CdSe quantum dots incurred hemoglobin RNA transcription inhibition

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CdSe quantum dots incurred hemoglobin RNA transcription inhibition in embryonic erythroid precursors and compromised embryonic development in mice under low-dose exposure Zhe Wang, Wei Liu, Shuping Zhang, Jie Zhang, and Sijin Liu ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04581 • Publication Date (Web): 29 Jan 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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ACS Sustainable Chemistry & Engineering

CdSe quantum dots incurred hemoglobin RNA transcription inhibition in embryonic erythroid precursors and compromised embryonic development in mice under low-dose exposure

Zhe Wang1,# ,*, Wei Liu2,3,#, Shuping Zhang4, Jie Zhang2,3, Sijin Liu2,3

1. School of Public Health, Xinxiang Medical University, No.601 Jinsui Avenue, Xinxiang, Henan 453003, China. 2. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, No.18 Shuangqing Road, Beijing 100085, China 3. University of Chinese Academy of Sciences, No.19 Yuquan Road, Beijing 100049, China. 4. Institute for Medical Engineering and Science, Massachusetts Institute of Technology, No.77 Massachusetts Avenue, Cambridge 02139, MA, USA.

*: Correspondence to Zhe Wang, Ph.D Email: [email protected]

#: These authors equally contribute to this work.

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Abstract Sustainable development of nanotechnology should comprise maximum benefits and minimum potential risks to the environment and human health. Despite quantum dots (QDs) being widely acknowledged as promising nanomaterials in various application fields, there are still knowledge gaps regarding their safe use in nanomedicine and their environmental health and safety. Given that previous investigations mostly elaborated the direct effects of nanomaterials on cells and organs at high doses, little consideration has been taken to look into the transportation-associated toxicity upon low-dose exposure to engineered nanomaterials. In the current study, we first uncovered that CdSe QDs did not elicit significant gross toxicity in mice upon acute and chronic low-dose exposure. Further, our chronic exposure results displayed that QD particles could accumulate in the embryonic hematopoietic organ fetal liver on embryonic day 14.5 (E14.5), leading to a great reduction of hemoglobin mRNA transcription in fetal liver cells. Mechanistic investigations revealed that the diminished globin RNA synthesis due to the significant inhibition on RNA polymerase activity, at least partially, accounted for hypoproduction of hemoglobin in embryonic erythroid precursors. Compromised embryonic erythropoiesis eventually resulted in embryonic developmental retardation upon chronic low-dose QD exposure. Of note, Cd ions at comparable doses did not manifest such effects as observed for QDs, ruling out a direct attribution of QD-induced effects to Cd ions. This study would open a new path to understand the potential impacts of QDs on susceptible objects, such as developing embryos, due to transportation-associated secondary low-dose exposure.

Keywords: Quantum dots; Fetal livers; Globin mRNA transcription; RNA polymerase; Embryonic development.

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Introduction Sustainable development of nanotechnology should be able to maximize the pre-designed functions and minimize the potential risks to the environment and human beings for engineered nanomaterials (ENMs)

1, 2

. With the increasing development of

nanotechnology, a variety of ENMs have been applied in medical products and commercial consumers

3, 4

. Quantum dots (QDs) are a class of fluorescent semiconductor

nanoparticles with dimensions smaller than the Bohr radius 5. QDs exhibit distinct physicochemical and optical properties, such as high quantum yield, narrow emission bands, broad absorption spectra, high molar extinction coefficients, and high resistance to photobleaching 6, 7. Because of these novel characteristics, QDs have been widely used in biomedical and biological research, especially in biosensing and bioimaging

8-10

.

Additionally, due to the large surface area and flexible surface for a variety of conjugations, QDs also displays promising prospects of application in drug delivery and cancer therapy 8. It has been estimated that the annual production of QDs was 0.6-55 tons worldwide in 2012 11. The wide usage of QDs in consumer and clinical products will inevitably raise the health risk from exposure to QDs in daily life. It is thus crucial to comprehensively understand the potential toxicity of QDs and corresponding mechanisms to promote the safe applications of QDs. Many studies have reported the potential hazards of QDs to various cells and organisms. For instance, the cytotoxicity of CdSe and CdSe/ZnS QDs to human fibroblasts and tumor cells have shown that QDs could give rise to increased cell detachment and impaired cell currents 12. Our recent studies have also demonstrated that QDs could impair lobular morphology and decrease the macrophagic ability to erythrophagocytize, due to their hepatic deposition in mice 13, 14. Given that the content of nanomaterials in nanoproducts is low, the environmental and health safety (EHS) from 3



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long-term exposure to low-dose nanomaterials should be particularly considered

15

.

However, most of the previous studies focused on the toxic effects of QDs on cells and organs at acute high doses, which can not address the chronic effects under environmental exposure scenarios. Fetuses are known to be more sensitive to environmental pollutants than adults, and it has been reported that some nanomaterials can induce developmental impairments in animals

16-20

. Although some studies have reported the embryotoxicity of

QDs to various aquatic organisms, such as zebrafish, rainbow trout and minnow

21

, the

current understanding of long-term effects of QDs on reproductivity is still limited, especially regarding the potential developmental toxicity of QDs to mammalian animals. The primary objective of the current study was to assess embryonic development in mice after chronic exposure to CdSe QDs. Specifically, we embarked on the developmental toxicity of QDs at low doses, where QDs would not induce overt toxicity to maternal mice upon QD exposure prior to pregnancy. Even though, no significant gross toxicity was found in mice upon acute and chronic low-dose exposure, QDs could accumulate in the embryonic hematopoietic organ fetal liver, leading to diminished hemoglobin mRNA expression in fetal liver cells. Importantly, our findings unearthed that the significant inhibition on RNA polymerase activity accounted for the repression of globin mRNA transcription. As a consequence, the inhibitory effects of QDs on RNA synthesis resulted in a mild embryonic developmental retardation.

Materials and methods Nanoparticle characterization Polyethylene glycol (PEG)-coated CdSe QD particles were purchased from Wuhan Jiayuan Quantum Dots Co., LTD., China. The QD solution was stored in the dark at 4°C. QDs were characterized by transmission electron microscopy (TEM, Hitachi H-7500, 4


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Japan). The elemental composition in the QD samples was determined by energy dispersive X-ray spectroscopy (EDS) equipped with TEM. Hydrodynamic diameter and zeta-potential of QDs were determined with a dynamic light scattering (DLS) instrument (Malvern, U.K). Prior to animal administration, QD particles were dissolved in phosphate buffered saline (PBS) buffer. Animal experiments All animal care and experimental protocols were approved by the Committee of Animal Care at the RCEES, Chinese Academy of Sciences. Male and female BALB/c mice were purchased from the Vital River Laboratories, China and housed under sterile conditions. Regarding the acute exposure, 6-week-old female mice were administrated intraperitoneally with 60 ng/kg body weight (equivalent to 20 nM Cd), 300 ng/kg body weight (equivalent to 100 nM Cd) and 600 ng/kg body weight (equivalent to 200 nM Cd) CdSe QDs in 200 µL PBS for 48 h. As Cd ion control, one group of mice was administrated with 360 ng/kg body weight (equivalent to 200 nM Cd) CdCl2 in 200 µL PBS. The control mice received PBS only. The body weights of mice were measured before and after treatment. After exposure for 48 h, peripheral blood was collected for hematological analysis. Organs and leg bones were collected and weighted, followed by histological examination. For the assessment of long-term exposure, 6-week-old mice were administrated intraperitoneally with 60 ng/kg body weight (equivalent to 20 nM Cd) and 300 ng/kg body weight (equivalent to 100 nM Cd) CdSe QDs, and CdCl2 at 180 ng/kg body weight (equivalent to 100 nM Cd) in 200 µL PBS three times a week for 40 days. The body weights of mice were measured every five or seven days, and the activity as well as diets was monitored at each time point of QD injection. Male and female mice were mated immediately after the final injection. Pregnant mice were anaesthetized and organs 5



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including liver, kidney and spleen were collected from maternal mice on embryonic day 14.5 (E14.5). Maternal peripheral blood was collected for hematological analysis. Meanwhile, embryos were also collected. Fetal livers were carefully dissociated from each embryo and collected for further analysis. With respect to the work on assessing the survival rate of embryos, fetuses were given birth. Hematological analysis Hematological analysis was performed as described previously

22, 23

. Mice were

anesthetized with CO2 at the end of animal experiments. Blood was withdrawn and collected into heparinized tubes (BD Biosciences). The complete blood count (CBC) analysis was subsequently performed on a MEK-7222 K automated hematology analyzer (Nihon Kohden, Japan). Histological examination After mice were sacrificed, the liver, kidney and spleen were collected and fixed in 10% formaldehyde in PBS. Fixed tissues were embedded in paraffin blocks, and then cut into 5 µm thick sections onto slides. Sections were stained with hematoxylin and eosin (H&E), and examined under an optical microscope (Carl Zeiss, Inc., Germany). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) assessment Serum concentrations of ALT and AST were assayed using commercial kits according to the manufacturer's protocol (Qiaodu, Shanghai, China). Determination of cadmium mass Cadmium mass in fetal livers was assessed using the inductively coupled plasma mass spectrometry (ICP-MS) method as described previously 24. Samples were quantified by weight and digested with strong oxidation-acid solution (a mix of nitric acid and hydrogen peroxide with a proportion of 3:2) at 180°C for 20 min by microwave assisted digestion (MAD, Mars5 HP500, CEM Corporation, USA). Cadmium content was then 6


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quantified using ICP-MS (Agilent 7500, USA). Real-time qRT-PCR analysis Levels of globin mRNAs in fetal livers were analyzed by qRT-PCR. Total RNAs from fetal liver cells were extracted with Trizol reagent according to the manufacturer's instructions (Invitrogen, USA). 2 µg of total RNAs from different samples was reverse-transcribed into cDNA with M-MLV reverse transcriptase (Promega). Expression levels of globin genes were assessed using a standard SYBR Green System. eIF2α was used as an internal control. The primer sequences for PCR are shown as follows, α-globin, forward:

5’-CACCACCAAGACCTACTTTCC-3’,

5’-CAGTGGCTCAGGAGCTTGA-3’;

reverse:

β-globin,

forward:

5’-GGAAAGGTGAACGCCGATGAA-3’,

reverse:

5’-GGGTCCAAGGGTAGACAACC-3’;

eIF2α,

forward:

5’-GGAAGCAATCAAATGTGAGGACA-3’,

reverse:

5’-GCACCGTATCCAGGTCTCTTG-3’. Run-off transcription assay Run off transcription assay was carried out using an in vitro transcription system according to the manufacturer's instructions (Promega). Briefly, CdSe QDs was added into the transcription buffer (40 mM Tris-HCl, pH 7.9, 6 mM MgCl2, 2 mM spermidine, 10 mM NaCl) with various concentrations. RNA synthesis was initiated with the addition of 4 µCi [α-32P]25 UTP. After reaction for 3 h at 37°C, DNA template was digested using 1 U RNase-free DNase (Promega). Radio-labeled nascent transcripts were quantified by slot blot hybridization according to the protocol as described in our recent study

18

. mRNA

transcripts from runoff were used as hybridization probes. Linearized plasmid DNA was fixed onto nitrocellulose membranes and used as hybridization templates. Statistical analysis 7



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All results were presented as means ± SEM. Statistical analysis was performed using the SPSS 13.0 software. The difference between two groups was assessed using an independent T-test. P < 0.05 indicated statistical significance.

Results Characterization of QDs We here employed PEG-coated CdSe QD particles in this study. Prior to exposure experiments, the physicochemical properties of QDs were thoroughly characterized. As shown in Fig. 1A, the TEM assessment showed that the QD particles were homogeneously dispersed with sphere-like shape. And the calculated average diameter of QDs was about 10 nm (Fig. 1A). The EDS spectrum manifested that elements of C, O, Se, S, Zn, and Cd were present in the QD samples (Fig. 2B). The element Cu was also observed in the EDS spectrum, being ascribed to the copper mesh during sample preparation (Fig. 2B). Further, the hydrodynamic diameter of QDs in water and PBS was approximately 25 nm (Fig. 1B). Additionally, the zeta-potential analysis showed that QDs were similarly negatively charged in water and PBS (Fig. 1B). No significant toxicity to mice upon acute exposure of QDs Nanomaterial administration in vivo might cause adverse effects on organisms

26, 27

.

Thus, we first investigated the toxic effects on maternal mice with the acute exposure to QDs with a range of doses in order to simulate the low-dose exposure, where no observable adverse effects could be defined in maternal mice. As shown in Fig. 2A, the body weight of mice was not altered after exposure to QDs at 60, 300 and 600 ng/kg for 48 h, compared to the control mice. Regardless of exposure routes, nanomaterials are translocated into the circulatory system and transported to internal organs with the aid of 8


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blood cells

28

. The latter is certainly the first line of defense against foreign particles.

Therefore, we collected maternal peripheral blood and conducted CBC analysis to test whether QDs could pose adverse influence on blood cells. As shown in Fig. 2B, the total number of white blood cells (WBC) was increased by 12-23% in QD-treated mice in a dose-dependent matter compared to the control mice, indicating that QDs caused slight systemic inflammation. The total number of red blood cells (RBC) and the concentration of hemoglobin (HGB) were not significantly changed for QD-treated mice (Fig. 2C&D). These results suggested that acute exposure to current doses of QDs did not induce significant toxicity to blood cells. Given that previous studies showed that QDs predominantly accumulated in liver, spleen, kidney and bone marrow

13, 29

, this preferential accumulation may cause

impairments to these organs. We thus collected and weighted these organs from maternal mice upon treatment with QDs. There were no significant alterations to the relative weight of these organs from mice treated with 60, 300 and 600 ng/kg body weight QDs after the acute exposure compared to those from the control mice (Fig. 3A). We also conducted histological examination of these organs. No noticeable tissue changes were observed in the liver, kidney and spleen based on histological examination, except for slight hepatoxicity at 600 ng/kg body weight only, as evidenced by morphological alternations to the hepatic lobules including disordered hepatic cords (denoted by yellow arrows) (Fig. 3B). Considering the detoxication function of heavy metals performed by hepatocytes, we further examined whether QDs could disturb the physiological function of liver cells. The ALT and AST are mainly produced in the liver, and will leak from hepatocytes into blood once the liver is damaged

30, 31

. As shown in Fig. 3C, serum ALT levels were increased

approximately by 60% in 600 ng/kg QD-treated mice compared to the control mice (P

CdSe quantum dots incurred hemoglobin RNA transcription inhibition

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