Background Al2O3 nanoparticles (AlNPs) are widely used in optoelectronics, pigments, catalysts, and ceramic industry because of its unique physical and chemical properties. AlNPs can be released into the environment directly or indirectly during processes such as product manufacturing, processing, and waste discharge. However, available toxicological information is insufficient to assess the potential risks of AlNPs to aquatic organisms, occupationally exposed populations, and the public.
Objective We aims to explore the neurotoxicity of AlNPs on zebrafish larvae and the role of specifically knocked down mTOR gene in the relevant toxic effects.
Methods Zebrafish embryos were exposed to 6.25, 12.5, 25.0, 50.0, and 100 mg/L AlNPs at 6 hours post-fertilization, with 60 fertilized eggs in each dose group. The testing indicators of larvae included motor behavioral indicators (locomotor activity in darkness, thigmotaxis, light-evoked startle response, and darkness-evoked escape response), oxidative stress indicators (superoxide dismutase activity and lactate dehydrogenase activity), and mTOR and Beclin1 mRNA expression changes. Another batch of embryos were divided into blank control group, negative control group, AINPs group, mTOR knockdown group, and mTOR knockdown+AINPs group. In 20-60 min after fertilization, the zebrafish eggs were exposed to 100 mg/L AlNPs and were injected with antisense oligonucleotides to specifically knock down mTOR gene under the microscope. The behavioral changes of larvae were detected on the 6th day after fertilization.
Results With increasing exposure dose of AINPs, the spontaneous movement velocity of zebrafish in darkness gradually decreased. The velocity in the 100 mg/L group was lower than that in the control group (P=0.003). Thigmotaxis was significantly lower in the 25, 50, and 100mg/L AlNPs groups (P < 0.05). Light-evoked startle response was attenuated in the 50 and 100 mg/L groups (P < 0.05). Darkness-evoked escape response was attenuated in the 25, 50, and 100 mg/L groups (P < 0.001). The activity of superoxide dismutase was decreased in each exposed group (P < 0.001); the activity of lactate dehydrogenase was decreased in the groups exposed to AlNPs greater than or equal to 12.5 mg/L (P < 0.05). The expression of mTOR gene was down-regulated in the 25, 50, and 100 mg/L groups of AlNPs (P < 0.05), and the expression of Beclin1 gene was up-regulated in the 100 mg/L concentration group (P=0.003). The mTOR knockdown+AINPs group's zebrafish light-evoked startle response and darkness-evoked escape response were attenuated compared with the AINPs group (Ps < 0.05).
Conclusion Exposure to AlNPs during embryo and larvae development may cause changes in motor behavior and oxidative stress in zebrafish larvae, which may be related to decreased expression of mTOR gene. Specific knockdown of mTOR gene could enhance the toxic effect of AlNPs on zebrafish larvae.
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