Aspect ratio plays a role in the hazard potential of CeO2 nanoparticles in mouse lung and zebrafish gastrointestinal tract

Abstract

We have previously demonstrated that there is a relationship between the aspect ratio (AR) of CeO2 nanoparticles and in vitro hazard potential. CeO2 nanorods with AR ≥ 22 induced lysosomal damage and progressive effects on IL-1β production and cytotoxicity in the human myeloid cell line, THP-1. In order to determine whether this toxicological paradigm for long aspect ratio (LAR) CeO2 is also relevant in vivo, we performed comparative studies in the mouse lung and gastrointestinal tract (GIT) of zebrafish larvae. Although oropharyngeal aspiration could induce acute lung inflammation for CeO2 nanospheres and nanorods, only the nanorods with the highest AR (C5) induced significant IL-1β and TGF-β1 production in the bronchoalveolar lavage fluid at 21 days but did not induce pulmonary fibrosis. However, after a longer duration (44 days) exposure to 4 mg/kg of the C5 nanorods, more collagen production was seen with CeO2 nanorods vs nanospheres after correcting for Ce lung burden. Using an oral-exposure model in zebrafish larvae, we demonstrated that C5 nanorods also induced significant growth inhibition, a decrease in body weight, and delayed vertebral calcification. In contrast, CeO2 nanospheres and shorter nanorods had no effect. Histological and transmission electron microscopy analyses showed that the key injury mechanism of C5 was in the epithelial lining of the GIT, which demonstrated blunted microvilli and compromised digestive function. All considered, these data demonstrate that, similar to cellular studies, LAR CeO2 nanorods exhibit more toxicity in the lung and GIT, which could be relevant to inhalation and environmental hazard potential.

Figure 1

Physicochemical characterization of CeO₂ nanospheres and nanorods. (A) Representative TEM images show the primary size, shape, and AR of CeO₂ (C1, C2, C3, C4 and C5). (B) Table summarizing the diameter, length, and AR of CeO₂ based on the TEM analysis, as well as the hydrodynamic diameter and surface charge of nanoparticles suspended in distilled water, cell culture medium (DMEM) and zebrafish growth medium (Holtfreter’s medium).

Figure 2

Acute pulmonary effects of CeO₂ nanoparticles in C57BL/6 mice. The dose-dependent experiment was carried out in mice exposed to CeO₂ nanoparticles C5 at 0.5, 1.0, 2.0 and 4.0 mg/kg by oropharyngeal aspiration. There were 6 animals per group. Animals were euthanized after 40 hr, and BALF was collected to determine neutrophil cell counts (A), LIX (B) and IL-1β (C) levels. Animals exposed to 5.0 mg/kg QTZ were used as positive control. The experiment was reproduced a second time; * p < 0.05 compared to control; # p < 0.05 for pairwise comparisons as shown.

Figure 3

Acute pulmonary effects of CeO₂ nanoparticles in mice. The comparison experiment was carried out in C57BL/6 mice exposed to CeO₂ nanoparticles C1, C2, C3 and C5 at 2.0 mg/Kg by oropharyngeal aspiration. There were 6 animals per group. Mice were sacrificed at 40 hr and the neutrophil cell counts (A), LIX (B) and IL -1β (C) levels in BALF were determined. QTZ at 5.0 mg/Kg was used as a positive control. The experiment was reproduced a second time; * p < 0.05 compared to control; # p < 0.05 for pairwise comparisons as shown.

Figure 4

Sub-chronic pulmonary effects of CeO₂ nanoparticles at 21 days. The experiment was performed as in Figure 3, except that the mice were sacrificed 21 days after the oropharyngeal aspiration. BALF was collected to determine the TGF-β1 (A) level. (B) The total collagen content of the lung tissues was determined by the Sircol collagen kit (Biocolor Ltd., Carrickfergus, U.K.). QTZ at 5.0 mg/kg was treated as positive control. * p < 0.05 compared to control.

Figure 5

Sub-chronic pulmonary effects of CeO₂ nanoparticles at 44 days. (A) TGF-β1 levels in BALF and (B) total collagen content of the lungs of mice receiving 4 mg/Kg CeO₂ nanoparticles. The animals were sacrificed after 44 days and all lung tissues were collected to determine the total collagen as described in Figure 4. (C) Lung sectioning and staining with Masson’s trichrome. Areas of concentrated blue staining represents collagen deposition sites. QTZ at 5.0 mg/kg served as positive control. (D) ICP-OES analysis to determine elemental Ce content in the lungs of mice receiving the same dose of C1 and C5 nanoparticles, followed by sacrifice after 44 days. (E) Comparative analysis of the collagen content in the lung of C1 and C5 exposed mice after correction for Ce content. This was accomplished by normalizing the total collagen content to the total elemental Ce content and expressed as collagen/Ce in lung. * p < 0.05 compared to control.

Figure 5

Sub-chronic pulmonary effects of CeO₂ nanoparticles at 44 days. (A) TGF-β1 levels in BALF and (B) total collagen content of the lungs of mice receiving 4 mg/Kg CeO₂ nanoparticles. The animals were sacrificed after 44 days and all lung tissues were collected to determine the total collagen as described in Figure 4. (C) Lung sectioning and staining with Masson’s trichrome. Areas of concentrated blue staining represents collagen deposition sites. QTZ at 5.0 mg/kg served as positive control. (D) ICP-OES analysis to determine elemental Ce content in the lungs of mice receiving the same dose of C1 and C5 nanoparticles, followed by sacrifice after 44 days. (E) Comparative analysis of the collagen content in the lung of C1 and C5 exposed mice after correction for Ce content. This was accomplished by normalizing the total collagen content to the total elemental Ce content and expressed as collagen/Ce in lung. * p < 0.05 compared to control.

Figure 5

Sub-chronic pulmonary effects of CeO₂ nanoparticles at 44 days. (A) TGF-β1 levels in BALF and (B) total collagen content of the lungs of mice receiving 4 mg/Kg CeO₂ nanoparticles. The animals were sacrificed after 44 days and all lung tissues were collected to determine the total collagen as described in Figure 4. (C) Lung sectioning and staining with Masson’s trichrome. Areas of concentrated blue staining represents collagen deposition sites. QTZ at 5.0 mg/kg served as positive control. (D) ICP-OES analysis to determine elemental Ce content in the lungs of mice receiving the same dose of C1 and C5 nanoparticles, followed by sacrifice after 44 days. (E) Comparative analysis of the collagen content in the lung of C1 and C5 exposed mice after correction for Ce content. This was accomplished by normalizing the total collagen content to the total elemental Ce content and expressed as collagen/Ce in lung. * p < 0.05 compared to control.

Figure 6

Pulse-exposure of zebrafish larvae to CeO₂ nanoparticles to assess the effect of AR on larval development. (A) Diagram showing the stepwise pulse-exposure protocol. Zebrafish larvae at 5 dpf were incubated in CeO₂ nanoparticle suspensions in petri dishes. Thirty larvae were exposed on each occasion to 25 μg/mL nanoparticles for 6 hr. The larvae were carefully and thoroughly washed before returning to standard aquarium tanks for regular feeding and water circulation. The same batch of larvae was used for secondary and tertiary exposures at 8 and 11 dpf. The survival rate of larvae was monitored daily and the overall health status of the larvae assessed on 14 dpf based on morphology features, body length and weight, number of calcified vertebrae and digestive function. Larvae were also randomly selected for histology and TEM analyses. (B) The survival rate of untreated larvae or larvae exposed to C1, C3, C5 and AgNPs. Only the larvae exposed to AgNPs (positive control) showed decreased survival. (C) Average larval length at 14 dpf. Larvae exposed to C5 and AgNPs showed significantly reduced length. (D) Average larval weight showed that C5 and AgNPs exposures resulted in significantly lower body weight. (E) Use of the number of calcified vertebrae as assessed by calcein staining. The representative fluorescence images show that control or larvae exposed to C1 and C3 exhibit 25 calcified vertebrae at 14 dpf. By contrast, larvae exposed to C5 and AgNPs showed ~17 and ~9 calcified vertebrae, respectively. Three images were captured and blended to cover the total body length, as indicated by the dashed line s. * p < 0.05 compared to control. Scale bar: 1 mm.

Figure 6

Pulse-exposure of zebrafish larvae to CeO₂ nanoparticles to assess the effect of AR on larval development. (A) Diagram showing the stepwise pulse-exposure protocol. Zebrafish larvae at 5 dpf were incubated in CeO₂ nanoparticle suspensions in petri dishes. Thirty larvae were exposed on each occasion to 25 μg/mL nanoparticles for 6 hr. The larvae were carefully and thoroughly washed before returning to standard aquarium tanks for regular feeding and water circulation. The same batch of larvae was used for secondary and tertiary exposures at 8 and 11 dpf. The survival rate of larvae was monitored daily and the overall health status of the larvae assessed on 14 dpf based on morphology features, body length and weight, number of calcified vertebrae and digestive function. Larvae were also randomly selected for histology and TEM analyses. (B) The survival rate of untreated larvae or larvae exposed to C1, C3, C5 and AgNPs. Only the larvae exposed to AgNPs (positive control) showed decreased survival. (C) Average larval length at 14 dpf. Larvae exposed to C5 and AgNPs showed significantly reduced length. (D) Average larval weight showed that C5 and AgNPs exposures resulted in significantly lower body weight. (E) Use of the number of calcified vertebrae as assessed by calcein staining. The representative fluorescence images show that control or larvae exposed to C1 and C3 exhibit 25 calcified vertebrae at 14 dpf. By contrast, larvae exposed to C5 and AgNPs showed ~17 and ~9 calcified vertebrae, respectively. Three images were captured and blended to cover the total body length, as indicated by the dashed line s. * p < 0.05 compared to control. Scale bar: 1 mm.

Figure 6

Pulse-exposure of zebrafish larvae to CeO₂ nanoparticles to assess the effect of AR on larval development. (A) Diagram showing the stepwise pulse-exposure protocol. Zebrafish larvae at 5 dpf were incubated in CeO₂ nanoparticle suspensions in petri dishes. Thirty larvae were exposed on each occasion to 25 μg/mL nanoparticles for 6 hr. The larvae were carefully and thoroughly washed before returning to standard aquarium tanks for regular feeding and water circulation. The same batch of larvae was used for secondary and tertiary exposures at 8 and 11 dpf. The survival rate of larvae was monitored daily and the overall health status of the larvae assessed on 14 dpf based on morphology features, body length and weight, number of calcified vertebrae and digestive function. Larvae were also randomly selected for histology and TEM analyses. (B) The survival rate of untreated larvae or larvae exposed to C1, C3, C5 and AgNPs. Only the larvae exposed to AgNPs (positive control) showed decreased survival. (C) Average larval length at 14 dpf. Larvae exposed to C5 and AgNPs showed significantly reduced length. (D) Average larval weight showed that C5 and AgNPs exposures resulted in significantly lower body weight. (E) Use of the number of calcified vertebrae as assessed by calcein staining. The representative fluorescence images show that control or larvae exposed to C1 and C3 exhibit 25 calcified vertebrae at 14 dpf. By contrast, larvae exposed to C5 and AgNPs showed ~17 and ~9 calcified vertebrae, respectively. Three images were captured and blended to cover the total body length, as indicated by the dashed line s. * p < 0.05 compared to control. Scale bar: 1 mm.

Figure 7

Confocal-Raman microscopy and ICP-OES analyses of CeO₂ uptake in zebrafish embryos and larvae after nanoparticle exposure. (A) Confocal Raman microscopy analysis was conducted on the C5 exposed larvae. The signature Raman scattering peak of CeO₂ (centered at 464 cm⁻¹) confirmed the presence of C5 inside the GIT. Raman spectra collected from the skin (green dot) or the blood vessels (red dot) did not show any signature peak for CeO₂. Developing larvae were anesthetized and placed on a glass slide with concavity wells. The excitation laser beam (514 nm) was programmed to scan across the lateral view of developing larvae as indicated by the dashed line. Raman scattering spectra were collected across this line at three spots (red dot = skin, green = blood vessel, blue = GIT). Among these, only the blue spot showed a Raman signature similar to that of CeO₂ nanoparticles (provided in the insert). (B) Groups (n = 50) of embryos (24 and 72 hpf) and larvae (120 hpf) were incubated with 25 μg/mL CeO₂ (C1 and C5) for 6 hr. The embryos and larvae were thoroughly washed before acid digestion and assessment of the amount (μg) of Ce by ICP-OES. The elemental Ce content in 24 and 72 hpf embryos were 1.5–3.0 μg/g of embryos, which is close to the detection limit of ICP-OES (indicated in grey dash line). This quantity went up to 14.52 and 14,34 μg/g of larvae exposed to C1 and C5 respectively if the exposure was performed at 120 hpf. However, incubation of these larvae for an additional 24 hr (i.e., up to 144 hpf) showed a significant reduction in the Ce content upon elimination from the gut (* p < 0.05). There was no statistical significant difference between uptake of C1 and C5 in any time point.

Figure 7

Confocal-Raman microscopy and ICP-OES analyses of CeO₂ uptake in zebrafish embryos and larvae after nanoparticle exposure. (A) Confocal Raman microscopy analysis was conducted on the C5 exposed larvae. The signature Raman scattering peak of CeO₂ (centered at 464 cm⁻¹) confirmed the presence of C5 inside the GIT. Raman spectra collected from the skin (green dot) or the blood vessels (red dot) did not show any signature peak for CeO₂. Developing larvae were anesthetized and placed on a glass slide with concavity wells. The excitation laser beam (514 nm) was programmed to scan across the lateral view of developing larvae as indicated by the dashed line. Raman scattering spectra were collected across this line at three spots (red dot = skin, green = blood vessel, blue = GIT). Among these, only the blue spot showed a Raman signature similar to that of CeO₂ nanoparticles (provided in the insert). (B) Groups (n = 50) of embryos (24 and 72 hpf) and larvae (120 hpf) were incubated with 25 μg/mL CeO₂ (C1 and C5) for 6 hr. The embryos and larvae were thoroughly washed before acid digestion and assessment of the amount (μg) of Ce by ICP-OES. The elemental Ce content in 24 and 72 hpf embryos were 1.5–3.0 μg/g of embryos, which is close to the detection limit of ICP-OES (indicated in grey dash line). This quantity went up to 14.52 and 14,34 μg/g of larvae exposed to C1 and C5 respectively if the exposure was performed at 120 hpf. However, incubation of these larvae for an additional 24 hr (i.e., up to 144 hpf) showed a significant reduction in the Ce content upon elimination from the gut (* p < 0.05). There was no statistical significant difference between uptake of C1 and C5 in any time point.

Figure 8

Histological and TEM analysis of the GIT. (A) Histopathology analysis shows the structural damage by C5 (but not C1) to the GIT. C5 exposed larvae showed desquamation of enterocytes and damage to the epithelial lining (marked by *). The control larvae and C1 exposed larvae showed normal enterocyte histology and an intact lining. (B) TEM analysis of a thin GIT section reveals ultrastructural damage by C5. This included blunting or loss of microvilli (marked by black arrows). (C) TEM analysis of the GIT immediately following CeO₂ exposure for 6 hr. While C1 agglomerates could be seen to loosely adhere to the tips of the microvilli, C5 bundles could be seen to pierce through the microvilli, disrupting their integrity (marked by red arrows). Mv= microvilli. Scale bar: 1 μm.

Figure 9

The digestive function of larvae exposed to C5 was significantly reduced compared to control or C1/C3 exposed larvae. (A) Explanation of the principle of digestive function testing. Quantification of the fluorescence intensity of the intramolecular-quenched protein, EnzChek, at 624 nm following experimental digestion by trypsin. (B) Left panel: representative fluorescent images of larvae fed with EnzChek. The fluorescence intensity of the digested peptides in the GIT of larvae exposed to C5 is significantly reduced compared to the fluorescence intensity in larvae exposed to C1 and C3. Right: Average ± SD of the fluorescence intensity extracted from larvae in each group. While fluorescence intensity was significant (p < 0.05) reduced for C5 exposed larvae, no decrease was seen for C1 and C3 exposed larvae.

Figure 9

The digestive function of larvae exposed to C5 was significantly reduced compared to control or C1/C3 exposed larvae. (A) Explanation of the principle of digestive function testing. Quantification of the fluorescence intensity of the intramolecular-quenched protein, EnzChek, at 624 nm following experimental digestion by trypsin. (B) Left panel: representative fluorescent images of larvae fed with EnzChek. The fluorescence intensity of the digested peptides in the GIT of larvae exposed to C5 is significantly reduced compared to the fluorescence intensity in larvae exposed to C1 and C3. Right: Average ± SD of the fluorescence intensity extracted from larvae in each group. While fluorescence intensity was significant (p < 0.05) reduced for C5 exposed larvae, no decrease was seen for C1 and C3 exposed larvae.