Carcinogenic Risk of 2,6-Di-tert-Butylphenol and Its Quinone Metabolite 2,6-DTBQ Through Their Interruption of RARβ: In Vivo, In Vitro, and In Silico Investigations

Abstract

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.

Dissecting the Enantioselective Neurotoxicity of Isocarbophos: Chiral Insight from Cellular, Molecular, and Computational Investigations

“…In fact, almost all the processes of life activity are continuously changing in organisms, and the inquisition of these physiological and pathological phenomena from a dynamic scale can unveil their intrinsic rules. , Meanwhile, the general opinions believe that biomolecular functions depend mainly on its conformations, or that conformational changes determine its biological functions . Moreover, biomacromolecular conformations are not completely rigid in the body; instead it usually has a high degree of inherent flexibility, which has been proved by some experimental techniques such as nuclear magnetic resonance, computational chemistry, and X-ray diffraction crystallography. , However, the physiological significance of biomolecular conformational flexibility has not yet been fully clarified.…”

Promotion of Bladder Cancer Cell Metastasis by 2-Mercaptobenzothiazole via Its Activation of Aryl Hydrocarbon Receptor Transcription: Molecular Dynamics Simulations, Cell-Based Assays, and Machine Learning-Driven Prediction

“…37 The MIE between pollutants as exogenous ligands and nuclear receptors is critical for the elucidation of the molecular mechanism of disrupting the biological effects of pollutants. 38,39 The potent interaction of MBT with hAhR LBD can be characterized as a MIE. Generally, the crystal structures of many nuclear receptors, including hAhR LBD, have missing amino acids, affecting the modeling of molecular interactions.…”

Bisphenol A Analogs Induce Cellular Dysfunction in Human Trophoblast Cells in a Thyroid Hormone Receptor-Dependent Manner: In Silico and In Vitro Analyses

“…The toxicological framework of the adverse outcome pathway (AOP) is an effective tool for identifying the hazard mechanisms of chemicals and for performing health risk assessments . By characterizing the interactions between pollutants and target biological macromolecules, that is, deciphering the molecular initiation events (MIEs) of AOPs and exploring their relationships with key events (KEs), the adverse outcomes (AOs) triggered by pollutants can be revealed . Determining the interaction mechanisms between pollutants and target biomacromolecules is very important for the identification of MIEs.…”

“…17 By characterizing the interactions between pollutants and target biological macromolecules, that is, deciphering the molecular initiation events (MIEs) of AOPs and exploring their relationships with key events (KEs), the adverse outcomes (AOs) triggered by pollutants can be revealed. 18 Determining the interaction mechanisms between pollutants and target biomacromolecules is very important for the identification of MIEs. As the main targets of exogenous ligand-binding TRs, ligand-binding domain (LBD) conformational changes interfere with transcriptional regulation downstream of TRs.…”