Materials

The cytotoxicity of two substances with a proven harmful effect on reproduction and/or endocrine-disrupting metabolism was assessed—individually and in two-component mixtures in different molar proportions. The test substances methyl 4-hydroxybenzoate (methyl paraben) (MePB) (CAS 99-76-3) and dibutyl phthalate (DBP) (CAS 84-74-2) were purchased from Merck Millipore and Sigma-Aldrich (Sigma Chemical Company, St. Louis, MO, USA).

Cell culture and reagents

The studies were performed on human epidermoid (skin) carcinoma cell line A431 (ATCC® CRL-1555). Cell line was purchased from LGC Standards, which distributes ATCC (American Type Cultures Collection) cultures.

A431 (ATCC® CRL-1555) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 1% antibiotic–antimycotic: penicillin G sodium, streptomycin sulphate, amphotericin B and fungizone (Sigma-Aldrich) and supplemented with 10% of foetal bovine serum obtained from Gibco BRL (Life Technologies Ltd., Paisley, Scotland) in sterile tissue culture flasks (Nunc, Roskilde, Denmark). Cells were maintained in monolayer cultures at 37 °C in a humidified atmosphere (5% CO2) and to detach them from the culture flasks, 0.25% trypsin–EDTA (Gibco) was used. Cells were checked for Mycoplasma sp. infection using a Myco-Alert™ PLUS Mycoplasma Detection Kit (Lonza, Walkersville, Inc.).

Before starting the experiments, cell viability in the suspension was assessed using a trypan blue dye exclusion test [22]. Suspensions were dispensed into a glass Bürker chamber and viable (trypan blue negative) and dead (trypan blue positive) cells were counted. The evaluation was carried out using a light microscope with inverted optics (Nikon TMS-F, Japan), employing a 100-fold magnification. Cell suspensions in which the number of faulty cells did not exceed 5% were used for further investigation.

In order to increase the solubility of the test compounds, which would allow the cells to be exposed to higher concentrations of xenobiotics, DMSO was used as their solvent, after determination of its non-toxic concentration to the exposed cells.

Cell cultures were seeded at a density of 1 × 105 viable cells/ml with 100 µl of medium in each well of the 96-well microplates (Nunc, USA) and cultured overnight to allow adherence and recovery from exposure to trypsin. Next, the medium was aspirated from the cells and a solution of the tested compound (100 µl) was added to each well of the microplate. Each sample was applied in nine replications. Control cells were incubated in a nutrient medium without xenobiotics. Microplates were placed in a CO2 incubator for 24 h, after which the assays were performed.

The exposure of A431 cells to mixtures containing the following concentrations were tested.

85% MePB and 15% DBP (stock solution contained 340 mM MePB and 60 mM DBP in 5 ml DMSO)

75% MePB and 25% DBP (3:1) (stock solution contained 300 mM MePB and 100 mM DBP in 5 ml DMSO)

50% MePB and 50% DBP (1:1) (stock solution contained 200 mM MePB and 200 mM DBP in 5 ml DMSO)

25% MePB and 75% DBP (1:3) (stock solution contained 100 mM MePB and 300 mM DBP in 5 ml DMSO)

15% MePB and 85% DBP (stock solution contained 60 mM MePB and 340 mM DBP in 5 ml DMSO).

Cytotoxicity tests

The following methods were used to evaluate the toxic effects of the test compounds and their binary mixtures on cells in vitro.

MTT tetrazole salt reduction test determining the metabolic activity of cells (MTT test);

Neutral Red Uptake Assay to assess the integrity of cell membranes (Neutral Red Uptake Assay, NRU test) according to INVITTOX Protocol No 17 and No 64, respectively.

Clonogenic assay determining the ability of cells to proliferation (CFEA test).

MTT assay

The in vitro cytotoxicity of single substances and their binary mixtures was evaluated using MTT assay as described by Mossman [23]. This assay is based on the assessment of the cells metabolic activity expressed by the ability to absorb yellow MTT tetrazolium salt and reducing it by mitochondrial succinate dehydrogenase to an insoluble formazan compound.

In the first step, cells were seeded at a density of 10,000 cells/well in a 96-well culture plate (Nunc) and cultured overnight (37 °C, 5% CO2). Then non-attached cells were aspirated and the rest of the cells were treated with various concentrations of the test substance/mixture and after removing the medium, medium-containing MTT (0.5 mg/ml in Hank’s buffered saline) was added to each well (100 µl). After 3 h incubation, supernatants were removed and the purple-blue formazan product was dissolved in 100 µl of DMSO (Gibco). Absorption was measured using an ELISA microplate reader (at 570/620 nm), after shaking the plate for 3 min before the measurement. On the basis of the results, it was determined that the concentration of the test substance/mixture caused a decrease in metabolic activity of cells by 50% (IC50) compared to the control. Cytotoxicity assays were performed in three iterations. IC50 values for each tested compound/mixture were calculated by a computer program using interpolation curves (four logistics) KCjunior (version 5), BioTek Instruments, Inc.

NRU assay

The principle of the NRU assay is based on the ability of live, intact cells to absorb the dye, neutral red (3-amino-7-dimethylamino-2-methyl phenazine hydrochloride) accumulated in the lysosomes. Similarly to the MTT test, cells were first seeded at a density of 10,000 cells/well in a 96-well culture plate (Nunc) and cultured overnight (37 °C, 5% CO2). Then, non-attached cells were aspirated and the rest of the cells were treated with various concentrations of the test substance medium-containing neutral red dye (100 µg/cm3) added to each well (100 µl). After 3 h incubation, the supernatants were removed and the cells were treated with 100 µl of solution containing 50% ethanol, 49% distillate water and 1% glacial acetic acid. The cell membranes were destroyed in order to release the dye. The concentration of the dye was determined spectrophotometrically using an ELISA microplate reader (at a wavelength of 540/450 nm). On the basis of the results, it was determined that the concentration of test substance inhibited the ability of cells to absorb the dye by 50% (IC50) compared to the control.

Based on the MTT and NRU tests for the tested equimolar (1:1) binary mixture, the synergy index (SI) was calculated [24], defined as:

where, EA+B(obs.) is the observed/real effect of the A + B mixture, EA+B(calc.) is the calculated/expected effect of the A + B mixture assessed on the basis of the effects of individual substances.

The theoretical IC50 values for the two-component mixture were calculated as the sum of the products of the mean IC50 values determined for each compound separately and the percentage of the compound in the mixture.

When synergy is estimated on the basis of IC50 values that are inversely proportional to the toxic effect of the substance, a synergistic effect is found when SI < 1.

CFEA test

Clonogenic assay was used to determine the ability of a cell to proliferate indefinitely, thereby retaining its reproductive ability to form a large colony or a clone. This cell is then said to be clonogenic [25]. Clonogenic assay, also known as colony-forming efficiency assay (CFEA), is based on the ability of a single cell to grow into a colony [26], where the colony is defined as to consist of at least 50 cells [27].

The clonogenic assay was conducted according to the procedure described by Franken et al. [27] and adapted from Kruszewski et al. [28]. Cells in the exponential growth phase were harvested and seeded in a Petri dish of 60 × 15 mm (21 cm2) (Iwaki Cell Biology, Japan) at a density of 500 cells/dish in 5 ml of medium-containing tested substance/mixture in appropriate concentrations. Each experiment was performed in three independent replicates. Cells were exposed to the tested substance/mixture for 7 days. After this period, the medium was removed, and the cells were washed with PBS. Then, the colonies were fixed with ethanol (Sigma-Aldrich), stained with Giemza solution (0.4%, Sigma-Aldrich) and counted using a stereomicroscope (IUL, Spain). The plating efficiency (PE) and surviving fraction (SF) were calculated, as below:

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$$\text\left(\text\right)=\frac}\times \text}$$

The SF for the control cells were understood to be equal to 1.

Visualisation of morphological changes in exposed cells using HTM

Morphological changes in the cells caused by the test compounds individually and their mixtures were assessed by holotomographic microscopy (HTM). To visualise mitochondrial structures, the cells were incubated with MitoView (Biotium) dye at 100 nM, while nuclei were stained with DAPI reagent (1: 1000, Sigma). The cells were incubated with the above-mentioned dyes for 15 min at 37 °C. After staining the cell structures, observations of the cells were carried out under a holotomographic microscope.

Apoptosis markers assessement

Assessment of caspase-3/7 activity was performed for 24 h using a IncuCyte® S3 Live-Cell Analysis System as well as using a fluorescence image cytometer NucleoCounter® NC-3000™ to determine caspase-3/7 enzymatic activity by FLICA (Fluorochrome-Labelled Inhibitors of Caspases).

The translocation of phosphatidylserine in the cells was also assessed using NC 3000 on the basis of binding the fluorescently labelled Annexin V protein.

Caspase 3/7 assay using IncuCyte

A431 cells were seeded at a density of 5000 cells per well in 96-well plates (Nunc) and incubated overnight at 37 °C in a humidified atmosphere (5% CO2). Next, the cells were exposed to different concentrations of the tested substance/mixture for 24 h. Next, fresh medium containing 1 × IncuCyte Caspase-3/7 Apoptosis Assay Reagent (Sartorius) were added. Before starting the scanning, plates were incubated (37 °C, 5% CO2) for 15 min. Cells treated with staurosporine (0.1 nM, Sigma-Aldrich) were used as a positive control. At each time point, five images were taken per well in both brightfield and FITC channels. The images were analysed for the number of green objects (fluorescing cells) per well by the algorithm in the IncuCyte S3 Software (v2018B). Each experiment was performed in three independent replications.

Evaluation of two apoptosis markers in cells exposed to the tested substances/mixtures using Nucleocounter NC 3000

The translocation of phosphatidylserine in cells was also assessed using Nucleocounter NC 3000 on the basis of binding the fluorescently labelled Annexin V protein.

Measuring the binding of Annexin V conjugated with fluorescein-FITC isotiocyanate allows the detection of conformational changes of the cell membrane and loss of asymmetry in membrane lipid distribution, which is associated with phosphatidylserine translocation (the main component of membrane phospholipids) to the outer membrane layer without impinging on its integrity (an early marker of apoptosis). In order to differentiate apoptotic cells from necrotic cells, DNA staining with propidium iodide (PI) was used, which penetrates the damaged membrane of dead cells but does not penetrate the intact membranes. The total cell population was also labelled using a Hoechst 33342 reagent for the detection of all cells.

Determination of the enzymatic activity of caspase-3/7 by the FLICA method is used to detect apoptosis based on the assessment of caspase activity in situ in cells. The assay involves the use of a carboxyfluorescein-labelled (FAM) four-amino acid (Asp-Glu-Val-Asp) substrate that is conjugated with a fluoromethyl ketone residue (FMK), which determines its penetration through the cell membrane into the cell interior and irreversible binding of the specific amino acid sequence to the active centre of the caspase. Any unbound inhibitor is washed away and as a result labelled, and only cells in which the caspase has been activated are detected using a fluorescence reader [29].

The w/v or v/v solutions of the tested substances were prepared immediately prior to exposure of the cells. The solutions of each xenobiotic were prepared in two concentrations corresponding to 1/4 and 1/10 of the average IC50 determined experimentally. The level of apoptosis was tested in at least three replications.

The cells were suspended in the culture medium at the concentration required for the assay, and then placed in a Petri dish—vol. 5 ml. After 24 h incubation (37 °C, 5% CO2, 95% humidity), during which the cells were stuck to the bottom of the dish, the medium was aspirated and a solution of the test substance of a certain concentration in the culture medium was added to each plate. The dishes were placed in a CO2 incubator for 24 h, after which tests were performed. Negative control were non-exposed cells, while positive control cells were exposed to 0.1 nM staurosporine (the concentration of the compound was chosen experimentally).

Annexin V test

After incubation with the test compound, the cells were harvested and their concentration determined, followed by centrifugation at 400×g for 5 min. The pellet was resuspended in 300 μl PBS and centrifuged again under the same conditions. Then 2 ÷ 4 × 105 cells were suspended in 100 μl working buffer, in which the Annexin V binding occurred and 2 μl of the Annexin V-FITC complex was added (labelling was performed under conditions that took into account the photosensitivity of the complex). Cells were then labelled with 2 μl of Hoechst 33342 reagent at 500 μg/ml and incubated at 37 °C for 15 min. in the heating block. After incubation, the cells were centrifuged at 400×g for 5 min. After removal of the supernatant, the pellet was resuspended in 300 μl working buffer, mixed and centrifuged. The procedure was repeated twice. The obtained cell pellet was resuspended in 100 μl of working buffer, 2 μl of 500 μg/ml propidium iodide solution was added, immediately placed on NC-Slide A2™ and analysed using a NucleoCounter NC-3000 with fluorescence microscope.

Determination of caspase-3/7 activity

After the incubation with the test compound/mixture, the cells were harvested and brought to a concentration of 2 ÷ 5 × 106 cells/ml. Then 5 μl of FLICA reagent (previously reconstructed by the addition of 50 μl DMSO) diluted in PBS in a ratio of 1: 5, was added to the cell suspension (93 μl). Then 2 μl of Hoechst 33342 reagent at a concentration of 500 μg/ml was added and incubated at 37 °C for 60 min. in the heating block. After incubation, the cells were washed twice with 400 μl washing buffer, followed by centrifugation at 400×g for 5 min. After the final rinse, the cell pellet was resuspended in 100 μl washing buffer, supplemented with propidium iodide (PI), so as to obtain a concentration of 10 μg/ml, immediately placed 30 μl in the NC-Slide A2™ slide and analysed using an image cytometer NucleoCounter NC-3000 with fluorescence microscope. Based on fluorescence measurements, the percentage of cells without apoptosis, early and late apoptotic and necrotic was calculated.

Statistical analysis

In the statistical analysis, firstly, descriptive statistics as well as one-sample t-test and one-sample Wilcoxon test were used. Listed tests were applied to compare the results of MTT, NRU and CFEA with theoretical values of these parameters at different levels of exposure to specific substances (DBP and MePB), with a different composition of mixtures. Additionally, using one-way ANOVA, a comparison was made of the results for Annexin V and caspases for subgroups distinguished by the composition of mixtures and two control groups. Effect size was measured by the partial eta-square (\(^\)). In the statistical analysis, the commonly significance level of α = 0.05 was applied. The calculations were performed in the IBM® SPSS® Statistics 25.0 statistical package.