Di-(2-ethylhexyl)phthalate (DEHP) is an endocrine-disrupting chemical (EDC), widely used as a plasticiser. Developmental exposure to EDCs could alter epigenetic programming and result in adult-onset disease. We investigated whether DEHP exposure during development affects glucose homoeostasis in the F1 offspring as a result of impaired insulin signal transduction in gastrocnemius muscle. Pregnant Wistar rats were administered DEHP (0, 1, 10 and 100 mg/kg per day) from embryonic days 9–21 orally. DEHP-exposed offspring exhibited elevated blood glucose, impaired serum insulin, glucose tolerance and insulin tolerance, along with reduced insulin receptor, glucose uptake and oxidation in the muscle at postnatal day 60. The levels of insulin signalling molecules and their phosphorylation were down-regulated in DEHP-exposed offspring. However, phosphorylated IRS1Ser636/639, which impedes binding of downstream effectors and the negative regulator (PTEN) of PIP3, was increased in DEHP-exposed groups. Down-regulation of glucose transporter 4 (Glut4 (Slc2a4)) gene expression and increased GLUT4Ser488 phosphorylation, which decreases its intrinsic activity and translocation towards the plasma membrane, were recorded. Chromatin immunoprecipitation assays detected decreased MYOD binding and increased histone deacetylase 2 interaction towards Glut4, indicative of the tight chromatin structure at the Glut4 promoter. Increased DNMTs and global DNA methylation levels were also observed. Furthermore, methylation of Glut4 at the MYOD-binding site was increased in DEHP-exposed groups. These findings indicate that, gestational DEHP exposure predisposes F1 offspring to glucometabolic dysfunction at adulthood by down-regulating the expression of critical genes involved in the insulin signalling pathway. Furthermore, DEHP-induced epigenetic alterations in Glut4 appear to play a significant role in disposition towards this metabolic abnormality.
Parsanathan Rajesh and Karundevi Balasubramanian
Palaniappan Murugesan, Muthusamy Balaganesh, Karundevi Balasubramanian and Jagadeesan Arunakaran
Polychlorinated biphenyls (PCBs) are ubiquitous and persistent environmental contaminants that disturb normal endocrine functions, including gonadal functions in humans and mammals. In the present study, we examined the direct effects of PCB on rat Leydig cells in vitro. Adult Leydig cells were purified by Percoll gradient centrifugation method and the purity of Leydig cells was also determined by 3β-hydroxysteroid dehydrogenase (3β-HSD) staining method. Purified Leydig cells were exposed to different concentrations (10− 10–10− 7 M) of PCB (Aroclor 1254) for 24 h under basal and LH-stimulated conditions. After the experimental period, cultured media were collected and used for the assay of testosterone and estradiol. The treated cells were used for the quantification of cell-surface LH receptors and activities of steroidogenic enzymes, such as cytochrome P450 side-chain cleavage enzyme (P450scc), 3β-HSD, and 17β-hydroxysteroid dehydrogenase (17β-HSD). Leydig cellular enzymatic antioxidants, such as superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, γ-glutamyl transpeptidase, glutathione-S-transferase, and nonenzymatic antioxidants, such as vitamins C and E, were assayed. Lipid peroxidation (LPO) and reactive oxygen species (ROS) were also estimated in Leydig cells. In addition, total RNA was isolated from control and Aroclor 1254-exposed Leydig cells to monitor the steady-state mRNA levels by reverse transcription(RT)-PCR for steroidogenic acute-regulatory (StAR) protein, cytochrome P450scc, 3β-HSD, and 17β-HSD. Our results indicated that Aroclor 1254 (10− 9, 10− 8, and 10− 7 M) treatments significantly inhibit basal and LH-stimulated testosterone and estradiol production. In addition, the activities of steroidogenic enzymes, enzymatic and nonenzymatic antioxidants were significantly diminished in a dose-dependent manner. However, LPO and ROS were elevated in a dose-dependent manner under basal and LH-stimulated conditions. RT-PCR analysis of StAR mRNA level showed a decrease only in 10− 7 M dose of Aroclor 1254 treatment, while cytochrome P450scc, 3β-HSD, and 17β-HSD mRNAs were drastically decreased in both 10− 8 and 10− 7 M Aroclor 1254 treatment. These findings suggest that PCBs can act directly on Leydig cells to diminish testosterone production by inhibiting gene expression of steroidogenic enzymes and antioxidant system.