Assessment of poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride-induced developmental neurotoxicity via oxidative stress mechanism: Integrative approaches with neuronal cells and zebrafish

Poly(hexamethylenebicyanoguanide-hexamethylenediamine) hydrochloride (PHMB) is a biocide with a broad spectrum of antibacterial activity. Its use as a disinfectant and preservative in consumer products results in human exposure to PHMB. Toxicity studies on PHMB mainly focus on systemic toxicity or skin irritation; however, its effects on developmental neurotoxicity (DNT) and the underlying mechanisms are poorly understood. In this study, the DNT effects of PHMB were evaluated using IMR-32 and SH-SY5Y cell lines and zebrafish. In both cell lines, PHMB concentrations ≥ 10 µM reduced neurite outgrowth, and cytotoxicity was observed at concentrations up to 40 µM. PHMB regulated expression of neurodevelopmental genes and induced reactive oxygen species (ROS) production and mitochondrial dysfunction. Treatment with N-acetylcysteine reversed the toxic effects of PHMB. Toxicity tests on zebrafish embryos showed that PHMB reduced viability and heart rate and caused irregular hatching. PHMB concentrations of 1–4 µM reduced the width of the brain and spinal cord of transgenic zebrafish and attenuated myelination processes. Furthermore, PHMB modulated expression of neurodevelopmental genes in zebrafish and induced ROS accumulation. These results suggested that PHMB exerted DNT effects in vitro and in vivo through a ROS-dependent mechanism, highlighting the risk of PHMB exposure. Human exposure to PHMB is unavoidable because PHMB is readily used as a biocide in pharmaceutical, industrial, and consumer products. As the DNT of PHMB remains uncharacterized, it is necessary to investigate its DNT mechanisms. In this study, the integrative results in cells and zebrafish suggest that PHMB affects neurodevelopment through oxidative stress. In particular, it is noteworthy that in zebrafish, developmental neurotoxic effects were shown at much lower concentrations than those demonstrated by in vitro studies. This study provides a theoretical basis for the chemical management and risk assessment of PHMB.