Altered Gene expression of ABC transporters, nuclear receptors and oxidative stress signaling in zebrafish embryos exposed to CdTe quantum dots☆
Graphical abstract
Introduction
Cd-based quantum dots (QDs) are widely applied for industrial and medical purposes, due to their outstanding electrical and optical properties (Matea et al., 2017). Unfortunately, such increase in applications leads to elevated levels of QDs released into water bodies, where toxicity may occur due to uptake by aquatic organisms (Hu et al., 2017). The reported adverse effects of QDs include cell death and apoptosis, oxidative stress damage, as well as altered neurobehaviors (Tang et al., 2013; Xiao et al., 2016). Therefore, it is important to determine the detoxification systems against QDs and to identify any convenient biomarkers for early signaling of exposure in aquatic organisms.
Recently, adenosine triphosphate-binding cassette (ABC) transporters, including multi-resistance associated protein (Mrps) and P-glycoprotein (Pgp) were found to be involved in the efflux and detoxification of nanoparticles. These proteins used to be highly expressed in human and various fishes (Kim et al., 2017; Luckenbach et al., 2014; Pedersen et al., 2017), conferring resistance to the toxicity of environmental toxicants. Further experiments employing mouse hepatic cells revealed a significant up-regulation of Mrp5, which pumped 51–52% of the intracellular nanoparticles in the form of glutathione (GSH)-conjugation. Similarly, a surface charge-dependent transport of polystyrene nanoparticles by Pgp was found in Caco-2 cells (Bhattacharjee et al., 2013). More importantly, both Al-Hajaj et al. (2011) and our lab (Chen et al., 2016) have reported that ABC transporters like Pgp and Mrps contributed significantly to the efflux of QDs in human liver and kidney cells. Nevertheless, such results need confirmation from in vivo studies and mechanism investigation.
Due to the protection role of ABC transporters in human and animals, their modulators, including nuclear receptors and oxidative stress signaling, were considered as potential biomarkers for exposure to xenobiotics (Vahdati Hassani et al., 2018; Wang et al., 2012). It has been widely recognized in mammalian cells that the expression of metabolic enzymes and ABC transporters are modulated by nuclear receptors like pregnane X receptor (PXR), aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), and peroxisome proliferator-activated receptor (PPAR) (Cuperus et al., 2014; Jigorel et al., 2006). On the other hand, elevated oxidative stress, represented as reduction of glutathione (GSH) level, superoxide dismutase (SOD) activity and glutathione s-transferase (gst) expression, were able to activate E2-related factor 2 (Nrf2) and promote the gene expression of pgp and breast cancer resistance protein (bcrp) in colon cancer cells subsequently (Pinzon-Daza et al., 2017). These nuclear receptors and transcriptional factors sense environmental xenobiotics, subsequently up-regulating the expression of metabolic enzymes and ABC transporters that detoxify these xenobiotics. In particular, the transcriptional changes of nuclear receptors and oxidative stress signaling are often earlier and more sensitive to contamination than transporter activities, suggesting their utility as “exposure” rather than “effect” biomarkers (De Iuliis et al., 2015; Du et al., 2017). Still, whether intrinsic modulatory effects of nuclear receptors and oxidative stress signaling are conserved in fishes, especially upon the exposure of CdTe QDs, has not yet been investigated.
As an ideal animal model for investigating developmental toxicity, zebrafish embryos have been widely used to study the toxicity of QDs, using developmental defects and altered neurobehaviors as endpoints (Zhang et al., 2012). In previous studies, analogs of ABC transporters, nuclear receptors (except for CAR), and oxidative stress signaling have been found in zebrafish embryos (Fischer et al., 2013; Hahn et al., 2015; Schaaf, 2017; Yin et al., 2016). It was still unclear, however, the exact detoxification mechanism in zebrafish embryos against QD exposure.
Therefore, the main objective of this work is to evaluate the possible involvement of three major ABC transporters, Pgp, Mrp1 and Mrp2, in the excretion of two distinct QDs, mercaptopropionic acid (MPA)CdTe and MPA-CdSCdTe QD in zebrafish embryos. Furthermore, we aim to clarify whether nuclear receptors (pxr, ahr1a, ahr1b, ppar-α, and ppar-β), and oxidative stress signaling (GSH, SOD, gst, nrf2) are involved in the modulation of ABC transporters. In this experiment, two typical QDs were applied to get more accurate results. They have same coatings and much smaller sizes (<10 nm) than chorionic pores (0.3–1 μm) (Lin et al., 2013). Moreover, core-shell structure of MPA-CdSCdTe QDs improved the stability of QDs, thus reducing the inference of released Cd2+ and more accurately reflect the effects of QDs themselves (Hardman, 2006). The effects of ABC transporter activities on the toxicity of QDs and involvement of free Cd2+ in the action of QDs were analyzed, as well.
Section snippets
Chemicals
MPA-CdSe and MPA-CdSCdTe QDs powders were purchased from Suzhou Xingshuo Nanotech Co., Ltd. (Suzhou, China). Kits for GSH level and SOD activity were obtained from Beyotime Institute of Biotechnology (Nantong, China). MK571 and reversin 205 (REV) were obtained from Sigma-Aldrich (St Louis, MO, USA). RNA isolation kit was obtained from Axygen Scientific Inc. (Union City, CA, USA). Reagents for the first strand cDNA synthesis and real-time PCR were obtained from Thermo Fisher Scientific (Waltham,
Characterization and accumulation of QDs
As shown in Fig. 1, MPA-CdTe QDs and MPA-CdSCdTe QDs were both monodispersed with diameters of 4.15 ± 0.93 nm and 8.12 ± 1.45 nm. These QDs would accumulate in zebrafish embryos after 24 h exposures (Fig. 2A–D). The green fluorescence from accumulated MPA-CdTe QDs remained primarily within in the yolk sac. The red fluorescence from MPA-CdSCdTe QDs, however, was observed over the entire chorion surface, with only a portion of them entered the yolk sac of zebrafish embryos. Such fluorescence
Discussion
In recent years, CdTe QDs are found in increasing levels in waste water samples from laboratories and industries that synthesize or use CdTe QDs, posing a significant threat to aquatic organisms (Gupta et al., 2017). After a long-term investigation on the toxicity of CdTe QDs, there is limited but growing evidence linking efflux and detoxification of QDs to ABC transporter in mammalian cells (Al-Hajaj et al., 2011; Chen et al., 2016). However, whether such observation is also true for aquatic
Conclusion
Overall, MPA-CdTe QDs and MPA-CdSCdTe QDs caused significant toxicity in zebrafish embryos under the protection of chorion. Such toxicity could be reduced by up-regulation of ABC transporters including mrp1 and mrp2. Nuclear receptors (pxr, ahr1b, and ppar-β) and oxidative stress signaling (nrf2) might be involved in the regulation of mrps. Future work is needed to confirm this phenomenon and illustrate the respective role of each system by employing gene-knockout techniques. In addition,
Conflicts of interest
The authors did not report any conflict of interest.
Acknowledgement
This work was supported by grants from National Science and Technology Major Project of China (2017ZX10302301-003) and National Key R&D Program of China (2017YFF0108600), National Post-Doctor Science Foundation of China (No. 2017M621825) and Post-Doctor Foundation of Jiangsu province (No. 1701034C), National Natural Science Foundation of China (21876198).
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2022, Aquatic ToxicologyCitation Excerpt :Therefore, transporter-mediated efflux might also occur in nanoparticles smaller than 20 nm. On the other hand, transcription factors like E2-related factor 2 (Nrf2) and pregnane X receptor (Pxr) in zebrafish embryos were believed to sense environmental xenobiotics and up-regulate the expressions of ABC transporters that pumping these xenobiotics out (Hu et al., 2019; Liu et al., 2019; Tian et al., 2019). However, whether the above modulation mechanisms were applicable for the interactions between ABC transporters and nanoparticles remained to be clarified.
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This paper has been recommended for acceptance by Dr. David Carpenter.
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These authors contributed equally to this work and should be considered co-first authors.