J BIOCHEM MOLECULAR TOXICOLOGY Volume 28, Number 11, 2014
Toxic Effects of Paraquat on Cytokine Expression in Common Carp, Cyprinus carpio L. Junguo Ma, Xiaoyu Li, Yuanyuan Li, Yao Li, and Daichun Niu College of Life Science, Henan Normal University, Xinxiang, Henan 453007, People’s Republic of China; E-mail: [email protected] Received 6 March 2014; revised 26 May 2014; accepted 5 June 2014
ABSTRACT: In this study, the acute toxicity of paraquat (PQ) in common carp was determined. Then, the contents and mRNA levels of the cytokines interleukin-1β (IL-1β), interferon-γ (IFN-γ ), and tumor necrosis factor-α (TNF-α) were evaluated following subacute exposure to PQ. The results show that the LC50 of PQ for common carp at 72 and 96 h was 15.962 and 15.106 mg/L, respectively. Moreover, after 7 days of subacute PQ exposure, the IL-1β content in the fish liver and kidney increased, although it decreased in spleen. However, changes in the IFN-γ content showed an irregular trend. The TNF-α content increased in the liver and spleen but decreased in the kidney. Additionally, PQ exposure also induced alterations in the mRNA levels of IL-1β, IFN-γ , and TNF-α. These results suggest that PQ exposure may result in suppression or excessive activation of the immune system in treated fish and lead to immune dysfunction and reduced imC 2014 Wiley Periodicals, Inc. J. Biochem. Mol. munity. Toxicol. 28:501–509, 2014; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21590
Paraquat; Common Carp; Acute Toxicity; Cytokine; Immunotoxicity
KEYWORDS:
INTRODUCTION
water solubility and extensive usage, many cases of acute poisoning and death have been reported in humans over the past few decades [3]. Pulmonary fibrosis is a major hallmark and a leading cause of death in PQ poisoning [4]. PQ toxicity has been found to be mediated by the production of free radicals [5], which cause oxidative damage to cells. The extensive use of PQ in agricultural practice throughout the world may compromise the integrity of biological systems in fish. PQ can enter bodies of water as a result of rain and soil leaching when used in the vicinity of aquatic ecosystems [6, 7]. PQ toxicity studies have been predominantly performed in mammals [4, 8], whereas their possible immunotoxic effect on fish has received relatively little attention [7, 9, 10]. The fish immune system is the first defense against exogenous stresses, such as environmental chemicals [11, 12]. It is more sensitive to xenobiotics and reacts more rapidly than other systems, which can be an early warning sign or index of environmental toxicant stress [13, 14]. Common carp (Cyprinus carpio L.) is a fish species that is distributed worldwide and is also frequently adopted as an animal model for toxicological testing to determine the toxicity of chemicals in the aquatic environment [15]. This study aimed to determine the acute toxicity and immunotoxic effects of PQ on common carp.
Paraquat (PQ) is a nonselective herbicide used worldwide, and it has been demonstrated to be highly toxic to animals and humans [1, 2]. Owing to its high
MATERIALS AND METHODS Correspondence to: Xiaoyu Li. Contract Grant Sponsor: National Science Foundation of China. Contract Grant Number: 31172415. Contract Grant Sponsor: Innovation Scientists and Technicians Troop Construction Projects of Henan Province. Contract Grant Sponsor: Plan for Scientific Innovation Talent of Henan Province. Contract Grant Number: 134200510014. Contract Grant Sponsor: Key Subjects of Biology and Ecology in Henan Province, China. C 2014 Wiley Periodicals, Inc.
PQ, Kits, and Chemicals PQ (1-1 -dimethyl-4,4 -bipyridinium dichloride) was purchased from the Anhui Fengle Agrochemical (Hefei, Anhui, People’s Republic of China) as a commercial formulation (200 g/L, w/v). PQ was first dissolved in distilled water to make a stock solution and then further diluted with dechlorinated tap water to obtain the experimental concentration. 501
502
MA ET AL.
The kits for interleukin-1β (IL-1β), interferon-γ (IFN-γ ), and tumor necrosis factor-α (TNF-α) assays were all purchased from the Shanghai Boyao Biotechnology (Shanghai, People’s Republic of China). Other reagents used in this study were purchased from Sigma (St. Louis, MO) and were of analytical grade.
Fish The common carp (8.14 ± 1.37 g mean body weight) used in this study was originally obtained from the Feilong aquarium fishery (Xinxiang, Henan, People’s Republic of China). The fish were raised in a 200L tank under laboratory conditions for 2 weeks before testing. During acclimation, the fish were fed ad libitum with commercial food from the Feilong aquarium fishery in the amount of 1–1.5% fish body weight per day. The water temperature was maintained at 25 ± 2°C, and the dissolved oxygen level was 7.0 mg/L. The fish were exposed to a 16-h light/8-h dark photoperiod, and the tank water was partially changed every day with aerated tap water (total water hardness of 340 mg/L, pH 7.6, turbidity 1.5 nephelometric turbidity unit (NTU), total dissolved solid content 660 mg/L). The fish were handled according to the guidelines described in the China Law for Animal Health Protection and Instructions for Granting Permits for Animal Experimentation for Scientific Purposes (Ethics approval No. SCXK (YU) 2005-0001).
LC50 Determination and Subacute Exposure of PQ The LC50 of PQ in common carp was determined according to the Spearman–K¨arber method [16]. Briefly, acute toxicity testing was carried out by 96 h of PQ exposure in 30-L glass jars containing variable PQ concentrations: 13.44, 14.933, 16.597, 18.436, 20.485, 22.761, and 25.29 mg/L. One control group was designated and exposed to aerated tap water. The various concentrations of PQ for acute exposure were selected according to the result of our preliminary acute toxicity test and the Spearman-K¨arber method [16]. A total of 40 fish were randomly divided into eight groups (seven treatment groups and one control group with five fish in each group) and were exposed to the PQ solution under semistatic conditions for 96 h. During exposure, no food was provided, saturated oxygen was maintained in the solution for all groups, and the water and PQ solution were completely changed daily. Each test was conducted in duplicate. During the testing period, fish behavior was observed and the number of deaths in every group was recorded. After 96 h of PQ exposure, the LC50 was calculated using SPSS 13.0 software.
Volume 28, Number 11, 2014
On the basis of LC50 value obtained from the acute toxicity test as described above, two concentrations of PQ, 1.596 (1/10 of 72-h LC50 ) and 3.192 mg/L (1/5 of 72-h LC50 ), were selected for the subacute exposure PQ dose. For subacute exposure, 54 fish were randomly divided into three groups (18 fish in each group): two PQ exposure groups and one aerated tap water control group. The fish were exposed to PQ solution under semistatic conditions for 7 days. Food was not provided during this time, but saturated oxygen was maintained in the solution. Each test was conducted in duplicate. The water and PQ solution were completely changed every day. None of the fish died during the testing period. After the first day, the third day, and seventh day of PQ exposure, six fish were randomly selected from each group, anesthetized with 100 mg/L of MS-222 (Tricaine), and dissected to collect the liver, kidney, and spleen. The tissues were excised and immediately placed onto an ice-cold plate, washed in a physiological saline solution (PBS), and immediately frozen in liquid nitrogen. The tissues were stored at –80°C until use in assays.
Sample Preparation and Cytokine Content Assay Preweighed tissues (0.1 g of liver, kidney, or spleen) were homogenized in cold PBS (pH 7.2) at a 1/10 (w/v) ratio. Then, each homogenate was centrifuged at 3000 × g for 10 min at 4°C. The supernatants obtained were stored at –20°C for cytokine assays. In addition, 1–2 g of each tissue was sampled and stored at –80°C for RNA isolation. The content of the cytokines IL-1β, IFN-γ , and TNF-α in the fish liver, kidney, and spleen was determined using kits from Shanghai Boyao Biotechnology according to manufacturer’s instructions.
Cytokine Transcription Analysis Total RNA was isolated from 1–2 g of frozen tissue using a TRIzon Reagent kit (Cwbiotech, Beijing, People’s Republic of China) according to manufacturer’s instructions. The concentration and purity of the total RNA was determined spectrophotometrically according to the method described by Sambrook and Russell [17]. A total of 2 μg of RNA was used as a template for the first-strand cDNA synthesis, which was performed with the HiFi-MMLV cDNA kit (Cwbiotech, Beijing, People’s Republic of China) with oligo (dT) as the primer. Quantitative real-time PCR (qPCR) was performed to detect the expression of IL-1β, IFN-γ , and TNF-α mRNA in the fish liver, kidney, and spleen using the J Biochem Molecular Toxicology
DOI 10.1002/jbt
Volume 28, Number 11, 2014
PARAQUAT IMMUNOTOXICITY ON COMMON CARP
TABLE 1. The Primers Used for qPCR Primers IL-1β F IL-1β R IFN-γ 1 F IFN-γ 1 R IFN-γ 2 F IFN-γ 2 R TNF-1,2,3α F TNF-1,2,3α R β-Actin F β- Actin R
Sequences 5 -GCTGGAGCAATGCAATACAAA-3
5 -AGGTAGAGGTTGCTGTTGGAA-3 5 -TGCACTTGTCAGTCGCTGCT-3 5 -TGTACTTGTGTCTCAGTATTT-3 5 -TCTTGAGGAACCTGAGCAGAA-3 5 -TGTGCAAGTCTTTCCTTTGTAG-3 5 -ACAACAATCAGGAAGGTGGAA-3 5 -TGGAAAGACACCTGGCTGTA-3 5 -GCTATGTGGCTCTTGACTTCG-3 5 -CCGTCAGGCAGCTGATAGCT-3
Abbreviations: F, forward; R, reverse.
UItraSYBR Mixture (with ROX) (Cwbiotech, Beijing, People’s Republic of China) according to manufacturer’s instructions. The qPCR primers were designed based on the sequences retrieved from GenBank: IL-1β (AJ245635), IFN-γ 1 (AM261214), IFN-γ 2 (AM168523), TNF-1α (AJ311800), TNF-2α (AJ311801), and TNF-3α (AB112424) (Table 1). β-Actin (accession no. M24113) was chosen as an internal standard. The amount of target mRNA, normalized to β-actin mRNA, was calculated using the formula 2−Ct [18], where the Ct value was determined by subtracting the average βactin Ct value from the average target gene Ct value as follows: Ct = (Ct (target) – Ct (β-actin)) target – (Ct (target) – Ct (β-actin)) standard. The threshold cycle (Ct ) was determined for each sample using the exponential growth phase and the baseline signals from the plot of fluorescence versus cycle number.
Statistical Analyses The data were analyzed using a one-way analysis of variance (Tukey) followed by a least significant difference determination using SPSS 13.0 for Windows. A p value of less than 0.05 was considered statistically significant.
RESULTS LC50 of PQ for Common Carp The results of the acute toxicity test showed that the 72-h LC50 and 96-h LC50 values for PQ in common carp were 15.962 mg/L (with a 95% confidence interval (CI) of 15.018–17.004 mg/L) and 15.106 mg/L (with a 95% CI of 14.113–6.139 mg/L) respectively. Additionally, we also observed that the behavior of fish in the treatment group was different from that of fish in the control group. For example, the respiratory rate of the treated fish increased. Additionally, in the early phase of exposure, the fish swam quickly or jumped; however, they gradually became dull and weak and J Biochem Molecular Toxicology
DOI 10.1002/jbt
503
swam feebly and slowly, all of which are obvious signs of toxicity. In the later stages of testing, hemorrhage or decay occurred in the gills or fins of some fish exposed to high concentrations of PQ (e.g., 25.29 mg/L), and finally, they sank to the bottom of the jar and died.
Content of IL-1β in the Liver, Kidney, and Spleen of Common Carp after Subacute Exposure to PQ for 7 Days Data related to the IL-1β content in the fish liver, kidney, and spleen are shown in Figure 1. The liver IL-1β content increased after 1 or 3 d of PQ exposure but remained unchanged at the end of the test when compared to the control (Figure 1A). The IL-1β content in the fish kidney showed similar changes after 7 days of PQ exposure, except a decrease was observed at 3 d of testing in the higher dose group (3.192 mg/L) (Figure 1B). However, in the spleen, no change in the IL-1β content was found between the treatment and control groups from 1 to 3 d of exposure, although a significant decrease was observed at the end of the test (Figure 1C).
IFN-γ Changes in the IFN-γ content in the liver, kidney, and spleen after PQ exposure were rather irregular (Figure 2). In the liver, exposure to PQ at a lower concentration (1.596 mg/L) caused a decrease in the IFNγ content over shorter exposure durations (from 1 to 3 d) and an increase over longer exposure durations (7 d), whereas the higher concentration of PQ (3.192 mg/L) first induced a decrease at the 1-day interval followed by an increase at 3 d with a final decrease at 7 d (Figure 2A). However, in the kidney, the IFN-γ content in the treated fish was higher than that in the controls, except for a decrease in the 3.192-mg/L group at the end of the test (Figure 2B). With regard to the spleen, the IFN-γ content was decreased in all treatment groups compared to the control group; however, an increase was observed at 3 d of PQ exposure in the 3.192-mg/L group (Figure 2C).
TNF-α Unlike IFN-γ , the change in the TNF-α content in the tissues of common carp after PQ exposure was relatively regular. For example, in all groups, TNF-α increased in the liver throughout the period of exposure (Figure 3A), and almost all groups showed a decrease in the kidney (Figure 3B). In the spleen, an initial increase
504
MA ET AL.
Volume 28, Number 11, 2014
160
A
**
140
** **
120 IL-1β ng/L)
**
100
0
80
1.596 mg/L
60
3.192 mg/L
40 20 0 1
140
3 Exposure time (day)
B
**
120 IL-1β (ng/L)
7
**
100
**
80
0
*
1.596 mg/L
60
3.192 mg/L
40 20 0 1
140
3 Exposure time (day)
7
C
IL-1β ng/L)
120 100 80
**
60
**
0 1.596 mg/L 3.192 mg/L
40 20 0 1
3 Exposure time (day)
7
FIGURE 1. IL-1β content in the (A) liver, (B) kidney, and (C) spleen of common carp exposed to 1.596 or 3.192 mg/L PQ for 7 d. The protocols for PQ exposure and the determination of IL-1β content are described in the section Sample Preparation and Cytokine Content Assay. The experiment was performed in duplicate, and the data are shown as the mean ± SD. Asterisks denote a response that is significantly different from the control (*p < 0.05, **p < 0.01).
was observed (from 1 to 3 d) followed by a decrease (7 d) (Figure 3C).
IL-1β mRNA Expression in the Liver, Kidney, and Spleen of Common Carp after Subacute Exposure to PQ for 7 Days The changes in IL-1β transcription were comparatively different between the three fish tissues after PQ exposure. IL-1β expression in the liver was first downregulated (1 d) but was later upregulated (3–7 d)
compared to the control (Figure 4A), whereas in the spleen, IL-1β was first upregulated and subsequently downregulated (Figure 4C). However, in the kidney, IL-1β transcription was enhanced as a whole in the treatment groups compared with the control group (Figure 4B).
TNF-α mRNA Levels Liver TNF-α expression was markedly downregulated after 1 d of PQ exposure and was later J Biochem Molecular Toxicology
DOI 10.1002/jbt
Volume 28, Number 11, 2014
PARAQUAT IMMUNOTOXICITY ON COMMON CARP
900 800
A *
IFN-γ (ng/L)
1.596 mg/L
**
*
**
600
0
**
700
505
3.192 mg/L *
500 400 300 200 100 0 1
3
7
Exposure time (day)
900
B
**
800 ng/L) IFN-γ
700 600 400
0
** **
1.596 mg/L
**
3.192 mg/L
500
**
300 200 100 0 1
3
7
Exposure time (day)
900
C
ng/L) IFN-γ
700 600 400
500
0
**
800
1.596 mg/L **
** **
**
1
3
3.192 mg/L **
300 200 100 0 7
Exposure time (day)
FIGURE 2. Content of IFN-γ in the (A) liver, (B) kidney, and (C) spleen of common carp exposed to 1.596 or 3.192 mg/L PQ for 7 d. The protocols for PQ exposure and the IFN-γ content assay are described in the section Sample Preparation and Cytokine Content Assay. All the experiments were performed in duplicate, and the data are shown as the mean ± SD. Asterisks denote a response that is significantly different from the control (*p < 0.05, **p < 0.01).
upregulated (3–7 d) compared to that of the control (Figure 4D). However, kidney TNF-α expression in the PQ-treated groups was downregulated during the duration of the test (except for the 3.192-mg/L group at 7 d) (Figure 4E). Regarding the spleen, TNF-α expression in the treatment groups was upregulated during the early stages of exposure (1 d) but was downregulated during the later stages (from 3 to 7 d, except for the 3.192-mg/L group) (Figure 4F).
J Biochem Molecular Toxicology
DOI 10.1002/jbt
IFN-γ mRNA Levels IFN-γ mRNA expression in the liver, kidney, and spleen of common carp after PQ exposure is shown in Figure 5. Liver IFN-γ 1 and IFN-γ 2 showed a similar trend of transcriptional change, i.e., downregulation at 1 d of exposure, upregulation in the higher PQ concentration group (3.192 mg/L) at 3 d of exposure, and downregulation in the higher concentration group at the end of the test compared to
506
MA ET AL.
Volume 28, Number 11, 2014
400
A
**
ng/L˅
350 **
300
** **
250
**
TNF-α
0 **
200
1.596 mg/L 3.192 mg/L
150 100 50 0 1
3
7
Exposure time (day)
400
B
TNF-α
ng/L)
350
*
**
300 250
0 **
200
1.596 mg/L
**
3.192 mg/L
150 100 50 0 1
3
7
Exposure time (day)
350
TNF-α
ng/L)
300
C **
250
**
** **
**
200
0
**
1.596 mg/L
150
3.192 mg/L
100 50 0 1
3
7
Exposure time (day)
FIGURE 3. Content of TNF-α in the (A) liver, (B) kidney, and (C) spleen of common carp exposed to 1.596 or 3.192 mg/L PQ for 7 d. The protocols for PQ exposure and the determination of TNF-α content are described in the section Sample Preparation and Cytokine Content Assay. The experiment was performed in duplicate, and the data are shown as the mean ± SD. Asterisks denote a response that is significantly different from the control (*p < 0.05, **p < 0.01).
that of the control group (Figure 5A). The changes in IFN-γ 1 and IFN-γ 2 mRNA levels in the kidney were similar to those in the liver, except for the upregulation observed in the 1.596-mg/L group at 1 d (Figure 5B). In addition, IFN-γ transcription in the spleen from the treated groups was downregulated at 1 d, promoted at 3 d, and downregulated again at 7 d (Figure 5C).
DISCUSSION The results of acute toxicity testing in this study revealed that PQ is highly toxic to common carp according to the PQ 96-h LC50 (15.106 mg/L) and the toxicity classification of pesticides [19]. The PQ 96-h LC50 for common carp approaches the same order of magnitude observed in other types of fish (e.g., 20 mg/L in
J Biochem Molecular Toxicology
DOI 10.1002/jbt
Volume 28, Number 11, 2014
1.2
A
1.4
** **
1.2
* 0
1
1.596 mg/L
0.8
3.192 mg/L
**
0.6
**
0.4
B
**
**
1 **
0.8
1
1.596 mg/L 3.192 mg/L
0.4
3
1
7
3
Exposure time (day) **
1.8
**
1.2 1
**
0.8
0 **
**
1.596 mg/L 3.192 mg/L
0.6
**
0.4
.
1.6 1.4
TNFα relative expression
IL-1β relative expression .
C
1.2
0.2
D
**
** 0
1
1.596 mg/L
0.8 0.4
**
3
7
1
3
Exposure time (day) 1.8
*
E **
** **
0.8
1.6
0 1.596 mg/L
0.6 **
0.4
7
Exposure time (day)
3.192 mg/L
**
0.2
TNF α relative expression .
TNFα relative expression .
3.192 mg/L
**
0.6
0
1
1
**
0.2
0
1.2
7
Exposure time (day)
1.4
1.4
0
0.6
0
0
1.6
*
0.2
0.2
1.8
**
507
.
.
1.4
IL-1β relative expression
1.6
IL-1β relative expression
1.8
PARAQUAT IMMUNOTOXICITY ON COMMON CARP
F
**
** **
1.4 1.2
0
**
1
1.596 mg/L
0.8
3.192 mg/L
**
0.6
**
0.4 0.2 0
0 1
3
7
Exposure time (day)
1
3
7
Exposure time (day)
FIGURE 4. IL-1β and TNF-α mRNA levels in the liver, kidney, and spleen of common carp exposed to 1.596 or 3.192 mg/L of PQ for 7 d. IL-1β mRNA expression was determined by qPCR as described in the section Cytokine Transcription Analysis. Asterisks denote a response that is significantly different from the control (*p < 0.05, **p < 0.01). The level of IL-1β is shown in the (A) liver, (B) kidney, and (C) spleen, and the level of TNF-α is shown in the (D) liver, (E) kidney, and (F) spleen.
Nile tilapia [9] and 52.48 mg/L in Cnesterodon decemmaculatus [20]). IL-1β is a key mediator in response to microbial invasion, tissue injury, and xenobiotics; it can stimulate immune responses by activating lymphocytes and inducing the release of other cytokines that can activate macrophages and natural killer (NK) cells [21]. Wang et al. [15] reported that IL-1β expression was upregulated in the kidney and spleen of common carp after exposure to atrazine, chlorpyrifos, or a mixture of both. Our results indicate that PQ increases the content of IL-1β in the liver and kidney at early time points following exposure but decreases it in the spleen at a later time, suggesting that IL-1β in different fish organs may respond variably to PQ. Meanwhile, IL-1β gene transcription in the three tissues evaluated was also inconsistent. For example, liver IL-1β expression was initially downregulated and subsequently upregulated, whereas in the spleen, it was initially upregulated and later downregulated. Contrary to what was observed in the liver and spleen, IL-1β transcription in
J Biochem Molecular Toxicology
DOI 10.1002/jbt
the kidney was promoted throughout the experiment. Additionally, we found a remarkable discordance between the IL-1β content and transcription in the fish liver after 1 d of PQ treatment (i.e., a significant increase in the IL-1β content but a dramatic downregulation of IL-1β gene expression). IFN-γ is constitutively produced by NK cells of the innate immune system. It stimulates macrophagemediated phagocytosis and the production of proinflammatory cytokines and antimicrobial oxygen radicals by macrophages [22–25]. In this study, PQ exposure changed the mRNA expression level or content of IFN-γ in the fish liver, kidney, and spleen. For example, the IFN-γ content was increased in the kidney and decreased in the spleen, while IFN-γ transcription was promoted in the liver and kidney but inhibited in the spleen. These alterations may affect IFN-γ expression and disturb its function. TNF-α is a proinflammatory cytokine involved in inflammation, apoptosis, cell proliferation, and the stimulation of various aspects of the immune system
MA ET AL.
A
** **
.
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
Volume 28, Number 11, 2014
0 1.596 mg/L 3.192 mg/L ** **
**
**
1
3
IFN γ2 relative expression
IFN γ1 relative expression .
508
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
**
0 1.596 mg/L **
** **
1
7
3
2
** **
1.5
**
0 *
1 0.5
1.596 mg/L 3.192 mg/L
**
0 1
3
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
** ** *
3.192 mg/L
1
3
7
Exposure time (day)
**
1.8
**
1.6
* 0 1.596 mg/L
**
3.192 mg/L ** **
**
IFN γ2 relative expression .
IFN γ1 relative expression .
1.596 mg/L
**
**
7
C
0
*
Exposure time (day) 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
7
Exposure time (day)
IFN γ2 relative expression .
IFN γ1 relative expression .
B
3.192 mg/L
**
Exposure time (day) 2.5
**
**
1.4
*
1.2 0
1
1.596 mg/L
0.8
3.192 mg/L
**
0.6
**
0.4 0.2 0
1
3
7
1
3
Exposure time (day)
7
Exposure time (day)
FIGURE 5. The mRNA levels of IFN-γ 1 and IFN-γ 2 in the (A) liver, (B) kidney, and (C) spleen of common carp exposed to 1.596 or 3.192 mg/L PQ for 7 d. The mRNA levels of IFN-γ 1 and IFN-γ 2 were determined by qPCR as described in the section Cytokine Transcription Analysis. Asterisks denote a response that is significantly different from the control (*p < 0.05, **p < 0.01).
[26]. There have been several reports regarding the upregulation of TNF-α expression observed in common carp fed with Spirulina [27] or treated with yeast extract compared to control fish [21]. In our study, we found a remarkable increase in the TNF-α content in the fish liver and spleen (after 1–3 d of PQ exposure), as shown in Figure 3, and upregulation of TNF-α gene expression in the liver (3–7 d) and spleen (1 d) (Figure 4), suggesting that PQ exposure affects the expression or function of TNF-α. Cytokines are important regulators of the immune system in teleost fish and are mainly responsible for host innate defense mechanisms [28]. Therefore, cytokines are important conventional terminal indexes of innate immunity for evaluating fish immunotoxicity [21]. Our results reveal that PQ exposure not only changes the content of cytokines but also alters their gene expression in the immune-associated organs of common carp. This may cause immune dysfunction and reduced immunity.
REFERENCES 1. Mandel JS, Adami H-O, Cole P. Paraquat and Parkinson’s disease: an overview of the epidemiology and a review of two recent studies. Regul Toxicol Pharmacol 2012;62:385– 392. 2. Suntres ZE. Role of antioxidants in paraquat toxicity. Toxicology 2012;180:65–77. 3. Corsinia E, Sokooti M, Galli CL, Moretto A, Colosio C. Pesticide induced immunotoxicity in humans: a comprehensive review of the existing evidence. Toxicology 2013;307:123–135. 4. Amirshahrokhi K. Anti-inflammatory effect of thalidomide in paraquat-induced pulmonary injury in mice. Int Immunopharmacol 2013;17:210–215. 5. Mussi MA, Calcaterra NB. Paraquat-induced oxidative stress response during amphibian early embryonic development. Comp Biochem Physiol C 2010;151:240–247. 6. Sun F, Chen HS. Monitoring of pesticide chlorpyrifos residue in farmed fish: investigation of possible sources. Chemosphere 2008;71:1866–1869. 7. Salazar-Lugoa R, Estrella A, Oliveros A, Rojas-Villarroel E, Villalobos de BL, Lemus M. Paraquat and temperature affect nonspecific immune response of Colossoma
J Biochem Molecular Toxicology
DOI 10.1002/jbt
Volume 28, Number 11, 2014
8.
9.
10.
11.
12. 13. 14. 15.
16. 17.
PARAQUAT IMMUNOTOXICITY ON COMMON CARP
macropomum. Environ Toxicol Pharmacol 2009;27:321– 326. Riahi B, Rafatpanah H, Mahmoudi M, Memar B, Brook A, Tabasi N, Karimi G. Immunotoxicity of paraquat after subacute exposure to mice. Food Chem Toxicol 2010;48:1627–1631. Figueiredo-Fernandes A, Fonta´ınhas-Fernandes A, Peixoto F, Rocha E, Reis-Henriques MA. Effects of gender and temperature on oxidative stress enzymes in Nile tilapia Oreochromis niloticus exposed to paraquat. Pestic Biochem Physiol 2006;85:97–103. Tukmechi A, Rezaee J, Nejati V, Sheikhzadeh N. Effect of acute and chronic toxicity of paraquat on immune system and growth performance in rainbow trout, Oncorhynchus mykiss. Aquac Res 2013; doi: 10.1111/are.12118 Burns-Naas LA, Meade BJ, Munson AE. Toxic responses of the immune system. In: Klaassen CD, editor. Casarett & Doull’s toxicology –the basic science of poisons. New York: McGraw Hill; 2001. pp 419–471. Galloway T, Handy R. Immunotoxicity of organophosphorous pesticides. Ecotoxicology 2003;12:345–363. Wester PW, Vehtaak AD, van Muiswinkel WB. Fish as biomarkers in immunotoxicology. Toxicology 1994;86:213–232. Colosio C, Birindelli S, Corsini E, Galli CL, Maroni M. Low level exposure to chemicals and immune system. Toxicol Appl Pharmacol 2005;207:320–328. Wang X, Xing HJ, Li XL, Xu SW, Wang XL. Effects of atrazine and chlorpyrifos on the mRNA levels of IL-1 and IFN-γ 2b in immune organs of common carp. Fish Shellfish Immunol 2011;31:126–133. K¨arber G. Beitrag zur kollektiven behandlung pharmakologischer reihenversuche. Arch Exp Pathol Pharmakol 1931;162:480–483. Sambrook J, Russel DW. Molecular cloning: a laboratory manual, 3rd edition. New York: Cold Spring Harbor Laboratory; 2001. Vol 1, pp 13–17.
J Biochem Molecular Toxicology
DOI 10.1002/jbt
509
18. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real- time quantitative PCR and the 2 −Ct method. Methods 2001;25:402–408. 19. Ecobichon DJ. Toxic effects of pesticides. In: Klaassen CD, editor. Casarett & Doull’s toxicology – the basic science of poisons. New York: McGraw Hill ; 2001. pp 763–802. 20. Di Marzio WD, Alberdi JL, S´aenz ME, Tortorelli MC. EfR Formulation) on survival fects of paraquat (Osaquat and total cholinesterase activity in male and female adults of Cnesterodon decemmaculatus (Pisces, Poeciliidae). Environ Toxicol Water Qual 1998;13:55–59. 21. Biswas G, Korenaga H, Takayama H, Kono T, Shimokawa H, Sakai M. Cytokine responses in the common carp, Cyprinus carpio L. treated with baker’s yeast extract. Aquaculture 2012;356–357:169–175. 22. Robertsen B. The interferon system of teleost fish. Fish Shellfish Immunol 2006;20:172–191. 23. Grayfer L, Belosevic M. Molecular characterization, expression and functional analysis of goldfish (Carassius aurutus L.) interferon gamma. Dev Comp Immunol 2009;33:235–246. 24. Kitao Y, Kono T, Korenaga H, Iizasa T, Nakamura K, Savan R, Sakai M. Characterization and expression analysis of type I interferon in common carp Cyprinus carpio L. Mol Immunol 2009;46:2548–2556. 25. Zou J, Secombes CJ. Teleost fish interferons and their role in immunity. Dev Comp Immunol 2011;35:1376–1387. 26. Savan R, Sakai M. Presence of multiple isoforms of TNF alpha in carp (Cyprinus carpio L.): genomic and expression analysis. Fish Shellfish Immunol 2004;17:87–94. 27. Watanuki H, Ota K, Tassakka ACMAR, Kato T, Sakai M. Immunostimulant effects of dietary Spirulina platensis on carp, Cyprinus carpio. Aquaculture 2006;258:157–163. 28. Huttenhuis HBT, Taverne-Thiele AJ, Grou CPO, Bergsma J, Saeij JPJ, Nakayasu C, Rombout JHWM. Ontogeny of the common carp (Cyprinus carpio L.) innate immune system. Dev Comp Immunol 2006;30:557–574.
Toxic Effects of Paraquat on Cytokine Expression in Common Carp, Cyprinus carpio L. Junguo Ma, Xiaoyu Li, Yuanyuan Li, Yao Li, and Daichun Niu College of Life Science, Henan Normal University, Xinxiang, Henan 453007, People’s Republic of China; E-mail: [email protected] Received 6 March 2014; revised 26 May 2014; accepted 5 June 2014
ABSTRACT: In this study, the acute toxicity of paraquat (PQ) in common carp was determined. Then, the contents and mRNA levels of the cytokines interleukin-1β (IL-1β), interferon-γ (IFN-γ ), and tumor necrosis factor-α (TNF-α) were evaluated following subacute exposure to PQ. The results show that the LC50 of PQ for common carp at 72 and 96 h was 15.962 and 15.106 mg/L, respectively. Moreover, after 7 days of subacute PQ exposure, the IL-1β content in the fish liver and kidney increased, although it decreased in spleen. However, changes in the IFN-γ content showed an irregular trend. The TNF-α content increased in the liver and spleen but decreased in the kidney. Additionally, PQ exposure also induced alterations in the mRNA levels of IL-1β, IFN-γ , and TNF-α. These results suggest that PQ exposure may result in suppression or excessive activation of the immune system in treated fish and lead to immune dysfunction and reduced imC 2014 Wiley Periodicals, Inc. J. Biochem. Mol. munity. Toxicol. 28:501–509, 2014; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.21590
Paraquat; Common Carp; Acute Toxicity; Cytokine; Immunotoxicity
KEYWORDS:
INTRODUCTION
water solubility and extensive usage, many cases of acute poisoning and death have been reported in humans over the past few decades [3]. Pulmonary fibrosis is a major hallmark and a leading cause of death in PQ poisoning [4]. PQ toxicity has been found to be mediated by the production of free radicals [5], which cause oxidative damage to cells. The extensive use of PQ in agricultural practice throughout the world may compromise the integrity of biological systems in fish. PQ can enter bodies of water as a result of rain and soil leaching when used in the vicinity of aquatic ecosystems [6, 7]. PQ toxicity studies have been predominantly performed in mammals [4, 8], whereas their possible immunotoxic effect on fish has received relatively little attention [7, 9, 10]. The fish immune system is the first defense against exogenous stresses, such as environmental chemicals [11, 12]. It is more sensitive to xenobiotics and reacts more rapidly than other systems, which can be an early warning sign or index of environmental toxicant stress [13, 14]. Common carp (Cyprinus carpio L.) is a fish species that is distributed worldwide and is also frequently adopted as an animal model for toxicological testing to determine the toxicity of chemicals in the aquatic environment [15]. This study aimed to determine the acute toxicity and immunotoxic effects of PQ on common carp.
Paraquat (PQ) is a nonselective herbicide used worldwide, and it has been demonstrated to be highly toxic to animals and humans [1, 2]. Owing to its high
MATERIALS AND METHODS Correspondence to: Xiaoyu Li. Contract Grant Sponsor: National Science Foundation of China. Contract Grant Number: 31172415. Contract Grant Sponsor: Innovation Scientists and Technicians Troop Construction Projects of Henan Province. Contract Grant Sponsor: Plan for Scientific Innovation Talent of Henan Province. Contract Grant Number: 134200510014. Contract Grant Sponsor: Key Subjects of Biology and Ecology in Henan Province, China. C 2014 Wiley Periodicals, Inc.
PQ, Kits, and Chemicals PQ (1-1 -dimethyl-4,4 -bipyridinium dichloride) was purchased from the Anhui Fengle Agrochemical (Hefei, Anhui, People’s Republic of China) as a commercial formulation (200 g/L, w/v). PQ was first dissolved in distilled water to make a stock solution and then further diluted with dechlorinated tap water to obtain the experimental concentration. 501
502
MA ET AL.
The kits for interleukin-1β (IL-1β), interferon-γ (IFN-γ ), and tumor necrosis factor-α (TNF-α) assays were all purchased from the Shanghai Boyao Biotechnology (Shanghai, People’s Republic of China). Other reagents used in this study were purchased from Sigma (St. Louis, MO) and were of analytical grade.
Fish The common carp (8.14 ± 1.37 g mean body weight) used in this study was originally obtained from the Feilong aquarium fishery (Xinxiang, Henan, People’s Republic of China). The fish were raised in a 200L tank under laboratory conditions for 2 weeks before testing. During acclimation, the fish were fed ad libitum with commercial food from the Feilong aquarium fishery in the amount of 1–1.5% fish body weight per day. The water temperature was maintained at 25 ± 2°C, and the dissolved oxygen level was 7.0 mg/L. The fish were exposed to a 16-h light/8-h dark photoperiod, and the tank water was partially changed every day with aerated tap water (total water hardness of 340 mg/L, pH 7.6, turbidity 1.5 nephelometric turbidity unit (NTU), total dissolved solid content 660 mg/L). The fish were handled according to the guidelines described in the China Law for Animal Health Protection and Instructions for Granting Permits for Animal Experimentation for Scientific Purposes (Ethics approval No. SCXK (YU) 2005-0001).
LC50 Determination and Subacute Exposure of PQ The LC50 of PQ in common carp was determined according to the Spearman–K¨arber method [16]. Briefly, acute toxicity testing was carried out by 96 h of PQ exposure in 30-L glass jars containing variable PQ concentrations: 13.44, 14.933, 16.597, 18.436, 20.485, 22.761, and 25.29 mg/L. One control group was designated and exposed to aerated tap water. The various concentrations of PQ for acute exposure were selected according to the result of our preliminary acute toxicity test and the Spearman-K¨arber method [16]. A total of 40 fish were randomly divided into eight groups (seven treatment groups and one control group with five fish in each group) and were exposed to the PQ solution under semistatic conditions for 96 h. During exposure, no food was provided, saturated oxygen was maintained in the solution for all groups, and the water and PQ solution were completely changed daily. Each test was conducted in duplicate. During the testing period, fish behavior was observed and the number of deaths in every group was recorded. After 96 h of PQ exposure, the LC50 was calculated using SPSS 13.0 software.
Volume 28, Number 11, 2014
On the basis of LC50 value obtained from the acute toxicity test as described above, two concentrations of PQ, 1.596 (1/10 of 72-h LC50 ) and 3.192 mg/L (1/5 of 72-h LC50 ), were selected for the subacute exposure PQ dose. For subacute exposure, 54 fish were randomly divided into three groups (18 fish in each group): two PQ exposure groups and one aerated tap water control group. The fish were exposed to PQ solution under semistatic conditions for 7 days. Food was not provided during this time, but saturated oxygen was maintained in the solution. Each test was conducted in duplicate. The water and PQ solution were completely changed every day. None of the fish died during the testing period. After the first day, the third day, and seventh day of PQ exposure, six fish were randomly selected from each group, anesthetized with 100 mg/L of MS-222 (Tricaine), and dissected to collect the liver, kidney, and spleen. The tissues were excised and immediately placed onto an ice-cold plate, washed in a physiological saline solution (PBS), and immediately frozen in liquid nitrogen. The tissues were stored at –80°C until use in assays.
Sample Preparation and Cytokine Content Assay Preweighed tissues (0.1 g of liver, kidney, or spleen) were homogenized in cold PBS (pH 7.2) at a 1/10 (w/v) ratio. Then, each homogenate was centrifuged at 3000 × g for 10 min at 4°C. The supernatants obtained were stored at –20°C for cytokine assays. In addition, 1–2 g of each tissue was sampled and stored at –80°C for RNA isolation. The content of the cytokines IL-1β, IFN-γ , and TNF-α in the fish liver, kidney, and spleen was determined using kits from Shanghai Boyao Biotechnology according to manufacturer’s instructions.
Cytokine Transcription Analysis Total RNA was isolated from 1–2 g of frozen tissue using a TRIzon Reagent kit (Cwbiotech, Beijing, People’s Republic of China) according to manufacturer’s instructions. The concentration and purity of the total RNA was determined spectrophotometrically according to the method described by Sambrook and Russell [17]. A total of 2 μg of RNA was used as a template for the first-strand cDNA synthesis, which was performed with the HiFi-MMLV cDNA kit (Cwbiotech, Beijing, People’s Republic of China) with oligo (dT) as the primer. Quantitative real-time PCR (qPCR) was performed to detect the expression of IL-1β, IFN-γ , and TNF-α mRNA in the fish liver, kidney, and spleen using the J Biochem Molecular Toxicology
DOI 10.1002/jbt
Volume 28, Number 11, 2014
PARAQUAT IMMUNOTOXICITY ON COMMON CARP
TABLE 1. The Primers Used for qPCR Primers IL-1β F IL-1β R IFN-γ 1 F IFN-γ 1 R IFN-γ 2 F IFN-γ 2 R TNF-1,2,3α F TNF-1,2,3α R β-Actin F β- Actin R
Sequences 5 -GCTGGAGCAATGCAATACAAA-3
5 -AGGTAGAGGTTGCTGTTGGAA-3 5 -TGCACTTGTCAGTCGCTGCT-3 5 -TGTACTTGTGTCTCAGTATTT-3 5 -TCTTGAGGAACCTGAGCAGAA-3 5 -TGTGCAAGTCTTTCCTTTGTAG-3 5 -ACAACAATCAGGAAGGTGGAA-3 5 -TGGAAAGACACCTGGCTGTA-3 5 -GCTATGTGGCTCTTGACTTCG-3 5 -CCGTCAGGCAGCTGATAGCT-3
Abbreviations: F, forward; R, reverse.
UItraSYBR Mixture (with ROX) (Cwbiotech, Beijing, People’s Republic of China) according to manufacturer’s instructions. The qPCR primers were designed based on the sequences retrieved from GenBank: IL-1β (AJ245635), IFN-γ 1 (AM261214), IFN-γ 2 (AM168523), TNF-1α (AJ311800), TNF-2α (AJ311801), and TNF-3α (AB112424) (Table 1). β-Actin (accession no. M24113) was chosen as an internal standard. The amount of target mRNA, normalized to β-actin mRNA, was calculated using the formula 2−Ct [18], where the Ct value was determined by subtracting the average βactin Ct value from the average target gene Ct value as follows: Ct = (Ct (target) – Ct (β-actin)) target – (Ct (target) – Ct (β-actin)) standard. The threshold cycle (Ct ) was determined for each sample using the exponential growth phase and the baseline signals from the plot of fluorescence versus cycle number.
Statistical Analyses The data were analyzed using a one-way analysis of variance (Tukey) followed by a least significant difference determination using SPSS 13.0 for Windows. A p value of less than 0.05 was considered statistically significant.
RESULTS LC50 of PQ for Common Carp The results of the acute toxicity test showed that the 72-h LC50 and 96-h LC50 values for PQ in common carp were 15.962 mg/L (with a 95% confidence interval (CI) of 15.018–17.004 mg/L) and 15.106 mg/L (with a 95% CI of 14.113–6.139 mg/L) respectively. Additionally, we also observed that the behavior of fish in the treatment group was different from that of fish in the control group. For example, the respiratory rate of the treated fish increased. Additionally, in the early phase of exposure, the fish swam quickly or jumped; however, they gradually became dull and weak and J Biochem Molecular Toxicology
DOI 10.1002/jbt
503
swam feebly and slowly, all of which are obvious signs of toxicity. In the later stages of testing, hemorrhage or decay occurred in the gills or fins of some fish exposed to high concentrations of PQ (e.g., 25.29 mg/L), and finally, they sank to the bottom of the jar and died.
Content of IL-1β in the Liver, Kidney, and Spleen of Common Carp after Subacute Exposure to PQ for 7 Days Data related to the IL-1β content in the fish liver, kidney, and spleen are shown in Figure 1. The liver IL-1β content increased after 1 or 3 d of PQ exposure but remained unchanged at the end of the test when compared to the control (Figure 1A). The IL-1β content in the fish kidney showed similar changes after 7 days of PQ exposure, except a decrease was observed at 3 d of testing in the higher dose group (3.192 mg/L) (Figure 1B). However, in the spleen, no change in the IL-1β content was found between the treatment and control groups from 1 to 3 d of exposure, although a significant decrease was observed at the end of the test (Figure 1C).
IFN-γ Changes in the IFN-γ content in the liver, kidney, and spleen after PQ exposure were rather irregular (Figure 2). In the liver, exposure to PQ at a lower concentration (1.596 mg/L) caused a decrease in the IFNγ content over shorter exposure durations (from 1 to 3 d) and an increase over longer exposure durations (7 d), whereas the higher concentration of PQ (3.192 mg/L) first induced a decrease at the 1-day interval followed by an increase at 3 d with a final decrease at 7 d (Figure 2A). However, in the kidney, the IFN-γ content in the treated fish was higher than that in the controls, except for a decrease in the 3.192-mg/L group at the end of the test (Figure 2B). With regard to the spleen, the IFN-γ content was decreased in all treatment groups compared to the control group; however, an increase was observed at 3 d of PQ exposure in the 3.192-mg/L group (Figure 2C).
TNF-α Unlike IFN-γ , the change in the TNF-α content in the tissues of common carp after PQ exposure was relatively regular. For example, in all groups, TNF-α increased in the liver throughout the period of exposure (Figure 3A), and almost all groups showed a decrease in the kidney (Figure 3B). In the spleen, an initial increase
504
MA ET AL.
Volume 28, Number 11, 2014
160
A
**
140
** **
120 IL-1β ng/L)
**
100
0
80
1.596 mg/L
60
3.192 mg/L
40 20 0 1
140
3 Exposure time (day)
B
**
120 IL-1β (ng/L)
7
**
100
**
80
0
*
1.596 mg/L
60
3.192 mg/L
40 20 0 1
140
3 Exposure time (day)
7
C
IL-1β ng/L)
120 100 80
**
60
**
0 1.596 mg/L 3.192 mg/L
40 20 0 1
3 Exposure time (day)
7
FIGURE 1. IL-1β content in the (A) liver, (B) kidney, and (C) spleen of common carp exposed to 1.596 or 3.192 mg/L PQ for 7 d. The protocols for PQ exposure and the determination of IL-1β content are described in the section Sample Preparation and Cytokine Content Assay. The experiment was performed in duplicate, and the data are shown as the mean ± SD. Asterisks denote a response that is significantly different from the control (*p < 0.05, **p < 0.01).
was observed (from 1 to 3 d) followed by a decrease (7 d) (Figure 3C).
IL-1β mRNA Expression in the Liver, Kidney, and Spleen of Common Carp after Subacute Exposure to PQ for 7 Days The changes in IL-1β transcription were comparatively different between the three fish tissues after PQ exposure. IL-1β expression in the liver was first downregulated (1 d) but was later upregulated (3–7 d)
compared to the control (Figure 4A), whereas in the spleen, IL-1β was first upregulated and subsequently downregulated (Figure 4C). However, in the kidney, IL-1β transcription was enhanced as a whole in the treatment groups compared with the control group (Figure 4B).
TNF-α mRNA Levels Liver TNF-α expression was markedly downregulated after 1 d of PQ exposure and was later J Biochem Molecular Toxicology
DOI 10.1002/jbt
Volume 28, Number 11, 2014
PARAQUAT IMMUNOTOXICITY ON COMMON CARP
900 800
A *
IFN-γ (ng/L)
1.596 mg/L
**
*
**
600
0
**
700
505
3.192 mg/L *
500 400 300 200 100 0 1
3
7
Exposure time (day)
900
B
**
800 ng/L) IFN-γ
700 600 400
0
** **
1.596 mg/L
**
3.192 mg/L
500
**
300 200 100 0 1
3
7
Exposure time (day)
900
C
ng/L) IFN-γ
700 600 400
500
0
**
800
1.596 mg/L **
** **
**
1
3
3.192 mg/L **
300 200 100 0 7
Exposure time (day)
FIGURE 2. Content of IFN-γ in the (A) liver, (B) kidney, and (C) spleen of common carp exposed to 1.596 or 3.192 mg/L PQ for 7 d. The protocols for PQ exposure and the IFN-γ content assay are described in the section Sample Preparation and Cytokine Content Assay. All the experiments were performed in duplicate, and the data are shown as the mean ± SD. Asterisks denote a response that is significantly different from the control (*p < 0.05, **p < 0.01).
upregulated (3–7 d) compared to that of the control (Figure 4D). However, kidney TNF-α expression in the PQ-treated groups was downregulated during the duration of the test (except for the 3.192-mg/L group at 7 d) (Figure 4E). Regarding the spleen, TNF-α expression in the treatment groups was upregulated during the early stages of exposure (1 d) but was downregulated during the later stages (from 3 to 7 d, except for the 3.192-mg/L group) (Figure 4F).
J Biochem Molecular Toxicology
DOI 10.1002/jbt
IFN-γ mRNA Levels IFN-γ mRNA expression in the liver, kidney, and spleen of common carp after PQ exposure is shown in Figure 5. Liver IFN-γ 1 and IFN-γ 2 showed a similar trend of transcriptional change, i.e., downregulation at 1 d of exposure, upregulation in the higher PQ concentration group (3.192 mg/L) at 3 d of exposure, and downregulation in the higher concentration group at the end of the test compared to
506
MA ET AL.
Volume 28, Number 11, 2014
400
A
**
ng/L˅
350 **
300
** **
250
**
TNF-α
0 **
200
1.596 mg/L 3.192 mg/L
150 100 50 0 1
3
7
Exposure time (day)
400
B
TNF-α
ng/L)
350
*
**
300 250
0 **
200
1.596 mg/L
**
3.192 mg/L
150 100 50 0 1
3
7
Exposure time (day)
350
TNF-α
ng/L)
300
C **
250
**
** **
**
200
0
**
1.596 mg/L
150
3.192 mg/L
100 50 0 1
3
7
Exposure time (day)
FIGURE 3. Content of TNF-α in the (A) liver, (B) kidney, and (C) spleen of common carp exposed to 1.596 or 3.192 mg/L PQ for 7 d. The protocols for PQ exposure and the determination of TNF-α content are described in the section Sample Preparation and Cytokine Content Assay. The experiment was performed in duplicate, and the data are shown as the mean ± SD. Asterisks denote a response that is significantly different from the control (*p < 0.05, **p < 0.01).
that of the control group (Figure 5A). The changes in IFN-γ 1 and IFN-γ 2 mRNA levels in the kidney were similar to those in the liver, except for the upregulation observed in the 1.596-mg/L group at 1 d (Figure 5B). In addition, IFN-γ transcription in the spleen from the treated groups was downregulated at 1 d, promoted at 3 d, and downregulated again at 7 d (Figure 5C).
DISCUSSION The results of acute toxicity testing in this study revealed that PQ is highly toxic to common carp according to the PQ 96-h LC50 (15.106 mg/L) and the toxicity classification of pesticides [19]. The PQ 96-h LC50 for common carp approaches the same order of magnitude observed in other types of fish (e.g., 20 mg/L in
J Biochem Molecular Toxicology
DOI 10.1002/jbt
Volume 28, Number 11, 2014
1.2
A
1.4
** **
1.2
* 0
1
1.596 mg/L
0.8
3.192 mg/L
**
0.6
**
0.4
B
**
**
1 **
0.8
1
1.596 mg/L 3.192 mg/L
0.4
3
1
7
3
Exposure time (day) **
1.8
**
1.2 1
**
0.8
0 **
**
1.596 mg/L 3.192 mg/L
0.6
**
0.4
.
1.6 1.4
TNFα relative expression
IL-1β relative expression .
C
1.2
0.2
D
**
** 0
1
1.596 mg/L
0.8 0.4
**
3
7
1
3
Exposure time (day) 1.8
*
E **
** **
0.8
1.6
0 1.596 mg/L
0.6 **
0.4
7
Exposure time (day)
3.192 mg/L
**
0.2
TNF α relative expression .
TNFα relative expression .
3.192 mg/L
**
0.6
0
1
1
**
0.2
0
1.2
7
Exposure time (day)
1.4
1.4
0
0.6
0
0
1.6
*
0.2
0.2
1.8
**
507
.
.
1.4
IL-1β relative expression
1.6
IL-1β relative expression
1.8
PARAQUAT IMMUNOTOXICITY ON COMMON CARP
F
**
** **
1.4 1.2
0
**
1
1.596 mg/L
0.8
3.192 mg/L
**
0.6
**
0.4 0.2 0
0 1
3
7
Exposure time (day)
1
3
7
Exposure time (day)
FIGURE 4. IL-1β and TNF-α mRNA levels in the liver, kidney, and spleen of common carp exposed to 1.596 or 3.192 mg/L of PQ for 7 d. IL-1β mRNA expression was determined by qPCR as described in the section Cytokine Transcription Analysis. Asterisks denote a response that is significantly different from the control (*p < 0.05, **p < 0.01). The level of IL-1β is shown in the (A) liver, (B) kidney, and (C) spleen, and the level of TNF-α is shown in the (D) liver, (E) kidney, and (F) spleen.
Nile tilapia [9] and 52.48 mg/L in Cnesterodon decemmaculatus [20]). IL-1β is a key mediator in response to microbial invasion, tissue injury, and xenobiotics; it can stimulate immune responses by activating lymphocytes and inducing the release of other cytokines that can activate macrophages and natural killer (NK) cells [21]. Wang et al. [15] reported that IL-1β expression was upregulated in the kidney and spleen of common carp after exposure to atrazine, chlorpyrifos, or a mixture of both. Our results indicate that PQ increases the content of IL-1β in the liver and kidney at early time points following exposure but decreases it in the spleen at a later time, suggesting that IL-1β in different fish organs may respond variably to PQ. Meanwhile, IL-1β gene transcription in the three tissues evaluated was also inconsistent. For example, liver IL-1β expression was initially downregulated and subsequently upregulated, whereas in the spleen, it was initially upregulated and later downregulated. Contrary to what was observed in the liver and spleen, IL-1β transcription in
J Biochem Molecular Toxicology
DOI 10.1002/jbt
the kidney was promoted throughout the experiment. Additionally, we found a remarkable discordance between the IL-1β content and transcription in the fish liver after 1 d of PQ treatment (i.e., a significant increase in the IL-1β content but a dramatic downregulation of IL-1β gene expression). IFN-γ is constitutively produced by NK cells of the innate immune system. It stimulates macrophagemediated phagocytosis and the production of proinflammatory cytokines and antimicrobial oxygen radicals by macrophages [22–25]. In this study, PQ exposure changed the mRNA expression level or content of IFN-γ in the fish liver, kidney, and spleen. For example, the IFN-γ content was increased in the kidney and decreased in the spleen, while IFN-γ transcription was promoted in the liver and kidney but inhibited in the spleen. These alterations may affect IFN-γ expression and disturb its function. TNF-α is a proinflammatory cytokine involved in inflammation, apoptosis, cell proliferation, and the stimulation of various aspects of the immune system
MA ET AL.
A
** **
.
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
Volume 28, Number 11, 2014
0 1.596 mg/L 3.192 mg/L ** **
**
**
1
3
IFN γ2 relative expression
IFN γ1 relative expression .
508
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
**
0 1.596 mg/L **
** **
1
7
3
2
** **
1.5
**
0 *
1 0.5
1.596 mg/L 3.192 mg/L
**
0 1
3
2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
** ** *
3.192 mg/L
1
3
7
Exposure time (day)
**
1.8
**
1.6
* 0 1.596 mg/L
**
3.192 mg/L ** **
**
IFN γ2 relative expression .
IFN γ1 relative expression .
1.596 mg/L
**
**
7
C
0
*
Exposure time (day) 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
7
Exposure time (day)
IFN γ2 relative expression .
IFN γ1 relative expression .
B
3.192 mg/L
**
Exposure time (day) 2.5
**
**
1.4
*
1.2 0
1
1.596 mg/L
0.8
3.192 mg/L
**
0.6
**
0.4 0.2 0
1
3
7
1
3
Exposure time (day)
7
Exposure time (day)
FIGURE 5. The mRNA levels of IFN-γ 1 and IFN-γ 2 in the (A) liver, (B) kidney, and (C) spleen of common carp exposed to 1.596 or 3.192 mg/L PQ for 7 d. The mRNA levels of IFN-γ 1 and IFN-γ 2 were determined by qPCR as described in the section Cytokine Transcription Analysis. Asterisks denote a response that is significantly different from the control (*p < 0.05, **p < 0.01).
[26]. There have been several reports regarding the upregulation of TNF-α expression observed in common carp fed with Spirulina [27] or treated with yeast extract compared to control fish [21]. In our study, we found a remarkable increase in the TNF-α content in the fish liver and spleen (after 1–3 d of PQ exposure), as shown in Figure 3, and upregulation of TNF-α gene expression in the liver (3–7 d) and spleen (1 d) (Figure 4), suggesting that PQ exposure affects the expression or function of TNF-α. Cytokines are important regulators of the immune system in teleost fish and are mainly responsible for host innate defense mechanisms [28]. Therefore, cytokines are important conventional terminal indexes of innate immunity for evaluating fish immunotoxicity [21]. Our results reveal that PQ exposure not only changes the content of cytokines but also alters their gene expression in the immune-associated organs of common carp. This may cause immune dysfunction and reduced immunity.
REFERENCES 1. Mandel JS, Adami H-O, Cole P. Paraquat and Parkinson’s disease: an overview of the epidemiology and a review of two recent studies. Regul Toxicol Pharmacol 2012;62:385– 392. 2. Suntres ZE. Role of antioxidants in paraquat toxicity. Toxicology 2012;180:65–77. 3. Corsinia E, Sokooti M, Galli CL, Moretto A, Colosio C. Pesticide induced immunotoxicity in humans: a comprehensive review of the existing evidence. Toxicology 2013;307:123–135. 4. Amirshahrokhi K. Anti-inflammatory effect of thalidomide in paraquat-induced pulmonary injury in mice. Int Immunopharmacol 2013;17:210–215. 5. Mussi MA, Calcaterra NB. Paraquat-induced oxidative stress response during amphibian early embryonic development. Comp Biochem Physiol C 2010;151:240–247. 6. Sun F, Chen HS. Monitoring of pesticide chlorpyrifos residue in farmed fish: investigation of possible sources. Chemosphere 2008;71:1866–1869. 7. Salazar-Lugoa R, Estrella A, Oliveros A, Rojas-Villarroel E, Villalobos de BL, Lemus M. Paraquat and temperature affect nonspecific immune response of Colossoma
J Biochem Molecular Toxicology
DOI 10.1002/jbt
Volume 28, Number 11, 2014
8.
9.
10.
11.
12. 13. 14. 15.
16. 17.
PARAQUAT IMMUNOTOXICITY ON COMMON CARP
macropomum. Environ Toxicol Pharmacol 2009;27:321– 326. Riahi B, Rafatpanah H, Mahmoudi M, Memar B, Brook A, Tabasi N, Karimi G. Immunotoxicity of paraquat after subacute exposure to mice. Food Chem Toxicol 2010;48:1627–1631. Figueiredo-Fernandes A, Fonta´ınhas-Fernandes A, Peixoto F, Rocha E, Reis-Henriques MA. Effects of gender and temperature on oxidative stress enzymes in Nile tilapia Oreochromis niloticus exposed to paraquat. Pestic Biochem Physiol 2006;85:97–103. Tukmechi A, Rezaee J, Nejati V, Sheikhzadeh N. Effect of acute and chronic toxicity of paraquat on immune system and growth performance in rainbow trout, Oncorhynchus mykiss. Aquac Res 2013; doi: 10.1111/are.12118 Burns-Naas LA, Meade BJ, Munson AE. Toxic responses of the immune system. In: Klaassen CD, editor. Casarett & Doull’s toxicology –the basic science of poisons. New York: McGraw Hill; 2001. pp 419–471. Galloway T, Handy R. Immunotoxicity of organophosphorous pesticides. Ecotoxicology 2003;12:345–363. Wester PW, Vehtaak AD, van Muiswinkel WB. Fish as biomarkers in immunotoxicology. Toxicology 1994;86:213–232. Colosio C, Birindelli S, Corsini E, Galli CL, Maroni M. Low level exposure to chemicals and immune system. Toxicol Appl Pharmacol 2005;207:320–328. Wang X, Xing HJ, Li XL, Xu SW, Wang XL. Effects of atrazine and chlorpyrifos on the mRNA levels of IL-1 and IFN-γ 2b in immune organs of common carp. Fish Shellfish Immunol 2011;31:126–133. K¨arber G. Beitrag zur kollektiven behandlung pharmakologischer reihenversuche. Arch Exp Pathol Pharmakol 1931;162:480–483. Sambrook J, Russel DW. Molecular cloning: a laboratory manual, 3rd edition. New York: Cold Spring Harbor Laboratory; 2001. Vol 1, pp 13–17.
J Biochem Molecular Toxicology
DOI 10.1002/jbt
509
18. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real- time quantitative PCR and the 2 −Ct method. Methods 2001;25:402–408. 19. Ecobichon DJ. Toxic effects of pesticides. In: Klaassen CD, editor. Casarett & Doull’s toxicology – the basic science of poisons. New York: McGraw Hill ; 2001. pp 763–802. 20. Di Marzio WD, Alberdi JL, S´aenz ME, Tortorelli MC. EfR Formulation) on survival fects of paraquat (Osaquat and total cholinesterase activity in male and female adults of Cnesterodon decemmaculatus (Pisces, Poeciliidae). Environ Toxicol Water Qual 1998;13:55–59. 21. Biswas G, Korenaga H, Takayama H, Kono T, Shimokawa H, Sakai M. Cytokine responses in the common carp, Cyprinus carpio L. treated with baker’s yeast extract. Aquaculture 2012;356–357:169–175. 22. Robertsen B. The interferon system of teleost fish. Fish Shellfish Immunol 2006;20:172–191. 23. Grayfer L, Belosevic M. Molecular characterization, expression and functional analysis of goldfish (Carassius aurutus L.) interferon gamma. Dev Comp Immunol 2009;33:235–246. 24. Kitao Y, Kono T, Korenaga H, Iizasa T, Nakamura K, Savan R, Sakai M. Characterization and expression analysis of type I interferon in common carp Cyprinus carpio L. Mol Immunol 2009;46:2548–2556. 25. Zou J, Secombes CJ. Teleost fish interferons and their role in immunity. Dev Comp Immunol 2011;35:1376–1387. 26. Savan R, Sakai M. Presence of multiple isoforms of TNF alpha in carp (Cyprinus carpio L.): genomic and expression analysis. Fish Shellfish Immunol 2004;17:87–94. 27. Watanuki H, Ota K, Tassakka ACMAR, Kato T, Sakai M. Immunostimulant effects of dietary Spirulina platensis on carp, Cyprinus carpio. Aquaculture 2006;258:157–163. 28. Huttenhuis HBT, Taverne-Thiele AJ, Grou CPO, Bergsma J, Saeij JPJ, Nakayasu C, Rombout JHWM. Ontogeny of the common carp (Cyprinus carpio L.) innate immune system. Dev Comp Immunol 2006;30:557–574.
