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Paraquat Induces Apoptosis through a Mitochondria-Dependent Pathway in RAW264.7 Cells.

Jang YJ, Won JH, Back MJ, Fu Z, Jang JM, Ha HC, Hong S, Chang M, Kim DK - Biomol Ther (Seoul) (2015)

Bottom Line: Additionally, PQ increased the cleaved form of caspase-3, an apoptotic marker.In conclusion, PQ induces apoptosis in RAW264.7 cells through a ROS-mediated mitochondrial pathway.Thus, our study improves our knowledge of PQ-induced toxicity, and may give us a greater understanding of how PQ affects the immune system.

View Article: PubMed Central - PubMed

Affiliation: Department of Health, Social, and Clinical Pharmacy, College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea.

ABSTRACT
Paraquat dichloride (N,N-dimethyl-4-4'-bipiridinium, PQ) is an extremely toxic chemical that is widely used in herbicides. PQ generates reactive oxygen species (ROS) and causes multiple organ failure. In particular, PQ has been reported to be an immunotoxic agrochemical compound. PQ was shown to decrease the number of macrophages in rats and suppress monocyte phagocytic activity in mice. However, the effect of PQ on macrophage cell viability remains unclear. In this study, we evaluated the cytotoxic effect of PQ on the mouse macrophage cell line, RAW264.7 and its possible mechanism of action. RAW264.7 cells were treated with PQ (0, 75, and 150 μM), and cellular apoptosis, mitochondrial membrane potential (MMP), and intracellular ROS levels were determined. Morphological changes to the cell nucleus and cellular apoptosis were also evaluated by DAPI and Annexin V staining, respectively. In this study, PQ induced apoptotic cell death by dose-dependently decreasing MMP. Additionally, PQ increased the cleaved form of caspase-3, an apoptotic marker. In conclusion, PQ induces apoptosis in RAW264.7 cells through a ROS-mediated mitochondrial pathway. Thus, our study improves our knowledge of PQ-induced toxicity, and may give us a greater understanding of how PQ affects the immune system.

No MeSH data available.


Related in: MedlinePlus

Effects of PQ on apoptosis-associated proteins in RAW 264.7 cells. Cells were treated with PQ for 24 h, and then harvested for western blot to examine the protein levels of cytochrome c (Fig. 7A) and caspase-3 (Fig. 7B). Densitometry was performed following normalization to β-actin. Data represent the mean ± SD of at least three independent experiments. ***p<0.001 indicates a significant difference as compared to the control group.
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f7-bt-23-407: Effects of PQ on apoptosis-associated proteins in RAW 264.7 cells. Cells were treated with PQ for 24 h, and then harvested for western blot to examine the protein levels of cytochrome c (Fig. 7A) and caspase-3 (Fig. 7B). Densitometry was performed following normalization to β-actin. Data represent the mean ± SD of at least three independent experiments. ***p<0.001 indicates a significant difference as compared to the control group.

Mentions: To determine if loss of MMP results in the release of cytochrome c, we evaluated cytochrome c content. After PQ treatment, mitochondria were separated from the cytosol, and cytochrome c was analyzed by western blot. As shown in Fig. 7A, cytochrome c appeared in the cytosol after PQ treatment. However, cytochrome c content in the mitochondria was not altered. Depolarization of MMP triggers the release of cytochrome c from the mitochondria into the cytoplasm. Cytochrome c oxidase activity was identical in the mitochondrial extracts, and was absent in the cytosolic extracts. Caspase proteins play a pivotal role downstream of the Bcl-2 family by initiating cellular breakdown during apoptosis. In the intrinsic cell death pathway, which is mediated by mitochondria, cytochrome c activates caspase-9, which then activates caspase-3. To determine whether caspase-3 is activated after PQ treatment, the cells were harvested for western blot after exposure to PQ. As shown in Fig 7B, PQ increased the protein levels of cleaved caspase-3, indicating that PQ promotes the release of cytochrome c from the mitochondria, thereby activating caspase-3.


Paraquat Induces Apoptosis through a Mitochondria-Dependent Pathway in RAW264.7 Cells.

Jang YJ, Won JH, Back MJ, Fu Z, Jang JM, Ha HC, Hong S, Chang M, Kim DK - Biomol Ther (Seoul) (2015)

Effects of PQ on apoptosis-associated proteins in RAW 264.7 cells. Cells were treated with PQ for 24 h, and then harvested for western blot to examine the protein levels of cytochrome c (Fig. 7A) and caspase-3 (Fig. 7B). Densitometry was performed following normalization to β-actin. Data represent the mean ± SD of at least three independent experiments. ***p<0.001 indicates a significant difference as compared to the control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556199&req=5

f7-bt-23-407: Effects of PQ on apoptosis-associated proteins in RAW 264.7 cells. Cells were treated with PQ for 24 h, and then harvested for western blot to examine the protein levels of cytochrome c (Fig. 7A) and caspase-3 (Fig. 7B). Densitometry was performed following normalization to β-actin. Data represent the mean ± SD of at least three independent experiments. ***p<0.001 indicates a significant difference as compared to the control group.
Mentions: To determine if loss of MMP results in the release of cytochrome c, we evaluated cytochrome c content. After PQ treatment, mitochondria were separated from the cytosol, and cytochrome c was analyzed by western blot. As shown in Fig. 7A, cytochrome c appeared in the cytosol after PQ treatment. However, cytochrome c content in the mitochondria was not altered. Depolarization of MMP triggers the release of cytochrome c from the mitochondria into the cytoplasm. Cytochrome c oxidase activity was identical in the mitochondrial extracts, and was absent in the cytosolic extracts. Caspase proteins play a pivotal role downstream of the Bcl-2 family by initiating cellular breakdown during apoptosis. In the intrinsic cell death pathway, which is mediated by mitochondria, cytochrome c activates caspase-9, which then activates caspase-3. To determine whether caspase-3 is activated after PQ treatment, the cells were harvested for western blot after exposure to PQ. As shown in Fig 7B, PQ increased the protein levels of cleaved caspase-3, indicating that PQ promotes the release of cytochrome c from the mitochondria, thereby activating caspase-3.

Bottom Line: Additionally, PQ increased the cleaved form of caspase-3, an apoptotic marker.In conclusion, PQ induces apoptosis in RAW264.7 cells through a ROS-mediated mitochondrial pathway.Thus, our study improves our knowledge of PQ-induced toxicity, and may give us a greater understanding of how PQ affects the immune system.

View Article: PubMed Central - PubMed

Affiliation: Department of Health, Social, and Clinical Pharmacy, College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea.

ABSTRACT
Paraquat dichloride (N,N-dimethyl-4-4'-bipiridinium, PQ) is an extremely toxic chemical that is widely used in herbicides. PQ generates reactive oxygen species (ROS) and causes multiple organ failure. In particular, PQ has been reported to be an immunotoxic agrochemical compound. PQ was shown to decrease the number of macrophages in rats and suppress monocyte phagocytic activity in mice. However, the effect of PQ on macrophage cell viability remains unclear. In this study, we evaluated the cytotoxic effect of PQ on the mouse macrophage cell line, RAW264.7 and its possible mechanism of action. RAW264.7 cells were treated with PQ (0, 75, and 150 μM), and cellular apoptosis, mitochondrial membrane potential (MMP), and intracellular ROS levels were determined. Morphological changes to the cell nucleus and cellular apoptosis were also evaluated by DAPI and Annexin V staining, respectively. In this study, PQ induced apoptotic cell death by dose-dependently decreasing MMP. Additionally, PQ increased the cleaved form of caspase-3, an apoptotic marker. In conclusion, PQ induces apoptosis in RAW264.7 cells through a ROS-mediated mitochondrial pathway. Thus, our study improves our knowledge of PQ-induced toxicity, and may give us a greater understanding of how PQ affects the immune system.

No MeSH data available.


Related in: MedlinePlus