Cr(VI)-induced overactive mitophagy contributes to mitochondrial loss and cytotoxicity in L02 hepatocytes

Hexavalent chromium [Cr(VI)] has aroused the main interest of environmental health researchers due to its high toxicity. Liver is the main target organ of Cr(VI), and the purpose of this study was to explore whether mitophagy contributes to Cr(VI)-induced hepatotoxicity and to demonstrate the potential mechanisms. Cr(VI) exposure induced mitochondrial loss, energy metabolism disorders and cell apoptosis, which were associated with the occurrence of excessive mitophagy characterized by the increased number of green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) puncta and lysosomal colocalization with mitochondria. In addition, the suppression of mitophagy by autophagy-related 5 (ATG5) siRNA can effectively inhibit Cr(VI)-induced mitochondrial loss and cytotoxicity. In summary, we reached the conclusion that Cr(VI)-induced overactive mitophagy contributes to mitochondrial loss and cytotoxicity in L02 hepatocytes, which will further reveal the possible mechanisms of Cr(VI)-induced hepatotoxicity, and provide a new experimental basis for the study of the health hazard effects of chromium.


Introduction
Hexavalent chromium [Cr(VI)] has aroused the main interest of environmental health researchers, who believe that past exposures will affect community health [1]. This toxic substance enters the domestic environment when the facilities discharge polluted air, which then contaminates the nearby soil and water. The main non-occupational exposure occurs by drinking the polluted water. At present, water contamination caused by Cr(VI) is a worldwide problem, making this an issue of great public health significance. An ecological mortality study conducted in Greece revealed that the standardized mortality ratio (SMR) for primary liver cancer was statistically significantly elevated due to the water contamination with Cr(VI) [2]. Study on Cr(VI)-induced hepatotoxicity is the hot topic in the field of toxicology. At present, a large number of studies [3,4] have confirmed that Cr(VI) can cause liver injury, and the mechanism involved have also been clarified.
Mitochondria are not only "stabilizers" for maintaining normal physiological functions of cells, but also "effectors" for external stimuli acting on cells [5]. It is known that mitochondria are involved in the regulation of important biological processes including reactive oxygen species (ROS) production, intracellular calcium level stabilization, cell apoptosis, and autophagy cascade signaling initiation [6]. Mitochondrial loss caused by various factor will have a serious impact on cells, resulting in energy metabolism disorders, the cellular environment imbalance, and even death [7]. The heavy metals with hepatotoxicity, such as cadmium, also exhibit mitochondrial toxicity. It has been confirmed that cadmium exposure caused mitochondrial loss in hepatocytes, which is manifested by the decrease of both mitochondrial DNA copy number and the expression of respiratory chain components, thus aggravating the energy metabolism disorder and causing cell death [8]. It is not clear whether Cr(VI) causes mitochondrial loss. Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200262/886457/bcj-2020-0262.pdf by guest on 01 July 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200262 Autophagy is a non-selective kinetic process of mass degradation of cytoplasmic macromolecules and organelles in lysosomes, which plays an important role in the degradation of many intracellular contents such as peroxisome, endoplasmic reticulum and mitochondria [9]. Autophagy is a dynamic biological process in which the cell encapsulates the target to be degraded into autophagosomes (APs) through the cup-shaped double membrane structure of unknown origin, and then transports it to the lysosomal cavity to form autolysosomes (ALs) and degrades it. Since ALs are composed of single membrane, it is difficult to distinguish them from APs with double membrane structure under electron microscope, so they are collectively called autophagic vacuoles (AVS) [10]. Correspondingly, the special process of clearing mitochondria from cells by autophagy is called "mitophagy" [11]. Mitophagy is involved in many pathological processes, such as infection, cancer, aging and neurodegenerative diseases, and is closely related to cell differentiation, survival and homeostasis [12]. Under physiological conditions, mitophagy plays an advantageous role in clearing damaged mitochondria and ensuring mitochondrial quality [13]; however, the abnormal and persistent activation of mitophagy can stimulate the degradation of mitochondria, leading to energy metabolism disorders and cell death [14].
In this study, we explored whether Cr(VI) causes mitochondrial loss and its related mechanisms, and we also proposed that Cr(VI)-induced hepatotoxicity may arise from mitochondria loss due to abnormal mitophagy, which will further reveal the possible mechanisms of Cr(VI)-induced hepatotoxicity, and provide a new experimental basis for the study of the health hazard effects of chromium.

Cell culture
Human L02 hepatocytes were obtained from the Experimental Central of Xiangya Hospital of Central South University and grown in RPMI 1640 medium (Gibco, Invitrogen Corporation, Carlsbad, CA, USA), which was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin solution. Cultured cells were incubated at 37°C in a humidified atmosphere of 5% CO 2 .

Quantitative real-time PCR (qPCR) analysis
The total RNA was isolated using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA) Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200262/886457/bcj-2020-0262.pdf by guest on 01 July 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200262 after chemicals treatment. The cDNAs were synthesized using the RT-PCR kit (Takara, Japan).

Mitochondrial DNA analyses
Total DNA was extracted from the treated cells using the DNA extraction kit (NEP002-1, Dingguo, China) according to the manufacturer's instructions. The relative mtDNA copy were performed in triplicate. The relative mtDNA copy number was calculated as described for the relative RNA quantification. chemiluminescence kit (Thermo, USA, 34095). Densitometric analysis of the bands was performed using Image J software (National Institutes of Health, USA).

ROS level analysis
The intracellular ROS accumulation was detected by Reactive Oxygen Species Assay Kit.

Determination of mitochondria mass
The L02 hepatocytes were seeded in a 96-well plate at a final density of 0.5 × 10 4 cells/well, and then were exposed to 5 μM 10-N-nonyl-acridine orange (NAO) (Invitrogen, A1372) for 30 min. The hepatocytes were washed twice with PBS. The fluorescence was then measured at the emission of 530 nm and the excitation of 485 nm using the Microplate Reader. The cellular fluorescence intensity was expressed as percentage of the control (% of control).

Detection of mitochondrial respiration
Mitochondrial respiration was determined using a Clark-type oxygen electrode (Rank Brothers Ltd, Cambridge, England). Briefly, the mitochondrial suspension (1 mg/ml) were added with the buffer (2 mM K 2 HPO 4 , 130 mM KCl, 3 mM HEPES, 2 mM MgCl 2 , 1 mM EGTA), followed by the addition of 5 mM glutamate-malate. State 4 respiration (ATP independent respiration) was monitored in the absence of ADP after the addition of 2 μg/ml oligomycin, and state 3 respiration (phosphorylating respiration) was measured after the addition of 2 μl ADP (27 mM) to examine maximal rate of coupled ATP synthesis. The respiratory control ratio (RCR) was calculated as the ratio between state 3/state 4 respiration.
100 μl ATP detection buffer was added to 100 μl samples, and then the luminescence of samples 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).

Caspase activity assay
The caspase 3/9 Activity Assay Kit (Beyotime Institute of Biotechnology, China, C1116, C1158) was used to detect of caspase-3/9 activities. The standard curve between caspase activities (0, 10, 20, 50, 100 and 200 μM) and absorbance was established. In brief, L02 hepatocytes were seed onto the 60 mm plates and given indicated treatments. Then, the cells were collected and incubated with Ac-DEVD-pNA (2mM) (for Caspase-3 activity) or Ac-LEHD-pNA(2mM) (for Caspase-9 activity) for 1 h at 37 °C in the dark. The absorbance was recorded at 405 nm. Caspase activities of the measured samples were extrapolated from the caspase activity standard curve.

Detection of cell apoptosis
The commercial Annexin V-FITC Apoptosis Detection Kit (Invitrogen Corporation, Carlsbad, CA, V13241) was used. The cells were treated with different chemicals and re-suspended in 100 μl of 1×binding buffer containing 5 μl Annexin-V-FITC and 1 μl propidium iodide (PI) for 30 min. Then 400 μl binding buffer was added to stop staining. The flow cytometric analysis was performed. Data analysis was performed using Flowjo 7.6 software.

Transmission electron microscope (TEM) analysis
For the TEM analysis, the L02 hepatocytes were treated with different concentrations of [16].

Statistics analysis
All results were presented as the mean ± SD from three independent experiments unless otherwise indicated. Difference of the data from different groups was performed using one-way analysis of variance. The Student t-test was used to evaluate the significant differences between the experimental values of the 2 samples being compared, and p < 0.05 was considered to be statistically significant.

Cr(VI) exposure induced mitochondrial loss
Evidence suggested that mitochondria are the most vulnerable target of major environmental pollutants such as heavy metals [17]. We first determined the activities of MRCCs to confirm whether Cr(VI) exposure (0, 8, 16 μM) could induce mitochondrial inhibition. As shown in Figure Figure   1E, the green fluorescence increased in a dose-dependent manner. The quantitative result of ROS level was shown in Figure 1F. Mitochondrial quality decline is one of the core indicators of mitochondrial loss. Figure 1G shows a significant reduction in mitochondrial mass after exposure to Cr(VI). After treatment with the highest dose of Cr(VI) for 24 h, the quality of mitochondria decreased by 40%.

Cr(VI) exposure induced energy metabolism disorders and cell death in L02 hepatocytes
We examined whether mitochondrial respiration was affected in Cr(VI)-treated hepatocytes.
The effect of Cr(VI) on the mitochondrial respiratory capacity was evaluated by determining State 3 (coupled to ADP phosphorylation) and State 4 (after ADP phosphorylation) respiration.
As shown in Figure 2A,

Cr(VI) triggered mitophagy in the hepatocytes
The autophagic vacuoles (AVs) including autophagosomes (APs) and autolysosomes (ALs) that observed by transmission electron microscopy (TEM) are the gold indicators of autophagy. Compared with the control group, we found that Cr(VI) treatment significantly increased the number of AVs in the hepatocytes in a dose-dependent manner ( Figure 3A). The average number of AVs was shown in Figure 3B. LC3 is an important cytoskeleton protein in cells, and the transformation of Lc3-I to LC3-II is a key event that indicates the occurrence of autophagy. As shown in Figure 3C & Figure 3D, Cr(VI) significantly increased the expression of LC3-II. Mitophagy is a specific autophagy process that represents an important way to control the quality of mitochondria. Mitochondria  mitochondria, so as to achieve the purpose of degradation of mitochondria. We measured the number of APs containing mitochondria by transfecting GFP-LC3 plasmid into L02 hepatocytes and co-locating it with TOMM20-labeled mitochondria. Using laser confocal three-dimensional reconstruction technique, we found that compared with the control group, the number of positive cells containing APs that engulfed mitochondria was increased when exposed to Cr(VI) ( Figure 4A). The percentage of cells positive for APs was shown in Figure   4B. We also measured the number of ALs containing mitochondria by co-locating LysoTraker-labeled mitochondria and MitoTraker-labeled lysosomes, and found that the number of positive cells containing ALs that engulfed mitochondria was also increased after Cr(VI) treatment ( Figure 4C). The percentage of cells positive for ALs was shown in Figure   4D.

Cr(VI) affected the autophagic flow of the hepatocytes by increasing APs formation
The increase of APs containing mitochondria may be due to the enhancement of autophagic activity or the impairment of the degradation pathway of APs. We then examined how Cr(VI) affected the autophagic flow of the hepatocytes. Firstly, we detected the change of the level of SQSTM1/p62 protein, which was selectively incorporated into APs through direct binding with LC3, and effectively degraded by autophagic pathway. As shown in Figure 5A & Figure   5B, we observed a dose-dependent decrease in the level of SQSTM1 protein, which

Discussion
Mitochondria play an essential role in various cellular pathways including ATP generation, calcium homeostasis, apoptosis regulation and nucleotide synthesis. Mitochondria are known Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200262/886457/bcj-2020-0262.pdf by guest on 01 July 2020 as semi-autonomous organelles since they have their own DNA (mtDNA), which encodes for 13 essential subunits of MRCCI, III, IV, and V [18]. Mitochondrial quality control, which plays an important role in the stability of mitochondrial function, is crucial for cell fate determination, and mitophagy is one of several processes to maintain mitochondrial quality.
Once mitochondrial quality control is disrupted, dysfunctional mitochondria with inhibited ATP production and enhanced ROS production accumulate, affecting cell fate. Hence, mitochondrial dysfunction definitely leads to cell dysfunction including cell death, which contributes to the pathogenesis of various diseases [19]. ROS, which are composed of a group of highly reactive molecules, participate in a variety of intracellular activities including cellular growth, proliferation, aging and apoptosis in regular conditions [20]. Mitochondria are known as the main intracellular sources of ROS. We have demonstrated in our previous research that MRCC I is the main site of ROS generation in mitochondria, and also the main reduction site of Cr(VI) after entering mitochondria [15]. The over accumulation of ROS may cause mitochondrial depolarization, triggering mitophagy, a mechanism that plays an important role in eliminating ROS by inhibiting oxidative stress [21]. ROS-associated mitophagy is recognized as a negative feedback response, which means mitophagy is considered to be protective by removing the over-accumulated ROS, however, some researchers believe that ROS-mediated overactive or insufficient mitophagy leads to cytotoxicity even cell death.
We found in the present study that Cr(VI) induced hepatotoxicity in a dose-dependent manner. Cr(VI) could also reduce mitochondrial mass, decrease the protein content of MRCCs and the copy number of mtDNA, and inhibit ATP production. These mitochondrial loss phenomena are attributed to Cr(VI)-induced excessive mitophagy rather than the inhibition of mitochondrial biogenesis. Interestingly, we found that Cr(VI) had no significant effect on mitochondrial biogenesis, which further confirmed the important and specific role of mitophagy in Cr(VI)-induced mitochondrial loss in hepatocytes. can inhibit the synthesis of MRCC I protein, cause the death of dopamine neurons in substantia nigra, and induce the symptoms of Parkinson's disease [24].
Mitophagy is the mechanism of mitochondrial degradation under various stress conditions, which has been observed in many diseases related to mitochondrial dysfunction. It has been reported that 6-OHDA can activate extracellular signal-regulated kinases 1/2 (ERK1/2) pathway in SH-SY5Y (human neuroblastoma cells), lead to excessive mitophagy, mitochondrial loss and even cell death [25], suggesting that mitophagy-mediated mitochondrial degradation can regulate the quantity and quality of mitochondria. As a quality control mechanism, mitophagy is responsible for eliminating damaged mitochondria [26], but the role of mitophagy in cell death is still controversial. Inhibition of autophagy makes hepatocytes more susceptible to apoptosis, probably because damaged mitochondria cannot be removed in time, resulting in energy supply disorders and cell death. However, the abnormal activation of mitophagy can remove or degrade excessive mitochondria, which makes cell physiological activity unsustainable and leads to fatal consequences. Whether caspase is activated or not, mitochondrial autophagic death has been recognized as another form of cell death in addition to apoptosis and necrosis [27]. Mitophagy is confirmed to control the quality and the number of mitochondria, while the excessive mitophagy promoted can further aggravate mitochondrial loss, thus forming a vicious cycle that will not terminate until cell death. This may be why inhibition of mitophagy is protective at the initial stage of Cr(VI) exposure, whereas at the later stage (48 h) the accumulation of damaged mitochondria accelerated cell death.
In summary, we confirmed in the present study that Cr(VI) exposure induced mitochondrial loss, energy metabolism disorders and cell death in L02 hepatocytes. In addition, Cr(VI) affected the autophagic flow of the hepatocytes by increasing APs formation, and the excessive mitophagy promoted Cr(VI)-induced mitochondrial loss and cytotoxicity.
Based on the effect of Cr(VI) on mitophagy, choosing specific inhibitors of mitophagy can protect against Cr(VI)-induced hepatotoxicity and provide new clues to mitigate the health hazards caused by chromium.

Author Contributions
Yujing Zhang: methodology and writing-Original draft preparation; Huanfeng Bian: investigation and software; Yu Ma: data curation; Yuanyuan Xiao: writing-reviewing and editing; Fang Xiao: conceptualization and supervision.