2-乙基己基二苯基磷酸酯(EHDPP)是一种常见的阻燃剂和环境污染物,暴露人类的内分泌干扰潜力。它的诱变性,尤其是在新陈代谢之后,尚不清楚。在这项研究中,分子对接分析表明,除CYP1A1外,EHDPP是几种人类CYP酶的潜在底物。在V79衍生的细胞系中,遗传工程用于表达每个CYP,然而,EHDPP(6小时暴露/18小时恢复)在表达人CYP1A1的V79或V79衍生细胞中不诱导微核,在表达人CYP2E1、3A4和2B6的V79衍生细胞系中呈阳性。在人类肝癌(HepG2)细胞系中,EHDPP(48小时暴露)中度诱导微核,被1-氨基苯并三唑(ABT,60μM,CYPs抑制剂);用双酚AF预处理HepG2细胞,另一种有机污染物作为CYPs的诱导剂(0.1μM,16小时),EHDPP显着增强了微核形成,阈值从10μM降低到1.25μM。这个效果被ABT阻断了,酮康唑大幅减少(抑制CYP3A表达/活性),并被反式-1,2-二氯乙烯(选择性CYP2E1抑制剂)中度抑制。免疫荧光着丝粒蛋白B染色表明EHDPP诱导的表达人CYP2E1和3A4的V79来源细胞系微核主要为着丝粒阴性,在用双酚AF(用于诱导多个CYP)预处理的HepG2细胞中,纯着丝粒阴性。在双酚AF预处理的HepG2细胞中,EHDPP有效诱导DNA断裂,如γ-H2AX的彗星测定和Western印迹分析所示。总之,我们的研究表明EHDPP具有很强的致裂作用,在被几种人类CYP酶激活后,CYP3A4是一个主要的。
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.