PTU, carbenoxolone (CBX), anti-GAPDH and secondary antibodies for western blotting were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Anti-Cx32 antibody was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Cell culture reagents, Lipofectamine 2000 and calcein acetoxymethyl ester (Calcein-AM) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The Cell Counting kit-8 (CCK-8) was obtained from Dojindo (Mashikimachi, Kumamoto, Japan). The 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was from Beyotime Institute of Biotechnology (Haimen, China). All other reagents and chemicals were obtained from Sigma-Aldrich; Merck KGaA, unless otherwise stated.

The BRL-3A rat liver cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultivated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin at 37°C in an atmosphere containing 5% CO₂.

Direct toxicity was determined using a CCK-8 kit according to the manufacturer's instructions. First, BRL-3A cells were subjected to 0.6 and 0.8 mg/ml PTU for 24 h at 37°C, after which they were incubated with 10% (v/v) CCK-8 reagent at 37°C for 3 h. The absorbance was read using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at a wavelength of 450 nm. The cell viability was normalized against that of the vehicle control.

A standard colony-formation assay was used for detecting the cytotoxicity of PTU to BRL-3A cells (22). Briefly, following exposure to PTU at 0.6 and 0.8 mg/ml for 12 h, cells were rinsed with phosphate-buffered saline (PBS), harvested with trypsin, diluted and seeded into 6-well plates at a density of 500 cells/well. Cells were subsequently stained with 4% crystal violet at room temperature 5–7 days later. Colonies consisting of ≥50 cells were counted. The surviving fraction was evaluated by normalizing to the colony-forming efficiency of the vehicle-treated cells.

Cx32 expression was inhibited by siRNA transfection in BRL-3A cells. The siRNA sequences targeted against the rat Cx32 gene were as described previously (23,24): siRNA-1, 5′-CACCAACAACACATAGAAA-3′; siRNA-2, 5′-GCATCTGCATTATCCTCAA-3′; and siRNA-3, 5′-GCCTCTCACCTGAATACAA-3′. Cx32 siRNAs (50 nM) or the negative control siRNA (NC siRNA) were transiently transfected into BRL-3A cells using Lipofectamine 2000, according to the manufacturer's instructions. After 48 h incubation, western blotting and a ‘parachute’ assay, as described below, were performed to confirm Cx32 expression knockdown and GJIC inhibition.

The procedure of western blotting was performed as described previously (25). In brief, cell lysates were obtained in lysis buffer (P0013; Beyotime Institute of Biotechnology), sonicated and centrifuged at 14,167 × g at 4°C for 30 min. Protein concentration was determined by BCA assay. Cell lysates (20 µg per lane) were separated using SDS-PAGE in 10% Tris-glycine gels and transferred to nitrocellulose membranes. Following blocking with 5% skimmed dry milk in Tris buffered saline with Tween-20 (0.05% Tween-20) at room temperature for 1 h, the membranes were incubated with specific antibodies against Cx32 (sc-59948; dilution, 1:1,000) and GAPDH (G8795; dilution, 1:2,000) overnight at 4°C. Secondary antibodies (goat anti-mouse IgG-peroxidase conjugated; A4416; dilution, 1:4,000) were then added at room temperature for 1 h. The immunopositive protein bands were visualized using an Amersham Enhanced Chemiluminescence Detection kit (GE Healthcare Life Sciences, Little Chalfont, UK) and the intensities were detected by the Quantity One software (Bio-Rad, version 4.6.2).

A ‘parachute’ dye-coupling assay was performed to evaluate GJ function as previously described (26). Cells were seeded into 12-well plates and cultured to 80–90% confluency. Donor cells from one well were labeled with 5 µM Calcein-AM and trypsinized, diluted and seeded onto receiver cells at a ratio of 1:150 (donor/receiver). GJs formed between donor cells and receiver cells during a 4-h incubation at 37°C and were monitored using an Olympus IX71 fluorescence microscope (Olympus Corporation, Tokyo, Japan). Under each experimental condition (treatment of CBX or (si)RNA transfection), the average number of receiver cells containing calcein dye per donor cell was counted and normalized to that of the vehicle control.

Cell morphology was observed using a FEI Quanta-400 scanning electron microscope (SEM; Thermo Fisher Scientific, Inc.). Sterile slides were placed in 12-well plates and served as substrates. Cells were treated with vehicle control or PTU at 0.6 and 0.8 mg/ml for 24 h, rinsed with PBS and fixed in 4% paraformaldehyde for 1 h at room temperature. Following gradient dehydration in a series of ethanol from 30–100%, the samples were dried in a vacuum freeze-drying apparatus for 1 h and were treated with gold sputtering for SEM observation.

Reversed-phase high-performance liquid chromatography was adopted to measure the PTU content in BRL-3A cells (27). Briefly, following incubation with PTU at 0.6 and 0.8 mg/ml for 24 h, cells were washed with PBS three times, harvested by trypsinization, resuspended in PBS and counted. Five freeze-thaw cycles were used to lyse the cells. Methanol (HPLC-grade) was added for protein precipitation. Following centrifugation at 14,167 × g for 10 min at 4°C, the supernatant was collected and injected into a Shimadzu LC-20AD system (Shimadzu Corporation, Kyoto, Japan). The chromatographic conditions used were as follows: Application of a Luna C₁₈ column (250×4.6 mm; 5mm; Phenomenex, Torrance, CA, USA), methanol and water (40:60) was used as the mobile phase at a flow rate of 1.0 ml/min, which was detected at a wavelength of 272 nm with a column temperature of 30°C. The concentrations of PTU in the cell samples were calculated using a calibration curve method.

Following exposure to PTU at 0.6 and 0.8 mg/ml for 6 h, BRL-3A cells were labeled with DCFH-DA, which is hydrolyzed by esterase into DCFH without fluorescence. Intracellular ROS can oxidize non-fluorescent DCFH to fluorescent DCF (28). Formation of DCF was determined using a Perkin LS55 fluorescence spectrophotometer (PerkinElmer Inc., Waltham, MA, USA) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Fluorescence intensity was normalized to that of the vehicle control and was regarded as a measure of the intracellular ROS level. The DCF images were captured via fluorescence microscopy (Olympus IX71; Olympus Corporation).

The data were presented as the mean ± standard error and were analyzed using IBM SPSS Statistics 19.0 (Armonk, NY, USA). Statistical analysis was performed by Student's t-test or one-way analysis of variance followed by Dunnett's test. In all cases, P<0.05 was considered to indicate a statistically significant difference.

BRL-3A cells were cultured separately under two cell-density conditions for initial investigation of the influence of GJIC on PTU toxicity. At a high cell density (2×10⁴ cells per cm²), GJs formed efficiently as the cells had frequent contact with one another. At a low cell density (5×10³ cells per cm²), the wide dispersion of cells infrequently permitted the formation of GJs. A preliminary cell viability assay for concentrations screening was performed with PTU from 0.3 to 0.8 mg/ml (data not shown). The concentrations of 0.6 and 0.8 mg/ml PTU were chosen in the present study. PTU-induced cytotoxicity was assessed using a CCK-8 assay and a standard colony-formation assay. Fig. 1 illustrates that PTU treatment, at concentrations of 0.6 and 0.8 mg/ml, decreased the cell survival of BRL-3A cells under both culture conditions. However, the survival was significantly greater in the low-density condition compared with the high-density condition. Thus, the toxicity of PTU in BRL-3A cells was reduced under low cell-density conditions wherein there was a deficiency in GJIC.

To investigate whether the cell density-dependence of PTU toxicity in BRL-3A cells is associated with GJIC, an extensive GJ inhibitor, CBX (29), was adopted to manipulate GJ function. BRL-3A cells were pretreated with the CBX prior to exposure to PTU. In a subsequent ‘parachute’ assay, dye-coupling was significantly blocked by CBX pretreatment (Fig. 2A). Under high cell-density culture conditions, the cell survival was significantly increased following pretreatment with 100 µM CBX for 1 h at PTU concentrations of 0.6 and 0.8 mg/ml, as compared with PTU alone (Fig. 2B and C). These results indicate that the inhibition of GJs contributes to the decreased toxicity of PTU at high BRL-3A cell densities.

Cx32 is a key GJ constituent protein in liver cells (12). Specific knockdown of the Cx32 gene was conducted to confirm the effect of Cx32-GJ on PTU cytotoxicity. As indicated in Fig. 3A and B, the downregulation of Cx32 expression significantly suppressed the spread of calcein dye through GJs in siRNA-transfected cells. Furthermore, Cx32-knockdown (siRNA-1 transfection) increased the cell viability by factors of 1.31 and 1.29 in the presence of 0.6 and 0.8 mg/ml PTU, respectively, at high cell densities (Fig. 3C). Likewise, clonogenic capacity was improved at these two concentrations when Cx32-GJs were inhibited (Fig. 3D). The results indicate that Cx32-GJ serves an important role in the PTU-induced cytotoxicity of BRL-3A cells.

PTU has been demonstrated to induce necrosis in liver injury (8). To assess the necrosis of BRL-3A cells subjected to PTU in the absence of Cx32-GJ, SEM images were captured. Fig. 4 illustrates the typical morphological characteristics of cell necrosis, including cell swelling and spillovers of cellular content (as indicated by the arrows), which were clearly noted following PTU treatment, but were less obvious following siRNA-mediated Cx32-GJ inhibition. These results suggest that the suppression Cx32-GJ attenuates PTU hepatotoxicity, which is partly associated with a decrease in PTU-induced necrosis.

GJs facilitate direct intercellular communication between neighboring cells. PTU, as a small molecule, could be transmitted through GJs. To assess whether the PTU concentration in BRL-3A cells could be reduced by blocking Cx32-GJ function, the intracellular PTU content was determined. As indicated in Fig. 5, the level of cellular PTU [PTU (µg) per 10⁴ cells] significantly decreased when cells were transfected with Cx32 siRNA-1. The reduction of PTU uptake in BRL-3A cells between those with and without Cx32-GJ were significant at 0.6 and 0.8 mg/ml PTU. These results demonstrate that PTU accumulation is reduced when Cx32-GJ is downregulated.

To investigate the possibility of ROS transmission through GJs, intracellular ROS levels in the presence and absence of Cx32-GJ were detected. The results revealed that PTU-induced toxicity was accompanied by an increase in ROS production at concentrations ≤0.8 mg/ml. Fig. 6 illustrates that Cx32 siRNA-1 transfection markedly suppressed the PTU-induced increase in DCF fluorescence, which was quantified by fluorescence spectrophotometry. The levels of intracellular ROS were significantly reduced in the Cx32-knockdown group at PTU concentrations of 0.6 and 0.8 mg/ml. These findings suggest that the attenuation of PTU-induced toxicity by Cx32-GJ downregulation may be associated with the inhibition of ROS transmission.