2-Ethylhexyl
diphenyl phosphate (EHDPP) is a common
flame retardant and
environmental pollutant, exposing humans with endocrinal disrupting potentials. Its mutagenicity, especially following metabolism, remains unclear. In this study, molecular docking analysis indicated that EHDPP was a potential substrate for several human CYP
enzymes except for
CYP1A1. Among V79-derived cell lines genetically engineered for the expression of each CYP, EHDPP (6 h exposure/18 h recovery) did not induce micronuclei in the V79 or V79-derived cells expressing human
CYP1A1, however, it was positive in V79-derived cell lines expressing human
CYP2E1, 3A4, and 2B6. In a human
hepatoma (HepG2) cell line, EHDPP (48 h exposure) moderately induced micronuclei, which was blocked by
1-aminobenzotriazole (ABT, 60 μM, inhibitor of CYPs); pretreating HepG2 cells with
bisphenol AF, another organic
pollutant as inducer of CYPs (0.1 μM for 16 h), significantly potentiated micronuclei formation by EHDPP, threshold being decreased from 10 to 1.25 μM. This effect was blocked by ABT, drastically reduced by
ketoconazole (inhibiting
CYP3A expression/activity), and moderately inhibited by trans-1,2-dichloroethylene (selective
CYP2E1 inhibitor). Immunofluorescent
centromere protein B staining indicated that EHDPP-induced micronuclei in V79-derived cell lines expressing human
CYP2E1 and 3A4 were predominantly centromere-negative, and that in HepG2 cells pretreated with
bisphenol AF (for inducing multiple CYPs) were purely centromere-negative. In
bisphenol AF-pretreated HepG2 cells EHDPP potently induced DNA breaks, as indicated by the comet assay and Western blot analysis of γ-H2AX. In conclusion, our study suggests that EHDPP is potently clastogenic, following activation by several human CYP
enzymes,
CYP3A4 being a major one.