Effect of ethylparaben on the growth and development of Drosophila melanogaster on preadult
Introduction
Parabens are a class of compounds formed by the esterification of p-hydroxybenzoic acid with alcohols under the action of a catalyst, mainly including methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BP), and etc (Błędzka et al., 2014). Due to their broad spectrum of antibacterial properties, air stability, low toxicity and low cost, they are commonly used as preservatives in food (Myint et al., 2004), pharmaceutical (Lokhnauth and Snow, 2005), cosmetic (Rastogi et al., 1995), and personal care products (Guo et al., 2014). At present, parabens have been detected in rivers (Peng et al., 2008), agricultural water (Brausch and Rand, 2011), farm soil (Pérez et al., 2012), drinking water (Carmona et al., 2014), indoor dust (Wang et al., 2012), and aquatic organisms (Yamamoto et al., 2011).
Parabens have endocrine disrupting effects and act as pro-oxidants (Boberg et al., 2010; Costa et al., 2017; Kjaerstad et al., 2010), reducing the concentration of antioxidant enzymes (Silva et al., 2018) and causing oxidative damage (Chiho et al., 2006; Tayama et al., 2008). In addition, parabens may decrease cell proliferation rates (Martín et al., 2010) and increase apoptotic rates (Yang et al., 2018). Studies have shown that parabens inhibit mitochondrial respiration and oxidative phosphorylation, causing insufficient ATP supply. Moreover, low sperm motility and reducing fertility were observed after paraben exposure (Dambal et al., 2017; Riad et al., 2018; Tavares et al., 2009). Parabens exhibit weak estrogen effects by competitively binding to estrogen receptors (Boberg et al., 2010); in addition, they activate ERα and ERβ receptors (Gomez et al., 2005), and compete with human estrogen-related receptor gamma (EERγ) (Zhang et al., 2013).
Drosophila melanogaster has toxin metabolic pathways similar to that of humans and vertebrates, hence we used these flies to study the toxicity of parabens. In the current study, we found that parabens reduced the lifespan and fertility of D. melanogaster and affected malonyldialdehyde (MDA) levels and superoxide dismutase (SOD) activity (Chen et al., 2016; Li et al., 2015), affected the relative expression of genes (Liu et al., 2014; Xue et al., 2017). Fruit flies are fully metamorphic insects, as juvenile hormone (JH) and 20-hydroxyecdysone (20E) regulate synergistically D. melanogaster metamorphosis. The receptor for 20E is the ecdysone receptor (EcR), or EcR/USP (Koelle et al., 1991; Yao et al., 1993). The combination of 20E and its receptor can activate early response genes (Minakuchi et al., 2009). In the process of moulting and metamorphosis (Fig. 1), the JH titre is high. JH binds to its receptor (methoprene-tolerant, Met or germ cell-expressed Gce), leading to Krüppel-homolog 1 (Kr-h1) up-regulation and Broad (Br) inhibition. Therefore, D. melanogaster maintains the larval state after moulting (Abdou et al., 2011; Jindra et al., 2013; Minakuchi et al., 2009). When D. melanogaster develops into the third instar larvae, the JH titer is significantly reduced, and 20E induces the expression of Br. JH reduction and Br induction results in caspase gene (Drice, Dronc) and apoptosis activation gene (hid, rpr) up-regulation, triggering programmed apoptosis of cells and the transformation of D. melanogaster from larvae to prepupa (Abdou et al., 2011; Zhou and Riddiford, 2002). Adult size in D. melanogaster is determined by the final size achieved during the larval growth phase. Once the larvae reach critical weight, the JH titre will decline, the prothoracicotropic hormone (PTTH) will release and act on the prothoracic gland (PG), thereby promoting the synthesis of the 20E. In the absence of JH, 20E cause commitment to metamorphosis and cessation of feeding (Mirth et al., 2005). If the larva is malnourished before reaching the critical weight, low insulin signaling will prolong the developmental time, but it will not affect the attainment normal size (Hyun, 2013). If there is insufficient food after reaching the critical weight, larval size will decrease (Hyun, 2013).
The aims of this study are (1) to detect the effect of EP on the development time and larval area of D. melanogaster; (2) to analyze the interference of EP on the synthesis and secretion of JH and 20E in larvae; (3) to detect the relative expression of growth and development related genes (Met, Gce, EcR, Kr-h1, and Br). It is of great significance to reveal the molecular mechanism of esters on the growth and development of D. melanogaster, and to evaluate the safety of esters more objectively and comprehensively.
Section snippets
Fly strains and treatment
Canton S strain of D. melanogaster was used in the experiment. The cultivation methods and conditions of D. melanogaster were consistent with Liu et al. (2014). According to the national food additive standard (Ministry of Health of the People’s Republic of China, 2014), 700 mg/L is the maximum amount of EP food added, and 300 mg/L is the highest frequency of use. Therefore, 300 mg/L, 700 mg/L, and 1000 mg/L of EP (Shanghai Qingxi Chemical Technology Co., Ltd, Shanghai, China) were selected as
Effect of EP on preadult development period
To test the effect of EP on preadult development time, we treated eggs with different concentrations of EP. The 700 mg/L and 1000 mg/L EP groups significantly prolonged the development time of egg-prepupa, and egg-fly (P < 0.01). The development time of 300 mg/L EP group was slightly shorter than that of the control group, but the difference was not significant (Fig. 2). The development time of prepupa-fly in the control group and each EP treatment group was about 4 days, and there was no
Discussion
Parabens are widely used in preservatives, mainly in cosmetics (Gomez et al., 2005), pharmaceuticals (Moreta et al., 2015), food (Błędzka et al., 2014), and personal care products (Brausch and Rand, 2011; Guo et al., 2014). However, scientific reports concerning their endocrine disrupting potential has led to extensive discussion about the effects and safety of these ubiquitous chemicals.
Our experimental results showed that with the increase of EP concentration, the development time of egg to
Conclusion
The analysis of this study showed that a certain concentration of EP prolonged the developmental cycle of D. melanogaster larvae, reduced its individual size, disordered the expression of development-related genes, and decreased the titers of JH Ⅲ and 20E in the body. These processes are strictly regulated by various hormone pathways, indicating that EP may interfere with endocrine signaling to affect gene expression and hormone secretion, causing endocrine dysfunction and prolonging the
CRediT authorship contribution statement
Lin Gao: Conceptualization, Methodology, Supervision, Data curation, Writing - original draft, Writing - review & editing, Investigation. Yajuan Li: Conceptualization, Supervision, Methodology, Data curation, Writing - review & editing. Hongqin Xie: Methodology, Investigation. Yuan Wang: Data curation, Investigation. Haizhen Zhao: Data curation, Investigation. Min Zhang: Data curation, Investigation. Wei Gu: Conceptualization, Supervision, Writing - review & editing, Validation, Funding
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
We thank Dr. Lei Zhang of College of Life Sciences, Shaanxi Normal University for many helpful comments, suggestions, and revision on this manuscript. This work was supported by the Natural Science Foundation of Shaanxi Province, China (2017JM3032), and the Fundamental Research Funds for the Central Universities (GK201703040).
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These authors contributed equally to this project.