Ecotoxicology DOI 10.1007/s10646-014-1321-8
The enzyme toxicity and genotoxicity of chlorpyrifos and its toxic metabolite TCP to zebrafish Danio rerio Jun Wang • Jinhua Wang • Lusheng Zhu Hui Xie • Bo Shao • Xinxin Hou
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Accepted: 9 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Chlorpyrifos is a broad-spectrum organophosphorus insecticide (O,O-diethyl -O-3,5,6-trichloro-2-pyridyl phosphorothioate) that is used in numerous agricultural and urban pest controls. The primary metabolite of chlorpyrifos is 3,5,6-trichloro pyridine-2-phenol (TCP). Because of its strong water solubility and mobility, this harmful metabolite exists in the environment in a large amount. Although TCP has potentially harmful effects on organisms in the environment, few studies have addressed TCP pollution. Therefore, this study was undertaken to investigate the effect of chlorpyrifos and TCP on the microsomal cytochrome P450 content in the liver, on the activity of NADPH-P450 reductase and antioxidative enzymes [catalase (CAT) and superoxide dismutase (SOD)], and on reactive oxygen species (ROS) generation and DNA damage in zebrafish. Male and female zebrafish were separated and exposed to a control solution and three concentrations of chlorpyrifos (0.01, 0.1, 1 mg L-1) and TCP (0.01, 0.1, 0.5 mg L-1), respectively, sampled after 5, 10, 15, 20 and 25 days. The results indicated that the P450 content and the NADPH-P450 reductase and antioxidative enzyme (CAT and SOD) activities could be induced by chlorpyrifos and TCP. DNA damage of zebrafish was enhanced with increasing chlorpyrifos and TCP concentrations. Meanwhile, chlorpyrifos and TCP induced a significant increase of ROS generation in the zebrafish
J. Wang J. Wang (&) L. Zhu (&) H. Xie B. Shao X. Hou Key Laboratory of Agricultural Environment in the University of Shandong, College of Resources and Environment, Shandong Agriculture University, 61 Daizong Road, Taian 271018, China e-mail: [email protected] L. Zhu e-mail: [email protected]; [email protected]
hepatopancreas. In conclusion, this study proved that chlorpyrifos (0.01–1 mg L-1) and TCP (0.01–0.5 mg L-1) are both highly toxic to zebrafish. Keywords Cytochrome P450 NADPH-P450 reductase SOD CAT ROS SCGE
Introduction Chlorpyrifos is a broad-spectrum organophosphorus insecticide (O,O-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothioate) with numerous agricultural crop and urban pest control uses (David and Stuart 1998). Currently, chlorpyrifos is used worldwide as a substitute for some highly toxic pesticides, such as methamidophos and methyl parathion. During recent years, many countries have recognized the hazards of chlorpyrifos and have gradually restricted or banned their use. According to David and Stuart (1998), chlorpyrifos has been in priority lists of pesticides within the European Economic Community. It is also one of the 12 major environmental pollutants in Japan (Gamo et al. 2003). However, chlorpyrifos continues to be one of the most commonly used organophosphorus insecticides. The primary metabolite of chlorpyrifos is TCP, and its strong water solubility and high mobility results in significant amounts of harmful TCP residue in the soil and water. The aim of this study was to gain a more comprehensive understanding of the effects of chlorpyrifos and its metabolite TCP to the aquatic ecosystem. Moreover, the sensitivity of many different biomarkers was also tested in this study. In aquatic ecosystems, many species of animals, including fish, have been widely studied to reveal the toxicology of chlorpyrifos. So far, especially in recent
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years, experiments have shown that the zebrafish (Danio rerio) is a useful experimental model to evaluate the environmental impact of pollutants in the aquatic environment. It has been employed as an experimental species since the 1980s (Streisinger et al. 1981). In our laboratory, zebrafish have been used in the research of the toxicity of atrazine (Dong et al. 2009; Zhu et al. 2010). Additionally, the testing indexes contain SCGE, anti-oxidant enzymes, ROS and P450, among others. The potential impact of chlorpyrifos to aquatic ecosystems is considerable (David and Stuart 1998). Chlorpyrifos not only directly harms fish, but also indirectly produces bioaccumulation effects of the toxicant in different parts of the body (Rao et al. 2003). According to Levin et al. (2004), chlorpyrifos has an adverse impact on spatial discrimination learning and swimming activity. At the same time, it caused clearly detectable behavioral impairments in zebrafish. Sandah et al. (2005) indicated that there is also a close relationship between brain AChE inhibition and behavioral impairment due to exposure to chlorpyrifos in juvenile coho. The in vivo accumulation of chlorpyrifos would produce harmful effects in aquatic organisms and humans (Serrano et al. 1997). TCP is potentially harmful to organisms in the environment, although the environmental fate data on TCP are relatively scarce (Petty et al. 2001). Baskaran et al. (2003) found that, because the sorption of chlorpyrifos was about 100 times higher than that of TCP, TCP has a much greater leaching potential than does chlorpyrifos. Meeker et al. (2004, 2006) found that TCP may affect human male thyroid function and may be associated with DNA damage in human sperm. Oxidative stress is an important manifestation in fish. Dorval et al. (2003) found that the potential damage is caused by one of the following factors: the increase in ROS, the damage of antioxidant defense systems, or the inbility to repair oxidative damage. At present, the level of cytochrome P450 enzymes (CYPs) in fish is well-established as an induction model for environmental contamination biomonitoring and ecotoxicology assays (Li et al. 2008). Research on the toxicity of chlorpyrifos is common, but few studies have addressed the toxicity of TCP, which may be more toxic than chlorpyrifos. Therefore, the comparison of these two pollutants is necessary. Meanwhile, the zebrafish is rarely utilized as a model organism in chlorpyrifos toxicity studies. This research not only measured the toxicity of chlorpyrifos and TCP to enzymes, ROS generation and DNA damage in zebrafish, but also examined these indictors at the same time, which was rarely done in previous studies. The difference in the toxicity of chlorpyrifos and TCP was also definite, which will help to assess the toxicity of chlorpyrifos in the environment more accurately in the future.
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Materials and methods Chemicals Chlorpyrifos (97.8 % purity) and TCP (99.3 % purity) were purchased from U.S. Dima Inc. (New Haven, CT, USA). The compound 20 ,70 -dichlorofluorescin-diacetate (DCFH-DA) was purchased from Beyotime Institute of Biotechnology. All other chemicals and solvents of analytical purity were obtained from Sigma Chemical Co. and Shanghai Sangon Biological Engineering Technology and Service Co. Fish treatment and toxicity test The zebrafish (D. rerio) used in this study were bought from a tropical fish culture research institute. Adult males (mean body weight 0.27 ± 0.01 g and length 2.47 ± 0.03 cm) and females (mean body weight 0.33 ± 0.01 g and length 2.48 ± 0.02 cm) were used. The fish were acclimated in the laboratory for 2 weeks to ensure that they had no disease prior to the experiments. Males and females were housed separately in fish tanks. Both groups were exposed to the control and three different concentrations of chlorpyrifos (0.01, 0.1, 1 mg L-1) and TCP (0.01, 0.1, 0.5 mg L-1) and were sampled on days 5, 10, 15, 20 and 25. Approximately 200 zebrafish were allocated to each fish tank. Each sample was performed in triplicate. The fish water was dechlorinated under a constant day/night rhythm in large 40 L glass aquaria provided with a filter and continuously aerated. During the experimental period, a light: dark regimen of 12 h:12 h was appropriated for maintenance to ensure a constant temperature of 26 ± 1 °C; oxygen saturation exceeded 70 %; and pH values ranged from 7.4 to 8.1 (Diekmann and Hultsch 2004). The fish were fed twice a day with commercial fish food, but were starved for 24 h prior to testing to avoid the effects of feces in the course of the assay. Furthermore, 50 % of the fish water was changed every 2 days for the duration of the exposure period to keep the water clean and to maintain stable concentrations. Protein content The protein content was determined according to the Sigma Bradford method (Bradford 1976) using bovine serum albumin as a standard. The assay of cytochrome P450 enzymes Preparation of liver microsomes All procedures were performed at 4 °C using cold buffers and centrifuges to prevent breakdown of the enzymes. The
The enzyme toxicity and genotoxicity of chlorpyrifos
livers were promptly removed from the fish under ice-cold conditions and rinsed with ice-cold 0.15 M KCl to completely eliminate blood traces. Subsequently, the livers were homogenized in seven volumes of ice-cold homogenization buffer [0.1 M sodium phosphate buffer, pH 7.5, containing 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM dithiothreitol (DTT), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] supplemented with 10 % (v/v) glycerol using a glass-Teflon homogenizer. The obtained homogenate was first centrifuged at 13,000 g for 30 min at 4 °C, and the supernatant was centrifuged at 105,000 g for 1 h at 4 °C. The microsomal pellet was resuspended in the homogenization buffer supplemented with 20 % (v/v) glycerol for future use. Microsomal assays and enzyme activities The cytochrome P450 content was determined by means of a difference spectrum of dithionite-reduced CO according to the method described by Omura and Sato (1964a, b). The results were expressed in nmol mg-1 of protein with an extinction coefficient of 91 mM-1 cm-1 (450–490 nm). The NADPH-P450 reductase (NCR) activity was determined according to the method described by Williams and Kamin (1962) with slight modifications. The activity was calculated using an extinction coefficient of 19.1 mM-1 cm-1. The results were expressed in nmol mg-1 of protein.
522 nm using a fluorescence spectrophotometer (RF5301PC). Single-cell gell electrophoresis SCGE was performed according to the procedure described by Singh et al. (1988), with slight modifications. Approximately 50 cells per slide were randomly scored with an image analysis system attached to a fluorescent microscope (Olympus BX51) with appropriate filters. All steps had to be conducted under dim lighting to avoid additional nonspecific breakage of DNA. The Comet images were analyzed by the Comet Assay Software Project (CASP). The olive tail moment (OTM) was used to evaluate the extent of DNA damage, which is the product of the distance between the center of gravity of the head and the center of gravity of the tail and the percent of the DNA that is tail. Statistical analysis The data in this study were analyzed with the statistical package for social sciences (SPSS) program (Standard Version 11.5, SPSS Inc). Least significance difference (LSD) test was used to determine the differences between treatments and control. Analysis of variance (ANOVA) was employed to determine the differences between duration and concentrations. The probability level used for the statistical significance was p \ 0.01. All the values were presented as mean ± standard deviation (SD).
The assay of antioxidant enzymes The homogenate was centrifuged at 10,000 rpm at 4 °C for 10 min. The supernatant was used immediately for the assay of enzyme activity and protein determination. The SOD activity was determined by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium chloride (NBT), as described by Sun et al. (1988), with slight modification. One unit of SOD activity (U) was defined as the amount of enzyme required to cause 50 % inhibition of the NBT photoreduction rate, and the result was expressed as U mg-1 of protein. CAT activity was determined according to the method described by Xu et al. (1997), but with a slight modification. One unit of CAT activity was defined as the enzyme quantity required to consume half of the H2O2 in 100 s at 25 °C and was normalized to the protein concentration. The detection of ROS The ROS production was quantified by the DCFH-DA method (Lawler et al. 2003), with slight modification. Fluorescence of the samples was monitored at an excitation wavelength of 488 nm and an emission wavelength of
Results Effect of chlorpyrifos and TCP on cytochrome P450 content and NADPH-P450 reductase activity in zebrafish liver As shown in Fig. 1, with the same pollutants and the same corresponding concentrations, the P450 content in females was higher than in males. The P450 content first increased and then decreased with time. During the course of the experiment, the liver P450 content at all concentrations of chlorpyrifos and TCP treatment was higher than that observed in the controls in both male and female zebrafish. The greatest induction of P450 content was in the case of the 0.1 mg L-1 chlorpyrifos treatment. Compared to the control, the induction of chlorpyrifos and TCP on liver P450 content can be seen in the middle stage of exposure (10, 15 and 20 days). The P450 content reached a maximum at 15 and 10 days when induced by chlorpyrifos and TCP, respectively. When exposed continuously for 25 days, the P450 content was somewhat lower, and the induction showed a slower trend. For the zebrafish of the same sex, the P450 content was affected more by TCP than by chlorpyrifos at the
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Fig. 1 Effect of chlorpyrifos on the cytochrome P450 content of female (a) and male (b) zebrafish. Effect of TCP on the cytochrome P450 content of female (c) and male (d) zebrafish. Pr is the abbreviation of protein. Each bar is the mean of three replicates.
Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
corresponding concentrations. For example, in the 0.1 mg L-1 treatment, the P450 content of chlorpyrifos for both the male and female fish reached the maximum value at 15 days; the content values were 2.94- and 3.38-fold higher, respectively, when compared with the controls. For TCP, the P450 content concentration reached the maximum value at 10 days, and the content values were 3.82- and 5.18-fold higher than the controls, respectively. As shown in Fig. 2, through the experiment, the NCR activity of male and female fish treated with various concentrations was always higher than the control. During exposure, chlorpyrifos and TCP induced the NCR activity significantly. Meanwhile, the females showed higher NCR activity than did the males when exposed to the same pollutants and the same corresponding concentration treatments.
For females treated with chlorpyrifos, the NRC activity reached a maximum at 15 days. The content values were 2.77-, 2.95- and 2.48-fold higher than the control. When treated with TCP, the value went to a maximum at 20 days. The content values were 3.37-, 3.55- and 2.75-fold higher than the control. For males treated with chlorpyrifos, the NRC activity reached a maximum at 20 days. The content values were 1.74-, 2.02- and 1.71-fold higher than the control. The maximum was reached at 15 days when they were treated with TCP. The content values were 2.82-, 4.41- and 2.28fold higher than the control. Effect of chlorpyrifos and TCP on anti-oxidative enzymes activity in zebrafish liver. It can be seen from Fig. 3 that the SOD activity at approximately 1 mg L-1 of chlorpyrifos and 0.5 mg L-1 of TCP first increased and
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Fig. 2 Effect of chlorpyrifos on the NADPH-P450 reductase activity of female (a) and male (b) zebrafish. Effect of TCP on the NADPHP450 reductase activity of female (c) and male (d) zebrafish. Pr is the abbreviation of protein. Each bar is the mean of three replicates.
Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
then decreased consistently over time. In addition, the other groups also showed an increase. During exposure, chlorpyrifos and TCP obviously induced the SOD activity. Some sex differences of SOD in zebrafish were also observed. The SOD activity of the females was higher than that of males when exposed to the same pollutants and the same corresponding concentrations. For females that were treated with 0.01 mg L-1 chlorpyrifos and TCP, the SOD activity reached a maximum at 25 days. The content values were 1.78- and 3.09-fold higher than the control, respectively. The SOD activity of those treated with either chlorpyrifos or TCP at 0.1 mg L-1 also reached a maximum at 25 days. The content values were 2.71- and 3.80-fold higher than the control, respectively. Those treated with chlorpyrifos at 1 mg L-1 showed a maximum SOD activity at 20 days, while those treated with TCP at 0.5 mg L-1 showed a maximum at 15 days.
The content values were 3.97- and 4.35-fold higher than the control, respectively. For males treated with 0.01 mg L-1 chlorpyrifos and TCP, the SOD activity reached a maximum at 25 days. The content values were 3.18- and 5.43-fold higher than the control, respectively. The SOD activity of those treated with 0.1 mg L-1 chlorpyrifos and TCP also showed a maximum at 25 days. The content values were 5.16- and 6.22-fold higher than the control, respectively. Those that were treated with chlorpyrifos at 1 mg L-1 showed a maximum at 25 days, while those that were treated with TCP at 0.5 mg L-1 showed a maximum at 15 days. The content values were 5.12- and 7.65-fold higher than the control, respectively. It can be seen in Fig. 4 that the CAT activity induced by 0.01 and 0.5 mg L-1 chlorpyrifos increased with time. The other groups first showed an increase and then a consistent
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Fig. 3 Effect of chlorpyrifos on the SOD activity of female (a) and male (b) zebrafish. Effect of TCP on the SOD activity of female (c) and male (d) zebrafish. Pr is the abbreviation for protein. Each
bar is the mean of three replicates. Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
decrease over time. During exposure, chlorpyrifos and TCP clearly induced the effect on CAT activity. The CAT activity of the male and female fish that were treated by chlorpyrifos and TCP reflected some sex differences; the CAT activity of the females was higher than that of the males when exposed to the same pollutants at corresponding concentrations. For females that were treated with 0.01 mg L-1 chlorpyrifos and TCP, the CAT activity reached a maximum at 25 days. The content values were 2.86- and 3.34-fold higher than the control, respectively. The CAT activity at 0.1 mg L-1 was maximized at 25 days as well. The content values were 3.36- and 4.40-fold higher than the control, respectively. The fish that were treated with chlorpyrifos at 1 mg L-1 showed a maximum at 20 days, while those that were treated with TCP at 0.5 mg L-1 had a maximum at 25 days. The content values were 2.24- and 5.56-fold higher than the control. For males that were treated with 0.01 mg L-1 chlorpyrifos and TCP, the CAT activity was maximized at 25 days. The
content values were 3.12- and 3.67-fold higher than the control. The CAT activity at 0.1 mg L-1 also reached a maximum at 25 days. The content values were 3.68- and 4.18-fold higher than the control. The fish that were treated with 1 mg L-1 chlorpyrifos and 0.5 mg L-1 TCP showed a maximum CAT activity at 15 days. The content values were 2.61- and 3.07-fold higher compared with the control.
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Effect of chlorpyrifos and TCP on ROS As shown in Fig. 5, the ROS levels of the fish that were treated with 0.01 and 0.1 mg L-1 chlorpyrifos and TCP had a tendency to increase. Compared with the control group, the ROS levels of the fish that were exposed to other concentrations were higher and significantly different. It can be seen that the ROS levels of the zebrafish were significantly activated by chlorpyrifos and TCP. The chlorpyrifos and TCP in the male and female fish also reflected some sex differences, where the ROS levels of the females were higher than those of the males.
The enzyme toxicity and genotoxicity of chlorpyrifos
Fig. 4 Effect of chlorpyrifos on the CAT activity of female (a) and male (b) zebrafish. Effect of TCP on the CAT activity of female (c) and male (d) zebrafish. Pr is the abbreviation for protein. Each
bar is the mean of three replicates. Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
For zebrafish of the same sex, the ROS levels were affected more by the TCP than by the chlorpyrifos under corresponding conditions (0.01 mg L-1 chlorpyrifos and 0.01 mg L-1 TCP; 0.1 mg L-1 chlorpyrifos and 0.1 mg L-1 TCP; 1 mg L-1 chlorpyrifos and 0.5 mg L-1 TCP).
concentration, the DNA damage was greater for females than for males, and the DNA of cells affected by TCP had a higher comet OTM than did the DNA of cells affected by chlorpyrifos.
Discussion DNA Damage Induced by chlorpyrifos and TCP The cell viability following exposure was consistently 85 % for all treatments, which made it possible to perform SCGE. In the SCGE, the DNA migration of the cells was found to increase with an increase of chlorpyrifos and TCP concentration, independent of the sampling time. As shown in Fig. 6, the treatments with chlorpyrifos and TCP caused damage to the DNA of zebrafish. A significant increase in the OTM could be observed with increasing chlorpyrifos (0.01, 0.1, 1 mg L-1) and TCP concentration (0.01, 0.1, 0.5 mg L-1) on days 5, 10, 15, 20 and 25 in both males and females. When treated with the same
The aim of this study was to get a more comprehensive understanding of the toxicity of chlorpyrifos and its metabolite TCP and to explore the toxicity and the potential ecological risk of this pesticide on the aquatic ecosystem. Our data show that, for a given sex of fish and concentration of pollutant, the cytochrome P450 content and the NCR, ROS, CAT and SOD activities were more sensitive to TCP than to chlorpyrifos. As a result, the impact of TCP was more intense in zebrafish. Meanwhile, DNA damage showed the same phenomenon. We can arrive at the preliminary conclusion that TCP has greater
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Fig. 5 Effect of chlorpyrifos on the ROS level of female (a) and male (b) zebrafish. Effect of TCP on the ROS level of female (c) and male (d) zebrafish. Pr is the abbreviation for protein. Each bar is the mean
of three replicates. Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
genotoxicity than does chlorpyrifos to zebrafish. Because of this increased toxicity, this topic will require continued attention and further study.
long intervals. The reason for the return to basal levels could be that the organism has some adaptive responses or was damaged. Data in our study showed that chlorpyrifos and TCP treatments could induce a significant increase of SOD and CAT activity. This may be attributed to fact that the chlorpyrifos and TCP induced ROS generation, and SOD and CAT are the major ROS-scavenging mechanisms (Anderson et al. 1995). To keep the ROS production at steady-state concentration, the activities of CAT and SOD increased to eliminate the redundant ROS (Mittler 2002). However, compared to the low chlorpyrifos and TCP concentration, some high concentrations of chlorpyrifos and TCP (for example, CAT at 1 mg L-1 chlorpyrifos in females and at 0.5 mg L-1 TCP in males) could induce a significant decrease in the activities of SOD and CAT. This phenomenon can be interpreted as the excessive chlorpyrifos and TCP inducing the superfluous generation of ROS. ROS are capable of unrestricted oxidation of various cellular components and can lead to oxidative destruction of the cell (Mittler 2002) or can cause the anti-oxidative
Oxidative stress At present, cytochrome P450 enzymes (CYPs) in fish are a useful tool that is used in environmental contamination biomonitoring and ecotoxicology assays for xenobiotic metabolism (Li et al. 2008). Our experiments showed that the P450 content in zebrafish was impacted by chlorpyrifos and TCP in both males and females across all treatment concentrations; the trend of the stimulation showed an initial increase followed by a decrease over time. NCR is an electron donor to CYP, so its alteration would probably affect the functioning of the monooxygenase system (Dong et al. 2009). Our results demonstrated that the NCR activity was affected by the CYPs. The observed effects can be explained by the role of increased P450 enzyme activities in detoxification and the ultimate elimination of chlorpyrifos and TCP. The effects were found to decrease at
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Fig. 6 Effect of chlorpyrifos on the liver comet olive tails moment of female (a) and male (b) zebrafish. Effect of TCP on the liver comet olive tails moment of female (c) and male (d) zebrafish. Each bar is
the mean of three replicates. Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
capacity of the organism to be exceeded, leaving the enzymes exhausted (Wang et al. 2006). ROS are generated in all aerobic organisms during normal respiratory metabolism (Schnabel et al. 2006) and include a series of chemically reactive molecules derived from oxygen. ROS are known to affect cells by directly oxidizing and damaging diverse cellular components (e.g., proteins, lipids, and DNA) in different ways that can affect their function (Bokov and Chaudhuri 2004). The major ROS are superoxide, nitric oxide, hydroxyl radical, hydroperoxide, and peroxynitrite. The intracellular concentration of ROS can be modulated by controlling the sites and amounts of their synthesis, or it can be inactivated with antioxidant molecules and repair mechanisms directly. We can study the ROS by measuring the activity of different antioxidant enzymes, such as SOD, CAT, and peroxidases (Brandes and Janiszewski 2005). This study showed that the concentration of 0.01 mg L-1 chlorpyrifos did not induce a significant increase of ROS levels in zebrafish, which was different from the effects of TCP. This may be due to the effect of anti-oxidative enzymes,
which can prevent the increase of noxious ROS and keep the ROS at a normal level. Meanwhile, a significant increase of ROS in the liver of fish occurred at the other concentrations. This result is similar to many previous studies and can be interpreted as TCP and higher concentrations of chlorpyrifos acting as highly toxic external sources of pollutants, which causes ROS to be generated at such a high rate that they exceed the capacity of the organisms to eliminate it. The action of parental compounds or their metabolites may damage membrane lipids, DNA, proteins and carbohydrates directly, and the generation of ROS may result in the same damage indirectly (Oliveira et al. 2009). SCGE, which is widely used to evaluate the genotoxicity of chemicals in the environment, was used to assess the toxic effects on zebrafish embryos and liver (Deventer 1996; Sun et al., 2004; Kosmehl et al. 2008; Seitz et al. 2008). Therefore, we use SCGE as an index to evaluate the genotoxicity of chlorpyrifos and TCP. Our results showed that the DNA damage in the zebrafish hepatopancreas increased with increasing chlorpyrifos and TCP
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concentrations. In our laboratory, Zhu et al. (2010) found the same regularity when zebrafish were treated with 0.01–1 mg L-1 atrazine. According to Liu et al. (2009), 0.1–10 mg kg-1 endosulfan also has significant genotoxicity to the earthworm. Experiments indicate that chlorpyrifos and TCP-induced DNA damage in zebrafish and the levels of DNA damage imposed by chlorpyrifos and TCP could be quantified by SCGE. Few studies have been reported that deal with the mechanism of genotoxicity of chlorpyrifos. In previous reports, reasons for the genotoxicity of organophosphorous compounds, which would include chlorpyrifos, include alkylation (Rahman et al. 2002), phosphorylation and oxidative stress (Pin˜a-Guzma´n et al. 2006; Sharma et al. 2006). Alkylation can attack DNA bases and proteins, including single- and double-DNA strand breaks, crosslinks, chromosomal aberrations, and DNA base oxidation (Pin˜a-Guzma´n et al. 2006). Oxidative stress was related to chromatin cross-linking, DNA strand breaks, DNA base oxidation, chromosomal aberrations (Possamai et al. 2007), and lipid peroxidation (Debnath and Mandal 2000). Furthermore, the setting of the chlorpyrifos and TCP concentration was done according to the natural residual quantity in the natural environment, especially in the aquatic environment. Therefore, this research can provide help for future studies on chlorpyrifos toxicity in the natural aquatic environment.
reliable early-warning indicators of environmental pollution. According to this study, CYPs and SCGE are more sensitive than antioxidant enzyme activity and ROS generation. Chlorpyrifos and TCP can induce excess ROS generation, which could be eliminated through the increased activity of anti-oxidative enzymes (CAT, SOD). According to (Contardo-Jara et al., 2009), because the antioxidant enzymes (CAT, SOD) studied here are part of a vital defense mechanism against ROS-induced tissue damage, the increased production of ROS with a concomitant reduction of antioxidant enzyme activities could lead to oxidative stress, including enzyme inactivation, protein degradation, DNA damage and lipid peroxidation. Many other studies also showed that the DNA strand breaks are mainly attributable to ROS (O’Brien et al. 2003; Luo et al. 1996; Casadevall et al. 1999). Conversely, the DNA damage and the activity decrease of the antioxidant enzyme could induce the ROS generation. The study of the interactions among the four indexes could provide a basic theory to the future study of the toxicity mechanism of chlorpyrifos and TCP in organisms.
Conclusions (1)
Sex differences Many factors, such as sex, reproductive phase, developmental stage and diet, can affect enzyme activity. Sexbased differences in zebrafish are mainly regulated by hormones (Anderson et al. 1995). Data from the literature demonstrates that contradictory results indeed exist in fish species. Our study showed that, in control zebrafish, cytochrome P450 content and NCR, ROS, CAT and SOD activities in females are higher than in males, as is the degree of DNA damage. This is in accordance with the reported results of previous studies performed by Otte et al. (2008) and Sole´ et al. (2002). In our study, sex differences were observed in the chlorpyrifos- and TCP-induced enzyme activity and DNA damage. Additionally, because females are more susceptible than males to changes in enzyme activity and DNA damage following chlorpyrifos and TCP treatment and to ensure the results are clear and accurate, we can choose the female zebrafish as a biological indicator to test the pollution. Relationship This study indicated that the CYPs, antioxidant enzyme activity, ROS generation and DNA damage level are all
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(2)
(3)
(4)
(5)
By comparing the CYPs, DNA damage, and CAT, SOD and ROS activities induced by the same concentrations of chlorpyrifos and TCP in the zebrafish, we can see that the toxicity of TCP was more serious than that of chlorpyrifos. Chlorpyrifos and TCP significantly stimulate cytochrome P450 enzymes in zebrafish, even at a concentration as low as 0.01 mg L-1. Because of their sensitivity to low chlorpyrifos and TCP concentrations, CYPs and SCGE could be potential biomarkers to evaluate the effect of exposure and could serve as early-warning signals of aquatic contamination. CAT, SOD and ROS generation is also affected by the chlorpyrifos and TCP exposure. They generally increased after exposure to low concentrations of chlorpyrifos and TCP, but decreased significantly after exposure to high chlorpyrifos and TCP concentrations. Chlorpyrifos and TCP can induce significant DNA damage in zebrafish. The DNA damage was enhanced as the chlorpyrifos and TCP concentrations increased, with a clear dose–response relationship. The biotransformation activities caused by chlorpyrifos and TCP show different levels between male and female zebrafish. Generally, the value for females was larger than for males. However, after exposure to the
The enzyme toxicity and genotoxicity of chlorpyrifos
pollutants, the change regularity of these indictors was similar between males and females. Acknowledgments This study was supported by grants from the National Science Foundation of China (No. 41071164, 21277083, 21377075, and 41001152) and the Specialized Research Fund for the Doctoral Program of Higher Education (20113702110007). Conflict of interest of interest.
The authors declare that they have no conflict
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The enzyme toxicity and genotoxicity of chlorpyrifos and its toxic metabolite TCP to zebrafish Danio rerio Jun Wang • Jinhua Wang • Lusheng Zhu Hui Xie • Bo Shao • Xinxin Hou
•
Accepted: 9 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Chlorpyrifos is a broad-spectrum organophosphorus insecticide (O,O-diethyl -O-3,5,6-trichloro-2-pyridyl phosphorothioate) that is used in numerous agricultural and urban pest controls. The primary metabolite of chlorpyrifos is 3,5,6-trichloro pyridine-2-phenol (TCP). Because of its strong water solubility and mobility, this harmful metabolite exists in the environment in a large amount. Although TCP has potentially harmful effects on organisms in the environment, few studies have addressed TCP pollution. Therefore, this study was undertaken to investigate the effect of chlorpyrifos and TCP on the microsomal cytochrome P450 content in the liver, on the activity of NADPH-P450 reductase and antioxidative enzymes [catalase (CAT) and superoxide dismutase (SOD)], and on reactive oxygen species (ROS) generation and DNA damage in zebrafish. Male and female zebrafish were separated and exposed to a control solution and three concentrations of chlorpyrifos (0.01, 0.1, 1 mg L-1) and TCP (0.01, 0.1, 0.5 mg L-1), respectively, sampled after 5, 10, 15, 20 and 25 days. The results indicated that the P450 content and the NADPH-P450 reductase and antioxidative enzyme (CAT and SOD) activities could be induced by chlorpyrifos and TCP. DNA damage of zebrafish was enhanced with increasing chlorpyrifos and TCP concentrations. Meanwhile, chlorpyrifos and TCP induced a significant increase of ROS generation in the zebrafish
J. Wang J. Wang (&) L. Zhu (&) H. Xie B. Shao X. Hou Key Laboratory of Agricultural Environment in the University of Shandong, College of Resources and Environment, Shandong Agriculture University, 61 Daizong Road, Taian 271018, China e-mail: [email protected] L. Zhu e-mail: [email protected]; [email protected]
hepatopancreas. In conclusion, this study proved that chlorpyrifos (0.01–1 mg L-1) and TCP (0.01–0.5 mg L-1) are both highly toxic to zebrafish. Keywords Cytochrome P450 NADPH-P450 reductase SOD CAT ROS SCGE
Introduction Chlorpyrifos is a broad-spectrum organophosphorus insecticide (O,O-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothioate) with numerous agricultural crop and urban pest control uses (David and Stuart 1998). Currently, chlorpyrifos is used worldwide as a substitute for some highly toxic pesticides, such as methamidophos and methyl parathion. During recent years, many countries have recognized the hazards of chlorpyrifos and have gradually restricted or banned their use. According to David and Stuart (1998), chlorpyrifos has been in priority lists of pesticides within the European Economic Community. It is also one of the 12 major environmental pollutants in Japan (Gamo et al. 2003). However, chlorpyrifos continues to be one of the most commonly used organophosphorus insecticides. The primary metabolite of chlorpyrifos is TCP, and its strong water solubility and high mobility results in significant amounts of harmful TCP residue in the soil and water. The aim of this study was to gain a more comprehensive understanding of the effects of chlorpyrifos and its metabolite TCP to the aquatic ecosystem. Moreover, the sensitivity of many different biomarkers was also tested in this study. In aquatic ecosystems, many species of animals, including fish, have been widely studied to reveal the toxicology of chlorpyrifos. So far, especially in recent
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years, experiments have shown that the zebrafish (Danio rerio) is a useful experimental model to evaluate the environmental impact of pollutants in the aquatic environment. It has been employed as an experimental species since the 1980s (Streisinger et al. 1981). In our laboratory, zebrafish have been used in the research of the toxicity of atrazine (Dong et al. 2009; Zhu et al. 2010). Additionally, the testing indexes contain SCGE, anti-oxidant enzymes, ROS and P450, among others. The potential impact of chlorpyrifos to aquatic ecosystems is considerable (David and Stuart 1998). Chlorpyrifos not only directly harms fish, but also indirectly produces bioaccumulation effects of the toxicant in different parts of the body (Rao et al. 2003). According to Levin et al. (2004), chlorpyrifos has an adverse impact on spatial discrimination learning and swimming activity. At the same time, it caused clearly detectable behavioral impairments in zebrafish. Sandah et al. (2005) indicated that there is also a close relationship between brain AChE inhibition and behavioral impairment due to exposure to chlorpyrifos in juvenile coho. The in vivo accumulation of chlorpyrifos would produce harmful effects in aquatic organisms and humans (Serrano et al. 1997). TCP is potentially harmful to organisms in the environment, although the environmental fate data on TCP are relatively scarce (Petty et al. 2001). Baskaran et al. (2003) found that, because the sorption of chlorpyrifos was about 100 times higher than that of TCP, TCP has a much greater leaching potential than does chlorpyrifos. Meeker et al. (2004, 2006) found that TCP may affect human male thyroid function and may be associated with DNA damage in human sperm. Oxidative stress is an important manifestation in fish. Dorval et al. (2003) found that the potential damage is caused by one of the following factors: the increase in ROS, the damage of antioxidant defense systems, or the inbility to repair oxidative damage. At present, the level of cytochrome P450 enzymes (CYPs) in fish is well-established as an induction model for environmental contamination biomonitoring and ecotoxicology assays (Li et al. 2008). Research on the toxicity of chlorpyrifos is common, but few studies have addressed the toxicity of TCP, which may be more toxic than chlorpyrifos. Therefore, the comparison of these two pollutants is necessary. Meanwhile, the zebrafish is rarely utilized as a model organism in chlorpyrifos toxicity studies. This research not only measured the toxicity of chlorpyrifos and TCP to enzymes, ROS generation and DNA damage in zebrafish, but also examined these indictors at the same time, which was rarely done in previous studies. The difference in the toxicity of chlorpyrifos and TCP was also definite, which will help to assess the toxicity of chlorpyrifos in the environment more accurately in the future.
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Materials and methods Chemicals Chlorpyrifos (97.8 % purity) and TCP (99.3 % purity) were purchased from U.S. Dima Inc. (New Haven, CT, USA). The compound 20 ,70 -dichlorofluorescin-diacetate (DCFH-DA) was purchased from Beyotime Institute of Biotechnology. All other chemicals and solvents of analytical purity were obtained from Sigma Chemical Co. and Shanghai Sangon Biological Engineering Technology and Service Co. Fish treatment and toxicity test The zebrafish (D. rerio) used in this study were bought from a tropical fish culture research institute. Adult males (mean body weight 0.27 ± 0.01 g and length 2.47 ± 0.03 cm) and females (mean body weight 0.33 ± 0.01 g and length 2.48 ± 0.02 cm) were used. The fish were acclimated in the laboratory for 2 weeks to ensure that they had no disease prior to the experiments. Males and females were housed separately in fish tanks. Both groups were exposed to the control and three different concentrations of chlorpyrifos (0.01, 0.1, 1 mg L-1) and TCP (0.01, 0.1, 0.5 mg L-1) and were sampled on days 5, 10, 15, 20 and 25. Approximately 200 zebrafish were allocated to each fish tank. Each sample was performed in triplicate. The fish water was dechlorinated under a constant day/night rhythm in large 40 L glass aquaria provided with a filter and continuously aerated. During the experimental period, a light: dark regimen of 12 h:12 h was appropriated for maintenance to ensure a constant temperature of 26 ± 1 °C; oxygen saturation exceeded 70 %; and pH values ranged from 7.4 to 8.1 (Diekmann and Hultsch 2004). The fish were fed twice a day with commercial fish food, but were starved for 24 h prior to testing to avoid the effects of feces in the course of the assay. Furthermore, 50 % of the fish water was changed every 2 days for the duration of the exposure period to keep the water clean and to maintain stable concentrations. Protein content The protein content was determined according to the Sigma Bradford method (Bradford 1976) using bovine serum albumin as a standard. The assay of cytochrome P450 enzymes Preparation of liver microsomes All procedures were performed at 4 °C using cold buffers and centrifuges to prevent breakdown of the enzymes. The
The enzyme toxicity and genotoxicity of chlorpyrifos
livers were promptly removed from the fish under ice-cold conditions and rinsed with ice-cold 0.15 M KCl to completely eliminate blood traces. Subsequently, the livers were homogenized in seven volumes of ice-cold homogenization buffer [0.1 M sodium phosphate buffer, pH 7.5, containing 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM dithiothreitol (DTT), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] supplemented with 10 % (v/v) glycerol using a glass-Teflon homogenizer. The obtained homogenate was first centrifuged at 13,000 g for 30 min at 4 °C, and the supernatant was centrifuged at 105,000 g for 1 h at 4 °C. The microsomal pellet was resuspended in the homogenization buffer supplemented with 20 % (v/v) glycerol for future use. Microsomal assays and enzyme activities The cytochrome P450 content was determined by means of a difference spectrum of dithionite-reduced CO according to the method described by Omura and Sato (1964a, b). The results were expressed in nmol mg-1 of protein with an extinction coefficient of 91 mM-1 cm-1 (450–490 nm). The NADPH-P450 reductase (NCR) activity was determined according to the method described by Williams and Kamin (1962) with slight modifications. The activity was calculated using an extinction coefficient of 19.1 mM-1 cm-1. The results were expressed in nmol mg-1 of protein.
522 nm using a fluorescence spectrophotometer (RF5301PC). Single-cell gell electrophoresis SCGE was performed according to the procedure described by Singh et al. (1988), with slight modifications. Approximately 50 cells per slide were randomly scored with an image analysis system attached to a fluorescent microscope (Olympus BX51) with appropriate filters. All steps had to be conducted under dim lighting to avoid additional nonspecific breakage of DNA. The Comet images were analyzed by the Comet Assay Software Project (CASP). The olive tail moment (OTM) was used to evaluate the extent of DNA damage, which is the product of the distance between the center of gravity of the head and the center of gravity of the tail and the percent of the DNA that is tail. Statistical analysis The data in this study were analyzed with the statistical package for social sciences (SPSS) program (Standard Version 11.5, SPSS Inc). Least significance difference (LSD) test was used to determine the differences between treatments and control. Analysis of variance (ANOVA) was employed to determine the differences between duration and concentrations. The probability level used for the statistical significance was p \ 0.01. All the values were presented as mean ± standard deviation (SD).
The assay of antioxidant enzymes The homogenate was centrifuged at 10,000 rpm at 4 °C for 10 min. The supernatant was used immediately for the assay of enzyme activity and protein determination. The SOD activity was determined by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium chloride (NBT), as described by Sun et al. (1988), with slight modification. One unit of SOD activity (U) was defined as the amount of enzyme required to cause 50 % inhibition of the NBT photoreduction rate, and the result was expressed as U mg-1 of protein. CAT activity was determined according to the method described by Xu et al. (1997), but with a slight modification. One unit of CAT activity was defined as the enzyme quantity required to consume half of the H2O2 in 100 s at 25 °C and was normalized to the protein concentration. The detection of ROS The ROS production was quantified by the DCFH-DA method (Lawler et al. 2003), with slight modification. Fluorescence of the samples was monitored at an excitation wavelength of 488 nm and an emission wavelength of
Results Effect of chlorpyrifos and TCP on cytochrome P450 content and NADPH-P450 reductase activity in zebrafish liver As shown in Fig. 1, with the same pollutants and the same corresponding concentrations, the P450 content in females was higher than in males. The P450 content first increased and then decreased with time. During the course of the experiment, the liver P450 content at all concentrations of chlorpyrifos and TCP treatment was higher than that observed in the controls in both male and female zebrafish. The greatest induction of P450 content was in the case of the 0.1 mg L-1 chlorpyrifos treatment. Compared to the control, the induction of chlorpyrifos and TCP on liver P450 content can be seen in the middle stage of exposure (10, 15 and 20 days). The P450 content reached a maximum at 15 and 10 days when induced by chlorpyrifos and TCP, respectively. When exposed continuously for 25 days, the P450 content was somewhat lower, and the induction showed a slower trend. For the zebrafish of the same sex, the P450 content was affected more by TCP than by chlorpyrifos at the
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Fig. 1 Effect of chlorpyrifos on the cytochrome P450 content of female (a) and male (b) zebrafish. Effect of TCP on the cytochrome P450 content of female (c) and male (d) zebrafish. Pr is the abbreviation of protein. Each bar is the mean of three replicates.
Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
corresponding concentrations. For example, in the 0.1 mg L-1 treatment, the P450 content of chlorpyrifos for both the male and female fish reached the maximum value at 15 days; the content values were 2.94- and 3.38-fold higher, respectively, when compared with the controls. For TCP, the P450 content concentration reached the maximum value at 10 days, and the content values were 3.82- and 5.18-fold higher than the controls, respectively. As shown in Fig. 2, through the experiment, the NCR activity of male and female fish treated with various concentrations was always higher than the control. During exposure, chlorpyrifos and TCP induced the NCR activity significantly. Meanwhile, the females showed higher NCR activity than did the males when exposed to the same pollutants and the same corresponding concentration treatments.
For females treated with chlorpyrifos, the NRC activity reached a maximum at 15 days. The content values were 2.77-, 2.95- and 2.48-fold higher than the control. When treated with TCP, the value went to a maximum at 20 days. The content values were 3.37-, 3.55- and 2.75-fold higher than the control. For males treated with chlorpyrifos, the NRC activity reached a maximum at 20 days. The content values were 1.74-, 2.02- and 1.71-fold higher than the control. The maximum was reached at 15 days when they were treated with TCP. The content values were 2.82-, 4.41- and 2.28fold higher than the control. Effect of chlorpyrifos and TCP on anti-oxidative enzymes activity in zebrafish liver. It can be seen from Fig. 3 that the SOD activity at approximately 1 mg L-1 of chlorpyrifos and 0.5 mg L-1 of TCP first increased and
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Fig. 2 Effect of chlorpyrifos on the NADPH-P450 reductase activity of female (a) and male (b) zebrafish. Effect of TCP on the NADPHP450 reductase activity of female (c) and male (d) zebrafish. Pr is the abbreviation of protein. Each bar is the mean of three replicates.
Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
then decreased consistently over time. In addition, the other groups also showed an increase. During exposure, chlorpyrifos and TCP obviously induced the SOD activity. Some sex differences of SOD in zebrafish were also observed. The SOD activity of the females was higher than that of males when exposed to the same pollutants and the same corresponding concentrations. For females that were treated with 0.01 mg L-1 chlorpyrifos and TCP, the SOD activity reached a maximum at 25 days. The content values were 1.78- and 3.09-fold higher than the control, respectively. The SOD activity of those treated with either chlorpyrifos or TCP at 0.1 mg L-1 also reached a maximum at 25 days. The content values were 2.71- and 3.80-fold higher than the control, respectively. Those treated with chlorpyrifos at 1 mg L-1 showed a maximum SOD activity at 20 days, while those treated with TCP at 0.5 mg L-1 showed a maximum at 15 days.
The content values were 3.97- and 4.35-fold higher than the control, respectively. For males treated with 0.01 mg L-1 chlorpyrifos and TCP, the SOD activity reached a maximum at 25 days. The content values were 3.18- and 5.43-fold higher than the control, respectively. The SOD activity of those treated with 0.1 mg L-1 chlorpyrifos and TCP also showed a maximum at 25 days. The content values were 5.16- and 6.22-fold higher than the control, respectively. Those that were treated with chlorpyrifos at 1 mg L-1 showed a maximum at 25 days, while those that were treated with TCP at 0.5 mg L-1 showed a maximum at 15 days. The content values were 5.12- and 7.65-fold higher than the control, respectively. It can be seen in Fig. 4 that the CAT activity induced by 0.01 and 0.5 mg L-1 chlorpyrifos increased with time. The other groups first showed an increase and then a consistent
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Fig. 3 Effect of chlorpyrifos on the SOD activity of female (a) and male (b) zebrafish. Effect of TCP on the SOD activity of female (c) and male (d) zebrafish. Pr is the abbreviation for protein. Each
bar is the mean of three replicates. Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
decrease over time. During exposure, chlorpyrifos and TCP clearly induced the effect on CAT activity. The CAT activity of the male and female fish that were treated by chlorpyrifos and TCP reflected some sex differences; the CAT activity of the females was higher than that of the males when exposed to the same pollutants at corresponding concentrations. For females that were treated with 0.01 mg L-1 chlorpyrifos and TCP, the CAT activity reached a maximum at 25 days. The content values were 2.86- and 3.34-fold higher than the control, respectively. The CAT activity at 0.1 mg L-1 was maximized at 25 days as well. The content values were 3.36- and 4.40-fold higher than the control, respectively. The fish that were treated with chlorpyrifos at 1 mg L-1 showed a maximum at 20 days, while those that were treated with TCP at 0.5 mg L-1 had a maximum at 25 days. The content values were 2.24- and 5.56-fold higher than the control. For males that were treated with 0.01 mg L-1 chlorpyrifos and TCP, the CAT activity was maximized at 25 days. The
content values were 3.12- and 3.67-fold higher than the control. The CAT activity at 0.1 mg L-1 also reached a maximum at 25 days. The content values were 3.68- and 4.18-fold higher than the control. The fish that were treated with 1 mg L-1 chlorpyrifos and 0.5 mg L-1 TCP showed a maximum CAT activity at 15 days. The content values were 2.61- and 3.07-fold higher compared with the control.
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Effect of chlorpyrifos and TCP on ROS As shown in Fig. 5, the ROS levels of the fish that were treated with 0.01 and 0.1 mg L-1 chlorpyrifos and TCP had a tendency to increase. Compared with the control group, the ROS levels of the fish that were exposed to other concentrations were higher and significantly different. It can be seen that the ROS levels of the zebrafish were significantly activated by chlorpyrifos and TCP. The chlorpyrifos and TCP in the male and female fish also reflected some sex differences, where the ROS levels of the females were higher than those of the males.
The enzyme toxicity and genotoxicity of chlorpyrifos
Fig. 4 Effect of chlorpyrifos on the CAT activity of female (a) and male (b) zebrafish. Effect of TCP on the CAT activity of female (c) and male (d) zebrafish. Pr is the abbreviation for protein. Each
bar is the mean of three replicates. Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
For zebrafish of the same sex, the ROS levels were affected more by the TCP than by the chlorpyrifos under corresponding conditions (0.01 mg L-1 chlorpyrifos and 0.01 mg L-1 TCP; 0.1 mg L-1 chlorpyrifos and 0.1 mg L-1 TCP; 1 mg L-1 chlorpyrifos and 0.5 mg L-1 TCP).
concentration, the DNA damage was greater for females than for males, and the DNA of cells affected by TCP had a higher comet OTM than did the DNA of cells affected by chlorpyrifos.
Discussion DNA Damage Induced by chlorpyrifos and TCP The cell viability following exposure was consistently 85 % for all treatments, which made it possible to perform SCGE. In the SCGE, the DNA migration of the cells was found to increase with an increase of chlorpyrifos and TCP concentration, independent of the sampling time. As shown in Fig. 6, the treatments with chlorpyrifos and TCP caused damage to the DNA of zebrafish. A significant increase in the OTM could be observed with increasing chlorpyrifos (0.01, 0.1, 1 mg L-1) and TCP concentration (0.01, 0.1, 0.5 mg L-1) on days 5, 10, 15, 20 and 25 in both males and females. When treated with the same
The aim of this study was to get a more comprehensive understanding of the toxicity of chlorpyrifos and its metabolite TCP and to explore the toxicity and the potential ecological risk of this pesticide on the aquatic ecosystem. Our data show that, for a given sex of fish and concentration of pollutant, the cytochrome P450 content and the NCR, ROS, CAT and SOD activities were more sensitive to TCP than to chlorpyrifos. As a result, the impact of TCP was more intense in zebrafish. Meanwhile, DNA damage showed the same phenomenon. We can arrive at the preliminary conclusion that TCP has greater
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Fig. 5 Effect of chlorpyrifos on the ROS level of female (a) and male (b) zebrafish. Effect of TCP on the ROS level of female (c) and male (d) zebrafish. Pr is the abbreviation for protein. Each bar is the mean
of three replicates. Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
genotoxicity than does chlorpyrifos to zebrafish. Because of this increased toxicity, this topic will require continued attention and further study.
long intervals. The reason for the return to basal levels could be that the organism has some adaptive responses or was damaged. Data in our study showed that chlorpyrifos and TCP treatments could induce a significant increase of SOD and CAT activity. This may be attributed to fact that the chlorpyrifos and TCP induced ROS generation, and SOD and CAT are the major ROS-scavenging mechanisms (Anderson et al. 1995). To keep the ROS production at steady-state concentration, the activities of CAT and SOD increased to eliminate the redundant ROS (Mittler 2002). However, compared to the low chlorpyrifos and TCP concentration, some high concentrations of chlorpyrifos and TCP (for example, CAT at 1 mg L-1 chlorpyrifos in females and at 0.5 mg L-1 TCP in males) could induce a significant decrease in the activities of SOD and CAT. This phenomenon can be interpreted as the excessive chlorpyrifos and TCP inducing the superfluous generation of ROS. ROS are capable of unrestricted oxidation of various cellular components and can lead to oxidative destruction of the cell (Mittler 2002) or can cause the anti-oxidative
Oxidative stress At present, cytochrome P450 enzymes (CYPs) in fish are a useful tool that is used in environmental contamination biomonitoring and ecotoxicology assays for xenobiotic metabolism (Li et al. 2008). Our experiments showed that the P450 content in zebrafish was impacted by chlorpyrifos and TCP in both males and females across all treatment concentrations; the trend of the stimulation showed an initial increase followed by a decrease over time. NCR is an electron donor to CYP, so its alteration would probably affect the functioning of the monooxygenase system (Dong et al. 2009). Our results demonstrated that the NCR activity was affected by the CYPs. The observed effects can be explained by the role of increased P450 enzyme activities in detoxification and the ultimate elimination of chlorpyrifos and TCP. The effects were found to decrease at
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The enzyme toxicity and genotoxicity of chlorpyrifos
Fig. 6 Effect of chlorpyrifos on the liver comet olive tails moment of female (a) and male (b) zebrafish. Effect of TCP on the liver comet olive tails moment of female (c) and male (d) zebrafish. Each bar is
the mean of three replicates. Error bars represent standard deviation (SD). Different letters (a, b, c, and d) above columns indicate significant differences at p \ 0.05 level between treatments
capacity of the organism to be exceeded, leaving the enzymes exhausted (Wang et al. 2006). ROS are generated in all aerobic organisms during normal respiratory metabolism (Schnabel et al. 2006) and include a series of chemically reactive molecules derived from oxygen. ROS are known to affect cells by directly oxidizing and damaging diverse cellular components (e.g., proteins, lipids, and DNA) in different ways that can affect their function (Bokov and Chaudhuri 2004). The major ROS are superoxide, nitric oxide, hydroxyl radical, hydroperoxide, and peroxynitrite. The intracellular concentration of ROS can be modulated by controlling the sites and amounts of their synthesis, or it can be inactivated with antioxidant molecules and repair mechanisms directly. We can study the ROS by measuring the activity of different antioxidant enzymes, such as SOD, CAT, and peroxidases (Brandes and Janiszewski 2005). This study showed that the concentration of 0.01 mg L-1 chlorpyrifos did not induce a significant increase of ROS levels in zebrafish, which was different from the effects of TCP. This may be due to the effect of anti-oxidative enzymes,
which can prevent the increase of noxious ROS and keep the ROS at a normal level. Meanwhile, a significant increase of ROS in the liver of fish occurred at the other concentrations. This result is similar to many previous studies and can be interpreted as TCP and higher concentrations of chlorpyrifos acting as highly toxic external sources of pollutants, which causes ROS to be generated at such a high rate that they exceed the capacity of the organisms to eliminate it. The action of parental compounds or their metabolites may damage membrane lipids, DNA, proteins and carbohydrates directly, and the generation of ROS may result in the same damage indirectly (Oliveira et al. 2009). SCGE, which is widely used to evaluate the genotoxicity of chemicals in the environment, was used to assess the toxic effects on zebrafish embryos and liver (Deventer 1996; Sun et al., 2004; Kosmehl et al. 2008; Seitz et al. 2008). Therefore, we use SCGE as an index to evaluate the genotoxicity of chlorpyrifos and TCP. Our results showed that the DNA damage in the zebrafish hepatopancreas increased with increasing chlorpyrifos and TCP
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concentrations. In our laboratory, Zhu et al. (2010) found the same regularity when zebrafish were treated with 0.01–1 mg L-1 atrazine. According to Liu et al. (2009), 0.1–10 mg kg-1 endosulfan also has significant genotoxicity to the earthworm. Experiments indicate that chlorpyrifos and TCP-induced DNA damage in zebrafish and the levels of DNA damage imposed by chlorpyrifos and TCP could be quantified by SCGE. Few studies have been reported that deal with the mechanism of genotoxicity of chlorpyrifos. In previous reports, reasons for the genotoxicity of organophosphorous compounds, which would include chlorpyrifos, include alkylation (Rahman et al. 2002), phosphorylation and oxidative stress (Pin˜a-Guzma´n et al. 2006; Sharma et al. 2006). Alkylation can attack DNA bases and proteins, including single- and double-DNA strand breaks, crosslinks, chromosomal aberrations, and DNA base oxidation (Pin˜a-Guzma´n et al. 2006). Oxidative stress was related to chromatin cross-linking, DNA strand breaks, DNA base oxidation, chromosomal aberrations (Possamai et al. 2007), and lipid peroxidation (Debnath and Mandal 2000). Furthermore, the setting of the chlorpyrifos and TCP concentration was done according to the natural residual quantity in the natural environment, especially in the aquatic environment. Therefore, this research can provide help for future studies on chlorpyrifos toxicity in the natural aquatic environment.
reliable early-warning indicators of environmental pollution. According to this study, CYPs and SCGE are more sensitive than antioxidant enzyme activity and ROS generation. Chlorpyrifos and TCP can induce excess ROS generation, which could be eliminated through the increased activity of anti-oxidative enzymes (CAT, SOD). According to (Contardo-Jara et al., 2009), because the antioxidant enzymes (CAT, SOD) studied here are part of a vital defense mechanism against ROS-induced tissue damage, the increased production of ROS with a concomitant reduction of antioxidant enzyme activities could lead to oxidative stress, including enzyme inactivation, protein degradation, DNA damage and lipid peroxidation. Many other studies also showed that the DNA strand breaks are mainly attributable to ROS (O’Brien et al. 2003; Luo et al. 1996; Casadevall et al. 1999). Conversely, the DNA damage and the activity decrease of the antioxidant enzyme could induce the ROS generation. The study of the interactions among the four indexes could provide a basic theory to the future study of the toxicity mechanism of chlorpyrifos and TCP in organisms.
Conclusions (1)
Sex differences Many factors, such as sex, reproductive phase, developmental stage and diet, can affect enzyme activity. Sexbased differences in zebrafish are mainly regulated by hormones (Anderson et al. 1995). Data from the literature demonstrates that contradictory results indeed exist in fish species. Our study showed that, in control zebrafish, cytochrome P450 content and NCR, ROS, CAT and SOD activities in females are higher than in males, as is the degree of DNA damage. This is in accordance with the reported results of previous studies performed by Otte et al. (2008) and Sole´ et al. (2002). In our study, sex differences were observed in the chlorpyrifos- and TCP-induced enzyme activity and DNA damage. Additionally, because females are more susceptible than males to changes in enzyme activity and DNA damage following chlorpyrifos and TCP treatment and to ensure the results are clear and accurate, we can choose the female zebrafish as a biological indicator to test the pollution. Relationship This study indicated that the CYPs, antioxidant enzyme activity, ROS generation and DNA damage level are all
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(2)
(3)
(4)
(5)
By comparing the CYPs, DNA damage, and CAT, SOD and ROS activities induced by the same concentrations of chlorpyrifos and TCP in the zebrafish, we can see that the toxicity of TCP was more serious than that of chlorpyrifos. Chlorpyrifos and TCP significantly stimulate cytochrome P450 enzymes in zebrafish, even at a concentration as low as 0.01 mg L-1. Because of their sensitivity to low chlorpyrifos and TCP concentrations, CYPs and SCGE could be potential biomarkers to evaluate the effect of exposure and could serve as early-warning signals of aquatic contamination. CAT, SOD and ROS generation is also affected by the chlorpyrifos and TCP exposure. They generally increased after exposure to low concentrations of chlorpyrifos and TCP, but decreased significantly after exposure to high chlorpyrifos and TCP concentrations. Chlorpyrifos and TCP can induce significant DNA damage in zebrafish. The DNA damage was enhanced as the chlorpyrifos and TCP concentrations increased, with a clear dose–response relationship. The biotransformation activities caused by chlorpyrifos and TCP show different levels between male and female zebrafish. Generally, the value for females was larger than for males. However, after exposure to the
The enzyme toxicity and genotoxicity of chlorpyrifos
pollutants, the change regularity of these indictors was similar between males and females. Acknowledgments This study was supported by grants from the National Science Foundation of China (No. 41071164, 21277083, 21377075, and 41001152) and the Specialized Research Fund for the Doctoral Program of Higher Education (20113702110007). Conflict of interest of interest.
The authors declare that they have no conflict
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