Altered Gene expression of ABC transporters, nuclear receptors and oxidative stress signaling in zebrafish embryos exposed to CdTe quantum dots

Highlights

• Both MPA-CdTe and MPA-CdS-CdTe QDs exhibited significant accumulation and toxicity in zebrafish embryos.

• Chorion hindered the entry of CdTe QDs to zebrafish embryos.

• Up-regulation of mrp1 and mrp2 contributed actively to the detoxification of CdTe QDs.

• Nuclear receptors (pxr, ahr1b, and ppar-β) and oxidative stress signaling mediated the up-regulation of mrps.

• Alterations of pxr and nrf2 could be developed as toxic biomarkers of CdTe QDs.

Abstract

Adenosine triphosphate-binding cassette (ABC) transporters, including P-glycoprotein (Pgp) and multi-resistance associated proteins (Mrps), have been considered important participants in the self-protection of zebrafish embryos against environmental pollutants, but their possible involvement in the efflux and detoxification of quantum dots (QDs), as well as their regulation mechanism are currently unclear. In this work, gene expression alterations of ABC transporters, nuclear receptors, and oxidative stress signaling in zebrafish embryos after the treatment of mercaptopropionic acid (MPA)CdTe QDs and MPA-CdSCdTe QDs were investigated. It was observed that both QDs caused concentration-dependent delayed hatching effects and the subsequent induction of transporters like mrp1&2 in zebrafish embryos, indicating the protective role of corresponding proteins against CdTe QDs. Accompanying these alterations, expressions of nuclear receptors including the pregnane X receptor (pxr), aryl hydrocarbon receptor (ahr) 1b, and peroxisome proliferator-activated receptor (ppar)-β were induced by QDs in a concentration- and time-dependent manner. Moreover, elevated oxidative stress, reflected by the reduction of glutathione (GSH) level and superoxide dismutase (SOD) activities, as well as the dramatic induction of nuclear factor E2 related factor (nrf) 2, was also found. More importantly, alterations of pxr and nrf2 were more pronounced than that of mrps, and these receptors exhibited an excellent correlation with delayed hatching rate in the same embryos (R² > 0.8). Results from this analysis demonstrated that the induction of mrp1 and mrp2 could be important components for the detoxification of QDs in zebrafish embryos. These transporters could be modulated by nuclear receptors and oxidative stress signaling. In addition, up-regulation of pxr and nrf2 could be developed as toxic biomarkers of CdTe QDs.

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.