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Cytotoxic effects and apoptosis induction of enrofloxacin in hepatic cell line of grass carp (Ctenopharyngodon idellus)
Fish & Shellfish Immunology 47 (2015) 639e644
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Cytotoxic effects and apoptosis induction of enrofloxacin in hepatic cell line of grass carp (Ctenopharyngodon idellus) Bo Liu a, b, Yanting Cui b, Paul B. Brown c, Xianping Ge a, b, Jun Xie a, b, *, Pao Xu a, b, ** a
Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, 214081, China b Wuxi Fisheries College, Nanjing Agriculture University, Wuxi, 214081, China c Department of Forestry and Natural Resources, Purdue University, West Lafayette, 47907, Indiana, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 July 2015 Received in revised form 4 October 2015 Accepted 9 October 2015 Available online 22 October 2015
We determined the effect of enrofloxacin on the lactate dehydrogenase (LDH) release, reactive oxygen species (ROS), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), malondialdehyde (MDA), mitochondria membrane potential (DJm) and apoptosis in the hepatic cell line of grass carp (Ctenopharyngodon idellus). Cultured cells were treated with different concentrations of enrofloxacin (12.5e200 ug/mL) for 24 h. We found that the cytotoxic effect of enrofloxacin was mediated by apoptosis, and that this apoptosis occurred in a dose-dependent manner. The doses of 50,100 and 200 mg/mL enrofloxacin increased the LDH release and MDA concentration, induced cell apoptosis and reduced the DJm compared to the control. The highest dose of 200 ug/mL enrofloxacin also significantly induced apoptosis accompanied by DJm disruption and ROS generation and significantly reduced T-AOC and increased MDA concentration compared to the control. Our results suggest that the dose of 200 ug/mL enrofloxacin exerts its cytotoxic effect and produced ROS via apoptosis by affecting the mitochondria of the hepatic cells of grass carp. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Enrofloxacin Cytotoxicity Apoptosis ROS Grass carp Hepatic cell
1. Introduction Enrofloxacin, a second generation fluoroquinolone, is a synthetic chemotherapeutic agent used to treat various systemic bacterial infections, especially gram-negative bacteria in mammals [1,2] and aquatic animals [3e5]. Fluoroquinolones are among the antimicrobial chemotherapeutics frequently detected in the aquatic environment in relatively high concentrations ranging from ng/L to ug/L [6]. Some of fluoroquinolones have also been detected in surface water and sediments [6], ground water [6,7], and drinking water [6,8]. Moreover, its accumulation [9e11], toxicity [12e14] and resistance in pathogenic microorganisms [15,16] have also been major concerns. Previous studies demonstrated that fluoroquinolones impacted the immune system [17,18], led to oxidative stress [19], inhibited bacterial growth by inhibiting DNA gyrase
* Corresponding author. Present address: FFRC, CAFS, No. 9 Shanshui East Road, Wuxi, 214081, China. ** Corresponding author. Present address: FFRC, CAFS, No. 9 Shanshui East Road, Wuxi, 214081, China. E-mail addresses: [email protected] (B. Liu), [email protected] (J. Xie), [email protected] (P. Xu). http://dx.doi.org/10.1016/j.fsi.2015.10.007 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
[1,20], and induced cell apoptosis and DNA fragmentation [21]. However, the mechanisms by which enrofloxacin impacts the immune systems are not clear. Apoptosis, or programmed cell death, is characterized by cell shrinkage, chromatin condensation, formation of cytoplasmic blebs and apoptotic bodies [22]. There are several factors involved in apoptosis and diverse apoptotic stimuli converge on a common apoptotic pathway mediated by the mitochondria [23,24]. Apoptosis through the mitochondrial pathway is possibly preceded by the overproduction of reactive oxygen species (ROS). Then, the damage of oxidative stress may result in mitochondrial outer membrane permeabilization, thus leading to the release of proapoptotic proteins and caspase activation [25e27]. The pathways of apoptosis have been studied extensively, but the signaling pathway of enrofloxacin-induced apoptosis of the hepatocytes of grass carp remains poorly understood. Grass carp (Ctenopharyngodon idellus) is a herbivorous freshwater fish and widely distributed and cultivated in Asia, Europe, and North America. It is native to China with a long cultivation history and is currently the most extensively cultivated freshwater fish in China (507 million tons in 2013), and the largest fresh water fish production worldwide [28]. However, cultured grass carps in
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China as well as in other parts of the world have suffered from serious diseases problem caused by viral infection and bacterial pathogens, which have caused significant economic losses and impeded the sustainable development of grass carp cultivation industry [29]. In order to control bacterial disease, enrofloxacin is one of the effective antimicrobials and has been widely used in aquaculture in China [30]. Its mechanism of anti-bacteria might inhibit bacterial DNA gyrase and prevent DNA supercoiling and DNA synthesis of bacteria [1,20]. It can enter into the environment through the excretion of unmetabolised quinolones or the disposal of unused drugs and become a main source of pollution. Enrofloxacin can bioaccumulate in the fish, and the liver is the main detoxification organ, displaying high concentrations of enrofloxacin. The liver is the main defense against oxidative stress caused by excessive ROS and plays an important role in the metabolism of different nutrients and toxins [31,32]. The in vitro hepatocyte culture system of grass carp has been used as a tool for xenobiotic metabolism and toxicity studies [27,33e35]. Thus, it is important to examine the effects of enrofloxacin on oxidative damage, apoptosis and its potential mechanism in the grass carp liver. The aim of this study was to explore the effects of enrofloxacin on lactate dehydrogenase (LDH), release, ROS, superoxide dismutase (SOD), total antioxidant capacity (T-AOC), malondialdehyde (MDA), mitochondria membrane potential (DJm) and apoptosis in the hepatic cells of grass carp. These findings may provide information for the mechanisms involved in the apoptotic toxicity of enrofloxacin in fish cells. 2. Materials and methods 2.1. Materials The hepatic grass carp cell line (L8824) was obtained from Wuhan Cell Institutional Repository (Wuhan, China). Enrofloxacin (purity > 99%) was purchased from Feida Chemical Reagent Company (Xi'an, China). Kits measuring LDH release, SOD activity, TAOC, MDA and Annexin V/FITC were purchased from the Beyotime Institute of Biotechnology (Beyotime, Haimen, Jiangsu, PR China). Other drugs and reagents, such as Rhodamine 123, MEM medium, fetal calf serum, penicillin, streptomycin, tripure reagent, and PBS, were purchased from commercial suppliers and were of biochemical quality. 2.2. Cell culture The hepatic cells of grass carp were cultured using a previously described methods [27,34], with some modifications. Briefly, the hepatic cells were maintained in MEM medium (Sigma Chemical Co.), supplemented with 10% fetal calf serum (FCS, Sijiqing Biological Engineering Materials Co. Ltd., Hangzhou, Zhejiang, China), 100 U/mL penicillin, and 100 U/mL streptomycin, and in a humid atmosphere of 5% CO2 at 27 C. Following treatment, cells were harvested using 0.25% trypsin (0.2% EDTA; Gibco), centrifuged at 1000g for 5 min, and washed twice with PBS. Three replicates were separately performed for each treatment and control, and each replicate described below was performed using 3 samples from 3 flasks or plates, respectively. L8824 cells were plated in 96- or 6-well plates. Then, cells were treated with graded concentrations of enrofloxacin (12.5, 25, 50, 100 and 200 ug/mL) for 24 h [35]. Enrofloxacin was dissolved in 20 mg/mL of DMSO. The stock solution of enrofloxacin was diluted to the desired concentration immediately before use. Control cells were cultured in the same medium but without enrofloxacin. Vehicle cells were added to the medium in 0.3% DMSO.
2.3. LDH release assay LDH is a soluble cytosolic enzyme present in most eukaryotic cells that is released into the culture medium upon cell death due to plasma membrane damage. The increase in the LDH activity in culture supernatant is proportional to the number of lysed cells [34,36]. L8824 cells were seeded in 96-well (5 103 cells/well) culture plates at a final volume of 200 ml/well of culture medium, supplemented with 10% fetal calf serum, for 24 h. After cells were exposed to enrofloxacin for 24 h, LDH release was measured according to manufacturer's instructions (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). Briefly, the supernatants and cell lysates were transferred to 96-well plates and incubated with 1 mg/mL of NADH in pyruvate substrate solution for 15 min at 37 C and then with 2,4-dinitrophenylhydrazine for another 15 min at 37 C. The reaction was stopped by the addition of 0.4 M NaOH, and changes in absorbance were determined at 450 nm using a spectrophotometric microplate reader (BIO-RAD Model 3550, CA, USA).
2.4. ROS measurements Changes in intracellular ROS levels were determined by measuring the oxidative conversion of cell permeable 20 , 7'dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF) in a microplate reader (Fluoroskan Ascent FL, Thermo, USA). Cells in 6-well (1 106 cells/well) culture plates were incubated with enrofloxacin for 24 h. The cells were then incubated with DCFH-DA at for 20 min at 37 C and washed twice with serum-free medium. The distribution of DCF fluorescence in 20,000 cells was detected by fluorospectrophotometry at an excitation wavelength of 488 nm and an emission wavelength of 535 nm [34,37].
2.5. Hepatic T-AOC, SOD and MDA Cells were seeded in 6-well (1 106 cells/well) culture plates after enrofloxacin exposure and were evaluated for SOD activity, TAOC and MDA content according to manufacturer's instructions (Beyotime, Haimen, Jiangsu, China). SOD activity was determined using xanthine oxidase-derived superoxide and was then monitored at 450 nm [38]. One unit of SOD activity was defined as the quantity of SOD required for 50% inhibition [39]. SOD activity was then normalized to on a protein basis. T-AOC was measured with 2, 2'-azino-bis (3 ethylbenzthiazoline-6-sulfonic acid (ABTS) using a microplate reader (BIO-RAD Model 3550, CA, USA) at 414 nm [34]. The relative T-AOC values of the samples were normalized to protein concentration. Lipid peroxidation was evaluated indirectly by measuring MDA formation from the breakdown of polyunsaturated fatty acids, the thiobarbituric acid reactive substances at 532 nm [40].
2.6. Mitochondrial membrane potential
DJm was monitored using fluorescent rhodamine 123 dye (Beyotime, Haimen, Jiangsu, China), which preferentially localizes to active mitochondria based on highly negative DJm [37]. Rhodamine 123 (final concentration of 10 mM) was added to cells after enrofloxacin treatment for 24 h. After 30 min at 37 C, the cells were collected by pipetting, washed twice with PBS, and then analyzed by fluorospectrophotometry at an excitation wavelength of 488 nm and at emission wavelength of 535 nm.
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2.7. Flow cytometric detection of apoptotic cells Apoptotic cells were evaluated using Annexin V/FITC (Beyotime, Haimen, Jiangsu, PR China). In brief, Annexin Vþ/PI cells were considered apoptotic, while Annexin Vþ/PI þ cells were considered necrotic [34,41]. Cells were stained according to manufacturer's instructions and analyzed by flow cytometry (Cytomics FC 500, Beckman Coulter, USA). The cells were collected by centrifugation after enrofloxacin exposure and washed with PBS. The pellets were resuspended in the Annexin V-FITC staining reagent and fixed at 20e25 C for 10 min. The cells were then washed and resuspended in the PI staining reagent. Staining was stable at 4 C for 30 min [42]. Samples were then analyzed by flow cytometry. 2.8. Statistical analyses
Fig. 2. The effects of enrofloxacin on ROS in hepatic cell of grass carp. Note: Data are expressed as mean þ SE (n ¼ 9). Legends are the same as in Fig. 1.
All results were shown as the mean ± standard error (X±SE) and were analyzed using SPSS statistics software, version 16.0. Data were subjected to one-way analysis of variance and Duncan's multiple range tests. A P < 0.05 was considered to be statistically significant. 3. Results 3.1. Cytotoxicity of enrofloxacin on hepatic cell of grass carp L8824 cells were exposed to different concentrations of enrofloxacin for 24 h, and cytotoxicity was measured by LDH release. As shown in Fig. 1, the amount of LDH release significantly increased in a dose-dependent manner with exposure to 50, 100, and 200 mg/mL enrofloxacin compared to the control cells (P < 0.05). Furthermore, LDH release of cells treated with 200 mg/mL enrofloxacin was significantly higher than that of 12.5, 25, 50 ug/mL groups (P < 0.05). 3.2. Detection the effect of enrofloxacin on ROS on hepatic cell of grass carp Using the fluorescent dye DCFH-DA, we further measured ROS production changes of cells induced with enrofloxacin. As shown in Fig. 2, the cells exposed to 200 mg/mL enrofloxacin significantly increased ROS while those exposed to 12.5, or 25 mg/mL enrofloxacin significantly decreased (P < 0.05) compared to control cells. Additionally, ROS generation in cells treated with 50, 100, and 200 mg/mL enrofloxacin was significantly higher than that of 12.5, 25 mg/mL enrofloxacin (P < 0.05).
Fig. 1. The effects of enrofloxacin on LDH release in hepatic cell of grass carp. Note: Data are expressed as mean þ SE (n ¼ 9). The letters above the bars indicate significant differences (P < 0.05) in different groups calculated using Duncan's multiple range test.
Fig. 3. The effects of enrofloxacin on total antioxidant capacity (A), superoxide dismutase activity (B), and malonaldehyde (C) in hepatic cell of grass carp. .Note: Data are expressed as mean þ SE (n ¼ 9). Legends are the same as in Fig. 1.
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3.3. Antioxidant capacity of enrofloxacin on hepatic cell of grass carp
after 24 h of exposure to 12.5, 25, 50, 100 and 200 mg/mL enrofloxacin than that of control cells (Fig. 4C; P < 0.05).
We examined the effect of enrofloxacin on the anti-oxidization enzymes of hepatic cells and the results are shown in Fig. 3.T-AOC in the cells treated with 50, 100, and 200 mg/mL enrofloxacin was significantly lower than that of the control group (Fig. 3A; P < 0.05). SOD activity in cells treated with 25, 50, and 100 mg/mL enrofloxacin was significantly higher than that of the control group (Fig. 3B; P < 0.05). However, MDA in cells treated with 25, 50, 100, and 200 mg/mL enrofloxacin significantly increased compared to the control cells. (Fig. 3C; P < 0.05).
4. Discussion
3.4. Assessment of enrofloxacin-induced apoptosis We also examined the effect of enrofloxacin on apoptosis and
DJm in the hepatic cells of grass carp (Fig. 4A). We found that the percentage of apoptotic cells in cells treated with 25, 50, 100, and 200 mg/mL enrofloxacin was significantly higher compared to the control cells (Fig. 4A and B; P < 0.05). In addition, the percentage of apoptotic cells exposed to 50, 100, and 200 mg/mL enrofloxacin was significantly higher than those exposed to 12.5 or 25 mg/mL enrofloxacin (Fig. 4B; P < 0.05). DJm in L8824 cells was much lower
Fig. 4. The effects of enrofloxacin on apoptosis (A), quantitative analyses of apoptosis (B) and mitochondrial membrane potential (C) in hepatic cell of grass carp. Note: Data are expressed as mean þ SE (n ¼ 3). Legends are the same as in Fig. 1.
Enrofloxacin is a synthetic chemotherapeutic medicine that has been extensively used to control bacterial pathogens and its use is increasingly leading to potential drug resistance and this would have a negative effect on the subsequent disease control in aquaculture, which has led to widespread public concern of human health and food safety [43]. In the present study, the grass carp hepatic cell line L8824 was used to study the cytotoxic effects of enrofloxacin in vitro. Exposure to 50, 100 and 200 mg/mL enrofloxacin for 24 h significantly increased LDH release in a dosedependent manner, which was consistent with previous studies on the hepatocytes of grass carp exposed to 100 and 200 ug/mL enrofloxacin for 1 day [35], chicken tendon cell reacted with 50e300 ug/mL enrofloxacin for 3 days [44] and horse tendon cells treated with 50 and 100 ug/mL enrofloxacin for 3 days [45]. The different toxicity of enrofloxacin in vitro might be due to the reaction time among the drug, cell cultivation and the species of the cultured cells [46]. The cellular damage caused by enrofloxacin was further confirmed by apoptosis assays. In our study, we demonstrated that enrofloxacin-induced apoptosis in the hepatocytes of grass carp occurred in a dose-dependent manner. This was consistent with finding that enrofloxacin can trigger apoptosis in vitro. Lim et al. [21] observed that canine tendon cells and chondrocytes treated with 200 ug/mL enrofloxacin for 4 days exhibited apoptotic features and fragmentation of DNA. These data emphasize that enrofloxacin leads to unavoidable apoptotic cell death. The signaling mechanisms that regulate apoptosis are very complicated. Generally there are two main apoptotic pathways, the mitochondrial pathway and the death receptor pathway. Mitochondrial changes are regarded as the central role in the apoptosis pathway in many experimental systems, include the enhanced production of oxygen radicals and loss of DJm [47,48]. To understand the role of apoptosis in grass carp hepatic cells following exposure to enrofloxacin, we examined the changes of parameters such as ROS, SOD, T-AOC, MDA, DJm in the apoptotic cells in response to enrofloxacin at different concentrations. ROS are chemically reactive molecules containing superoxide anion radicals and hydrogen peroxide [49]. Studies have implicated ROS as central mediators in the induction of apoptosis by many diverse cytotoxic stimuli [50]. The mechanisms driving apoptosis in cells under oxidative stress may involve high ROS levels, which directly inhibit caspase activity, disrupt intracellular Ca2þ homeostasis, and lead to ATP depletion [51]. In the present study, the highest concentration of 200 mg/mL enrofloxacin significantly increased ROS compared to control cells. This result was consistent with earlier findings reported in the literature that in many cases, drug-induced apoptosis is accompanied by elevation of cellular levels of ROS [27,50,52e54]. As a result, our study indicated the ROS was probably responsible for enrofloxacin-induced apoptosis, that was, oxidative stress likely played a role in enrofloxacin-induced apoptosis in hepatic grass carp cells. ROS can rapidly be removed by enzymatic and non-enzymatic antioxidants, thereby maintaining a healthy pro-oxidant/ antioxidant homeostasis [55]. The activation of endogenous antioxidant enzymes and non-enzymatic antioxidants, such as SOD and T-AOC, respectively, has a protective role against the damage of lipid peroxidation [56,57]. Meanwhile, the generation of excessive ROS results in lipid oxidation and produces a large amount of aldehydes, alcohols and hydrocarbon of which MDA is a substance with strong biotoxicity, which may hurt organisms [58]. In rats,
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chloramphenicol induced lipid peroxidation and a significant decrease of glutathione-S-transferase, glutathione peroxidase and catalase activities in hepatic microsomal components [59,60]. In aquatic animal, the animals treated with enrofloxac caused the oxidative stress [61,62]. In the present study, we found that 50, 100, and 200 mg/mL enrofloxacin significantly reduced T-AOC and enhanced MDA. This may indicate a deficiency in some enzymatic antioxidants or severe oxidative injury [63]. Therefore, the oxidative effect of higher concentrations of enrofloxacin may be attributed to the loss of the affected cell's ability to maintain the activity of its radical-scavenging enzymes. In addition, the current study demonstrated that SOD significantly increased in the treatments of 25, 50 mg/mL enrofloxacin, which was the relationship of loss of the ROS in concentrations of 12.5 or 25 mg/mL enrofloxacin and contributed to attempt to maintain its redox balance. However, these mechanisms may not be able to compensate for the increased oxidative stress induced by the highest concentration of 200 mg/mL enrofloxacin in this study since ROS levels remained high. Loss of DJm has been an established indicator of mitochondrial damage in the progression of apoptosis [48]. To investigate the role of DJm in enrofloxacin-induced apoptosis, the mitochondriaspecific dye Rh123 was employed, and FACS analysis was conducted. Our data showed the concentrations of 12.5, 25, 50, 100 and 200 mg/mL enrofloxacin after 24 h exposure reduced the DJm in L8824 cells compared with the control cell and the DJm declined to approximately 60e75% of control levels within 24 h of treatment. It seemed that DJm decreased in a dose-dependent manner in enrofloxacin -treated cells and a drop in DJm was a responsible for the enhancement of enrofloxacin-induced apoptosis, which was consistent with previous studies that drug-induced apoptosis is accompanied by a fall in the DJm of grass carp cell [27,52,53].
[6] [7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15] [16] [17] [18]
5. Conclusion [19]
In conclusion, the present study demonstrated that enrofloxacin exhibited cytotoxic effects in hepatic cell line of grass carp because of the induction of apoptosis. Furthermore, we were able to ascertain that intracellular ROS and DJm played important roles in enrofloxacin-induced apoptosis. The concentration of 200 mg/mL enrofloxacin produced ROS, which altered the subcellular redox equilibrium, reduced DJm and subsequently triggered apoptosis in the hepatic cells of grass carp. This finding provides a new understanding of the cytotoxic effects on fish cells caused by enrofloxacin.
[21]
Acknowledgments
[25]
This work was supported by the Modern Agriculture Industrial Technology System special project-the National Technology System for Conventional Freshwater Fish Industries (CARS-46), the Three New Projects of Fishery in Jiangsu province, and Special Fund for Agro-Scientific Research in the Public Interest (D2013-5). References
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Full length article
Cytotoxic effects and apoptosis induction of enrofloxacin in hepatic cell line of grass carp (Ctenopharyngodon idellus) Bo Liu a, b, Yanting Cui b, Paul B. Brown c, Xianping Ge a, b, Jun Xie a, b, *, Pao Xu a, b, ** a
Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, 214081, China b Wuxi Fisheries College, Nanjing Agriculture University, Wuxi, 214081, China c Department of Forestry and Natural Resources, Purdue University, West Lafayette, 47907, Indiana, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 July 2015 Received in revised form 4 October 2015 Accepted 9 October 2015 Available online 22 October 2015
We determined the effect of enrofloxacin on the lactate dehydrogenase (LDH) release, reactive oxygen species (ROS), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), malondialdehyde (MDA), mitochondria membrane potential (DJm) and apoptosis in the hepatic cell line of grass carp (Ctenopharyngodon idellus). Cultured cells were treated with different concentrations of enrofloxacin (12.5e200 ug/mL) for 24 h. We found that the cytotoxic effect of enrofloxacin was mediated by apoptosis, and that this apoptosis occurred in a dose-dependent manner. The doses of 50,100 and 200 mg/mL enrofloxacin increased the LDH release and MDA concentration, induced cell apoptosis and reduced the DJm compared to the control. The highest dose of 200 ug/mL enrofloxacin also significantly induced apoptosis accompanied by DJm disruption and ROS generation and significantly reduced T-AOC and increased MDA concentration compared to the control. Our results suggest that the dose of 200 ug/mL enrofloxacin exerts its cytotoxic effect and produced ROS via apoptosis by affecting the mitochondria of the hepatic cells of grass carp. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Enrofloxacin Cytotoxicity Apoptosis ROS Grass carp Hepatic cell
1. Introduction Enrofloxacin, a second generation fluoroquinolone, is a synthetic chemotherapeutic agent used to treat various systemic bacterial infections, especially gram-negative bacteria in mammals [1,2] and aquatic animals [3e5]. Fluoroquinolones are among the antimicrobial chemotherapeutics frequently detected in the aquatic environment in relatively high concentrations ranging from ng/L to ug/L [6]. Some of fluoroquinolones have also been detected in surface water and sediments [6], ground water [6,7], and drinking water [6,8]. Moreover, its accumulation [9e11], toxicity [12e14] and resistance in pathogenic microorganisms [15,16] have also been major concerns. Previous studies demonstrated that fluoroquinolones impacted the immune system [17,18], led to oxidative stress [19], inhibited bacterial growth by inhibiting DNA gyrase
* Corresponding author. Present address: FFRC, CAFS, No. 9 Shanshui East Road, Wuxi, 214081, China. ** Corresponding author. Present address: FFRC, CAFS, No. 9 Shanshui East Road, Wuxi, 214081, China. E-mail addresses: [email protected] (B. Liu), [email protected] (J. Xie), [email protected] (P. Xu). http://dx.doi.org/10.1016/j.fsi.2015.10.007 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
[1,20], and induced cell apoptosis and DNA fragmentation [21]. However, the mechanisms by which enrofloxacin impacts the immune systems are not clear. Apoptosis, or programmed cell death, is characterized by cell shrinkage, chromatin condensation, formation of cytoplasmic blebs and apoptotic bodies [22]. There are several factors involved in apoptosis and diverse apoptotic stimuli converge on a common apoptotic pathway mediated by the mitochondria [23,24]. Apoptosis through the mitochondrial pathway is possibly preceded by the overproduction of reactive oxygen species (ROS). Then, the damage of oxidative stress may result in mitochondrial outer membrane permeabilization, thus leading to the release of proapoptotic proteins and caspase activation [25e27]. The pathways of apoptosis have been studied extensively, but the signaling pathway of enrofloxacin-induced apoptosis of the hepatocytes of grass carp remains poorly understood. Grass carp (Ctenopharyngodon idellus) is a herbivorous freshwater fish and widely distributed and cultivated in Asia, Europe, and North America. It is native to China with a long cultivation history and is currently the most extensively cultivated freshwater fish in China (507 million tons in 2013), and the largest fresh water fish production worldwide [28]. However, cultured grass carps in
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China as well as in other parts of the world have suffered from serious diseases problem caused by viral infection and bacterial pathogens, which have caused significant economic losses and impeded the sustainable development of grass carp cultivation industry [29]. In order to control bacterial disease, enrofloxacin is one of the effective antimicrobials and has been widely used in aquaculture in China [30]. Its mechanism of anti-bacteria might inhibit bacterial DNA gyrase and prevent DNA supercoiling and DNA synthesis of bacteria [1,20]. It can enter into the environment through the excretion of unmetabolised quinolones or the disposal of unused drugs and become a main source of pollution. Enrofloxacin can bioaccumulate in the fish, and the liver is the main detoxification organ, displaying high concentrations of enrofloxacin. The liver is the main defense against oxidative stress caused by excessive ROS and plays an important role in the metabolism of different nutrients and toxins [31,32]. The in vitro hepatocyte culture system of grass carp has been used as a tool for xenobiotic metabolism and toxicity studies [27,33e35]. Thus, it is important to examine the effects of enrofloxacin on oxidative damage, apoptosis and its potential mechanism in the grass carp liver. The aim of this study was to explore the effects of enrofloxacin on lactate dehydrogenase (LDH), release, ROS, superoxide dismutase (SOD), total antioxidant capacity (T-AOC), malondialdehyde (MDA), mitochondria membrane potential (DJm) and apoptosis in the hepatic cells of grass carp. These findings may provide information for the mechanisms involved in the apoptotic toxicity of enrofloxacin in fish cells. 2. Materials and methods 2.1. Materials The hepatic grass carp cell line (L8824) was obtained from Wuhan Cell Institutional Repository (Wuhan, China). Enrofloxacin (purity > 99%) was purchased from Feida Chemical Reagent Company (Xi'an, China). Kits measuring LDH release, SOD activity, TAOC, MDA and Annexin V/FITC were purchased from the Beyotime Institute of Biotechnology (Beyotime, Haimen, Jiangsu, PR China). Other drugs and reagents, such as Rhodamine 123, MEM medium, fetal calf serum, penicillin, streptomycin, tripure reagent, and PBS, were purchased from commercial suppliers and were of biochemical quality. 2.2. Cell culture The hepatic cells of grass carp were cultured using a previously described methods [27,34], with some modifications. Briefly, the hepatic cells were maintained in MEM medium (Sigma Chemical Co.), supplemented with 10% fetal calf serum (FCS, Sijiqing Biological Engineering Materials Co. Ltd., Hangzhou, Zhejiang, China), 100 U/mL penicillin, and 100 U/mL streptomycin, and in a humid atmosphere of 5% CO2 at 27 C. Following treatment, cells were harvested using 0.25% trypsin (0.2% EDTA; Gibco), centrifuged at 1000g for 5 min, and washed twice with PBS. Three replicates were separately performed for each treatment and control, and each replicate described below was performed using 3 samples from 3 flasks or plates, respectively. L8824 cells were plated in 96- or 6-well plates. Then, cells were treated with graded concentrations of enrofloxacin (12.5, 25, 50, 100 and 200 ug/mL) for 24 h [35]. Enrofloxacin was dissolved in 20 mg/mL of DMSO. The stock solution of enrofloxacin was diluted to the desired concentration immediately before use. Control cells were cultured in the same medium but without enrofloxacin. Vehicle cells were added to the medium in 0.3% DMSO.
2.3. LDH release assay LDH is a soluble cytosolic enzyme present in most eukaryotic cells that is released into the culture medium upon cell death due to plasma membrane damage. The increase in the LDH activity in culture supernatant is proportional to the number of lysed cells [34,36]. L8824 cells were seeded in 96-well (5 103 cells/well) culture plates at a final volume of 200 ml/well of culture medium, supplemented with 10% fetal calf serum, for 24 h. After cells were exposed to enrofloxacin for 24 h, LDH release was measured according to manufacturer's instructions (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). Briefly, the supernatants and cell lysates were transferred to 96-well plates and incubated with 1 mg/mL of NADH in pyruvate substrate solution for 15 min at 37 C and then with 2,4-dinitrophenylhydrazine for another 15 min at 37 C. The reaction was stopped by the addition of 0.4 M NaOH, and changes in absorbance were determined at 450 nm using a spectrophotometric microplate reader (BIO-RAD Model 3550, CA, USA).
2.4. ROS measurements Changes in intracellular ROS levels were determined by measuring the oxidative conversion of cell permeable 20 , 7'dichlorofluorescein diacetate (DCFH-DA) to fluorescent dichlorofluorescein (DCF) in a microplate reader (Fluoroskan Ascent FL, Thermo, USA). Cells in 6-well (1 106 cells/well) culture plates were incubated with enrofloxacin for 24 h. The cells were then incubated with DCFH-DA at for 20 min at 37 C and washed twice with serum-free medium. The distribution of DCF fluorescence in 20,000 cells was detected by fluorospectrophotometry at an excitation wavelength of 488 nm and an emission wavelength of 535 nm [34,37].
2.5. Hepatic T-AOC, SOD and MDA Cells were seeded in 6-well (1 106 cells/well) culture plates after enrofloxacin exposure and were evaluated for SOD activity, TAOC and MDA content according to manufacturer's instructions (Beyotime, Haimen, Jiangsu, China). SOD activity was determined using xanthine oxidase-derived superoxide and was then monitored at 450 nm [38]. One unit of SOD activity was defined as the quantity of SOD required for 50% inhibition [39]. SOD activity was then normalized to on a protein basis. T-AOC was measured with 2, 2'-azino-bis (3 ethylbenzthiazoline-6-sulfonic acid (ABTS) using a microplate reader (BIO-RAD Model 3550, CA, USA) at 414 nm [34]. The relative T-AOC values of the samples were normalized to protein concentration. Lipid peroxidation was evaluated indirectly by measuring MDA formation from the breakdown of polyunsaturated fatty acids, the thiobarbituric acid reactive substances at 532 nm [40].
2.6. Mitochondrial membrane potential
DJm was monitored using fluorescent rhodamine 123 dye (Beyotime, Haimen, Jiangsu, China), which preferentially localizes to active mitochondria based on highly negative DJm [37]. Rhodamine 123 (final concentration of 10 mM) was added to cells after enrofloxacin treatment for 24 h. After 30 min at 37 C, the cells were collected by pipetting, washed twice with PBS, and then analyzed by fluorospectrophotometry at an excitation wavelength of 488 nm and at emission wavelength of 535 nm.
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2.7. Flow cytometric detection of apoptotic cells Apoptotic cells were evaluated using Annexin V/FITC (Beyotime, Haimen, Jiangsu, PR China). In brief, Annexin Vþ/PI cells were considered apoptotic, while Annexin Vþ/PI þ cells were considered necrotic [34,41]. Cells were stained according to manufacturer's instructions and analyzed by flow cytometry (Cytomics FC 500, Beckman Coulter, USA). The cells were collected by centrifugation after enrofloxacin exposure and washed with PBS. The pellets were resuspended in the Annexin V-FITC staining reagent and fixed at 20e25 C for 10 min. The cells were then washed and resuspended in the PI staining reagent. Staining was stable at 4 C for 30 min [42]. Samples were then analyzed by flow cytometry. 2.8. Statistical analyses
Fig. 2. The effects of enrofloxacin on ROS in hepatic cell of grass carp. Note: Data are expressed as mean þ SE (n ¼ 9). Legends are the same as in Fig. 1.
All results were shown as the mean ± standard error (X±SE) and were analyzed using SPSS statistics software, version 16.0. Data were subjected to one-way analysis of variance and Duncan's multiple range tests. A P < 0.05 was considered to be statistically significant. 3. Results 3.1. Cytotoxicity of enrofloxacin on hepatic cell of grass carp L8824 cells were exposed to different concentrations of enrofloxacin for 24 h, and cytotoxicity was measured by LDH release. As shown in Fig. 1, the amount of LDH release significantly increased in a dose-dependent manner with exposure to 50, 100, and 200 mg/mL enrofloxacin compared to the control cells (P < 0.05). Furthermore, LDH release of cells treated with 200 mg/mL enrofloxacin was significantly higher than that of 12.5, 25, 50 ug/mL groups (P < 0.05). 3.2. Detection the effect of enrofloxacin on ROS on hepatic cell of grass carp Using the fluorescent dye DCFH-DA, we further measured ROS production changes of cells induced with enrofloxacin. As shown in Fig. 2, the cells exposed to 200 mg/mL enrofloxacin significantly increased ROS while those exposed to 12.5, or 25 mg/mL enrofloxacin significantly decreased (P < 0.05) compared to control cells. Additionally, ROS generation in cells treated with 50, 100, and 200 mg/mL enrofloxacin was significantly higher than that of 12.5, 25 mg/mL enrofloxacin (P < 0.05).
Fig. 1. The effects of enrofloxacin on LDH release in hepatic cell of grass carp. Note: Data are expressed as mean þ SE (n ¼ 9). The letters above the bars indicate significant differences (P < 0.05) in different groups calculated using Duncan's multiple range test.
Fig. 3. The effects of enrofloxacin on total antioxidant capacity (A), superoxide dismutase activity (B), and malonaldehyde (C) in hepatic cell of grass carp. .Note: Data are expressed as mean þ SE (n ¼ 9). Legends are the same as in Fig. 1.
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3.3. Antioxidant capacity of enrofloxacin on hepatic cell of grass carp
after 24 h of exposure to 12.5, 25, 50, 100 and 200 mg/mL enrofloxacin than that of control cells (Fig. 4C; P < 0.05).
We examined the effect of enrofloxacin on the anti-oxidization enzymes of hepatic cells and the results are shown in Fig. 3.T-AOC in the cells treated with 50, 100, and 200 mg/mL enrofloxacin was significantly lower than that of the control group (Fig. 3A; P < 0.05). SOD activity in cells treated with 25, 50, and 100 mg/mL enrofloxacin was significantly higher than that of the control group (Fig. 3B; P < 0.05). However, MDA in cells treated with 25, 50, 100, and 200 mg/mL enrofloxacin significantly increased compared to the control cells. (Fig. 3C; P < 0.05).
4. Discussion
3.4. Assessment of enrofloxacin-induced apoptosis We also examined the effect of enrofloxacin on apoptosis and
DJm in the hepatic cells of grass carp (Fig. 4A). We found that the percentage of apoptotic cells in cells treated with 25, 50, 100, and 200 mg/mL enrofloxacin was significantly higher compared to the control cells (Fig. 4A and B; P < 0.05). In addition, the percentage of apoptotic cells exposed to 50, 100, and 200 mg/mL enrofloxacin was significantly higher than those exposed to 12.5 or 25 mg/mL enrofloxacin (Fig. 4B; P < 0.05). DJm in L8824 cells was much lower
Fig. 4. The effects of enrofloxacin on apoptosis (A), quantitative analyses of apoptosis (B) and mitochondrial membrane potential (C) in hepatic cell of grass carp. Note: Data are expressed as mean þ SE (n ¼ 3). Legends are the same as in Fig. 1.
Enrofloxacin is a synthetic chemotherapeutic medicine that has been extensively used to control bacterial pathogens and its use is increasingly leading to potential drug resistance and this would have a negative effect on the subsequent disease control in aquaculture, which has led to widespread public concern of human health and food safety [43]. In the present study, the grass carp hepatic cell line L8824 was used to study the cytotoxic effects of enrofloxacin in vitro. Exposure to 50, 100 and 200 mg/mL enrofloxacin for 24 h significantly increased LDH release in a dosedependent manner, which was consistent with previous studies on the hepatocytes of grass carp exposed to 100 and 200 ug/mL enrofloxacin for 1 day [35], chicken tendon cell reacted with 50e300 ug/mL enrofloxacin for 3 days [44] and horse tendon cells treated with 50 and 100 ug/mL enrofloxacin for 3 days [45]. The different toxicity of enrofloxacin in vitro might be due to the reaction time among the drug, cell cultivation and the species of the cultured cells [46]. The cellular damage caused by enrofloxacin was further confirmed by apoptosis assays. In our study, we demonstrated that enrofloxacin-induced apoptosis in the hepatocytes of grass carp occurred in a dose-dependent manner. This was consistent with finding that enrofloxacin can trigger apoptosis in vitro. Lim et al. [21] observed that canine tendon cells and chondrocytes treated with 200 ug/mL enrofloxacin for 4 days exhibited apoptotic features and fragmentation of DNA. These data emphasize that enrofloxacin leads to unavoidable apoptotic cell death. The signaling mechanisms that regulate apoptosis are very complicated. Generally there are two main apoptotic pathways, the mitochondrial pathway and the death receptor pathway. Mitochondrial changes are regarded as the central role in the apoptosis pathway in many experimental systems, include the enhanced production of oxygen radicals and loss of DJm [47,48]. To understand the role of apoptosis in grass carp hepatic cells following exposure to enrofloxacin, we examined the changes of parameters such as ROS, SOD, T-AOC, MDA, DJm in the apoptotic cells in response to enrofloxacin at different concentrations. ROS are chemically reactive molecules containing superoxide anion radicals and hydrogen peroxide [49]. Studies have implicated ROS as central mediators in the induction of apoptosis by many diverse cytotoxic stimuli [50]. The mechanisms driving apoptosis in cells under oxidative stress may involve high ROS levels, which directly inhibit caspase activity, disrupt intracellular Ca2þ homeostasis, and lead to ATP depletion [51]. In the present study, the highest concentration of 200 mg/mL enrofloxacin significantly increased ROS compared to control cells. This result was consistent with earlier findings reported in the literature that in many cases, drug-induced apoptosis is accompanied by elevation of cellular levels of ROS [27,50,52e54]. As a result, our study indicated the ROS was probably responsible for enrofloxacin-induced apoptosis, that was, oxidative stress likely played a role in enrofloxacin-induced apoptosis in hepatic grass carp cells. ROS can rapidly be removed by enzymatic and non-enzymatic antioxidants, thereby maintaining a healthy pro-oxidant/ antioxidant homeostasis [55]. The activation of endogenous antioxidant enzymes and non-enzymatic antioxidants, such as SOD and T-AOC, respectively, has a protective role against the damage of lipid peroxidation [56,57]. Meanwhile, the generation of excessive ROS results in lipid oxidation and produces a large amount of aldehydes, alcohols and hydrocarbon of which MDA is a substance with strong biotoxicity, which may hurt organisms [58]. In rats,
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chloramphenicol induced lipid peroxidation and a significant decrease of glutathione-S-transferase, glutathione peroxidase and catalase activities in hepatic microsomal components [59,60]. In aquatic animal, the animals treated with enrofloxac caused the oxidative stress [61,62]. In the present study, we found that 50, 100, and 200 mg/mL enrofloxacin significantly reduced T-AOC and enhanced MDA. This may indicate a deficiency in some enzymatic antioxidants or severe oxidative injury [63]. Therefore, the oxidative effect of higher concentrations of enrofloxacin may be attributed to the loss of the affected cell's ability to maintain the activity of its radical-scavenging enzymes. In addition, the current study demonstrated that SOD significantly increased in the treatments of 25, 50 mg/mL enrofloxacin, which was the relationship of loss of the ROS in concentrations of 12.5 or 25 mg/mL enrofloxacin and contributed to attempt to maintain its redox balance. However, these mechanisms may not be able to compensate for the increased oxidative stress induced by the highest concentration of 200 mg/mL enrofloxacin in this study since ROS levels remained high. Loss of DJm has been an established indicator of mitochondrial damage in the progression of apoptosis [48]. To investigate the role of DJm in enrofloxacin-induced apoptosis, the mitochondriaspecific dye Rh123 was employed, and FACS analysis was conducted. Our data showed the concentrations of 12.5, 25, 50, 100 and 200 mg/mL enrofloxacin after 24 h exposure reduced the DJm in L8824 cells compared with the control cell and the DJm declined to approximately 60e75% of control levels within 24 h of treatment. It seemed that DJm decreased in a dose-dependent manner in enrofloxacin -treated cells and a drop in DJm was a responsible for the enhancement of enrofloxacin-induced apoptosis, which was consistent with previous studies that drug-induced apoptosis is accompanied by a fall in the DJm of grass carp cell [27,52,53].
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[15] [16] [17] [18]
5. Conclusion [19]
In conclusion, the present study demonstrated that enrofloxacin exhibited cytotoxic effects in hepatic cell line of grass carp because of the induction of apoptosis. Furthermore, we were able to ascertain that intracellular ROS and DJm played important roles in enrofloxacin-induced apoptosis. The concentration of 200 mg/mL enrofloxacin produced ROS, which altered the subcellular redox equilibrium, reduced DJm and subsequently triggered apoptosis in the hepatic cells of grass carp. This finding provides a new understanding of the cytotoxic effects on fish cells caused by enrofloxacin.
[21]
Acknowledgments
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This work was supported by the Modern Agriculture Industrial Technology System special project-the National Technology System for Conventional Freshwater Fish Industries (CARS-46), the Three New Projects of Fishery in Jiangsu province, and Special Fund for Agro-Scientific Research in the Public Interest (D2013-5). References
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