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Microcystin-LR Induced Apoptosis in Rat Sertoli Cells via the Mitochondrial Caspase-Dependent Pathway: Role of Reactive Oxygen Species

View Article: PubMed Central - PubMed

ABSTRACT

Microcystins (MCs), the secondary metabolites of blue-green algae, are ubiquitous and major cyanotoxin contaminants. Besides the hepatopancreas/liver, the reproductive system is regarded as the most important target organ for MCs. Although reactive oxygen species (ROS) have been implicated in MCs-induced reproductive toxicity, the role of MCs in this pathway remains unclear. In the present study, Sertoli cells were employed to investigate apoptotic death involved in male reproductive toxicity of microcystin-LR (MC-LR). After exposure to various concentrations of MC-LR for 24 h, the growth of Sertoli cells was concentration-dependently decreased with an IC50 of ~32 μg/mL. Mitochondria-mediated apoptotic changes were observed in Sertoli cells exposed to 8, 16, and 32 μg/mL MC-LR including the increased expression of caspase pathway proteins, collapse of mitochondrial membrane potential (MMP), and generation of ROS. Pretreatment with a global caspase inhibitor was found to depress the activation of caspases, and eventually increased the survival rate of Sertoli cells, implying that the mitochondrial caspases pathway is involved in MC-LR-induced apoptosis. Furthermore, N-acetyl-l-cysteine attenuated the MC-LR-induced intracellular ROS generation, MMP collapse and cytochrome c release, resulting in the inhibition of apoptosis. Taken together, the observed results suggested that MC-LR induced apoptotic death of Sertoli cells by the activation of mitochondrial caspases cascade, while its effects on the ROS-mediated signaling pathway may contribute toward the initiation of mitochondrial dysfunction.

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Effects of NAC pretreatment on distribution and protein expression of cytochrome c (Cyt c). (A) Rat Sertoli cells were treated with 32 μg/mL MC-LR or NAC + 32 μg/mL MC-LR. The expression of Cyt c and GAPDH in the mitochondrial fraction was tested by western blotting analysis; Cyt c and Hsp70 protein in the cytoplasmic fraction was tested by western blotting analysis. Cyto, cytosol; Mito, mitochondria; N+, 10 mM NAC+32 μg/ml MC-LR. GAPDH was used as the cytosolic control and mitochondria Hsp70 was used as the mitochondrial control. (B) Data were presented as mean ± SEM of three independent experiments. *P < 0.05 vs. control group; #P < 0.05 vs. 32 μg/mL MC-LR.
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Figure 7: Effects of NAC pretreatment on distribution and protein expression of cytochrome c (Cyt c). (A) Rat Sertoli cells were treated with 32 μg/mL MC-LR or NAC + 32 μg/mL MC-LR. The expression of Cyt c and GAPDH in the mitochondrial fraction was tested by western blotting analysis; Cyt c and Hsp70 protein in the cytoplasmic fraction was tested by western blotting analysis. Cyto, cytosol; Mito, mitochondria; N+, 10 mM NAC+32 μg/ml MC-LR. GAPDH was used as the cytosolic control and mitochondria Hsp70 was used as the mitochondrial control. (B) Data were presented as mean ± SEM of three independent experiments. *P < 0.05 vs. control group; #P < 0.05 vs. 32 μg/mL MC-LR.

Mentions: Mitochondria Cyt c, an important intermediate in intrinsic mitochondrial apoptosis, plays a well-documented role in activating caspase-9 and caspase-3 to execute cell death. We first separated the mitochondria from the cytoplasm of the cells. Then, the expressions of GAPDH in the mitochondrial fraction, and Hsp70 in the cytoplasm were detected. The results showed that there was no GAPDH band in the mitochondrial fraction nor as there Hsp70 band in the cytoplasm (Figure 7). This demonstrated that the subcellular fractionation was successful. As shown in Figure 7, in accordance with inhibition of ROS generation following co-treatment with MC-LR and NAC, Cyt c release in the mitochondria of Sertoli cells was down-regulated significantly (P < 0.05) compared with the non-NAC group. In addition, the activation of capase-3, caspase-9, cleaved-caspase-3, and −9 were reversed in the presence of NAC (Figure 8). These observations indicate that MC-LR-induced apoptosis can be inhibited by NAC through mitochondrial cascades.


Microcystin-LR Induced Apoptosis in Rat Sertoli Cells via the Mitochondrial Caspase-Dependent Pathway: Role of Reactive Oxygen Species
Effects of NAC pretreatment on distribution and protein expression of cytochrome c (Cyt c). (A) Rat Sertoli cells were treated with 32 μg/mL MC-LR or NAC + 32 μg/mL MC-LR. The expression of Cyt c and GAPDH in the mitochondrial fraction was tested by western blotting analysis; Cyt c and Hsp70 protein in the cytoplasmic fraction was tested by western blotting analysis. Cyto, cytosol; Mito, mitochondria; N+, 10 mM NAC+32 μg/ml MC-LR. GAPDH was used as the cytosolic control and mitochondria Hsp70 was used as the mitochondrial control. (B) Data were presented as mean ± SEM of three independent experiments. *P < 0.05 vs. control group; #P < 0.05 vs. 32 μg/mL MC-LR.
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Figure 7: Effects of NAC pretreatment on distribution and protein expression of cytochrome c (Cyt c). (A) Rat Sertoli cells were treated with 32 μg/mL MC-LR or NAC + 32 μg/mL MC-LR. The expression of Cyt c and GAPDH in the mitochondrial fraction was tested by western blotting analysis; Cyt c and Hsp70 protein in the cytoplasmic fraction was tested by western blotting analysis. Cyto, cytosol; Mito, mitochondria; N+, 10 mM NAC+32 μg/ml MC-LR. GAPDH was used as the cytosolic control and mitochondria Hsp70 was used as the mitochondrial control. (B) Data were presented as mean ± SEM of three independent experiments. *P < 0.05 vs. control group; #P < 0.05 vs. 32 μg/mL MC-LR.
Mentions: Mitochondria Cyt c, an important intermediate in intrinsic mitochondrial apoptosis, plays a well-documented role in activating caspase-9 and caspase-3 to execute cell death. We first separated the mitochondria from the cytoplasm of the cells. Then, the expressions of GAPDH in the mitochondrial fraction, and Hsp70 in the cytoplasm were detected. The results showed that there was no GAPDH band in the mitochondrial fraction nor as there Hsp70 band in the cytoplasm (Figure 7). This demonstrated that the subcellular fractionation was successful. As shown in Figure 7, in accordance with inhibition of ROS generation following co-treatment with MC-LR and NAC, Cyt c release in the mitochondria of Sertoli cells was down-regulated significantly (P < 0.05) compared with the non-NAC group. In addition, the activation of capase-3, caspase-9, cleaved-caspase-3, and −9 were reversed in the presence of NAC (Figure 8). These observations indicate that MC-LR-induced apoptosis can be inhibited by NAC through mitochondrial cascades.

View Article: PubMed Central - PubMed

ABSTRACT

Microcystins (MCs), the secondary metabolites of blue-green algae, are ubiquitous and major cyanotoxin contaminants. Besides the hepatopancreas/liver, the reproductive system is regarded as the most important target organ for MCs. Although reactive oxygen species (ROS) have been implicated in MCs-induced reproductive toxicity, the role of MCs in this pathway remains unclear. In the present study, Sertoli cells were employed to investigate apoptotic death involved in male reproductive toxicity of microcystin-LR (MC-LR). After exposure to various concentrations of MC-LR for 24 h, the growth of Sertoli cells was concentration-dependently decreased with an IC50 of ~32 &mu;g/mL. Mitochondria-mediated apoptotic changes were observed in Sertoli cells exposed to 8, 16, and 32 &mu;g/mL MC-LR including the increased expression of caspase pathway proteins, collapse of mitochondrial membrane potential (MMP), and generation of ROS. Pretreatment with a global caspase inhibitor was found to depress the activation of caspases, and eventually increased the survival rate of Sertoli cells, implying that the mitochondrial caspases pathway is involved in MC-LR-induced apoptosis. Furthermore, N-acetyl-l-cysteine attenuated the MC-LR-induced intracellular ROS generation, MMP collapse and cytochrome c release, resulting in the inhibition of apoptosis. Taken together, the observed results suggested that MC-LR induced apoptotic death of Sertoli cells by the activation of mitochondrial caspases cascade, while its effects on the ROS-mediated signaling pathway may contribute toward the initiation of mitochondrial dysfunction.

No MeSH data available.


Related in: MedlinePlus