Cross-species transcriptomic signatures identify mechanisms related to species sensitivity and common responses to nanomaterials.

Physico-chemical characteristics of engineered nanomaterials are known to be important in determining the impact on organisms but effects are equally dependent upon the characteristics of the organism exposed. Species sensitivity may vary by orders of magnitude, which could be due to differences in the type or magnitude of the biochemical response, exposure or uptake of nanomaterials. Synthesizing conclusions across studies and species is difficult as multiple species are not often included in a study, and differences in batches of nanomaterials, the exposure duration and media across experiments confound comparisons. Here three model species, Danio rerio, Daphnia magna and Chironomus riparius, that differ in sensitivity to lithium cobalt oxide nanosheets are found to differ in immune-response, iron-sulfur protein and central nervous system pathways, among others. Nanomaterial uptake and dissolution does not fully explain cross-species differences. This comparison provides insight into how biomolecular responses across species relate to the varying sensitivity to nanomaterials.

Expression Patterns of Energy-Related Genes in Single Cells Uncover Key Isoforms and Enzymes That Gain Priority Under Nanoparticle-Induced Stress.

Cellular responses to nanoparticles (NPs) have been largely studied in cell populations, providing averaged values that often misrepresent the true molecular processes that occur in the individual cell. To understand how a cell redistributes limited molecular resources to achieve optimal response and survival requires single-cell analysis. Here we applied multiplex single molecule-based fluorescence in situ hybridization (fliFISH) to quantify the expression of 10 genes simultaneously in individual intact cells, including glycolysis and glucose transporter genes, which are critical for restoring and maintaining energy balance. We focused on individual gill epithelial cell responses to lithium cobalt oxide (LCO) NPs, which are actively pursued as cathode materials in lithium-ion batteries, raising concerns about their impact on the environment and human health. We found large variabilities in the expression levels of all genes between neighboring cells under the same exposure conditions, from only a few transcripts to over 100 copies in individual cells. Gene expression ratios among the 10 genes in each cell uncovered shifts in favor of genes that play key roles in restoring and maintaining energy balance. Among these genes are isoforms that can secure and increase glycolysis rates more efficiently, as well as genes with multiple cellular functions, in addition to glycolysis, including DNA repair, regulation of gene expression, cell cycle progression, and proliferation. Our study uncovered prioritization of gene expression in individual cells for restoring energy balance under LCO NP exposures. Broadly, our study gained insight into single-cell strategies for redistributing limited resources to achieve optimal response and survival under stress.

Energy Starvation in Daphnia magna from Exposure to a Lithium Cobalt Oxide Nanomaterial.

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 nonspecific─nanotoxicity mechanism. To further explore metabolic impacts, we determined lithium cobalt oxide's (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 that 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 h 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 (γ-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 respond to energy starvation by altering metabolic pathways, both at the gene expression and metabolite levels. 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.

Protein Fe-S Centers as a Molecular Target of Toxicity of a Complex Transition Metal Oxide Nanomaterial with Downstream Impacts on Metabolism and Growth.

Oxidative stress is frequently identified as a mechanism of toxicity of nanomaterials. However, rarely have the specific underlying molecular targets responsible for these impacts been identified. We previously demonstrated significant negative impacts of transition metal oxide (TMO) lithium-ion battery cathode nanomaterial, lithium cobalt oxide (LCO), on the growth, development, hemoglobin, and heme synthesis gene expression in the larvae of a model sediment invertebrate Chironomus riparius. Here, we propose that alteration of the Fe-S protein function by LCO is a molecular initiating event leading to these changes. A 10 mg/L LCO exposure causes significant oxidation of the aconitase 4Fe-4S center after 7 d as determined from the electron paramagnetic resonance spectroscopy measurements of intact larvae and a significant reduction in the aconitase activity of larval protein after 48 h (p < 0.05). Next-generation RNA sequencing identified significant changes in the expression of genes involved in 4Fe-4S center binding, Fe-S center synthesis, iron ion binding, and metabolism for 10 mg/L LCO at 48 h (FDR-adjusted, p < 0.1). We propose an adverse outcome pathway, where the oxidation of metabolic and regulatory Fe-S centers of proteins by LCO disrupts metabolic homeostasis, which negatively impacts the growth and development, a mechanism that may apply for these conserved proteins across species and for other TMO nanomaterials.

Anionic nanoparticle-induced perturbation to phospholipid membranes affects ion channel function.

Understanding the mechanisms of nanoparticle interaction with cell membranes is essential for designing materials for applications such as bioimaging and drug delivery, as well as for assessing engineered nanomaterial safety. Much attention has focused on nanoparticles that bind strongly to biological membranes or induce membrane damage, leading to adverse impacts on cells. More subtle effects on membrane function mediated via changes in biophysical properties of the phospholipid bilayer have received little study. Here, we combine electrophysiology measurements, infrared spectroscopy, and molecular dynamics simulations to obtain insight into a mode of nanoparticle-mediated modulation of membrane protein function that was previously only hinted at in prior work. Electrophysiology measurements on gramicidin A (gA) ion channels embedded in planar suspended lipid bilayers demonstrate that anionic gold nanoparticles (AuNPs) reduce channel activity and extend channel lifetimes without disrupting membrane integrity, in a manner consistent with changes in membrane mechanical properties. Vibrational spectroscopy indicates that AuNP interaction with the bilayer does not perturb the conformation of membrane-embedded gA. Molecular dynamics simulations reinforce the experimental findings, showing that anionic AuNPs do not directly interact with embedded gA channels but perturb the local properties of lipid bilayers. Our results are most consistent with a mechanism in which anionic AuNPs disrupt ion channel function in an indirect manner by altering the mechanical properties of the surrounding bilayer. Alteration of membrane mechanical properties represents a potentially important mechanism by which nanoparticles induce biological effects, as the function of many embedded membrane proteins depends on phospholipid bilayer biophysical properties.

Interaction of Phosphate with Lithium Cobalt Oxide Nanoparticles: A Combined Spectroscopic and Calorimetric Study.

Adsorption of small ions such as phosphates to the surfaces of metal oxides can significantly alter the behavior of these materials, especially when present in the nanoscale form. Lithium cobalt oxide is a good model system for understanding small-molecule interactions with emerging nanomaterials because of its widespread use in lithium ion batteries and its known activity as a water oxidation catalyst. Here, we present a thermodynamic analysis of phosphate adsorption to LiCoO2 and corroborate the results with additional in situ techniques, including zeta potential measurements and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, at pH values relevant to potential environmental release scenarios. Flow microcalorimetry measurements of phosphate interaction with LiCoO2 at pH 7.4 show that there are two distinct exothermic processes taking place. Time-sequence in situ ATR-FTIR with two-dimensional correlation analysis reveals the spectroscopic signatures of these processes. We interpret the data as an interaction of phosphate with LiCoO2 that occurs through the release of two water molecules and is therefore, best described as a condensation process rather than a simple adsorption, consistent with prior studies, demonstrating that phosphate interaction with LiCoO2 is highly irreversible. Additional measurements for over longer times of 5 months show that phosphate adsorption terminates with one surface layer and that continued transformation over longer periods of time arises from H+/Li+ exchange and slow transformation to a cobalt hydroxide, with phosphate adsorbed to the surface only. To the best of our knowledge, this is the first time that flow microcalorimetry and two-dimensional correlation spectroscopy have been applied in tandem to clarify the specific chemical reactions that occur at the interface of solids and adsorbates.

Quantitative Mapping of Oxidative Stress Response to Lithium Cobalt Oxide Nanoparticles in Single Cells Using Multiplexed in Situ Gene Expression Analysis.

Engineered nanoparticles (NPs) can negatively impact biological systems through induced generation of reactive oxygen species (ROS). Overproduced ROS cause biochemical damage and hence need to be effectively buffered by a sophisticated cellular oxidative stress response system. How this complex cellular system, which consists of multiple enzymes, responds to NP-induced ROS is largely unknown. Here, we apply a single cell analysis to quantitatively evaluate 10 key ROS responsive genes simultaneously to understand how the cell prioritizes tasks and reallocates resources in response to NP-induced oxidative stress. We focus on rainbow trout gill epithelial cells-a model cell type for environmental exposure-and their response to the massive generation of ROS induced by lithium cobalt oxide (LCO) NPs, which are extensively used as cathode materials in lithium ion batteries. Using multiplexed fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) in single cells, we found a shift in the expression of oxidative stress response genes with initial increase in genes targeting superoxide species, followed by increase in genes targeting peroxide and hydroxyl species. In contrast, Li+ and Co2+, at concentrations expected to be shed from the NPs, did not induce ROS generation but showed a potent inhibition of transcription for all 10 stress response genes. Taken together, our findings suggest a "two-hit" model for LCO NP toxicity, where the intact LCO NPs induce high levels of ROS that elicit sequential engagement of stress response genes, while the released metal ions suppress the expression of these genes. Consequently, these effects synergistically drive the exposed cells to become more vulnerable to ROS stress and damage.

Impact of Phosphate Adsorption on Complex Cobalt Oxide Nanoparticle Dispersibility in Aqueous Media.

A commonly overlooked and largely unknown aspect of assessing the environmental and biological safety of engineered nanomaterials is their transformation in aqueous systems. Complex metal oxides are an important class of materials for catalysis, energy storage, and water purification. However, the potential impact of nano complex metal oxides on the environment upon improper disposal is not well understood. We present a comprehensive analysis of the interaction of an environmentally relevant oxyanion, phosphate, with a complex metal oxide nanomaterial, lithium cobalt oxide. Our results show that adsorption of phosphate to the surface of these materials drastically impacts their surface charge, rendering them more stable in aqueous systems. The adsorbed phosphate remains on the surface over significant periods of time, suggesting that desorption is not kinetically favored. The implications of this interaction may be increased dispersibility and bioavailability of these materials in environmental water systems.