Final Report: Nanoparticle Toxicity in Zebrafish

EPA Grant Number: R833339
Title: Nanoparticle Toxicity in Zebrafish
Investigators: Mayer, Gregory D. , Nadeau, Jay L. , Nohe, Anja , Smorodin, V.
Institution: University of Maine , McGill University
EPA Project Officer: Lasat, Mitch
Project Period: October 1, 2006 through September 30, 2009
Project Amount: $398,298
RFA: Exploratory Research: Nanotechnology Research Grants Investigating Environmental and Human Health Effects of Manufactured Nanomaterials: a Joint Research Solicitation-EPA, NSF, NIOSH, NIEHS (2006) RFA Text |  Recipients Lists
Research Category: Nanotechnology , Health Effects , Health , Safer Chemicals

Objective:

Investigate the toxicity of semiconductor nanostructures using zebrafish embryos. Monitor the effects of particle composition, size, and charge on uptake and accumulation of nanostructures in multiple tissues. Monitor the release of ions from the particles using a transgenic zebrafish model that expresses green fluorescent protein (GFP) in the presence of metal ions. Correlate these data to altered embryo development after particle exposure and extrapolate the effects to human health.

Summary/Accomplishments (Outputs/Outcomes):

Nanoparticle Synthesis

CdSe/ZnS QDs were synthesized using a published method (Asokan et al., 2005; Clarke et al., 2008). Briefly, cadmium oxide (CdO) (0.05 g, 0.39 mmol) and oleic acid (OA) (1.78 g, 6.3 mmol) were added into a three-neck flask containing 20 mL of ODE. This mix was degassed and heated under an atmosphere of nitrogen to 250°C. The mixture turned colorless around 150°C. Selenium (Se) precursor was prepared by mixing Se (0.016 g, 0.2 mmol), trioctylphoshine (TOP) (0.75 mL, 1.7 mmol), under inert atmosphere, followed by sonication until the solution became transparent. The zinc sulfide (ZnS) precursors were prepared as follows: hexamethyldisilathiane [(TMS)2S] (0.2 mL, 0.95 mmol), dimethylzinc [Zn(CH3)2] (0.3 mL of a 1 M solution in toluene) and TOP (0.75 mL, 1.7 mmol) were mixed under inert atmosphere and diluted to 5 mL with ODE. Once the CdO/OA/ODE mixture reached 250 °C, the heat was turned off, and the Se precursor was injected rapidly using a needled syringe. To stop further growth of the QDs, the reaction temperature was dropped rapidly to 100 °C, using a water bath at room temperature (rt). The ZnS precursor was injected at 175°C over a time course of 5 min. Afterward, the temperature was allowed to drop to 100°C and it was maintained at this temperature for 3 h. To purify the quantum dots, repeated extractions with equivolume hexanes/methanol were performed. Ethanol was added to the washed QDs in ODE/Hexane until the solution became cloudy and centrifuged for 5 min at 5000 g. The QDs formed a thick paste on the bottom of the tube. The purified QDs were finally stored in the toluene in air-tight vials until further use. Mercaptopropionic acid (MPA) was used to replace the OA surfactant by a thiol-exchange reaction. One milliliter of concentrated QDs (OD > 5 at first exciton peak) in toluene was diluted to 5 mL using methanol. MPA was added to a final concentration of 1 M and the pH was adjusted to 10 using tetramethylammonium hydroxide pentahydrate (TMAH). This solution was stirred overnight at room temperature. The thiol-modified QDs were separated from excess MPA ligand by precipitation and several washings with ethyl acetate. The QDs were dried at room temperature for 1 h under air and resuspended in Milli-Q water (Millipore).

Synthesis of CdTe

CdTe QDs were synthesized by a recently developed procedure (Kloper et al., 2007). CdO (0.026 g, 0.20 mmol) and OA (0.179 g, 0.63 mmol) were mixed in a three-neck flask with 10 mL of ODE. This mixture was degassed for 5 min and heated under nitrogen (N2) atmosphere to 220°C until the solution became colorless. In a separate vessel, the tellurium precursor (TOPTe) was prepared by mixing Te (0.01 g, 0.08 mmol) with TOP (0.415 g, 1.12 mmol) and 2 mL ODE under N2 atmosphere and vigorously stirring until solution became light yellow. Next, the temperature of the CdO-ODE mixture was increased to 310°C. Formation of a grey CdO precipitate was evident after prolonged heating (10-20 min) of the reaction mixture at 310°C. Immediately after formation of the CdO precipitate, the TOPTe precursor was rapidly injected using a needled syringe. To stop further growth of the QDs the reaction temperature was dropped rapidly to 25°C, using a water bath at room temperature. The CdTe Nps were purified and stored following the same procedure as for CdSe/ZnS. MPA was used to replace the OA surfactant by a thiol-exchange reaction. 200 µL of CdTe in toluene were mixed with 800 µL of toluene, 1000 µL of borate buffer (0.2 M at pH 9) and 1 µL of MPA. The solution was stirred vigorously and the mixture left to separate into two phases. 400 mL of the buffer solution containing the QDs were mixed with 200 µL of borate buffer (0.05 M at pH 9), transferred to a 10000 Molecular weight cutoff filter (vivaspin 500), and centrifuged at 2500 g for 13 min. The precipitate was washed three more times and stored in borate buffer (0.05 M at pH 9).

Synthesis of InP/ZnS

InP/ZnS QDs were synthesized using a method adapted from the literature (Chibli et al., 2011; Xu et al., 2008) with slight variation. Hexadecylamine (96 mg, 0.4 mmol), stearic acid (57 mg, 0.2 mmol), zinc undecylenate (260 mg, 0.6 mmol) and indium chloride (44 mg, 0.2 mmol) were mixed in octadecene (4 mL) under an inert atmosphere in a three-neck round bottom flask. A thermometer and condenser were mounted into the flask. The system was flushed with an inert gas (Ar or N2), the flask was heated to 270°C and tris-(trimethylsilyl)phosphine (2 mL of 0.1 M solution in octadecene) was rapidly added. The temperature dropped to 240°C upon the addition and was maintained for 20 min. The solution was cooled down in a water bath at 20°C. Zinc diethyldithiocarbamate (72 mg, 0.1 mmol) was added to the mixture at room temperature. The system was flushed with inert gas before heating at 180°C for 10 min and 240°C for 20 min. Timing started when the thermometer reached the desired temperature. The solution was cooled down to room temperature in a water bath at 20°C and toluene (8 mL) was added. The mixture was centrifuged at 2500 g for 5 min and the precipitate discarded. The particles were precipitated by adding ethanol (42 mL) to the orange solution and centrifuging at 2500 g for 20 min. The InP/ZnS was dissolved in toluene (3 mL).

To transfer the QDs into an aqueous environment, butanol (800 μL), borate buffer (1000 μL, 200 mM) at pH 9 and mercaptopropionic acid (8 μL, 10 μmol) were added to 200 μL of the QDs at 5 μM. The mixture was heated at 50°C for 15 min. The two phases were separated and the solution containing the InP/Zn-MPA was purified by washing/filtration. We used a 10000 Molecular weight cutoff filter (vivaspin 500), added borate buffer (50 mM, pH 9), and centrifuged at 2000 g for 13 min. This was repeated four times. The product was dissolved in borate buffer at the desired pH and stored at 4°C. Transmission electron microscopy (TEM) images of QDs were taken on a Philips CM200 [Figure 1 (1)]. Absorbance spectra were measured on a SpectraMax Plus plate reader and emission spectra on a SpectraMax Gemini (Molecular Devices, Sunnyvale, CA) [Figure 1 (2)]. Concentrations were determined using UV-vis spectroscopy with a NanoDrop ND-1000 Spectrophotometer. Solution concentrations were calculated from a basal absorbance of 0.1 signifying a concentration of 1 mM.

Quantum Dot Toxicity in vivo

Our data show quantum dot concentrations more than 1000-fold less than those used in our heavy metal salt exposures exert a similar toxicity. Thus, a more specific set of regulations governing semiconductor nanoparticle pollution should reflect proven toxic levels of the compound and not those of its individual constituents.

Our experiments utilizing the transgenic strain demonstrate the efficacy of the model to detect and quantify trace amounts of heavy metal ions liberated by semiconductor nanoparticles based on increased fluorescence. Simple detection of elevated heavy metals can be acquired through visual inspection of control and exposed groups. Of five metals, copper, silver and zinc would definitively be detected using our model.

Our experiments demonstrate that comparable concentrations of semiconductor nanoparticles and heavy metal salts elicit much different responses and are acutely toxic to zebrafish at different levels. Conversely, semiconductor nanoparticles and heavy metals do share some common toxicological properties. Both semiconductor nanoparticles and heavy metals are capable of bioaccumulation and exerting hepatotoxicity. However, both heavy metal species theoretically do so via different mechanisms. Heavy metals such as mercury and cadmium bioaccumulate due to their high affinity for available sulfhydryl groups present in nearly all proteins. Nanoparticles bioaccumulate via an unknown mechanism, but are designed to be aqueous molecules with low reactivity in a neutral environment to allow a simplified biological introduction and distribution. Our experiments show quantum dots form bulky crystalline aggregates that bind to and potentially puncture the cell membrane as a mode of toxicity indicating aggregated quantum dots may have increased hydrophobicity. It also has been suggested crystalline quantum dot structures become embedded in the plasma membrane creating a pore allowing solutes to escape and enter the cell freely. Ionic disturbances would disrupt osmotic homeostasis and quickly destroy the cell. Our images of quantum dot aggregates co-localized with the plasma membrane support this hypothesis. The embedding of quantum dot nanocrystals into the cellular membrane also may account for the observed bioaccumulation.

Images obtained during our confocal microscopy experiments show that over the course of quantum dot exposure, cells exhibit blebbing”, a characteristic frequently seen in apoptotic cells (data not shown). Our data suggest quantum dots may induce apoptosis, but our experiments involving antioxidants did not improve cellular proliferation eliminating lipid peroxidation as the primary source of toxicity. It can be argued that NAC may prevent lipid peroxidation from occurring, but it may simply hinder physical and chemical properties of the quantum dot surface that may bind to and activate the Fas receptor. Further experiments involving uncoated quantum dots, antioxidants and apoptosis identification methods such as TUNEL staining and/or caspase quantification should be performed to identify the direct mode of quantum dot toxicity.

Our research quantifying the release of free cadmium ions in solution after nanoparticle removal implies the time course of heavy metal release from semiconductors in the aquatic environment should be studied to evaluate environmental fate. The soluble free cadmium released from a 1 mM solution of quantum dots over a relatively short period of time was sufficient enough to elicit a response in our transgenic strain comparable to 2.5 mM CdCl2. Quantum dots are hypothesized to contain thousands of individual heavy metal atoms; therefore, the molar concentration of free heavy metal ions could become substantially greater than that of the nanoparticles. Our experiments studying heavy metal release from quantum dots only used the supernatant from a quantum dot solution stored in conditions without light and under low temperatures, thus demonstrating the heavy metal release in ideal conditions. Environmental conditions could presumably exacerbate the potential release of heavy metals through photoxidation and elevated temperatures. This suggests our experiments demonstrate what semiconductor breakdown could lead to in the environment on a much smaller scale.

Conclusions:

The conclusions of this work eliminate many formerly proposed modes of semiconductor nanoparticle toxicity, but do not precisely identify the source. However, our data do form a distinction between heavy metal and semiconductor nanoparticle toxicity. Experiments monitoring GFP expression in our heavy metal-responsive transgenic zebrafish strain exposed to quantum dots exhibit no observable increase in fluorescence. The use of heavy metal chelators in cell culture following nanoparticle exposure also did not show any rescue of cell proliferation. In addition, the use of antioxidants did not prevent toxicity stemming from oxidative damage suggested in prior quantum dot experiments. These data are indicative of semiconductors inducing a unique form of acute toxicity discernable from heavy metal toxicity. Taken together with the time-dependent release of heavy metals, semiconductor contamination cannot be viewed solely as heavy metal pollution, but as a unique form of aquatic contamination.

Cd2+ was more toxic than Zn2+, and the general trend of IC50-24 h values of QDs was determined to be CdTe < CdSe/ZnS or InP/ZnS, suggesting that ZnS-shelled CdSe/ZnS were more cytocompatible than bare core CdTe crystals. Intracellular and extracellular concentrations of Cd2+ revealed that core-only CdTe released more Cd2+ than core-shell CdSe/ZnS. The dose-dependent correlation between cell viability and intracellular [Cd2+] from CdTe was similar to Cd salts, implying that its cytotoxicity could be attributed to the toxic effect of Cd2+ that leached from the nanocrystalline particle. CdTe exposure induced expression of metal responsive genes including metallothionein (MT), metal response element-binding transcription factor (MTF-1), divalent metal transporter (DMT-1), zrt and irt like protein (ZIP-1) and the zinc transporter, ZnT-1 in a dose dependent manner similar to that of CdSO4 exposure. However, CdSe/ZnS and InP/ZnS altered gene expression of metal homeostasis genes in a manner different from that of the corresponding Cd or Zn salts. This implies that ZnS shells reduce QD toxicity attributed to the release of Cd2+, but do not eliminate toxic effects caused by the nanoparticles themselves.

Taken together, our results showed that cell viability was decreased, Cd was accumulated, and intracellular ROS generation was increased, which was associated with an induction of oxidative DNA damage by CdSO4 and CdTe QDs exposure in zebrafish hepatocytes. We presented for the first time that the mRNA expression of genes related to oxidative stress defense and DNA repair pathways were affected in general at both in vitro and in vivo levels, giving extra credit that both Cd2+ and QDs are genotoxic. Our study also revealed for the first time that cells exposed to CdTe QDs performed a different pattern of NER repair compared to Cd2+, supporting evidences that adverse effects caused by acute exposure of CdTe might be mediated through differing mechanisms than those resulting from ionic cadmium toxicity, and that studying the effects of the metallic components may be not enough to understand QD toxicities in aquatic vertebrates. Our study will facilitate exploring and providing new information regarding genotoxicity responsible not only for Cd-based QDs exposure in aquatic organisms, but also for human health by enhancing our understanding of QDs effects on toxicology, making it easy to access the risks regarding application of this new product.


Journal Articles on this Report : 9 Displayed | Download in RIS Format

Other project views: All 9 publications 9 publications in selected types All 9 journal articles
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Journal Article Chibli H, Carlini L, Park S, Dimitrijevic NM, Nadeau JL. Cytotoxicity of InP/ZnS quantum dots related to reactive oxygen species generation. Nanoscale 2011;3(6):2552-2559. R833339 (Final)
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  • Journal Article Cooper DR, Dimitrijevic NM, Nadeau JL. Photosensitization of CdSe/ZnS QDs and reliability of assays for reactive oxygen species production. Nanoscale 2010;2(1):114-121. R833339 (Final)
    R833323 (Final)
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  • Journal Article Dumas EM, Ozenne V, Mielke RE, Nadeau JL. Toxicity of CdTe quantum dots in bacterial strains. IEEE Transactions on Nanobioscience 2009;8(1):58-64. R833339 (Final)
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  • Journal Article Dumas E, Gao C, Suffern D, Bradforth SE, Dimitrijevic NM, Nadeau JL. Interfacial charge transfer between CdTe quantum dots and gram negative vs gram positive bacteria. Environmental Science & Technology 2010;44(4):1464-1470. R833339 (Final)
    R833323 (Final)
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  • Journal Article Park S, Chibli H, Wong J, Nadeau JL. Antimicrobial activity and cellular toxicity of nanoparticle-polymyxin B conjugates. Nanotechnology 2011;22(18):185101. R833339 (Final)
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  • Journal Article Park S, Chibli H, Nadeau J. Solubilization and bio-conjugation of quantum dots and bacterial toxicity assays by growth curve and plate count. Journal of Visualized Experiments 2012;(65):e3969 (7 pp.). R833339 (Final)
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  • Journal Article Tang S, Cai Q, Chibli H, Allagadda V, Nadeau JL, Mayer GD. Cadmium sulfate and CdTe-quantum dots alter DNA repair in zebrafish (Danio rerio) liver cells. Toxicology and Applied Pharmacology 2013;272(2):443-452. R833339 (Final)
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  • Journal Article Tang S, Allagadda V, Chibli H, Nadeau JL, Mayer GD. Comparison of cytotoxicity and expression of metal regulatory genes in zebrafish (Danio rerio) liver cells exposed to cadmium sulfate, zinc sulfate and quantum dots. Metallomics 2013;5(10):1411-1422. R833339 (Final)
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  • Journal Article Tang S, Wu Y, Ryan CN, Yu S, Qin G, Edwards DS, Mayer GD. Distinct expression profiles of stress defense and DNA repair genes in Daphnia pulex exposed to cadmium, zinc, and quantum dots. Chemosphere 2015;120:92-99. R833339 (Final)
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  • Supplemental Keywords:

    Quantum dot, nanoparticle, CdSe/ZnS, CdTe, InP/ZnS, zebrafish, daphnia, DNA repair, DNA damage, Health, Scientific Discipline, PHYSICAL ASPECTS, Risk Assessments, Physical Processes, Biology, exposure, nanotechnology, nanomaterials, nanoparticle toxicity

    Progress and Final Reports:

    Original Abstract
  • 2007
  • 2008