Poly(hexamethylenebiguanide) hydrochloride induces craniofacial cartilage defects and alters Wnt signaling related gene expression in zebrafish larvae.

Poly(hexamethylenebiguanide) hydrochloride (PHMB) is widely used as a broad spectrum antimicrobial preservative, but its effects on craniofacial cartilage development require further characterization. Here, zebrafish were used to evaluate the developmental effects of PHMB following embryonic exposure to 0.15, 0.35, or 0.55 mg/L from 6 to 72 hours post fertilization (hpf). PHMB exposed larvae exhibited craniofacial cartilage malformations alongside general developmental abnormalities and altered locomotor behavior. These phenotypic changes were accompanied by increased reactive oxygen species (ROS) associated fluorescence, reduced proliferating cell nuclear antigen (PCNA) fluorescence intensity, and increased TUNEL positivity in the craniofacial region. qRT-PCR analysis of pooled larval heads revealed downregulation of cartilage development related genes and altered expression of inflammation related genes. Changes were also observed in the expression of Wnt signaling related genes, including β-catenin, lef1, and axin2. Furthermore, in separate rescue experiments, astaxanthin (AST) and the Wnt signaling agonist BML-284 each partially alleviated the craniofacial abnormalities. Taken together, these findings provide experimental evidence of PHMB associated craniofacial developmental toxicity under laboratory conditions, accompanied by oxidative stress related changes and altered Wnt related gene expression. General developmental toxicity may contribute to the craniofacial phenotype, and the partial pharmacological rescue does not establish pathway specific causality.