James D. Ede

Widespread nanoparticle-assay interference: implications for nanotoxicity testing.

The evaluation of engineered nanomaterial safety has been hindered by conflicting reports demonstrating differential degrees of toxicity with the same nanoparticles. The unique properties of these materials increase the likelihood that they will interfere with analytical techniques, which may contribute to this phenomenon. We tested the potential for: 1) nanoparticle intrinsic fluorescence/absorbance, 2) interactions between nanoparticles and assay components, and 3) the effects of adding both nanoparticles and analytes to an assay, to interfere with the accurate assessment of toxicity. Silicon, cadmium selenide, titanium dioxide, and helical rosette nanotubes each affected at least one of the six assays tested, resulting in either substantial over- or under-estimations of toxicity. Simulation of realistic assay conditions revealed that interference could not be predicted solely by interactions between nanoparticles and assay components. Moreover, the nature and degree of interference cannot be predicted solely based on our current understanding of nanomaterial behaviour. A literature survey indicated that ca. 95% of papers from 2010 using biochemical techniques to assess nanotoxicity did not account for potential interference of nanoparticles, and this number had not substantially improved in 2012. We provide guidance on avoiding and/or controlling for such interference to improve the accuracy of nanotoxicity assessments.

Evaluating the Toxicity of Hydroxyapatite Nanoparticles in Catfish Cells and Zebrafish Embryos

The toxicity of needle-(nHA-ND) and rod-shaped (nHA-RD) hydroxyapatite (HA) nanoparticles is evaluated in vitro on catfish B-cells (3B11) and catfish T-cells (28s.3) and in vivo on zebrafish embryos to determine if biological effects are similar to the effects seen in mammalian in vitro systems. Neither nHA-ND nor nHA-RD affect cell viability at concentrations of 10 to 300 μg mL(-1) . However, 30 μg mL(-1) needle-shaped nHA lower metabolic activity of the cells. Axial deformations are seen in zebrafish exposed to 300 μg mL(-1) needle shaped nHA after 120 h. For the first time, nHA is reported to cause zebrafish hatching delay. The lowest concentration (3 μg mL(-1) ) of both types of nHA cause the highest hatching inhibition and needle-shaped nHA exposed zebrafish exhibit the lowest hatch at 72 h post fertilization.

Physicochemical characteristics of polymer-coated metal-oxide nanoparticles and their toxicological effects on zebrafish (Danio rerio) development.

Coated nanoparticles (NPs) will end up in the environment due to their proposed use in agricultural applications and may potentially cause toxic effects due to their unique properties. To determine the effects of coated NPs on zebrafish (Danio rerio) development, we tested aqueous poly(acrylic acid) (PAA)-coated metal-oxide NPs including TiO2, ZnO, Fe2O3, and CeO2, as well as the polymer coating alone (nanocapsule). Zebrafish embryos were exposed to NPs over a 72 h period at 1, 10, 50, 100, 200, 400, 800, 1200, 1600, and 2000 mg/L to measure various end points. We also ran free metal controls. Time-dependent changes in physicochemical properties of NPs were characterized using dynamic light scattering. Dissolution experiments over 72 h showed minimal free metals were present in stock suspensions and released from the NPs. Interestingly, nanocapsules (≥ 800 mg/L) cause inhibition of hatch, and we suggest that a low pH environment may explain this effect. This study has also demonstrated that CeO2 NPs and nanocapsules containing Nile red are able to traverse the chorion. Overall, our findings indicate that each NP type is stable and neither the NP or encapsulating PAA coating causes apparent toxicity to developing zebrafish.

Humic acid ameliorates nanoparticle-induced developmental toxicity in zebrafish

Many aquatic toxicity experiments are not performed under realistic environmental conditions. We examined several inorganic and organic nanoparticle (NP) formulations (citrate-capped silver NPs, carboxylic acid functionalized single-walled carbon nanotubes, mercaptoundecanoic acid functionalized cadmium selenide NPs, poly(acrylic acid) (PAA)-coated zinc oxide [ZnO] NPs, Nile red-loaded PAA nanocapsules, and uncoated sphere- and leaf-shaped ZnO NPs) to determine whether the presence of humic acid (HA) affects the physicochemical properties (e.g. size, zeta potential, and particle dissolution) of NPs in suspension and ameliorates noted effects on zebrafish (Danio rerio) development. We investigated the toxicological effects of these NPs with and without HA addition during early stages of zebrafish development by measuring survival, hatching success, length, head-tail angle, movement, and protease activity in the chorionic fluid, as well as NP interaction with the chorion. Though NP-induced effects on survival were not mitigated by the presence of HA, hatching inhibition and reduced head-tail angle in developing zebrafish caused by certain NPs were restored to near control values by HA addition. Interestingly, despite the ameliorating effects noted with the addition of HA, combined NP and HA treatments still resulted in reduced enzyme activity and NP interaction with the zebrafish embryo. We suggest that observed effects were NP-specific and not attributed to ionic metal species. In the interest of performing more environmentally representative toxicity studies, HA should be included in standardized laboratory nanotoxicity tests since it alters certain biological effects.

Regulation of plasma glucose and sulfate excretion in Pacific hagfish, Eptatretus stoutii is not mediated by 11-deoxycortisol.

The goal of this study was to identify whether Pacific hagfish (Eptatretus stoutii) possess glucocorticoid and mineralocorticoid responses and to examine the potential role(s) of four key steroids in these responses. Pacific hagfish were injected with varying amounts of cortisol, corticosterone or 11-deoxycorticosterone (DOC) using coconut oil implants and plasma glucose and gill total-ATPase activity were monitored as indices of glucocorticoid and mineralocorticoid responses. Furthermore, we also monitored plasma glucose and 11-deoxycortisol (11-DOC) levels following exhaustive stress (30 min of agitation) or following repeated infusion with SO42-. There were no changes in gill total-ATPase following implantation with any steroid, with only very small statistical increases in plasma glucose noted in hagfish implanted with either DOC (at 20 and 200mgkg-1 at 7 and 4days post-injection, respectively) or corticosterone (at 100mgkg-1 at 7days post-injection). Following exhaustive stress, hagfish displayed a large and sustained increase in plasma glucose. Repeated infusion of SO42- into hagfish caused increases in both plasma glucose levels and SO42- excretion rate suggesting a regulated glucocorticoid and mineralocorticoid response. However, animals under either condition did not show any significant increases in plasma 11-DOC concentrations. Our results suggest that while there are active glucocorticoid and mineralocorticoid responses in hagfish, 11-DOC does not appear to be involved and the identity and primary function of the steroid in hagfish remains to be elucidated.

Rosette nanotubes alter IgE-mediated degranulation in the rat basophilic leukemia (RBL)-2H3 cell line

In this study, the effects of rosette nanotube (RNT) exposure on immune cell viability and function were investigated in vitro using the rat basophilic leukemia (RBL)-2H3 cell line. RBL-2H3 viability was decreased in a dose- and time-dependent manner after lysine-functionalized RNT (K-RNT) exposure. In addition, K-RNTs had a significant effect on RBL-2H3 degranulation. When K-RNT exposure was concurrent with IgE sensitization, 50 and 100 mg l(-1) K-RNTs elicited a heightened degranulatory response compared with IgE alone. Exposure to 50 and 100 mg l(-1) K-RNTs also caused degranulation in RBL-2H3 cells not sensitized with IgE (0 ng ml(-1) IgE). Furthermore, in cells preexposed to K-RNTs for 2 h and subsequently washed, sensitized, and stimulated with IgE, a potentiated degranulatory response was observed. Using confocal laser scanning microscopy and a fluorescein isothiocyanate (FITC)-functionalized RNT construct (termed FITC(1)/TBL(19)-RNT), we demonstrated a strong and direct affiliation between RNTs and RBL-2H3 cell membranes. We also demonstrated cellular internalization of RNTs after 2 h of exposure. Together, these data demonstrate that RNTs may affiliate with the cellular membrane of RBL-2H3 cells and can be internalized. These interactions can affect viability and alter the ability of these cells to elicit IgE-FcεR mediated degranulation.

Polymer-Coated Metal-Oxide Nanoparticles Inhibit IgE Receptor Binding, Cellular Signaling, and Degranulation in a Mast Cell-like Cell Line

Previous reports have shown that nanoparticles (NPs) can both enhance and suppress immune effector functions; however the mechanisms that dictate these responses are still unclear. Here, the effects of polyacrylic acid (PAA) functionalized metal‐oxide NP are investigated on RBL‐2H3 (representative mammalian granulocyte‐like cell line) cell viability, cellular degranulation, immunoglobulin E (IgE) receptor binding, and cell signaling pathways related to immune function. The increasing development of PAA‐NPs as pesticide dispersants and as drug carriers in therapeutics necessitates their investigation for safe production. Using two in vitro experimental approaches, this study demonstrates that pre‐exposing RBL‐2H3 cells, or IgE antibodies, to PAA‐NPs (TiO2, CeO2, ZnO, Fe2O3, and PAA‐Capsules (NP coating control) over 24 h, significantly decrease the binding capacity of IgE for Fcε receptors, inhibit the phosphorylation of intracellular signaling proteins (e.g., MAPK ERK) that mediate degranulation, and inhibited RBL‐2H3 cell degranulation. In addition, and unlike the other NPs tested, PAA‐TiO2 significantly reduced RBL‐2H3 viability, in a time (4–24 h) and dose‐dependent manner (>50 μg mL−1). Together, these data demonstrate that PAA‐NPs at sub‐lethal doses can interact with cell surface structures, such as receptors, to suppress various stages of the RBL‐2H3 degranulatory response to external stimuli, and modify immune cell functions that can impact host‐immunity.

The effects of rosette nanotubes with different functionalizations on channel catfish (Ictalurus punctatus) lymphocyte viability and receptor function

The effects of self-assembled rosette nanotubes (RNTs) with different surface functionalizations (K-, TBL-, RGDSK1/TBL9-) on fish lymphocyte viability and effector function were examined using channel catfish (Ictalurus punctatus) as a model. Surface functionalization was an important determinant of nanotoxicity. Lysine-functionalized RNTs (K-RNTs) consistently caused the greatest decline in lymphocyte viability while aminobutyl-functionalized RNTs (TBL-RNTs) had the least effect on lymphocyte viability. This trend was conserved across the multiple cell lines examined (both B- and T-cells). However, the absolute change in viability was distinct for each type of lymphocyte studied. Following RNT exposure, the two channel catfish B-cell lines tested, 3B11 and 1G8, had similar toxicity profiles for each of the RNT functionalizations examined. This was in contrast to the T-cell line tested, 28S.3, which had more viable cells remaining in culture post RNT exposure and suggests differences in toxicity based on the lymphocyte examined (B- versus T-cells). Lastly, exposure of cells to K-, TBL- or Arg-Gly-Asp-Ser-Lys peptide-functionalized RNTs (RGDSK-RNTs), significantly reduced the ability of cells to phagocytose through the channel catfish immune receptor, leukocyte immune-type receptor (IpLITR). Sub-lethal levels of RNT exposure affected the ability of immune cells to elicit this effector response in vitro and was concentration- and functionalization-dependent. Together, these data demonstrate that distinct classes of fish lymphocytes respond differentially to RNT exposure and that RNT functionalization impacts both lymphocyte viability and effector functions.