Once Upon a Time, Microplastics in the Lab: First Glimpse of Their Impact on Ruminant Placenta and Mammary Gland Cells

Through forage consumption, ruminants are primarily exposed to microplastics (MPs, < 5 mm), becoming potential reservoirs for humans via milk and meat. Environmental contamination is mainly due to the use of plastic in agricultural practices (silage bags) and the withdrawal of polyethylene terephthalate (PET) materials (bottles, bags). To date, the health effects of these emerging contaminants are still poorly understood. Very recent studies have detected MPs in the human placenta [1], in follicular fluid [2], and in the semen of cattle [3]. Additionally, MPs act as a vehicle for harmful substances, such as phthalates and bisphenols, known as endocrine disruptors. Throughout the bloodstream, MPs can reach multiple organs, potentially accumulating in highly vascularized tissues, such as the mammary gland and placenta. Based on this hypothesis, here we tested the effect of PET-MPs in in vitro cellular systems of sheep placenta and bovine mammary gland, previously characterized in our laboratory of Veterinary Physiology. Virgin PET fragments (100-400 μm) from plastic processing waste were subjected to grinding cycles to simulate the mechanical stresses that MPs undergo in the environment. The fragments were separated into three fractions (< 30, 20-50, > 50 μm), and the smaller ones were tested in cellular systems. The morphology and size distribution of MPs were analyzed using Particle Flow Image Analysis, and structural modifications were assessed through X-ray diffraction. Term placental cells from ovine cotyledons (oTP) and bovine mammary epithelial cells (MAC-T) were exposed to 1, 5, and 50 μg/mL PET of both < 30 and 20-50 μm for 24h and 48h. Interaction between cells and MPs was checked by microscopy; cell viability (MTT; Resazurin assay) and migration (wound healing) were evaluated. Firstly, it was observed that PET MPs remain in contact with the cell monolayer, without floating or aggregating in the culture medium. MTT revealed that the size of the fragments has a different effect depending on the cell type. Cell viability was reduced in placental cells exposed to 50 μg/mL of 20-50 MPs (P < 0.05), while no effect was observed for MACT cells. Conversely, MPs < 30 μm seem to have a cytotoxic effect on oTP and MACT cells starting from 1 μg/mL at both 24 and 48h (P < 0.01). However, migration was not impacted by MPs exposure, although in 50 μg/mL cells physically interacting with PET particles. Our preliminary data suggest that smaller-sized MPs can alter cell viability, likely due to steric hindrance in the cell growth environment. We also suspect that chronic exposure (> 48 h), potentially mimicking normal contact, could have more pronounced effects on cellular functionality. Although the MPs used here are from common plastics, they are derived from virgin plastics. In that context, future experiments with "environmental" MPs will help assess their impact on cellular processes like proliferation, hormone production, and oxidative stress. [1] Ragusa et al. Plasticenta: First evidence of microplastics in human placenta. Environment International, 146: 106274, 2021. [2] Greghi et al. Microplastics are present in women’s and cows’ follicular fluid and polystyrene microplastics compromise bovine oocyte function in vitro. eLife 12, RP86791. 2023. [3] Greghi et al. Microplastics are detected in bull epididymal sperm and polystyrene microparticles impair sperm fertilization. Biology of Reproduction, 111: 1341–1351, 2024.