Elsevier

Toxicon

Volume 137, October 2017, Pages 106-113
Toxicon

Apigenin attenuates patulin-induced apoptosis in HEK293 cells by modulating ROS-mediated mitochondrial dysfunction and caspase signal pathway

https://doi.org/10.1016/j.toxicon.2017.07.018Get rights and content

Highlights

  • Apigenin attenuate patulin-induced nephrocyte in vivo.

  • ROS-mediated mitochondrial dysfunction regulation might be associated with the effect of apigenin.

  • Apigenin inhibited the caspase cascade signal pathway.

Abstract

Mycotoxins like patulin (PAT) are among the most significant food contaminant with regard to public health. This study aimed to evaluate the protective effect of apigenin (API), one of the most bioactive flavonoids in plant-derived food, on PAT-induced apoptosis in HEK293 cells. Cells were treated under basic conditions, 8 μM PAT without or with API (2.5, 5 and 10 μM) concomitantly for 10 h. API exerted renoprotective effect by inhibiting intracellular reactive oxygen species (ROS) accumulation, modulating oxidative phosphorylation especially elevating the expression of ATP synthase, re-establishing mitochondrial membrane potential (MMP) and maintaining higher intracellular ATP level, accompanied by p53, Bax downregulation and Bcl-2 upregulation. Thereby, cytochrome c release from mitochondria to cytoplasm was reduced, causing inhibition of initiator caspases-9 and executioner caspases (3, 6 and 7) expression and enzyme activities. Results revealed dietary apigenin attenuates patulin-induced apoptosis in HEK293 cells by modulating ROS-mediated mitochondrial dysfunction and caspase signal pathway.

Introduction

Patulin (PAT) is a polyketide lactone that was discovered to be a secondary metabolite produced by Penicillium expansum, an important postharvest pathogen of fruits, and other species of Penicillium, Aspergillus and Byssochlamys (McCormick, 2013). PAT has been shown to be immunotoxic, genotoxic, teratogenic and carcinogenic to several specials of animals, with kidney and liver as the major target organs (Boussabbeh et al., 2016a, Jin et al., 2016). Several major mechanisms have been shown to be involved in the toxicity of PAT: inhibition of DNA replication, RNA and protein biosynthesis (Arafat et al., 1985, Hatey and Moule, 1979, Lee and Roschenthaler, 1986), endoplasmic reticulum (ER) stress (Boussabbeh et al., 2015), interference with metabolic systems involving glutathione and thioredoxin systems, as well as reactive oxygen species (ROS) production and oxidative damage (Ayed-Boussema et al., 2013, Song et al., 2014b, Yang et al., 2011). Additionally, the cytotoxic effects of PAT have been shown to attributed to apoptosis, which was mediated through mitochondria pathway as revealed through the release of cytochrome c and then leading to enhancement of caspases activities (Wu et al., 2008). Moreover, p53, Bcl-2 family members and mitogen-activated protein kinase (MAPK) signaling pathway were involved in PAT triggered apoptotic process (Jin et al., 2016, Pillay et al., 2015). From a chemical point of view, PAT is a highly reactive molecule with a marked preference for sulfhydryl-containing compounds such as cysteine or gultathione in proteins. Previous studies in our laboratory have also demonstrated that exposure of HEK293 cells to PAT induced oxidative stress and mitochondria dependent apoptosis (Zhang et al., 2015).

Faced with the threat of health caused by PAT, the detoxification of PAT has been of major interest to researchers. Physical, chemical and biological approaches have been developed to reduce and/or eliminate the toxic effect of PAT and improve food safety (Karaca and Velioglu, 2009, McCormick, 2013). Some phytochemicals, including Vitamin E (Ayed-Boussema et al., 2013), ascorbic acid (Suzuki and Iwahashi, 2011), selenium (Song et al., 2014a), 6-gingerol (Yang et al., 2011), quercetin (Boussabbeh et al., 2016b), crocin (Boussabbeh et al., 2016a, Boussabbeh et al., 2015b) and green tea polyphenols (Song et al., 2014b) have been reported to protect animals and cultured cells against the toxic effects of PAT.

Apigenin (API) is a flavone subclass of flavonoid ubiquitously distributed in fruits, vegetables and herbs such as celery, parsley, perilla, oranges and grapefruits in the form of yellow pigment (Sharma et al., 2014). After dietary intake, API can cross blood-brain barrier and widely distribute in various tissues (Sharma et al., 2014). API has been shown to exhibit a variety of biological properties, including antioxidant (Sharma et al., 2014), anticancer (Saeed et al., 2015), hepatoprotective (Yang et al., 2013), renoprotective (Sharma et al., 2014) and neuroprotective (Wang et al., 2001). However, studies on API protecting cells or bodies against the deleterious effects of foodstuff contaminants are quite limited. And there is no available scientific literature concerning the ameliorative effect of API against PAT-induced toxicity. The present study which employed HEK293 cells as an in vitro model, was designed to elucidate the protective effects of API on the cell damage and apoptosis caused by PAT and the possible underlying mechanisms.

Section snippets

Cell culture and treatment condition

HEK293 cells were cultured in DMEM (Gibco, USA) with 10% FBS (Gibco) and 1% penicillin/streptomycin (Beyotime, China) at 37 °C in a humidified atmosphere with 5% CO2. For the treatment conditions, PAT was dissolved in sterile water to make a stock solution of 10 mM and further diluted to a final concentration of 8 μM with sterile water. API was dissolved in DMSO to make a stock solution of 5 mM and further diluted to final concentrations of 2.5, 5 and 10 μM with serum-free culture medium. The

API protects HEK293 cells against PAT-induced apoptosis

The cytoprotective effect of API on HEK293 cells was measured using MTT and LDH release assays. Both are indicative of cell viability whereas a decrease in crystal violet coloration measured by MTT assay is a sign of initial damage leading to cell detachment from the culture plate, but increase of LDH leakage in culture medium may be considered a marker indicative of necrosis. As shown in Fig. 1A, cell viability decreased to 71.4% when cells exposed 8 μM PAT for 10 h. A significant LDH leakage

Discussion

Recently, we have shown that patulin induced apoptosis in human embryonic kidney cells with oxidative stress and mitochondrial dysfunction involvement (Zhang et al., 2015). Here, we demonstrated that apigenin, one of the most bioactive flavonoids in plant-derived food, alleviates the ROS release and modulates the mitochondrial dysfunction and alterations of caspase cascade signals induced by PAT. This study established, for the first time, the renoprotective effect of API against PAT cellular

Conclusion

To summarise, our data revealed the mechanistic insights in the signaling pathways of API cytoprotection against the cell death process induced by PAT. We showed that API could significantly protect HEK293 cells against ROS-mediated mitochondria and caspase-dependent apoptotic pathway induced by PAT, providing a stronger evidence for a protective role of flavonoids against mycotoxin-drived toxicity in mammalian cells.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by grants from National Natural Science Foundation of China (NSFC, 31571928) and the Fundamental Research Funds for the Central Universities of China (2014YB021).

References (38)

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These authors contributed equally.

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