Thousands of contaminants are used worldwide and eventually released into the environment, presenting a challenge of health risk assessment. The identification of key toxic pathways and characterization of interactions with target biomacromolecules are essential for health risk assessments. The adverse outcome pathway (AOP) incorporates toxic mechanisms into health risk assessment by emphasizing the relationship among molecular initiating events (MIEs), key events (KEs), and adverse outcome (AO). Herein, we attempted the use of AOP to decipher the toxic effects of 2,6-di-tert-butylphenol (2,6-DTBP) and its para-quinone metabolite 2,6-di-tert-butyl-1,4-benzoquinone (2,6-DTBQ) based on integrated transcriptomics, molecular modeling, and cell-based assays. Through transcriptomics and quantitative real-time PCR validation, we identified retinoic acid receptor β (RARβ) as the key target biomacromolecule. The epigenetic analysis and molecular modeling revealed RARβ interference as one MIE, including DNA methylation and conformational changes. In vitro assays extended subsequent KEs, including altered protein expression of p-Erk1/2 and COX-2, and promoted cancer cell H4IIE proliferation and metastasis. These toxic effects altogether led to carcinogenic risk as the AO of 2,6-DTBP and 2,6-DTBQ, in line with chemical carcinogenesis identified from transcriptome profiling. Overall, our simplified AOP network of 2,6-DTBP and 2,6-DTBQ facilitates relevant health risk assessment.
Keywords: adverse outcome pathway; chemical carcinogenesis; emerging contaminants; molecular modeling; transcriptomics.