molecules Article
Curcumin Ameliorates Furazolidone-Induced DNA Damage and Apoptosis in Human Hepatocyte L02 Cells by Inhibiting ROS Production and Mitochondrial Pathway Chongshan Dai 1 , Daowen Li 1 , Lijing Gong 2 , Xilong Xiao 1, * and Shusheng Tang 1, * 1 2
*
College of Veterinary Medicine, China Agricultural University, 2 Yuanmingyuan West Road, Beijing 100193, China;
[email protected] (C.D.);
[email protected] (D.L.) Sport Science Research Center, Beijing Sport University, 48 Xinxi Road, Haidian District, Beijing 100084, China;
[email protected] Correspondences:
[email protected] (X.X.);
[email protected] (S.T.); Tel.: +86-6273-3377 (X.X.); +86-6273-4255 (S.T.)
Academic Editors: Luciano Saso, László Dux, Grzegorz Wegrzyn and Tamás Csont Received: 19 June 2016; Accepted: 10 August 2016; Published: 22 August 2016
Abstract: Furazolidone (FZD), a synthetic nitrofuran derivative, has been widely used as an antibacterial and antiprotozoal agent. Recently, the potential toxicity of FZD has raised concerns, but its mechanism is still unclear. This study aimed to investigate the protective effect of curcumin on FZD-induced cytotoxicity and the underlying mechanism in human hepatocyte L02 cells. The results showed that curcumin pre-treatment significantly ameliorated FZD-induced oxidative stress, characterized by decreased reactive oxygen species (ROS) and malondialdehyde formation, and increased superoxide dismutase, catalase activities and glutathione contents. In addition, curcumin pre-treatment significantly ameliorated the loss of mitochondrial membrane potential, the activations of caspase-9 and -3, and apoptosis caused by FZD. Alkaline comet assay showed that curcumin markedly reduced FZD-induced DNA damage in a dose-dependent manner. Curcumin pre-treatment consistently and markedly down-regulated the mRNA expression levels of p53, Bax, caspase-9 and -3 and up-regulated the mRNA expression level of Bcl-2. Taken together, these results reveal that curcumin protects against FZD-induced DNA damage and apoptosis by inhibiting oxidative stress and mitochondrial pathway. Our study indicated that curcumin may be a promising combiner with FZD to reduce FZD-related toxicity in clinical applications. Keywords: curcumin; furazolidone; oxidative stress; DNA damage; mitochondrial pathway
1. Introduction Furazolidone (FZD), a synthetic nitrofuran drug, has been used to treat the infections caused by bacteria and protozoa in human and animals [1,2]. Recent literatures report that FZD has novel applications in treating leukemia [3] or leishmaniasis [4]. In particular, FZD is usually used as a low-cost and effective drug to treat infections caused by Helicobacter pylori in human in developing countries, including China [5]. However, FZD is limited in the clinic due to its potential side effects, such as genotoxicity, hepatotoxicity, and carcinogenicity [5–7]. A pooled-data analysis reported that FZD-based regimens achieved low eradication rates for Helicobacter pylori infections at the current dosage regimen, but the incidence of severe side effects was observed when the dose was increased [8]. As a result, development of agents against FZD-related adverse effects is very urgent and it is a crucial strategy for optimizing potential antimicrobial activity and clinical usage of FZD.
Molecules 2016, 21, 1061; doi:10.3390/molecules21081061
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A previous study showed that oxidative stress may play a critical role in FZD-induced cytotoxicity and genotoxicity in human hepatoma (HepG2) cells [9]. Using the pig model the liver was suggested as the primary target organ of FZD metabolism; in addition, its metabolites 3-amino-2-oxazolidinone 2016, 21, 1061 2 of 15 couldMolecules accumulate in the liver [10]. The in vitro studies showed that FZD at concentrations of 4–10 µg/mL could increase the frequency of sister chromatid exchanges (SCE) in human lymphocytes A previous study showed that oxidative stress may play a critical role in FZD-induced cytotoxicity and increased SCE in was detected when mice cells were[9]. exposed topig FZD at the a dose mg/kg [11]. and genotoxicity human hepatoma (HepG2) Using the model liver of was30suggested Reactive oxygen species (ROS) are mainly generated by the mitochondria [12,13]. The mitochondrion as the primary target organ of FZD metabolism; in addition, its metabolites 3-amino-2-oxazolidinone is thecould major mediator of liver oxidative it is considered most vulnerable target in accumulate in the [10]. Thestress in vitroand studies showed that FZDasat the concentrations of 4–10 µg/mL could increase the frequency ofprocess sister chromatid (SCE) in human and increased the FZD-induced cytotoxicity [9,13]. exchanges Very recently, Deng etlymphocytes al. demonstrated that the SCE was detected when mice were exposed to FZD at a dose of(PI3K)/Akt 30 mg/kg [11]. Reactive oxygen mitochondrial pathway and phosphatidylinositol-3-kinase pathway playedspecies the critical are mainly generated thedeath mitochondria [12,13]. mitochondrion is the major mediator of roles (ROS) in FZD-induced apoptoticbycell in HepG2 cellsThe [14]. oxidative stress and it is considered as the most vulnerable target in the FZD-induced Curcumin, a natural polyphenol found in the spice turmeric, has manycytotoxicity biologicalprocess functions, [9,13]. Very recently, Deng et al. demonstrated that the mitochondrial pathway and phosphatidylinositolsuch as anti-inflammatory, anti-oxidative, anti-carcinogenic and immuno-regulatory abilities [15]. 3-kinase (PI3K)/Akt pathway played the critical roles in FZD-induced apoptotic cell death in HepG2 Manycells studies have demonstrated that curcumin could protect against DNA damage and [14]. oxidativeCurcumin, stress caused bypolyphenol some drugs environmental mutagens, including arsenic a natural found or in the spice turmeric, has many biological functions, such [16], acrylamide [17] and cisplatin [18], viaanti-carcinogenic scavenging ROS improving the abilities overall [15]. anti-oxidative as anti-inflammatory, anti-oxidative, andand immuno-regulatory Many ability. Curcumin administration showed could immense therapeutic effects against Helicobacter studies have demonstrated that curcumin protect against DNA damage and oxidative stresspylori caused some drugs or environmental arsenic [16], acrylamide [17] and cisplatin also infection inby mice and reduced the gastricmutagens, damageincluding due to infection [19]. In addition, curcumin [18],anti-parasitic via scavengingpotential, ROS and including improving trypanocidal the overall anti-oxidative ability. Curcumin showed and leishmanicidal activity,administration in several in vitro showed immense therapeutic effects against Helicobacter pylori infection in mice and reduced the gastricorally and in vivo models [20,21]. Human clinical trials showed that healthy human volunteers damage due to infection [19]. In addition, curcumin also showed anti-parasitic potential, including administered 500 mg of curcumin per day for 7 days showed significantly decreased levels of serum trypanocidal and leishmanicidal activity, in several in vitro and in vivo models [20,21]. Human lipid peroxide, a biomarker of oxidative stress [22]. Curcumin combination with some antibiotics clinical trials showed that healthy human volunteers orally administered 500 mg of curcumin per day and chemotherapy agents showed better therapeutic effect for infections and cancer compared to for 7 days showed significantly decreased levels of serum lipid peroxide, a biomarker of oxidative stress either[22]. oneCurcumin alone, which has raised wideantibiotics interest and in clinical practice [23–25]. Thus far, however, the combination with some chemotherapy agents showed better therapeutic potential preventive role of curcumin against FZD-induced adverse effects has not been effect for infections and cancer compared to either one alone, which has raised wide interest ininvestigated. clinical Therefore, the[23–25]. present study protective role ofrole curcumin on FZD-induced cytotoxicity practice Thus far,investigated however, thethe potential preventive of curcumin against FZD-induced adverse effects has nothepatocyte been investigated. Therefore, the aim present study investigated the protective role of and DNA damage using L02 cells, with the of providing a promising combination of curcumin on FZD-induced cytotoxicity and DNA damage FZD with curcumin regarding the in vitro toxicology aspect.using hepatocyte L02 cells, with the aim of providing a promising combination of FZD with curcumin regarding the in vitro toxicology aspect.
2. Results 2. Results
2.1. Curcumin Attenuates FZD Induced Cytotoxicity in L02 Cells 2.1. Curcumin Attenuates FZD Induced Cytotoxicity in L02 Cells
FZD treatment for 24 h reduced the cell viability of L02 cells in a dose-dependent manner. FZD treatment for 24 h reduced the cell viability of L02 cells in a dose-dependent manner. Compared to the negative control group (0.2% DMSO), FZD treatment at 10, 20, 40 and 60 µg/mL for Compared to the negative control group (0.2% DMSO), FZD treatment at 10, 20, 40 and 60 µg/mL for 24 h significantly decreased the cell viabilities to 89.4% (p < 0.05), 73.2% (p < 0.01), 53.4% (p < 0.01) and 24 h significantly decreased the cell viabilities to 89.4% (p < 0.05), 73.2% (p < 0.01), 53.4% (p < 0.01) and 42.6%42.6% (p < 0.01), respectively (Figure (p < 0.01), respectively (Figure1). 1).
Figure 1. Curcumin protects againstFZD FZDinduced induced cytotoxicity cytotoxicity ininL02 cells. Values were presented as as Figure 1. Curcumin protects against L02 cells. Values were presented mean ± SD, from five independent experiments. * p < 0.05, ** p < 0.01, compared to the negative control mean ± SD, from five independent experiments. * p < 0.05, ** p < 0.01, compared to the negative control group (0.2% DMSO); # p < 0.05, ## p < 0.01, compared to the FZD alone group. FZD, furazolidone. group (0.2% DMSO); # p < 0.05, ## p < 0.01, compared to the FZD alone group. FZD, furazolidone.
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However, curcumin pre-treatment, especially at the final concentrations of 2.5 and 5 µM, markedly However, curcumin thethe finalFZD concentrations of 2.5 and 5 µM,(Figure markedly attenuated FZD inducedpre-treatment, cytotoxicity, especially comparedat to alone treatment groups 1). attenuated FZD induced cytotoxicity, compared to the FZD alone treatment groups (Figure 1). There was no significant change in cell viability in curcumin alone treatment (at 1.25, 2.5 and 5 µM, There was nogroups significant change respectively) (Figure 1). in cell viability in curcumin alone treatment (at 1.25, 2.5 and 5 µM, respectively) groups (Figure 1). 2.2. Curcumin Suppresses FZD Induced Oxidative Stress in L02 Cells 2.2. Curcumin Suppresses FZD Induced Oxidative Stress in L02 Cells To investigate the protective effect of curcumin, the intracellular ROS and the levels of superoxide To investigate the protective of curcumin, theand intracellular ROS and (MDA) the levels of superoxide dismutase (SOD), catalase (CAT),effect glutathione (GSH) malondialdehyde were measured. dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) were measured. As shown in Figure 2, compared to the negative control group, FZD treatment at 40 µg/mL As for shown in Figure 2,increased compared the negative group,ROS FZDtotreatment at 404.5-fold µg/mL (pfor< 24 h 24 h significantly thetoproduction of control intracellular approximate 0.01) significantly the production of intracellular ROS to approximate (p < 0.01) (Figure 2A). (Figure 2A).increased Meanwhile, after FZD treatment, MDA level increased to 4.5-fold 3.1 nmol/mg protein (equal Meanwhile, after FZD treatment, MDA level increased to 3.1 nmol/mg protein (equal to 194% of control), to 194% of control), and the SOD, CAT activities and GSH levels decreased to 21.3 U/mg protein, and SOD, CAT and activities and GSH levels decreased to 58.7% 21.3 U/mg protein, 41.1 U/mg protein and 41.1 the U/mg protein 84.5 mg/g protein (equal to 56.2%, and 61.4% of control, respectively, all 84.5 mg/gcompared protein (equal 56.2%, group 58.7% (Figure and 61.4% of control, respectively, p < 0.01), compared to p < 0.01), to thetocontrol 2B–E). Moreover, curcumin all pre-treatment, especially the control group (Figure 2B–E). Moreover, curcumin pre-treatment, especially at 5 µM could at 5 µM could effectively inhibit the ROS production, decrease the levels of MDA, and increase the effectively the CAT ROS and production, decrease levels of MDA, and increase SOD activities ofinhibit SOD and GSH levels (all p
