IN VITRO ASSESSMENT OF CARBOXYLATED POLYSTYRENE NANOPLASTICS TOXICITY IN TM3 LEYDIG CELLS

Abstract

Pollution by plastic microparticles is a growing concern for both the environment and biological systems, including reproductive health. Nanoplastics can accumulate in the testis tissue and result in testosterone insufficiency and reproductive dysfunction in males, according to many in vivo studies. In the natural environment, plastic particles are rarely found in their original form because physical, chemical and biological processes lead to surface modifications that can alter their biological interactions. The present study aimed to evaluate the potential in vitro cytotoxicity of carboxylated polystyrene nanoplastics (PS-COOH NPs) at a size of 0.03 μm and dosages of 1, 10, 50, 100 and 200 µg/mL, representing environmentally weathered nanoplastics, on TM3 Leydig cells, with particular focus on cell viability, metabolism, oxidative stress, and steroidogenic function. TM3 Leydig cells were exposed to PS-COOH NPs for 24 h, followed by assessment of cell viability, proliferation and metabolic activity using MTT and Alamar Blue assays. Intracellular superoxide production as a marker of oxidative stress was measured by the nitroblue tetrazolium assay, and testosterone levels were determined using ELISA analysis. Our results demonstrated a concentration-dependent inhibition in cellular metabolic activity, with significant decline observed at concentrations 100 and 200 µg/mL. Increased superoxide radical production was detected at lower concentrations (1–50 µg/mL), indicating the induction of oxidative stress in metabolically active cells. Testosterone production was significantly suppressed at all tested concentrations, including those that did not markedly affect cell viability. The most significant reduction in testosterone levels was observed at the highest tested concentration of PS-COOH NPs. Overall, findings of this study suggest that carboxylated polystyrene nanoplastics may disrupt Leydig cell function by inducing oxidative stress and impairing steroidogenesis. These results highlight the potential endocrine-disrupting effects of environmentally relevant nanoplastics and emphasize the need for further studies.