Ringer s solution

24 h, 48 h or 72 h after treatment, supernatants of the cells were collected and frozen at −80 °C for further use. Then, cells were harvested using 0.05% Trypsin (PAN-Biotech GmbH, Aidenbach, Germany). 25 µL aliquots of the cells were either fixated in ethanol (70%) and frozen at −20 °C for cell cycle analysis or stained for cell viability measurement in flow cytometry. For that purpose, 10 µL of cells were stained with 10 µg Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA), 1 µL AnnexinA5-fluorescein isothiocyanate (FITC) or 1 µL AnnexinA5-allophycocyanin (APC, both from ImmunoTools GmbH, Friesoythe, Germany) and 66.6 ng propidium iodide (PI, Sigma-Aldrich Chemie GmbH, Munich, Germany) per 1 mL Ringer’s solution (Fresenius Kabi, Bad Homburg, Germany) for 30 min at 4 °C.
For cell cycle characterization, the ethanol-fixated cells were washed with PBS and incubated for 5 min with 500 µL DNA extraction buffer (19.2 mL 0.2 M Na₂HPO₄, 0.8 mL 0.1% Triton X-100 (Sigma-Aldrich Chemie GmbH, Munich, Germany), pH 7.8) following Riccardi et al. [30 (link)]. After that, cells were centrifuged, the supernatants were removed and DNA was stained with 300 µL of DNA staining buffer (20 µg/mL PI, 200 µg/mL RNAse A (Sigma-Aldrich Chemie GmbH, Munich, Germany) in PBS for 30 min at room temperature. PI fluorescence was analyzed by flow cytometry.

Rat sacrifice was performed through overdosing sodium pentobarbital via intraperitoneal administration. Directly after declaring termination of vital functions, the chest cavity was opened. The descending aorta and inferior vena cava were cannulated, blood was removed, and infusion of the body using large volumes of Ringer’s solution (Fresenius Kabi, Germany) was conducted in order to clean the protein material originating from the vascular bed away from the myocardium. Next, the heart and its main vessels were dissected, blot dried, and weighed. Using a stereoscopic microscope, the muscle tissue of the LV and RV free wall and interventricular septum were completely separated from each other and remaining heart structures and then weighed. Tissue samples were divided into adequately large sections and immediately frozen at –80 °C or fixed in 10% buffered paraformaldehyde solution.

Directly after declaring termination of vital functions, the chest cavity was opened. The inferior vena cava and descending aorta were cannulated, all blood was removed, and infusion of the body using large volumes of Ringer’s solution (Fresenius Kabi, Germany) was conducted in order to clean the organs, including the myocardium, from protein material originating from the vascular bed. Next, the heart, along with proximal parts of main vessels, was dissected, blotted dry, and weighted using an electronic laboratory scale (Ohaus PA224C, Switzerland). Using a stereoscopic microscope, the pulmonary trunk and ascending aorta were dissected, and the diameters of their lumen were measured. Then, the atria and main vessels were separated from ventricles, the mid-diameters of the ventricles were measured, and then the ventricles were weighed en bloc. Next, muscle tissue of the left and right ventricle free wall and interventricular septum were completely separated from each other and the remaining heart structures and were then weighed. The whole wall thicknesses of the left and right ventricle free wall and interventricular septum were measured in their middle sectors. Linear measurements were performed using 0.03-mm precision electronic calipers (YT-7201 YATO, Poland). Small tissue samples from the left ventricle were fixed in 10% buffered paraformaldehyde solution.

The second stage of the ex vivo model used standard electrophysiological methods (Fig. 1b). Isolated peripheral porcine axillary nerve trunks were harvested immediately post mortem and stored in Ringer’s solution (Fresenius Kabi). They were used for CAP recording after an initial storage period of at least two hours but no longer than 32 h. The isolated nerve specimens were placed on electrodes in a nerve chamber that could be closed with a lid to preserve humidity inside. The electrodes in the nerve chamber were used either as stimulation or recording locations. The stimulation electrodes were connected to an arbitrary waveform generator (Keysight 33512B), serving as nerve stimulator. The recording electrodes were connected to a differential head stage amplifier (npi electronic EXT-02F/2). In turn, this amplifier was connected to a storage oscilloscope for CAP recording.

The THP-1 cells were seeded in a density of 0.2 × 10⁶ cells/mL in cell culture medium containing SPION^Dex (50, 100 and 200 µg/mL) for 24 and 48 h. Then, SPION^Dex were washed several times with phosphate-buffered saline (PBS). Finally, the cell count in the cell pellets was determined using MUSE^® Cell Analyzer (Merck-Millipore, Billerica, MA, USA). After centrifugation, cell pellets were dissolved in 65% nitric acid for 15 min at 95 °C. After addition of 450 µL H₂O, the iron content was determined using atomic emission spectroscopy (MP-AES, 4200 device, Agilent Technologies, Santa Clara, CA, USA). An iron solution of 1000 g/L served as an external standard (Bernd Kraft, Duisburg, Germany). Measurement was performed at a wavelength of 371,993 nm.
To analyze cell viability, 50 µL aliquots of the THP-1 cells were stained with 250 µL of Ringer’s solution (Fresenius Kabi, Bad Homburg, Germany) containing 2 µL/mL Annexin A5-fluorescein isothiocyanate (AxV-Fitc, ImmunoTools, Friesoythe, Germany) and 66.6 ng/mL propidium iodide (Sigma-Aldrich, Taufkirchen, Germany) for 20 min at 4 °C. Then, cells were measured in a Gallios flow cytometer and data analyzed using Kaluza analysis software (1.3, 2.1.) (Beckman Coulter, Brea, CA, USA).

The acrylamide intake from hospital and home diets (µg/person/day) by the breastfeeding women and the study subjects’ exposure to acrylamide (µg/kg bw/day) were calculated—taking into account the results of the authors’ own studies on the average acrylamide content in food products in Poland.
The content of acrylamide in each product consumed (in µg/kg) was multiplied by the size of the consumed portion of the product (in kg) calculated based on 24 h recall. For the exposure evaluation, the sum of daily acrylamide intake from all products consumed was divided by the individual body weight (kg) of each study subject. Four of the subjects, at 2nd day postpartum, received only a drip on the day of the test. It included the following infusion liquids: Ringer’s solution, 0.95 NaCl, 5% glucose solution or multi-electrolyte solution, supplied by Fresenius Kabi (Poland). The analytically determined acrylamide content in the above solutions was below the limit of quantification (0.1 μg/L) [38 (link)]. For these persons, it was assumed that their acrylamide intake was 0.0 µg/day.