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Effects of atrazine and chlorpyrifos on cytochrome P450 in common carp liver
Chemosphere 104 (2014) 244–250
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Effects of atrazine and chlorpyrifos on cytochrome P450 in common carp liver Houjuan Xing a,b, Ziwei Zhang a, Haidong Yao a, Tao Liu a, Liangliang Wang a, Shiwen Xu c,⇑, Shu Li c,⇑ a
College of Animal Science and Technology, Northeast Agricultural University, 59 Mucai Street, Harbin 150030, PR China Animal Health Supervision Institute of Heilongjiang Province, 243 Haping Road, Xiangfang District, Harbin 150069, PR China c College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Harbin 150030, PR China b
h i g h l i g h t s The Cyt P450 system in carp liver was investigated by ATR, CPF and their mixture. Our aim was to evaluate the sub-chronic effects of ATR, CPF and their mixture on carp. This is the first report from pesticide residues in carp tissues by ATR, CPF and their mixture. The Cyt P450 system can be used as biomarker for monitoring ATR and CPF.
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Article history: Received 18 April 2013 Received in revised form 20 December 2013 Accepted 2 January 2014 Available online 14 February 2014 Keywords: CYP system Common carp Pesticide residue Chlorpyrifos Atrazine
a b s t r a c t Atrazine (ATR) and chlorpyrifos (CPF), widely used in agriculture, have resulted in a series of toxicological and environmental problems. We investigated the activities of the biotransformation enzymes ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-deethylase (PROD), total cytochrome P450 (CYP), CYP1A mRNA level and level of tissue ATR, CPF, and their metabolites in the liver of common carp (Cyprinus carpio L.) after a 40-d exposure to CPF and ATR, alone or in combination, and a 20-d recovery. In the present study, juvenile common carp was exposed to ATR (at concentrations of 4.28, 42.8 and 428 lg L1), CPF (1.16, 11.6 and 116 lg L1), and ATR/CPF mixture (at concentrations of 1.13, 11.3 and 113 lg L1). A general increasing trend for the activity of the biotransformation enzymes (EROD and PROD), CYP and CYP1A mRNA level was observed in the liver of common carp exposed to ATR, CPF and the ATR/CPF mixture. In addition, ATR, CPF, and their metabolites demonstrated a high accumulation in the liver. These results demonstrated that the CYP system in fish could be used as a biomarkers in evaluating the impact of ATR and CPF exposure on the common carp. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction With the growth of human activities, increasing numbers of chemicals are being released into the environment, many of which are hazardous to living organisms and ecosystems. The triazine herbicide atrazine (ATR) is used as a selective pre-emergence and post-emergence herbicide for the control of weeds in asparagus, maize, sorghum, sugarcane and pineapple. ATR is also used in forestry and for non-selective weed control on non-crop areas. ATR has been employed extensively in agriculture in the US and worldwide for over 40 years (Singh et al., 2011). According to USDA NASS (2008), in 2003 about 24.3 million kg of ATR was used on corn crops in the US. In China, where ATR is used as a ⇑ Corresponding authors. Tel./fax: +86 451 55190407. E-mail addresses: [email protected] (S. Xu), [email protected] (S. Li). http://dx.doi.org/10.1016/j.chemosphere.2014.01.002 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
broad-spectrum herbicide, which is applied to major field crops such as corn, wheat and soybeans, it has been reported that in 2008 the amounts of ATR applied to fields was at least 5000 t (Song et al., 2009). Several studies have already detected its presence in ground- and surface-waters at levels above the limits determined by local guidelines (Ren et al., 2002; Dong et al., 2006; Konstantinou et al., 2006; Gilliom, 2007; Davis et al., 2007). Chlorpyrifos (CPF), as an organophosphorus insecticide, is widely used to control a variety of pests on agricultural and animal farms (Whyatt et al., 2007; Saulsbury et al., 2009). Field surveys performed in many countries have shown that CPF is commonly reported as contaminant of surface and ground water (Banks et al., 2005; Du Preez et al., 2005). Several studies have been confirmed that ATR and CPF are toxic to fish (De Silva and Samayawardhena, 2005; Ali et al., 2008; Kavitha and Venkateswara Rao, 2008; Nwani et al., 2010).
H. Xing et al. / Chemosphere 104 (2014) 244–250
The cytochrome P450 (CYP) enzymes serve as terminal oxidases in the mixed-function oxidase system for metabolizing various endogenous substrates and xenobiotics including drugs, toxins and carcinogens (Dong et al., 2009). The CYP family, such as CYP1A, plays an important role in the detoxification mechanism. Although the expression of CYP1A is constitutively low, it is highly inducible in animal tissue by environmental contaminants. Accordingly, the induction of hepatic CYP1A mRNA in fish by certain classes of xenobiotics, including pesticides, has been suggested as an early warning system, a most sensitive biological response for assessing environmental contamination (Coimbra et al., 2007; Fu et al., 2013). Compared to the protein levels of CYP1A, the induction of CYP1A can occur within hours of exposure and the level of CYP1A mRNA decreases when induction response is eliminated. Moreover, the real-time quantitative polymerase chain reaction developed recently is at least 100-fold more sensitive than Northern or slot blotting in measuring CYP1A RNA (Cousinou et al., 2000; An et al., 2011). EROD activity was used as a marker of CYP1A function. The liver is considered as the major organ for CYP-mediated biotransformation (Tabrez et al., 2010). Many studies have reported CYP1A mRNA level and CYP-dependent enzymatic activity in fish liver in response to the presence of different pollutants in the aquatic environment (Moore et al., 2003; Binelli et al., 2006; Santos and Martincez, 2012). Fish species are widely used as biological monitors of variations in the environmental levels of anthropogenic pollutants. The present paper is a contribution to the assessment of the toxicity and effects of ATR and CPF based-pesticides on the common carp (Cyprinus carpio L.). The common carp was selected as the test fish because although it is one of the most economically important freshwater fishes of the world (Li et al., 2010), there is a scarcity of information regarding the toxicity of ATR and CPF on freshwater fishes. To improve our understanding of the toxicity mechanisms and responses of different organs to environmentally relevant concentrations of ATR and CPF, a better understanding of the CYP system is required. In this study, we examined the changes in the transcription of the CYP1A gene, activity of CYP enzymes and level of tissue ATR, CPF, and their metabolites in the liver of common carp exposed to ATR and CPF alone and in combination. These results will enable us to further understand the hepatic detoxification mechanisms affected by ATR and CPF exposure in common carp. 2. Materials and methods 2.1. Chemicals The Folin Ciocalteau reagent, ethoxyresorufine, pentoxyresorufin, ATR (purity 98.0%) and CPF (purity 99.5%) were purchased from Sigma–Aldrich Chemical Co. (USA). All others reagents were obtained from Reanal, Budapest, Hungary. All chemicals were of analytical grade. Stock solutions of ATR and CPF were prepared in analytical grade acetone (purity 99%). All working solutions were taken from this stock solution. The concentration of acetone was kept at <0.05% in all pesticide solutions used. 2.2. Animals and treatments Juvenile 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 laboratory tanks (90 55 45 cm) with continuous aeration. Acclimatization to experimental conditions was performed for 15 d using dechlorinated tap water with 230 mg L1 CaCO3, 42.5 ± 1.2 mg L1 Ca, a dissolved oxygen concentration >7 mg L1,
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and pH 7.4 ± 0.2. The water temperature was adjusted to 20 ± 1 °C, and the photoperiod was 12 h light and 12 h dark. Commercial food was given once a day until satiation. No mortality was observed either in the control animals or in any of the treatment groups. 2.3. Toxicity test Experimental fish were randomly divided into eleven groups: three ATR treatment groups (4.28, 42.8 and 428 lg L1), three CPF treatment groups (1.16, 11.6 and 116 lg L1), three mixturetreatment groups (ATR/CPF) (1.13, 11.3 and 113 lg L1), one solvent control (acetone) group, and one water control group. The binary mixtures were composed of a 1:1 mass ratio of ATR and CPF. Each treated group has 20 fish and 2 replicates. The concentrations used in the present study are about 1/500, 1/50 and 1/5 of the 96 h LC50s (unpublished data). In China, the commercial solutions used as herbicides and insecticides contain 400 g L1 ATR and 380 g L1 CPF, respectively. In addition, ATR and CPF are stable in water and have a long half-life, so we can easily speculate that the dose we chose could be found in the environment. The fish were exposed under semi-static conditions for 40 d to water and pesticide, which were completely replaced once every 2 d by transferring fish to freshly prepared pesticide solutions. At the end of the exposure, five fish were killed in each group, and the liver was excised immediately on an ice-cold plate and washed in physiological saline solution (0.86% NaCI). The tissues were divided into two portions, one for further protein and enzyme analysis and a second to be stored at 80 °C for RNA examination. Experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) and were approved by a local ethics committee. 2.4. Recovery test Ten exposed fish from each batch were kept in pesticide-free water for 20 d in a large fresh 200-L glass aquarium provided with a filter and continuous aeration. The conditions in the recovery experiments were identical to those in the exposure experiments. In the present study, there were 10 fish per group killed at the two sampling events, and 5 fish per group were used in official test. The remaining 5 fish were used in preliminary experiment and served as standby tissues. 2.5. Determination of the CYP system 2.5.1. Preparation of microsomes and determination of enzymatic activities The microsomes were prepared in the following way: the fish were sacrificed and the gall bladders were removed to avoid contaminating the livers with bile. The method used as described by Forlin (1980) with some slight modifications. After weighing, the liver was chopped with a pair of scissors and homogenized in a Braun 853302/4 homogenizer with 1:5 w/v of ice-cold phosphate buffer (0.1 M K-phosphate, pH 7.4) containing 0.15 M KCl. The homogenate was centrifuged with a Sorvall RC 5B Plus centrifuge at 10 000 g for 20 min at 4 °C and the supernatant obtained was ultracentrifuged with a Sorvall Ultra Pro 80 at 105 000 g for 65 min, also at 4 °C. The pellet (after 105 000 g centrifugation) was resuspended in 1 mL phosphate buffer containing 20% (vol.) glycerol, the suspension was homogenized and the resultant microsomal fraction was stored at 80 °C until the determination the enzymatic activities. 7-ethoxyresorufin-O-dealkylase (EROD) activity was determined by the method described before (Burke et al., 1985), with slight modifications. Briefly, one milliliter of the reaction mixture
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containing one milligram of protein, 10 mM glucose-6-phosphate (G-6-P), 10 mM MgCl2, and 2.5 lM ethoxyresorufin in 0.1 M KPB, pH 7.4, was preincubated for 5 min at 37 °C. The reaction was started by adding 20 lL of a mixture of 50 mM reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) and 200 U mL1 of glucose-6-phosphate dehydrogenase (G-6-PDH). After incubation for 5 min, the reaction was terminated by adding 4 mL of cold methanol. The mixture was centrifuged at 3000 g for 5 min, and the supernatant methanol layer was collected for measurement of resorufin. Assays were conducted using a fluorescence spectrophotometer set at wavelengths of 530 nm excitation and 590 nm emission with a cuvette stirring function. 7-pentoxyresorufin–O-dealkylase (PROD) activity was measured exactly in the same manner as described for the ethoxyresorufin assay, except that the substrate was pentoxyresorufin. The enzymatic activities are shown in nmol min1 mg1 of protein. The total CYP protein contents were determined as the dithionite-reduced CO complex, as described by Omura and Sato (1964). The results are expressed in nmol mg1 of protein, with an extinction coefficient of 91 mM1cm1 (450–490 nm). The protein concentration of the microsomes was measured by the method of Lowry et al. (1951).
2.5.2. Determination of gene expression Primer design: The primers for real-time amplification of the CYP1A and b-actin cDNA were designed using Oligo_6.0 Software (Molecular Biology Insights, Cascade, CO) based on the deposited sequences in GenBank under the accession numbers AB048939 and AF057040. The following primers were used for CYP1A: forward 50 - ATT TCA TTC CCA AAG ACA CCT G-30 ; and reverse 50 CAA AAA CCA ACA CCT TCT CTC C-30 . The following primers were used for b-actin: forward 50 -GAT GGA CTC TGG TGA TGG TGT GAC-30 ; and reverse 50 - TTT CTC TTT CGG CTG TGG TGG TG-30 . The amplification product sizes were CYP1A, 159 bp; and b-actin, 167 bp. The PCR products were electrophoresed on two percent agarose gels, extracted, cloned into the pMD18-T vector (Takara, Ohtsu, Japan), and sequenced. Total RNA isolation and reverse transcription: Total RNA was isolated from the livers of each fish using Trizol reagent according to the manufacturer’s instructions (Invitrogen). The reverse transcription reactions (40 lL) consisted of 10 lg of total RNA, 1 lL of Moloney murine leukemia virus reverse transcriptase, 1 lL of RNase inhibitor, 4 lL of deoxynucleoside triphosphate, 2 lL of Oligo dT, 4 lL of dithiothreitol, and 8 lL of 5 reverse transcriptase buffer. The reverse transcription procedure used was based on instructions from the manufacturer (Invitrogen). Real-time quantitative reverse transcription PCR: Real-time quantitative reverse transcription PCR was used to detect the expression of the CYP1A gene using SYBR Premix Ex Taq (Takara, Shiga, Japan). Real-time PCR was conducted on each the liver tissues. Reaction mixtures were incubated in an ABI PRISM 7500 real-time PCR system (Applied Biosystems, Foster City, CA). The following program was used: 1 cycle at 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and at 60 °C for 34 s. Dissociation curves were analyzed with Dissociation Curve 1.0 software (Applied Biosystems) for each PCR reaction to detect and eliminate the possible primer–dimer and nonspecific amplifications. The results (fold changes) are expressed as 2DDCt, where CtCYP1A and
DDCt ¼ ðCtCYPIA Ctb-actin Þt ðCtCYPIA Ctb-actin Þc Ctb-actin are the cycle thresholds for the carp CYP1A and b-actin genes in the different treated groups, respectively, t indicates the treatment group; and c denotes the control group.
2.6. Analysis of tissue pesticide content 2.6.1. Analysis of tissue ATR content analysis Stored liver samples were cut and homogenized using a glass– Teflon homogenizer (Heidolph S01 10R2RO). The homogenate was extracted three times using 20 mL of 50% aqueous acetone by shaking the suspension for 60 min. The extracts were filtered and combined, and then 3 g of NaCI were added successively to each sample and dissolved by shaking the suspension. The filtrate was re-extracted three times with 30 mL of trichloromethane. The extracts were combined, passed through anhydrous Na2SO4 columns, and collected. The eluates were concentrated into a triangular flask by rotary evaporation, dissolved with 1.5 mL of methanol, and filtered. The concentrations of ATR in the extracts were analyzed with an Agilent high-performance liquid chromatography (HPLC) system. HPLC was performed with Waters equipment, equipped with a diode array using an ODS 5-micron Hypersil capillary column (250 mm long, 4.6 mm in diameter) folˇ oz and Rosés (2000). Molowing the procedures described by Mun bile phases used in the isocratic elution were distilled with water and methanol (v/v: 20/80). The column head temperature was 25 °C and the flow rate was held constant at 1 mL min1. The volume injected was 10 lL. The eluents were monitored by UV detection of a wavelength of 222 nm for ATR, atrazine-2-hydroxy, and atrazine–desethyl.
2.6.2. Analysis of tissue CPF content analysis For the analysis of CPF content, approximately 2 g (wet wt) of stored soft tissues samples was weighed in a centrifuge tube (50 mL). Each sample received 1 g of anhydrous Na2SO4 and 10 mL of acetidin and was homogenized using an IKA homogenizer at 6000 r min1 for 2 min. Then, the tool bit was washed using 10 mL of acetidin. The homogenate was centrifuged at 5000 r min1 for 5 min to obtain the supernatant. The residue in the centrifuge tube was re-extracted once with 10 mL of acetidin and centrifuged. The two supernatants were concentrated in a rotary evaporation flask by rotary evaporation at 40 °C, and the precipitate was dissolved with 5 mL of acetonitrile by convolution agitate and supersonic ablution. The extracts were passed through Acor aluminum oxide columns and collected. The eluates were evaporated to dryness under a nitrogen stream at 50 °C. The residue was finally dissolved in 2 mL of N-hexane and filtered for the HPLC analysis of the concentrations of CPF in extracts. The HPLC procedure was similar to the method used by Abu-Qare and Abou-Donia (2001). Briefly, mobile phases used in the isocratic elution were distilled with water and acetonitrile (v/v: 40/60). The column head temperature was 25 °C, and the flow rate was held constant at 1 mL min1. The volume injected was 10 lL. The eluents were monitored by UV detection at a wavelength of 230 nm for CPF and chlorpyrifos-oxon.
2.7. Statistical analysis Statistical analysis of all data was performed using SPSS for Windows (version 13, SPSS Inc., Chicago, IL). One-way ANOVA with a post hoc test was used to elucidate if there were significant differences between the treatment groups and the control group. The difference between the treatment groups and the recovery groups was assessed by using Paired t-test. All expressed data were collected in quintuple from at least five fish and were expressed as mean ± standard deviation. Differences were considered to be significant at P < 0.05.
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3. Results 3.1. Responses of the activities of EROD and PROD and the level of CYP in common carp exposed to ATR, CPF and their mixture The activity of EROD and PROD in the liver tissue is shown in Fig. 1. The activity of EROD and PROD was significantly higher (P < 0.05) at all groups exposed to ATR, CPF and the ATR/CPF combination compared with the control group except for the groups treated with the low doses of pesticides (1.13, 1.16 and 4.28 lg L1). The activity of EROD and PROD increased after exposure and reached their highest level at the high-dose groups for each pesticide. During the recovery period, the activity of EROD and PROD at all treated groups tended to increase with higher concentration of ATR or CPF or the ATR/CPF mixture, but no significant difference was observed between exposure and recovery groups. The CYP content in the liver tissue for each group is shown in Fig. 2. After exposure, the liver CYP content increased with increasing concentration of pesticides. The CYP content at all groups exposed to ATR, CPF and the ATR/CPF combination was significantly higher (P < 0.05) than that observed in the control fish. After the recovery period, CYP content of all treatment groups was still significantly higher (P < 0.05) compared with the control group. The CYP content of all recovery groups, except for the 42.8, 428, 11.3 and 113 lg L1, had significantly decreased (P < 0.05) compared to the exposed groups.
Fig. 2. Effects of ATR, CPF and ATR/CPF mixture on the cytochrome P450 content of common carp liver. Each value represented the mean ± SD of 5 individuals. * Significant differences (P < 0.05) between the control group and the exposure group. #Significant difference (P < 0.05) between the exposure group and the recovery group at the same concentration.
3.2. Quantification of CYP1A expression in the liver of common carp after exposure to ATR, CPF, and their mixture The effect of ATR, CPF and their mixture on the mRNA level of CYP1A in the liver is presented in Fig. 3. Compared with the control group, a significantly increase (P < 0.05) in the mRNA level of the CYP1A gene in the liver was observed in the fish exposed to ATR, CPF and ATR/CPF mixture. For the each treatment group, a significantly increase (P < 0.05) in the mRNA level of CYP1A gene in the liver was observed when exposed fish were compared to recovery-treated fish (P < 0.05) but the mRNA level at all recovery groups was still higher than those from the control fish. 3.3. Accumulations of ATR, CPF, and their metabolites in liver tissue The concentrations of ATR and its metabolites (atrazine-2-hydroxy and atrazine–desethyl) in fish liver are summarized in Table 1. ATR and its metabolites (atrazine–desethyl and
Fig. 1. Effects of ATR, CPF and ATR/CPF mixture on the EROD (a) and PROD (b) activities of common carp liver. Each value represented the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the control group and the exposure group. #Significant difference (P < 0.05) between the exposure group and the recovery group at the same concentration.
Fig. 3. Relative mRNA expression of the CYP1A gene in common carp liver after exposure to ATR, CPF and their mixture for the 40 d and a recovery period. Each value represented the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the control group and the exposure group. # Significant differences (P < 0.05) between the exposure group and the recovery group at an identical concentration.
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Table 1 Concentrations of ATR and its metabolites in the livers from the common carp after exposure and recovery treatments. ATR (mg kg1)
Treatment groups
Atrazine–desethyl (mg kg1)
Atrazine-2-hydroxy (mg kg1)
Exposure
Recovery
Exposure
Recovery
Exposure
Recovery
Control
0 4.28
n.d. 0.15 ± 0.006
n.d. 0.07 ± 0.002*
n.d. 0.02 ± 0.002
n.d. 0.07 ± 0.002*
n.d. 0.27 ± 0.005
n.d. 0.09 ± 0.002*
Atrazine (lg L1)
42.8 428
1.64 ± 0.012 2.29 ± 0.014
0.18 ± 0.005* 0.23 ± 0.005*
2.12 ± 0.014 4.64 ± 0.025
0.43 ± 0.007* 0.43 ± 0.006*
1.10 ± 0.009 2.29 ± 0.015
0.54 ± 0.008* 0.59 ± 0.008*
Mixture of atrazine and chlorpyrifos (lg L1)
1.13 11.3 113
0.16 ± 0.004 1.26 ± 0.009 2.29 ± 0.021
0.09 ± 0.003* 0.26 ± 0.005* 0.29 ± 0.006*
0.43 ± 0.004 2.42 ± 0.015 3.16 ± 0.016
0.02 ± 0.002* 0.78 ± 0.008* 0.43 ± 0.006*
0.69 ± 0.006 1.20 ± 0.008 2.17 ± 0.015
0.10 ± 0.002* 0.39 ± 0.001* 0.44 ± 0.007*
Note: Values are the means ± SD (n = 5). n.d. indicates not detected. * Significant differences (P < 0.05) between the exposure group and the recovery group at an identical concentration.
atrazine-2-hydroxy) were not detected in the liver of common carp that were cultivated in pesticide-free control water. As the pesticide concentration in the water increased, the accumulations of ATR and its metabolites in the liver significantly increased. For ATR and its metabolites, atrazine-2-hydroxy had the highest concentrations in the liver at the treated groups (4.28 and 1.13 lg L1), and atrazine–desethyl had the highest concentrations in the other treated groups. After recovery for 20 d, the accumulations of ATR and its metabolites in the liver were significantly lower (P < 0.05) than compared with that in the corresponding exposure group; however, the accumulations of these pesticides in the liver were still detected. The concentrations of CPF and its metabolite (chlorpyrifosoxon) in fish liver are summarized in Table 2. CPF and chlorpyrifos-oxon were not detected in the liver of common carp that were cultivated in pesticide-free control water. With increasing pesticide concentration in the water, the accumulations of CPF and chlorpyrifos-oxon in the liver were significantly elevated. Chlorpyrifos-oxon accumulations in the liver at 1.13 and 116 lg L1 groups was higher than the CPF accumulations. After recovery for 20 d, the accumulations of CPF and chlorpyrifos-oxon in the liver were significantly lower (P < 0.05) than that in the corresponding exposure group; however, the accumulations of these pesticides in the tissues were still detected. 4. Discussion The liver is primarily responsible for the metabolism of toxic substances, including ATR and CPF. In particular the liver is the major site of CYP expression in teleost fish. Therefore, the effect of ATR and CPF on liver-detoxifying enzymes is very important. In the present study, we investigated the changes in CYP1A gene transcription, EROD and PROD activities, CYP content and accumulations of ATR, CPF and their metabolites in the liver of common carp exposed to ATR, CPF and an ATR/CPF mixture.
4.1. Accumulations of ATR, CPF, and their metabolites in response to ATR and CPF exposure in common carp liver Pesticide pollution in the environment has been increasing due to their extensive use in agriculture. Agricultural runoff and irrigation water are the major sources of these contaminants in the aquatic environment. Alterations in the chemical composition of natural aquatic environments can affect the freshwater fauna, particularly fish (Oruc, 2010; Jin et al., 2010; Xing et al., 2012a). The results of the present study indicated that the accumulations of ATR, CPF, and their metabolites were detected in the liver of common carp exposed to ATR, CPF and an ATR/CPF mixture, suggesting that ATR, CPF and their metabolites were present in the organism because ATR and CPF have low solubility in water (Kidd and James, 1991; Powell et al., 2011). ATR can be found at levels as high as 21 ppb in groundwater, 42 ppb in surface waters, 102 ppb in river basins in agricultural areas, and up to 224 ppb in Midwestern streams during May–August (Kolpin et al., 1998). Del Prado Lu (2010) reported the presence of CPF (0.07 mg L1) residues in river water samples in agricultural areas. The present study has also demonstrated that the concentrations of ATR, CPF, and their metabolites were increased with increasing concentrations of ATR, CPF and the ATR/CPF combination. The results could be related to the lipophilic nature of ATR and CPF thus enhancing their bioaccumulation. Alternatively, the relatively high accumulations of ATR and CPF by the common carp could be due to their relatively readily reabsorption from the gastrointestinal tract and to their characteristic distribution in the liver (Dong et al., 2009). The metabolism of poisons in vivo was usually presented curve mode, therefore, a 10 or 100 times higher concentration of the pesticides in the exposure treatments do not give 10 or 100 times increase of the pesticides in the liver in this manuscript. After recovery for 20 d, accumulations of ATR, CPF, and their metabolites in the liver were significantly lower (P < 0.05) compared with that in the corresponding exposure group, but there compounds were still
Table 2 Concentrations of CPF and its metabolites in the livers from common carp after exposure and recovery. CPF (mg kg1)
Treatment groups
Chlorpyrifos-oxon (mg kg1)
Exposure
Recovery
Exposure
Recovery
Control
0
n.d.
n.d.
n.d.
n.d.
Chlorpyrifos (lg L1)
1.16 11.6 116
0.85 ± 0.006 2.64 ± 0.021 3.33 ± 0.025
0.09 ± 0.002* 0.39 ± 0.004* 1.04 ± 0.011*
0.65 ± 0.005 2.28 ± 0.023 3.47 ± 0.026
0.07 ± 0.002* 0.40 ± 0.003* 0.54 ± 0.007*
Mixture of atrazine and chlorpyrifos (lg L1)
1.13 11.3 113
0.58 ± 0.008 0.69 ± 0.009 2.17 ± 0.022
0.11 ± 0.003* 0.46 ± 0.006* 0.84 ± 0.009*
1.25 ± 0.013 0.52 ± 0.007 1.46 ± 0.014
0.10 ± 0.002* 0.46 ± 0.007* 1.08 ± 0.012*
Note: Values are the means ± SD (n = 5). n.d. indicates not detected. * Significant differences (P < 0.05) between the exposure group and the recovery group at an identical concentration.
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markedly detected, suggesting that the dissipation of ATR, CPF, and their metabolites in the organism is a long-term process. High concentrations of ATR, CPF, and their metabolites in the liver of common carp indicated that possible tissue damage could exist (Adesiyan et al., 2011; Xing et al., 2012b). 4.2. Responses of the CYP1A gene and enzyme activities to ATR and CPF exposure in common carp liver In fish, many environmental pollutants act as CYP1A inducers, and CYP1A has been recognized as biomarker for the assessment of aquatic pollution. Additionally, CYP1A induction is closely related to detrimental effects such as apoptosis and embryonic mortality in exposed fish (Dong et al., 2002). Thus, the interaction of pharmaceutical compounds with CYP1A enzyme is likely to have a significant toxicological relevance in fish. Environmental pollutants can induce CYP1A expression and the induction response can be monitored at the transcriptional level by measuring the change in the CYP1A mRNA level (Cousinou et al., 2000; Rees and Li, 2004). Currently, there is limited information available regarding the toxicity of ATR and CPF on freshwater fish. Little is known about the involvement of CYP1A in ATR and CPF biotransformation in fish. Chang et al. (2005) reported that 7 ppb ATR could induced CYP1A1 mRNA level in common carp exposure after 4 d. The present study demonstrated that exposure to ATR, CPF and an ATR/CPF mixture elicited a conspicuous induction of mRNA expression patterns and EROD activity in carp liver for CYP1A, which is important in fish hepatic antidotal function. Salaberria et al. (2009) found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. These results showed that ATR can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked. In our previous study, a significantly alteration in glutathione S-transferase and antioxidant enzymes was observed from the liver of the same carp (Xing et al., 2012a,c). The results of these studies (CYP1A, glutathione S-transferase and antioxidant enzymes) indicated that the liver of carp was injured exposed to ATR and CPF alone and in combination. The induction of fish CYP1A is typically assessed as liver microsomal EROD activity. Torre et al. (2011) reported that alteration of EROD activity and CYP1A mRNA level by musk xylene in PLHC-1 and RTG-2 fish cell lines was different. In the present study, the EROD activity increased about 2-fold using the highest concentration of pesticides. However, the CYP1A mRNA level increased 6–7-fold at the same exposure. As we all know, the chemical nature of enzyme is the protein. It is called translation that the process of transformation of RNA to protein, and a variety of factors can interfere with this process. Therefore, alteration of enzyme activity and mRNA level are not always consistent. The result only indicated pesticides (ATR and CPF) can induce CYP1A expression. However, whether the CYP1A induction directly affects the increase of the total CYP is needed to be further studied. In contrast to mammals, studies examining the toxicity of ATR and CPF in aquatic vertebrates have been limited, particularly on the CYP system. A study by Ugazio et al. (1993) found a significant induction of the activity of PROD in young male rats following a single oral dose (430 mg kg1) of ATR treatment. A significant increase was also observed in microsomal PROD activity in rats after CPF treatment (Atterberry et al., 1997). Dong et al. (2009) reported that CYP enzymes in fish exposed to ATR were significantly increased. In our study, the increase in the PROD activity was quite modest in the liver rising approximately only around 2-fold. CYP content was found to be enhanced roughly by 3.5-fold over control as a result of ATR/CPF mixture treatment. The results on hepatic
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EROD and PROD activities and CYP content were in agreement with previous studies (Ugazio et al., 1993; Atterberry et al., 1997; Dong et al., 2009. These results indicated that ATR and CPF could interact with hepatic microsomal CYP in fish. 5. Conclusion The present study indicates important biochemical effects of ATR and CPF in carp liver microsomes, and suggests that exposure to ATR and CPF may cause marked changes in the CYP system in fish. In addition, the present work also demonstrated that ATR, CPF, and their metabolites become highly accumulated in the liver of the common carp. The experimental results demonstrated that the CYP system can be used as biomarkers in evaluating the impact of ATR and CPF exposure on the common carp and common carp could be used as a useful tool in the research of the toxicology of ATR and CPF. Acknowledgements The authors would like to thank Ying Han and Hui Zhang at the College of Animal Science and Technology, Northeast Agricultural University for their assistance. China Postdoctoral Science Foundation (Project No. 2012M511437) and Postdoctoral Foundation of Heilongjiang Province (Project No. LBH-Z10269) supported this study. References Abu-Qare, A.W., Abou-Donia, M.B., 2001. Development of a high-performance liquid chromatographic method for the quantification of chlorpyrifos, pyridostigmine bromide, N,N-diethyl-m-toluamide and their metabolites in rat plasma and urine. J. Chromatogr. B Biomed. Sci. Appl. 754, 533–538. Adesiyan, A.C., Oyejola, T.O., Abarikwu, S.O., Oyeyemi, M.O., Farombi, E.O., 2011. Selenium provides protection to the liver but not the reproductive organs in an atrazine-model of experimental toxicity. Exp. Toxicol. Pathol. 63, 201–207. Ali, D., Nagpure, N.S., Kumar, S., Kumar, R., Kushwaha, B., 2008. Genotoxicity assessment of acute exposure of chlorpyrifos to freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis. Chemosphere 71, 1823–1831. An, L., Hu, J., Yang, M., Zheng, B., Wei, A., Shang, J., Zhao, X., 2011. CYP1A mRNA expression in redeye mullets (Liza haematocheila) from Bohai Bay, China. Mar. Pollut. Bull. 62, 718–725. Atterberry, T.T., Burnett, W.T., Chambers, J.E., 1997. Age-related differences in parathion and chlorpyrifos toxicity in male rats: target and nontarget esterase sensitivity and cytochrome P450-mediated metabolism. Toxicol. Appl. Pharmacol. 147, 411–418. Banks, K.E., Hunter, D.H., Wachal, D.J., 2005. Chlorpyrifos in surface waters before and after a federally mandated ban. Environ. Int. 31, 351–356. Binelli, A., Ricciardi, F., Riva, C., Provini, A., 2006. New evidences for old biomarkers: effects of several xenobiotics on EROD and AChE activities in Zebra mussel (Dreissena polymorpha). Chemosphere 62, 510–519. Burke, M.D., Thompson, S., Elcombe, C.R., Halpert, J., Haaparanta, T., Mayer, R.T., 1985. Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem. Pharmacol. 34, 3337–3345. Chang, L.W., Toth, G.P., Gordon, D.A., Graham, D.W., Meier, J.R., Knapp, C.W., DeNoyelles, F.J., Campbell, S., Lattier, D.L., 2005. Responses of molecular indicators of exposure in mesocosms: common carp (Cyprinus carpio) exposed to the herbicides alachlor and atrazine. Environ. Toxicol. Chem. 24, 190–197. Coimbra, A.M., Figueiredo-Fernandes, A., Reis-Henriques, M.A., 2007. Nile tilapia (Oreochromis niloticus), liver morphology, CYP1A activity and thyroid hormones after Endosulfan dietary exposure. Pest Biochem. Physiol. 89, 230–236. Cousinou, M., Nilsen, B., Lopez-Barea, J., Dorado, G., 2000. New methods to use fish cytochrome P4501A to assess marine organic pollutants. Sci. Total Environ. 247, 213–225. Davis, R.K., Pederson, D.T., Blum, D.A., Carr, J.D., 2007. Atrazine in a stream-aquifer system: estimation of aquifer properties from atrazine concentration profiles. Ground Water Monit. Remediat. 13, 134–141. De Silva, P.M., Samayawardhena, L.A., 2005. Effects of chlorpyrifos on reproductive performances of guppy (Poecilia reticulata). Chemosphere 58, 1293–1299. Del Prado Lu, J.L., 2010. Multipesticide residue assessment of agricultural soil and water in major farming areas in Benguet, Philippines. Arch. Environ. Contam. Toxicol. 59, 175–181. Dong, W., Teraoka, H., Yamazaki, K., Tsukiyama, S., Imani, S., Imagawa, T., Stegeman, J.J., Peterson, R.E., Hiraga, T., 2002. 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity
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Effects of atrazine and chlorpyrifos on cytochrome P450 in common carp liver Houjuan Xing a,b, Ziwei Zhang a, Haidong Yao a, Tao Liu a, Liangliang Wang a, Shiwen Xu c,⇑, Shu Li c,⇑ a
College of Animal Science and Technology, Northeast Agricultural University, 59 Mucai Street, Harbin 150030, PR China Animal Health Supervision Institute of Heilongjiang Province, 243 Haping Road, Xiangfang District, Harbin 150069, PR China c College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Harbin 150030, PR China b
h i g h l i g h t s The Cyt P450 system in carp liver was investigated by ATR, CPF and their mixture. Our aim was to evaluate the sub-chronic effects of ATR, CPF and their mixture on carp. This is the first report from pesticide residues in carp tissues by ATR, CPF and their mixture. The Cyt P450 system can be used as biomarker for monitoring ATR and CPF.
a r t i c l e
i n f o
Article history: Received 18 April 2013 Received in revised form 20 December 2013 Accepted 2 January 2014 Available online 14 February 2014 Keywords: CYP system Common carp Pesticide residue Chlorpyrifos Atrazine
a b s t r a c t Atrazine (ATR) and chlorpyrifos (CPF), widely used in agriculture, have resulted in a series of toxicological and environmental problems. We investigated the activities of the biotransformation enzymes ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-deethylase (PROD), total cytochrome P450 (CYP), CYP1A mRNA level and level of tissue ATR, CPF, and their metabolites in the liver of common carp (Cyprinus carpio L.) after a 40-d exposure to CPF and ATR, alone or in combination, and a 20-d recovery. In the present study, juvenile common carp was exposed to ATR (at concentrations of 4.28, 42.8 and 428 lg L1), CPF (1.16, 11.6 and 116 lg L1), and ATR/CPF mixture (at concentrations of 1.13, 11.3 and 113 lg L1). A general increasing trend for the activity of the biotransformation enzymes (EROD and PROD), CYP and CYP1A mRNA level was observed in the liver of common carp exposed to ATR, CPF and the ATR/CPF mixture. In addition, ATR, CPF, and their metabolites demonstrated a high accumulation in the liver. These results demonstrated that the CYP system in fish could be used as a biomarkers in evaluating the impact of ATR and CPF exposure on the common carp. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction With the growth of human activities, increasing numbers of chemicals are being released into the environment, many of which are hazardous to living organisms and ecosystems. The triazine herbicide atrazine (ATR) is used as a selective pre-emergence and post-emergence herbicide for the control of weeds in asparagus, maize, sorghum, sugarcane and pineapple. ATR is also used in forestry and for non-selective weed control on non-crop areas. ATR has been employed extensively in agriculture in the US and worldwide for over 40 years (Singh et al., 2011). According to USDA NASS (2008), in 2003 about 24.3 million kg of ATR was used on corn crops in the US. In China, where ATR is used as a ⇑ Corresponding authors. Tel./fax: +86 451 55190407. E-mail addresses: [email protected] (S. Xu), [email protected] (S. Li). http://dx.doi.org/10.1016/j.chemosphere.2014.01.002 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
broad-spectrum herbicide, which is applied to major field crops such as corn, wheat and soybeans, it has been reported that in 2008 the amounts of ATR applied to fields was at least 5000 t (Song et al., 2009). Several studies have already detected its presence in ground- and surface-waters at levels above the limits determined by local guidelines (Ren et al., 2002; Dong et al., 2006; Konstantinou et al., 2006; Gilliom, 2007; Davis et al., 2007). Chlorpyrifos (CPF), as an organophosphorus insecticide, is widely used to control a variety of pests on agricultural and animal farms (Whyatt et al., 2007; Saulsbury et al., 2009). Field surveys performed in many countries have shown that CPF is commonly reported as contaminant of surface and ground water (Banks et al., 2005; Du Preez et al., 2005). Several studies have been confirmed that ATR and CPF are toxic to fish (De Silva and Samayawardhena, 2005; Ali et al., 2008; Kavitha and Venkateswara Rao, 2008; Nwani et al., 2010).
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The cytochrome P450 (CYP) enzymes serve as terminal oxidases in the mixed-function oxidase system for metabolizing various endogenous substrates and xenobiotics including drugs, toxins and carcinogens (Dong et al., 2009). The CYP family, such as CYP1A, plays an important role in the detoxification mechanism. Although the expression of CYP1A is constitutively low, it is highly inducible in animal tissue by environmental contaminants. Accordingly, the induction of hepatic CYP1A mRNA in fish by certain classes of xenobiotics, including pesticides, has been suggested as an early warning system, a most sensitive biological response for assessing environmental contamination (Coimbra et al., 2007; Fu et al., 2013). Compared to the protein levels of CYP1A, the induction of CYP1A can occur within hours of exposure and the level of CYP1A mRNA decreases when induction response is eliminated. Moreover, the real-time quantitative polymerase chain reaction developed recently is at least 100-fold more sensitive than Northern or slot blotting in measuring CYP1A RNA (Cousinou et al., 2000; An et al., 2011). EROD activity was used as a marker of CYP1A function. The liver is considered as the major organ for CYP-mediated biotransformation (Tabrez et al., 2010). Many studies have reported CYP1A mRNA level and CYP-dependent enzymatic activity in fish liver in response to the presence of different pollutants in the aquatic environment (Moore et al., 2003; Binelli et al., 2006; Santos and Martincez, 2012). Fish species are widely used as biological monitors of variations in the environmental levels of anthropogenic pollutants. The present paper is a contribution to the assessment of the toxicity and effects of ATR and CPF based-pesticides on the common carp (Cyprinus carpio L.). The common carp was selected as the test fish because although it is one of the most economically important freshwater fishes of the world (Li et al., 2010), there is a scarcity of information regarding the toxicity of ATR and CPF on freshwater fishes. To improve our understanding of the toxicity mechanisms and responses of different organs to environmentally relevant concentrations of ATR and CPF, a better understanding of the CYP system is required. In this study, we examined the changes in the transcription of the CYP1A gene, activity of CYP enzymes and level of tissue ATR, CPF, and their metabolites in the liver of common carp exposed to ATR and CPF alone and in combination. These results will enable us to further understand the hepatic detoxification mechanisms affected by ATR and CPF exposure in common carp. 2. Materials and methods 2.1. Chemicals The Folin Ciocalteau reagent, ethoxyresorufine, pentoxyresorufin, ATR (purity 98.0%) and CPF (purity 99.5%) were purchased from Sigma–Aldrich Chemical Co. (USA). All others reagents were obtained from Reanal, Budapest, Hungary. All chemicals were of analytical grade. Stock solutions of ATR and CPF were prepared in analytical grade acetone (purity 99%). All working solutions were taken from this stock solution. The concentration of acetone was kept at <0.05% in all pesticide solutions used. 2.2. Animals and treatments Juvenile 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 laboratory tanks (90 55 45 cm) with continuous aeration. Acclimatization to experimental conditions was performed for 15 d using dechlorinated tap water with 230 mg L1 CaCO3, 42.5 ± 1.2 mg L1 Ca, a dissolved oxygen concentration >7 mg L1,
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and pH 7.4 ± 0.2. The water temperature was adjusted to 20 ± 1 °C, and the photoperiod was 12 h light and 12 h dark. Commercial food was given once a day until satiation. No mortality was observed either in the control animals or in any of the treatment groups. 2.3. Toxicity test Experimental fish were randomly divided into eleven groups: three ATR treatment groups (4.28, 42.8 and 428 lg L1), three CPF treatment groups (1.16, 11.6 and 116 lg L1), three mixturetreatment groups (ATR/CPF) (1.13, 11.3 and 113 lg L1), one solvent control (acetone) group, and one water control group. The binary mixtures were composed of a 1:1 mass ratio of ATR and CPF. Each treated group has 20 fish and 2 replicates. The concentrations used in the present study are about 1/500, 1/50 and 1/5 of the 96 h LC50s (unpublished data). In China, the commercial solutions used as herbicides and insecticides contain 400 g L1 ATR and 380 g L1 CPF, respectively. In addition, ATR and CPF are stable in water and have a long half-life, so we can easily speculate that the dose we chose could be found in the environment. The fish were exposed under semi-static conditions for 40 d to water and pesticide, which were completely replaced once every 2 d by transferring fish to freshly prepared pesticide solutions. At the end of the exposure, five fish were killed in each group, and the liver was excised immediately on an ice-cold plate and washed in physiological saline solution (0.86% NaCI). The tissues were divided into two portions, one for further protein and enzyme analysis and a second to be stored at 80 °C for RNA examination. Experiments were carried out in accordance with the European Communities Council Directive (86/609/EEC) and were approved by a local ethics committee. 2.4. Recovery test Ten exposed fish from each batch were kept in pesticide-free water for 20 d in a large fresh 200-L glass aquarium provided with a filter and continuous aeration. The conditions in the recovery experiments were identical to those in the exposure experiments. In the present study, there were 10 fish per group killed at the two sampling events, and 5 fish per group were used in official test. The remaining 5 fish were used in preliminary experiment and served as standby tissues. 2.5. Determination of the CYP system 2.5.1. Preparation of microsomes and determination of enzymatic activities The microsomes were prepared in the following way: the fish were sacrificed and the gall bladders were removed to avoid contaminating the livers with bile. The method used as described by Forlin (1980) with some slight modifications. After weighing, the liver was chopped with a pair of scissors and homogenized in a Braun 853302/4 homogenizer with 1:5 w/v of ice-cold phosphate buffer (0.1 M K-phosphate, pH 7.4) containing 0.15 M KCl. The homogenate was centrifuged with a Sorvall RC 5B Plus centrifuge at 10 000 g for 20 min at 4 °C and the supernatant obtained was ultracentrifuged with a Sorvall Ultra Pro 80 at 105 000 g for 65 min, also at 4 °C. The pellet (after 105 000 g centrifugation) was resuspended in 1 mL phosphate buffer containing 20% (vol.) glycerol, the suspension was homogenized and the resultant microsomal fraction was stored at 80 °C until the determination the enzymatic activities. 7-ethoxyresorufin-O-dealkylase (EROD) activity was determined by the method described before (Burke et al., 1985), with slight modifications. Briefly, one milliliter of the reaction mixture
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containing one milligram of protein, 10 mM glucose-6-phosphate (G-6-P), 10 mM MgCl2, and 2.5 lM ethoxyresorufin in 0.1 M KPB, pH 7.4, was preincubated for 5 min at 37 °C. The reaction was started by adding 20 lL of a mixture of 50 mM reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) and 200 U mL1 of glucose-6-phosphate dehydrogenase (G-6-PDH). After incubation for 5 min, the reaction was terminated by adding 4 mL of cold methanol. The mixture was centrifuged at 3000 g for 5 min, and the supernatant methanol layer was collected for measurement of resorufin. Assays were conducted using a fluorescence spectrophotometer set at wavelengths of 530 nm excitation and 590 nm emission with a cuvette stirring function. 7-pentoxyresorufin–O-dealkylase (PROD) activity was measured exactly in the same manner as described for the ethoxyresorufin assay, except that the substrate was pentoxyresorufin. The enzymatic activities are shown in nmol min1 mg1 of protein. The total CYP protein contents were determined as the dithionite-reduced CO complex, as described by Omura and Sato (1964). The results are expressed in nmol mg1 of protein, with an extinction coefficient of 91 mM1cm1 (450–490 nm). The protein concentration of the microsomes was measured by the method of Lowry et al. (1951).
2.5.2. Determination of gene expression Primer design: The primers for real-time amplification of the CYP1A and b-actin cDNA were designed using Oligo_6.0 Software (Molecular Biology Insights, Cascade, CO) based on the deposited sequences in GenBank under the accession numbers AB048939 and AF057040. The following primers were used for CYP1A: forward 50 - ATT TCA TTC CCA AAG ACA CCT G-30 ; and reverse 50 CAA AAA CCA ACA CCT TCT CTC C-30 . The following primers were used for b-actin: forward 50 -GAT GGA CTC TGG TGA TGG TGT GAC-30 ; and reverse 50 - TTT CTC TTT CGG CTG TGG TGG TG-30 . The amplification product sizes were CYP1A, 159 bp; and b-actin, 167 bp. The PCR products were electrophoresed on two percent agarose gels, extracted, cloned into the pMD18-T vector (Takara, Ohtsu, Japan), and sequenced. Total RNA isolation and reverse transcription: Total RNA was isolated from the livers of each fish using Trizol reagent according to the manufacturer’s instructions (Invitrogen). The reverse transcription reactions (40 lL) consisted of 10 lg of total RNA, 1 lL of Moloney murine leukemia virus reverse transcriptase, 1 lL of RNase inhibitor, 4 lL of deoxynucleoside triphosphate, 2 lL of Oligo dT, 4 lL of dithiothreitol, and 8 lL of 5 reverse transcriptase buffer. The reverse transcription procedure used was based on instructions from the manufacturer (Invitrogen). Real-time quantitative reverse transcription PCR: Real-time quantitative reverse transcription PCR was used to detect the expression of the CYP1A gene using SYBR Premix Ex Taq (Takara, Shiga, Japan). Real-time PCR was conducted on each the liver tissues. Reaction mixtures were incubated in an ABI PRISM 7500 real-time PCR system (Applied Biosystems, Foster City, CA). The following program was used: 1 cycle at 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and at 60 °C for 34 s. Dissociation curves were analyzed with Dissociation Curve 1.0 software (Applied Biosystems) for each PCR reaction to detect and eliminate the possible primer–dimer and nonspecific amplifications. The results (fold changes) are expressed as 2DDCt, where CtCYP1A and
DDCt ¼ ðCtCYPIA Ctb-actin Þt ðCtCYPIA Ctb-actin Þc Ctb-actin are the cycle thresholds for the carp CYP1A and b-actin genes in the different treated groups, respectively, t indicates the treatment group; and c denotes the control group.
2.6. Analysis of tissue pesticide content 2.6.1. Analysis of tissue ATR content analysis Stored liver samples were cut and homogenized using a glass– Teflon homogenizer (Heidolph S01 10R2RO). The homogenate was extracted three times using 20 mL of 50% aqueous acetone by shaking the suspension for 60 min. The extracts were filtered and combined, and then 3 g of NaCI were added successively to each sample and dissolved by shaking the suspension. The filtrate was re-extracted three times with 30 mL of trichloromethane. The extracts were combined, passed through anhydrous Na2SO4 columns, and collected. The eluates were concentrated into a triangular flask by rotary evaporation, dissolved with 1.5 mL of methanol, and filtered. The concentrations of ATR in the extracts were analyzed with an Agilent high-performance liquid chromatography (HPLC) system. HPLC was performed with Waters equipment, equipped with a diode array using an ODS 5-micron Hypersil capillary column (250 mm long, 4.6 mm in diameter) folˇ oz and Rosés (2000). Molowing the procedures described by Mun bile phases used in the isocratic elution were distilled with water and methanol (v/v: 20/80). The column head temperature was 25 °C and the flow rate was held constant at 1 mL min1. The volume injected was 10 lL. The eluents were monitored by UV detection of a wavelength of 222 nm for ATR, atrazine-2-hydroxy, and atrazine–desethyl.
2.6.2. Analysis of tissue CPF content analysis For the analysis of CPF content, approximately 2 g (wet wt) of stored soft tissues samples was weighed in a centrifuge tube (50 mL). Each sample received 1 g of anhydrous Na2SO4 and 10 mL of acetidin and was homogenized using an IKA homogenizer at 6000 r min1 for 2 min. Then, the tool bit was washed using 10 mL of acetidin. The homogenate was centrifuged at 5000 r min1 for 5 min to obtain the supernatant. The residue in the centrifuge tube was re-extracted once with 10 mL of acetidin and centrifuged. The two supernatants were concentrated in a rotary evaporation flask by rotary evaporation at 40 °C, and the precipitate was dissolved with 5 mL of acetonitrile by convolution agitate and supersonic ablution. The extracts were passed through Acor aluminum oxide columns and collected. The eluates were evaporated to dryness under a nitrogen stream at 50 °C. The residue was finally dissolved in 2 mL of N-hexane and filtered for the HPLC analysis of the concentrations of CPF in extracts. The HPLC procedure was similar to the method used by Abu-Qare and Abou-Donia (2001). Briefly, mobile phases used in the isocratic elution were distilled with water and acetonitrile (v/v: 40/60). The column head temperature was 25 °C, and the flow rate was held constant at 1 mL min1. The volume injected was 10 lL. The eluents were monitored by UV detection at a wavelength of 230 nm for CPF and chlorpyrifos-oxon.
2.7. Statistical analysis Statistical analysis of all data was performed using SPSS for Windows (version 13, SPSS Inc., Chicago, IL). One-way ANOVA with a post hoc test was used to elucidate if there were significant differences between the treatment groups and the control group. The difference between the treatment groups and the recovery groups was assessed by using Paired t-test. All expressed data were collected in quintuple from at least five fish and were expressed as mean ± standard deviation. Differences were considered to be significant at P < 0.05.
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3. Results 3.1. Responses of the activities of EROD and PROD and the level of CYP in common carp exposed to ATR, CPF and their mixture The activity of EROD and PROD in the liver tissue is shown in Fig. 1. The activity of EROD and PROD was significantly higher (P < 0.05) at all groups exposed to ATR, CPF and the ATR/CPF combination compared with the control group except for the groups treated with the low doses of pesticides (1.13, 1.16 and 4.28 lg L1). The activity of EROD and PROD increased after exposure and reached their highest level at the high-dose groups for each pesticide. During the recovery period, the activity of EROD and PROD at all treated groups tended to increase with higher concentration of ATR or CPF or the ATR/CPF mixture, but no significant difference was observed between exposure and recovery groups. The CYP content in the liver tissue for each group is shown in Fig. 2. After exposure, the liver CYP content increased with increasing concentration of pesticides. The CYP content at all groups exposed to ATR, CPF and the ATR/CPF combination was significantly higher (P < 0.05) than that observed in the control fish. After the recovery period, CYP content of all treatment groups was still significantly higher (P < 0.05) compared with the control group. The CYP content of all recovery groups, except for the 42.8, 428, 11.3 and 113 lg L1, had significantly decreased (P < 0.05) compared to the exposed groups.
Fig. 2. Effects of ATR, CPF and ATR/CPF mixture on the cytochrome P450 content of common carp liver. Each value represented the mean ± SD of 5 individuals. * Significant differences (P < 0.05) between the control group and the exposure group. #Significant difference (P < 0.05) between the exposure group and the recovery group at the same concentration.
3.2. Quantification of CYP1A expression in the liver of common carp after exposure to ATR, CPF, and their mixture The effect of ATR, CPF and their mixture on the mRNA level of CYP1A in the liver is presented in Fig. 3. Compared with the control group, a significantly increase (P < 0.05) in the mRNA level of the CYP1A gene in the liver was observed in the fish exposed to ATR, CPF and ATR/CPF mixture. For the each treatment group, a significantly increase (P < 0.05) in the mRNA level of CYP1A gene in the liver was observed when exposed fish were compared to recovery-treated fish (P < 0.05) but the mRNA level at all recovery groups was still higher than those from the control fish. 3.3. Accumulations of ATR, CPF, and their metabolites in liver tissue The concentrations of ATR and its metabolites (atrazine-2-hydroxy and atrazine–desethyl) in fish liver are summarized in Table 1. ATR and its metabolites (atrazine–desethyl and
Fig. 1. Effects of ATR, CPF and ATR/CPF mixture on the EROD (a) and PROD (b) activities of common carp liver. Each value represented the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the control group and the exposure group. #Significant difference (P < 0.05) between the exposure group and the recovery group at the same concentration.
Fig. 3. Relative mRNA expression of the CYP1A gene in common carp liver after exposure to ATR, CPF and their mixture for the 40 d and a recovery period. Each value represented the mean ± SD of 5 individuals. *Significant differences (P < 0.05) between the control group and the exposure group. # Significant differences (P < 0.05) between the exposure group and the recovery group at an identical concentration.
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Table 1 Concentrations of ATR and its metabolites in the livers from the common carp after exposure and recovery treatments. ATR (mg kg1)
Treatment groups
Atrazine–desethyl (mg kg1)
Atrazine-2-hydroxy (mg kg1)
Exposure
Recovery
Exposure
Recovery
Exposure
Recovery
Control
0 4.28
n.d. 0.15 ± 0.006
n.d. 0.07 ± 0.002*
n.d. 0.02 ± 0.002
n.d. 0.07 ± 0.002*
n.d. 0.27 ± 0.005
n.d. 0.09 ± 0.002*
Atrazine (lg L1)
42.8 428
1.64 ± 0.012 2.29 ± 0.014
0.18 ± 0.005* 0.23 ± 0.005*
2.12 ± 0.014 4.64 ± 0.025
0.43 ± 0.007* 0.43 ± 0.006*
1.10 ± 0.009 2.29 ± 0.015
0.54 ± 0.008* 0.59 ± 0.008*
Mixture of atrazine and chlorpyrifos (lg L1)
1.13 11.3 113
0.16 ± 0.004 1.26 ± 0.009 2.29 ± 0.021
0.09 ± 0.003* 0.26 ± 0.005* 0.29 ± 0.006*
0.43 ± 0.004 2.42 ± 0.015 3.16 ± 0.016
0.02 ± 0.002* 0.78 ± 0.008* 0.43 ± 0.006*
0.69 ± 0.006 1.20 ± 0.008 2.17 ± 0.015
0.10 ± 0.002* 0.39 ± 0.001* 0.44 ± 0.007*
Note: Values are the means ± SD (n = 5). n.d. indicates not detected. * Significant differences (P < 0.05) between the exposure group and the recovery group at an identical concentration.
atrazine-2-hydroxy) were not detected in the liver of common carp that were cultivated in pesticide-free control water. As the pesticide concentration in the water increased, the accumulations of ATR and its metabolites in the liver significantly increased. For ATR and its metabolites, atrazine-2-hydroxy had the highest concentrations in the liver at the treated groups (4.28 and 1.13 lg L1), and atrazine–desethyl had the highest concentrations in the other treated groups. After recovery for 20 d, the accumulations of ATR and its metabolites in the liver were significantly lower (P < 0.05) than compared with that in the corresponding exposure group; however, the accumulations of these pesticides in the liver were still detected. The concentrations of CPF and its metabolite (chlorpyrifosoxon) in fish liver are summarized in Table 2. CPF and chlorpyrifos-oxon were not detected in the liver of common carp that were cultivated in pesticide-free control water. With increasing pesticide concentration in the water, the accumulations of CPF and chlorpyrifos-oxon in the liver were significantly elevated. Chlorpyrifos-oxon accumulations in the liver at 1.13 and 116 lg L1 groups was higher than the CPF accumulations. After recovery for 20 d, the accumulations of CPF and chlorpyrifos-oxon in the liver were significantly lower (P < 0.05) than that in the corresponding exposure group; however, the accumulations of these pesticides in the tissues were still detected. 4. Discussion The liver is primarily responsible for the metabolism of toxic substances, including ATR and CPF. In particular the liver is the major site of CYP expression in teleost fish. Therefore, the effect of ATR and CPF on liver-detoxifying enzymes is very important. In the present study, we investigated the changes in CYP1A gene transcription, EROD and PROD activities, CYP content and accumulations of ATR, CPF and their metabolites in the liver of common carp exposed to ATR, CPF and an ATR/CPF mixture.
4.1. Accumulations of ATR, CPF, and their metabolites in response to ATR and CPF exposure in common carp liver Pesticide pollution in the environment has been increasing due to their extensive use in agriculture. Agricultural runoff and irrigation water are the major sources of these contaminants in the aquatic environment. Alterations in the chemical composition of natural aquatic environments can affect the freshwater fauna, particularly fish (Oruc, 2010; Jin et al., 2010; Xing et al., 2012a). The results of the present study indicated that the accumulations of ATR, CPF, and their metabolites were detected in the liver of common carp exposed to ATR, CPF and an ATR/CPF mixture, suggesting that ATR, CPF and their metabolites were present in the organism because ATR and CPF have low solubility in water (Kidd and James, 1991; Powell et al., 2011). ATR can be found at levels as high as 21 ppb in groundwater, 42 ppb in surface waters, 102 ppb in river basins in agricultural areas, and up to 224 ppb in Midwestern streams during May–August (Kolpin et al., 1998). Del Prado Lu (2010) reported the presence of CPF (0.07 mg L1) residues in river water samples in agricultural areas. The present study has also demonstrated that the concentrations of ATR, CPF, and their metabolites were increased with increasing concentrations of ATR, CPF and the ATR/CPF combination. The results could be related to the lipophilic nature of ATR and CPF thus enhancing their bioaccumulation. Alternatively, the relatively high accumulations of ATR and CPF by the common carp could be due to their relatively readily reabsorption from the gastrointestinal tract and to their characteristic distribution in the liver (Dong et al., 2009). The metabolism of poisons in vivo was usually presented curve mode, therefore, a 10 or 100 times higher concentration of the pesticides in the exposure treatments do not give 10 or 100 times increase of the pesticides in the liver in this manuscript. After recovery for 20 d, accumulations of ATR, CPF, and their metabolites in the liver were significantly lower (P < 0.05) compared with that in the corresponding exposure group, but there compounds were still
Table 2 Concentrations of CPF and its metabolites in the livers from common carp after exposure and recovery. CPF (mg kg1)
Treatment groups
Chlorpyrifos-oxon (mg kg1)
Exposure
Recovery
Exposure
Recovery
Control
0
n.d.
n.d.
n.d.
n.d.
Chlorpyrifos (lg L1)
1.16 11.6 116
0.85 ± 0.006 2.64 ± 0.021 3.33 ± 0.025
0.09 ± 0.002* 0.39 ± 0.004* 1.04 ± 0.011*
0.65 ± 0.005 2.28 ± 0.023 3.47 ± 0.026
0.07 ± 0.002* 0.40 ± 0.003* 0.54 ± 0.007*
Mixture of atrazine and chlorpyrifos (lg L1)
1.13 11.3 113
0.58 ± 0.008 0.69 ± 0.009 2.17 ± 0.022
0.11 ± 0.003* 0.46 ± 0.006* 0.84 ± 0.009*
1.25 ± 0.013 0.52 ± 0.007 1.46 ± 0.014
0.10 ± 0.002* 0.46 ± 0.007* 1.08 ± 0.012*
Note: Values are the means ± SD (n = 5). n.d. indicates not detected. * Significant differences (P < 0.05) between the exposure group and the recovery group at an identical concentration.
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markedly detected, suggesting that the dissipation of ATR, CPF, and their metabolites in the organism is a long-term process. High concentrations of ATR, CPF, and their metabolites in the liver of common carp indicated that possible tissue damage could exist (Adesiyan et al., 2011; Xing et al., 2012b). 4.2. Responses of the CYP1A gene and enzyme activities to ATR and CPF exposure in common carp liver In fish, many environmental pollutants act as CYP1A inducers, and CYP1A has been recognized as biomarker for the assessment of aquatic pollution. Additionally, CYP1A induction is closely related to detrimental effects such as apoptosis and embryonic mortality in exposed fish (Dong et al., 2002). Thus, the interaction of pharmaceutical compounds with CYP1A enzyme is likely to have a significant toxicological relevance in fish. Environmental pollutants can induce CYP1A expression and the induction response can be monitored at the transcriptional level by measuring the change in the CYP1A mRNA level (Cousinou et al., 2000; Rees and Li, 2004). Currently, there is limited information available regarding the toxicity of ATR and CPF on freshwater fish. Little is known about the involvement of CYP1A in ATR and CPF biotransformation in fish. Chang et al. (2005) reported that 7 ppb ATR could induced CYP1A1 mRNA level in common carp exposure after 4 d. The present study demonstrated that exposure to ATR, CPF and an ATR/CPF mixture elicited a conspicuous induction of mRNA expression patterns and EROD activity in carp liver for CYP1A, which is important in fish hepatic antidotal function. Salaberria et al. (2009) found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. These results showed that ATR can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked. In our previous study, a significantly alteration in glutathione S-transferase and antioxidant enzymes was observed from the liver of the same carp (Xing et al., 2012a,c). The results of these studies (CYP1A, glutathione S-transferase and antioxidant enzymes) indicated that the liver of carp was injured exposed to ATR and CPF alone and in combination. The induction of fish CYP1A is typically assessed as liver microsomal EROD activity. Torre et al. (2011) reported that alteration of EROD activity and CYP1A mRNA level by musk xylene in PLHC-1 and RTG-2 fish cell lines was different. In the present study, the EROD activity increased about 2-fold using the highest concentration of pesticides. However, the CYP1A mRNA level increased 6–7-fold at the same exposure. As we all know, the chemical nature of enzyme is the protein. It is called translation that the process of transformation of RNA to protein, and a variety of factors can interfere with this process. Therefore, alteration of enzyme activity and mRNA level are not always consistent. The result only indicated pesticides (ATR and CPF) can induce CYP1A expression. However, whether the CYP1A induction directly affects the increase of the total CYP is needed to be further studied. In contrast to mammals, studies examining the toxicity of ATR and CPF in aquatic vertebrates have been limited, particularly on the CYP system. A study by Ugazio et al. (1993) found a significant induction of the activity of PROD in young male rats following a single oral dose (430 mg kg1) of ATR treatment. A significant increase was also observed in microsomal PROD activity in rats after CPF treatment (Atterberry et al., 1997). Dong et al. (2009) reported that CYP enzymes in fish exposed to ATR were significantly increased. In our study, the increase in the PROD activity was quite modest in the liver rising approximately only around 2-fold. CYP content was found to be enhanced roughly by 3.5-fold over control as a result of ATR/CPF mixture treatment. The results on hepatic
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EROD and PROD activities and CYP content were in agreement with previous studies (Ugazio et al., 1993; Atterberry et al., 1997; Dong et al., 2009. These results indicated that ATR and CPF could interact with hepatic microsomal CYP in fish. 5. Conclusion The present study indicates important biochemical effects of ATR and CPF in carp liver microsomes, and suggests that exposure to ATR and CPF may cause marked changes in the CYP system in fish. In addition, the present work also demonstrated that ATR, CPF, and their metabolites become highly accumulated in the liver of the common carp. The experimental results demonstrated that the CYP system can be used as biomarkers in evaluating the impact of ATR and CPF exposure on the common carp and common carp could be used as a useful tool in the research of the toxicology of ATR and CPF. Acknowledgements The authors would like to thank Ying Han and Hui Zhang at the College of Animal Science and Technology, Northeast Agricultural University for their assistance. China Postdoctoral Science Foundation (Project No. 2012M511437) and Postdoctoral Foundation of Heilongjiang Province (Project No. LBH-Z10269) supported this study. References Abu-Qare, A.W., Abou-Donia, M.B., 2001. Development of a high-performance liquid chromatographic method for the quantification of chlorpyrifos, pyridostigmine bromide, N,N-diethyl-m-toluamide and their metabolites in rat plasma and urine. J. Chromatogr. B Biomed. Sci. Appl. 754, 533–538. Adesiyan, A.C., Oyejola, T.O., Abarikwu, S.O., Oyeyemi, M.O., Farombi, E.O., 2011. Selenium provides protection to the liver but not the reproductive organs in an atrazine-model of experimental toxicity. Exp. Toxicol. Pathol. 63, 201–207. Ali, D., Nagpure, N.S., Kumar, S., Kumar, R., Kushwaha, B., 2008. Genotoxicity assessment of acute exposure of chlorpyrifos to freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis. Chemosphere 71, 1823–1831. An, L., Hu, J., Yang, M., Zheng, B., Wei, A., Shang, J., Zhao, X., 2011. CYP1A mRNA expression in redeye mullets (Liza haematocheila) from Bohai Bay, China. Mar. Pollut. Bull. 62, 718–725. Atterberry, T.T., Burnett, W.T., Chambers, J.E., 1997. Age-related differences in parathion and chlorpyrifos toxicity in male rats: target and nontarget esterase sensitivity and cytochrome P450-mediated metabolism. Toxicol. Appl. Pharmacol. 147, 411–418. Banks, K.E., Hunter, D.H., Wachal, D.J., 2005. Chlorpyrifos in surface waters before and after a federally mandated ban. Environ. Int. 31, 351–356. Binelli, A., Ricciardi, F., Riva, C., Provini, A., 2006. New evidences for old biomarkers: effects of several xenobiotics on EROD and AChE activities in Zebra mussel (Dreissena polymorpha). Chemosphere 62, 510–519. Burke, M.D., Thompson, S., Elcombe, C.R., Halpert, J., Haaparanta, T., Mayer, R.T., 1985. Ethoxy-, pentoxy- and benzyloxyphenoxazones and homologues: a series of substrates to distinguish between different induced cytochromes P-450. Biochem. Pharmacol. 34, 3337–3345. Chang, L.W., Toth, G.P., Gordon, D.A., Graham, D.W., Meier, J.R., Knapp, C.W., DeNoyelles, F.J., Campbell, S., Lattier, D.L., 2005. Responses of molecular indicators of exposure in mesocosms: common carp (Cyprinus carpio) exposed to the herbicides alachlor and atrazine. Environ. Toxicol. Chem. 24, 190–197. Coimbra, A.M., Figueiredo-Fernandes, A., Reis-Henriques, M.A., 2007. Nile tilapia (Oreochromis niloticus), liver morphology, CYP1A activity and thyroid hormones after Endosulfan dietary exposure. Pest Biochem. Physiol. 89, 230–236. Cousinou, M., Nilsen, B., Lopez-Barea, J., Dorado, G., 2000. New methods to use fish cytochrome P4501A to assess marine organic pollutants. Sci. Total Environ. 247, 213–225. Davis, R.K., Pederson, D.T., Blum, D.A., Carr, J.D., 2007. Atrazine in a stream-aquifer system: estimation of aquifer properties from atrazine concentration profiles. Ground Water Monit. Remediat. 13, 134–141. De Silva, P.M., Samayawardhena, L.A., 2005. Effects of chlorpyrifos on reproductive performances of guppy (Poecilia reticulata). Chemosphere 58, 1293–1299. Del Prado Lu, J.L., 2010. Multipesticide residue assessment of agricultural soil and water in major farming areas in Benguet, Philippines. Arch. Environ. Contam. Toxicol. 59, 175–181. Dong, W., Teraoka, H., Yamazaki, K., Tsukiyama, S., Imani, S., Imagawa, T., Stegeman, J.J., Peterson, R.E., Hiraga, T., 2002. 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity
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