Nano-TiO aggravates bioaccumulation and developmental neurotoxicity of difenoconazole in zebrafish larvae via oxidative stress and apoptosis: Protective role of vitamin C.

Titanium dioxide nanoparticles (n-TiO) could enhance the bioavailability and toxicity of coexisting organic contaminants in the aquatic environment. This study attempted to investigate the combined effects of n-TiO and difenoconazole (DIF) on the neurodevelopment of zebrafish and the underlying mechanisms. In this study, zebrafish embryos were exposed to n-TiO (100 μg/L), DIF (0, 0.1 and 0.5 mg/L) and their mixtures from 4 to 96 h post fertilization (hpf) and neurotoxicity was evaluated. Our results indicated that n-TiO adsorbed DIF into the brain of zebrafish and significantly enhanced the bioaccumulation of DIF and n-TiO in the 0.5 mg/L co-exposure group. 100 μg/L n-TiO was not developmentally toxic to the zebrafish larvae, but it exacerbated DIF-induced neurobehavioral alterations in the zebrafish larvae. n-TiO also aggravated DIF-induced suppression of central nervous system (CNS) neurogenesis in Tg (HuC:egfp) zebrafish, motor neuron axon length in Tg (hb9:egfp) zebrafish, and downregulation of neurodevelopmental genes (elavl3, ngn1, gap43, gfap and mbp). In addition, DIF elevated oxidative stress by accumulation of reactive oxygen species (ROS) and inhibition of antioxidant enzymes, and triggered apoptosis by upregulation of p53, bax, bcl-2 and caspase-3, which were markedly intensified in the presence of n-TiO. Moreover, vitamin C (VC) ameliorated n-TiO/DIF-induced abnormal locomotor behaviors and neurotoxicity by inhibiting oxidative stress and apoptosis, indicating that oxidative stress and apoptosis are involved in n-TiO/DIF-induced neurotoxicity. Taken together, our data indicated that n-TiO enhanced the accumulation of DIF and heightened oxidative stress and apoptosis, thereby inducing neurotoxicity. This study exemplifies the importance of the toxicity assessment of chemical mixtures and novel insights to mitigate their combined toxicity.