This article was downloaded by: [UNSW Library] On: 24 August 2015, At: 19:57 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG
Toxicology Mechanisms and Methods Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/itxm20
Curcumin attenuates quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells a
a
a
a
Chongshan Dai , Shusheng Tang , Daowen Li , Kena Zhao & Xilong Xiao
a
a
College of Veterinary Medicine, China Agricultural University, Beijing, PR China Published online: 26 May 2015.
Click for updates To cite this article: Chongshan Dai, Shusheng Tang, Daowen Li, Kena Zhao & Xilong Xiao (2015): Curcumin attenuates quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells, Toxicology Mechanisms and Methods To link to this article: http://dx.doi.org/10.3109/15376516.2015.1045659
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2015.1045659
RESEARCH ARTICLE
Curcumin attenuates quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells Chongshan Dai, Shusheng Tang, Daowen Li, Kena Zhao, and Xilong Xiao
Downloaded by [UNSW Library] at 19:57 24 August 2015
College of Veterinary Medicine, China Agricultural University, Beijing, PR China
Abstract
Keywords
Quinocetone (QCT), a new quinoxaline 1,4-dioxides, has been used as antimicrobial feed additive in China. Potential genotoxicity of QCT was concerned as a public health problem. This study aimed to investigate the protective effect of curcumin on QCT-induced oxidative stress and genotoxicity in human hepatocyte L02 cells. Cell viability and intracellular reactive oxygen species (ROS), biomarkers of oxidative stress including superoxide dismutase (SOD) activity and glutathione (GSH) level were measured. Meanwhile, comet assay and micronucleus assay were carried out to evaluate genotoxicity. The results showed that, compared to the control group, QCT at the concentration ranges of 2–16 mg/mL significantly decreased L02 cell viability, which was significantly attenuated with curcumin pretreatment (2.5 and 5 mM). In addition, QCT significantly increased cell oxidative stress, characterized by increases of intracellular ROS level, while decreased endogenous antioxidant biomarkers GSH level and SOD activity (all p50.05 or 0.01). Curcumin pretreatment significantly attenuated ROS formation, inhibited the decreases of SOD activity and GSH level. Furthermore, curcumin significantly reduced QCT-induced DNA fragments and micronuclei formation. These data suggest that curcumin could attenuate QCT-induced cytotoxicity and genotoxicity in L02 cells, which may be attributed to ROS scavenging and anti-oxidative ability of curcumin. Importantly, consumption of curcumin may be a plausible way to prevent quinoxaline 1,4-dioxides-mediated oxidative stress and genotoxicity in human or animals.
Curcumin, genotoxicity, oxidative stress, quinocetone, ROS
Introduction Quinoxaline 1,4-dioxides (QdNOs), a class of quinoxaline derivatives consisting of one or two acyclic chains moiety combined with quinoxaline ring (Figure 1A), have been considered as important biologically active compounds and effectively used as antibacterial, anticancer and antiprotozoal drugs or growth promoters (Carta et al., 2002; Diab-Assef et al., 2002; Gali-Muhtasib et al., 2001, 2004, 2005; Guo et al., 2012; He et al., 2013; Mu et al., 2014; Wang et al., 2015). Olaquindox (OLA) and carbadox (CBX) are wellknown members of QdNOs with promoting animal growth, however, they have been forbidden to use as animal growth promoters in 1999 by Commission of the European Community because of their genotoxicity and no threshold for the safe use (Ihsan et al., 2013; Wang et al., 2015). Quinocetone (QCT; Figure 1B) is one of the QdNOs family and it has been widely used to promote animal growth in food producing animals including chicken, pig, fish and goats, as a replacement for OLA and CBX in China (Li et al., 2014). Address for correspondence: Xilong Xiao, College of Veterinary Medicine, China Agricultural University, 2 Yuanmingyuan West Road, Beijing 100193, PR China. Tel: +86 10 6273 3857. Fax: +86 10 6273 1032. E-mail: [email protected]
History Received 7 February 2015 Revised 12 March 2015 Accepted 10 April 2015 Published online 21 May 2015
In addition, it is also used as an antimicrobial agent against Salmonella, Escherichia coli, Brachyspira hyodysenteriae and other gram-negative bacterial infections (Ihsan et al., 2013; Wang et al., 2011; Yang et al., 2013). However, recently available data illustrated that QCT could induce genotoxic and cytoxic effects in several in vitro; high doses of QCT (at least 4–10-folds) can induce pathological (such as liver and renal tissues)/behavioral alterations in rats or mice (Chen et al., 2009; Ihsan et al., 2013; Jin et al., 2009; Wang et al., 2010; Zhang et al., 2013). Food safety of QCT concerned potential toxicity to human should be evaluated because quinocetone residues and metabolites in animal tissues may endanger consumer health by bioaccumulation. Therefore, the risk assessment of QCT is very important and necessary. In our previous study, it was shown that QCT can induce marked cytoxicity and genotoxicity in African green monkey cell lines (Vero cells) and human hepatoma cells (HepG2) (Chen et al., 2009; Jin et al., 2009). Our previous study showed that reactive oxygen species (ROS) and caspasedependent apoptosis participated into QCT-mediated cell toxicity in HepG2 cell (Zhang et al., 2013). Yang et al. (2013) demonstrated that QCT can trigger oxidative stress and induce cytotoxicity and genotoxicity in human peripheral lymphocytes. Clearly, excessive ROS generation-mediated oxidative
2
C. Dai et al.
Toxicol Mech Methods, Early Online: 1–7
Downloaded by [UNSW Library] at 19:57 24 August 2015
Figure 1. Structure of quinoxalines (A), quinocetone (B) and curcumin (C).
stress plays a key role in QCT-induced DNA injury and genotoxicity, albeit the precise mechanism is still far from clear. Curcumin (diferuloylmethane) (Figure 1C), a natural polyphenol in the spice turmeric, exhibits antioxidant (Gao et al., 2013), anti-inflammatory (Menon & Sudheer, 2007) and anti-carcinogenic properties (Brouet & Ohshima, 1995). It has been reported that curcumin can protect against DNA damage induced by drug or environmental mutagens including arsenic (Gao et al., 2013), acryl amide (Cao et al., 2008), benz(a)pyren (Zhu et al., 2014) and cisplatin (Waseem & Parvez, 2013) based on its suppression of ROS generation in vitro and in vivo studies. In animal production, dietary supplementations with curcumin can improve immunity and the ability to adapt to environmental stress without any adverse effect (Oh et al., 2013; Rajput et al., 2013). In human clinical trials, dietary supplementation of curcumin can attenuate exercise-induced oxidative stress by increasing blood antioxidant capacity (Takahashi et al., 2014). Additionally, curcumin at low doses could significantly reduce the chemotherapeutic agent-induced micronucleus formation (Celik et al., 2013). Therefore, in the present study, we investigated the protective effect of curcumin on QCT-induced genotoxicity and cytotoxicity in human hepatocyte L02 cells.
Materials and methods Chemical and regents QCT (C18Hl4N2O3, CAS NO. 81810-66-4; purity 98%) was purchased from Zhongmu Pharmaceutical Co. Ltd. (Wuxue, PR China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Gibco, Grand Island, NY). 3-(4,5-Dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT), cytochalasin B, triton X-100, agarose and propidium iodide (PI) were purchased from Sigma–Aldrich (St. Louis, MO). Curcumin (purity 98%) was purchased from Aladdin Reagent Co., Ltd (Shanghai, China). Curcumin and QCT were respectively prepared as a 20 mM and 16 mg/mL stock solutions in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO), and
stored at 20 C in the dark. For each experiment, curcumin and QCT were diluted with cell culture medium to the concentration indicated with a final DMSO concentration of 0.1% (vol/vol) in the dark. Other reagents were of analytical reagent grade. Cell culture L02 cell, a normal human hepatocyte cell line from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), was employed because liver is the major organ to metabolize QCT. Cells were cultured in DMEM containing with 2% L -glutamine, 10% FBS, 100 U/mL (100 units/mL, Gibco) and streptomycin (100 mg/mL, Gibco) at 37 C in a humidified incubator (5% CO2). The medium was renewed every 2 days until the cells were grown to confluence. Cell viability assay MTT assay was used to measure the cytotoxicity of QCT as described previously with modifications (Zhang et al., 2013). L02 cells were plated in a 96-well micro-titer plate at a density of 1 104 cells per well in a final volume of 100 mL DMEM. After pretreatment with curcumin (the final concentration was 2.5 and 5 mM, respectively) for 2 h, the medium was replaced with fresh medium containing QCT (the final concentration was from 2 to 16 mg/mL). The cells in the control group were pre-treated with 0.1% DMSO, 2.5 or 5 mM curcumin for 2 h, then replaced with fresh medium containing 0.1% DMSO. After incubation for 4 or 24 h, the medium was discarded, 100 mL serum-free DMEM containing 10 mL MTT (5 mg/mL) was added and followed by incubation for 4 h at 37 C. Finally, the medium was discarded and added 100 mL DMSO. After incubation for 20 min at room temperature, the absorbance was read at 490 nm in a micro-plate reader (Molecular Devices, Sunnyvale, CA). Cells incubated with 0.1% DMSO were used as the control. Measurement of intracellular ROS production, superoxide dismutase activity and glutathione level The production of intracellular ROS was measured using the ROS-specific fluorescent dye 2,7-dichlorofluorescein
Curcumin attenuates QCT-induced oxidative stress
Downloaded by [UNSW Library] at 19:57 24 August 2015
DOI: 10.3109/15376516.2015.1045659
diacetate (DCFH-DA) (Beyotime, Haimen, China). L02 cells were seeded at an initial density of 1 104 cells/mL in 96well plates. The cells were pretreated with curcumin (2.5 and 5 mM, respectively) for 2 h at 37 C, then the medium was discarded and replaced with 200 mL DMEM with QCT at different final concentrations (8 and 16 mg/mL, respectively) for incubation. After 4 h exposure to QCT, the cells were washed with PBS, then 200 mL DMEM was added and loaded with 10 mM DCFH-DA at 37 C for 30 min in the darkness, then rinsed three times with PBS, and measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm using a microplate fluorescence reader (MD, Spectramax M3). To further examine the role of oxidative stress on QCTinduced cytoxicity, genotoxicity and the protection of curcumin, intracellular total superoxide dismutase (SOD) activity and glutathione (GSH) level as the biomarkers of oxidative stress were determined using commercial kits (Nanjing Jiancheng Institute of Biological Engineering). Briefly, L02 cells were seeded at a density of 1 105 into 12-well plate and incubated for 24 h at 37 C. Then cells were washed with PBS and pretreated with curcumin at 2.5 and 5 mM for 2 h, then washed twice with PBS, and incubated without (0.1% DMSO) or with QCT (8 and 16 mg/mL) in fresh DMEM for 4 h. After the desired time of treatment, cells were harvested, washed with PBS and homogenized in 10 mM phosphate buffer containing 0.15 M KCl, 0.1 mM EDTA, 1 mM DTT and 0.1 mM phenylmethylsulfonylfluoride (pH 7.4) at 4 C, then centrifuged at 3000 g (4 C) for 15 min. The supernatant was collected for examination. Protein concentrations in the supernatant were measured using the BCA protein assay (Wuhan Boster Bio-engineering Limited Co., Wuhan, China). Comet assay The comet assay was carried out under alkaline conditions as our previous study (Jin et al., 2009). L02 cells were seeded in a 24-well plate at a density of 1 104 cells/well. After 24 h of growth, the cells were pretreated with curcumin (final concentration was 2.5 and 5 mM, respectively) for 2 h, then discarded and replaced with 500 mL DMEM with QCT at 8 and 16 mg/mL, respectively for 4 h incubation. The corresponding control groups were exposed with 0.1% DMSO, QCT or curcumin for equal time. Then, cells were washed twice with PBS and trypsinized for 2 min then suspended in 80 mL PBS. To avoid artifacts resulting from cell death, the cell suspensions (40 mL) were mixed with trypan blue solution (40 mL, 0.5% in PBS) to examine the cell viability. Only the viabilities of cell suspensions were more than 80%, they were used for examining DNA damage. Then, 20 mL of the cell suspensions was mixed with 60 mL of 0.8% LMA and placed on frosted slides pre-layered with 0.8% NMA. Then, the cover slip was removed and the slides were immersed in lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10 and 10% DMSO with 1% Triton X-100) in darkness at 4 C for 1 h. After lysis, the slides were placed in alkaline solution (1 mM Na2-EDTA and 300 mM NaOH, pH 13) for 20 min at room temperature to allow DNA unwinding. Electrophoresis were performed in alkaline solution for 30 min at 25 V. Cells were
3
neutralized using 0.4 M Tris-HCl (pH 7.5) and stained with 50 mL of ethidium bromide (10 mg/mL). Slides were viewed at 40 magnification using fluorescent microscopy (Leica, Omachi, Japan). At least 100 randomly selected cells (30 or 40 cells from each of the three replicated slides) were quantitatively analyzed using Comet Assay Software Project casp-1.2.2 (University of Wroclaw, Poland). Micronucleus assay L02 cells growing in the logarithmic phase were seeded at a density of 1 105 into 12-well plate and incubated for 24 h at 37 C. After this period, the cells were washed with PBS and pretreated with curcumin at 2.5 and 5 mM for 2 h, then washed twice with PBS and incubated with QCT (1 and 2 mg/mL) in fresh DMEM for 24 h, then cells were washed twice with PBS and incubated with DMEM containing cytochalasin B (the final concentration was 4.5 mg/mL) for 20 h. Fixation and slide preparation were carried out according to the conventional techniques. Statistical analysis Each experiment was performed at least three times in triplicate and all data are presented as mean ± SD unless specified. Statistical analysis was conducted using SPSS V16.0 (SPSS Inc., Chicago, IL) and figures were prepared using Graphpad Prism 5.0 (Graph Pad Software, Inc., La Jolla, CA). Data from the control and treatment groups were analyzed with one-way analysis of variance (ANOVA), followed by LSD post-hoc test. A p value 50.05 was considered significant.
Results Effects of curcumin on QCT-induced cytotoxicity in L02 cells The cytotoxicity of QCT exposed to L02 cells for 4 and 24 h was examined. At 4 h, cell viabilities had no significant changes in the QCT 1, 2 and 4 mg/mL groups and a slight cytotoxicity was shown in the QCT 8 and 16 mg/mL groups, the cell viabilities decreased to 88% (p50.05) and 81% (p50.01), respectively. Furthermore, L02 cells were treated with QCT for 24 h, the cytotoxicity was significantly decreased in a dose-dependent manner and the IC50 (inhibitory concentration 50%) value was 7.04 ± 1.21 mg/mL, which was significantly improved by pre-treatment with curcumin at the dose of 2.5 and 5 mM, the IC50s were significantly increased to 12.16 ± .06, 15.42 ± 1.64 mg/mL (both p50.01) (Figure 2). Compared to the control group, pretreatment of curcumin at 2.5 and 5 mM for 2 h did not change the cell viability did not significant change in the curcumin control group (Figure 2). Effects of curcumin on QCT-induced ROS formation and the biomarkers of oxidative stress in L02 cells To explore the protective effects of curcumin, intracellular ROS formation and biomarkers of oxidative stress including GSH and SOD were examined, as shown in Figure 3. L02 cells were treated with QCT at high doses of 8 and 16 mg/mL for a 4 h-term observation. Compared to those untreated
4
C. Dai et al.
Downloaded by [UNSW Library] at 19:57 24 August 2015
control, there were no marked changes of intracellular ROS formation, SOD activity and GSH level after incubation with 2.5 and 5 mM curcumin. Intracellular ROS formation significantly increased in the cells treated with the 8 and 16 mg/mL QCT groups (both p50.01), meanwhile, SOD activity significantly decreased to 84.1% and 76.0% (both p50.01), GSH level significantly decreased to 76.3% and 62.1% (both
Figure 2. Effects of curcumin on quinocetone (QCT)-induced cytotoxicity in L02 cells determined by MTT. Values were presented as mean ± SD, from three independent experiments (n ¼ 3). *p50.05, **p50.01, compared to the vehicle control group; #p50.05, ##p50.01, compared to the QCT alone group.
Toxicol Mech Methods, Early Online: 1–7
p50.01), compared to the control. Pretreatment of curcumin at 2.5 and 5 mM significantly decreased QCT caused by the formation of intracellular ROS (Figure 3A), increased SOD activity (Figure 3B) and GSH level (Figure 3C) (all p50.05 or 0.01), compared to that in the QCT alone group. Effects of curcumin on QCT-induced DNA strand breaks in L02 cells QCT can significantly induce DNA strand breaks in L02 cells, as shown in Figure 4. Before beginning to carry out comet assay test, cell viability was examined using trypan blue stain. In all the groups, cell viabilities were more than 80%. Subsequently, cells were used to evaluate DNA damage. Compared to the control, at the QCT 8 and 16 mg/mL, the percentage (%) tail DNA increased to 21.7% and 32.3% (both p50.01), significant decreases of % tail DNA were detected when L02 cell were pretreated with curcumin at 2.5 mM (decreased to 15.7% and 22.0%) (both p50.05) and 5 mM (decreased to 12.5% and 18.4%) (both p50.01), respectively (Figure 4A); the tail length increased to 42.4 and 54.0 mm, which were significantly decreased by curcumin pretreatment (decreased to 31.0 and 37.7 mm at curcumin 2.5 mM pretreatment group; decreased to 25.4 and 31.5 mm at curcumin 5 mM pretreatment group, respectively) (all p50.05 or 0.01) (Figure 4B); the comet tail moment values increased to 9.2 mm (&35-folds) and 17.6 mm (&67-folds) (both p50.01), which were decreased in the pretreatment of curcumin at
Figure 3. Effects of curcumin on quinocetone (QCT)-induced ROS production and oxidative stress in L02 cells. (A) represents ROS production, ROSspecific fluorescent dye 2,7-dichlorofluorescein diacetate was used to measure the production of intracellar ROS. (B) and (C) represent SOD activity and GSH level, respectively. Values were presented as mean ± SD, from three independent experiments (n ¼ 3). *p50.05, **p50.01, compared to the vehicle control group; #p50.05, ##p50.01, compared to the QCT alone group.
Downloaded by [UNSW Library] at 19:57 24 August 2015
DOI: 10.3109/15376516.2015.1045659
Curcumin attenuates QCT-induced oxidative stress
5
Figure 4. Effects of curcumin on QCT-induced DNA strand-breaks in L02 cells. (A) % tail DNA, (B) tail length, (C) tail moment. DNA damage was measured in the comet assay in L02 cells treated with QCT (0, 8 and 16 mg/mL, respectively) or QCT plus curcumin (2.5 and 5 mM, respectively) for 4 h. Data were presented as mean ± SD, from three independent experiments (n ¼ 3). *p50.05, **p50.01, compared to the vehicle control group; #p50.05, ##p50.01, compared to the QCT alone group.
2.5 mM (decreased to 4.6 and 8.1 mm, respectively) and 5 mM (decreased to 3.1 and 5.9 mm, respectively) (all p50.01). Effects of curcumin on the frequencies of QCT-induced MN Compare to that non-treated cells, QCT treatment at the doses of 1 and 2 mg/mL for 24 h, the number of MN significantly increased to 36.7% and 49.0% (both p50.01), which were decreased to 30.4% and 38.2% (both p50.05) when cell was pretreated with curcumin at 2.5 mM, further decreased to 26.3% and 30.7% (both p50.01) when cell was pretreated with curcumin at 5 Mm (Figure 5). Only pretreatment of curcumin at dose of 2.5 and 5 mg/mL did not induce an increase in the frequency of MN in that of the negative control.
Discussion QCT has been widely used as growth-promoting feed additives in food-producing animals in China (Ihsan et al., 2013; Li et al., 2014). QCT-related toxicity to animal and human has been widely concerned in China. Recent data reported that QCT could induce genotoxicity in vitro studies using various type cell lines including Vero (Chen et al., 2009), HepG2 (Zhang et al., 2013, 2014) and rat and porcine primary hepatocyte cells (Wang et al., 2015). Also, long-term with high-dose exposure of QCT can induce marked genotoxicity in rats and mice (Ihsan et al., 2013;
Figure 5. Effects of curcumin on QCT-induced micronuclei in L02 cells. Three independent experiments were carried out and 1000 binucleated cells were scored per treatment of each experiment. Values were presented as mean ± SD. *p50.05, **p50.01, compared to the vehicle control group; #p50.05, ##p50.01, compared to the QCT alone group.
Wang et al., 2011). Recently, Yang et al. (2013) demonstrated that QCT caused marked cytotoxicity and genotoxicity in human peripheral lymphocytes. In the present study, human normal hepatocyte L02 cell was used to investigate the QCTinduced cytotoxicity and genotoxicity and the protective effect of curcumin, a natural antioxidant widely used in medicine and animal production field.
Downloaded by [UNSW Library] at 19:57 24 August 2015
6
C. Dai et al.
Our results showed that QCT treatment significantly decreased L02 cell viability in a dose-dependent manner (Figure 2), which is consistent with the previous studies (Zhang et al., 2012). Notably, pretreatment of curcumin can significantly increase the tolerance of cell to QCT exposure (Figure 2). Curcumin at high doses imposed oxidative stress and damaged DNA, but low levels of curcumin does not induce DNA damage in vitro study and may play an antioxidant role in carcinogenesis (Cao et al., 2007, 2008). In the present study, we found that there are no any marked ROS formation and DNA injury in the pretreatment of curcumin at 2.5 and 5 mM control groups (Figures 2–5). It indicated that low levels of curcumin can attenuate QCTinduced cytotoxicity. Our previous studies have illustrated that ROS is a key mediator in cell death induced by some members of QdNOs (Zhang et al., 2013). Indeed, chemicals that induced excess ROS production could cause various types of toxicity, including genotoxicity and cell necrosis or apoptotic death (a program cell death). In the previous study, we have illustrated that QdNOs family members inducing OLA and QCT can induce cell apoptosis (Zhang et al., 2013; Zou et al., 2011). ROS-mediated oxidative stress has been proven to be involved in mutation, chromosome aberration, tumor promotion and cancer development. These adverse effects can be suppressed by antioxidants via ROS scavenging (Cao et al., 2008; Dopp et al., 2005). For curcumin, a polyphenol derived from the herbal remedy and dietary spice, turmeric, is well known for its antioxidant properties (Menon & Sudheer, 2007; Zhu et al., 2014). Curcumin can suppress the generation of ROS to attenuate carcinogen-induced genotoxicity, such as Benzo(a)pyrene and acrylamide (Cao et al., 2008; Zhu et al., 2014). It is all evidenced to be associated with the main metabolic pathway of QdNOs metabolism including QCT, i.e. N-oxide reduction (Wang et al., 2015). NADPH coenzyme was found to be involved in and to play important roles in the progress of Noxide reduction (Wang et al., 2015). GSH is one of the most important antioxidants in cell, and its depletion is closely related to NADPH (Cao et al., 2008; Wang et al., 2015). SOD catalyses the dismutation of superoxide anion into oxygen and hydrogen peroxide (Dai et al., 2014). Correspondingly, superoxide anion and hydrogen peroxide were major sources of QdNOs-induced ROS (Wang et al., 2015). This present study found that QCT treatment markedly decreases SOD activity (Figure 3B) and GSH level (Figure 3C) with the increase of intracellular ROS (Figure 3A). These changes can be attenuated by pretreatment with curcumin. As reported, curcumin could interact directly with the superoxide anion and hydroxyl peroxide as an oxygen radical scavenger (Biswas et al., 2005). Thus, it is very reasonable to assume that the chemopreventive effects of curcumin on QCTinduced cytotoxicity and genotoxicity could be mainly attributed to the ability to scavenge QCT-induced free radicals. Comet assay and micronucleus test are two important experimental methods in chemical compounds-induced genotoxicity. Our previous study has showed that QCT can cause DNA double-strand breaks and micronucleus formation by using comet assay and micronucleus test in HepG2 and Vero cell (Chen et al., 2009; Jin et al., 2009). They were also used
Toxicol Mech Methods, Early Online: 1–7
in the present study and the results showed the same findings in L02 cell (Figures 4 and 5). Wang et al. (2011) reported that ROS participated in the DNA damaging action of QCT and antioxidants, such as Pu-erh black tea decreased the potential of QCT to damage DNA in rat kidneys. Previous study proved that curcumin can act as an antigenotoxic agent at low doses by decreasing exogenous carcinogens including acrylamideand cyclophosphamide-induced DNA frequency of MN (Cao et al., 2007, 2008). Consequently, in the present study, we found that curcumin can protect against QCT-induced double-strand breaks (Figure 4) and increases of MN (Figure 5). That revealed that curcumin can indeed inhibit QCT caused genotoxicity. In conclusion, our results revealed that QCT can cause apparent cytotoxicity and genotoxicity in normal human hepatocyte L02 cell and natural antioxidant curcumin can significantly attenuate QCT-induced cytotoxicity and genotoxicity. This founding provides important information for preventing QCT and other QdNOs family members-induced cytotoxicity and genotoxicity.
Declaration of interest The authors declare that there are no conflicts of interest. This study was supported by Chinese Universities Scientific Fund (Award number 2015DY003) and National Natural Science Foundation of China (Award number 31372486).
References Biswas SK, McClure D, Jimenez LA, et al. (2005). Curcumin induces glutathione biosynthesis and inhibits NF-kappaB activation and interleukin-8 release in alveolar epithelial cells: mechanism of free radical scavenging activity. Antioxid Redox Signal 7:32–41. Brouet I, Ohshima H. (1995). Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochem Biophys Res Commun 206:533–40. Cao J, Jiang LP, Liu Y, et al. (2007). Curcumin-induced genotoxicity and antigenotoxicity in HepG2 cells. Toxicon 49:1219–22. Cao J, Liu Y, Jia L, et al. (2008). Curcumin attenuates acrylamideinduced cytotoxicity and genotoxicity in HepG2 cells by ROS scavenging. J Agric Food Chem 56:12059–63. Carta A, Paglietti G, Rahbar Nikookar ME, et al. (2002). Novel substituted quinoxaline 1,4-dioxides with in vitro antimycobacterial and anticandida activity. Eur J Med Chem 37:355–66. Celik A, Eke D, Ekinci SY, Yildirim S. (2013). The protective role of curcumin on perfluorooctane sulfonate-induced genotoxicity: single cell gel electrophoresis and micronucleus test. Food Chem Toxicol 53: 249–55. Chen Q, Tang S, Jin X, et al. (2009). Investigation of the genotoxicity of quinocetone, carbadox and olaquindox in vitro using Vero cells. Food Chem Toxicol 47:328–34. Dai C, Li J, Tang S, et al. (2014). Colistin-induced nephrotoxicity in mice involves the mitochondrial, death receptor, and endoplasmic reticulum pathways. Antimicrob Agents Chemother 58:4075–85. Diab-Assef M, Haddadin MJ, Yared P, et al. (2002). Quinoxaline 1,4dioxides: hypoxia-selective therapeutic agents. Mol Carcinog 33: 198–205. Dopp E, Yadav S, Ansari FA, et al. (2005). ROS-mediated genotoxicity of asbestos-cement in mammalian lung cells in vitro. Part Fibre Toxicol 2:9. Gali-Muhtasib H, Sidani M, Geara F, et al. (2004). Quinoxaline 1,4dioxides are novel angiogenesis inhibitors that potentiate antitumor effects of ionizing radiation. Int J Oncol 24:1121–31. Gali-Muhtasib HU, Diab-Assaf M, Haddadin MJ. (2005). Quinoxaline 1,4-dioxides induce G2/M cell cycle arrest and apoptosis in human colon cancer cells. Cancer Chemother Pharmacol 55:369–78.
Downloaded by [UNSW Library] at 19:57 24 August 2015
DOI: 10.3109/15376516.2015.1045659
Gali-Muhtasib HU, Haddadin MJ, Rahhal DN, Younes IH. (2001). Quinoxaline 1,4-dioxides as anticancer and hypoxia-selective drugs. Oncol Rep 8:679–84. Gao S, Duan X, Wang X, et al. (2013). Curcumin attenuates arsenic-induced hepatic injuries and oxidative stress in experimental mice through activation of Nrf2 pathway, promotion of arsenic methylation and urinary excretion. Food Chem Toxicol 59: 739–47. Guo W, Hao H, Dai M, et al. (2012). Development of quinoxaline 1,4dioxides resistance in Escherichia coli and molecular change under resistance selection. PLoS One 7:e43322. He Q, Fang B, Su Y, et al. (2013). Simultaneous determination of quinoxaline-1,4-dioxides in feeds using molecularly imprinted solidphase extraction coupled with HPLC. J Sep Sci 36:301–10. Ihsan A, Wang X, Zhang W, et al. (2013). Genotoxicity of quinocetone, cyadox and olaquindox in vitro and in vivo. Food Chem Toxicol 59: 207–14. Jin X, Chen Q, Tang SS, et al. (2009). Investigation of quinocetoneinduced genotoxicity in HepG2 cells using the comet assay, cytokinesis-block micronucleus test and RAPD analysis. Toxicol In Vitro 23: 1209–14. Li J, Huang L, Wang X, et al. (2014). Metabolic disposition and excretion of quinocetone in rats, pigs, broilers, and carp. Food Chem Toxicol 69:109–19. Menon VP, Sudheer AR. (2007). Antioxidant and anti-inflammatory properties of curcumin. Adv Exp Med Biol 595:105–25. Mu P, Zheng M, Xu M, et al. (2014). N-oxide reduction of quinoxaline1,4-dioxides catalyzed by porcine aldehyde oxidase SsAOX1. Drug Metab Dispos 42:511–9. Oh J, Hristov AN, Lee C, et al. (2013). Immune and production responses of dairy cows to postruminal supplementation with phytonutrients. J Dairy Sci 96:7830–43. Rajput N, Naeem M, Ali S, et al. (2013). The effect of dietary supplementation with the natural carotenoids curcumin
Curcumin attenuates QCT-induced oxidative stress
7
and lutein on broiler pigmentation and immunity. Poult Sci 92: 1177–85. Takahashi M, Suzuki K, Kim HK, et al. (2014). Effects of curcumin supplementation on exercise-induced oxidative stress in humans. Int J Sports Med 35:469–75. Wang D, Zhong Y, Luo X, et al. (2011). Pu-erh black tea supplementation decreases quinocetone-induced ROS generation and oxidative DNA damage in Balb/c mice. Food Chem Toxicol 49:477–84. Wang X, Zhang H, Huang L, et al. (2015). Deoxidation rates play critical role in DNA damage of important synthetic drugs, quinoxaline 1,4-dioxides. Chem Res Toxicol 28:470–81. Wang X, Zhang W, Wang Y, et al. (2010). Acute and sub-chronic oral toxicological evaluations of quinocetone in Wistar rats. Regul Toxicol Pharmacol 58:421–7. Waseem M, Parvez S. (2013). Mitochondrial dysfunction mediated cisplatin induced toxicity: modulatory role of curcumin. Food Chem Toxicol 53:334–42. Yang W, Fu J, Xiao X, et al. (2013). Quinocetone triggers oxidative stress and induces cytotoxicity and genotoxicity in human peripheral lymphocytes of both genders. J Sci Food Agric 93:1317–25. Zhang C, Wang C, Tang S, et al. (2013). TNFR1/TNF-alpha and mitochondria interrelated signaling pathway mediates quinocetoneinduced apoptosis in HepG2 cells. Food Chem Toxicol 62:825–38. Zhang K, Ban M, Zhao Z, et al. (2012). Cytotoxicity and genotoxicity of 1,4-bisdesoxyquinocetone, 3-methylquinoxaline-2-carboxylic acid (MQCA) in human hepatocytes. Res Vet Sci 93:1393–401. Zhang K, Zheng W, Zheng H, et al. (2014). Identification of oxidative stress and responsive genes of HepG2 cells exposed to quinocetone, and compared with its metabolites. Cell Biol Toxicol 30:313–29. Zhu W, Cromie MM, Cai Q, et al. (2014). Curcumin and vitamin E protect against adverse effects of benzo[a]pyrene in lung epithelial cells. PLoS One 9:e92992. Zou J, Chen Q, Jin X, et al. (2011). Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells. Toxicology 285: 104–13.
Toxicology Mechanisms and Methods Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/itxm20
Curcumin attenuates quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells a
a
a
a
Chongshan Dai , Shusheng Tang , Daowen Li , Kena Zhao & Xilong Xiao
a
a
College of Veterinary Medicine, China Agricultural University, Beijing, PR China Published online: 26 May 2015.
Click for updates To cite this article: Chongshan Dai, Shusheng Tang, Daowen Li, Kena Zhao & Xilong Xiao (2015): Curcumin attenuates quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells, Toxicology Mechanisms and Methods To link to this article: http://dx.doi.org/10.3109/15376516.2015.1045659
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
http://informahealthcare.com/txm ISSN: 1537-6516 (print), 1537-6524 (electronic) Toxicol Mech Methods, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2015.1045659
RESEARCH ARTICLE
Curcumin attenuates quinocetone-induced oxidative stress and genotoxicity in human hepatocyte L02 cells Chongshan Dai, Shusheng Tang, Daowen Li, Kena Zhao, and Xilong Xiao
Downloaded by [UNSW Library] at 19:57 24 August 2015
College of Veterinary Medicine, China Agricultural University, Beijing, PR China
Abstract
Keywords
Quinocetone (QCT), a new quinoxaline 1,4-dioxides, has been used as antimicrobial feed additive in China. Potential genotoxicity of QCT was concerned as a public health problem. This study aimed to investigate the protective effect of curcumin on QCT-induced oxidative stress and genotoxicity in human hepatocyte L02 cells. Cell viability and intracellular reactive oxygen species (ROS), biomarkers of oxidative stress including superoxide dismutase (SOD) activity and glutathione (GSH) level were measured. Meanwhile, comet assay and micronucleus assay were carried out to evaluate genotoxicity. The results showed that, compared to the control group, QCT at the concentration ranges of 2–16 mg/mL significantly decreased L02 cell viability, which was significantly attenuated with curcumin pretreatment (2.5 and 5 mM). In addition, QCT significantly increased cell oxidative stress, characterized by increases of intracellular ROS level, while decreased endogenous antioxidant biomarkers GSH level and SOD activity (all p50.05 or 0.01). Curcumin pretreatment significantly attenuated ROS formation, inhibited the decreases of SOD activity and GSH level. Furthermore, curcumin significantly reduced QCT-induced DNA fragments and micronuclei formation. These data suggest that curcumin could attenuate QCT-induced cytotoxicity and genotoxicity in L02 cells, which may be attributed to ROS scavenging and anti-oxidative ability of curcumin. Importantly, consumption of curcumin may be a plausible way to prevent quinoxaline 1,4-dioxides-mediated oxidative stress and genotoxicity in human or animals.
Curcumin, genotoxicity, oxidative stress, quinocetone, ROS
Introduction Quinoxaline 1,4-dioxides (QdNOs), a class of quinoxaline derivatives consisting of one or two acyclic chains moiety combined with quinoxaline ring (Figure 1A), have been considered as important biologically active compounds and effectively used as antibacterial, anticancer and antiprotozoal drugs or growth promoters (Carta et al., 2002; Diab-Assef et al., 2002; Gali-Muhtasib et al., 2001, 2004, 2005; Guo et al., 2012; He et al., 2013; Mu et al., 2014; Wang et al., 2015). Olaquindox (OLA) and carbadox (CBX) are wellknown members of QdNOs with promoting animal growth, however, they have been forbidden to use as animal growth promoters in 1999 by Commission of the European Community because of their genotoxicity and no threshold for the safe use (Ihsan et al., 2013; Wang et al., 2015). Quinocetone (QCT; Figure 1B) is one of the QdNOs family and it has been widely used to promote animal growth in food producing animals including chicken, pig, fish and goats, as a replacement for OLA and CBX in China (Li et al., 2014). Address for correspondence: Xilong Xiao, College of Veterinary Medicine, China Agricultural University, 2 Yuanmingyuan West Road, Beijing 100193, PR China. Tel: +86 10 6273 3857. Fax: +86 10 6273 1032. E-mail: [email protected]
History Received 7 February 2015 Revised 12 March 2015 Accepted 10 April 2015 Published online 21 May 2015
In addition, it is also used as an antimicrobial agent against Salmonella, Escherichia coli, Brachyspira hyodysenteriae and other gram-negative bacterial infections (Ihsan et al., 2013; Wang et al., 2011; Yang et al., 2013). However, recently available data illustrated that QCT could induce genotoxic and cytoxic effects in several in vitro; high doses of QCT (at least 4–10-folds) can induce pathological (such as liver and renal tissues)/behavioral alterations in rats or mice (Chen et al., 2009; Ihsan et al., 2013; Jin et al., 2009; Wang et al., 2010; Zhang et al., 2013). Food safety of QCT concerned potential toxicity to human should be evaluated because quinocetone residues and metabolites in animal tissues may endanger consumer health by bioaccumulation. Therefore, the risk assessment of QCT is very important and necessary. In our previous study, it was shown that QCT can induce marked cytoxicity and genotoxicity in African green monkey cell lines (Vero cells) and human hepatoma cells (HepG2) (Chen et al., 2009; Jin et al., 2009). Our previous study showed that reactive oxygen species (ROS) and caspasedependent apoptosis participated into QCT-mediated cell toxicity in HepG2 cell (Zhang et al., 2013). Yang et al. (2013) demonstrated that QCT can trigger oxidative stress and induce cytotoxicity and genotoxicity in human peripheral lymphocytes. Clearly, excessive ROS generation-mediated oxidative
2
C. Dai et al.
Toxicol Mech Methods, Early Online: 1–7
Downloaded by [UNSW Library] at 19:57 24 August 2015
Figure 1. Structure of quinoxalines (A), quinocetone (B) and curcumin (C).
stress plays a key role in QCT-induced DNA injury and genotoxicity, albeit the precise mechanism is still far from clear. Curcumin (diferuloylmethane) (Figure 1C), a natural polyphenol in the spice turmeric, exhibits antioxidant (Gao et al., 2013), anti-inflammatory (Menon & Sudheer, 2007) and anti-carcinogenic properties (Brouet & Ohshima, 1995). It has been reported that curcumin can protect against DNA damage induced by drug or environmental mutagens including arsenic (Gao et al., 2013), acryl amide (Cao et al., 2008), benz(a)pyren (Zhu et al., 2014) and cisplatin (Waseem & Parvez, 2013) based on its suppression of ROS generation in vitro and in vivo studies. In animal production, dietary supplementations with curcumin can improve immunity and the ability to adapt to environmental stress without any adverse effect (Oh et al., 2013; Rajput et al., 2013). In human clinical trials, dietary supplementation of curcumin can attenuate exercise-induced oxidative stress by increasing blood antioxidant capacity (Takahashi et al., 2014). Additionally, curcumin at low doses could significantly reduce the chemotherapeutic agent-induced micronucleus formation (Celik et al., 2013). Therefore, in the present study, we investigated the protective effect of curcumin on QCT-induced genotoxicity and cytotoxicity in human hepatocyte L02 cells.
Materials and methods Chemical and regents QCT (C18Hl4N2O3, CAS NO. 81810-66-4; purity 98%) was purchased from Zhongmu Pharmaceutical Co. Ltd. (Wuxue, PR China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Gibco, Grand Island, NY). 3-(4,5-Dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT), cytochalasin B, triton X-100, agarose and propidium iodide (PI) were purchased from Sigma–Aldrich (St. Louis, MO). Curcumin (purity 98%) was purchased from Aladdin Reagent Co., Ltd (Shanghai, China). Curcumin and QCT were respectively prepared as a 20 mM and 16 mg/mL stock solutions in dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO), and
stored at 20 C in the dark. For each experiment, curcumin and QCT were diluted with cell culture medium to the concentration indicated with a final DMSO concentration of 0.1% (vol/vol) in the dark. Other reagents were of analytical reagent grade. Cell culture L02 cell, a normal human hepatocyte cell line from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), was employed because liver is the major organ to metabolize QCT. Cells were cultured in DMEM containing with 2% L -glutamine, 10% FBS, 100 U/mL (100 units/mL, Gibco) and streptomycin (100 mg/mL, Gibco) at 37 C in a humidified incubator (5% CO2). The medium was renewed every 2 days until the cells were grown to confluence. Cell viability assay MTT assay was used to measure the cytotoxicity of QCT as described previously with modifications (Zhang et al., 2013). L02 cells were plated in a 96-well micro-titer plate at a density of 1 104 cells per well in a final volume of 100 mL DMEM. After pretreatment with curcumin (the final concentration was 2.5 and 5 mM, respectively) for 2 h, the medium was replaced with fresh medium containing QCT (the final concentration was from 2 to 16 mg/mL). The cells in the control group were pre-treated with 0.1% DMSO, 2.5 or 5 mM curcumin for 2 h, then replaced with fresh medium containing 0.1% DMSO. After incubation for 4 or 24 h, the medium was discarded, 100 mL serum-free DMEM containing 10 mL MTT (5 mg/mL) was added and followed by incubation for 4 h at 37 C. Finally, the medium was discarded and added 100 mL DMSO. After incubation for 20 min at room temperature, the absorbance was read at 490 nm in a micro-plate reader (Molecular Devices, Sunnyvale, CA). Cells incubated with 0.1% DMSO were used as the control. Measurement of intracellular ROS production, superoxide dismutase activity and glutathione level The production of intracellular ROS was measured using the ROS-specific fluorescent dye 2,7-dichlorofluorescein
Curcumin attenuates QCT-induced oxidative stress
Downloaded by [UNSW Library] at 19:57 24 August 2015
DOI: 10.3109/15376516.2015.1045659
diacetate (DCFH-DA) (Beyotime, Haimen, China). L02 cells were seeded at an initial density of 1 104 cells/mL in 96well plates. The cells were pretreated with curcumin (2.5 and 5 mM, respectively) for 2 h at 37 C, then the medium was discarded and replaced with 200 mL DMEM with QCT at different final concentrations (8 and 16 mg/mL, respectively) for incubation. After 4 h exposure to QCT, the cells were washed with PBS, then 200 mL DMEM was added and loaded with 10 mM DCFH-DA at 37 C for 30 min in the darkness, then rinsed three times with PBS, and measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm using a microplate fluorescence reader (MD, Spectramax M3). To further examine the role of oxidative stress on QCTinduced cytoxicity, genotoxicity and the protection of curcumin, intracellular total superoxide dismutase (SOD) activity and glutathione (GSH) level as the biomarkers of oxidative stress were determined using commercial kits (Nanjing Jiancheng Institute of Biological Engineering). Briefly, L02 cells were seeded at a density of 1 105 into 12-well plate and incubated for 24 h at 37 C. Then cells were washed with PBS and pretreated with curcumin at 2.5 and 5 mM for 2 h, then washed twice with PBS, and incubated without (0.1% DMSO) or with QCT (8 and 16 mg/mL) in fresh DMEM for 4 h. After the desired time of treatment, cells were harvested, washed with PBS and homogenized in 10 mM phosphate buffer containing 0.15 M KCl, 0.1 mM EDTA, 1 mM DTT and 0.1 mM phenylmethylsulfonylfluoride (pH 7.4) at 4 C, then centrifuged at 3000 g (4 C) for 15 min. The supernatant was collected for examination. Protein concentrations in the supernatant were measured using the BCA protein assay (Wuhan Boster Bio-engineering Limited Co., Wuhan, China). Comet assay The comet assay was carried out under alkaline conditions as our previous study (Jin et al., 2009). L02 cells were seeded in a 24-well plate at a density of 1 104 cells/well. After 24 h of growth, the cells were pretreated with curcumin (final concentration was 2.5 and 5 mM, respectively) for 2 h, then discarded and replaced with 500 mL DMEM with QCT at 8 and 16 mg/mL, respectively for 4 h incubation. The corresponding control groups were exposed with 0.1% DMSO, QCT or curcumin for equal time. Then, cells were washed twice with PBS and trypsinized for 2 min then suspended in 80 mL PBS. To avoid artifacts resulting from cell death, the cell suspensions (40 mL) were mixed with trypan blue solution (40 mL, 0.5% in PBS) to examine the cell viability. Only the viabilities of cell suspensions were more than 80%, they were used for examining DNA damage. Then, 20 mL of the cell suspensions was mixed with 60 mL of 0.8% LMA and placed on frosted slides pre-layered with 0.8% NMA. Then, the cover slip was removed and the slides were immersed in lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10 and 10% DMSO with 1% Triton X-100) in darkness at 4 C for 1 h. After lysis, the slides were placed in alkaline solution (1 mM Na2-EDTA and 300 mM NaOH, pH 13) for 20 min at room temperature to allow DNA unwinding. Electrophoresis were performed in alkaline solution for 30 min at 25 V. Cells were
3
neutralized using 0.4 M Tris-HCl (pH 7.5) and stained with 50 mL of ethidium bromide (10 mg/mL). Slides were viewed at 40 magnification using fluorescent microscopy (Leica, Omachi, Japan). At least 100 randomly selected cells (30 or 40 cells from each of the three replicated slides) were quantitatively analyzed using Comet Assay Software Project casp-1.2.2 (University of Wroclaw, Poland). Micronucleus assay L02 cells growing in the logarithmic phase were seeded at a density of 1 105 into 12-well plate and incubated for 24 h at 37 C. After this period, the cells were washed with PBS and pretreated with curcumin at 2.5 and 5 mM for 2 h, then washed twice with PBS and incubated with QCT (1 and 2 mg/mL) in fresh DMEM for 24 h, then cells were washed twice with PBS and incubated with DMEM containing cytochalasin B (the final concentration was 4.5 mg/mL) for 20 h. Fixation and slide preparation were carried out according to the conventional techniques. Statistical analysis Each experiment was performed at least three times in triplicate and all data are presented as mean ± SD unless specified. Statistical analysis was conducted using SPSS V16.0 (SPSS Inc., Chicago, IL) and figures were prepared using Graphpad Prism 5.0 (Graph Pad Software, Inc., La Jolla, CA). Data from the control and treatment groups were analyzed with one-way analysis of variance (ANOVA), followed by LSD post-hoc test. A p value 50.05 was considered significant.
Results Effects of curcumin on QCT-induced cytotoxicity in L02 cells The cytotoxicity of QCT exposed to L02 cells for 4 and 24 h was examined. At 4 h, cell viabilities had no significant changes in the QCT 1, 2 and 4 mg/mL groups and a slight cytotoxicity was shown in the QCT 8 and 16 mg/mL groups, the cell viabilities decreased to 88% (p50.05) and 81% (p50.01), respectively. Furthermore, L02 cells were treated with QCT for 24 h, the cytotoxicity was significantly decreased in a dose-dependent manner and the IC50 (inhibitory concentration 50%) value was 7.04 ± 1.21 mg/mL, which was significantly improved by pre-treatment with curcumin at the dose of 2.5 and 5 mM, the IC50s were significantly increased to 12.16 ± .06, 15.42 ± 1.64 mg/mL (both p50.01) (Figure 2). Compared to the control group, pretreatment of curcumin at 2.5 and 5 mM for 2 h did not change the cell viability did not significant change in the curcumin control group (Figure 2). Effects of curcumin on QCT-induced ROS formation and the biomarkers of oxidative stress in L02 cells To explore the protective effects of curcumin, intracellular ROS formation and biomarkers of oxidative stress including GSH and SOD were examined, as shown in Figure 3. L02 cells were treated with QCT at high doses of 8 and 16 mg/mL for a 4 h-term observation. Compared to those untreated
4
C. Dai et al.
Downloaded by [UNSW Library] at 19:57 24 August 2015
control, there were no marked changes of intracellular ROS formation, SOD activity and GSH level after incubation with 2.5 and 5 mM curcumin. Intracellular ROS formation significantly increased in the cells treated with the 8 and 16 mg/mL QCT groups (both p50.01), meanwhile, SOD activity significantly decreased to 84.1% and 76.0% (both p50.01), GSH level significantly decreased to 76.3% and 62.1% (both
Figure 2. Effects of curcumin on quinocetone (QCT)-induced cytotoxicity in L02 cells determined by MTT. Values were presented as mean ± SD, from three independent experiments (n ¼ 3). *p50.05, **p50.01, compared to the vehicle control group; #p50.05, ##p50.01, compared to the QCT alone group.
Toxicol Mech Methods, Early Online: 1–7
p50.01), compared to the control. Pretreatment of curcumin at 2.5 and 5 mM significantly decreased QCT caused by the formation of intracellular ROS (Figure 3A), increased SOD activity (Figure 3B) and GSH level (Figure 3C) (all p50.05 or 0.01), compared to that in the QCT alone group. Effects of curcumin on QCT-induced DNA strand breaks in L02 cells QCT can significantly induce DNA strand breaks in L02 cells, as shown in Figure 4. Before beginning to carry out comet assay test, cell viability was examined using trypan blue stain. In all the groups, cell viabilities were more than 80%. Subsequently, cells were used to evaluate DNA damage. Compared to the control, at the QCT 8 and 16 mg/mL, the percentage (%) tail DNA increased to 21.7% and 32.3% (both p50.01), significant decreases of % tail DNA were detected when L02 cell were pretreated with curcumin at 2.5 mM (decreased to 15.7% and 22.0%) (both p50.05) and 5 mM (decreased to 12.5% and 18.4%) (both p50.01), respectively (Figure 4A); the tail length increased to 42.4 and 54.0 mm, which were significantly decreased by curcumin pretreatment (decreased to 31.0 and 37.7 mm at curcumin 2.5 mM pretreatment group; decreased to 25.4 and 31.5 mm at curcumin 5 mM pretreatment group, respectively) (all p50.05 or 0.01) (Figure 4B); the comet tail moment values increased to 9.2 mm (&35-folds) and 17.6 mm (&67-folds) (both p50.01), which were decreased in the pretreatment of curcumin at
Figure 3. Effects of curcumin on quinocetone (QCT)-induced ROS production and oxidative stress in L02 cells. (A) represents ROS production, ROSspecific fluorescent dye 2,7-dichlorofluorescein diacetate was used to measure the production of intracellar ROS. (B) and (C) represent SOD activity and GSH level, respectively. Values were presented as mean ± SD, from three independent experiments (n ¼ 3). *p50.05, **p50.01, compared to the vehicle control group; #p50.05, ##p50.01, compared to the QCT alone group.
Downloaded by [UNSW Library] at 19:57 24 August 2015
DOI: 10.3109/15376516.2015.1045659
Curcumin attenuates QCT-induced oxidative stress
5
Figure 4. Effects of curcumin on QCT-induced DNA strand-breaks in L02 cells. (A) % tail DNA, (B) tail length, (C) tail moment. DNA damage was measured in the comet assay in L02 cells treated with QCT (0, 8 and 16 mg/mL, respectively) or QCT plus curcumin (2.5 and 5 mM, respectively) for 4 h. Data were presented as mean ± SD, from three independent experiments (n ¼ 3). *p50.05, **p50.01, compared to the vehicle control group; #p50.05, ##p50.01, compared to the QCT alone group.
2.5 mM (decreased to 4.6 and 8.1 mm, respectively) and 5 mM (decreased to 3.1 and 5.9 mm, respectively) (all p50.01). Effects of curcumin on the frequencies of QCT-induced MN Compare to that non-treated cells, QCT treatment at the doses of 1 and 2 mg/mL for 24 h, the number of MN significantly increased to 36.7% and 49.0% (both p50.01), which were decreased to 30.4% and 38.2% (both p50.05) when cell was pretreated with curcumin at 2.5 mM, further decreased to 26.3% and 30.7% (both p50.01) when cell was pretreated with curcumin at 5 Mm (Figure 5). Only pretreatment of curcumin at dose of 2.5 and 5 mg/mL did not induce an increase in the frequency of MN in that of the negative control.
Discussion QCT has been widely used as growth-promoting feed additives in food-producing animals in China (Ihsan et al., 2013; Li et al., 2014). QCT-related toxicity to animal and human has been widely concerned in China. Recent data reported that QCT could induce genotoxicity in vitro studies using various type cell lines including Vero (Chen et al., 2009), HepG2 (Zhang et al., 2013, 2014) and rat and porcine primary hepatocyte cells (Wang et al., 2015). Also, long-term with high-dose exposure of QCT can induce marked genotoxicity in rats and mice (Ihsan et al., 2013;
Figure 5. Effects of curcumin on QCT-induced micronuclei in L02 cells. Three independent experiments were carried out and 1000 binucleated cells were scored per treatment of each experiment. Values were presented as mean ± SD. *p50.05, **p50.01, compared to the vehicle control group; #p50.05, ##p50.01, compared to the QCT alone group.
Wang et al., 2011). Recently, Yang et al. (2013) demonstrated that QCT caused marked cytotoxicity and genotoxicity in human peripheral lymphocytes. In the present study, human normal hepatocyte L02 cell was used to investigate the QCTinduced cytotoxicity and genotoxicity and the protective effect of curcumin, a natural antioxidant widely used in medicine and animal production field.
Downloaded by [UNSW Library] at 19:57 24 August 2015
6
C. Dai et al.
Our results showed that QCT treatment significantly decreased L02 cell viability in a dose-dependent manner (Figure 2), which is consistent with the previous studies (Zhang et al., 2012). Notably, pretreatment of curcumin can significantly increase the tolerance of cell to QCT exposure (Figure 2). Curcumin at high doses imposed oxidative stress and damaged DNA, but low levels of curcumin does not induce DNA damage in vitro study and may play an antioxidant role in carcinogenesis (Cao et al., 2007, 2008). In the present study, we found that there are no any marked ROS formation and DNA injury in the pretreatment of curcumin at 2.5 and 5 mM control groups (Figures 2–5). It indicated that low levels of curcumin can attenuate QCTinduced cytotoxicity. Our previous studies have illustrated that ROS is a key mediator in cell death induced by some members of QdNOs (Zhang et al., 2013). Indeed, chemicals that induced excess ROS production could cause various types of toxicity, including genotoxicity and cell necrosis or apoptotic death (a program cell death). In the previous study, we have illustrated that QdNOs family members inducing OLA and QCT can induce cell apoptosis (Zhang et al., 2013; Zou et al., 2011). ROS-mediated oxidative stress has been proven to be involved in mutation, chromosome aberration, tumor promotion and cancer development. These adverse effects can be suppressed by antioxidants via ROS scavenging (Cao et al., 2008; Dopp et al., 2005). For curcumin, a polyphenol derived from the herbal remedy and dietary spice, turmeric, is well known for its antioxidant properties (Menon & Sudheer, 2007; Zhu et al., 2014). Curcumin can suppress the generation of ROS to attenuate carcinogen-induced genotoxicity, such as Benzo(a)pyrene and acrylamide (Cao et al., 2008; Zhu et al., 2014). It is all evidenced to be associated with the main metabolic pathway of QdNOs metabolism including QCT, i.e. N-oxide reduction (Wang et al., 2015). NADPH coenzyme was found to be involved in and to play important roles in the progress of Noxide reduction (Wang et al., 2015). GSH is one of the most important antioxidants in cell, and its depletion is closely related to NADPH (Cao et al., 2008; Wang et al., 2015). SOD catalyses the dismutation of superoxide anion into oxygen and hydrogen peroxide (Dai et al., 2014). Correspondingly, superoxide anion and hydrogen peroxide were major sources of QdNOs-induced ROS (Wang et al., 2015). This present study found that QCT treatment markedly decreases SOD activity (Figure 3B) and GSH level (Figure 3C) with the increase of intracellular ROS (Figure 3A). These changes can be attenuated by pretreatment with curcumin. As reported, curcumin could interact directly with the superoxide anion and hydroxyl peroxide as an oxygen radical scavenger (Biswas et al., 2005). Thus, it is very reasonable to assume that the chemopreventive effects of curcumin on QCTinduced cytotoxicity and genotoxicity could be mainly attributed to the ability to scavenge QCT-induced free radicals. Comet assay and micronucleus test are two important experimental methods in chemical compounds-induced genotoxicity. Our previous study has showed that QCT can cause DNA double-strand breaks and micronucleus formation by using comet assay and micronucleus test in HepG2 and Vero cell (Chen et al., 2009; Jin et al., 2009). They were also used
Toxicol Mech Methods, Early Online: 1–7
in the present study and the results showed the same findings in L02 cell (Figures 4 and 5). Wang et al. (2011) reported that ROS participated in the DNA damaging action of QCT and antioxidants, such as Pu-erh black tea decreased the potential of QCT to damage DNA in rat kidneys. Previous study proved that curcumin can act as an antigenotoxic agent at low doses by decreasing exogenous carcinogens including acrylamideand cyclophosphamide-induced DNA frequency of MN (Cao et al., 2007, 2008). Consequently, in the present study, we found that curcumin can protect against QCT-induced double-strand breaks (Figure 4) and increases of MN (Figure 5). That revealed that curcumin can indeed inhibit QCT caused genotoxicity. In conclusion, our results revealed that QCT can cause apparent cytotoxicity and genotoxicity in normal human hepatocyte L02 cell and natural antioxidant curcumin can significantly attenuate QCT-induced cytotoxicity and genotoxicity. This founding provides important information for preventing QCT and other QdNOs family members-induced cytotoxicity and genotoxicity.
Declaration of interest The authors declare that there are no conflicts of interest. This study was supported by Chinese Universities Scientific Fund (Award number 2015DY003) and National Natural Science Foundation of China (Award number 31372486).
References Biswas SK, McClure D, Jimenez LA, et al. (2005). Curcumin induces glutathione biosynthesis and inhibits NF-kappaB activation and interleukin-8 release in alveolar epithelial cells: mechanism of free radical scavenging activity. Antioxid Redox Signal 7:32–41. Brouet I, Ohshima H. (1995). Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages. Biochem Biophys Res Commun 206:533–40. Cao J, Jiang LP, Liu Y, et al. (2007). Curcumin-induced genotoxicity and antigenotoxicity in HepG2 cells. Toxicon 49:1219–22. Cao J, Liu Y, Jia L, et al. (2008). Curcumin attenuates acrylamideinduced cytotoxicity and genotoxicity in HepG2 cells by ROS scavenging. J Agric Food Chem 56:12059–63. Carta A, Paglietti G, Rahbar Nikookar ME, et al. (2002). Novel substituted quinoxaline 1,4-dioxides with in vitro antimycobacterial and anticandida activity. Eur J Med Chem 37:355–66. Celik A, Eke D, Ekinci SY, Yildirim S. (2013). The protective role of curcumin on perfluorooctane sulfonate-induced genotoxicity: single cell gel electrophoresis and micronucleus test. Food Chem Toxicol 53: 249–55. Chen Q, Tang S, Jin X, et al. (2009). Investigation of the genotoxicity of quinocetone, carbadox and olaquindox in vitro using Vero cells. Food Chem Toxicol 47:328–34. Dai C, Li J, Tang S, et al. (2014). Colistin-induced nephrotoxicity in mice involves the mitochondrial, death receptor, and endoplasmic reticulum pathways. Antimicrob Agents Chemother 58:4075–85. Diab-Assef M, Haddadin MJ, Yared P, et al. (2002). Quinoxaline 1,4dioxides: hypoxia-selective therapeutic agents. Mol Carcinog 33: 198–205. Dopp E, Yadav S, Ansari FA, et al. (2005). ROS-mediated genotoxicity of asbestos-cement in mammalian lung cells in vitro. Part Fibre Toxicol 2:9. Gali-Muhtasib H, Sidani M, Geara F, et al. (2004). Quinoxaline 1,4dioxides are novel angiogenesis inhibitors that potentiate antitumor effects of ionizing radiation. Int J Oncol 24:1121–31. Gali-Muhtasib HU, Diab-Assaf M, Haddadin MJ. (2005). Quinoxaline 1,4-dioxides induce G2/M cell cycle arrest and apoptosis in human colon cancer cells. Cancer Chemother Pharmacol 55:369–78.
Downloaded by [UNSW Library] at 19:57 24 August 2015
DOI: 10.3109/15376516.2015.1045659
Gali-Muhtasib HU, Haddadin MJ, Rahhal DN, Younes IH. (2001). Quinoxaline 1,4-dioxides as anticancer and hypoxia-selective drugs. Oncol Rep 8:679–84. Gao S, Duan X, Wang X, et al. (2013). Curcumin attenuates arsenic-induced hepatic injuries and oxidative stress in experimental mice through activation of Nrf2 pathway, promotion of arsenic methylation and urinary excretion. Food Chem Toxicol 59: 739–47. Guo W, Hao H, Dai M, et al. (2012). Development of quinoxaline 1,4dioxides resistance in Escherichia coli and molecular change under resistance selection. PLoS One 7:e43322. He Q, Fang B, Su Y, et al. (2013). Simultaneous determination of quinoxaline-1,4-dioxides in feeds using molecularly imprinted solidphase extraction coupled with HPLC. J Sep Sci 36:301–10. Ihsan A, Wang X, Zhang W, et al. (2013). Genotoxicity of quinocetone, cyadox and olaquindox in vitro and in vivo. Food Chem Toxicol 59: 207–14. Jin X, Chen Q, Tang SS, et al. (2009). Investigation of quinocetoneinduced genotoxicity in HepG2 cells using the comet assay, cytokinesis-block micronucleus test and RAPD analysis. Toxicol In Vitro 23: 1209–14. Li J, Huang L, Wang X, et al. (2014). Metabolic disposition and excretion of quinocetone in rats, pigs, broilers, and carp. Food Chem Toxicol 69:109–19. Menon VP, Sudheer AR. (2007). Antioxidant and anti-inflammatory properties of curcumin. Adv Exp Med Biol 595:105–25. Mu P, Zheng M, Xu M, et al. (2014). N-oxide reduction of quinoxaline1,4-dioxides catalyzed by porcine aldehyde oxidase SsAOX1. Drug Metab Dispos 42:511–9. Oh J, Hristov AN, Lee C, et al. (2013). Immune and production responses of dairy cows to postruminal supplementation with phytonutrients. J Dairy Sci 96:7830–43. Rajput N, Naeem M, Ali S, et al. (2013). The effect of dietary supplementation with the natural carotenoids curcumin
Curcumin attenuates QCT-induced oxidative stress
7
and lutein on broiler pigmentation and immunity. Poult Sci 92: 1177–85. Takahashi M, Suzuki K, Kim HK, et al. (2014). Effects of curcumin supplementation on exercise-induced oxidative stress in humans. Int J Sports Med 35:469–75. Wang D, Zhong Y, Luo X, et al. (2011). Pu-erh black tea supplementation decreases quinocetone-induced ROS generation and oxidative DNA damage in Balb/c mice. Food Chem Toxicol 49:477–84. Wang X, Zhang H, Huang L, et al. (2015). Deoxidation rates play critical role in DNA damage of important synthetic drugs, quinoxaline 1,4-dioxides. Chem Res Toxicol 28:470–81. Wang X, Zhang W, Wang Y, et al. (2010). Acute and sub-chronic oral toxicological evaluations of quinocetone in Wistar rats. Regul Toxicol Pharmacol 58:421–7. Waseem M, Parvez S. (2013). Mitochondrial dysfunction mediated cisplatin induced toxicity: modulatory role of curcumin. Food Chem Toxicol 53:334–42. Yang W, Fu J, Xiao X, et al. (2013). Quinocetone triggers oxidative stress and induces cytotoxicity and genotoxicity in human peripheral lymphocytes of both genders. J Sci Food Agric 93:1317–25. Zhang C, Wang C, Tang S, et al. (2013). TNFR1/TNF-alpha and mitochondria interrelated signaling pathway mediates quinocetoneinduced apoptosis in HepG2 cells. Food Chem Toxicol 62:825–38. Zhang K, Ban M, Zhao Z, et al. (2012). Cytotoxicity and genotoxicity of 1,4-bisdesoxyquinocetone, 3-methylquinoxaline-2-carboxylic acid (MQCA) in human hepatocytes. Res Vet Sci 93:1393–401. Zhang K, Zheng W, Zheng H, et al. (2014). Identification of oxidative stress and responsive genes of HepG2 cells exposed to quinocetone, and compared with its metabolites. Cell Biol Toxicol 30:313–29. Zhu W, Cromie MM, Cai Q, et al. (2014). Curcumin and vitamin E protect against adverse effects of benzo[a]pyrene in lung epithelial cells. PLoS One 9:e92992. Zou J, Chen Q, Jin X, et al. (2011). Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells. Toxicology 285: 104–13.
