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Full length article
Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.) Q6
Dechun Chen a, b, Ziwei Zhang a, Haidong Yao a, Yang Liang a, Houjuan Xing a, c, *, Shiwen Xu a, * a b c
Q2
College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Harbin 150030, PR China Department of Biological Engineering, Jilin Engineering Vocational College, 1299 Changfa Road, Siping 136001, China Animal Health Supervision Institute of Heilongjiang Province, 243 Haping Road, Xiangfang District, Harbin 150069, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 September 2014 Received in revised form 11 January 2015 Accepted 22 January 2015 Available online xxx
Atrazine (ATR) and chlorpyrifos (CPF) are the most common agrochemical in the freshwater ecosystems of the world. This study assessed the effects of ATR (4.28, 42.8 and 428 mg/L), CPF (1.16, 11.6 and 116 mg/L) and combined ATR/CPF (1.13, 11.3 and 113 mg/L) on common carp head kidneys and spleens following 40 d exposure and 40 d recovery treatments. Nitric oxide (NO) content, activities of anti hydroxyl radical (AHR), anti superoxide anion (ASA), peroxidase (POD) and inducible nitric oxide synthase (iNOS), and the mRNA levels of the autophagy genes (LC3-II, dynein, TOR) were determined. The results indicate that the antioxidant enzyme (AHR, ASA, POD and iNOS) activities and NO content in the head kidney and spleen of the common carp increased significantly after a 40 d exposure to ATR and CPF alone or in combination. The mRNA levels of LC3-II and dynein in common carp increased significantly after exposure to ATR and CPF alone, or in combination. Moreover, the mRNA levels of LC3-II and dynein decreased significantly after a 40-d recovery. However, the mRNA levels of TOR gene for all decreased significantly at the end of the exposure and the recovery. To our knowledge, this is the first study to report the oxidative stress-induced autophagic effects in the common carp by exposure to ATR, CPF and the ATR/CPF combination. The information presented in the present study may be helpful to understanding the mechanisms of autophagy induced by ATR, CPF and the ATR/CPF combination in fish. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Oxidative stress Autophagy Atrazine Chlorpyrifos Common carp Immune organ
1. Introduction The triazine herbicide atrazine (ATR) and the organophosphorus insecticide (OPs) chlorpyrifos (CPF) are extensively applied in agriculture all over the world. ATR is principally used for control of certain annual broadleaf and grass weeds. It was found in many surface and ground waters in China [1,2]. It has been shown that ATR could cause biochemical and histopathological changes [3,4], genotoxicity [5], endocrine disruption [6], oxidative stress [7] in fish. CPF has a wide application in agricultural pest control and could induce oxidative stress [7]. Autophagy plays an essential role in differentiation and development, as well as in cellular response to stress. This pathway can
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* Corresponding authors. College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Harbin 150030, PR China. Tel./fax: þ86 451 55190407. E-mail addresses: [email protected] (H. Xing), [email protected] (S. Xu).
be stimulated by multiple forms of cellular stress, including nutrient or growth factor deprivation, hypoxia, reactive oxygen species, DNA damage, protein aggregates, damaged organelles, or intracellular pathogens [8]. To date, more than 30 ATG (autophagyrelated) genes required for autophagy and its related pathways have been identified [9]. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. Recent studies have regarded LC3- accumulation as marker of autophagy [10]. Mammalian target of rapamycin (mTOR) is activated in the presence of growth factors and abundant cellular nutrients such as amino acids. mTOR is widely used as an inhibitor of the initiation step of macroautophagy [11]. It also inhibits autophagy under nutrient rich condition. In contrast, the down-regulation of mTOR activity by rapamycin is shown to enhance cell viability under ER stress [12,13]. However, little is known about the effects of pesticide on autophagy in the common carp. Many freshwater ecosystems are contaminated with industrial, domestic and agricultural chemicals, such as herbicides and
http://dx.doi.org/10.1016/j.fsi.2015.01.014 1050-4648/© 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Chen D, et al., Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.), Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.01.014
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insecticides, which are ubiquitous [14]. Alterations in the chemical composition of natural aquatic environments may affect the freshwater fauna, particularly fish. Common carp (Cyprinus carpio L.), which belong to higher trophic levels in the biosphere and food chain, is commonly selected as experimental models exposed to environmental pollution for the evaluation of the health of aquatic ecosystems [4]. Oxidative stress-induced autophagy under starvation and ischemia/reperfusion conditions was quite established [15]. Pesticides may cause oxidative stress leading to the generation of free radicals and alterations in antioxidants or free oxygen radical scavenging enzyme systems [16]. The previous study in our laboratory also suggested that ATR, CPF and the ATR/CPF combination could significantly change antioxidant enzymes activity in the brain and kidney of the common carp [4]. However, study on autophagic cell death by oxidative stress in the immune organ of common carp was unknown after exposure to ATR, CPF and the ATR/CPF combination. 2. Materials and methods 2.1. Chemicals ATR (purity 98.0%) and CPF (purity 99.5%) were purchased from SigmaeAldrich Chemical Co. (USA). The stock solution of ATR and CPF were prepared in analytical grade (99% purity) acetone as a carrier solvent. All working solutions were taken from this stock solution. The concentration of acetone was kept less than 0.05% in all pesticide and herbicide solutions. 2.2. Fish All procedures used in the present study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University in China. Common carp (mean body length, 12.5 ± 1.29 cm; mean body weight, 190 ± 10 g) used in this study were bought from an aquarium specializing in freshwater fish species and maintained in the laboratory tanks (90 55 45 cm) with continuous aeration. Acclimatization to experimental conditions for 15 d was done using dechlorinated tap water (CaCO3: 230 mg/L, Ca: 42.5 ± 1.2 mg/L, the dissolved oxygen concentration remained above 7 mg/L and pH 7.4 ± 0.2). Water temperature was adjusted at 20 ± 1 C and photoperiod was 12 h light and 12 h dark. Commercial food was given once a day until satiation. No mortality was observed either in control animals or in any of the treatment groups. 2.3. Experimental design 2.3.1. Toxicity test The fish model was developed as previously described [4] 0.220 fish were randomly divided into eleven groups: three ATR treatment groups (4.28, 42.8, 428 mg/L), three CPF treatment groups (1.16, 11.6, 116 mg/L), three ATR/CPF treatment groups (1.13, 11.3, 113 mg/L), one solvent control (acetone) and one water control. Each
treatment group contained 20 fish and two replicates. The binary mixtures comprised a 1:1 mass ratio of ATR and CPF. The fish were exposed under semi-static conditions for 40 d, where the water and herbicide/pesticide were completely replaced once every 2 d by transferring the fish into freshly prepared herbicide/pesticide solutions. Other conditions for fish acclimation were consistent with the previous description (2.2). At the end of the exposure, fish were sacrificed by decapitation and bled. Then the head kidney and spleen were excised immediately on an icecold plate washed in physiological saline solution. The tissues were divided into three portions: the first fortion was homogenized for antioxidant enzyme analysis, the second fortion was fixed for electron microscopy, and the third fortion was stored at 80 C for mRNA isolation. 2.3.2. Recovery test Ten exposed fish from each batch were kept in pesticide-free water for 40 d in a large fresh 200 L glass aquarium provided with a filter and continuous aeration. The conditions in the recovery experiments were the same as that in the exposure experiments. 2.4. Gene expression analysis Total RNA was isolated from the head kidney and spleen of each fish using Trizol reagent according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). The RNA concentrations were determined using GeneQuant 1300 (GE Healthcare Biosciences, Piscataway, NJ). The reverse transcription reaction (40 mL) consisted of the following: 10 mg total RNA, 1 mL Moloney murine leukemia virus reverse transcriptase (200 U/mL), 1 mL RNase inhibitor (40 U/mL), 4 mL deoxynucleoside triphosphate (10 mM), 2 mL Oligo dT (50 mM), 4 mL dithiothreitol (0.1 M), and 8 mL 5 reverse transcriptase buffer. The reverse transcription procedure was performed according to the manufacturer's instructions (Invitrogen). The reverse transcription products (cDNA) were then stored at 20 C for PCR. These synthesized cDNAs were used for PCR reaction of LC3-II, dynein, TOR and b-actin by PCR Supermix with primers at a final concentration of 50 nM. Primer Premier Software 5.0 (PREMIER Biosoft International, USA) was employed to design specific primers for LC3-II, dynein, TOR and b-actin based on known sequences (Table 1). b-actin, a house-keeping gene, was used as an internal reference. BLASTX and BLASTN were used to determine PCR assay specificity. The reaction specificity of each assay was verified by observing a single peak in the melting curve. The quantitative realtime RT-PCR (qPCR) work was conducted according to the MIQE guidelines. qPCR was used to detect the mRNA levels of the LC3-II, dynein, TOR and b-actin genes in the head kidney and spleen by using SYBR Premix Ex Taq (Takara), and qPCR work was performed on an ABI PRISM 7500 Detection System (Applied Biosystems, USA). The program was one cycle at 95 C for 30 s and 40 cycles at 95 C for 5 s and at 61 C for 34 s. Dissociation curves were analyzed with
Table 1 Real-time PCR primer sequences and product.
Q4 0
0
Gene
Accession number
Sequences (5 / 3 )
Product size, bp
b-actin
M24113.1
167 bp
LC3-II
NM199604.1
Dynein
NM001079990.1
TOR
FJ899680.1
Forward: ATG GAC TCT GGT GAT GGT GTG AC Reverse: TTT CTC TTT CGG CTG TGG TGG TG Forward: GGA ACA GCA TCC AAG CAA GA Reverse: TCA GAA ATG GCG GTG GAC A Forward: AAT CAG TGA GCC CAC CCA GT Reverse: AGC ACC CAT GAA CCG AATC T Forward: CCA CAA CGC AGC CAA CAA Reverse: CCC TCG TGC CAC ATT TCAT
219 bp 135 bp 129 bp
Please cite this article in press as: Chen D, et al., Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.), Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.01.014
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Dissociation Curve 1.0 software (Applied Biosystems) for each PCR reaction to detect and eliminate the possible primer-dimer and nonspecific amplifications. The magnitude of change in gene expression relative to acetone control was determined by the 2DDCt method of Livak and Schmittgen [17]. 2.5. Antioxidant enzyme activity and NO The head kidney and spleen homogenates were determined for Nitric oxide (NO) content and enzyme activities like anti hydroxyl radical (AHR), anti superoxide anion (ASA), peroxidase (POD) and inducible nitric oxide synthase (iNOS). NO content and iNOS activity were determined by the method of Zhang et al. [18]. The hydroxyl radical (OH.) inhibition ability in immune organs was measured spectrophotometrically [19]. ASA activity was measured at 550 nm after incubation at 37 C for 40 min. POD activity was measured at 420 nm after incubation at 37 C for 30 min. 2.6. Transmission electron microscopy analysis For electron microscopy, tissue specimens were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 3 h at 4 C, washed in the same buffer for 1 h at 4 C and postfixed with 1% osmium tetroxide in sodium phosphate buffer for 1 h at 4 C. The tissues were then dehydrated in graded series of ethanol, starting at 50% each step for 10 min, after two changes in propylene oxide. The tissue specimens were embedded in araldite. Ultrathin sections were stained with Mg-uranyl acetate and lead citrate for transmission electron microscopy evaluation.
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expressed as the mean ± standard deviation. P < 0.05 was considered significant. 3. Results No mortality was recorded in any of the treatment groups during our semi-static experiments when compared with the controls. We also observed no differences between the control group of tap water and the control group of tap water plus acetone. 3.1. The expression of LC3, dynein and TOR in the head kidney and spleen The effects of ATR, CPF and their combination on the mRNA levels of LC3, dynein and TOR are shown in immune organs of common carp (Figs. 1e3). Compared to the control group, the mRNA levels of LC3 and dynein genes for all of the treatment groups increased significantly (P < 0.05) in the head kidney and spleen after 40 d of exposure. In contrary, the TOR mRNA level in all the treatment groups were significantly lower (P < 0.05) than those in the solvent control at the end of exposure. After 40 d of recovery, the mRNA levels of LC3 and dynein genes decreased significantly (P < 0.05) in all of the treatment groups in the head kidney and spleen compared to corresponding exposure group. However, the TOR mRNA level was increased significantly (P < 0.05) in the 11.6 and 116 mg/L CPF and all ATR/CPF groups in the head kidney and in the 428 mg/L ATR, 11.6 mg/L CPF and 11.3 and 113 mg/L ATR/CPF groups in the spleen compared to corresponding exposure group after 40 d recovery. 3.2. Antioxidant enzyme activity and NO
2.7. Statistical analysis The statistical analysis of all of the data was performed using SPSS for Windows (version 13, SPSS Inc., Chicago, IL). One-way ANOVA was used to identify significant differences between the treatment groups and the solvent control group. The difference between the exposure group and the recovery group at the same concentration was assessed using a paired t-test. The data were
3.2.1. AHR, ASA and POD activity The activities of AHR, ASA, POD, iNOS and NO content in the head kidney and spleen of common carp are summarized in Tables 2 and 3. Compared with the control group, the activities of AHR, ASA and POD decreased in a dose-dependent manner in the head kidney and spleen of fish exposed to ATR, CPF and the ATR/CPF combination. The AHR activity for all of the treatment groups in
Fig. 1. Effects of ATR, CPF and the ATR/CPF combination on the mRNA levels of LC3 gene in the head kidney (As shown in A, B and C) and spleen (As shown in D, E and F) of common carp. The mRNA level from the control group was used as the reference values in panels AeF. Each value represented the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the control and the exposure groups. # Significant differences (P < 0.05) between the treatment and the recovery groups at the same concentration.
Please cite this article in press as: Chen D, et al., Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.), Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.01.014
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Fig. 2. Effects of ATR, CPF and ATR/CPF combination on the mRNA levels of dynein gene in the head kidney (As shown in A, B and C) and spleen (As shown in D, E and F) of common carp. The mRNA level from the control group was used as the reference values in panels AeF. Each value represented the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the control and the exposure groups. # Significant differences (P < 0.05) between the treatment and the recovery groups at the same concentration.
both head kidney and spleen decreased significantly (P < 0.05) than those in the solvent control at the end of the exposure, except for the 4.28 mg/L ATR and 1.16 and 11.6 mg/L CPF groups. After 40 d of recovery, the AHR activity increased significantly (P < 0.05) in the 428 mg/L ATR, 116 mg/L CPF, 11.3 and 113 mg/L ATR/CPF groups in the head kidney and in the 42.8 and 428 mg/L ATR, 116 mg/L CPF and 11.3 and 113 mg/L ATR/CPF groups in the spleen compared to corresponding exposure group.
At the end of the exposure, the ASA activity for all of the treatment groups decreased significantly (P < 0.05) compared to the control group, except for the 1.16 mg/L CPF group in the head kidney and 4.28 mg/L ATR and 1.16 mg/L groups in the spleen. After 40 d of recovery, the ASA activity increased significantly (P < 0.05) in the 42.8 and 428 mg/L ATR, 11.6 and 116 mg/L CPF, and 11.3 and 113 mg/L ATR/CPF groups in the head kidney and spleen compared to corresponding exposure group.
Fig. 3. Effects of ATR, CPF and ATR/CPF mixture on the mRNA levels of TOR gene in the head kidney (As shown in A, B and C) and spleen (As shown in D, E and F) of common carp. The mRNA level from the control group was used as the reference values in panels AeF. Each value represented the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the control and the exposure groups. # Significant differences (P < 0.05) between the treatment and the recovery groups at the same concentration.
Please cite this article in press as: Chen D, et al., Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.), Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.01.014
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AHR (U/mg protein)
ASA (U/g protein)
Exposure recovery Control Atrazine (mg L1)
Chlorpyrifos (mg L1)
Mixture of atrazine and chlorpyrifos (mg L1)
0 4.28 42.8 428 1.16 11.6 116 1.13 11.3 113
443.4 413.6 362.5 309.1 412.0 391.8 369.5 411.3 342.4 300.2
± ± ± ± ± ± ± ± ± ±
12.5 11.9 18.8a 11.5a 15.4 14.4 17.9a 18.5 12.4a 15.9a
Exposure recovery 449.9 433.9 413.4 377.2 442.6 421.1 380.9 424.3 389.1 366.9
± ± ± ± ± ± ± ± ± ±
17.9 10.2 12.8 16.7b 13.4 17.1 15.7b 13.3 13.8b 11.9b
73.16 61.20 49.37 33.21 68.74 59.85 41.37 58.34 43.68 31.49
± ± ± ± ± ± ± ± ± ±
1.23 2.93a 1.53a 2.74a 1.56 1.38a 1.42a 2.57a 1.32a 1.84a
Exposure recovery 76.86 68.67 56.59 48.45 73.41 66.40 57.91 69.97 54.21 48.75
± 1.92 ± 2.94 ± 1.96b ± 1.76b ± 1.27 ± 2.87b ±0 .67b ± 1.33 ± 1.32b ± 1.97b
8.17 7.82 5.89 4.42 7.25 6.26 5.44 6.19 4.22 3.31
± 0.48 ± 0.55 þ 0.65a ± 0.33a ± 0.65 ± 0.32a ± 0.43a ± 0.46a ± 0.38a ± 0.24a
8.15 8.05 7.13 6.23 8.14 7.43 6.05 6.91 6.09 5.16
Q5
NO (mmol/g protein)
POD (U/mg protein)
iNOS (U/mg protein)
Exposure recovery ± ± ± ± ± ± ± ± ± ±
0.67 0.67 0.51b 0.47b 0.79 0.57 0.57b 0.46 0.56b 0.45b
41.03 57.53 86.93 97.32 48.95 71.41 89.27 56.02 94.85 113.3
± ± ± ± ± ± ± ± ± ±
0.53 0.78a 1.36a 0.60a 0.67a 0.81a 0.89a 1.21a 0.87a 1.23a
Exposure recovery 43.19 49.29 58.71 64.10 46.21 51.19 63.96 47.84 61.13 69.24
± ± ± ± ± ± ± ± ± ±
0.74 0.95b 1.43b 1.09b 1.25 1.80b 1.65b 1.50 0.82b 1.49b
3.36 4.06 5.06 7.96 4.02 4.69 6.91 4.33 5.04 8.68
± 0.35 ± 0.67 þ 0.71a ± 0.38a ± 0.22 ± 0.29a ± 0.48a ± 0.30a ± 0.35a ± 0.32a
3.51 3.86 3.92 5.44 3.61 3.74 4.94 4.11 4.02 5.76
± ± ± ± ± ± ± ± ± ±
0.38 0.14 0.31b 0.37b 0.27 0.31b 0.15b 0.37 0.34b 0.25b
± ± ± ± ± ± ± ± ± ±
0.29 0.34 0.28b 0.33b 0.23 0.35 0.21b 0.22 0.39b 0.35b
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Note: values are means ± SD (n ¼ 10). a Significant difference (P < 0.05) between the control and exposure groups. b Significant difference (P < 0.05) between the exposure and recovery groups of the same concentration.
Table 3 AHR, ASA, POD, NO and iNOS levels in spleen tissues of common carp after exposure (atrazine, chlorpyrifos and their combination) and during recovery treatment. Treatment groups
Control Atrazine (mg L1)
Chlorpyrifos (mg L1)
Mixture of atrazine and chlorpyrifos (mg L1)
0 4.28 42.8 428 1.16 11.6 116 1.13 11.3 113
AHR (U/mg protein)
ASA (U/g protein)
POD (U/mg protein)
NO (mmol/g protein)
iNOS (U/mg protein)
Exposure recovery
Exposure recovery
Exposure recovery
Exposure recovery
Exposure recovery
315.2 284.2 231.4 211.5 304.7 283.8 246.6 280.5 243.9 212.4.2
± ± ± ± ± ± ± ± ± ±
10.5 10.7 11.9a 8.63a 9.6 10.1 7.63a 9.5a 8.6a 9.9a
315.6 320.2 283.8 277.6 311.5 298.6 283.1 305.9 274.0 243.4
± ± ± ± ± ± ± ± ± ±
10.7 8.7 9.5b 9.4b 10.6 9.6 8.1b 10.5 6.6b 9.4b
41.42 36.69 27.34 17.45 38.66 29.77 21.85 33.65 20.57 19.98
± ± ± ± ± ± ± ± ± ±
1.31 1.30 1.33a 1.67a 1.72 1.54a 1.46a 1.18a 1.19a 1.12a
48.57 48.67 41.79 32.75 45.71 43.20 36.91 49.17 30.61 29.77
± ± ± ± ± ± ± ± ± ±
1.12 1.19 1.92b 1.74b 1.25 1.89b 1.67b 1.32 1.34b 1.94b
3.10 2.84 2.28 1.62 3.00 2.76 2.21 2.61 2.43 1.48
± 0.29 ± 0.19 þ 0.16a ± 0.13a ± 0.27 ± 0.16 ± 0.15a ± 0.19 ± 0.16a ± 0.14a
3.15 3.06 2.61 2.18 3.37 2.81 2.74 2.98 2.81 2.03
± ± ± ± ± ± ± ± ± ±
0.31 0.29 0.17b 0.19b 0.27 0.19 0.18b 0.18 0.18b 0.17b
12.34 18.97 26.91 36.77 16.40 21.01 29.11 18.54 28.12 41.43
± ± ± ± ± ± ± ± ± ±
0.78 0.58a 0.75a 0.62a 0.57 1.21a 1.28a 1.21a 0.77a 1.66a
13.78 15.85 18.38 22.48 13.89 17.26 20.19 15.78 19.72 23.71
± ± ± ± ± ± ± ± ± ±
0.51 1.28 1.52b 1.58b 1.21b 1.45 1.36b 1.32 0.82b 1.49b
3.53 4.45 5.30 6.82 4.21 4.53 6.85 4.57 5.95 7.25
± ± ± ± ± ± ± ± ± ±
0.31 0.39a 0.30a 0.51a 0.32 0.42a 0.19a 0.34a 0.31a 0.52a
3.66 4.00 4.31 4.88 3.69 4.17 4.65 3.57 4.34 4.74
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Table 2 AHR, ASA, POD, NO and iNOS levels in head kidney of common carp after exposure (atrazine, chlorpyrifos and their combination) and during recovery treatment.
Note: values are means ± SD (n ¼ 10). a Significant difference (P < 0.05) between the control and exposure groups. b Significant difference (P < 0.05) between the exposure and recovery groups of the same concentration.
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Compared to the control group, the POD activity for all of the treatment groups decreased significantly (P < 0.05) at the end of the exposure, except for the 4.28 mg/L ATR and 1.16 mg/L CPF groups in the head kidney and 4.28 mg/L ATR, 1.16 mg/L CPF and 1.13 mg/L ATR/CPF groups in the spleen. After 40 d of recovery, the POD activity increased significantly (P < 0.05) in the 42.8 and 428 mg/L ATR, 116 mg/L CPF, and 11.3 and 113 mg/L ATR/CPF groups in both head kidney and spleen compared to corresponding exposure group. 3.2.2. iNOS activity and NO At the end of the exposure, a significant increase (P < 0.05) in the NO concent and iNOS activity was observed in the head kidney and spleen of fish exposed to ATR, CPF and the ATR/CPF combination compared to the control group, except for the 1.16 mg/L CPF group in the spleen. After 40 d of recovery, the NO concent for all of the treatment groups decreased significantly (P < 0.05) compared to
corresponding exposure group, except for the 1.16 mg/L CPF and 1.13 mg/L ATR/CPF groups in the head kidney and the 4.28 mg/L ATR, 11.6 mg/L CPF and 1.13 mg/L ATR/CPF groups in the spleen. The iNOS activity for all of the treatment groups increased significantly (P < 0.05) compared to corresponding exposure group, except for the 4.28 mg/L ATR, 1.16 mg/L CPF and 1.13 mg/L ATR/CPF groups in the head kidney and the 4.28 mg/L ATR, 1.16 and 11.6 mg/L CPF, and 1.13 mg/L ATR/CPF groups in the spleen.
3.3. Analysis of electron microscopy Head kidney and spleen tissues from the control group showed no autophagic features under transmission electron microscopy (Fig. 4). In the exposure and the recovery groups, characteristic autophagic vacuolar organelles were observed. In some
Fig. 4. Electron microscopy representative images from the head kidney (AeG) and spleen (HeN). (B) (L) Autophagosome (black arrows); (C) Autolysosome; (D) Phagophores (blue arrows); Amphisomes (black arrows); (I) (J) (M) Vacuolization of mitochondria.
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Fig. 4. (continued).
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autolysosomes, organelles such as mitochondria and other cytoplasmic elements were detected. 4. Discussion Oxidative stress could induce autophagy under certain conditions such as ischemia and reperfusion [20]. The conditions that regulate the activity of the autophagic process are also associated with changes in the production of reactive oxygen/nitrogen species (ROS/RNS) in cells [21]. It is known that ATR, CPF and their combination probably produced damages on immune organ in the carp [22,23]. A study reported that two ancient processes, autophagy and innate immunity, together through a shared signaling pathway [24]. The immune system utilizes autophagic degradation of cytoplasmic material and regulates adaptive immunity [25]. Thus, we investigate that autophagy and oxidative stress responses in the immune organ of common carp exposed to ATR, CPF and their combination. Because oxidative stress is evoked by many chemicals including some pesticides, pro-oxidant factors' action in fish organism can be used to assess specific area pollution or world sea pollution [26]. Immunotoxic effects of ATR and CPF on carp may be related with lipid peroxidation and releasing of reactive oxygen species (ROS) after their exposure [7]. The activities of antioxidant enzymes have been shown to be sensitive indicators of increased oxidative stress [23]. Pesticides may cause oxidative stress that leads to the generation of free radicals and alterations in antioxidants or free oxygen radical scavenging enzyme systems [27,28]. The changes in antioxidant enzymes have been observed after actuation of pesticides deltamethrin and cypermethrin [29]. Bipyridyl herbicides were able to form redox cycles and thereby cause oxidative stress [30]. ATR sublethal concentrations could induce oxidative stress in bluegill sunfish [32]. In a previous study, the activities of SOD, GSHPx and CAT decreased after exposure to ATR and CPF in the carp [4]. However, low concentrations of simazine do not cause oxidative stress in carps during sub-chronic tests [31]. In this study, the activities of AHR, ASA and POD were significantly induced in the immune organ of common carp exposed to ATR, CPF and their combination. The changes of antioxidant activity indicates a response against increased ROS production in pesticide toxicity. Many environmental contaminants, including pesticides, have the potential to induce production of NO [33]. NO is an important signaling molecule involved in the regulation of diverse physiological and pathophysiological mechanisms in nervous, cardiovascular, and immunological systems [34]. A number of previous reports demonstrated that ATR and CPF could induce the NO production and the iNOS mRNA level [35,36]. The present study demonstrates that NO content and iNOS activity are markedly upregulated in the head kidney and spleen of common carp exposed to ATR and CPF. The results obtained in this study are in accordance with the previous reports that ATR and CPF upregulates iNOS mRNA level and NO production in the brain of common carp and aorta of rat [34,37]. Thus, these results indicate that iNOS involves in the process of immunotoxicity induced by ATR and CPF. The function of autophagy is generally thought to be a natural process to maintain cellular homeostasis as well as a cellular response to starvation, infection or disease progression [38]. LC3 is widely used to monitor autophagy [39]. Dynein motor machinery plays a role in the delivery of autophagosome contents to lysosomes, in the process of autophagosome-lysosome fusion [40]. TOR negatively regulates the induction of autophagy. TOR is the first molecule that is identified as a pivotal player in the starvationsignaling pathway of autophagy [41]. In the present study, the results from transmission electron microscopy and qPCR confirmed ATR and CPF can induce autophagy in common carp. After human
SH-SY5Y cells exposed to paraquat, inhibition of the cytoplasmic accumulation of autophagic vacuoles as well as the recruitment of LC3 fusion protein to the vacuoles and an increase in mTOR phosphorylation were observed [42]. Cadmium can induce autophagy that formation of autophagosomes and processing of LC3 in MES-13 cells [43]. Cd-mediated ROS generation eventually induces autophagy through the down-regulation of mTOR in skin epidermal cells [44]. So we think that autophagy plays an important role in carp immunotoxicity by ATR, CPF and the ATR/CPF combination. In addition, Our results from oxidative-stress, histological parameters and autophagy genes also indicate that the joint toxicity of ATR/CPF combination is higher than ATR or CPF alone. Oxidative stress plays an important role in mediating autophagy during ischemia/reperfusion (I/R), and that activation of autophagy gene LC-II through oxidative stress mediates myocardial injury in response to I/R in the mouse heart [45]. Oxidative modification of the NOSs can cause disruption of electron transfer within the enzymes, inhibiting the formation of NO and generating O 2 [46]. In a biological setting, it is then likely that the cell is responding to the combined effects of ROS/RNS acting at different sites within the autophagic process [21]. Autophagy is regulated by ROS through the Atg1eAtg13 complex, which is inhibited by mTOR activation. Atg8epro-LC3 is cleaved by Atg4, modified by phosphatidylethanolamine (PE) to become LC3-II and inserted into the autophagosomes [20]. Autophagy signaling LC3-II is upregulated in response to denervation-induced oxidative stress in skeletal muscle [47]. Our results indicate that oxidative stress plays an important role in inducing autophagy and provides a novel mechanism for cell death. In conclusion, the results of this study indicate that autophagy is involved in oxidative stress and immune organ damage induced by ATR, CPF, and their combination. To our knowledge, this is the first work to report the effects of ATR, CPF and their combination on the mRNA levels of autophagy-related genes in the immune organ of carp. Thus, the information presented in this study is helpful to understand the mechanism of ATR-, CPF- and ATR/CPFcombination-induced immunotoxicity in fish. Acknowledgment China Postdoctoral Science Foundation (Project No. 2012M511437) and the Foundation of the Postdoctoral Research from Heilongjiang Province (Project No. LBH-Q14146) supported the study. The authors thank the members of the veterinary internal medicine laboratory in the College of Veterinary Medicine, Northeast Agricultural University. References [1] Jin R, Ke J. Impact of atrazine disposal on the water resources of the Yang river in Zhangjiakou area in China. Bull Environ Contam Toxicol 2002;68:893e900. [2] Gu JG, Fan Y, Gu JD. Biodegradability of atrazine, cyanazine and dicamba under methanogenic condition in three soils of China. Chemosphere 2003;52: 1515e21. [3] Paulino M, Souza N, Fernandes M. Subchronic exposure to atrazine induces biochemical and histopathological changes in the gills of a neotropical freshwater fish, Prochilodus lineatus. Ecotoxicol Environ Saf 2012;80:6e13. [4] Xing H, Li S, Wang Z, Gao X, Xu S, Wang X. Histopathological changes and antioxidant response in brain and kidney of common carp exposed to atrazine and chlorpyrifos. Chemosphere 2012;88:377e83. [5] de Campos Ventura B, de Angelis DdF, Marin-Morales MA. Mutagenic and genotoxic effects of the Atrazine herbicide in Oreochromis niloticus (Perciformes, Cichlidae) detected by the micronuclei test and the comet assay. Pestic Biochem Physiol 2008;90:42e51. [6] Moore A, Waring CP. Mechanistic effects of a triazine pesticide on reproductive endocrine function in mature male Atlantic Salmon (Salmo salar L.) Parr. Pestic Biochem Physiol 1998;62:41e50. [7] Xing H, Li S, Wang Z, Gao X, Xu S, Wang X. Oxidative stress response and histopathological changes due to atrazine and chlorpyrifos exposure in common carp. Pestic Biochem Physiol 2012;103:74e80.
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Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.) Q6
Dechun Chen a, b, Ziwei Zhang a, Haidong Yao a, Yang Liang a, Houjuan Xing a, c, *, Shiwen Xu a, * a b c
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College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Harbin 150030, PR China Department of Biological Engineering, Jilin Engineering Vocational College, 1299 Changfa Road, Siping 136001, China Animal Health Supervision Institute of Heilongjiang Province, 243 Haping Road, Xiangfang District, Harbin 150069, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 September 2014 Received in revised form 11 January 2015 Accepted 22 January 2015 Available online xxx
Atrazine (ATR) and chlorpyrifos (CPF) are the most common agrochemical in the freshwater ecosystems of the world. This study assessed the effects of ATR (4.28, 42.8 and 428 mg/L), CPF (1.16, 11.6 and 116 mg/L) and combined ATR/CPF (1.13, 11.3 and 113 mg/L) on common carp head kidneys and spleens following 40 d exposure and 40 d recovery treatments. Nitric oxide (NO) content, activities of anti hydroxyl radical (AHR), anti superoxide anion (ASA), peroxidase (POD) and inducible nitric oxide synthase (iNOS), and the mRNA levels of the autophagy genes (LC3-II, dynein, TOR) were determined. The results indicate that the antioxidant enzyme (AHR, ASA, POD and iNOS) activities and NO content in the head kidney and spleen of the common carp increased significantly after a 40 d exposure to ATR and CPF alone or in combination. The mRNA levels of LC3-II and dynein in common carp increased significantly after exposure to ATR and CPF alone, or in combination. Moreover, the mRNA levels of LC3-II and dynein decreased significantly after a 40-d recovery. However, the mRNA levels of TOR gene for all decreased significantly at the end of the exposure and the recovery. To our knowledge, this is the first study to report the oxidative stress-induced autophagic effects in the common carp by exposure to ATR, CPF and the ATR/CPF combination. The information presented in the present study may be helpful to understanding the mechanisms of autophagy induced by ATR, CPF and the ATR/CPF combination in fish. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Oxidative stress Autophagy Atrazine Chlorpyrifos Common carp Immune organ
1. Introduction The triazine herbicide atrazine (ATR) and the organophosphorus insecticide (OPs) chlorpyrifos (CPF) are extensively applied in agriculture all over the world. ATR is principally used for control of certain annual broadleaf and grass weeds. It was found in many surface and ground waters in China [1,2]. It has been shown that ATR could cause biochemical and histopathological changes [3,4], genotoxicity [5], endocrine disruption [6], oxidative stress [7] in fish. CPF has a wide application in agricultural pest control and could induce oxidative stress [7]. Autophagy plays an essential role in differentiation and development, as well as in cellular response to stress. This pathway can
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* Corresponding authors. College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Harbin 150030, PR China. Tel./fax: þ86 451 55190407. E-mail addresses: [email protected] (H. Xing), [email protected] (S. Xu).
be stimulated by multiple forms of cellular stress, including nutrient or growth factor deprivation, hypoxia, reactive oxygen species, DNA damage, protein aggregates, damaged organelles, or intracellular pathogens [8]. To date, more than 30 ATG (autophagyrelated) genes required for autophagy and its related pathways have been identified [9]. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. Recent studies have regarded LC3- accumulation as marker of autophagy [10]. Mammalian target of rapamycin (mTOR) is activated in the presence of growth factors and abundant cellular nutrients such as amino acids. mTOR is widely used as an inhibitor of the initiation step of macroautophagy [11]. It also inhibits autophagy under nutrient rich condition. In contrast, the down-regulation of mTOR activity by rapamycin is shown to enhance cell viability under ER stress [12,13]. However, little is known about the effects of pesticide on autophagy in the common carp. Many freshwater ecosystems are contaminated with industrial, domestic and agricultural chemicals, such as herbicides and
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Please cite this article in press as: Chen D, et al., Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.), Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.01.014
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insecticides, which are ubiquitous [14]. Alterations in the chemical composition of natural aquatic environments may affect the freshwater fauna, particularly fish. Common carp (Cyprinus carpio L.), which belong to higher trophic levels in the biosphere and food chain, is commonly selected as experimental models exposed to environmental pollution for the evaluation of the health of aquatic ecosystems [4]. Oxidative stress-induced autophagy under starvation and ischemia/reperfusion conditions was quite established [15]. Pesticides may cause oxidative stress leading to the generation of free radicals and alterations in antioxidants or free oxygen radical scavenging enzyme systems [16]. The previous study in our laboratory also suggested that ATR, CPF and the ATR/CPF combination could significantly change antioxidant enzymes activity in the brain and kidney of the common carp [4]. However, study on autophagic cell death by oxidative stress in the immune organ of common carp was unknown after exposure to ATR, CPF and the ATR/CPF combination. 2. Materials and methods 2.1. Chemicals ATR (purity 98.0%) and CPF (purity 99.5%) were purchased from SigmaeAldrich Chemical Co. (USA). The stock solution of ATR and CPF were prepared in analytical grade (99% purity) acetone as a carrier solvent. All working solutions were taken from this stock solution. The concentration of acetone was kept less than 0.05% in all pesticide and herbicide solutions. 2.2. Fish All procedures used in the present study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University in China. Common carp (mean body length, 12.5 ± 1.29 cm; mean body weight, 190 ± 10 g) used in this study were bought from an aquarium specializing in freshwater fish species and maintained in the laboratory tanks (90 55 45 cm) with continuous aeration. Acclimatization to experimental conditions for 15 d was done using dechlorinated tap water (CaCO3: 230 mg/L, Ca: 42.5 ± 1.2 mg/L, the dissolved oxygen concentration remained above 7 mg/L and pH 7.4 ± 0.2). Water temperature was adjusted at 20 ± 1 C and photoperiod was 12 h light and 12 h dark. Commercial food was given once a day until satiation. No mortality was observed either in control animals or in any of the treatment groups. 2.3. Experimental design 2.3.1. Toxicity test The fish model was developed as previously described [4] 0.220 fish were randomly divided into eleven groups: three ATR treatment groups (4.28, 42.8, 428 mg/L), three CPF treatment groups (1.16, 11.6, 116 mg/L), three ATR/CPF treatment groups (1.13, 11.3, 113 mg/L), one solvent control (acetone) and one water control. Each
treatment group contained 20 fish and two replicates. The binary mixtures comprised a 1:1 mass ratio of ATR and CPF. The fish were exposed under semi-static conditions for 40 d, where the water and herbicide/pesticide were completely replaced once every 2 d by transferring the fish into freshly prepared herbicide/pesticide solutions. Other conditions for fish acclimation were consistent with the previous description (2.2). At the end of the exposure, fish were sacrificed by decapitation and bled. Then the head kidney and spleen were excised immediately on an icecold plate washed in physiological saline solution. The tissues were divided into three portions: the first fortion was homogenized for antioxidant enzyme analysis, the second fortion was fixed for electron microscopy, and the third fortion was stored at 80 C for mRNA isolation. 2.3.2. Recovery test Ten exposed fish from each batch were kept in pesticide-free water for 40 d in a large fresh 200 L glass aquarium provided with a filter and continuous aeration. The conditions in the recovery experiments were the same as that in the exposure experiments. 2.4. Gene expression analysis Total RNA was isolated from the head kidney and spleen of each fish using Trizol reagent according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). The RNA concentrations were determined using GeneQuant 1300 (GE Healthcare Biosciences, Piscataway, NJ). The reverse transcription reaction (40 mL) consisted of the following: 10 mg total RNA, 1 mL Moloney murine leukemia virus reverse transcriptase (200 U/mL), 1 mL RNase inhibitor (40 U/mL), 4 mL deoxynucleoside triphosphate (10 mM), 2 mL Oligo dT (50 mM), 4 mL dithiothreitol (0.1 M), and 8 mL 5 reverse transcriptase buffer. The reverse transcription procedure was performed according to the manufacturer's instructions (Invitrogen). The reverse transcription products (cDNA) were then stored at 20 C for PCR. These synthesized cDNAs were used for PCR reaction of LC3-II, dynein, TOR and b-actin by PCR Supermix with primers at a final concentration of 50 nM. Primer Premier Software 5.0 (PREMIER Biosoft International, USA) was employed to design specific primers for LC3-II, dynein, TOR and b-actin based on known sequences (Table 1). b-actin, a house-keeping gene, was used as an internal reference. BLASTX and BLASTN were used to determine PCR assay specificity. The reaction specificity of each assay was verified by observing a single peak in the melting curve. The quantitative realtime RT-PCR (qPCR) work was conducted according to the MIQE guidelines. qPCR was used to detect the mRNA levels of the LC3-II, dynein, TOR and b-actin genes in the head kidney and spleen by using SYBR Premix Ex Taq (Takara), and qPCR work was performed on an ABI PRISM 7500 Detection System (Applied Biosystems, USA). The program was one cycle at 95 C for 30 s and 40 cycles at 95 C for 5 s and at 61 C for 34 s. Dissociation curves were analyzed with
Table 1 Real-time PCR primer sequences and product.
Q4 0
0
Gene
Accession number
Sequences (5 / 3 )
Product size, bp
b-actin
M24113.1
167 bp
LC3-II
NM199604.1
Dynein
NM001079990.1
TOR
FJ899680.1
Forward: ATG GAC TCT GGT GAT GGT GTG AC Reverse: TTT CTC TTT CGG CTG TGG TGG TG Forward: GGA ACA GCA TCC AAG CAA GA Reverse: TCA GAA ATG GCG GTG GAC A Forward: AAT CAG TGA GCC CAC CCA GT Reverse: AGC ACC CAT GAA CCG AATC T Forward: CCA CAA CGC AGC CAA CAA Reverse: CCC TCG TGC CAC ATT TCAT
219 bp 135 bp 129 bp
Please cite this article in press as: Chen D, et al., Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.), Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.01.014
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Dissociation Curve 1.0 software (Applied Biosystems) for each PCR reaction to detect and eliminate the possible primer-dimer and nonspecific amplifications. The magnitude of change in gene expression relative to acetone control was determined by the 2DDCt method of Livak and Schmittgen [17]. 2.5. Antioxidant enzyme activity and NO The head kidney and spleen homogenates were determined for Nitric oxide (NO) content and enzyme activities like anti hydroxyl radical (AHR), anti superoxide anion (ASA), peroxidase (POD) and inducible nitric oxide synthase (iNOS). NO content and iNOS activity were determined by the method of Zhang et al. [18]. The hydroxyl radical (OH.) inhibition ability in immune organs was measured spectrophotometrically [19]. ASA activity was measured at 550 nm after incubation at 37 C for 40 min. POD activity was measured at 420 nm after incubation at 37 C for 30 min. 2.6. Transmission electron microscopy analysis For electron microscopy, tissue specimens were fixed with 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for 3 h at 4 C, washed in the same buffer for 1 h at 4 C and postfixed with 1% osmium tetroxide in sodium phosphate buffer for 1 h at 4 C. The tissues were then dehydrated in graded series of ethanol, starting at 50% each step for 10 min, after two changes in propylene oxide. The tissue specimens were embedded in araldite. Ultrathin sections were stained with Mg-uranyl acetate and lead citrate for transmission electron microscopy evaluation.
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expressed as the mean ± standard deviation. P < 0.05 was considered significant. 3. Results No mortality was recorded in any of the treatment groups during our semi-static experiments when compared with the controls. We also observed no differences between the control group of tap water and the control group of tap water plus acetone. 3.1. The expression of LC3, dynein and TOR in the head kidney and spleen The effects of ATR, CPF and their combination on the mRNA levels of LC3, dynein and TOR are shown in immune organs of common carp (Figs. 1e3). Compared to the control group, the mRNA levels of LC3 and dynein genes for all of the treatment groups increased significantly (P < 0.05) in the head kidney and spleen after 40 d of exposure. In contrary, the TOR mRNA level in all the treatment groups were significantly lower (P < 0.05) than those in the solvent control at the end of exposure. After 40 d of recovery, the mRNA levels of LC3 and dynein genes decreased significantly (P < 0.05) in all of the treatment groups in the head kidney and spleen compared to corresponding exposure group. However, the TOR mRNA level was increased significantly (P < 0.05) in the 11.6 and 116 mg/L CPF and all ATR/CPF groups in the head kidney and in the 428 mg/L ATR, 11.6 mg/L CPF and 11.3 and 113 mg/L ATR/CPF groups in the spleen compared to corresponding exposure group after 40 d recovery. 3.2. Antioxidant enzyme activity and NO
2.7. Statistical analysis The statistical analysis of all of the data was performed using SPSS for Windows (version 13, SPSS Inc., Chicago, IL). One-way ANOVA was used to identify significant differences between the treatment groups and the solvent control group. The difference between the exposure group and the recovery group at the same concentration was assessed using a paired t-test. The data were
3.2.1. AHR, ASA and POD activity The activities of AHR, ASA, POD, iNOS and NO content in the head kidney and spleen of common carp are summarized in Tables 2 and 3. Compared with the control group, the activities of AHR, ASA and POD decreased in a dose-dependent manner in the head kidney and spleen of fish exposed to ATR, CPF and the ATR/CPF combination. The AHR activity for all of the treatment groups in
Fig. 1. Effects of ATR, CPF and the ATR/CPF combination on the mRNA levels of LC3 gene in the head kidney (As shown in A, B and C) and spleen (As shown in D, E and F) of common carp. The mRNA level from the control group was used as the reference values in panels AeF. Each value represented the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the control and the exposure groups. # Significant differences (P < 0.05) between the treatment and the recovery groups at the same concentration.
Please cite this article in press as: Chen D, et al., Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.), Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.01.014
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Fig. 2. Effects of ATR, CPF and ATR/CPF combination on the mRNA levels of dynein gene in the head kidney (As shown in A, B and C) and spleen (As shown in D, E and F) of common carp. The mRNA level from the control group was used as the reference values in panels AeF. Each value represented the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the control and the exposure groups. # Significant differences (P < 0.05) between the treatment and the recovery groups at the same concentration.
both head kidney and spleen decreased significantly (P < 0.05) than those in the solvent control at the end of the exposure, except for the 4.28 mg/L ATR and 1.16 and 11.6 mg/L CPF groups. After 40 d of recovery, the AHR activity increased significantly (P < 0.05) in the 428 mg/L ATR, 116 mg/L CPF, 11.3 and 113 mg/L ATR/CPF groups in the head kidney and in the 42.8 and 428 mg/L ATR, 116 mg/L CPF and 11.3 and 113 mg/L ATR/CPF groups in the spleen compared to corresponding exposure group.
At the end of the exposure, the ASA activity for all of the treatment groups decreased significantly (P < 0.05) compared to the control group, except for the 1.16 mg/L CPF group in the head kidney and 4.28 mg/L ATR and 1.16 mg/L groups in the spleen. After 40 d of recovery, the ASA activity increased significantly (P < 0.05) in the 42.8 and 428 mg/L ATR, 11.6 and 116 mg/L CPF, and 11.3 and 113 mg/L ATR/CPF groups in the head kidney and spleen compared to corresponding exposure group.
Fig. 3. Effects of ATR, CPF and ATR/CPF mixture on the mRNA levels of TOR gene in the head kidney (As shown in A, B and C) and spleen (As shown in D, E and F) of common carp. The mRNA level from the control group was used as the reference values in panels AeF. Each value represented the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the control and the exposure groups. # Significant differences (P < 0.05) between the treatment and the recovery groups at the same concentration.
Please cite this article in press as: Chen D, et al., Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.), Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.01.014
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AHR (U/mg protein)
ASA (U/g protein)
Exposure recovery Control Atrazine (mg L1)
Chlorpyrifos (mg L1)
Mixture of atrazine and chlorpyrifos (mg L1)
0 4.28 42.8 428 1.16 11.6 116 1.13 11.3 113
443.4 413.6 362.5 309.1 412.0 391.8 369.5 411.3 342.4 300.2
± ± ± ± ± ± ± ± ± ±
12.5 11.9 18.8a 11.5a 15.4 14.4 17.9a 18.5 12.4a 15.9a
Exposure recovery 449.9 433.9 413.4 377.2 442.6 421.1 380.9 424.3 389.1 366.9
± ± ± ± ± ± ± ± ± ±
17.9 10.2 12.8 16.7b 13.4 17.1 15.7b 13.3 13.8b 11.9b
73.16 61.20 49.37 33.21 68.74 59.85 41.37 58.34 43.68 31.49
± ± ± ± ± ± ± ± ± ±
1.23 2.93a 1.53a 2.74a 1.56 1.38a 1.42a 2.57a 1.32a 1.84a
Exposure recovery 76.86 68.67 56.59 48.45 73.41 66.40 57.91 69.97 54.21 48.75
± 1.92 ± 2.94 ± 1.96b ± 1.76b ± 1.27 ± 2.87b ±0 .67b ± 1.33 ± 1.32b ± 1.97b
8.17 7.82 5.89 4.42 7.25 6.26 5.44 6.19 4.22 3.31
± 0.48 ± 0.55 þ 0.65a ± 0.33a ± 0.65 ± 0.32a ± 0.43a ± 0.46a ± 0.38a ± 0.24a
8.15 8.05 7.13 6.23 8.14 7.43 6.05 6.91 6.09 5.16
Q5
NO (mmol/g protein)
POD (U/mg protein)
iNOS (U/mg protein)
Exposure recovery ± ± ± ± ± ± ± ± ± ±
0.67 0.67 0.51b 0.47b 0.79 0.57 0.57b 0.46 0.56b 0.45b
41.03 57.53 86.93 97.32 48.95 71.41 89.27 56.02 94.85 113.3
± ± ± ± ± ± ± ± ± ±
0.53 0.78a 1.36a 0.60a 0.67a 0.81a 0.89a 1.21a 0.87a 1.23a
Exposure recovery 43.19 49.29 58.71 64.10 46.21 51.19 63.96 47.84 61.13 69.24
± ± ± ± ± ± ± ± ± ±
0.74 0.95b 1.43b 1.09b 1.25 1.80b 1.65b 1.50 0.82b 1.49b
3.36 4.06 5.06 7.96 4.02 4.69 6.91 4.33 5.04 8.68
± 0.35 ± 0.67 þ 0.71a ± 0.38a ± 0.22 ± 0.29a ± 0.48a ± 0.30a ± 0.35a ± 0.32a
3.51 3.86 3.92 5.44 3.61 3.74 4.94 4.11 4.02 5.76
± ± ± ± ± ± ± ± ± ±
0.38 0.14 0.31b 0.37b 0.27 0.31b 0.15b 0.37 0.34b 0.25b
± ± ± ± ± ± ± ± ± ±
0.29 0.34 0.28b 0.33b 0.23 0.35 0.21b 0.22 0.39b 0.35b
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Note: values are means ± SD (n ¼ 10). a Significant difference (P < 0.05) between the control and exposure groups. b Significant difference (P < 0.05) between the exposure and recovery groups of the same concentration.
Table 3 AHR, ASA, POD, NO and iNOS levels in spleen tissues of common carp after exposure (atrazine, chlorpyrifos and their combination) and during recovery treatment. Treatment groups
Control Atrazine (mg L1)
Chlorpyrifos (mg L1)
Mixture of atrazine and chlorpyrifos (mg L1)
0 4.28 42.8 428 1.16 11.6 116 1.13 11.3 113
AHR (U/mg protein)
ASA (U/g protein)
POD (U/mg protein)
NO (mmol/g protein)
iNOS (U/mg protein)
Exposure recovery
Exposure recovery
Exposure recovery
Exposure recovery
Exposure recovery
315.2 284.2 231.4 211.5 304.7 283.8 246.6 280.5 243.9 212.4.2
± ± ± ± ± ± ± ± ± ±
10.5 10.7 11.9a 8.63a 9.6 10.1 7.63a 9.5a 8.6a 9.9a
315.6 320.2 283.8 277.6 311.5 298.6 283.1 305.9 274.0 243.4
± ± ± ± ± ± ± ± ± ±
10.7 8.7 9.5b 9.4b 10.6 9.6 8.1b 10.5 6.6b 9.4b
41.42 36.69 27.34 17.45 38.66 29.77 21.85 33.65 20.57 19.98
± ± ± ± ± ± ± ± ± ±
1.31 1.30 1.33a 1.67a 1.72 1.54a 1.46a 1.18a 1.19a 1.12a
48.57 48.67 41.79 32.75 45.71 43.20 36.91 49.17 30.61 29.77
± ± ± ± ± ± ± ± ± ±
1.12 1.19 1.92b 1.74b 1.25 1.89b 1.67b 1.32 1.34b 1.94b
3.10 2.84 2.28 1.62 3.00 2.76 2.21 2.61 2.43 1.48
± 0.29 ± 0.19 þ 0.16a ± 0.13a ± 0.27 ± 0.16 ± 0.15a ± 0.19 ± 0.16a ± 0.14a
3.15 3.06 2.61 2.18 3.37 2.81 2.74 2.98 2.81 2.03
± ± ± ± ± ± ± ± ± ±
0.31 0.29 0.17b 0.19b 0.27 0.19 0.18b 0.18 0.18b 0.17b
12.34 18.97 26.91 36.77 16.40 21.01 29.11 18.54 28.12 41.43
± ± ± ± ± ± ± ± ± ±
0.78 0.58a 0.75a 0.62a 0.57 1.21a 1.28a 1.21a 0.77a 1.66a
13.78 15.85 18.38 22.48 13.89 17.26 20.19 15.78 19.72 23.71
± ± ± ± ± ± ± ± ± ±
0.51 1.28 1.52b 1.58b 1.21b 1.45 1.36b 1.32 0.82b 1.49b
3.53 4.45 5.30 6.82 4.21 4.53 6.85 4.57 5.95 7.25
± ± ± ± ± ± ± ± ± ±
0.31 0.39a 0.30a 0.51a 0.32 0.42a 0.19a 0.34a 0.31a 0.52a
3.66 4.00 4.31 4.88 3.69 4.17 4.65 3.57 4.34 4.74
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Table 2 AHR, ASA, POD, NO and iNOS levels in head kidney of common carp after exposure (atrazine, chlorpyrifos and their combination) and during recovery treatment.
Note: values are means ± SD (n ¼ 10). a Significant difference (P < 0.05) between the control and exposure groups. b Significant difference (P < 0.05) between the exposure and recovery groups of the same concentration.
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Compared to the control group, the POD activity for all of the treatment groups decreased significantly (P < 0.05) at the end of the exposure, except for the 4.28 mg/L ATR and 1.16 mg/L CPF groups in the head kidney and 4.28 mg/L ATR, 1.16 mg/L CPF and 1.13 mg/L ATR/CPF groups in the spleen. After 40 d of recovery, the POD activity increased significantly (P < 0.05) in the 42.8 and 428 mg/L ATR, 116 mg/L CPF, and 11.3 and 113 mg/L ATR/CPF groups in both head kidney and spleen compared to corresponding exposure group. 3.2.2. iNOS activity and NO At the end of the exposure, a significant increase (P < 0.05) in the NO concent and iNOS activity was observed in the head kidney and spleen of fish exposed to ATR, CPF and the ATR/CPF combination compared to the control group, except for the 1.16 mg/L CPF group in the spleen. After 40 d of recovery, the NO concent for all of the treatment groups decreased significantly (P < 0.05) compared to
corresponding exposure group, except for the 1.16 mg/L CPF and 1.13 mg/L ATR/CPF groups in the head kidney and the 4.28 mg/L ATR, 11.6 mg/L CPF and 1.13 mg/L ATR/CPF groups in the spleen. The iNOS activity for all of the treatment groups increased significantly (P < 0.05) compared to corresponding exposure group, except for the 4.28 mg/L ATR, 1.16 mg/L CPF and 1.13 mg/L ATR/CPF groups in the head kidney and the 4.28 mg/L ATR, 1.16 and 11.6 mg/L CPF, and 1.13 mg/L ATR/CPF groups in the spleen.
3.3. Analysis of electron microscopy Head kidney and spleen tissues from the control group showed no autophagic features under transmission electron microscopy (Fig. 4). In the exposure and the recovery groups, characteristic autophagic vacuolar organelles were observed. In some
Fig. 4. Electron microscopy representative images from the head kidney (AeG) and spleen (HeN). (B) (L) Autophagosome (black arrows); (C) Autolysosome; (D) Phagophores (blue arrows); Amphisomes (black arrows); (I) (J) (M) Vacuolization of mitochondria.
Please cite this article in press as: Chen D, et al., Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.), Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.01.014
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Fig. 4. (continued).
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autolysosomes, organelles such as mitochondria and other cytoplasmic elements were detected. 4. Discussion Oxidative stress could induce autophagy under certain conditions such as ischemia and reperfusion [20]. The conditions that regulate the activity of the autophagic process are also associated with changes in the production of reactive oxygen/nitrogen species (ROS/RNS) in cells [21]. It is known that ATR, CPF and their combination probably produced damages on immune organ in the carp [22,23]. A study reported that two ancient processes, autophagy and innate immunity, together through a shared signaling pathway [24]. The immune system utilizes autophagic degradation of cytoplasmic material and regulates adaptive immunity [25]. Thus, we investigate that autophagy and oxidative stress responses in the immune organ of common carp exposed to ATR, CPF and their combination. Because oxidative stress is evoked by many chemicals including some pesticides, pro-oxidant factors' action in fish organism can be used to assess specific area pollution or world sea pollution [26]. Immunotoxic effects of ATR and CPF on carp may be related with lipid peroxidation and releasing of reactive oxygen species (ROS) after their exposure [7]. The activities of antioxidant enzymes have been shown to be sensitive indicators of increased oxidative stress [23]. Pesticides may cause oxidative stress that leads to the generation of free radicals and alterations in antioxidants or free oxygen radical scavenging enzyme systems [27,28]. The changes in antioxidant enzymes have been observed after actuation of pesticides deltamethrin and cypermethrin [29]. Bipyridyl herbicides were able to form redox cycles and thereby cause oxidative stress [30]. ATR sublethal concentrations could induce oxidative stress in bluegill sunfish [32]. In a previous study, the activities of SOD, GSHPx and CAT decreased after exposure to ATR and CPF in the carp [4]. However, low concentrations of simazine do not cause oxidative stress in carps during sub-chronic tests [31]. In this study, the activities of AHR, ASA and POD were significantly induced in the immune organ of common carp exposed to ATR, CPF and their combination. The changes of antioxidant activity indicates a response against increased ROS production in pesticide toxicity. Many environmental contaminants, including pesticides, have the potential to induce production of NO [33]. NO is an important signaling molecule involved in the regulation of diverse physiological and pathophysiological mechanisms in nervous, cardiovascular, and immunological systems [34]. A number of previous reports demonstrated that ATR and CPF could induce the NO production and the iNOS mRNA level [35,36]. The present study demonstrates that NO content and iNOS activity are markedly upregulated in the head kidney and spleen of common carp exposed to ATR and CPF. The results obtained in this study are in accordance with the previous reports that ATR and CPF upregulates iNOS mRNA level and NO production in the brain of common carp and aorta of rat [34,37]. Thus, these results indicate that iNOS involves in the process of immunotoxicity induced by ATR and CPF. The function of autophagy is generally thought to be a natural process to maintain cellular homeostasis as well as a cellular response to starvation, infection or disease progression [38]. LC3 is widely used to monitor autophagy [39]. Dynein motor machinery plays a role in the delivery of autophagosome contents to lysosomes, in the process of autophagosome-lysosome fusion [40]. TOR negatively regulates the induction of autophagy. TOR is the first molecule that is identified as a pivotal player in the starvationsignaling pathway of autophagy [41]. In the present study, the results from transmission electron microscopy and qPCR confirmed ATR and CPF can induce autophagy in common carp. After human
SH-SY5Y cells exposed to paraquat, inhibition of the cytoplasmic accumulation of autophagic vacuoles as well as the recruitment of LC3 fusion protein to the vacuoles and an increase in mTOR phosphorylation were observed [42]. Cadmium can induce autophagy that formation of autophagosomes and processing of LC3 in MES-13 cells [43]. Cd-mediated ROS generation eventually induces autophagy through the down-regulation of mTOR in skin epidermal cells [44]. So we think that autophagy plays an important role in carp immunotoxicity by ATR, CPF and the ATR/CPF combination. In addition, Our results from oxidative-stress, histological parameters and autophagy genes also indicate that the joint toxicity of ATR/CPF combination is higher than ATR or CPF alone. Oxidative stress plays an important role in mediating autophagy during ischemia/reperfusion (I/R), and that activation of autophagy gene LC-II through oxidative stress mediates myocardial injury in response to I/R in the mouse heart [45]. Oxidative modification of the NOSs can cause disruption of electron transfer within the enzymes, inhibiting the formation of NO and generating O 2 [46]. In a biological setting, it is then likely that the cell is responding to the combined effects of ROS/RNS acting at different sites within the autophagic process [21]. Autophagy is regulated by ROS through the Atg1eAtg13 complex, which is inhibited by mTOR activation. Atg8epro-LC3 is cleaved by Atg4, modified by phosphatidylethanolamine (PE) to become LC3-II and inserted into the autophagosomes [20]. Autophagy signaling LC3-II is upregulated in response to denervation-induced oxidative stress in skeletal muscle [47]. Our results indicate that oxidative stress plays an important role in inducing autophagy and provides a novel mechanism for cell death. In conclusion, the results of this study indicate that autophagy is involved in oxidative stress and immune organ damage induced by ATR, CPF, and their combination. To our knowledge, this is the first work to report the effects of ATR, CPF and their combination on the mRNA levels of autophagy-related genes in the immune organ of carp. Thus, the information presented in this study is helpful to understand the mechanism of ATR-, CPF- and ATR/CPFcombination-induced immunotoxicity in fish. Acknowledgment China Postdoctoral Science Foundation (Project No. 2012M511437) and the Foundation of the Postdoctoral Research from Heilongjiang Province (Project No. LBH-Q14146) supported the study. The authors thank the members of the veterinary internal medicine laboratory in the College of Veterinary Medicine, Northeast Agricultural University. References [1] Jin R, Ke J. Impact of atrazine disposal on the water resources of the Yang river in Zhangjiakou area in China. Bull Environ Contam Toxicol 2002;68:893e900. [2] Gu JG, Fan Y, Gu JD. Biodegradability of atrazine, cyanazine and dicamba under methanogenic condition in three soils of China. Chemosphere 2003;52: 1515e21. [3] Paulino M, Souza N, Fernandes M. Subchronic exposure to atrazine induces biochemical and histopathological changes in the gills of a neotropical freshwater fish, Prochilodus lineatus. Ecotoxicol Environ Saf 2012;80:6e13. [4] Xing H, Li S, Wang Z, Gao X, Xu S, Wang X. Histopathological changes and antioxidant response in brain and kidney of common carp exposed to atrazine and chlorpyrifos. Chemosphere 2012;88:377e83. [5] de Campos Ventura B, de Angelis DdF, Marin-Morales MA. Mutagenic and genotoxic effects of the Atrazine herbicide in Oreochromis niloticus (Perciformes, Cichlidae) detected by the micronuclei test and the comet assay. Pestic Biochem Physiol 2008;90:42e51. [6] Moore A, Waring CP. Mechanistic effects of a triazine pesticide on reproductive endocrine function in mature male Atlantic Salmon (Salmo salar L.) Parr. Pestic Biochem Physiol 1998;62:41e50. [7] Xing H, Li S, Wang Z, Gao X, Xu S, Wang X. Oxidative stress response and histopathological changes due to atrazine and chlorpyrifos exposure in common carp. Pestic Biochem Physiol 2012;103:74e80.
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Please cite this article in press as: Chen D, et al., Effects of atrazine and chlorpyrifos on oxidative stress-induced autophagy in the immune organs of common carp (Cyprinus carpio L.), Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.01.014
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