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p53 activation contributes to patulin-induced nephrotoxicity via modulation of reactive oxygen species generation.

Jin H, Yin S, Song X, Zhang E, Fan L, Hu H - Sci Rep (2016)

Bottom Line: Patulin is a major mycotoxin found in fungal contaminated fruits and their derivative products.To the best of our knowledge, this is the first report addressing the functional role of p53 in patulin-induced oxidative stress.The findings of the present study provided novel insights into understanding mechanisms behind oxidative stress in response to patulin exposure.

View Article: PubMed Central - PubMed

Affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, Department of Nutrition and Health, College of Food Science and Nutritional Engineering, China Agricultural University, No17 Qinghua East Road, Haidian District, Beijing 100083, China.

ABSTRACT
Patulin is a major mycotoxin found in fungal contaminated fruits and their derivative products. Previous studies showed that patulin was able to induce increase of reactive oxygen species (ROS) generation and oxidative stress was suggested to play a pivotal role in patulin-induced multiple toxic signaling. The objective of the present study was to investigate the functional role of p53 in patulin-induced oxidative stress. Our study demonstrated that higher levels of ROS generation and DNA damage were induced in wild-type p53 cell lines than that found in either knockdown or knockout p53 cell lines in response to patulin exposure, suggesting p53 activation contributed to patulin-induced ROS generation. Mechanistically, we revealed that the pro-oxidant role of p53 in response to patulin was attributed to its ability to suppress catalase activity through up-regulation of PIG3. Moreover, these in vitro findings were further validated in the p53 wild-type/knockout mouse model. To the best of our knowledge, this is the first report addressing the functional role of p53 in patulin-induced oxidative stress. The findings of the present study provided novel insights into understanding mechanisms behind oxidative stress in response to patulin exposure.

No MeSH data available.


Related in: MedlinePlus

p53 exerted pro-apoptotic activity through a transcriptional mechanism.(A). Effects of p53 inhibition by RNAi on patulin-induced Bax up-regulations, p38 phosphorylation and PARP1 cleavages. The cells were transfected with p53 siRNA using INTERFER siRNA transfection agent. At 24 h post-transfection, the cells were treated with 7 μM patulin for 24 h and then Bax, p38 phosphorylation and PARP cleavages were analyzed by western blotting. (B). Effects of p53 knockdown on patulin-induced apoptosis. The cells were transfected with p53 siRNA using INTERFER siRNA transfection agent. At 24 h post-transfection, the cells were treated with 7 μM patulin for 30 h and then apoptosis was measured by Annexin V staining. (C). Patulin induced apoptosis in p53 knockout/wild type MEF cells. The cells were treated with various patulin concentrations for 18 h and then apoptosis was measured by Annexin V staining. (D). PAT induced nuclear morphological changes in p53 knockout/wild type MEF cells. The cells were treated with 7 μM patulin for 12 h and then the nuclei were stained with DAPI. (E). Effects of p53 inhibitor pifithrin-α on patulin-induced apoptosis measured by Annexin V staining. (F). Effects of p53 inhibitor pifithrin-μ on patulin-induced apoptosis measured by Annexin V staining. (G). Effects of p53 inhibitor pifithrin-α on patulin-induced Bax up-regulations. The cells were treated with patulin in the presence or absence of pifithrin-α for 24 h and then Bax expression was analyzed by western blotting. n = 3, *P < 0.05, **P < 0.01. (The blots shown are representative of three independent experiments).
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f5: p53 exerted pro-apoptotic activity through a transcriptional mechanism.(A). Effects of p53 inhibition by RNAi on patulin-induced Bax up-regulations, p38 phosphorylation and PARP1 cleavages. The cells were transfected with p53 siRNA using INTERFER siRNA transfection agent. At 24 h post-transfection, the cells were treated with 7 μM patulin for 24 h and then Bax, p38 phosphorylation and PARP cleavages were analyzed by western blotting. (B). Effects of p53 knockdown on patulin-induced apoptosis. The cells were transfected with p53 siRNA using INTERFER siRNA transfection agent. At 24 h post-transfection, the cells were treated with 7 μM patulin for 30 h and then apoptosis was measured by Annexin V staining. (C). Patulin induced apoptosis in p53 knockout/wild type MEF cells. The cells were treated with various patulin concentrations for 18 h and then apoptosis was measured by Annexin V staining. (D). PAT induced nuclear morphological changes in p53 knockout/wild type MEF cells. The cells were treated with 7 μM patulin for 12 h and then the nuclei were stained with DAPI. (E). Effects of p53 inhibitor pifithrin-α on patulin-induced apoptosis measured by Annexin V staining. (F). Effects of p53 inhibitor pifithrin-μ on patulin-induced apoptosis measured by Annexin V staining. (G). Effects of p53 inhibitor pifithrin-α on patulin-induced Bax up-regulations. The cells were treated with patulin in the presence or absence of pifithrin-α for 24 h and then Bax expression was analyzed by western blotting. n = 3, *P < 0.05, **P < 0.01. (The blots shown are representative of three independent experiments).

Mentions: Having established the pro-oxidant role of p53 activation in patulin-induced oxidative stress, we next investigated the role of p53 in patulin-induced apoptosis. As shown in Fig. 5A, when p53 was inactivated by RNAi, Bax up-regulation, p38 phosphorylation and PARP cleavages induced by patulin were significantly decreased in HEK293 cells. Accordingly, apoptosis induction by patulin was also decreased under the condition of p53 inactivation in HEK293 cells (Fig. 5B). In addition, a significant decreased apoptosis induction result was also observed in p53 knockout MEF cells in comparison with that found in p53 wt MEF cells measured by annexin v staining (Fig. 5C) and nuclear DAPI staining (Fig. 5D). These results suggested that p53-mediated Bax activation might contribute to apoptosis induction by patulin. We further validated the pro-apoptotic role of p53 by measuring apoptosis in the presence of pifithrin-alpha23 or pifithrin-mu2425, the two chemical inhibitors of p53 that can block p53-dependent transcriptional activation and inhibit transcriptional-independent p53 binding to mitochondria respectively. As shown in Fig. 5E,F, treatment with pifithrin-alpha led to a significant suppression of patulin-induced apoptosis, whereas pifithrin-mu failed to offer any protective effect on apoptosis induction by patulin. In support the protective effect of pifithrin-alpha, Bax induction by patulin was also blocked in the presence of pifithrin-alpha (Fig. 5G) but not in the presence of pifithrin-mu (data not shown). These results suggested that p53 activation contributed to apoptosis in response to patulin through its transcriptional mechanism.


p53 activation contributes to patulin-induced nephrotoxicity via modulation of reactive oxygen species generation.

Jin H, Yin S, Song X, Zhang E, Fan L, Hu H - Sci Rep (2016)

p53 exerted pro-apoptotic activity through a transcriptional mechanism.(A). Effects of p53 inhibition by RNAi on patulin-induced Bax up-regulations, p38 phosphorylation and PARP1 cleavages. The cells were transfected with p53 siRNA using INTERFER siRNA transfection agent. At 24 h post-transfection, the cells were treated with 7 μM patulin for 24 h and then Bax, p38 phosphorylation and PARP cleavages were analyzed by western blotting. (B). Effects of p53 knockdown on patulin-induced apoptosis. The cells were transfected with p53 siRNA using INTERFER siRNA transfection agent. At 24 h post-transfection, the cells were treated with 7 μM patulin for 30 h and then apoptosis was measured by Annexin V staining. (C). Patulin induced apoptosis in p53 knockout/wild type MEF cells. The cells were treated with various patulin concentrations for 18 h and then apoptosis was measured by Annexin V staining. (D). PAT induced nuclear morphological changes in p53 knockout/wild type MEF cells. The cells were treated with 7 μM patulin for 12 h and then the nuclei were stained with DAPI. (E). Effects of p53 inhibitor pifithrin-α on patulin-induced apoptosis measured by Annexin V staining. (F). Effects of p53 inhibitor pifithrin-μ on patulin-induced apoptosis measured by Annexin V staining. (G). Effects of p53 inhibitor pifithrin-α on patulin-induced Bax up-regulations. The cells were treated with patulin in the presence or absence of pifithrin-α for 24 h and then Bax expression was analyzed by western blotting. n = 3, *P < 0.05, **P < 0.01. (The blots shown are representative of three independent experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829895&req=5

f5: p53 exerted pro-apoptotic activity through a transcriptional mechanism.(A). Effects of p53 inhibition by RNAi on patulin-induced Bax up-regulations, p38 phosphorylation and PARP1 cleavages. The cells were transfected with p53 siRNA using INTERFER siRNA transfection agent. At 24 h post-transfection, the cells were treated with 7 μM patulin for 24 h and then Bax, p38 phosphorylation and PARP cleavages were analyzed by western blotting. (B). Effects of p53 knockdown on patulin-induced apoptosis. The cells were transfected with p53 siRNA using INTERFER siRNA transfection agent. At 24 h post-transfection, the cells were treated with 7 μM patulin for 30 h and then apoptosis was measured by Annexin V staining. (C). Patulin induced apoptosis in p53 knockout/wild type MEF cells. The cells were treated with various patulin concentrations for 18 h and then apoptosis was measured by Annexin V staining. (D). PAT induced nuclear morphological changes in p53 knockout/wild type MEF cells. The cells were treated with 7 μM patulin for 12 h and then the nuclei were stained with DAPI. (E). Effects of p53 inhibitor pifithrin-α on patulin-induced apoptosis measured by Annexin V staining. (F). Effects of p53 inhibitor pifithrin-μ on patulin-induced apoptosis measured by Annexin V staining. (G). Effects of p53 inhibitor pifithrin-α on patulin-induced Bax up-regulations. The cells were treated with patulin in the presence or absence of pifithrin-α for 24 h and then Bax expression was analyzed by western blotting. n = 3, *P < 0.05, **P < 0.01. (The blots shown are representative of three independent experiments).
Mentions: Having established the pro-oxidant role of p53 activation in patulin-induced oxidative stress, we next investigated the role of p53 in patulin-induced apoptosis. As shown in Fig. 5A, when p53 was inactivated by RNAi, Bax up-regulation, p38 phosphorylation and PARP cleavages induced by patulin were significantly decreased in HEK293 cells. Accordingly, apoptosis induction by patulin was also decreased under the condition of p53 inactivation in HEK293 cells (Fig. 5B). In addition, a significant decreased apoptosis induction result was also observed in p53 knockout MEF cells in comparison with that found in p53 wt MEF cells measured by annexin v staining (Fig. 5C) and nuclear DAPI staining (Fig. 5D). These results suggested that p53-mediated Bax activation might contribute to apoptosis induction by patulin. We further validated the pro-apoptotic role of p53 by measuring apoptosis in the presence of pifithrin-alpha23 or pifithrin-mu2425, the two chemical inhibitors of p53 that can block p53-dependent transcriptional activation and inhibit transcriptional-independent p53 binding to mitochondria respectively. As shown in Fig. 5E,F, treatment with pifithrin-alpha led to a significant suppression of patulin-induced apoptosis, whereas pifithrin-mu failed to offer any protective effect on apoptosis induction by patulin. In support the protective effect of pifithrin-alpha, Bax induction by patulin was also blocked in the presence of pifithrin-alpha (Fig. 5G) but not in the presence of pifithrin-mu (data not shown). These results suggested that p53 activation contributed to apoptosis in response to patulin through its transcriptional mechanism.

Bottom Line: Patulin is a major mycotoxin found in fungal contaminated fruits and their derivative products.To the best of our knowledge, this is the first report addressing the functional role of p53 in patulin-induced oxidative stress.The findings of the present study provided novel insights into understanding mechanisms behind oxidative stress in response to patulin exposure.

View Article: PubMed Central - PubMed

Affiliation: Beijing Advanced Innovation Center for Food Nutrition and Human Health, Department of Nutrition and Health, College of Food Science and Nutritional Engineering, China Agricultural University, No17 Qinghua East Road, Haidian District, Beijing 100083, China.

ABSTRACT
Patulin is a major mycotoxin found in fungal contaminated fruits and their derivative products. Previous studies showed that patulin was able to induce increase of reactive oxygen species (ROS) generation and oxidative stress was suggested to play a pivotal role in patulin-induced multiple toxic signaling. The objective of the present study was to investigate the functional role of p53 in patulin-induced oxidative stress. Our study demonstrated that higher levels of ROS generation and DNA damage were induced in wild-type p53 cell lines than that found in either knockdown or knockout p53 cell lines in response to patulin exposure, suggesting p53 activation contributed to patulin-induced ROS generation. Mechanistically, we revealed that the pro-oxidant role of p53 in response to patulin was attributed to its ability to suppress catalase activity through up-regulation of PIG3. Moreover, these in vitro findings were further validated in the p53 wild-type/knockout mouse model. To the best of our knowledge, this is the first report addressing the functional role of p53 in patulin-induced oxidative stress. The findings of the present study provided novel insights into understanding mechanisms behind oxidative stress in response to patulin exposure.

No MeSH data available.


Related in: MedlinePlus