Battistoni M et al. (2020), Effect of nano-encapsulation of β-carotene on X...

XB-ART-56930

Toxicol Rep 2020 Jan 01;7:510-519. doi: 10.1016/j.toxrep.2020.04.004.

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Effect of nano-encapsulation of β-carotene on Xenopus laevis embryos development (FETAX).

Battistoni M , Bacchetta R , Di Renzo F , Metruccio F , Menegola E .

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Vitamin A plays a vital role during embryo development as most precursor of embryonic retinoic acid, a key morphogen during embryogenesis. Carotenoids, including β-carotene, are important vegetal source of Vitamin A and in contrast to teratogenic potential of animal-derived retinoids, β-carotene is usually considered freed from embryotoxic effects and supplements in pregnancy with β-carotene are suggested. The aim of the present work is to evaluate the effect of bulk and nano-encapsulated β-carotene on embryo development, by using the animal model Frog Embryo Teratogenesis Assay: Xenopus- FETAX. Xenopus laevis embryos were exposed from late gastrula till pharyngula (the phylotypic stage for vertebrates) to the concentrations of BULK β-carotene 150-3000 ng/mL and NANO β-carotene 0.75-30 ng/mL. At pharyngula stage, some embryos were processed for whole mount neural crest cell immunostaining, while others embryos were allowed to develop till tadpole for morphological and histological evaluation of neural crest cells-derived structures. In this model, the nano-encapsulated β-carotene induced specific malformations at craniofacial and eye level, while the bulk formulation only induced developmental delays. Finally, the applied alternative animal model resulted a rapid and sensitive screening method able to re-evaluate the teratogenic profile of nano-encapsulated micronutrients.

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Species referenced: Xenopus laevis
GO keywords: response to beta-carotene
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	graphical abstract
	Fig. 1. Morphological appearance of NF stage 46 larvae before (A-B-C-D) and after fixation and acridine orange staining according to Zucker et al. [51] (A’-B’-C’-D’). Magnification 20X (A-B-C-D); magnification 32X (A’-B’-C’-D’). A: lateral view of a control larva with normal phenotype. Note the dorsal neural tube (NT), the well-expanded gill basket (GB), the normal eye (), the intestine (I) and the oral opening (arrow). A': frontal view of a larva with normal phenotype stained with acridine orange. The naris (o) are well distinguishable, as well as the well-expanded oral cavity (‘’). B: lateral view of a NANO β-carotene 3 ng/mL larva with malformed phenotype with round-shape head (NT). B': frontal view of a malformed larva with round-shape head stained with acridine orange. The nasal pits (o) are well formed but in a different inclination due to the round-shape head. The oral cavity present marked reduction accompanied by oedema (arrow). C: lateral view of a larva exposed to NANO β-carotene 7,5 ng/mL with severely malformed phenotype with round-shape and coerced head. Note the extremely reduced oral cavity (>), the abnormal shape of the eye (<<) and the presence of oedema in the cardiac region (). C': frontal view of a severely malformed larva with round-shape and coerced head stained with acridine orange. Note the nasal pits barely visible (o), the abnormal eyes (<<) and the altered oral cavity (arrow). D: lateral view of a larva exposed to NANO β-carotene 30 ng/mL with extremely malformed phenotype with funnel shaped oral cavity (>). Note the not linear and swollen neural tube (NT), the abnormal eye with half-moon shape (<<) and the oedema in the cardiac region (*). D': frontal view of an extremely malformed larva with funnel shaped oral cavity stained with acridine orange. Note the irregular nasal pits (o), the half-moon shape eye (>>) and the oral cavity, almost a small split (arrow).
	Fig. 2. Evaluation of the benchmark doses (BMDs) for Benchmark-response (BMR) at 50 % of the different categories (malformations, severe malformations and extreme malformations) observed in larvae exposed to NANO β-carotene. Malformations: continuous dose-response curve; Severe malformations: dashed dose-response curve; Extreme malformations: dotted dose-response curve.
	Fig. 3. Evaluation of the relative potency factor (RPF) of the NANO formulation (dose-response curve with crosses) in respect to the BULK form (dose-response curve with triangles) of β-carotene. RPF was calculated at the effective concentration at 50 % (EC50). RPF of the nano-encapsulated β-carotene was 6*10−4 (BMD modelling by using PROAST software).
	Fig. 4. Frontal histological sections of NF stage 46 control and exposed larvae. A: Dorsal view of a control larva with normal phenotype. Note the well-expanded ethmoid cartilage (E) in the anterior portion of the head and laterally to the olfactory mucosa (o). The eyes () and the well-developed intestine (I) are visible. Magnification 25 × .A': Detail of the eye: note the pigmented layer () and the photoreceptor, bipolar cell and multipolar layers of the retina (>) Magnification 400 × . B: Dorsal view of a larva exposed to NANO β-carotene 3 ng/mL with malformed phenotype. Note the anterior schisis (black arrow) and the moderate oedema in the mesenchyme of the oral cavity (). Magnification 25 × .B': Detail of the eye: note the slight folding at the level of the layers of the retina (<). Magnification 100 × . C: Dorsal view of a larva exposed to NANO β-carotene 7,5 ng/mL with severely malformed phenotype. Note the anterior schisis (§) and the opened branchial basket (). Magnification 25 × .C': Detail of the eye: note the prominent folding of different layers of the retina (>>) and the abnormal shape of the crystalline (arrow). Magnification 100 × . D: Dorsal view of a larva exposed to NANO β-carotene 7,5 ng/mL with extremely malformed phenotype. Note the ethmoid cartilage with elongated and flattened shape (E) and the oedematous mesenchyme (*). Magnification 25 × . D': Detail of the anterior portion: note the disorganized tissues at the level of the nasal pits (>) and at the eye level with no centrally positioned crystalline (§) and very folded retinal layer separated by the pigmented layer (#). Magnification 100 × .
	Fig. 5. Morphological appearance of a NF stage 37 control larva before (A) and after fixation and acridine orange staining according to Zucker et al. [51] (B, C), with the corresponding histological sections (D, D’, E, E’). A: Lateral view of a control larva with normal phenotype. Note the well-developed eye (). Magnification 10 × . B: Lateral view of a control larva with normal phenotype stained with acridine orange. Note the eye pigment circularly positioned and the centrally positioned crystalline ().Magnification 40 × . C: Frontal view of a control larva with normal phenotype stained with acridine orange. Note the round-opened stomodeum (s) and the forebrain bulges (t). Magnification 40 × . D: Dorsal section of a control larva. Note the optical nerve (>) and the eye structure with centrally positioned crystalline and retinal layer already differentiated (). Magnification 100 × . D': Detail of the eye. Note the crystalline with different layers () and the regularly organized retinal layers (>). Magnification 400 × .E: Ventral section of a control larva. Note the well-introflexed stomodeum (>>). Magnification 100 × .E': Detail of E. Note the stomodeal ectoderm (black >) and the pharyngeal endoderm (red >) which are about to merge. Magnification 400 × .
	Fig. 6. Morphological appearance of NF stage 37 larvae exposed to NANO β-carotene before (A) and after fixation and acridine orange staining according to Zucker et al. [51] (B, C), with the corresponding histological sections (D, D’, E, E’). A: Lateral view of a larva exposed to NANO β-carotene 15 ng/mL with alteration at the cephalic portion. Note the half-shape of the eye (>). Magnification 10 × .B: Lateral view of a larva with alteration at the cephalic portion stained with acridine orange. Note the pigmented layers in the upper side of the eye (>). It is not possible to note the crystalline. Magnification 40 × . C: Frontal view of a larva with alteration at the cephalic portion stained with acridine orange. Note the narrowed stomodeum (<). It is not possible to note the forebrain bulges. Magnification 40 × . D: Dorsal section of a larva exposed to NANO β-carotene 15 ng/mL. Note the compromised eye structure with reduced crystalline (>) and with over folded retinal layers (arrow). Magnification 100 × .D': Detail of the eye. Note the reduced crystalline (>) and the disorganized layers of the retina (*). Magnification 400 × . E: Ventral section of a larva exposed to NANO β-carotene 30 ng/mL. Note the reduced stomodeum (>>). Magnification 100 × .E': Detail of the reduced stomodeum (>). Magnification 400 × .
	Fig. 7. X. laevis embryos at NF stage 26 immunostained with antibody anti-CRABP (C-D'). Magnification: 25X, 40 × .A, A’: X. laevis control embryo. Note the immunostained areas at the level of the oral cavity (o), of the frontal region (§), of the otic vesicle (>), of the optic vesicle (*), and at the level of the branchial arches with separate neural crest migratory flows (>>). B, B': X. laevis embryos exposed to NANO-β carotene 30 ng/mL. Note the altered migration of the neural crest cells: the migration streams appear fused forming a ventrally indistinct immunostained mass with fusion regions (<<).

References [+] :

Allam, The spectrum of median craniofacial dysplasia. 2011, Pubmed