Differential sensitivity of hemocyte subpopulations (Mytilus edulis) to aged polyethylene terephthalate micro- and nanoplastic particles

Abstract

Bivalve hemocytes, particularly granulocytes and hyalinocytes, play a crucial role in cell-mediated immunity. However, their interactions with aged plastic particles, exhibiting altered properties that more closely resemble those in natural environments, remain largely underexplored. This study assesses the differential responses of hemocyte subpopulations (Mytilus edulis) to chemically aged polyethylene terephthalate (PET) microplastic (MPs) and nanoplastic (NPs) particles across multiple cellular effect endpoints. Particle characteristics were analyzed using Single Particle Extinction and Scattering, Raman Spectroscopy, Scanning Electron Microscopy, and Dynamic Light Scattering. In vitro experiments with aged PET MPs (1.9 µm) and NPs (0.68 µm) were conducted at three internally relevant concentrations: 10 (C1), 10³ (C2), and 10⁵ particles/mL (C3). Cellular responses were assessed by measuring lysosomal content stability, reactive oxygen species (ROS) production, cellular mortality, and morphological parameters using flow cytometry at 6, 12, 24, and 48 hours. Our findings provide mechanistic insights into the differential sensitivities of granulocytes and hyalinocytes to aged PET, influenced by particle size and concentration. Specifically, aged PET MPs and NPs induce distinct size and concentration-dependent patterns of lysosomal destabilization, coinciding with the loss of functional integrity. Elevated ROS levels were observed only in granulocytes and hyalinocytes exposed to high concentrations of aged PET NPs, underscoring the effects on oxidative stress. Both aged PET MPs and NPs induce significant increases in cellular mortality, particularly after 24 h of exposure at high concentrations. These findings reveal the complex cellular mechanisms underlying hemocyte functional impairment following exposure to aged PET particles under environmentally and biologically relevant conditions.

Keywords: Aged PET, Hemocytes; Flow cytometry; Functional assays; Immunotoxicity; In-vitro.