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ARE is the potential pathway to protect Sprague–Dawley rats against oxidative stress induced by quinocetone
Regulatory Toxicology and Pharmacology 66 (2013) 279–285
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Nrf2/ARE is the potential pathway to protect Sprague–Dawley rats against oxidative stress induced by quinocetone Miao Yu a,b, Mengjing Xu a,b, Yang Liu a,b, Wei Yang a,b, Ying Rong a,b, Ping Yao a,b, Hong Yan b, Di Wang a,b,⇑, Liegang Liu a,b,⇑ a Department of Nutrition and Food Hygiene, Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China b Ministry of Education Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China
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Article history: Received 12 January 2013 Available online 26 April 2013 Keywords: Quinocetone Nephrotoxicity ROS DNA Damage Nrf2
a b s t r a c t 3-methyl-2-quinoxalin benzenevinylketo-1, 4-dioxide (Quinocetone, QCT) is a newly used veterinary drug which has been proven to promote feed efficiency and growth of animals; however, its potential toxicity can’t be ignored. Therefore, the present study was aimed to investigate the nephrotoxicity of QCT and the oxidative stress induced by it. Sprague–Dawley rats (SD rats) were randomly divided into four groups with doses of 2400, 800, 50 and 0 mg/kg/day with administration of QCT for 4 weeks. Results proved that QCT could induce nephrotoxicity and this phenomenon had dose dependent manner. Simultaneously, this phenomenon was accompanied by intracellular reactive oxygen species (ROS) accumulation, enhanced lipid peroxidation and inhibited antioxidant system, i.e. glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GSH). Additionally, the higher expression of Nrf2 in QCT treated groups illustrated that QCT-induced oxidative stress would be partly mitigated by the induction of phase II detoxifying enzymes via increasing Nrf2 expression. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction 3-methyl-2-quinoxalin benzenevinylketo-1,4-dioxide (Quinocetone, QCT) is a newly used veterinary drug in P.R. China which has been proven to promote feed efficiency and growth of animals (Li et al., 2007). Furthermore, QCT can inhibit many kinds of intestinal pathogens, such as Staphylococcus aureus, Esherichia coli, Clay burt’s coli, Dysentery bacterium, etc. (Zhou, 2005). Nevertheless, recent studies found that QCT induced a lot of adverse effects both
Abbreviations: QCT, quinocetone; ROS, reactive oxygen species; SD rats, Sprague–Dawley rats; QdNOs, quinoxaline-1,4-dioxides family; GST, glutathione S-transferase; HO-1, heme oxygenase-1; GPx, glutathione peroxidase; GCL, glutamate cysteine ligase; PRX I, peroxiredoxin I; H-QCT, high QCT group (2400 mg/kg/ day); M-QCT, media QCT group (800 mg/kg/day); L-QCT, low QCT group (50 mg/kg/ day); BUN, blood urea nitrogen; Cr, creatinine; MFD, mean fluorescence density; GSH, glutathione reductase; MDA, malonaldehyde; SOD, superoxide dismutase; CAT, catalase; OTM, olive tail moment; TBS, tris base solution; BSA, bovine serum albumin; PI, propidium iodide; FITC, fluorescein isothiocyanate; SEM, standard error of the mean; ANOVA, one-way analysis of variance for homogeneity; Keap1, kelch-like ECH-associated protein 1; ARE, antioxidant response element. ⇑ Corresponding authors at: Department of Nutrition and Food Hygiene, Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China. Fax: +86 27 83650522. E-mail addresses: [email protected] (D. Wang), [email protected] (L. Liu). 0273-2300/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yrtph.2013.04.005
in vivo and in vitro. In Vero cells, the cell viability result indicated that QCT could lead to severe inhibitory effects in both dose and time dependent manner and meanwhile comet assay illustrated that QCT enhanced DNA damage (Chen et al., 2009). Similarly in human hepatoma cells, the cell viability test and comet assay indicated that QCT inhibited cell proliferation and induced significant DNA fragment migration (Jin et al., 2009). On the other hand, in vivo tests indicated that, when feeding QCT (1800 mg/kg) to Wistar rats, body weights, feed efficiency, fetal body lengths, tail lengths, litter weights and number of viable fetuses all significantly decreased (Wang et al., 2012). Evaluating sub-chronic oral toxicological test in Wistar rats with dose of 1800 mg/kg/day, researchers discovered that QCT could induce renal damage (Wang et al., 2010). Previous literatures mainly mentioned about the adverse effects of QCT. However, related toxicological mechanisms are not yet clear. Some members of QdNOs (i.e. olaquindox, mequindox and cyadox) have been proven to induce DNA damage in vitro. It was associated with oxidative stress due to excessive ROS generation (Huang et al., 2010). As the member of QdNOs, in our prior study, we already demonstrated that QCT would induce ROS generation and oxidative DNA damage as well. Simultaneously, we found the antioxidant supplementation could attenuate this phenomenon (Wang et al., 2011). In this study, we aimed to find out the cell defense system to protect itself from oxidative damage.
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The Nrf2-Keap1 system is a major cellular defense mechanism against oxidative stress. Nrf2 activates genes that encode phase II detoxifying enzymes and antioxidant enzymes, such as GST, HO1, GPx, GCL and PRX I, which play crucial roles in cellular defense by improving the removal of ROS (Pedruzzi et al., 2012). In addition, a recent study showed that ginsenoside Rb1 attenuated acute renal injury induced by intestinal ischemia reperfusion by activating the Nrf2/ARE pathway (Sun et al., 2012). Consequently, this time we focused on considering nephrotoxicity of QCT and the oxidative stress induced by it. Simultaneously, we discussed the cell defense system which could mitigate or repair ROS-induced cell damage. 2. Materials and methods 2.1. Materials QCT (C18H14N2O3, purity > 98%) was provided by Hubei Zhongmu Anda Pharmaceutical Co., Ltd. (Wuxue, Hubei, China). The chemical structures of QCT and its metabolisms had been reported in detail (Shen et al., 2010). 2.2. Animals and treatment A total of 40 male SD rats were procured from Sino-British Sippr/ BK (Shanghai, China) and used after 1 week acclimatization. In order to ensure the health of each animal, the body weights and detailed physical examinations were recorded twice during the acclimation period. An independent cage was applied for every single animal. Feed and tap water were provided freely during the non-exposure periods. A Specific-Pathogen Free (SPF) level room in which the light–dark cycle (12–12 h, lights on 7:00–19:00), ventilation (airexchange rate of 18 times per hour), temperature (23 ± 2 °C) and relative humidity (55 ± 5%) were strictly controlled was supplied for the animals during the study. The cages and the chip bedding were exchanged twice a week. The use of animals in this study was in accordance with ‘‘Guidelines for the Care and Use of Laboratory Animals, 1996’’ that prepared by National Institute of Health. 2.3. Study design The SD rats were randomly divided into four groups and treated with QCT by gavage. The doses of QCT used in the present study were calculated according to the LD50 given in the previous study (Wang et al., 2010). They were 2400 mg/kg/day (H-QCT), 800 mg/kg/day (M-QCT), 50 mg/kg/day (L-QCT) and 0 mg/kg/day (control) respectively. Clinical observations were recorded daily, food consumption and body weights were measured weekly. Serum from blood samples was collected and stored at 80 °C after the 4-week administration. In each group, sections of one side kidney were placed in 10% neutral buffered formalin and stained with hematoxylin and eosin (H&E). Sections of another side kidney were washed with ice cold isotonic saline. Finally all sections were stored at 80 °C after been drying out (by blotting between two pieces of filter paper). 2.4. BUN and Cr levels in serum Serum was centrifuged at 3500g for 10 min at 4 °C (Eppendorf centrifuge 5804R, Germany). Then the BS-200 automatic biochemistry analyzer (Mindary Co., Ltd.) was used to measure the BUN and Cr levels in serum. 2.5. ROS in renal cells Dihydroethidium (Invitrogen, Corporation) was used to measure ROS in the renal cells (Carter et al., 1994). 10 lm cross-
sections of unfixed, frozen kidney tissues were incubated with 5 lmol dihydroethidium (diluted in PBS) at 37 °C for 15 min. Using a Nikon 2000S fluorescence microscope (Nikon, Melville, NY) to observe the slides that were washed twice by ice-cold PBS. Three samples of kidney in each group were detected and the MFD was calculated as fluorescence intensity per unit area and assayed using Image-pro Plus 5.0 (Media Cybernetics, Corporation). 2.6. Antioxidant capacity biomarkers in blood Carefully weighing the sections of kidneys which had been stored at 80 °C, then the sections were homogenized in ice cold 50 mmol/l phosphate buffer (containing 0.1 mmol/l EDTA). In order to remove all the cell debris and nuclei, the homogenate was centrifuged at 3500g for 10 min at 4 °C (Eppendorf centrifuge 5804R, Germany). The final supernatant was stored at 20 °C for kinds of biochemical assays. Then the protein concentrations of the tissue homogenate samples were measured according to the prior study (Lowry et al., 1951). The levels of GSH and MDA and the activities of SOD, GPx, CAT and GST were measured using commercial assay kits (Nanjing Jiancheng Institute, China). All the procedures were performed in accordance with the manufacturer’s instructions. The levels of GSH and MDA were expressed as mg/g protein and nmol/mg protein respectively. Besides, the activities of SOD, GPx, CAT and GST were all expressed as units/mg protein. 2.7. Comet assay in vivo The Comet assay was mainly performed according to our prior study (Wang et al., 2011). The isolation of nephric cells was basically conducted (Sasaki et al., 1997). Each cell suspension was chosen three slides to prepare. The microscope slides were first layered with 0.8% normal melting-point agarose (Sigma, USA) and the cell suspensions that embedded in a layer of 0.5% of low melting-point agarose (Sigma, USA) were placed onto it. Then the slides were immersed in a lysing solution for 1 h at 4 °C, afterwards they were removed and placed on a horizontal gel electrophoresis unit (filled with freshly prepared alkaline electrophoresis buffer, 1 mM EDTA and 300 mM NaOH, pH > 13). In this unit, DNA was unwound and shown as single-strand breaks. Next, electrophoresis was conducted for 20 min at 0–4 °C by applying an electric current of 0.7 V/cm (25 V/300 mA). After electrophoresis, the slides were immediately neutralized with 0.4 M Tris (pH 7.5) and then they were air-dried and stored at room temperature until scored for DNA migration. After being stained with ethidium bromide, 150 randomly selected cells were analyzed under a Nikon 2000S fluorescence microscope (Nikon, Melville, NY) (magnification 400) equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm. The microscope was connected through a camera to a computer-based image analysis system (Comet Assay IV software, Perspective Instruments). Three samples of kidney in each group were detected. The OTM (Olive Tail Moment) and Tail DNA% were calculated by Comet Assay Software Project (CASP) 1. 2. 2 (University of Wroclaw, Poland). OTM was first presented by Olive, which was calculated by multiplying the percent of DNA (fluorescence) in the tail by the length of the tail. And the tail length is measured between the edge of Comet head and the end of the Comet tail (Olive et al., 1990; Nesslany et al., 2007). 2.8. Nrf2 expression by confocal microscopy The Nrf2 expression in kidney was determined with primary antibody rabbit polyclonal IgG Nrf2 [Cat No. sc-13032] and goat anti-rabbit–FITC-conjugated secondary antibody and confocal microscopy (Sriram et al., 2009). Briefly, at first the renal sections
M. Yu et al. / Regulatory Toxicology and Pharmacology 66 (2013) 279–285
were incubated with TBS (at a concentration of 1 mg/ml with 3% BSA) overnight at 4 °C, and the sections were washed three times using TBS. After that, the sections were incubated with secondary antibody for 2 h at room temperature before counterstaining with PI. FITC and PI were excited at 488 nm and 529 nm respectively for observation under a confocal microscopy (Carl Zeiss LSM510 META, Oberkochen, Germany). 2.9. Statistical analysis Variances in data for ROS, GSH, MDA, GPx, SOD, CAT and GST were presented as mean ± SEM. If the variances were homogeneous, ANOVA was used. If the variances were heterogeneous, the Kruskal–Wallis non-parametric ANOVA was applied. When statistically significant differences were indicated, the Dunnett’s multiple tests were employed for comparisons between control and treated groups. Since the OTM and tail DNA% frequencies did not follow a Gaussian distribution, the statistical significance of differences in the median values between each group versus the control was determined with the non-parametric Mann–Whitney U-test. All data were analyzed by SPSS 12.0 statistical software (SPSS Inc., Chicago, IL) and a difference was considered significant when P < 0.05. 3. Results During the study, all rats survived and no obviously significant changes were observed in mortality, illness and clinical signs. 3.1. Body weight and food consumption Throughout the whole study, the levels of mean body weight and food consumption of SD rats were shown in Figs. 1 and 2 respectively. H-QCT had significant decrease in body weight and food consumption compared with control from week 1 to week 4. In the same way, M-QCT had a significant difference in body weight throughout the whole study. However, significant difference appeared at the first week when compared M-QCT with control in food consumption, then this influence disappeared from week 2 to the end of the study. However, 50 mg/kg/day QCT had not been observed any influence on body weight and food consumption throughout the study period. Furthermore, significant differences was discovered in body weight and food consumption when compared any two QCT-treated groups (except L-QCT and M-QCT in food consumption).
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3.2. Histopathological changes in the kidney The sections stained with H&E were shown in Fig. 3. Histology of renal tissue from the control showed normal architecture (A). Increased Bowman’s capsule space, slight glomerulus degenerative atrophy, inflammatory cell infiltration, congested glomerular capillaries, vesicle pole damage and constriction of distal tubule were observed in renal tissue of rats in H-QCT (B). As for M-QCT and L-QCT, renal was shown little difference with control. 3.3. Biochemistry levels in serum BUN and Cr levels in serum reflect the function of kidney (Chen et al., 2013). By detecting BUN and Cr levels in serum of the rats, significant increases of Cr were observed in both H-QCT and MQCT when compared with control (P < 0.05) (Table 1), while no significant differences were observed when comparing all other groups with control respectively. 3.4. ROS in renal cells In order to observe oxidative stress, the level of ROS in renal cells was measured (Fig. 4.). Significant increase of ROS was observed in H-QCT and M-QCT when compared with control respectively (P < 0.05). This phenomenon directly reflected the upregulated oxidative stress in these two QCT-treated groups. 3.5. Antioxidant capacity biomarkers in blood Significant differences in the levels of MDA (both H-QCT and MQCT) and GSH (H-QCT) as well as in the activities of CAT (both HQCT and M-QCT), GPx (H-QCT) and GST (both H-QCT and M-QCT) were observed when compared with control (Fig. 5.). Moreover, significant differences were also discovered when compared MQCT with H-QCT in MDA, CAT and GST respectively. However, no significant difference in SOD activity was found among any QCT group and control. Moreover, no influence on antioxidant capacity biomarkers in blood of SD rats in 50 mg/kg/day QCT group had been observed during the whole study. 3.6. Comet assay in vivo Comet assay reflects the DNA damage of renal cells (Singh et al., 1988). As we described before, alkaline solution was used to distinguish DNA single-strand breaks. Therefore, from Table 2, we found a significant increase in OTM and tail DNA% values comparing
Fig. 1. Mean body weights of SD rats following dietary exposure to quinocetone in 4 weeks.
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Fig. 2. Mean feed consumption of SD rats following dietary exposure to quinocetone in 4 weeks.
Fig. 3. Histopathological changes in the kidney of SD rats at 400. Control (A) compared with H-QCT (B).
Table 1 Renal damage measured by BUN and Cr in serum.
3.7. Nrf2 expression by confocal microscopy
Group
BUN (lmol/L) Cr (mmol/L)
H-QCT
M-QCT
L-QCT
Control
4.61 ± 0.43 55.11 ± 4.1⁄
5.24 ± 0.64 55.00 ± 3.08⁄
5.29 ± 0.46 46.21 ± 7.87
4.99 ± 0.55 49.26 ± 4.61
The values were expressed as means ± SEM (n = 10). Significant statistical difference was indicated by: ⁄ P < 0.05.
Through the confocal microscopy (Fig. 6), it was obvious that the level of Nrf2 expression in H-QCT was much higher than in control. Meanwhile we noticed that the structure of kidney in control was easily to be distinguished, which means kidney in control was still in normal shape. While kidney in H-QCT lost its common shape that made it difficult to be observed.
4. Discussion H-QCT with control. However, M-QCT and L-QCT had not been observed any significant difference in OTM and tail DNA% values.
Since QCT was found having the ability of promoting the growth of animals, it was soon widely used as a veterinary drug in
Fig. 4. ROS in renal cells of SD rats following dietary exposure to quinocetone in 4 weeks. Significant statistical difference was indicated by: treated group compared with control.
⁄⁄
means P < 0.05 when QCT
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Fig. 5. Antioxidant capacity biomarkers in blood of SD rats. Significant statistical difference was indicated by: control. NN means P < 0.05 when M-QCT compared with H-QCT.
P.R. China to promote the growth and feed efficiency of animals (Li et al., 2007). QCT was supposed to replace carbadox and olaquindox which had lots of side effects on animals as a new promising veterinary drug. However, as the toxicity of other QdNOs (such as carbadox, olaquindox and cyadox) has already been reported (Chen et al., 2009; Wang et al., 2011), the toxicity of QCT was particularly concerned. In the former study (Wang et al., 2010), it was proven that the no-observed-adverse-effect level of quinocetone was considered to be 300 mg/kg diet. In P.R. China, the dose of QCT used as veterinary drug is below 300 mg/kg, and no side effects are observed up to now. However, in our study, we used relatively higher doses to induce the negative effects. We aimed to seek out the potential damage of QCT and present corresponding measures to protect animals. In previous studies, QCT was reported to cause genotoxicity in Vero cells (Chen et al., 2009), cytotoxicity in porcine adrenocortical cells (Huang et al., 2010) and oxidative DNA damage in Balb/c mice (Wang et al., 2011). In the present study, we investigated the nephrotoxicity of QCT and its probable mechanism. Table 2 DNA damage measured by the comet assay in isolated renal cells after administration of QCT. Group
H-QCT M-QCT L-QCT **
OTM
Tail DNA%
Median
P25–P75
Median
P25–P75
3.96** 0.36 0.29
3.29–4.71 0.16–0.63 0.01–0.90
29.18** 3.34 2.86
22.40–37.02 1.46–8.35 0.07–8.70
P < 0.01, significant different (Mann–Whitney U -test) versus vehicle control.
⁄⁄
283
means P < 0.05 when QCT treated group compared with
Kidney is an extremely important organ that maintains the internal environment of the body. Nevertheless, kidney can be damaged by kinds of factors, such as drugs, chemicals, acute injures, etc. Besides of the levels of BUN and Cr in serum, we also used SOD, CAT and other antioxidant capacity biomarkers in blood to detect oxidative stress and nephrotoxicity (Waring and Moonie, 2011). These biomarkers are more sensitive than BUN and Cr. In present study, we focused on the effects caused by different doses of QCT. Taking 50 mg/kg/day QCT orally had little adverse effects on SD rats for all indexes we measured. The reason was that the no-observed-adverse-effect level of quinocetone in Wistar rats was considered to be 300 mg/kg/day (Wang et al., 2010). QCT-induced kidney injure could lead to disturbance of internal environment. This disturbance eventually resulted in the lower body weight and food consumption in M-QCT and H-QCT. Cr and BUN, as the products of metabolism, are mainly eliminated from the body by the glomerular filtration. As to say the levels of them reflect the function of kidney. The levels of Cr in H-QCT and M-QCT were significantly higher than control (P < 0.05), which represented the decreased kidney function. No significant difference was discovered in BUN. This mainly because of its relatively lower sensitivity. In other words, BUN may be able to maintain in normal range in the early stage of renal impairment, and the measurable changes can only be detected in the late stage of renal impairment. ROS, such as superoxide (O2 ), hydrogen peroxide (H2O2) and hydroxyl radicals (OH), is a series of reactive oxygen species produced by aerobic cells in metabolic processes. Accumulative evidence has shown that for lots of factors, inducing the generation of ROS is the key process to cause renal impairment (Liu et al.,
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Fig. 6. Analysis of Nrf2 expression in kidney of control (D–F) and H-QCT (A–C) by confocal microscopy at 400. Green represented Nrf2-FITC stained sections (A and D), red represented images of PI (B and E). C was the overlay of A and B, meanwhile F was the overlay of D and E. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2010; Shah and Iqbal, 2010) and DNA damage (Ahamed and Siddiqui, 2007). As a radical scavenger, the significant decrease of GSH in H-QCT reflected the ROS generation. Simultaneously, the significant increase of MDA (final products of peroxidation) also confirmed this theory. Additionally, significant differences between H-QCT and control in OTM and Tail DNA% could directly reflect DNA damage in renal cells. In brief, when the oxidative stress is activated, increasing ROS causes organ impairment and DNA damage. Nevertheless, cells have various defense systems to mitigate or repair ROS-induced cell damage. As already stated, GSH is one of the antioxidant enzymes which can scavenge ROS. Furthermore, many other enzymes can scavenge ROS as well. Consequently, induction of phase II detoxifying enzymes or antioxidative enzymes is one pivotal defense system to mitigate or repair ROS-induced cell damage (Zhang and Gordon, 2004). As a widely known antioxidative pathway, Nrf2/ARE is a major cellular defense mechanism which prevents damage from ROS generation through induction of antioxidative phase II enzymes such as GST and NAD(P)H, etc. (Boddupalli et al., 2012; Boettler et al., 2012). Under normal circumstances, Nrf2 and the Keap1 which acts as a negative regulator of Nrf2 are both presented in the cytosol. When electrophiles or ROS are generating, Nrf2 dissociates from Keap1 and translocates into the nucleus where it dimerizes with small Maf binding proteins. Binding of these heterodimers to ARE ultimately activates ARE-dependent gene expression (Kobayashi and Yamamoto, 2005; Li and Kong, 2009). Subsequently, the phase II detoxifying enzymes or antioxidative enzymes are generated and these enzymes eventually protect cells from ROS generation. In present study, the higher expression of Nrf2 in H-QCT illustrated that QCT-induced oxidative stress would be partly mitigated by the induction of phase II detoxifying enzymes via increasing Nrf2 expression.
As mentioned, SOD, CAT and GPx are very important antioxidant enzymes. The SOD decomposes superoxide radicals (O2 ) and produces H2O2. H2O2 is subsequently removed to water by CAT in the peroxisomes, or by GPx oxidizing GSH in the cytosol (Droge, 2002; Lee and Choi, 2003). When the expression of Nrf2 is up-regulated, the activity of these antioxidant enzymes should increase as well (Jung and Kwak, 2010; Ishikado et al., 2012). However, in present study, the activities of antioxidant enzymes (GPx, CAT and GST) decreased instead. The reason might be that in one hand the intermediate products of lipid peroxidation directly decreased the activities of GPx, CAT and GST, and in the other hand these intermediate products damaged cell structure that continuous activation of the Nrf2/ARE pathway was suppressed in the late stage of oxidative stress. We will verify this idea in the future with more experiments.
5. Conclusion Production of ROS is the key mediator of QCT-induced nephrotoxicity and DNA damage. Furthermore, on one hand, the enzymes activities are correlated with nephrotoxicity. On the other hand, induction of phase II detoxifying enzymes or antioxidative enzymes is one of pivotal mechanisms that mitigate or repair ROS-induced organ and cell damage. With present evidence, we thought the Nrf2/ARE pathway might be a major cellular defense mechanism which could protect cell damage against ROS generation. Nevertheless, in this manuscript the current use of QCT lacked a specific standard to limit its usage. Furthermore, different animals exist metabolic differences, it’s necessary to establish dose limits of QCT for different animals. Above all, in further study, we desire to precede our research on the Nrf2/ARE pathway and try our best to solve these problems and exhibit its molecular mechanism clearly.
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Nrf2/ARE is the potential pathway to protect Sprague–Dawley rats against oxidative stress induced by quinocetone Miao Yu a,b, Mengjing Xu a,b, Yang Liu a,b, Wei Yang a,b, Ying Rong a,b, Ping Yao a,b, Hong Yan b, Di Wang a,b,⇑, Liegang Liu a,b,⇑ a Department of Nutrition and Food Hygiene, Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China b Ministry of Education Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China
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Article history: Received 12 January 2013 Available online 26 April 2013 Keywords: Quinocetone Nephrotoxicity ROS DNA Damage Nrf2
a b s t r a c t 3-methyl-2-quinoxalin benzenevinylketo-1, 4-dioxide (Quinocetone, QCT) is a newly used veterinary drug which has been proven to promote feed efficiency and growth of animals; however, its potential toxicity can’t be ignored. Therefore, the present study was aimed to investigate the nephrotoxicity of QCT and the oxidative stress induced by it. Sprague–Dawley rats (SD rats) were randomly divided into four groups with doses of 2400, 800, 50 and 0 mg/kg/day with administration of QCT for 4 weeks. Results proved that QCT could induce nephrotoxicity and this phenomenon had dose dependent manner. Simultaneously, this phenomenon was accompanied by intracellular reactive oxygen species (ROS) accumulation, enhanced lipid peroxidation and inhibited antioxidant system, i.e. glutathione S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GSH). Additionally, the higher expression of Nrf2 in QCT treated groups illustrated that QCT-induced oxidative stress would be partly mitigated by the induction of phase II detoxifying enzymes via increasing Nrf2 expression. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction 3-methyl-2-quinoxalin benzenevinylketo-1,4-dioxide (Quinocetone, QCT) is a newly used veterinary drug in P.R. China which has been proven to promote feed efficiency and growth of animals (Li et al., 2007). Furthermore, QCT can inhibit many kinds of intestinal pathogens, such as Staphylococcus aureus, Esherichia coli, Clay burt’s coli, Dysentery bacterium, etc. (Zhou, 2005). Nevertheless, recent studies found that QCT induced a lot of adverse effects both
Abbreviations: QCT, quinocetone; ROS, reactive oxygen species; SD rats, Sprague–Dawley rats; QdNOs, quinoxaline-1,4-dioxides family; GST, glutathione S-transferase; HO-1, heme oxygenase-1; GPx, glutathione peroxidase; GCL, glutamate cysteine ligase; PRX I, peroxiredoxin I; H-QCT, high QCT group (2400 mg/kg/ day); M-QCT, media QCT group (800 mg/kg/day); L-QCT, low QCT group (50 mg/kg/ day); BUN, blood urea nitrogen; Cr, creatinine; MFD, mean fluorescence density; GSH, glutathione reductase; MDA, malonaldehyde; SOD, superoxide dismutase; CAT, catalase; OTM, olive tail moment; TBS, tris base solution; BSA, bovine serum albumin; PI, propidium iodide; FITC, fluorescein isothiocyanate; SEM, standard error of the mean; ANOVA, one-way analysis of variance for homogeneity; Keap1, kelch-like ECH-associated protein 1; ARE, antioxidant response element. ⇑ Corresponding authors at: Department of Nutrition and Food Hygiene, Hubei Key Laboratory of Food Nutrition and Safety, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Road, Wuhan 430030, China. Fax: +86 27 83650522. E-mail addresses: [email protected] (D. Wang), [email protected] (L. Liu). 0273-2300/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yrtph.2013.04.005
in vivo and in vitro. In Vero cells, the cell viability result indicated that QCT could lead to severe inhibitory effects in both dose and time dependent manner and meanwhile comet assay illustrated that QCT enhanced DNA damage (Chen et al., 2009). Similarly in human hepatoma cells, the cell viability test and comet assay indicated that QCT inhibited cell proliferation and induced significant DNA fragment migration (Jin et al., 2009). On the other hand, in vivo tests indicated that, when feeding QCT (1800 mg/kg) to Wistar rats, body weights, feed efficiency, fetal body lengths, tail lengths, litter weights and number of viable fetuses all significantly decreased (Wang et al., 2012). Evaluating sub-chronic oral toxicological test in Wistar rats with dose of 1800 mg/kg/day, researchers discovered that QCT could induce renal damage (Wang et al., 2010). Previous literatures mainly mentioned about the adverse effects of QCT. However, related toxicological mechanisms are not yet clear. Some members of QdNOs (i.e. olaquindox, mequindox and cyadox) have been proven to induce DNA damage in vitro. It was associated with oxidative stress due to excessive ROS generation (Huang et al., 2010). As the member of QdNOs, in our prior study, we already demonstrated that QCT would induce ROS generation and oxidative DNA damage as well. Simultaneously, we found the antioxidant supplementation could attenuate this phenomenon (Wang et al., 2011). In this study, we aimed to find out the cell defense system to protect itself from oxidative damage.
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The Nrf2-Keap1 system is a major cellular defense mechanism against oxidative stress. Nrf2 activates genes that encode phase II detoxifying enzymes and antioxidant enzymes, such as GST, HO1, GPx, GCL and PRX I, which play crucial roles in cellular defense by improving the removal of ROS (Pedruzzi et al., 2012). In addition, a recent study showed that ginsenoside Rb1 attenuated acute renal injury induced by intestinal ischemia reperfusion by activating the Nrf2/ARE pathway (Sun et al., 2012). Consequently, this time we focused on considering nephrotoxicity of QCT and the oxidative stress induced by it. Simultaneously, we discussed the cell defense system which could mitigate or repair ROS-induced cell damage. 2. Materials and methods 2.1. Materials QCT (C18H14N2O3, purity > 98%) was provided by Hubei Zhongmu Anda Pharmaceutical Co., Ltd. (Wuxue, Hubei, China). The chemical structures of QCT and its metabolisms had been reported in detail (Shen et al., 2010). 2.2. Animals and treatment A total of 40 male SD rats were procured from Sino-British Sippr/ BK (Shanghai, China) and used after 1 week acclimatization. In order to ensure the health of each animal, the body weights and detailed physical examinations were recorded twice during the acclimation period. An independent cage was applied for every single animal. Feed and tap water were provided freely during the non-exposure periods. A Specific-Pathogen Free (SPF) level room in which the light–dark cycle (12–12 h, lights on 7:00–19:00), ventilation (airexchange rate of 18 times per hour), temperature (23 ± 2 °C) and relative humidity (55 ± 5%) were strictly controlled was supplied for the animals during the study. The cages and the chip bedding were exchanged twice a week. The use of animals in this study was in accordance with ‘‘Guidelines for the Care and Use of Laboratory Animals, 1996’’ that prepared by National Institute of Health. 2.3. Study design The SD rats were randomly divided into four groups and treated with QCT by gavage. The doses of QCT used in the present study were calculated according to the LD50 given in the previous study (Wang et al., 2010). They were 2400 mg/kg/day (H-QCT), 800 mg/kg/day (M-QCT), 50 mg/kg/day (L-QCT) and 0 mg/kg/day (control) respectively. Clinical observations were recorded daily, food consumption and body weights were measured weekly. Serum from blood samples was collected and stored at 80 °C after the 4-week administration. In each group, sections of one side kidney were placed in 10% neutral buffered formalin and stained with hematoxylin and eosin (H&E). Sections of another side kidney were washed with ice cold isotonic saline. Finally all sections were stored at 80 °C after been drying out (by blotting between two pieces of filter paper). 2.4. BUN and Cr levels in serum Serum was centrifuged at 3500g for 10 min at 4 °C (Eppendorf centrifuge 5804R, Germany). Then the BS-200 automatic biochemistry analyzer (Mindary Co., Ltd.) was used to measure the BUN and Cr levels in serum. 2.5. ROS in renal cells Dihydroethidium (Invitrogen, Corporation) was used to measure ROS in the renal cells (Carter et al., 1994). 10 lm cross-
sections of unfixed, frozen kidney tissues were incubated with 5 lmol dihydroethidium (diluted in PBS) at 37 °C for 15 min. Using a Nikon 2000S fluorescence microscope (Nikon, Melville, NY) to observe the slides that were washed twice by ice-cold PBS. Three samples of kidney in each group were detected and the MFD was calculated as fluorescence intensity per unit area and assayed using Image-pro Plus 5.0 (Media Cybernetics, Corporation). 2.6. Antioxidant capacity biomarkers in blood Carefully weighing the sections of kidneys which had been stored at 80 °C, then the sections were homogenized in ice cold 50 mmol/l phosphate buffer (containing 0.1 mmol/l EDTA). In order to remove all the cell debris and nuclei, the homogenate was centrifuged at 3500g for 10 min at 4 °C (Eppendorf centrifuge 5804R, Germany). The final supernatant was stored at 20 °C for kinds of biochemical assays. Then the protein concentrations of the tissue homogenate samples were measured according to the prior study (Lowry et al., 1951). The levels of GSH and MDA and the activities of SOD, GPx, CAT and GST were measured using commercial assay kits (Nanjing Jiancheng Institute, China). All the procedures were performed in accordance with the manufacturer’s instructions. The levels of GSH and MDA were expressed as mg/g protein and nmol/mg protein respectively. Besides, the activities of SOD, GPx, CAT and GST were all expressed as units/mg protein. 2.7. Comet assay in vivo The Comet assay was mainly performed according to our prior study (Wang et al., 2011). The isolation of nephric cells was basically conducted (Sasaki et al., 1997). Each cell suspension was chosen three slides to prepare. The microscope slides were first layered with 0.8% normal melting-point agarose (Sigma, USA) and the cell suspensions that embedded in a layer of 0.5% of low melting-point agarose (Sigma, USA) were placed onto it. Then the slides were immersed in a lysing solution for 1 h at 4 °C, afterwards they were removed and placed on a horizontal gel electrophoresis unit (filled with freshly prepared alkaline electrophoresis buffer, 1 mM EDTA and 300 mM NaOH, pH > 13). In this unit, DNA was unwound and shown as single-strand breaks. Next, electrophoresis was conducted for 20 min at 0–4 °C by applying an electric current of 0.7 V/cm (25 V/300 mA). After electrophoresis, the slides were immediately neutralized with 0.4 M Tris (pH 7.5) and then they were air-dried and stored at room temperature until scored for DNA migration. After being stained with ethidium bromide, 150 randomly selected cells were analyzed under a Nikon 2000S fluorescence microscope (Nikon, Melville, NY) (magnification 400) equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm. The microscope was connected through a camera to a computer-based image analysis system (Comet Assay IV software, Perspective Instruments). Three samples of kidney in each group were detected. The OTM (Olive Tail Moment) and Tail DNA% were calculated by Comet Assay Software Project (CASP) 1. 2. 2 (University of Wroclaw, Poland). OTM was first presented by Olive, which was calculated by multiplying the percent of DNA (fluorescence) in the tail by the length of the tail. And the tail length is measured between the edge of Comet head and the end of the Comet tail (Olive et al., 1990; Nesslany et al., 2007). 2.8. Nrf2 expression by confocal microscopy The Nrf2 expression in kidney was determined with primary antibody rabbit polyclonal IgG Nrf2 [Cat No. sc-13032] and goat anti-rabbit–FITC-conjugated secondary antibody and confocal microscopy (Sriram et al., 2009). Briefly, at first the renal sections
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were incubated with TBS (at a concentration of 1 mg/ml with 3% BSA) overnight at 4 °C, and the sections were washed three times using TBS. After that, the sections were incubated with secondary antibody for 2 h at room temperature before counterstaining with PI. FITC and PI were excited at 488 nm and 529 nm respectively for observation under a confocal microscopy (Carl Zeiss LSM510 META, Oberkochen, Germany). 2.9. Statistical analysis Variances in data for ROS, GSH, MDA, GPx, SOD, CAT and GST were presented as mean ± SEM. If the variances were homogeneous, ANOVA was used. If the variances were heterogeneous, the Kruskal–Wallis non-parametric ANOVA was applied. When statistically significant differences were indicated, the Dunnett’s multiple tests were employed for comparisons between control and treated groups. Since the OTM and tail DNA% frequencies did not follow a Gaussian distribution, the statistical significance of differences in the median values between each group versus the control was determined with the non-parametric Mann–Whitney U-test. All data were analyzed by SPSS 12.0 statistical software (SPSS Inc., Chicago, IL) and a difference was considered significant when P < 0.05. 3. Results During the study, all rats survived and no obviously significant changes were observed in mortality, illness and clinical signs. 3.1. Body weight and food consumption Throughout the whole study, the levels of mean body weight and food consumption of SD rats were shown in Figs. 1 and 2 respectively. H-QCT had significant decrease in body weight and food consumption compared with control from week 1 to week 4. In the same way, M-QCT had a significant difference in body weight throughout the whole study. However, significant difference appeared at the first week when compared M-QCT with control in food consumption, then this influence disappeared from week 2 to the end of the study. However, 50 mg/kg/day QCT had not been observed any influence on body weight and food consumption throughout the study period. Furthermore, significant differences was discovered in body weight and food consumption when compared any two QCT-treated groups (except L-QCT and M-QCT in food consumption).
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3.2. Histopathological changes in the kidney The sections stained with H&E were shown in Fig. 3. Histology of renal tissue from the control showed normal architecture (A). Increased Bowman’s capsule space, slight glomerulus degenerative atrophy, inflammatory cell infiltration, congested glomerular capillaries, vesicle pole damage and constriction of distal tubule were observed in renal tissue of rats in H-QCT (B). As for M-QCT and L-QCT, renal was shown little difference with control. 3.3. Biochemistry levels in serum BUN and Cr levels in serum reflect the function of kidney (Chen et al., 2013). By detecting BUN and Cr levels in serum of the rats, significant increases of Cr were observed in both H-QCT and MQCT when compared with control (P < 0.05) (Table 1), while no significant differences were observed when comparing all other groups with control respectively. 3.4. ROS in renal cells In order to observe oxidative stress, the level of ROS in renal cells was measured (Fig. 4.). Significant increase of ROS was observed in H-QCT and M-QCT when compared with control respectively (P < 0.05). This phenomenon directly reflected the upregulated oxidative stress in these two QCT-treated groups. 3.5. Antioxidant capacity biomarkers in blood Significant differences in the levels of MDA (both H-QCT and MQCT) and GSH (H-QCT) as well as in the activities of CAT (both HQCT and M-QCT), GPx (H-QCT) and GST (both H-QCT and M-QCT) were observed when compared with control (Fig. 5.). Moreover, significant differences were also discovered when compared MQCT with H-QCT in MDA, CAT and GST respectively. However, no significant difference in SOD activity was found among any QCT group and control. Moreover, no influence on antioxidant capacity biomarkers in blood of SD rats in 50 mg/kg/day QCT group had been observed during the whole study. 3.6. Comet assay in vivo Comet assay reflects the DNA damage of renal cells (Singh et al., 1988). As we described before, alkaline solution was used to distinguish DNA single-strand breaks. Therefore, from Table 2, we found a significant increase in OTM and tail DNA% values comparing
Fig. 1. Mean body weights of SD rats following dietary exposure to quinocetone in 4 weeks.
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Fig. 2. Mean feed consumption of SD rats following dietary exposure to quinocetone in 4 weeks.
Fig. 3. Histopathological changes in the kidney of SD rats at 400. Control (A) compared with H-QCT (B).
Table 1 Renal damage measured by BUN and Cr in serum.
3.7. Nrf2 expression by confocal microscopy
Group
BUN (lmol/L) Cr (mmol/L)
H-QCT
M-QCT
L-QCT
Control
4.61 ± 0.43 55.11 ± 4.1⁄
5.24 ± 0.64 55.00 ± 3.08⁄
5.29 ± 0.46 46.21 ± 7.87
4.99 ± 0.55 49.26 ± 4.61
The values were expressed as means ± SEM (n = 10). Significant statistical difference was indicated by: ⁄ P < 0.05.
Through the confocal microscopy (Fig. 6), it was obvious that the level of Nrf2 expression in H-QCT was much higher than in control. Meanwhile we noticed that the structure of kidney in control was easily to be distinguished, which means kidney in control was still in normal shape. While kidney in H-QCT lost its common shape that made it difficult to be observed.
4. Discussion H-QCT with control. However, M-QCT and L-QCT had not been observed any significant difference in OTM and tail DNA% values.
Since QCT was found having the ability of promoting the growth of animals, it was soon widely used as a veterinary drug in
Fig. 4. ROS in renal cells of SD rats following dietary exposure to quinocetone in 4 weeks. Significant statistical difference was indicated by: treated group compared with control.
⁄⁄
means P < 0.05 when QCT
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Fig. 5. Antioxidant capacity biomarkers in blood of SD rats. Significant statistical difference was indicated by: control. NN means P < 0.05 when M-QCT compared with H-QCT.
P.R. China to promote the growth and feed efficiency of animals (Li et al., 2007). QCT was supposed to replace carbadox and olaquindox which had lots of side effects on animals as a new promising veterinary drug. However, as the toxicity of other QdNOs (such as carbadox, olaquindox and cyadox) has already been reported (Chen et al., 2009; Wang et al., 2011), the toxicity of QCT was particularly concerned. In the former study (Wang et al., 2010), it was proven that the no-observed-adverse-effect level of quinocetone was considered to be 300 mg/kg diet. In P.R. China, the dose of QCT used as veterinary drug is below 300 mg/kg, and no side effects are observed up to now. However, in our study, we used relatively higher doses to induce the negative effects. We aimed to seek out the potential damage of QCT and present corresponding measures to protect animals. In previous studies, QCT was reported to cause genotoxicity in Vero cells (Chen et al., 2009), cytotoxicity in porcine adrenocortical cells (Huang et al., 2010) and oxidative DNA damage in Balb/c mice (Wang et al., 2011). In the present study, we investigated the nephrotoxicity of QCT and its probable mechanism. Table 2 DNA damage measured by the comet assay in isolated renal cells after administration of QCT. Group
H-QCT M-QCT L-QCT **
OTM
Tail DNA%
Median
P25–P75
Median
P25–P75
3.96** 0.36 0.29
3.29–4.71 0.16–0.63 0.01–0.90
29.18** 3.34 2.86
22.40–37.02 1.46–8.35 0.07–8.70
P < 0.01, significant different (Mann–Whitney U -test) versus vehicle control.
⁄⁄
283
means P < 0.05 when QCT treated group compared with
Kidney is an extremely important organ that maintains the internal environment of the body. Nevertheless, kidney can be damaged by kinds of factors, such as drugs, chemicals, acute injures, etc. Besides of the levels of BUN and Cr in serum, we also used SOD, CAT and other antioxidant capacity biomarkers in blood to detect oxidative stress and nephrotoxicity (Waring and Moonie, 2011). These biomarkers are more sensitive than BUN and Cr. In present study, we focused on the effects caused by different doses of QCT. Taking 50 mg/kg/day QCT orally had little adverse effects on SD rats for all indexes we measured. The reason was that the no-observed-adverse-effect level of quinocetone in Wistar rats was considered to be 300 mg/kg/day (Wang et al., 2010). QCT-induced kidney injure could lead to disturbance of internal environment. This disturbance eventually resulted in the lower body weight and food consumption in M-QCT and H-QCT. Cr and BUN, as the products of metabolism, are mainly eliminated from the body by the glomerular filtration. As to say the levels of them reflect the function of kidney. The levels of Cr in H-QCT and M-QCT were significantly higher than control (P < 0.05), which represented the decreased kidney function. No significant difference was discovered in BUN. This mainly because of its relatively lower sensitivity. In other words, BUN may be able to maintain in normal range in the early stage of renal impairment, and the measurable changes can only be detected in the late stage of renal impairment. ROS, such as superoxide (O2 ), hydrogen peroxide (H2O2) and hydroxyl radicals (OH), is a series of reactive oxygen species produced by aerobic cells in metabolic processes. Accumulative evidence has shown that for lots of factors, inducing the generation of ROS is the key process to cause renal impairment (Liu et al.,
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Fig. 6. Analysis of Nrf2 expression in kidney of control (D–F) and H-QCT (A–C) by confocal microscopy at 400. Green represented Nrf2-FITC stained sections (A and D), red represented images of PI (B and E). C was the overlay of A and B, meanwhile F was the overlay of D and E. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2010; Shah and Iqbal, 2010) and DNA damage (Ahamed and Siddiqui, 2007). As a radical scavenger, the significant decrease of GSH in H-QCT reflected the ROS generation. Simultaneously, the significant increase of MDA (final products of peroxidation) also confirmed this theory. Additionally, significant differences between H-QCT and control in OTM and Tail DNA% could directly reflect DNA damage in renal cells. In brief, when the oxidative stress is activated, increasing ROS causes organ impairment and DNA damage. Nevertheless, cells have various defense systems to mitigate or repair ROS-induced cell damage. As already stated, GSH is one of the antioxidant enzymes which can scavenge ROS. Furthermore, many other enzymes can scavenge ROS as well. Consequently, induction of phase II detoxifying enzymes or antioxidative enzymes is one pivotal defense system to mitigate or repair ROS-induced cell damage (Zhang and Gordon, 2004). As a widely known antioxidative pathway, Nrf2/ARE is a major cellular defense mechanism which prevents damage from ROS generation through induction of antioxidative phase II enzymes such as GST and NAD(P)H, etc. (Boddupalli et al., 2012; Boettler et al., 2012). Under normal circumstances, Nrf2 and the Keap1 which acts as a negative regulator of Nrf2 are both presented in the cytosol. When electrophiles or ROS are generating, Nrf2 dissociates from Keap1 and translocates into the nucleus where it dimerizes with small Maf binding proteins. Binding of these heterodimers to ARE ultimately activates ARE-dependent gene expression (Kobayashi and Yamamoto, 2005; Li and Kong, 2009). Subsequently, the phase II detoxifying enzymes or antioxidative enzymes are generated and these enzymes eventually protect cells from ROS generation. In present study, the higher expression of Nrf2 in H-QCT illustrated that QCT-induced oxidative stress would be partly mitigated by the induction of phase II detoxifying enzymes via increasing Nrf2 expression.
As mentioned, SOD, CAT and GPx are very important antioxidant enzymes. The SOD decomposes superoxide radicals (O2 ) and produces H2O2. H2O2 is subsequently removed to water by CAT in the peroxisomes, or by GPx oxidizing GSH in the cytosol (Droge, 2002; Lee and Choi, 2003). When the expression of Nrf2 is up-regulated, the activity of these antioxidant enzymes should increase as well (Jung and Kwak, 2010; Ishikado et al., 2012). However, in present study, the activities of antioxidant enzymes (GPx, CAT and GST) decreased instead. The reason might be that in one hand the intermediate products of lipid peroxidation directly decreased the activities of GPx, CAT and GST, and in the other hand these intermediate products damaged cell structure that continuous activation of the Nrf2/ARE pathway was suppressed in the late stage of oxidative stress. We will verify this idea in the future with more experiments.
5. Conclusion Production of ROS is the key mediator of QCT-induced nephrotoxicity and DNA damage. Furthermore, on one hand, the enzymes activities are correlated with nephrotoxicity. On the other hand, induction of phase II detoxifying enzymes or antioxidative enzymes is one of pivotal mechanisms that mitigate or repair ROS-induced organ and cell damage. With present evidence, we thought the Nrf2/ARE pathway might be a major cellular defense mechanism which could protect cell damage against ROS generation. Nevertheless, in this manuscript the current use of QCT lacked a specific standard to limit its usage. Furthermore, different animals exist metabolic differences, it’s necessary to establish dose limits of QCT for different animals. Above all, in further study, we desire to precede our research on the Nrf2/ARE pathway and try our best to solve these problems and exhibit its molecular mechanism clearly.
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