Cytotoxicity and genotoxicity of cadmium oxide nanoparticles evaluated using in vitro assays

Journal Pre-proof Cytotoxicity and genotoxicity of cadmium oxide nanoparticles evaluated using in vitro assays Es¸ref Demir, Taichun Qin, Yan Li, Yongbin Zhang, Xiaoqing Guo, Taylor Ingle, Jian Yan, Annamaria Ioana Orza, Alexandru Biris, Suman Ghorai, Tong Zhou, Tao Chen

PII:

S1383-5718(20)30019-X

DOI:

https://doi.org/10.1016/j.mrgentox.2020.503149

Reference:

MUTGEN 503149

To appear in: Mutagenesis

Mutation Research - Genetic Toxicology and Environmental

Received Date:

12 June 2019

Revised Date:

28 November 2019

Accepted Date:

10 December 2019

Please cite this article as: Demir E, Qin T, Li Y, Zhang Y, Guo X, Ingle T, Yan J, Orza AI, Biris A, Ghorai S, Zhou T, Chen T, Cytotoxicity and genotoxicity of cadmium oxide nanoparticles evaluated using in vitro assays, Mutation Research - Genetic Toxicology and Environmental Mutagenesis (2020), doi: https://doi.org/10.1016/j.mrgentox.2020.503149

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

Cytotoxicity and genotoxicity of cadmium oxide nanoparticles evaluated using in vitro assays Running Title: Genotoxicity and cytotoxicity of CdO NPs Eşref Demir1,8, Taichun Qin1,9, Yan Li1,10, Yongbin Zhang2, Xiaoqing Guo1, Taylor Ingle3, Jian Yan1, Annamaria Ioana Orza4,5, Alexandru Biris6, Suman Ghorai3, Tong Zhou7, and Tao Chen1, * 1

Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, USA 2

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Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA 3

Office of Scientific Coordination, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, USA 4

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Department of Radiology and Imaging Sciences and Center for Systems Imaging, Emory University School of Medicine, Atlanta, Georgia, USA CellaCurre LLC., 3630 Peachtree Rd, Atlanta, GA, 30326

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Center for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, Little Rock, AR, USA Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, MD, USA

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Present Address: Antalya Bilim University, Faculty of Engineering, Department of Material Science and Nanotechnology Engineering, Antalya, TURKEY 9

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Present Address: Office of Global Regulatory Operations and Policy, U.S. Food and Drug Administration, Los Angeles, CA, USA Present Address: Exploratory Medicine & Pharmacology, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285 U.S.A. Author: 3900 NCTR Rd, Jefferson, AR 72079, USA

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*Corresponding

: 870-543-7682, E-mail: [email protected]

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Phone : 870-543-7954 Fax

FDA Disclaimer: The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration.

Highlights 

Cellular uptake of CdO NPs was detected using TEM coupled with EDS

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CdO NPs were cytotoxic and genotoxic in mammalian cells 1

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CdO NPs decreased cell viability and ATP contents and increased LDH leakage

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CdO NPs induced DNA damage and chromosomal breaks in TK6 cells

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CdO NPs induced mutations in mouse lymphoma cells

Abstract Cadmium oxide nanoparticles (CdO NPs) are among the most industrially used metal

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oxide NPs. They have been widely used for industrial application, such as paint pigments and electronic devices, and medical therapeutics. With increasing use of CdO NPs and concerns for their potential adverse effects on the environment and public health, evaluation of the

cytotoxicity and genotoxicity of CdO NPs becomes very important. To date, there is a limited

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understanding of the potential hazard brought by CdO NPs and a lack of information and

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research, particularly on the genotoxicity assessment of these NPs. In this study, 10 nm CdO core-PEG stabilized NPs were synthesized, characterized and used for evaluation of CdO

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NPs’ cytotoxicity and genotoxicity. Release of cadmium ions (Cd+2) from the CdO NPs in cell culture medium, cellular uptake of the NPs, and the endotoxin content of the particles

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were measured prior to the toxicity assays. Cytotoxicity was evaluated using the MTS assay, ATP content detection assay, and LDH assay. Genotoxicity was assessed using the Ames test,

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Comet assay, micronucleus assay, and mouse lymphoma assay. The cytotoxicity of cadmium chloride (CdCl2) was also evaluated along with that of the CdO NPs. The results showed that

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endotoxin levels within the CdO NPs were below the limit of detection. CdO NPs induced concentration-dependent cytotoxicity in TK6 and HepG2 cells with the MTS, ATP and LDH assays. Although the genotoxicity of CdO NPs was negative in the Ames test, positive results were obtained with the micronucleus, Comet, and mouse lymphoma assays. The negative response of CdO NPs with the Ames test may be the result of unsuitability of the assay for measuring NPs, while the positive responses from other genotoxicity assays suggest that CdO 2

NPs can induce chromosomal damage, single or double strand breaks in DNA, and mutations. The toxicity of the CdO NPs results from the NPs themselves and not from the released Cd+2, because the ions released from the NPs were minimal. These results demonstrate that CdO NPs are cytotoxic and genotoxic and provide new insights into risk assessment of CdO NPs for human exposure and environmental protection.

Keywords: Cadmium oxide nanoparticles, Cadmium ion, Cytotoxicity, Genotoxicity, DNA

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damage, Mutant frequency, Cell uptake, Endotoxin

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1. Introduction

With at least one dimension of less than 100 nm, nanomaterials (NMs) are defined by

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their small size. Due to their novel physicochemical properties, they are increasingly used in diverse areas of industry, including textiles, finishes, cosmetics, sunscreens, dietary

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supplements, medical devices, prescription drugs, and electronics. Thus, NMs are being introduced into the environment, and humans are being exposed to NMs at increasing rates.

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For these reasons, nanotoxicology is being developed as a novel field to study the potential risk of NMs and their mechanisms of action [1-3].

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Cadmium (Cd) is a well-known toxic element and environmental pollutant [4].

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Ecotoxicity and physicochemical results indicate that metallic Cd is noteworthy among harmful metallic particles in terms of cytotoxicity and bio-accessibility [5]. Cadmium oxide nanoparticles (CdO NPs) have been used as the starting material in the manufacture of quantum dots, which are gaining favour in both medical diagnostic imaging and targeted therapeutics [6, 7]. The release of Cd NPs into the environment could result in their accumulation in the food chain and increased human exposure. Therefore, it is very important

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to understand toxicity of CdO NPs, especially their genotoxicity, to protect the environment and reduce harm from their exposure. Many researchers have investigated the genotoxicity and cytotoxicity of NMs, including CdO NPs. The toxicity of CdO NPs has been tested by different in vitro and in vivo studies [4, 5, 8-12] . Their results show that CdO NPs have adverse effects on living organisms, and eukaryotic systems are generally more sensitive than prokaryotic cells [12]. Despite the many applications of CdO NPs, data related to the genotoxic potential of

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CdO NPs are lacking [13]. To evaluate the cytotoxicity and genotoxicity of CdO NPs, we synthesized 10 nm CdO NPs and characterized the particles in this study. The NPs were

evaluated in three cell lines, the human lymphoblastoid TK6 cells, human liver hepatocellular

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carcinoma cells (HepG2), and L5178Y/Tk+/−-3.7.2C mouse lymphoma cells. The TK6 and HepG2 cells were used for the cytotoxicity assays to compare the toxic effects of the

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nanoparticles on suspension and adherent cells. The TK6 cells were used for the

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micronucleus assay and the Comet assay since the cells have extensively been used for genotoxicity study. The mouse lymphoma cells were used for mouse lymphoma assay

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because the cell line is required for the assay. Three commonly used assays for cytotoxicity measurement were applied including the MTS assay, LDH assay, and ATP content detection

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assay. The genotoxicity of CdO NPs was assessed using four standard assays including the Ames test, the in vitro micronucleus (MN) assay, the in vitro Comet assay and the mouse

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lymphoma assay (MLA). To understand the mechanism, release of Cd ions from the CdO NPs, cytotoxicity of CdCl2, and uptake of CdO NPs were also studied.

2. Materials and methods 2.1. Synthesis of CdO NPs

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The synthesis of 10 nm CdO core-PEG stabilized NPs was conducted in two steps: the precipitation of cadmium oxide from a solution of Cd (acac)2 (0.04 mol) with NaOH (0.04 mol), followed by a thermal seed mediated synthesis. For the seed mediated synthesis, 10 ml of the resulting NPs were injected into a 50 ml solution of ethylene glycol and the temperature was set to 250°C. When the temperature was reached, an aqueous solution of PEG3000 (100 mg/ml, 12.2 μl, 4.06 x 10-4 mol) and Cd (acac)2, (0.04 mol) were simultaneously added dropwise. The color of the reaction mixture changed from colorless to

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yellowish, to red. Throughout the entire reaction N2 gas was bubbled through the reaction solution.

2.2. Characterization of the CdO NP and dispersion procedure

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The sizes and components of the CdO NPs were characterized by transmission

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electron microscopy (TEM, Jeol JEM-2100 LaB6, 200 kV), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS) (Hitachi S-415 electron

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Microscope, Tokyo, Japan at 25 kV). X-ray Diffraction (XRD) measurements were performed on a D8 advance diffractometer (Bruker, AXS, Germany) with a goniometer

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radius 217.5 nm, Gobel mirror parallel beam optics 2 Sollers slits and 0.2 mm receiving slit. The instrumental broadening was determined using LaB6 powder (NIST SRM 660). The

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samples were prepared on the same day and immediately analysed.

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2.3. Hydrodynamic size of CdO NPs To characterize the selected NPs, dynamic light scattering (DLS) was used for

hydrodynamic size measurement. CdO NPs were suspended in 10% DMSO in water at 1 mg/ml concentration and measured using Zetasizer (Malvern, Worcestershire, UK). Viscosity value of the suspension was adjusted to account for hydrodynamic size. Briefly, the CdO NPs (0.625-10 µg/ml) were measured following the dilution of the particle stock suspension to 50

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µg/ml in the cell culture medium (RPMI 1640). These dilutions were briefly vortexed and sonicated for 5 min. For the size measurement, 60 µl of the diluted dispersion of the NPs was transferred to a cuvette for dynamic size measurement. The concentration of the samples and experimental methods were optimized to assure the quality of the data. The sizes were measured at least three times. The data were calculated as the average size of NPs. 2.4. Release of Cd+2 from the CdO NPs in cell culture medium An Ultra-15 K filter (Amicon, Carrigtwohill, CO, USA) was used to separate Cd+2

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from the CdO NPs per the standard protocol described in the instruction manual

(www.millipore.com). After incubating 2.5, 5 and 10 μg/ml CdO NPs with 7 ml cell culture

medium (RPMI 1640, 10% FBS, and 1% PS) at 37⁰C for 24 h, the medium was added to the

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internal filter and centrifuged at 4000 g for 30 min at room temperature. The filtered medium

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was collected for measurement of Cd+2 concentration. In addition, 1 mg/ml CdO NPs stock suspension was filtered to measure ion concentration in the stock suspension. The samples

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were stored at 4°C until processing. The medium samples were acid digested in a CEM MARS Express digestion system with 2:1 HNO3:HCl. Metal analysis was performed using a

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PerkinElmer's NexION 2000 series inductively coupled plasma mass spectrometry (ICP-MS) with indium as an internal standard.

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2.5. Endotoxin assay for NPs

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To test if NPs were contaminated with endotoxin, the endotoxin content was measured using the chromogenic Limulus amebocyte lysate (LAL) assay (Lonza (QCL1000TM), Inc., Walkersville, MD, USA) per the standard protocol described in the instruction manual and previous studies [14]. Briefly, all glassware was rendered pyrogen free by heating overnight at 200°C. Then, 50 µl of test sample or standard were added to the test well in the 96-well plate at 37°C. At least three wells were used for each sample. The linearity of

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the standard was verified using lipopolysaccharide (LPS) supplied in the kit. For each assay, a standard curve (the concentration ranged from 0.1 to 1 EU/ml endotoxin) was generated over the concentration range 0.182-1.047333 EU/ml and referenced to control standard endotoxin (Escherichia coli E50-640). Endotoxin standards and serial dilutions of sample were assayed in pyrogen-free microtiter plates (Costar No. 3596; Corning, Inc., Corning, NY, USA) in a BioTek Synergy 2 microplate reader (BioTek, Winooski, VT, USA) at 37°C. Absorbance measurements were taken at 405-410 nm. Commercially available endotoxin

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standard (lipopolysaccharide, LPS; 0.5 EU/ml) and LAL water were used as positive and negative controls, respectively. The percentage of the recovery of spiked value was

The recovery of spiked value (%) =

a-b x 100 c

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calculated as following (www.lonza.com/qcl1000).

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where a is the amount of endotoxin found in spiked sample, b is the amount of endotoxin found in sample and c is the amount of added endotoxin. The recovery of spiked values was calculated as 102.2, 102.6, 103.6, 103.2, 101.2 and 99.4% for CdO NPs (0.625, 1.25, 2.5, 5,

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7.5 and10 µg/ml) and 101.8, 102.2, 102.6, 102.8, 101 and 98.8% for CdCl2 (0.625, 1.25, 2.5,

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5, 7.5 and10 µg/ml), respectively.

2.6. Cell lines and treatment

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Three cell lines were used in this study; TK6, HepG2 and mouse lymphoma cells.

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Cell lines were routinely cultured under standard culturing conditions (37°C, 5% CO2 in a humidified environment). TK6 cells were maintained in Roswell Park Memorial Institute (RPMI)1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA) and 1% penicillin/streptomycin (Life Technologies). HepG2 cells were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 100 µg/ml

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gentamicin. Mouse lymphoma cells were thawed and seeded at a concentration of 1.33 × 105 cells/ml in RPMI 1640 supplemented with 10% heat-inactivated horse serum, 2 mg/ml sodium bicarbonate, 10 ml/L nonessential amino acids, 200 UI/ml penicillin, 5 µg/ml streptomycin, 2.5 µg/ml amphotericin B, 0.02% wt/vol L-glutamine, 0.02 mg/ml sodium pyruvate, 0.05% vol/vol pluronic F68 solution. Before the treatment, the CdO NPs or CdCl2 were vortexed and bath sonicated for 5 min to disperse them throughout the suspension or solution. Working suspension of CdO NPs

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or solution of CdCl2 were prepared by diluting the stock suspension or solution in distilled water. Cells were cultured in the treatment medium (culture medium with different concentrations of CdO NPs).

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For the MTS, ATP, and LDH assays, cells were seeded into 96-well plates at density

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of 1x105, 1x106, and 2x105 cells/200 µl well, respectively. In the cytotoxicity assays, TK6 and HepG2 cells were treated in triplicate with serial concentrations of CdO NPs (0, 0.3125,

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0.625, 1.25, 2.5. 5, 7.5 and 10 µg/ml) for 24 h. The selections of these concentrations were based on previously published data [9]. The choice of the incubation period of 24 h was

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based on literature data [5, 15, 16].

The TK6 cells were treated in triplicate with serial concentrations of CdO NPs (0, 1,

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2.5, 5 and 7.5 μg/ml) for the MN assay. X-rays generated by an RS-2000 Biological Irradiator (Rad Source Technologies, Suwanee, GA, USA) at 0.75 Gy were used as the positive control

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(PC), while the vehicle controls (NC) were treated with water only. The cells were then incubated with gentle shaking for 28 h so that the cells in the vehicle controls would go through 1.5-2.0 cell divisions. Four concentrations (0.625, 1.25, 2.5 and 5 µg/ml) of CdO NPs were tested for the Comet assay. Aliquots of 10 µl of each CdO NP working suspension were added to the

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cultures for 4 h at 37°C. Distilled water was used as NCs, while hydrogen peroxide (H2O2, 100 µM) was used as a PC for the genotoxicity and cytotoxicity assays. Three and/or four independent replicates were conducted for each concentration in each experiment. 2.7. Cellular uptake of CdO NPs evaluated using transmission electron microscopy (TEM) TK6 cells exposed to CdO NPs (5 µg/ml) for 4 hours were fixed in 2% (w/v) paraformaldehyde (EMS, Hatfield, PA) and 2.5% (v/v) glutaraldehyde (Merck, Darmstadt, Germany) in 0.1 M cacodylate buffer (Sigma-Aldrich, Steinheim, Germany) at pH=7.4. Cells

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were then processed as described previously [17]. Briefly, TK6 cells post-fixed in 1%

osmium tetroxide were dehydrated through a graded ethanol series, cleared in acetone, and

infiltrated and embedded in Spurr's resin (Polysciences, Inc., Warrington, PA) in a BEEMTM

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embedding capsule. Thick sections of 1 µm were cut for area selection and thin sections of

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700-800 Å were cut on an ultramicrotome (Leica EM UC7, Wetzlar, Germany) and placed on 200 mesh copper grids and viewed at an accelerating voltage of 80 kV on a FEI Tecnai™ G2

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Spirit BioTWIN transmission microscope. For elemental analysis, the intracellular localization of CdO NP was confirmed by HAADF STEM detector and energy dispersive X-

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ray spectroscopy (EDX) with an Oxford X-Max 65T SDD EDS detector. The grids were not stained to allow for better visualization of the CdO NPs and to ensure the absence of stain

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artifacts due to staining with lead citrate and uranyl acetate.

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2.8. MTS assay

The MTS assay is a variation of the MTT assay but with several advantages when

compared with the MTT assay, such as requiring no volatile organic solvent to solubilize the formazan product; also, the reactions can be read and returned to the incubator for further color development. Cell viability was detected using an MTS cell proliferation colorimetric assay kit (BioVision, CA, USA) as previously described [18]. TK6 and HepG2 cells in the

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treatment medium were plated in 96-well microplates and were cultured at 37°C in a humidified atmosphere for 24 h. The cells were then treated with CdO NPs and CdCl2 for 24 h. Following incubation, 20 µl/well of MTS reagent was added and incubated for 4 h at 37°C in standard culture conditions. Absorbance of soluble dye was measured at 490 nm with a BioTek Synergy 2 microplate reader (BioTek, Winooski, VT, USA). 2.9. ATP assay The ATP content detection assay is a method used to determine the number of viable

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cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. The intracellular ATP content was measured using an ATP

colorimetric/fluorometric assay kit (BioVision, CA, USA) per the manufacturer’s instructions

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and previous studies (Nguyen et al. 2015). Following treatments of TK6 and HepG2 cells, the

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cells were incubated with somatic cell ATP releasing reagent. After that, ATP measurements were determined on fluorescence at Ex/Em = 535/587 nm using a BioTek Synergy 2

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microplate reader (BioTek, Winooski, VT, USA).

2.10. Lactate dehydrogenase (LDH) activity release assay

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Lactate dehydrogenase (LDH), a cytoplasmic enzyme, was released into the medium when the cell was lysed. The release of cytoplasmic LDH, used as an indicator of cell

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membrane damage, was measured in the culture medium using a LDH-cytotoxicity

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colorimetric assay kit II (BioVision, CA, USA) per the manufacturer’s instructions. Absorbance at 450 nm was measured using a BioTek Synergy 2 microplate reader (BioTek, Winooski, VT, USA). Data from control and treated TK6 and HepG2 cells were calculated as percent LDH leakage (100xLDH activity in medium/total LDH activity) and expressed as the mean ± SD, using at least triplicate wells per concentration. Hydrogen peroxide (H2O2) can serve as a positive control for the LDH assay [19].

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2.11. Ames test The Ames test was performed as previously described [20]. Tester strains used in the Ames test were Salmonella typhimurium TA98, TA100, and TA102. A pre-incubation assay using 6.3, 12.5, 25, 50 μg/plate without S9 activation was conducted according to guidelines by Organization for Economic Cooperation and Development (OECD) with modification [21]. For pre-incubation, the tester strains were incubated in nutrient broth II with CdO NPs for 4 h at 37°C with shaking at 80 rpm. Before incubation, the CdO NPs were dispersed by

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vortexing for 5 min followed by 10 min of bath sonication in a Branson B2510 (Branson Ultrasonics, Danbury, CT, USA) with 100W output power and 42 kHz frequency. The

negative control was 100 μl H2O per plate for all the tester strains. The positive controls were

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2-nitrofluorene (3 μg per plate) for TA98, nitrofurantoin (5 μg per plate) for TA100 and

mitomycin (0.2 μg per plate) for TA102. . After 4 h incubation, top agar was added to the

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treatment mixture, which was then poured on a minimal glucose agar plate. The plate was

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then incubated for 48 h at 37°C. After the incubation, the plates were observed, and the colonies formed were counted using a Quebec Darkfield Colony Counter.

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For the Ames test, positive and negative responses were defined as described previously [22]. When treatments caused a dose-dependent response and at least one

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treatment group induced two-fold or more change in the number of revertant colonies over the control, the testing agents were considered as positive. A negative response was defined

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as a no dose-dependent increase in the number of revertant colonies. 2.12. In vitro micronucleus assay by flow cytometry

At the end of the treatment, the TK6 cells were collected and washed twice with PBS. Cytotoxicity was estimated by relative population doubling (RPD), as recommended by the OECD [23]. Cells were counted using the TC10™ Automated Cell Counter (Bio-Rad, Hercules, CA, USA) with trypan blue staining. The MN analysis was performed as described 11

in the High Content Protocol 1 in the instruction manual for the In Vitro MicroFlow® Kit (Litron Laboratories, Rochester, NY, USA). The samples were analysed using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). We counted 10,000 healthy nuclei, and information on cytotoxicity, apoptosis, MN events, and hypodiploid features was obtained from the flow cytometer as previously described [22]. 2.13. Comet assay After treatment of TK6 cells, cell viability was tested using trypan blue dye exclusion

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to avoid false positive responses. Ten µl 0.4% trypan blue was added to 10 µl of cell culture, and a 10 µl mixture of the trypan blue/cell was added on a microscope slide [24]. Cells were counted using the TC10™ Automated Cell Counter (Bio-Rad, Hercules, CA, USA) with

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trypan blue staining.

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The Comet assay was performed with a kit purchased from Trevigen Inc. (Gaithersburg, MD, USA) and the assay was conducted per the method described by the

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manufacturer’s instruction manual. The slides were then immersed in a pre-chilled lysis solution and placed back in the dark refrigerator for at least 1 h. The lysis buffer was then

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removed, and the slides were immersed in alkaline unwinding solution for 30 min at 4°C in the dark at room temperature. The slides then were removed from the alkaline unwinding

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solution and electrophoresed at 1 V/cm for 30 min in the dark. The analysis war conducted using a system comprised of a Nikon 501 fluorescent microscope and Comet IV digital

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imaging software (Perceptive Instruments, Wiltshire, UK). The Comet tail lengths were digitally analysed and scored based on tail length, width and intensity. Percent (%) DNA in the tail, defined as the fraction of DNA in the tail divided by the total amount of DNA associated with a cell multiplied by 150, was used as the parameter for DNA damage analysis using the software.

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2.14. Mouse lymphoma thymidine-kinase assay (MLA) Mouse lymphoma cells were treated with different concentrations of CdO NPs and 4nitroquinoline-1-oxide (4-NQO, 0.1 µg/ml) as a positive control. Following a 2-day expression period, the treated cultures were divided into two parts to perform mutant selection experiments using the microwell version of the MLA [25]. In the selection plates, 3 µg/ml of trifluorothymidine (TFT) was added to the cells in the cloning medium for mutant enumeration, and the cells were seeded into four 96-well flat-bottom microtiter plates using

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200 µL per well and a final density of 2,000 cells/well. For the determination of plating

efficiency, the cultures were adjusted to 8 cells/ml medium and aliquoted in 200 µL per well into two 96-well flat-bottom microtiter plates. In the soft-agar version, 1 µg/ml of TFT was

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added in the cloning medium to enumerate the mutants.

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All 96-well plates were incubated at 37°C in a humidified incubator with 5% CO2 in air. After 11 days of incubation, the colonies were counted, and mutant colonies were

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categorized as small or large [25]. For the microwell version, counting was performed visually, and the small colonies were defined as those smaller than 25% of the diameter of the

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well. Mutant frequencies (MFs) were calculated using the Poisson distribution. Mutant colonies approximately <0.6 mm in diameter were counted as small-colony mutants.

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Cytotoxicity was measured using relative total growth (RTG) that includes a measure of growth during treatment, expression, and cloning [25].

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The data evaluation criteria developed by the MLA expert workgroup of the

international workgroup for genotoxicity tests (IWGT) were used to determine whether a response was positive or negative [26, 27]. Positive responses are defined as those where the induced MF in one or more treated cultures exceeds the global evaluation factor (GEF) of 126 mutants per 106 cells and there is also a dose related increase in MF.

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2.15. Statistical analysis The ANOVA analysis was applied using SigmaPlot version 11.0 (SPSS, Chicago, IL, USA) for the three cytotoxicity assays, the MN assay and the Comet assay. p< 0.05 was used to identify statistically significant differences.

3. Results

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3.1. NP characterization High resolution transmission electron microscopy (HR-TEM) and energy dispersive

X-ray (EDS) spectroscopy were used to analyze the 10 nm CdO NPs. The TEM image of the

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10 nm dry CdO NPs is shown in Fig. 1A. The prepared CdO NPs are spherical in shape,

having an average size of approximately 16.3 nm in diameter. The CdO NPs exhibited a

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broad size distribution, ranging from appropriately 10 nm to 32 nm. Histogram of particle

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diameter analysis is also shown in Fig. 1A. All the data fit in the histograms were done using a Lorentz fit function. Moreover, TEM/EDS analysis results were assembled (STEM

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HAADF image of a CdO NP along with elemental maps of Cd and O, as well as EDS spectroscopy results), clearly showing the presence of Cd in the sample. The EDS analysis of this CdO NP revealed an atomic ratio between Cd:O is ~1:9 (0.20:1.80) (Fig. 1B). The

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extremely high concentration of O is due to the background while the Co, Cu and Zn resulted

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from the grid for the TEM.

TEM was used for directly measuring the core size of the single NPs based on the

projected area; while DLS was used for measuring the hydrodynamic diameter of the NPs based on the translational diffusion area of the particle being measured. DLS results suggested that the CdO nanoparticles formed agglomerates in suspension. DLS is sensitive to a small amount of the particle agglomerates in the suspension. The Z-average size of the 14

particles or particle aggregates were measured in 10% DMSO in water suspension as 440 nm with a PdI of 0.314, indicating particle polydispersity and instability. The relative high value of the hydrodynamic diameter indicates the tendency of CdO NPs to aggregate/agglomerate. To avoid aggregation, all subsequent experiments were carried out using freshly sonicated NPs dispersions. 3.2. The endotoxin level of CdO NPs The endotoxin levels of CdO NPs were evaluated in the absence and presence of 0.5

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EU/ml added endotoxin (LPS) using LAL assay (Fig. 2). The endotoxin levels in all

concentrations of the test chemicals were below the detectable level (0.182 EU/ml). Thus, the

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CdO NPs were not contaminated with endotoxin. 3.3 Cellular uptake

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TEM was used to determine the internalization of CdO NPs in TK6 cells. The untreated TK6 cells are round in shape. Cell structure is well preserved in the treated cells

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and the nucleolus within the nucleus is clearly visible (Fig. 3A). TEM images showed the internalization of CdO NPs in TK6 cells after 4 h of exposure. Cellular uptake of CdO NPs

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was observed in the cells treated with 5 µg/ml of CdO NPs. CdO NPs were present inside of the cells in the cytoplasm (Fig. 3B-C). HAADF STEM spectrum image indicates the presence

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of high contrast NPs (Fig. 3D). The presence of CdO NP aggregate was confirmed by reading

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the energy dispersive X-ray spectroscopy (EDS) spectrum at localized area, which resulted in clear peaks for Cadmium (Cd) and Oxygen (O) in the given spectrum image (Fig. 3E). 3.4. Release of Cd+2 from CdO NPs The culture medium with 2.5, 5 and 10 µg/ml CdO NPs was incubated for 24 h at the cell culture condition. After the incubation, the CdO NPs were filtered out before the measurement. The concentrations of the released Cd+2 were 0.622, 1.097, and 2.158 ng/ml as 15

measured by ICP-MS, or 0.02488, 0.02194, and 0.02158 % of 2.5, 5 and 10 µg/ml CdO NPs, respectively (Fig. 4). In addition, the concentration of the released Cd+2 from the 1 mg/ml CdO NPs stock suspension was also measured and was 0.97976 µg/ml, about 0.1 % of the CdO NPs in the stock suspension (Fig. 4), suggesting that CdO NPs are not prone to release Cd+2 when diluted in the culture medium. 3.5. MTS assay The cytotoxic effects of CdO NPs and CdCl2 were examined by the MTS assay. After

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treatment for 24 h, the viability of the HepG2 cells treated with 1.25 µg/ml CdO NPs was

reduced to 64% when compared with the concurrent control cells (Fig. 5B). Furthermore, the viability of the TK6 cells treated with 1.25 µg/ml CdO NPs was reduced to 40% (Fig. 5A).

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Similar results for CdCl2 treatment were also observed for both TK6 and HepG2 cell cultures

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(Fig. 5A-B). These cytotoxic effects were concentration-dependent. 3.6. ATP levels

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The cytotoxicity of the 10 nm CdO NPs and CdCl2 were assessed using an ATP colorimetric/fluorometric assay kit, which calculates the amount of ATP in cells. Cells were

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lysed to release ATP, followed by a fluorescence reaction to produce a stable fluorescent signal proportional to the ATP contents. The effects of 0.625-10 µg/ml CdO NPs and CdCl2

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were tested on intracellular ATP concentration. When compared with the negative control,

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the ATP concentrations in CdO NPs and CdCl2-treated cells were significantly reduced (Fig. 6A-B). The data show that CdO NPs and CdCl2 are toxic in a concentration-dependent manner.

3.7. LDH release The cytotoxicity of the 10 nm CdO NPs and CdCl2, as determined using the LDHcytotoxicity colorimetric assay kit II, is shown in Fig 7A-B. The LDH activity levels were 16

increased with the treatment concentrations of CdO NPs and CdCl2. As shown in Figure 7B, a significant increase in LDH activity was observed at 2.5, 5 and 10 µg/ml of CdO NPs and CdCl2 in HepG2 cells. At the highest concentration (10 µg/ml), the treatment resulted in LDH release to 123% and 128% over the control for CdO and CdCl2, respectively. Similarly, a significant increase in LDH activity was observed at the same concentrations following CdO NPs and CdCl2 in TK6 cells (Fig. 7A). The highest concentrations of CdO NPs and CdCl2 resulted in a very high LDH release up to 189% and 160%, respectively. As shown in Fig.

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7A-B, the cytotoxicity increased in a concentration-dependent manner. 3.8. Ames test

The results for the Ames test are shown in Fig. 8. The bacteria were treated with CdO

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NPs at 6.3, 12.5, 25 and 50 μg/plate for three different strains (TA98, TA100, and TA102).

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There was no significant difference in mutagenicity from the negative control in all the three strains, whereas the positive control chemicals for the strains significantly increased the

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mutant frequencies over the negative control (Fig. 8). The results from the positive and negative controls in this study were similar to our historical data for positive and negative

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controls. Thus, the CdO NPs were not mutagenic in the Ames test. According to the OECD Guideline [21], a negative result should be confirmed using the highest concentration up to 5

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mg per plate. The highest concentration in this study was 50 μg/ml because the 10 nm CdO NP was very toxic to the test strains. Treatments at doses higher than 50 μg/ml resulted in

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cell death.

3.9. Micronuclei assay Treatment of CdO NPs produced a dose-dependent cytotoxicity and micronucleus induction (Fig. 9). The RPDs in 2.5 μg/ml or high dose CdO NP treatment groups increased significantly over the negative control and the concentration at which 50 ± 5% RPD induced

17

was 7.5 μg/ml of CdO NPs, suggesting that the NPs were very toxic (Fig 9). When compared with the control, a significant increase in micronuclei was also observed at 5, 7.5 and 10 μg/ml CdO NPs treatment groups, indicating that CdO NPs are genotoxic. X-rays at 0.75 Gy were used as the positive control and they caused high induction in micronuclei at a relatively low level of cytotoxicity (Fig. 9). 3.10. Comet assay Cell viability in TK6 cells treated with CdO NPs was evaluated using the Trypan blue

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dye exclusion assay. If cell viability is too low, the treatment can produce a false positive

result in the Come assay. As shown in Fig. 10A, CdO NP treatment caused cytotoxicity in

TK6 cells in a concentration dependent manner. At 5 µg/ml concentration of CdO NPs, the

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cell viability was approximately 63% (Fig. 10A). In the Comet assay, all treatments of CdO

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NPs resulted in a significant induction of DNA damage in the cells in a dose-dependent manner. At the concentration 0.625 μg/ml, at which the CdO NPs did not significantly reduce

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the cell viability, CdO NPs increased DNA damage about 4-fold over the negative control. The DNA damage even reached to 8-fold over the control at the highest dose. H2O2 was used

10B).

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3.11. MLA assay

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as the positive control and it induced a significant induction of DNA breaks in the assay (Fig.

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The MLA of the CdO NPs showed a positive result. Mouse lymphoma cells were treated with CdO NPs at concentrations of 0.1, 0.25, 0.5, 0.75 and 1 μg/ml. The highest treatment concentration induced a RTG higher than 20% so that the mutant frequency data can be used to evaluate the mutagenicity of the NPs [25]. The treatment of CdO NPs induced a concentration-related increase in mutant frequency, which inversely correlated with a decrease of RTG. The treatment of CdO NPs at the highest concentration of 0.75 μg/ml was

18

associated with the lowest RTG (22.6±15) % and produced the highest MF induction (480 ± 142x 10-6), about 5-fold induction over the control (Fig. 11). The positive control, 4-NQO, induced a high MF at 949 x 10-6 with an RTG at 66%. The results suggest that CdO NPs are mutagenic in the mouse lymphoma cells.

4. Discussion

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There is increasing concern regarding adverse effects of metal and metal oxide NPs on the environment and public health. The information on toxicity of CdO NPs, especially on their genotoxicity, is lacking. Cd is widely used in industry and is one of the important

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environmental pollutants that can have adverse effects in humans via the food chain [28]. Cd is a human carcinogen defined by the International Agency for Research on Cancer (IARC)

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[29, 30]. Although potential health effects of NPs have been intensively studied over the past few decades, the detailed mechanisms underlying NP toxicity and genotoxicity are still

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poorly understood. Thus, evaluation of genotoxicity and cytotoxicity of NMs is a crucial part of risk assessment and regulation of NMs.

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Different toxicity assays for assessing NMs can generate results through different mechanisms [31, 32]. Therefore, it has been suggested that different methodologies should be

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used to compare the toxicity of different types of NMs and this practice has been used by

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researchers. Lanone et al. [33] evaluated the toxic effects of 24 manufactured NPs using MTT and neutral red staining. Xu et al. [34] studied the cytotoxicity of both nano and microsized aluminium oxide NPs using three different viability assays (CCK-8, MTT, and LDH), and suggested that the LDH assay was more suitable for assessing cell viability of NMs than the CCK-8 and MTT assays. In this study, we examined cytotoxicity of CdO NPs using three assays that can assess cell viability, ATP production, and membrane disruption. All three

19

assays were positive to the exposure of CdO NPs in mammalian cells, suggesting that CdO NPs can reduce cell viability, decrease cellular ATP production, and increase cell membrane disruption. Our results also suggest that TK6 cells are more sensitive to the cytotoxicity of CdO than HepG2 cells. TK6 cells showed lower viability than HepG2, suggesting that different cell lines respond differently to NPs. HepG2 cells are human liver hepatocellular cells and can produce more metallothionein, a metal sequestering protein. Metallothionein can

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efficiently reduce toxicity of metals by binding to them. This could explain the higher tolerance of HepG2 cells to CdO NPs in this study.

We also evaluated DNA and chromosome damage caused by CdO NPs using the

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Comet assay and micronucleus assay, and mutagenicity of the particles using the Ames test

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and MLA. Many studies have demonstrated that NPs, especially metal oxide NPs, can induce DNA damage and mutations [24, 35]. The Comet assay is a sensitive method for evaluating

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single and double DNA breaks, while the micronucleus assay can detect chromosome breaks. In this study, CdO NPs showed clastogenicity in the TK6 cells. The NPs induced high fold

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changes over their concurrent controls in DNA damage and chromosome breaks in the Comet and micronucleus assays, respectively. Furthermore, the CdO NPs induced mutations in

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mouse lymphoma cells. The concentration-related increases in MFs were associated with concentration-related increases in cytotoxicity. Contrary to what was observed in MLA, CdO

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NPs (0-50 µg per plate) were negative in the Ames test. The results are consistent with many others that demonstrate NPs are generally negative in the Ames test [22, 36-38], further suggesting that the Ames test is not suitable to measure mutagenicity of NPs. To understand the mechanism underlying toxicity of CdO NPs, we measured Cd+2 released from CdO NPs in cell culture medium by ICP-MS, examined the cell uptake of CdO NPs using TEM coupled with EDX, and determined the cytotoxicity of CdCl2 along with the 20

measurement of CdO NPs using the MTS, ATP content detection and LDH assays (Figs. 57). Although CdCl2 caused cytotoxicity at similar concentrations as these for CdO NPs, there was no toxicity at 0.3 µg/ml CdCl2 which was much higher than the ion concentrations released by the CdO NPs incubated in cell culture medium for 24 h (0.002158 µg/ml Cd+2 was released from 10 µg/ml CdO NPs). Therefore, the Cd ions released from CdO NPs in cell culture medium were minimal, and no cytotoxicity of CdO NPs was found at this concentration (Figs. 5, 6 and 7). Thus, the toxicity of CdO NPs should result from the

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nanoparticles themselves. The microscopic examination of cells showed that the CdO NPs were inside the

treated cells. Since NPs are very small, they can diffuse through the cell membrane or enter

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the cell via endocytosis [39-41]. Studies on other metal oxide NPs suggest that size is an

important parameter for toxicity of NPs and smaller NPs are more toxic [42-44]. The small

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particles have relatively large surface areas that offer more chance for interaction between the

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molecules of particles and biomaterials within cells [45-48]. Cd is a toxic metal material and the mechanism of the toxicity is by causing mitochondrial dysfunction [49, 50] and

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increasing membrane leakage [9, 15] in mammalian cells. Similarly, our results demonstrated that CdO NPs can enter cells, leading to LDH leakage and ATP content decrease possibly via

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membrane leakage and mitochondrial dysfunction. The cytotoxicity can eventually decrease cell viability. Also, mitochondrial and membrane damage by CdO NPs can increase oxidative

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stress in cells and contribute to the genotoxicity of the NPs. Considering that CdO NPs are used as the starting material in the manufacture of

quantum dots and widely applied for medical diagnostic imaging and therapeutics, humans may be exposed to the nanoparticles through inhalation, the skin (dermal), and food chain in environment, which will pose health risk. So far, only a few studies on CdO NPs have been conducted in a limited number of test systems for their potential ability to cause cytotoxicity, 21

most of which are not the standard assays required by the regulatory agencies. Therefore, the results on CdO NPs’ cytotoxicity and genotoxicity obtained from this study using standard assays can help us to assess human health risks of CdO NPs and to apply for regulatory decision making. In summary, CdO NPs were both cytotoxic and genotoxic. The treatment of the NPs resulted in decreased cell viability, reduced ATP content and increased LDH leakage in both TK6 and HepG2 cells. The NPs produced DNA breaks, chromosome damage in TK6 cells

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and induced mutations in mouse lymphoma cells. The cell uptake results showed that the NPs can enter mammalian cells. Data from experiments on Cd ions release from CdO NPs and cytotoxicity of CdCl2 suggest that the toxicity of the CdO NPs resulted from the NPs

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themselves, but not the released Cd ions because the ions released from the NPs were

minimal. These results provide insights into cytotoxicity and genotoxicity in human and

Conflict of interest statement

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rodent cell lines, which can be used for risk assessment of CdO NPs.

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Acknowledgments

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The authors declare that there are no conflicts of interest.

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Eşref Demir, Taichun Qin, Yan Li, and Suman Ghorai were supported by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science Education (ORISE) through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration.

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References [1] R. Landsiedel, M.D. Kapp, M. Schulz, K. Wiench, F. Oesch, Genotoxicity investigations on nanomaterials: methods, preparation and characterization of test material, potential artifacts and limitations--many questions, some answers, Mutat Res, 681 (2009) 241-258. [2] N. Singh, B. Manshian, G.J. Jenkins, S.M. Griffiths, P.M. Williams, T.G. Maffeis, C.J. Wright, S.H. Doak, NanoGenotoxicology: the DNA damaging potential of engineered

ro of

nanomaterials, Biomaterials, 30 (2009) 3891-3914.

[3] J. Ai, E. Biazar, M. Jafarpour, M. Montazeri, A. Majdi, S. Aminifard, M. Zafari, H.R. Akbari, H.G. Rad, Nanotoxicology and nanoparticle safety in biomedical designs, Int J

-p

Nanomedicine, 6 (2011) 1117-1127.

[4] T.S. Nawrot, J.A. Staessen, H.A. Roels, E. Munters, A. Cuypers, T. Richart, A. Ruttens,

re

K. Smeets, H. Clijsters, J. Vangronsveld, Cadmium exposure in the population: from health

lP

risks to strategies of prevention, Biometals, 23 (2010) 769-782. [5] S. Goix, T. Leveque, T.T. Xiong, E. Schreck, A. Baeza-Squiban, F. Geret, G. Uzu, A.

na

Austruy, C. Dumat, Environmental and health impacts of fine and ultrafine metallic particles: assessment of threat scores, Environ Res, 133 (2014) 185-194.

ur

[6] L.A. Bentolila, X. Michalet, F.F. Pinaud, J.M. Tsay, S. Doose, J.J. Li, G. Sundaresan, A.M. Wu, S.S. Gambhir, S. Weiss, Quantum dots for molecular imaging and cancer

Jo

medicine, Discov Med, 5 (2005) 213-218. [7] P. Chan, T. Yuen, F. Ruf, J. Gonzalez-Maeso, S.C. Sealfon, Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization, Nucleic Acids Res, 33 (2005) e161.

23

[8] G. Fotakis, J.A. Timbrell, In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride, Toxicol Lett, 160 (2006) 171-177. [9] M. Niitsuya, M. Watanabe, M. Okada, H. Shinji, T. Satoh, Y. Aizawa, Y.C. Cho, M. Kotani, Magnetometric evaluation of cadmium oxide-induced toxicity to pulmonary alveolar macrophages of Syrian golden hamsters, J Toxicol Environ Health A, 66 (2003) 365-378. [10] S.T. Hossain, S.K. Mukherjee, Toxicity of cadmium sulfide (CdS) nanoparticles against

ro of

Escherichia coli and HeLa cells, J Hazard Mater, 260 (2013) 1073-1082. [11] S.T. Hossain, S.K. Mukherjee, CdO nanoparticle toxicity on growth, morphology, and cell division in Escherichia coli, Langmuir, 28 (2012) 16614-16622.

-p

[12] J.L. Blum, J.Q. Xiong, C. Hoffman, J.T. Zelikoff, Cadmium associated with inhaled

cadmium oxide nanoparticles impacts fetal and neonatal development and growth, Toxicol

re

Sci, 126 (2012) 478-486.

lP

[13] X. Zhu, E. Hondroulis, W. Liu, C.Z. Li, Biosensing approaches for rapid genotoxicity and cytotoxicity assays upon nanomaterial exposure, Small, 9 (2013) 1821-1830. [14] A. Lankoff, M. Arabski, A. Wegierek-Ciuk, M. Kruszewski, H. Lisowska, A. Banasik-

na

Nowak, K. Rozga-Wijas, M. Wojewodzka, S. Slomkowski, Effect of surface modification of silica nanoparticles on toxicity and cellular uptake by human peripheral blood lymphocytes in

ur

vitro, Nanotoxicology, 7 (2013) 235-250.

Jo

[15] S.M. Hussain, K.L. Hess, J.M. Gearhart, K.T. Geiss, J.J. Schlager, In vitro toxicity of nanoparticles in BRL 3A rat liver cells, Toxicol In Vitro, 19 (2005) 975-983. [16] L. Zapor, Evaluation of the toxic potency of selected cadmium compounds on A549 and CHO-9 cells, Int J Occup Saf Ergon, 20 (2014) 573-581. [17] T. Mussa, C. Rodriguez-Carino, A. Sanchez-Chardi, M. Baratelli, M. Costa-Hurtado, L. Fraile, J. Dominguez, V. Aragon, M. Montoya, Differential interactions of virulent and non-

24

virulent H. parasuis strains with naive or swine influenza virus pre-infected dendritic cells, Vet Res, 43 (2012) 80. [18] X.L. Li, Y.S. Wong, G. Xu, J.C. Chan, Selenium-enriched Spirulina protects INS-1E pancreatic beta cells from human islet amyloid polypeptide-induced apoptosis through suppression of ROS-mediated mitochondrial dysfunction and PI3/AKT pathway, Eur J Nutr, 54 (2015) 509-522. [19] A. Irfan, M. Cauchi, W. Edmands, N.J. Gooderham, J. Njuguna, H. Zhu, Assessment of

ro of

temporal dose-toxicity relationship of fumed silica nanoparticle in human lung A549 cells by conventional cytotoxicity and (1)H-NMR-based extracellular metabonomic assays, Toxicol Sci, 138 (2014) 354-364.

-p

[20] F. Meng, J. Yan, Y. Li, P.P. Fu, L.H. Fossom, R.K. Sood, D.J. Mans, P.I. Chu, M.M.

Moore, T. Chen, Genotoxicity of 2-bromo-3'-chloropropiophenone, Toxicol Appl Pharmacol,

re

270 (2013) 158-163.

lP

[21] OECD, OECD GUIDELINE FOR TESTING OF CHEMICALS: Bacterial Reverse Mutation Test, http://www.oecd.org/dataoecd/18/31/1948418.pdf, (1997). [22] Y. Li, D.H. Chen, J. Yan, Y. Chen, R.A. Mittelstaedt, Y.B. Zhang, A.S. Biris, R.H.

na

Heflich, T. Chen, Genotoxicity of silver nanoparticles evaluated using the Ames test and in vitro micronucleus assay, Mutat Res-Gen Tox En, 745 (2012) 4-10.

ur

[23] OECD, In vitro Mammalian Cell Micronucleus Test (Mnvit). OECD Guideline for

Jo

Testing of Chemicals, No. 487 (2014). [24] Z. Magdolenova, M. Drlickova, K. Henjum, E. Runden-Pran, J. Tulinska, D. Bilanicova, G. Pojana, A. Kazimirova, M. Barancokova, M. Kuricova, A. Liskova, M. Staruchova, F. Ciampor, I. Vavra, Y. Lorenzo, A. Collins, A. Rinna, L. Fjellsbo, K. Volkovova, A. Marcomini, M. Amiry-Moghaddam, M. Dusinska, Coating-dependent induction of

25

cytotoxicity and genotoxicity of iron oxide nanoparticles, Nanotoxicology, 9 Suppl 1 (2015) 44-56. [25] T. Chen, M.M. Moore, Screening for chemical mutagens using the mouse lymphoma assay, in: Z. Yan, G.W. Caldwell (Eds.) Optimization in Drug Discovery: In-vitro Methods, Humana Press, Totowa, NJ, 2004, pp. 337-352. [26] M.M. Moore, M. Honma, J. Clements, G. Bolcsfoldi, B. Burlinson, M. Cifone, J. Clarke, P. Clay, R. Doppalapudi, M. Fellows, B. Gollapudi, S. Hou, P. Jenkinson, W. Muster, K.

ro of

Pant, D.A. Kidd, E. Lorge, M. Lloyd, B. Myhr, M. O'Donovan, C. Riach, L.F. Stankowski, Jr., A.K. Thakur, F. Van Goethem, I. Mouse Lymphoma Assay Workgroup, Mouse

lymphoma thymidine kinase gene mutation assay: meeting of the International Workshop on

-p

Genotoxicity Testing, San Francisco, 2005, recommendations for 24-h treatment, Mutat Res, 627 (2007) 36-40.

re

[27] M.M. Moore, M. Honma, J. Clements, G. Bolcsfoldi, B. Burlinson, M. Cifone, J. Clarke,

lP

R. Delongchamp, R. Durward, M. Fellows, B. Gollapudi, S. Hou, P. Jenkinson, M. Lloyd, J. Majeska, B. Myhr, M. O'Donovan, T. Omori, C. Riach, R. San, L.F. Stankowski, Jr., A.K. Thakur, F. Van Goethem, S. Wakuri, I. Yoshimura, Mouse lymphoma thymidine kinase gene

na

mutation assay: follow-up meeting of the International Workshop on Genotoxicity Testing-Aberdeen, Scotland, 2003--Assay acceptance criteria, positive controls, and data evaluation,

ur

Environ Mol Mutagen, 47 (2006) 1-5.

Jo

[28] J.R. Larison, G.E. Likens, J.W. Fitzpatrick, J.G. Crock, Cadmium toxicity among wildlife in the Colorado Rocky Mountains, Nature, 406 (2000) 181-183. [29] IARC, Beryllium, cadmium, mercury, and exposures in the glass manufacturing industry, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 58 (1993) 119-238.

26

[30] M. Waisberg, P. Joseph, B. Hale, D. Beyersmann, Molecular and cellular mechanisms of cadmium carcinogenesis, Toxicology, 192 (2003) 95-117. [31] A. Kroll, M.H. Pillukat, D. Hahn, J. Schnekenburger, Interference of engineered nanoparticles with in vitro toxicity assays, Arch Toxicol, 86 (2012) 1123-1136. [32] T. Xia, R.F. Hamilton, J.C. Bonner, E.D. Crandall, A. Elder, F. Fazlollahi, T.A. Girtsman, K. Kim, S. Mitra, S.A. Ntim, G. Orr, M. Tagmount, A.J. Taylor, D. Telesca, A. Tolic, C.D. Vulpe, A.J. Walker, X. Wang, F.A. Witzmann, N. Wu, Y. Xie, J.I. Zink, A. Nel,

ro of

A. Holian, Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: the NIEHS Nano GO Consortium, Environ Health Perspect, 121 (2013) 683-690.

-p

[33] S. Lanone, F. Rogerieux, J. Geys, A. Dupont, E. Maillot-Marechal, J. Boczkowski, G.

Lacroix, P. Hoet, Comparative toxicity of 24 manufactured nanoparticles in human alveolar

re

epithelial and macrophage cell lines, Part Fibre Toxicol, 6 (2009) 14.

lP

[34] L. Xu, Q.L. Zhang, F.P. Gao, J.S. Nie, Q. Niu, [Cell toxicity assessment methodologies applied in the study of the toxicity of nano-alumina to nerve cells], Zhonghua Yu Fang Yi Xue Za Zhi, 44 (2010) 785-789.

na

[35] A.M. Schrand, T. Powell, T. Robertson, S.M. Hussain, Assessment of Carbon- and Metal-Based Nanoparticle DNA Damage with Microfluidic Electrophoretic Separation

ur

Technology, J Nanosci Nanotechnol, 15 (2015) 1053-1059.

Jo

[36] E.R. Kisin, A.R. Murray, M.J. Keane, X.C. Shi, D. Schwegler-Berry, O. Gorelik, S. Arepalli, V. Castranova, W.E. Wallace, V.E. Kagan, A.A. Shvedova, Single-walled carbon nanotubes: Geno- and cytotoxic effects in lung fibroblast V79 cells, J Toxicol Env Heal A, 70 (2007) 2071-2079.

27

[37] D.B. Warheit, R.A. Hoke, C. Finlay, E.M. Donner, K.L. Reed, C.M. Sayes, Development of a base set of toxicity tests using ultrafine TiO2 particles as a component of nanoparticle risk management, Toxicol Lett, 171 (2007) 99-110. [38] R.S. Woodruff, Y. Li, J. Yan, M. Bishop, M.Y. Jones, F. Watanabe, A.S. Biris, P. Rice, T. Zhou, T. Chen, Genotoxicity evaluation of titanium dioxide nanoparticles using the Ames test and Comet assay, J Appl Toxicol, 32 (2012) 934-943. [39] R. Pan, G. Liu, Y. Li, Y. Wei, S. Li, L. Tao, Size-dependent endocytosis and a dynamic-

ro of

release model of nanoparticles, Nanoscale, 10 (2018) 8269-8274. [40] C.Y. Dombu, M. Kroubi, R. Zibouche, R. Matran, D. Betbeder, Characterization of endocytosis and exocytosis of cationic nanoparticles in airway epithelium cells,

-p

Nanotechnology, 21 (2010) 355102.

[41] Y.J. Ma, H.C. Gu, Study on the endocytosis and the internalization mechanism of

re

aminosilane-coated Fe3O4 nanoparticles in vitro, J Mater Sci Mater Med, 18 (2007) 2145-

lP

2149.

[42] H.L. Karlsson, J. Gustafsson, P. Cronholm, L. Moller, Size-dependent toxicity of metal oxide particles--a comparison between nano- and micrometer size, Toxicol Lett, 188 (2009)

na

112-118.

[43] J. Pelka, H. Gehrke, M. Esselen, M. Turk, M. Crone, S. Brase, T. Muller, H. Blank, W.

ur

Send, V. Zibat, P. Brenner, R. Schneider, D. Gerthsen, D. Marko, Cellular uptake of platinum

Jo

nanoparticles in human colon carcinoma cells and their impact on cellular redox systems and DNA integrity, Chem Res Toxicol, 22 (2009) 649-659. [44] P.J. Moos, K. Chung, D. Woessner, M. Honeggar, N.S. Cutler, J.M. Veranth, ZnO particulate matter requires cell contact for toxicity in human colon cancer cells, Chem Res Toxicol, 23 (2010) 733-739.

28

[45] M. Abdelhalim, Alterations in Gold Nanoparticle Levels Are Size Dependent, with the Smaller Ones Inducing the Most Toxic Effects and Related to the Time of Exposure of the Gold Nanoparticles, West Indian Med J, 65 (2015) 87-92. [46] N. Hoshyar, S. Gray, H. Han, G. Bao, The effect of nanoparticle size on in vivo pharmacokinetics and cellular interaction, Nanomedicine (Lond), 11 (2016) 673-692. [47] S. Zhang, A. Nelson, P.A. Beales, Freezing or wrapping: the role of particle size in the mechanism of nanoparticle-biomembrane interaction, Langmuir, 28 (2012) 12831-12837.

ro of

[48] C. Carlson, S.M. Hussain, A.M. Schrand, L. K. Braydich-Stolle, K.L. Hess, R.L. Jones, J.J. Schlager, Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species, The Journal of Physical Chemistry B, 112 (2008) 13608-13619.

-p

[49] L. Braydich-Stolle, S. Hussain, J.J. Schlager, M.C. Hofmann, In vitro cytotoxicity of nanoparticles in mammalian germline stem cells, Toxicol Sci, 88 (2005) 412-419.

re

[50] F. Tian, D. Cui, H. Schwarz, G.G. Estrada, H. Kobayashi, Cytotoxicity of single-wall

Jo

ur

na

lP

carbon nanotubes on human fibroblasts, Toxicol In Vitro, 20 (2006) 1202-1212.

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Figure Legends Fig. 1. Characterization of CdO NPs. (A) Typical TEM images of the 10 nm CdO NPs and size distribution histogram. (B) EDS spectrum shows the chemical composition of CdO NPs in the dispersion suspension by TEM. (C) Hydrodynamic size (nm) of CdO NPs in cell culture medium (7.5 µg/ml) at 4 h of the incubation. (D) Size distribution by DLS characterization. Fig. 2. Endotoxin level (EU/ml) in different concentrations (from 0.625 to 10 µg/ml) of CdO

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NPs detected using chromogenic Limulus amebocyte lysate (LAL) assay. Each point

represents a mean of 3 replicates ± standard error. Assay was performed according to the kit protocol (Lonza, QCL-1000TM). Lipopolysaccharide (LPS)(0.5 EU/ml) was used as a positive

< 0.001 when compared with the negative control (LAL reagent water) using the

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***p

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control. # The endotoxin level of CdO NPs was below the limit of detection (0.182 EU/ml).

Student’s t-test.

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Fig. 3. Representative TEM figures of CdO NPs uptake by TK6 cells. (A) Untreated TK6 cell: No CdO NPs are found. (B) Exposed cells: CdO NPs are observed in the cytoplasm. (C)

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Detail of figure B at higher magnification. (D) STEM spectrum image indicates the presence of high contrast NPs in figure B. (E) Energy dispersive X-rays microanalysis (EDS) spectrum

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B.

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shows the chemical composition of the CdO NPs indicated by the arrow and red ring in figure

Fig. 4. Release of cadmium ion (Cd+2) from CdO NPs in culture medium and stock suspension. The percent of Cd+2 released from a stock suspension of 1 mg/ml CdO NPs and from cell culture medium after incubating with the 2.5, 5 and 10 μg/ml CdO NPs at 37°C for 24 h.

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Fig. 5. Cell viability measured using MTS assay. TK6 (A) and HepG2 (B) cells (1x105) were treated with different concentrations of the CdO NPs and CdCl2 and cultured in a final volume of 200 µl medium/well at 37°C for 24 h. Each data point represents a mean of 4 replicates. H2O2 (100 µM) was used as the positive control (PC). ** and *** indicate p < 0.01 and p < 0.001, respectively, when compared with the negative control (NC) using the Student's t-test. Fig. 6. Cytotoxicity of CdO NPs measured using ATP assay. TK6 (A) and HepG2 (B) cells

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(1x106) were treated with different concentrations of the NPs and CdCl2 and cultured in a

final volume of 100 µl medium/well at 37°C for 24 h. Each data point represents a mean of 3 replicates. H2O2 (100 µM) was used as the positive control (PC). ** and *** indicate p < 0.01

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and p < 0.001, respectively, when compared with the negative control (NC) using the

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Student's t-test.

Fig. 7. Cytotoxicity of CdO NPs and CdCl2 measured using LDH assay. TK6 (A) and HepG2

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(B) cells (2 x105) were treated with different concentrations of the CdO NPs and CdCl2 and cultured in 96-well plates in 100 µl of culture medium/well at 37°C for 24 h. Each point

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represents a mean of 3 replicates. H2O2 (100 µM) was used as the positive control (PC). * p < 0.05 when compared with the negative control (NC) using the Student's t-test.

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Fig. 8. Mutagenicity of CdO NPs evaluated using the Ames test. Three strains of Salmonella typhimurium TA98, TA100, and TA102 were used in this test. The values represent the

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number of revertant colonies from three independent experiments. The negative control was 100 μl H2O per plate for all the tester strains. The positive controls were 2-nitrofluorene (3 μg per plate) for TA98, nitrofurantoin (5 μg per plate) for TA100 and mitomycin (0.2 μg per plate) for TA102. Values in parentheses correspond to the concentrations of the positive control chemicals (μg/plate).

31

Fig. 9. Cytotoxicity and micronucleus induction in TK6 cells by CdO NPs. TK6 cells were treated in triplicate with serials of concentrations of CdO NPs at 1, 2.5, 5, 7.5, and 10 μg/ml for 4 h. After the treatment, the treated cells were replaced with fresh medium and cultured for another 24 h before their micronuclei were measured by flow cytometry analysis. Relative population doubling was used to measure cytotoxicity (A). Micronucleus induction is expressed as number of micronuclei per 10,000 cells measured (B). H2O was used as negative control and X-rays were used as the positive control. All the values are expressed as mean ±

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SD. The assays were performed in triplicate in three independent experiments. ** and *** indicate p < 0.01 and p < 0.001, respectively, when compared with the negative control.

Fig. 10. Effects of CdO NPs on DNA damage in TK6 cells evaluated using the Comet assay.

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After treatment with CdO NPs (0.625-5 µg/ml) for 4 h, the cells were collected for the assay. DNA damage was measured as % DNA in the tail. (A) Effects of CdO NPs on cell viability.

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The viability analysis was conducted with the Trypan blue dye exclusion assay. (B) Effects of

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CdO NPs on DNA damage. Hydrogen peroxide (H2O2, 100 µM) was used as the positive control (PC). *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001 vs. the control,

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respectively, when compared to the negative control (NC) using the Student's t-test. Fig. 11. Mutagenicity of CdO NPs evaluated using mouse lymphoma assay. Mouse

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lymphoma cells were treated with different concentrations of CdO NPs for 4 h. Cytotoxicity was assessed using the relative total growth (RTG) (% of negative control). Each data point is

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the mean ± S.D. (n=3). The mutation frequency (MF) was significantly increased by the NPs in a dose-dependent manner.

32

ro of

-p

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lP

na

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33

ro of

-p

re

lP

na

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34

ro of

-p

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lP

na

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35

ro of

-p

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lP

na

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36

Fig. 2

0.6

***

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0.4

0.3

0.2 Limit of detection (#)

0.1

-p

Endotoxin activity (EU/ml)

0.5

NC

0.625

1.25

2.5

re

0 5

7.5

Jo

ur

na

lP

CdO NPs (µg/ml)

37

10

PC

E

A

C D

38

ro of

-p

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lP

na

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Jo Fig. 3.

B

Fig. 4.

0.1

0 CdO NPs 2.5 µg/ml

CdO NPs 5 µg/ml

CdO NPs 10 µg/ml

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ur

na

lP

re

-p

CdO NPs 1 mg/ml

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Cd+2 Release (%)

0.2

39

Fig. 5. A.

TK6 cells

CdO NPs CdCl2

75

**

50

***

*** *** *** ****** ****** ******

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Cell viability (% of control)

100

25

0 NC

0.3125

0.625

1.25

5

2.5

10

re

-p

Concentrations (µg/ml)

PC

lP

100

na

50

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0

CdCl2

******

75

25

CdO NPs

HepG2 cells

*** ****** *** *** *** *** ***

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Cell viability (% of control)

B.

NC

0.3125

0.625

1.25

2.5

5

Concentrations (µg/ml)

40

10

PC

Fig. 6. A.

TK6 cells

CdO NPs

** **

75

** **

** **

** **

**

50

**

25

0 1.25

0.625

5

2.5

10

re

Concentrations (µg/ml)

lP

B.

HepG2 cells

100

**

***

CdO NPs CdCl2

*** *** ***

******

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50

25

**

na

75

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ATP production (% of control)

PC

-p

NC

CdCl2

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ATP production (% of control)

100

0

NC

0.625

1.25

5

2.5

Concentrations (µg/ml)

41

10

PC

Fig. 7. A. TK6 cells

200

*

CdO NPs

*

CdCl2

100

50

0 NC

1.25

0.625

5

2.5

10

PC

re

-p

Concentrations (µg/ml)

ro of

LDH (% of control)

* *

*

*

*

150

*

*

*

lP

B.

200

na

* *

* *

CdCl2 * *

* *

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100

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LDH (% of control)

150

50

CdO NPs

HepG2 cells

0

NC

0.625

1.25

5

2.5

Concentrations (µg/ml)

42

10

PC

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lP

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Fig. 9. A.

**

100

*** ***

75

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*** 50

***

25

0 NC

1

2.5

5

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Relative population doubling (%)

125

7.5

10

PC

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Concentration ( g/ml)

lP

B.

800 400

na

***

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1000

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Micronuclei per 10,000 cells

1200

***

300

***

200

**

100 0 NC

1

2.5

5

7.5

Concentration ( g/ml)

44

10

PC

Fig. 10.

100

*

75

*

**

**

5

ro of

Cell viability (% of living cells in total cells)

A.

50

25

NC

0.0625

1.25

2.5

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Concentration (µg/ml)

lP

B.

35

na

30

20 15 10

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25

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DNA damage (% of DNA in tail)

PC

-p

0

***

*** ***

***

***

5 0

NC

0.625

1.25

2.5

Concentration (μg/ml)

45

5

PC

ro of

-p

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lP

na

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Jo Fig. 11.

46