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Blockage of ROS-ERK-DLP1 signaling and mitochondrial fission alleviates Cr(VI)-induced mitochondrial dysfunction in L02 hepatocytes
Ecotoxicology and Environmental Safety 186 (2019) 109749
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Blockage of ROS-ERK-DLP1 signaling and mitochondrial fission alleviates Cr (VI)-induced mitochondrial dysfunction in L02 hepatocytes
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Yujing Zhang, Yu Ma, Ningjuan Liang, Yuehui Liang, Chan Lu, Fang Xiao∗ Department of Health Toxicology, Xiangya School of Public Health, Central South University, Changsha, 410078, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hexavalent chromium [Cr(VI)] Mitochondrial dysfunction Reactive oxygen species (ROS) Extracellular regulated protein kinases (ERK) Dynamic-like protein 1 (DLP1)
Hexavalent chromium [Cr(VI)] is a common heavy metal pollutant widely used in various industrial fields. It is well known that mitochondria are the most vulnerable targets of heavy metals, but the key molecule/event that directly mediated mitochondrial dysfunction after Cr(VI) exposure is still unclear. The present study was aimed to explore whether Cr(VI) exposure could affect the mitochondrial fission/fusion process, and whether the related abnormal mitochondrial dynamics have been implicated in Cr(VI)-induced mitochondrial dysfunction. We found that the mitochondrial dysfunction caused by Cr(VI) exposure was characterized by decreased mitochondrial respiratory chain complex (MRCC) I/II activities and levels, collapsed mitochondrial membrane potential (MMP), depleted ATP, and increased reactive oxygen species (ROS) level. Cr(VI) induced abnormal mitochondrial fission/fusion events, the antioxidant Nacetyl-L-cysteine (NAC) restored the abnormal mitochondrial function as well as the fission/fusion dynamics. ROS was the up-stream regulator of extracellular regulated protein kinases (ERK) signaling, and the application of a specific ERK1/2 inhibitor PD98059 confirmed that activation of ERK1/2 signaling was associated with the abnormal mitochondrial fission/fusion and mitochondrial dysfunction. We also demonstrated that treatment with dynamic-like protein 1 (DLP1)-siRNA rescued mitochondrial dysfunction in Cr(VI)-exposed L02 hepatocytes. We reached the conclusion that blockage of ROS-ERK-DLP1 signaling and mitochondrial fission alleviates Cr(VI)-induced mitochondrial dysfunction in L02 hepatocytes, which may provide the new avenue for developing effective strategies to protect against Cr(VI)induced hepatotoxicity.
1. Introduction Hexavalent chromium [Cr(VI)] is a common heavy metal pollutant widely used in electroplating, metallurgy, tanning and other industrial fields (Jobby et al., 2018). The international agency for research on cancer (IARC) has identified Cr(VI) as a human carcinogen in 1990. Cr (VI) can be dispersed through air, soil and water. In addition to occupational exposure, food and/or drinking water are also important exposure routes of Cr(VI) (Huang et al., 2017; Yatera et al., 2018). In 2007, the US national toxicology research program (NTP) conducted a two-year study which confirmed that Cr(VI) ingestion through the digestive tract caused increased tumor incidence in mice and rats (Stout et al., 2009). Epidemiological and animal studies have evidenced that both acute and chronic Cr(VI) exposure can contribute to organs damage, including liver and kidney (Bosgelmez and Guvendik, 2017; Cengiz et al., 2016). Our previous reports also proved that Cr(VI) could induce cell apoptosis in hepatocytes through ER stress- and/or
mitochondrial-dependent pathway (Yi et al., 2016; Zhang et al., 2017). Evidence suggested that disturbed bioenergetic function, especially mitochondrial dysfunction, played a central role in Cr(VI)-induced cytotoxicity. It is well known that mitochondria are the most vulnerable targets of heavy metals, but the key molecule/event that directly mediated mitochondrial dysfunction after Cr(VI) exposure is still unclear. Mitochondria are critical for cell proliferation, survival, and death. Mitochondria are dynamic cytoplasmic organelles, they undergo coordinated and continuous cycles of fission and fusion in order to maintain their morphology, size, and distribution. The fission/fusionrelated mitochondrial morphological changes are important for many cellular physiological processes including cell cycle, innate immunity, autophagy, calcium homeostasis, and apoptosis. Dynamic-like protein 1 (DLP1), also called dynamic-related protein 1 (DRP1) (Wang et al., 2008), is a key molecule for mitochondrial fission regulation, directly or indirectly acting on other fission/fusion proteins, and its translocation
∗ Corresponding author. Department of Health Toxicology, Xiangya School of Public Health, Central South University, NO.238 Shangmayuanling Road, Kaifu District, Changsha, Hunan 410078, China. E-mail address: [email protected] (F. Xiao).
https://doi.org/10.1016/j.ecoenv.2019.109749 Received 5 July 2019; Received in revised form 23 September 2019; Accepted 1 October 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
Ecotoxicology and Environmental Safety 186 (2019) 109749
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Fig. 1. Mitochondrial dysfunction in Cr(VI)-exposed L02 hepatocytes. L02 hepatocytes were treated with different concentrations of Cr(VI) (0, 8, 16 μM) for 24 h. (A) The enzyme activities of MRCC I–V were determined using commercial kits. (B) The protein levels of MRCC I and II were analyzed by western blotting. The density of the immunoreactive bands was quantified using Image J software. (C) Mitotracker (Green) staining was used to show mitochondria. Images with TMRM staining indicated the change of MMP. (D) ATP content was determined by ATP Bioluminescence Assay Kit. (E) ROS was detected by fluorescent microscope using DCFH-DA staining. DAPI staining was used to show nucleus. (F) Cell apoptosis was detected by FITC-labeled Annexin V/PI apoptosis detection kit. Each experiment was repeated as least 3 times. *p < 0.05 vs. control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
from cytoplasm to mitochondria initiates the fission/fusion events (Smirnova et al., 2001). The inhibition of DLP1 during mitochondrial division leads to mitochondrial fragmentation, which prevents it from correctly localizing to dendrites or axons, thereby inhibiting the formation and function of synapse (Li et al., 2004). Mitochondrial fusion can be simply regarded as the fusion of two organelles, but the fusion process needs to go through two steps, namely outer membrane fusion and inner membrane fusion. The proteins mainly regulating mitochondrial outer membrane fusion are mitochondrial fusion proteins Mitofusins 1 and 2 (Mfn1 and Mfn2) (Youle and van der Bliek, 2012). It is confirmed that Mfn2 gene knockout led to decreased ATP synthesis, collapsed mitochondrial membrane potential (MMP), and reduced oxidation of glucose, pyruvate and fatty acids, resulting in
mitochondrial dysfunction (Chen et al., 2005). An emerging literature has focused on environmental contaminants exposure-induced altered mitochondrial dynamics. Evidence suggested that metals such as Cadmium (Xu et al., 2013) and Manganese could induce increased DLP1 expression as well as mitochondria fragmentation. It is still not clear whether Cr(VI) exposure could affect the mitochondrial fission/fusion process, and whether the related abnormal mitochondrial dynamics have been implicated in Cr(VI)-induced mitochondrial dysfunction. Therefore, the present study aimed to demonstrate the effects and the underlying mechanisms of Cr(VI) on mitochondrial dynamics-related mitochondrial dysfunction during hepatotoxicity in vitro, which may provide the new avenue for developing effective strategies to protect against Cr(VI)-induced hepatotoxicity. 2
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Fig. 2. Abnormal mitochondrial fission/fusion events in Cr(VI)-exposed L02 hepatocytes. L02 hepatocytes were treated with PBS or 16 μM Cr(VI) for 24 h. (A) The cells were labeled with Mitotracker Red for visualization of mitochondrial morphology. Hoechst 33342 was used to stain the nucleus. (B) Mitochondrial length and density were quantified by the investigator blinded to the treatment groups using Image J software. (C) Mitochondrial protein was extracted and the protein levels of DLP1 and Mfn2 were analyzed by western blotting. *p < 0.05 vs. control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2. Materials and methods
(Amersham, Piscataway, NJ, USA). The density of the immunoreactive bands was analyzed using Image J software. The primary antibodies for MRCC I subunit NDUFS3 (MS110) and II subunit 70 KDa Fp (MS204) were purchased from MitoScience (Eugene, OR, USA). Antibodies for DLP1 (D8H5) (#5391), Mfn2 (D1E9) (#11925), p-ERK1/2 (D13.14.4E) (#4370), ERK1/2 (L34F12) (#4696), Hsp60 (D6F1) (#12165), and β-actin (#4967) were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibody for Goat Anti-Rabbit IgG (H + L) HRP (S0001) was purchased from Affinity Biosciences (Shanghai, China), and Anti-mouse IgG, HRPlinked Antibody (#7076P2) was purchased from Cell Signaling Technology (Danvers, MA, USA).
2.1. Cell culture and treatment Human L02 hepatocytes, purchased from the Experimental Central of Xiangya Hospital of Central South University, were cultured in RPMI 1640 medium (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution in a humidified 5% CO2 incubator at 37 °C. The hepatocytes were treated with different doses of potassium dichromate (K2Cr2O7, Sigma, St. Louis, MO). 2.2. Determination of mitochondrial respiratory chain complexes (MRCCs) activities
2.4. Detection of MMP
The hepatocytes were treated with different concentrations of Cr (VI) (0, 8, 16 μM) for 24 h. The enzyme activities of MRCC I (nicotinamide adenine dinucleotide (NADH)-ubiquinone reductase), II (succinate dehydrogenase), III (ubiquinol: cytochrome c (Cyt c) reductase), IV (Cyt c oxidase), and V (ATP synthase) were determined as previously described (Xiao et al., 2019).
The cells were treated with the indicated concentrations of different chemicals. The fluorescent dye tetramethylrhodamine methyl ester (TMRM) and Mito Tracker Green were used to detect the MMP. In brief, the hepatocytes were incubated with TMRM (10 μg/mL) (Beyotime Institute of Biotechnology, Shanghai, China) at 37 °C for 5 min, and then washed 3 times with phosphate buffer saline (PBS). Mito Tracker Green was then added to the culture. After the incubation for another 45 min at 37 °C, cells were rinsed with PBS. The hepatocytes were then observed using a fluorescence microscope (Olympus, Tokyo, Japan).
2.3. Western blots The cells were treated with the indicated concentrations of different chemicals. The specific ERK1/2 inhibitor PD98059 was applied for 2 h before Cr(VI) treatment. Mitochondrial protein was extracted using Cytoplasmic and Mitochondrial Protein Extraction Kit (Sangon Biotech, Shanghai, China). Bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to detect the protein concentration. Proteins were separated by 12% Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene difluoride (PVDF) transfer membranes (Millipore, Billerica, MA). Then, the blots were blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibody at 4 °C. Then, the blots were incubated with secondary antibodies for 1 h at room temperature. The immunoreactive bands were detected using an Enhanced Chemiluminescence (ECL) Detection Kit
2.5. ATP content analysis ATP content was determined by ATP Bioluminescence Assay Kit (Roche, Mannheim, Germany) according to manufacturer's instructions. Briefly, 1 × 106 cells were homogenized with 200 μL lysis buffer supplied with the ATP assay kit and then centrifugated at 12,000×g for 5 min at 4 °C, and the supernatant was transferred to a new tube. 100 μl ATP detection buffer was added to 100 μl sample, and then the luminescence of the culture was measured in a luminescence plate reader with the integration time of 10 s. The ATP standard curve was prepared from a known amount (0.01–10 μM). 3
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Fig. 3. Effect of NAC on mitochondrial function in Cr(VI)-exposed hepatocytes. The hepatocytes were pretreated with NAC for 1 h before Cr(VI) treatment for another 24 h. (A) ROS was detected using DCFH-DA staining. The mean DCF fluorescence was measured by fluorescent microscope. (B) The enzyme activities of MRCC I and II were determined using commercial kits. (C) The protein levels of MRCC I and II were analyzed by western blotting. (D) The MMP-dependent fluorescent indicator TMRM was used to detect the change of MMP. (E) The ATP content was measured by fluorescence microplate reader using commercial kit. (F) Annexin V/PI assay was used to detect the cell apoptosis. *p < 0.05 vs. control group. #p < 0.05 vs. 16 μM Cr(VI) treatment group.
product of DCF diacetate (DCFH-DA). Briefly, the cells were loaded with 10 μM DCFH-DA (Beyotime Institute of Biotechnology, Shanghai, China) at 37 °C for 40 min. Then the cells were washed twice with PBS and incubated with DAPI for another 45 min in the dark at 37 °C. The fluorescence signals were observed using a fluorescence microscope
2.6. Measurement of reactive oxygen species (ROS) level The cells were treated with the indicated concentrations of different chemicals. The intracellular ROS level was assayed by detecting the fluorescent intensity of 2′, 7′-dichlorofluorescein (DCF), the oxidized 4
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Fig. 4. Effect of NAC on mitochondrial fission/fusion in Cr(VI)-exposed hepatocytes. The hepatocytes were pretreated with NAC for 1 h before Cr(VI) treatment for another 24 h. (A) Mitotracker Red and Hoechst 33342 were used for visualization of mitochondrial morphology. (B) Mitochondrial length and density were quantified by the investigator blinded to the treatment groups using Image J software. (C) Mitochondrial protein was extracted and analyzed by western blotting to detect the expression of mitochondrial fission/fusion proteins. (D) Whole cell lysates were collected and analyzed by western blotting to exam the levels of P-ERK and T-ERK. *p < 0.05 vs. control group. #p < 0.05 vs. 16 μM Cr(VI) treatment group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2.8. Measurement of mitochondrial morphology change
(Olympus, Tokyo, Japan).
The L02 hepatocytes were exposed to PBS or 16 μM Cr(VI) for 24 h with or without Nacetyl-L-cysteine (NAC) pretreatment for 1 h. After the indicated treatment, the hepatocytes were stained with 10 nM Mitotracker Red (Invitrogen Life Technologies, CA, USA) for 45 min at 37 °C. Then, the cells were washed twice with PBS and incubated with Hoechst 33,342 for another 45 min in the dark at 37 °C. Live-cell fluorescence images were captured with a fluorescence microscope. More than 20 clearly identifiable mitochondria from each treatment group were randomly selected. Mitochondrial length and density were quantified by the investigator blinded to the treatment groups using Image J software.
2.7. Cell apoptosis detection Cell apoptosis was detected by fluorescein isothiocyanate (FITC)labeled Annexin V/propidium iodide (PI) Apoptosis Detection Kit (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's instructions. Briefly, L02 hepatocytes were harvested, washed with icecold PBS, re-suspended in 500 μl binding buffer supplemented with 5 μl Annexin-V-FITC and 1 μl PI for 30 min at room temperature. The flow cytometric analysis was performed immediately. Data acquisition and analysis were performed using FlowJo 7.6 software (TreeStar, San Carlos, CA).
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Fig. 5. Activation of ERK1/2 signaling was associated with the abnormal mitochondrial fission/fusion and mitochondrial dysfunction. The cells were incubated with PD98059 for 2 h before Cr(VI) treatment for another 24 h. (A) Protein levels of P-ERK and T-ERK were determined using western blotting. (B) The fluorescent indicator TMRM was used to detect the change of MMP. (C) The ATP content was measured using commercial kit. (D) Annexin V/PI assay was used to detect the apoptotic cells. (E) DLP1 and Mfn2 were analyzed by western blotting and then were quantified. *p < 0.05 vs. control group. #p < 0.05 vs. 16 μM Cr(VI) treatment group.
Student-Newman-Keuls (SNK) test. Statistical significance was defined as p < 0.05. All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS), version 19.0.
2.9. Transient transfection of small interfering RNA (siRNA) L02 hepatocytes were transfected with siRNA targeting DLP1 or non-targeted siRNA duplexes (Ribobio, Guangzhou, China) using lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA) according to manufacturer's protocol.
3. Results 3.1. Mitochondrial dysfunction in Cr(VI)-exposed L02 hepatocytes
2.10. Statistics analysis L02 hepatocytes were treated with different concentrations of Cr (VI) (0, 8, 16 μM) for 24 h. In order to determine whether Cr(VI) could influence mitochondrial function, we first evaluated the key enzymes associated with respiratory chain, MRCCs. As shown in Fig. 1A and
All data are expressed as mean ± SD from 3 independent experiments. Significant difference among the groups was evaluated by oneway analysis of variance (ANOVA). Post-hoc test was analyzed by 6
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Fig. 6. Treatment with DLP1-siRNA rescued mitochondrial dysfunction in Cr(VI)-exposed L02 hepatocytes. L02 hepatocytes were transfected with DLP1 siRNA or control siRNA for 24 h, and then were treated with PBS or 16 μM Cr(VI) for another 24 h. (A) Western blotting was used to assess the knockdown efficiency of DLP1-siRNA. (B) The fluorescent indicator TMRM was used to detect the change of MMP. (C) The ATP content was measured using commercial kit. (D) Annexin V/ PI assay was used to detect the apoptotic cells. #p < 0.05 vs. 16 μM Cr(VI) treatment group.
3.2. Abnormal mitochondrial fission/fusion events in Cr(VI)-exposed L02 hepatocytes
compared to the control, the hepatocytes had a significant decrease in MRCC I and II activities. No significant change in MRCC III, IV, or V activity was found after Cr(VI) exposure. Western blotting result in Fig. 1B also confirmed that Cr(VI) could down-regulated the protein expression levels of MRCC I and II. Decreased MMP is a biomarker of mitochondrial dysfunction, and the degree of cell damage increases with the reduction of MMP. The hepatocytes were treated with Mito Tracker green and TMRM (red) to monitor MMP. The intensity of the red fluorescence was significantly decreased in L02 hepatocytes after Cr (VI) treatment in a concentration-dependent manner, indicating the collapse of MMP (Fig. 1C). Similarly, Cr(VI) induced the dysfunction of cellular energy metabolism since ATP levels were also reduced in the hepatocytes (Fig. 1D). It has been confirmed that mitochondria are the major source of ROS production and ROS overproduction is an important indicator of mitochondrial dysfunction. Thus, we measured the formation of intracellular ROS using DAPI and DCFH stain. Fig. 1E showed that Cr(VI) significantly increased the green fluorescence signals in a concentration-dependent manner, indicating that mitochondrial ROS production closely correlated with mitochondrial dysfunction. We also used Annexin V-FITC/PI staining to detect cell apoptosis (Fig. 1F) and found Cr(VI) increased the proportion of both early and late apoptotic cells.
Mitochondrial fission/fusion are essential for the maintenance of mitochondrial shape and mitochondrial homeostasis. We then evaluated the changes in mitochondrial morphology in L02 hepatocytes exposed to Cr(VI). Representative pictures for Mito Tracker (red) staining showed that mitochondria were tubular, netlike, elongated and regularly distributed in control group, while mitochondria were punctuated, shorter, fragmented, and collapsed away from the mitochondrial network in Cr(VI)-exposed cells (Fig. 2A). Accordingly, mitochondrial density was decreased and mitochondrial length was shorter in Cr(VI)-exposed cells than in the control cells (Fig. 2B). DLP1 is a key player and regulator in mitochondrial fission whose translocation from cytoplasm to mitochondria initiates the fission process, and Mfn2 is known to control the fusion process. Mitochondrial fractions were isolated to determine the fission protein DLP1 and the fusion protein Mfn2. As shown in Fig. 2C, DLP1 was increased while Mfn2 was decreased in mitochondrial fraction of Cr(VI)-exposed hepatocytes as compared to non-treated mitochondrial fraction, confirming that both fission/fusion proteins were changed in a direction favoring the fission process in Cr(VI)-exposed cells.
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3.3. Effect of NAC on mitochondrial function and fission/fusion in Cr(VI)exposed hepatocytes
3.5. Treatment with DLP1-siRNA rescued mitochondrial dysfunction in Cr (VI)-exposed L02 hepatocytes
Previous evidence suggest that increased mitochondrial ROS-associated oxidative stress is a key initiator of mitochondrial dysfunction and abnormal changes in mitochondrial morphology. We then explored if antioxidant treatment could restore the abnormal mitochondrial function as well as the fission/fusion dynamics. The hepatocytes were pretreated with NAC for 1 h before Cr(VI) treatment for another 24 h. The results in Fig. 3A showed NAC pretreatment markedly inhibited Cr (VI)-induced ROS formation as shown by reduced DCFH green fluorescence signals. Such antioxidant treatment significantly restored mitochondrial function and energy metabolism as evidenced by increased MRCC I/II activities (Fig. 3B) and protein expression levels (Fig. 3C), MMP (as determined by TMRM staining) (Fig. 3D), and ATP content (Fig. 3E), and decreased proportion of both early and late apoptotic cells (Fig. 3F) in Cr(VI)-treated L02 hepatocytes. The protective effect of NAC indicated the involvement of oxidative stress in Cr(VI)-induced mitochondrial dysfunction. We further assayed the effect of NAC on mitochondrial morphology, density, and length. As shown in Fig. 4A, NAC pre-treatment largely reversed the abnormal mitochondrial morphology induced by Cr(VI), suggesting the antioxidant could protect mitochondrial morphology from damage and maintain it in a normal and regular state. Accordingly, both mitochondria density and length were significantly increased in Cr(VI) group pretreated with NAC compared to that of the Cr (VI) alone treatment group (Fig. 4B). NAC pretreatment significantly suppressed the increase of DLP1 expression and alleviated the decrease of Mfn2 expression caused by Cr(VI) (Fig. 4C). The above data revealed that antioxidant could reverse impaired mitochondrial fission/fusion dynamics and recover mitochondrial dysfunction. We further determined the effect of oxidative stress on ERK1/2 activation and confirmed that ROS was the up-stream regulator of ERK1/2 signaling. As shown in Fig. 4D, Cr(VI) significantly increased the ERK1/2 phosphorylation (p-ERK) level but had no obvious effect on total ERK1/2 expression. NAC pretreatment suppressed the increase of p-ERK1/2 expression, which is consistent with the previous evidence that oxidative stress could activate the MAP kinase such as ERK1/2 and p38.
The perturbed balance of mitochondrial fission/fusion process is the key mechanism for mitochondrial dysfunction, we then explored whether the inhibition of mitochondrial division could rescue Cr(VI)-induced mitochondrial function defects. We constructed the siRNA-targeted DLP1 to block DLP1 expression in L02 hepatocytes. Western blotting was used to assess the knockdown efficiency of DLP1-siRNA. As shown in Fig. 6A, DLP1 protein expression was significantly reduced in cells transfected with DLP1-siRNA than in cells transfected with the control siRNA, and there was a marked reduction in DLP1 protein expression following DLP1 knockdown and Cr(VI) treatment. As shown in Fig. 6B-C, DLP1-siRNA transfection rescued mitochondrial dysfunction by increasing the MMP and ATP levels. To demonstrate the role of DLP1 in cell apoptosis caused by Cr(VI), we detected the proportion of apoptotic cells and found DLP1-siRNA could partially inhibit Cr(VI)induced cell apoptosis (Fig. 6D). 4. Discussion Mitochondria, the highly dynamic organelle, are the main targets of many toxins and environmental pollutants (Firdaus et al., 2018). The alterations in mitochondrial morphology and dynamics, which obviously affect almost all aspects of mitochondrial function, are reported to be associated with the progression of various diseases (Maycotte et al., 2017; Vasquez-Trincado et al., 2016). Although abnormalities in mitochondrial function in Cr(VI)-induced hepatotoxicity are well documented, the related mechanisms and the effective strategy to rescue mitochondrial dysfunction remain elusive. Particularly, the signaling pathway associated with fission/fusion process and its relationship between mitochondrial bioenergy and mitochondrial function in L02 hepatocytes exposed to Cr(VI) have not been fully elucidated. Cr(VI) induced mitochondrial damage, which was characterized by the decreased expression of MRCC I and II, the accumulation of intracellular ROS, the collapse of MMP, and the depletion of ATP. It is known that changes of mitochondrial morphology and fission/fusion events could affect cell apoptosis (Xie et al., 2018). The present study showed marked changes in mitochondrial morphology and fission/fusion balance in L02 hepatocytes exposed to Cr(VI). We also observed that the abnormalities in mitochondrial structure and function were associated with changed expression and distribution of DLP1. Taking these results together, we recognized that mitochondrial dysfunction, especially the altered mitochondrial morphology and fission/fusion, was essential in promoting Cr(VI)-induced apoptosis in L02 hepatocytes. Mitochondria are not only the main sources of ROS, but also the key targets of ROS (Resseguie et al., 2015). Our previous study indicated that Cr(VI)-induced hepatotoxicity is associated with oxidative stress (Zhang et al., 2017), which is mediated by ROS. Intracellular ROS accumulation could destroy cell integrity, leading to damage of various organs such as the liver (Muriel and Gordillo, 2016). The present study showed that Cr(VI)-induced mitochondrial dysfunction was accompanied by the accumulation of ROS. NAC, the ROS scavenger, was used in our study to investigated the role of ROS in Cr(VI)-induced mitochondrial dysfunction. Indeed, pretreatment with NAC significantly blunted ROS accumulation and augmented MRCC I and II expression, MMP, and ATP content in L02 hepatocytes treated with Cr(VI). In this study, we also found that cell apoptosis induced by Cr(VI) was almost completely reversed by the presence of NAC. Although there is abundant evidence that the disruption of mitochondrial fission/fusion status is related to ROS accumulation in many diseases, to the best of our knowledge, this is the first time that we have demonstrated the relationship between ROS production and mitochondrial fission/fusion events after Cr(VI) exposure. NAC pretreatment alleviated abnormal mitochondrial morphology via rescuing the altered protein expression levels of DLP1 and Mfn2, suggesting that increased oxidative stress is
3.4. Activation of ERK1/2 signaling was associated with the abnormal mitochondrial fission/fusion and mitochondrial dysfunction The activation of ERK1/2 has been shown to be associated with abnormal mitochondrial morphology and function (Wainstein and Seger, 2016). To explore the possible role of ERK1/2 activation in Cr (VI)-induced mitochondrial dysfunction, we applied the specific ERK1/ 2 inhibitor PD98059 in the present study. As shown in Fig. 5A, PD98059 inhibited the increase of p-ERK1/2 expression, indicating that ERK1/2 phosphorylation was largely abolished. The total ERK1/2 was not significantly altered. To determine if ERK1/2 activation was directly related to mitochondrial dysfunction, we examined the effect of PD98059 on MMP. The result in Fig. 5B showed that PD98059 exposure caused a significantly higher intensity of TMRM fluorescence in combination treatment cells than in Cr(VI) alone treatment cells. Similarly, PD98059 treatment alleviated Cr(VI)-induced ATP depletion in the L02 hepatocytes (Fig. 5C). Fig. 5D also demonstrated that Cr(VI)-induced cell apoptosis could be partially abolished by PD98059. These results revealed that in Cr(VI)-exposed hepatocytes, the activation of ERK1/2 signaling caused by oxidative stress was associated with mitochondrial dysfunction. Furthermore, we immunoblotted mitochondrial fractions for fission/fusion proteins expression as described above. Consistently, PD98059 treatment reversed DLP1 and Mfn2 expression levels compared to Cr(VI) alone treatment cells (Fig. 5E), suggesting that blockage of ERK1/2 activation rescued Cr(VI)-induced abnormal mitochondrial dynamics. 8
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Science Foundation of China (NO. 81773478) and Natural Science Foundation of Hunan Province, China (NO. 2019JJ40402).
responsible for the perturbation of mitochondrial dynamics and the subsequent apoptosis in the cells exposed to Cr(VI). The signal transduction mediated by mitogen activated protein kinase (MAPK) pathway has attracted extensive attention in regulating cell growth, apoptosis, and various diseases (Huang et al.,2019; Zhang et al., 2019). Extracellular regulated protein kinases (ERK), a class of extracellular regulatory protein kinases, including ERK1 and ERK2, are critical to transfer signals from surface receptors to the nucleus. Inhibition of ERK activation not only attenuates abnormal mitochondrial morphsology and function, but also restores the balance of mitochondrial division and fusion, and it is believed that oxidative stress-mediated ERK signaling plays a role in regulating mitochondrial fission/fusion events (Gan et al., 2014a). In the current study, our data demonstrated that ERK1/2 phosphorylation was significantly increased in Cr(VI)-exposed L02 hepatocytes, and was partially rescued by application of the ERK inhibitor (PD98059). The collapsed MMP, decreased ATP levels, and increased cell apoptosis caused by Cr(VI) were also partially abolished by PD98059 pretreatment, indicating that ERK activation was involved in Cr(VI)-induced apoptosis through mitochondrial-dependent pathway. In addition, evidence suggests that ERK activation is involved in regulating mitochondrial fission and DLP1 translocation through α-synuclein-mediated mitochondrial dynamics changes (Gui et al., 2012). We demonstrated in the present study that ERK inhibition restored the changes in mitochondrial morphology and the expressions of DILP1 and Mfn2, clearly indicating the involvement of ERK activation in Cr(VI)-mediated mitochondrial dynamics abnormalities. It is well known that the increase of DLP1 recruitment to the surface of mitochondria could contribute to DLP1-dependent mitochondrial division and fragmentation (Schauss et al., 2006). Therefore, the differential expression of DLP1 as well as other fusion proteins (such as Mfn2), is likely responsible for the fragmentation of mitochondria observed in L02 hepatocytes exposed to Cr(VI). Both pharmacological blockade of mitochondrial division using inhibitor of DLP1 GTPase (mdivi-1) and knockdown of DLP1 expression significantly restored mitochondrial morphology and function (Gan et al., 2014b). Our data revealed that transfection with DLP1 siRNA significantly attenuated Cr (VI)-induced increased DLP1 expression and abnormal mitochondrial morphology. DLP1 siRNA, which alleviated the mitochondrial fragmentation, could partly recover the decrease in MMP and ATP level, confirming that mitochondria fragmentation was an upstream event. We also found that the cell apoptosis induced by Cr(VI) could be abolished by DLP1 inhibition, which strongly supported the conclusion that Cr(VI)-induced apoptosis was at least partially DLP1-dependent. In summary, our data demonstrated that Cr(VI) significantly inhibited mitochondrial function, accompanied by changes in mitochondrial morphology and fission/fusion events. We provided substantial evidence that the ROS accumulation and ERK pathway activation were involved in the translocation of DLP1 to mitochondria in Cr(VI)-exposed L02 hepatocytes. We confirmed that increased ROS accumulation activated ERK transduction, disrupted mitochondrial fission/fusion balance, and augmented DLP1 recruitment to mitochondria, resulting in mitochondrial fragmentation. These findings provided the molecular evidence that Cr(VI) induced mitochondrial dysfunction and cell apoptosis through ROS-ERK-DLP1 signaling pathway in L02 hepatocytes.
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Res. (Camb.) 6, 324–332. Huang, Z., Wu, L.M., Zhang, J.L., Sabri, A., Wang, S.J., Qin, G.J., Guo, C.Q., Wen, H.T., Du, B.B., Zhang, D.H., Kong, L.Y., Tian, X.Y., Yao, R., Li, Y.P., Liang, C., Li, P.C., Wang, Z., Guo, J.Y., Li, L., Dong, J.Z., Zhang, Y.Z., 2019. DUSP12 regulates hepatic lipid metabolism through inhibition of lipogenesis and ASK1 pathways. Hepatology. https://doi.org/10.1002/hep. 30597. Jobby, R., Jha, P., Yadav, A.K., Desai, N., 2018. Biosorption and biotransformation of hexavalent chromium [Cr(VI)]: a comprehensive review. Chemosphere 207, 255–266. Li, Z., Okamoto, K., Hayashi, Y., Sheng, M., 2004. The importance of dendritic mitochondria in the morphogenesis and plasticity of spines and synapses. Cell 119, 873–887. Maycotte, P., Marin-Hernandez, A., Goyri-Aguirre, M., Anaya-Ruiz, M., Reyes-Leyva, J., CortesHernandez, P., 2017. Mitochondrial dynamics and cancer. Tumour Biol. 39 1010428317698391. Muriel, P., Gordillo, K.R., 2016. Role of oxidative stress in liver Health and disease. Oxid. Med. Cell Longevity 2016, 9037051. Resseguie, E.A., Staversky, R.J., Brookes, P.S., O'Reilly, M.A., 2015. Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction. Redox Biol. 5, 176–185. Schauss, A.C., Bewersdorf, J., Jakobs, S., 2006. Fis1p and Caf4p, but not Mdv1p, determine the polar localization of Dnm1p clusters on the mitochondrial surface. J. Cell Sci. 119, 3098–3106. Smirnova, E., Griparic, L., Shurland, D.L., van der Bliek, A.M., 2001. Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Mol. Biol. Cell 12, 2245–2256. Stout, M.D., Herbert, R.A., Kissling, G.E., Collins, B.J., Travlos, G.S., Witt, K.L., Melnick, R.L., Abdo, K.M., Malarkey, D.E., Hooth, M.J., 2009. Hexavalent chromium is carcinogenic to F344/N rats and B6C3F1 mice after chronic oral exposure. Environ. Health Perspect. 117, 716–722. Vasquez-Trincado, C., Garcia-Carvajal, I., Pennanen, C., Parra, V., Hill, J.A., Rothermel, B.A., Lavandero, S., 2016. Mitochondrial dynamics, mitophagy and cardiovascular disease. J. Physiol. Lond. 594, 509–525. Wainstein, E., Seger, R., 2016. The dynamic subcellular localization of ERK: mechanisms of translocation and role in various organelles. Curr. Opin. Cell Biol. 39, 15–20. Wang, X., Su, B., Fujioka, H., Zhu, X., 2008. Dynamin-like protein 1 reduction underlies mitochondrial morphology and distribution abnormalities in fibroblasts from sporadic Alzheimer's disease patients. Am. J. Pathol. 173, 470–482. Xiao, Y., Zeng, M., Yin, L., Li, N., Xiao, F., 2019. Clusterin increases mitochondrial respiratory chain complex I activity and protects against hexavalent chromium-induced cytotoxicity in L-02 hepatocytes. Toxicol. Res. 8, 15–24. Xie, L.L., Shi, F., Tan, Z.Q., Li, Y.S., Bode, A.M., Cao, Y., 2018. Mitochondrial network structure homeostasis and cell death. Cancer Sci. 109, 3686–3694. Xu, S., Pi, H., Chen, Y., Zhang, N., Guo, P., Lu, Y., He, M., Xie, J., Zhong, M., Zhang, Y., Yu, Z., Zhou, Z., 2013. Cadmium induced Drp1-dependent mitochondrial fragmentation by disturbing calcium homeostasis in its hepatotoxicity. Cell Death Dis. 4, e540. Yatera, K., Morimoto, Y., Ueno, S., Noguchi, S., Kawaguchi, T., Tanaka, F., Suzuki, H., Higashi, T., 2018. Cancer risks of hexavalent chromium in the respiratory tract. J. UOEH 40, 157–172. Yi, X., Zhang, Y., Zhong, C., Zhong, X., Xiao, F., 2016. The role of STIM1 in the Cr(vi)-induced [Ca2+]i increase and cell injury in L-02 hepatocytes. Metallomics 8, 1273–1282. Youle, R.J., van der Bliek, A.M., 2012. Mitochondrial fission, fusion, and stress. Science 337, 1062–1065. Zhang, G.Y., He, J.P., Ye, X.F., Zhu, J., Hu, X., Shen, M.Y., Ma, Y.R., Mao, Z.M., Song, H.D., Chen, F.L., 2019. beta-Thujaplicin induces autophagic cell death, apoptosis, and cell cycle arrest through ROS-mediated Akt and p38/ERK MAPK signaling in human hepatocellular carcinoma. Cell Death Dis. 10. Zhang, Y.J., Xiao, F., Liu, X.M., Liu, K.H., Zhou, X.X., Zhong, C.G., 2017. Cr(VI) induces cytotoxicity in vitro through activation of ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction via the PI3K/Akt signaling pathway. Toxicol. In Vitro 41, 232–244.
Declaration of competing interest The authors have no conflicts of interest to declare. Acknowledgements We thank all of the individuals in this laboratory for their valuable suggestions. The current work was supported by the National Natural
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Blockage of ROS-ERK-DLP1 signaling and mitochondrial fission alleviates Cr (VI)-induced mitochondrial dysfunction in L02 hepatocytes
T
Yujing Zhang, Yu Ma, Ningjuan Liang, Yuehui Liang, Chan Lu, Fang Xiao∗ Department of Health Toxicology, Xiangya School of Public Health, Central South University, Changsha, 410078, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Hexavalent chromium [Cr(VI)] Mitochondrial dysfunction Reactive oxygen species (ROS) Extracellular regulated protein kinases (ERK) Dynamic-like protein 1 (DLP1)
Hexavalent chromium [Cr(VI)] is a common heavy metal pollutant widely used in various industrial fields. It is well known that mitochondria are the most vulnerable targets of heavy metals, but the key molecule/event that directly mediated mitochondrial dysfunction after Cr(VI) exposure is still unclear. The present study was aimed to explore whether Cr(VI) exposure could affect the mitochondrial fission/fusion process, and whether the related abnormal mitochondrial dynamics have been implicated in Cr(VI)-induced mitochondrial dysfunction. We found that the mitochondrial dysfunction caused by Cr(VI) exposure was characterized by decreased mitochondrial respiratory chain complex (MRCC) I/II activities and levels, collapsed mitochondrial membrane potential (MMP), depleted ATP, and increased reactive oxygen species (ROS) level. Cr(VI) induced abnormal mitochondrial fission/fusion events, the antioxidant Nacetyl-L-cysteine (NAC) restored the abnormal mitochondrial function as well as the fission/fusion dynamics. ROS was the up-stream regulator of extracellular regulated protein kinases (ERK) signaling, and the application of a specific ERK1/2 inhibitor PD98059 confirmed that activation of ERK1/2 signaling was associated with the abnormal mitochondrial fission/fusion and mitochondrial dysfunction. We also demonstrated that treatment with dynamic-like protein 1 (DLP1)-siRNA rescued mitochondrial dysfunction in Cr(VI)-exposed L02 hepatocytes. We reached the conclusion that blockage of ROS-ERK-DLP1 signaling and mitochondrial fission alleviates Cr(VI)-induced mitochondrial dysfunction in L02 hepatocytes, which may provide the new avenue for developing effective strategies to protect against Cr(VI)induced hepatotoxicity.
1. Introduction Hexavalent chromium [Cr(VI)] is a common heavy metal pollutant widely used in electroplating, metallurgy, tanning and other industrial fields (Jobby et al., 2018). The international agency for research on cancer (IARC) has identified Cr(VI) as a human carcinogen in 1990. Cr (VI) can be dispersed through air, soil and water. In addition to occupational exposure, food and/or drinking water are also important exposure routes of Cr(VI) (Huang et al., 2017; Yatera et al., 2018). In 2007, the US national toxicology research program (NTP) conducted a two-year study which confirmed that Cr(VI) ingestion through the digestive tract caused increased tumor incidence in mice and rats (Stout et al., 2009). Epidemiological and animal studies have evidenced that both acute and chronic Cr(VI) exposure can contribute to organs damage, including liver and kidney (Bosgelmez and Guvendik, 2017; Cengiz et al., 2016). Our previous reports also proved that Cr(VI) could induce cell apoptosis in hepatocytes through ER stress- and/or
mitochondrial-dependent pathway (Yi et al., 2016; Zhang et al., 2017). Evidence suggested that disturbed bioenergetic function, especially mitochondrial dysfunction, played a central role in Cr(VI)-induced cytotoxicity. It is well known that mitochondria are the most vulnerable targets of heavy metals, but the key molecule/event that directly mediated mitochondrial dysfunction after Cr(VI) exposure is still unclear. Mitochondria are critical for cell proliferation, survival, and death. Mitochondria are dynamic cytoplasmic organelles, they undergo coordinated and continuous cycles of fission and fusion in order to maintain their morphology, size, and distribution. The fission/fusionrelated mitochondrial morphological changes are important for many cellular physiological processes including cell cycle, innate immunity, autophagy, calcium homeostasis, and apoptosis. Dynamic-like protein 1 (DLP1), also called dynamic-related protein 1 (DRP1) (Wang et al., 2008), is a key molecule for mitochondrial fission regulation, directly or indirectly acting on other fission/fusion proteins, and its translocation
∗ Corresponding author. Department of Health Toxicology, Xiangya School of Public Health, Central South University, NO.238 Shangmayuanling Road, Kaifu District, Changsha, Hunan 410078, China. E-mail address: [email protected] (F. Xiao).
https://doi.org/10.1016/j.ecoenv.2019.109749 Received 5 July 2019; Received in revised form 23 September 2019; Accepted 1 October 2019 0147-6513/ © 2019 Elsevier Inc. All rights reserved.
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Fig. 1. Mitochondrial dysfunction in Cr(VI)-exposed L02 hepatocytes. L02 hepatocytes were treated with different concentrations of Cr(VI) (0, 8, 16 μM) for 24 h. (A) The enzyme activities of MRCC I–V were determined using commercial kits. (B) The protein levels of MRCC I and II were analyzed by western blotting. The density of the immunoreactive bands was quantified using Image J software. (C) Mitotracker (Green) staining was used to show mitochondria. Images with TMRM staining indicated the change of MMP. (D) ATP content was determined by ATP Bioluminescence Assay Kit. (E) ROS was detected by fluorescent microscope using DCFH-DA staining. DAPI staining was used to show nucleus. (F) Cell apoptosis was detected by FITC-labeled Annexin V/PI apoptosis detection kit. Each experiment was repeated as least 3 times. *p < 0.05 vs. control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
from cytoplasm to mitochondria initiates the fission/fusion events (Smirnova et al., 2001). The inhibition of DLP1 during mitochondrial division leads to mitochondrial fragmentation, which prevents it from correctly localizing to dendrites or axons, thereby inhibiting the formation and function of synapse (Li et al., 2004). Mitochondrial fusion can be simply regarded as the fusion of two organelles, but the fusion process needs to go through two steps, namely outer membrane fusion and inner membrane fusion. The proteins mainly regulating mitochondrial outer membrane fusion are mitochondrial fusion proteins Mitofusins 1 and 2 (Mfn1 and Mfn2) (Youle and van der Bliek, 2012). It is confirmed that Mfn2 gene knockout led to decreased ATP synthesis, collapsed mitochondrial membrane potential (MMP), and reduced oxidation of glucose, pyruvate and fatty acids, resulting in
mitochondrial dysfunction (Chen et al., 2005). An emerging literature has focused on environmental contaminants exposure-induced altered mitochondrial dynamics. Evidence suggested that metals such as Cadmium (Xu et al., 2013) and Manganese could induce increased DLP1 expression as well as mitochondria fragmentation. It is still not clear whether Cr(VI) exposure could affect the mitochondrial fission/fusion process, and whether the related abnormal mitochondrial dynamics have been implicated in Cr(VI)-induced mitochondrial dysfunction. Therefore, the present study aimed to demonstrate the effects and the underlying mechanisms of Cr(VI) on mitochondrial dynamics-related mitochondrial dysfunction during hepatotoxicity in vitro, which may provide the new avenue for developing effective strategies to protect against Cr(VI)-induced hepatotoxicity. 2
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Fig. 2. Abnormal mitochondrial fission/fusion events in Cr(VI)-exposed L02 hepatocytes. L02 hepatocytes were treated with PBS or 16 μM Cr(VI) for 24 h. (A) The cells were labeled with Mitotracker Red for visualization of mitochondrial morphology. Hoechst 33342 was used to stain the nucleus. (B) Mitochondrial length and density were quantified by the investigator blinded to the treatment groups using Image J software. (C) Mitochondrial protein was extracted and the protein levels of DLP1 and Mfn2 were analyzed by western blotting. *p < 0.05 vs. control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2. Materials and methods
(Amersham, Piscataway, NJ, USA). The density of the immunoreactive bands was analyzed using Image J software. The primary antibodies for MRCC I subunit NDUFS3 (MS110) and II subunit 70 KDa Fp (MS204) were purchased from MitoScience (Eugene, OR, USA). Antibodies for DLP1 (D8H5) (#5391), Mfn2 (D1E9) (#11925), p-ERK1/2 (D13.14.4E) (#4370), ERK1/2 (L34F12) (#4696), Hsp60 (D6F1) (#12165), and β-actin (#4967) were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibody for Goat Anti-Rabbit IgG (H + L) HRP (S0001) was purchased from Affinity Biosciences (Shanghai, China), and Anti-mouse IgG, HRPlinked Antibody (#7076P2) was purchased from Cell Signaling Technology (Danvers, MA, USA).
2.1. Cell culture and treatment Human L02 hepatocytes, purchased from the Experimental Central of Xiangya Hospital of Central South University, were cultured in RPMI 1640 medium (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution in a humidified 5% CO2 incubator at 37 °C. The hepatocytes were treated with different doses of potassium dichromate (K2Cr2O7, Sigma, St. Louis, MO). 2.2. Determination of mitochondrial respiratory chain complexes (MRCCs) activities
2.4. Detection of MMP
The hepatocytes were treated with different concentrations of Cr (VI) (0, 8, 16 μM) for 24 h. The enzyme activities of MRCC I (nicotinamide adenine dinucleotide (NADH)-ubiquinone reductase), II (succinate dehydrogenase), III (ubiquinol: cytochrome c (Cyt c) reductase), IV (Cyt c oxidase), and V (ATP synthase) were determined as previously described (Xiao et al., 2019).
The cells were treated with the indicated concentrations of different chemicals. The fluorescent dye tetramethylrhodamine methyl ester (TMRM) and Mito Tracker Green were used to detect the MMP. In brief, the hepatocytes were incubated with TMRM (10 μg/mL) (Beyotime Institute of Biotechnology, Shanghai, China) at 37 °C for 5 min, and then washed 3 times with phosphate buffer saline (PBS). Mito Tracker Green was then added to the culture. After the incubation for another 45 min at 37 °C, cells were rinsed with PBS. The hepatocytes were then observed using a fluorescence microscope (Olympus, Tokyo, Japan).
2.3. Western blots The cells were treated with the indicated concentrations of different chemicals. The specific ERK1/2 inhibitor PD98059 was applied for 2 h before Cr(VI) treatment. Mitochondrial protein was extracted using Cytoplasmic and Mitochondrial Protein Extraction Kit (Sangon Biotech, Shanghai, China). Bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to detect the protein concentration. Proteins were separated by 12% Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene difluoride (PVDF) transfer membranes (Millipore, Billerica, MA). Then, the blots were blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibody at 4 °C. Then, the blots were incubated with secondary antibodies for 1 h at room temperature. The immunoreactive bands were detected using an Enhanced Chemiluminescence (ECL) Detection Kit
2.5. ATP content analysis ATP content was determined by ATP Bioluminescence Assay Kit (Roche, Mannheim, Germany) according to manufacturer's instructions. Briefly, 1 × 106 cells were homogenized with 200 μL lysis buffer supplied with the ATP assay kit and then centrifugated at 12,000×g for 5 min at 4 °C, and the supernatant was transferred to a new tube. 100 μl ATP detection buffer was added to 100 μl sample, and then the luminescence of the culture was measured in a luminescence plate reader with the integration time of 10 s. The ATP standard curve was prepared from a known amount (0.01–10 μM). 3
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Fig. 3. Effect of NAC on mitochondrial function in Cr(VI)-exposed hepatocytes. The hepatocytes were pretreated with NAC for 1 h before Cr(VI) treatment for another 24 h. (A) ROS was detected using DCFH-DA staining. The mean DCF fluorescence was measured by fluorescent microscope. (B) The enzyme activities of MRCC I and II were determined using commercial kits. (C) The protein levels of MRCC I and II were analyzed by western blotting. (D) The MMP-dependent fluorescent indicator TMRM was used to detect the change of MMP. (E) The ATP content was measured by fluorescence microplate reader using commercial kit. (F) Annexin V/PI assay was used to detect the cell apoptosis. *p < 0.05 vs. control group. #p < 0.05 vs. 16 μM Cr(VI) treatment group.
product of DCF diacetate (DCFH-DA). Briefly, the cells were loaded with 10 μM DCFH-DA (Beyotime Institute of Biotechnology, Shanghai, China) at 37 °C for 40 min. Then the cells were washed twice with PBS and incubated with DAPI for another 45 min in the dark at 37 °C. The fluorescence signals were observed using a fluorescence microscope
2.6. Measurement of reactive oxygen species (ROS) level The cells were treated with the indicated concentrations of different chemicals. The intracellular ROS level was assayed by detecting the fluorescent intensity of 2′, 7′-dichlorofluorescein (DCF), the oxidized 4
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Fig. 4. Effect of NAC on mitochondrial fission/fusion in Cr(VI)-exposed hepatocytes. The hepatocytes were pretreated with NAC for 1 h before Cr(VI) treatment for another 24 h. (A) Mitotracker Red and Hoechst 33342 were used for visualization of mitochondrial morphology. (B) Mitochondrial length and density were quantified by the investigator blinded to the treatment groups using Image J software. (C) Mitochondrial protein was extracted and analyzed by western blotting to detect the expression of mitochondrial fission/fusion proteins. (D) Whole cell lysates were collected and analyzed by western blotting to exam the levels of P-ERK and T-ERK. *p < 0.05 vs. control group. #p < 0.05 vs. 16 μM Cr(VI) treatment group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
2.8. Measurement of mitochondrial morphology change
(Olympus, Tokyo, Japan).
The L02 hepatocytes were exposed to PBS or 16 μM Cr(VI) for 24 h with or without Nacetyl-L-cysteine (NAC) pretreatment for 1 h. After the indicated treatment, the hepatocytes were stained with 10 nM Mitotracker Red (Invitrogen Life Technologies, CA, USA) for 45 min at 37 °C. Then, the cells were washed twice with PBS and incubated with Hoechst 33,342 for another 45 min in the dark at 37 °C. Live-cell fluorescence images were captured with a fluorescence microscope. More than 20 clearly identifiable mitochondria from each treatment group were randomly selected. Mitochondrial length and density were quantified by the investigator blinded to the treatment groups using Image J software.
2.7. Cell apoptosis detection Cell apoptosis was detected by fluorescein isothiocyanate (FITC)labeled Annexin V/propidium iodide (PI) Apoptosis Detection Kit (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's instructions. Briefly, L02 hepatocytes were harvested, washed with icecold PBS, re-suspended in 500 μl binding buffer supplemented with 5 μl Annexin-V-FITC and 1 μl PI for 30 min at room temperature. The flow cytometric analysis was performed immediately. Data acquisition and analysis were performed using FlowJo 7.6 software (TreeStar, San Carlos, CA).
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Fig. 5. Activation of ERK1/2 signaling was associated with the abnormal mitochondrial fission/fusion and mitochondrial dysfunction. The cells were incubated with PD98059 for 2 h before Cr(VI) treatment for another 24 h. (A) Protein levels of P-ERK and T-ERK were determined using western blotting. (B) The fluorescent indicator TMRM was used to detect the change of MMP. (C) The ATP content was measured using commercial kit. (D) Annexin V/PI assay was used to detect the apoptotic cells. (E) DLP1 and Mfn2 were analyzed by western blotting and then were quantified. *p < 0.05 vs. control group. #p < 0.05 vs. 16 μM Cr(VI) treatment group.
Student-Newman-Keuls (SNK) test. Statistical significance was defined as p < 0.05. All statistical analyses were performed using the Statistical Program for Social Sciences (SPSS), version 19.0.
2.9. Transient transfection of small interfering RNA (siRNA) L02 hepatocytes were transfected with siRNA targeting DLP1 or non-targeted siRNA duplexes (Ribobio, Guangzhou, China) using lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA) according to manufacturer's protocol.
3. Results 3.1. Mitochondrial dysfunction in Cr(VI)-exposed L02 hepatocytes
2.10. Statistics analysis L02 hepatocytes were treated with different concentrations of Cr (VI) (0, 8, 16 μM) for 24 h. In order to determine whether Cr(VI) could influence mitochondrial function, we first evaluated the key enzymes associated with respiratory chain, MRCCs. As shown in Fig. 1A and
All data are expressed as mean ± SD from 3 independent experiments. Significant difference among the groups was evaluated by oneway analysis of variance (ANOVA). Post-hoc test was analyzed by 6
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Fig. 6. Treatment with DLP1-siRNA rescued mitochondrial dysfunction in Cr(VI)-exposed L02 hepatocytes. L02 hepatocytes were transfected with DLP1 siRNA or control siRNA for 24 h, and then were treated with PBS or 16 μM Cr(VI) for another 24 h. (A) Western blotting was used to assess the knockdown efficiency of DLP1-siRNA. (B) The fluorescent indicator TMRM was used to detect the change of MMP. (C) The ATP content was measured using commercial kit. (D) Annexin V/ PI assay was used to detect the apoptotic cells. #p < 0.05 vs. 16 μM Cr(VI) treatment group.
3.2. Abnormal mitochondrial fission/fusion events in Cr(VI)-exposed L02 hepatocytes
compared to the control, the hepatocytes had a significant decrease in MRCC I and II activities. No significant change in MRCC III, IV, or V activity was found after Cr(VI) exposure. Western blotting result in Fig. 1B also confirmed that Cr(VI) could down-regulated the protein expression levels of MRCC I and II. Decreased MMP is a biomarker of mitochondrial dysfunction, and the degree of cell damage increases with the reduction of MMP. The hepatocytes were treated with Mito Tracker green and TMRM (red) to monitor MMP. The intensity of the red fluorescence was significantly decreased in L02 hepatocytes after Cr (VI) treatment in a concentration-dependent manner, indicating the collapse of MMP (Fig. 1C). Similarly, Cr(VI) induced the dysfunction of cellular energy metabolism since ATP levels were also reduced in the hepatocytes (Fig. 1D). It has been confirmed that mitochondria are the major source of ROS production and ROS overproduction is an important indicator of mitochondrial dysfunction. Thus, we measured the formation of intracellular ROS using DAPI and DCFH stain. Fig. 1E showed that Cr(VI) significantly increased the green fluorescence signals in a concentration-dependent manner, indicating that mitochondrial ROS production closely correlated with mitochondrial dysfunction. We also used Annexin V-FITC/PI staining to detect cell apoptosis (Fig. 1F) and found Cr(VI) increased the proportion of both early and late apoptotic cells.
Mitochondrial fission/fusion are essential for the maintenance of mitochondrial shape and mitochondrial homeostasis. We then evaluated the changes in mitochondrial morphology in L02 hepatocytes exposed to Cr(VI). Representative pictures for Mito Tracker (red) staining showed that mitochondria were tubular, netlike, elongated and regularly distributed in control group, while mitochondria were punctuated, shorter, fragmented, and collapsed away from the mitochondrial network in Cr(VI)-exposed cells (Fig. 2A). Accordingly, mitochondrial density was decreased and mitochondrial length was shorter in Cr(VI)-exposed cells than in the control cells (Fig. 2B). DLP1 is a key player and regulator in mitochondrial fission whose translocation from cytoplasm to mitochondria initiates the fission process, and Mfn2 is known to control the fusion process. Mitochondrial fractions were isolated to determine the fission protein DLP1 and the fusion protein Mfn2. As shown in Fig. 2C, DLP1 was increased while Mfn2 was decreased in mitochondrial fraction of Cr(VI)-exposed hepatocytes as compared to non-treated mitochondrial fraction, confirming that both fission/fusion proteins were changed in a direction favoring the fission process in Cr(VI)-exposed cells.
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3.3. Effect of NAC on mitochondrial function and fission/fusion in Cr(VI)exposed hepatocytes
3.5. Treatment with DLP1-siRNA rescued mitochondrial dysfunction in Cr (VI)-exposed L02 hepatocytes
Previous evidence suggest that increased mitochondrial ROS-associated oxidative stress is a key initiator of mitochondrial dysfunction and abnormal changes in mitochondrial morphology. We then explored if antioxidant treatment could restore the abnormal mitochondrial function as well as the fission/fusion dynamics. The hepatocytes were pretreated with NAC for 1 h before Cr(VI) treatment for another 24 h. The results in Fig. 3A showed NAC pretreatment markedly inhibited Cr (VI)-induced ROS formation as shown by reduced DCFH green fluorescence signals. Such antioxidant treatment significantly restored mitochondrial function and energy metabolism as evidenced by increased MRCC I/II activities (Fig. 3B) and protein expression levels (Fig. 3C), MMP (as determined by TMRM staining) (Fig. 3D), and ATP content (Fig. 3E), and decreased proportion of both early and late apoptotic cells (Fig. 3F) in Cr(VI)-treated L02 hepatocytes. The protective effect of NAC indicated the involvement of oxidative stress in Cr(VI)-induced mitochondrial dysfunction. We further assayed the effect of NAC on mitochondrial morphology, density, and length. As shown in Fig. 4A, NAC pre-treatment largely reversed the abnormal mitochondrial morphology induced by Cr(VI), suggesting the antioxidant could protect mitochondrial morphology from damage and maintain it in a normal and regular state. Accordingly, both mitochondria density and length were significantly increased in Cr(VI) group pretreated with NAC compared to that of the Cr (VI) alone treatment group (Fig. 4B). NAC pretreatment significantly suppressed the increase of DLP1 expression and alleviated the decrease of Mfn2 expression caused by Cr(VI) (Fig. 4C). The above data revealed that antioxidant could reverse impaired mitochondrial fission/fusion dynamics and recover mitochondrial dysfunction. We further determined the effect of oxidative stress on ERK1/2 activation and confirmed that ROS was the up-stream regulator of ERK1/2 signaling. As shown in Fig. 4D, Cr(VI) significantly increased the ERK1/2 phosphorylation (p-ERK) level but had no obvious effect on total ERK1/2 expression. NAC pretreatment suppressed the increase of p-ERK1/2 expression, which is consistent with the previous evidence that oxidative stress could activate the MAP kinase such as ERK1/2 and p38.
The perturbed balance of mitochondrial fission/fusion process is the key mechanism for mitochondrial dysfunction, we then explored whether the inhibition of mitochondrial division could rescue Cr(VI)-induced mitochondrial function defects. We constructed the siRNA-targeted DLP1 to block DLP1 expression in L02 hepatocytes. Western blotting was used to assess the knockdown efficiency of DLP1-siRNA. As shown in Fig. 6A, DLP1 protein expression was significantly reduced in cells transfected with DLP1-siRNA than in cells transfected with the control siRNA, and there was a marked reduction in DLP1 protein expression following DLP1 knockdown and Cr(VI) treatment. As shown in Fig. 6B-C, DLP1-siRNA transfection rescued mitochondrial dysfunction by increasing the MMP and ATP levels. To demonstrate the role of DLP1 in cell apoptosis caused by Cr(VI), we detected the proportion of apoptotic cells and found DLP1-siRNA could partially inhibit Cr(VI)induced cell apoptosis (Fig. 6D). 4. Discussion Mitochondria, the highly dynamic organelle, are the main targets of many toxins and environmental pollutants (Firdaus et al., 2018). The alterations in mitochondrial morphology and dynamics, which obviously affect almost all aspects of mitochondrial function, are reported to be associated with the progression of various diseases (Maycotte et al., 2017; Vasquez-Trincado et al., 2016). Although abnormalities in mitochondrial function in Cr(VI)-induced hepatotoxicity are well documented, the related mechanisms and the effective strategy to rescue mitochondrial dysfunction remain elusive. Particularly, the signaling pathway associated with fission/fusion process and its relationship between mitochondrial bioenergy and mitochondrial function in L02 hepatocytes exposed to Cr(VI) have not been fully elucidated. Cr(VI) induced mitochondrial damage, which was characterized by the decreased expression of MRCC I and II, the accumulation of intracellular ROS, the collapse of MMP, and the depletion of ATP. It is known that changes of mitochondrial morphology and fission/fusion events could affect cell apoptosis (Xie et al., 2018). The present study showed marked changes in mitochondrial morphology and fission/fusion balance in L02 hepatocytes exposed to Cr(VI). We also observed that the abnormalities in mitochondrial structure and function were associated with changed expression and distribution of DLP1. Taking these results together, we recognized that mitochondrial dysfunction, especially the altered mitochondrial morphology and fission/fusion, was essential in promoting Cr(VI)-induced apoptosis in L02 hepatocytes. Mitochondria are not only the main sources of ROS, but also the key targets of ROS (Resseguie et al., 2015). Our previous study indicated that Cr(VI)-induced hepatotoxicity is associated with oxidative stress (Zhang et al., 2017), which is mediated by ROS. Intracellular ROS accumulation could destroy cell integrity, leading to damage of various organs such as the liver (Muriel and Gordillo, 2016). The present study showed that Cr(VI)-induced mitochondrial dysfunction was accompanied by the accumulation of ROS. NAC, the ROS scavenger, was used in our study to investigated the role of ROS in Cr(VI)-induced mitochondrial dysfunction. Indeed, pretreatment with NAC significantly blunted ROS accumulation and augmented MRCC I and II expression, MMP, and ATP content in L02 hepatocytes treated with Cr(VI). In this study, we also found that cell apoptosis induced by Cr(VI) was almost completely reversed by the presence of NAC. Although there is abundant evidence that the disruption of mitochondrial fission/fusion status is related to ROS accumulation in many diseases, to the best of our knowledge, this is the first time that we have demonstrated the relationship between ROS production and mitochondrial fission/fusion events after Cr(VI) exposure. NAC pretreatment alleviated abnormal mitochondrial morphology via rescuing the altered protein expression levels of DLP1 and Mfn2, suggesting that increased oxidative stress is
3.4. Activation of ERK1/2 signaling was associated with the abnormal mitochondrial fission/fusion and mitochondrial dysfunction The activation of ERK1/2 has been shown to be associated with abnormal mitochondrial morphology and function (Wainstein and Seger, 2016). To explore the possible role of ERK1/2 activation in Cr (VI)-induced mitochondrial dysfunction, we applied the specific ERK1/ 2 inhibitor PD98059 in the present study. As shown in Fig. 5A, PD98059 inhibited the increase of p-ERK1/2 expression, indicating that ERK1/2 phosphorylation was largely abolished. The total ERK1/2 was not significantly altered. To determine if ERK1/2 activation was directly related to mitochondrial dysfunction, we examined the effect of PD98059 on MMP. The result in Fig. 5B showed that PD98059 exposure caused a significantly higher intensity of TMRM fluorescence in combination treatment cells than in Cr(VI) alone treatment cells. Similarly, PD98059 treatment alleviated Cr(VI)-induced ATP depletion in the L02 hepatocytes (Fig. 5C). Fig. 5D also demonstrated that Cr(VI)-induced cell apoptosis could be partially abolished by PD98059. These results revealed that in Cr(VI)-exposed hepatocytes, the activation of ERK1/2 signaling caused by oxidative stress was associated with mitochondrial dysfunction. Furthermore, we immunoblotted mitochondrial fractions for fission/fusion proteins expression as described above. Consistently, PD98059 treatment reversed DLP1 and Mfn2 expression levels compared to Cr(VI) alone treatment cells (Fig. 5E), suggesting that blockage of ERK1/2 activation rescued Cr(VI)-induced abnormal mitochondrial dynamics. 8
Ecotoxicology and Environmental Safety 186 (2019) 109749
Y. Zhang, et al.
Science Foundation of China (NO. 81773478) and Natural Science Foundation of Hunan Province, China (NO. 2019JJ40402).
responsible for the perturbation of mitochondrial dynamics and the subsequent apoptosis in the cells exposed to Cr(VI). The signal transduction mediated by mitogen activated protein kinase (MAPK) pathway has attracted extensive attention in regulating cell growth, apoptosis, and various diseases (Huang et al.,2019; Zhang et al., 2019). Extracellular regulated protein kinases (ERK), a class of extracellular regulatory protein kinases, including ERK1 and ERK2, are critical to transfer signals from surface receptors to the nucleus. Inhibition of ERK activation not only attenuates abnormal mitochondrial morphsology and function, but also restores the balance of mitochondrial division and fusion, and it is believed that oxidative stress-mediated ERK signaling plays a role in regulating mitochondrial fission/fusion events (Gan et al., 2014a). In the current study, our data demonstrated that ERK1/2 phosphorylation was significantly increased in Cr(VI)-exposed L02 hepatocytes, and was partially rescued by application of the ERK inhibitor (PD98059). The collapsed MMP, decreased ATP levels, and increased cell apoptosis caused by Cr(VI) were also partially abolished by PD98059 pretreatment, indicating that ERK activation was involved in Cr(VI)-induced apoptosis through mitochondrial-dependent pathway. In addition, evidence suggests that ERK activation is involved in regulating mitochondrial fission and DLP1 translocation through α-synuclein-mediated mitochondrial dynamics changes (Gui et al., 2012). We demonstrated in the present study that ERK inhibition restored the changes in mitochondrial morphology and the expressions of DILP1 and Mfn2, clearly indicating the involvement of ERK activation in Cr(VI)-mediated mitochondrial dynamics abnormalities. It is well known that the increase of DLP1 recruitment to the surface of mitochondria could contribute to DLP1-dependent mitochondrial division and fragmentation (Schauss et al., 2006). Therefore, the differential expression of DLP1 as well as other fusion proteins (such as Mfn2), is likely responsible for the fragmentation of mitochondria observed in L02 hepatocytes exposed to Cr(VI). Both pharmacological blockade of mitochondrial division using inhibitor of DLP1 GTPase (mdivi-1) and knockdown of DLP1 expression significantly restored mitochondrial morphology and function (Gan et al., 2014b). Our data revealed that transfection with DLP1 siRNA significantly attenuated Cr (VI)-induced increased DLP1 expression and abnormal mitochondrial morphology. DLP1 siRNA, which alleviated the mitochondrial fragmentation, could partly recover the decrease in MMP and ATP level, confirming that mitochondria fragmentation was an upstream event. We also found that the cell apoptosis induced by Cr(VI) could be abolished by DLP1 inhibition, which strongly supported the conclusion that Cr(VI)-induced apoptosis was at least partially DLP1-dependent. In summary, our data demonstrated that Cr(VI) significantly inhibited mitochondrial function, accompanied by changes in mitochondrial morphology and fission/fusion events. We provided substantial evidence that the ROS accumulation and ERK pathway activation were involved in the translocation of DLP1 to mitochondria in Cr(VI)-exposed L02 hepatocytes. We confirmed that increased ROS accumulation activated ERK transduction, disrupted mitochondrial fission/fusion balance, and augmented DLP1 recruitment to mitochondria, resulting in mitochondrial fragmentation. These findings provided the molecular evidence that Cr(VI) induced mitochondrial dysfunction and cell apoptosis through ROS-ERK-DLP1 signaling pathway in L02 hepatocytes.
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Declaration of competing interest The authors have no conflicts of interest to declare. Acknowledgements We thank all of the individuals in this laboratory for their valuable suggestions. The current work was supported by the National Natural
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