**4. Discussion**

We demonstrated in the present study that MRCC I inhibition led to cytotoxicity in L02 hepatocytes, which was characterized by the increase of ALT/AST leakage, the activation of caspase-3, the inhibition of cell proliferation, and the induction of apoptosis. We then confirmed that Cr(VI) targeted MRCC I to induce ROS accumulation and then triggered mitochondrial-related cytotoxicity. Cr(VI) caused a marked increase in mPTP opening rate and decrease in MMP, declined the mtDNA copy number, mitochondrial mass, and ATP level, increased the mitochondrial release of apoptosis-inducing factors, and eventually triggered apoptosis. The pretreatment of Glu/Mal significantly alleviated Cr(VI)-induced cytotoxicity, while the cotreatment of ROT obviously aggravated Cr(VI)-induced cytotoxicity.

In theory, all four complexes (I–IV) of the ETC could generate ROS; evidence suggests that MRCC I and III are the main sites of ROS formation [13,14]. However, the ability of MRCC III to produce ROS has also been questioned, while MRCC I is considered to be the relevant major site of superoxide formation. It is reported that the formation of ROS can not be detected until MRCC III was inhibited by up to 71 ± 4%; in contrast, the small-extent deactivation of MRCC I (16 ± 2%) could lead to a significant increase of ROS generation [15]. It is also demonstrated that the production rate of endogenous H2O2 was much lower when Glu/Mal, the substrates of MRCC I were used as respiratory fuel; the application of ROT, the inhibitor of MRCC I, led to a significantly increasing H2O2 release [16]. ROT can interrupt the transfer of electrons to CoQ and enhance ROS production. The amount of ROS produced by various stimulation determines whether ROS play profitable or detrimental roles. In the physiological state, it was believed that ROS appear to be important second messengers that mediate di fferent cell signaling pathways, while the overburdened ROS are exclusively harmful to the cells [17]. The burst generation of ROS leads to irreversible damage to mitochondria, DNA damage, lipid peroxidation, ATP exhaustion, and, eventually, cell death [18]. Although the precise mechanisms involved in ROS burst-generation and ROS-related cytotoxicity are still not clear, the application of specific ROS inhibitors such as NAC and Trolox to reduce the over-production of ROS under pathological conditions has been shown to ameliorate various diseases mediated by oxidative stress [19].

The mtDNA copy number in each mitochondrion is constant, thus, the total number of mtDNA copies reflects the total number of mitochondria in cells [20]. mtDNA is located near the ETC and its repair mechanism is incomplete, thus, mtDNA is more susceptible to damage when exposed to oxidative stress compared with the nuclear DNA [21]. The opening of mPTP represents the abrupt change in permeability of the mitochondrial inner membrane, allowing not only protons but also various ions and solutes of up to 1.5 kDa in size to freely pass through the membrane [22]. Apoptosis is initiated by the change of mitochondrial membrane permeability transition (MPT), and the opening of the non-specific mega-channel mPTP enables the membrane permeability suddenly increase, resulting in the release of the apoptosis executors including apoptosis-inducing factor (AIF), cytochrome c (Cyt c), and endonuclease G from the mitochondrial matrix to cytoplasm. The increase of mPTP opening may lead to the flow back of protons from mitochondrial membrane space to matrix, thus inhibiting MMP and ATP production, resulting in metabolic abnormalities and cytotoxicity [23]. Moreover, Cr(VI) exposure triggered over-opening of mPTP, dropped MMP, decreased mtDNA number, declined ATP synthesis, indicating that Cr(VI) can directly disrupt the structure and function of mitochondria and induce cytotoxicity. As one of the early events of apoptosis induced by various stimuli, the increase of intracellular ROS can trigger Cyt c first detaches from cardiolipin and then being released into the cytoplasm. Cr(VI) exposure disrupted mitochondrial redox homeostasis and accumulated excessive ROS, then mitochondrial function such as MMP and mPTP was severely a ffected, thus, L02 hepatocytes undergo mitochondria-mediated apoptosis.

Cr(VI) induced excessive mitochondrial fragmentation and mitochondrial Drp1 translocation, the application of Drp1-siRNA alleviated Cr(VI)-induced ALT/AST leakage, caspase-3 activation, and apoptosis induction. The cytotoxicity in Drp1-si plus Cr(VI) treatment group was alleviated by the application of Glu/Mal, and aggravated by the application of ROT. Drp1 siRNA can further promote the inhibition of Glu/Mal on Cr(VI)-induced cytotoxicity, and also can further alleviate the

aggravation of ROT on Cr(VI)-induced cytotoxicity. The dynamic network of mitochondria can meet the energy and metabolic needs of cells. The intracellular mitochondrial morphology network represents a perfect balance between the fusion/fission events [24]. Mitochondrial dynamics is mainly regulated by Drp1, mitofusin (Mfn) 1 and Mfn2, and other mitochondrial fusion and fission-related proteins [25]. Once the damage progressed to the irreversible stage, mitochondria will reveal excessive fission and fragmentation, mass decrease, as well as membrane integrity loss. The application of Drp1 siRNA or Mdivi-1 (a specific Drp1 inhibitor) can inhibit the conversion to punctate mitochondrial phenotype, weaken the insertion and oligomerization of pro-apoptotic Bax protein, thus attenuating cell apoptosis, reducing transient focal ischemia-induced infarct and neurological deficits. Drp1 activity could be altered under all kinds of stimulation, leading to mitochondrial dynamics abnormality and cell damage [26]. It is reported that oxidative stress can enhance the activity of Drp1 and promote mitochondrial fragmentation and dysfunction mediated by Drp1, while the application of antioxidants can restore mitochondrial morphology [27,28]. Evidence suggested that the upstream transcription activator of Drp1 is p53, which could promote Drp1 transcription by binding to its promoter [29]. In the present study, we confirmed that Cr(VI)-induced Drp1 modulation was dependent on MRCC I inhibition-mediated ROS production. Taken together, this study demonstrated that Drp1-mediated mitochondrial fragmentation contributes to Cr(VI)-induced MRCC I-dependent cytotoxicity.
