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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 MC-LR on ROS content in rat Sertoli cells were observed by fluorescence microscope (200×) and monitored by measuring the DCF fluorescence intensity via Flow cytometry. (A) Control group; (B) 8 μg/mL MC-LR; (C) 16 μg/mL MC-LR; (D) 32 μg/mL MC-LR, (E) 10 mM NAC + 32 μg/mL MC-LR; Bar = 50 μm. (F) The qualitative representative images. Fluorescence intensity is 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 4: Effects of MC-LR on ROS content in rat Sertoli cells were observed by fluorescence microscope (200×) and monitored by measuring the DCF fluorescence intensity via Flow cytometry. (A) Control group; (B) 8 μg/mL MC-LR; (C) 16 μg/mL MC-LR; (D) 32 μg/mL MC-LR, (E) 10 mM NAC + 32 μg/mL MC-LR; Bar = 50 μm. (F) The qualitative representative images. Fluorescence intensity is 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, which are both a major source of intracellular ROS and a primary target for ROS, play a key role in the regulation of apoptosis. To determine the involvement of ROS during MC-LR-induced apoptosis, Sertoli cells were exposed to 8~32 μg/mL of MC-LR for 24 h, and the intensity of DCF fluorescence was observed via fluorescence microscope. As shown in Figures 4A–D, MC-LR treatment induced intracellular ROS fluorescence intensity dose-dependently. However, when cells were treated with a mixture of MC-LR and NAC (with 1 h of preincubation), ROS intensity was decreased as compared to cells that were treated with MC-LR alone (Figure 4E). Moreover, ROS expression levels were detected by flow cytometer, and the results showed that ROS level was increased dose-dependently after MC-LR treatment, but decreased when cells were treated with a mixture of MC-LR and NAC (Figure 4F). Meanwhile, the inhibitory effect of NAC on MC-LR-induced apoptosis was also confirmed. As shown in Figures 5A–C, MC-LR treatment induced apoptosis obviously, however, when cells were treated with a mixture of MC-LR and NAC (with 1 h of preincubation), apoptosis ratio was decreased as compared to cells that were treated with MC-LR alone. The percent of early apoptosis cells was remarkably reduced (Figure 5D).


Microcystin-LR Induced Apoptosis in Rat Sertoli Cells via the Mitochondrial Caspase-Dependent Pathway: Role of Reactive Oxygen Species
Effects of MC-LR on ROS content in rat Sertoli cells were observed by fluorescence microscope (200×) and monitored by measuring the DCF fluorescence intensity via Flow cytometry. (A) Control group; (B) 8 μg/mL MC-LR; (C) 16 μg/mL MC-LR; (D) 32 μg/mL MC-LR, (E) 10 mM NAC + 32 μg/mL MC-LR; Bar = 50 μm. (F) The qualitative representative images. Fluorescence intensity is 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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Related In: Results  -  Collection

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Figure 4: Effects of MC-LR on ROS content in rat Sertoli cells were observed by fluorescence microscope (200×) and monitored by measuring the DCF fluorescence intensity via Flow cytometry. (A) Control group; (B) 8 μg/mL MC-LR; (C) 16 μg/mL MC-LR; (D) 32 μg/mL MC-LR, (E) 10 mM NAC + 32 μg/mL MC-LR; Bar = 50 μm. (F) The qualitative representative images. Fluorescence intensity is 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, which are both a major source of intracellular ROS and a primary target for ROS, play a key role in the regulation of apoptosis. To determine the involvement of ROS during MC-LR-induced apoptosis, Sertoli cells were exposed to 8~32 μg/mL of MC-LR for 24 h, and the intensity of DCF fluorescence was observed via fluorescence microscope. As shown in Figures 4A–D, MC-LR treatment induced intracellular ROS fluorescence intensity dose-dependently. However, when cells were treated with a mixture of MC-LR and NAC (with 1 h of preincubation), ROS intensity was decreased as compared to cells that were treated with MC-LR alone (Figure 4E). Moreover, ROS expression levels were detected by flow cytometer, and the results showed that ROS level was increased dose-dependently after MC-LR treatment, but decreased when cells were treated with a mixture of MC-LR and NAC (Figure 4F). Meanwhile, the inhibitory effect of NAC on MC-LR-induced apoptosis was also confirmed. As shown in Figures 5A–C, MC-LR treatment induced apoptosis obviously, however, when cells were treated with a mixture of MC-LR and NAC (with 1 h of preincubation), apoptosis ratio was decreased as compared to cells that were treated with MC-LR alone. The percent of early apoptosis cells was remarkably reduced (Figure 5D).

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