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High-throughput phenotypic profiling assays, popular for their ability to characterize alternations in single-cell morphological feature data, have been useful in recent years for predicting cellular targets and mechanisms of action (MoAs) for different chemicals and novel drugs. However, this approach has not been extensively used in environmental toxicology due to the lack of studies and established methods for performing this kind of assay in environmentally relevant species. Here, we developed a multiplexed algal cytological imaging (MACI) assay, based on the subcellular structures of the unicellular microalgae, Raphidocelis subcapitata, a toxicology and ecological model species. Several different herbicides and antibiotics with unique MoAs were exposed to R. subcapitata cells, and MACI was used to characterize cellular impacts by measuring subtle changes in their morphological features, including metrics of area, shape, quantity, fluorescence intensity, and granularity of individual subcellular components. This study demonstrates that MACI offers a quick and effective framework for characterizing complex phenotypic responses to environmental chemicals that can be used for determining their MoAs and identifying their cellular targets in plant-type organisms. 

Currently, there is a lack of knowledge of how complex metal oxide nanomaterials, like LiCoO2 (LCO) nanosheets, interact with eukaryotic green algae. Previously, LCO was reported to cause a number of physiological impacts to Raphidocelis subcapitata including endpoints related to growth, reproduction, pigment & lipid biosynthesis, and carbon biomass assimilation. Furthermore, LCO was proven to physically enter the cells, thus indicating the possibility for it to directly interact with key subcellular components. However, the mechanisms through which LCO interacts with these key subcellular components is still unknown. This study assesses the interactions of LCO at the biointerface of R. subcapitata using a novel multiplexed algal cytological imaging (MACI) assay and machine learning in order to predict its phytotoxic mechanism of action (MoA). Algal cells were exposed to varying concentrations of LCO, and their phenotypic profiles were compared to that of cells treated with reference chemicals which had already established MoAs. Hierarchical clustering and machine learning analyses indicated photosynthetic electron transport to be the most probable phytotoxic MoA of LCO. Additionally, single-cell chlorophyll fluorescence results demonstrated an increase in irreversibly oxidized photosystem II proteins. Lastly, LCO-treated cells were observed to have less nuclei/cell and less DNA content/nucleus when compared to non-treated cell controls. 

Complex metal oxide nanomaterials, like LiCoO2 (LCO) nanosheets, are among the most widespread classes of nanomaterials on the market. Their ubiquitous application in battery storage technology drives their production to rates of environmental significance without sufficient infrastructure for proper disposal/recycling, thus posing a risk to ecosystem health and sustainability. This study assesses the general toxicological impacts of LCO when exposed to Raphidocelis subcapitata; physiological endpoints relating to growth and energy production are considered. Algal growth inhibition was significantly increased at concentrations as low as 0.1 µg·mL?1, while exhibiting an EC50 of 0.057 µg·mL?1. The average biovolume of cells were significantly enlarged at 0.01 µg·mL?1, thus indicating increased instances of cell cycle arrest in LCO-treated cells. Additionally, LCO-treated cells produced significantly less carbon biomass while significantly overproducing neutral lipid content starting at 0.1 µg·mL?1, thus indicating interference with CO2 assimilation chemistry and/or carbon partitioning. However, the relative abundance of chlorophyll was significantly increased, likely to maximize light harvesting and compensate for photosynthetic interference. Cells that were treated with dissolved Li+/Co2+ ions did not significantly impact any of the endpoints tested, suggesting LCO phytotoxicity is mainly induced through nano-specific mechanisms rather than ion-specific. These results indicate that this type of nanomaterial can significantly impact the way this algae proliferates, as well as the way it produces and stores its energy, even at lower, sublethal, concentrations. Furthermore, impairments to crucial cellular pathways, like carbon assimilation, could potentially cause implications at the ecosystem level. Thus, in future work, it will be important to characterize the molecular mechanisms of LCO at the nano-bio interface.This article is protected by copyright. All rights reserved. Environ Toxicol Chem 2023;00:0?0. ? 2023 SETAC. 

Growing evidence across organisms points to altered energy metabolism as an adverse outcome of metal oxide nanomaterial toxicity, with a mechanism of toxicity potentially related to the redox chemistry of processes involved in energy production. Despite this evidence, the significance of this mechanism has gone unrecognized in nanotoxicology due to the field’s focus on oxidative stress as a universal—but non-specific—nanotoxicity mechanism. To further explore metabolic impacts, we determined LCO’s effects on these pathways in the model organism Daphnia magna through global gene expression analysis using RNA-Seq and untargeted metabolomics by direct-injection mass spectrometry. Our results show a sublethal 1 mg/L 48 h exposure of D. magna to LCO nanosheets causes significant impacts on metabolic pathways versus untreated controls, while exposure to ions released over 48 hr does not. Specifically, transcriptomic analysis using DAVID indicated significant enrichment (Benjamini-adjusted p ≤0.0.5) in LCO-exposed animals for changes in pathways involved in the cellular response to starvation (25 genes), mitochondrial function (70 genes), ATP-binding (70 genes), oxidative phosphorylation (53 genes), NADH dehydrogenase activity (12 genes), and protein biosynthesis (40 genes). Metabolomic analysis using MetaboAnalyst indicated significant enrichment (gamma-adjusted p < 0.1) for changes in amino acid metabolism (19 metabolites) and starch, sucrose, and galactose metabolism (7 metabolites). Overlap of significantly impacted pathways by RNA-Seq and metabolomics suggests amino acid breakdown and increased sugar import for energy production. Results indicate that LCO-exposed Daphnia are responding to energy starvation by altering metabolic pathways, both at the gene expression and metabolite level. These results support altered energy production as a sensitive nanotoxicity adverse outcome for LCO exposure and suggest negative impacts on energy metabolism as an important avenue for future studies of nanotoxicity, including for other biological systems and for metal oxide nanomaterials more broadly.