Objective Di(2-ethylhexyl) phthalate (DEHP), a representative plasticizer, is commonly used in plastcs producton and widely present in environment. Epidemiological studies have proved that DEHP is associated with the occurrence and development of diabetes, but the mechanism research is exiguous. The current study is conducted to explore the oxidatve stress and apoptosis in rat islet β cells (INS-1 cells) induced by DEHP.
Methods INS-1 cells were cultured in vitro, following exposure to 30, 60, and 120 μmol/L DEHP for 24 h. MTT assay was used to measure the cell proliferation inhibition rate, and flow cytometry for cell apoptosis. Cellular reactve oxygen species (ROS) generaton was detected by 2', 7'-dichlorodi-hydrofluorescein diacetate (DCFH-DA) method, the actvity of superoxide dismutase (SOD) by water soluble tetrazolium-8 (WST-8) method, reduced and oxidized glutathione (GSH and GSSG) by 5, 5'-dithiobis-(2-nitrobenzoic acid) (DTNB) method, malondialdehyde (MDA) by thiobarbituric acid (TBA) method, and mitochondrial membrane potential (MMP) by JC-1 staining. The cell apoptosis associated proteins, including Bax, Bcl-2, cytochrome C (Cyt-C), Caspase-3, and Caspase-9, were detected by Western blot afer exposure to 60, 120, and 240μmol/L DEHP for 24h.
Results The MTT and flow cytometry results showed that DEHP induced cell proliferation inhibition, and 120 μmol/L DEHP caused apoptosis (P < 0.05). Compared with the control group, the cellular ROS fluorescence intensites in the 30, 60, and 120 μmol/L DEHPexposed cells increased by 13.9%, 20.7%, and 33.7%, respectvely, and the MDA increased by 42.9%, 44.8%, and 67.7%, respectvely (P < 0.01); the cellular GSH in the 60 and 120 μmol/L DEHP-exposed cells decreased by 9.2% and 54.8%, the GSSG increased by 101.2% and 444.1%, and the GSH/GSSG decreased by 54.8% and 91.7%, respectvely (P < 0.05); the cellular SOD actvity in the 120 μmol/L DEHPexposed cells decreased by 26.4% (P < 0.05); the MMP red-to-green fluorescence ratos in the 30, 60, and 120 μmol/L DEHP-exposed cells decreased by 10.6%, 36.8% and 38.4%, respectvely (P < 0.01). The results of Western blot showed that compared with the control group, 60, 120, and 240 μmol/L DEHP treatments increased Cyt-C expression levels; 120 and 240 μmol/L DEHP treatments increased Bax/Bcl-2; 240μmol/L DEHP treatment increased Caspase-3 and Caspase-9 expression levels (P < 0.05).
Conclusion DEHP may promote oxidatve stress, reduce MMP, and induce cell apoptosis by actvatng proteins related to mitochondrial pathway.
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