Tire rubber derivative 6PPD and 6PPD-Q induce lipid accumulation in hepatocytes through ERRγ pathway

Abstract

N-phenyl-N′-(1,3-dimethylbutyl)-p-phenylenediamine (6PPD) is a tire rubber antioxidant that can be oxidized to form derivative 6PPD-quinone (6PPD-Q). 6PPD and 6PPD-Q have been detected in human urine with concentrations reaching nanomolar levels. However, their human adverse effects and toxic mechanisms are not explicit. This study elucidated their lipid metabolism disruption effects on hepatic cells (HepG2) and revealed a potential molecular mechanism via estrogen-related receptor γ (ERRγ) pathway. The results of fluorescence competitive binding assay showed that 6PPD and 6PPD-Q could bind to ERRγ with an IC₅₀ (the concentration of a ligand competing 50 % probe from ERRγ) of 9 nmol/L and 6 nmol/L, respectively. The reporter gene assay indicated that 6PPD-Q activated ERRγ in a concentration-dependent mode with the lowest observable effective concentration (LOEC) of 100 nmol/L and the maximum induction rate of 320 %, while 6PPD displayed weak ERRγ activity. The molecular docking showed that the binding energies of 6PPD/6PPD-Q with agonistic ERRγ were much lower than those with antagonistic ERRγ, implying that 6PPD/6PPD-Q tended to display ERRγ agonistic binding mode. The BODIPY fluorescence staining and triglyceride (TG) assay revealed that 6PPD and 6PPD-Q promoted lipid accumulation and TG production in HepG2 cells with LOEC of 10 nmol/L and 100 nmol/L, respectively. The ERRγ antagonist (GSK5182) co-exposure reversed the lipid induction effects of 6PPD/6PPD-Q, which confirmed the regulatory roles of ERRγ. Overall, our study revealed a novel endocrine disruption mechanism of 6PPD and 6PPD-Q via ERRγ and their hepatic lipid-inducing risks, providing novel information for their health hazard evaluation.