Article pubs.acs.org/est
Toxicogenomic Responses of Zebrafish Embryos/Larvae to Tris(1,3dichloro-2-propyl) Phosphate (TDCPP) Reveal Possible Molecular Mechanisms of Developmental Toxicity Jie Fu,† Jian Han,‡ Bingsheng Zhou,‡ Zhiyuan Gong,ζ Eduarda M. Santos,§ Xiaojing Huo,ζ Weiling Zheng,ζ Hongling Liu,† Hongxia Yu,† and Chunsheng Liu*,† †
State Key Laboratory of Pollution Control and Resource Reuse & School of the Environment, Nanjing University, Nanjing, Jiangsu 210023, China ‡ State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei 430072, China ζ Department of Biological Sciences, National University of Singapore, 119077 Singapore § Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, Devon EX4 4PS, U.K. S Supporting Information *
ABSTRACT: Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is frequently present in indoor dust and can be detected in human milk. In order to evaluate the effects of TDCPP on vertebrate development, zebrafish embryos/larvae were used as an animal model to examine developmental phenotypes and explore possible mechanisms of toxicity by employing microarrays and iTRAQ labeling quantitative proteomics. The results demonstrated that treatment with TDCPP (3 μM) from 0.75 h postfertilization (hpf) inhibited cell rearrangement at 4 hpf, caused delay in epiboly at 5.7 and 8.5 hpf, and led to abnormal development (e.g., short tail, reduced body size) and lethality between 14 and 45 hpf, which might be related with altered expression of genes regulating embryogenesis. Furthermore, trunk curvature was observed as the main phenotype in 96 hpf zebrafish larvae exposed to 1 or 3 μM TDCPP, possibly by changing somite formation and expression of proteins related to fast muscle and cartilage development. Collectively, our results suggest that exposure to TDCPP causes developmental toxicity in vertebrates and warrant the need for studies to evaluate the potential health risks of TDCPP to developing human embryos/infants/children, due to its frequent presence in indoor dust and potential for human exposure.
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INTRODUCTION
out pentabrominated diphenyl ether (pentaBDE), the production of TDCPP has increased gradually.1 Environmental monitoring demonstrated that TDCPP was present in more than 96% of the indoor dust samples examined in the United States, with concentrations ranging from 1.50, P < 0.05). We found no over-representation of gene ontology terms or pathways among the list of differentially expressed genes (DAVID Bioinformatics Resources 6.7, http:// david.abcc.ncifcrf.gov/), possibly because the number of differentially expressed genes was limited. Therefore, we focused our further work on the changes in expression of 8 genes because of their known involvement in embryogenesis. Exposure to 3 μM TDCPP significantly down-regulated the expression of kazald2 (kazal-type serine peptidase inhibitor domain 2), pck2 (phosphoenolpyruvate carboxykinase 2), cdx4 and slc38a4 (solute carrier family 38, member 4) by 1.6-, 1.6-, 1.5-, and 1.5-fold compared with the control, while the expression of igf bp1a (insulin-like growth factor-binding protein 1a), egln3 (egl nine homologue 3), chka (choline kinase alpha), and aldocb (aldolase C, fructose-bisphosphate, b) was significantly up-regulated by 3.9-, 2.7-, 1.6-, and 1.7-fold, respectively. These results were further validated by qRT-PCR and Western blotting (Figure 2). TDCPP Bioaccumulation in Zebrafish Larvae. Using UPLC-ESI−MS/MS, we quantified the concentrations of TDCPP in 96-hpf larvae. The deuterated tributyl phosphate (d27-TBP) was used as internal standard (IS). The total ion chromatogram (TIC) and mass spectra of TDCPP and d27TBP are shown in Figures S2 and S3 (see the Supporting Information). Rates of recovery for standard compounds ranged between 102%−115% and 73−85% for TDCPP and d27-TBP, respectively. The coefficient of correlation (R2) of the standard curves (1−500 μg/L) for TDCPP and d27-TBP was 0.9996 and 0.9939, respectively. The instrumental detection limit for TDCPP and d27-TBP was 0.29 and 0.006 μg/L, respectively. The method detection limit of quantification for TDCPP was 25 μg/kg dry weight (dw). Mean concentrations of TDCPP in larvae in the 0.2, 1, and 3 μM TDCPP exposure groups were 6373.1, 26716.7, and 86610.0 μg/kg dw, respectively. No TDCPP was detected in the control groups (Figure S4, see the Supporting Information). TDCPP Exposure Increases Malformation in Zebrafish Larvae. TDCPP exposure increased the incidence of malformations (trunk curvature) in 96 hpf larvae. Exposure to 1 or 3 μM TDCPP significantly increased the rate of occurrence of malformations by 6.3% and 18.0%, respectively, while no significant alteration in the proportion of embryos
nonfat milk in TBST (Tris-buffered saline with 0.05% Tween 20) and incubated with mouse cdx4 monoclonal antibody (1:300 dilution) for 8 h at 4 °C. The membrane was then washed with TBST and then incubated with secondary antibody (goat antimouse IgG-B, 1:3000 dilution) (Santa Cruz, CA, USA). The quantification of the relative expression of cdx4 was performed by using Gene Snap software (Syngene, America). For each treatment (solvent control and 3 μM TDCPP) 4 biological replicates were analyzed, and each replicate contained 20 4-hpf embryos. Quantitative Proteomic Analysis. Quantitative proteomic analysis was performed in 96-hpf larvae exposed to the solvent alone, 0.2, 1, or 3 μM TDCPP, according to the methodology described in previous studies, with some modifications,14−16 and the full details were given in the Supporting Information. Given that the iTRAQ 8-plex used in the present study can technically only measure 8 samples in one run, we adopted a strategy to pool larvae from 2 beakers to obtain each biological replicate for this analysis. Therefore, each measured sample was comprised of 100 zebrafish larvae originated from 2 independent exposure chambers, and two samples were analyzed for each treatment. All MS/MS data were acquired with Xcalibur 2.1 software (Thermo-Fisher Scientific). Once data were acquired, all data files were submitted to the Protein Pilot 4.0 software (AB Sciex, Foster City, CA, USA) for peptide identification using the following parameters: Cys alkylation: MMT(C), ID focus: biological modification, Digestion: typsin, Database: zebrafish, Search effort: thorough ID, unused protein score >1.3 (>95% confidence). Relative quantification of proteins was performed with the Protein Pilot 4.0 software (AB Sciex, Foster City, CA, USA) using the Paragon algorithm as described in detail in the Electronic Supplementary Information (ESI)† “Protein ID/QT Methods”. Statistically significant differences between control and exposure groups were determined by use of One-Way Analysis Of Variance (ANOVA) followed by Tukey’s multiple range test. Protein expressions were considered statistically significant if the P-value 1.50 for up-regulation and 1.50 for up-regulation and
