Male Sprague-Dawley (SD) 8-week-old rats were obtained from the animal center of Central South University (Changsha, Hunan, China). The rats were clinically normal, without infection or inflammation, and were housed in clean cages with free access to water and food. The animals were maintained in a 12-h light/dark cycle at a temperature of 22±2°C and a humidity of 55±5%. The study was conducted with the approval of the Animal Care and Use Committee of Central South University.

Vit C, 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT), Hoechst 33258, Coomassie Brilliant Blue (G-250) and rhodamine 123 (Rh123) were purchased from Sigma (St. Louis, MO, USA). RPMI-1640 culture medium, fetal bovine serum (FBS) and trypsin-EDTA (0.25%) were obtained from Gibco-BRL (Gaithersburg, MD, USA). Potassium dichromate (K₂Cr₂O₇) was obtained from Changsha Chemical Reagents Company (Changsha, Hunan, China) and was used as the standard reagent. All other chemicals and solvents were of analytical grade, HPLC grade or the optimum pharmaceutical grade.

The preparation of rat PBLs was performed as previously described with slight modifications (22,23). A peripheral blood sample (20 ml) was drawn from SD rats and collected in a heparinized tube. As the lymphocytes mainly comprised of mononuclear cell fractions, we immediately (within 2 h) isolated the PBLs by density gradient centrifugation (1,200 × g, 20 min) using Gradiaol L (Aqua-Medic, Kobylnica, Poland). The lymphocytes were then collected, and washed twice with 0.9% NaCl solution and twice with transport solution (20 mM HEPES, 150 mM NaCl, 5 mM KCl, 5 mM MgSO₄, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 2 mM pyruvate, pH 7.4). An adequate volume of transport solution was added to the isolated PBLs to obtain the density of 10⁶ cells/ml.

An MTT assay was performed to evaluate the cell viability as previously described; however, with slight modifications (24). The PBLs were seeded in each well of a 96-well plate with 100 μl medium containing 10⁴ cells. K₂Cr₂O₇, vit C and the different combinations of the two chemicals of indicated final concentrations were added to each well, respectively. Control cells and medium controls without cells received dimethylsulfoxide (DMSO) only. Following incubation at 37°C for the indicated time period, the PBLs were treated with 5 μl 5 mg/ml MTT 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 at 570 nm on a Versamax multiwell enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

The assay for measuring DPC formation was based on the binding of SDS to proteins and its lack of binding to DNA, according to the K-SDS assay developed by Zhitkovich and Costa (25). Protein-bound DNA is easily separated from free DNA, as protein-linked DNA precipitates with SDS, while free DNA remains in the supernatant when the cation is altered from Na to K. This method provided a direct measurement of DPC formation by analyzing the quantity of DNA in the SDS pellet. Following chemical exposure, the PBLs were washed and then resuspended in 100 μl ice-cold PBS. The suspensions were lysed with 500 μl lysis buffer [1% SDS, 20 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.5] at −70°C overnight. The frozen tubes were thawed at 65°C in a water bath for 10 min, and then the DNA was sheared by passing 10 times through a syringe with a 21-gauge needle (foam must be avoided). Then, 100 μl precipitation solution (2.5 M KCl, 10 mM Tris-HCl, pH 7.4) was added and vortexed for 10 sec. The tubes were incubated at 65°C for 10 min in a water bath, cooled on ice and then centrifuged at 6,000 × g for 5 min. Subsequently, the pellet was resuspended by adding 1 ml scavenging buffer solution (0.1 M KCl, 0.1 mM EDTA, 20 mM Tris-HCl, pH 7.4) and heating for 5 min at 65°C. The heating step was then repeated and the pellet was washed three times. The pellet was resuspended in 500 μl scavenging buffer solution, heated to 65°C and then digested with 500 μl 0.4 mg/ml protease K at 50°C for 3 h. Subsequently, the pellet was incubated with 1 ml freshly prepared fluorescent dye (Hoechst 33258) to the final concentration of 200 mg/ml, for 30 min in the dark. A fluorescence spectrophotometer with an excitation wavelength of 360 nm and an emission wavelength of 450 nm was used to assay the DPC coefficient. The DPC coefficient was calculated as the ratio of the percentage of DNA involved in DPCs to the percentage of total DNA (DNA involved in DPCs and the unbound fraction of DNA).

The PBLs were lysed using the Mammalian Cell Lysis kit, which was purchased from Sigma-Aldrich (St. Louis, MO, USA). The total protein content of the PBLs was isolated and the protein concentrations were determined using the Bicinchoninic Acid (BCA) Protein Assay kit (Sigma-Aldrich). The measurement of MDA content was performed using the MDA Content Detection Assay kit (Jiancheng Institute of Biotechnology, Nanjing, Jiangsu, China) according to the manufacturer’s instructions.

The PBLs were collected following chemical exposure and were then processed for mitochondrial isolation as previously described (26). The cell pellets were resuspended with solution 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 PMSF, pH 7.5). The PBLs were homogenized by syringe homogenization and then centrifuged twice at 750 × g for 10 min at 4°C. The supernatants were collected and centrifuged at 10,000 × g for 15 min at 4°C, to obtain the mitochondrial pellets. Mitochondrial protein concentrations were estimated using Coomassie Brilliant Blue (G-250) according to the method developed by Bradford (27). The opening of the PTP was determined using a fluorescence spectrophotometer, with a wavelength of 520 nm for the excitation and emission, at 25°C.

According to the method previously described by Emaus et al(28), with certain modifications, the MMP was monitored by the changes in the fluorescence of a specific fluorescent cationic dye, Rh123, the uptake of which by mitochondria is strongly dependent on the transmembrane potential. The absorbance was recorded by a fluorescence spectrophotometer at an excitation wavelength of 495 nm and an emission wavelength of 535 nm, at 25°C. For each test, Rh123 (final concentration, 30 μM) was added to the mitochondrial suspension.

The mitochondrial ROS production was determined spectrofluorimetrically, by detecting the fluorescence intensity of 2′,7′-dichlorofluorescein (DCF), the oxidized product of the membrane permeable fluoroprobe 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; Molecular Probes, Carlsbad, CA, USA). Briefly, following chemical exposure, 2×10⁶ PBLs were collected and incubated with 10 μM CM-H₂DCFDA solution for 1 h at 37°C, in the dark. Fluorescence was measured with a fluorescence spectrometer with an excitation wavelength of 488 nm and an emission wavelength of 535 nm. The cellular ROS levels were considered to be directly proportional to fluorescence intensity.

Western blot analysis was performed using the WesternBreeze Chemiluminescent Immunodetection kit (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Sample proteins (40 μg) were separated by electrophoresis on 10% SDS polyacrylamide gels (SDS-PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary antibodies overnight at 4°C, blocked with 4% non-fat milk and then incubated for 1 h with secondary antibodies at room temperature. The membranes were developed with the detection system and exposed to films. The primary antibody for p53 (PAb 240) (ab26) was purchased from Abcam (Cambridge, MA, USA) and β-actin was purchased from Cell Signaling Technology, Inc. (no. 4967; Danvers, MA, USA).

Statistical analysis was performed using one-way analysis of variance (ANOVA), in the Statistical Package for the Social Sciences (SPSS) v15.0 software (SPSS, Inc., Chicago, IL, USA), to assess the significance of the differences between groups. The results are expressed as mean ± standard deviation and were calculated from quantitative data obtained from three replicate experiments. P<0.05 was considered to indicate a statistically significant result.

To investigate whether Cr(VI) affected the viability of PBLs, the response of cells to different doses (25, 50, 100, 200 and 400 μM) of Cr(VI) was tested using the MTT assay. As shown in Fig. 1A, Cr(VI) decreased the viability of PBLs in a dose-response manner. A concentration of 100 μM Cr(VI) caused ~30% inhibition of cell viability, and this Cr(VI) concentration was used for the following experiment. We then sought to determine whether different time-order treatments of vit C with Cr(VI) altered the inhibition of cell viability in the PBLs. In the vit C alone treatment group, >95% of the cells were alive, indicating that the concentrations of 100 and 200 μM vit C were not the cytotoxic concentrations in PBLs following treatment for 6 h. Both vit C pre- and co-treatment protected PBLs from the deleterious effects of Cr(VI) toxicity, and resulted in higher viability compared with treatment with Cr(VI) alone (P<0.05), particularly in the 200-μM vit C groups. While co-treatment with vit C and Cr(VI) demonstrated the optimal protective effect on cell viability, vit C post-treatment did not rescue the decreased cell viability compared with treatment with Cr(VI) alone (P>0.05) (Fig. 1B). The results indicated that when PBLs were exposed to Cr(VI) together with different time orders of vit C treatments, vit C pre- and co-treatment exerted a protective effect against Cr(VI)-induced inhibition of cell viability.

Previous studies on Cr(VI)-induced toxicity suggested the potential utility of DPC lesions as biomarkers of exposure to Cr(VI) and its compounds (29). Treatment of PBLs with various concentrations of Cr(VI) (0, 25, 50, 100, 200 and 400 μM) for 6 h resulted in a significant increase in DPCs, in a dose-dependent manner (Fig. 2A). The DPC coefficients of the treatment groups receiving vit C alone (100 and 200 μM) were not significantly different compared with that of the control group (P>0.05). However, compared with the treatment groups receiving vit C alone, more DPC formation occurred in the Cr(VI) treatment groups that were pre-treated with vit C (P<0.05). Additionally, while there was no significant difference in DPC formation between the Cr(VI) treatment groups that received vit C post-treatment and the treatment group that received Cr(VI) alone (P>0.05), the combination treatment of Cr(VI) with vit C demonstrated a protective effect against DPC occurrence (P<0.05), particularly when the treatment concentration of vit C was 200 μM (Fig. 2B). The results indicated that when PBLs were exposed to Cr(VI) together with different time orders of vit C treatments, only vit C co-treatment exerted a protective effect against the Cr(VI)-induced increase in DPC formation.

In addition to the opening status of the PTP and the maintenance of the MMP, mitochondrial damage may also be evaluated by the content of MDA (30). As shown in Fig. 3, Cr(VI) treatment resulted in a significant increase in the MDA content and the open PTP rate; however, a decrease in MMP (signified by an increase in the light density of Rh123; P<0.05). The results indicated that when PBLs were exposed to Cr(VI) together with different time orders of vit C treatments, while the Cr(VI) treatment groups that received vit C post-treatment did not rescue mitochondrial damage, vit C pre-treatment and co-treatment demonstrated a protective effect against Cr(VI)-induced mitochondrial damage, particularly when the treatment concentration of vit C was 200 μM.

As ROS play a central role in Cr(VI)-induced cytotoxicity in different types of cells, we measured the ROS levels in PBLs exposed to various concentrations of Cr(VI) (25, 50, 100, 200 and 400 μM). By utilizing the oxidant-sensitive fluorogenic probe, CM-H₂DCFDA, we identified that Cr(VI) stimulation induced significantly higher values of DCF fluorescence in a dose-dependent manner compared with the control group (P<0.05), suggesting a greater quantity of cellular ROS were generated in the Cr(VI) treatment groups (Fig. 4A, upper panel). When detected under the fluorescence microscope, we observed that Cr(VI) (100 μM) induced a significantly higher level of fluorescence signals compared with the control group (Fig. 4A, bottom panel). We then determined the ROS levels in the groups of different time-order treatments of vit C with Cr(VI) in PBLs. As shown in Fig. 4B, the cells treated with 100 μM Cr(VI) and vit C exhibited up to a ~4-fold increase in ROS levels when compared with the control. The ROS levels of the treatment groups receiving vit C alone (100 and 200 μM) were not significantly different (P>0.05). Compared with the treatment groups receiving Cr(VI) alone, while the Cr(VI) plus vit C post-treatment group demonstrated no significant change in ROS levels (P>0.05), the Cr(VI) groups receiving vit C pre- and co-treatment demonstrated significantly lower ROS levels (P<0.05). As the changes in the ROS levels in the groups of different time-order treatments of vit C with Cr(VI) are similar to the changes in MDA content, open PTP rate, and MMP (Fig. 3), we concluded that cellular ROS levels were correlated with Cr(VI)-induced mitochondrial damage.

A previous study suggested a possible correlation between p53 expression and DPC formation following formaldehyde exposure (31). Thus, we detected p53 expression levels using western blot analysis. Cr(VI) induced higher p53 expression levels. The p53 expression levels of the treatment groups receiving vit C alone (100 and 200 μM) were slightly decreased when compared with that of the control group. Compared with the treatment group receiving Cr(VI) alone, the p53 levels in the vit C pre-treatment plus Cr(VI) groups were significantly increased. Additionally, while the Cr(VI) plus vit C post-treatment groups revealed no significant change in p53 levels compared with the treatment groups receiving vit C alone, the Cr(VI) and vit C co-treatment groups demonstrated a significant decrease in p53 levels (Fig. 5). The changes in p53 expression in the groups of different time-order treatments of vit C with Cr(VI) corresponded with the changes in the DPC coefficient (Fig. 2B); thus, we concluded that p53 expression was correlated with the Cr(VI)-induced increase of DPC.