Key events relating to homeostasis and regeneration of freshwater planarians (Dugesia Japonica) after exposure to various ZnO-forms

Abstract

This study aims to investigate the toxicity and underlying mechanisms of ZnO nanoparticles (ZnO NPs), bulk ZnO (ZnO MPs), and zinc ions (Zn^2 +) on Dugesia japonica planarians, with a focus on their bioaccumulation, transformation, and associated biological effects. Using advanced techniques such as synchrotron X-ray fluorescence (XRF), X-ray Absorption Near Edge Structure (XANES) and single particle ICP-MS (sp-ICP-MS), we measured the accumulation, distribution, and transformation of these materials in planarians. All treatments caused significant Zn accumulation: ZnO NPs increased Zn by 120-fold, ZnO MPs by 100-fold, and Zn²⁺ by 430-fold. XANES and sp-ICP-MS analysis confirmed that ZnO NPs remained largely in particulate form (40–60 %) following uptake by planarians. Toxicity tests revealed that all treatments impaired blastema growth, locomotion, stem cell proliferation, differentiation, and neural regeneration. ZnO MPs exhibited higher toxicity than ZnO NPs, while Zn²⁺ resulted in elevated oxidative stress. ZnO NPs induced severe energy damage and triggered cell apoptosis, whereas ZnO MPs caused more pronounced necrosis cell death. Transcriptomic and proteomic analyses showed that all treatments disrupted pathways related to oxidative stress response, energy metabolism and cell apoptosis. ZnO NPs primarily affected the membrane integrity pathway, ZnO MPs altered cell homeostasis and membrane potential, while Zn²⁺ exposure triggered metal ion-specific cellular reactions. These molecular and cellular changes collectively explain the observed phenotypic outcomes, which align with the Adverse Outcome Pathway framework. The findings provide insights into the environmental risks of different ZnO forms and highlight their distinct toxicity mechanisms.