Oocyte exposure to ZnO nanoparticles inhibits early embryonic development through the γ-H2AX and NF-κB signaling pathways

Abstract

The impacts of zinc oxide nanoparticles on embryonic development following oocyte stage exposure are unknown and the underlying mechanisms are sparsely understood. In the current investigation, intact nanoparticles were detected in ovarian tissue in vivo and cultured cells in vitro under zinc oxide nanoparticles treatment. Zinc oxide nanoparticles exposure during the oocyte stage inhibited embryonic development. Notably, in vitro culture data closely matched in vivo embryonic data, in that the impairments caused by Zinc oxide nanoparticles treatment passed through cell generations; and both gamma-H2AX and NF-kappaB pathways were involved in zinc oxide nanoparticles caused embryo-toxicity. Copper oxide and silicon dioxide nanoparticles have been used to confirm that particles are important for the toxicity of zinc oxide nanoparticles. The toxic effects of zinc oxide nanoparticles emanate from both intact nanoparticles and Zn²⁺. Our investigation along with others suggests that zinc oxide nanoparticles are toxic to the female reproductive system [ovaries (oocytes)] and subsequently embryo-toxic and that precaution should be taken regarding human exposure to their everyday use.

INTRODUCTION

Concerns over the embryo-toxicity of nanoparticles (NPs) have increased exponentially over recent years and numerous articles have been published [1–71]. Many animal models have been used to test NPs embryo-toxicity; these include zebrafish [2–8, 10, 11, 14–17, 20–22, 24–28, 31, 33, 35, 37, 38, 41, 43, 45–47, 49–51, 53–57, 59, 62, 64, 66–70], mice [1, 9, 12, 13, 42, 48, 52, 63, 71], rat [18, 19, 30, 32], chicken [36, 44], oryzias latipes [23, 29, 34, 58], Xenopus laevis [7, 60], sea urchin [40] Mytilus galloprovincialis (Lmk) [39], snail [61], and oyster [65]. Many different types of NPs have been tested using these animal models. The most popular NPs are silver (Ag) NPs [1, 3, 8, 12, 13, 16, 22–26, 28, 32, 33, 34, 37, 38, 41, 43, 48, 49, 55, 56, 65, 66, 70], then zinc oxide (ZnO) NPs [3, 7, 14, 15, 18–20, 26, 40, 47, 62], gold (Au) NPs [3, 29, 30, 45, 46, 50–52, 55, 66, 67], titanium dioxide (TiO2) NPs [9, 16, 17, 21, 25, 28, 31, 39, 63, 69], silicon dioxide (SiO2) NPs [3, 26, 35, 58, 63], Cd NPs [3, 26, 53, 61], chitosan NPs [4, 42, 57], quantum tots [11, 60, 68], Cu NPs [25, 27, 64], platinium (Pt) NPs [44, 55] and other NPs including CoFe2O4, selenium, diamond, Ni, F2O3, and lead NPs [2, 5, 6, 10, 26, 27, 36, 45, 54, 59, 69, 71]. Although many articles have been published exploring the embryo-toxicity of NPs, only their toxic effects on different embryos have been investigated and very few studies have examined the underlying mechanisms.

Austin et al. found that intravenous injection of Ag NPs (10 nm) into pregnant mice resulted in notable silver accumulation in the maternal liver, spleen, and visceral yolk sac, and might suppress embryonic growth [1]. Parivar et al. observed that TiO₂ NPs caused significant changes in chondrocytes in the following developmental stages: resting, proliferating, hypertrophy, degenerating, perichondrium, and mesenchymal cells [9]. Park et al. investigated the effects of chitosan nanoparticles on mouse embryonic development, and indicated that they lowered the expression of both trophectoderm-associated genes and pluripotent marker genes. Yamashita et al. found that SiO₂ and TiO₂ nanoparticles caused pregnancy complications in mice with both types being found in the placenta, fetal liver, and fetal brain [63]. Hong et al. found significant reductions in fetal weights along with an increase in abnormalities after administration of ZnO NPs at a rate of 400 mg/kg/day; however, no significant difference was found in the Zn content of fetal tissue between the control and 400 mg/kg/day groups [18, 19]. Tsyganova et al. reported that Au NPs penetrated the rat placental barrier in vivo; however, no morphological changes took place in the liver, kidneys, spleen, and brain of fetuses [30]. Prasek et al. found that Pt NPs did not inhibit the growth and development of chicken embryos; however, it did induce apoptosis and cause a reduction in the number of proliferating cells in brain tissue [44]. In almost all published articles based on studies of nanoparticle embryo-toxicity, the embryos were tested directly (fertilized eggs of aquatic animals, or chickens) or indirectly (pregnant mouse or rat).

Although ZnO NPs are useful and novel material for a broad range of aspects, many reports have indicated that they may have adverse effects on organisms [72–77] and specifically on the reproductive systems [78, 79]. Nanoparticle safety, however, is not fully understood, particularly the effects and underlying mechanisms of ZnO NPs on embryonic development following oocyte stage exposure. Our results demonstrated that ZnO NPs inhibited embryo development during oocyte stage exposure, and that the γ-H2AX and NF-κB pathways might be crucial in this inhibition. Our investigation, along with others, suggests that ZnO NPs are toxic to female reproductive systems [ovaries (oocytes)] and are subsequently embryo-toxic. Precautions should therefore be taken in situations of human exposure.

RESULTS

Characterization of ZnO, CuO, and SiO2 NPs

ZnO, CuO, and SiO₂ NPs were manufactured by Beijing DK Nano Technology Co. LTD (Beijing, China). The ultra-structure of ZnO NPs and their characterization in cells and tissues have been published in our recent articles [Supplementary Figure 1] [80, 81]. Morphologically, the ZnO NPs were nearly spherical with a milky white color. The size, surface area, and density were approximately 30 nm, 50 m²/g, and 5.606 g/cm³, respectively (Supplementary Figure 1). The hydrodynamic diameter, polydispersity index and zeta potential for ZnO NPs in phosphate buffered saline (PBS) was 148 ± 26 nm, 0.089 μ2/T², or −27.6 ± 2.3 mV respectively. The morphology and size of CuO and SiO₂ NPs were determined using transmission electron microscopy (TEM) and X-ray Diffractometer (Figure 1). The CuO NPs were nearly spherical and black in color. The size, surface area, and density were approximately 30 nm, 13.1 m²/g, and 6.4 g/cm³, respectively (Figure 1A and 1B). The SiO₂ NPs were nearly spherical and white in color. Their size, surface area, and density were approximately 30 nm, 600 m²/g, and 2.4 g/cm³, respectively (Figure 1C and 1D). The hydrodynamic diameter, polydispersity index and zeta potential for CuO NPs in phosphate buffered saline (PBS) was 189 ± 42 nm, 0.072 μ2/T², or −43.2 ± 1.7 mV respectively. The hydrodynamic diameter, polydispersity index and zeta potential for SiO₂ NPs in phosphate buffered saline (PBS) was 137 ± 31 nm, 0.104 μ2/T², or −25.4 ± 2.1 mV respectively.

Figure 1. Characterization of CuO and SiO₂ NPs.

(A) TEM image of CuO NPs. (B) XRD image of CuO NPs. (C) TEM image of SiO₂ NPs. (D) XRD image of SiO₂ NPs.

Inhibition of chicken embryonic development by ZnO NPs

Previously, we found that 10–200 mg/kg (diet) ZnO NPs or 10–200 mg/kg (diet) ZnSO₄ treatments did not decrease hen body weight or egg production [80]. Intact NPs were detected in ZnO NP treated hen ovarian tissues (Supplementary Figure 1). ZnO NP or ZnSO₄ treatments had little effect on Zn content in ovarian tissues compared to control treatments (Supplementary Figure 2). The major difference between ZnO NPs and ZnSO₄ treatments was that the ZnO-NP-200 mg/kg treatment significantly decreased egg yolk lipid content when compared with the ZnSO_4-200 mg/kg treatment [80]. Therefore, ZnSO₄-200 mg/kg and ZnO-NP-200 mg/kg treatments were used in the current investigation. The concentrations of ZnO NPs or ZnSO₄ used in our studies were based on the diet. If the concentration of 200 mg/kg of diet was calculated based on animal body weight (BW), it was calculated to be around 20 mg/kg BW. Therefore, the concentration was lower than that used in other embryo-toxic studies (200–400 mg/kg BW) [18, 19].

After a 24-wk trial, hens were artificial inseminated. Before hatching, 200 eggs per treatment were used to determination the fertilization rate; this was approximately 98% and it was the same for control, ZnSO₄-200 mg/kg and ZnO-NP-200 mg/kg treatments (Figure 2A). However, 7 d after hatching, ZnO-NP-200 mg/kg increased embryonic developmental failure rate (lethality) by 15% when compared with the ZnSO₄-200 mg/kg treatment or control, which suggested that the ZnO-NP-200 mg/kg treatment inhibited chicken embryonic development (Figure 2B).

Figure 2. Inhibition of chicken embryonic development by ZnO NPs.

(A) Fertilization rate; (B) Embryo development failure rate; (C) Decrease in γ-H2AX by ZnO NPs using WB analysis; (D) Elevation of the protein levels of de-phosphorylation enzymes PP4C, PP4R3β, PP4R2, PP2ACα, and PP2A by ZnO NPs using WB analysis; (E) Reduction in NF-κB major component p65 and increase in A20 by ZnO NPs treatment using WB analysis; (F) Decrease in PCNA while elevation in caspase 8 by ZnO NPs treatment using WB analysis; N³5.

Blockage of γ-H2AX and NF-κB pathways by ZnO NPs in chicken embryos

In order to explore the underlying mechanism of ZnO NP inhibition of chicken embryonic development, the protein levels of many pathways were investigated in 3 d and 5 d embryos. It was found that the protein level of γ-H2AX in embryos was significantly decreased by the ZnO-NP-200 mg/kg treatment (Figure 2C). Phosphorylation enzymes were responsible for the de-phosphorylation of γ-H2AX; therefore, these phosphorylation enzymes were subsequently monitored. It was found that the protein levels of phosphorylation enzymes PP4C, PP4R3β, PP4R2, PP2ACα, and PP2A in embryos were increased by ZnO NPs treatment (Figure 2D). Another important factor, NF-κB, was also reduced by ZnO NPs (Figure 2E). ZnO NPs decreased both NF-κB and γ-H2AX protein levels, which suggested that this treatment might decrease cell proliferation or increase apoptosis. Protein levels of PCNA and apoptosis markers were analyzed by WB. ZnO NPs treatment dramatically reduced PCNA protein levels and increased caspase-8 in chicken embryos (Figure 2F).

Disturbance in the γ-H2AX pathway due to ZnO NPs in ovarian cells (CKO-K1 cells)

To verify the phenomenon seen in embryos, hamster ovarian cells (CHO-K1) were cultured and treated with ZnSO₄-12.5 μg/mL or ZnO-NP-12.5 μg/mL for 24 h. After the 24 h treatment, cells [passage 0 (P₀)] were collected and half were used for analysis and half were passaged. Following passage and 24 h of growth, the cells [passage 1 (P₁)] were collected and half were used for analysis and half were passaged again. Subsequently, using the same method, P₂ and P₃ cells were collected. Intact NPs were found in ZnO NPs treated CHO-K1 cells (P₀) and confirmed by EDS (Figure 3A and 3B). The cellular concentration of Zn was increased by ZnSO₄ and ZnO NPs treatments (P₀ cells). Further, the concentration of Zn was higher in the ZnO-NP-12.5 μg/mL treatment than that in the ZnSO₄-12.5 μg/mL treatment (Figure 3C). However, these two treatments produced a similar cell growth inhibition of approximately 15% (Figure 3D). ZnSO₄ did not stimulate γ-H2AX protein level; however, ZnO NPs significantly increased the protein level of γ-H2AX after 24 h of treatment (P₀ cells). However, after the passages, γ-H2AX was dramatically reduced (Figure 3E). This indicated that ZnO NPs might cause DNA damage after 24 h treatment. Therefore, the DNA damage related proteins ATM (Ataxia telangiectasia mutated), ATR (ATM- and Rad3-related), and DNA-PK (DNA-dependent protein kinase) were monitored. It was found that ATM and DNA-PK were elevated after a 24-h ZnO NP treatment (P₀ cells; Figure 4A and 4B); however, they were not changed in P₂ or P₃ cells. ATR was not altered by these treatments. Subsequently, TUNEL assay and apoptotic markers were used to monitor apoptosis. More positive cells were detected in ZnO NPs treated cells than that in the control or ZnSO₄ treatment (Figure 5A) by TUNEL assay. Furthermore, the p53 protein level was also increased by ZnO NPs after 24 h treatment (P₀; Figure 5B). In P₀ cells, γ-H2AX was increased to repair the DNA damage. However, γ-H2AX was decreased gradually from P₁ to P₃ cells to lower than that in the control treatment, which indicated that γ-H2AX itself might also be suppress by ZnO NPs. Phosphorylation enzymes were also determined and it was found that PP4C and PP6C were gradually elevated from P₁ to P₃ cells and PP2ACα was also increased from P₀ to P₃ cells in ZnO NPs treatment (Figure 6A–6C). The proliferation of the cells was determined by EdU assay and it was found that EdU positive cells were decreased in P₀ and P₁ cells (Figure 6D) due to ZnO NPs treatment. However, ZnSO₄ did not induce DNA damage, apoptosis rate, or γ-H2AX protein level.

Figure 3. ZnO NPs in CHO-K1 cells and the impacts on cell growth and γ-H2AX.

(A) TEM photo of ZnO NPs in CHO-K1 cells indicated by the red arrow; (B) EDS image of ZnO NPs in CHO-K1 cells, three Zn peaks shown; (C) Concentration of Zn in ZnO NPs or ZnSO₄ treated CHO-K1, a,b,c, same letter means no difference, different letters mean the groups have difference, p < 0.05; (D) Effects of ZnO NP and ZnSO₄ on CHO-K1 cell growth; (E) Alteration of γ-H2AX by ZnO NPs using WB analysis; N ≥ 3.

Figure 4. IHF images for ATM and DNA-PK in ZnO NPs treated CHO-K1 cells.

(A) Increase in the protein level of ATM in P₀ and P₁ cells by ZnO NPs treatment. Red: ATM staining; Blue: DAPI staining for nucleus; (B) Elevation of the protein levels of DNA-PK in P₀ and P₁ cells by ZnO NPs treatment. Red: DNA-PK staining; Blue: DAPI staining for nucleus. Scale bar: 25 mm; N ≥ 3. ^a-c Means not sharing a common superscript are different.

Figure 5. IHF images for TUNEL assay and p53 levels in ZnO NPs treated CHO-K1 cells.

(A) Increase in the TUNEL positive cells in P₀ and P₁ cells by ZnO NPs treatment. Red: TUNEL staining; Blue: DAPI staining for nucleus; (B) Elevation of the protein levels of p53 in P₀ and P₁ cells by ZnO NPs treatment. Red: p53 staining; Blue: DAPI staining for nucleus. Scale bar: 25 μm; N ≥ 3. ^a-c Means not sharing a common superscript are different.

Figure 6. IHF images for de-phosphorylation enzymes PP4C, PP6C, PP2ACα and EdU analysis after ZnO NPs treatment.

(A) IHF staining for PP4C, increase in the PP4C protein levels in P₁, P₂ and P₃ cells. Red: PP4C staining; Blue: DAPI staining for nucleus; (B) IHF staining for PP6C, elevation of the PP6C protein levels in P₁, P₂ and P₃ cells. Red: PP6C staining; Blue: DAPI staining for nucleus; (C) IHF staining for PP2ACα, increase in the PP2ACα protein levels in P₀, P₁, P₂ and P₃ cells. Red: PP2ACα staining; Blue: DAPI staining for nucleus; (D) EdU analysis, decrease in the EdU positive cells in P₀ cells. Green: P EdU staining; Blue: DAPI staining for nucleus; Scale bar: 25 mm; N ≥ 3. ^a-c Means not sharing a common superscript are different.

Inhibition of the NF-κB pathway by ZnO NPs in CKO-K1 cells

Protein levels of NF-κB (p65) were also detected using WB. It was found that NF-κB (p65) was significantly and similarly decreased by the ZnO NPs treatment of P₀ to P₃ cells. However, ZnSO₄ did not alter NF-κB in all these passaged cells (Figure 7A). A20 (TNFAIP3) was found to be an inhibitor of NF-κB, and it was found that ZnO NPs dramatically increased A20 in all passaged cells (P₀ to P₃; Figure 7B). However, A20 was not altered in ZnSO₄ treated cells. The data here might suggest that ZnO NPs treatment blocked the NF-κB pathway through A20.

Figure 7. NF-κB major component p65 and A20 in ZnO NPs treated CHO-K1 cells.

(A) reduction in NF-κB major component p65 by ZnO NPs treatment; (B) Increase in A20 by ZnO NPs in CHO-K1 cells using IHF staining; Scale bar: 25 mm; N ≥ 3. ^a-b Means not sharing a common superscript are different.

Impairment of γ-H2AX not NF-κB by CuO or SiO2 NPs in CHO-K1 cells

It is still not clear, and it is even controversial whether the toxic effects of ZnO NPs come from Zn²⁺ or intact nanoparticles. The properties of CuO NPs and ZnO NPs are similar as they can be dissolved in acidic or neutral solutions to release ionic forms of Zn or Cu. However, SiO₂ NPs differ in that they cannot be dissolved in acidic or neutral solutions. Therefore CuO NPs and SiO₂ NPs were used to verify the impact of ZnO NPs. If CuO and SiO₂ NPs produce similar effects to ZnO NPs, this would suggest that the effects of the latter come from intact nanoparticles; if similar results are not achieved, it would suggest that the effects of ZnO NPs come from Zn²⁺. Intact CuO NPs were detected in treated cells (Figure 8A) and confirmed by EDS (Figure 8B). After a 24-h treatment, the copper content in the CuO-NP-5 μg/mL treatment was much higher than that in the control cells (Figure 8C). Figure 8E showed intact SiO₂ NPs in CHO-K1 cells after a 24-h treatment and this was confirmed by EDS (Figure 8F). The content of Si in the SiO₂-NP-5 μg/mL treatment was higher than that in the control cells (Figure 8G). Cell growth inhibition was similar (16%) for both CuO-NP-5 μg/mL and SiO₂-NP-5 μg/mL treatments (Figure 8D and 8H). γ-H2AX was elevated by CuO-NP-5 μg/mL and SiO₂-NP-5μg/mL after 24 h treatments (P₀ cells); subsequently it was gradually decreased during passages (from P₁ to P₃ cells; Figure 8I). However, NF-κB was not altered by the CuO-NP-5 μg/mL or SiO₂-NP-5 μg/mL treatment, and a similar level was maintained during passages (from P₀ to P₃ cells; Figure 8J).

Figure 8. Impairment of γ-H2AX not NF-κB by CuO or SiO2 NPs in CHO-K1 cells.

(A) TEM photo of CuO NPs in CHO-K1 cells indicated by the red arrow; (B) EDS image of CuO NPs in CHO-K1 cells, two Cu peaks have shown; (C) Concentration of Cu in CuO NPs treated CHO-K1, *p < 0.05; (D) Effects of CuO NPs on CHO-K1 cell growth; (E) TEM photo of SiO₂ NPs in CHO-K1 cells indicated by the red arrow; (F) EDS image of SiO₂ NPs in CHO-K1 cells; (G) Concentration of Si in SiO₂ NPs treated CHO-K1, *p < 0.05; (H) Effects of SiO₂ NPs on CHO-K1 cell growth; (I) Decrease in γ-H2AX by CuO and SiO₂ NPs using WB analysis; (J) No alteration in NF-κB major component p65 by CuO NPs or SiO₂ NPs treatment using WB analysis; N ≥ 3.

Since γ-H2AX was altered by CuO-NP-5 μg/mL and SiO₂-NP-5 μg/mL treatments, ATM, ATR and DNA-PK were determined in CHO-K1 cells after treatment. It was found that ATM and DNA-PK were increased in P₀ cells in both CuO-NP-5 μg/mL and SiO₂-NP-5 μg/mL treatments (Figure 9A and 9B). TUNEL positive cells were found at higher levels in P₀ and P₁ cells, in both the CuO-NP-5 μg/mL and SiO₂-NP-5 μg/mL treatments, than that in the control treatment (Figure 9C). The protein level of p53 was higher in P₀ and P₁ cells in the CuO-NP-5 μg/mL treatment than that in the control treatment, while it was higher in P₀ cells in the SiO₂-NP-5μg/mL treatment than that in the control (Figure 9D).

Figure 9. IHF images for ATM, DNA-PK, TUNEL assay and p53 levels in CuO and SiO2 NPs treated CHO-K1 cells.

(A) Increase in the protein levels of ATM in P₀ and P₁ cells. Red: ATM staining; Blue: DAPI staining for nucleus; (B) Elevation of the protein levels of DNA-PK in P₀ and P₁ cells. Red: DNA-PK staining; Blue: DAPI staining for nucleus. Scale bar: 25 μm. (C) Increase in TUNEL positive cells in P₀ and P₁ cells. Red: TUNEL staining; Blue: DAPI staining for nucleus; (D) Elevation of the protein levels of p53 in P₀ and P₁ cells. Red: p53 staining; Blue: DAPI staining for nucleus. Scale bar: 25 μm; N ≥ 3. ^a-b Means not sharing a common superscript are different.

Protein levels of PP4C, PP6C, and PP2ACα were elevated in both CuO-NP-5 μg/mL and SiO₂-NP-5 μg/mL treatments (Figure 10A–10C). EdU positive cells were decreased in P₀ cells in both CuO-NP-5 μg/mL and SiO₂-NP-5 μg/mL treatments (Figure 10D). Altogether, ZnO-NP-12.5 μg/mL, CuO-NP-5 μg/mL, and SiO₂-NP-5 μg/mL treatments altered γ-H2AX in a similar pattern in the four passages cells (P₀ to P₃); however, only the ZnO-NP-12.5 μg/mL treatment decreased NF-κB in the four passages cells (P₀ to P₃). These findings suggest that intact NPs from ZnO, CuO, and SiO₂ NP treatments block the γ-H2AX pathway while just Zn²⁺ upsets the NF-κB pathway. Clearly, the toxic effects of ZnO NPs emanate from both intact nanoparticles and Zn²⁺. These in vitro culture findings matched well with in vivo embryonic data, suggesting that impairment caused by ZnO NP treatment can pass through cell generations, and that the γ-H2AX and NF-κB pathways were involved in the embryo-toxicity of ZnO NPs.

Figure 10. IHF images for de-phosphorylation enzymes PP4C, PP6C, PP2ACα and EdU analysis in CuO and SiO2 NPs treated cells.

(A) IHF staining for PP4C, increase in the PP4C protein levels in P₁, P₂ and P₃ cells. Red: PP4C staining; Blue: DAPI staining for nucleus; (B) IHF staining for PP6C, elevation of the PP6C protein levels in P₁, P₂ and P₃ cells. Red: PP6C staining; Blue: DAPI staining for nucleus; (C) IHF staining for PP2ACα, increase in the PP2ACα protein level in P₀, P₁, P₂ and P₃ cells. Red: PP2ACα staining; Blue: DAPI staining for nucleus; (D) EdU analysis, decrease in the EdU positive cells in P₀ cells. Scale bar: 25 μm; N ≥ 3. ^a-d Means not sharing a common superscript are different.

DISCUSSION

Researchers have explored the effects of nanoparticles on embryo development; however, these studies have only investigated the effects (phenomenon) on embryos [1–71], and not on oocytes. Therefore, the impacts of nanoparticles on embryonic development due to oocyte stage exposure are as yet unknown, and the underlying mechanisms are sparsely understood. ZnO NPs are commonly used in almost every aspect of our lives especially in sunscreen, cosmetics, and biocides [72–76]. Even though many reports indicate that ZnO NPs cause adverse effects on reproductive systems and embryonic development [3, 7, 14, 15, 18–20, 26, 40, 47, 62], their safety is not fully understood, particularly the impacts and underlying mechanisms of ZnO NPs on embryonic development due to oocyte stage exposure.

In the current investigation, hens were exposed to ZnO NPs, and after fertilization their impacts on embryonic development and the underlying mechanisms were explored. Results indicated that ZnO NPs inhibited embryonic development by increasing embryo lethality; however, the fertilization rate was not suppressed. Further mechanistic study found that γ-H2AX in embryos was decreased with ZnO NPs treatment. It has been reported that nanoparticles can induce ROS and DNA damage, which consequently activate ATM to induce H2AX phosphorylation (γ-H2AX) [82–85]. ZnO NPs induce DNA damage and increase γ-H2AX to inhibit cell growth [82, 85]. However, many recent investigations suggest that γ-H2AX is not simply a specific DNA DSB marker and its role is not restricted to the DNA damage response [86]. Reports suggest that it involves a multitude of biological processes during cell division [86, 87], stem cell biology [88–90], angiogenesis [91–93], and aging [94–96]. The de-phosphorylation of γ-H2AX by phosphatases is an important event in complete DNA repair after exogenous DNA damage [97–100]. Two families of phosphatases are involved in the de-phosphorylation of γ-H2AX: firstly, the PP2A family of serine/threonine phosphatases including four distinct catalytic proteins PP2ACα, PP2ACβ, PP4C, and PP6C [97, 99], and secondly the PP4 phosphatase complex containing PP4C, PP4R2, and PP4R3β [97, 99]. In the current study, these phosphatases were determined, and it was found that PP4C, PP4R3β, PP4R2, PP2ACα, and PP2A were elevated by ZnO NPs in embryos (Figure 2D). Transcription factor NF-κB (nuclear factor-kappa B) is a critical regulator of multiple biological functions: cell growth, cell survival, innate and adaptive immunity, and others [101–104]. In the current study it was found that ZnO NPs treatment decreased NF-κB in embryos. Vyas et al. (2015) found that iron oxide nanoparticles could enhance the effects of the anticancer drug, doxorubicin, through elevation in apoptosis and down regulation of both pro-inflammatory mediators IL-6 and NF-κB [105]. Sarkar et al. (2015) reported that Ag NPs exposure suppressed mycobacterium tuberculosis-induced expression of a subset of NF-κB mediated genes to suppress mycobacterium tuberculosis-induced NF-κB activation and host immune responses in macrophage cells [106]. A20 (TNFAIP3) has been originally described as a zinc finger protein which is a TNF-inducible gene and acts to protect cells from TNF cytotoxicity [107, 108]. During later experiments, it has been found that A20 acts as a negative regulator of NF-κB signaling involving IL-1b, TNFα, TRAF1/TRAF2, RIP/TRAF2, and other factors [109–111]. Kim and Jeong (2015) reported that ZnO NPs induced the expression of A20 and reduce NF-κB activation to increase anti-inflammatory effects [112]. Feng et al. (2016) found that A20 suppressed the expression of inflammatory cytokines induced by Ag NPs [113]. In the current study, ZnO NP treatment elevated A20 and decreased NF-κB in embryos. Since the NF-κB pathway is very important for cell survival and growth, the blockage of this pathway might be one of the major reasons for the inhibition of embryonic development caused by ZnO NPs.

ZnO NPs exposure may damage DNA replication and repairmen machinery in hen oocytes, which impairs the embryos to inhibit embryonic development. In order to confirm this phenomenon, normal hamster ovarian cells (CHO-K1) were used to determine the impact of ZnO NPs exposure on γ-H2AX and NF-κB pathways after a 24-h treatment and three cell passages. γ-H2AX was elevated after a 24-h ZnO NP exposure, but it was gradually decreased during progressive cell passages to a level much lower than that in the control treatment; this indicated that ZnO NP treatment caused DNA damage and DNA repairmen/replication machinery failure. Furthermore, the phosphatases PP4C, PP6C, and PP2ACα were induced in P₂ and P₃ cells, which coordinated well with changes of γ-H2AX. NF-κB was diminished after a 24-h ZnO NPs exposure and remained at similar low levels during cell passages while A20 was present at higher levels in all passaged cells than in the control treatment. These in vitro results verified that ZnO NPs treatment blocked both γ-H2AX and NF-κB pathways in vivo (embryos).

ZnO NPs can be endocytosed into cells and dissolved to release Zn²⁺ to significantly increase intracellular Zn²⁺ levels [114, 115]. It is known that locally high levels of intracellular Zn²⁺ have toxic effects [114, 115]. Furthermore, ZnO NPs internalized into cells remain as intact NPs for a long time and these induce different toxic effects when compared with Zn²⁺ [116–119]. We have already found that ZnO NPs and ZnSO₄ (sole Zn²⁺ provider) produced a significantly different impact on gene and protein expression in vitro and in vivo with intact NPs being detected in cells and animal tissues [117–119]. Intact ZnO NPs can be found in hen ovarian, liver, spleen, and uterine tissues [80, 81, 117–119]. In order to investigate whether the blockage of γ-H2AX and NF-κB pathways by ZnO NP treatment was due to intact NPs or Zn²⁺, CuO NPs and SiO₂ NPs were used in the current investigation. The size of ZnO, CuO, and SiO2 NPs is similar at around 30 nm. The properties of CuO NPs are similar to ZnO NPs in that they can be dissolved in cells, animal tissues, or even solutions [25, 64]. However, SiO₂ NPs are different from ZnO and CuO NPs in that SiO₂ NPs cannot be dissolved in vivo or in vitro culture [63]. If ZnO, CuO, and SiO₂ NPs produce similar effects on γ-H2AX and NF-κB pathways, it would suggest that these effects are from intact NPs; if not, the effects would emanate from Zn²⁺. In the current study it was found that γ-H2AX was increased in CHO-K1 cells after 24 h CuO NP and SiO NP treatments, and it was decreased with progressive cell passages; a similar trend was seen with ZnO NP treatment. This indicated that γ-H2AX blockage was most likely caused by intact NPs (ZnO, CuO, or SiO₂ NPs). However, NF-κB was not altered by CuO NPs or SiO₂ NPs treatments, which suggested that NF-κB blockage was due to Zn²⁺. It seems clear that the toxic effects of ZnO NPs emanate from both intact NPs and Zn²⁺. The blockage of γ-H2AX and NF-κB pathways might increase apoptosis and reduce cell proliferation as indicated by the apoptosis markers and cell proliferating assay in ZnO NP, CuO NP, and SiO NP treatments.

CONCLUSIONS

In summary, intact NPs were detected in ovarian tissue in vivo and cultured cells in vitro under ZnO NPs treatments. ZnO NPs treatment inhibited embryo development following oocyte stage exposure, and the γ-H2AX and NF-κB pathways involve in the inhibition of embryo development. Notably, the in vitro cultural data closely matched in vivo embryonic data indicating that impairments caused by ZnO NPs treatment can pass through cell generations, and that γ-H2AX and NF-κB pathways were involved in the embryotoxicity of ZnO NPs. The current study clearly shows that the toxic effects of ZnO NPs emanate from both intact NPs and Zn²⁺. This study, along with others, suggests that ZnO NPs are toxic to the female reproductive system [ovaries (oocytes)] and subsequently embryo-toxic. Therefore, precautions should be taken against human exposure during daily life.

MATERIALS AND METHODS

Characterization of ZnO, CuO and SiO₂ NPs

ZnO NPs were synthesized by Beijing DK Nano Technology Co. LTD (Beijing, China) as reported in our recent publications [80, 81]. The characteristics of ZnO NPs (morphology, size, agglomeration, etc.) were determined by transmission electron microscopy (TEM; JEM-2100F, JEOL Inc., Japan) and dynamic light scattering (DLS) particle size analyzer (Nano-Zetasizer-HT, Malvern Instruments, Malvern, UK). CuO and SiO2 NPs were manufactured by Beijing DK Nano Technology Co. LTD (Beijing, China) too. The morphology and size of CuO and SiO₂ NPs were characterized using transmission electron microscopy (TEM; JEM-2100F, JEOL Inc., Japan) and a D8 Advance Powder X-ray Diffractometer (Bruker AXS, Karlsruhe, Germany). The hydrodynamic diameter, polydispersity index and zeta potential were determined in phosphate buffered saline (PBS) after 30 min sonication.

Animal study design (diets and treatments) and sample collection

All animal experimental procedures followed the regulations of the animal ethics committee of the Qingdao Agricultural University and were reported in our recent publication [80]. All hens (Jinghong-1 strain) were housed in a ventilated and conventional caged commercial poultry house with a lighting program of 16:8 light/dark and ad-lib food and water. The formulation of the basal diet (corn-soybean base) has been previously reported [Supplementary Table 1] [80, 118]. The experimental feeding time was from 6 wks to 32 wks of age (Supplementary Figure 3A). ZnO NPs or ZnSO₄ was added into the diet and the concentration of Zn (mg/kg) addition was based on diet. The two treatments were ZnSO₄-200 mg/kg and ZnO-NP-200 mg/kg of diet. A total of 400 pullets were randomly assigned to the two treatments, with five replicates per treatment and forty animals per replicate. After 24 wks treatments, the hens were artificially inseminated with fresh, diluted semen 0.03 mL/hen providing about 210 million sperm (Supplementary Figure 3A). Eggs were collected and stored at 13°C and 75% relative humidity for 5 days until placed in incubators. The fertilization rate was detected by propidium iodide staining of the germinal disc. Egg yolks were separated from the albumen and placed into 0.9% NaCl solution. After visual fertility examination germinal discs seeming to be infertile were removed from the vitelline membrane, put into 0.9% NaCl solution and stained on a slide with propidium iodide (PI; P4170, Sigma-Aldrich Ltd). In case of fertile egg, propidium iodide stains the nucleus of the dividing embryonic cells which appear in lighting points [120, 121]. The embryonic development failure rate was determined by candling eggs after 7 d of incubation and breaking out eggs to differentiate early dead from infertile [120–122]. At 3 d and 5 d incubation, 50 embryos/treatment were collected and stored at −80°C. At the age of 30, 31 and 32 wks, the experiments were repeated three times.

CHO-K1 cell culture

The Hamster Ovarian cell line CHO-K1 was obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco Invitrogen Corporation, USA) at 37°C in 5% CO₂ [123].

In vitro cytotoxicity assays

CHO-K1 cells were plated in 96-well plates at a density of 5000 cells/well. After 24h cell attachment and growth, the cells were treated with different concentrations (based on Zn, Cu or Si; 0.1, 1.0, 5.0, 7.5, 10.0, 12.5 15.0, 20.0, 30.0 and 50.0 μg/mL) of ZnO NPs, ZnSO₄ (Cat. No: Z1001, Sigma-Aldrich Co. LLC in China, Beijing, P.R. China), CuO NPs, and SiO₂ NPs for 24 h. After 24 h treatment, the cells were washed with fresh basic medium (No FBS or antibiotics) and then the cell viability was determined by the reported method using a colorimetric assay with MTT [3-(4, 5)-dimethylthiazol-2, 5-diphenyltetrazolium bromide; Cat. No: M5655, Sigma-Aldrich [81].

In vitro mechanism study

CHO-K1 were cultured and treated with ZnSO₄, ZnO NPs, CuO NPs or SiO₂ NPs for 24 h (Supplementary Figure 3B). After 24h treatment, the cells (passage 0 (P₀)) were collected and half were used for analysis and half were passaged. Following passage and 24 h growth the cells (passage 1 (P₁)) were collected and half were used for analysis and half were passaged. Similarly, P₂ and P₃ cells were collected (Supplementary Figure 3B). The cells collected were for detection of NPs presentation in cells, cellular element concentrations, protein levels by western blotting or IHF analysis.

Detection of ZnO NPs in cultural cells using transmission electron microscopy (TEM) and energy disperse spectroscopy (EDS)

Sample preparation procedures for detecting nanoparticles are reported in our recent publication [80, 81]. Briefly, cell samples were collected and fixed for 2 h in 2% glutaraldehyde made in sodium phosphate buffer (pH 7.2). Specimens were then washed extensively to remove the excess fixative and subsequently post-fixed in 1% OsO₄ for 1 h in the dark. Specimens were then dehydrated in an increasingly graded series of ethanol and infiltrated with increased concentrations of Spur's embedding medium in propylene epoxide. Subsequently the specimens were polymerized in embedding medium for 12 h at 37°C, 12 h at 45°C, and 48 h at 60°C. Fifty nanometer sections were cut on a Leica Ultracut E equipped with a diamond knife (Diatome, Hatfield, PA), and collected on form var-coated, carbon-stabilized Mo grids. The section-containing grids were stained with uranyl acetate, air dried overnight, and imaged on a JEM-2010F TEM (JEOL Ltd., Japan). The presence of ZnO NPs in the tissues was confirmed by using X-Max^N 80 TLE EDS (Oxford Instruments, U.K.).

Measurement of Zn concentration in cultured cells

After 24 h treatment with ZnO NPs, ZnSO₄, CuO NPs or SiO₂ NPs, cells (2 × 10⁶ cells/treatment) were lysed in 500 μL of 0.4% Triton X-100 in PBS, then the lysate was diluted to 2mL with 0.1% Triton X-100. Samples were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES, Optima 2100, Perkin-Elmer, Shelton, CT, USA) [81]. The voltage for the ion lens was set at 6 V; the gas flow rate in the spray chamber was 0.88 L/min; the power output for the RF generator was 1100 W; the auxiliary gas flow rate was 1.2 L/min; and the nebulizer gas flow rate of the plasma was 16 L/min. All the certified reference materials (in solution) were purchased from the National Institute of Metrology (Beijing, China). Blank controls underwent the same procedures. All procedures were performed in triplicate [81].

EdU (5-Ethynyl-2′-deoxyuridine) cell proliferation assay

After 22 h ZnSO₄, ZnO NPs, CuO NPs and SiO₂ NPs treatment, a Click-iT 5-ethynyl-2′-deoxyuridine (EdU) kit (Molecular Probes, Carlsbad, CA, USA) was used to measure cell proliferation according to the manufacturer's procedures [124]. Cells were labeled with EdU (10 μmol/L) for 2 h. After fixation (4% paraformaldehyde, 60 minutes) and transparency (0.5% Triton X-l00, 30 minutes) treatment, the cells were incubated with Click-iT^® reaction cocktail, followed by 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. After rinsing three times, cells were observed under an inverted fluorescence microscope with three random fields of view. The stained sections were visualized with a Nikon Eclipse TE2000-U fluorescence microscope (Nikon, Inc., Melville, NY), and the captured fluorescent images were analyzed using MetaMorph software [118].

TUNEL assay

The apoptotic cells were determined by the Dead End Colorimetric TUNEL assay kit (Promega, Madison, WI, USA) with CHO-K1 cells after ZnSO₄, ZnO NPs, CuO NPs and SiO₂ NPs treatments according to the manufacturer's protocols. This assay is detecting the DNA fragmentation by labeling the terminal end of nucleic acids. The stained sections were visualized with a Nikon Eclipse TE2000-U fluorescence microscope (Nikon, Inc., Melville, NY), and the captured fluorescent images were analyzed using MetaMorph software [118].

Western blotting

Embryo and CHO-K1 cell samples were lysed in RIPA buffer containing a protease inhibitor cocktail from Sangong Biotech, Ltd. (Shanghai, China). Protein concentration was determined using a BCA kit (Beyotime Institute of Biotechnology, Shanghai, PR China) [81]. The information for the primary antibodies (Abs) is present in Supplementary Table 2. GAPDH and Actin were used as loading controls. Secondary donkey anti-goat Ab (Cat no. A0181) was purchased from Beyotime Institute of Biotechnology, and goat anti-rabbit (Cat no.: A24531) Abs were bought from Novex^® by Life Technologies (USA). Fifty micrograms of total protein per sample were loaded onto 10% SDS polyacrylamide electrophoresis gels. The gels were transferred to a polyvinylidene fluoride (PVDF) membrane at 300 mA for 2.5 h at 4°C. Subsequently, the membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature (RT), followed by three washes with 0.1% Tween-20 in TBS (TBST). The membranes were incubated with primary Abs diluted at 1:500 in TBST with 1% BSA overnight at 4°C. After three washes with TBST, the blots were incubated with the HRP-labeled secondary goat anti-rabbit or donkey anti-goat Ab respectively for 1 h at RT. After three washes, the blots were imaged [81].

Immunofluorescent staining

The collected cells were fixed in 4% paraformaldehyde for 1 h, then the cells were spread onto poly-L-lysine coated microscope slides and air-dry. After three washings with PBS (5 min each) cells were incubated with 2% (v/v) Triton X-100 in PBS for 1 h at RT. Then, after three washes with PBS, the cells were blocked with 1% (wt/v) BSA and 1% goat serum in PBS for 30 min at RT, then incubation with primary antibodies diluted in blocking solution overnight at 4°C. The information for the primary antibodies (Abs) is present in Supplementary Table 2. The following morning, after three washes with PBS Tween 20 (0.5%) the slides were incubated with Alexa Fluor 546 goat anti-rabbit IgG (1:200) for 30 min in darkness at RT. The negative controls samples were incubated with secondary antibody and without primary antibody. Slides were washed with PBS Tween-20 three times and then incubated with DAPI (4.6-diamidino-2-phenylindole hydrochloride, 100 ng/ml) as nuclear stain for 5 min. After brief wash with ddH₂O, the slides were covered with an anti-fading mounting medium (Vector, Burlingame, USA). Fluorescent images were obtained with Leica Laser Scanning Confocal Microscope (LEICA TCS SP5 II, Germany) [118].

Statistical analyses

The data were statistically analyzed using SPSS statistical software (IBM Co., NY, USA) and ANOVA. Comparisons between groups were tested by One-Way ANOVA analysis and the LSD test. All the groups were compared with each other for every parameter (mean ± SE). Differences were considered significant at P < 0.05.

Abbreviations

Ag

silver

ATCC

American Type Culture Collection

ATM

Ataxia telangiectasia mutated

ATR

ATM- and Rad3-related

Au

gold

CuO

cupric oxide

DAPI

4′,6-diamidino-2-phenylindole

DLS

dynamic light scattering

DMEM

Dulbecco's modified Eagle's medium

DNA-PK

DNA-dependent protein kinase

DSB

double strand break

EDS

Energy Disperse Spectroscopy

EdU

5-Ethynyl-2′-deoxyuridine

FBS

Fetal bovine serum

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

HRP

horse radish peroxidase

IKK

IκB kinase

LPS

lipopolysaccharide

MTT

3-(4, 5)-dimethylthiazol-2, 5-diphenyltetrazolium bromide

NaCl

sodium chloride

ND

diamond nanoparticle

NF-κB

Nuclear factor kappa-light-chain-enhancer of activated B

NPs

nanoparticles

PCNA

Proliferating Cell Nuclear Antigen

PP2A

Protein phosphatase 2A

PP2AC

Protein phosphatase2A homologues

PP4C

Protein phosphatase 4 catalytic subunit

PP4R2

Protein phosphatase 4 regulator subunit 2

PP4R3

Protein phosphatase 4 regulatory subunit 3 beta

PP6C

Protein phosphatase 6 catalytic subunit

Pt

platinium

PVDF

polyvinylidene fluoride

ROS

reactive oxygen species

RT

room temperature

SiO₂

silicon dioxide

TBS

Tris buffer saline

TBST

Tris buffer saline Tween 20

TEM

transmission electron microscopy

TiO₂

titanium dioxide

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick end labeling

VYS

visceral yolk sac

ZnO

Zinc oxide

ZnSO₄

Zinc sulfide