Chemicals and materials

Mixtures of SCCPs (C10–13 63.0% Cl w/w, 100 µg/ml cyclohexane), MCCPs (C14–17 52.0% Cl and 57.0% Cl w/w, both 100 µg/ml cyclohexane) and PCB 166 (10 µg/ml isooctane; used as the internal standard in the analysis of CPs) were obtained from LGC Standards (Teddington, UK). The CPs mixtures were chosen for the experiments as they more closely represent the CPs contaminating the environment and biota than PCA standards. Dichloromethane (≥ 99.8%), isooctane (≥ 99.8%), diethyl ether (≥ 99.7%), florisil (particle size 0.15–0.25 mm; baked at 600 °C for 4 h before use), and sulfuric acid (≥ 98.0%) were all purchased from Merck (Darmstadt, Germany). Acetonitrile (≥ 99.9%) and n-hexane (≥ 97.0%) were obtained from Honeywell Riedel-de Haën™ (Charlotte, USA). Sodium sulfate (≥ 99.0%; baked at 600 °C for 6 h before its use) was obtained from Penta s.r.o. (Prague, Czech Republic). Deionized water was prepared using a Milli-Q water purification system (Merck, Darmstadt, Germany). Technical gasses (nitrogen 5.0 and 4.0, helium 6.0, and methane 5.5) were supplied by SIAD (Prague, Czech Republic).

Mouse 3T3-L1 embryonic fibroblasts were purchased from the American Type Culture Collection (USA, Manassas, VA). The rest of the material and chemicals was from Gibco (USA, Carlsbad, CA) unless otherwise indicated.

Cell culture and differentiation to adipocytes

Mouse 3T3-L1 embryonic fibroblasts were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM; cat. no. 21969035) containing 4.5 g/l d-glucose and 1 mmol/l sodium pyruvate and supplemented with 4 mmol/l l-glutamine, 10% fetal bovine serum (FBS) and the mixture of 100 U/ml penicillin and 100 µg/ml streptomycin sulfate in the humidified atmosphere with 5% CO2 at 37 °C.

Non-differentiated 3T3-L1 cells are referred to as preadipocytes. The 3T3-L1 cells after differentiation protocol are referred to as adipocytes.

The cells were differentiated into mature adipocytes as described previously (Skop et al. 2014; Vacurova et al. 2022). Briefly, the cells were seeded in 6-well plates at 1·105 cells/well density and incubated for 48 h to reach an absolute confluence. Then, the routine DMEM with 10% calf serum (CS) instead of FBS was applied for another 48 h. Differentiation of 3T3-L1 into adipocytes was started (day 0) by adding differentiation medium containing routine DMEM with a cocktail of differentiation inducers 0.5 mmol/l 3-isobutyl-1-methylxanthine (Merck, Darmstadt, Germany), 1 μmol/l dexamethasone (Merck), 1.7 μmol/l insulin (Merck, Darmstadt, Germany), and 25 μmol/l HEPES for another 48 h (day 0–2). Then, the differentiation medium was switched for the second differentiation medium containing DMEM with 1.7 μmol/l insulin and 25 μmol/l HEPES. This medium was changed every 48 h for 6 days (2–8). The differentiation and experiment time schedule is in Fig. 1.

Fig. 1

Scheme of differentiation—CS (calf serum), Dex (dexamethasone), DMEM (Dulbecco’s modified Eagle’s medium), DMI (differentiation medium I), DMII (differentiation medium II), FBS (fetal bovine serum), IBMX (3-isobutyl-1-methylxanthine), Ins (insulin)

Preparation of medium with CPs

The mixture of SCCPs (63.0% Cl content), MCCPs (52.0% Cl content) and MCCPs 57.0% Cl content) was prepared in a ratio of 2:1:1 (w/w/w). Cyclohexane was evaporated by a gentle stream of nitrogen, and the mixture was then dissolved in methanol and added to the culture medium, reaching constant 0.1% methanol in the medium. Each experiment listed below was performed in routine DMEM without phenol red and with 20% FBS. After the experiment cells and media were harvested by pipetting out the medium, washing cells with Dulbecco’s Phosphate-Buffered Saline (Biosera, Nuaille, France; PBS), and scraping off the cells. Harvested cells and media were stored at − 20 °C.

Measurement of viability

Mouse 3T3-L1 cells were seeded in 96-well plates at the concentration of 5·103 cells/well. Experiment was performed on both preadipocytes and mature adipocytes. At day 8, mixture of CPs was added in concentrations 0–20,000 ng/ml. Medium with/without CPs was changed every 24 h and viability was measured on day 1, 3 and 7 after the first addition of CPs.

Cell viability was measured as described previously (Trnovska et al. 2021) using the Cell Proliferation Reagent WST-1 (Roche Diagnostics GmbH, Mannheim, Germany). Absorbance was measured at 450 nm with reference absorbance at 650 nm. The experiment time schedule is in Fig. 2A.

Fig. 2

Schemes of experiments—CPs (chlorinated paraffins), DMI (differentiation medium I), DMII (differentiation medium II), DMEM (Dulbecco’s modified Eagle’s medium)

Measurement of differences in rate accumulation of CPs in time

Mouse 3T3-L1 cells were seeded in 6-well plates at 1·105 cells/well. Cells were differentiated to adipocytes. At day 8, mixture of CPs was added in two concentrations—120 and 1200 ng/ml. After this one-time addition cells and media were harvested at 2, 4, 6, 8, 12 and 24 h after the first addition of CPs. Also, experimental group without any addition of CPs was harvested. The experiment time schedule is in Fig. 2 B.

Measurement of cellular lipids content and chlorine levels/carbon chain length on CPs accumulation

Mouse 3T3-L1 cells were seeded in 6-well plates at 1·105 cells/well. Experiment was performed on preadipocytes as well as on the mature adipocytes. At day 8, mixture of CPs was added to the preadipocytes/adipocytes in two concentrations—50 and 500 ng/ml. Medium with CPs was changed every 12 h and cells and media were harvested for total of 6 days every 24 h after the first addition of CPs. The experiment time schedule is in Fig. 2C.

For the measurement of lipid content, both cells and media from separate experiment with the same time layout were used. Prior to the triacylglycerols measurement, cells were scraped to in PBS and then lysed by sonication for 15 s. Triacylglycerols (TAG) were quantified using an enzymatic kit (Erba-Lachema). A total volume of 10 μl of cell lysate or medium was used for each measurement on 96-well plate. Subsequently, 200 μl of reaction mix from the kit was added to the well, and the cells were incubated at 37 °C for 30 min. Absorbance was then measured at 540 nm wavelength. For quantification, a TAG stand was used. Using this method, CPs level could be adjusted for cellular lipid content.

Measurement of distribution pattern of CPs

Mouse 3T3-L1 cells were seeded in 6-well plates at 1·105 cells/well. Experiment was performed on preadipocytes as well as on the mature adipocytes that were differentiated using described differentiation protocol. At day 8, a mixture of CPs was added to the preadipocytes/adipocytes in two concentrations—120 and 1200 ng/ml. Medium with CPs was changed every 48 h and cells and media were harvested at day 2, 4 and 6 after the first addition of CPs. The experiment time schedule is in Fig. 2D.

Analysis of adsorption/evaporation rate of CPs in wells

CPs were added to the medium in the 6-well plates without any cells seeded. The plates were then incubated under the same condition as in previous experiments—humidified atmosphere with 5% CO2 at 37 °C for 48 h. After this period, volume of medium was measured and CPs in this volume were quantified.

Sample preparation for the analysis of CPs

Before extracting CPs from the cells, the harvested cultures were lyophilized using Freeze Dryer ALPHA 2–4 LDplus (Martin Christ, Osterode am Hartz, Germany). Then, PCB 166 in isooctane (0.25 ml; 5 ng/ml) was added, and the samples were thoroughly vortexed. To release all CPs from the cells and to hydrolyze the lipids, 50 µl of concentrated sulfuric acid was added, and the samples were vortexed again for 30 s. After centrifugation (3186 × g for 2 min), the upper organic layer was transferred into a vial and further analyzed by gas chromatography coupled with high-resolution mass spectrometry operated in negative chemical ionization mode (GC-NCI-HRMS).

Samples of culture medium (1 ml of medium collected after cell incubation with CPs was pipetted into a 15 ml centrifuge tube) were mixed with 1 ml of acetonitrile to precipitate the proteins, and the CPs were then extracted with 3 ml n-hexane:diethyl ether (9:1, v/v), shaken for 2 min and centrifuged at 3186 × g for 2 min. The upper organic layer (2.4 ml) was transferred into a 5 ml vial, and the extraction was repeated once more with a 1.5 ml n-hexane:diethyl ether (9:1, v/v) addition to the original tube. After the second extraction, 1.8 ml of the organic phase was pooled with the first aliquot in the vial. It was then concentrated to 0.2–0.3 ml under a gentle stream of nitrogen. The residual water and other possible coextracts were removed from the combined extract by solid-phase extraction on a multilayer column (from bottom to the top filled with glass wool, 0.4 g florisil, and 1 g anhydrous sodium sulfate). The CPs were eluted by n-hexane:dichloromethane (3:1, v/v), the obtained eluate was evaporated by a vacuum rotary evaporator, and the residual solvent was dried under a gentle stream of nitrogen. The sample was then dissolved in a 0.25 ml syringe standard (PCB 166, 5 ng/ml of isooctane), and the CPs were analyzed by GC-NCI-HRMS.

GC–NCI–HRMS analysis

The analysis of SCCPs and MCCPs was carried out on a gas chromatograph Agilent 7890B coupled with Agilent 7200B quadrupole-time of flight mass spectrometer (GC/Q-TOF system; both Agilent Technologies, Santa Clara, USA) operated in a negative chemical ionization mode with resolution of < 10,000 (FWHM at 185 m/z) [11].

The monitored masses are described elsewhere (Tomasko et al. 2023). The standards of CPs are described by a total concentration of all components, with no further information about the concentrations of individual compounds. The content of congener groups (described by a molecular formula) can be described by internal normalization (ratio of congener group area divided by a total area of either total CPs or only SCCPs or MCCPs). In this study, CPs profiles were made for SCCPs and MCCPs separately, as the response factors for SCCPs and MCCPs differs strongly on the used GC–MS instrumentation. On the one hand, the concentrations should not be derived from relative abundances without proper calibration by standards and also the comparison of profiles obtained in different laboratories might be hardly comparable, due to the influence of instrumentation and measurement conditions on CPs signals. On the other hand, the changes in CPs profiles obtained under the same conditions might hint a change in CPs composition, which would be too slight to be seen on a change of total concentrations.

The quantification was done by external calibration curve method. The calibration standard was prepared from a mixture in methanol used for spiking of cell media and it was diluted to levels 5, 10, 20, 50, 200, 500, and 1000 ng/ml isooctane. The total responses of either SCCPs or MCCPs were used. For the quantification of CPs in adipocytes and preadipocytes, matrix calibration points were prepared (by extracting blank cells) because of strong matrix effects. CPs in media were quantified by solvent calibration points, which was considered sufficient according to the validation parameters (see QA/QC section). The CPs are usually quantified by special procedures because of the nature of CPs composition. In this case, we were quantifying mixtures of known origin and composition, which have not changed significantly during the experiments (in the point of view of instrument response).

Determination of CPs and their possible biotransformation products

The data obtained by GC–NCI–HRMS were processed with MassHunter Quantitative Analysis software (B.10.1.733.0; Agilent Technologies, Santa Clara, USA). The monitored exact masses have been described elsewhere (Tomasko et al. 2021). Potential biotransformation products were screened in the measured data of preadipocytes and adipocytes after 7 days. The possible transformation products were selected according to He et al. (2021), i.e., CPs with shorter carbon chains, alcohols, ketones, and carboxylic acids. The above study used a direct MS (without chromatographic separation) to monitor the [M-Cl]− ions. The exact masses were therefore recalculated to [M–Cl]− formations limited to 600 m/z.

QA/QC in the analysis of CPs

Before starting the experiments, the used materials were tested for the presence of CPs (DMSO, cells, fetal bovine serum, medium), and no SCCPs and MCCPs were detectable.

During the experiments, procedural blanks were analyzed in each batch—apart from the analysis of the control samples (blank medium and cells without exposure to CPs). The procedural blanks for the analysis of cells were all without any CPs contamination. The procedural blanks for the analysis of medium contained small amounts of CPs, just below the limits of quantification. The areas of detected CPs in blanks were subtracted from the samples.

The recovery and repeatability of the method for the analysis of CPs in media were evaluated by analyzing spiked samples at two different concentrations (10 and 100 ng/ml of media for SCCPs and 20 and 200 ng/ml media for MCCPs) in six parallel determinations (each level). The recoveries were 79% and 95% (SCCPs) and 99% and 119% (MCCPs). The repeatability (expressed as relative standard deviations—RSD) was 9% and 5% (SCCPs) and 9% and 7% (MCCPs).

Statistical analysis

The data are expressed as the mean ± SD. Differences among variables were evaluated by one-way ANOVA. Statistical analysis was performed using Graph Prism 9.0.0 (GraphPad, CA, USA). The differences between CPs profiles were evaluated by paired t-tests using MS Excel (Microsoft, USA). Differences were considered statistically significant at p < 0.05 in both cases.

Heat maps were created using the ggplot function [H. Wickham. ggplot2], and linear regression analysis was performed using the lm function, both in R [R Core Team (2023)]. First, linear regression between time (days) and the relative quantity changes of each congener was performed to assess whether some congeners accumulate more than others. Slopes from these regressions were recorded and used in the next step, where these slopes of relative quantity changes were modeled using linear regression with the formula: slope (relative quantity change over time) ~ length (number of carbon atoms) + chlorine content (number of chlorine atoms).