Ismail T et al. (2025), In vivo analyses of embryotoxicity and teratoge...

XB-ART-61526

Ecotoxicol Environ Saf 2025 Sep 01;304:119111. doi: 10.1016/j.ecoenv.2025.119111.

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In vivo analyses of embryotoxicity and teratogenicity of gold nanoparticles: Emphasis on the surface chemistry and toxicological responses.

Ismail T , Jeong YG , Lee HK , Lee H , Kim Y , Lee JY , Kim SH , Ryu HY , Kwon TK , Park TJ , Kwon T , Khang D , Lee HS .

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The surface chemistry and physical characteristics of gold nanoparticles (AuNPs) influence their biological interactions and toxicological responses. However, the toxicological effects of surface charge on embryonic development remain poorly understood. In this study, we investigated the in vivo developmental toxicity and teratogenicity of differentially charged AuNPs during early embryogenesis of Xenopus laevis - a sensitive and ecologically relevant animal model for developmental toxicology. Our study indicated that cationic AuNPs induced significant embryotoxicity and teratogenicity including lethality, phenotypical abnormalities and disruption of gene expression associated with liver, digestive tract, neural, and eye development. In contrast, such effects, including lethality and malformations associated with changes in gene expression were not observed in embryos exposed to anionic AuNPs. In addition, cationic AuNPs affected ciliogenesis by reducing the number of multiciliated cells and disturbing cilia-driven fluid flow, a critical endpoint in nanoparticle-induced toxicity. Furthermore, gene expression profiles suggested that necroptosis might be the mechanism of cell death in embryos exposed to cationic AuNPs. Notably, the surface charge dependent AuNPs exposure leading to impaired ciliogenesis and activation of necroptosis during embryogenesis represents significant endpoints in nanotoxicology. Unlike previous studies focusing on zebrafish or rodents, this study provides the first systematic evaluation in X. laevis embryos with identical nanoparticle cores but distinct surface chemistries. Our study underscores the significance of nanoparticle surface functionalization in determining developmental toxicity and pinpoints the ecological risks imposed by cationic AuNPs during early embryonic development in aquatic systems.

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Species referenced: Xenopus laevis
GO keywords: cytosolic ciliogenesis [+]

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	Fig. 1. Synthetic and physicochemical characterization of cationic (CTAB-capped) and anionic (citrate-capped) gold nanoparticles (AuNPs). a.3D chemical presentation of anionic AuNPs functionalized with citrate acetate trihydrate (Graphical representation show elements as follows; Grey: Carbon, White: Hydrogen, Red: Oxygen). b.3D chemical presentation of cationic AuNPs functionalized with cetyltrimethyl ammonium bromide (Graphical representation show elements as follows; Grey: Carbon, White: Hydrogen, Red: Oxygen, Blue: Nitrogen). c.TEM images and corresponding EDS elemental mapping of anionic (left) and cationic (right) AuNPs. TEM images show that both cationic and anionic AuNPs exhibit a spherical morphology with uniform size distribution (scale bar: 20 nm). EDS mapping for Au (cyan) confirms the elemental composition of the nanoparticles, while nitrogen (N, red) signals are detected only on the surface of cationic AuNPs, consistent with CTAB capping, and absent in citrate-functionalized anionic AuNPs. d.UV–vis absorption spectra of cationic AuNPs and anionic AuNPs exhibiting resonance peaks at approximately 520–530 nm. e.Electric Potential (ζ) was measured immediately after dispersion in DI water and FETAX solution. Cationic AuNPs exhibited values of + 28 mV in DI water and + 18 mV in FETAX solution, while anionic AuNPs showed –31 mV in DI water and –23 mV in FETAX solution, thereby establishing stable charge profiles for subsequent assessment of charge-dependent developmental toxicity. f. FT-IR spectra display distinct absorption bands for CTAB- and citrate-capped AuNPs, confirming the presence of ligand-specific functional groups. Citrate-capped AuNPs show prominent O–H and carboxylate signals, whereas CTAB-capped AuNPs exhibit characteristic C–H and C–N vibrations, enabling differentiation of surface chemistries. g.Hydrodynamic size analysis showed stable distributions for both AuNP types in DI water, with minimal change at 6 h. In FETAX solution, anionic AuNPs exhibited a bimodal profile at 6 h, with the secondary peak indicating aggregation, while cationic AuNPs maintained a narrow size range near 100–150 nm.
	Fig. 2. Comparative embryotoxicity of differentially charged AuNPs to Xenopus embryos. a Cationic AuNPs exposure led to 100 % lethality at 625 µg/mL, whereas the lethality observed for anionic AuNPs was 35 % at 625 μg/mL. LC50 values calculated for cationic AuNPs was 70.85 µg/mL but not determined (n.d.) for anionic AuNPs. All values are expressed as mean ± standard error of mean (SE). b Comparative embryotoxicity of cationic and anionic AuNPs expressed in terms of percentage malformation rate. Exposure to cationic AuNPs resulted in 100 % malformed embryos in the case of 125 µg/mL and 625 µg/mL treatment groups, while anionic AuNPs exposure resulted in 32 % malformed embryos in 625 µg/mL treatment group. The calculated EC50 value for cationic AuNPs was 42.63 µg/mL but not determined (n.d.) for anionic AuNPs. All values are expressed as mean ± standard error. c Statistical analysis indicated that cationic AuNPs exposure resulted in a significant reduction of total body length compared to unexposed embryos, even at the lowest exposed concentration of 1 µg/mL. In contrast, exposure to anionic AuNPs did not cause a significant reduction in total body length, but at the concentration of 125 µg/mL of anionic AuNPs, a reduction in body length was observed (P < 0.05, **P < 0.0001, not significant (n.s.), ANOVA + Dunnett’s method). d Lateral view of embryos exposed to differentially charged AuNPs showed that cationic AuNPs exposure resulted in total body length reduction and small-sized heads in Xenopus larvae after 96 h. Embryos exposed to anionic AuNPs did not show significant total body length reduction and small-sized heads compared to cationic AuNPs-exposed embryos. (Scale bar = 500μm). e Cationic AuNPs exposure resulted in small-sized heads, malformed eyes, and reduced intraocular distance compared to control embryos. Embryos exposed to cationic AuNPs were classified as moderately affected (showing less severe abnormal phenotypes) and severely affected (showing more severe abnormal phenotypes). Anionic AuNPs did not induce such malformations. (Scale bar = 500μm). f Graphical representation showing the percentage of embryos with head and eye defects in cationic and anionic AuNPs-exposed embryos compared to control embryos (P < 0.05, **P < 0.01, n.s.(not significant), ANOVA + Dunnett’s method; n = no of embryos).
	Fig. 3. Cationic-AuNPs perturbed the expression of organ specific genes during Xenopus embryonic development. a AuNPs exposure altered the expression of organ-specific transcripts as analyzed by WISH analyses. Lateral and dorsal views of embryos clearly showed that cationic AuNPs induced the upregulation of the neural-specific transcript (sox3). In contrast, the expression of pax6 (eye- & brain-specific), ldlrap1 (liver-specific), darmin (intestine-specific) was downregulated in cationic-AuNPs exposed embryos compared to control embryos, indicated by red arrows. The expression of ldlrap1 and darmin was also reduced in anionic AuNPs-treated embryos, showed by red arrows. (Scale bar = 500 μm). b A graph showed that cationic AuNPs induced the significant upregulation of sox3 and significantly downregulated the expression of pax6, ldlrap1, and darmin. The expression of ldlrap1 and darmin was also downregulated in anionic AuNPs-exposed embryos (P < 0.05, *P < 0.01, n.s. (not significant), ANOVA + Dunnett’s method). c Cationic AuNPs affected the expression of genes associated with organogenesis. Cationic AuNPs-exposed embryos led to upregulation of genes associated with organogenesis, with Log2FC values of 3.27–6.59. Moreover, one gene with Log2FC value of −2.3 was downregulated after cationic AuNPs exposure.
	Fig. 4. Cationic AuNPs induced cell death in developing Xenopus embryos a TUNEL assay was performed for early- and late-stage Xenopus embryos after exposure to differentially charged AuNPs. Blue dots (TUNEL-positive cells) were clearly evident in cationic AuNPs-exposed embryos in both early- and late-stage embryos, indicating cell death, while only a negligible number of blue dots (TUNEL-positive cells) were seen in early-stage anionic AuNPs-exposed embryos and no dead cells were observed in late-stage anionic AuNPs-treated embryos. (Scale bar = 500μm). b Statistical analysis showed a significant number of TUNEL-positive cells in cationic AuNPs-treated embryos compared to control embryos (***P < 0.001, n.s. (Not significant), ANOVA + Dunnett’s method). c Cationic AuNPs led to upregulation of cell death-associated genes. Log2FC values of genes associated with the cell death pathway showed upregulation in cationic AuNPs-treated embryos, with Log2FC values ranging from 2.2 to 4.6.
	Fig. 5. Cationic AuNPs affected the ciliogenesis during Xenopus embryogenesis. a Cationic AuNPs-exposed embryos were treated with acetylated tubulin antibodies and analyzed under a confocal microscope to analyze the effects of AuNPs on the number of MCCs. Cationic AuNPs exposure resulted in reduced number of MCCs in Xenopus embryos compared to control embryos. Representative images are shown at resolution of 200X and 600X. (Scale bar = 40μm) b Statistical analysis showed a significant reduction in cationic AuNPs-exposed embryos compared to control embryos. However, anionic AuNPs-exposed embryos did not show significant reduction compared to control embryos (*P < 0.001, P < 0.01, n.s. (not significant), ANOVA + Dunnett’s method).

	Fig. 1. Synthetic and physicochemical characterization of cationic (CTAB-capped) and anionic (citrate-capped) gold nanoparticles (AuNPs). a.3D chemical presentation of anionic AuNPs functionalized with citrate acetate trihydrate (Graphical representation show elements as follows; Grey: Carbon, White: Hydrogen, Red: Oxygen). b.3D chemical presentation of cationic AuNPs functionalized with cetyltrimethyl ammonium bromide (Graphical representation show elements as follows; Grey: Carbon, White: Hydrogen, Red: Oxygen, Blue: Nitrogen). c.TEM images and corresponding EDS elemental mapping of anionic (left) and cationic (right) AuNPs. TEM images show that both cationic and anionic AuNPs exhibit a spherical morphology with uniform size distribution (scale bar: 20 nm). EDS mapping for Au (cyan) confirms the elemental composition of the nanoparticles, while nitrogen (N, red) signals are detected only on the surface of cationic AuNPs, consistent with CTAB capping, and absent in citrate-functionalized anionic AuNPs. d.UV–vis absorption spectra of cationic AuNPs and anionic AuNPs exhibiting resonance peaks at approximately 520–530 nm. e.Electric Potential (ζ) was measured immediately after dispersion in DI water and FETAX solution. Cationic AuNPs exhibited values of + 28 mV in DI water and + 18 mV in FETAX solution, while anionic AuNPs showed –31 mV in DI water and –23 mV in FETAX solution, thereby establishing stable charge profiles for subsequent assessment of charge-dependent developmental toxicity. f. FT-IR spectra display distinct absorption bands for CTAB- and citrate-capped AuNPs, confirming the presence of ligand-specific functional groups. Citrate-capped AuNPs show prominent O–H and carboxylate signals, whereas CTAB-capped AuNPs exhibit characteristic C–H and C–N vibrations, enabling differentiation of surface chemistries. g.Hydrodynamic size analysis showed stable distributions for both AuNP types in DI water, with minimal change at 6 h. In FETAX solution, anionic AuNPs exhibited a bimodal profile at 6 h, with the secondary peak indicating aggregation, while cationic AuNPs maintained a narrow size range near 100–150 nm.
	Fig. 2. Comparative embryotoxicity of differentially charged AuNPs to Xenopus embryos. a Cationic AuNPs exposure led to 100 % lethality at 625 µg/mL, whereas the lethality observed for anionic AuNPs was 35 % at 625 μg/mL. LC50 values calculated for cationic AuNPs was 70.85 µg/mL but not determined (n.d.) for anionic AuNPs. All values are expressed as mean ± standard error of mean (SE). b Comparative embryotoxicity of cationic and anionic AuNPs expressed in terms of percentage malformation rate. Exposure to cationic AuNPs resulted in 100 % malformed embryos in the case of 125 µg/mL and 625 µg/mL treatment groups, while anionic AuNPs exposure resulted in 32 % malformed embryos in 625 µg/mL treatment group. The calculated EC50 value for cationic AuNPs was 42.63 µg/mL but not determined (n.d.) for anionic AuNPs. All values are expressed as mean ± standard error. c Statistical analysis indicated that cationic AuNPs exposure resulted in a significant reduction of total body length compared to unexposed embryos, even at the lowest exposed concentration of 1 µg/mL. In contrast, exposure to anionic AuNPs did not cause a significant reduction in total body length, but at the concentration of 125 µg/mL of anionic AuNPs, a reduction in body length was observed (P < 0.05, **P < 0.0001, not significant (n.s.), ANOVA + Dunnett’s method). d Lateral view of embryos exposed to differentially charged AuNPs showed that cationic AuNPs exposure resulted in total body length reduction and small-sized heads in Xenopus larvae after 96 h. Embryos exposed to anionic AuNPs did not show significant total body length reduction and small-sized heads compared to cationic AuNPs-exposed embryos. (Scale bar = 500μm). e Cationic AuNPs exposure resulted in small-sized heads, malformed eyes, and reduced intraocular distance compared to control embryos. Embryos exposed to cationic AuNPs were classified as moderately affected (showing less severe abnormal phenotypes) and severely affected (showing more severe abnormal phenotypes). Anionic AuNPs did not induce such malformations. (Scale bar = 500μm). f Graphical representation showing the percentage of embryos with head and eye defects in cationic and anionic AuNPs-exposed embryos compared to control embryos (P < 0.05, **P < 0.01, n.s.(not significant), ANOVA + Dunnett’s method; n = no of embryos).
	Fig. 3. Cationic-AuNPs perturbed the expression of organ specific genes during Xenopus embryonic development. a AuNPs exposure altered the expression of organ-specific transcripts as analyzed by WISH analyses. Lateral and dorsal views of embryos clearly showed that cationic AuNPs induced the upregulation of the neural-specific transcript (sox3). In contrast, the expression of pax6 (eye- & brain-specific), ldlrap1 (liver-specific), darmin (intestine-specific) was downregulated in cationic-AuNPs exposed embryos compared to control embryos, indicated by red arrows. The expression of ldlrap1 and darmin was also reduced in anionic AuNPs-treated embryos, showed by red arrows. (Scale bar = 500 μm). b A graph showed that cationic AuNPs induced the significant upregulation of sox3 and significantly downregulated the expression of pax6, ldlrap1, and darmin. The expression of ldlrap1 and darmin was also downregulated in anionic AuNPs-exposed embryos (P < 0.05, *P < 0.01, n.s. (not significant), ANOVA + Dunnett’s method). c Cationic AuNPs affected the expression of genes associated with organogenesis. Cationic AuNPs-exposed embryos led to upregulation of genes associated with organogenesis, with Log2FC values of 3.27–6.59. Moreover, one gene with Log2FC value of −2.3 was downregulated after cationic AuNPs exposure.
	Fig. 4. Cationic AuNPs induced cell death in developing Xenopus embryos a TUNEL assay was performed for early- and late-stage Xenopus embryos after exposure to differentially charged AuNPs. Blue dots (TUNEL-positive cells) were clearly evident in cationic AuNPs-exposed embryos in both early- and late-stage embryos, indicating cell death, while only a negligible number of blue dots (TUNEL-positive cells) were seen in early-stage anionic AuNPs-exposed embryos and no dead cells were observed in late-stage anionic AuNPs-treated embryos. (Scale bar = 500μm). b Statistical analysis showed a significant number of TUNEL-positive cells in cationic AuNPs-treated embryos compared to control embryos (***P < 0.001, n.s. (Not significant), ANOVA + Dunnett’s method). c Cationic AuNPs led to upregulation of cell death-associated genes. Log2FC values of genes associated with the cell death pathway showed upregulation in cationic AuNPs-treated embryos, with Log2FC values ranging from 2.2 to 4.6.
	Fig. 5. Cationic AuNPs affected the ciliogenesis during Xenopus embryogenesis. a Cationic AuNPs-exposed embryos were treated with acetylated tubulin antibodies and analyzed under a confocal microscope to analyze the effects of AuNPs on the number of MCCs. Cationic AuNPs exposure resulted in reduced number of MCCs in Xenopus embryos compared to control embryos. Representative images are shown at resolution of 200X and 600X. (Scale bar = 40μm) b Statistical analysis showed a significant reduction in cationic AuNPs-exposed embryos compared to control embryos. However, anionic AuNPs-exposed embryos did not show significant reduction compared to control embryos (*P < 0.001, P < 0.01, n.s. (not significant), ANOVA + Dunnett’s method).