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Int J Environ Res Public Health
2015 Jul 28;128:8828-48. doi: 10.3390/ijerph120808828.
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Do Nanoparticle Physico-Chemical Properties and Developmental Exposure Window Influence Nano ZnO Embryotoxicity in Xenopus laevis?
Bonfanti P
,
Moschini E
,
Saibene M
,
Bacchetta R
,
Rettighieri L
,
Calabri L
,
Colombo A
,
Mantecca P
.
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The growing global production of zinc oxide nanoparticles (ZnONPs) suggests a realistic increase in the environmental exposure to such a nanomaterial, making the knowledge of its biological reactivity and its safe-by-design synthesis mandatory. In this study, the embryotoxicity of ZnONPs (1-100 mg/L) specifically synthesized for industrial purposes with different sizes, shapes (round, rod) and surface coatings (PEG, PVP) was tested using the frog embryo teratogenesis assay-Xenopus (FETAX) to identify potential target tissues and the most sensitive developmental stages. The ZnONPs did not cause embryolethality, but induced a high incidence of malformations, in particular misfolded gut and abdominal edema. Smaller, round NPs were more effective than the bigger, rod ones, and PEGylation determined a reduction in embryotoxicity. Ingestion appeared to be the most relevant exposure route. Only the embryos exposed from the stomodeum opening showed anatomical and histological lesions to the intestine, mainly referable to a swelling of paracellular spaces among enterocytes. In conclusion, ZnONPs differing in shape and surface coating displayed similar toxicity in X. laevis embryos and shared the same target organ. Nevertheless, we cannot exclude that the physico-chemical characteristics may influence the severity of such effects. Further research efforts are mandatory to ensure the synthesis of safer nano-ZnO-containing products.
Figure 1. Physical and chemical characterization of ZnO nanoparticles. SEM and TEM images of sZnO (smaller, round) (a,b) and bZnO (bigger, rod) (c,d). XRD analysis of dry sZnO (e) and bZnO (f); the main planes for zincite crystal are reported.
Figure 2. Comparative FETAX results after exposure of embryos to 1–100 mg/L of sZnO and bZnO. (a) Malformation rates; (b) growth retardation. Dark grey = sZnO-exposed larvae; light grey = bZnO-exposed larvae; bars = SEM; * statistically different from the control (p < 0.05, ANOVA + Fisher LSD method).
Figure 3. Xenopus laevis larvae at the end of the FETAX test. (a) Lateral and (b) ventral view of a control; (c) lateral and (d) ventral view of an embryo exposed to 50 mg /L sZnO; (e) lateral and (f) ventral view of an embryo exposed to 50 mg/L bZnO. The treated larvae show abnormal gut coiling (arrow head), abdominal and cardiac edemas (empty arrow) and a slight dorsal tail flexure. (b,d,f) Original magnification: 4×. Bars = 1 mm.
Figure 4. Comparative FETAX malformation percentages after exposure of embryos to nude and polymer-coated sZnO and bZnO at 50 mg/L. Dark grey = sZnO-exposed larvae; light grey = bZnO-exposed larvae. Bars = SEM; * statistically different from the control at p < 0.001; ** statistically different from the corresponding nude nanoparticles at p < 0.05; # statistically different from the corresponding PVP-coated bZnO at p < 0.05,ANOVA + Fisher LSD method.
Figure 5. Percentages of malformed embryos after exposure to sZnO and bZnO at 50 mg/L in different developmental windows. Dark grey = sZnO-exposed larvae; light grey = bZnO-exposed larvae. Stages 8–46, exposure during the whole embryogenesis as in the FETAX protocol; Stages 8–39, exposure from blastula to the stomodeum opening (two days 8 hpf); Stages 39–46, exposure from stomodeum opening to the end of the primary organogenesis. * Statistically different from control at p < 0.001, ANOVA + Fisher LSD method.
Figure 6. SOD enzymatic activity in embryos exposed to nude and polymer-coated sZnO and bZnO 50 mg/L during different developmental stages. Dark grey = sZnO-exposed larvae; light grey = bZnO-exposed larvae.
Figure 7. Light (a–d) and electron microscopy (e–h) imaging of the X. laevis small intestine. Transversal sections at the level of an intestinal loop of a control (a), bZnO (b) and sZnO (c,d) exposed embryos. Magnification of the sZnO intestinal loop (d) shows the swelling of paracellular spaces between cells (empty arrow) and detachment in some regions of epithelial cells from basal lamina (*). These damages are more evident in the detail of the junctional complex between two enterocytes (g) (black arrow) and of the basal portion (h) (*) of sZnO-exposed embryos in comparison to the control (e) (black arrow) and (f) (*). ► = brush border; gl = gut lumen.
Figure S1. Levels of dissolved ions from sZnO and bZnO after 24 h and 96 h from the beginning of the FETAX assay measured by ICP-OES.
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