YTAAP-13098; No. of pages: 7; 4C: Toxicology and Applied Pharmacology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap

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Leah J. Smith a,b,c, Amie L. Holmes a,b,c, Sanjeev Kumar Kandpal d, Michael D. Mason d, Tongzhang Zheng e, John Pierce Wise Sr. a,b,c,⁎

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Article history: Received 28 January 2014 Revised 1 May 2014 Accepted 5 May 2014 Available online xxxx

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Keywords: Cobalt Cobalt chloride Cobalt oxide Cytotoxicity Genotoxicity

Wise Laboratory of Environmental and Genetic Toxicology, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300, USA Maine Center for Environmental Toxicology and Health, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300, USA Department of Applied Medical Science, University of Southern Maine, 96 Falmouth St., P.O. Box 9300, Portland, ME 04101-9300, USA d Department of Chemical & Biological Engineering, University of Maine, Orono, ME, USA e Department of Environmental Health Sciences, Yale School of Public Health, New Haven, CT, USA

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a b s t r a c t

Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity. © 2014 Published by Elsevier Inc.

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The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells

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Introduction

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The demand for cobalt is rising worldwide. Cobalt is increasingly being used in the production of rechargeable batteries, super-alloys and magnetic products due to its enhanced electrode conductivity, anti-corrosive, high melting point and magnetic properties, (CDI, 2013). Based on World Bureau of Metal Statistics, the demand for cobalt has increased by 15% worldwide from 2010 to 2011 alone (CDI, 2013). As cobalt is increasingly used in manufacturing, the potential for exposure of both industrial workers and the general population is also rising. The International Agency for Research on Cancer (IARC) has classified cobalt as a Group 2B possible human carcinogen (IARC, 2006). Epidemiological studies on the carcinogenicity of cobalt are limited and inconclusive, partially due to co-exposure with established carcinogens, such as nickel and chromium (IARC, 2006). However, human studies do suggest a correlation between cobalt exposure and lung diseases, including lung cancer, asthma and alveotitis (Mur et al., 1987; Sauni et al., 2010; Van Cutsem et al., 1987). Additionally, animal studies conducted by the

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⁎ Corresponding author at: P.O. Box 9300, 96 Falmouth St., Portland, ME 04104-9300, USA. E-mail address: [email protected] (J.P. Wise).

National Toxicology Program (1998) demonstrated an increased incidence of alveolar/bronchiolar neoplasms in murine models as a result of cobalt inhalation exposure (ATSDR, 2004; IARC, 2006; NTP, 1998). The potential mechanisms of cobalt-induced lung carcinogenesis remain unknown. Studies indicate that cobalt is genotoxic to human and rodent cells, inducing chromosome aberrations, single and double strand breaks, sister chromatid exchanges and micronuclei (reviewed in ATSDR, 2004; Beyersmann and Hartwig, 2008; Lison et al., 2001). However, despite the fact that the lung is the major target organ, little is known about the genotoxic effects of cobalt in human lung cells. Only one study has investigated the genotoxicity of cobalt in human lung cells and found that exposure to soluble cobalt ions induces DNA double strand breaks in the cancer-derived H460 human lung epithelial cell line (Pastel et al., 2012). Thus far, no studies have investigated cobalt-induced genotoxicity in a non-cancerous human lung cell model. Both soluble and particulate cobalt compounds are used in industry, but the role of solubility in cobalt-induced genotoxicity remains unknown. Studies with other metals, such as nickel and chromium, indicate that solubility can play an important role in the potency of metal-induced genotoxicity and carcinogenicity. To date, no studies have compared the potency of soluble and particulate cobalt compounds

http://dx.doi.org/10.1016/j.taap.2014.05.002 0041-008X/© 2014 Published by Elsevier Inc.

Please cite this article as: Smith, L.J., et al., The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.002

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L.J. Smith et al. / Toxicology and Applied Pharmacology xxx (2014) xxx–xxx

Cytotoxicity assay

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Materials and methods

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Chemicals and reagents

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Clastogenicity assay

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A 50:50 mixture of Dulbecco's minimal essential medium and Ham's F-12 (DMEM/F-12) was purchased from Mediatech Inc. (Herndon, VA). Sodium pyruvate, penicillin/streptomycin, Gluta-Gro, trypsin/EDTA, phosphate buffered saline (PBS), propidium iodide (PI), and Gurr's buffer were purchased from Life Technologies Corp (Carlsbad, CA). Acetic acid, crystal violet, and methanol were purchased from J.T. Baker (Phillipsburg, NJ). Cosmic calf serum (CCS) was purchased from Hyclone (Logan, UT). Cobalt (II) chloride hexahydrate, cobalt (II) oxide, potassium chloride (KCl), and demecolchicine were purchased from Sigma/Aldrich (St. Louis, MO). Giesma stain was purchased from Biomedical Specialties Inc. (Santa Monica, CA). Sodium dodecyl sulfate (SDS) was purchased from American Bioanalytical (Natick, MA). Tissue culture dishes, flasks and plastic ware were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ).

Cytotoxicity was determined by a clonogenic assay as previously described (Wise et al., 2002). Briefly, cells were seeded at a density of 9000 cells/cm2 in a tissue-culture dish, and allowed to grow for 48 h. The cultures were then treated for 24 h with cobalt compounds. Cells were then removed from the dish and reseeded at colony forming density (1000 cells per dish). Colonies were allowed to grow for 14 days; fixed with 100% methanol; stained with crystal violet; and the colonies with at least 50 cells were counted. There were four dishes per treatment group and each experiment was repeated at least three times. Results are expressed as relative survival reflecting the number of colonies within a treatment group divided by the negative control.

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Cells and cell culture

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A human lung fibroblast cell line, WTHBF-6, was used as the cell model for this study. WTHBF-6 cells are an hTERT-immortalized clonal cell line derived from primary human bronchial fibroblasts. They exhibit a stable, normal diploid karyotype and have similar clastogenic and cytotoxic responses to metals as their parent cells (Wise et al., 2004). WTHBF6 cells were cultured as sub-confluent monolayers in DMEM/F-12 supplemented with 15% cosmic calf serum, 100 u/ml penicillin/100 ug/ml streptomycin, 2 mM gluta-GRO, and 0.1 mM sodium pyruvate and incubated in 5% CO2 at 37 °C. Media were replaced with fresh, warm media every two days. Cells were subcultured every three to four days using 0.25% trypsin/1 mM EDTA solution.

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Cobalt preparation

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130 131

Cobalt oxide (CAS #1307-96-6) was used as a representative particulate cobalt compound and was administered as a suspension in water, as previously described (Wise et al., 2002). Briefly, cobalt oxide was suspended in cold sterile-filtered water and spun overnight with a magnetic stir bar. Dilutions were made from the stock using a vortex mixer and appropriate volumes were dispensed into cell cultures. Cobalt chloride hexahydrate (CAS #7791-13-1) was used as a representative soluble cobalt compound. Stock cobalt chloride solutions were filtered through a 0.2 μM filter, and then appropriate dilutions were made with sterile-filtered water and administered to the cells. It is important to choose treatment doses relevant to actual exposure concentrations that humans might encounter. The Occupational Safety and Health Administration's permissible exposure limit (OSHA PEL) for cobalt is 100 μg/m 3 . A human exposed to 100 μg/m 3 for eight hours with an average daily air consumption of 20 m3 (6.67 m 3 in eight hours) could potentially be exposed to up to approximately 667 μg of cobalt each day occupationally. Treatment dilution concentrations were chosen to be less than the OSHA PEL for cobalt, which is presumed to be the amount of cobalt a human would occupationally be exposed to in a 24 hour period.

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Particle size distribution

Soluble cobalt is more cytotoxic to human lung cells than particulate cobalt 187

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Cobalt oxide particle size distributions were determined using a Malvern-2000S (Mastersizer) on the basis of number, volume and

Exposure to particulate or soluble cobalt induced a concentration- 188 dependent increase in cytotoxicity in human lung fibroblasts (Fig. 1). 189

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113 114 115 116 117 118 119 120 121 122 123 Q4 124 125 126 127 128 129

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Intracellular and extracellular cobalt ion measurements

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Intracellular cobalt ion levels were determined using inductively coupled plasma optical emission spectroscopy (ICP-OES) as previously described (Holmes et al., 2005). Briefly, cells were seeded at a density of 9000 cells/cm2 in a tissue-culture dish, and allowed to grow for 48 h. The cultures were then treated with varying concentrations of cobalt chloride for 24 h or cobalt oxide for 0 h or 24 h exposure periods. After treatment, cells were harvested and placed in hypotonic solution followed by 2% SDS to degrade the cellular membrane. This solution was sheered through an 18 gauge needle and filtered through a 0.2 μm filter. Samples were diluted in 2% nitric acid and cobalt ion concentrations were measured by ICP-OES as previously described (Holmes et al., 2005). The 0 h treatment for particulate cobalt was performed to account for the possibility that cobalt particles may have passed through the 0.2 μm filter. This potential confounding factor was accounted for by subtracting the 0 h cobalt ion levels from the 24 h cobalt ion levels. The corrected intracellular concentrations were converted from ug/L to μM by dividing by the volume of the sample, the atomic weight of the chemical, the number of cells in the sample and the average cell volume (determined to be 1.125 pl by a Beckman Coulter Multisizer 3).

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Statistics

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Cobalt-induced clastogenicity was measured using the chromosome aberration assay, as previously described (Wise et al., 2002). Briefly, cells were seeded at a density of 9000 cells/cm2 in a tissue-culture dish, and allowed to grow for 48 h. The cultures were then treated with varying concentrations of cobalt chloride or cobalt oxide for 24 h exposure periods and harvested for metaphases. One hundred metaphases per data point were analyzed in each experiment and each experiment was repeated at least three times. Metaphases were analyzed for chromatid breaks, isochromatid breaks, chromatid exchanges, dicentrics, double minutes, acentric fragments, fragmented chromosomes and centromere spreading.

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scattering intensity. Particle sizes ranged from 0.27 μm to 3.56 μm, 135 with an average particle size of 1 μm. 136

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in human lung cells. Accordingly, the objective of this study was to determine the cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells and to investigate the role of solubility in cobalt-induced toxicity.

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163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181

Student's t-tests were conducted to determine statistical signifi- 183 cance between data points. Statistical significance was determined to 184 be a p value less than 0.05. 185 Results

Please cite this article as: Smith, L.J., et al., The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.002

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L.J. Smith et al. / Toxicology and Applied Pharmacology xxx (2014) xxx–xxx

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Cobalt Oxide Concentration (ug/cm²)

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Cobalt Chloride Concentration (uM)

Fig. 2. Exposure to particulate and soluble cobalt increases intracellular cobalt ion concentrations in human lung cells. This figure shows that exposure to particulate or soluble cobalt increases intracellular cobalt ion levels in a concentration-dependent manner. Data represent an average of three independent experiment ± standard error of the mean. *Statistically significant compared to control (p b 0.05). A) Cobalt oxide. B) Cobalt chloride.

cobalt oxide induced 10, 13, 20, 26 and 57% of metaphases with damage and 11, 16, 24, 40 and 147 aberrations in 100 metaphases. The highest concentration (10 μg/cm2 cobalt oxide) induced no metaphases indicating cell cycle arrest. Exposure to 50, 100, 175, and 250 μM of cobalt chloride induced 8, 6, 14, and 16% of metaphases with damage and 8, 8, 16 and 22 aberrations in 100 metaphases, respectively. No metaphases were observed after exposure to 500 μM cobalt chloride, indicating cell cycle delay. The most common aberrations observed for both particulate and soluble cobalt were chromatid lesions (Tables 1 and 2).

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Relative Survival (Percent of Control)

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Relative Survival (Percent of Control)

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Relative Survival (Percent of Control)

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Exposure to particulate or soluble cobalt induced concentrationdependent increases in chromosome damage in human lung fibroblast cells (Fig. 4). For example, exposure to 0.1, 0.5, 1, 2.5 and 5 μg/cm2

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Intracellular Cobalt Concentration (uM)

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Intracellular Cobalt Concentration (uM)

Particulate and soluble cobalt induce similar levels of genotoxicity

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For particulate cobalt, exposure to 0.1, 0.5, 1, and 5 μg/cm2 cobalt oxide for 24 h reduced relative survival to 85, 61, 55, and 8%, respectively (Fig. 1A). For soluble cobalt, exposure to 100, 175, 250, and 500 μM cobalt chloride reduced relative survival to 76, 49, 29, and 4% (Fig. 1B). Since soluble and particulate cobalt cannot be directly compared based on administered concentration, we sought to determine the intracellular cobalt levels after exposure to cobalt oxide and cobalt chloride in order to compare the relative toxicity of these compounds. Intracellular cobalt ion levels increased in a concentration-dependent manner after exposure to soluble and particulate cobalt (Fig. 2). For example, exposure to 0.5, 1, and 5 μg/cm2 cobalt oxide for 24 h produced intracellular cobalt ion levels of 833, 1753, and 9820 μM (Fig. 2A), respectively, while a 24 h exposure to 100, 250 or 500 μM cobalt chloride induced intracellular cobalt ion levels of 445, 1693, and 4999 μM, respectively (Fig. 2B). Based on intracellular cobalt concentrations, cobalt chloride induced more cytotoxicity than cobalt oxide (Fig. 3). At intracellular cobalt concentrations below 1000 μM, cobalt oxide and cobalt chloride induced similar levels of cytotoxicity, but as intracellular cobalt levels increased, cobalt chloride induced more cytotoxicity than cobalt oxide. For example, cobalt chloride induced 29% relative survival at an intracellular cobalt level of approximately 1700 μM while cobalt oxide only induced 55% relative survival (Fig. 3).

190 191

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Cobalt Oxide Cobalt Chloride

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Cobalt Chloride Concentration (uM) Fig. 1. Particulate and soluble cobalt induces cytotoxicity in human lung cells. This figure shows that exposure to particulate or soluble cobalt for 24 h induces a concentrationdependent decrease in relative survival. Data represent an average of three independent experiment ± standard error of the mean. *Statistically significant compared to control (p b 0.05). Results are expressed as relative survival reflecting the number of colonies within a treatment group divided by the negative control.

0 0

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Intracellular Cobalt Concentration (uM) Fig. 3. Cobalt chloride induces more cytotoxicity than cobalt oxide in human lung cells. This figure shows that at similar intracellular cobalt ion levels, cobalt chloride induces more cytotoxicity than cobalt oxide. Data represent an average of three independent experiment ± standard error of the mean.

Please cite this article as: Smith, L.J., et al., The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.002

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L.J. Smith et al. / Toxicology and Applied Pharmacology xxx (2014) xxx–xxx

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ion level of 140 μM (Table 4). Extracellular levels in the top and bottom wells were similar indicating the difference in intracellular ion levels was due to particle-cell contact. Consistent with the ion uptake results, particle-cell contact was also required for cobalt oxide-induced cytotoxicity and genotoxicity. Cells at the bottom well exposed to both cobalt oxide particles and cobalt ions exhibited a significantly greater amount of cytotoxicity and chromosome damage compared to cells on the top well that were exposed to only the cobalt ions generated from the particulate treatment (Table 3). For example, cells at the bottom well exposed to 5 μg/cm2 cobalt oxide exhibited 14% relative survival, 45% of metaphases with damage and 103 total aberrations while cells on the top well only exhibited 80% relative survival, 8.7% of metaphases with damage and 10 total aberrations in 100 metaphases (Table 3).

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Total Aberrations in 100 Metaphases

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Percent of Metaphases with Damage

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Total Aberrations in 100 Metaphases

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Discussion

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Cobalt Chloride Concentration (uM)

237 238 239 240 241 242 243 244 245 246 247 248 Q5 249 250 251 252 253 Q6 254 255

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235 236

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Particle internalization is required for particulate cobalt-induced cytotoxicity and genotoxicity

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Based on intracellular cobalt levels, exposure to soluble or particulate cobalt induced relatively similar levels of genotoxicity (Fig. 5). For example, cells with an intracellular cobalt ion concentration of approximately 1700 μM exhibited 15% of metaphases with damage and 21 total aberrations after cobalt chloride exposure and 16% of metaphases with damage and 20 aberrations in 100 metaphases following cobalt oxide exposure (Fig. 5). Interestingly, exposure to cobalt chloride induced cell cycle arrest at much lower intracellular cobalt concentrations compared to cobalt oxide. For example, an intracellular cobalt level of 5000 μM after cobalt chloride exposure resulted in cell cycle delay (no metaphases) while metaphases with significant chromosome damage were observed up to an intracellular cobalt ion level of 9800 μM after exposure to cobalt oxide (Fig. 5).

Considering the differences observed between particulate and soluble-induced cytotoxicity and genotoxicity, we sought to determine the importance of particle-cell contact in cobalt oxide-induced cytotoxicity and genotoxicity. We treated cells grown in a transwell dish which contains two growing surfaces separated by a 0.2 μm membrane. The membrane allows for nutrient and ion exchange but does not allow the cobalt oxide particles or cells to pass through to the upper growing surface. Thus, cells at the bottom of the dish are exposed to particles and cobalt ions while cells on the top well of the dish are exposed to only cobalt ions. We first investigated cobalt uptake and found that particle-cell contact was required to generate the high intracellular cobalt levels observed after cobalt oxide exposure. Cells at the bottom well exposed to 5 μg/cm2 cobalt oxide exhibited an intracellular cobalt ion level of 6854 μM while cells on the top well only had an intracellular cobalt

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Fig. 4. Particulate and soluble cobalt induces clastogenicity in human lung cells. This figure shows that a 24 h exposure to particulate or soluble cobalt induces a concentrationdependent increase in percent of metaphase with chromosome damage and total aberrations in 100 metaphases. Data represent an average of three independent experiment ± standard error of the mean. *Statistically significant compared to control (p b 0.05). A) Cobalt oxide. B) Cobalt Chloride.

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With the advancement of cobalt mining in the United States and the growing demand for cobalt in general, cobalt inhalation is a growing health concern. Due to lack of conclusive human epidemiological studies, cobalt is listed as a possible carcinogen by IARC, but animal and cell culture studies indicate that cobalt is carcinogenic (Abbracchio et al., 1982; ATSDR, 2004; Costa et al., 1982; Doran et al., 1998; IARC, 2006; NTP, 1998). The potential mechanisms involved in cobalt-induced carcinogenicity remain unknown. Studies show that cobalt is cytotoxic and genotoxic to mammalian cells (reviewed in ATSDR, 2004; Beyersmann and Hartwig, 2008; Lison et al., 2001) but few studies have investigated cobalt's effect in its target cell, human lung cells, and no studies have investigated the role of solubility in cobalt-induced toxicity. In this study, we show that both particulate and soluble cobalt are cytotoxic and genotoxic to human lung fibroblasts with soluble cobalt inducing more cytotoxicity than particulate cobalt but similar levels of genotoxicity. These are the first data to report the cytotoxic effects of cobalt ions in karyotypically normal human lung cells and the first to report on the cytotoxic effects of particulate cobalt in a human lung cell model. Two previous studies investigated the cytotoxic effects of cobalt ions in a karyotypically abnormal cancer-derived human lung epithelial model, H460, and found a similar concentration-dependent decrease in relative survival (Green et al, 2013; Pastel et al., 2012). Despite the altered karyotype, exposure to 100 μM cobalt chloride for 24 h induced 78% relative survival in H460 cells and 76% relative survival in human lung fibroblasts (Pastel et al., 2012; Fig. 1B). The mechanism of cytotoxicity is likely via apoptosis rather than necrosis and involves the activation of caspases 3 and 7 (Green et al., 2013; Pastel et al., 2012). Our data show that both particulate and soluble cobalt are cytotoxic to human lung cells which are consistent with previous data in other mammalian cell models (Akbar et al., 2011; Allen et al, 1997; Catelas et al., 2005; Colognato et al., 2008; Green et al, 2013; Pastel et al., 2012; Ponti et al., 2009). To our knowledge, this is the first study to directly investigate the ability of soluble or particulate cobalt to induce chromosomal aberrations, looking at a full spectrum of damage, in general and more specifically in human lung cells, an important target cell for cobalt exposure. Our data are consistent with previous studies indicating that cobalt induces DNA double strand breaks and micronuclei (Beyersmann and Hartwig, 2008; Costa et al., 1982; Pastel et al., 2012). Cobalt primarily induced simple chromatid lesions and induced minimal to no complex lesions such as dicentrics and chromatid exchanges (Tables 1 and 2) which are consistent with a previous report that used M-FISH to investigate chromosomal translocations and dicentrics in human skin fibroblasts treated with cobalt chloride (Figgitt et al., 2010). The mechanism of cobalt-induced chromosome damage remains unknown. The literature suggests that there are two potential mechanisms for cobalt-induced genotoxicity; a direct oxidative DNA damaging effect and an indirect effect through inhibition of DNA repair (Beyersmann and Hartwig, 2008; Lison et al., 2001). Cobalt ions can induce reactive oxygen species through a Fenton-type reaction that can directly break the

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Chromosome Damage

B

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160

Percent of Metaphases with Damage

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180

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Chromosome Damage

A

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4

Please cite this article as: Smith, L.J., et al., The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.002

256 Q7 257 Q8 258 259 260 Q9 261 262 263 264 265 Q10 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319

L.J. Smith et al. / Toxicology and Applied Pharmacology xxx (2014) xxx–xxx t1:1 t1:2

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Table 1 Spectrum of chromosome aberrations induced by cobalt oxide in human lung fibroblast cells a. Cobalt oxide concentration (μg/cm2)

Chromatid break and gap

Isochromatid break and gap

Chromatid exchange

Dicentric

t1:3

Double minutes

Acentric fragment

Centromere spreading

Total damage

t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10

0 0.1 0.5 1 2.5 5 10

4.4 ± 1.0 9.0 ± 3.5 12 ± 3.8 22 ± 7.2 36 ± 9.2 132 ± 16 NM b

0.2 ± 0.3 ± 1.3 ± 0.3 ± 2.0 ± 5.0 ± NM

0±0 0±0 0.3 ± 0.3 0±0 0±0 0.7 ± 0.3 NM

0± 0± 0± 0± 0± 0± NM

0.2 ± 0.3 ± 0.7 ± 1.3 ± 1.0 ± 5.3 ± NM

0.2 ± 1.0 ± 0.7 ± 0.3 ± 0.3 ± 3.7 ± NM

0±0 0±0 1.3 ± 1.3 0±0 0.3 ± 0.3 0.3 ± 0.3 NM

5.0 ± 1.1 11 ± 3.8 16 ± 3.5 24 ± 7.2 40 ± 8.3 147 ± 18 NM

t1:11 t1:12

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0.2 0.3 0.3 0.9 0.6 1.7

0.2 0.6 0.3 0.3 0.3 1.2

Data represent the average of three experiments ± the standard error of the mean. NM: no metaphases.

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DNA backbone producing a DNA double strand break (Kawanishi et al., 1994; Lloyd et al., 1997; Nackerdien et al., 1991). Alternatively, cobalt can also inhibit the repair of endogenous and exogenous DNA damage 323 Q11 by interfering with the repair machinery (Beyersmann and Hartwig, 324 2008). Either mechanism may be involved in cobalt-induced chromo325 some damage. 326 Using intracellular cobalt ion levels to compare particulate and 327 soluble-induced cytotoxicity and genotoxicity, we find that cobalt chlo328 ride is more cytotoxic than cobalt oxide but induces similar levels of 329 genotoxicity to human lung cells (Figs. 3 and 5). However, cobalt chlo330 ride induces cell cycle arrest at lower intracellular cobalt concentrations 331 compared to cobalt oxide (Fig. 5). To date no studies have compared the 332 cytotoxic or genotoxic effects of cobalt ions and micron-sized cobalt 333 particles. One study compared cobalt metal and cobalt ions and found 334 that cobalt metal and cobalt chloride induced similar levels of strand 335 breaks but the comparison was based on Co-equivalent/ml instead of 336 intracellular cobalt levels (DeBoeck et al., 1998). Other studies have 337 compared the effects of cobalt ions with cobalt nanoparticles and 338 found that cobalt nanoparticles induced more cytotoxicity than cobalt 339 ions while cobalt ions induced more micronuclei but fewer strand 340 Q12 breaks than cobalt nanoparticles (Colognato et al., 2009; Ponti et al., 341 2009). Thus, it is apparent that cobalt nanoparticles behave differently 342 inside the cell compared to cobalt microparticles. 343 The reasons for the difference in cytotoxicity and genotoxicity be344 tween particulate and soluble cobalt are unknown. One possibility is a 345 particle effect since soluble and particulate cobalt exhibit different 346 mechanisms of cobalt uptake. For particulate cobalt, uptake of cobalt 347 into the cell and the subsequent cytotoxicity and genotoxicity observed 348 require particle-cell contact (Table 3) indicating that the primary mech349 anism for cobalt uptake is from the internal dissolution of phagocytized 350 particles rather than uptake of extracellular cobalt ions. This is support351 ed by previous studies showing that cobalt ions utilize cellular 352 transporters, such as calcium channels or the divalent metal ion trans353 porter, to enter the cell while particulate cobalt is transported into the 354 cell via phagocytosis (Kreyling, 1992; Lundborg et al., 1992; Simonsen 355 et al., 2011, 2012). Once the particles are taken into the cell, the vacuoles 356 likely associate with lysosomes and dissolve inside the cell producing 357 the high intracellular cobalt concentration observed in the cells. The co358 balt ions produced from the dissolved particles and/or the intact

particles are then responsible for particulate cobalt-induced cytotoxicity and genotoxicity (Table 3). This is contrary to lead chromate, a model of particulate Cr(VI), which does not require particle-cell contact and rather extracellular dissolution and subsequent uptake of the chromate anion into the cell is required for clastogenicity (Xie et al., 2004). Interestingly, similar to cobalt, particle internalization is necessary for particulate nickel-induced chromosome damage and more cytotoxicity is observed after exposure to soluble nickel compared to particulate nickel (Sen and Costa, 1985). This difference in cytotoxicity is attributed to an increase in nickel ions distributed throughout the cytoplasm while particulate nickel specifically dissolves in the perinuclear regions of the cell (Sen and Costa, 1985). This overload in nickel ions in the cytoplasm subsequently induces cytotoxicity via a nongenotoxic mechanism. It is possible that for particulate cobalt internal particle dissolution is localized to the perinuclear regions of the cell resulting in a more direct administration of the cobalt ions to the DNA while exposure to soluble cobalt results in ionic cobalt being more uniformly distributed throughout the cell. This overload of cobalt in the cytoplasm may lead to increased cytotoxicity due to non-genotoxic mechanisms, such as metabolic disruption. In support of this hypothesis, cobalt ions have a high affinity for sulfhydryl groups which can result in the inhibition of critical enzymes involved in mitochondrial respiration and inhibition of ATP synthesis has been observed after soluble cobalt exposure (Bragadin et al., 2007; Simonsen et al., 2012). Additionally, this cytoplasmic disruption and increased cytotoxicity may be responsible for the cell cycle arrest at much lower intracellular cobalt concentrations. Another possibility, albeit untested, is that the particle may be interfering with apoptotic or cell cycle arrest machinery. In conclusion, particulate and soluble cobalt are cytotoxic and genotoxic to human lung cells with soluble cobalt inducing more cytotoxicity and cell cycle arrest but similar genotoxicity. Particle-cell contact is required for particulate cobalt-induced cytotoxicity and genotoxicity indicating that particulate and soluble cobalt compounds exhibit different mechanisms of toxicity and solubility plays a clear role in cobalt-induced lung toxicity. Considering this data, it is important to assess particulate cobalt compounds as an individual class of compounds that may have different carcinogenic potentials compared to soluble cobalt compounds. Future work is aimed at assessing the carcinogenicity of particulate cobalt compounds.

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Table 2 Spectrum of chromosome aberrations induced by cobalt chloride in human lung fibroblast cells a. Cobalt chloride concentration (μM)

Chromatid break and gap

Isochromatid break and gap

Chromatid exchange

Dicentric

t2:3

Double minutes

Acentric fragment

Centromere spreading

Total damage

t2:4 t2:5 t2:6 t2:7 t2:8 t2:9

0 50 100 175 250 500

1.0 ± 0.6 7.0 ± 3.1 5.0 ± 1.0 12 ± 1.0 18 ± 4.1 NM b

0±0 1.7 ± 0.3 1.3 ± 0.9 1.5 ± 0.5 3.0 ± 0.6 NM

0±0 0±0 0±0 0.5 ± 0.5 0±0 NM

0±0 0±0 0.3 ± 0.3 0.5 ± 0.5 0±0 NM

0± 0± 0± 0± 0± NM

0±0 0±0 0.3 ± 0.3 1.0 ± 0 1.0 ± 0.6 NM

0±0 0±0 1.3 ± 1.3 0±0 0±0 NM

1.0 ± 0.6 8.7 ± 3.3 8.3 ± 2.6 16 ± 1.5 22 ± 4.1 NM

t2:10 t2:11

a b

0 0 0 0 0

Data represent the average of three experiments ± the standard error of the mean. NM: no metaphases.

Please cite this article as: Smith, L.J., et al., The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.002

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Abbracchio, M.P., Heck, J.D., Costa, M., 1982. The phagocytosis and transforming activity of crystalline metal sulfide particles are related to their negative surface charge. Carcinogenesis 3, 175–180. Agency for Toxic Substances, Disease Registry (ATSDR), 2004. Toxicological Profile for Cobalt. , U.S. Department of Health and Human Services, Public Health Service, Atlanta, GA. Akbar, M., Brewer, J.M., Grant, M.H., 2011. Effect of chromium and cobalt ions on primary human lymphocytes in vitro. J. Immunotoxicol. 8, 140–149. Allen, M.J., Myer, B.J., Millett, P.J., Rushton, N., 1997. The effects of particulate cobalt, chromium and cobalt-chromium alloy on human osteoblast-like cells in vitro. J. Bone Joint Surg. (Br.) 79, 475–482. Beyersmann, D., Hartwig, A., 2008. Carcinogenic metal compounds: recent insights into molecular and cellular mechanisms. Arch. Toxicol. 82, 493–512. Bragadin, M., Toninello, A., Mancon, M., Manente, S., 2007. The interaction of cobalt (II) with mitochondria from rat liver. J. Biol. Inorg. Chem. 12, 631–635. Catelas, I., Petit, A., Vali, H., Fragiskatos, C., Meilleur, R., Zukor, D.J., Antoniou, J., Huk, O.L., 2005. Quantitative analysis of macrophage apoptosis vs. necrosis induced by cobalt and chromium ions in vitro. Biomaterials 26, 2241–2453. Cobalt Development Institute (CDI), 2013. Rechargeable Batteries. CDI Website http:// www.thecdi.com/index.php (Accessed March 27, 2013). Colognato, R., Bonelli, A., Ponti, J., Farina, M., Bergamaschi, E., Sabbioni, E., Migliore, L., 2008. Comparative genotoxicity of cobalt nanoparticles and ions on human peripheral leukocytes in vitro. Mutagenesis 23, 377–382. Costa, M., Heck, J.D., Robison, S.H., 1982. Selective phagocytosis of crystalline metal sulfide particles and DNA strand breaks as a mechanism for the induction of cellular transformation. Cancer Res. 42, 2757–2763. DeBoeck, M., Lison, D., Kirsch-Volders, M., 1998. Evaluation of the in vitro direct and indirect genotoxic effects of cobalt compounds using the alkaline comet assay. Influence of interdonor and interexperimental variability. Carcinogenesis 19 (11), 2021–2029. Doran, A., Law, F.C., Allen, M.J., Rushton, N., 1998. Neoplastic transformation of cells by soluble but not particulate forms of metals used in orthopaedic implants. Biomaterials 19, 751–759. Figgitt, M., Newson, R., Leslie, I.J., Fisher, J., Ingham, E., Case, C.P., 2010. The genotoxicity of physiological concentrations of chromium (Cr(III) and Cr(VI)) and cobalt (Co(II)): an in vitro study. Mutat. Res. 688, 53–61. Green, S.E., Luczak, M.W., Morse, J.L., DeLoughery, Z., Zhitkovich, A., 2013. Uptake, p53 pathway activation, and cytotoxic responses for Co(II) and Ni(II) in human lung cells: implications for carcinogenicity. Toxicol. Sci. 136, 467–477. Holmes, A.L., Wise, S.S., Xie, H., Gordon, N., Thompson, W.D., Wise Sr., J.P., 2005. Lead ions do not cause human lung cells to escape chromate-induced cytotoxicity. Toxicol. Appl. Pharmacol. 203, 167–176. International Agency for Research on Cancer (IARC), 2006. Cobalt in hard metals and cobalt sulfate, gallium arsenide, indium phosphide and vanadium pentoxide. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. , volume 86. , World Health Organization, Lyon, France. Kawanishi, S., Inoue, S., Yamamoto, K., 1994. Active oxygen species in DNA damage induced by carcinogenic metal compounds. Environ. Health Perspect. 102, 17–20. Kreyling, W.G., 1992. Intracellular particle dissolution in alveolar macrophages. Environ. Health Perspect. 97, 121–126. Lison, D., DeBoeck, M., Verougstraete, V., Kirsch-Volder, M., 2001. Update on the genotoxicity and carcinogenicity of cobalt compounds. Occup. Environ. Med. 58, 619–625. Lloyd, D.R., Phillips, D.H., Carmichael, P.L., 1997. Generation of putative intrastrand crosslinks and strand breaks in DNA by transition metal ion-mediated oxygen radical attack. Chem. Res. Toxicol. 10, 393–400. Lundborg, M., Falk, R., Johansson, A., Kreyling, W., Camner, P., 1992. Phagolysosomal pH and dissolution of cobalt oxide particles by alveolar marcrophages. Environ. Health Perspect. 97, 153–157. Mur, J.M., Moulin, J.J., Charruyer-Seinerra, M.P., Lafitte, J., 1987. A cohort mortality study among cobalt and sodium workers in an electrochemical plant. Am. J. Ind. Med. 11, 75–81. Nackerdien, Z., Kasprzak, K.S., Rao, G., Halliwell, B., Dizdaroglu, M., 1991. Nickel(II)- and cobalt(II)-dependent damage by hydrogen peroxide to the DNA bases in isolated human chromatin. Cancer Res. 51, 5837–5842.

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O

180

Cobalt Oxide

160

Cobalt Chloride

R O

140 120 100 80 60

P

Total Aberrations in 100 Metaphases

406

F

Intracellular Cobalt Concentration (uM)

B

References Cobalt Oxide

40 20 0

5000

10000

D

0 15000

Intracellular Cobalt Concentration (uM)

R

E

C

T

Fig. 5. Particulate and soluble cobalt induce similar levels of chromosome damage in human lung cells. This figure shows that at similar intracellular cobalt ion levels, cobalt oxide and cobalt chloride induce similar levels of chromosome damage. However, cobalt chloride induces cell cycle arrest observed as no metaphases at a much lower intracellular cobalt ion level compared to cobalt oxide. Data represent an average of three independent experiment ± standard error of the mean. A) Percent of metaphases with damage. B) Total aberrations in 100 metaphases.

E

Percent of Metaphases with Damage

A

398 Q13 Conflict of interest 399

O

R

The authors declare that there are no conflicts of interest.

400 Q14 Acknowledgments 401 402

t3:1 t3:2

Table 3 Role of particle-cell contact in human lung fibroblast cellsa.

N

U

403 404

C

405

We would like to thank Shouping Huang and Chris Gianios for the administrative and technical support and Kellie Joyce for the scientific support. This work was supported by ARO Grant #W911NF-09-1-0296 (J.P.W.), and the Maine Center for Toxicology and Environmental Health.

t3:3

Cobalt oxide concentration (μg/cm2)

Well orientation

Intracellular cobalt (μM)

Extracellular cobalt (μM)

Relative survival

Percent of metaphases with damage

Total damage

t3:4 t3:5 t3:6 t3:7

0 0 5 5

Bottom Top Bottom b Top c

0±0 0±0 6,854 ± 1897 d,e 140 ± 26d,e

0±0 0±0 54 ± 2.8 d 58 ± 2.2 d

100 ± 0 100 ± 0 13.9 ± 2.7 d,e 79 ± 12.1

4.3 ± 0.9 6.0 ± 1.0 45 ± 5.7 d,e 8.7 ± 1.3

5.3 ± 0.7 6.7 ± 1.7 103 ± 29 d,e 10.3 ± 1.7

t3:8 t3:9 t3:10 t3:11 t3:12

a b c d e

Data represent the average of three experiments ± the standard error of the mean. Bottom well of the transwell dish: cells experienced particle-cell contact. Top well of the transwell dish: cells were only exposed to cobalt ions released from the particles. Statistically different compared to control (p b 0.05). Statistically different compared to top well (p b 0.05).

Please cite this article as: Smith, L.J., et al., The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.002

L.J. Smith et al. / Toxicology and Applied Pharmacology xxx (2014) xxx–xxx National Toxicology Program (NTP), 1998. Toxicology and carcinogenesis studies of cobalt sulfate heptahydrate (CAS No. 10026-24-1) in F344/N rats and B6C3F1 mice (inhalation studies). Technical Report Series No. 471. NIH Publication No. 98-3961. U.S. Department of Health and Human Services, Public Health Services, National Institutes of Health, Research Triangle Park, NC. Pastel, E., Lynch, C., Ruff, V., Reynolds, M., 2012. Co-exposure to nickel and cobalt chloride enhances cytotoxicity and oxidative stress in human lung epithelial cells. Toxicol. Appl. Pharmacol. 258, 367–375. Ponti, J., Sabbioni, E., Munaro, B., Broggi, F., Marmorato, P., Franchini, F., Colognato, R., Rossi, F., 2009. Genotoxicity and morphological transformation induced by cobalt nanoparticles and cobalt chloride: an in vitro study in Balb/3 T3 mouse fibroblasts. Mutagenesis 24, 439–445. Sauni, R., Linna, A., Oksa, P., Nordman, H., Tupperainen, M., Uitti, J., 2010. Cobalt asthma — a case series from a cobalt plant. Occup. Med. 60, 301–306. Sen, P., Costa, M., 1985. Induction of chromosomal damage in Chinese hamster ovary cells by soluble and particulate nickel compounds: preferential fragmentation of the heterochromatic long arm of the X-chromosome by carcinogenic crystalline NiS particles. Cancer Res. 45, 2320–2325.

Simonsen, L.O., Brown, A.M., Harbak, H., Kristensen, B.I., Bennekou, P., 2011. Cobalt uptake and binding in human red blood cells. Blood Cells Mol. Dis. 46, 266–276. Simonsen, L.O., Harbak, H., Bennekou, P., 2012. Cobalt metabolism and toxicology — a brief update. Sci. Total Environ. 432, 210–215. Van Cutsem, E., Ceuppens, J.L., Lacquet, L.M., Demedts, M., 1987. Combined asthma and alveolitis induced by cobalt in a diamond polisher. Eur. J. Respir. Dis. 70, 54–61. Wise, J.P., Wise, S.S., Little, J.E., 2002. The cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human lung cells. Mutat. Res. 517, 221–229. Wise, S.S., Elmore, L.W., Holt, S.E., Little, J.E., Antonucci, P.G., Bryant, B.H., Wise Sr., J.P., 2004. Telomerase-mediated lifespan extension of human bronchial cells does not affect hexavalent chromium-induced cytotoxicity or genotoxicity. Mol. Cell. Biochem. 255, 103–111. Xie, H., Holmes, A.L., Wise, S.S., Gordon, N., Wise Sr., J.P., 2004. Lead chromate-induced chromosome damage requires extracellular dissolution to liberate chromium ions but does not require particle internalization or intracellular dissolution. Chem. Res. Toxicol. 17, 1362–1367.

F

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7

U

N C O

R

R

E

C

T

E

D

P

R O

O

507

Please cite this article as: Smith, L.J., et al., The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells, Toxicol. Appl. Pharmacol. (2014), http://dx.doi.org/10.1016/j.taap.2014.05.002

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