Titanium dioxide nanoparticles (n-TiO₂) could enhance the bioavailability and toxicity of coexisting organic contaminants in the aquatic environment. This study attempted to investigate the combined effects of n-TiO₂ and difenoconazole (DIF) on the neurodevelopment of zebrafish and the underlying mechanisms. In this study, zebrafish embryos were exposed to n-TiO₂ (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-TiO₂ adsorbed DIF into the brain of zebrafish and significantly enhanced the bioaccumulation of DIF and n-TiO₂ in the 0.5 mg/L co-exposure group. 100 μg/L n-TiO₂ was not developmentally toxic to the zebrafish larvae, but it exacerbated DIF-induced neurobehavioral alterations in the zebrafish larvae. n-TiO₂ 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-TiO₂. Moreover, vitamin C (VC) ameliorated n-TiO₂/DIF-induced abnormal locomotor behaviors and neurotoxicity by inhibiting oxidative stress and apoptosis, indicating that oxidative stress and apoptosis are involved in n-TiO₂/DIF-induced neurotoxicity. Taken together, our data indicated that n-TiO₂ 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.

Background: Lysine-Specific Demethylase 1A (Kdm1a) is the first discovered histone lysine-specific demethylase, and mutations in kdm1a have been detected in neurodevelopmental disorders. However, the effect of kdm1a on neurobehaviors and the underlying mechanisms remain largely unknown. Methods: In this study, kdm1a deficient zebrafish were constructed using (clustered regularly interspaced short palindromic repeat) Clustered Regularly Interspaced Short Palindromic Repeats/CRISPRassociated protein 9 (CRISPR/Cas9) and the neurodevelopment was systematically assessed by a series of behavioral tests. Results: We found that kdm1a knockout zebrafish exhibited developmental toxicity and abnormal neurobehaviors, including locomotor abnormalities, and learning and memory deficits. Kdm1a deficiency suppressed central nervous system (CNS) neurogenesis in Tg (HuC:egfp) zebrafish, reduced motor neuron axon length in Tg (hb9:egfp) zebrafish and downregulated the expression of neurodevelopment related genes at 96 hours post fertilization (hpf). In addition, the expression of genes related to autophagy and apoptosis increased significantly in kdm1a knockout zebrafish. Conclusions: These results indicated that kdm1a deficiency induced locomotor abnormalities and learning and memory deficits in zebrafish larvae accompanied by activation of autophagy and apoptosis. These findings indicate a key role of kdm1a in neurodevelopment, providing novel insights into the mechanisms underlying the neurodevelopmental disorders.