Combined Effects of Chronic Graphene Oxide Exposure and High-Fat Diet-Induced Obesity on Neurotoxicity in Zebrafish: Behavioral, Histopathological, and HIF-1-Mediated Metabolic Pathway Changes.

Graphene oxide (GO) as typical 2D nanomaterials (NMs) have various applications, but their long-term neurological effects, particularly under health conditions like obesity, are unknown. This study investigated the chronic neurotoxicity of GO in zebrafish, both alone and in combination with high-fat diet (HFD)-induced obesity. Zebrafish were exposed to 0.01 or 0.1 mg/L GO for up to 3 months to establish a chronic exposure regimen. Subsequently, the co-exposure group was treated with HFD and 0.1 mg/L GO for 3 months. Our results showed that chronic exposure to GO induced subtle alterations in locomotor behavior, and HFD + GO co-exposure resulted in neurobehavioral impairment similar to HFD alone. Histopathological analysis revealed significant brain damage particularly in the HFD + GO co-exposure group. RNA-sequencing data revealed that co-exposure uniquely perturbed the inositol phosphate metabolism pathway, as well as 13 gene ontology terms related to metabolic processes, including phosphatidylinositol, glycerophospholipid, glycerolipid, and purine nucleotide metabolism. We further verified that the co-exposure upregulated the protein levels of HIF-1α, LDHA, and caspase-3, as well as key genes involved in glycolysis. This study demonstrates that chronic low-dose GO exposure can induce neurobehavioral changes and neuropathology in zebrafish, and HFD-induced obesity exacerbated HIF-1-mediated metabolic pathway changes, suggesting a combined or additive effect on specific neuropathological and molecular endpoints. These findings highlight the importance of considering underlying metabolic conditions, such as obesity, for the assessment of environmental health risks of NMs.