Chemicals and reagents
Four PAHs, which have not been explored for their effects on pregnancy, were selected for experimentation on cell viability and necrosis in a placental cell line treated with the selected PAHs. In addition, 4,4-9(fluorenylidene)diphenol, a BPA substitute, which was recently used in BPA-free products, was also studied for its effect on cell viability and necrosis in a placental cell line.
PAHs used in this experiment are as follows:
1. Anthracene (Sigma-Aldrich, St Louis, MO USA, #07, 671-100 mg)
2. Benzo[k]fluoranthene (Sigma-Aldrich, St Louis, MO USA, #03323-10 mg)
3. Benzo[a]pyrene (Sigma-Aldrich, St Louis, MO USA, #51968-50 mg)
4. 4,4-(9-Fluorenylidene)diphenol (BPA substitute) (Sigma-Aldrich, St Louis, MO USA, #39981-25 g)
Cell culture and treatment
The human placental HTR-8/SVneo cell line was provided by Queen’s University (Ontrio, Canada). HTR-8/SVneo cells were seeded in 100-mm culture plates at a density of 1 × 106, followed by starvation for 24 h to increase the effectiveness of the substance to be treated. After the cells were grown up to 80% confluency in the plate containing RPMI 1640 medium without fetal bovine serum (Gibco, CAT NO. #21875034), the serum-free medium was removed, the cells were washed lightly with phosphate-buffered saline to remove all traces of the medium, and were then treated with trypsin/EDTA. After treatment with trypsin/EDTA for 1 min, the plate was tapped lightly on one side for the detachment of the cells from the bottom of the plate. After removing the cells, 10 mL of serum-free medium was evenly sprayed into the plate to remove all remaining cells from the bottom, and the solution containing the cells was collected, and transferred to a 15-mL Eppendorf tube. The solution containing the cells was centrifuged at 200 × g for 10 min (Centrifuge 5810r, Eppendorf). After centrifugation, we the supernatant was discarded and 4 mL of RPMI 1640 medium was added to the pellet. Then, 1 mL of cell suspension was transferred into a microtube, and the cells were detached once again, following which, 20 µL of the cell suspension was transferred into another microtube. After the sufficient mixing of the cell suspension, 20 µL of the cell suspension was injected into the narrow groove of the prepared hemocytometer using a pipette. We counted the cells in 4 squares of the hemocytometer mounted on a microscope at ×10 magnification. The diluted cells of 100 µL each were transferred in three compartments of 96-well microtiter plates in triplicate. Only the cell-free medium (100 µL) was separately added into the three compartments and was used as a blank. The seeded plates were incubated for 12 h at 37°C in 5% CO2. After confirming that the cells were attached to the plate using a microscope, the cells were treated with each PAH separately and cultured.
Dose response
Prior to the experiment, we attempted to determine the effective concentration of each PAH to use in time-response experiments with PAHs. The prepared cells were incubated for 24 h at predetermined concentrations (Table 1) [8–10]. After dissolving PAHs in dimethyl sulfoxide, they were diluted with RPMI 1640 medium at different concentrations. The cells were cultured with 0 µg/mL, 0.05 µg/mL, 0.5 µg/mL, 5 µg/mL, and 50 µg/mL of anthrane; 0 µg/mL, 0.3 µg/mL, 3 µg/mL, 30 µg/mL, and 300 µg/mL of benzo[k]fluoranthene; 0 µg/mL, 0.1 µg/mL, 1 µg/mL, 10 µg/mL, and 100 µg/mL of benzo[a]pyrene; 0 µg/mL, 0.005 µg/mL, 0.05 µg/mL, 0.5 µg/mL, and 5 µg/mL of 4,4-(9-Fluorenylidene)diphenol. Cell growth and viability were examined by measuring absorbance using the XTT assay (XTT Cell Proliferation Assay Kit, American Type Culture Collection, Manassas, VA, USA) at 450–650 nm. The concentration of PAH showing a statistically significant change in optical density (OD) was compared with the respective control.
Table 1
Absorbance of HTR8/SVneo according to the exposure dose of the endocrine disruptors
Materials | Doses (µg/mL) | O.D. (Individuals) | O.D. (Total, n = 4) |
Anthracene | 0 | 1.502 | 1.534 | 1.572 | 1.510 | 1.530 ± 0.029 |
0.05 | 1.500 | 1.520 | 1.566 | 1.475 | 1.515 ± 0.036 |
0.5 | 1.469 | 1.501 | 1.508 | 1.400 | 1.470 ± 0.046* |
5 | 1.414 | 1.469 | 1.453 | 1.345 | 1.420 ± 0.051** |
50 | 1.385 | 1.419 | 1.399 | 1.260 | 1.366 ± 0.067** |
Benzo[k]fluoranthene (B[k]F) | 0 | 1.543 | 1.555 | 1.570 | 1.552 | 1.555 ± 0.010 |
0.3 | 1.569 | 1.574 | 1.564 | 1.560 | 1.567 ± 0.006 |
3 | 1.546 | 1.539 | 1.567 | 1.542 | 1.549 ± 0.012 |
30 | 1.540 | 1.524 | 1.549 | 1.539 | 1.538 ± 0.010* |
300 | 1.510 | 1.508 | 1.483 | 1.500 | 1.500 ± 0.011** |
Benzo[a]pyrene (B[a]P) | 0 | 1.560 | 1.575 | 1.522 | 1.547 | 1.551 ± 0.021 |
0.1 | 1.505 | 1.592 | 1.500 | 1.541 | 1.535 ± 0.039 |
1 | 1.582 | 1.547 | 1.510 | 1.538 | 1.544 ± 0.027 |
10 | 1.481 | 1.502 | 1.494 | 1.504 | 1.495 ± 0.010** |
100 | 1.205 | 1.464 | 1.470 | 1.429 | 1.392 ± 0.117* |
Fluorene-9-bisphenol (BHPF) | 0 | 1.541 | 1.509 | 1.512 | 1.572 | 1.534 ± 0.027 |
0.005 | 1.550 | 1.481 | 1.515 | 1.571 | 1.529 ± 0.037 |
0.05 | 1.521 | 1.479 | 1.473 | 1.599 | 1.518 ± 0.054 |
0.5 | 1.500 | 1.452 | 1.465 | 1.555 | 1.493 ± 0.043 |
5 | 1.401 | 1.429 | 1.391 | 1.462 | 1.421 ± 0.029** |
Total values are mean ± SEM. |
Significantly different from control; p < 0.05*, p < 0.005* |
Time response
The most effective concentration confirmed for each PAH in the dose–response experiment was used to incubate the prepared cells for 24, 48, and 72 h (5% CO2, humidified atmosphere at 37°C). The OD value for each incubation time was determined using the XTT assay.
XTT assay
An XTT solution was prepared by dissolving 1 mL of activation reagent (sterile solution containing N-methyl dibenzopyrazine methyl sulfate) with 5 mL of XTT reagent at 37°C immediately before use. Then, 50 µL of the solution was added to each well, incubated at 37°C in a CO2 incubator for 3 h, and the plate was slowly shaken manually until the solution turned orange. The absorbance of the wells containing cells and blank background control was measured at 450–500 nm using a microtiter plate reader. The absorbance of the cell-containing wells and control wells was also measured at 630–690 nm to assess non-specific readings. We determined the average value from the triplicate readings and subtracted the average value for the blank wells as well as the average value of the non-specific readings. When performing the XTT assay, the following parameters were used:
Specific absorbance filter: 475 nm
Non-Specific absorbance filter: 660 nm
The specific absorbance of the sample was calculated using the following formula:
Specific absorbance = A475nm (Test) − A475nm (Blank) − A660nm (Test)
Statistical analysis
Each experimental group was conducted with the minimum number (n = 3) required for statistical analysis. The results were analyzed using t-tests; p < 0.05 was considered statistically significant.