Chemosphere 100 (2014) 42–49

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Airborne quinones induce cytotoxicity and DNA damage in human lung epithelial A549 cells: The role of reactive oxygen species Yu Shang a, Ling Zhang a, Yuting Jiang a, Yi Li b,⇑, Ping Lu c a

Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, China Chinese Academy of Meteorological Sciences, Beijing 100081, China c Center for Spatial Information Science and Sustainable Development Applications, College of Surveying and Geo-Informatics, Tongji University, Shanghai 200092, China b

h i g h l i g h t s  Different airborne quinones exerted different biological responses in A549 cells. 2+

 Quinones caused cell membrane damage, cell death, DNA damge, and Ca

release.

 These biological responses could be abolished by the treatment of NAC.  Quinone-induced in vitro responses is mediated by elevated production of ROS.  Quinones participate in adverse health effects of particles through ROS generation.

a r t i c l e

i n f o

Article history: Received 5 August 2013 Received in revised form 13 December 2013 Accepted 20 December 2013 Available online 27 January 2014 Keywords: Airborne quinones cytotoxicity DNA damage ROS formation Ca2+

a b s t r a c t Ambient particulate matter (PM) is associated with adverse health effects. Quinones present in PM are hypothesized to contribute to these harmful effects through the generation of reactive oxygen species (ROS). However, whether the ROS induced by quinones is involved in mediating DNA damage as well as other biological responses in pulmonary cells is less well known. In this study, the toxic effects of five typical airborne quinones, including 1,2-naphthoquinone, 2-methylanthraquinone, 9,10-phenanthrenequinone, 2-methyl-1,4-naphthoquinone, and acenaphthenequinone, on cytotoxicity, DNA damage, intracellular calcium homeostasis, and ROS generation, were studied in human lung epithelial A549 cells. An antioxidant N-acetylcysteine (NAC) was used to examine the involvement of ROS in adverse biological responses induced by quinones. The quinones caused a concentration-dependent viability decrease, cellular LDH release, DNA damage, and ROS production in A549 cells. 1,2-Naphthoquinone, but not the other four quinones, increased intracellular calcium (Ca2+) levels in a dose-dependent manner. These toxic effects were abolished by administration of NAC, suggesting that ROS played a key role in the observed toxic effects of quinones in A549 cells. These results emphasize the importance of quinones in PM on the adverse health effects of PMs, which has been underestimated in the past few years, and highlight the need, when evaluating the effects on health and exposure management, to always consider their qualitative chemical compositions in addition to the size and concentration of PMs. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Ambient particulate matter (PM) is associated with cardiopulmonary morbidity and mortality (Brunekreef and Holgate, 2002). However, the underlying mechanisms remain poorly Abbreviations: ACQ, acenaphthenequinone; DMSO, dimethyl sulfoxide; LDH, lactate dehydrogenase; LMA, low melting agarose; MAQ, 2-methylanthraquinone; MNQ, 2-methyl-1,4-naphthoquinone; MTT, 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5di-phenytetrazoliumromide; NAC, N-acetylcysteine; NMA, normal melting agarose; 1,2-NQ, 1,2-naphthoquinone; OS, oxidative stress; PM, particulate matter; PQ, 9,10phenanthrenequinone; ROS, reactive oxygen species; tBHP, tert-butyl hydroperoxide. ⇑ Corresponding author. Tel./fax: +86 10 58993139. E-mail address: [email protected] (Y. Li). 0045-6535/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.12.079

understood because of their complex and heterogeneous properties. Previous studies have indicated that PM interacts with biological systems through direct generation of reactive oxygen species (ROS) and further results in oxidative stress (OS) (Li et al., 2008). Quinones are typical oxygenated derivatives of polycyclic aromatic hydrocarbons (PAHs) in air (Walgraeve et al., 2010). Recently, quinones are hypothesized to contribute to these adverse health effects caused by PM because of their high redox potency in producing ROS (Li et al., 2012) and are believed to some extent to be more toxic than their parent PAHs (Sidhu et al., 2005). Most airborne quinones are polycyclic aromatic chemicals, such as anthraquinone, naphthoquinone, and phenanthrenequinone. They are released during the incomplete combustion process of

Y. Shang et al. / Chemosphere 100 (2014) 42–49

fossil fuels and/or formed via atmospheric photochemical conversions from PAHs (Walgraeve et al., 2010). Quinones could also be formed in vivo from PAHs by enzymes (Walgraeve et al., 2010). The reported concentrations of quinones are typically from pg m3 to several ng m3 in air (Walgraeve et al., 2010). A study in Birmingham, in UK, measured 11 quinones during January 2010 (Alam et al., 2013), including the most abundant 9,10-phenanthrenequinone (6.1 ng m3), 1,2-naphthoquinone (3.0 ng m3), 1,4-naphthoquinone (2.1 ng m3), and 2-methylanthraquinone (2.8 ng m3). The ability of ROS production are usually used to assess the following toxic effects resulting from PM exposure. Although most studies are focused on ROS generation through the Fenton reaction of transition metals, several studies have focused on the mechanisms caused by quinones present in PM. Using the dithiothreitol (DTT) assay, Chung et al. (2006) found that ROS generation from PM showed a strong positive correlation with quinones detected in particles. Recently, through electron spin resonance and capillary electrophoresis, the semiquinone radical and OH were detected directly in a physiological buffer solution, providing direct evidence for the redox cycling hypothesis of ROS generation by quinones (Li et al., 2012). Additionally, 1,4-naphthoquinone (1,4-NQ) was found to cause significant cell death in Ana-1 macrophages through the generation of ROS via redox cycling (Shang et al., 2012). Calcium ion (Ca2+) is a second messenger and plays important roles in the regulation of many physiological processes in cells. Recent studies suggest that Ca2+ may participate in cytotoxicity and inflammation induced by ambient particles (Happo et al., 2013). Sakamoto et al. (2007) determined that PM10 increased cellular Ca2+, which is associated with IL-1b and IL-8 production in human bronchial epithelial cells. Until now, it is at least partially clear that airborne quinones contribute to the oxidative potential of PM. However, whether quinone-induced adverse biological responses are correlated with irregular ROS generation is still not well documented. In this study, we investigated the cytotoxicity and DNA damage in human lung epithelial A549 cells exposed to five typical airborne quinones, including 1,2-naphthoquinone (1,2-NQ), 2-methyl-1,4-naphthoquinone (MNQ), 2-methylanthraquinone (MAQ), acenaphthenequinone (ACQ), and 9,10-phenanthrenequinone (PQ). To explore the mechanisms of quinone-induced cellular biological responses, their ability to elevate Ca2+ and generate ROS was also examined. The protective role of NAC towards ROS generation induced by quinones was also studied. These results should contribute to better understanding of the potential cellular mechanisms of potential toxic effects of human exposure to airborne quinones. 2. Materials and methods 2.1. Materials A549 cells (type II pulmonary epithelial cell line) were from American Tissue Type Culture Collection (USA). RPMI 1640 medium and fetal bovine serum (FBS) were from Gibco (UK). Lactate Dehydrogenase (LDH) Assay Kit was from Nanjing Jiancheng Bio-engineering Research Institute (Nanjing, China). Dimethyl sulfoxide (DMSO), trypsin, 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5di-phenytetrazoliumromide (MTT), and 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA) were from Sigma (USA). 1,2-NQ, MAQ, PQ, MNQ, and ACQ were from Sigma–Aldrich (USA). All other reagents were analytical grade chemicals from Sinopharm Chemical Reagent Company (Shanghai, China). 2.2. Cell culture and treatment A549 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and penicillin/streptomycin (100 units mL1) in an atmosphere of 5% CO2 and 100% relative humidity at 37 °C.

43

Quinones were dissolved in DMSO and working solutions were freshly diluted by RPMI 1640 with 2% FBS. The final concentration of DMSO was 0.1% (v/v). Treatment dosages were 5, 10, 15, 20 lM for 1,2-NQ and MNQ; 10, 20, 40, 60 lM for MAQ and ACQ; 1, 2, 3, 4 lM for PQ. The cells were exposed for 24 h except for Ca2+ and ROS measurements, which were 1 h. DMSO-treated cells (0.1%, v/ v) served as control. Tert-butyl hydroperoxide (tBHP) was employed as positive control in Comet assay and ROS measurement. NAC is a cell-permeable thiol antioxidant involved in the induction of glutathione synthesis and scavenging of ROS, which inhibits the effects of endogenous ROS. To determine the involvement of ROS in the biological responses of quinones, A549 cells were treated with quinones with or without NAC. A 10 mM concentration was chosen based on the viability of A549 cells caused by NAC. A549 cells treated with 10 mM NAC for 24 h showed no significant decrease in viability. NAC was dissolved in PBS, and the filter-sterilized NAC was freshly diluted in culture medium before use. 2.3. MTT assay Cells were plated in 96-well plates (5  103 cells/well) and cultured for 24-h to allow cell adhesion. Subsequently, the culture medium was replaced with fresh medium containing various concentrations of quinones with or without 10 mM NAC. After the 24h treatment, cells were incubated with 100 lL of freshly prepared MTT (1 mg mL1) in medium for 4 h in the dark at 37 °C. The formazan crystals formed in the cells were solubilized by 100 lL DMSO. Optical density (OD) was read using a Multi scan Mk3 plate reader (Thermo Electron Corporation, USA) at 570 nm. Based on the concentration–response curves, LC50 values (lethal concentration where cell viability was reduced by 50%) were determined. 2.4. LDH release LDH release was assessed to indicate plasma membrane damage. A549 cells were plated in 24-well plates (1  104 cells/well) and cultured for 24 h to allow cell adhesion. Subsequently, the culture medium was replaced with fresh medium containing quinones with or without 10 mM NAC. After 24 h, LDH in culture medium was measured according to the manufacturer’s procedure as previously described (Shang et al., 2013). LDH catalyzes the conversion of lactic acid into pyroracemic acid, which reacts with 2,4-dinitrophenylhydrazine to form brownish red dinitrophenylhydrazone. Subsequently, LDH was assayed by colorimetry at 400 nm using a Quant microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA). LDH activity was calculated according to the following formula: LDH activity (U/L) = [(ODtreated  ODcontrol)/(ODstandard  ODblank)]  standard concentration  dilution factor  1000. The control was culture medium without cells. The standard was sodium pyruvate, and the blank was distilled water. 2.5. Comet assay DNA damage was evaluated by alkaline single cell gel electrophoresis (Comet assay) according to the recommended procedure (Tice et al., 2000). Briefly, cells were plated in 35-mm culture dishes and treated with quinones with or without NAC. Following treatment, cells were resuspended in low melting agarose (LMA) and placed on slides coated with 1% normal melting agarose (NMA), and LMA was then added as the top layer. Cells were lysed in cold (4 °C) lysis buffer (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1% Triton X, and 10% DMSO, pH 10.0) for 1 h. After lysis, the slides were subjected to horizontal gel electrophoresis in cold (4 °C) alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA, pH 12.5) at 25 V and 300 mA for 20 min. The slides were

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PQ (µM) Fig. 1. The effects of five quinones with or without 10 mM NAC on the viability of A549 cells after 24-h treatment, as measured by MTT assay. All values are expressed as mean ± SEM. Experiments were repeated three times with six duplicates in each. Control was cells treated with 0.1% DMSO (v/v). *P < 0.05 and **P < 0.01, compared with control; #P < 0.05 and ##P < 0.01, quinones treated groups compared with quinones + NAC treated groups.

then soaked twice with neutralization buffer (0.4 M Trizma base, pH 7.5, 4 °C) for 8 min and air-dried. DNA was stained with PI (20 lg mL1) and analyzed using a fluorescence microscope (Olympus BX-51; Olympus, Japan). Three hundred randomly captured cells from each sample were analyzed using CASP software (University of Wroclaw, Poland). We chose DNA percentage in the tail (%Tail DNA) as the metric for DNA migration (Tice et al., 2000). 2.6. ROS measurement H2DCFDA diffuses through cell membranes and is deacetylated by intracellular esterases to non-fluorescent 20 ,70 -dichlorodihydrofluorescein (DCFH). In the presence of ROS, DCFH is oxidized to the highly fluorescent 20 ,70 -dichlorodihydrofluorescein (DCF). The ROS was measured exactly according to the method as we have published before (Shang et al., 2013). Briefly, A549 cells were seeded

in 35-mm culture dishes at a density of 2  104 cells per dishes and treated with quinones with or without NAC for 1 h. Subsequently, cells were washed twice with warm D-Hank’s solution and incubated with H2DCFDA (5 lM in D-Hank’s solution) at 37 °C for 30 min in the dark. The images of cells were shown in Fig. s1 in the supporting information (SI). The fluorescence was evaluated under a fluorescence microscope (Olympus BX-51; Olympus, Japan). The intensity of fluorescence was analyzed by Image-pro Plus 6, and the fluorescence of the control group was normalized to 100%. 2.7. Intracellular Ca2+ determination Fluo-3/AM, which is a probe for Ca2+ measurement, can freely enter cells (Takahashi et al., 1999). Inside cells, Fluo-3/AM is hydrolyzed by cytosolic esterases to Fluo-3, which is effectively combined with Ca2+ to form Fluo-3-Ca2+, and the fluorescence

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Y. Shang et al. / Chemosphere 100 (2014) 42–49

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PQ (μM) Fig. 2. Effects of five quinones with or without NAC on the LDH release in the culture medium of A549 cells after 24-h treatment. All values are expressed as mean ± SEM. Experiments were repeated three times with two duplicates in each. Control was cells treated with 0.1% DMSO (v/v) only. *P < 0.05 and **P < 0.01, compared with control; #P < 0.05 and ##P < 0.01, quinones treated groups compared with quinones + NAC treated groups.

intensity of Fluo-3-Ca2+ is used to measure the level of Ca2+. Briefly, A549 cells were plated onto glass coverslips in triplicate and treated with quinones for 1 h. Cells were then washed with warm DHank’s solution and loaded with 10 lM Fluo 3/AM at 37 °C for 30 min. Subsequently, cells were washed with D-Hank’s solution to eliminate extracellular Fluo-3/AM and incubated at 37 °C for an additional 30 min. Fluorescence images were recorded with a fluorescence microscope (Olympus BX-51, Japan) at an emission wavelength of 528 nm, and the fluorescence of the control group was normalized to 100%.

2.8. Data analysis Data were analyzed with SPSS software (ver. 16.0). All data are expressed as the mean ± standard error of the mean (SEM) and

were compared using one-way analysis of variance (ANOVA). In all cases, P < 0.05 was considered statistically significant.

3. Results and discussion 3.1. Cell viability and plasma membrane damage Chemical structures and detailed characteristics of the five quinones are shown in Table s1 (SI). A549 cells were chosen based on previous results (seen in SI). The viability of A549 cells after incubation with 1,2-NQ, MNQ, MAQ, ACQ, and PQ for 24 h was assayed by MTT method (Fig. 1). A549 cells treated with each quinone showed significantly decreased viability in a concentration-dependent manner (one-way ANOVA analysis, P < 0.05). The 24-h LC50 values were determined to be 13.3 lM for 1,2-NQ, 18.4 lM for

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Y. Shang et al. / Chemosphere 100 (2014) 42–49

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The alkaline version of Comet assay was used to evaluate genotoxic effects of these quinones. Fig. 3A summarizes the %Tail DNA in A549 cells after treatment with a range of concentrations of quinones for 24 h. These results indicate that 1,2-NQ and MNQ caused significant DNA damage in A549 cells in a concentration-dependent manner (one-way ANOVA test, P < 0.01). The %Tail DNA increased from 5.0% to 12.8% and 5.6% to 9.8% when concentrations of 1,2-NQ and MNQ, respectively, increased from 0 to 20 lM. MAQ (0–60 lM), ACQ (0–60 lM), and PQ (0–4 lM) did not change the level of DNA tail moment in the A549 cells. In addition, Fig. 3B shows that NAC significantly reduced DNA damage in A549 cells caused by 1,2-NQ and MNQ. These results suggest that ROS formation is involved in DNA damage induced by 1,2-NQ and MNQ. 3.4. ROS formation

4

0

et al., 2013). Administration of 10 mM NAC significantly reduced the acute cytotoxic effects of quinones, which is indicated by the significantly increased viability and decreased LDH level in the presence of NAC (Figs. 1 and 2). These results strongly implicate that ROS formation and possible oxidative stress are involved in the cytotoxicity of these quinones.

0

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Fig. 3. Effects of (A) five quinones and (B) 1,2-NQ and MNQ with 10 mM NAC on DNA damage in A549 cells after 24-h treatment, measured by Comet assay. All values are expressed as mean ± SEM. Experiments were repeated three times with two duplicates in each. Control was cells treated with 0.1% DMSO (v/v); tBHP (200 lM) was served as the positive control. *P < 0.05 and **P < 0.01, compared with control; #P < 0.05 and ##P < 0.01, quinones treated groups compared with quinones + NAC treated groups.

MNQ, 46.8 lM for MAQ, 88.8 lM for ACQ, and 3.3 lM for PQ. These results indicate the different cytotoxicity of the five quinones. PQ had the lowest LC50, indicating it had the greatest acute cytotoxic effects on A549 cells, followed by 1,2-NQ, MNQ, MAQ and ACQ. The decreased viability caused by quinones has been investigated in many cell types (Klaus et al., 2010; Nishiyama et al., 2010). The previously reported LC50 values of PQ and ACQ in A549 cells (Das et al., 2012) are identical to the data of the present study. The LC50 of MNQ and MAQ were similar to our previously reported results of 1,4-NQ and AQ in A549 cells, respectively (Shang et al., 2013), probably because of their similar chemical structures. LDH is a stable cytoplasmic enzyme in cells and is rapidly released into culture medium upon damage of the plasma membrane of cells. In addition to viability, LDH leakage in cell culture medium was measured as another indicator of cell toxicity after exposure to quinones for 24-h (Fig. 2). Release of LDH was increased in a dosedependent manner after treatment with the five quinones (oneway ANOVA test, P < 0.05). Statistically significant increases of LDH were always observed in the highest two or three doses. 3.2. NAC eliminates quinone cytotoxicity NAC is a thiol antioxidant that acts as a store of cysteine in cells and a scavenger of radicals by directly interacting with ROS (Du

In addition to supporting the hypothesis that ROS formation may be one of the mechanisms causing cytotoxicity of quinones, excessive production of ROS was determined. In Fig. 4A, treatment of A549 cells with 1,2-NQ caused a dose-dependent increase in ROS formation up to 10 lM (by 4.5-fold) and was significant (P < 0.05) above 5 lM. However, above 10 lM, there was a dose-dependent decline in ROS levels. A similar response of ROS formation was observed in previously published studies (Klaus et al., 2010; Shang et al., 2012, 2013). MNQ, MAQ, ACQ, and PQ increased ROS levels in a dose-dependent manner (ANOVA test, P < 0.01) (Fig. 4B–E). MAQ exposure significantly increased ROS only at the highest dose of 60 lM. Fig. 4 also shows NAC significantly prevents ROS formation induced by quinones, additionally supporting the hypothesis that excessive production of ROS in A549 cells may be one of the mechanisms causing cytotoxicity and genotoxic effects of the quinones tested in this study. The intracellular ROS production by quinones (Qs) is thought to be a consequence of O2 reduction by semiquinone intermediates (Q). The higher the electron affinity (one-electron reduction potential, E(Q/Q)) is, the easier the quinone is to undergo one-electron reduction. The amount of ROS induced by the five quinones were in accordance with their E(Q/Q) values (the E(Q/ Q) values of ACQ, 1,2-NQ, PQ, MNQ, MAQ are 81, 89, 124, 203 and