Zebrafish as a model system to study toxicology

Authors


Abstract

Monitoring and assessing the effects of contaminants in the aquatic eco-environment is critical in protecting human health and the environment. The zebrafish has been widely used as a prominent model organism in different fields because of its small size, low cost, diverse adaptability, short breeding cycle, high fecundity, and transparent embryos. Recent studies have demonstrated that zebrafish sensitivity can aid in monitoring environmental contaminants, especially with the application of transgenic technology in this area. The present review provides a brief overview of recent studies on wild-type and transgenic zebrafish as a model system to monitor toxic heavy metals, endocrine disruptors, and organic pollutants for toxicology. The authors address the new direction of developing high-throughput detection of genetically modified transparent zebrafish to open a new window for monitoring environmental pollutants. Environ Toxicol Chem 2014;33:11–17. © 2013 SETAC

INTRODUCTION

Zebrafish (Danio rerio), the cyprinid schooling teleost, is an ideal model for studying genetics. In 1981, Streisinger et al. [1] reported methods to produce homozygous diploid zebrafish by using hydrostatic pressure or heat shock. This study greatly promoted the development of genetic analyses in zebrafish [1]. In 1996, Driever et al. [2] and Haffter et al. [3] reported a large-scale genetic screen for mutation in zebrafish. They obtained numerous mutations and identified the mutation genes and their functions in zebrafish embryogenesis. These results provided a powerful basis for development studies using zebrafish as a model organism. The zebrafish has many advantages as a model organism, such as small size, ex utero development of the embryo, short reproductive cycle, and transparent embryos [4]. In addition, the zebrafish shares a high degree of homology with the human genome [5]. Thus, the zebrafish is becoming a powerful model organism for studying genetics [6-8], development [9-11], environmental toxicology [11-15], pharmacology [16, 17], DNA damage repair [18-20], cancer [4, 21], and other diseases [22-24].

Zebrafish could be used for studies on eco-environmental monitoring and multitudinous pollutant evaluations, such as toxic heavy metals, endocrine disruptors, and organic pollutants [25-27]. However, new biotechnologies can now improve detection sensitivity. Recently, with the development of transgenic technology [28], especially the applications of both luciferase (LUC) and green fluorescent protein (GFP) reporters in this field [29], monitoring efficiency and sensitivity have been dramatically improved. The present review focuses on recent studies on the monitoring of environmental pollutants using both wild-type and transgenic zebrafish. The potential applications of the zebrafish model in toxicology also are predicted.

APPLICATIONS OF WILD-TYPE ZEBRAFISH FOR ENVIRONMENTAL MONITORING

Environmental pollution, especially water pollution, is a serious issue throughout the world. Water pollution not only affects the survival and reproduction of aquatic organisms but also adversely impacts human health through bioconcentration. Sensitivity to different contaminants makes the zebrafish an ideal model organism for environmental monitoring. The characteristic changes in morphology, gene expression, behavior, and physiology were observed as biological indicators. The International Organization for Standardization (ISO) first published the zebrafish toxicity test in 1984 [30]. Thereafter, multiple countries promulgated their own toxicity test standards by using zebrafish according to ISO 7346-1996, such as the British standard BS/EN/ISO 7346-3-1998, the German standard DIN/EN/ISO 7346-3-1998, and the Chinese standard GB/T13267-91. In brief, the pollutants detected in in vivo studies with wild-type zebrafish can be classified mainly as toxic heavy metals, endocrine disruptors, and organic pollutants (Table 1).

Table 1. Applications of wild-type zebrafish for environmental monitoring
ClassesContaminantsBiomarkers/endpointsReference
  1. BDE = brominated diphenyl ether; PBDE = polybrominated diphenyl ether; TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Heavy metalsCadmium acetate, lead acetate, mercury chloride, zinc chlorideAcetylcholinesterase activity, gene expression pattern, antioxidant competence in the brain[33]
 Mercury chlorideAdenosine deaminase activity, gene expression[34]
 Cd2+mRNA expression of smtB and mt2[35]
 Cadmium, zinc,Superoxide dismutase, catalase, acetylcholinesterase[36]
 Cu2+, Hg2+, Cd2+, Zn2+Metallothionein (zMT) gene expression[37]
 ArsenicDvr1 expression[40]
 Nano-scale TiO2, ZnO and their bulk formsAcute toxicity, oxidative stress, oxidative damage[43]
Endocrine disruptorsBDE-47Behavior[45]
 PBDEsRetinoid content, gene transcription levels[48]
 DE-71Thyroid hormone levels, gene transcription[49]
 TCDDTCDD-induced transcriptional changes[51]
 Bisphenol A and triclocarbanBrain-specific expression of aromatase[55]
 Bisphenol A, endosulfan, heptachlor, methoxychlor, tetrabromobisphenol AVitellogenin expression[53]
Organic pollutantsChlorpyrifos and nickel chlorideBehavior[64]
 ChlorpyrifosBehavior[62]
 Perfluorononanoic acidTranscriptional expression of FABPs[70]
 Methyl parathionMembrane protein[84]

Monitoring toxic heavy metals with wild-type zebrafish

The most common method of monitoring toxic heavy metals is toxicity experiments with both embryos and adult zebrafish [31, 32]. Some heavy metals can inhibit the activity of enzymes [33, 34] or affect genes expression [35]. Toxic heavy metals could be detected indirectly in the aquatic environment by monitoring enzymes' activity or biomarker gene expression in wild-type embryos or adult zebrafish. For example, Ling et al. [36] found that the activities of some enzymes, including superoxide dismutase, catalase, and acetylcholinesterase, were affected by exposure to Cd, Zn, or methyl parathion. These 3 enzymes can act as biomarkers for joint pollution detection. However, superoxide dismutase activity is only sensitive to Cd exposure [36]. Chan et al. [37] found that the gene expression of metallothionein in zebrafish (mt) was induced when exposed to Hg2+, Cu2+, Zn2+, and Cd2+, respectively. Hg2+ potently induces the highest mt mRNA in vivo, and Cd2+ was the most potent inducer in vitro. Therefore, the mt mRNA level in zebrafish can be chosen as a marker to monitor Hg2+ and Cd2+ in water [37]. Some heavy metals also cause toxic effects on the development of zebrafish embryos [38, 39]. Li et al. [40] demonstrated that dvr1, the gene functioning for the left–right asymmetry of zebrafish embryogenesis, is closely related to arsenic-mediated embryo toxicity. The dvr1 was significantly down-regulated when zebrafish embryos were exposed to arsenic compounds. Therefore, the dvr1 level in zebrafish can be used to monitor inorganic arsenic compounds [40].

Use of nanomaterials is increasing exponentially, leading to major concerns about nanomaterial hazard on aquatic organisms. Therefore, studying the toxicology of nanomaterials and monitoring these substances have become topics of interest in recent years. There are some common features in the sublethal responses to nanometals. For example, nanometals can result in a series of sublethal effects, including respiratory toxicity, trace elements disturbances in tissues, inhibition of Na(+)K(+)–adenosine triphosphatase, and oxidative stress. However, some nanometals showed more toxicity to zebrafish embryos compared with their bulk counterparts [41-43]. George et al. developed a high-throughput assessment system with the combination of in silico data analysis and in vivo zebrafish embryo screening, which could be used to perform the rapid screening of the toxicology of nanomaterials [44].

Monitoring endocrine disruptors with wild-type zebrafish

Polybrominated diphenyl ethers (PBDEs), dioxin, bisphenol A (BPA), and their derivatives are the most common endocrine disruptors. They can interfere with natural hormones by binding to estrogen or androgen receptors with or without activating them. Endocrine disruptors can affect the synthesis, release, transport, metabolism, and combination of endocrine substances, thereby leading to reproductive problems, birth defects, developmental abnormalities, metabolic disorders, and cancer. Besides the traditional methods of chemical analysis, some biological technologies have been developed, such as gonadal morphology and histological comparative analysis, messenger RNA (mRNA), and protein levels of chorionic gonadotropin or vitellogenin (vtg). Zebrafish can be used as a model organism not only to detect the concentration of endocrine disruptors in the aquatic environment but also to assess their toxic effects in reproductive and nervous systems. Therefore, we can finally achieve direct monitoring and early warnings of endocrine disruptors with this model organism.

Polybrominated diphenyl ethers are a group of ubiquitous pollutants in the environment that could disrupt behavior and cause developmental toxicity in zebrafish [45, 46]. In addition, exposure to PBDEs could cause adverse effects on the neurodevelopment of offspring [47]. Chen et al. [48] indicated that DE-71, a commercial PBDE mixture, disrupted the transport, storage, and metabolism of retinoid in zebrafish at environmentally relevant concentrations, indicating that retinoid levels in zebrafish are sensitive to PBDEs. A significant up-regulation of the transcription of corticotrophin-releasing hormone and thyroid-stimulating hormone genes occurred in a concentration-dependent manner, when zebrafish larvae are exposed to up to 10 mg/L DE-71 [49]. Therefore, these 2 genes could be chosen as sensitive biomarkers to monitor DE-71.

Dioxin, a strongly toxic substance in the reproductive system, can be produced naturally or through anthropogenic processes, such as waste incineration. Dioxin exposure to humans or other model organisms can cause chloracne, cancers, hepatotoxicity, diabetes, immune changes of gonadal, pulmonary tuberculosis, and other complications such as diabetes, skewed sex ratio, and infertility. Dioxin pollution is a direct threat to the environment and human health [50]. To study the molecular mechanism in reproductive toxicity, Heiden et al. [51] evaluated the transcription of the related genes in zebrafish ovary and found that 2,3,7,8-tetrachlorodibenzo-p-dioxin inhibited follicle maturation by impairing gonadotropin responsiveness and disturbing estradiol biosynthesis and estrogen-regulated signal transduction. These physiological changes could effectively reflect the presence of endocrine disruptors [51]. Recently, by detecting the expression of specific genes in zebrafish, Pelayo et al. [52] provided a zebrafish scale assay to determine the presence of dioxin-like compounds. This method could directly monitor contaminants in natural water samples without the treatment of preconcentration [52].

Bisphenol A is a common endocrine disruptor with embryo toxicity and teratogenicity [53, 54]. Chung et al. [55] reported that BPA exposure induced strong brain-specific overexpression of aromatase in early zebrafish embryos. Gibert et al. [54] showed that BPA induced severe malformations of the otic vesicle. Saili et al. [56] found that low-dose BPA exposure led to larval hyperactivity or learning deficits in adult zebrafish. Therefore, using the methods described above as a tool to detect the presence of BPA is feasible. In addition, Chow et al. [53] assessed vtg gene expression after acutely exposing zebrafish embryos and larvae to BPA, endosulfan, heptachlor, Methoxychlor, and tetrabromobisphenol A. The results showed that vitellogenin 1 (vtg1) mRNA expression was a sensitive biomarker for monitoring these compounds [53].

Monitoring organic pollutants with wild-type zebrafish

In recent years, organic pollutants have been detected frequently in the environment, raising concerns on the hazard of organic pollutants. As major organic pollutants, polyaromatic hydrocarbon pollutants (PAHs) have posed a serious threat to organism survival in water, soil, and sediments. Common PAHs, such as naphthalene, dichlorophenol, and aromatic pesticides, interfere with functions of the endocrine system by affecting the development of embryos, damaging DNA, and inducing oxidative stress in zebrafish [57, 58]. Not only do PAHs possess developmental toxicity in zebrafish embryos [59], but they also affect the cardiac development and expression of the related genes [60]. Peddinghaus et al. [61] provided a method to determine the hazard potential by combining zebrafish embryo toxicity tests with analytical quantification. In addition, the toxicity of different types of pesticides can be evaluated by studying the toxic effects of pesticides on zebrafish and their bioconcentration [62, 63]. Kienle et al [64] investigated the toxic effects of chlorpyrifos and nickel chloride on zebrafish larvae and found that, compared with developmental or survival parameters, behavior could be a sensitive endpoint for chlorpyrifos exposure in zebrafish [64].

Recently, more and more attention has been paid to organic chiral pollutants because of the different toxicity of enantiomers. Although the physical and chemical properties are the same, enantiomers show different physiological, biochemical, and toxic effects. Discovery of those differences will help us to avoid the poisonous enantiomers and to reduce eco-environmental pollution. Liu's group isolated a variety of pyrethroid insecticide enantiomers and analyzed their aquatic toxicity. The results showed enantioselectivity in the acute toxicity and developmental toxicity [65-67]. Jin et al. [68] found the different expression levels of vtg1, estrogen receptor alpha, and CYP19b after exposing zebrafish embryos and larvae to 4 permethrin isomers. Therefore, estrogenic activity of these 4 isomers could be evaluated by detecting the levels of expression of these genes [68].

Perfluorochemicals, a new persistent organic pollutant, threaten human health because of their bioaccumulation and difficult degradation. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) could affect embryonic development in zebrafish [69]. Zhang et al. reported that PFOA exposure changed the expression of the fatty acid–binding protein gene and affected the levels of liver triglyceride [70]. Du et al. [71] used a combination of in vivo and in vitro testing to study the endocrinal disruption of PFOS. The results showed that PFOS was similar to the estrogen receptor agonist and thyroid hormone receptor antagonist. Perfluorooctane sulfonate could interfere with steroidogenesis and change the endocrine-related gene expression in zebrafish embryos. In particular, PFOS promotes the expression of the early thyroid development genes (hhex and pax8) in a concentration-dependent manner, which could provide a feasible method for monitoring these pollutants [71].

In addition, excessive uses of antibiotics in aquaculture lead to environmental pollution and have significant toxic effects on aquatic organisms. Recent reports indicate that some antibiotics interfere with the zebrafish immune system and embryonic development [72]. To study the inflammatory effects of the antibiotics in water, Barros et al [73] analyzed the responses of chronic exposure to oxytetracycline in zebrafish. They found that oxytetracycline can cause a widespread inflammation process after 48 h of exposure [73].

APPLICATIONS OF TRANSGENIC ZEBRAFISH FOR ENVIRONMENTAL MONITORING

Although the wild-type zebrafish are widely used to monitor the toxicity of environmental contaminants, they have multiple disadvantages, such as low detection sensitivity and cumbersome statistical experiments. Recently, transgenic zebrafish was successfully used to improve these deficiencies. After the transgenic fish was first developed by Zhu et al. [74], it has gradually become an optimal tool in different research fields. The transgenic zebrafish is developed and reported to be a more advanced system for monitoring environmental pollutants [29, 75]. With the successful development of transgenic fish expressing fluorescent protein, the use of zebrafish as a model organism for environmental monitoring becomes more simple and convenient. Fluorescent protein as the report gene can be expressed in heterologous cells and easily detected without any toxicity. Therefore, fluorescent protein has been widely used to study transgenic animals. Response elements can drive the expression of reporter genes when exposed to environmental pollutants [26, 28]. Manipulation of transgenic zebrafish to monitor environmental pollution is described in Figure 1.

Figure 1.

The manipulation of transgenic zebrafish for environmental monitoring. The target plasmids with response elements were constructed to drive the expression of different fluorescent reporter proteins. The plasmids were injected into zebrafish embryos. After screening the positive transgenic zebrafish in offspring, the stably transgenic zebrafish can be exposed to different environmental pollutants and used for monitoring aquatic hazard by observing the fluorescence. RFP = red fluorescent protein; GFP = green fluorescent protein; YFP = yellow fluorescent protein; CFP = cyan fluorescent protein. [Color figure can be seen in the online version of this article, available at wileyonlinelibrary.com]

Several transgenic methods were reported to introduce the target DNA into zebrafish, such as microinjection, electroporation, particle gun bombardment, liposome-mediated gene transfer, and sperm-mediated gene transfer. Because of its highest survival rate, microinjection is the most commonly used method to produce transgenic zebrafish [11]. Currently, researchers have developed a variety of response elements to detect different specific environmental pollutants (Table 2). After microinjected recombinant constructed plasmids, the zebrafish line stably inherited fluorescent protein gene could be obtained by screening.

Table 2. Response elements for biological monitoring of environmental pollution in fish
Activating agentsResponse elementReferences
  1. TCDD = 2,3,7,8 – tetrachlorodibenzo-p-dioxin ; EREs = estrogen response elements; vtg = vitellogenin; AhREs = aryl hydrocarbon hydroxylase responsive elements; EpREs = electrophile response elements; MREs = metal response elements; hsp70 = heat shock protein 70; RARE = retinoic acid response elements; RXRE = retinoid X response elements.
Estrogens (E2, EE2, NP)EREs[85-87]
Ethinylestradiol, methyltestosteronevtg[79, 88, 89]
TCDD; dioxinAhREs[28, 90, 92-94]
Quinines, potent electrophilic oxidantsEpREs[28, 91]
Mercury, copper, nickel, cadmium, and zincMREs hsp70[28, 76]
RetinoidsRARE, RXRE[28]

Toxic heavy metals monitoring by transgenic zebrafish

To monitor the toxic heavy metals, the common method is to detect metal response elements (MREs) or heat shock protein (hsp70) promoter inducible expression of GFP or LUC in transgenic zebrafish. In 2002, Blechinger et al. [76] developed a transgenic zebrafish line with hsp70 promoter–GFP for monitoring heavy metal Cd in water pollution. The GFP signal could be clearly observed even exposed to 0.2 µM Cd solution using this transgenic zebrafish line. However, the hsp70 promoter could be induced not only by heavy metals, but also by other environmental changes, such as temperature change [76]. Nebert et al. [28, 77] made a recombinant promoter specifically induced by toxic heavy metals, with mMREd5mt1 (a concatamer of 5 mouse MREd9 sequences fused to the minimal mouse mt1 promoter) to drive the LUC expression in zebrafish cell cultures. The metal response element can be further optimized to improve monitoring sensitivity.

Monitoring endocrine disruptors with transgenic zebrafish

The endocrine disruptors in the aquatic environment are commonly monitored using a comparative analysis method to check gonadal morphology and histology or to detect the expression of chorionic gonadotrophin and vtg gene at both transcriptional and translational levels. However, these detection techniques need special instruments and relatively complex experimental operation. Transgenic technology has been used in recent years to remedy these deficiencies. For example, some researchers managed to develop a transgenic fish line to determine the presence of endocrine disruptors by detecting GFP driven by estrogen-inducing promoter. In 2005, Kurauchi et al. [78] generated a transgenic medaka strain to detect hormone analogs by harboring the GFP gene under the control of chorionic gonadotrophin promoter in the medaka line. However, low detectable GFP expression was seen in the juvenile (14 d after hatching) or adult transgenic medaka. In the same year, Zeng et al.[79] detected the presence of endocrine disruptors with the transgenic medaka strain in which medaka vtg1 gene promoter regulated the GFP reporter gene. Chen et al. [80] identified and cloned the vtg promoter from zebrafish and then developed a transgenic zebrafish line that was sensitive to estrogen-like substances. The promoters of chorionic gonadotrophin and vgt1 genes are both tissue-specific, and they can only drive GFP gene expression in liver. Because of the restrictions of the tissue-specific promoter, the sensitivity for monitoring endocrine disruptors is low. In addition, the medaka or zebrafish that were used, were not entirely transparent, resulting in insufficient monitoring sensitivity.

Monitoring aromatic hydrocarbon with transgenic zebrafish

The traditional methods to detect toxic effects of aromatic hydrocarbon pollutants include in vitro and in vivo tests. Toxic effects on the whole organism cannot be fully reflected by an in vitro test. In addition, traditional methods have some defects, including complicated operations, high costs, and instability in low-concentration exposure. Therefore, detecting the toxic effects of aromatic hydrocarbon pollutants by tiered tests is popular in current research. However, the limitations of the tiered test are that it is time-consuming and labor-intensive, and it requires professional personnel. Transgenic fish for monitoring aromatic hydrocarbon pollutants gradually came into being because of these difficulties. In 2001, Mattingly et al. [80] confirmed that human aryl hydrocarbon response elements can be recognized by the transcription factor of zebrafish to regulate GFP expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin [81]. Payne [82] observed that the activity of cunner CYP1A1 could be induced by oil, and the oil pollution in the sea can be monitored by detecting the activity of benzopyrene hydroxylase. With the inspiration of these studies, Nebert et al. [77] have developed a transgenic zebrafish line, in which the expression of the LUC reporter gene was driven by a human Ah response element promoter to detect organic pollutants of the aromatics family in the aquatic environment. Unfortunately, the LUC reporter gene in the transgenic zebrafish cannot be inherited to the F2 generation [77].

In summary, many achievements have resulted from using transgenic zebrafish to study environmental pollutants. However, some inadequacies exist. Therefore, further exploration of new ways to broaden the applications of this method is necessary.

NEW DIRECTIONS USING TRANSGENIC ZEBRAFISH TO MONITOR ENVIRONMENTAL POLLUTANTS

Transparent zebrafish to improve the detective sensitivity

Wild-type zebrafish embryos are transparent, whereas the adult fish are opaque because of color stripes. The fluorescence intensity of transgenic adult zebrafish induced by environmental pollutants is weak and insensitive. To address this, the transparent zebrafish would be an ideal model organism. In 2008, White et al. [83] constructed a transparent zebrafish line named casper, which is a double mutant of nacre and roy genes [83]. The casper fish was completely transparent, and its tissues and organs—such as gut, heart, kidney, and gallbladder—could be directly observed using a stereomicroscope or the naked eye. The main reason for its transparency was that it lost melanocyte and iridophores, which are responsible for light absorption and reflection, respectively. The transparent casper line provided broader prospects for environmental monitoring [83].

Studies exploring new response elements or biomarkers and high-throughput detection using transparent transgenic zebrafish

Discovered response elements are still not enough to specifically detect different pollutants. Therefore, we should focus on the development of new response elements, recombination of existing response elements, and optimization of the number of response element repeats to achieve highly sensitive monitoring of multiple pollutants at the same time. Development of the zebrafish gene chip technology makes it easy to find novel response elements. By analyzing the response elements map driven by special pollutants, we can identify the different expression pattern and then subclone the specific marker elements to produce chip or array for high-throughput biological monitoring. Recently, most of the transgenic zebrafish for environmental monitoring could specifically identify only 1 or a class of pollutants in the environment. Detecting mixture pollutants at the same time is not easy. However, this problem can be solved by combinational expression of different fluorescent protein genes (GFP, red fluorescent protein [RFP], cyan fluorescent protein [CFP], and yellow fluorescent protein [YFP]), which were driven by different specific response elements in zebrafish. Therefore, the development of high-throughput multiplex detection of genetically modified transparent zebrafish can open a new window for monitoring environmental pollutants.

CONCLUSION

Zebrafish as a model organism for environmental monitoring has low cost, ease of breeding, and other advantages. With the creation of various transgenic zebrafish lines, monitoring sensitivity has been continuously improved. Biological monitoring technology can directly reflect the toxic effects of pollutants on organisms and biological health, which is different from conventional chemical methods. Environmental pollution is a global problem and the pollutants now show diversity and complexity. Some emerging pollutants, such as synthetic materials, pesticides, pharmaceuticals, veterinary drugs, and cosmetics, have been released into the water and threaten human health via the ecology cycle. Harm may be prevented by monitoring environmental pollutants. To avoid further deterioration of water quality, we should develop efficient, cost-effective transgenic zebrafish to perform highly sensitive and more efficient monitoring of the environmental pollutants in water.

Acknowledgment

Y.-J. Dai and Y.-F. Jia contributed equally to this manuscript. The present work was supported by the Chongqing Application Development Projects in China (Studies on the monitoring of emerging and toxic pollutants in water using transparent and transgenic zebrafish) and the National Natural Science Foundation of China (Grant No. 30700607). We acknowledge the researchers whose biomonitoring works are not cited in the present study because of space limitations.

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