Comparative metal oxide nanoparticle toxicity using embryonic zebrafish

AbstractEngineered metal oxide nanoparticles (MO NPs) are finding increasing utility in the medical field as anticancer agents. Before validation of in vivo anticancer efficacy can occur, a better understanding of whole-animal toxicity is required. We compared the toxicity of seven widely used semiconductor MO NPs made from zinc oxide (ZnO), titanium dioxide, cerium dioxide and tin dioxide prepared in pure water and in synthetic seawater using a five-day embryonic zebrafish assay. We hypothesized that the toxicity of these engineered MO NPs would depend on physicochemical properties. Significant agglomeration of MO NPs in aqueous solutions is common making it challenging to associate NP characteristics such as size and charge with toxicity. However, data from our agglomerated MO NPs suggests that the elemental composition and dissolution potential are major drivers of toxicity. Only ZnO caused significant adverse effects of all MO particles tested, and only when prepared in pure water (point estimate median lethal concentration=3.5–9.1mg/L). This toxicity was life stage dependent. The 24h toxicity increased greatly (∼22.7 fold) when zebrafish exposures started at the larval life stage compared to the 24h toxicity following embryonic exposure. Investigation into whether dissolution could account for ZnO toxicity revealed high levels of zinc ion (40–89% of total sample) were generated. Exposure to zinc ion equivalents revealed dissolved Zn2+ may be a major contributor to ZnO toxicity

Influence of ovarian stage on transcript profiles in fathead minnow (Pimephales promelas) ovary tissue.

Interpretation of toxicogenomic experiments conducted with ovary tissue from asynchronous-spawning small fish species is complicated by background variation in the relative abundance and proportion of follicles at different stages within the ovary tissue sample. This study employed both real-time quantitative polymerase chain reaction and a 15,000 gene oligonucleotide microarray to examine variation in the fathead minnow (Pimephales promelas) ovarian transcriptional profile as a function of quantitative and qualitative differences in ovarian histology. The objectives were to provide data that could potentially aid interpretation of future toxicogenomics experiments, identify putative stage-related transcriptional markers, and generate insights into basic biological regulation of asynchronous oocyte development. Multiple lines of evidence from the present study indicate that variation in the transcriptional profile is primarily dependent on the relative abundance of previtellogenic versus vitellogenic follicles in the ovary. Due to the relatively small proportions of mature ovulated follicles or atretic follicles in the overall follicle population, few potential transcriptional markers of maturation, ovulation, or atresia could be identified. However, among the 460 differentially expressed genes identified in the present study, several targets, including HtrA serine peptidase 3 (htra3), tissue inhibitor of metalloproteinase 3 (timp3), aquaporin 8 (aqp8), transgelin 2 like (tagln2), Nedd4 family interacting protein 2 (ndfip2), chemokine ligand 12a (cxcl12a), midkine-related growth factor (mdka), and jagged 1b (jag 1b) exhibited responses and functional properties that support them as candidate molecular markers of significant shift in gross ovarian stage. Genes associated with a diversity of functions including cellular development, morphogenesis, coated vesicle transport, sexual reproduction, and neuron development, among others, were statistically enriched within the list of 460 genes differentially expressed among different ovarian classes. Overall, results of this study provide insights into background variation in ovary transcript profiles that should aid and enhance the interpretation of toxicogenomic data generated in experiments conducted with small, asynchronous-spawning fish species

Ankley, Influence of ovarian stage on transcript profiles in fathead minnow (Pimephales promelas) ovary tissue, Aquat. Toxicol. 98

a b s t r a c t Interpretation of toxicogenomic experiments conducted with ovary tissue from asynchronous-spawning small fish species is complicated by background variation in the relative abundance and proportion of follicles at different stages within the ovary tissue sample. This study employed both real-time quantitative polymerase chain reaction and a 15,000 gene oligonucleotide microarray to examine variation in the fathead minnow (Pimephales promelas) ovarian transcriptional profile as a function of quantitative and qualitative differences in ovarian histology. The objectives were to provide data that could potentially aid interpretation of future toxicogenomics experiments, identify putative stage-related transcriptional markers, and generate insights into basic biological regulation of asynchronous oocyte development. Multiple lines of evidence from the present study indicate that variation in the transcriptional profile is primarily dependent on the relative abundance of previtellogenic versus vitellogenic follicles in the ovary. Due to the relatively small proportions of mature ovulated follicles or atretic follicles in the overall follicle population, few potential transcriptional markers of maturation, ovulation, or atresia could be identified. However, among the 460 differentially expressed genes identified in the present study, several targets, including HtrA serine peptidase 3 (htra3), tissue inhibitor of metalloproteinase 3 (timp3), aquaporin 8 (aqp8), transgelin 2 like (tagln2), Nedd4 family interacting protein 2 (ndfip2), chemokine ligand 12a (cxcl12a), midkine-related growth factor (mdka), and jagged 1b (jag 1b) exhibited responses and functional properties that support them as candidate molecular markers of significant shift in gross ovarian stage. Genes associated with a diversity of functions including cellular development, morphogenesis, coated vesicle transport, sexual reproduction, and neuron development, among others, were statistically enriched within the list of 460 genes differentially expressed among different ovarian classes. Overall, results of this study provide insights into background variation in ovary transcript profiles that should aid and enhance the interpretation of toxicogenomic data generated in experiments conducted with small, asynchronous-spawning fish species. Published by Elsevier B.V

Toward safer multi-walled carbon nanotube design: Establishing a statistical model that relates surface charge and embryonic zebrafish mortality

<p>Given the increased utility and lack of consensus regarding carbon nanotube (CNT) environmental and human health hazards, there is a growing demand for guidelines that inform safer CNT design. In this study, the zebrafish (<i>Danio rerio</i>) model is utilized as a stable, sensitive biological system to evaluate the bioactivity of systematically modified and comprehensively characterized multi-walled carbon nanotubes (MWNTs). MWNTs were treated with strong acid to introduce oxygen functional groups, which were then systematically thermally reduced and removed using an inert temperature treatment. While 25 phenotypic endpoints were evaluated at 24 and 120 hours post-fertilization (hpf), high mortality at 24 hpf prevented further resolution of the mode of toxicity leading to mortality. Advanced multivariate statistical methods are employed to establish a model that identifies those MWNT physicochemical properties that best estimate the probability of observing an adverse outcome. The physicochemical properties considered in this study include surface charge, percent surface oxygen, dispersed aggregate size and morphology and electrochemical activity. Of the five physicochemical properties, surface charge, quantified as the point of zero charge (PZC), was determined as the best predictor of mortality at 24 hpf. From a design perspective, the identification of this property–hazard relationship establishes a foundation for the development of design guidelines for MWNTs with reduced hazard.</p

Use of chemical mixtures to differentiate mechanisms of endocrine action in a small fish model.

Various assays with adult fish have been developed to identify potential endocrine-disrupting chemicals (EDCs) which may cause toxicity via alterations in the hypothalamic-pituitary-gonadal (HPG) axis. These assays can be sensitive and highly diagnostic for key mechanisms such as agonism of the estrogen and androgen receptors (ERs, ARs) and inhibition of steroid synthesis. However, most of the tests do not unambiguously identify AR antagonists. The purpose of this work was to explore the utility of a mixture test design with the fathead minnow (Pimephales promelas) for detecting different classes of EDCs including AR antagonists. Adults of both sexes were exposed via the water to EDCs with diverse mechanisms of action in the absence or presence of 17beta-trenbolone (TB), a potent AR agonist which masculinizes female fathead minnows. Similar to previous studies with the model AR antagonists flutamide and vinclozolin, exposure of females to the AR antagonist cyproterone acetate in the presence of TB decreased expression of an easily-observed masculinization response, nuptial tubercle formation. Mixture studies with TB and the model ER agonists, 17alpha-ethinylestradiol and bisphenol A, also showed inhibition of tubercle formation in the females, but unlike the AR antagonists, the estrogens markedly induced synthesis of vitellogenin (VTG: egg yolk protein), particularly in males. The ER agonists also offset TB-induced depressions in plasma VTG concentrations in female fish. Additional mixture experiments were conducted with TB and triclocarban, an anti-microbial reported to enhance AR-mediated responses, or ammonia, a "negative control" with no known direct effects on HPG function. Neither chemical affected VTG status in males or females in the absence or presence of TB; however, both slightly enhanced TB-induced tubercle formation in females. Based on studies described herein and elsewhere with the fathead minnow, a TB co-exposure assay appears to be an effective approach for clearly identifying AR antagonists as well as potential EDCs with other relevant mechanisms of action

Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz.

The vertebrate hypothalamic-pituitary-gonadal (HPG) axis is controlled through various feedback mechanisms that maintain a dynamic homeostasis in the face of changing environmental conditions, including exposure to chemicals. We assessed the effects of prochloraz on HPG axis function in adult fathead minnows (Pimephales promelas) at multiple sampling times during 8-day exposure and 8-day depuration/recovery phases. Consistent with one mechanism of action of prochloraz, inhibition of cytochrome P450 (CYP) 19 aromatase activity, the fungicide depressed ex vivo ovarian production and plasma concentrations of 17beta-estradiol (E2) in female fish. At a prochloraz water concentration of 30 microg/l, inhibitory effects on E2 production were transitory and did not persist during the 8-day exposure phase. At 300 microg/l prochloraz, inhibition of E2 production was evident throughout the 8-day exposure but steroid titers recovered within 1 day of cessation of exposure. Compensation or recovery of steroid production in prochloraz-exposed females was accompanied by upregulation of several ovarian genes associated with steroidogenesis, including cyp19a1a, cyp17 (hydroxylase/lyase), cyp11a (cholesterol side-chain cleavage), and follicle-stimulating hormone receptor. In male fathead minnows, the 8-day prochloraz exposure decreased testosterone (T) production, possibly through inhibition of CYP17. However, as for E2 in females, ex vivo testicular production and plasma concentrations of T recovered within 1 day of stopping exposure. Steroidogenic genes upregulated in testis included cyp17 and cyp11a. These studies demonstrate the adaptability of the HPG axis to chemical stress and highlight the need to consider the dynamic nature of the system when developing approaches to assess potential risks of endocrine-active chemicals

Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz.

The vertebrate hypothalamic-pituitary-gonadal (HPG) axis is controlled through various feedback mechanisms that maintain a dynamic homeostasis in the face of changing environmental conditions, including exposure to chemicals. We assessed the effects of prochloraz on HPG axis function in adult fathead minnows (Pimephales promelas) at multiple sampling times during 8-day exposure and 8-day depuration/recovery phases. Consistent with one mechanism of action of prochloraz, inhibition of cytochrome P450 (CYP) 19 aromatase activity, the fungicide depressed ex vivo ovarian production and plasma concentrations of 17beta-estradiol (E2) in female fish. At a prochloraz water concentration of 30 microg/l, inhibitory effects on E2 production were transitory and did not persist during the 8-day exposure phase. At 300 microg/l prochloraz, inhibition of E2 production was evident throughout the 8-day exposure but steroid titers recovered within 1 day of cessation of exposure. Compensation or recovery of steroid production in prochloraz-exposed females was accompanied by upregulation of several ovarian genes associated with steroidogenesis, including cyp19a1a, cyp17 (hydroxylase/lyase), cyp11a (cholesterol side-chain cleavage), and follicle-stimulating hormone receptor. In male fathead minnows, the 8-day prochloraz exposure decreased testosterone (T) production, possibly through inhibition of CYP17. However, as for E2 in females, ex vivo testicular production and plasma concentrations of T recovered within 1 day of stopping exposure. Steroidogenic genes upregulated in testis included cyp17 and cyp11a. These studies demonstrate the adaptability of the HPG axis to chemical stress and highlight the need to consider the dynamic nature of the system when developing approaches to assess potential risks of endocrine-active chemicals