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FoxO pathway mediates glycolytic metabolism in HepG2 cells exposed to triclosan (TCS)
Environment International 136 (2020) 105428
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PI3K/Akt/FoxO pathway mediates glycolytic metabolism in HepG2 cells exposed to triclosan (TCS) ⁎
Jing Ana, Huixin Hea, Weiwei Yaoa, Yu Shanga, Yun Jiangc, , Zhiqiang Yub,
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a
Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, PR China State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, PR China c Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, PR China b
A R T I C LE I N FO
A B S T R A C T
Handling Editor: Da Chen
Triclosan (TCS) has been widely used as an antibacterial agent for the last several decades in personal care products. The toxicological effect of TCS has attracted more and more attention of researchers. The purpose of this study is to evaluate the cytotoxic effects of TCS in HepG2 cells and to elucidate the molecular mechanism focusing on regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/forkhead box O (FoxO) pathway in the glycolytic metabolism. In this study, we evaluated the adverse effect of TCS exposure on cell viability, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and mitochondrial membrane potential (MMP). In addition, the glycolysis process in HepG2 cells exposed to TCS was examined in terms of glucose consumption, lactate production and ATP generation. Furthermore, Affymetrix Human U133 plus 2.0 gene chips and gene function enrichment analysis were conducted to screen differential expression genes (DEGs) and potential signaling pathway. Expressions of the glycolysis-related proteins were measured and quantified with Western Blotting. The results showed that TCS could suppress the cell viability, induce oxidative stress, and cause mitochondrial damage. In addition, TCS exposure promoted the glycolysis process, as manifested by accelerated conversion of glucose to lactate and increased energy release. Western Blotting results confirmed that the expression levels of glycolysis related proteins were significantly elevated. The PI3K/Akt/ FoxO pathway was identified to play a pivot role in TCS-induced glycolysis, which was further confirmed by inhibitor tests using specific inhibitors LY294002 and MK2206. In general, TCS can induce oxidative stress, cause oxidative damages and promote glycolysis in HepG2 cells, which was mediated by the PI3K/Akt/FoxO pathway.
Keywords: Glycolysis Molecular mechanism PI3K/Akt/FoxO pathway
1. Introduction As a broad-spectrum antibacterial agent, triclosan (2,4,4-trichloro2-hydroxydiphenyl ether, TCS) has been widely used in personal care products for several decades. From 2008 to 2010, TCS has been found in 93% of liquid, gel, or foam soaps (FDA, 2013). TCS is usually discharged into sewerage system and then enters downstream water bodies (Bedoux et al., 2012). Presently, TCS has been detected in the influent and effluent of wastewater treatment plants, sludge, rivers, estuaries, and sediments (Chalew and Halden, 2009; Chu and Metcalfe, 2007; Lee et al., 2019; Zhu et al., 2019). Extensive application of TCS has further resulted in its wide existence in a variety of environmental media including air, water, sediments, sludge, as well as organisms (Buth et al., 2010; Davis et al., 2012; Escarrone et al., 2016; Karthikraj et al., 2019; Reichert et al., 2019).
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TCS exposure in human occurs mainly via contacting personal care products containing TCS or water and digesting food products contaminated by TCS (Geens et al., 2009; Husøy et al., 2019). Calafat et al. (2008) found that TCS was detected in 74.6% of 2517 urine samples in U.S. population with concentrations ranging from 7.9 nM to 13.1 μM (Calafat et al., 2008). A recent study in Queensland, Australia, showed that all of 2400 urine samples displayed detectable levels of TCS from 0.08 to 0.71 µM (Heffernan et al., 2015). With the increased consumption and elevated bioaccumulation level, TCS might pose a potential hazard to the ecological environment and human health, although it was generally considered to be a low-toxic compound (Johnson et al., 2016). In addition, TCS may degraded to some carcinogenic by-products such as dioxins, chloroforms, phenols and methyl triclosan (MTCS), which make it necessary to clarify the toxicity of TCS (DeLorenzo et al., 2008; Mezcua et al., 2004). In recent years, the
Corresponding authors. E-mail addresses: [email protected] (Y. Jiang), [email protected] (Z. Yu).
https://doi.org/10.1016/j.envint.2019.105428 Received 26 August 2019; Received in revised form 20 November 2019; Accepted 16 December 2019 0160-4120/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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experiments (Wang et al., 2019b). TCS was diluted with dimethyl sulfoxide (DMSO, Sigma, MO, USA), and the control group was with DMSO (0.1%, v/v) only.
effects of TCS on the ecological environment and human health have become one of the research focuses in the world. Experimental studies have shown that liver is one of the main target organs for TCS exposure. Escarrone et al. (2016) performed a bioaccumulation study in guppy that revealed the bioconcentration factor of TCS in liver was much higher than that in gill, gonads, brain and muscle tissues. The concentrations of TCS detected in human tissue of Belgium were highest in liver with 3.14 ng/g, followed by 0.61 ng/g in adipose tissue and 0.03 ng/g in brain (Geens et al., 2012). Several studies have shown that TCS is capable of interfering with liver function. Recently, Tang et al (2018) demonstrated that TCS exposure to mice for 13 weeks caused increased liver weight, elevated serum alanine aminotransferase and alkaline phosphatase levels, and hepatocyte hypertrophy. Furthermore, long-term TCS exposure in mice resulted in liver fibrogenesis (Yueh et al., 2014) and even hepatocellular carcinoma (HCC) (Rodricks et al., 2010), indicating its potential carcinogenic effect. At present, the US Environmental Protection Agency (USEPA) has identified TCS as a potential carcinogen. For tumor cells, glycolysis pathway was principally used to metabolize glucose and generate ATP, and the metabolic shift from oxidative phosphorylation to glycolysis is considered to be a major feature of tumors (Upadhyay et al., 2013). Physiologically, enhanced glycolysis can meet the increased energy needs of rapid cell proliferation, and on the other hand glycolysis may provide a favorable microenvironment for carcinogenesis and progression of HCC (Shang et al., 2016). Previous studies have shown that TCS can downshift oxidative phosphorylation in the rat liver mitochondria, upshift the rate of glucose consumption in mammalian cells (Ajao et al., 2015), impair energy and amino acid metabolism in developing zebrafish embryos (Fu et al., 2019). The latest data of Zhang et al. (2019) suggested that TCS could promote the progression of HCC by accelerating energy metabolism and enhancing antioxidant defenses system. To date, the promotion of glycolysis induced by TCS and its effects on glucose metabolism and hepatocyte toxicity have not been well elucidated. In this study, we report on the glycolysis induced by TCS exposure and to explore the underlying molecular regulatory mechanism. The human hepatocellular carcinoma cells (HepG2) were chosen as the experimental model system, which was widely applied for hepatotoxicity studies of xenobiotics. Affymetrix HumanU133 plus 2.0 chips were used to investigate the widespread changes in transcriptome profile of HepG2 cells induced by TCS. The gene function enrichment analysis and signaling pathways analysis were further conducted to evaluate the metabolic-related biological functions. The purpose of this study was to screen and validate the pivotal gene marker of TCS-induced hepatotoxicity. Our results will help to provide new insights into the hepatotoxicity of TCS and provide theoretical basis for further clarifying the toxicological characterization and risk assessment of TCS.
2.2. Biological effects induced by TCS Biological effects responding to TCS exposure were measured as described in our previous studies (Wang et al., 2019b). HepG2 cells during the exponential growth phase were treated with 5, 10, 20 and 40 μM of TCS for 24 h. Cell viability was measured with Cell Counting Kit-8 (CCK-8) kit (Dojindo, Kumamoto, Japan); HepG2 cells were incubated with 2′-7′-dichlorofluorescein diacetate (DCF-DA, Sigma, MO, USA) for 30 min, and then intracellular reactive oxygen species (ROS) level was determined based on fluorescence intensity of 2′,7′-dichlorofluorescein (DCF); The activity of superoxide dismutase (SOD) was examined by SOD kit (Beyotime, Shanghai, China); Detection of mitochondrial membrane potential (MMP) levels in HepG2 cells was conducted using Rhodamine 123 (Sigma, MO, USA) as a fluorescent probe. For biological effects detection, all experiments were performed at least thrice with more than three parallel samples. 2.3. The detection of glucose and energy metabolism After treatment with TCS (10, 20 μM) for 24 h, the glucose and energy metabolism in HepG2 cells were measured as described below. The medium supernatants were harvested to measure the contents of glucose or lactate with a spectrophotometer using corresponding commercial kits (Jiancheng, Nanjing, China). The glucose and lactate concentrations were determined with OD values at 505 nm and 530 nm, respectively. HepG2 cells were collected and homogenized with 100 ml of icecold homogenization buffer. It was then centrifuged at 10,000 g for 10 min at 4 °C. The lysate supernatant was used for ATP measurement using a system bioluminescence detection kit (Beyotime, Shanghai, China). According to the kit instructions, the intracellular ATP level was determined by bioluminescence using a multi-function microplate luminometer. For metabolism examination, all experiments were performed at least thrice with more than three parallel samples. 2.4. Microarray chip The method of chip microarray analysis has been described in detail in the previous article (Kong et al., 2019; An et al., 2017). Before hybridization, total RNA from HepG2 cells exposed with 20 μM of TCS for 6 h was isolated with TRIZOL reagent (Life technologies, Carlsbad, CA, USA) and qualified with Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, USA). Qualified RNA samples were then amplified, labeled and purified using RNeasy micro kit (QIAGEN, GmBH, Germany). The quality inspection results are shown in Table 1. RIN: RNA Integrity Number. The RIN value is directly proportional to RNA integrity and RIN > =6.0 is the criteria value for chip analysis. Array hybridization and washing were performed with GeneChip Hybridization, Wash and Stain Kit (Affymetrix, Santa Clara, CA, USA) according to the procedures provided by the manufacturer. Fold change (FD) > =2 or < =1/2 were used as criteria to screen differential expression genes (DEGs). Moreover, the function DEGs was annotated, and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG)
2. Materials and methods 2.1. Cell culture and treatment HepG2 were obtained from ATCC (American Type Culture Collection), which were typically cultured in Dulbecco's modified eagle medium (DMEM) and 10% FBS (fetal bovine serum) (Invitrogen, Paisley, U.K.) as previous described by Wang et al. (Wang et al., 2019b). TCS (CAS: 3380-34-5, Dr. Ehrensorfer, Germany, purity > 97%) concentrations selected in this study were based on our preliminary Table 1 Quality report of total RNA samples after TCS exposure in HepG2 cells. Samples
Concentration(ng/μL)
Volume (μL)
Total amount(μg)
A260/A280
RIN
28S/18S
Results
Control TCS (20 μM)
548.3 1302.6
200 100
109.66 130.26
1.96 2
9.5 9.6
2 1.9
A3 A3
2
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heme oxygenase-1 (HO-1), which has a positive effect on removing excess reactive oxygen species (Wang et al., 2019b). It is well established that oxidative stress is a pivotal mechanism underlining the toxicity of most xenobiotics and ROS overproduction can induce oxidative damage, destroy homeostasis and interfere with cell growth (Park et al., 2017). Several researches have reported that TCS can induce oxidative stress and lipid oxidative damages in aquatic organisms, accompanying with activation of antioxidant system to maintain redox balance (Parenti et al., 2019). To further investigate the effect of TCS on oxidative stress in mammal cells, the ROS generation and SOD activity were measured in this study. After treatment with various concentrations of TCS, the cellular ROS levels dose-dependently increased to 122.4 ± 0.3%, 138.0 ± 1.5%, 175.2 ± 11.4%, 206.4 ± 13.5%, respectively, compared with the control groups (Fig. 1B). In addition, as an antioxidant enzyme, SOD mainly functions to remove cellular superoxide free radicals, whose activity can indirectly reflect the antioxidant capacity. The results of enzyme activity assay shown in Fig. 1C indicated that TCS caused reduction of SOD activity in HepG2 cells from 100% (control group) to 88.5 ± 2.6%, 76.6 ± 3.3%, 64.6 ± 3.9% and 59.1 ± 3.8% respectively, in a concentration dependent manner. These data further confirmed the activation of oxidative stress response and oxidative damage might play a role in TCS induced cytotoxicity in HepG2 cells. After treatment with 5, 10, 20 and 40 μM of TCS for 24 h, the MMP of HepG2 cells reduced to 89.3 ± 4.3%, 79.9 ± 7.1%, 68.3 ± 5.1%, and 56.5 ± 4.2%, respectively, that of control groups (Fig. 1D). Maintenance of MMP is a prerequisite for electron transport chain and ATP synthesis during oxidative phosphorylation process. Excessive ROS can depress MMP and impair cellular ATP production, resulting in mitochondrial dysfunction (Redza-Dutordoir and Averill-Bates, 2016). Reduction of MMP by TCS was consistent with previous reports on TCS toxicity (Shim et al., 2016), indicating that TCS could impair mitochondrial function in HepG2 cells as mitochondrial uncoupler. Collectively, TCS could induce ROS overproduction, SOD reduction and MMP loss in HepG2 cells, suggesting that oxidative damage was involved in TCS-induced cytotoxicity.
database were utilized to perform functional classification, enrichment analysis and pathway mapping. Data were analyzed using Student's ttest (p < 0.05) using Bonferroni multiple test calibration to minimize false positive selection. Three biological replicates were established in the microarray experiment. 2.5. Western Blotting Western Blotting was conducted according to our previous study (Shang et al., 2019). After 24 h of treatment with TCS (10, 20 μM), cells were lysed on ice using mammalian protein extraction reagent and total protein was collected by centrifugation. According to the abundance of different proteins detected in pre-experiments, 60–80 µg of total protein samples were separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membrane (Sigma, MO, USA). The membrane was blotted with special primary antibody (at 4 °C, overnight) and then incubated with corresponding secondary antibody (at 25 °C, 1 h). Detection was performed using enhanced chemiluminescence system (Pierce, Socochim SA, Lausanne, Switzerland). In this experiment, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference. Three replicates were established in this experiment, and the mean of replicates is used as the quantitated result. The primary antibodies used in this experiment were as follows: anti-hexokinase 2 (HK2), anti-phosphofructokinase (PFK), anti-pyruvate kinase isoform M2 (PKM2), anti-lactate dehydrogenase A (LDHA), anti-pyruvate dehydrogenase (PDH), anti-phosphor-protein kinase B (p-Akt), anti-Akt, anti-phosphor-forkhead box O (p-FoxO), and anti-FoxO were purchased from Cell Signaling (MA, USA); anti-GAPDH was purchased from Abcam (Cambridgeshire, UK). The secondary antibodies, anti-mouse immunoglobulin G (IgG) and anti-rabbit IgG, were purchased from Zhong Shan Bio Tech (Beijing, China). 2.6. Statistical analysis The mean values for continuous variables of each experiment were used for statistical analyses. Results were presented as means ± s.e.m, and statistically evaluated using one-way ANOVA followed by Tukey's post hoc test. The significance thresholds were deemed as statistically significant (p < 0.05) and highly significant (p < 0.01).
3.2. TCS treatment increases glycolysis and promote energy metabolism in HepG2 cells Based on the results of biological effects, the moderate doses (10 and 20 μM) of TCS were chosen for the subsequent experiments. Fig. 2 indicated the metabolic effects and related protein expression in HepG2 cells treated with TCS (10 and 20 μM) for 24 h. Compared with the control group, glucose consumption in the 20 µM TCS-treated group was approximately 9.3% higher (Fig. 2A), accompanied by a 26.2% increase in lactic acid production (Fig. 2B), indicating that TCS can promote glycolysis process. The main energy source of cells is obtained through glucose metabolism including oxidative phosphorylation or glycolysis, the latter is a common biological process for all organisms during glucose catabolism. During this process, glucose is decomposed into pyruvate and further reduced to lactate under anaerobic or anoxic conditions, accompanying with ATP production. It is worth noting that most tumor cells mainly produce ATP through glycolysis even under aerobic conditions. The diversion of glucose catabolism away from mitochondria oxidative phosphorylation into glycolysis in hepatocellular carcinoma facilitates the progression of tumors through more sufficient ATP and suitable microenvironment (Shang et al., 2016). Zhang et al. reported that glucose transport deficiency by mitochondrial disorders induced ATP production through glycolysis (Zhang et al., 2007). Our results also suggested that TCS exposure of 10 and 20 μM increased the ATP production by 8.9% and 29.7%, respectively, compared with the control group (Fig. 2C). This mechanism is partially due to the injury of mitochondrial respiration and enhancement of the glycolytic pathway. Western Blotting was further performed to investigate the
3. Results and discussion 3.1. TCS treatment inhibits cell proliferation, increases ROS accumulation in HepG2 cells The biotoxicity of TCS focused on aquatic organisms had been evaluated using representative biological bacteria, plants, fish, birds, and protozoa (Lyndall et al. 2010; Fuchsman et al. 2010; Price et al., 2010). For mammals, the most prominent toxicity of TCS is proved to endocrine disruption effects and carcinogenic potential, for instance TCS exposure could induce hepatocellular carcinomas in mice (Rodricks et al., 2010; Tang et al., 2018; Wang et al, 2018; Yueh et al., 2014). In this study, the cell viability of HepG2 cells after TCS exposure was examined using CCK8 assay. As shown in Fig. 1A, after treated with 5, 10, 20 and 40 μM of TCS for 24 h, the cell viability decreased to 96.0 ± 1.4%, 94.0 ± 4.6%, 89.8 ± 4.8%, 64.6 ± 9.2% (p < 0.05), respectively, as compared with the control group. This result is consistent with our previous MTT test (Wang et al., 2019b). The LC50 value of TCS for 24 h treatment detected in this study was 58.5 μM, which was comparable to the results of Ma et al (2013) with a LC50 value 66.5 μM. In other words, TCS is generally a low toxic substance, which may cause acute toxicity only under comparatively high concentrations. And the main health risks of TCS may come from long-term persistent exposure and its bioaccumulation characteristic. Our previous study found that TCS induced enhanced expression of 3
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Fig. 1. Biological effects of TCS in HepG2 cells. HepG2 cells were treated with TCS (5, 10, 20 and 40 μM) for 24 h. (A): Cell viability was determined by CCK assay. (B): The intracellular ROS level was measured by DCF fluorescence intensity. (C): SOD activity was detected with commercially SOD detection kit. (D): MMP was evaluated with Rhodamine 123 fluorescent probe assay. In the control group (C), HepG2 cells were treated only with DMSO (0.1%, v/v). *p < 0.05, **p < 0.01, vs. C group.
Fig. 2. Metabolic effects and related protein expression in HepG2 cells treated with TCS. HepG2 cells were treated with TCS (10 and 20 μM) for 24 h. (A): Glucose uptake and (B): Lactate release were measured with commercially available assay kits; (C): Intracellular ATP level was measured with bioluminescence detection kit; (D): The expression of proteins were examined by Western Blotting. (E): Quantization of protein expression levels. In the control group (C), HepG2 cells were treated with DMSO (0.1%, v/v) only. *p < 0.05, **p < 0.01, vs. C group. 4
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expression of glycolytic key enzymes. As shown in Fig. 2D, 10 μM of TCS treatment had no effect on expression of HK2 and PFK, and 20 μM of TCS significantly promoted the protein expression levels of HK2 and PFK, with 50.7% and 39.2% higher expression level than control groups (p < 0.05), respectively. PKM2 was more sensitive than HK2 and PFK, with the expression levels increased by 69.0% and 121.4% after 10 and 20 μM treatments, (p < 0.05), respectively. It is well known that three rate-limiting enzymes including HK2, PFK, and PKM2 jointly control the process of glycolytic metabolic flow. The phosphorylation of glucose at the C6 site catalyzed by HK2 can produce glucose 6-phosphate (G-6-P) (Gong et al., 2012). And then PFK is responsible for phosphorylation of 6-phosphate fructose to generate fructose 1,6-diphosphate (Park et al., 2013). Excessive activation of PFK will break the balance between glycolysis and glycogen synthesis, and promote consumption of glucose in cytoplasm. This metabolic reprogramming will help cancer cells adapt to micro environmental disturbances. M2 isoform of pyruvate kinases (PK) is in charge of catalyzing phosphoenolpyruvate (PEP) to produce ATP and pyruvate (Wu et al., 2016). The up-regulated expression of PKM2 acts as a predictor of survival and recurrence (Chen et al., 2015). These results obviously confirmed the promoting effect of TCS on glycolysis. Furthermore, as shown in Fig. 3D and 3E, 20 μM of TCS promoted the expression of LDHA by 30.1% (p < 0.05). Meanwhile, 10 and 20 μM of TCS could increase the expression of PDH by 86.9% and 161.5% (p < 0.05), respectively (Fig. 2D). In the process of glucose metabolism, pyruvic acid is further decomposed into lactate under the control of LDHA, and up-regulated LDHA had a positive effect in promoting glycolysis (Fantin et al., 2006; Le et al., 2010). Activation of
LDHA can also influence PDH expression, which catalyzes the irreversible decarboxylation of pyruvate to acetyl-CoA, thereby controlling the carbohydrate-derived fuel to enter mitochondria for complete oxidation. Deregulation of PDH is associated with suppression of mitochondrial energy supply. In summary, the results of metabolic effects and Western Blotting proved that TCS could promote decomposition of glucose into lactic acid and production of ATP through glycolysis metabolism. To our knowledge, this is firstly demonstrating the induction of glycolysis by TCS. Taken together, TCS treatment could inhibit overall cell proliferation through increased oxidative stress damages in HepG2 cells. On the other hand, the enhanced glycolysis of survived cells under TCS exposure accelerated the glucose decomposition and induced ATP production. Thus, the enhanced glycolysis seemed to be an adapitve response of HepG2 cells against TCS induced oxicative stress. The cell proliferation inhibition would be the dominant outcome during the acute short-time TCS exposure. However, a long-term exposure of TCS could cause abnormally rapid proliferation due to enhanced glycolysis (Shang et al., 2016; Zhang et al., 2019), which might partially explain the carcinogenic mechanism of TCS.
3.3. Extensive transcriptomic changes triggered by TCS To screen and filter the sensitive target biomarkers and explore the molecular toxicological mechanisms of TCS, Affymetrix Human U133 plus 2.0 array chips which spotted total 54,613 genes were recruited to analyze the overall transcriptomic changes in HepG2 cells treated with TCS. Considering the results of biological effects and protein changes, only 20 μM of TCS exposure group was retained in the chip experiment,
Fig. 3. PI3K/Akt pathway activation induced by TCS exposure. HepG2 cells were treated with TCS (0 and 20 μM) for 6 h, Affymetrix Human U133 plus 2.0 array chips were conducted. (A): GO terms related to metabolism after TCS treatment. (B): Top 25 pathways enriched based on DEGs count. (C): HepG2 cells were treated with TCS (10 and 20 μM) for 24 h. Western Blotting was used to detect the expression of Akt, p-Akt, FoxO and p-FoxO in HepG2 cells. (D): The quantization of protein expression levels. The ratios of p-Akt/Akt, p-FoxO/FoxO of every group were calculated with GAPDH as an internal reference. In the control group (C), HepG2 cells were treated only with DMSO (0.1%, v/v). *p < 0.05, **p < 0.01, vs. C group. 5
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Fig. 4. The effect of PI3K/Akt pathway on glycolysis induced by TCS in HepG2 cells. HepG2 cells were pretreated with LY294002 (30 μM) and MK2206 (30 μM) for 3 h, and then treated with 20 μM of TCS for 24 h. (A): The activation of PI3K/Akt was examined with Western Blotting. (B): The quantization of PI3K/Akt protein expression levels. (C): glucose uptake and (D): lactate release were measured with commercially available assay kits; (E): intracellular ATP level was measured with bioluminescence detection kit; (F): The expression of glycolysis related proteins were examined by Western Blotting. (G): The quantization of glycolysis related protein expression levels. In the control group (C), HepG2 cells were treated only with DMSO (0.1%, v/v). *p < 0.05, **p < 0.01, vs. C group. # p < 0.05, # # p < 0.01, vs. TCS (20 μM) treated group.
metabolism were picked out. There was dozens of DEGs in each GO term, and then we further analyzed and merged the genes involved in the eight biological function terms. In this study, three common pivot genes were highlighted. Phosphatidylinositol 3-kinase (PI3K) is located at the most upstream of the PI3K/Akt pathway. Robey and Hay reported that PI3K/Akt can accelerate the binding of HK2 to voltagedependent anion channels in mitochondria to promote glycolysis (Robey and Hay, 2006), and participate in glycolysis by regulating hypoxia inducible factor 1 (HIF-1) activity in cancer cells (Majumder et al., 2004). The abnormally altered expression of HIF-1 is closely related to enhanced glycolysis in tumor cells (Lum et al., 2007). Pyruvate dehydrogenase kinase 1 (PDK1) can inactivate PDH to prevent tricarboxylic acid cycle and promote the glycolysis (Shoshan-Barmatz et al., 2009). In addition, PDK1 can act as a direct target of HIF-1 and
and three biological replicates were set up to confirm the credibility of the results. Compared with control group, totally 1664 genes were deferentially expressed, of which 946 (56.9%) genes were up-regulated and 718 (43.1%) genes down-regulated. Such widespread mRNA changes induced by TCS in mammal cells have not been reported yet. Previous gene expression profiling researches mainly focused on aquatic organisms and prokaryotes (Chuanchuen and Schweizer, 2012; Haggard et al., 2016; Lenahan et al., 2014; Wang et al., 2019a). After that, differential expression gene annotation and enrichment analysis were conducted to investigate the distribution of differential genes in GO, and elucidate the manifestation of gene function (Ashburner et al., 2000). In this study, the “metabolism” and “glycolysis” were used as keywords to narrow down the enriched GO terms. As shown in Fig. 3A, 8 biological process GO terms related to 6
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pathway in TCS-induced glycolysis. PI3K/Akt pathway participated in TCS toxicology by regulating downstream genes such as FoxO. At present, we can't exclude the participation of other downstream molecules of PI3K/Akt pathway, which need to be verified by more extensive examination and deeper investigation.
regulates glycolysis (Wang et al., 2015). Moreover, the results of KEGG enrichment analysis indicated that a total of 324 KEGG pathways were enriched, and Fig. 3B displayed the top 25 signaling pathways. Based on the DEGs count enriched in each pathway, PI3K/Akt/FoxO pathway was most significantly activated. Combined with GO enrichment and KEGG pathway analysis, it was suggested that the PI3K regulatory pathways are mostly relevant to TCS-induced metabolism mechanism. The data of Western Blotting shown in Fig. 3C and 3D, displayed that the ratio of p-Akt/Akt increased by 64.6% and 111.9% in 10 and 20 μM TCS treated groups, respectively, compared with the control groups. In addition, as a transcription factor, FoxO can be directly phosphorylated by Akt (Tzivion et al., 2011). The ratio of p-FoxO/FoxO was also 42.1% and 107.4% higher in 10 and 20 μM TCS exposure groups than that in control group, which further confirmed the activation of PI3K/Akt under the TCS treatment. Our results illustrated the activation of the PI3K/Akt/FoxO pathway at both transcription and translation level.
5. Author statement Jing An and Huixin He carried out the in vitro study, analyzed and interpreted the data and drafted the manuscript. Weiwei Yao and Yu Shang helped to finish the in vitro experiments and microarray assay. Yun Jiang and Zhiqiang Yu designed and supervised the in vitro study. Zhiqiang Yu revised the drafted manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
3.4. Effects of PI3K/Akt pathway on the glycolysis induced by TCS
Acknowledgments
To clarify the regulation role of the PI3K/Akt pathway in the glycolysis triggered by TCS, HepG2 cells were pretreated with specific PI3K inhibitor (LY294002, 30 μM) and specific Akt inhibitor (MK2206, 30 μM) for 3 h, and then treated with 20 μM of TCS. Glycolysis and energy metabolism were detected by the same method as described above and related protein expression was assessed by Western Blotting. The dose and pretreatment time of inhibitors were determined according to our preliminary experiments. As shown in Fig. 4 A and B, both inhibitors could significantly reduce the high level of Akt phosphorylation induced by TCS, even to the background levels (p < 0.05). In addition, LY294002 and MK2206 could alleviate FoxO phosphorylation by 44.3% and 31.5% (p < 0.05), respectively, as compared with the TCS treated group (no inhibitors). Outcomes of glycolysis metabolism showed that the application of LY294002 and MK2206 could depress glucose consumption and lactic acid production by 16.3%, 28.3% (Fig. 4C, p < 0.05) and 13.4%, 11.3% (Fig. 4D, p < 0.05), respectively. A similar effect was also observed in ATP generation with 14.0%, 15.6% reduction (Fig. 4E, p < 0.05), respectively, as compared with the TCS treated group (no inhibitors). Further analysis with Western Blotting showed that both inhibitors reversed the up-expression of key glycolytic enzymes HK2, PFKP, LDHA, PDH and PKM2 induced by TCS (Fig. 4F and G). Almost all protein expression is reduced to the background level or even lower (p < 0.05). In particular, MK2206 reduced expressions of PFK, PKM2 and PDH by 38.1%, 53.7% and 52.4%, respectively, compared with TCS (20 μM) treatment group. The expressions of PFK and PKM2 even reduced by 8.0% and 4.1%, respectively, compared with control group. All of these results suggested that inhibition of PI3K/Akt pathway activity can significantly inhibit the glycolytic effects induced by TCS.
The study was supported by Joint NSFC-ISF Research Program, jointly funded by the National Natural Science Foundation of China and the Israel Science Foundation (41561144007); National Natural Science Foundation of China (81403237); Key Research Program of Frontier Sciences of the Chinese Academy of Sciences (QYZDJ-SSW-DQC01802); Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Z134); Shanghai Promoting TCM 3Year Action Program (ZY(2018-2020)-RCPY-2005); and Innovative Research Team in University (IRT13078). References Ajao, C., Andersson, M.A., Teplova, V.V., Nagy, S., Gahmberg, C.G., Andersson, L.C., Hautaniemi, M., Kakasi, B., Roivainen, M., Salkinoja-Salonen, M., 2015. Mitochondrial toxicity of triclosan on mammalian cells. Toxicol. Rep. 2, 624–637. An, J., Zhou, Q., Qian, G.R., Wang, T., Wu, M.Y., Zhu, T., Qiu, X.H., Shang, Y., Shang, J., 2017. Comparison of gene expression profiles induced by fresh or ozone-oxidized black carbon particles in A549 cells. Chemosphere 180, 212–220. Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M., Sherlock, G., 2000. Gene ontology: tool for the unification of biology. Gene Ontology Consortium. Nat. Genet. 25 (1), 25–29. Bedoux, G., Roig, B., Thomas, O., Dupont, V., Le Bot, B., 2012. Occurrence and toxicity of antimicrobial triclosan and by-products in the environment. Environ. Sci. Pollut. Res. Int. 19 (4), 1044–1065. Buth, J.M., Steen, P.O., Sueper, C.H., Vikesland, P.J., Arnold, W.A., 2010. Dioxinphotoproducts of triclosan and its chlorinated derivatives in sediment cores. Environ. Sci. Technol. 44 (12), 4545–4551. Calafat, A.M., Ye, X., Wong, L.Y., Reidy, J.A., Needham, L.L., 2008. Urinary concentrations of triclosan in the US population: 2003–2004. Environ. Health Perspect. 116 (3), 303–307. Chalew, T.E.A., Halden, R.U., 2009. Environmental exposure of aquatic and terrestrial biota to triclosan and triclocarban. J. Am. Water Works Assoc. 45 (1), 4–13. Chen, Z., Lu, X., Wang, Z., Jin, G., Wang, Q., Chen, D., Chen, T., Li, J., Fan, J., Cong, W., Gao, Q., He, X., 2015. Co-expression of PKM2 and TRIM35 predicts survival and recurrence in hepatocellular carcinoma. Oncotarget 6 (4), 2538–2548. Chu, S., Metcalfe, C.D., 2007. Simultaneous determination of triclocarban and triclosan in municipal biosolids by liquid chromatography tandem mass spectrometry. J. Chromatogr. A 1164 (1–2), 212–218. Chuanchuen, R., Schweizer, H.P., 2012. Global transcriptional responses to triclosan exposure in Pseudomonas aeruginosa. Int. J. Antimicrob. Agents 40 (2), 114–122. Davis, E.F., Klosterhaus, S.L., Stapleton, H.M., 2012. Measurement of flame retardants and triclosan in municipal sewage sludge and biosolids. Environ. Int. 40, 1–7. DeLorenzo, M.E., Keller, J.M., Arthur, C.D., Finnegan, M.C., Harper, H.E., Winder, V.L., Zdankiewicz, D.L., 2008. Toxicity of the antimicrobial compound triclosan and formation of the metabolite methyl-triclosan in estuarine systems. Environ. Toxicol. 23 (2), 224–232. Escarrone, A.L., Caldas, S.S., Primel, E.G., Martins, S.E., Nery, L.E., 2016. Uptake, tissue distribution and depuration of triclosan in the guppy Poecilia vivipara acclimated to freshwater. Sci. Total Environ. 560–561, 218–224. Fantin, V.R., St-Pierre, J., Leder, P., 2006. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer
4. Conclusions It is well known that mitochondria provide energy for cell growth through aerobic respiration. In this study, the MMP reduction by TCS exposure indicated the mitochondrial dysfunction and aerobic respiration interference in HepG2 cell. Pyruvic acid is the end product of the glycolytic pathway, which is further converted to lactic acid in the cytoplasm to release energy. The accumulation of lactic acid content suggested an increase of glycolysis process induced by TCS. Up-regulated proteins expression related to glycolysis confirmed the enhancement of glycolysis triggered by TCS. The comprehensive analysis of gene expression, gene function and signal pathway through Affymetrix Human U133 plus 2.0 array chips, and the data of Western Blotting revealed that the PI3K/Akt pathway was closely associated with enhanced glycolysis after TCS exposure. Inhibitor experiments further proved the indispensable role of PI3K/Akt 7
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PI3K/Akt/FoxO pathway mediates glycolytic metabolism in HepG2 cells exposed to triclosan (TCS) ⁎
Jing Ana, Huixin Hea, Weiwei Yaoa, Yu Shanga, Yun Jiangc, , Zhiqiang Yub,
T
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a
Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, PR China State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, PR China c Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 201203, PR China b
A R T I C LE I N FO
A B S T R A C T
Handling Editor: Da Chen
Triclosan (TCS) has been widely used as an antibacterial agent for the last several decades in personal care products. The toxicological effect of TCS has attracted more and more attention of researchers. The purpose of this study is to evaluate the cytotoxic effects of TCS in HepG2 cells and to elucidate the molecular mechanism focusing on regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/forkhead box O (FoxO) pathway in the glycolytic metabolism. In this study, we evaluated the adverse effect of TCS exposure on cell viability, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) activity and mitochondrial membrane potential (MMP). In addition, the glycolysis process in HepG2 cells exposed to TCS was examined in terms of glucose consumption, lactate production and ATP generation. Furthermore, Affymetrix Human U133 plus 2.0 gene chips and gene function enrichment analysis were conducted to screen differential expression genes (DEGs) and potential signaling pathway. Expressions of the glycolysis-related proteins were measured and quantified with Western Blotting. The results showed that TCS could suppress the cell viability, induce oxidative stress, and cause mitochondrial damage. In addition, TCS exposure promoted the glycolysis process, as manifested by accelerated conversion of glucose to lactate and increased energy release. Western Blotting results confirmed that the expression levels of glycolysis related proteins were significantly elevated. The PI3K/Akt/ FoxO pathway was identified to play a pivot role in TCS-induced glycolysis, which was further confirmed by inhibitor tests using specific inhibitors LY294002 and MK2206. In general, TCS can induce oxidative stress, cause oxidative damages and promote glycolysis in HepG2 cells, which was mediated by the PI3K/Akt/FoxO pathway.
Keywords: Glycolysis Molecular mechanism PI3K/Akt/FoxO pathway
1. Introduction As a broad-spectrum antibacterial agent, triclosan (2,4,4-trichloro2-hydroxydiphenyl ether, TCS) has been widely used in personal care products for several decades. From 2008 to 2010, TCS has been found in 93% of liquid, gel, or foam soaps (FDA, 2013). TCS is usually discharged into sewerage system and then enters downstream water bodies (Bedoux et al., 2012). Presently, TCS has been detected in the influent and effluent of wastewater treatment plants, sludge, rivers, estuaries, and sediments (Chalew and Halden, 2009; Chu and Metcalfe, 2007; Lee et al., 2019; Zhu et al., 2019). Extensive application of TCS has further resulted in its wide existence in a variety of environmental media including air, water, sediments, sludge, as well as organisms (Buth et al., 2010; Davis et al., 2012; Escarrone et al., 2016; Karthikraj et al., 2019; Reichert et al., 2019).
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TCS exposure in human occurs mainly via contacting personal care products containing TCS or water and digesting food products contaminated by TCS (Geens et al., 2009; Husøy et al., 2019). Calafat et al. (2008) found that TCS was detected in 74.6% of 2517 urine samples in U.S. population with concentrations ranging from 7.9 nM to 13.1 μM (Calafat et al., 2008). A recent study in Queensland, Australia, showed that all of 2400 urine samples displayed detectable levels of TCS from 0.08 to 0.71 µM (Heffernan et al., 2015). With the increased consumption and elevated bioaccumulation level, TCS might pose a potential hazard to the ecological environment and human health, although it was generally considered to be a low-toxic compound (Johnson et al., 2016). In addition, TCS may degraded to some carcinogenic by-products such as dioxins, chloroforms, phenols and methyl triclosan (MTCS), which make it necessary to clarify the toxicity of TCS (DeLorenzo et al., 2008; Mezcua et al., 2004). In recent years, the
Corresponding authors. E-mail addresses: [email protected] (Y. Jiang), [email protected] (Z. Yu).
https://doi.org/10.1016/j.envint.2019.105428 Received 26 August 2019; Received in revised form 20 November 2019; Accepted 16 December 2019 0160-4120/ © 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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experiments (Wang et al., 2019b). TCS was diluted with dimethyl sulfoxide (DMSO, Sigma, MO, USA), and the control group was with DMSO (0.1%, v/v) only.
effects of TCS on the ecological environment and human health have become one of the research focuses in the world. Experimental studies have shown that liver is one of the main target organs for TCS exposure. Escarrone et al. (2016) performed a bioaccumulation study in guppy that revealed the bioconcentration factor of TCS in liver was much higher than that in gill, gonads, brain and muscle tissues. The concentrations of TCS detected in human tissue of Belgium were highest in liver with 3.14 ng/g, followed by 0.61 ng/g in adipose tissue and 0.03 ng/g in brain (Geens et al., 2012). Several studies have shown that TCS is capable of interfering with liver function. Recently, Tang et al (2018) demonstrated that TCS exposure to mice for 13 weeks caused increased liver weight, elevated serum alanine aminotransferase and alkaline phosphatase levels, and hepatocyte hypertrophy. Furthermore, long-term TCS exposure in mice resulted in liver fibrogenesis (Yueh et al., 2014) and even hepatocellular carcinoma (HCC) (Rodricks et al., 2010), indicating its potential carcinogenic effect. At present, the US Environmental Protection Agency (USEPA) has identified TCS as a potential carcinogen. For tumor cells, glycolysis pathway was principally used to metabolize glucose and generate ATP, and the metabolic shift from oxidative phosphorylation to glycolysis is considered to be a major feature of tumors (Upadhyay et al., 2013). Physiologically, enhanced glycolysis can meet the increased energy needs of rapid cell proliferation, and on the other hand glycolysis may provide a favorable microenvironment for carcinogenesis and progression of HCC (Shang et al., 2016). Previous studies have shown that TCS can downshift oxidative phosphorylation in the rat liver mitochondria, upshift the rate of glucose consumption in mammalian cells (Ajao et al., 2015), impair energy and amino acid metabolism in developing zebrafish embryos (Fu et al., 2019). The latest data of Zhang et al. (2019) suggested that TCS could promote the progression of HCC by accelerating energy metabolism and enhancing antioxidant defenses system. To date, the promotion of glycolysis induced by TCS and its effects on glucose metabolism and hepatocyte toxicity have not been well elucidated. In this study, we report on the glycolysis induced by TCS exposure and to explore the underlying molecular regulatory mechanism. The human hepatocellular carcinoma cells (HepG2) were chosen as the experimental model system, which was widely applied for hepatotoxicity studies of xenobiotics. Affymetrix HumanU133 plus 2.0 chips were used to investigate the widespread changes in transcriptome profile of HepG2 cells induced by TCS. The gene function enrichment analysis and signaling pathways analysis were further conducted to evaluate the metabolic-related biological functions. The purpose of this study was to screen and validate the pivotal gene marker of TCS-induced hepatotoxicity. Our results will help to provide new insights into the hepatotoxicity of TCS and provide theoretical basis for further clarifying the toxicological characterization and risk assessment of TCS.
2.2. Biological effects induced by TCS Biological effects responding to TCS exposure were measured as described in our previous studies (Wang et al., 2019b). HepG2 cells during the exponential growth phase were treated with 5, 10, 20 and 40 μM of TCS for 24 h. Cell viability was measured with Cell Counting Kit-8 (CCK-8) kit (Dojindo, Kumamoto, Japan); HepG2 cells were incubated with 2′-7′-dichlorofluorescein diacetate (DCF-DA, Sigma, MO, USA) for 30 min, and then intracellular reactive oxygen species (ROS) level was determined based on fluorescence intensity of 2′,7′-dichlorofluorescein (DCF); The activity of superoxide dismutase (SOD) was examined by SOD kit (Beyotime, Shanghai, China); Detection of mitochondrial membrane potential (MMP) levels in HepG2 cells was conducted using Rhodamine 123 (Sigma, MO, USA) as a fluorescent probe. For biological effects detection, all experiments were performed at least thrice with more than three parallel samples. 2.3. The detection of glucose and energy metabolism After treatment with TCS (10, 20 μM) for 24 h, the glucose and energy metabolism in HepG2 cells were measured as described below. The medium supernatants were harvested to measure the contents of glucose or lactate with a spectrophotometer using corresponding commercial kits (Jiancheng, Nanjing, China). The glucose and lactate concentrations were determined with OD values at 505 nm and 530 nm, respectively. HepG2 cells were collected and homogenized with 100 ml of icecold homogenization buffer. It was then centrifuged at 10,000 g for 10 min at 4 °C. The lysate supernatant was used for ATP measurement using a system bioluminescence detection kit (Beyotime, Shanghai, China). According to the kit instructions, the intracellular ATP level was determined by bioluminescence using a multi-function microplate luminometer. For metabolism examination, all experiments were performed at least thrice with more than three parallel samples. 2.4. Microarray chip The method of chip microarray analysis has been described in detail in the previous article (Kong et al., 2019; An et al., 2017). Before hybridization, total RNA from HepG2 cells exposed with 20 μM of TCS for 6 h was isolated with TRIZOL reagent (Life technologies, Carlsbad, CA, USA) and qualified with Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, USA). Qualified RNA samples were then amplified, labeled and purified using RNeasy micro kit (QIAGEN, GmBH, Germany). The quality inspection results are shown in Table 1. RIN: RNA Integrity Number. The RIN value is directly proportional to RNA integrity and RIN > =6.0 is the criteria value for chip analysis. Array hybridization and washing were performed with GeneChip Hybridization, Wash and Stain Kit (Affymetrix, Santa Clara, CA, USA) according to the procedures provided by the manufacturer. Fold change (FD) > =2 or < =1/2 were used as criteria to screen differential expression genes (DEGs). Moreover, the function DEGs was annotated, and the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG)
2. Materials and methods 2.1. Cell culture and treatment HepG2 were obtained from ATCC (American Type Culture Collection), which were typically cultured in Dulbecco's modified eagle medium (DMEM) and 10% FBS (fetal bovine serum) (Invitrogen, Paisley, U.K.) as previous described by Wang et al. (Wang et al., 2019b). TCS (CAS: 3380-34-5, Dr. Ehrensorfer, Germany, purity > 97%) concentrations selected in this study were based on our preliminary Table 1 Quality report of total RNA samples after TCS exposure in HepG2 cells. Samples
Concentration(ng/μL)
Volume (μL)
Total amount(μg)
A260/A280
RIN
28S/18S
Results
Control TCS (20 μM)
548.3 1302.6
200 100
109.66 130.26
1.96 2
9.5 9.6
2 1.9
A3 A3
2
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heme oxygenase-1 (HO-1), which has a positive effect on removing excess reactive oxygen species (Wang et al., 2019b). It is well established that oxidative stress is a pivotal mechanism underlining the toxicity of most xenobiotics and ROS overproduction can induce oxidative damage, destroy homeostasis and interfere with cell growth (Park et al., 2017). Several researches have reported that TCS can induce oxidative stress and lipid oxidative damages in aquatic organisms, accompanying with activation of antioxidant system to maintain redox balance (Parenti et al., 2019). To further investigate the effect of TCS on oxidative stress in mammal cells, the ROS generation and SOD activity were measured in this study. After treatment with various concentrations of TCS, the cellular ROS levels dose-dependently increased to 122.4 ± 0.3%, 138.0 ± 1.5%, 175.2 ± 11.4%, 206.4 ± 13.5%, respectively, compared with the control groups (Fig. 1B). In addition, as an antioxidant enzyme, SOD mainly functions to remove cellular superoxide free radicals, whose activity can indirectly reflect the antioxidant capacity. The results of enzyme activity assay shown in Fig. 1C indicated that TCS caused reduction of SOD activity in HepG2 cells from 100% (control group) to 88.5 ± 2.6%, 76.6 ± 3.3%, 64.6 ± 3.9% and 59.1 ± 3.8% respectively, in a concentration dependent manner. These data further confirmed the activation of oxidative stress response and oxidative damage might play a role in TCS induced cytotoxicity in HepG2 cells. After treatment with 5, 10, 20 and 40 μM of TCS for 24 h, the MMP of HepG2 cells reduced to 89.3 ± 4.3%, 79.9 ± 7.1%, 68.3 ± 5.1%, and 56.5 ± 4.2%, respectively, that of control groups (Fig. 1D). Maintenance of MMP is a prerequisite for electron transport chain and ATP synthesis during oxidative phosphorylation process. Excessive ROS can depress MMP and impair cellular ATP production, resulting in mitochondrial dysfunction (Redza-Dutordoir and Averill-Bates, 2016). Reduction of MMP by TCS was consistent with previous reports on TCS toxicity (Shim et al., 2016), indicating that TCS could impair mitochondrial function in HepG2 cells as mitochondrial uncoupler. Collectively, TCS could induce ROS overproduction, SOD reduction and MMP loss in HepG2 cells, suggesting that oxidative damage was involved in TCS-induced cytotoxicity.
database were utilized to perform functional classification, enrichment analysis and pathway mapping. Data were analyzed using Student's ttest (p < 0.05) using Bonferroni multiple test calibration to minimize false positive selection. Three biological replicates were established in the microarray experiment. 2.5. Western Blotting Western Blotting was conducted according to our previous study (Shang et al., 2019). After 24 h of treatment with TCS (10, 20 μM), cells were lysed on ice using mammalian protein extraction reagent and total protein was collected by centrifugation. According to the abundance of different proteins detected in pre-experiments, 60–80 µg of total protein samples were separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membrane (Sigma, MO, USA). The membrane was blotted with special primary antibody (at 4 °C, overnight) and then incubated with corresponding secondary antibody (at 25 °C, 1 h). Detection was performed using enhanced chemiluminescence system (Pierce, Socochim SA, Lausanne, Switzerland). In this experiment, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference. Three replicates were established in this experiment, and the mean of replicates is used as the quantitated result. The primary antibodies used in this experiment were as follows: anti-hexokinase 2 (HK2), anti-phosphofructokinase (PFK), anti-pyruvate kinase isoform M2 (PKM2), anti-lactate dehydrogenase A (LDHA), anti-pyruvate dehydrogenase (PDH), anti-phosphor-protein kinase B (p-Akt), anti-Akt, anti-phosphor-forkhead box O (p-FoxO), and anti-FoxO were purchased from Cell Signaling (MA, USA); anti-GAPDH was purchased from Abcam (Cambridgeshire, UK). The secondary antibodies, anti-mouse immunoglobulin G (IgG) and anti-rabbit IgG, were purchased from Zhong Shan Bio Tech (Beijing, China). 2.6. Statistical analysis The mean values for continuous variables of each experiment were used for statistical analyses. Results were presented as means ± s.e.m, and statistically evaluated using one-way ANOVA followed by Tukey's post hoc test. The significance thresholds were deemed as statistically significant (p < 0.05) and highly significant (p < 0.01).
3.2. TCS treatment increases glycolysis and promote energy metabolism in HepG2 cells Based on the results of biological effects, the moderate doses (10 and 20 μM) of TCS were chosen for the subsequent experiments. Fig. 2 indicated the metabolic effects and related protein expression in HepG2 cells treated with TCS (10 and 20 μM) for 24 h. Compared with the control group, glucose consumption in the 20 µM TCS-treated group was approximately 9.3% higher (Fig. 2A), accompanied by a 26.2% increase in lactic acid production (Fig. 2B), indicating that TCS can promote glycolysis process. The main energy source of cells is obtained through glucose metabolism including oxidative phosphorylation or glycolysis, the latter is a common biological process for all organisms during glucose catabolism. During this process, glucose is decomposed into pyruvate and further reduced to lactate under anaerobic or anoxic conditions, accompanying with ATP production. It is worth noting that most tumor cells mainly produce ATP through glycolysis even under aerobic conditions. The diversion of glucose catabolism away from mitochondria oxidative phosphorylation into glycolysis in hepatocellular carcinoma facilitates the progression of tumors through more sufficient ATP and suitable microenvironment (Shang et al., 2016). Zhang et al. reported that glucose transport deficiency by mitochondrial disorders induced ATP production through glycolysis (Zhang et al., 2007). Our results also suggested that TCS exposure of 10 and 20 μM increased the ATP production by 8.9% and 29.7%, respectively, compared with the control group (Fig. 2C). This mechanism is partially due to the injury of mitochondrial respiration and enhancement of the glycolytic pathway. Western Blotting was further performed to investigate the
3. Results and discussion 3.1. TCS treatment inhibits cell proliferation, increases ROS accumulation in HepG2 cells The biotoxicity of TCS focused on aquatic organisms had been evaluated using representative biological bacteria, plants, fish, birds, and protozoa (Lyndall et al. 2010; Fuchsman et al. 2010; Price et al., 2010). For mammals, the most prominent toxicity of TCS is proved to endocrine disruption effects and carcinogenic potential, for instance TCS exposure could induce hepatocellular carcinomas in mice (Rodricks et al., 2010; Tang et al., 2018; Wang et al, 2018; Yueh et al., 2014). In this study, the cell viability of HepG2 cells after TCS exposure was examined using CCK8 assay. As shown in Fig. 1A, after treated with 5, 10, 20 and 40 μM of TCS for 24 h, the cell viability decreased to 96.0 ± 1.4%, 94.0 ± 4.6%, 89.8 ± 4.8%, 64.6 ± 9.2% (p < 0.05), respectively, as compared with the control group. This result is consistent with our previous MTT test (Wang et al., 2019b). The LC50 value of TCS for 24 h treatment detected in this study was 58.5 μM, which was comparable to the results of Ma et al (2013) with a LC50 value 66.5 μM. In other words, TCS is generally a low toxic substance, which may cause acute toxicity only under comparatively high concentrations. And the main health risks of TCS may come from long-term persistent exposure and its bioaccumulation characteristic. Our previous study found that TCS induced enhanced expression of 3
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Fig. 1. Biological effects of TCS in HepG2 cells. HepG2 cells were treated with TCS (5, 10, 20 and 40 μM) for 24 h. (A): Cell viability was determined by CCK assay. (B): The intracellular ROS level was measured by DCF fluorescence intensity. (C): SOD activity was detected with commercially SOD detection kit. (D): MMP was evaluated with Rhodamine 123 fluorescent probe assay. In the control group (C), HepG2 cells were treated only with DMSO (0.1%, v/v). *p < 0.05, **p < 0.01, vs. C group.
Fig. 2. Metabolic effects and related protein expression in HepG2 cells treated with TCS. HepG2 cells were treated with TCS (10 and 20 μM) for 24 h. (A): Glucose uptake and (B): Lactate release were measured with commercially available assay kits; (C): Intracellular ATP level was measured with bioluminescence detection kit; (D): The expression of proteins were examined by Western Blotting. (E): Quantization of protein expression levels. In the control group (C), HepG2 cells were treated with DMSO (0.1%, v/v) only. *p < 0.05, **p < 0.01, vs. C group. 4
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expression of glycolytic key enzymes. As shown in Fig. 2D, 10 μM of TCS treatment had no effect on expression of HK2 and PFK, and 20 μM of TCS significantly promoted the protein expression levels of HK2 and PFK, with 50.7% and 39.2% higher expression level than control groups (p < 0.05), respectively. PKM2 was more sensitive than HK2 and PFK, with the expression levels increased by 69.0% and 121.4% after 10 and 20 μM treatments, (p < 0.05), respectively. It is well known that three rate-limiting enzymes including HK2, PFK, and PKM2 jointly control the process of glycolytic metabolic flow. The phosphorylation of glucose at the C6 site catalyzed by HK2 can produce glucose 6-phosphate (G-6-P) (Gong et al., 2012). And then PFK is responsible for phosphorylation of 6-phosphate fructose to generate fructose 1,6-diphosphate (Park et al., 2013). Excessive activation of PFK will break the balance between glycolysis and glycogen synthesis, and promote consumption of glucose in cytoplasm. This metabolic reprogramming will help cancer cells adapt to micro environmental disturbances. M2 isoform of pyruvate kinases (PK) is in charge of catalyzing phosphoenolpyruvate (PEP) to produce ATP and pyruvate (Wu et al., 2016). The up-regulated expression of PKM2 acts as a predictor of survival and recurrence (Chen et al., 2015). These results obviously confirmed the promoting effect of TCS on glycolysis. Furthermore, as shown in Fig. 3D and 3E, 20 μM of TCS promoted the expression of LDHA by 30.1% (p < 0.05). Meanwhile, 10 and 20 μM of TCS could increase the expression of PDH by 86.9% and 161.5% (p < 0.05), respectively (Fig. 2D). In the process of glucose metabolism, pyruvic acid is further decomposed into lactate under the control of LDHA, and up-regulated LDHA had a positive effect in promoting glycolysis (Fantin et al., 2006; Le et al., 2010). Activation of
LDHA can also influence PDH expression, which catalyzes the irreversible decarboxylation of pyruvate to acetyl-CoA, thereby controlling the carbohydrate-derived fuel to enter mitochondria for complete oxidation. Deregulation of PDH is associated with suppression of mitochondrial energy supply. In summary, the results of metabolic effects and Western Blotting proved that TCS could promote decomposition of glucose into lactic acid and production of ATP through glycolysis metabolism. To our knowledge, this is firstly demonstrating the induction of glycolysis by TCS. Taken together, TCS treatment could inhibit overall cell proliferation through increased oxidative stress damages in HepG2 cells. On the other hand, the enhanced glycolysis of survived cells under TCS exposure accelerated the glucose decomposition and induced ATP production. Thus, the enhanced glycolysis seemed to be an adapitve response of HepG2 cells against TCS induced oxicative stress. The cell proliferation inhibition would be the dominant outcome during the acute short-time TCS exposure. However, a long-term exposure of TCS could cause abnormally rapid proliferation due to enhanced glycolysis (Shang et al., 2016; Zhang et al., 2019), which might partially explain the carcinogenic mechanism of TCS.
3.3. Extensive transcriptomic changes triggered by TCS To screen and filter the sensitive target biomarkers and explore the molecular toxicological mechanisms of TCS, Affymetrix Human U133 plus 2.0 array chips which spotted total 54,613 genes were recruited to analyze the overall transcriptomic changes in HepG2 cells treated with TCS. Considering the results of biological effects and protein changes, only 20 μM of TCS exposure group was retained in the chip experiment,
Fig. 3. PI3K/Akt pathway activation induced by TCS exposure. HepG2 cells were treated with TCS (0 and 20 μM) for 6 h, Affymetrix Human U133 plus 2.0 array chips were conducted. (A): GO terms related to metabolism after TCS treatment. (B): Top 25 pathways enriched based on DEGs count. (C): HepG2 cells were treated with TCS (10 and 20 μM) for 24 h. Western Blotting was used to detect the expression of Akt, p-Akt, FoxO and p-FoxO in HepG2 cells. (D): The quantization of protein expression levels. The ratios of p-Akt/Akt, p-FoxO/FoxO of every group were calculated with GAPDH as an internal reference. In the control group (C), HepG2 cells were treated only with DMSO (0.1%, v/v). *p < 0.05, **p < 0.01, vs. C group. 5
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Fig. 4. The effect of PI3K/Akt pathway on glycolysis induced by TCS in HepG2 cells. HepG2 cells were pretreated with LY294002 (30 μM) and MK2206 (30 μM) for 3 h, and then treated with 20 μM of TCS for 24 h. (A): The activation of PI3K/Akt was examined with Western Blotting. (B): The quantization of PI3K/Akt protein expression levels. (C): glucose uptake and (D): lactate release were measured with commercially available assay kits; (E): intracellular ATP level was measured with bioluminescence detection kit; (F): The expression of glycolysis related proteins were examined by Western Blotting. (G): The quantization of glycolysis related protein expression levels. In the control group (C), HepG2 cells were treated only with DMSO (0.1%, v/v). *p < 0.05, **p < 0.01, vs. C group. # p < 0.05, # # p < 0.01, vs. TCS (20 μM) treated group.
metabolism were picked out. There was dozens of DEGs in each GO term, and then we further analyzed and merged the genes involved in the eight biological function terms. In this study, three common pivot genes were highlighted. Phosphatidylinositol 3-kinase (PI3K) is located at the most upstream of the PI3K/Akt pathway. Robey and Hay reported that PI3K/Akt can accelerate the binding of HK2 to voltagedependent anion channels in mitochondria to promote glycolysis (Robey and Hay, 2006), and participate in glycolysis by regulating hypoxia inducible factor 1 (HIF-1) activity in cancer cells (Majumder et al., 2004). The abnormally altered expression of HIF-1 is closely related to enhanced glycolysis in tumor cells (Lum et al., 2007). Pyruvate dehydrogenase kinase 1 (PDK1) can inactivate PDH to prevent tricarboxylic acid cycle and promote the glycolysis (Shoshan-Barmatz et al., 2009). In addition, PDK1 can act as a direct target of HIF-1 and
and three biological replicates were set up to confirm the credibility of the results. Compared with control group, totally 1664 genes were deferentially expressed, of which 946 (56.9%) genes were up-regulated and 718 (43.1%) genes down-regulated. Such widespread mRNA changes induced by TCS in mammal cells have not been reported yet. Previous gene expression profiling researches mainly focused on aquatic organisms and prokaryotes (Chuanchuen and Schweizer, 2012; Haggard et al., 2016; Lenahan et al., 2014; Wang et al., 2019a). After that, differential expression gene annotation and enrichment analysis were conducted to investigate the distribution of differential genes in GO, and elucidate the manifestation of gene function (Ashburner et al., 2000). In this study, the “metabolism” and “glycolysis” were used as keywords to narrow down the enriched GO terms. As shown in Fig. 3A, 8 biological process GO terms related to 6
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pathway in TCS-induced glycolysis. PI3K/Akt pathway participated in TCS toxicology by regulating downstream genes such as FoxO. At present, we can't exclude the participation of other downstream molecules of PI3K/Akt pathway, which need to be verified by more extensive examination and deeper investigation.
regulates glycolysis (Wang et al., 2015). Moreover, the results of KEGG enrichment analysis indicated that a total of 324 KEGG pathways were enriched, and Fig. 3B displayed the top 25 signaling pathways. Based on the DEGs count enriched in each pathway, PI3K/Akt/FoxO pathway was most significantly activated. Combined with GO enrichment and KEGG pathway analysis, it was suggested that the PI3K regulatory pathways are mostly relevant to TCS-induced metabolism mechanism. The data of Western Blotting shown in Fig. 3C and 3D, displayed that the ratio of p-Akt/Akt increased by 64.6% and 111.9% in 10 and 20 μM TCS treated groups, respectively, compared with the control groups. In addition, as a transcription factor, FoxO can be directly phosphorylated by Akt (Tzivion et al., 2011). The ratio of p-FoxO/FoxO was also 42.1% and 107.4% higher in 10 and 20 μM TCS exposure groups than that in control group, which further confirmed the activation of PI3K/Akt under the TCS treatment. Our results illustrated the activation of the PI3K/Akt/FoxO pathway at both transcription and translation level.
5. Author statement Jing An and Huixin He carried out the in vitro study, analyzed and interpreted the data and drafted the manuscript. Weiwei Yao and Yu Shang helped to finish the in vitro experiments and microarray assay. Yun Jiang and Zhiqiang Yu designed and supervised the in vitro study. Zhiqiang Yu revised the drafted manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
3.4. Effects of PI3K/Akt pathway on the glycolysis induced by TCS
Acknowledgments
To clarify the regulation role of the PI3K/Akt pathway in the glycolysis triggered by TCS, HepG2 cells were pretreated with specific PI3K inhibitor (LY294002, 30 μM) and specific Akt inhibitor (MK2206, 30 μM) for 3 h, and then treated with 20 μM of TCS. Glycolysis and energy metabolism were detected by the same method as described above and related protein expression was assessed by Western Blotting. The dose and pretreatment time of inhibitors were determined according to our preliminary experiments. As shown in Fig. 4 A and B, both inhibitors could significantly reduce the high level of Akt phosphorylation induced by TCS, even to the background levels (p < 0.05). In addition, LY294002 and MK2206 could alleviate FoxO phosphorylation by 44.3% and 31.5% (p < 0.05), respectively, as compared with the TCS treated group (no inhibitors). Outcomes of glycolysis metabolism showed that the application of LY294002 and MK2206 could depress glucose consumption and lactic acid production by 16.3%, 28.3% (Fig. 4C, p < 0.05) and 13.4%, 11.3% (Fig. 4D, p < 0.05), respectively. A similar effect was also observed in ATP generation with 14.0%, 15.6% reduction (Fig. 4E, p < 0.05), respectively, as compared with the TCS treated group (no inhibitors). Further analysis with Western Blotting showed that both inhibitors reversed the up-expression of key glycolytic enzymes HK2, PFKP, LDHA, PDH and PKM2 induced by TCS (Fig. 4F and G). Almost all protein expression is reduced to the background level or even lower (p < 0.05). In particular, MK2206 reduced expressions of PFK, PKM2 and PDH by 38.1%, 53.7% and 52.4%, respectively, compared with TCS (20 μM) treatment group. The expressions of PFK and PKM2 even reduced by 8.0% and 4.1%, respectively, compared with control group. All of these results suggested that inhibition of PI3K/Akt pathway activity can significantly inhibit the glycolytic effects induced by TCS.
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4. Conclusions It is well known that mitochondria provide energy for cell growth through aerobic respiration. In this study, the MMP reduction by TCS exposure indicated the mitochondrial dysfunction and aerobic respiration interference in HepG2 cell. Pyruvic acid is the end product of the glycolytic pathway, which is further converted to lactic acid in the cytoplasm to release energy. The accumulation of lactic acid content suggested an increase of glycolysis process induced by TCS. Up-regulated proteins expression related to glycolysis confirmed the enhancement of glycolysis triggered by TCS. The comprehensive analysis of gene expression, gene function and signal pathway through Affymetrix Human U133 plus 2.0 array chips, and the data of Western Blotting revealed that the PI3K/Akt pathway was closely associated with enhanced glycolysis after TCS exposure. Inhibitor experiments further proved the indispensable role of PI3K/Akt 7
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