EXPLORING THE NEUROTOXIC POTENTIAL OF LDPE MICROPLASTICS: EVIDENCE IN VITRO AND IN VIVO ASSESSMENT

Marilena MARRAUDINO^1,2, Elena RICCI^1,2, Silvia CHASSEUR^1,2, Daniela RASA'¹, Riccardo ROLANDO¹, Stefano GOTTI^1,2, Marina BOIDO^1,2 | ¹Neuroscience Institute Cavalieri Ottolenghi (NICO), Orbassano (TO), Italy; ²Department of Neuroscience ‘Rita Levi Montalcini’, University of Torino, Torino, Italy

The widespread use of plastics has led to significant environmental pollution, as large amounts of plastic waste are often buried, burned, or discarded into rivers. Since plastics break down slowly, they create microplastics (MPs) that accumulate in the environment, forming a new type of pollutant. In animal studies, oral intake of MPs results in their buildup in various organs, causing metabolic disorders, oxidative stress, decreased reproductive health, and neurotoxicity. The brain is especially vulnerable to MPs, as they can cross the blood-brain barrier (BBB) and trigger neurobehavioral changes. Recent human research has found that a notable accumulation of low-density polyethylene (LDPE), a versatile and widely used polymer, has been observed in the brains of decedents diagnosed with documented dementia. In this study, we aimed to investigate the neurotoxic effects of MPs-LDPE both in vitro and in vivo. The murine MN-like cell line NSC-34 was treated with <100 μm MPs-LDPE (0.5, 1, 5, 10, 50, and 100 μg/mL), with fluorescent yields measured using Nile Red. Cells were tested in two states: undifferentiated for 4 days and differentiated for 4 days, 48 hours, or 24 hours. Quantitative analysis of cell viability, assessed by the MTS assay and maintained under standard growth conditions for 48 hours, showed a more pronounced toxic effect in differentiated cells compared to undifferentiated ones, especially at concentrations of 50 and 100 μg/mL. Additionally, at these concentrations, Neurotrack analysis indicated that the cells exhibited a significant reduction in neurite length. Adult male CD1 mice were chronically treated orally with a control solution (CON) and with a solution containing a suspension of the same MPs used in vitro at a dose of 50 μg/mL. A group of animals was sacrificed after 45 days of treatment (T1), and a second group after 90 days (T2). The animals were subjected to the initial treatment (T0) and before sacrifice to the NOR test (Novel Object Recognition) and the Morris test, two behavioral tests used to assess memory and cognition in rodents. The NOR test, which evaluates short-term memory through preference for new objects over familiar ones, showed at T1 a 20% decrease in exploratory activity in the LDPE-treated mice and a significant loss of interest in the new object. The Morris Water Maze, which assesses spatial memory, particularly the ability to learn and remember the location of a hidden platform, revealed a significant increase in latency in LDPE-treated mice at T1 in entering the quadrant containing the platform. Analyses at T2 are ongoing, as well as the analysis of brain tissues collected at sacrifice. Our results showed that MPs-LDPE has a dose-dependent toxic effect on NSC-34 cells, making differentiated cells more vulnerable at higher concentrations. In vivo, exposure as early as 45 days caused significant cognitive changes in the animals, affecting both recognition and spatial memory. Ongoing analysis will help clarify how the neurotoxic effect develops with long-term treatment using LDPE microplastics.

References

1. Nihart AJ, et al. Nat Med. 2025 Apr;31(4):1367. doi: 10.1038/s41591-025-03675-x. 
2. Zhang Y, et al. Ecotoxicol Environ Saf. 2024 Jun 15;278:116393. doi: 10.1016/j.ecoenv.2024.116393. 
3. Liang Y, et al. Exp Ther Med. 2022 Aug 19;24(4):630. doi: 10.3892/etm.2022.11567.