DOI: 10.25345/c5qv3ch7f
title: MassIVE MSV000100812 - The influence of microplastic PET on the functions of the adrenal cortex in immature gilts
title: Plastics and their products in our everyday environment have become a global problem and an integral part of life, and the extent of exposure and potential toxic effects of these pollutants on several human organs are becoming increasingly evident. We hypothesize that polyethylene terephthalate (PET) disrupts the function of the adrenal cortex in immature piglets. The study was approved by the Local Ethics Committee of the University of Warmia and Mazury in Olsztyn (decision No. 10/2020 of 26 February 2020) and conducted in accordance with the provisions of the European Union directive on the ethical use of experimental animals (EU Directive 2010/63/EU for animal experiments). All animals were housed in breeding pens under standard laboratory conditions (temperature was maintained at 20-22 degree Celsius, and humidity ranged from 55 % to 60 %) and were provided with free access to fresh water (ad libitum) and an age-appropriate feed mixture. All plastic items were removed from the animals environment. The equipment on the farm where the gilts were raised before the experiment was also made of stainless steel. The experiment lasted 4 weeks and was conducted on 8-week-old (Pietrain x Duroc) immature gilts (n = 15) with an estimated body weight of 20 kg. The animals were divided into three groups: 1) control group (CTRL; n = 5) which received empty gelatin capsules per os; 2) experimental group (LD; n = 5) which received a low dose of PET MPs (0.1 g/pig/day in gelatin capsules) per os; 3) experimental group (HD; n = 5) which received a high dose of PET MPs (1 g/pig/day in gelatin capsules) per os. The microplastics used for the experiment is a semi-crystalline polyethylene terephthalate powder (Goodfellow Cambridge Ltd., Huntingdon, England). The particles have different shapes (spherical, fibrous, irregular) with the average length of the larger side of the molecule 153.09 um. The applied doses were selected based on weekly human consumption of MPs reported in the literature (0.1-5 g of MPs per week) as well the previous toxicological studies of mice and aquatic organisms (Deng et al., 2017a, Senathirajah et al., 2021). The low dose was adjusted to the weight of gilts, whereas the high dose was 10 times higher than the low dose to determine the effect of MP accumulation (calculated on a logarithmic scale) (Yang et al., 2019). Immediately after euthanasia the adrenals were isolated and frozen in liquid nitrogen. In the laboratory of the Department of Animal Anatomy and Physiology, the adrenal cortex was mechanically separated from the medulla under a stereomicroscope and stored -80 degree Celsius for further analyses. Protein extraction was performed using T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, Merck Group, Darmstadt, Germany), according to the manufacturers instructions. Protein concentration was determined using the bicinchoninic acid (BCA) assay (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific). Samples containing 150 ug of total protein were transported to the Department of Biochemistry and Molecular Biology, University of Southern Denmark. For proteomic analysis, 50 ug of protein per sample was reduced with dithiothreitol (DTT) and alkylated with iodoacetamide (IAA) (both from Sigma-Aldrich), followed by digestion with sequencing-grade modified trypsin (Sigma-Aldrich) at a dose of 2.5 ug. Digestion was performed overnight at 37 degree Celsius. The reaction was terminated by the addition of 20% trifluoroacetic acid (TFA) (Sigma-Aldrich), and the pH was adjusted to pH 3. Peptides were desalted using Evotips according to the manufacturers protocol. Peptide separation was performed using an Evosep One LC system (Evosep ApS, Odense, Denmark) coupled to an Orbitrap Astral mass spectrometer (Thermo Fisher Scientific). 0.1 ug of peptides was loaded per Evotip. LC MS/MS data acquisition was performed in data-independent acquisition (DIA) mode, and data analysis was conducted using the directDIA workflow implemented in Spectronaut software (Biognosys, Zurich, Switzerland). The MS RAW-files were analyzed using Spectronaut v20.3 (Biognosys AG, Schlieren) with directDIA using default search settings against a FAST file containing the pig proteome (UniProt, 22,804 sequences, downloaded November 2025).