas platyrhyncha) brain.

Excess molybdenum (Mo) and cadmium (Cd) are harmful to animals, but neurotoxicity caused by Mo and Cd co-exposure in ducks is yet unknown. To assess joint impacts of Mo and Cd on autophagy via calcium homeostasis and unfolded protein response (UPR) in duck brain, 40 healthy 7-day-old ducks (Anas platyrhyncha) were assigned to 4 groups at random and fed diets supplemented with different doses of Mo or/and Cd for 16 weeks, respectively. Brain tissues were excised for experiment. Results exhibited that Mo or/and Cd disturbed calcium homeostasis by decreased ATPase activities and increased calcium (Ca) content, and upregulated calcium homeostasis-related factors Ca2+/CAM-dependent kinase IIɑ (CaMKIIɑ), calcineurin (CaN), inositol-1,4,5-trisphosphate receptor (IP3R), and calreticulin (CRT) expression levels. Meanwhile, the upregulation of UPR-related factor expression levels indicated that Mo or/and Cd activated UPR. Moreover, Mo or/and Cd triggered autophagy through promoting the number of autophagosomes and LC3II immunofluorescence intensity and altering autophagy key factor expression levels. Correlation analysis showed that there were obvious connections among Ca2+ homeostasis, endoplasmic reticulum (ER) stress, and autophagy induced by Mo or/and Cd. Thence, it can be speculated that autophagy initiated by Mo or/and Cd may be associated with interfering Ca2+ homeostasis and triggering UPR.

In vivo assessment of molybdenum and cadmium co-induce nephrotoxicity via causing calcium homeostasis disorder and autophagy in ducks (Anas platyrhyncha).

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 CaMKKß, CaMKIIɑ, CaN, IP3R, GRP78, GRP94, CRT mRNA levels and CaMKIIɑ, CaN, IP3R 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.

New insights into crosstalk between pyroptosis and autophagy co-induced by molybdenum and cadmium in duck renal tubular epithelial cells.

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-1ß, IL-18 mRNA levels, NLRP3, Caspase-1 p20, ASC, GSDMD protein and ROS levels, and NO, LDH, IL-1ß, 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.

In vivo assessment of molybdenum and cadmium co-induce nephrotoxicity via NLRP3/Caspase-1-mediated pyroptosis in ducks.

Excessive molybdenum (Mo) and cadmium (Cd) cause toxic effects on animals, but their joint effects on pyroptosis in kidney of ducks remain unclear. 160 healthy 7-day-old ducks were randomly divided into four groups which were fed with basal diet containing different dosages of Mo or/and Cd for 16 weeks. On the 4th, 8th, 12th and 16th weeks, kidney tissue and serum were collected. The results showed that Mo or/and Cd could significantly elevate their contents in kidney, disturb the homeostasis of trace elements, cause renal function impairment and histological abnormality, and oxidative stress as accompanied by increasing hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations and decreasing glutathione peroxidase (GSH-Px), catalase (CAT) and total-superoxide dismutase (T-SOD) activities. Simultaneously, Mo or/and Cd could markedly increase interleukin-1ß (IL-1ß), interleukin-18 (IL-18) contents and the expression levels of pyroptosis-related genes (NOD-like receptor protein-3 (NLRP3), Caspase-1, apoptosis-associated speck-like protein (ASC), NIMA-related kinase 7 (NEK7), Gasdermin A (GSDMA), Gasdermin E (GSDME), IL-1ß and IL-18) and proteins (NLRP3, Caspase-1 p20, ASC and Gasdermin D (GSDMD)). Moreover, the changes of above these indicators were more obvious in combined group. Taken together, the results illustrate that Mo and Cd might synergistically lead to oxidative stress and induce pyroptosis via NLRP3/Caspase-1 pathway, whose mechanism is somehow related to Mo and Cd accumulation in duck kidneys.

Cadmium and molybdenum co-induce pyroptosis via ROS/PTEN/PI3K/AKT axis in duck renal tubular epithelial cells.

Cadmium (Cd) and excess molybdenum (Mo) are harmful to animals, but the combined nephrotoxic mechanism of Cd and Mo in duck remains poorly elucidated. To assess joint effects of Cd and Mo on pyroptosis via ROS/PTEN/PI3K/AKT axis in duck renal tubular epithelial cells, cells were cultured with 3CdSO4·8H2O (4.0 µM), (NH4)6Mo7O24·4H2O (500.0 µM), MCC950 (10.0 µM), BHA (100.0 µM) and combination of Cd and Mo or Cd, Mo and MCC950 or Cd, Mo and BHA for 12 h, and the joint cytotoxicity was explored. The results manifested that toxicity of non-equitoxic binary mixtures of Mo and Cd exhibited synergic interaction. Mo or/and Cd elevated ROS level, PTEN mRNA and protein levels, and decreased PI3K, AKT and p-AKT expression levels. Simultaneously, Mo or/and Cd upregulated ASC, NLRP3, NEK7, Caspase-1, GSDMA, GSDME, IL-18 and IL-1ß mRNA levels and Caspase-1 p20, NLRP3, ASC, GSDMD protein levels, increased the percentage of pyroptotic cells, LDH, NO, IL-18 and IL-1ß releases as well as relative conductivity. Moreover, NLRP3 inhibitor MCC950 and ROS scavenger BHA could ameliorate the above changed factors induced by Mo and Cd co-exposure. Collectively, our results reveal that combination of Mo and Cd synergistically cause oxidative stress and trigger pyroptosis via ROS/PTEN/PI3K/AKT axis in duck tubular epithelial cells.

Inhibition of autophagy aggravates molybdenum-induced mitochondrial dysfunction by aggravating oxidative stress in duck renal tubular epithelial cells.

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 (NH4)6Mo7O24.4H2O (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 (H2O2) 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.

Inhibition of ROS/NLRP3/Caspase-1 mediated pyroptosis alleviates excess molybdenum-induced apoptosis in duck renal tubular epithelial cells.

OBJECTIVE: Excess molybdenum (Mo) is harmful to the body, and the kidney is the vital target organ for Mo exposure. This study focused on the impacts of excess Mo on pyroptosis and the relationship between pyroptosis and apoptosis in kidney. METHODS: The duck renal tubular epithelial cells were treated with (NH4)6Mo7O24·4H2O (0, 480, 720 and 960 µM Mo), N-acetyl-L-cysteine (NAC) (100 µM), Z-YVAD-fluoromethylketone (YVAD) (10 µM) and the combination of Mo and NAC or YVAD for 12 h. The LDH release and IL-1ß, IL-18 contents of cell supernatant were detected by LDH and ELISA kits. The MMP and ROS level were measured using MMP and ROS kits by flow cytometry. The apoptotic rate of cell was detected by AO/EB counterstaining. Pyroptosis and apoptosis-related factors mRNA and protein levels were assayed by real-time qPCR and western blot, respectively. RESULTS: Excessive Mo markedly increased LDH, IL-18, IL-1ß releases and induced overproduction of ROS, pyroptosis-related factors mRNA and protein levels. NAC and YVAD dramatically decreased pyroptosis induced by Mo. Simultaneously, YVAD significantly changed apoptosis-related factors mRNA and protein levels, and reduced cell apoptotic rate. CONCLUSION: Excessive Mo exposure can induce pyroptosis by the ROS/NLRP3/Caspase-1 pathway in duck renal tubular epithelial cells, and restraining pyroptosis of Caspase-1 dependence might weaken excess Mo-induced apoptosis. The study provides theoretical basis for excess Mo exposure nephrotoxic researches on waterfowl and the interplay between pyroptosis and apoptosis highlights a new sight into the mechanism of Mo-induced nephrotoxicity.

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.

Inhibition of autophagy enhances cadmium-induced apoptosis in duck renal tubular epithelial cells.

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 3CdSO4·8H2O (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.

Inhibition of ROS/NLRP3/Caspase-1 mediated pyroptosis attenuates cadmium-induced apoptosis in duck renal tubular epithelial cells.

Cadmium (Cd) is an occupational and environmental pollutant, which mainly causes nephrotoxicity by damaging renal proximal tubular cells. To evaluate the effects of Cd on pyroptosis and the relationship between pyroptosis and apoptosis in duck renal tubular epithelial cells, the cells were cultured with 3CdSO4·8H2O (0, 2.5, 5.0, or 10.0 µM Cd), N-acetyl-L-cysteine (NAC) (100.0 µM), Z-YVAD-FMK (10.0 µM) or the combination of Cd and NAC or Z-YVAD-FMK for 12 h, and then cytotoxicity was assessed. The results evidenced that Cd significantly increased the releases of interleukin-18 (IL-18) and interleukin-1ß (IL-1ß), lactate dehydrogenase (LDH) and nitric oxide (NO), relative conductivity and cellular reactive oxygen species (ROS) level. Simultaneously, Cd also markedly upregulated NLRP3, Caspase-1, ASC, NEK7, IL-1ß and IL-18 mRNA levels and NLRP3, Caspase-1 p20, GSDMD and ASC protein levels. Additionally, NAC notably improved the changes of above indicators induced by Cd. Combined treatment with Cd and Z-YVAD-FMK remarkably elevated Bcl-2 mRNA and protein levels, inhibited p53, Bax, Bak-1, Cyt C, Caspase-9 and Caspase-3 mRNA levels and p53, Bax, Bak-1, Caspase-9/cleaved Caspase-9 and Caspase-3/cleaved Caspase-3 protein levels, increased mitochondrial membrane potential (MMP), decreased apoptosis ratio and cell damage compared to treatment with Cd alone. Taken together, Cd exposure induces duck renal tubular epithelial cell pyroptosis through ROS/NLRP3/Caspase-1 signaling pathway, and inhibiting Caspase-1 dependent pyroptosis attenuates Cd-induced apoptosis.

Molybdenum and cadmium co-induce oxidative stress and apoptosis through mitochondria-mediated pathway in duck renal tubular epithelial cells.

High doses of molybdenum (Mo) and cadmium (Cd) cause adverse reactions on animals, but the joint toxic effects of Mo and Cd on duck renal tubular epithelial cells are not fully illustrated. To investigate the combined effects of Mo and Cd on oxidative stress and mitochondrial apoptosis in primary duck renal tubular epithelial cells, the cells were either treated with (NH4)6Mo7O24·4H2O (480, 960 µM Mo), 3CdSO4·8H2O (2.5, 5.0 µM Cd) or combination of Mo and Cd for 12 h, and then the joint cytotoxicity was evaluated. The results demonstrated that Mo or/and Cd exposure could induce release of intracellular lactate dehydrogenase, reactive oxygen species generation, acidification, increase levels of malondialdehyde and [Ca2+]i, decrease levels of glutathione, glutathione peroxidase, catalase, superoxide dismutase, total antioxidant capacity, Na+/K+-ATPase, Ca2+-ATPase, and mitochondrial membrane potential; upregulate mRNA levels of Caspase-3, Bak-1, Bax, and cytochrome C, inhibit Bcl-2 mRNA level, and induce cell apoptosis in a dose-dependent manner. Furthermore, the changes of these indicators in co-treated groups were more remarkable. The results indicated that exposure to Mo or/and Cd could induce oxidative stress and apoptosis via the mitochondrial pathway in duck renal tubular epithelial cells and the two metals may have a synergistic effect.

Cadmium induces cytotoxicity through oxidative stress-mediated apoptosis pathway in duck renal tubular epithelial cells.

Cadmium (Cd) is a well studied nephrotoxic metal element. To investigate the effects of Cd-induced cytotoxicity on oxidative stress-mediated apoptosis in primary renal tubular epithelial cells of duck. Shaoxing duck (Anas platyrhyncha) renal tubular epithelial cells were cultured in medium in absence and presence of 3CdSO4·8H2O (1.25, 2.5, 5.0 µM Cd), in N-acetyl-l-cysteine (NAC) (100 µM), and the combination of Cd and NAC for 12 h. After 12 h exposure, morphologic observation and function, reactive oxygen species (ROS) level, antioxidant indices, the activity of ATPase, intracellular pH and [Ca2+]i, mitochondrial membrane potential (MMP), and apoptosis-related genes mRNA were determined. The results showed that Cd exposure could induce release of intracellular lactate dehydrogenase (LDH), simultaneously, enhance the ROS generation, acidification, malondialdehyde (MDA) and [Ca2+]i, decrease glutathione (GSH), Na+, K+-ATPase, Ca2+-ATPase, catalase (CAT), superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) activities as well as MMP, upregulated Bak-1, Bax and Caspase-3 mRNA expression, inhibited Bcl-2 mRNA expression, and induced cell apoptosis. The toxicity of Cd to cells showed a dose-dependent manner. Antioxidant NAC could efficiently alleviate Cd-induced the cytotoxicity. Taken together, these results suggest that Cd exposure cause cytotoxicity through oxidative stress-mediated apoptosis pathway in duck renal tubular epithelial cells.

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.

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.

Molybdenum and Cadmium exposure influences the concentration of trace elements in the digestive organs of Shaoxing duck (Anas platyrhyncha).

To investigate the toxic effects of Molybdenum (Mo) and Cadmium (Cd) on trace elements in digestive organs of Shaoxing duck (Anas platyrhyncha), 120 Shaoxing ducks were randomly divided into control group and 5 treatment groups which were treated with a commercial diet containing different dosages of Mo and Cd. On the 60th and 120th days, the beak, esophagus, glandular stomach, muscular stomach, small intestine, large intestine and feces were collected to determine contents of Mo, Cd, copper (Cu), iron (Fe), zinc (Zn) and selenium (Se), then correlation analysis was performed. The results showed that Cd content in digestive organs significantly increased in co-treated groups compared to single treated groups and Mo concentration increased in Mo-treated groups compared to control group, whereas Cu, Fe, Zn and Se concentrations in digestive organs decreased in co-treated groups. Furthermore, Cd and Mo were mainly accumulated in the small intestine and esophagus, respectively. There was a strongly positive correlation between Cd and Mo while they had negative correlation with Cu, Fe, Zn and Se, respectively. In feces, Mo and Fe contents in high dose of Mo group and high Mo combined with Cd group were significantly higher than those in control group, and Cu content in all treated groups significantly increased and Cd, Zn and Se concentrations had no difference. The results indicated that dietary Mo or/and Cd might disturb homeostasis of trace elements in digestive organs of Shaoxing duck. Moreover, the two elements presented a synergistic relationship.