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Assessment of immunotoxicity of dibutyl phthalate using live zebrafish embryos Hai Xu a, *, Xing Dong a, Zhen Zhang a, Ming Yang b, Xiangyang Wu a, Hongcui Liu c, Qiaocong Lao c, Chunqi Li c a b c
School of Environmental and Safety Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu Province, China School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, China Hunter Biotechnology, Inc., Hangzhou 311231, Zhejiang Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 January 2015 Received in revised form 16 April 2015 Accepted 27 April 2015 Available online xxx
This study set out to understand the immune-toxic effects of dibutyl phthalate (DBP) using transgenic, albino or AB line zebrafish. Zebrafish embryos were exposed to different concentrations of DBP, and the immune cells formation, phagocytosis ability were measured after a short-term exposure to DBP for 6 h post-fertilization (hpf) to 72 or 96 hpf. Exposure to DBP was found to inhibit the neutrophils and macrophage formation in a concentration-dependent manner. The ability of macrophage phagocytosis was all decreased after exposure to DBP, indicating the occurrence of immunotoxicity. The respiratory burst was induced, and the transcription levels of T/B cell-related genes rag1/2 were up-regulated. The overall results indicate that DBP in aquatic environment greatly influence the immune system in fish, and zebrafish embryos can serve as a reliable model for the developmental immunotoxicity of toxicchemicals. © 2015 Published by Elsevier Ltd.
Keywords: Immune cells Dibutyl phthalate Phthalate esters Zebrafish
1. Introduction Dibutyl phthalate (DBP) is a member of a class of compounds called phthalate esters (PAEs), which widely used as plasticizer in a variety of consumer products such as sealants, paints, adhesives, cosmetics and food packaging [1,2]. According to a reported data from European Chemicals Agency (ECHA), DBP is identified as a substance of very high concern (SVHC) due to its high volume, wide dispersive uses and toxic properties [3]. The report indicated the total manufactured poundage of DBP was less than 22 million pounds in 2007 and it has been ubiquitously diffused in the environment via the manufacturing process. A large number of studies have detected DBP contamination in various aquatic environments at mg/L to even mg/L or mg/kg [4e7], therefore, its detrimental effects on aquatic animals have also raised great concern about the healthy and sustainable development of aquatic organisms. In the past decades, much attention has been given to the finding that PAEs adversely affect multiple biological processes, including growth, metabolism and endocrine system function, in * Corresponding author. School of Environmental Safety and Engineering, Jiangsu University, Xuefu Road 301, Zhenjiang 212013, Jiangsu Province, China. E-mail address: [email protected] (H. Xu).
mammals and teleosts [8]. However, study on their detrimental effects on immune system in fish is still scarce. The immune system including in fish consists of a variety of immune defense mechanisms. Chemicals can impair the immune defense of exposed organisms through these mechanisms, including interference with signaling pathways in immune cells, suppressing immune functions such as oxidative burst activity, induction of apoptosis, and so on. There is evidence that some PAEs can damage immune system in aquatic organisms. For example, Sung et al. reported eight PAEs, including DBP, could damage haemocytes of prawns and increase expression of immune mediators of shrimp [9,10]. Our previous study also indicated DBP could impact immune-related gene expression [11]. Therefore, it is of great importance to explore the immunotoxicity in aquatic organisms to monitor and predict the toxicological risk of PAEs. The zebrafish model has been suggested recently as a suitable organism for studying immune-relevant processes. Zebrafish embryos possess a functional innate immune system after one day of development, comprised primarily of embryonic neutrophils and macrophages in the embryonic blood circulation [12]. To address the question if DBP exposure could modulate immune cells, we decide to analyze neutrophils and macrophage using transgenic and albino zebrafish. The transgenic zebrafish express fluorescent
http://dx.doi.org/10.1016/j.fsi.2015.04.033 1050-4648/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: H. Xu, et al., Assessment of immunotoxicity of dibutyl phthalate using live zebrafish embryos, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.033
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reporter gene in individual neutrophils of specific lineages, whereas the albino zebrafish are optically transparent during whole developmental stage which is conductive to detect macrophages. On the other hand, the adaptive immune system is not functionally active in zebrafish during the first 3 weeks of development [13], depends on T and B lymphocytes which are specific for particular antigens. To evaluate if DBP could affect the adaptive immune system, we detected the expression of T/B cells relevant gene recombination activating gene 1 and 2 (rag1/2). Additionally, respiratory burst was assayed in live embryos. Our results suggest that DBP have the potential to effect on immune system in zebrafish during the early developmental stage. Our works should provide new insights into the toxicological effects of the PAEs on the immune response of fish embryo. 2. Materials and methods 2.1. Animals and embryo collection Three lines of zebrafish were used in this study: wild-type AB line, albino type (alb-1/alb-1) [14], and Tg(mpx:GFP). All fish were cultured at 28 ± 0.5 C in a 14 h light: 10 h dark cycle in a flowthrough system in dechlorinated tap water and fed with brine shrimp (Artemia nauplii) twice daily. Zebrafish embryos were obtained from spawning adults in tanks overnight with a sex ratio of 1:1. Embryos were collected within an hour of fertilization and rinsed in aquarium water (dissolved oxygen 6.8e8.3 mg/L, pH 6.9e7.2). Embryonic development was monitored and staged according to the previously described method [15]. The zebrafish facility at Hunter Biotechnology, Inc. is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. 2.2. DBP exposure DBP (CAS#84-74-2, purity > 99%, SigmaeAldrich) was dissolved in dimethyl sulfoxide (DMSO) to obtain stock solution of 10 g/L and stored at 4 C. Working solution was prepared by dilution of the stock solution immediately prior to experimental use. Normal embryos at 6 h post-fertilization (hpf) were randomly distributed into 6-well plate (10 embryos per well with 5 ml solution) and exposed to DBP at 0.02, 0.2, 2 mM, with triplicate at each treatment concentration. The concentrations were selected according to previous studies [11]. The final concentration of DMSO was 0.1% across all treatment groups. The solvent control group (vehicle) received 0.1% DMSO (v/v), while the aqueous control received dechlorinated tap water only. All tested embryos were growing at 28 ± 0.5 C in a 14 h light: 10 h dark cycle. The exposure was performed semi-statically, and fresh solutions were replaced completely every 24 h. 2.3. Neutrophils formation Neutrophils formtion was addressed by analyzing with a fluorescence stereoscope, the displacement of GFP positive cells in Tg(mpx:GFP) from the caudal hematopoietic tissue to the tail at 72 hpf. Quantification of fluorescence was accomplished using an inverse thresholding function and particle counting with ImageJ (NIH, Bethesda, MD). Fluorescence in DBP-treated zebrafish was compared with control zebrafish treated with carrier (DMSO) alone. 2.4. Macrophage formation Macrophage formation was detected by analyzing with a fluorescence stereoscope, the macrophage in albino zebrafish embryos were stained with neutral red at 96 hpf. Quantification of
fluorescence was accomplished using an inverted fluorescence microscope and particle counting with ImageJ (NIH, Bethesda, MD). Images were analyzed for total fluorescent area above threshold in head area. Fluorescence in DBP-treated zebrafish was compared with control zebrafish treated with carrier (DMSO) alone. 2.5. Macrophage phagocytosis assay To determine whether DBP exposure affect the ability of macrophage phagocytosis, albino line zebrafish embryos were microinjected with India ink at 72 hpf, and thereafter exposed to DBP at above concentrations until 96 hpf. Then, embryos were stained with neutral red, washed and imaged with 1 h of staining. Quantification of fluorescence was accomplished using an inverted fluorescence microscope and particle counting with ImageJ (NIH, Bethesda, MD). Images were analyzed for total fluorescent area above threshold in head area. Fluorescence in DBP-treated zebrafish was compared with control zebrafish treated with carrier (DMSO) alone. 2.6. Respiratory burst assay using whole zebrafish embryos The method for measuring the respiratory burst was modified from Hermann et al. [16]. Briefly, AB line zebrafish embryos were exposed by static immersion from 6 to 96 hpf at DBP concentrations of 0.02, 0.2, and 2 mM. Then, the production of reactive oxygen species (ROS) was measured in whole embryos, using 96-well microplates in which each well contained one embryo in 100 mL of egg water. To each well containing one embryo, 100 mL of 1 mg/mL dihydrodichlorofluorescein diacetate (H2DCFDA) in 20 ng/mL phorbol-12-myristate-13-acetate (PMA) were added to each well to a final concentration of 500 ng/mL H2DCFDA and 10 ng/mL PMA. Fluorescence was measured in a microplate reader with excitation and emission filters set at 485 and 535 nm, respectively. Data from eight individual embryos were averaged for each group. Data were also normalized to background fluorescence to permit calculation of fold induction. 2.7. Analysis of T/B cell relevant gene expression after exposure of fish to DBP For real-time quantitative PCR analysis, the total RNA was extracted from 20 homogenized zebrafish larvae using the RNAprep pure Tissure kit (TIANGEN Biotech, Shanghai, China) according to the manufacturer's protocol. The quality and quantity of RNA were detemined by UV spectrophotometry and by 1% agarose gel electrophoresis. For each sample, cDNA was synthesized from 100 ng total RNA using PrimeScript™ RT Reagent Kit (TaKaRa). The primers of rag1, rag2 and b-actin were designed by Primer 5.0. The primer sequences for rag1: forward-ggaaggactgagagagagggcgc, reverseaggaccagatccgtgcttctcg. The primer sequences for rag2: forwardgctcatgtccaactgggatatttgg, reverse-ctcgtggataccgcaagattc. The primer sequences for b-actin: forward-tcgagcaggagatgggaacc, reverse-ctcgtggataccgcaagattc. Quantitative real-time PCR was performed using the SYBE® Premix Ex Taq™ II kit (TaKaRa) and a CFX96™ Real-Time PCR Detection System (Bio-Rad, USA). The reaction mix consisted of 10 mL of SYBE II Mix, 0.5 mM of each primer, and 1 mL of cDNA. The PCR reaction cycle was as follows: 95 C for 3 min, followed by 40 cycles of 95 C for 10 s, 60 C for 30 s, and the fluorescent signal were measured at the annealing/extension step. Melt curve analyses were performed to validate the specificity of the PCR amplicons. A Ct-based relative quantification with efficiency correction normalizing to b-actin was calculated by the 2 DDCt method [17].
Please cite this article in press as: H. Xu, et al., Assessment of immunotoxicity of dibutyl phthalate using live zebrafish embryos, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.033
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Fig. 1. The developmental effects of DBP on zebrafish embryos. Yolk sac resorption and swim bladder inflation were inhibited in the 2 mM DBP-treated groups. Y: yolk sac; S: swim bladder.
Fig. 2. Neutrophils migration in zebrafish embryos exposed to DBP. Incubation of Tg(mpx:GFP) transgenic larvae (that express GFP exclusively in neutrophils) in DBP induce a progressive migration of neutrophils from to the caudal hematopoietic tissue to the entire tail. (A) Close up of the tail of control and experimental embryos at 72 hpf. (B) Quantification of neutrophils migration into the selected area. (C) The numbers of neutrophils that presented the phenotype shown is expressed as a percentage. For all experiments, at least 25 larvae were used for each condition. * indicated significant difference at p < 0.05, and ** indicated significant difference at p < 0.01.
Please cite this article in press as: H. Xu, et al., Assessment of immunotoxicity of dibutyl phthalate using live zebrafish embryos, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.033
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2.8. Statistical analysis Data are shown as the mean ± standard error of the mean (S.E.M.). All data were checked for normality and homogeneity of variance using KolmogoroveSmirnov one-sample test and Levene's test. The average fluorescent area was determined and compared between compound treated and vehicles with two-sample T-test assuming unequal variances. Intergroup differences were assessed using one-way analysis of variance (ANOVA) followed by Dunette's test, using SPSS Statistics 18 (SPSS Inc., Chicago, IL, USA). The level for statistical significance was set at p < 0.05 or 0.01 and indicated by “*” or “**”. 3. Results and discussion There were no statistically significant differences in hatching and survival rates among the treatment groups. However, some developmental abnormalities, such as yolk sac resorption and swim bladder inflation were inhibited in the 2 mM DBP-treated groups (Fig. 1). These results suggested DBP induced severe developmental toxicity in live embryos at high concentration. 3.1. DBP affects innate immune cells in zebrafish embryos To evaluate the possibilities that DBP inhibited neutrophils formation, we took advantage of the Tg(mpx:GFP) transgenic line, referred to as mpx from this point forward, which expresses GFP
under the control of the myeloperoxidase entire regulatory region and allows tracking individual immune cells in live animals. We monitored the number of GFP-positive heterophil granulocytes bodies in the tail of mpx zebrafish at 72 hpf utilizing a fluorescence stereoscope. We focused on the larval tail because in this region neutrophils are normally restricted to the caudal hematopoietic tissue and only a few are circulating. As illustrated in Fig. 2, exposure from 6 to 72 hpf to DBP at 0.2 and 2 mM had significantly inhibited the apparent density of neutrophils bodies. Quantification of the number of GFP-positive neutrophile granulocytes per hemisegment confirmed that DBP can alter the density of neutrophils in the tail of zebrafish. As shown in Fig. 3, exposure to DBP caused a decrease in macrophage formation in a concentration-dependent manner. The quantitative number of macrophage was greatly decreased in 0.2 and 2 mM DBP group, respectively. Phagocytosis reflects the ability of disease resistance and immune response in animals. We evaluate the phagocytotic ability of macrophage using ink phagocytosis test. Our results showed that the phagocytosis of phagocytes was greatly reduced in 0.2 and 2 mM DBP (Fig. 4). Quantification of the number of ink signals confirmed that DBP can inhibit the phagocytotic ability of macrophage in the head of zebrafish. In some instances, low exposure concentrations or short exposure times can result in an immuno-stimulatory or immunosuppressive effect; in the present study, acute DBP exposure reduced the phagocytotic ability of macrophage in zebrafish
Fig. 3. Macrophages formation in zebrafish embryos exposed to DBP. Zebrafish were stained with neutral red, and dorsal images of the head were acquired using a fluorescent scope with the same exposure time and gain. (A) Close up of the head of control and experimental embryos at 72 hpf. (B) Quantification of macrophages formation in the selected area. (C) The numbers of macrophages that presented the phenotype shown is expressed as a percentage. For all experiments, at least 30 larvae were used for each condition. *** indicated significant difference at p < 0.001.
Please cite this article in press as: H. Xu, et al., Assessment of immunotoxicity of dibutyl phthalate using live zebrafish embryos, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.033
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Fig. 4. Macrophage phagocytosis in zebrafish embryos exposed to DBP. Zebrafish were stained with neutral red, and dorsal images of the head were acquired using a fluorescent scope with the same exposure time and gain. (A) Ink signals of the head of control and experimental embryos at 96 hpf. (B) Quantification of ink signals in the selected area. (C) The numbers of ink signals that presented the phenotype shown is expressed as a percentage. For all experiments, at least 30 larvae were used for each condition. * indicated significant difference at p < 0.05, ** indicated significant difference at p < 0.01, *** indicated significant difference at p < 0.001.
embryos. A short-term, low-dose suppression of phagocytosis has also been reported in aquatic organisms response to contaminants including pesticides [18] and polycyclic aromatic hydrocarbons (PAHs) [19,20]. According to previous studies, phagocytotic processes are dependent upon the membrane properties of immune cells; environmental pollutants can interfere with the fluidity of cell membranes [21,22], restricting the deformation of the membrane essential to the phagocytic endocytosis process [23]. The reduced phagocytosis observed here, suggesting that reduced cell membranes may have contributed towards the lower phagocytotic ability. 3.2. Respiratory burst in live zebrafish embryos The embryos were stimulated with PMA, to induce ROS production via the respiratory burst. The results showed that PMAinduced ROS was inhibited in 0.2 mM DBP-treated groups compared to vehicle. However, there was a slight increase in 2 mM DBP treatments (Fig. 5). The respiratory burst response, a measure of the immune health of an organism, is often used to assess the immune-toxic effects of environmental toxicants. Both stimulatory and inhibitory effects of
these substances on respiratory burst activity have been described. The heavy metal arsenic, when administered to zebrafish, decreased respiratory burst activity [24]; however, the endocrine disruptor bisphenol A and nonylphenol-exposed zebrafish showed an increase in ROS production [25,26]. The proliferation of ROS has been suggested as a mechanism of contaminant toxicity in exposed organisms, with previous studies suggesting the involvement of ROS as a critical mediator in toxin-induced immune insult. Our results showed that DBP exerts seemingly consistent effects on ROS production in connection with the respiratory burst response as well. In our previous study, a slight increase was detected in ROS production by biochemical assay in zebrafish embryos exposed to DBP. The present study showed that DBP induces ROS production in whole live zebrafish larvae. 3.3. T/B cell relevant gene expression Rag1 and rag2 play an essential role in developing adaptive immunity, since rag genes catalyze the rearrangement of immunoglobulin genes in immature B lymphocytes and of T cell receptor genes in immature T lymphocytes and are therefore appropriate
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Fig. 5. Respiratory burst activity of PMA-stimulated and DBP-treated whole zebrafish embryos, measured by oxidation of H2DCFDA to DCF. (A) Relative fluorescence units in zebrafish embryos. (B) Fold induction of respiratory burst. As fold-induction of respiratory burst activity of DBP-treated embryos, the embryos were pretreated with 0.02, 0.2 and 2 mM DBP prior to adding of PMA and H2DCFDA.
Fig. 6. Expression of rag1 (A) and rag2 (B) in zebrafish larvae after exposure to various concentrations of DBP. The results are means ± S.E.M. of triplicate samples. ** indicated significant difference at p < 0.01.
markers to follow the development of organs containing these cells. Consistent with their essential function in the immune system, they are highly conserved among different species. Currently, rag genes were only identified as lymphoid-specific genes in zebrafish. Our previous studies have reported DBP could disrupt the transcript levels of innate immune genes such as IFNg and IL1b in zebrafish embryos [11]. In order to complement our previous results, we decided to monitor at molecular level the progression of the immune disruption by evaluating several adaptive immune markers rag1 and rag2 by qPCR. We evaluated the well-known immune markers at 72 hpf. The results showed that a significant increase in the transcript levels of rag1 at the highest exposure concentration; however, no significant changes in the rag2 expression in all treated groups (Fig. 6). This finding is interesting because DBP has been shown to be estrogenic [27]. It is unclear whether DBP effects rag1 expression via an estrogen-dependent pathway and needs further study. In fact, estrogens are known to be involved in immune process [28]. Therefore, the induction of rag1 gene expression in this study indicated that the adaptive immune process has been already triggered at high dose of DBP. 4. Conclusions In the present study, we assess the immune-toxicological effects of DBP using wild-type, transgenic and albino zebrafish. The exposure of DBP to zebrafish embryos during their early developmental stages led to the decrease of immune cell formation and function, increase of transcription levels of T/B cell-related gene.
Our results revealed a susceptibility of the immune system in zebrafish embryos to DBP, and thus, the transgenic and albino zebrafish should be useful for assessing immunotoxicity of PAEs in the aquatic environment. Acknowledgments This work was supported financially by the National Natural Science Foundation of China (31100376), China Postdoctoral Science Foundation (2013M541618), Research Foundation for Advanced Talents of Jiangsu University (13JDG014) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Q1 References [1] C.A. Staples, D.R. Peterson, T.F. Parkerton, W.J. Adams, The environmental fate of phthalate esters: a literature review, Chemosphere 35 (1997) 667e749. [2] T.F. Parkerton, W.J. Konkel, Application of quantitative structure-activity relationships for assessing the aquatic toxicity of phthalate esters, Ecotoxicol Environ Saf 45 (2000) 61e78. [3] European Chemicals Agency, Background document for dibutyl phthalate (DBP), 1 June 2009. [4] P.C. Huang, C.J. Tien, Y.M. Sun, C.Y. Hsieh, C.C. Lee, Occurrence of phthalates in sediment and biota: relationship to aquatic factors and the biota-sediment accumulation factor, Chemosphere 73 (2008) 539e544. [5] F. Zeng, J.X. Wen, K.Y. Cui, L.N. Wu, M. Liu, Y.J. Li, et al., Seasonal distribution of phthalate esters in surface water of the urban lakes in the subtropical city, Guangzhou, China, J Hazard Mater 169 (2009) 719e725. [6] W.J.G.M. Peijnenburg, J. Struijs, Occurrence of phthalate esters in the environment of the Netherlands, Ecotoxicol Environ Saf 63 (2006) 204e215.
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Contents lists available at ScienceDirect
Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short communication
Q3 Q2
Assessment of immunotoxicity of dibutyl phthalate using live zebrafish embryos Hai Xu a, *, Xing Dong a, Zhen Zhang a, Ming Yang b, Xiangyang Wu a, Hongcui Liu c, Qiaocong Lao c, Chunqi Li c a b c
School of Environmental and Safety Engineering, Jiangsu University, Zhenjiang 212013, Jiangsu Province, China School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, China Hunter Biotechnology, Inc., Hangzhou 311231, Zhejiang Province, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 January 2015 Received in revised form 16 April 2015 Accepted 27 April 2015 Available online xxx
This study set out to understand the immune-toxic effects of dibutyl phthalate (DBP) using transgenic, albino or AB line zebrafish. Zebrafish embryos were exposed to different concentrations of DBP, and the immune cells formation, phagocytosis ability were measured after a short-term exposure to DBP for 6 h post-fertilization (hpf) to 72 or 96 hpf. Exposure to DBP was found to inhibit the neutrophils and macrophage formation in a concentration-dependent manner. The ability of macrophage phagocytosis was all decreased after exposure to DBP, indicating the occurrence of immunotoxicity. The respiratory burst was induced, and the transcription levels of T/B cell-related genes rag1/2 were up-regulated. The overall results indicate that DBP in aquatic environment greatly influence the immune system in fish, and zebrafish embryos can serve as a reliable model for the developmental immunotoxicity of toxicchemicals. © 2015 Published by Elsevier Ltd.
Keywords: Immune cells Dibutyl phthalate Phthalate esters Zebrafish
1. Introduction Dibutyl phthalate (DBP) is a member of a class of compounds called phthalate esters (PAEs), which widely used as plasticizer in a variety of consumer products such as sealants, paints, adhesives, cosmetics and food packaging [1,2]. According to a reported data from European Chemicals Agency (ECHA), DBP is identified as a substance of very high concern (SVHC) due to its high volume, wide dispersive uses and toxic properties [3]. The report indicated the total manufactured poundage of DBP was less than 22 million pounds in 2007 and it has been ubiquitously diffused in the environment via the manufacturing process. A large number of studies have detected DBP contamination in various aquatic environments at mg/L to even mg/L or mg/kg [4e7], therefore, its detrimental effects on aquatic animals have also raised great concern about the healthy and sustainable development of aquatic organisms. In the past decades, much attention has been given to the finding that PAEs adversely affect multiple biological processes, including growth, metabolism and endocrine system function, in * Corresponding author. School of Environmental Safety and Engineering, Jiangsu University, Xuefu Road 301, Zhenjiang 212013, Jiangsu Province, China. E-mail address: [email protected] (H. Xu).
mammals and teleosts [8]. However, study on their detrimental effects on immune system in fish is still scarce. The immune system including in fish consists of a variety of immune defense mechanisms. Chemicals can impair the immune defense of exposed organisms through these mechanisms, including interference with signaling pathways in immune cells, suppressing immune functions such as oxidative burst activity, induction of apoptosis, and so on. There is evidence that some PAEs can damage immune system in aquatic organisms. For example, Sung et al. reported eight PAEs, including DBP, could damage haemocytes of prawns and increase expression of immune mediators of shrimp [9,10]. Our previous study also indicated DBP could impact immune-related gene expression [11]. Therefore, it is of great importance to explore the immunotoxicity in aquatic organisms to monitor and predict the toxicological risk of PAEs. The zebrafish model has been suggested recently as a suitable organism for studying immune-relevant processes. Zebrafish embryos possess a functional innate immune system after one day of development, comprised primarily of embryonic neutrophils and macrophages in the embryonic blood circulation [12]. To address the question if DBP exposure could modulate immune cells, we decide to analyze neutrophils and macrophage using transgenic and albino zebrafish. The transgenic zebrafish express fluorescent
http://dx.doi.org/10.1016/j.fsi.2015.04.033 1050-4648/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: H. Xu, et al., Assessment of immunotoxicity of dibutyl phthalate using live zebrafish embryos, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.033
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reporter gene in individual neutrophils of specific lineages, whereas the albino zebrafish are optically transparent during whole developmental stage which is conductive to detect macrophages. On the other hand, the adaptive immune system is not functionally active in zebrafish during the first 3 weeks of development [13], depends on T and B lymphocytes which are specific for particular antigens. To evaluate if DBP could affect the adaptive immune system, we detected the expression of T/B cells relevant gene recombination activating gene 1 and 2 (rag1/2). Additionally, respiratory burst was assayed in live embryos. Our results suggest that DBP have the potential to effect on immune system in zebrafish during the early developmental stage. Our works should provide new insights into the toxicological effects of the PAEs on the immune response of fish embryo. 2. Materials and methods 2.1. Animals and embryo collection Three lines of zebrafish were used in this study: wild-type AB line, albino type (alb-1/alb-1) [14], and Tg(mpx:GFP). All fish were cultured at 28 ± 0.5 C in a 14 h light: 10 h dark cycle in a flowthrough system in dechlorinated tap water and fed with brine shrimp (Artemia nauplii) twice daily. Zebrafish embryos were obtained from spawning adults in tanks overnight with a sex ratio of 1:1. Embryos were collected within an hour of fertilization and rinsed in aquarium water (dissolved oxygen 6.8e8.3 mg/L, pH 6.9e7.2). Embryonic development was monitored and staged according to the previously described method [15]. The zebrafish facility at Hunter Biotechnology, Inc. is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. 2.2. DBP exposure DBP (CAS#84-74-2, purity > 99%, SigmaeAldrich) was dissolved in dimethyl sulfoxide (DMSO) to obtain stock solution of 10 g/L and stored at 4 C. Working solution was prepared by dilution of the stock solution immediately prior to experimental use. Normal embryos at 6 h post-fertilization (hpf) were randomly distributed into 6-well plate (10 embryos per well with 5 ml solution) and exposed to DBP at 0.02, 0.2, 2 mM, with triplicate at each treatment concentration. The concentrations were selected according to previous studies [11]. The final concentration of DMSO was 0.1% across all treatment groups. The solvent control group (vehicle) received 0.1% DMSO (v/v), while the aqueous control received dechlorinated tap water only. All tested embryos were growing at 28 ± 0.5 C in a 14 h light: 10 h dark cycle. The exposure was performed semi-statically, and fresh solutions were replaced completely every 24 h. 2.3. Neutrophils formation Neutrophils formtion was addressed by analyzing with a fluorescence stereoscope, the displacement of GFP positive cells in Tg(mpx:GFP) from the caudal hematopoietic tissue to the tail at 72 hpf. Quantification of fluorescence was accomplished using an inverse thresholding function and particle counting with ImageJ (NIH, Bethesda, MD). Fluorescence in DBP-treated zebrafish was compared with control zebrafish treated with carrier (DMSO) alone. 2.4. Macrophage formation Macrophage formation was detected by analyzing with a fluorescence stereoscope, the macrophage in albino zebrafish embryos were stained with neutral red at 96 hpf. Quantification of
fluorescence was accomplished using an inverted fluorescence microscope and particle counting with ImageJ (NIH, Bethesda, MD). Images were analyzed for total fluorescent area above threshold in head area. Fluorescence in DBP-treated zebrafish was compared with control zebrafish treated with carrier (DMSO) alone. 2.5. Macrophage phagocytosis assay To determine whether DBP exposure affect the ability of macrophage phagocytosis, albino line zebrafish embryos were microinjected with India ink at 72 hpf, and thereafter exposed to DBP at above concentrations until 96 hpf. Then, embryos were stained with neutral red, washed and imaged with 1 h of staining. Quantification of fluorescence was accomplished using an inverted fluorescence microscope and particle counting with ImageJ (NIH, Bethesda, MD). Images were analyzed for total fluorescent area above threshold in head area. Fluorescence in DBP-treated zebrafish was compared with control zebrafish treated with carrier (DMSO) alone. 2.6. Respiratory burst assay using whole zebrafish embryos The method for measuring the respiratory burst was modified from Hermann et al. [16]. Briefly, AB line zebrafish embryos were exposed by static immersion from 6 to 96 hpf at DBP concentrations of 0.02, 0.2, and 2 mM. Then, the production of reactive oxygen species (ROS) was measured in whole embryos, using 96-well microplates in which each well contained one embryo in 100 mL of egg water. To each well containing one embryo, 100 mL of 1 mg/mL dihydrodichlorofluorescein diacetate (H2DCFDA) in 20 ng/mL phorbol-12-myristate-13-acetate (PMA) were added to each well to a final concentration of 500 ng/mL H2DCFDA and 10 ng/mL PMA. Fluorescence was measured in a microplate reader with excitation and emission filters set at 485 and 535 nm, respectively. Data from eight individual embryos were averaged for each group. Data were also normalized to background fluorescence to permit calculation of fold induction. 2.7. Analysis of T/B cell relevant gene expression after exposure of fish to DBP For real-time quantitative PCR analysis, the total RNA was extracted from 20 homogenized zebrafish larvae using the RNAprep pure Tissure kit (TIANGEN Biotech, Shanghai, China) according to the manufacturer's protocol. The quality and quantity of RNA were detemined by UV spectrophotometry and by 1% agarose gel electrophoresis. For each sample, cDNA was synthesized from 100 ng total RNA using PrimeScript™ RT Reagent Kit (TaKaRa). The primers of rag1, rag2 and b-actin were designed by Primer 5.0. The primer sequences for rag1: forward-ggaaggactgagagagagggcgc, reverseaggaccagatccgtgcttctcg. The primer sequences for rag2: forwardgctcatgtccaactgggatatttgg, reverse-ctcgtggataccgcaagattc. The primer sequences for b-actin: forward-tcgagcaggagatgggaacc, reverse-ctcgtggataccgcaagattc. Quantitative real-time PCR was performed using the SYBE® Premix Ex Taq™ II kit (TaKaRa) and a CFX96™ Real-Time PCR Detection System (Bio-Rad, USA). The reaction mix consisted of 10 mL of SYBE II Mix, 0.5 mM of each primer, and 1 mL of cDNA. The PCR reaction cycle was as follows: 95 C for 3 min, followed by 40 cycles of 95 C for 10 s, 60 C for 30 s, and the fluorescent signal were measured at the annealing/extension step. Melt curve analyses were performed to validate the specificity of the PCR amplicons. A Ct-based relative quantification with efficiency correction normalizing to b-actin was calculated by the 2 DDCt method [17].
Please cite this article in press as: H. Xu, et al., Assessment of immunotoxicity of dibutyl phthalate using live zebrafish embryos, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.033
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Fig. 1. The developmental effects of DBP on zebrafish embryos. Yolk sac resorption and swim bladder inflation were inhibited in the 2 mM DBP-treated groups. Y: yolk sac; S: swim bladder.
Fig. 2. Neutrophils migration in zebrafish embryos exposed to DBP. Incubation of Tg(mpx:GFP) transgenic larvae (that express GFP exclusively in neutrophils) in DBP induce a progressive migration of neutrophils from to the caudal hematopoietic tissue to the entire tail. (A) Close up of the tail of control and experimental embryos at 72 hpf. (B) Quantification of neutrophils migration into the selected area. (C) The numbers of neutrophils that presented the phenotype shown is expressed as a percentage. For all experiments, at least 25 larvae were used for each condition. * indicated significant difference at p < 0.05, and ** indicated significant difference at p < 0.01.
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2.8. Statistical analysis Data are shown as the mean ± standard error of the mean (S.E.M.). All data were checked for normality and homogeneity of variance using KolmogoroveSmirnov one-sample test and Levene's test. The average fluorescent area was determined and compared between compound treated and vehicles with two-sample T-test assuming unequal variances. Intergroup differences were assessed using one-way analysis of variance (ANOVA) followed by Dunette's test, using SPSS Statistics 18 (SPSS Inc., Chicago, IL, USA). The level for statistical significance was set at p < 0.05 or 0.01 and indicated by “*” or “**”. 3. Results and discussion There were no statistically significant differences in hatching and survival rates among the treatment groups. However, some developmental abnormalities, such as yolk sac resorption and swim bladder inflation were inhibited in the 2 mM DBP-treated groups (Fig. 1). These results suggested DBP induced severe developmental toxicity in live embryos at high concentration. 3.1. DBP affects innate immune cells in zebrafish embryos To evaluate the possibilities that DBP inhibited neutrophils formation, we took advantage of the Tg(mpx:GFP) transgenic line, referred to as mpx from this point forward, which expresses GFP
under the control of the myeloperoxidase entire regulatory region and allows tracking individual immune cells in live animals. We monitored the number of GFP-positive heterophil granulocytes bodies in the tail of mpx zebrafish at 72 hpf utilizing a fluorescence stereoscope. We focused on the larval tail because in this region neutrophils are normally restricted to the caudal hematopoietic tissue and only a few are circulating. As illustrated in Fig. 2, exposure from 6 to 72 hpf to DBP at 0.2 and 2 mM had significantly inhibited the apparent density of neutrophils bodies. Quantification of the number of GFP-positive neutrophile granulocytes per hemisegment confirmed that DBP can alter the density of neutrophils in the tail of zebrafish. As shown in Fig. 3, exposure to DBP caused a decrease in macrophage formation in a concentration-dependent manner. The quantitative number of macrophage was greatly decreased in 0.2 and 2 mM DBP group, respectively. Phagocytosis reflects the ability of disease resistance and immune response in animals. We evaluate the phagocytotic ability of macrophage using ink phagocytosis test. Our results showed that the phagocytosis of phagocytes was greatly reduced in 0.2 and 2 mM DBP (Fig. 4). Quantification of the number of ink signals confirmed that DBP can inhibit the phagocytotic ability of macrophage in the head of zebrafish. In some instances, low exposure concentrations or short exposure times can result in an immuno-stimulatory or immunosuppressive effect; in the present study, acute DBP exposure reduced the phagocytotic ability of macrophage in zebrafish
Fig. 3. Macrophages formation in zebrafish embryos exposed to DBP. Zebrafish were stained with neutral red, and dorsal images of the head were acquired using a fluorescent scope with the same exposure time and gain. (A) Close up of the head of control and experimental embryos at 72 hpf. (B) Quantification of macrophages formation in the selected area. (C) The numbers of macrophages that presented the phenotype shown is expressed as a percentage. For all experiments, at least 30 larvae were used for each condition. *** indicated significant difference at p < 0.001.
Please cite this article in press as: H. Xu, et al., Assessment of immunotoxicity of dibutyl phthalate using live zebrafish embryos, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.04.033
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Fig. 4. Macrophage phagocytosis in zebrafish embryos exposed to DBP. Zebrafish were stained with neutral red, and dorsal images of the head were acquired using a fluorescent scope with the same exposure time and gain. (A) Ink signals of the head of control and experimental embryos at 96 hpf. (B) Quantification of ink signals in the selected area. (C) The numbers of ink signals that presented the phenotype shown is expressed as a percentage. For all experiments, at least 30 larvae were used for each condition. * indicated significant difference at p < 0.05, ** indicated significant difference at p < 0.01, *** indicated significant difference at p < 0.001.
embryos. A short-term, low-dose suppression of phagocytosis has also been reported in aquatic organisms response to contaminants including pesticides [18] and polycyclic aromatic hydrocarbons (PAHs) [19,20]. According to previous studies, phagocytotic processes are dependent upon the membrane properties of immune cells; environmental pollutants can interfere with the fluidity of cell membranes [21,22], restricting the deformation of the membrane essential to the phagocytic endocytosis process [23]. The reduced phagocytosis observed here, suggesting that reduced cell membranes may have contributed towards the lower phagocytotic ability. 3.2. Respiratory burst in live zebrafish embryos The embryos were stimulated with PMA, to induce ROS production via the respiratory burst. The results showed that PMAinduced ROS was inhibited in 0.2 mM DBP-treated groups compared to vehicle. However, there was a slight increase in 2 mM DBP treatments (Fig. 5). The respiratory burst response, a measure of the immune health of an organism, is often used to assess the immune-toxic effects of environmental toxicants. Both stimulatory and inhibitory effects of
these substances on respiratory burst activity have been described. The heavy metal arsenic, when administered to zebrafish, decreased respiratory burst activity [24]; however, the endocrine disruptor bisphenol A and nonylphenol-exposed zebrafish showed an increase in ROS production [25,26]. The proliferation of ROS has been suggested as a mechanism of contaminant toxicity in exposed organisms, with previous studies suggesting the involvement of ROS as a critical mediator in toxin-induced immune insult. Our results showed that DBP exerts seemingly consistent effects on ROS production in connection with the respiratory burst response as well. In our previous study, a slight increase was detected in ROS production by biochemical assay in zebrafish embryos exposed to DBP. The present study showed that DBP induces ROS production in whole live zebrafish larvae. 3.3. T/B cell relevant gene expression Rag1 and rag2 play an essential role in developing adaptive immunity, since rag genes catalyze the rearrangement of immunoglobulin genes in immature B lymphocytes and of T cell receptor genes in immature T lymphocytes and are therefore appropriate
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Fig. 5. Respiratory burst activity of PMA-stimulated and DBP-treated whole zebrafish embryos, measured by oxidation of H2DCFDA to DCF. (A) Relative fluorescence units in zebrafish embryos. (B) Fold induction of respiratory burst. As fold-induction of respiratory burst activity of DBP-treated embryos, the embryos were pretreated with 0.02, 0.2 and 2 mM DBP prior to adding of PMA and H2DCFDA.
Fig. 6. Expression of rag1 (A) and rag2 (B) in zebrafish larvae after exposure to various concentrations of DBP. The results are means ± S.E.M. of triplicate samples. ** indicated significant difference at p < 0.01.
markers to follow the development of organs containing these cells. Consistent with their essential function in the immune system, they are highly conserved among different species. Currently, rag genes were only identified as lymphoid-specific genes in zebrafish. Our previous studies have reported DBP could disrupt the transcript levels of innate immune genes such as IFNg and IL1b in zebrafish embryos [11]. In order to complement our previous results, we decided to monitor at molecular level the progression of the immune disruption by evaluating several adaptive immune markers rag1 and rag2 by qPCR. We evaluated the well-known immune markers at 72 hpf. The results showed that a significant increase in the transcript levels of rag1 at the highest exposure concentration; however, no significant changes in the rag2 expression in all treated groups (Fig. 6). This finding is interesting because DBP has been shown to be estrogenic [27]. It is unclear whether DBP effects rag1 expression via an estrogen-dependent pathway and needs further study. In fact, estrogens are known to be involved in immune process [28]. Therefore, the induction of rag1 gene expression in this study indicated that the adaptive immune process has been already triggered at high dose of DBP. 4. Conclusions In the present study, we assess the immune-toxicological effects of DBP using wild-type, transgenic and albino zebrafish. The exposure of DBP to zebrafish embryos during their early developmental stages led to the decrease of immune cell formation and function, increase of transcription levels of T/B cell-related gene.
Our results revealed a susceptibility of the immune system in zebrafish embryos to DBP, and thus, the transgenic and albino zebrafish should be useful for assessing immunotoxicity of PAEs in the aquatic environment. Acknowledgments This work was supported financially by the National Natural Science Foundation of China (31100376), China Postdoctoral Science Foundation (2013M541618), Research Foundation for Advanced Talents of Jiangsu University (13JDG014) and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Q1 References [1] C.A. Staples, D.R. Peterson, T.F. Parkerton, W.J. Adams, The environmental fate of phthalate esters: a literature review, Chemosphere 35 (1997) 667e749. [2] T.F. Parkerton, W.J. Konkel, Application of quantitative structure-activity relationships for assessing the aquatic toxicity of phthalate esters, Ecotoxicol Environ Saf 45 (2000) 61e78. [3] European Chemicals Agency, Background document for dibutyl phthalate (DBP), 1 June 2009. [4] P.C. Huang, C.J. Tien, Y.M. Sun, C.Y. Hsieh, C.C. Lee, Occurrence of phthalates in sediment and biota: relationship to aquatic factors and the biota-sediment accumulation factor, Chemosphere 73 (2008) 539e544. [5] F. Zeng, J.X. Wen, K.Y. Cui, L.N. Wu, M. Liu, Y.J. Li, et al., Seasonal distribution of phthalate esters in surface water of the urban lakes in the subtropical city, Guangzhou, China, J Hazard Mater 169 (2009) 719e725. [6] W.J.G.M. Peijnenburg, J. Struijs, Occurrence of phthalate esters in the environment of the Netherlands, Ecotoxicol Environ Saf 63 (2006) 204e215.
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