Interaction of TiO2 nanoparticles with lung fluid proteins and the resulting macrophage inflammatory response.

Inhalation is a major exposure route to nanoparticles. Following inhalation, nanoparticles first interact with the lung lining fluid, a complex mixture of proteins, lipids, and mucins. We measure the concentration and composition of lung fluid proteins adsorbed on the surface of titanium dioxide (TiO2) nanoparticles. Using proteomics, we find that lung fluid results in a unique protein corona on the surface of the TiO2 nanoparticles. We then measure the expression of three cytokines (interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and macrophage inflammatory protein 2 (MIP-2)) associated with lung inflammation. We find that the corona formed from lung fluid leads to elevated expression of these cytokines in comparison to bare TiO2 nanoparticles or coronas formed from serum or albumin. These experiments show that understanding the concentration and composition of the protein corona is essential for understanding the pulmonary response associated with human exposure to nanoparticles.

Use of Magnetic Modulation of Nitrogen-Vacancy Center Fluorescence in Nanodiamonds for Quantitative Analysis of Nanoparticles in Organisms.

The fluorescence intensity emitted by nitrogen-vacancy (NV) centers in diamond nanoparticles can be readily modulated by the application of a magnetic field using a small electromagnet. By acquiring interleaved images acquired in the presence and absence of the magnetic field and performing digital subtraction, the fluorescence intensity of the NV nanodiamond can be isolated from scattering and autofluorescence even when these backgrounds are changing monotonically during the experiments. This approach has the potential to enable the robust identification of nanodiamonds in organisms and other complex environments. Yet, the practical application of magnetic modulation imaging to realistic systems requires the use of quantitative analysis methods based on signal-to-noise considerations. Here, we describe the use of magnetic modulation to analyze the uptake of diamond nanoparticles from an aqueous environment into Caenorhabditis elegans, used here as a model system for identification and quantification of nanodiamonds in complex matrices. Based on the observed signal-to-noise ratio of sets of digitally subtracted images, we show that nanodiamonds can be identified on an individual pixel basis with a >99.95% confidence. To determine whether surface functionalization of the nanodiamond significantly impacted uptake, we used this approach to analyze the presence of nanodiamonds in C. elegans that had been exposed to these functionalized nanodiamonds in the water column, with uptake likely occurring by ingestion. In each case, the images show a significant nanoparticle uptake. However, differences in uptake between the three ligands were not outside of the experimental error, indicating that additional factors beyond the surface charge are important factors controlling uptake. Analysis of the number of pixels above the threshold in individual C. elegans organisms revealed distributions that deviate significantly from a Poisson distribution, suggesting that uptake of nanoparticles may not be a statistically independent event. The results presented here demonstrate that magnetic modulation combined with quantitative analysis of the resulting images can be used to robustly characterize nanoparticle uptake into organisms.

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.

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.

Next-Generation Complex Metal Oxide Nanomaterials Negatively Impact Growth and Development in the Benthic Invertebrate Chironomus riparius upon Settling.

Most studies of nanomaterial environmental impacts have focused on relatively simple first-generation nanomaterials, including metals or metal oxides (e.g., Ag, ZnO) for which dissolution largely accounts for toxicity. Few studies have considered nanomaterials with more complex compositions, such as complex metal oxides, which represent an emerging class of next-generation nanomaterials used in commercial products at large scales. Importantly, many nanomaterials are not colloidally stable in aqueous environments and will aggregate and settle, yet most studies use pelagic rather than benthic-dwelling organisms. Here we show that exposure of the model benthic species Chironomus riparius to lithium cobalt oxide (Li xCo1- xO2, LCO) and lithium nickel manganese cobalt oxide (Li xNi yMn zCo1- y- zO2, NMC) at 10 and 100 mg·L-1 caused 30-60% declines in larval growth and a delay of 7-25 d in adult emergence. A correlated 41-48% decline in larval hemoglobin concentration and related gene expression changes suggest a potential adverse outcome pathway. Metal ions released from nanoparticles do not cause equivalent impacts, indicating a nanospecific effect. Nanomaterials settled within 2 days and indicate higher cumulative exposures to sediment organisms than those in the water column, making this a potentially realistic environmental exposure. Differences in toxicity between NMC and LCO indicate compositional tuning may reduce material impact.

Low-dose metformin exposure causes changes in expression of endocrine disruption-associated genes.

The presence of intersex fish in watersheds around the world is a warning of the presence of anthropogenic endocrine-disrupting compounds (EDCs) being deposited into the aquatic environment. The anti-diabetic drug metformin is among the most prevalent and ubiquitous of the myriad pharmaceuticals found in wastewater effluent and watersheds worldwide. In addition to its prescription for type-2 diabetes, metformin is indicated as a treatment in cancers and the endocrine disorder polycystic ovarian syndrome (PCOS). Our previous research found evidence of endocrine-disruption following Pimephales promelas (fathead minnow) exposure to metformin at an environmentally relevant concentration. However, the mechanism of action leading to these impacts is unknown. Although metformin does not structurally resemble classical EDCs, there's an increasing recognition that endocrine disruption may occur by mechanisms other than classical endocrine receptor binding, and metformin's off-label use for treating endocrine-related disorders such as PCOS indicates its potential interaction with the endocrine system. To further explore metformin's mechanism of action as an EDC, we measured expression of numerous endocrine-related genes in male fathead minnows exposed to metformin at a low-dose similar to that found in wastewater effluent and the environment (40 µg L-1) for a full year (early development to adulthood) and discovered significant upregulation of the AR (3.6 ±â€¯0.9-fold), 3ß-HSD (3.9 ±â€¯0.8-fold), 17ß-HSD (17 ±â€¯4-fold), CYP19A1 (40 ±â€¯20-fold), and SULT2A1 (2.3 ±â€¯0.4-fold) genes in exposed male gonad. We also found a significant correlation between expression of 3ß-HSD, 17ß-HSD, and CYP19A1 in testis of metformin-treated male fish and the degree of intersex occurring in their gonads. These results provide additional evidence of the endocrine disrupting impact of the drug metformin and insight into the potential mechanisms by which metformin may influence the endocrine system in aquatic organisms.

Intracellular disulfide reduction by phosphine-borane complexes: Mechanism of action for neuroprotection.

Phosphine-borane complexes are novel cell-permeable drugs that protect neurons from axonal injury in vitro and in vivo. These drugs activate the extracellular signal-regulated kinases 1/2 (ERK1/2) cell survival pathway and are therefore neuroprotective, but do not scavenge superoxide. In order to understand the interaction between superoxide signaling of neuronal death and the action of phosphine-borane complexes, their biochemical activity in cell-free and in vitro assays was studied by electron paramagnetic resonance (EPR) spectrometry and using an intracellular dithiol reporter that becomes fluorescent when its disulfide bond is cleaved. These studies demonstrated that bis(3-propionic acid methyl ester) phenylphosphine-borane complex (PB1) and (3-propionic acid methyl ester) diphenylphosphine-borane complex (PB2) are potent intracellular disulfide reducing agents which are cell permeable. EPR and pharmacological studies demonstrated reducing activity but not scavenging of superoxide. Given that phosphine-borane complexes reduce cell injury from mitochondrial superoxide generation but do not scavenge superoxide, this implies a mechanism where an intracellular superoxide burst induces downstream formation of protein disulfides. The redox-dependent cleavage of the disulfides is therefore a novel mechanism of neuroprotection.

Emerging wastewater contaminant metformin causes intersex and reduced fecundity in fish.

The occurrence of intersex fish, where male reproductive tissues show evidence of feminization, have been found in freshwater systems around the world, indicating the potential for significant endocrine disruption across species in the ecosystem. Estrogens from birth control medications in wastewater treatment plant effluent have been cited as the likely cause, but research has shown that endocrine disruption is not solely predictable based on hormone receptor interactions. Many other non-hormone pharmaceuticals are found in effluent at concentrations orders of magnitude higher than estrogens, yet there is little data indicating the impacts of these other medications. The widely prescribed anti-diabetic metformin is among the most abundant of pharmaceuticals found in effluent and is structurally dissimilar from hormones. However, we show here that exposing fathead minnows (Pimephales promelas) to a concentration of metformin found in wastewater effluent causes the development of intersex gonads in males, reduced size of treated male fish, and reduction in fecundity for treated pairs. Our results demonstrate that metformin acts as an endocrine disruptor at environmentally relevant concentrations.

Metformin exposure at environmentally relevant concentrations causes potential endocrine disruption in adult male fish.

Pharmaceuticals and personal care products (PPCPs) are emerging contaminants that have been found ubiquitously in wastewater and surface waters around the world. A major source of these compounds is incomplete metabolism in humans and subsequent excretion in human waste, resulting in discharge into surface waters by wastewater treatment plant (WWTP) effluent. One pharmaceutical found in particularly high abundance in recent WWTP effluent and surface water studies is metformin, one of the world's most widely prescribed antidiabetic drugs. Interactions between insulin signaling and steroidogenesis suggest potential endocrine-disrupting effects of metformin found in the aquatic environment. Adult fathead minnows (Pimephales promelas) were chronically exposed to metformin for 4 wk, at 40 µg/L, a level similar to the average found in WWTP effluent in Milwaukee, Wisconsin, USA. Genetic endpoints related to metabolism and endocrine function as well as reproduction-related endpoints were examined. Metformin treatment induced significant up-regulation of messenger ribonucleic acid (mRNA) encoding the egg-protein vitellogenin in male fish, an indication of endocrine disruption. The present study, the first to study the effects of environmentally relevant metformin exposure in fathead minnows, demonstrates the need for further study of the endocrine-disrupting effects of metformin in aquatic organisms.

Quantitative in vivo redox sensors uncover oxidative stress as an early event in life.

Obstacles in elucidating the role of oxidative stress in aging include difficulties in (1) tracking in vivo oxidants, in (2) identifying affected proteins, and in (3) correlating changes in oxidant levels with life span. Here, we used quantitative redox proteomics to determine the onset and the cellular targets of oxidative stress during Caenorhabditis elegans' life span. In parallel, we used genetically encoded sensor proteins to determine peroxide levels in live animals in real time. We discovered that C. elegans encounters significant levels of oxidants as early as during larval development. Oxidant levels drop rapidly as animals mature, and reducing conditions prevail throughout the reproductive age, after which age-accompanied protein oxidation sets in. Long-lived daf-2 mutants transition faster to reducing conditions, whereas short-lived daf-16 mutants retain higher oxidant levels throughout their mature life. These results suggest that animals with improved capacity to recover from early oxidative stress have significant advantages later in life.