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Fenbuconazole exposure impacts the development of zebrafish embryos
Ecotoxicology and Environmental Safety 158 (2018) 293–299
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Fenbuconazole exposure impacts the development of zebrafish embryos b
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Yuqiong Wu , Qihong Yang , Meng Chen , Ying Zhang , Zhenghong Zuo , Chonggang Wang a b c
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State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China Wuyi University, College of Tea and Food Science, Wuyishan, Fujian 354300, China Key Laboratory of Ministry of Education for Subtropical Wetland Ecosystem Research, Xiamen University, Xiamen, Fujian 361005, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Triazole fungicide Fish Embryonic development Mechanism
Fenbuconazole (FBZ), a triazole-containing fungicide, is widely used in agriculture and horticulture. In the present study, the development and cardiac functioning were observed and determined in zebrafish embryos exposed to FBZ at 5, 50 and 500 ng/L nominal concentrations for 72 h. The results showed that 500 ng/L FBZ significantly increased pericardial edema rate, spine curvature rate, disturbed cardiac function, and led a shortened lower jaw. The transcription of genes such as tbx5, nkx2.5, tnnt2, gata4, bmp2b, myl7 was altered, which might be responsible for the cardiac developmental and functioning defects in the larvae. The deformation in bone development might be related with the impaired transcription levels of shh and bmp2b. The transcription of cyp26a1 (encoding retinoic acid metabolism enzyme) was significantly up-regulated in the 500 ng/L group, which might be a reason causing the teratogenic effect of FBZ. These results suggest that FBZ could have toxic effects on embryonic development, which should be considered in the risk evaluation of FBZ application.
1. Introduction Among pesticides currently in use, triazole-containing fungicides are one of the most widely applied (Konwick et al., 2006; Goetz et al., 2007). Fenbuconazole (FBZ) is used in agriculture and horticulture against powdery mildew of vegetables, cereal and fruits (Holb and Schnabel, 2007; Sobeiha et al., 2009). Since its long biodegradation half-life with 61-day under field soil under anaerobic conditions (Pesticide Properties Data Base), FBZ could persist in the environment for a long period. After 190 d of incubation with soil, only a small amount of total FBZ (∼46.8% or ∼36.4% for Langfang or Changsha soil, respectively) disappeared (Li et al., 2012). The detected enantiomers of FBZ range from 10.49 to 23.54 μg/kg in soil samples collected from Langfang, China (Li et al., 2012). Thus, FBZ may contaminate aquatic environments through run-off. FBZ concentration detected in two waterworks of Xiamen city, China ranged from 0.22 to 6.98 ng/L (our unpublished data), indicating that the Jiulong river, from which these two waterworks got water source, was contaminated by FBZ. There is concern regarding potential exposure of fishes from its residues in aquatic environment (Li et al., 2012). The detected residues of FBZ in fruits such as oranges, pears, peaches and grapes range from 0.11 to 0.34 mg/kg (EFSA, 2015). People are also probably exposed to FBZ via food uptake. The WHO (1997) established an Acceptable Daily Intake for humans of 0.03 mg/kg/day, and maximum admissible concentration in drinking water is 0.1 μg/L (EFSA, 2015). Due to its wildly
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Corresponding author. E-mail address: [email protected] (C. Wang).
https://doi.org/10.1016/j.ecoenv.2018.04.048 Received 12 February 2018; Received in revised form 16 April 2018; Accepted 21 April 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
use, the toxic effects need to be adequately researched. Existing reports show that FBZ seems to be low toxicity to animals. Acute 96 h LC50 of FBZ to fish is 1.5 mg/L; acute oral LD50 to mammals is > 2000 mg/kg (Pesticide Properties Data Base: Fenbuconazole). FBZ is not genotoxic (Federal Register, 2002), studies in rodent show that it does not induce liver tumor but is associated with a slight increase in the incidence of liver adenomas (Lassalle et al., 2015). Mice treated with FBZ exhibit a reversible change of liver including increased liver weight and histological damages (Juberg et al., 2006). FBZ perturbs thyroid-pituitary functioning and induces thyroid tumors in rodent (Hurley et al., 1998). However, there are few studies concerning the developmental effects of FBZ on fishes. As a vertebrate model, zebrafish (Danio rerio) has numerous advantages such as small size, ease of culture, high fecundity, morphological and physiological similarities to mammals. The optical clarity of the chorion is in favor of the observation of embryogenesis and the assessment of endpoints of toxicity (Yang et al., 2009; He et al., 2014). The molecular weight (336.82 Da) of FBZ much lower than the threshold (3000 Da) (Braunbeck et al., 2015) allows its passage across the chorion. The rapid development of zebrafish involves all organic effects, and the genetic basis of development has been extensively studied, providing many benefits to mechanism assay (Fernández et al., 2018). These characteristics suggest that zebrafish embryos/larvae are an ideal research model for investigating the developmental effects of chemicals. In the present study, the influence of FBZ on the
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Software (Nikon, Tokyo, Japan), the number of frames between cardiac contraction initiations was calculated to obtain a mean and standard deviations (SD) for each larva. Since a regular rhythm would have a low SD, this SD is a measure of heart rate irregularity. Three larvae from each replicate were assessed to get a mean for this replicate. The means from five replicates were used for analysis.
development of zebrafish embryos was researched, and the mechanisms involved were investigated. 2. Materials and methods 2.1. Zebrafish maintenance and embryos collection All fish experiments followed the ethical guidelines of Xiamen University. Twenty wild-type TU zebrafish were housed in 20 L tanks with dechlorinated and aerated freshwater in a recirculation system at 28 ± 1 °C, under a normal photoperiod at 14:10 h light: dark. The pH and dissolved oxygen were 7.2–7.3 and 7–8 mg/L respectively. Fish were fed with live brine shrimp and commercial fish diet twice daily. Sexually mature fish without any signs of disease were selected as breeders. Adult fish were mated at a ratio of 1:2 (female: male). Spawned eggs were collected within 0.5 h. Fertilized eggs were washed with zebrafish facility water and randomly distributed to multiple petri dishes for exposure experiments.
2.4. Whole mount Alcian-blue staining
2.2. Embryonic exposure and sampling
Fifty larvae from each replicate were pooled into a subsample. Total RNA was isolated by a Trizol Kit (TaKaRa, Dalian, China). The mRNA expression was determined based on the previously described method (Huang et al., 2012). QPCR analysis was performed on an Mx3000P Real-Time PCR system (Stratagene, La Jolla, CA, USA) using the Brilliant SYBR Green QPCR reagent kit (Stratagene) following the manufacturer's protocol. Gene expression levels were normalized to zebrafish gapdh. The real time quantitative PCR primers (Table S1) were designed using Primer Premier 5.0. Threshold cycles (Ct) and dissociation curves analysis of the amplification products were determined with MxPro software (Stratagene), to confirm that only one PCR product was amplified and detected. The Relative Expression Software Tool (RESTMCS_-version 2) was used to calculate the relative expression of target gene mRNA (Pfaffl et al., 2002). Three housekeeping genes (gapdh, αtubulin, β-actin) were analyzed for suitable reference gene, the results showed that FBZ exposure did not alter the expression of gapdh (supplemental material). Therefore, gene expression levels were normalized to zebrafish gapdh.
Exposed larvae were randomly chosen in each treatment for Alcianblue staining to observe the craniofacial cartilage development. Alcianblue staining was followed the method described by Walker and Kimmel (2007). Stained larvae were observed using a Leica M165FC stereo fluorescence microscope, and the morphology of the craniofacial cartilage was analyzed following the method of Carvan et al. (2004). Three larvae from each replicate were assessed to get a mean for each replicate. 2.5. Real-time quantitative PCR (qPCR)
FBZ (purity > 98%) was purchased from Agro-Environmental Protection Institute, Ministry of Agriculture, China. It was dissolved in acetone at analytic grade to reach stock concentrations of 5, 50 and 500 μg/mL. FBZ exposure solutions were obtained by adding 1 μL the stock concentration to 1 L zebrafish culture medium (3.5 g/L NaCl, 0.05 g/L NaHCO3, 0.05 g/LKCl, 0.05 g/L CaCl2). Eggs were collected after oviposition and remove coagulated eggs and debris. Eggs at 0.5–1 h post-fertilization (hpf) were exposed to nominal concentrations of FBZ (0, 5, 50 and 500 ng/L). One hundred fertilized eggs were cultured in 30 mL exposure solution in petri dish at 28 ± 1 °C. Similar criterion was applied to solvent control group, which received an equal volume of acetone (1 μl/L). There were five replicates for each treatment. The test solutions were renewed twice daily, and dead embryos were removed. The development of the embryos and mortality was monitored with an Olympus SZ51 stereo microscope every 24 h. The rates of malformation, such as dorsal curvature, pericardial edema were calculated as: malformation rate = malformed embryos number / survival embryos number × 100%; the hatching success (embryonic membrane opening and larva swimming up) of the embryo were assessed: hatched rate = hatched embryos number / total embryos number × 100%. After exposure for 72 h, the embryos were collected for analysis. The remainder larvae after exposure for 72 h were transferred to clean water. Since the remaining amount of fish is small, they were put together from the five replicates and cultured in an aquatic system. Larvae within 7-day post-fertilization (dpf) occupied 10 mL water per fish in beak; and at 30 dpf, 400 mL in transparent fish tank. Fish at 60 dpf were maintained in the Aquatic Habitats Zebrafish System up to 90 days and occupied 1 L water per fish. The culture conditions were the same as those mentioned above. The numbers of remainder adult fish at 90 d were 28–47 in each treatment, whose morphological changes were observed and the occurrence rates of deformation were recorded.
2.6. Determination of FBZ in exposure solutions Exposure solutions, freshly made up with the stock solutions, were collected three times at random for determination. The FBZ concentrations were measured based on the method of Zhang et al. (2017) with slight modification. Briefly, 1 L exposure solution was added with simeconazole (purity > 97%) (Witega Laboratories Berlin-Adlershof GmbH, Berlin, Germany) as a surrogate, and was extracted using a liquid-liquid extraction method with 50 mL CH2Cl2 in a separatory funnel. The organic phase was collected and dried with anhydrous sodium sulfate, and the extracts were concentrated to dryness under a rotary evaporator, and then were diluted with acetone/n-hexane (1:1) solution. FBZ concentration was detected using a GC/MS/MS system (Agilent Technology, USA) following the description of Zhang et al. (2017). The recoveries of FBZ were 94% ± 0.6% (n = 3) and the limit of detection was 3.0 ng/L. The detected concentration of FBZ in the exposure medium was 0, 4.96 ± 0.09, 48.29 ± 0.41 and 473.58 ± 9.13 ng/L in the each group.
2.3. Cardiac function analysis The heart rate and cardiac arrhythmia of the exposed larvae were assayed based on the method of Incardona et al. (2009). The end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV) and cardiac output (CO) in the larvae were measured and assessed from 20-s video segments collected from individual embryos following the method of Chen et al. (2008). SV was calculated by using the equations SV = EDV－ESV, and CO was calculated as: CO = SV ×HR. Cardiac arrhythmia was obtained by determining the interbeat variability (Incardona et al., 2009). Using NIS-Elements Imaging
2.7. Data processing Results are reported as means ± SE (standard error). After the homogeneity of variances being determinated, the data were statistically analyzed with one-way analysis of variance (ANOVA) followed by the Duncan test via SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). A value of P < 0.05 was used to indicate significant difference. 294
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Fig. 1. Developmental effects of zebrafish embryos exposed to fenbuconazole. A. Survival rate at 72 h; B. Hatching rate at 48 h. C. Pericardial edema rate at 72 h; D. Spinal deformation rate at 72 h. Results are reported as mean ± SE (n = 5). Means of exposures not sharing a common letter are significantly different at P < 0.05 as assessed by one-way ANOVA followed by the Duncan test.
3. Results
including the neurocranium bones (ethmoid plate and ceratohyal) and the craniofacial bones [Meckel's cartilage (lower jaw), and ceratobranchial (branchial arches)] (Fig. 4). The lengths of palatoquadrate and mandibular arch length were significantly decreased in the FBZ treated fish; the lengths of ethmoid plate and ceratohyal were significantly reduced in the 500 ng/L treatment; the lower jaw length was significantly decreased, while the lower jaw width was significantly increased in the 500 ng/L group compared to the control (Table 1).
3.1. Solvent effects Since the solvent treatment showed no significant alteration in all tested indices compared to the blank control, the change fold of these indices was calculated compared to the solvent treatment. 3.2. Developmental effects of FBZ
3.5. Quantitative analysis of transcript levels of selected genes
The survival rate of larvae exposure to FBZ for 72 h (Fig. 1A) and the hatching rate at 48 hpf (Fig. 1B) showed no significant change in the all treatments compared to the solvent control (Fig. 1A). Pericardial edema rate and spinal curvature rate were significantly increased (by 1.59 and 2.50-fold) in the 500 ng/L group (Fig. 1C and D).
A total of 11 genes were selected for analysis by qPCR: FBZ exposure did not cause significant alteration in the mRNA levels of genes myh6 and ihh. The mRNA levels of nkx2.5 (1.56 and 1.83-fold), gata4 (1.85, 1.54-fold) and cdh2 (1.58 and1.61-fold) was significantly up-regulated in the 50 and 500 ng/L groups compared to the control, the mRNA expression of tnnt2 (1.85-fold) was significantly up-regulated in the 50 ng/L group, the transcription levels of cyp26a1 (1.61-fold), myl7 (2.68-fold), bmp2b (1.86 -fold) were significantly up-regulated in the 500 ng/L group, and that of shh (3.22, 3.33 and 3.52-fold) was significantly up-regulated in the 5, 50 and 500 ng/L groups. However, that of tbx5 was significantly down-regulated (by 39%) in the 500 ng/L group (Fig. 5).
3.3. Effects on cardiac development and function Obvious abnormalities in the heart morphology, including cardiac looping defects, were observed in the larvae treated with FBZ for 72 h (Fig. 2). Untreated fish showed a normal looped atria and ventricles, while FBZ-treated larvae displayed a separated atria and ventricles without overlapping (Fig. 2). FBZ-treated larvae showed no significant change in EDV and ESV compared to the solvent control (Fig. 3A). A significant reduction in SV and CO (by 41% and 27%) was observed in the 500 ng/L group (Fig. 3B and C). The heart rate was significantly increased (by 1.22-fold) in the 500 ng/L group (Fig. 3D). A very low interbeat variability ( ± 3.5 ms) was showed in the control group, while the 500 ng/L FBZ-treated larvae displayed a significant increased irregularity of the rhythm with a mean interbeat variability ( ± 5.7 ms) (Fig. 3E).
3.6. Deformities in adults after embryonic exposure to fenbuconazole for 72 h An increased degree of jaw deformities and spinal curvature was observed at 90 days old adults after embryonic exposure to FBZ, and jaw deformity mainly exhibited a longer jaw than the normal fish (Fig. 6A). The percentages of elongated jaw and spinal curvature were increased with increased FBZ concentration (Fig. 6B).
3.4. Effects on craniofacial cartilage development The larvae from the solvent group displayed a normal skeleton
Fig. 2. Effects of fenbuconazole on the larval heart morphology. Fertilized embryos were exposed to fenbuconazole for 72 h. The heart malformation was enhanced with increasing concentration of fenbuconazole. Abbreviations: A, Atrium; V, Ventricle. Scale bar = 100 µm. 295
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Fig. 3. Cardiac function in zebrafish larvae after exposure to fenbuconazole for 72 h. A. Volume of the ventricle at end-diastole (EDV) and end-systole (ESV); B. Stroke volume; C. Cardiac output; D. Heart rate; E. Cardiac arrhythmia. Each bar indicates the mean ± SE (n = 5). Means of exposures not sharing a common letter are significantly different at P < 0.05 as assessed by one-way ANOVA followed by the Duncan test.
Fig. 4. Craniofacial skeletal defects in zebrafish larvae after exposure to fenbuconazole for 72 h. Ventral views of larval heads (A, B, C, D, E). Lateral views of larval heads (a, b, c, d, e). Control embryos displayed a normal craniofacial cartilage; exposure to fenbuconazole led a shorter lower jaw. Abbreviations: mc, Meckel´s cartilage (lower jaw); cb, ceratobranchial; ch, ceratohyal;ep, ethmoid plate (neurocranium). A, Mandibular arch length; B, Lower jaw width; C, Lower jaw (Meckel's cartilage) length. Scale bar = 100 µm.
increased and hatchability is decreased with concentrations > 600 μg/ L (Chu et al., 2016). Exposure to 1–15 mg/L five triazole fungicides (myclobutanil, fluconazole, flusilazole, triflumizole and epoxiconazole) alone for 72 h increases malformation and mortality, and decreases body length, body weight, and heart rate in rare minnow (Gobiocypris rarus) embryos in a concentration-dependent manner (Zhu et al., 2014). Exposure of zebrafish embryos to difenoconazole (DFZ) (0.5–3 mg/L)
4. Discussion The embryonic developmental stage of fish is vulnerable to chemicals. Some investigations have revealed the adverse developmental effects of triazole fungicides on fishes. Embryonic exposure to triadimenol within 3 μg/L and 3 mg/L causes no developmental deformities in hatchlings of medaka fish (Oryzias latipes), while hatching time is
Table 1 The morphometric parameters of the craniofacial skeleton in zebrafish larvae exposed to fenbuconazole for 72 h. Group
Morphometric parameters (μm) pq
blank solvent 5 ng/L 50 ng/L 500 ng/L
179.8 181.0 154.6 150.0 122.6
ep ± ± ± ± ±
1.6 2.1 4.8 5.6 3.0
a a b b c
ch a
107.8 ± 2.3 106.8 ± 1.4 a 108.2 ± 3.0 a 105.2 ± 1.7 a 95.8 ± 1.6 b
173.6 170.8 172.6 166.4 151.6
hm ± ± ± ± ±
2.2 2.4 2.5 1.5 2.4
a
105.0 101.2 104.6 100.6 101.8
a a a b
A ± ± ± ± ±
2.1 4.0 1.8 1.3 1.8
a a a a a
385.8 386.2 364.0 350.6 288.0
B ± ± ± ± ±
a
4.6 4.0a 8.0b 8.8b 6.4c
130.8 132.8 136.4 140.8 162.2
C ± ± ± ± ±
a
1.2 1.8a 1.9ab 1.8ab 2.6b
85.0 85.8 83.6 71.8 64.2
± ± ± ± ±
2.6a 1.6a 2.3a 1.5ab 1.6b
Data are presented as mean ± S.E. (n = 5). Means of exposures not sharing a common letter are significantly different at p < 0.05 as assessed by one-way ANOVA followed by the Duncan test. Pq: palatoquadrate; ep: ethmoid plate; ch: ceratohyal; hm: hyomandibular; A: Mandibular arch length; B: Lower jaw width; C: Lower jaw (Meckel's cartilage) length. 296
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Fig. 5. The transcription of genes in zebrafish larvae after embryonic exposure to fenbuconazole for 72 h. Values were normalized against gapdh. Results are reported as mean ± SE (n = 5). Means of exposures not sharing a common letter are significantly different at P < 0.05 as assessed by one-way ANOVA followed by the Duncan test.
2016). In zebrafish embryos exposed to DFZ (0.5 mg/L), the transcription of genes related to embryonic development, such as gh, tyr, igf1, bmp2 and bmp4, is decreased, and genes related to hatching (he1a), retinoic acid metabolism (cyp26a1) and lipid homeostasis (hmgcra, cyp51, fas, acc1 and ppara1) are up-regulated (Mu et al., 2016). Zebrafish cardiac development and morphogenesis are regulated by some critical genes and factors. Tbx5 and Nkx2.5, two crucial transcription factors, can affect conduction system development and cardiac morphogenesis (Takeuchi and Bruneau, 2009; Balci and Akdemir, 2011). Tbx5 is required for heart development (Hiroi et al., 2001) and cardiac conduction system function (Moskowitz et al., 2004) and for contracting cardiomyocytes differentiation (Takeuchi and Bruneau, 2009). Nkx2.5 plays a key role in the initiation of cardiogenic differentiation program and determination of myocardial cell fate (Balci and Akdemir, 2011). In zebrafish, Bmp2b promotes myocardial differentiation via regulating nkx2.5 expression (Reiter et al., 2001). Gata4 has an obvious function in cardiac morphogenesis, loss of Gata4 destroys late cardiac morphogenetic movements (Holtzinger and Evans, 2005). Both cardiac troponin T2 (encoded by tnnt2) and myosin light polypeptide 7 (encode by myl7) are necessary for heart muscle differentiation and functioning in zebrafish (Maves et al., 2009). Cadherin2 is a cell-adhesion molecule playing an important role in zebrafish cardiovascular development, the disruption of cadherin2 function impairs myocardiocyte differentiation and physiological cardiovascular performance (Bagatto et al., 2006). Some studies showed that contaminants such as polycyclic aromatic hydrocarbons (Zhang et al., 2012b) and hexabromocyclododecane (Wu et al., 2013) resulted in the defects of cardiac development and function in zebrafish embryos through disturbing the transcription of these genes. In the present study, the mRNA levels of genes including tbx5, nkx2.5, tnnt2, gata4, bmp2b, myh6, myl7, were disturbed by FBZ exposure, which would be responsible for the cardiac developmental and functioning defects. The development of endochondral bone is regulated by many signaling molecules including hedgehog (Hh) family and bone morphogenetic proteins (BMPs) (Havens et al., 2008; Mak et al., 2008; Reddi, 1994). Hedgehog (Hh) family, including three members: Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog, play an important role in endochondral bone formation during embryonic development (Nie et al., 2005). Hh signaling plays multiple roles in the development of the anterior craniofacial skeleton (Eberhart et al., 2006). Shh is required to specify the movements of progenitors of two main cartilage elements (the paired trabeculae and the midline ethmoid) at the midline, and to induce them to form cartilage (Wada et al., 2005). BMPs are key regulators in bone formation in vertebrates. BMP2 is directly involved in the early formation of the dorsum (Rafael et al., 2006). Photolytic compounds of pesticide methoprene induce malformation of zebrafish by depressing Shh expression (Smith et al., 2003). A down-regulation of Shh expression in rockfish embryos might be associated with craniofacial skeleton defects induced by tributyltin (Zhang et al., 2012a). Spine curvature is occurred with down-regulation
Fig. 6. Deformities in adult zebrafish after embryonic exposure to fenbuconazole for 72 h. A. Showing tail curvature and longer jaw (arrows); B. The percentages of the deformities. Scale bar = 5 mm.
induces abnormal developmental symptoms, including hatching inhibition, decreased spontaneous movement, heart rate reduction, and morphological malformation (Mu et al., 2013, 2015). Triadimenfon (TDF) can disrupt embryonic thyroid endocrine function in zebrafish (Cao et al., 2016). Exposure of zebrafish embryos to hexaconazole (HEX) (0.625, 1.25 and 2.5 mg/L) and tebuconazole (1, 2 and 4 mg/L) significantly decreases whole body levels of thyroxine (T4) and significantly increases triiodothyronine (T3) levels (Yu et al., 2013). In the present study, the observed effect concentration of FBZ for morphological malformation was 500 ng/L, which was much lower than that of the previous studies, suggesting that the developmental toxicity of FBZ should be highly concerned. Some studies concerning the mechanisms by which triazole fungicides influence the development of fish are available. Six triazole fungicides, including flusilazole, triticonazole, cyproconazole, myclobutanil, HEX and TDF result in a different degree of regulation of genes related to developmental toxicity, steroid biosynthesis and retinol metabolism in the zebrafish embryos (Hermsen et al., 2012). Zebrafish embryos treated with 2.5, 5 and 10 μg/mL TDF for 24 h and 72 h exhibit malformations such as smaller head, shorter tail, and cardiac edema, and the differential expression of some genes involved in many metabolic/signaling pathways are exploited using RNA-seq (Hsu et al., 297
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of bmp2 and bmp4 in zebrafish embryos after 1 mg/L polychlorinated biphenyls exposure (Ju et al., 2011). Exposure to 0.5 and 2.0 mg/L DFZ for 96 h reduces the mRNA levels of bmp2 and bmp4, which may have a negative effect on bone development (Mu et al., 2016). In the present study, the deformation in the bone development might be related with the impaired transcription levels of Shh and bmp2. However, it is interesting that the jaw (Meckel's cartilage) length was decreased in the larvae after exposure to DFZ while was increased in the adults. To our knowledge, it is the first time to observe this reversal outcome in adults resulted from the developmental effects of fish embryos. This was similar to the “catch-up growth” phenomenon, which is a compensatory acceleration in growth and development in animals following release from stressed states or adverse environments. The mechanism involved needs to be further investigated. CYP26A1 is an important retinoic acid metabolism enzyme, the upregulation of cyp26a1 always causes the decrease of retinoic acid (Chen et al., 2009; Sun et al., 2015). Hepatic trans-retinoic acid levels are decreased by TDF, PCZ or myclobutanil, while cyp26a1 gene is overexpressed in the livers of TDF or PCZ-treated mice (Chen et al., 2009). The expression level of cyp26a1 is up-regulated in zebrafish embryos exposed to TDF (10 μg/mL) from fertilization to 72 hpf (Hsu et al., 2016). The mRNA of cyp26a1 in zebrafish embryos is significantly increased after exposure to flusilazole and cyproconazole (Hermsen et al., 2011). Pericardial edema of zebrafish embryos is occurred with the upregulation of cyp26a1 (Wang et al., 2013). Zebrafish embryos exposed to 2.0 mg/L DFZ exhibits a significant correlation between increased transcription of cyp26a11 and the rate of pericardial edema (Mu et al., 2016). In flounder (Paralichthys olivaceus) at the hatching period, retinoic acid treatment disturbs pharyngeal cartilage development by affecting the expression of Shh at the pharyngeal endoderm (Suzuki et al., 1998, 1999). In this study, FBZ exposure up-regulated the transcription of cyp26a1, which was in line with the previous results, suggesting that the teratogenic effect of FBZ may relate to the increase in cyp26a1 transcription. In summary, our results showed that waterborne exposure to FBZ at low concentrations could cause adverse effects on zebrafish embryos development, including pericardial edema, cardiac dysfunction and teratogenic effects. qPCR analysis showed that FBZ exposure altered the transcription of genes related to cardiac development and function, and bone formation. The morphological, functional and molecular results indicated that the significant effects almost appeared in the 500 ng/L treatment, which would provide a referential concentration for FBZ risk assessment.
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Acknowledgments This work was supported by the National Natural Science Foundation of China (41376118). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2018.04.048. References Braunbeck, T., Kais, B., Lammer, E., Otte, J., Schneider, K., Stengel, D., Strecker, R., 2015. The fish embryo test (FET): origin, applications, and future. Environ. Sci. Pollut. Res. 22, 16247–16261. Bagatto, B., Francl, J., Liu, B., Liu, Q., 2006. Cadherin2 (N-cadherin) plays an essential role in zebrafish cardiovascular development. BMC Dev. Biol. 6, 23. Balci, M.M., Akdemir, R., 2011. NKX2.5 mutations and congenital heart disease: is it a marker of cardiac anomalies? Int. J. Cardiol. 147, 44–45. Cao, C., Wang, Q., Jiao, F., Zhu, G., 2016. Impact of co-exposure with butachlor and triadimefon on thyroid endocrine system in larval zebrafish. Exp. Toxicol. Pathol. 68, 463–469. Chen, P.J., Padgett, W.T., Moore, T., Winnik, W., Lambert, G.R., Thai, S.F., Hester, S.D., Nesnow, S., 2009. Three conazoles increase hepatic microsomal retinoic acid
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C.J., 2016. The developmental effect of difenoconazole on zebrafish embryos: a mechanism research. Environ. Pollut. 212, 18–26. Mu, X.Y., Pang, S., Sun, X.Z., Gao, J.J., Chen, J.Y., Chen, X.F., Li, X.F., Wang, C.J., 2013. Evaluation of acute and developmental effects of difenoconazole via multiple stage zebrafish assays. Environ. Pollut. 175, 147–157. Nie, X., Luukko, K., Kvinnsland, I.H., Kettunen, P., 2005. Developmentally regulated expression of Shh and Ihh in the developing mouse cranial base: comparison with Sox9 expression. Anat. Rec. A: Discov. Mol. Cell Evol. Biol. 286, 891–898. Pesticide Properties Data Base, 〈http://sitem.herts.ac.uk/aeru/ppdb/en/Reports/293. htm〉. Pfaffl, M.W., Horgan, G.W., Dempfle, L., 2002. Relative expression software tool (REST ©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30 (9), e36. Rafael, M.S., Laizé, V., Cancela, M.L., 2006. Identification of Sparus aurata bone morphogenetic protein 2: molecular cloning, gene expression and in silico analysis of protein conserved features in vertebrates. Bone 39, 1373–1381. Reddi, A.H., 1994. Bone and cartilage differentiation. Curr. Opin. Genet. Dev. 4, 737–744. Reiter, J.F., Verkade, H., Stainier, D.Y.R., 2001. Bmp2b and Oep promote early myocardial differentiation through their regulation of gata5. Dev. Biol. 234, 330–338. Smith, G.D., Wilburn, C., McCarthy, R.M., 2003. Methoprene photolytic compounds disrupt zebrafish development, producing phenocopies of mutants in the sonic hedgehog signaling pathway. Mar. Biotechnol. 5, 201–212. Sobeiha, A.K., Abdel-Megeed, M.I., Dahroug, S.M.A., Eklo, O.M., Hanafi, A., 2009. Disappearance of azoxystrobin, cyprodinil, fludioxonil, fenhexamid and myclobutanil in strawberry fruits under field conditions. Arab Univ. J. Agric. Sci. 17, 239–245. Sun, B., Song, S., Hao, C.Z., Huang, W.X., Liu, C.C., Xie, H.L., Lin, B., Cheng, M.S., Zhao, D.M., 2015. Molecular recognition of CYP26A1 binding pockets and structure-activity relationship studies for design of potent and selective retinoic acid metabolism blocking agents. J. Mol. Graph. Model 56, 10–19. Suzuki, T., Oohara, I., Kurokawa, T., 1998. Hoxd-4 expression during pharyngeal arch development in flounder (Paralichthys olivaceus) embryos and effects of retinoic acid on expression. Zool. Sci. 15, 57–67. Suzuki, T., Oohara, I., Kurokawa, T., 1999. Retinoic acid given at late embryonic stage depresses sonic hedgehog and Hoxd-4 expression in the pharyngeal area and induces
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Fenbuconazole exposure impacts the development of zebrafish embryos b
a
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a
a
Yuqiong Wu , Qihong Yang , Meng Chen , Ying Zhang , Zhenghong Zuo , Chonggang Wang a b c
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State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China Wuyi University, College of Tea and Food Science, Wuyishan, Fujian 354300, China Key Laboratory of Ministry of Education for Subtropical Wetland Ecosystem Research, Xiamen University, Xiamen, Fujian 361005, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Triazole fungicide Fish Embryonic development Mechanism
Fenbuconazole (FBZ), a triazole-containing fungicide, is widely used in agriculture and horticulture. In the present study, the development and cardiac functioning were observed and determined in zebrafish embryos exposed to FBZ at 5, 50 and 500 ng/L nominal concentrations for 72 h. The results showed that 500 ng/L FBZ significantly increased pericardial edema rate, spine curvature rate, disturbed cardiac function, and led a shortened lower jaw. The transcription of genes such as tbx5, nkx2.5, tnnt2, gata4, bmp2b, myl7 was altered, which might be responsible for the cardiac developmental and functioning defects in the larvae. The deformation in bone development might be related with the impaired transcription levels of shh and bmp2b. The transcription of cyp26a1 (encoding retinoic acid metabolism enzyme) was significantly up-regulated in the 500 ng/L group, which might be a reason causing the teratogenic effect of FBZ. These results suggest that FBZ could have toxic effects on embryonic development, which should be considered in the risk evaluation of FBZ application.
1. Introduction Among pesticides currently in use, triazole-containing fungicides are one of the most widely applied (Konwick et al., 2006; Goetz et al., 2007). Fenbuconazole (FBZ) is used in agriculture and horticulture against powdery mildew of vegetables, cereal and fruits (Holb and Schnabel, 2007; Sobeiha et al., 2009). Since its long biodegradation half-life with 61-day under field soil under anaerobic conditions (Pesticide Properties Data Base), FBZ could persist in the environment for a long period. After 190 d of incubation with soil, only a small amount of total FBZ (∼46.8% or ∼36.4% for Langfang or Changsha soil, respectively) disappeared (Li et al., 2012). The detected enantiomers of FBZ range from 10.49 to 23.54 μg/kg in soil samples collected from Langfang, China (Li et al., 2012). Thus, FBZ may contaminate aquatic environments through run-off. FBZ concentration detected in two waterworks of Xiamen city, China ranged from 0.22 to 6.98 ng/L (our unpublished data), indicating that the Jiulong river, from which these two waterworks got water source, was contaminated by FBZ. There is concern regarding potential exposure of fishes from its residues in aquatic environment (Li et al., 2012). The detected residues of FBZ in fruits such as oranges, pears, peaches and grapes range from 0.11 to 0.34 mg/kg (EFSA, 2015). People are also probably exposed to FBZ via food uptake. The WHO (1997) established an Acceptable Daily Intake for humans of 0.03 mg/kg/day, and maximum admissible concentration in drinking water is 0.1 μg/L (EFSA, 2015). Due to its wildly
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Corresponding author. E-mail address: [email protected] (C. Wang).
https://doi.org/10.1016/j.ecoenv.2018.04.048 Received 12 February 2018; Received in revised form 16 April 2018; Accepted 21 April 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
use, the toxic effects need to be adequately researched. Existing reports show that FBZ seems to be low toxicity to animals. Acute 96 h LC50 of FBZ to fish is 1.5 mg/L; acute oral LD50 to mammals is > 2000 mg/kg (Pesticide Properties Data Base: Fenbuconazole). FBZ is not genotoxic (Federal Register, 2002), studies in rodent show that it does not induce liver tumor but is associated with a slight increase in the incidence of liver adenomas (Lassalle et al., 2015). Mice treated with FBZ exhibit a reversible change of liver including increased liver weight and histological damages (Juberg et al., 2006). FBZ perturbs thyroid-pituitary functioning and induces thyroid tumors in rodent (Hurley et al., 1998). However, there are few studies concerning the developmental effects of FBZ on fishes. As a vertebrate model, zebrafish (Danio rerio) has numerous advantages such as small size, ease of culture, high fecundity, morphological and physiological similarities to mammals. The optical clarity of the chorion is in favor of the observation of embryogenesis and the assessment of endpoints of toxicity (Yang et al., 2009; He et al., 2014). The molecular weight (336.82 Da) of FBZ much lower than the threshold (3000 Da) (Braunbeck et al., 2015) allows its passage across the chorion. The rapid development of zebrafish involves all organic effects, and the genetic basis of development has been extensively studied, providing many benefits to mechanism assay (Fernández et al., 2018). These characteristics suggest that zebrafish embryos/larvae are an ideal research model for investigating the developmental effects of chemicals. In the present study, the influence of FBZ on the
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Software (Nikon, Tokyo, Japan), the number of frames between cardiac contraction initiations was calculated to obtain a mean and standard deviations (SD) for each larva. Since a regular rhythm would have a low SD, this SD is a measure of heart rate irregularity. Three larvae from each replicate were assessed to get a mean for this replicate. The means from five replicates were used for analysis.
development of zebrafish embryos was researched, and the mechanisms involved were investigated. 2. Materials and methods 2.1. Zebrafish maintenance and embryos collection All fish experiments followed the ethical guidelines of Xiamen University. Twenty wild-type TU zebrafish were housed in 20 L tanks with dechlorinated and aerated freshwater in a recirculation system at 28 ± 1 °C, under a normal photoperiod at 14:10 h light: dark. The pH and dissolved oxygen were 7.2–7.3 and 7–8 mg/L respectively. Fish were fed with live brine shrimp and commercial fish diet twice daily. Sexually mature fish without any signs of disease were selected as breeders. Adult fish were mated at a ratio of 1:2 (female: male). Spawned eggs were collected within 0.5 h. Fertilized eggs were washed with zebrafish facility water and randomly distributed to multiple petri dishes for exposure experiments.
2.4. Whole mount Alcian-blue staining
2.2. Embryonic exposure and sampling
Fifty larvae from each replicate were pooled into a subsample. Total RNA was isolated by a Trizol Kit (TaKaRa, Dalian, China). The mRNA expression was determined based on the previously described method (Huang et al., 2012). QPCR analysis was performed on an Mx3000P Real-Time PCR system (Stratagene, La Jolla, CA, USA) using the Brilliant SYBR Green QPCR reagent kit (Stratagene) following the manufacturer's protocol. Gene expression levels were normalized to zebrafish gapdh. The real time quantitative PCR primers (Table S1) were designed using Primer Premier 5.0. Threshold cycles (Ct) and dissociation curves analysis of the amplification products were determined with MxPro software (Stratagene), to confirm that only one PCR product was amplified and detected. The Relative Expression Software Tool (RESTMCS_-version 2) was used to calculate the relative expression of target gene mRNA (Pfaffl et al., 2002). Three housekeeping genes (gapdh, αtubulin, β-actin) were analyzed for suitable reference gene, the results showed that FBZ exposure did not alter the expression of gapdh (supplemental material). Therefore, gene expression levels were normalized to zebrafish gapdh.
Exposed larvae were randomly chosen in each treatment for Alcianblue staining to observe the craniofacial cartilage development. Alcianblue staining was followed the method described by Walker and Kimmel (2007). Stained larvae were observed using a Leica M165FC stereo fluorescence microscope, and the morphology of the craniofacial cartilage was analyzed following the method of Carvan et al. (2004). Three larvae from each replicate were assessed to get a mean for each replicate. 2.5. Real-time quantitative PCR (qPCR)
FBZ (purity > 98%) was purchased from Agro-Environmental Protection Institute, Ministry of Agriculture, China. It was dissolved in acetone at analytic grade to reach stock concentrations of 5, 50 and 500 μg/mL. FBZ exposure solutions were obtained by adding 1 μL the stock concentration to 1 L zebrafish culture medium (3.5 g/L NaCl, 0.05 g/L NaHCO3, 0.05 g/LKCl, 0.05 g/L CaCl2). Eggs were collected after oviposition and remove coagulated eggs and debris. Eggs at 0.5–1 h post-fertilization (hpf) were exposed to nominal concentrations of FBZ (0, 5, 50 and 500 ng/L). One hundred fertilized eggs were cultured in 30 mL exposure solution in petri dish at 28 ± 1 °C. Similar criterion was applied to solvent control group, which received an equal volume of acetone (1 μl/L). There were five replicates for each treatment. The test solutions were renewed twice daily, and dead embryos were removed. The development of the embryos and mortality was monitored with an Olympus SZ51 stereo microscope every 24 h. The rates of malformation, such as dorsal curvature, pericardial edema were calculated as: malformation rate = malformed embryos number / survival embryos number × 100%; the hatching success (embryonic membrane opening and larva swimming up) of the embryo were assessed: hatched rate = hatched embryos number / total embryos number × 100%. After exposure for 72 h, the embryos were collected for analysis. The remainder larvae after exposure for 72 h were transferred to clean water. Since the remaining amount of fish is small, they were put together from the five replicates and cultured in an aquatic system. Larvae within 7-day post-fertilization (dpf) occupied 10 mL water per fish in beak; and at 30 dpf, 400 mL in transparent fish tank. Fish at 60 dpf were maintained in the Aquatic Habitats Zebrafish System up to 90 days and occupied 1 L water per fish. The culture conditions were the same as those mentioned above. The numbers of remainder adult fish at 90 d were 28–47 in each treatment, whose morphological changes were observed and the occurrence rates of deformation were recorded.
2.6. Determination of FBZ in exposure solutions Exposure solutions, freshly made up with the stock solutions, were collected three times at random for determination. The FBZ concentrations were measured based on the method of Zhang et al. (2017) with slight modification. Briefly, 1 L exposure solution was added with simeconazole (purity > 97%) (Witega Laboratories Berlin-Adlershof GmbH, Berlin, Germany) as a surrogate, and was extracted using a liquid-liquid extraction method with 50 mL CH2Cl2 in a separatory funnel. The organic phase was collected and dried with anhydrous sodium sulfate, and the extracts were concentrated to dryness under a rotary evaporator, and then were diluted with acetone/n-hexane (1:1) solution. FBZ concentration was detected using a GC/MS/MS system (Agilent Technology, USA) following the description of Zhang et al. (2017). The recoveries of FBZ were 94% ± 0.6% (n = 3) and the limit of detection was 3.0 ng/L. The detected concentration of FBZ in the exposure medium was 0, 4.96 ± 0.09, 48.29 ± 0.41 and 473.58 ± 9.13 ng/L in the each group.
2.3. Cardiac function analysis The heart rate and cardiac arrhythmia of the exposed larvae were assayed based on the method of Incardona et al. (2009). The end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV) and cardiac output (CO) in the larvae were measured and assessed from 20-s video segments collected from individual embryos following the method of Chen et al. (2008). SV was calculated by using the equations SV = EDV－ESV, and CO was calculated as: CO = SV ×HR. Cardiac arrhythmia was obtained by determining the interbeat variability (Incardona et al., 2009). Using NIS-Elements Imaging
2.7. Data processing Results are reported as means ± SE (standard error). After the homogeneity of variances being determinated, the data were statistically analyzed with one-way analysis of variance (ANOVA) followed by the Duncan test via SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). A value of P < 0.05 was used to indicate significant difference. 294
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Fig. 1. Developmental effects of zebrafish embryos exposed to fenbuconazole. A. Survival rate at 72 h; B. Hatching rate at 48 h. C. Pericardial edema rate at 72 h; D. Spinal deformation rate at 72 h. Results are reported as mean ± SE (n = 5). Means of exposures not sharing a common letter are significantly different at P < 0.05 as assessed by one-way ANOVA followed by the Duncan test.
3. Results
including the neurocranium bones (ethmoid plate and ceratohyal) and the craniofacial bones [Meckel's cartilage (lower jaw), and ceratobranchial (branchial arches)] (Fig. 4). The lengths of palatoquadrate and mandibular arch length were significantly decreased in the FBZ treated fish; the lengths of ethmoid plate and ceratohyal were significantly reduced in the 500 ng/L treatment; the lower jaw length was significantly decreased, while the lower jaw width was significantly increased in the 500 ng/L group compared to the control (Table 1).
3.1. Solvent effects Since the solvent treatment showed no significant alteration in all tested indices compared to the blank control, the change fold of these indices was calculated compared to the solvent treatment. 3.2. Developmental effects of FBZ
3.5. Quantitative analysis of transcript levels of selected genes
The survival rate of larvae exposure to FBZ for 72 h (Fig. 1A) and the hatching rate at 48 hpf (Fig. 1B) showed no significant change in the all treatments compared to the solvent control (Fig. 1A). Pericardial edema rate and spinal curvature rate were significantly increased (by 1.59 and 2.50-fold) in the 500 ng/L group (Fig. 1C and D).
A total of 11 genes were selected for analysis by qPCR: FBZ exposure did not cause significant alteration in the mRNA levels of genes myh6 and ihh. The mRNA levels of nkx2.5 (1.56 and 1.83-fold), gata4 (1.85, 1.54-fold) and cdh2 (1.58 and1.61-fold) was significantly up-regulated in the 50 and 500 ng/L groups compared to the control, the mRNA expression of tnnt2 (1.85-fold) was significantly up-regulated in the 50 ng/L group, the transcription levels of cyp26a1 (1.61-fold), myl7 (2.68-fold), bmp2b (1.86 -fold) were significantly up-regulated in the 500 ng/L group, and that of shh (3.22, 3.33 and 3.52-fold) was significantly up-regulated in the 5, 50 and 500 ng/L groups. However, that of tbx5 was significantly down-regulated (by 39%) in the 500 ng/L group (Fig. 5).
3.3. Effects on cardiac development and function Obvious abnormalities in the heart morphology, including cardiac looping defects, were observed in the larvae treated with FBZ for 72 h (Fig. 2). Untreated fish showed a normal looped atria and ventricles, while FBZ-treated larvae displayed a separated atria and ventricles without overlapping (Fig. 2). FBZ-treated larvae showed no significant change in EDV and ESV compared to the solvent control (Fig. 3A). A significant reduction in SV and CO (by 41% and 27%) was observed in the 500 ng/L group (Fig. 3B and C). The heart rate was significantly increased (by 1.22-fold) in the 500 ng/L group (Fig. 3D). A very low interbeat variability ( ± 3.5 ms) was showed in the control group, while the 500 ng/L FBZ-treated larvae displayed a significant increased irregularity of the rhythm with a mean interbeat variability ( ± 5.7 ms) (Fig. 3E).
3.6. Deformities in adults after embryonic exposure to fenbuconazole for 72 h An increased degree of jaw deformities and spinal curvature was observed at 90 days old adults after embryonic exposure to FBZ, and jaw deformity mainly exhibited a longer jaw than the normal fish (Fig. 6A). The percentages of elongated jaw and spinal curvature were increased with increased FBZ concentration (Fig. 6B).
3.4. Effects on craniofacial cartilage development The larvae from the solvent group displayed a normal skeleton
Fig. 2. Effects of fenbuconazole on the larval heart morphology. Fertilized embryos were exposed to fenbuconazole for 72 h. The heart malformation was enhanced with increasing concentration of fenbuconazole. Abbreviations: A, Atrium; V, Ventricle. Scale bar = 100 µm. 295
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Fig. 3. Cardiac function in zebrafish larvae after exposure to fenbuconazole for 72 h. A. Volume of the ventricle at end-diastole (EDV) and end-systole (ESV); B. Stroke volume; C. Cardiac output; D. Heart rate; E. Cardiac arrhythmia. Each bar indicates the mean ± SE (n = 5). Means of exposures not sharing a common letter are significantly different at P < 0.05 as assessed by one-way ANOVA followed by the Duncan test.
Fig. 4. Craniofacial skeletal defects in zebrafish larvae after exposure to fenbuconazole for 72 h. Ventral views of larval heads (A, B, C, D, E). Lateral views of larval heads (a, b, c, d, e). Control embryos displayed a normal craniofacial cartilage; exposure to fenbuconazole led a shorter lower jaw. Abbreviations: mc, Meckel´s cartilage (lower jaw); cb, ceratobranchial; ch, ceratohyal;ep, ethmoid plate (neurocranium). A, Mandibular arch length; B, Lower jaw width; C, Lower jaw (Meckel's cartilage) length. Scale bar = 100 µm.
increased and hatchability is decreased with concentrations > 600 μg/ L (Chu et al., 2016). Exposure to 1–15 mg/L five triazole fungicides (myclobutanil, fluconazole, flusilazole, triflumizole and epoxiconazole) alone for 72 h increases malformation and mortality, and decreases body length, body weight, and heart rate in rare minnow (Gobiocypris rarus) embryos in a concentration-dependent manner (Zhu et al., 2014). Exposure of zebrafish embryos to difenoconazole (DFZ) (0.5–3 mg/L)
4. Discussion The embryonic developmental stage of fish is vulnerable to chemicals. Some investigations have revealed the adverse developmental effects of triazole fungicides on fishes. Embryonic exposure to triadimenol within 3 μg/L and 3 mg/L causes no developmental deformities in hatchlings of medaka fish (Oryzias latipes), while hatching time is
Table 1 The morphometric parameters of the craniofacial skeleton in zebrafish larvae exposed to fenbuconazole for 72 h. Group
Morphometric parameters (μm) pq
blank solvent 5 ng/L 50 ng/L 500 ng/L
179.8 181.0 154.6 150.0 122.6
ep ± ± ± ± ±
1.6 2.1 4.8 5.6 3.0
a a b b c
ch a
107.8 ± 2.3 106.8 ± 1.4 a 108.2 ± 3.0 a 105.2 ± 1.7 a 95.8 ± 1.6 b
173.6 170.8 172.6 166.4 151.6
hm ± ± ± ± ±
2.2 2.4 2.5 1.5 2.4
a
105.0 101.2 104.6 100.6 101.8
a a a b
A ± ± ± ± ±
2.1 4.0 1.8 1.3 1.8
a a a a a
385.8 386.2 364.0 350.6 288.0
B ± ± ± ± ±
a
4.6 4.0a 8.0b 8.8b 6.4c
130.8 132.8 136.4 140.8 162.2
C ± ± ± ± ±
a
1.2 1.8a 1.9ab 1.8ab 2.6b
85.0 85.8 83.6 71.8 64.2
± ± ± ± ±
2.6a 1.6a 2.3a 1.5ab 1.6b
Data are presented as mean ± S.E. (n = 5). Means of exposures not sharing a common letter are significantly different at p < 0.05 as assessed by one-way ANOVA followed by the Duncan test. Pq: palatoquadrate; ep: ethmoid plate; ch: ceratohyal; hm: hyomandibular; A: Mandibular arch length; B: Lower jaw width; C: Lower jaw (Meckel's cartilage) length. 296
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Fig. 5. The transcription of genes in zebrafish larvae after embryonic exposure to fenbuconazole for 72 h. Values were normalized against gapdh. Results are reported as mean ± SE (n = 5). Means of exposures not sharing a common letter are significantly different at P < 0.05 as assessed by one-way ANOVA followed by the Duncan test.
2016). In zebrafish embryos exposed to DFZ (0.5 mg/L), the transcription of genes related to embryonic development, such as gh, tyr, igf1, bmp2 and bmp4, is decreased, and genes related to hatching (he1a), retinoic acid metabolism (cyp26a1) and lipid homeostasis (hmgcra, cyp51, fas, acc1 and ppara1) are up-regulated (Mu et al., 2016). Zebrafish cardiac development and morphogenesis are regulated by some critical genes and factors. Tbx5 and Nkx2.5, two crucial transcription factors, can affect conduction system development and cardiac morphogenesis (Takeuchi and Bruneau, 2009; Balci and Akdemir, 2011). Tbx5 is required for heart development (Hiroi et al., 2001) and cardiac conduction system function (Moskowitz et al., 2004) and for contracting cardiomyocytes differentiation (Takeuchi and Bruneau, 2009). Nkx2.5 plays a key role in the initiation of cardiogenic differentiation program and determination of myocardial cell fate (Balci and Akdemir, 2011). In zebrafish, Bmp2b promotes myocardial differentiation via regulating nkx2.5 expression (Reiter et al., 2001). Gata4 has an obvious function in cardiac morphogenesis, loss of Gata4 destroys late cardiac morphogenetic movements (Holtzinger and Evans, 2005). Both cardiac troponin T2 (encoded by tnnt2) and myosin light polypeptide 7 (encode by myl7) are necessary for heart muscle differentiation and functioning in zebrafish (Maves et al., 2009). Cadherin2 is a cell-adhesion molecule playing an important role in zebrafish cardiovascular development, the disruption of cadherin2 function impairs myocardiocyte differentiation and physiological cardiovascular performance (Bagatto et al., 2006). Some studies showed that contaminants such as polycyclic aromatic hydrocarbons (Zhang et al., 2012b) and hexabromocyclododecane (Wu et al., 2013) resulted in the defects of cardiac development and function in zebrafish embryos through disturbing the transcription of these genes. In the present study, the mRNA levels of genes including tbx5, nkx2.5, tnnt2, gata4, bmp2b, myh6, myl7, were disturbed by FBZ exposure, which would be responsible for the cardiac developmental and functioning defects. The development of endochondral bone is regulated by many signaling molecules including hedgehog (Hh) family and bone morphogenetic proteins (BMPs) (Havens et al., 2008; Mak et al., 2008; Reddi, 1994). Hedgehog (Hh) family, including three members: Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog, play an important role in endochondral bone formation during embryonic development (Nie et al., 2005). Hh signaling plays multiple roles in the development of the anterior craniofacial skeleton (Eberhart et al., 2006). Shh is required to specify the movements of progenitors of two main cartilage elements (the paired trabeculae and the midline ethmoid) at the midline, and to induce them to form cartilage (Wada et al., 2005). BMPs are key regulators in bone formation in vertebrates. BMP2 is directly involved in the early formation of the dorsum (Rafael et al., 2006). Photolytic compounds of pesticide methoprene induce malformation of zebrafish by depressing Shh expression (Smith et al., 2003). A down-regulation of Shh expression in rockfish embryos might be associated with craniofacial skeleton defects induced by tributyltin (Zhang et al., 2012a). Spine curvature is occurred with down-regulation
Fig. 6. Deformities in adult zebrafish after embryonic exposure to fenbuconazole for 72 h. A. Showing tail curvature and longer jaw (arrows); B. The percentages of the deformities. Scale bar = 5 mm.
induces abnormal developmental symptoms, including hatching inhibition, decreased spontaneous movement, heart rate reduction, and morphological malformation (Mu et al., 2013, 2015). Triadimenfon (TDF) can disrupt embryonic thyroid endocrine function in zebrafish (Cao et al., 2016). Exposure of zebrafish embryos to hexaconazole (HEX) (0.625, 1.25 and 2.5 mg/L) and tebuconazole (1, 2 and 4 mg/L) significantly decreases whole body levels of thyroxine (T4) and significantly increases triiodothyronine (T3) levels (Yu et al., 2013). In the present study, the observed effect concentration of FBZ for morphological malformation was 500 ng/L, which was much lower than that of the previous studies, suggesting that the developmental toxicity of FBZ should be highly concerned. Some studies concerning the mechanisms by which triazole fungicides influence the development of fish are available. Six triazole fungicides, including flusilazole, triticonazole, cyproconazole, myclobutanil, HEX and TDF result in a different degree of regulation of genes related to developmental toxicity, steroid biosynthesis and retinol metabolism in the zebrafish embryos (Hermsen et al., 2012). Zebrafish embryos treated with 2.5, 5 and 10 μg/mL TDF for 24 h and 72 h exhibit malformations such as smaller head, shorter tail, and cardiac edema, and the differential expression of some genes involved in many metabolic/signaling pathways are exploited using RNA-seq (Hsu et al., 297
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of bmp2 and bmp4 in zebrafish embryos after 1 mg/L polychlorinated biphenyls exposure (Ju et al., 2011). Exposure to 0.5 and 2.0 mg/L DFZ for 96 h reduces the mRNA levels of bmp2 and bmp4, which may have a negative effect on bone development (Mu et al., 2016). In the present study, the deformation in the bone development might be related with the impaired transcription levels of Shh and bmp2. However, it is interesting that the jaw (Meckel's cartilage) length was decreased in the larvae after exposure to DFZ while was increased in the adults. To our knowledge, it is the first time to observe this reversal outcome in adults resulted from the developmental effects of fish embryos. This was similar to the “catch-up growth” phenomenon, which is a compensatory acceleration in growth and development in animals following release from stressed states or adverse environments. The mechanism involved needs to be further investigated. CYP26A1 is an important retinoic acid metabolism enzyme, the upregulation of cyp26a1 always causes the decrease of retinoic acid (Chen et al., 2009; Sun et al., 2015). Hepatic trans-retinoic acid levels are decreased by TDF, PCZ or myclobutanil, while cyp26a1 gene is overexpressed in the livers of TDF or PCZ-treated mice (Chen et al., 2009). The expression level of cyp26a1 is up-regulated in zebrafish embryos exposed to TDF (10 μg/mL) from fertilization to 72 hpf (Hsu et al., 2016). The mRNA of cyp26a1 in zebrafish embryos is significantly increased after exposure to flusilazole and cyproconazole (Hermsen et al., 2011). Pericardial edema of zebrafish embryos is occurred with the upregulation of cyp26a1 (Wang et al., 2013). Zebrafish embryos exposed to 2.0 mg/L DFZ exhibits a significant correlation between increased transcription of cyp26a11 and the rate of pericardial edema (Mu et al., 2016). In flounder (Paralichthys olivaceus) at the hatching period, retinoic acid treatment disturbs pharyngeal cartilage development by affecting the expression of Shh at the pharyngeal endoderm (Suzuki et al., 1998, 1999). In this study, FBZ exposure up-regulated the transcription of cyp26a1, which was in line with the previous results, suggesting that the teratogenic effect of FBZ may relate to the increase in cyp26a1 transcription. In summary, our results showed that waterborne exposure to FBZ at low concentrations could cause adverse effects on zebrafish embryos development, including pericardial edema, cardiac dysfunction and teratogenic effects. qPCR analysis showed that FBZ exposure altered the transcription of genes related to cardiac development and function, and bone formation. The morphological, functional and molecular results indicated that the significant effects almost appeared in the 500 ng/L treatment, which would provide a referential concentration for FBZ risk assessment.
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