Effects of di-(2-ethylhexyl) phthalate and its metabolites on transcriptional activity via human nuclear receptors and gene expression in HepaRG cells

Di-(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in polyvinyl chloride products. DEHP exposure in humans is of great concern due to its endocrine-disrupting properties. In this study, we characterized the agonistic activities of DEHP and its five metabolites, mono-(2-ethylhexyl) phthalate (MEHP), 5OH-MEHP, 5oxoMEHP, 5cx-MEPP and 2cx-MMHP against human nuclear receptors, peroxisome proliferator-activated receptor alpha (PPAR alpha), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) using transactivation assays. In the PPAR alpha assay, the order of the agonistic activity was MEHP >> 5cx-MEPP >5OH-MEHP, 5oxo-MEHP >2cxMMHP > DEHP, with DEHP significantly inhibiting MEHP-induced PPAR alpha agonistic activity. This finding was compared to the results from in silico docking simulation. In the PXR assay, DEHP showed PXR agonistic activity more potent than that of MEHP, whereas the other metabolites showed little activity. In the CAR assay, none of the tested compounds showed agonistic activity. Moreover, the expression levels of PPAR alpha-, PXR-, and CARtarget genes in HepaRG cells exposed to DEHP or MEHP were investigated using qRT-PCR analysis. As a result, exposure to these compounds significantly upregulated PXR/CAR target genes (CYP3A4 and CYP2B6), but not PPAR alpha target genes (CYP4A11, etc.) in HepaRG cells. Taken together, these results suggest that direct PXR and/or indirect CAR activation by several DEHP metabolites may be involved in the endocrine disruption by altering hormone metabolism.