Transcriptomic responses predict the toxic effect of parental co-exposure to dibutyl phthalate and diisobutyl phthalate on the early development of zebrafish offspring
Introduction
Phthalate esters (PAEs) have been classified as endocrine-disrupting compounds (EDCs) (Xu et al., 2020). PAEs are frequently used in the production of polyvinylchloride (PVC) plastic, cosmetics and food packaging materials. Since PAEs are added to polymers without covalent bonds, they can slowly be released into the environment during production and consumption (Benjamin et al., 2017). Di-n-butyl phthalate (DBP) is one of the common PAEs widely used in the production of resins, polymers and adhesives (Liao et al., 2010). It has been found in surface water, atmosphere, and soil. For example, in surface water of the East London harbor, concentrations of DBP were reported to range between 1.0 and 1028.1 μg L−1 (Fatoki and Noma 2002). In atmospheric particles in the vicinity of Chaohu Lake in China, the detected concentration of DBP ranged from 304-499 pg cm−3 (He et al., 2019). Concentration of DBP in agricultural soils in Shandong Peninsula in China were between 0 and 9.855 mg kg−1 (Li et al., 2016). The United States Environmental Protection Agency (USEPA) and China have listed DBP as a priority controllable hazardous substance due to its toxicity to organisms (Liao et al., 2010). As a result, alternative compounds with similar application characteristics begin to be widely used. Diisobutyl phthalate (DiBP) is an isomer of DBP, which has been used as a substitute for DBP to improve the flexibility and durability of industrial and consumer products. Biomonitoring studies showed that there has been an increase in DiBP exposures in recent years (Yost et al., 2019). In aquatic environments, it has been reported that the concentration of DiBP can reach μg L−1 levels (He et al., 2019).
PAEs can accumulate in aquatic organisms from polluted water and through the food chain. For example, DBP and DiBP have been detected in fish of the Asan Lake of Korea (Lee et al., 2019). It has been found that food fish sold in Hong Kong supermarkets contained 0.16-0.94 μg g−1 wet weight (ww) of DBP and 0.43-2.08 μg/g wet weight of DiBP (Cheng et al., 2013). Moreover, the detected concentration of DBP in wild marine fish from the East China Sea was 78.7 ng g−1 (ww), which is much higher than concentrations reported in wild marine fish from the United States and Canada (Hu et al., 2016). Therefore, considering the accumulation of DBP and DiBP in aquatic organisms, further studies are needed to evaluate their potential risks.
The accumulation of DBP and DiBP in parents may affect the development of offspring. For example, human exposure to DBP during pregnancy was shown to induce abnormal development of germ cells in male offspring (Arbuckle et al., 2016). Moreover, testosterone levels in fetal testes were reduced after maternal exposure of rats to DBP from gestation day (GD) 12-19 (Lehmann et al., 2004). Similar to DBP, exposure of GD 0-21 pregnant mice to DiBP (450 mg kg−1 per day) decreased the testosterone concentrations of serum and testes in offspring (Wang et al., 2017). These studies indicated that DBP and DiBP might undergo maternal transfer to the embryo. Compared to mammals, studies on parental transmission for DBP and DiBP in aquatic organisms are scarce, and the potential adverse effects on fish offspring remain unclear.
In fish, parental transfer of some reproductive toxicants, such as polybrominated diphenyl ethers (PBDEs), bisphenol A (BPA) and organochlorine pesticides (OPs) have been observed to interfere with the developmental of their offspring (Chen et al., 2012; Dong et al., 2018; Peng et al., 2012). PAEs are lipophilic and thus accumulate in lipids. In fact, maternally deposited lipids are important nutrition source for early embryo development of fish offspring, and PAEs can be maternally transferred to the embryo through the yolk (Fraher et al., 2016; Qiu et al., 2019b).
There are a variety of PAE compounds in waters, and aquatic organisms are generally exposed to the mixtures of these compounds. Previous studies have shown that exposure to mixtures of PAEs may result in synergistic, additive, or antagonistic effects on different endpoints in aquatic organisms (Chen et al., 2014; Chen et al., 2015). Furthermore, zebrafish embryos exposed to mixtures of DBP (5 μgL−1 and 500 μg/L) and DEP (5 μg/L and 500 μg/L) for 96h had a reduction of acetylcholinesterase activity embryos, showing their potential for causing neurotoxicity (Xu et al., 2013). Considering DiBP is a substitute for DBP, it is reasonable to assume that there is a simultaneous existence of DBP and DiBP in aquatic environments (Cheng et al., 2013; Lee et al., 2019). However, despite increasing awareness of the combined toxicity of DBP and DiBP, there is limited knowledge about their potential interactions and their parental transfer effect. Therefore, we hypothesise that parental exposure to DBP and DiBP may influence the early development of offspring in fish.
Our previous study suggested that combined exposure to DBP and DiBP exerted potential toxicity to ovaries of adult zebrafish at the molecular level (Chen et al., 2019). In the present study, we further studied the effect of parental co-exposure to DBP and DiBP on early development of zebrafish offspring. Our objectives were to investigate whether parental co-exposure could affect the reproduction of zebrafish and pose adverse effects to the offspring, and to assess the interactive effects between DBP and DiBP, and to reveal the potential toxic mechanisms through transcriptomic analysis.
Section snippets
Chemicals
DBP (CAS#84-74-2, purity > 99%), DiBP (CAS#84-69-5, purity > 99%) and the solvent dimethyl sulfoxide (DMSO) (CAS#67-68-5, purity ≥99.5%) were obtained from Sigma-Aldrich. Analytical grade chemical reagents were used throughout the experiment.
Zebrafish maintenance and exposure
The laboratory animal care and use guidelines released by the National Institutes of Health in China were strictly respected during the experiment. The experiment was authorized by the Animal Protection and Utilization Committee in Jiangsu University
Analysis of DBP and DiBP in exposure solutions and F1 embryos
The measured DBP and DiBP concentrations after exposure solution renewal (T0) were 90.5%-99.9% and 95.0%-98.5% of the nominal concentrations, respectively (Table S3). DBP concentrations in the F1 embryos after parental exposure to 11, 113 and 1133 μg L−1 were 12.7±1.3, 64.9±2.5, 113.8±0.7 ng g−1 wet weight, respectively (Fig. 2). Concentrations of DiBP in the F1 embryos after parental exposure to 10, 103 and 1038 μg L−1 were 9.4±3.1, 53.7±0.5, 99.2±2.3 ng g−1 wet weight, respectively. Measured
Discussion
DBP and DiBP are ubiquitous reproductive toxicants that have caused widespread concern about their potential toxic effects in offspring. In this study, 4-month old zebrafish were used to evaluate the combined effect of parental DBP and DiBP exposure on their reproduction and the early development of their offspring. Data showed that while exposure to the greatest concentrations of the individual compounds significantly inhibited fecundity, no comparable effects occurred when parent fish were
Conclusion
This study demonstrated that parental co-exposure to DBP and DiBP could induce early developmental toxicity of zebrafish offspring. The transcriptomic response analysis based on GO an KEGG revealed that changes of molecular functions involving unfolded protein binding, E-box binding and photoreceptor activity in F1 larvae from parental co-exposure may produce adverse effects on eye development in offspring. Different from F1 larvae from parental co-exposure, the effects on gene transcripts of
Author contributions
Hui Chen: Writing, Conceptualization, Methodology, Software, Investigation, Writing Original Draft, Data Curation
Weiwei Feng: Writing: Review & Editing.
Kun Chen: Validation, Formal analysis, Visualization, Software.
Xuchun Qiu: Validation, Formal analysis, Visualization.
Hai Xu: Resources, Writing - Review & Editing, Supervision
Guanghua Mao: Writing: Review & Editing
Ting Zhao: Writing: Review & Editing
Xiangyang Wu: Resources, Writing - Review & Editing, Supervision, Data Curation.
Liuqing Yang:
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
Priority Academic Program Development of Jiangsu Higher Education Institutions and Collaborative Innovation Center of Technology and Material of Water Treatment. This work was financially supported by State Key Laboratory of Environmental Chemistry and Ecotoxicology Open Fund KF 2017-19.
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