Developmental and neurobehavioral toxicity of 2,2'-methylenebis(6-tert-butyl-4-methylphenol) (antioxidant AO2246) during the early life stage of zebrafish.

ABSTRACT

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.