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Cellular, Subcellular, and Bioenergetic Effects of Functionalized Nanoplastics and Emerging Plasticizers on Rainbow Trout Gill Epithelial Cells
Summary
Scientists tested tiny plastic particles (nanoplastics) and plastic softening chemicals on fish gill cells to understand how these pollutants might harm living tissue at the cellular level. They found that positively charged plastic particles were the most damaging, disrupting the cell's "power plants" (mitochondria) and reducing their ability to produce energy—even at doses too low to kill the cells outright. This matters because it shows plastic pollution can cause hidden cellular damage before any visible harm appears, and since fish are a common entry point for microplastics into the food chain, understanding these effects helps scientists predict risks that could
Nanoplastics are emerging contaminants of concern in freshwater ecosystems and have been shown to be ingested and assimilated tissues of fish and other organisms. Understanding the nano-bio interactions and potential impacts of these pollutants is essential. While previous work has focused on traditional whole organism endpoints of toxicity (i.e., reproduction, survival and growth) at lethal concentrations, there remains a critical gap in understanding the sublethal effects and mechanisms of action in environmentally relevant models. For many pollutants we are just beginning to understand how pollutants act through impacts on cellular metabolism and morphology. Additionally, plasticizers within plastics are an additional understudied aspect in plastic pollution. Plasticizers represent a major class of additives that are not covalently bonded to the polymer matrix and are prone to leaching into aquatic environments. While legacy plasticizers have been extensively researched, emerging alternative replacements remain poorly characterized and the cellular effects of combined nanoplastic-plasticizer exposure remain unclear. In this dissertation, we utilized pristine polystyrene spheres using three model particles of differing functional groups representing positive, negative and neutral surfaces charges since surface charge is well-established as an important factor in determining biological impacts. RTgill-W1 (Oncorhynchus mykiss, Rainbow trout gill epithelia), were used as a model to investigate sensitive cellular impacts. By employing high-content imaging, phenotypic profiling, and mitochondrial bioenergetic analyses we found that surface chemistry played a major role in cellular and subcellular responses. With a traditional cytotoxicity assay, we determined that PS-NH2 NPs (amine-functionalized polystyrene nanoplastics) significantly decreased cell viability compared to its neutral and negatively charged counterparts. Additionally, distinct phenotypic responses at sublethal concentrations suggested early nano-bio interactions in subcellular compartments where no cytotoxicity was observed due to impacts to cellular actin and the mitochondria. The results suggested that the mitochondria may be a primary target of NP-induced stress and provided the rationale for further investigation even though multiple organelles were affected. Subsequent analyses demonstrated that PS-NH2 NPs disrupted mitochondrial bioenergetics. By conducting the Seahorse XF Mito Stress Test, a high-throughput respirometer, we found that PS-NH2 NPs decreased basal and maximal respiration, altered ATP-linked respiration (Adenosine Triphosphate) and induced changes to mitochondrial morphology indicating disruption of mitochondrial function. These findings expand mechanistic knowledge of PS NP toxicity and suggest that the mitochondria may play a key role in governing nanoplastic-induced cellular responses in particles of cationic surface chemistry. These results present opportunities for further study of identifying which specific complex of the electron transport chain is impaired and if increase in proton leakage is associated with a reduction in the cardiolipin content of the inner mitochondrial membrane allowing the exploration of novel mechanisms enhancing our understanding in nano-mito interactions. The second knowledge gap is the need to better characterize the cellular and subcellular effects of emerging alternative plasticizers, and of that combined nano-plasticizer exposure. The current work investigated how RTgill-W1 are perturbed by replacement compounds and will be paired with polyethylene terephthalate nanoplastics (nanoPET) to compare effects between nanoPET with and without the selected additive of bis(2-ethylhexyl) adipate (DOA) by applying phenotypic profiling. The results demonstrated that replacement plasticizers and NPs with additive are not biologically inert, generating distinct phenotypic fingerprints and shared subcellular targets. Combined nanoPET-DOA treatments revealed both additive-driven and particle specific effects, with mitochondrial responses dominated by the plasticizers and other features reflecting particle-specific effects.