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The toxicity of chlorpyrifos on the early life stage of zebrafish: A survey on the endpoints at development, locomotor behavior, oxidative stress and immunotoxicity

https://doi.org/10.1016/j.fsi.2015.01.010Get rights and content

Highlights

  • Effects of CPF on the different endpoints in early life stage of zebrafish are evaluated.

  • CPF decreases the body length, hatchability, heart rate and caused the increase of abnormalities.

  • CPF alters the motor behavior, activity of AChE and mRNA levels of genes related to neurotoxicity.

  • CPF affects the activities of SOD, CAT, GPX and the transcriptional levels of their respective genes.

  • CPF increases the mRNA levels of genes related to the innate immune system in zebrafish larvae.

Abstract

Chlorpyrifos (CPF) is one of the most toxic pesticides in aquatic ecosystem, but its toxicity mechanisms to fish are still not fully understood. This study examined the toxicity targets of CPF in early life stage of zebrafish on the endpoints at developmental toxicity, neurotoxicity, oxidative stress and immunotoxicity. Firstly, CPF exposure decreased the body length, inhibited the hatchability and heart rate, and resulted in a number of morphological abnormalities, primarily spinal deformities (SD) and pericardial edema (PE), in larval zebrafish. Secondly, the free swimming activities and the swimming behaviors of the larvae in response to the stimulation of light-to-dark photoperiod transition were significantly influenced by the exposure to 100 and 300 μg/L CPF. In addition, the activity of acetylcholinesterase (AChE) and the transcription of some genes related to neurotoxicity were also influenced by CPF exposure. Thirdly, CPF exposure induced oxidative stress in the larval zebrafish. The malondialdehyde (MDA) levels increased and the glutathione (GSH) contents decreased significantly in a concentration-dependent manner after the exposure to CPF for 96 hours post fertilization (hpf). CPF affected not only the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione S-transferase (GST), but also the transcriptional levels of their respective genes. Finally, the mRNA levels of the main cytokines including tumor necrosis factor α (Tnfα), interferon (Ifn), interleukin-1 beta (Il-1β), interleukin 6 (Il6), complement factor 4 (C4) in the larvae increased significantly after the exposure to 100 or 300 μg/L CPF for 96 hpf, suggesting that the innate immune system disturbed by CPF in larvae. Taken together, our results suggested that CPF had the potential to cause developmental toxicity, behavior alterations, oxidative stress and immunotoxicity in the larval zebrafish.

Introduction

In the last decades, organophosphate pesticides are among the most widely used classes of pesticides. China, India and other developing countries have substantially increased production of organophosphate pesticides even in recent years [1]. As a well-known organophosphate pesticide, chlorpyrifos (CPF) is frequently and extensively used for controlling agriculture and house hold pests all over the world [1]. As reported, about 18,000 tones of CPF were consumed annually in China [2]. Runoff, erosion, leaching are the major routes of CPF entry into surface waters [3], [4], [5]. Thus, CPF is commonly monitored in groundwater and surface water [6], [7], [8]. Like other organophosphate pesticides, CPF also exerts its pharmacological activity primarily through the binding of the enzyme acetylcholinesterase (AChE) by phosphorylation, leading to the inhibition of this enzyme activity [9], [10]. As a result of widespread use of CPF, there is no doubt that the misuses of CPF and other organophosphate pesticides can have adverse effects on non-target organisms including aquatic vertebrates.

The toxicity of organophosphate pesticides have been linked to nausea, dizziness, confusion, increased heart rate, respiratory failure, and even death. A number of previous studies have indicated that CPF exposures are associated with a wide range of toxic effects including nephrotoxicity, oxidative stress, genotoxic and mutagenic effects, alterations in swimming performance, as well as effects on development, in different aquatic organisms [11], [12], [13], [14]. Recently, the toxicities of CPF have been reported on zebrafish, but they are mainly focused on the inhibition of the activity of AChE and their unpredictable consequences. For example, Levin et al. [15] reported that the 100 μg/L CPF administered to zebrafish embryos on Days 1–5 postfertilization caused significant slowing of swimming activity on Days 6 and 9 dpf and had persisting effects of impairing spatial discrimination and decreasing response latency in adulthood. Yen et al. [1] found that the AChE activity was significantly inhibited in larval zebrafish after exposure to 300 nM CPF for 5 dpf. However, the effects on oxidative stress and immunotoxicity and other toxicological endpoints of zebrafish in response to the CPF have received limited concern.

Zebrafish is an established powerful laboratory fish model with a well characterized genome allowing for the application of sophisticated molecular approaches to investigate mechanisms of toxicity [16], [17], [18], [19], [20], [21]. Recently, the expression of some genes was adopted as a powerful tool to analyze the neurotoxicity and immunotoxicity in zebrafish induced by different environmental chemicals. For example, in a recent study, Fan et al. [22] observed that the expression profiles of genes such as glial fibrillary acidic protein (Gfap), myelin basic protein (Mbp), Elval3, neurogenin 1 (Ngnl), sonic hedgehog a (Shha) may be useful biomarkers for rapid evaluation of the developmental neurotoxicity potential of chemicals in zebrafish. As reported, the cytokines, such as tumor necrosis factor (TNF), interleukins (IL), etc., have an important role in initiating responses once a pathogen penetrates the host [23]. And the transcriptional levels of these genes are also considered as effective biomarkers induced by environmental chemicals [24].

Although there are many papers on the biochemical and physiological influences of CPF on aquatic organisms, a survey of many different CPF toxicity endpoints including the locomotor behavior in response to light change stimulus, the transcription of several key genes related to neurotoxicity, innate immune system and oxidative stress have not been evaluated, to our knowledge, in a single study dealing with early life stage of zebrafish. Such an inclusive work would help to understand the mechanisms of CPF toxicity in fish comprehensively. In the present study, we thus attempted to compare CPF toxicity effects measured in the model of the early life stage of zebrafish at multiple endpoints on development, locomotor behavior, oxidative stress and immunotoxicity. Our results bring some insights into the toxicity mechanisms of CPF in the aquatic ecosystem.

Section snippets

Chemicals

CPF (Purity > 99%) was purchased from Sigma–Aldrich. The chemical was used as received. Stock solutions of CPF were prepared by dissolving it in DMSO. The commercial kits for determining malondialdehyde (MDA), glutathione (GSH) contents and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione S-transferase (GST), AChE activities were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Protein concentrations were determined by the

Developmental toxicity

The hatchabilities decreased in a concentration-dependent manner after the exposure to CPF for 60 hpf, being 27.4%, 42.9% and 51.7% lower after the treatment of 30, 100 and 300 μg/L CPF when compared with that of the control group, respectively (Fig. 1A). However, no significant difference in the hatchability was observed between any CPF treated group and the control group when the exposure period was extended to 96 h (Fig. 1B). The heart rates of larval zebrafish decreased significantly when

Discussion

The concern on the toxicity of pesticides including CPF in aquatic organisms has increasing in recently years [31], [32], [33]. The objective of the present study was to perform an examination of the aquatic toxicity of CPF on the early life stage of zebrafish. In a previous study, Watson et al. [34] found that high concentrations (10, 100 μM) of CPF, dichlorvos or diazinon exposure could cause the decrease of survival rates and heart rates, increase of morphological changes, i.e., kyphosis

Acknowledgments

This work was supported by the National Natural Science Foundation of China (21277128), the National Basic Research Program of China (No. 2010CB126100) and Program for Changjiang Scholars and Innovative Research Team in University (IRT13096).

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    These authors contributed equally to this work.

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