Ecotoxicology and Environmental Safety 127 (2016) 170–174
Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
The protective effect of blueberry anthocyanins against perfluorooctanoic acid-induced disturbance in planarian (Dugesia japonica) Zuoqing Yuan, Jianyong Zhang n, Changchao Tu, Zhijing Wang, Wenpeng Xin School of Life Sciences, Shandong University of Technology, No. 12 Zhangzhou Road, Zibo, Shandong 255049, China
art ic l e i nf o
a b s t r a c t
Article history: Received 30 October 2015 Received in revised form 22 January 2016 Accepted 25 January 2016 Available online 4 February 2016
The influence of blueberry anthocyanins on perfluorooctanoic acid (PFOA)-induced stress response in planarian mitochondria was investigated. PFOA at 15 mg/L and anthocyanins at 10 or 20 mg/L were individually and simultaneously administered to planarians for up to 10 d. The results showed PFOA treatment induced an increase in mitochondrial permeability transition pore opening and a decrease antioxidant capacity and enzyme activities. In anthocyanin treated animals, the activity of succinate dehydrogenase, cytochrome oxidase and monoamine oxidase increased, but mitochondrial permeability transition pore opening decreased and total antioxidant capacity increased. An improvement in abovementioned physiological and biochemical parameters was found in the combined PFOA and anthocyanin treated animals, in a dose-dependent manner. Anthocyanins attenuated the PFOA induced toxicity; antioxidant capacity and enzyme activities are involved in the protective mechanism of anthocyanins. & 2016 Elsevier Inc. All rights reserved.
Keywords: Dugesia japonica Perfluorooctanoic acid (PFOA) Anthocyanins Mitochondria Total antioxidant capacity Enzyme activities
1. Introduction Dugesia japonica is common in East Asia and widely distributed in China. In view of the planarian regeneration capacity and chemical sensitivity, D. japonica are used as model organisms in the research fields of regenerative medicine, stem cell biology, neurological disease and toxicology (Abril et al., 2010; Newmark and Alvarado, 2002; Kitamura et al., 1998; Wu et al., 2014; Zhang et al., 2013). Perfluorooctanoic acid (PFOA) is one of the major fully fluorinated organic compounds found in a variety of environmental matrices and biological samples worldwide (Lau et al., 2007). However, the mechanisms of action, the affected metabolic pathways and their resulting toxicological effects were mainly examined in rodents, and only a few studies have been performed on aquatic organisms, despite the aquatic environment being an important site for PFOA deposit. Many PFOA studies demonstrated that long-term treatment with PFOA results in hepatotoxicity, developmental toxicity, immunotoxicity, hormonal effects and carcinogenic potency (Andersen et al., 2008). Previously, we found that the enzyme activities of superoxidedismutase (SOD) and catalase (CAT) were altered in PFOA-exposed planarian, with obvious increase of apoptosis in brain, eye and parenchyma region (Yuan et al., 2015). Additionally, PFOA caused oxidative stress and n
Corresponding author. E-mail address: [email protected] (J. Zhang).
http://dx.doi.org/10.1016/j.ecoenv.2016.01.019 0147-6513/& 2016 Elsevier Inc. All rights reserved.
mitochondrial dysfunction in cells (Panaretakis et al., 2001), interfered with tissue metabolism by inducing mitochondrial permeability transition leading to inhibition of mitochondrial fatty acid β-oxidation (O’Brien and Wallace, 2004) and induced cell apoptosis via a p53-dependent mitochondrial pathway (Huang et al., 2013). These findings indicate that exposure to PFOA alters normal mitochondrial function or damages their structure. Anthocyanins (ANT) are a water-soluble natural pigment that appears as red, purple, and blue in plants and belongs to the flavonoid parent class of molecules. ANT are phytonutrients that have phenolic groups in their structures and have been widely studied due to their antioxidant activities (Williamson and Clifford, 2010). Blueberries (Vaccinium corymbosum L.) are recognized as a good source of ANT. Human studies demonstrated that blueberry anthocyanins provide and activate cellular antioxidant protection, prevent cellular oxidative stress, and consequently protect against oxidant-induced inflammatory cell damage and cytotoxicity (Harborne and Wiliam, 2000; Zafra-Stone et al., 2007; Tan et al., 2014). To examine the potential protective role of blueberry anthocyanins to planarian, we administered the blueberry anthocyanins to planarian under PFOA stress. Mitochondrial permeability transition pore (MPTP) opening, total antioxidant capacity (TAC), succinate dehydrogenase (SDH), cytochrome oxidase (COX) and monoamine oxidase (MAO) activities were then examined and compared. Although there are many studies investigating PFOA toxicity, little is known about the effect of PFOA on mitochondrial function
Z. Yuan et al. / Ecotoxicology and Environmental Safety 127 (2016) 170–174
at the biochemical level in planarian. Additionally, the mechanisms of action associated with ANT are not well defined. Investigating the properties of this phytonutrient is of great significance, because it is a natural compound found in fruits and vegetables and is present in the human diet in many countries. The aim of this study was to assess the toxic effects of PFOA and the protective effects of blueberry anthocyanins, under continuous exposure, on the MPTP opening, TAC, SDH, COX and MAO activities in this freshwater planarian. The results of this study provide a mechanistic understanding of PFOA toxicity, and helpful information toward establishing D. japonica as a research model for studying the toxicity of other environmental contaminants.
2. Materials and methods 2.1. Animals and chemicals The stock of D. japonica used in this study was obtained from a fountain in Quanhetou (Zibo, China) and acclimated in our laboratory for 42 weeks as described previously (Zhang et al., 2013). The animals were kept in autoclaved tap water at 20 °C. The water was aerated continuously and changed every 2 days until use. Size-selected intact planarians ( 41 cm TL) were used for this study. Planarians were starved for 1 week to create a uniform metabolic status before starting the experiments. PFOA was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). ANT were extracted and purified from blueberries and were commercially available from Reain Biotechnology Co. (Xian, China). 2.2. Exposure experiments Planarians 1.0–1.5 cm in length were used within 10 days for the experiments. Stock solutions of PFOA and blueberry anthocyanins were prepared on the day of the experiment and diluted to desired concentrations using autoclaved tap water. The planarians were continuously exposed to ANT and PFOA at different concentrations as follows: (1) control: incubated in autoclaved tap water; (2) PFOA-treatment: incubated in PFOA solution at 15 mg/L; (3) Anthocyanins treatment: incubated in solution containing ANT (10 mg/L); (4) PFOA þANT treatment: incubated in solution containing PFOA (15 mg/L) and ANT (10 mg/L or 20 mg/L, respectively). All experiments at the different concentrations were repeated in triplicate and each experiment involved 100 planarians. Planarians were sampled on days 1, 3, 7 and 10, and used for the desired analysis. 2.3. Isolation of mitochondria All of the procedures were carried out at 4 °C. 100 mg of the planarians undergoing the different treatments were washed with phosphate-buffered saline (PBS) buffer in 1.5 mL centrifugal tubes. Mitochondria were isolated from different treatments and different treatment days using a Mitochondria Isolation Kit (C3606; Beyotime Institute of Biotechnology, Shanghai, China). Isolation of mitochondria was repeated in triplicate and the isolated mitochondria were suspended in the storage solution from the Kit. Mitochondria solution (10 μL) was used for the determination of protein content by the Bradford method. 2.4. MPTP opening assay The isolated mitochondria (0.1 mL) were pre-incubated in a Quartz cup containing 2.9 mL medium (pH 7.4) consisting of sucrose (0.25 mM), KCl (10 mM), MgCl (5 mM), KH2PO4 (5 mM), Tris–HCl (10 mM), and succinate (10 mM) at 30 °C for 10 min. The
171
MPTP opening in the mitochondria from different groups was measured by monitoring the absorbance at 520 nm (A520) as described (Zhao et al., 2010).
MPTP opening (%) =
Ac − At Ac
where Ac represents the absorbance value of the control group and At is the absorbance value of treatment group. 2.5. Total antioxidant capacity assay Planarians (20 mg) from different treatment groups and different treatment days were homogenized using a grinding rod in a centrifugal tube on ice. After centrifugation at 12,000 g for 10 min at 4 °C, the supernatant was collected and stored at 20 °C. Total antioxidant capacity of the planarians was measured using the Total Antioxidant Capacity Kit (S0116; Beyotime Institute of Biotechnology, Shanghai, China) according to manufacturer instructions using the ferric-reducing antioxidant power method (FRAP) (Benzie and Strain, 1999). For the FRAP assay, total antioxidant capacity is represented by different concentrations of FeSO4 absorbance values. If the absorbance value of the planarian samples is same to the absorbance value of 1 mM FeSO4, and the total antioxidant capacity of the tissue samples was 1 mM. Sample solutions (20 μL) were added directly to the 96-well microplate followed by 180 μL of working FRAP solution. The mixtures were incubated in the dark for 30 min at 37 °C and absorbance readings were recorded at 593 nm (A593). The standard curve was constructed using iron (II) sulfate solution (150–1500 μM). All measurements were performed in triplicate and the mean values were calculated. 2.6. Enzymes activity assays The isolated mitochondria (10 μL) were used to determine the activities of SDH, COX and MAO. The activities of SDH, COX and MAO were measured using the Tissue SDH Assay Kit, COX Assay Kit and Tissue MAO Assay Kit (GenMed, Shanghai, China) according to manufacturer instructions, respectively. The enzyme activities were measured by ultraviolet-visible spectrophotometric (SP756 Spectrophotometer; Shanghai Spectrum Instruments, Shanghai, China) analysis at 595 nm. Measurements were collected at 5-min intervals and normalized to protein content. 2.7. Statistical analysis Data are presented as mean and standard deviation (SD). Statistical significance was determined by the analysis of variance using Statistical Package for the Social Sciences for Windows (SPSS, version 16.0; Chicago, IL, USA). Treatment groups were tested for differences from the control and PFOA groups using Dunnett’s t test. Statistically significant differences were determined at P o0.05.
3. Results 3.1. Effect of PFOA and blueberry anthocyanins on MPTP opening MPTP opening was investigated using the spectrophotometric method. Changes in the absorbance values were used to detect MPTP opening. As shown in Fig. 1, PFOA resulted in a significant increase in MPTP opening as compared to the control group (P o0.01). The MPTP opening slightly decreased in ANT treatment groups relative to the control. The treatment groups with exposure to PFOA and ANT exhibited rapidly decreasing absorbance values
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Fig. 1. Evaluation of MPTP opening in isolated mitochondria from planarian subjected to PFOA or blueberry anthocyanins-enriched extracts. The isolated mitochondria were preincubated in Quartz cups containing 2.9 mL medium. MPTP opening of mitochondria from different groups was measured by monitoring the absorbance at treatment times of 1, 3, 7 and 10 days, respectively. *Po 0.05 and ** Po 0.01 indicate differences as compared to the normal control group; # P o0.05 and ## P o 0.01 indicate differences as compared to the PFOA group.
as compared to the PFOA group, indicating that MPTP opening decreased. As shown in Fig. 1, 20 mg/L ANT was capable of protecting PFOA-induced MPTP opening (P o0.01). This indicated that the protective effects of ANT on MPTP was dose-dependent. From these data, we concluded that PFOA can induce MPTP opening, while ANT can inhibit MPTP opening and maintain the normal physiological function of mitochondria. 3.2. Effect of PFOA and blueberry anthocyanins on TAC The mean 7SD of TAC in different study groups are shown in Fig. 2. It was observed that TAC was affected by the PFOA. Studies confirmed that PFOA exposure caused a significant decrease in TAC compared to the control group (Po 0.01). Moreover, there was a significant increase in TAC when following ANT treatment as compared to the control group (Po 0.01). Although the ANT increased TAC levels under PFOA stress, the significant increases were only observed in the group treated with 20 mg/L ANT and PFOA (Po0.01). These data indicated that PFOA can decrease TAC, but ANT can increase antioxidant capacity and attenuate PFOA stress. 3.3. Effect of PFOA and blueberry anthocyanins on SDH, COX and MAO In the assay, the direct effects of PFOA and/or ANT on SDH, COX and MAO activity were measured (Fig. 3). PFOA affected enzyme activity (P o0.01), with the enzyme activities of SDH, COX and MAO significantly enhanced by the addition of ANT, while PFOA toxicity was significantly attenuated by the addition of 10 or 20 mg/L ANT. SDH activity significantly decreased in the PFOA
treatment group and significantly increased as compared to the control group on the first day (P o0.05). The SDH activity improved significantly in the PFOA and ANT group as compared with the PFOA group (Fig. 3A). COX activity was also enhanced in the different treatment groups along with the increased number of exposure days (Fig. 3B). Here, we found that COX activity was the highest in the ANT group, showing a significant increase (P o0.01). We also observed that ANT significantly attenuated the negative impact of PFOA during the entire process (P o0.01). Under PFOA exposure, MAO activity was markedly decreased (Fig. 3C), planarians from the ANT group demonstrated the highest MAO activity (Po 0.01). ANT (10 and 20 mg/L) significantly inhibited the decrease of MAO activity (P o0.01). Although ANT can increase MAO activity, there was no significant difference observed between ANT and PFOA mixture groups as compared to the control group.
4. Discussion This study focused on the toxic effects of PFOA and the protective role of blueberry anthocyanins. As in our previous report, we hypothesized that PFOA may result in reactive oxygen species (ROS) and cell apoptosis (Yuan et al., 2015). Here, we showed that PFOA treatment induced oxidative stress-mediated mitochondria dysfunction, while ANT effectively inhibited the toxic effects of PFOA and maintained normal cellular functionality. The MPTP has many functions, including maintaining matrix volume, regulating pH, acting as a redox sensor, protein import and allowing Ca2 þ release from the mitochondria (Belzacq et al., 2002). Under normal conditions, the MPTP remains closed, but
Fig. 2. Evaluation of total antioxidant capacity in planarian subjected to PFOA or blueberry anthocyanins-enriched extracts. Planarians from different treatments groups and different treatment days were homogenized. Total antioxidant capacity of planarians was measured using the total antioxidant capacity kit using the ferric-reducing antioxidant power method (FRAP) at treatment times of 1, 3, 7 and 10 days, respectively. *Po 0.05 and ** Po 0.01 indicate differences as compared to the normal control group; ## P o 0.01 indicate differences as compared to the PFOA group.
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maintaining optimal cellular function to help protect the planarian from oxidative injury. Research of the effects of blueberries on organisms showed that ANT is an important antioxidant capable of effectively preventing decline in TAC (Wu et al., 2002). ANT is also effective ROS and reactive nitrogen species scavengers (Kahkonen and Heinonen, 2003). We also observed that TAC in planarian tissues increased following the ANT administration. Although the mechanisms of ANT activity are not well defined, our studies have shown that ANT is capable of preventing deleterious effects induced by PFOA in planarian. The mechanisms that are responsible for PFOA toxicity have not been fully elucidated. The inhibition of SDH, COX and MAO appears to represent the mechanism by which PFOA produces its toxic effects, since such inhibition would lead to other observed PFOA-induced effects. SDH, COX and MAO localized to the mitochondrial membrane, and dysregulation of SDH, COX and MAO can result in oxidative stress. A broad spectrum of in vitro, in vivo, and human studies have demonstrated that blueberry anthocyanins provide and activate cellular antioxidant protection, inhibit inflammatory gene expression and consequently protect against oxidant-induced cell damage or cytotoxicity (Zafra-Stone et al., 2007). In conclusion, we described the protective effects of blueberry anthocyanins following PFOA-induced stress in planarian. Moreover, this protective effect may be mediated by the inhibition of MPTP opening, increasing TAC and enzyme activities. A potential mechanism by which ANT inhibit MPTP opening, increase TAC and enzyme activities includes the mitigation of ROS generation. The elucidation of additional ANT effects, as well as how ANTs attenuate ROS generation to modulate the MPTP, requires further investigation.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgments This research work was supported by the National Natural Science Foundation of China (Grant no. 31100377, 41201518).
References Fig. 3. Evaluation the activity of SDH (A), COX (B) and MAO (C) in planarian subjected to PFOA or blueberry anthocyanins-enriched extracts. The isolated mitochondria were used to determine the activities of of SDH, COX and MAO by kit. The enzyme activities were measured by ultraviolet-visible spectrophotometric analysis at treatment times of 1, 3, 7 and 10 days, respectively. *P o0.05 and ** Po 0.01 indicate differences as compared to the normal control group; # P o 0.05 and ## P o0.01 indicate differences as compared to the PFOA group.
then opens under conditions of cellular stress following accumulation of Ca2 þ or oxidative damage-generating ROS. Therefore, inhibiting MPTP opening is an important target for attenuating stress. Increasing evidence suggested that ROS can trigger MPTP opening during oxidative stress. And that transient MPTP opening can lead to the release of cytochrome c and initiation of the apoptotic cascade (Hausenloy et al., 2009). In this experiment, the effect of PFOA on MPTP opening and the protective effects of blueberry anthocyanins were investigated. We concluded that one of the protective mechanisms of ANT against PFOA stress was the inhibition of MPTP opening via mitigated ROS generation. Additionally, we believe that when oxidative stress accumulates, protection by endogenous antioxidants is necessary for
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Contents lists available at ScienceDirect
Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
The protective effect of blueberry anthocyanins against perfluorooctanoic acid-induced disturbance in planarian (Dugesia japonica) Zuoqing Yuan, Jianyong Zhang n, Changchao Tu, Zhijing Wang, Wenpeng Xin School of Life Sciences, Shandong University of Technology, No. 12 Zhangzhou Road, Zibo, Shandong 255049, China
art ic l e i nf o
a b s t r a c t
Article history: Received 30 October 2015 Received in revised form 22 January 2016 Accepted 25 January 2016 Available online 4 February 2016
The influence of blueberry anthocyanins on perfluorooctanoic acid (PFOA)-induced stress response in planarian mitochondria was investigated. PFOA at 15 mg/L and anthocyanins at 10 or 20 mg/L were individually and simultaneously administered to planarians for up to 10 d. The results showed PFOA treatment induced an increase in mitochondrial permeability transition pore opening and a decrease antioxidant capacity and enzyme activities. In anthocyanin treated animals, the activity of succinate dehydrogenase, cytochrome oxidase and monoamine oxidase increased, but mitochondrial permeability transition pore opening decreased and total antioxidant capacity increased. An improvement in abovementioned physiological and biochemical parameters was found in the combined PFOA and anthocyanin treated animals, in a dose-dependent manner. Anthocyanins attenuated the PFOA induced toxicity; antioxidant capacity and enzyme activities are involved in the protective mechanism of anthocyanins. & 2016 Elsevier Inc. All rights reserved.
Keywords: Dugesia japonica Perfluorooctanoic acid (PFOA) Anthocyanins Mitochondria Total antioxidant capacity Enzyme activities
1. Introduction Dugesia japonica is common in East Asia and widely distributed in China. In view of the planarian regeneration capacity and chemical sensitivity, D. japonica are used as model organisms in the research fields of regenerative medicine, stem cell biology, neurological disease and toxicology (Abril et al., 2010; Newmark and Alvarado, 2002; Kitamura et al., 1998; Wu et al., 2014; Zhang et al., 2013). Perfluorooctanoic acid (PFOA) is one of the major fully fluorinated organic compounds found in a variety of environmental matrices and biological samples worldwide (Lau et al., 2007). However, the mechanisms of action, the affected metabolic pathways and their resulting toxicological effects were mainly examined in rodents, and only a few studies have been performed on aquatic organisms, despite the aquatic environment being an important site for PFOA deposit. Many PFOA studies demonstrated that long-term treatment with PFOA results in hepatotoxicity, developmental toxicity, immunotoxicity, hormonal effects and carcinogenic potency (Andersen et al., 2008). Previously, we found that the enzyme activities of superoxidedismutase (SOD) and catalase (CAT) were altered in PFOA-exposed planarian, with obvious increase of apoptosis in brain, eye and parenchyma region (Yuan et al., 2015). Additionally, PFOA caused oxidative stress and n
Corresponding author. E-mail address: [email protected] (J. Zhang).
http://dx.doi.org/10.1016/j.ecoenv.2016.01.019 0147-6513/& 2016 Elsevier Inc. All rights reserved.
mitochondrial dysfunction in cells (Panaretakis et al., 2001), interfered with tissue metabolism by inducing mitochondrial permeability transition leading to inhibition of mitochondrial fatty acid β-oxidation (O’Brien and Wallace, 2004) and induced cell apoptosis via a p53-dependent mitochondrial pathway (Huang et al., 2013). These findings indicate that exposure to PFOA alters normal mitochondrial function or damages their structure. Anthocyanins (ANT) are a water-soluble natural pigment that appears as red, purple, and blue in plants and belongs to the flavonoid parent class of molecules. ANT are phytonutrients that have phenolic groups in their structures and have been widely studied due to their antioxidant activities (Williamson and Clifford, 2010). Blueberries (Vaccinium corymbosum L.) are recognized as a good source of ANT. Human studies demonstrated that blueberry anthocyanins provide and activate cellular antioxidant protection, prevent cellular oxidative stress, and consequently protect against oxidant-induced inflammatory cell damage and cytotoxicity (Harborne and Wiliam, 2000; Zafra-Stone et al., 2007; Tan et al., 2014). To examine the potential protective role of blueberry anthocyanins to planarian, we administered the blueberry anthocyanins to planarian under PFOA stress. Mitochondrial permeability transition pore (MPTP) opening, total antioxidant capacity (TAC), succinate dehydrogenase (SDH), cytochrome oxidase (COX) and monoamine oxidase (MAO) activities were then examined and compared. Although there are many studies investigating PFOA toxicity, little is known about the effect of PFOA on mitochondrial function
Z. Yuan et al. / Ecotoxicology and Environmental Safety 127 (2016) 170–174
at the biochemical level in planarian. Additionally, the mechanisms of action associated with ANT are not well defined. Investigating the properties of this phytonutrient is of great significance, because it is a natural compound found in fruits and vegetables and is present in the human diet in many countries. The aim of this study was to assess the toxic effects of PFOA and the protective effects of blueberry anthocyanins, under continuous exposure, on the MPTP opening, TAC, SDH, COX and MAO activities in this freshwater planarian. The results of this study provide a mechanistic understanding of PFOA toxicity, and helpful information toward establishing D. japonica as a research model for studying the toxicity of other environmental contaminants.
2. Materials and methods 2.1. Animals and chemicals The stock of D. japonica used in this study was obtained from a fountain in Quanhetou (Zibo, China) and acclimated in our laboratory for 42 weeks as described previously (Zhang et al., 2013). The animals were kept in autoclaved tap water at 20 °C. The water was aerated continuously and changed every 2 days until use. Size-selected intact planarians ( 41 cm TL) were used for this study. Planarians were starved for 1 week to create a uniform metabolic status before starting the experiments. PFOA was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). ANT were extracted and purified from blueberries and were commercially available from Reain Biotechnology Co. (Xian, China). 2.2. Exposure experiments Planarians 1.0–1.5 cm in length were used within 10 days for the experiments. Stock solutions of PFOA and blueberry anthocyanins were prepared on the day of the experiment and diluted to desired concentrations using autoclaved tap water. The planarians were continuously exposed to ANT and PFOA at different concentrations as follows: (1) control: incubated in autoclaved tap water; (2) PFOA-treatment: incubated in PFOA solution at 15 mg/L; (3) Anthocyanins treatment: incubated in solution containing ANT (10 mg/L); (4) PFOA þANT treatment: incubated in solution containing PFOA (15 mg/L) and ANT (10 mg/L or 20 mg/L, respectively). All experiments at the different concentrations were repeated in triplicate and each experiment involved 100 planarians. Planarians were sampled on days 1, 3, 7 and 10, and used for the desired analysis. 2.3. Isolation of mitochondria All of the procedures were carried out at 4 °C. 100 mg of the planarians undergoing the different treatments were washed with phosphate-buffered saline (PBS) buffer in 1.5 mL centrifugal tubes. Mitochondria were isolated from different treatments and different treatment days using a Mitochondria Isolation Kit (C3606; Beyotime Institute of Biotechnology, Shanghai, China). Isolation of mitochondria was repeated in triplicate and the isolated mitochondria were suspended in the storage solution from the Kit. Mitochondria solution (10 μL) was used for the determination of protein content by the Bradford method. 2.4. MPTP opening assay The isolated mitochondria (0.1 mL) were pre-incubated in a Quartz cup containing 2.9 mL medium (pH 7.4) consisting of sucrose (0.25 mM), KCl (10 mM), MgCl (5 mM), KH2PO4 (5 mM), Tris–HCl (10 mM), and succinate (10 mM) at 30 °C for 10 min. The
171
MPTP opening in the mitochondria from different groups was measured by monitoring the absorbance at 520 nm (A520) as described (Zhao et al., 2010).
MPTP opening (%) =
Ac − At Ac
where Ac represents the absorbance value of the control group and At is the absorbance value of treatment group. 2.5. Total antioxidant capacity assay Planarians (20 mg) from different treatment groups and different treatment days were homogenized using a grinding rod in a centrifugal tube on ice. After centrifugation at 12,000 g for 10 min at 4 °C, the supernatant was collected and stored at 20 °C. Total antioxidant capacity of the planarians was measured using the Total Antioxidant Capacity Kit (S0116; Beyotime Institute of Biotechnology, Shanghai, China) according to manufacturer instructions using the ferric-reducing antioxidant power method (FRAP) (Benzie and Strain, 1999). For the FRAP assay, total antioxidant capacity is represented by different concentrations of FeSO4 absorbance values. If the absorbance value of the planarian samples is same to the absorbance value of 1 mM FeSO4, and the total antioxidant capacity of the tissue samples was 1 mM. Sample solutions (20 μL) were added directly to the 96-well microplate followed by 180 μL of working FRAP solution. The mixtures were incubated in the dark for 30 min at 37 °C and absorbance readings were recorded at 593 nm (A593). The standard curve was constructed using iron (II) sulfate solution (150–1500 μM). All measurements were performed in triplicate and the mean values were calculated. 2.6. Enzymes activity assays The isolated mitochondria (10 μL) were used to determine the activities of SDH, COX and MAO. The activities of SDH, COX and MAO were measured using the Tissue SDH Assay Kit, COX Assay Kit and Tissue MAO Assay Kit (GenMed, Shanghai, China) according to manufacturer instructions, respectively. The enzyme activities were measured by ultraviolet-visible spectrophotometric (SP756 Spectrophotometer; Shanghai Spectrum Instruments, Shanghai, China) analysis at 595 nm. Measurements were collected at 5-min intervals and normalized to protein content. 2.7. Statistical analysis Data are presented as mean and standard deviation (SD). Statistical significance was determined by the analysis of variance using Statistical Package for the Social Sciences for Windows (SPSS, version 16.0; Chicago, IL, USA). Treatment groups were tested for differences from the control and PFOA groups using Dunnett’s t test. Statistically significant differences were determined at P o0.05.
3. Results 3.1. Effect of PFOA and blueberry anthocyanins on MPTP opening MPTP opening was investigated using the spectrophotometric method. Changes in the absorbance values were used to detect MPTP opening. As shown in Fig. 1, PFOA resulted in a significant increase in MPTP opening as compared to the control group (P o0.01). The MPTP opening slightly decreased in ANT treatment groups relative to the control. The treatment groups with exposure to PFOA and ANT exhibited rapidly decreasing absorbance values
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Fig. 1. Evaluation of MPTP opening in isolated mitochondria from planarian subjected to PFOA or blueberry anthocyanins-enriched extracts. The isolated mitochondria were preincubated in Quartz cups containing 2.9 mL medium. MPTP opening of mitochondria from different groups was measured by monitoring the absorbance at treatment times of 1, 3, 7 and 10 days, respectively. *Po 0.05 and ** Po 0.01 indicate differences as compared to the normal control group; # P o0.05 and ## P o 0.01 indicate differences as compared to the PFOA group.
as compared to the PFOA group, indicating that MPTP opening decreased. As shown in Fig. 1, 20 mg/L ANT was capable of protecting PFOA-induced MPTP opening (P o0.01). This indicated that the protective effects of ANT on MPTP was dose-dependent. From these data, we concluded that PFOA can induce MPTP opening, while ANT can inhibit MPTP opening and maintain the normal physiological function of mitochondria. 3.2. Effect of PFOA and blueberry anthocyanins on TAC The mean 7SD of TAC in different study groups are shown in Fig. 2. It was observed that TAC was affected by the PFOA. Studies confirmed that PFOA exposure caused a significant decrease in TAC compared to the control group (Po 0.01). Moreover, there was a significant increase in TAC when following ANT treatment as compared to the control group (Po 0.01). Although the ANT increased TAC levels under PFOA stress, the significant increases were only observed in the group treated with 20 mg/L ANT and PFOA (Po0.01). These data indicated that PFOA can decrease TAC, but ANT can increase antioxidant capacity and attenuate PFOA stress. 3.3. Effect of PFOA and blueberry anthocyanins on SDH, COX and MAO In the assay, the direct effects of PFOA and/or ANT on SDH, COX and MAO activity were measured (Fig. 3). PFOA affected enzyme activity (P o0.01), with the enzyme activities of SDH, COX and MAO significantly enhanced by the addition of ANT, while PFOA toxicity was significantly attenuated by the addition of 10 or 20 mg/L ANT. SDH activity significantly decreased in the PFOA
treatment group and significantly increased as compared to the control group on the first day (P o0.05). The SDH activity improved significantly in the PFOA and ANT group as compared with the PFOA group (Fig. 3A). COX activity was also enhanced in the different treatment groups along with the increased number of exposure days (Fig. 3B). Here, we found that COX activity was the highest in the ANT group, showing a significant increase (P o0.01). We also observed that ANT significantly attenuated the negative impact of PFOA during the entire process (P o0.01). Under PFOA exposure, MAO activity was markedly decreased (Fig. 3C), planarians from the ANT group demonstrated the highest MAO activity (Po 0.01). ANT (10 and 20 mg/L) significantly inhibited the decrease of MAO activity (P o0.01). Although ANT can increase MAO activity, there was no significant difference observed between ANT and PFOA mixture groups as compared to the control group.
4. Discussion This study focused on the toxic effects of PFOA and the protective role of blueberry anthocyanins. As in our previous report, we hypothesized that PFOA may result in reactive oxygen species (ROS) and cell apoptosis (Yuan et al., 2015). Here, we showed that PFOA treatment induced oxidative stress-mediated mitochondria dysfunction, while ANT effectively inhibited the toxic effects of PFOA and maintained normal cellular functionality. The MPTP has many functions, including maintaining matrix volume, regulating pH, acting as a redox sensor, protein import and allowing Ca2 þ release from the mitochondria (Belzacq et al., 2002). Under normal conditions, the MPTP remains closed, but
Fig. 2. Evaluation of total antioxidant capacity in planarian subjected to PFOA or blueberry anthocyanins-enriched extracts. Planarians from different treatments groups and different treatment days were homogenized. Total antioxidant capacity of planarians was measured using the total antioxidant capacity kit using the ferric-reducing antioxidant power method (FRAP) at treatment times of 1, 3, 7 and 10 days, respectively. *Po 0.05 and ** Po 0.01 indicate differences as compared to the normal control group; ## P o 0.01 indicate differences as compared to the PFOA group.
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maintaining optimal cellular function to help protect the planarian from oxidative injury. Research of the effects of blueberries on organisms showed that ANT is an important antioxidant capable of effectively preventing decline in TAC (Wu et al., 2002). ANT is also effective ROS and reactive nitrogen species scavengers (Kahkonen and Heinonen, 2003). We also observed that TAC in planarian tissues increased following the ANT administration. Although the mechanisms of ANT activity are not well defined, our studies have shown that ANT is capable of preventing deleterious effects induced by PFOA in planarian. The mechanisms that are responsible for PFOA toxicity have not been fully elucidated. The inhibition of SDH, COX and MAO appears to represent the mechanism by which PFOA produces its toxic effects, since such inhibition would lead to other observed PFOA-induced effects. SDH, COX and MAO localized to the mitochondrial membrane, and dysregulation of SDH, COX and MAO can result in oxidative stress. A broad spectrum of in vitro, in vivo, and human studies have demonstrated that blueberry anthocyanins provide and activate cellular antioxidant protection, inhibit inflammatory gene expression and consequently protect against oxidant-induced cell damage or cytotoxicity (Zafra-Stone et al., 2007). In conclusion, we described the protective effects of blueberry anthocyanins following PFOA-induced stress in planarian. Moreover, this protective effect may be mediated by the inhibition of MPTP opening, increasing TAC and enzyme activities. A potential mechanism by which ANT inhibit MPTP opening, increase TAC and enzyme activities includes the mitigation of ROS generation. The elucidation of additional ANT effects, as well as how ANTs attenuate ROS generation to modulate the MPTP, requires further investigation.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgments This research work was supported by the National Natural Science Foundation of China (Grant no. 31100377, 41201518).
References Fig. 3. Evaluation the activity of SDH (A), COX (B) and MAO (C) in planarian subjected to PFOA or blueberry anthocyanins-enriched extracts. The isolated mitochondria were used to determine the activities of of SDH, COX and MAO by kit. The enzyme activities were measured by ultraviolet-visible spectrophotometric analysis at treatment times of 1, 3, 7 and 10 days, respectively. *P o0.05 and ** Po 0.01 indicate differences as compared to the normal control group; # P o 0.05 and ## P o0.01 indicate differences as compared to the PFOA group.
then opens under conditions of cellular stress following accumulation of Ca2 þ or oxidative damage-generating ROS. Therefore, inhibiting MPTP opening is an important target for attenuating stress. Increasing evidence suggested that ROS can trigger MPTP opening during oxidative stress. And that transient MPTP opening can lead to the release of cytochrome c and initiation of the apoptotic cascade (Hausenloy et al., 2009). In this experiment, the effect of PFOA on MPTP opening and the protective effects of blueberry anthocyanins were investigated. We concluded that one of the protective mechanisms of ANT against PFOA stress was the inhibition of MPTP opening via mitigated ROS generation. Additionally, we believe that when oxidative stress accumulates, protection by endogenous antioxidants is necessary for
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