3.1 Leaching of virgin and recycled PVC
The PVC MPs used for leaching were received from Potch Plastics (North-West, South Africa) a leading recycler and compounder of various plastic materials, in pellet form. Three variants of virgin PVC MPs, as well as three variants of recycled PVC MPs were used (Figure 3-1).
The additive content of the PVC could not be divulged, as it is understood to be an industry secret.
However, the intended use of the various PVCs was disclosed. All the PVC manufactured by this factory is used in the shoe industry. PVC A is particularly used for the straps of sandals, whilst PVCs B-E are used in the production of shoe soles. Furthermore, PVC A is also used for the manufacturing of clear PVC tubing. PVC D and E are both recycled from mines, PVC E however is more pliable, and PVC D is harder and more ridged.
Leaching was done in 10 mL Pyrex© (borosilicate tempered glass) tubes, where 8 g of each PVC MPs type was covered in ethanol (EtOH; Sigma-Aldrich CAT # 270741-1L) (Figure 3-2). Since the final volume was adjusted to 2 mL, the exact volume added was not noted. Ethanol was used Figure 3-1: The polyvinyl chloride microplastics used. A. Clear PVC pellets; B. Tan PVC
pellets; C. White PVC pellets; D. Recycled PVC from mines (ridged); E. Semi- recycled PVC from mines (pliable); F. Ground unrecycled PVC from mines.
PVC F is the unrecycled counterpart of PVC E.
to create environmentally realistic leaching conditions. This is to mimic the natural leaching of microplastic in an aquatic environment. Water was not used due to the higher risk of bacterial growth from the plastic in the water that kills the cells and provides unreliable results. The sterilisation of the plastic by UV or heat exposure would start with the weathering process the results would not be of ‘virgin’ plastic. Likewise, the sterilisation of the leachates by heat, could alter the chemical integrity of the leachate. Previous studies have found that EtOH makes no noticeable difference to the compounds that are leached (Chen et al., 2019). The PVC MPs were leached at 4°C and 30°C for 24, 48, and 96 hours respectively, resulting in 6 leached samples per plastic type. These leaching conditions were decided on in an attempt to cover as many environmental conditions as possible in the Austral seasons of South Africa. These conditions include the difference in water temperature across the seasons (temperature), and water movement due to rain fall (time). After leaching, the PVC MPs were removed from the EtOH using a metal spatula pre-rinsed with EtOH to prevent contamination of the extract. The EtOH containing the leachates was evaporated using a gentle stream and steady flow of N2. Once evaporated to near dryness, the contents of the tubes were reconstituted in 2 mL EtOH and stored in glass amber vials at 4°C until use (Figure 3-2).
Figure 3-2: A diagrammatic representation of the leaching procedure.
The 36 different exposure groups of leachates from the PVC MPs (4 g/mL) were serially diluted in a 3:1 ratio in EtOH (Table 3:1). These samples were kept at 4°C in 2 mL amber glass vials (ALWSA Cat # C0000753) until used in the bio-assays.
3.2 Maintenance of cells 3.2.1 MDA-kb2 cell line
The parent cell line MDA-MB-453 that had been stably transfected with the murine mammalian tumour virus (MMTV)-luciferase neo-reporter-construct into the MDA-kb2 cell line (Wilson et al., 2002) was gifted to our research group by Saskatchewan University (Canada). The cells were grown in tissue culture dishes (TPP, Separations) in Leibovitz L-15 media with phenol red (Sigma CAT # L4386-50L) and supplemented with 10% foetal bovine serum (FBS; Capricorn CAT # FBS- GI-12A). The MDA-kb2 cells were cultured with a 1% antibiotic antimycotic mix (Biowest CAT # L0010100). They were incubated at 37°C in a humidified environment, and received no additional CO2.
3.2.2 T47D-KBluc cell line
The T47D-KBluc breast cancer cell line that had been stably transfected with pEREtata-Luc (Legler et al., 1999) was purchased from the American Type Culture Collection (ATCC). The cells were grown in Corning® tissue culture dishes in Roswell Park Memorial Institute (RPMI) growth media (Sigma-Aldrich CAT # R8755-10X1L) with 2.5 g/L D-glucose (Sigma CAT # G7021-1KG), 10 mM HEPES (Corning CAT # 25-060-C1), 1 mM sodium pyruvate (Sigma CAT # 58636- 100ML), 1.5 g/L sodium bicarbonate (Sigma CAT # 55761-1KG) and 10% FBS. The media was made in high-performance liquid chromatography (HPLC) grade water that is free from organic and inorganic compounds (Honeywell CAT # 7732-18-5). They were incubated at 37°C in humidified air supplemented with 5% CO2.
3.2.3 HuTu-80 cell line
HuTu-80 cells (HTB-40TM), human duodenum adenocarcinoma cells, were obtained from the ATCC. They were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM), low glucose (Sigma CAT # D2902-10X1L), and supplemented with 10% FBS. They were incubated at 37°C and received an additional 5% CO2.
3.2.4 General maintenance
Cells were started up from cryostorage, where the cells were stored in a freeze medium which is the growth medium also containing dimethyl sulphoxide at 40 µl/mL (DMSO; Honeywell CAT # 34869-1L). They were stored in cryovials (Corning CAT # 430488). All cell work was completed under sterile conditions, within a laminar flow (Labotech Bioflow II). Cells were inspected under a
phase-contrast microscope. Cell growth media was replaced every 2–3 days, and the cells were passaged when the tissue culture dishes were confluent. Confluent plates were washed with Dulbecco’s Phosphate Buffer Saline (DPBS)(Sigma CAT # D5652-10L) three times to remove cell debris, and 1.5 mL enzyme trypsin-EDTA x10 (Biowest CAT # M500TG100B) was used to loosen the cells from the tissue culture dishes and the resuspended cells were then transferred to new dishes.
3.3 Preparation of exposure concentrations 3.3.1 Leachates
The leachates from the plastic (4 g/mL) were serially diluted in a 3:1 ratio. The dilutions were prepared as follows: 200 μL of the 4 g/mL stock was added to 600 μL EtOH in amber glass vials.
This was repeated several times resulting in the exposure concentrations for the respective assays (Table 3-1).
3.3.2 Reference compounds
The reference compounds used were testosterone (Sigma CAT # 86500-1G) (AR agonist), flutamide (Sigma CAT # F9397-1G) (AR antagonist), dexamethasone (Sigma CAT # D4902- 100MG) (GR agonist), β-oestradiol (E2; Sigma CAT # E1132-1VL) (ER agonist) and ICI-1782,780 (hereafter referred to only as ICI) (European Pharmacopoeia Reference Standards CAT # Y0001399) (ER antagonist) for each respective receptor. The reference compounds were made up in EtOH at specific concentrations and serially diluted (Table 3-1). The concentration given to the various cell lines differed based on the cytotoxicity data as well as data from previous trials (data not shown) in our laboratory.
Table 3-1: Exposure concentrations for each of the compounds.
Compound Exposure concentrations
Testosterone 0.8; 4.7; 14.2; 28.3; 56.7; 85 ng/mL Flutamide 0.02; 0.06; 0.19; 0.56; 1.67; 5 µg/mL Dexamethasone 0.9; 4.5; 22.5; 112.5; 562.5; 843.8 ng/mL Oestradiol 0.17; 0.34; 0.68; 1.37; 2.73; 5.45 pg/mL ICI 0.002; 0.005; 0.015; 0.047; 0.14; 0.42 µg/mL
PVC concentrations per cell line Hutu-80 0.04; 0.16; 0.63; 2.5; 10; 40 mg/mL MDA-kb2 0.16; 0.61; 2.4; 9.8; 39.1; 156.3 µg/mL T47D-KBluc 0.001; 0.04; 0.16; 2.4; 9.8 µg/mL
3.4 Reporter gene assays
The assays were performed in 96-well microtitre, white-walled, clear, flat bottom plates (Bio- Greiner One & Corning). The cell media was supplemented with charcoal dextran treated (cdt) FBS (Hyclone, Separations CAT # SH30068.03) containing little to no hormones, preventing false-positive results. For the MDA-kb2 and T47D-KBluc cell lines, the inner 60 wells were seeded with 250 μL and 100 µL of stripped media respectively (Figure 3-3). They were seeded at densities of 120 000 and 100 000 cells/mL respectively. The outer 36 wells received 250 μL of PBS (Figure 3-3), to establish the same microenvironment across all cell-containing wells.
The plates were dosed in triplicates. Meaning that three plates were dosed in the same manner (Figure 3-3). The three repeats of each exposure were one on each plate, therefore only three repeats in total.
PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS
PBS L L L L L L BC L L RC PBS
PBS L L L L L L BC L L RC PBS
PBS L L L L L L BC L L RC PBS
PBS L L L L L L SC L L RC PBS
PBS L L L L L L SC L L RC PBS
PBS L L L L L L SC L L RC PBS
PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS PBS
Figure 3-3: An example of the plate layout including the Dulbecco’s Phosphate Buffer Saline (PBS) filled wells, and the wells dosed with leachates (L), blank control (BC), solvent control (SC), and the relevant reference compound (RC) used as a positive control.
3.4.1 AR activation assay using MDA-kb2 cell line
After 48 hours of initial incubation to ensure proper attachment of cells, they were dosed with 2.5 µL of the leachates and testosterone as a positive control. The plates were incubated for a further 48 hours.
3.4.2 GR activation assay using MDA-kb2 cell line
Since the MDA-kb2 cell line has both AR and GR a second assay is performed when activation is measured. This assay uses an AR antagonist to inhibit the AR. This is done in order to
determine how much of the activation measured is due to the binding of the GR and not the AR (Wilson et al., 2002).
The GR activation assay is seeded with a background of 0.6 µg/mL flutamide on day 1 and incubated for 48 h. After the initial incubation, the cell-containing wells were dosed with 2.5 µL of the leachates and dexamethasone as a positive control. The plates were incubated for a further 48 hours.
3.4.3 AR inhibition assay using MDA-kb2 cell line
When seeding the AR inhibition assay, cells were treated with 0.283 ng/mL testosterone to activate a small portion of the receptors before exposure to the leachates. This concentration represents the EC50 of the testosterone dose-response curve (Fig. 4-8). The plates were incubated for 48 hours, after which the inner wells were dosed with 2.5 µL of the leachates and flutamide as a positive control. The plates were incubated for a further 48 hours
3.4.4 ER activation assay using T47D-KBluc cell line
When seeding the ER activation assay, the wells are treated with 12.14 ng/mL ICI as a background. This was done due to a pilot assay indicating a background activation that was not due to the dosed E2. The ICI was added to compete for binding before adding E2 that would bind only to the open receptors. After an incubation period of 24 hours, the media within the wells was replaced (Legler et al., 1998). The cell-containing wells were dosed with 2.5 µL of the leachates and E2 as a positive control before being incubated for 24 hours.
3.4.5 ER inhibition assay using T47D-KBluc cell line
After 24 hours of initial incubation, the media was removed from the cell containing wells and replaced with clean media containing hormone-free FBS (Legler et al., 1998). The media was enriched with 5.45 pg/mL E2 to excite the cells within the wells, as with the AR inhibition assay.
The E2 concentration of 5.45 pg/mL was used based on trial experiments in our laboratory, of which the results are not shown. The cells were then dosed in the same manner as the other assays, and ICI was used as a positive control. The plates were incubated for a further 24 hours.
3.4.6 Reading the plates
After the final incubation of each of the preceding assays, the plates were washed with DPBS containing Mg2+ and Ca2+. Cell lysis buffer (Promega CAT # ADE3971) was added to cell- containing wells before the plates were stored at -80⁰C for rapid freezing to lyse the cells. Once frozen the plates were thawed and the luminescence was quantified using the Berthold multimode microplate reader. The plate reader injected 100 μL luciferase assay reagent (LAR) containing
20 mM tricine (Sigma-Aldrich CAT # T5816-25G), 1.07 mM Mg(CO3)2Mg(OH)2·7H2O (Sigma- Aldrich CAT # M5671-500G), 2.67 Mm MgSO4·7H2O (Sigma-Aldrich CAT # 23.239-1), 0.1 mM EDTA (ethylene-diamine-tetra-acetic acid)-disodium salt (Sigma-Aldrich CAT # 318884-500ML), 33.3 mM dithiothreitol (DTT; Melford CAT # D11000-5.0), 270 μM coenzyme A (Melford CAT # MEL-C70800-0.1), 530 μM ATP (Melford CAT # B3003) and 470 μM beetle luciferin (Promega CAT # E160C) (Villeneuve et al., 1999). The light produced in each well was recorded as relative light units (RLUs), and the number of light produced in directly proportional to the agonistic effects within the cells (Denison et al., 2004).
Table 3-2: A summary of the cell lines used, their respective receptors, assays, seeding densities, and reference compounds.
Cell line Receptor Assay Seeding density
Exposure period
Reference compounds
MDA-kb2 AR Activation
120 000 48 hr
Testosterone
Inhibition Flutamide
GR Activation Dexamethasone
T47D ER Activation
100 000 24 hr Oestradiol
Inhibition ICI
HuTu-80 No receptor Cytotoxicity 80 000 72 hr None
AR: androgen receptor; GR: glucocorticoid receptor; ER: oestrogen receptor
3.5 Viability assay (MTT)
The 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyl-tetrazolium bromide (MTT) (Sigma CAT # M5655- 1G) assay was performed to determine the viability of the cells after exposures. This was done to ensure that the inhibition that could be measured in the inhibition reporter gene assays was in fact due to inhibition of the receptor and not cell death. An MTT assay is performed parallel to each reporter gene assay and its respective cell line. Cytotoxicity was however, also measured as an end point using the HuTu-80 cell line. An MTT assay is a colorimetric assay that relies on metabolically active cells transforming a yellow tetrazolium salt to a purple formazan crystal. The formation of the purple crystal can be quantified by measuring optical density. The optical density within the well is directly proportional to the viability of the cells.
The cells were seeded and exposed under the same conditions as that of the reporter gene assays to determine whether potential negative results from the reporter gene assay may have been due to cytotoxicity. Additional negative control cells were seeded and killed with methanol after exposure.
The MTT solution is made up in nutrient media, cell line dependant, at 0.5 mg/mL and 100 µL is given to each of the cell containing wells. The plates were incubated for 30 minutes to allow the
metabolically active cells to reduce the salts. The MTT is removed from the wells, and the crystals that have formed were solubilized in 200 µL DMSO and left to dissolve for 20 mins before the absorbance was measured at 560 nm using a multimode microplate reader. The absorbance measured is directly proportional to the viability of the cells (Ulukaya et al., 2008).
The viability assays were seeded slightly differently. In addition to the inner 60 wells, an extra column on the 96 well plate was seeded to allow for methanol (MeOH; Honeywell CAT # 230-4) killed cells. Methanol is used to kill the cells, in doing so it allows for an optical density that can be subtracted in the final calculations to eliminate the optical density that is measured due to the media and such. The absorbance value of which is subtracted from the values measured in the other wells, thereby removing any background absorbance. Resulting in 66 cell-containing wells.
PBS was added to the remaining wells. After seeding, the plates were incubated to allow for cell attachment before exposure to the compounds. Each cell line had a positive control (PC) (reference compounds), a solvent control (EtOH), and a blank control. The solvent control is used to control potential solvent-related effects. The leachates and controls were dosed in triplicate across three 96-well plates.
3.6 Data processing and statistical analysis
The luminescence data was exported to Microsoft Excel to determine the testosterone-, flutamide, dexamethasone-, oestrogen-, and ICI- equivalents (TTEQ, FluEQ, DexaEQ, EEQ, and ICI-EQ).
The mean, standard deviation, and coefficient of variation values were calculated for the RLUs for each of the exposures. The concentrations of the various compounds were logarithmically transformed, and a sigmoidal dose-response was created. From these dose-response curves the EC50 and IC50 for the various positive controls were determined and the leachates’ responses interpolated in response to the positive control, where the TTEQ, FluEQ, DexaEQ, EEQ and ICI- EQ’s for each compound with a quantifiable response was obtained (Escher et al., 2018). The bioanalytical equivalents were back-calculated based on the initial quantity of plastic extracted.
The viability assay results were exported to Microsoft Excel to determine the viability of the cells after exposure to the compounds. The mean, standard deviation, and CV values were calculated for all the exposures. The mean absorbance of the MeOH killed cells was subtracted from the absorbance measured in each exposure well to remove any background noise. The %viability was determined for each well based on the mean of the SC, which is considered 100% viable. A reduction of cell viability by more than 30% is considered cytotoxic (ISO, 2009). Therefore, the cytotoxicity of the compounds was assessed comparatively considering cell viability above 80%
to be non-cytotoxic, with 80%-60% weak, 60%-40% moderate and below 40% strong cytotoxicity respectively (López-García et al., 2014).