Copper Oxide and Zinc Oxide Nanomaterials Act as Inhibitors of

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Copper Oxide and Zinc Oxide Nanomaterials Act as Inhibitors of Multidrug Resistance Transport in Sea Urchin Embryos: Their Role as Chemosensitizers Bing Wu, Cristina Torres Duarte, Bryan J. Cole, and Gary Cherr Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00345 • Publication Date (Web): 07 Apr 2015 Downloaded from http://pubs.acs.org on April 12, 2015

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Environmental Science & Technology

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Copper Oxide and Zinc Oxide Nanomaterials Act as

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Inhibitors of Multidrug Resistance Transport in Sea

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Urchin Embryos: Their Role as Chemosensitizers

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Bing Wu, †,‡ Cristina Torres-Duarte, ‡ Bryan J. Cole, ‡ Gary N. Cherr‡,§,

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†

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Environment, Nanjing University, Nanjing, 210023, P.R. China

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‡

Bodega Marine Laboratory, University of California, Davis, California, USA

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§

Departments of Environmental Toxicology and Nutrition, University of California,

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Davis, California, USA

State Key Laboratory of Pollution Control and Resource Reuse, School of the

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Gary N. Cherr

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Bodega Marine Laboratory

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University of California Davis

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PO Box 247

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2099, Westside Road,

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Bodega Bay, CA 94951 USA

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Bodega Bay, CA 94923 USA

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Phone: 001-707-875-2051

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FAX: 001-707-875-2009

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E-mail:[email protected]

Corresponding Author

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Notes

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The authors declare no competing financial interest.

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ACS Paragon Plus Environment


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ABSTRACT

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The ability of engineered nanomaterials (NMs) to act as inhibitors of ATP-binding

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cassette (ABC) efflux transporters in embryos of white sea urchin (Lytechinus pictus)

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was studied. Nano-copper oxide (nano-CuO), nano-zinc oxide (nano-ZnO), and their

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corresponding metal ions (CuSO4 and ZnSO4) were used as target chemicals. The

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results showed that nano-CuO, nano-ZnO, CuSO4 and ZnSO4, even at relatively low

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concentrations (0.5ppm), significantly increased calcein-AM (CAM, an indicator of

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ABC transporter activity) accumulation in sea urchin embryos at different stages of

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development. Exposure to nano-CuO, a very low solubility NM, at increasing times

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after fertilization (>30min) decreased CAM accumulation, but nano-ZnO (much more

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soluble NM) did not, indicating that metal ions could cross the hardened fertilization

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envelope, but not undissolved metal oxide NMs. Moreover, non-toxic levels (0.5ppm)

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of nano-CuO and nano-ZnO significantly increased developmental toxicity of

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vinblastine (an established ABC transporter substrate) and functioned as

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chemosensitizers. The multidrug resistance associated protein (MRP, one of ABC

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transporters) inhibitor MK571 significantly increased copper concentrations in

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embryos, indicating ABC transporters are important in maintaining low intracellular

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copper levels. We show that low concentrations of nano-CuO and nano-ZnO can

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make embryos more susceptible to other contaminants, representing a potent

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amplification of nanomaterial-related developmental toxicity.

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KEYWORDS: Sea

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Nano-ZnO, Developmental toxicity

urchin,

ABC transporter, Chemosensitizer, Nano-CuO,

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TOC Art

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INTRODUCTION

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Large-scale production and application of metal oxide nanomaterials (NMs) in

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several industries has led to environmental concern in estuarine and marine

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environments1, 2. Nano-zinc oxide (nano-ZnO) and nano-copper oxide (nano-CuO) are

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two common metal oxide NMs. Nano-ZnO is used as an ultraviolet light scattering

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additive in cosmetics such as sunscreens, toothpastes and beauty products, and is

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widely used in rubber manufacturing, production of solar cells and liquid crystal

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displays, pigments, chemical fibers, and textiles3, 4. The most important and unique

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application of nano-CuO is in electronics and technology (semiconductors, electronic

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chips, heat transfer nanofluids), as nano-CuO has excellent thermophysical properties5.

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Nano-CuO has also been used in air and liquid filtration, wood preservation, and

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bioactive coatings, including antifouling paints

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nano-CuO have been assessed in aquatic organisms

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have assessed impacts of metal oxide NMs on marine embryos1, 8.

6, 7

. The toxicity of nano-ZnO and 8-16

. However, very few studies

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Developmental toxicity caused by metal oxide NMs has received some recent

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attention, although not in marine developing systems. For example, Zhao et al.17

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found that nano-ZnO and Zn2+ elicited embryonic toxicity in fish by increasing

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reactive oxidative species (ROS) and/or compromising the cellular oxidative stress

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response. Nations et al.18 found gastrointestinal, spinal, and other abnormalities in the

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freshwater amphibian, Xenopus laevis, occurred following nano-CuO and nano-ZnO

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exposures. The minimum concentration to inhibit growth of tadpoles exposed to

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nano-CuO or nano-ZnO was very high (10 ppm). Most studies on developmental

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toxicity induced by metal oxide NMs have focused on morphological abnormalities 8.

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In general, relatively high levels of NMs were required to observe these effects.

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One unexamined aspect of metal oxide NMs toxicity is their potential to act as

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chemosensitizers, which are compounds that increase the toxicity of other chemicals

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through their interaction with or inhibition of toxicant defense mechanisms19-22. In

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embryos, ATP-binding cassette (ABC) efflux transporters are responsible for

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multidrug resistance22-28 and play important roles in protection from contaminants.

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ABC transporters are known to efflux numerous compounds that include hormones,

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sterols, lipids, phospholipids, oligopeptides, nucleotides, chloride ions, heavy metals

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that have been conjugated to metallothionein or glutathione, and a wide range of

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moderately hydrophobic xenobiotics28-31. This protective role as the first line of

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defense against xenobiotics32 is similar to the protective functions ascribed to these

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transporters in somatic cells and consistent with their expression in the adult blood–

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brain barrier, blood–testis barrier, and in intestinal epithelia33, 34. The inhibition of

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ABC transporter activities can increase the accumulation of other toxic chemicals that

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are substrates for efflux transporters, thus rendering embryos susceptible to these

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chemicals.

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Aquatic ecosystems, including estuarine and near shore marine environments, are

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expected to be one of the most heavily impacted by NM pollution 1, 2. Sea urchins are

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key species in coastal environments and are a major model system in elucidating a

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variety of classic developmental problems. In this study, the potential of nano-CuO

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and nano-ZnO to inhibit efflux transporters, and thus act as chemosensitizers, was

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studied in embryos of the white sea urchin (Lytechinus pictus). This research provides

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a new endpoint of chemosensitization via indirect NM toxicity.

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MATERIALS AND METHODS

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Reagents

Potassium

chloride

(KCl),

dimethyl

5


sulphoxide

(DMSO),


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ethylhexadecyldimethylammonium bromide, 1,10-phenanthroline, hydrogen peroxide

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and sodium hydroxide were purchased from Sigma-Aldrich (St. Louis, MO). ZnSO4

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and CuSO4 were obtained from Sigma-Aldrich (St. Louis, MO). Hydrochloric acid

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trace metal grade was purchased from Fisher Scientific (Fair Lawn, NJ). Cell dyes

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Calcien-AM

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mitochondrial membrane potential probe JC1 (5,5′,6,6′-tetrachloro-1,1′,3,3′

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-tetraethylbenzimidazolylcarbocyanine

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Technologies (Carlsbad, CA). DMSO was used as a solvent for all cell dye solutions.

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Vinblastine and MK571 were obtained from Cayman Chemical (Ann Arbor, MI).

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Paraformaldehyde was purchased from Electron Microscopy Sciences (Hatfield, PA).

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Nanomaterials

(CAM),

2’,7’-Dichlorofluorescin

iodide)

diacetate

were

(DCFH)

purchased

and

from

the

Life

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Nano-ZnO was purchased from Meliorum Technologies (Rochester, NY).

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Nano-CuO was obtained from Sigma-Aldrich (St. Louis, MO). Nano-ZnO and

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nano-CuO have been previously characterized by Keller et al.9 and Adeleye et al.35,

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respectively. All chemicals solutions were freshly prepared for each analysis. Their

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stock solutions were at a concentration of 1000 ppm prepared in ddH2O, sonicated

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(B-2200R-1, Fisher Scientific, USA) for 30 min, then 10 ppm alginate (final

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concentration) was added. All stocks were diluted into desired exposure

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concentrations in filtered seawater (FSW) containing 0.2 ppm alginate (final

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concentration)36. Six concentrations (0, 0.5, 1, 2, 5 and 10 ppm) for nano metal oxides

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and soluble metal ions were used for all analyses. To determine the copper

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concentration in copper oxide nanoparticle suspensions, samples were acidified to pH

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1.7 with trace metal grade 6N HCl and analyzed with the method described below.

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Sea urchin embryo culture

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Adult white sea urchins (Lytechinus pictus) were kept at the University of

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California Davis Bodega Marine Laboratory (Bodega Bay, CA) in flow-through

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seawater tanks. Gametes were collected and fertilized as described previously8. Prior

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to use in experiments, eggs were washed three times in 0.2 µm FSW containing 0.2

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ppm alginate (final concentration). Sperm was stored dry at 4oC until use.

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Fertilization success was checked under the microscope and only batches of embryos

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exhibiting >90% successful fertilization were used for subsequent experiment. All

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experiments were conducted at 14oC.

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Cytotoxicity assays

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Sea urchin embryos at 30min post-fertilization were exposed to target metals in

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96-well plate (100 embryos per well). To analyze the mitochondrial membrane

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potential stability after 3.5 h exposure to target metals, 0.2 µg/ml JC1 (final

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concentration) was added to the each well and incubated for 1h. Then the green

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(Ex:485nm, Em:535nm) and red (Ex:530nm, Em:630nm) fluorescence values were

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measured. For analysis of ROS production after 3.75 h exposure of target metals, 1.0

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µM DCF (final concentration) was added to each well, and incubated for 45 min. The

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DCF fluorescence values were measured at 485nm (excitation wavelength) and

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535nm (emission wavelengths). The fluorescence was measured using a TECAN

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GENios microplate reader (Maennedorf, Switzerland). Each combination of target

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chemical concentration and probe assay were internally replicated in 8 wells of each

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plate. Each exposure plate was replicated 3X for each target chemical using a

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different batch of embryos collected from different sea urchins.

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Cellular efflux pump inhibition assay

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Cellular efflux pump inhibition was measured by CAM accumulation. Embryos

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at cleavage and blastula stages were used. For the cleavage stage, sea urchin embryos

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(100 embryos per well) were exposed to target metals in 96-well plate. For

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comparison among different exposure concentrations, the embryos at 30min

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post-fertilization were used. For analysis on the influence of the fertilization envelope

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on CAM accumulation, the embryos at 5, 15, 30, 60 and 90 min post-fertilization

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were used. After 3 h exposure to target metals, 0.5 µM CAM (final concentration) was

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added into each well. Then the plates were incubated for 1.5 h at 14oC, and measured

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in plate reader. The excitation wavelength and emission wavelengths for CAM were

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485nm and 535nm, respectively. For the fluorescence imaging, the same exposure

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approaches were applied, but 6-well plates were used. Fluorescence imaging was

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performed using a scanning laser confocal microscope (Fluoview 500, Olympus,

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Japan).

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For the embryos at blastula stage, 50 embryos per well at 20 h post-fertilization

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were added to a 96-well plate. The exposure procedures and CAM measurement were

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performed as described above. The embryos were fixed with 1% paraformaldehyde in

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FSW immediately before plate reader measurements were made such that blastulae

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were in a monolayer on the bottom of the wells.

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Vinblastine assay

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The vinblastine (0-0.75µM dose response) assay was performed in 24-well plates

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at a density of 100 embryos/ml. Embryos at 30min post-fertilization were exposed to

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0.5 ppm nano-CuO, nano-ZnO and ZnSO4, and 0.2ppm CuSO4. When embryos in the

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control group developed to the 4-cell stage, vinblastine (0-0.75µM) with or without

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5µM MK571 (positive control) were added. When embryos in the control group

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developed to the 8-cell stage (>90%), the embryos in all groups were fixed using 1%

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paraformaldehyde. Then the percentage of 8-cell embryos in treated groups was

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counted. Each combination of target chemical and vinblastine concentration was

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internally replicated in 2 wells of each plate. Each exposure was replicated 3X for

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each target chemical using different batches of embryos collected from sea urchins.

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Copper measurement in sea urchin embryos

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Embryos (200 embryos/ml) at the blastula stage were exposed to CuSO4 and

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nano-CuO at 1ppm, with or without 10µM MK571, in 6-well plates. After 4.5h

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exposure, embryos were collected from the plates and washed three times with

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Ca2+-Mg2+-free artificial seawater (10 mM EDTA, pH 5.0), and then once with FSW,

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all at 14oC. Embryos were then homogenized in FSW using a probe sonicator. The pH

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was adjusted to 1.7 using 6 M copper-free HCl. Samples were incubated overnight at

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4°C, and centrifuged at 14,000 g for 10 min. The copper concentration in the

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supernatant was measured using a modified method by Durand et al. 37. The method is

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based on the light-emitting catalytic decomposition of hydrogen peroxide by

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copper(II)-1,10-phenanthroline complex. Standard solutions were prepared by

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diluting Certified Copper Standard solution (Sigma-Aldrich, St. Luis MO) in FSW

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acidified to pH 1.7 with copper-free 6N HCl. Standard solutions and samples are

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placed in a 96-well plate, and a freshly made solution of reagents was added. Final

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mixture contains 7.7mM ethylhexadecyldimethylammonium bromide, 0.43 µM

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tetraethylenepentamine, 29mM NaOH, 2.9mM 1,10-phenanthroline and 4% hydrogen

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peroxide. Light emission was detected using a TECAN GENios microplate reader

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(Maennedorf, Switzerland) in the luminescence mode. Concentration was determined

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by comparison with a standard curve conducted for each plate.

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Localization of nano-CuO in blastula stage embryos was conducted according to

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the method of Jose et al.38. Briefly, hatched blastula embryos were incubated in

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nano-CuO or CuSO4 (1ppm) for 4.5h, washed as described above with

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Ca2+-Mg2+-free artificial seawater (10 mM EDTA, pH 5), and fixed in 3%

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paraformaldehyde in FSW for 18 h at 40C.

After washing, blastula were incubated

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in 10µM Hoechst 33342 (Sigma-Aldrich, St. Luis MO), mounted on slides and

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compressed under coverglass, then viewed using scanning laser confocal microscopy

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using both interference contrast optics (transmitted light) and excitation at 405nm

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(blue diode laser) with a 60X water immersion lens on an Olympus BX61WI fixed

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stage upright microscope.

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RESULTS

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Characterization of nano-CuO and nano-ZnO

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Particles used in this study have been previously characterized9,

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. Major

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physicochemical properties are presented in Table 1. Both NMs rapidly aggregate in

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high ionic strength media, such as seawater, reaching a stable size in 60 min or less of

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around 400 nm for nano-ZnO and around 600 nm for nano-CuO. Sedimentation rates

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are concentration and ionic strength dependent. When a 10 ppm solution is made at

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high ionic strength, 90% of nano-ZnO particles remain in suspension after 6 h, while

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60% remains in suspension for a 50 ppm solution. Nano-CuO has a higher

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sedimentation rate, with less than 25% of nanoparticles remaining in suspension after

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6 h for a 50 ppm solution35.

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Nano-ZnO undergoes rapid dissolution in FSW, being almost complete within

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10-20 h for a 1 ppm solution8, 9. On the other hand, Adeleye et al.35 reported very low

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dissolution rates of less than 4% for a 10 ppm solution of nano-CuO after 90 days at

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pH 7 (100 mM NaCl).

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The dispersed copper oxide nanoparticles were analyzed to determine the copper

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content. The copper concentration was between typically between 69% to 77% of the

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nominal concentrations used (i.e. 10 ppm of nano-CuO is equivalent to 6.9-7.7 ppm of

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Cu2+).

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ROS generation in cleavage stage embryos

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The cell dye DCFH-DA was used to assess the generation of ROS. Nano-CuO at

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relatively low concentration (0.5, 1, and 2 ppm) caused a significant increase in DCF

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fluorescence values (Figure 1A), indicating an increase in intracellular ROS. However,

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higher concentrations of nano-CuO (5 and 10ppm) and CuSO4 (0.5-10ppm) did not

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cause an increase in DCF fluorescence values, and in fact, these fluorescence values

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decreased. For nano-ZnO and ZnSO4, we found that they have similar effects of ROS

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generation. A significant increase in DCF fluorescence values was found at 0.5, 1, 2,

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and 5ppm, but not at 10ppm. (Figure 1B).

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NMs induce changes in mitochondrial membrane potential in cleavage stage

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embryos

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Mitochondrial membrane potential was determined by cell dye JC1. A decreasing

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ratio of red/green fluorescence values from JC1 indicates a loss of mitochondrial

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membrane potential, and can be indicative of apoptosis. Nano-CuO did not cause

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changes in mitochondrial membrane potential (Figure 1C), but CuSO4 significantly

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decreased the ratio of red and green fluorescence values of JC1 at 1, 2, and 5ppm. For

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nano-ZnO, this NM caused a significant decrease in the ratio of red and green

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fluorescence values at 1, 2, and 5ppm (Figure 1D). ZnSO4 also decreased the ratio at

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0.5, 1, and 2ppm.

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Increased Calcien-AM (CAM) accumulation in sea urchin embryos treated with

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NMs

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Plasma membrane ABC transporter activity was assessed by CAM dye

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accumulation. CAM is an established indicator of ABC transporter efflux activity39, 40.

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CAM is not fluorescent and is membrane permeable, but once inside of cells, CAM is

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cleaved by esterases to calcein, which is fluorescent, is membrane impermeable, and

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is poorly effluxed from cells41, 42. Thus, the level of CAM accumulation can indicate

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the relative activity of ABC transporters. Two developmental stages (cleavage and

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blastula stages) of sea urchin embryos were utilized in this study. Both stages showed

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two important developmental stages of sea urchin embryos. At the cleavage stage (2-8

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cell), sea urchin embryos are surrounded by the fertilization envelope and cannot

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swim, but at the blastula stage, embryos hatch and can swim.

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At the cleavage stage, sea urchin embryos were exposed to nano-CuO, CuSO4,

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nano-ZnO and ZnSO4 at 30 min post-fertilization. After a 4.5h exposure, nano-CuO,

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CuSO4, nano-ZnO and ZnSO4 significantly increased CAM accumulation (p

Copper Oxide and Zinc Oxide Nanomaterials Act as Inhibitors of

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