DEHP induces ferroptosis in testicular interstitial cells by inhibiting Fto expression

Objective To explore the role and mechanism of RNA demethylase fat mass and obesity-associated protein (FTO) in the ferroptosis in testicular interstitial cells induced by di (2-ethylhexyl) phthalate (DEHP). Methods Forty 3-week-old C57BL/6 male mice were randomly divided into a control group (corn oil) and 3 dosed DEHP treatment groups (5, 250 and 500 mg/kg), and received an intragastric infusion of corresponding agents for 35 d, respectively. After mouse testicular interstitial TM3 cells was treated with 0, 100, 200 and 400 μmol/L mono-2-ethylhexyl phthalate (MEHP) for 24 h, corresponding plasmids were transfected to construct Fto overexpressing TM3 cells. Serum testosterone level was detected by ELISA, expression of testicular proteins was detected with immunohistochemical assay, and contents of Fe2+, malondialdehyde (MDA) and lipid peroxides in the testicle were detected by colorimetry. Methylated RNA immunoprecipitation, RT-PCR, and Western blotting were used to detect the level of N6-methyladenosine (m6A) modification. Results In the mice exposed to 250 and 500 mg/kg DEHP, the serum testosterone level was significantly reduced (P < 0.01), contents of Fe2+, MAD and lipid peroxides in testicular tissue were obviously increased (P < 0.01), and protein levels of RNA demethylase FTO, and ferroptosis related molecules ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) were significantly down-regulated (P < 0.05), while those of transferrin receptor (TFRC), ferroportin (FPN), cyclooxygenase-2 (COX-2), and acyl-CoA synthetase long-chain family member 4 (ACSL4) were notably up-regulated (P < 0.05). MEHP treatment for 24 h resulted in remarkably decreased cell viability in the TM3 cells, increased production of intracellular reactive oxygen species (ROS), reduced mitochondrial membrane potential (MMP) (P < 0.01), down-regulated mRNA and protein levels of Fto (P < 0.01), and the changes in other ferroptosis related proteins were consistent with the trend in testicular tissue, indicating ferroptosis in testicular interstitial cells. Intervention with ferroptosis inhibitor Fer-1 or overexpression of Fto significantly inhibited MEHP-induced toxicity and ferroptosis in TM3 cells (P < 0.05), and overexpression of Fto reduced the m6A modification of Gpx4 and Fth1 mRNA (P < 0.05). Conclusion Abnormal m6A modification of Gpx4 and Fth1 caused by inhibiting FTO expression may be the mechanism of ferroptosis in testicular interstitial cells induced by DEHP.