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Nanotoxicology
2020 Mar 01;142:196-213. doi: 10.1080/17435390.2019.1685695.
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Iron nanoparticle bio-interactions evaluated in Xenopus laevis embryos, a model for studying the safety of ingested nanoparticles.
Bonfanti P
,
Colombo A
,
Saibene M
,
Fiandra L
,
Armenia I
,
Gamberoni F
,
Gornati R
,
Bernardini G
,
Mantecca P
.
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Iron nanoparticles (NPs) have been proposed as a tool in very different fields such as environmental remediation and biomedical applications, including food fortification against iron deficiency, even if there is still concern about their safety. Here, we propose Xenopus laevis embryos as a suitable model to investigate the toxicity and the bio-interactions at the intestinal barrier of Fe3O4 and zerovalent iron (ZVI) NPs compared to Fe(II) and (III) salts in the 5 to 100 mg Fe/L concentration range using the Frog Embryo Teratogenesis Assay in Xenopus (FETAX). Our results demonstrated that, at concentrations at which iron salts induce adverse effects, both iron NPs do not cause acute toxicity or teratogenicity even if they accumulate massively in the embryogut. Prussian blue staining, confocal and electron microscopy allowed mapping of iron NPs in enterocytes, along the paracellular spaces and at the level of the basement membrane of a well-preserved intestinal epithelium. Furthermore, the high bioaccumulation factor and the increase in embryo length after exposure to iron NPs suggest greater iron intake, an essential element for organisms. Together, these results improve the knowledge on the safety of orally ingested iron NPs and their interaction with the intestinal barrier, useful for defining the potential risks associated with their use in food/feed fortification.
Figure 1. Transmission electron microscopy of iron NPs. The analysis revealed the large heterogeneity in the NP size and shape. Scale bar: ZVI NPs 100 nm; Fe3O4 NPs 50 nm.
Figure 2. Dissolution of iron NPs in FETAX medium. Measures were performed at 25 and 100 mg Fe/L NPs at 24 h by ICP-OES after removal of NPs by ultrafiltration. Histograms represent the mean concentrations of soluble Fe in NP suspensions (n = 3). Bars = standard error of the mean (SEM).
Figure 3. FETAX results after exposure to iron NPs and Fe (II/III) salts. (A) Comparative embryotoxicity expressed as mortality and malformed embryo rates. (B) Effect on growth expressed as head-tail length of stage 46 X. laevis embryos. All values are given as mean ± SEM of three independent assays. In (A): (*) statistically different from control; (#) statistically different from the corresponding concentration of iron salt or NPs (p < 0.05, ANOVA + Fisher LSD Method). In (B): (+) statistically significant increase versus control; (−) statistically significant decrease versus control (p < 0.05, ANOVA + Dunn's test).
Figure 4. Xenopus laevis embryos at the end of the FETAX test. Ventral views of a control (A) and embryos exposed to 25 mg Fe/L of FeSO4 (B), FeCl3 (C), ZVI NPs (D) and Fe3O4 NPs (E). FeSO4 treated embryos show abnormal gut coiling (arrow) and the presence of dark material in the intestinal loops is appreciable in NP treated embryos (D and E). Bars = 500 µm.
Figure 5. Histological transversal sections of stage 46 X. laevis embryos at level of abdominal region. Low (A–E) and high magnification (F–L) of a control (A and F) and embryos exposed to 25 mg Fe/L of FeSO4 (B and G), FeCl3 (C and H), ZVI NPs (D and I) and Fe3O4 NPs (E and L). In all treated embryos is appreciable the presence of material in the intestinal loops related to iron salts (B, C and G, H) and NPs (D, E and I, L). Damages at brush border (black arrow) and yolk platelets accumulation (arrowhead) in enterocytes are visible. Bars = 100 µm (low magnification) and 20 µm (high magnification).
Figure 6. Histological transversal sections at abdominal region of stage 46 X. laevis embryos stained with Perl’s protocol. Low (A–E) and high magnification (F–L) of a control (A and F) and embryos exposed to 25 mg Fe/L of FeSO4 (B), FeCl3 (C), ZVI NPs (D) and Fe3O4 NPs (E). In all treated embryos, the presence of blue material in the intestinal loops related to iron in form of Fe(III) is appreciable. In embryos treated with FeCl3 (H), ZVI NPs (I) and Fe3O4 NPs (L), blue staining evidenced that iron adheres to the brush border (black arrow) Bars = 100 µm (low magnification) and 10 µm (high magnification).
Figure 7. Laser scanning confocal microscopy in reflection mode on histological transversal sections at the level of small intestine of stage 46 X. laevis embryos. Control (A), and exposed to 25 mg Fe/L of ZVI NPs (B) and Fe3O4 NPs (C) embryos. Figure C is representative of an intestinal tract in which the nanoparticles have accumulated in the lumen. In treated embryos, NP reflection is visible in white color. (*) intestinal lumen; (>) brush border; () basement membrane; (←) serous membrane of the abdominal cavity.
Figure 8. Electron microscopy imaging of the X. laevis small intestine. Apical and basal region of enterocytes of a control embryo (A and B, respectively) and embryos exposed to 25 mg Fe/L of ZVI NPs (C, E, G) or Fe3O4 NPs (D, F, H). NPs aggregates in treated embryos are clearly visible near to the microvilli (mv), yolk platelets (yp) and basement membrane (bm).
Figure 9. Electron microscopy imaging of the X. laevis liver. Bile canaliculus (bc) and hepatic sinusoid (hs) of control (A) and exposed to 25 mg Fe/L of Fe3O4 NPs (B–D) embryos. NPs aggregates in treated embryos are clearly visible in sinusoidal space and within hepatocytes (B, C). (D) High magnification of C.
Figure 10. Total iron concentrations measured by ICP-OES in stage 46 embryos exposed during FETAX to Fe(II/III) salts and iron NPs at the nominal concentration of 25 mg Fe/L, with the correspondent bioconcentration factors (BCF). Data are mean ± SE (n=three replicates per concentration; fifty embryos per replicate). (BCF, mL/g wet-bw)=iron concentration in embryos (mg/Kg wet-bw/nominal iron concentration in treatment solutions at 25 mg/L). (*) statistically different from control; (#) statistically different from the other iron treatments (p < 0.05, ANOVA + Fisher LSD Method).
Figure S1. Time course of pH values of treatment solutions containing FeCl3 and FeSO4 at concentrations of 25 and 100mg Fe/L over 24 h. Each point represents the mean ± SEM of three independent assays.
Figure S2. Time course of iron NP sedimentation in FETAX solution over 24 h. Each point represents the mean ± SEM of three independent assays.
