David Boyle

Aquatic toxicity of manufactured nanomaterials: challenges and recommendations for future toxicity testing

Environmental context The increased use of nanomaterials in industrial and consumer products requires robust strategies to identify risks when they are released into the environment. Aquatic toxicologists are beginning to possess a clearer understanding of the chemical and physical properties of nanomaterials in solution, and which of the properties potentially affect the health of aquatic organisms. This review highlights the main challenges encountered in aquatic nanotoxicity testing, provides recommendations for overcoming these challenges, and discusses recent studies that have advanced our understanding of the toxicity of three important OECD nanomaterials, titanium dioxide, zinc oxide and silver nanomaterials. Abstract Aquatic nanotoxicologists and ecotoxicologists have begun to identify the unique properties of the nanomaterials (NMs) that potentially affect the health of wildlife. In this review the scientific aims are to discuss the main challenges nanotoxicologists currently face in aquatic toxicity testing, including the transformations of NMs in aquatic test media (dissolution, aggregation and small molecule interactions), and modes of NM interference (optical interference, adsorption to assay components and generation of reactive oxygen species) on common toxicity assays. Three of the major OECD (Organisation for Economic Co-operation and Development) priority materials, titanium dioxide (TiO2), zinc oxide (ZnO) and silver (Ag) NMs, studied recently by the Natural Sciences and Engineering Research Council of Canada (NSERC), National Research Council of Canada (NRC) and the Business Development Bank of Canada (BDC) Nanotechnology Initiative (NNBNI), a Canadian consortium, have been identified to cause both bulk effect, dissolution-based (i.e. free metal), or NM-specific toxicity in aquatic organisms. TiO2 NMs are most toxic to algae, with toxicity being NM size-dependent and principally associated with binding of the materials to the organism. Conversely, dissolution of Zn and Ag NMs and the subsequent release of their ionic metal counterparts appear to represent the primary mode of toxicity to aquatic organisms for these NMs. In recent years, our understanding of the toxicological properties of these specific OECD relevant materials has increased significantly. Specifically, researchers have begun to alter their experimental design to identify the different behaviour of these materials as colloids and, by introducing appropriate controls and NM characterisation, aquatic nanotoxicologists are now beginning to possess a clearer understanding of the chemical and physical properties of these materials in solution, and how these materials may interact with organisms. Arming nanotoxicologists with this understanding, combined with knowledge of the physics, chemistry and biology of these materials is essential for maintaining the accuracy of all future toxicological assessments.

The effectiveness of low dose tranexamic acid in primary cardiac surgery.

PurposeThis randomized controlled clinical trial compared three doses of tranexamic acid (TA) in primary cardiac surgery in terms of blood loss and transfusion requirements.MethodsPatients presenting for primary coronary artery bypass grafting (CABG) and/or valve replacement were randomly assigned to one of three TA regimens: 20 mg·kg−1 (LD), 50 mg·kg−1 (MD), and 100 mg·kg−1 (HD). All participants and staff were blinded to the allocation. Haemoglobin (Hgb), haematocrit and platelet count were determined preoperatively, after bypass, at CCA arrival, and 12 and 24 hr after surgery. Coagulation parameters were measured before and after surgery. Blood loss was measured intraoperatively and for 24 hr postoperatively following a standardized protocol. Blood products were administered in a standardized fashion.ResultsTwo hundred twenty patients completed the trial over 10 months: 74 in LD, 75 in MD and 72 in HD dose groups. All patient groups were comparable; similar procedures were performed in each group. No differences were identified for blood loss intra-operatively (490 ± 232 ml, 523 ± 413 ml, 488 ± 357 ml, respectively), 24 hr post-operatively (543 ± 223 ml, 544 ± 231, 458 ± 210 ml, respectively), and overall (1032 ± 358 ml, 1067 ± 502 ml, 946 ± 459 ml, respectively). Blood product administration was similar in the three groups. No differences in postoperative complications were found.ConclusionsThis study demonstrates the equivalency of the three doses of TA in primary cardiac surgical procedures. The use of low dose (20 mg·kg−1) TA results in comparable outcomes, without additional complications.RésuméObjectifCet essai clinique contrôlé randomisé a comparé trois doses d’acide tranexamique (AT), lors d’une cardiochirurgie primaire, en termes de pertes sanguines et de besoins transfusionnels.MéthodesDes patients qui se présentent pour un pontage aorto-coronaire primaire et / ou pour un remplacement valvulaire ont reçu de façon aléatoire l’un des trois régimes d’AT: 20 mg·kg−1 (dose faible DF), 50 mg·kg−1 (dose moyenne DM) et 100 mg·kg−1 (dose élevée DE). La répartition dans les groupes s’est faite à l’insu des participants et du personnel. Les taux d’hémoglobine (Hb) et d’hématocrites ainsi que le décompte des plaquettes ont été faits avant l’opération, après le pontage, à l’arrivée à l’unité des soins intensifs coronariens et, 12h et 24h après la chirurgie. Les paramètres de coagulation ont été mesurés avant et après la chirurgie. Les pertes sanguines ont été mesurées pendant l’opération et pendant les 24 heures qui ont suivi l’intervention, selon un protocole standardisé. Les produits sanguins ont été administrés de façon classique.RésultatsLessai a exigé 10 mois et la participation de deux cent vingt patients: 74 dans le groupe à DF, 75 à DM et 72 à DE. Tous les groupes de patients étaient semblables; des traitements similaires ont été réalisés dans chaque groupe. Aucune différence de perte sanguine n’a été identifiée pendant l’opération (490 ml ± 232 ml, 523 ml ± 413 ml, 488 ml ± 357 ml respectivement) et au total (1032 ml ± 358 ml, 1067 ml ± 502 ml, 946 ml ± 459 ml respectivement). L’administration de produit sanguin était similaire dans les trois groupes. Aucune différence n’a été constatée dans les complications postopératoires.ConclusionCette étude démontre des effets équivalents de trois doses d’AT dans la conduite de la cardiochirurgie primaire. L’utilisation d’une dose faible (20 mg·kg−1) d’AT amène des résultats comparables à l’utilisation d’une dose moyenne ou élevée sans complications supplémentaires.

The role of acid-sensing ion channels in epithelial Na+ uptake in adult zebrafish (Danio rerio)

ABSTRACT Acid-sensing ion channels (ASICs) are epithelial Na+ channels gated by external H+. Recently, it has been demonstrated that ASICs play a role in Na+ uptake in freshwater rainbow trout. Here, we investigate the potential involvement of ASICs in Na+ transport in another freshwater fish species, the zebrafish (Danio rerio). Using molecular and histological techniques we found that asic genes and the ASIC4.2 protein are expressed in the gill of adult zebrafish. Immunohistochemistry revealed that mitochondrion-rich cells positive for ASIC4.2 do not co-localize with Na+/K+-ATPase-rich cells, but co-localize with cells expressing vacuolar-type H+-ATPase. Furthermore, pharmacological inhibitors of ASIC and Na+/H+-exchanger significantly reduced uptake of Na+ in adult zebrafish exposed to low-Na+ media, but did not cause the same response in individuals exposed to ultra-low-Na+ water. Our results suggest that in adult zebrafish ASICs play a role in branchial Na+ uptake in media with low Na+ concentrations and that mechanisms used for Na+ uptake by zebrafish may depend on the Na+ concentration in the acclimation medium. Summary: Acid-sensing ion channels in zebrafish gill epithelium are involved in Na+ uptake.

Dietary selenomethionine influences the accumulation and depuration of dietary methylmercury in zebrafish (Danio rerio).

Methylmercury (MeHg) is a toxicant of concern for aquatic food chains. In the present study, the assimilation and depuration of dietary MeHg and the influence of dietary selenium on MeHg toxicokinetics was characterised in zebrafish (Danio rerio). In a triplicate tank experimental design (n=3 tanks per treatment group), adult zebrafish were exposed to dietary MeHg (as methylmercury-cysteine) at 5 and 10 μg/g and with or without selenium (as selenomethionine) supplemented to the diets at a concentration of 5 μg/g for 8 weeks followed by a 4-week depuration period. Methylmercury accumulated in muscle, liver and brain of zebrafish; with higher mercury concentrations in liver and brain than in muscle following 8 weeks of exposure. In muscle, the mercury concentrations were 3.4±0.2 and 6.4±0.1 μg/g ww (n=3) in zebrafish fed the 5 and 10 μg Hg/g diets, respectively. During the depuration period, mercury concentrations were significantly reduced in muscle in both the 5 and 10 μg Hg/g diet groups with a greater reduction in the high dose group. After depuration, the mercury concentrations were 2.4±0.1 and 4.0±0.3 μg/g ww (n=3) for zebrafish fed the 5 and 10 μg Hg/g diets, respectively. Data also indicated that supplemented dietary selenium reduced accumulation of MeHg and enhanced the elimination of MeHg. Lower levels of mercury were found in muscle of zebrafish fed MeHg and SeMet compared with fish fed only MeHg after 8 weeks exposure; the mercury concentrations in muscle were 5.8±0.2 and 6.4±0.1 μg/g ww (n=3) for zebrafish fed the 10 μg Hg/g+5 μg Se/g diet and the 10 μg Hg/g diet, respectively. Furthermore, the elimination of MeHg from muscle during the 4-week depuration period was significantly greater in the fish fed the diet containing SeMet compared to a control diet; the mercury concentrations were 3.3±0.1 and 4.0±0.3 μg/g ww (n=3) for zebrafish fed the 5 μg Se/g and the control diets, respectively. In summary, dietary SeMet reduces the accumulation and enhances the elimination of dietary MeHg in muscle of zebrafish.

Mechanisms of Cl− uptake in rainbow trout: Cloning and expression of slc26a6, a prospective Cl−/HCO3− exchanger

In fresh waters, fishes continuously acquire ions to offset diffusive losses to a more dilute ambient environment and to maintain acid-base status. The objectives of the present study were to clone slc26a6, a prospective Cl(-)/HCO3(-) exchanger from rainbow trout, investigate its expression patterns in various tissues, at different developmental stages and after differential salinity exposure, and probe the mechanisms of Cl(-) uptake in rainbow trout embryos during development using a pharmacological inhibitor approach combined with (36)Cl(-) unidirectional fluxes. Results showed that the cloned gene encoded a 783 amino acid protein with conserved domains characteristic of the SLC26a family of anion exchange proteins. Phylogenetic analysis of this sequence against all subfamilies of the SLC26a family demonstrated that this translated protein shared a common ancestor with other actinopterygii and mammalian SLC26a6 isoforms and thus confirmed the identity of the cloned gene. Expression of slc26a6 was detected in all tissues and developmental stages assayed but was highest in the gill of juvenile trout. In trout embryos, Cl(-) uptake increased significantly post-hatch and was demonstrated to be mediated via an anion exchanger specific (DIDS sensitive) pathway that was also sensitive to hypercapnia. This parallels well with the predicted function of slc26a6, and the detection of the transcript in embryos and tissues of trout. In conclusion, this study is the first report of slc26a6 in rainbow trout and functional and expression analyses indicate its likely involvement in Cl(-)/HCO3(-) exchange in two life stages of rainbow trout.

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.

Characterization of developmental Na(+) uptake in rainbow trout larvae supports a significant role for Nhe3b.

Developing freshwater fish must compensate for the loss of ions, including sodium (Na(+)), to the environment. In this study, we used a radiotracer flux approach and pharmacological inhibitors to investigate the role of sodium/hydrogen exchange proteins (Nhe) in Na(+) uptake in rainbow trout (Oncorhynchus mykiss) reared from fertilization in soft water (0.1mM Na(+)). For comparison, a second group of embryos/larvae reared in hard water (2.2mM Na(+), higher pH and [Ca(2+)]) were also included in the experiment but were fluxed in soft water, only. Unidirectional rates of Na(+) uptake increased throughout development and were significantly higher in embryos/larvae reared in soft water. However, the mechanisms of Na(+) uptake in both groups of larvae were not significantly different, either in larvae immediately post-hatch or later in development: the broad spectrum Na(+) channel blocker amiloride inhibited 85-90% of uptake and the Nhe-inhibitor EIPA also caused near maximal inhibitions of Na(+) uptake. These data indicated Na(+) uptake was Nhe-mediated in soft water. A role of Nhe3b (but not Nhe2 or Nhe3a) in Na(+) uptake in soft water was also supported through gene expression analyses: expression of nhe3b increased throughout development in whole embryos/larvae in both groups and was significantly higher in those reared in soft water. This pattern of expression correlated well with measurements of Na(+) uptake. Together these data indicate that in part, rainbow trout embryos/larvae reared in low Na(+) soft water maintained Na(+) homeostasis by an EIPA sensitive component of Na(+) uptake, and support a primary role for Nhe3b.

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.