Toxicon 174 (2020) 19–25
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Microcystin-LR induced oxidative stress, inflammation, and apoptosis in alveolar type II epithelial cells of ICR mice in vitro Shengzheng Zhong a, Ying Liu b, Fang Wang a, Zaiwei Wu a, Sujuan Zhao a, * a b
School of Public Health, Anhui Medical University, Hefei, 230032, China Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Microcystin-LR Alveolar type II epithelial cells Inflammation Apoptosis Oxidative stress
Previous studies have shown that microcystin-LR (MC-LR) produced by toxic cyanobacterial blooms could inflict damage to the lung. However, the mechanisms underlying MC-induced pulmonary toxicity are not fully described. In this study, the primary’ fetal alveolar type II epithelial cells (AEC II) from ICR mice, which are involved in formation of bioactive component of pulmonary epithelium and secretion of pulmonary surfactants, were exposed to MC-LR at different concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20 μg/mL) for different time (12, 24, 36 h). Results showed that the viabilities of AEC II exposed to 10 and 20 μg MC-LR/mL were significantly decreased compared with the control group. Furthermore, MC-LR exposure resulted in overproduction of reac tive oxygen species (ROS) and induced a significant reduction in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Expressions of apoptosis-related proteins including bax, cyt-c, and caspase-9 were signif icantly up-regulated by exposure to 2.5, 5, 10, or 20 μg MC-LR/mL. When exposed to 5, 10, or 20 μg MC-LR/mL, expressions of proteins involved in inflammatory, p-65 and iNOS were significantly greater than those of the controls. In conclusion, inflammation and apoptosis might be responsible for MC-LR-induced pulmonary injury.
1. Introduction In the recent decades, cyanobacterial blooms have been increased around the world, and aquatic ecosystems contaminated by toxic cya nobacteria have raised public health concerns in many countries (Fer guson, 1999). MCs are a family of toxins produced by freshwater species of bloom-forming cyanobacteria, primarily Microcystis aeruginosa. Microcystin-leucine arginine (MC-LR) is one of the most common and toxic congeners among the 100 known MC isoforms (Yang et al., 2018). Previous studies showed that the concentration of MCLR reached up to 35.8 μg/L in October of 2008 in the Taihu Lake (Wang et al., 2010) and the maximum value found in the Chaohu Lake of China in 2012 was 17.6 μg/L (Niu et al., 2018). The environmental concentrations of dis solved MCs in lakes are far beyond the guidelines which set by WHO (1 μg/L for MCLR), which poses a potential threat to animals and human beings. There had been evidences suggesting that MCs caused death of animals that consumed water polluted by blue-green algae (Duy et al., 2000; Harding et al., 1995). There is also strong and increasing evidence for their adverse impacts on humans (Svircev et al., 2017). In Caruaru, Brazil, 100 of 131 patients who recieved routine renal dialysis treatment and used MCs polluted water developed acute liver failure, and 52 of
them died (Jochimsen et al., 1998). The mechanisms of MC toxicity have not been fully elucidated. MCLR is a highly specific and potent inhibitor of protein phosphatases 1 and 2A (Honkanen et al., 1990), which are involved in the regulation of phosphorylation of proteins (Bøe et al., 1991). It is well known that liver is the target organ of MC (Bhattacharya et al., 1996; Hooser et al., 1989). Apart from liver, MC-LR also cause toxicity to many organs, such as brain, kidney and testis (Jayaraj and Rao, 2006; Myhre et al., 2018; Zhao et al., 2016). Pulmonary injury induced by MCs is also a cause for concern. In 1989, two recruits in England developed severe pneumonia after swal lowing water containing massive Microcystis aeruginosa (Turner et al., 1990). Concentrations of MCs were highest in kidney, followed by lung in Wistar rats via intravenous injection (Wang et al., 2008). MCs can induce apoptosis and inflammation, as well as a rapid increase in lung impedance (Soares et al., 2007). Furthermore, results of several studies showed that the lung injury induced by MC-LR was associated with the dysfunction of immune cells and the production of inflammatory factors (Benson et al., 2005; Hermansky et al., 2010; Marcelino et al., 2004). However, the mechanism by which MC-LR causes lung injury is not clear.
* Corresponding author. E-mail address: [email protected]
(S. Zhao). https://doi.org/10.1016/j.toxicon.2019.12.152 Received 28 April 2019; Received in revised form 28 November 2019; Accepted 19 December 2019 Available online 23 December 2019 0041-0101/© 2019 Elsevier Ltd. All rights reserved.
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In this study, we investigated the effect of MC-LR on AEC II in vitro, which is quite important for the formation of bioactive component of pulmonary epithelium and the secretion of pulmonary surfactants (Rooney et al., 1994). The results will provide a better understanding of how the MC-LR exerts adverse effects in lungs.
three freeze-thaw cycles ( 80 � C and 37 � C, respectively), the cells were centrifuged at 10,000�g for 20 min at 4 � C and the culture supernatants were collected. In the assays for Western blot, the cells were seeded in 6-well culture plates at a density of 1 � 106 cells/well and were treated with 6 different doses (0, 1.25, 2.5, 5, 10, 20 μg/mL) of MC-LR for 24 h.
2. Materials and methods
2.5. MTT assay
2.1. Chemicals and reagents
The MTT assay was performed to identify the cell viability as described in a previous article (Pang et al., 2004). After the incubation in MC-LR, 10 μL MTT solution (5 mg/mL, 0.5%MTT) was added to each well and the cells were continuously incubated for 4 h. Then, the su pernatant was discarded and 150 μL dimethyl sulfoxide (DMSO) was added to each well. When the crystals were dissolved sufficiently, the plate was incubated for 4 h in a 37 � C incubator. The cell viability was measured by an enzyme-linked immune sorbent assay (ELISA) reader at 570 nm.
MC-LR (purity of �95%) purified by high-performance liquid chro matography (HPLC) was purchased from Taiwan Algal Science Inc. All other chemicals and reagents were purchased from conventional com mercial suppliers. 2.2. Animals and treatment Male or female 6-7-week-old ICR mice were obtained from the Central Institute of Anhui Animals in Anhui, China. Protocols for animal care and experimental management were approved by the Anhui Med ical University Animal Experimentation Committee. Mice were kept on a 12 h light/12 h dark cycle at a room, where the temperature was 22 � 2 � C and the mice had free access to food and water. Mice were put in cages to be mated and the female-male proportion was 2:1 in each cage. On the 18th day of gestation, after sacrificing the pregnant mice through CO2 inhalation, the fetuses were decapitated under sterile conditions. Then, fetal lungs were removed and placed in sterile phosphate-buffered saline (PBS) pre-cooled rapidly.
2.6. DCFH-DA assay The DCFH-DA assay was conducted to determine the levels of ROS. Having been incubated in different doses of MC-LR for different time, the cells were washed twice in EBSS (Eargle’s Balanced Salt Solution) and incubated in a 25 μM solution of DCFH-DA (in DMSO) for 30 min. Following the incubation, the ROS levels were measured with a micro plate reader at 488 nm excitation wavelength and 525 nm emission wavelength to detect fluorescence.
2.3. Isolation and purity of AEC II
2.7. Assays for SOD and GSH-Px
The isolation of AEC II was performed using the method of Cao et al. (2013), who had proved the validity of the method. Specifically, the lungs tissues separated from non-lung tissues were added to a 7 mL EP tube containing sterile PBS pre-heated to 37 � C, washed 3 times in PBS and cut into pieces rapidly (about 1200 times in 5 min). The lung tissues cut into pieces were rinsed 3 times in PBS and the 0.25% trypsin was used to digest cells at a temperature of 37 � C for 15–20 min. Dulbecco’s Modified Eagle Medium (DMEM), which contained 10% fetal calf serum (FBS), 100 units/mL of penicillin and 100 units/mL of streptomycin, was added to EP tube to cease the digestion. The lung homogenate was filtered through 150 μm and centrifuged for 5 min at 1000 r/min. After discarding the supernatant, the precipitate was resuspended with DMEM and cultured in a 100 mm Petri dish in an incubator with 95% air and 5% CO2 at 37 � C for 2 h. The cell suspension was transferred into another prepared Petri dish and continuously cultured in incubator for 24 h. Then, the medium was renewed every two days. When the cells grew to 5–6 � 106 cells/mL, they were harvested with 0.25% trypsin in 0.02% EDTA and obtained after centrifuging at 1400 r/min for 5 min. Ac cording to the following disparate experiments and analyses, the cells were made to cell suspension of different concentration with PBS.
According to the manufacture’s protocol, SOD levels were measured with kits purchased from Jiancheng Biochemical Inc in Nanjing, China. GSH-Px content was assayed using commercially available detection kits (Jiancheng Biochemical Inc, Nanjing, China) and the assay was based on the reaction with the thiol-specific reagent dithio-nitrobenzoic acid. The product was measured at 412 nm with a plate reader. 2.8. Western blotting The isolation of total proteins from the treated cells was imple mented with a total protein extraction kit (Beyotime, China) on the basis of manufacturer’s protocol. The concentrations of protein were deter mined by Nanodrop One (Thermo, USA). Western blotting analysis was performed according to our laboratory experience (Zhao et al., 2018). After electrophoresis, the PVDF membrane was blocked in TBS con taining 5% skim milk for 3 h and incubated in primary antibodies at 4 � C overnight. After 4 washes for 10 min in TBST, the PVDF membrane was incubated in matched secondary antibodies at room temperature for at least 1.5 h. After incubation, chemiluminescence imaging analysis sys tem (Tanon, China) was used to visualize and analyze protein bands. The primary antibodies employed in this analysis were listed below: rabbit anti-bax, rabbit anti-Cyt c, rabbit anti-iNOS, rabbit anti-p-65, mouse anti-bcl-2, mouse anti-caspase-9, and mouse anti-β-actin (CST, MA, USA).
2.4. Cell culture and treatment In MTT and DCFH-DA assays, AEC II cells were seeded in a 96-well cell culture plate at a density of 1 � 105 cells/well. After an incuba tion with 5% CO2 at 37 � C for 2 h, cells were treated with 7 different doses (0, 0.625, 1.25, 2.5, 5, 10, 20 μg/mL) of MC-LR for different time (12, 24 and 36 h) in triplicate. In the assays for SOD and GSH-Px, the cells seeded in a 24-well cell culture plate were cultured in DMEM supplemented with 10% FCS at a density of 2.5 � 105 cells/well and incubated with 5% CO2 at 37 � C for 24 h. Then, the cells were harvested after the removal of the medium. The cells alive were incubated in increasing densities of 0, 0.625, 1.25, 2.5, 5, 10, 20 μg/mL MC-LR solutions respectively for 12, 24 or 36 h, during which the culture media wasn’t renewed. After incubation and
2.9. Statistical analysis All analyses of data were conducted with SPSS 23.0 and all experi mental results were expressed as the mean � standard deviation. Normality was confirmed by the Kolmogorov-Smirnov test and homo geneity of variance was confirmed by use of Levine’s test. All graphic data analyses were performed by Graphpad prism 6.0. The significance of difference between control and MC-LR exposure groups was analyzed with one-way analysis of variance (ANOVA). 20
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3. Results 3.1. Reduced cell viability by MC-LR There were significant decreases in cell viability after 12 h and 24 h MC-LR exposures at the doses of 10 and 20 μg/mL (Fig. 1). Compared to the control, the cell viability decreased dramatically upon 36 h expo sures to MC-LR at the doses of 2.5, 5, 10 and 20 μg/mL (P < 0.01). 3.2. Over-production of ROS There was an obvious elevation in ROS levels after 12 h, 24 h and 36 h MC-LR exposures (Fig. 2). Except for the groups of 1.25 μg/mL for 12 h and 24 h, and 0.625 μg/mL for 36 h, ROS levels were significantly increased in all other groups (P < 0.05). 3.3. The inhibition of SOD and GSH-Px A downward trend of SOD activity was observed among the groups treated with the increased concentrations of MC-LR when compared to
Fig. 2. ROS levels are expressed as DCF fluorescence intensity in AECIIexposed to different concentrations of MC-LR for 12 h, 24 h, and 36 h. Each value is mean � S. D. *P < 0.05, **P < 0.01.
the control (Fig. 3A). The GSH-Px levels also decreased in a dosedependent manner after 12 h, 24 h and 36 h MC-LR exposure respec tively (Fig. 3B). 3.4. The changed protein expressions related to apoptosis and inflammation Following exposures to MC-LR, no significant differences between the control and MC-LR-treated AEC II were observed in the protein expression of Bcl-2 (Fig. 4). Compared to the control, the expressions of Bax in 5, 10 and 20 μg/mL exposure groups increased significantly (P < 0.01). The expressions of Cyt c appeared significant increases in all experimental groups (P < 0.05). There is significant elevation of the expressions of Caspase-9 upon exposure to MC-LR at the doses of 2.5–20 μg/mL (P < 0.05). In addition, the protein expressions related to inflammation also had been altered after exposure to MC-LR. The sig nificant increase of expression of p-65 occurred at the doses of 2.5, 5, 10 and 20 μg/mL (P < 0.01), while iNOS also increased significantly at the dose of 5, 10 and 20 μg/mL (P < 0.01).
Fig. 1. The effect of MC-LR on cell viability. The AECII cells were exposed to MC-LR at different doses for 12 h, 24 h, and 36 h. The levels of cell viability were normalized to the control. The * represents the differences compared with the corresponding control are statistically significant, *P < 0.05, **P < 0.01. 21
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Fig. 3. Alternations of antioxidant enzymes (SOD, GSH-Px) in AECII after exposure to MC-LR at different concentrations for 12 h, 24 h and 36 h (A) The activity of SOD; (B) The activity of GSH-Px. The values are expressed as mean � SD (N ¼ 6). *P < 0.05, **P < 0.01.
counteracted by the endogenous antioxidant defense system (D’Autr� eaux and Toledano, 2007; Mittler et al., 2011). The detection of SOD and GSH-Px levels can provide an indication of the antioxidant status and serve as biomarkers of oxidative stress. Endogenous ROS originated mainly from mitochondrial respiratory chain, and partly generated from enzyme system like nitric oxide synthetase (NOS) (Cao et al., 1988; Shaul, 2002). SOD is one of the major scavenger of ROS and the disproportionation reaction of SOD catalyzed oxygen radical (O2� ) with formation of the end-product, hydrogen peroxide (H2O2), which could be reduced to water by GSH-Px (McCord and Fridovich, 1969; Chance et al., 1979). Our data revealed that the ROS production in AEC II cells rose with an increase of MC-LR exposure dose. However, the activities of SOD and GSH-Px showed a significant reduction after MC-LR exposure. These results indicated that MC-LR could induce the oxidative stress in AEC II cells and the antioxidant capacity was greatly influenced by increasing doses of MC-LR. Oxidative stress can activate a variety of transcription factors, which lead to the differential expression of some genes involved in inflam matory pathways. The inflammation triggered by oxidative stress is the cause of many chronic diseases (Berlett and Stadtman, 1997). Many
Accumulating evidences have indicated that the lung might be another target organ of MC (Benson et al., 2005; Soares et al., 2007). It is well known that MC-LR can induce both acute and subacute toxic effects on the lung via intraperitoneal or intravenous injection (Ito et al., 2001). However, the possible mechanism causing the harmful effects of MC-LR on lung has not been clarified well. In this study, we found that MC-LR exposure could significantly reduce the viabilities of AEC II with the increasing doses and prolonged exposure time. It has been documented that MC-LR can inhibit cell growth and reduce cell viability (Mcdermott et al., 1998). In accordance with our study, one previous study also revealed that the cell viability of AEC II was significantly decreased after exposure to 50 and 500 nM MC-LR for 12 h (Wang et al., 2016). Recent studies have documented that MC-LR could induce excessive ROS, which consequently resulted in hydropic degeneration of mito chondria (Jiang et al., 2013; Meng et al., 2015; Zhi-Quan and Bo, 2008). In this study, we detected the intracellular ROS levels, which play a key role in oxidative stress. Oxidative stress and excessive ROS can be 22
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Fig. 4. Expression profiles of tested proteins in AECII cells exposed to each treatment. (A) Western blot of expression of relevant proteins; (B) The expression levels of proteins were quantified by densitometry and normalized to the expression of β-actin, including Bax, Bcl-2, Cyt c, Caspase-9, p-65 and iNOS. Data are presented as presented as the mean � sd. and analyzed by one-way ANOVA combined with a Turkey’s test. *P < 0.05, **P < 0.01vs control. 23
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acute studies demonstrated the inflammation induced by MC-LR in the lung (Carvalho et al., 2010; Casquilho et al., 2011; Toygar et al., 2015). In order to investigate the inflammation in AEC II, we detected the protein expressions of iNOS and p-65. We found that the expressions of iNOS and p-65 protein levels were promoted in the AEC II after MC-LR exposure. The dimer of p50/p65 is the most important nuclear factor-κB (NF-κB) almost existing in all types of cells (Boudoulas et al., 2017). NF-κB, the foremost regulatory factor in inflammation, can regulate the expression of inflammatory factor (Gerry and Leake, 2014; Mercurio and Manning, 1999). As the marker of inflammatory and oxidative stress, iNOS can be activated by NF-κB in inflammation process. The excessive iNOS could elicit the over-production of NO and the latter could induce €rstermann and Sessa, 2012). Coincidentally, our the oxidative stress (Fo present work demonstrated that MC-LR exposure induced NF-κB-me diated inflammation in AEC II cells. Amounting data showed that increased apoptosis was due to increase in oxidative stress and inflammation (Buttke and Sandstrom, 1994; Ratan et al., 2010). We detected the apoptosis related proteins in AEC II in this study. The results showed that the protein expressions of Cyt c, Bax and caspase-9 increased significantly upon MC-LR exposure, whereas the expression of Bcl-2 had no any significant changes. These data suggested that oxidative stress and ROS induced by MC-LR expo sure might result in apoptosis in AEC II cells. A previous study showed that the increase of ROS could induce the release of Cyt c (Nakamura and Sakamoto, 2001) and many studies demonstrated that ROS could induce apoptosis in various cell types (Pan et al., 1999; Efferth et al., 2007; Sharma et al., 2012). Besides, the promotion of Bax can induce the release of cytochrome C (Cyt c) from mitochondria to cytoplasm, consequently inducing apoptosis with the activation of caspase families (Salakou et al., 2007; Abdel-Latif et al., 2010; Xu et al., 2010). There fore, oxidative stress and inflammation induced apoptosis may be the main reason for pulmonary toxicity after MC-LR exposure. Taken together, results in this study showed that MC-LR exposure could lead to oxidative stress and inflammatory response, as well as cell apoptosis in AEC II cells in vitro. This study may improve understanding of the toxic effects of MC-LR on the respiratory system. Humans may result in lung injury by routine intake of MC-LR-contaminated foods or drinking water. There is a limitation that cells were directly exposed to MC-LR in vitro which can’t truly represent the effects in vivo. Further research is need.
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Author contributions section Author Contributions: Shengzheng Zhong and Ying Liu carried out the experiments. Fang Wang and Zaiwei Wu analyzed the data. Sujuan Zhao conceived and designed the experiments and wrote the article. All authors carefully reviewed the manuscript. Ethical statement All protocols for animal care and experimental management were approved by the Anhui Medical University Animal Experimentation Committee. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgments This work was funded by National Natural Science Foundation of China (31971521) and the Project Foundation for the Young Talents in Colleges of Anhui Province (Grand Number: gxyq2019011). We also want to thanks the platform of pathogenesis and precision therapy for major autoimmune diseases, the platform of environmental exposure and life health research, and the translational medicine project of 24
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