The L-02 hepatocyte line was provided by the China Center for Type Culture Collection of Wuhan University. The MRCC substrates (glutamate/malate, succinate, coenzyme Q, vitamin C), rotenone (ROT) and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 culture medium, fetal bovine serum (FBS), and trypsin-EDTA (0.25%) were obtained from Gibco (Gaithersburg, MD, USA). Potassium dichromate (K₂Cr₂O₇) was obtained from Changsha Chemical Reagents Co. (Changsha, China).

L-02 hepatocytes were cultured in RPMI-1640 medium supplemented with 10% (vol/vol) FBS, 2 mM L-glutamine, and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin) at 37°C under a humidified atmosphere of 5% CO₂. The medium was changed every other day.

MTT assay was performed to evaluate cell viability as previously described (18). The cells in exponential growth were seeded in 96-well plates with 100 μl of medium containing 10⁴ cells. K2Cr₂O₇ solution of indicated final concentrations (0, 2, 8, 32, 128 and 512 μM) was added. Control cells and medium controls without cells received DMSO without K₂Cr₂O₇. After incubation at 37°C in a 5% CO2 saturated atmosphere for an indicated time period, the cells were treated with 5 μl 5 mg/ml of MTT solution for an additional 4 h at 37°C, and then lysed in phosphate-buffered saline (PBS, pH 7.4) containing 20% sodium dodecyl sulfate (SDS) and 50% N, N-dimethylformamide (pH 4.5). The absorbance was read on a multiwell ELISA reader Versamax (Molecular Devices, Sunnyvale, CA, USA) at 570 nm for each well.

Intracellular ROS production was determined by detecting the fluorescent intensity of 2′,7′-dichlorofluorescein (DCF), the oxidized product of the fluoroprobe 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Molecular Probes, USA). Briefly, 2×10⁶ cells were collected by centrifugation and were then incubated with 10 μM CM-H2DCFDA in PBS for 40 min at 37°C in the dark. After incubation, the cells were split into two parts. One part was checked with a fluorescence microscope equipped with a Leica DC 100 digital camera. The other part was measured with a flow cytometer with excitation at 488 nm and emission at 535 nm. The amount of ROS production was considered directly proportional to the fluorescence intensity.

L-02 hepatocytes were lysed using a Mammalian Cell Lysis kit from Sigma-Aldrich. Western blotting was performed with the WesternBreeze Chemiluminescent Immunodetection protocol (Invitrogen, Carlsbad, CA, USA). Proteins were separated by electrophoresis on 10% sodium dodecyl sulfatepolyacrylamide gels (SDS-PAGE), and were then transferred to polyvinylidene fluoride (PVDF) membranes by electroelution. The membranes were incubated with a primary antibody overnight at 4°C following blocking with 4% non-fat milk. Membranes were then incubated for 1 h at room temperature with secondary antibodies, developed with a detection system and then exposed onto films.

The primary antibodies for MRCC including Complex I subunit NDUFS3 (MS110), Complex II subunit 70 kDa Fp (MS204), Complex III subunit core 2 (MS304), Complex IV subunit II (MS405), and ATP synthase subunit α (MS502) were purchased from MitoScience (Eugene, OR, USA). Antibodies for SOD 1 (#2770), catalase (#8841), Trx (C63C6) (#2429), caspase-3 (#9662), heat shock protein (HSP)70 (#4872), HSP90 (E289) (#4877), and β-actin (#4967) were purchased from Cell Signaling Technology (Danvers, MA, USA).

The mitochondria were isolated as previously described with slight modifications (19). Cells were washed twice with cold PBS, and resuspended with 5 ml buffer A (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, pH 7.5). Cells were homogenized and centrifuged twice at 750 x g for 10 min. Mitochondria pellets were obtained after centrifugation at 10,000 x g for 15 min.

The activities of MRCC were determined using Mitochondrial Respiratory Chain Complexes Activity Assay kits (Genmed Scientifics, Inc., Shanghai, China). All assays were performed in a final volume of 1 ml using a UV-9100 spectrophotometer. To establish the optimum conditions for the release of complexes, the mitochondria were freeze-thawed three times at 20/−20°C in hypotonic media (25 mM potassium phosphate, 5 mM MgCl₂, pH 7.2) before the determination. The activity of MRCC I [nicotinamide adenine dinucleotide (NADH) coenzyme Q (CoQ) oxidoreductase, expressed as nmol oxidized NADH/min/mg protein] was measured following the oxidation of NADH at 340 nm. The activity of MRCC II (succinate: 2,6-dichlorophenolindophenol (DCIP) oxireductase, expressed as nmol reduced DCIP/min/mg protein) was measured following the reduction of DCIP at 600 nm. The activity of MRCC III (ubiquinol: cytochrome c (Cyt c) reductase, expressed as nmol reduced Cyt c/min/mg protein) was measured following the reduction of Cyt c at 550 nm. The activity of MRCC IV (Cyt c oxidase, expressed as nmol oxidized Cyt c/min/mg protein) was measured following the oxidation of Cyt c at 550 nm. All measurements were performed in triplicate.

Cr(VI) reduction was determined colorimetrically with a spectrophotometer using the S-diphenylcarbazide (DPC) (Nacalai Tesque, Inc., Japan) method (20). The mitochondria isolated from hepatocytes were pretreated with the MRCC I substrates 10 mM glutamate/10 mM malate (Glu/Mal), MRCC II substrate 10 mM succinate (Suc), MRCC III substrate 5 μM CoQ, or MRCC IV substrate 2 mM vitamin C (Vit C) for 10 min prior to 32 μM Cr(VI) treatment. Cr(VI) reduction rate was measured at different time points (5, 30 and 60 min). After 3 freeze and thaw cycles, the mitochondria treatment suspensions (2 ml) were centrifuged for 5 min at 15,000 x g. The supernatant was added with 20 μl H₂SO₄ and 20 μl H₃PO₄, mixed and then added with 80 μl DPC (0.076 x g DPC previously dissolved in 20 ml of 95% ethanol). Twenty minutes later, the absorbance of the color produced was measured at 540 nm. Cr(VI) concentration in the sample was calculated from a standard curve using K₂Cr₂O₇ as a standard.

Caspase-3 activity was detected using a Caspase-3 colorimetric assay kit (Millipore, Billerica, MA, USA). Briefly, L-02 hepatocytes were harvested and washed twice with ice-cold PBS. Then cell pellets were incubated with lysis buffer (50 mM Tris-HCl, 1 mM EDTA, and 10 mM ethyleneglycoltetraacetic acid, pH 7.4) for 30 min on ice. After centrifugation at 13,000 x g at 4°C for 5 min, the supernatants were collected and added with caspase-3 substrate Ac-DEVD-pNA to the final concentration of 100 μM. The samples were incubated at 37°C for 1 h and the alternative activity of caspase-3 was described as the cleavage of the colorimetric substrate by measuring the absorbance at 405 nm.

L-02 hepatocytes were harvested by trypsinization and washed with PBS. Washed cells were treated with FITC-conjugated Annexin V (0.5 μg/ml final concentration) and propidium iodide (PI, 1 μg/ml final concentration). After incubation for 20 min at room temperature, the apoptosis was determined by flow cytometry and analyzed by the CellQuest software. For each measurement, 20,000 cells were analyzed. PI was added to a sample to distinguish early apoptotic cells (Annexin V-positive, PI-negative) and late apoptotic cells (positive for both Annexin V and PI).

Statistical analysis was performed using SPSS 15.0 one-way analysis of variance (ANOVA) to assess the significance of differences between groups. The acceptance level of significance was P<0.05. Results are expressed as mean ± SD.

L-02 hepatocytes exposed to varying doses of Cr(VI) (2–512 μM) over a 24 h period and a concentration-dependent loss of cell viability was observed in Fig. 1. The Cr(VI) concentration that required for 50% inhibition of cell viability (IC₅₀) was 38.97 μM. Therefore, we chose two concentrations of Cr(VI) (16, 32 μM) for the following experiments.

ROS play a critical role in mediating the cytotoxicity induced by Cr(VI), but the targets by which Cr(VI) induces ROS accumulation are unknown. To identify the targets for Cr(VI)-induced ROS accumulation, we first measured ROS levels. L-02 hepatocytes exposed to 16 or 32 μM Cr(VI) for 24 h were analyzed for ROS production utilizing the oxidant-sensitive fluorogenic probe CM-H₂DCFDA. Cr(VI) stimulation induced higher levels of fluorescence signal in a dose-dependent manner, indicating the generation of a large amount of intracellular ROS in the Cr(VI) treatment groups (Fig. 2A). Quantitative analysis by flow cytometry showed that the ROS levels were about 3-fold higher in the 16 μM Cr(VI) treatment group, and were about 5-fold higher in the 32 μM Cr(VI) treatment group compared with the control group (Fig. 2B). It has been reported that downregulation of antioxidative proteins results in ROS accumulation (15). Therefore, we investigated the effect of Cr(VI) on the expression of antioxidative proteins such as SOD, catalase and Trx. It was revealed that Cr(VI) had no significant effect on the three proteins (Fig. 2C). Therefore, we reached the conclusion that Cr(VI) does not cause ROS overproduction by downregulating the antioxidative proteins.

Inhibiting the activity of MRCC promotes ROS accumulation (13). Thus we speculated that Cr(VI) may induce ROS accumulation by inhibiting MRCC activity. We measured the effect of Cr(VI) (16, 32 μM) on different MRCC activities. Cr(VI) significantly inhibited MRCC I and slightly inhibited MRCC II at a dose of 32 μM, but had no effect on MRCC III and IV (Fig. 3A), indicating that MRCC I as well as MRCC II may be targets of Cr(VI) in mediating ROS accumulation. This was further explored by measuring the activities of MRCC I and II at different time points after 32 μM Cr(VI) exposure. MRCC I activity was significantly inhibited as early as 30 min after Cr(VI) treatment (Fig. 3B). In contrast, the activity of MRCC II was not altered until 120 min after Cr(VI) exposure, indicating that MRCC I is the target of Cr(VI) in promoting ROS production, and the decrease in MRCC II activity may be viewed as the result of inhibited MRCC I activity. The data were further confirmed by western blotting. Cr(VI) significantly decreased the expression of MRCC I (subunit NDUF S3), but had no effect on the expression levels of the left three complexes (Fig. 3C).

Based on the previous results, we inferred that Cr(VI) accepted the electrons and was reduced at the MRCC I. Mitochondria can reduce Cr(VI) using MRCC substrates as electron donors (21). The mitochondria isolated from hepatocytes were pretreated with the MRCC I substrates glutamate/malate (Glu/Mal), the MRCC II substrate succinate (Suc), the MRCC III substrate CoQ, or the MRCC IV substrate Vit C prior to Cr(VI) treatment. We then measured the Cr(VI) reduction rate in mitochondria at different time points. Cr(VI) reduction rate significantly increased in the mitochondria treated with Glu/Mal, indicating that Cr(VI) reduction occurs at the MRCC I (Fig. 3D).

Our results suggested that Cr(VI) acts as an MRCC I inhibitor. Thus we used the specific MRCC I inhibitor rotenone (ROT) as a positive control to confirm the functional role of Cr(VI). ROT at 5 μM for 24 h resulted in the accumulation of 3.6-fold higher ROS compared with control (Fig. 2A and B). Caspase-3 activity was determined after different doses of Cr(VI) exposure in L-02 hepatocytes. Similar to ROT, Cr(VI) significantly increased caspase-3 activity in a dose-dependent manner (Fig. 4A), again confirming that Cr(VI) targets MRCC I to induce the activation of caspase-3. Western blotting results also revealed that Cr(VI) exposure induced the increased expression of cleaved caspase-3 (17/19 kDa) and decreased expressions of full length caspase-3 (35 kDa), thus leading to the activation of caspase-3 (Fig. 4B). Occurrence of apoptosis was assayed by flow cytometry. The results of Annexin V-FITC and PI staining showed that Cr(VI) induced apoptosis in a dose-dependent manner (Fig. 4C). The percentages of both early apoptotic cells (LR, as reflected in the lower-right-hand quadrant, Annexin V positive) and late apoptotic cells (UR, depicted in the upper-right-hand quadrant, positive for both Annexin V and PI) were significantly increased in the Cr(VI) treatment groups compared with control (LR, 5%; UR, 6%).

HSP has been identified as caspase-3 negative regulator and mediates the prevention of apoptosis (22,23). Thus, we determined whether Cr(VI) also mediated the inhibition of HSP to induce caspase-3 activation. The levels of HSP70, HSP90 were decreased following Cr(VI) treatment in a dose-dependent manner (Fig. 4D).

In order to confirm that Cr(VI)-induces caspase-3 activation is dependent on ROS function, we used NAC to inhibit ROS. The hepatocytes were exposed to Cr(VI) (16, 32 μM) in the presence of 10 mM NAC for 24 h. The production of ROS was blocked in each group, which confirmed the specificity of NAC (Fig. 5A). Western blot analysis revealed that NAC blocked the activation of caspase-3 and the inhibition of HSP70 and HSP90 (Fig. 5B), suggesting that Cr(VI) activated caspase-3 by inducing ROS-dependent decrease of HSP70 and HSP90. ROS is essential in Cr(VI)-induced caspase-3 activation.