Polychlorinated biphenyls (PCBs) are a class of persistent organic chemicals whose slow biodegradation in the environment and high lipophilicity allow them to bioaccumulate and reach high concentrations in food, mainly fish, meat, and dairy products (1). Although PCB levels are constantly decreasing in the environment (2), the risk is still present in Croatia due to leakage from power and industrial plants destroyed in war operations between 1991 and 1995 (3) and inadequate landfill disposal of transformers and capacitors.
One of the most persistent among them is the di-
PCBs are known to disrupt the endocrine, immune, nervous, and reproductive systems (13, 14), but because most such studies have been carried out with Aroclor mixtures, little is known about the exact mechanisms of action of individual PCBs, particularly with respect to the ovaries. Sechman et al. (15) have reported that PCB 153 disrupts follicular steroidogenesis, oestrogen receptor expression, and recruitment of small yellow follicles into the preovulatory hierarchy. Wojtowicz et al. (16) reported various disruptive effects on oestradiol, progesterone, and testosterone secretion from ovarian cells
In our previous study we reported the molecular mechanisms of one of the most toxic PCB congeners – dioxin-like PCB 77 in Chinese hamster ovary K1 cells (13). The aim of this study was to expand research to PCB 153, as it differs from PCB 77 in the degree of chlorination, planarity, and activity.
The Chinese hamster ovary cell line (CHO-K1) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco’s MEM F-12 (DMEM F-12), heat inactivated foetal bovine serum (FBS), and trypsin/EDTA solution (0.25 % trypsin with EDTA 4Na) were supplied by GIBCO (Paisley, UK). PCB 153 (2,2’,4,4’,5,5’-hexachlorobiphenyl, CAS 35065-27-1), Trypan Blue dye (CAS 72-57-1), MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 h-tetrazolium bromide, CAS 298-93-1), and propidium iodide (PI, CAS 25535-16-4) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Coomassie brilliant blue R-250 (CAS 6104-59-2) was purchased from LKB (Bromma, Sweden). Neutral Red dye (CAS 553-24-2) and Muse™ Annexin V & Dead Cell Kit (cat #MCH100105) were supplied by Merck (Billerica, MA, USA). The 2’,7’-dichlorofluorescin diacetate (DCFDA) Cellular ROS Detection Assay Kit (cat #ab113851) was obtained from Abcam (Cambridge, UK). DMSO (CAS 67-68-5) and ethanol (CAS 64-17-5) were purchased from Kemika (Zagreb, Croatia).
Adherent, epithelial-like CHO-K1 cells were grown in DMEM F-12 supplemented with 10 % (v/v) FBS in a 5 % CO2 humidified environment at 37 °C. Antibiotics were not used during the study. In order to perform cell viability analysis, exponentially growing cells were trypsinised and seeded in 6-well plates at an initial concentration of 2x104 cells/mL in 2 mL of culture medium. After an overnight incubation, cells were treated with 10 μL/mL of different PCB dilutions in DMSO to obtain the desired final concentration (10, 25, 50, 75, or 100 μmol/L) in culture medium for 6, 24, 48, and 72 h. Samples treated with 10 μL/mL DMSO were used as controls.
Viability and proliferation endpoints were quantified with a battery of
The CHO-K1 cells were exposed to 10–100 μmol/L PCB 153, and cell proliferation and viability were determined after 6, 24, 48, and 72 h of treatment. The assays were conducted as described earlier (20) with minor modifications. For each assay and each time point at least two experiments were performed, and within a single experiment each PCB 153 concentration was tested in triplicate.
Percentages of live, apoptotic, and dead cells after treatment with PCB 153 were determined using the Muse™ Annexin V & Dead Cell Kit and the Muse™ Cell Analyzer (Merck) following the manufacturer’s protocol. Briefly, after 6 and 48 h of exposure to PCB 153 (10, 50, and 100 μmol/L), both floating and adherent CHO-K1 cells (plated at a density of 2x104 cells/mL) were collected, centrifuged at 600
Cellular DNA content and cell cycle progression upon PCB 153 (50 μmol/L) treatment were determined by flow cytometry using propidium iodide (PI). The cells were collected by centrifugation, washed with PBS, centrifuged again, and fixed in ice-cold 70 % ethanol at indicated time points (after 6, 16, 24, 48, and 72 h of PCB treatment). The samples were stored at -20 °C. On the day of analysis, cell samples were centrifuged and washed twice in PBS, resuspended in a solution of RNAse (0.1 mg/mL) and PI (0.02 mg/mL) in PBS, and incubated in the dark at room temperature for at least 30 min. Cytometry was performed on a BD FACSort flow cytometer using CellQuest™, and data analysed with the ModFit LT™ software (Topsham, ME, USA).
Reactive oxygen species (ROS) formation was determined spectrofluorimetrically with the DCFDA Cellular ROS Detection Assay Kit following the manufacturer’s protocol. CHO-K1 cells were seeded in 96-well black plates (1x105 cells/mL; 100 μL per well). After overnight incubation, cells were stained with DCFDA for 45 min, then washed with buffer, and exposed to 10–100 μmol/L PCB 153 at 37 °C for 3 h. Fluorescence was measured with a Varian Cary Eclipse Spectrofluorimeter (Palo Alto, USA) at excitation / emission wavelengths of 485 nm / 535 nm. Data are reported as percentages of control (cells treated with 10 μL/mL DMSO) ± SEM (n=5).
A two-tailed Student’s
Figure 1 (A–D) summarises the results of four
The fact that the NR and TB assays showed almost complete loss of cell viability at 48 h and 72 h of exposure to the highest PCB 153 dose (100 μmol/L) suggests that the main cellular targets of its cytotoxicity are the cell membrane and lysosomes (especially at prolonged incubation). This has been confirmed by lower IC50 values obtained with these two bioassays than with the MTT and KB assays (Table 1). Variations in IC50 values between the four assays suggest that combining multiple assays with different endpoints (such as membrane integrity, lysosomal activity, total cellular protein content, and mitochondrial function) can provide valuable information about possible mechanisms of action of the tested compound. Compared to the IC50 values obtained for planar dioxin-like PCB 77 from our previous study (13), non-planar di-
Inhibition concentrations (μmol/L) determined with four different cytotoxicity assays after 6, 24, 48, and 72 h of CHO-K1 cell exposure to PCB 153
PCB 153 (μmol/L) | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
6 h | 24 h | 48 h | 72 h | |||||||||
Endpoint | IC20 | IC50 | IC80 | IC20 | IC50 | IC80 | IC20 | IC50 | IC80 | IC20 | IC50 | IC80 |
TB | 32.7 | 66.3 | 92.6 | 23.3 | 64.5 | ND | 28.5 | 66.1 | 98.3 | 20.8 | 55.5 | 88.6 |
NR | 61.5 | ND | ND | 22.6 | 65.9 | ND | 34.0 | 65.2 | 89.3 | 21.7 | 51.0 | 78.3 |
KB | 61.0 | ND | ND | 77.4 | ND | ND | 73.0 | 95.9 | ND | 85.2 | ND | ND |
MTT | 19.7 | 58.4 | 98.6 | 67.8 | ND | ND | 63.4 | ND | ND | 34.8 | 86.7 | ND |
All presented values were determined with polynomial interpolation from dose–response curves. TB – Trypan Blue; NR – Neutral Red; KB – Kenacid Blue; ND – not determined
Figure 2 shows the distribution of apoptotic cells in the first 6 h of exposure to PCB 153, established with the cytofluorimetric analysis using annexin V and 7-AAD as markers. Significant differences from control started with doses ≥50 μmol/L. At 50 μmol/L significantly higher was the fraction of cells in early apoptosis (p<0.025), while at 100 μmol/L both early and late apoptotic cell fractions significantly increased (p<0.01 and p<0.001, respectively). After 48 h, apoptosis gave way to necrosis (Figure 3). The observed decrease in the number of viable cells was concentration-dependent. Our results in ovarian cells are consistent with related literature data (22–25). Considering the consistent apoptosis induction in cells of reproductive tissues, the inhibitory effect of PCBs should also be investigated on cancer cell lines. To date, there are many papers addressing the carcinogenicity of PCBs, but data regarding antiproliferative effects are scarce. An exception is the study by Oenga et. al. (26), who showed effective inhibition of E2-stimulated breast cancer cell proliferation by PCBs (in the following order: PCB 81>PCB 126= PCB 169>PCB 77).
Figure 4 shows representative DNA histograms (A) and the distribution of cells across the G0 (sub G1), G0/G1, S, and G2/M phases of the cell cycle (average of two experiments) (B). Treatment with 50 μmol/L PCB 153 disturbed the normal cell cycle progression in a time-dependent manner, and the most significant changes were observed at 72 h of exposure. Time dependency was also evident in the increase in the apoptotic peak at 48 h (Figure 4A). We reported similar findings for PCB 77 earlier (13) and Venkatesha et al. (5) also reported that PCB 153 inhibited the entry of MCF-10A human mammary epithelial cells into the S-phase, while the proportion of G1 cells remained high (90–95 %).
PCB 153 showed dose-dependent induction of oxidative stress in CHO-K1 cells (Figure 5), except for the lowest tested dose. Our findings are in line with numerous findings that apoptosis induced by PCBs is mediated in part by elevated levels of ROS (27–30).
We confirmed the hypothesis that intracellular effects of PCB congeners vary by their structure and planarity. IC50 values for PCB 153 were higher than those previously determined for PCB 77 (13), which correlates with their LD50 values