Effects of QDs exposure on the reproductive and embryonic developmental toxicity in mice at various pregnancy stages

Abstract

Quantum dots (QDs) have recently attracted considerable attention in the biomedical fields because of their unique and excellent optical properties. However, information on their health effects, particularly in the reproductive system, is limited. The present study focuses on the effects of intravenous injection of CdSe/ZnS QDs on the reproductive system and embryo development at various stages of pregnancy in mice. The CdSe/ZnS QDs intravenously injected in mice during pregnancy accumulated in the maternal liver, uterus and placenta. This accumulation affected the growth and development of the embryo during the early and middle stages of pregnancy. Moreover, genotoxicity to the placenta after exposure to CdSe/ZnS QDs was demonstrated by the increased expression levels of genes related to oxidative stress and apoptosis and the reduced expression levels of genes related to the nutrient and waste transportation. Alterations in the gene expression levels have hindered the transport of metabolites across the placenta, which in turn affected the ability of the fetus to obtain nutrients.

Introduction

Semiconductor quantum dots (QDs) with specific size-tunable optical and electronic properties, such as narrow and symmetric emission spectra, resistance against photobleaching, size-tunable light emission, narrow emission spectra and long-term stability, have been used in various fields, including biomedical imaging, drug delivery and electronic industries [1–3]. Examples of these QDs are CdTe- and CdSe-core QDs, which are widely investigated because of their excellent electronic and fluorescence emissions [4, 5]. However, the potential release of toxic metals from QDs is a major biosafety issue because of the presence of Cd or other toxic heavy metals. Many studies have demonstrated the toxicity of different QDs in in vitro cell cultures [6–9]. Furthermore, some in vivo studies indicated that the QDs can cause nephrotoxicity, neurotoxicity, pulmonary toxicity and hepatotoxicity [10–12]. Therefore, aside from the in vitro toxicity studies, in vivo toxicity evaluations of the biodistribution, absorption and metabolism of QDs are necessary. Studies on the reproductive organ toxicity of QDs, such as impairment of ovarian function [13] and testicular structural destruction and dysfunction [14], have been conducted. However, studies on the placenta toxicity and embryonic developmental toxicity of QDs are limited.

Placenta, the organ associated with pregnancy, consists mainly of trophoblast cells from special blastocyst that make up the “placenta barrier” [15], which functions in the selective transfer of substances between the mother and the fetus depending on its permeability. During the various stages of pregnancy, changes in placental structure, function and blood stream occur [16], thereby altering placental uptake and transport. Developmental changes in the placenta occur from embryonic day (E) 3.5, and the transformation in the placental blood supply and the formation of villi occur on E8.5 in mice [17, 18]. Nutrition in the blood results from direct material exchange between the fetus and the mother.

Based on the stages of organogenesis and metabolic maturation, the fetus exhibits significant changes resulting from the invasion of foreign substances during its entire development process [19]. Hence, when assessing nanoparticle transplacental transport and fetotoxicity during pregnancy, the periods of fetus–placental development must be considered carefully. Mice have always been used as animal models for toxicological evaluations [20]. In the present study, a suspension of CdSe/ZnS QDs was administrated intravenously with successive injections at three different stages of pregnancy in mice in early pregnancy at gestation day (GD)2-GD4, middle pregnancy at GD7-GD9 and late pregnancy at GD15-GD17 to investigate possible delivery into the placenta and its effects on fetal development. The effect of the method of delivery through intravenous administration on the assimilation and exposure to CdSe/ZnS QDs was evaluated. Inductively coupled plasma-mass spectrometry (ICP-MS) and histopathology were performed to measure the Cd contents in tissues and to evaluate the toxicity effects, respectively. RT-qPCR was performed to assess the effects of exposure to CdSe/ZnS QDs on the expression levels of the genes for apoptosis, oxidative stress, inflammatory reaction and nutrient transportation. This study aimed to provide biosafety information during pregnancy for the biomedical applications of nanomaterials.

Experimental Section

Particle characterization

In this study, the nanoparticles CdSe QDs coated with ZnS and prepared as an aqueous solution at 0.2 μM were purchased from Ocean NanoTech LLC, USA. UV-Vis absorption spectra were measured with a Hewlett Packard 8453 UV-Vis absorption spectrophotometer (Palo Alto, CA), and photoluminescence spectra were analyzed with a Perkin Elmer Lambda LS 50B luminescence spectrometer (Waltham, MA). The hydrodynamic diameter distributions of CdSe/ZnS QDs were determined through dynamic light scattering (DLS) using a Zetatrac Ultra 151 (Montgomeryville, PA). The CdSe/ZnS QDs were characterized by using a JEOL (Peabody, MA) transmission electron microscope (TEM).

For comparison, the effect of bulk-size Cd species was evaluated using CdCl₂ (Damao Chemical Reagent Factory, Tianjin, China), which was diluted with deionized water and used at a final concentration of 2.0 μM. The concentration of the QDs used in these studies was calculated following previously published methods [21, 22].

Animals

Wild-type Kunming mice (aged 8–10 weeks) were obtained from the Experimental Animal Center of Nanchang University, China, and acclimatized at animal facilities. All mice were held in a room with 12-h light/12-h dark cycle and were provided adequate supply of water and food. The temperature and relative humidity were maintained at 22 ± 2°C and 50 ± 5%, respectively. All experimental procedures were authorized by the Animal Care Review Committee (Approval No. 0064257), Nanchang University, Jiangxi, China, following Institutional Animal Care Committee guidelines. After 1 week of acclimation, the females were mated with the males, and the presence of white vaginal plugs was recorded as GD0. The gestating mice were randomly divided into seven groups (N = 8), namely, CdSe/ZnS QD (early pregnancy), CdSe/ZnS QD (middle pregnancy), CdSe/ZnS QD (late pregnancy), CdCl₂ (early pregnancy), CdCl₂ (middle pregnancy), CdCl₂(late pregnancy) and control groups. Each group was injected with the respective test and control treatments at 100 μl/mice per day, and the control group was treated with normal saline. Injections were conducted once daily for 3 days during early gestation (GD2–GD4), middle gestation (GD7–GD9) and late gestation (GD15–GD17). The animals were weighed and observed daily, and all were sacrificed at GD18. The following were collected: blood, serum and tissues (liver, uterus and placenta) and fetus. The following data were recorded for each mouse: the development of the fetus in terms of various appendages, length of body and tail and weight.

Cd content analysis

Approximately 0.1–0.5 g of tissues (liver, uterus and placenta) and fetus were digested with 10 ml of HNO₃ and 2 ml of HCl. The mixtures were heated to 230°C for 1 h, and the temperature was increased to 280°C until the samples were digested to almost dry. After cooling to room temperature, double distilled water was added to a final volume of 25 ml. The Cd content of these tissues was analyzed by ICP-MS (Thermo Elemental X7; Thermo Electron Co., USA).

Histopathological examination

The collected liver, uterus and placenta from pregnant mice were immersed and immediately fixed in neutral 10% formalin solution, embedded in paraffin blocks before slicing (5 μm thick) and mounted onto glass slides. Hematoxylin-eosin (HE) was used to stain the tissue sections on glass slides, which were observed under an optical microscope (Tokyo, Japan).

Real-time quantitative polymerase chain reaction (RT-qPCR) analysis

The levels of mRNA expression in the placenta were evaluated by RT-qPCR. According to the manufacturer’s protocol, AxyPrep Multisource Total RNA Miniprep Kit (Axygen Scientific, USA) was used to extract the placental total RNA, which was reverse transcribed into cDNA and then subjected to RT-qPCR reaction following the procedures used in a previous study [23]. The RT-qPCR data for each gene product were normalized against GAPDH levels, and threshold cycle (Ct) values were used to calculate the expression of each gene by using the 2^−△△Ct method.

Statistical analysis

The results were shown as means ± standard error (SE) by using one-way analysis of variance (ANOVA) with SPSS 22 software. Dunnett’s test was carried out for each dataset compared with the control data. Statistical significance was accepted at levels of 0.05 (P < 0.05, ^*) and 0.01 (P < 0.01, ^**).

Results

Characterization of CdSe/ZnS QDs

The UV-Vis absorption spectrum of the CdSe/ZnS QDs demonstrated a maximum peak at 540 nm (Fig. 1A and B). DLS suggested that the particles were well resolved in water with an approximate hydrodynamic diameter of 20 nm, which is slightly larger than the corresponding size (14 nm) determined by TEM (Fig. 1C).

Figure 1.

Characterization of CdSe/ZnS QDs. (A) UV-visible light absorption (peak at 540 nm), (B) emission spectra of CdSe/ZnS QDs (peak at 540 nm) and (C) the hydrodynamic size (20 nm) of CdSe/ZnS QDs in the water at pH of 7.0 and TEM image of CdSe/ZnS QDs (8–10 nm).

Body weight changes in pregnant mice

The effects of the CdSe/ZnS QDs treatments were evaluated using the possible changes in body weight. The body weight of the pregnant mice significantly decreased from GD3 to GD18 in the early pregnancy stage (P < 0.05 or P < 0.01) compared with the control group. The same observation was recorded from GD8 to GD18 in the middle pregnancy stage (P < 0.05), whereas no significant changes were observed in the late pregnancy stage. However, in the CdCl₂ treatment groups, no significant changes were detected in the three stages of pregnancy (Fig. 2).

Figure 2.

Percentage of body weight gain in pregnant mice after intravenous injection of CdSe/ZnS QDs, CdCl₂ or saline for three consecutive days in the early pregnancy (GD2–GD4), the middle pregnancy (GD7–GD9) and the late pregnancy (GD15–GD17), respectively. (Mean ± SE), ^*P < 0.05, ^**P < 0.01 versus the control group.

Fetus development

The results indicated that the fetal weight and lengths of body and tail were significantly decreased in the CdSe/ZnS QDs treatment groups in the early and middle pregnancy stages compared with the control group (P < 0.05), whereas no significant changes were observed in the late pregnancy stage. However, among the CdCl₂ treatment groups, no significant changes were observed in the three stages of pregnancy (Fig. 3).

Figure 3.

The effects of intravenous injection of CdSe/ZnS QDs, CdCl₂ or saline in early pregnancy (GD2–GD4), the middle pregnancy (GD7–GD9) and the late pregnancy (GD15–GD17) on the development of fetus. (A) Weight of the fetus, (B) weight of the placenta, (C) fetal body length and (D) fetal tail length. (Mean ± SE), ^*P < 0.05, ^**P < 0.01 versus the control group.

Cd element content

One way to evaluate the migration of QDs was to detect the Cd levels in the collected organs (liver, uterus, placenta and fetus) using ICP-MS. The results (Table 1) suggested that the Cd contents in the liver, uterus and placenta were significantly higher in the CdCl₂ treatment groups (P < 0.05) and in the CdSe/ZnS QDs treatment groups (P < 0.01). Interestingly, no significant difference was observed in the Cd contents among the fetuses in all experimental groups compared with the control group.

Table 1.

The mean and standard error (μg/g) (Mean ± SE) of cadmium contents in liver, uterus, placenta and fetus in pregnant mice at GD18

Group/tissue	Liver	Uterus	Placenta	Fetus
Control	0.0369 ± 0.0021	0.0229 ± 0.0098	0.0107 ± 0.0075	0.0146 ± 0.0044
CdCl2 (early)	0.0491 ± 0.0020*	0.0305 ± 0.0028	0.0206 ± 0.0060*	0.0082 ± 0.0012
CdSe/ZnS QDs (early)	4.1282 ± 0.5727**	0.1768 ± 0.0059**	0.1160 ± 0.0197**	0.0145 ± 0.0038
CdCl2 (middle)	0.0433 ± 0.0007*	0.0329 ± 0.0025*	0.0316 ± 0.0018*	0.0076 ± 0.0028
CdSe/ZnS QDs (middle)	2.1742 ± 0.6628**	0.1648 + 0.0706**	0.1963 ± 0.0035**	0.0081 ± 0.0041
CdCl2 (late)	0.0419 ± 0.0192*	0.0393 ± 0.0112*	0.0252 ± 0.0195*	0.0121 ± 0.0082
CdSe/ZnS QDs (late)	2.6883 ± 0.6193**	0.1542 ± 0.0045**	0.1211 ± 0.0259**	0.0112 ± 0.0041

Pregnant mice were treated intravenously with CdSe/ZnS QDs, CdCl₂ or saline on three consecutive days in early gestation (GD2–GD4), middle gestation (GD7–GD9) or late gestation (GD15–GD17), respectively, (n = 3). ^*P < 0.05, ^**P < 0.01 versus the control group.

Histopathological evaluation

The effects of the QD treatments were evaluated in the liver, uterus and placenta by histopathological examination as shown in Fig. 4. After treatment with CdSe/ZnS QDs or CdCl₂, in the early and middle stages of pregnancy, the histopathological changes in the liver exhibited hepatocyte arrangement disorder, cell edema, and slight cell vacuole degeneration; in the uterus showed that disintegration of uterine glands and disorder of epithelial cells; in the placenta showed that vasodilation and hyperemia, red blood cells exudation, incomplete intercellular connection and cell necrosis.

Figure 4.

Histopathologic evaluation of the liver, uterus and placenta from pregnant mice treated with CdSe/ZnS QDs, CdCl₂ and water. In the liver, black arrows indicate hepatocyte arrangement disorder, red arrows indicate cell edema and slight cell vacuole degeneration and white arrows indicate inflammatory cells. In the uterus, black arrows indicate disintegration of uterine glands and disorder of epithelial cells. In the placenta, black arrows indicate incomplete intercellular connection, red arrows indicate vasodilation and hyperemia and red blood cell exudation and white arrows indicate cell necrosis.

Gene expression profile

The genotoxicity in the placenta of mice exposed to CdSe/ZnS QDs in the three stages of pregnancy was evaluated using gene expression profiles. Several genes associated with oxidative stress (Gpx-2 and SOD), apoptosis (Bax, Caspase9 and Caspase3) and nutrient transportation (eNOS, GluT1 and IGF-2) were selected for RT-qPCR assays. The expression levels of SOD, Gpx-2, Bax, Caspase9, Caspase3, eNOS, GluT1 and IGF-II were 0.61 ± 0.05, 1.00 ± 0.02, 1.31 ± 0.03, 1.20 ± 0.02, 1.44 ± 0.03, 0.85 ± 0.03, 0.50 ± 0.01 and 0.92 ± 0.01, respectively, in the early pregnancy in the CdCl₂ treatment group, most of them have no significant changes than the control. The expressions of the above genes in the early pregnancy in the CdSe/ZnS QD treatment group were 0.56 ± 0.01, 0.57 ± 0.04, 1.55 ± 0.04, 1.72 ± 0.06, 1.71 ± 0.03, 0.72 ± 0.01, 0.33 ± 0.02 and 0.73 ± 0.01, respectively. The Bax, Caspase9 and Caspase3 were obviously upregulated, whereas the SOD, Gpx-2, eNOS, GluT1 and IGF-2 were obviously downregulated than the control group (P < 0.05 or 0.01). The expression levels of those above genes were 0.78 ± 0.00, 0.79 ± 0.01, 1.00 ± 0.06, 1.09 ± 0.11, 1.06 ± 0.03, 0.96 ± 0.05, 0.69 ± 0.01 and 0.76 ± 0.01, respectively, in the middle pregnancy in the CdCl₂ treatment group, most of them have no significant changes. The expressions of above genes in the middle pregnancy in the CdSe/ZnS QD treatment group were 0.75 ± 0.03, 0.53 ± 0.05, 1.43 ± 0.03, 1.30 ± 0.07, 1.85 ± 0.07, 0.65 ± 0.02, 0.30 ± 0.01 and 0.49 ± 0.01, respectively. The Bax, Caspase9 and Caspase3 were obviously upregulated, whereas the SOD, Gpx-2, eNOS, GluT1 and IGF-2 were obviously downregulated (P < 0.05 or 0.01). Similarly, the expression levels of Bax, Caspase9, Caspase3, eNOS, GluT1 and IGF-II were 0.94 ± 0.06, 0.88 ± 0.02, 0.9 ± 0.02, 0.79 ± 0.01, 0.86 ± 0.05, 0.81 ± 0.04, 0.62 ± 0.01 and 0.89 ± 0.03, respectively, in the last pregnancy in the CdCl₂ treatment group, most of them have no significant changes than the control. The expression of the above genes in the last pregnancy in the CdSe/ZnS QD treatment group were 0.43 ± 0.02, 0.59 ± 0.06, 1.1 ± 0.06, 0.9 ± 0.04, 1.2 ± 0.08, 0.51 ± 0.02, 0.23 ± 0.01 and 0.82 ± 0.01, respectively, and the SOD, Gpx-2, eNOS and GluT1 were obviously downregulated (P < 0.05 or 0.01).

Discussion

With the rapid development of nanotechnology, the potential risks of nanoparticle exposure to the human body, especially reproductive toxicity, cannot be ignored. One of the ways by which such reproductive toxicity could be evaluated is to monitor the potential effects during pregnancy. In this study, we assessed the possible effects and the potential mechanism underlying the toxicity of CdSe/ZnS QDs during the three stages of pregnancy periods (early, middle and late) in mice.

Body weight is one of the general toxicity indicators used for potentially toxic chemicals [24, 25]; in this study the effect of CdSe/ZnS QD on the rate of weight growth of pregnant mice was evaluated. The results indicated that the rate of weight growth of pregnant mice decreased significantly in the early and middle pregnancy, especially in the early stage in the CdSe/ZnS QD treatment group (Fig. 2). The delay in weight gain may be due to delayed embryonic implantation or fetal development disorder [26].

To investigate whether the CdSe/ZnS QDs could accumulate in tissues, the Cd content in liver, uterus, placenta and fetus was measured. The results showed that there were higher level Cd in the liver, uterus and placenta of the mice in the experimental groups compared with the control group, especially in the CdSe/ZnS QDs groups. From these observations, the nanosize CdSe/ZnS QDs are allowed for higher penetration ability into the tissues compared with the bulk-size materials. This finding is similar to the results of a previous study on the effects of silver nanoparticles on breastfed mice offspring [27]. However, no obvious changes were detected in the fetal Cd content among the CdSe/ZnS QD, CdCl₂ and control groups (Table 1), which indicated that nanoparticles could penetrate the maternal tissues but not the placental barrier. This might be closely related to the placental barrier function; the placenta is an important barrier between the fetus and the mother to maintain fetal intrauterine nutrition and development. This is similar to the results of previous studies [28]. The accumulation of Cd may cause tissue damage. Histopathological examination of the CdSe/ZnS QDs and CdCl₂ treatment groups in the liver showed hepatocyte arrangement disorder, cell edema and slight cell vacuole degeneration indicating liver damage, which is similar to the results of Hong et al. [29]; in the uterus, disintegration of uterine glands and disorder of epithelial cells indicating uterus damage were seen; in the placenta, vasodilation and hyperemia, red blood cells exudation, incomplete intercellular connection and cell necrosis, indicating CdSe/ZnS QDs affected the placenta structure, were seen, which is similar to the previous study [30].

Although QDs did not accumulate in the fetus in this study, some studies showed that nanoparticles can indirectly cause fetal injury through destroying the placental barrier [31]. Therefore, the pregnancy outcomes (the average body weight, body length and tail length of a single fetus and the average weight of the placenta) at different stages were evaluated in this study. Those results showed that the average body weight, body length and tail length of a single fetus decreased significantly in the early and middle pregnancy in the CdSe/ZnS QDs treatment groups, especially in the early pregnancy, but no significant change in the late pregnancy (Fig. 3). Such finding might be attributed to the maturation time of the placental barrier function. Studies showed that the barrier function of the placenta matures at around E10, thereby resulting in greater restriction on the flow of blood into the placenta and reducing the influx of the nanoparticles and decreasing fetal exposure during E9.5–E11.5 [19]. When the placental barrier function is not yet mature, the nanoparticles in the blood stream can penetrate the placenta and cause fetal toxicity.

To further evaluate the effects of exposure to CdSe/ZnS QDs, the genotoxicity in the placenta during the three stages of pregnancy was explored by evaluating the expression levels of several biomarker genes. Previous studies have shown that the toxicity from exposure nanoparticles may be caused by ROS and apoptosis [32], and ROS plays an important role in promoting apoptosis. In addition, the placental damage can affect nutrient transport, and the normal development of fetus is closely related to the transport of nutrients, and alteration of nutrient transport factors in placenta can cause abnormal development of offspring [33–35]. Thereby, the changes in the expression of genes regulating oxidative stress (SOD and Gpx-2), apoptosis (Bax, Caspase-3 and Caspase-9) and nutrient transport (eNOS, Glu-T1 and IGF-II) were studied.

In vivo toxicity studies indicated that disproportionate generation of reactive oxygen species (ROS) posed serious problems to homeostasis and caused ischemia-reperfusion injury, inflammatory disorders and infections [36]. SOD and Gpx-2 provide major antioxidant defenses against ROS through the removal of free radicals from the system and inhibition of self-oxidation, thereby ultimately protecting cells from oxidative damage [37, 38]. In this study, SOD and Gpx-2 decreased significantly in the CdSe/ZnS QD treatment groups in all stages of pregnancy (Fig. 5). Those results demonstrated that CdSe/ZnS QDs could lead to the reduction of antioxidants, thereby causing oxidative damage in the placenta. Bax is a pro-apoptotic gene; Caspase-9 serves as an internal promoter of caspase, which activates the mitochondrial membrane and the release of cytochrome C; Caspase-3 is an effector caspase that regulates the division of many cellular proteins together with other effector caspases [39, 40]. In the CdSe/ZnS QDs treatment groups, Bax, Caspase-3 and Caspase-9 were upregulated in the early and middle pregnancy stages, but no significant increase was observed in the late pregnancy stage, which indicated that the CdSe/ZnS QDs might induce apoptosis in the placenta.

Figure 5.

Gene expression changes in the placenta in the pregnant mice. Relative gene expression levels were determined using the critical threshold (Ct) number and calculated using the 2^−△△Ct method, with GAPDH as the reference gene for all experimental groups. (Mean ± SE) ^*P < 0.05, ^**P < 0.01 versus the control group.

eNOS is considered an important regulator of placental vascular development. It is found in the vascular endothelium and trophoblast at varying degrees during pregnancy. In eNOS knockout mice, the increase in the placental hypoxic zone is obvious [41, 42]. Glucose is the main substrate for fetal growth. It passes through the placenta through Glu-T1 glucose transporter. The change in Glu-T1 expression is very important for fetal growth [33]. The main function of IGF-II is to promote cell proliferation, differentiation and metabolism; hence, it participates in placental and embryonic growth [34] and destroys the IGF-II gene, which can cause growth restriction in mice [35]. In the present study, eNOS, Glu-T1 and IGF-II showed different degrees of downregulation in the different stages of pregnancy. Hence, the accumulation of QDs in the placenta could hinder the transport of nutrients and excretion of waste products between the mother and the fetus, which may eventually lead to fetal intrauterine growth retardation. The absence of significant changes in the same genes observed in the late pregnancy stage could be attributed to the maturity of the protective function of the placenta [19] and the shorter period of time for the QDs to affect the late pregnancy until birth.

Conclusion

In this study, the reproductive toxicity of CdSe/ZnS QDs by tail vein injection at various stages of pregnancy was investigated. The results show that after the mice treated with CdSe/ZnS QDs, CdSe/ZnS QDs could accumulate in the organs (liver, uterus and placenta) of the mice, and might cause the damage of the organs, which might lead to the abnormal development of fetus. These observations were attributed to CdSe/ZnS QDs triggering excessive ROS that led to apoptosis and affecting the transport of placental nutrients. Furthermore, all results indicated that exposure to CdSe/ZnS QDs at different stages of pregnancy could cause reproductive toxicity of pregnant mice and the adverse effects on fetal growth and development in early and middle pregnant stages possibly resulted from the mediation of indirect factors that damage the placenta and not from direct exposure of the fetal tissues. Hence, future investigations on the mechanism of toxicity of the QDs are needed to determine how the QDs manifest genotoxicity.

Data Availability

The data used to support the findings of this study are included within the article.

Funding

This work was supported by Natural Science Foundation of China (81560537 and 81771658).

Conflicts of Interest

The authors declare that they have no competing interests.