Intensive epidermal adsorption and specific venous deposition of carboxyl quantum dots in zebrafish early-life stages

Chemosphere 184 (2017) 44e52

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Intensive epidermal adsorption and specific venous deposition of carboxyl quantum dots in zebrafish early-life stages Li Qiang Chen a, *, Cheng Zhi Ding a, Jian Ling b a

Institute of International Rivers and Eco-security, Yunnan Key Laboratory of International Rivers and Trans-boundary Eco-security, Yunnan University, Kunming, 650091, People's Republic of China b School of Chemical Science and Technology, Yunnan University, Kunming, 650091, People's Republic of China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Carboxyl-QDs exhibited an intensive adsorption and accumulation in the gill and intestinal tract in larvae.
Carboxyl-QDs were specifically deposited in the veins and capillary network system, but not in arteries.
The exact tissue condition strongly affected the adsorption, uptake and distribution of carboxyl-QDs in zebrafish body.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 April 2017 Received in revised form 26 May 2017 Accepted 30 May 2017 Available online 31 May 2017

To properly assess the environmental risk of quantum dots (QDs), it is necessary to determine their fate in living organisms, including adsorption, distribution and bioaccumulation under representative environmental or physiological conditions. We comprehensively investigated the fate of QDs with carboxyl terminal functional groups (carboxyl-QDs) in zebrafish (Danio rerio) embryo and larvae subjected to either waterborne exposure or cardiovascular system microinjection. On waterborne exposure, carboxylQDs exhibited an intensive adsorption and accumulation in the chorion of embryos, and their predominate target organs were the gill and intestinal tract in larvae. On microinjection, carboxyl-QDs were rapidly delivered into the cardiovascular system and specifically deposited in veins and the capillary network system of zebrafish larvae, but not in the arterial system. Taken together, we found that the exact tissue condition including epidermal structures, mucus secretion and vascular microstructures strongly affected the adsorption, uptake and distribution of carboxyl-QDs in zebrafish. This work highlights the intensive tissue epidermal adsorption and accumulation of carboxyl-QDs and their specific vein and capillary deposition in the cardiovascular system in zebrafish early-life stages. © 2017 Elsevier Ltd. All rights reserved.

Handling Editor: David Volz Keywords: Biodistribution Carboxyl-QDs Cardiovascular system Zebrafish Fluorescence imaging

1. Introduction Along with the rapid development of nanotechnology, a large

* Corresponding author. E-mail address: [email protected] (L.Q. Chen). http://dx.doi.org/10.1016/j.chemosphere.2017.05.173 0045-6535/© 2017 Elsevier Ltd. All rights reserved.

number of nanoparticles (NPs) have inevitably been released into the environment, which has raised multiple concerns regarding the unforeseen health and environmental hazards of NPs (Maynard et al., 2006; Nel et al., 2006; Wiesner et al., 2006; Hirano, 2009; Mahapatra et al., 2013). Many studies on the environmental fate, behavior, and toxicity of NPs have been carried out to investigate

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potential hazards, but the results are far from conclusive (Handy et al., 2008a, 2008b; Maurer-Jones et al., 2013). It is known that multiple factors influence the environmental and biological fate as well as the toxicity of NPs. The physicochemical properties of NPs, such as size (Chithrani et al., 2006; Ofek Bar-Ilan et al., 2009; Zhao and Wang, 2012), shape and surface chemistry (Zhu et al., 2010; Helle et al., 2013), play a critical role in determining the behavior, fate and toxicity of NPs in the biological and environmental systems (Xu et al., 2010; Duan and Li, 2013). Moreover, the specific environmental conditions such as pH (Domingos et al., 2011), salinity (Kashiwada, 2006) and organic matter content (Quigg et al., 2013), as well as the exact microstructures and physiological states of tissues (Praetner et al., 2010; Qu et al., 2011), are also crucial for the fate of NPs. Quantum dots, a type of semiconductor NPs, have been considered as alternatives of conventional fluorescent dyes for biomedical imaging, cancer cell detection and cardiovascular disease diagnosis (Michalet et al., 2005). Because most QDs contain cadmium as a constituent component, which is a highly reactive and potentially toxic metal, there is increasing concern about the health impacts and environmental risks associated with exposure to QDs (Yong et al., 2013). It has been demonstrated by in vivo studies that QDs intravenously injected into mouse body will accumulate in liver, spleen, kidney and brain, and eventually cause tissue injury (Hoshino et al., 2011; Clift and Stone, 2012). Moreover, once released into the environment, QDs may inevitably interact with microorganisms, invertebrates, fish and mammals, and thereby exhibit potential toxicity to these organisms and consequently human beings through food chains (Farre et al., 2009; Ma and Lin, 2013). However, the manner in which specific tissue conditions influence QDs in vivo behavior and fate is still unclear (Su et al., 2011; Clift and Stone, 2012). The zebrafish (Danio rerio) is a well-established and widely used vertebrate model in developmental toxicity and has recently been shown to have utility in assessing the toxicity of NPs (Lin et al., 2013). More importantly, zebrafish embryos are transparent throughout every developmental stage and develop outside their mothers (Ko et al., 2011), allowing direct observation of the entire cardiovascular system (Yozzo et al., 2013), as well as the distribution of QDs in all internal organs without disturbing the embryos. Although various toxic effects caused by QDs exposure in zebrafish have been revealed (King-Heiden et al., 2009; Zhang et al., 2012a, 2012b; Wiecinski et al., 2013), the fate of QDs in vivo and their behaviors in the cardiovascular system of zebrafish remain largely unknown. The objective of the present work is to investigate the fate and behaviors of QDs with carboxyl terminal functional groups (carboxyl-QDs) in zebrafish early-life stages using fluorescence imaging paired with metal analysis. Carboxyl-QDs, as a most widely used type of QDs, was chosen as the representative QD due to its optical stability, specific surface activity, and high bioavailability (Hardman, 2006; Geys et al., 2008; Yong et al., 2013). To this end, the adsorption and distribution of QDs in zebrafish through waterborne exposure and cardiovascular system microinjection were fully investigated. The results highlight the intensive epidermal adsorption and accumulation of carboxyl-QDs in zebrafish and their specific vein and capillary deposition in the cardiovascular system. 2. Experimental 2.1. QDs and characterization Commercial carboxyl-QDs were purchased from Wuhan Jiayuan Quantum Dots Co., Ltd, Wuhan, China, which are provided in a

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200 mL tubes. This type of QDs consists of a semiconductor CdSe core encapsulated with a ZnS shell (CdSe/ZnS). Batches were stored at 4  C and consumed within 4 weeks. The fluorescence and plasma resonance light scattering properties of carboxyl-QDs were measured by a fluorescence spectrophotometer F-7000 (Hitachi, Japan). The hydrodynamic diameters and zeta potentials of the QDs were measured using a Zetasizer Nano ZS (Malvern Instruments, UK). The detailed physicochemical properties of the QDs are listed in Fig. S1 and Table S1. 2.2. Zebrafish maintain and exposures Raising, spawning, and maintaining of wild-type AB stain and transgenic zebrafish lines were performed as described previously (Kimmel et al., 1995). To locate the QDs in specific tissue and cardiovascular system, a transgenic zebrafish line Tg (kdrl: GFP) that expresses green fluorescent protein in the vascular system was chosen. Briefly, zebrafish were maintained at 28  C on a 14:10 h's light/dark cycle. One day before experiment, we placed 2 pairs of mature zebrafish into a breeding tank and allow them breeding to get the embryos. All embryos were collected and transferred to a Petri dish, then washed with egg water (1.0 mM NaCl in DI water) to remove the debris on the chorion. At 4 h post fertilization (hpf), healthy embryos were incubated with a given concentration of QDs (1, 4, 8 nM) in a multi-well plate. Then, the hatching rate, abnormalities and heart rate of the embryos after waterborne exposure to carboxyl-QDs for 5 d were recorded and analyzed. All studies were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Southwest University, China. 2.3. Body cadmium contents measurement For aquatic exposure, fertilized eggs (4 hpf) were collected form wild-type AB strain zebrafish and distributed in 96-well cell culture plates with one embryo per well and hold at 28  C. Carboxyl-QDs exposure suspension was freshly prepared in the embryo water without any sonication. For body cadmium content measurements, collected embryos (chorion intact and chorion removed, n ¼ 12 embryos per sample) after aquatic exposure for 24 h, were separately washed with deionized water three times, and then the samples were homogenized in 7 N HNO3. The homogenate was transferred to Teflon vials, and 15.8 N HNO3 was added to each sample. Embryos and larvae samples were digested in HNO3 overnight at 90  C. Following digestion, all samples were sonicated for 5 min and then 30% H2O2 was added. The samples were stirred and mixed for 6 h. Each sample was combined with internal standard solution and centrifuged at 1500  g for 10 min to remove undissolved particulates. The supernatant was transferred to a fresh tube and diluted. Then, the samples were analyzed using inductively coupled plasma mass spectroscopy (ICP-MS, PerkinElmer, Inc.) and compared to a cadmium standard curve to determine the concentration. 2.4. Microinjection A transgenic zebrafish line Tg (kdrl: GFP) that expresses GFP in the endothelial vascular system was used for microinjection. Three groups of transgenic zebrafish larvae (each group having 30 larvae) were injected with carboxyl-QDs at 72 hpf. The method for microinjection of QDs into the zebrafish cardiovascular system was performed as described previously with minor modification (Rieger et al., 2005). Briefly, zebrafish larvae were anesthetized in 0.01% tricaine in a 30% Danieau medium, and embedded on their backs in 1.2% ultra-low gelling agarose. A glass capillary filled with a 100 nM

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QD suspension was inserted into the atrium or ventricle of the beating heart. Then, about 2.0 nL of QD suspension was delivered into the pierced heart chamber with the assistance of air pressure. Successful injection was confirmed immediately using fluorescence microscopy. 2.5. Fluorescence imaging In the initial, triplicate experiments (n ¼ 12 embryos/ treatment/ replicate) embryos were exposed to carboxyl-QDs beginning at 4 hpf for 24 h. Then, the chorion adsorption of QDs on the embryos was examined by fluorescence microscopy. For the concentrationdependent experiments, spawned zebrafish embryos (n ¼ 12 embryos/ treatment/ replicate) were exposed to carboxyl-QDs for 5 d in the concentrations of 1, 4 and 8 nM, and then the hatched larvae were imaged by confocal microscopy. For investigation the distribution and accumulation of QDs in larvae, the embryos at 4 hpf were exposed to 4 nM QD solution, then the larvae will be examined at 6 dpf using fluorescence microscopy. Fluorescent images were acquired on an Olympus IX-81 inverted fluorescence microscope equipped with an Olympus IX2-DSU confocal scanning system and a Rolera-MGi electron-multiplying charge-coupled device as we reported before (Chen et al., 2012a, 2012b). QDs were excited at 340e390 nm using a (DM565 dichroic mirror) and detected with a barrier filter (595e615 nm). The GFP was excited at 488 nm and detected at 500e520 nm. For

the imaging of QDs, the exposure time was 100 ms and the CCD gain value was 300. For GFP imaging, the exposure time was 100 ms and the CCD gain value was decreased to 100. Time-lapse video was recorded at an overall frame interval of 5 s with an image acquisition of 512  512 pixels. For z-axis scanning, the micrographs were taken at 1 mm intervals, while the focal plane was moved in incremental steps from the bottom to the top of the larvae. A sequence of 50 images was obtained for three-dimensional (3D) reconstruction using in vivo 3.2/3D Analyzer Suite 6.2 software (Medical Cybernetics, Inc.). 3. Results and discussion 3.1. Concentration-dependent adsorption and uptake of carboxylQDs in zebrafish embryos Fig. 1a shows the concentration-dependent adsorption and accumulation of carboxyl-QDs on zebrafish chorionic embryos after 24 h waterborne exposure. The red fluorescence from QDs was observed over the entire chorion surface, and the fluorescence intensity of each group exhibited a dependency on the concentration. When the concentration of carboxyl-QDs increased from 1 to 8 nM, the basic fluorescence intensity of embryos accordingly increased from 4500 ± 560 to 6800 ± 930, as shown in Fig. 1b. It was also found that a large amount of carboxyl-QDs adsorbed on the surface of chorion and forming many large aggregates (Fig. S2).

Fig. 1. The concentration-dependent adsorption and uptake of carboxyl-QDs in zebrafish embryos. (a) The micrographs indicate that the adsorption of carboxyl-QDs in embryos exhibit a concentration dependency. QDs are shown in red. (b) Concentration-dependent fluorescence intensity increase from embryos that exposed to 1, 4, and 8 nM of carboxylQDs for 24 h. (c) Cadmium body burden of embryos exposed to different concentrations of QDs for 24 h. The bars and error bars represent the means and standard deviation (for n ¼ 12) of the means. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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To determine the extent to which carboxyl-QDs can penetrate the chorion, the cadmium content in the embryo was monitored using ICP-MS. As shown in Fig. 1c, the cadmium content in embryos with the chorion intact also demonstrated a dependency on the QD concentrations. With exposure to increasing concentrations from 1 to 8 nM, the mean cadmium burden of embryos with chorion intact increased from 1200 to 4200 mg kg1 wet weight, respectively. In contrast, the cadmium content in the embryos with chorion removed maintained a low level for all concentrations. There was only 28 mg kg1 wet weight of cadmium burden in average in the embryos with chorion removed, even when they were exposed to QDs at a higher concentration of 8 nM. Despite the low content, the results still reveal that a small amount of cadmium (either QDs or Cd2þ) penetrated the chorion and entered into the embryo. In comparison of the body burdens of cadmium, results indicate that the cadmium contents in the embryos with chorion intact were almost 1500-fold higher than that of the embryos with chorion removed, illustrating the chorion has an extremely high capacity to absorb carboxyl-QDs. Zebrafish embryos are protected during development by the chorion, which serves as a barrier against exposure to environmental NPs (Lin et al., 2013). Considering the protective effect of the chorion, two possible pathways for carboxyl-QDs to influence the embryo developmental process are suggested. For the first pathway, carboxyl-QDs tend to aggregate and adhere to the chorion surface (as we observed in Fig. 1 and Fig. S2), preventing the transport of oxygen/carbon dioxide from the embryo by blocking the chorionic pores (Cheng et al., 2007). A number of NPs, such as carbon nanotubes (Cheng et al., 2007), graphene oxide (Chen et al., 2012a) and silica NPs (Kashiwada, 2006), may affect embryonic development via this pathway. For the second pathway, carboxylQDs (or their dissolved ions) can pass into the embryo by crossing the chorion through chorionic pores and injure the embryo directly (Shaw and Handy, 2011). We have detected the presence of cadmium inside of all exposed embryos (Fig. 1c), which

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identified the possibility of this pathway. It is expected that metal NPs of smaller size or those comprised of soluble metal ions, such as gold NPs (Browning et al., 2009) and silver NPs (Lee et al., 2007), would cause adverse effects on embryonic development through this pathway. Alternatively, QDs could inhibit the embryonic hatching process via direct interaction with functional biomolecules that distribute inside of the chorion, such as hatching enzymes (Ong et al., 2014). 3.2. Distribution and uptake of carboxyl-QDs in larvae Larvae exhibited a strong adsorption and accumulation of carboxyl-QDs in the epidermal of organs after exposure to 4 nM carboxyl-QDs for 6 d. Fig. 2aed shows that red fluorescence of QDs was mainly detected in organs including the mouth, gill, fins, intestine and genital openings. In addition, Z-axis scanning images also revealed that a certain amount of QDs scattering on the surface of the skin were accumulated in the cheek, operculum and abdominal skin of larvae (Fig. S3, S4). Carboxyl-QDs did not exhibit significant negative effects on the hatching rate and heart rate of the embryos, but increased the abnormalities, such as malformed tail, yolk sac malformations and pericardial edema, when their concentration increases upon 4 nM (Fig. S5). The gills, a priority organ in its contact with xenobiotics (Kashiwada, 2006), showed a significant accumulation of carboxylQDs（Fig. 2a）. Due to the gill respiration, a large number of carboxyl-QDs are supposed to flow through the gill filaments with water flow. In this case, QDs hold a very high probability to make collisions with gill surface and ultimately adhere to the mucus layer of gill surface. This result is in agreement with previous observation on AgNPs (Osborne et al., 2015), titanium dioxide NPs (Federici et al., 2007) and carbon nanotubes (Cheng et al., 2007), which were found to accumulate in the gill of fish after waterborne exposure. The higher level of carboxyl-QDs in the gills also can be explained by the physico-chemical nature of the mucus layers that

Fig. 2. Distribution of carboxyl-QDs in larvae at 6 dpf after aquatic exposure. Microphotographs show that QDs were adsorbed and aggregated in (a) the gill and oropharyngeal cavity, (b) genital opening, (c) tail fins, and (d) intestine of zebrafish larvae. (eeg) Fluorescence and differential interference contrast images exhibit the specific location of carboxylQDs in the intestine. Larvae were exposed to 4 nM QD solutions for 6 d. QDs are shown in red and the green fluorescent protein (GFP) in the vascular system is shown in green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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cover the gill surfaces. This layer, which covers the epithelial cells of the gill, consists of multiple proteins and is enriched in cations such as Ca2þ, Mg2þ and Naþ, essentially making the mucus a viscous solution for adsorption and aggregation of anionic NPs (Handy et al., 2008c; Shaw and Handy, 2011), such as carboxyl-QDs. Thus, we speculated that negatively charged carboxyl-QDs might experience a strong electrostatic attraction with the mucus layer resulting in heavy adsorption and aggregation and finally, adherence to the gill surface. In detailed examination of the distribution of the carboxyl-QDs in the larvae, we found that the intestine is the predominant site for QDs uptake and accumulation in vivo. Overlap images of the fluorescence channel and the differential interference contrast channel clearly indicate that carboxyl-QDs were distributed along the elongated intestine with a strong red fluorescence emission （Fig. 2eeg）. Our results are in line with the previous reports, suggest that the uptake of NPs via the intestine may contribute much to their internal distributions in fish (Handy et al., 2008c; Osborne et al., 2015). Similar to titanium dioxide NPs, carboxylQDs may enter into the digestive tract of fish through the action of swallowing after waterborne exposure (Federici et al., 2007). There are also some evidences have showed that QDs can enter into intestines via predation of QD-exposed microorganisms(Werlin et al., 2011) or zooplankton (Lewinski et al., 2011). In analyzing the distribution characteristics of carboxyl-QD in zebrafish larvae, we found that QDs tend to accumulate in the specific organs that are rich in mucus secretion, such as the gill and intestine, and scatter over the areas comprised of a rough surface with little mucus covering. This result suggests that both the epidermal structures and mucus secretion of the tissues play a curial role for carboxyl-QD adsorption and aggregation. What is

more important, QDs may go through the thin epidermis of gill and intestine via a very similar pathway of AgNPs (Shaw and Handy, 2011; Osborne et al., 2015), and would have access to all the internal organs via the cardiovascular system. 3.3. Biodistribution and accumulation of QDs in the cardiovascular system To further investigate the distribution and accumulation of carboxyl-QDs in the cardiovascular system of zebrafish, microinjection technique was used to simulate the situation of QDs get into the blood circulation system. Up to 100 nM carboxyl-QDs could be injected into zebrafish larvae, with 83.3% of larvae showing no significant malformations or developmental problems, as shown in Fig. S6. After 1 h of heart micro-injection, carboxyl-QDs were found to be distributed and accumulated in the vasculature system of larvae as shown in Fig. 3a. Surprisingly, it is observed that carboxylQDs specifically deposited in veins and the capillary network system, but not in the arterial system (Fig. 3b, c, d). In the head region, carboxyl-QDs showed a centralized distribution in the primordial hindbrain channel (PHBC), primordial midbrain channel (PMBC) and primary head sinus (PHS) as shown in Fig. 3b. In the trunk, QDs exhibited a stable distribution along the vessel wall of the posterior cardinal vein (PCV), caudal vein (CV) and postcapillary network, while none of them were observed in the caudal artery (CA) or dorsal aorta (DA), as shown in Fig. 3c and d. The specific venous and capillary deposition of carboxyl-QDs can be explained by the unique vascular physio-anatomy of zebrafish. Firstly, the special endothelium structure of veins (rough surface, thin and poor flexibility) compared with arteries suggests much greater association of carboxyl-QDs with the endothelial wall

Fig. 3. Distribution of carboxyl-QDs in the cardiovascular system of zebrafish larvae after 1 h of microinjection. (a) Entire body fluorescence images show the distribution of QDs in the vasculature of larvae. (b) In the head, QDs exhibit a centralized distribution in the primordial hindbrain channel (PHBC), primordial midbrain channel (PMBC) and primary head sinus (PHS). (c, d) In the trunk and tail, carboxyl-QDs exhibit a stable distribution along the vessel wall of the posterior cardinal vein (PCV), caudal vein (CV) and postcapillary network, however few QDs were observed in the caudal artery (CA) or dorsal aorta (DA). Owing to the association of QDs (red) with vasculatures (green), the overlap exhibits a yellow fluorescence. Bar is 500 mm in image (a) and 200 mm in image (b, c, d), respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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(Praetner et al., 2010; Rehberg et al., 2012). Secondly, the lower flow rates and the large volume of blood carried in the veins relative to those of arteries make it more conducive to the adhesion and deposition of QDs. Finally, capillaries possess an abundance of branches, large surface area and narrow lumen, which facilitate QD deposition greatly (Isogai et al., 2001; Rehberg et al., 2010; Wang et al., 2012). In contrast to the distribution of carboxyl-QDs observed in the present study, streptavidin-conjugated QDs exhibited a non-specific distribution pattern in the cardiovascular system of zebrafish and spread into the entire vascular system, including arteries, veins, and the capillary network (Rieger et al., 2005). We speculate that both the varied surface modification of QDs and the various dosages injected are attributed to the distinct distribution patterns of these two types of QDs in the cardiovascular system. It has been identified that the surface chemistry of QDs and the microstructure of the capillary endothelium that can strongly affect the distribution and deposition behavior of QDs in murine microvessels (Praetner et al., 2010; Rehberg et al., 2010, 2012). 3.4. Transport, behavior and fate of carboxyl-QDs in the cardiovascular system The brightness and photostability of carboxyl-QDs in zebrafish

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larvae prompted us to investigate the transport, behavior and fate of QDs in the vasculature system over an extended period. After 3 d of injection, most of the carboxyl-QDs remained in the vascular system of larvae, distributed in the venous system of the head, trunk and tail. Specially, we performed z-axis scanning to reveal the distribution of carboxyl-QDs in the head section of larvae in greater detail (Movie S1). As shown in Fig. 4a 3D-distribution pattern of carboxyl-QDs in the cardiovascular system was constructed, which revealed that QDs were mainly deposited in the heart, CCV, PHS, PCV and PHBC after 3 d of injection. Note that the colocalization level between the heart and other vascular systems was quite different. Specifically, the heart showed red fluorescence (Fig. 4b), while other vascular systems, such as the PCV and PHBC, which usually accumulate more QDs, exhibited yellow fluorescence (Fig. 4c and d). The heart of zebrafish contains many valves, a bicuspid flap like structure, to maintain the flow of blood in the right direction (Lawson and Weinstein, 2002; Wang et al., 2012). The unique structural nature of heart valves facilitates the deposition of foreign particulates, such as QDs, in the heart. This observation suggests that, in contrast to the heart, the carboxyl-QDs in the veins may incorporate with the vascular epidermal cells. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.chemosphere.2017.05.173. We also attempted to monitor the dynamic behavior of QDs in

Fig. 4. A three-dimensional (3D) reconstruction pattern showing the precise distribution of carboxyl-QDs in the cardiovascular system after 3 d of microinjection. (a) 3D reconstruction pattern images of the cardiovascular system in the head and trunk. The distributions of QDs in the (b) heart, (c) PCV, and (d) PHBC were revealed using colocalization analysis. QDs are shown in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. Distribution of carboxyl-QDs in cardiovascular system after 6 d of microinjection. (a) The distribution of QDs in the head. A few QDs were found to be transported into the anterior cerebral vein (ACeV) and PHS. (b) The distribution of QDs in abdominal cavity and heart. (c, d) In the trunk and tail, most of QDs exhibited a stable distribution along the PCV and CV. The overlap of QDs (red) and vasculatures (green) show yellow fluorescence. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

the cardiovascular system using real-time imaging techniques. It was observed that the red QDs remained nearly stationary and were fixed to the endothelial wall, probably through phagocytosis within the reticular cells, while the blood cells slowly flowed through the vein at 3 d of injection (Movie S2). Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.chemosphere.2017.05.173. Next, we attempted to monitor the residence status of carboxylQDs in the cardiovascular system after 6 d of injection. In general, the distribution pattern of carboxyl-QDs in zebrafish larvae after 6 d injection was quite similar to results following 3 d, as shown in Fig. 5aed. In the head, the PHS was still the dominant vein for QD residence. As a slight variation, a minor concentration of QDs was found to be transported into the anterior cerebral vein, a vein that parallels the anterior cerebral artery and drains into the basal vein in the brain (Fig. 5a). While a small number of QDs were in residence in the endothelium of the heart, the quantity of QDs was significantly reduced after 6 d relative to that of 3 d (Fig. 5b). This observation suggests that the adsorption of carboxyl-QDs in the endothelium of the heart was not solidly adhered, and was easily removed by blood flow. In the trunk and tail, most carboxyl-QDs exhibited a stable distribution along the PCV and CV (Fig. 5c), and only a few QDs were found to enter into the intersegmental vessel (Se). In addition to the venous system, a large number of QDs remained deposited in the postcapillary network (Fig. 5d). In these regions, the extravasations of QDs are quite efficient due to the openings in discontinuous or fenestrated capillaries. These structures possess enhanced permeability, a phenomenon referred to as enhanced permeation retention effect (Isogai et al., 2001; Lawson and Weinstein, 2002). This result is in accord with previous reports indicating that the majority of QDs injected intravenously in the rat bloodstream were likely to be cleared from the bloodstream and be transported into the tissue of the liver and spleen (Chen et al., 2008; Choi et al., 2009; Su et al., 2011). Of course, it is reasonable to assume that the degradation and elimination of carboxyl-QDs that have accumulated in veins or organs is inevitable (Wang et al., 2012; Xu and Chen, 2012), and the

degradation and elimination rates of carboxyl-QDs in zebrafish warranted further investigation. Nevertheless, a large of number of QDs still detained in the PCV and CV after 6 d of injection, which demonstrates that many QDs are associated with vascular endothelium in the venous system for a longer time, and cannot be eliminated from the circulatory system, which pose a great potential for long-time health risk. 4. Conclusions In the present work, we comprehensively investigated the fate of carboxyl-QDs in the early-life stages of zebrafish subjected to either waterborne exposure or cardiovascular system microinjection. For aquatic exposures, the main target organs for adsorption or accumulation of carboxyl-QDs are gill, fins and intestine. For exposure by microinjection, the venous system and capillary network, but not arteries system are the predominate targets for deposition of QDs. The exact tissue condition, such as epidermal structures, mucus secretion and vascular microstructures strongly affected the adsorption, uptake and distribution of carboxyl-QDs in zebrafish. Two possible pathways for carboxyl-QD uptake or accumulation are identified: (1) through the mouth via dietary exposure, and (2) through the gill via the respiration route. In both pathways, carboxyl-QDs can be transmitted across the epithelial of target organs and may finally have access to all the internal organs via the cardiovascular system, which pose a great potential for health risk. Conflict of interest statement The authors declare no conflict interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 21567029 and 21307105).

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