背景:2,2'-亚甲基双(4-甲基-6-叔丁基苯酚)(AO2246)是一种合成的酚类抗氧化剂,广泛用于食品包装袋和化妆品中。最近,在孕妇和哺乳期妇女的血浆和母乳样品中检测到AO2246的浓度出乎意料地很高。因此,必须进行彻底的调查,以评估AO2246对生物群的有害影响。
目的:研究AO2246对斑马鱼的发育和行为毒性,以及这些影响的分子机制。
方法:将斑马鱼胚胎以0.05至10μM的浓度暴露于AO2246,受精后(dpf)长达6天。孵化率,存活率,心率,和身体长度进行了测量。进行了运动行为和电生理学分析。使用了两种荧光标记的转基因斑马鱼系(内皮Tg和巨噬细胞/小胶质细胞Tg)。进行RNA测序。
结果:AO2246的96小时LC50值为3μM。AO2246的暴露导致孵化率和心率均显着降低。运动行为的分析表明,暴露于AO2246剂量超过2μM的幼虫的总距离和平均速度均显着降低。电生理记录表明,在3μM的浓度下,刺突活性显着降低,相对于控制条件。以3μM的AO2246的施用引起了巨噬细胞/小胶质细胞转基因斑马鱼系中中脑小胶质细胞的形态反应和免疫改变,表明神经系统疾病对行为缺陷的潜在贡献。RNA测序分析显示在高AO2246浓度下基因表达谱发生了改变,特别是与神经元功能相关的通路失调。
结论:本研究表明,AO2246暴露会引起斑马鱼幼虫的发育和神经行为毒性。具体来说,发现暴露于AO2246会导致神经元电生理活动和神经系统疾病的紊乱,最终导致斑马鱼幼虫的运动行为受损。
BACKGROUND: 2,2\'-Methylenebis (4-methyl-6-tert-butylphenol) (AO2246) is a synthetic phenolic antioxidant extensively used in food packaging bags and cosmetics. Recently, AO2246 was detected with unexpectedly high concentrations in plasma and breast milk samples from pregnant and lactating women. Hence, it is essential to conduct a thorough investigation to evaluate the detrimental effects of AO2246 on biota.
OBJECTIVE: To investigate the developmental and behavioral toxicity of AO2246 in zebrafish, as well as the molecular mechanisms underlying these effects.
METHODS: Zebrafish embryos were exposed to AO2246 at concentrations ranging from 0.05 to 10 μM for up to 6 days postfertilization (dpf). Hatching rate, survival rate, heart rate, and body length were measured. Locomotor behavioral and electrophysiologal analyses were performed. Two fluorescence-labeled transgenic zebrafish lines (endothelium-Tg and macrophage/microglia-Tg) were employed. RNA sequencing was carried out.
RESULTS: AO2246 has a 96-hour LC50 value of 3 μM. The exposure of AO2246 resulted in a significant reduction in both hatching rate and heart rate. Analysis of locomotor behavior demonstrated that larvae exposed to AO2246 doses exceeding 2 μM exhibited a significant decrease in both total distance and mean velocity. Electrophysiological recordings demonstrated a noteworthy reduction in spike activity at a concentration of 3 μM, relative to control conditions. The administration of AO2246 at 3 μM elicited morphological reactivity and immune alteration of the midbrain microglia in the macrophage/microglia-transgenic zebrafish line, indicating a potential contribution of neurological disorders to behavioral defects. RNA sequencing analysis revealed altered gene expression profiles at high AO2246 concentrations, particularly the dysregulation of pathways associated with neuronal function.
CONCLUSIONS: The present study demonstrates that AO2246 exposure elicits developmental and neurobehavioral toxicity in zebrafish larvae. Specifically, exposure to AO2246 was found to cause disturbances in neuronal electrophysiological activity and neurological disorders, which ultimately led to the impairment of locomotor behavior in zebrafish larvae.