Riesova B et al. (2025), Developmental toxicity of fluconazole and 1,2,4...

XB-ART-61631

Sci Rep 2025 Dec 06; doi: 10.1038/s41598-025-30992-5.

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Developmental toxicity of fluconazole and 1,2,4-triazole in Xenopus laevis.

Riesova B, Maia LA, Hesova R, Peskova N, Marsalek P, Blahova J, Lakdawala P, Harnos J.

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Fluconazole (FLU) is a widely used antifungal agent frequently detected in surface waters because of its extensive use in medicine, agriculture, and personal care products.Despite concerns about its persistence and developmental toxicity in aquatic species, its effects on amphibians remain poorly understood. This study aimed to assess the developmental and molecular effects of FLU and its structural core, 1,2,4-triazole (TRI), in amphibian embryos. Xenopus laevis embryos were exposed to FLU or TRI and evaluated for mortality, hatching rate, heart rate, body length, malformation incidence, and changes in gene expression. Even at low micromolar concentrations, both azoles altered the expression of Wnt- and BMP-associated genes, indicating disruption of these signaling pathways. At higher micromolar concentrations, these molecular changes were accompanied by early signs of developmental abnormalities, which intensified at the highest doses. Observed phenotypes included reduced head size, altered skin pigmentation, prolonged body length, changes in heart rate, and mild digestive tract malformations. These findings demonstrate that even the core structural motif TRI can disrupt key developmental signaling pathways in vertebrate embryos, underscoring the need for closer monitoring of azole compounds in aquatic environments. Given the fundamental role of these pathways in vertebrate development, the results raise concerns about potential risks from long-term or prenatal exposure to azoles, in both environmental and clinical contexts.

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Species referenced: Xenopus laevis
Genes referenced: chrd ctnnb1 nog tbxt tll2
GO keywords: eye development [+]

Phenotypes: Xla wt + 1,2,4-triazole [1000ug/L](Fig. 2. r6-7; Fig. 3) [+]

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	Fig. 1. Representative images of Xenopus laevis embryos exposed to fluconazole (FLU). Embryos were treated from stage NF 3 to NF 45 with increasing concentrations of FLU (1, 100, and 1000ug/L). Image acquisition and scoring followed FETAX criteria for morphological endpoints. Phenotypic abnormalities were observed at all tested concentrations, with most prominent features including reduced head size, eye deformities, craniofacial malformations, altered pigmentation, and gut abnormalities. Images were captured at NF stage 45 using a stereomicroscope. Scale bar: 500um.
	Fig. 2. Representative images of Xenopus laevis embryos exposed to 1,2,4-triazole (TRI). Embryos were treated from stage NF 3 to NF 45 with 1, 100, and 1000g/L TRI. Phenotype scoring adhered to FETAX guidelines. A concentration-dependent increase in morphological abnormalities was observed, including smaller heads, eye spacing defects, changes in pigmentation, and intestinal malformations. Images were taken at stage NF 45. Scale bar: 500m.
	Fig. 3. Detailed analysis of malformation incidence in embryos treated with FLU and TRI. (AB) Bar plots show the percentage of embryos with developmental malformations at 120 hpf across increasing concentrations of FLU and TRI. While FLU caused a plateau in malformation incidence (~40%) starting from 1ug/L, TRI exhibited a gradual, concentration-dependent increase in malformations, reaching~25% at 1000ug/L. Data represent 24 embryos per each experimental condition. Red lines represent type of dependency. (C) Incidence of malformations in Xenopus laevis embryos following exposure to FLU and TRI. The table presents the incidence of the main morphological alterations observed. Color intensity indicates a qualitative assessment of severity (dark gray: severe; gray: moderate; light gray: mild). Representative images of each phenotype are highlighted on the left. FLU exposure had a stronger effect on Xenopus development compared to TRI.
	Fig. 4. Morphometric and physiological endpoints in embryos exposed to FLU and TRI. (A) Body length measurements of embryos at 120 hpf showed that FLU significantly increased embryo length at higher doses, while TRI had no measurable effect. The values 1, 10, and 1000 below the graphs correspond to compound concentrations expressed in ug/L. (B) Heartbeat analysis revealed that both compounds increased heart rate at lower concentrations, but TRI led to a drop at 1000g/L. The values 1, 10, and 1000 below the graphs correspond to compound concentrations expressed in ug/L. Data represent mean +/- SD from 12 individual embryos per group. See Material and Methods for more information about the measurement. Statistical significance was determined using ANOVA with Dunnetts test. , p<0.05, , p<0.01, **, p<0.001.
	Fig. 5. Gene expression analysis in Xenopus laevis embryos exposed to FLU and TRI. qPCR analysis of selected developmental genes (Beta-catenin, noggin, chordin, xbra, and xolloid) was performed at 120 hpf. The values 0.1 and 1.0 in the graph correspond to exposure concentrations of 0.1ug/L and 1.0ug/L, respectively. Significant upregulation was detected in embryos treated with both compounds, particularly TRI. These changes may reflect activation of Wnt signaling and modulation of BMP signaling. Data represent mean fold-change +/- SD from 710 embryos (ANOVA, Dunnetts test).
	Fig. 6. Summary of molecular and phenotypic effects of fluconazole (FLU) and 1,2,4-triazole (TRI) exposure on early Xenopus laevis development. Embryos were continuously exposed to FLU or TRI from NF stage 3 (upper row) to NF stage 45 (lower row) and subsequently analyzed, with embryos treated with vehicle solution serving as negative controls. Both azole compounds disrupt key embryonic signaling pathways, leading to upregulation (e.g., xbra, xolloid) and upward trends (e.g., β-catenin, chordin, noggin) in Wnt- and BMP-related genes, together with head and gut malformations, altered pigmentation, and increased heart rate. These findings highlight the developmental toxicity of azole compounds and their potential ecological impact on aquatic vertebrates. The azole ring is highlighted in light blue in the chemical structures of both compounds.