**1. Introduction**

Chromium (Cr) widely exists in the ecological environment and can be found in pigments, chrome-plated metals, cement, detergents, and industrial Cr waste dumps [1]. Cr has a variety of oxidation states (−2 to +6), but only trivalent chromium (Cr(III)) and hexavalent chromium (Cr(VI)) are stable. The increase in industrial use, coupled with improper disposal of Cr(VI)-related waste, has led to the serious increase of Cr(VI) levels in air, water, and soil, resulting in the pollution of the environment, even the food chain [2].

Mitochondria constantly undergo a dynamic fusion/fission process, which is mainly controlled by regulatory proteins such as mitofusins (Mfns) and dynamin-related protein 1 (Drp1) [3]. This kind of dynamic balance is essential to maintain constant changes in mitochondrial shape, size, and network. Mitochondrial fission process is mainly mediated by Drp1, which exists in the cytosol and translocates to the outer membrane of mitochondrial during fission [4]. Increasing evidence suggested that the

dynamic morphology of the mitochondrial network is very important. In the physiological state, the long, continuous tracks of fused mitochondria and branching networks are dominant and mainly regulated by Mitofusins (Mfns). However, upon exposure to various stresses, mitochondria undergo fission, networks become unraveled, and the fragmented morphology is more prominent (regulated Drp1). Preliminary studies have shown that Drp1 ablation declined Cyt c release and inhibited apoptosis [5]. In contrast, other studies revealed that blockage of Drp1 partially decreased [6] or had little e ffect [7] on Cyt c release, without a ffecting apoptosis.

The electron transport chain (ETC), which exists in the folded inner membranes of mitochondria, is mainly composed of four mitochondrial respiratory chain complexes (MRCC; I–IV) and two free-moving electron transfer carriers cytochrome c (cyt c) and ubiquinone. The four complexes are assembled into a specifically configured super-complex, which together with MRCC V (F1F0ATP synthase), becomes the basis of ATP generation during oxidative phosphorylation [8]. As the largest multi-subunit enzyme complex located in ETC, MRCC I is also called NADH-ubiquinone oxidoreductase, and its key role is to transfer electrons from NADH to ubiquinone [9]. The ETC is the key component of mitochondria and also known as the most important source of intracellular reactive oxygen species (ROS). ROS include oxygen-free radicals such as hydroxyl radical (OH) and superoxide anion radical (O2 −), and non-radical oxidants such as hydrogen peroxide (H2O2). Due to the existence of electron leakage, not all the electrons could be successfully transferred to the final electron acceptor, O2. Under normal conditions, 0.2–2% of the electrons do not follow the transmission order but directly leak out from ETC, and then interact with oxygen to produce ROS [10]. As a double-edged sword, ROS plays an important role in intracellular signaling pathways, but ROS accumulation can lead to cytotoxicity and even cell death.

Although it is believed that mitochondrial fragmentation is a common phenomenon in apoptosis, whether excessive fission is crucial for apoptosis progression remains controversial. Our previous studies [11,12] have demonstrated that Cr(VI) mainly targeted MRCC I and increased ROS generation to induce cytotoxicity. In addition, Cr(VI) could also cause both mitochondrial damage and apoptotic cell death during ROS-triggered cytotoxicity, but the related mechanism involved in MRCC I-dependent cytotoxicity was unclear. The present study will demonstrate the role of increased mitochondrial fragmentation mediated by Drp1 in Cr(VI)-induced MRCC I-dependent cytotoxicity, which will provide experimental evidence for further elucidating the cytotoxicity of Cr(VI).

#### **2. Materials and Methods**

#### *2.1. Cell Culture and Cell Counts*

Human L02 hepatocytes, obtained from Experimental Central of Xiangya Hospital of Central South University, Changsha, China, were cultured in 25 cm<sup>2</sup> culture flasks in the standard humidified incubator with the set of 5% CO2 and 37 ◦C. Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin (P/S) solution was used.

Under the optimal conditions, the doubling time of L02 hepatocytes was 18–24 h, and the cells were subcultured every 2.5–3 days (d). Cell numbers were measured and recorded everyday using a hemocytometer by trypan blue exclusion method.
