CEDIE   05498
CENTRO DE INVESTIGACIONES ENDOCRINOLOGICAS "DR. CESAR BERGADA"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Disruption of Sertoli cell intercellular junctions after exposure to MEHP : a potential role of oxidative stress
Autor/es:
SOBARZO CM; NOGUEIRA DE MORAIS, R; LUSTIG L; DENDUCHIS B; SCHTEINGART HF
Lugar:
Campinas
Reunión:
Simposio; IV International Symposium on Animal Biology of Reproduction; 2012
Institución organizadora:
Brazilian College of Animal Reproduction
Resumen:
Introduction Di-(2-ethylhexyl) phthalate (DEHP) is one of the phthalate esters plasticizers most commonly used as an additive in plastics industry. Its active metabolite, a Mono-(2-ethylhexyl) phthalate (MEHP) is a recognized cause of testicular atrophy and disruption of cellular redox state. A significant increase in testicular adherens components junctions, protein N-cadherin and a-catenin levels, has also been observed (1), after DEHP exposure. However increasing evidence suggests that environmental toxicants triggers oxidative stress in the testis. After this stress, the production of reactive oxygen species (ROS) leaves to lipid peroxidation of cell membranes. Besides we have previously described that Glutathione (GSH) levels are high in Sertoli cells (SC) (2). GSH is a non-protein thiol with an essential role in cell protection against oxidative stress. One of the precursors in intracellular synthesis, N-Acetyl-L-cysteine (NAC), is a reducing agent too, that may directly modify the activity of several proteins, that can reduce ROS and inhibit apoptosis in testicular germ cells in vitro. Moreover glutathione-S-Transferases, a family of detoxification isoenzymes present in the testis, which catalyzes the conjugation with GSH of many toxic substrates, such as MEHP. This conjugation reduces toxicity of MEHP and protects SC against oxidative stress.   Objective: to analyze MEHP effect on the integrity of intercellular junctions on Sertoli cell primary cultures and to evaluate the role of oxidative stress and Glutathione on these junction’s proteins changes.   Material and Methods SC were isolated from 20-days-old male rat testis and cultured during 5 days in chemically defined medium (2). Cells were exposed to two different concentrations of MEHP: 100 µM alone or MEHP plus N-Acetyl-cysteine (NAC 1mM) during the last 24 h of culture to evaluate junctions’ proteins; and MEHP 200 µM to perform GSH and Glutathione-S-transferases (G-S-T) assays. For lipoperoxide (LPO) assays, the  SC were exposed to MEHP (200 µM) during only the last hour of culture. The localization and expression of intercellular tight (occludin, claudin-11, zonula occludens-1(ZO-1)), adherens (N-cadherin, a and b-catenin) and gap (connexin 43 (Cx-43)) junctions’ proteins were evaluated by immunofluorescence (IF) and Western blot (Wb), (1). Intracellular GSH level and G-S-T activity were determined by spectrophotometry (2). LPO levels were quantified by the FOX2 method (3). Results were expressed as percentage of control value. Different letters means p<0.01 vs. control cells.   Results Fig 1.  Expression and localization of SC intercellular junctions’ proteins by IF. The  intercellular SC junctions showed a unmodified linear IF pattern for occludin, claudin-11, and connexin 43 Cx-43 either in controls as in MEHP treated cells for 24h. However, this treatment induced a delocalization of the IF signal from membrane to the cytoplasm for N-cadherin, á-catenin and ZO-1 proteins. Bars: 25 µm   Fig 2.  The Western blot analysis shows that MEHP increased N-cadherin, á-and b-catenin and ZO-1 protein expression and reduced Cx-43 expression. No changes in claudin-11 and occludin expression were found in MEHP treatment SC cultures, compared to controls. Fig 3. Exposure to MEHP reduced GSH levels in relation to controls and NAC treatment helped to prevent MEHP effects. NAC alone or in combination with MEHP, increased GSH levels in comparison to control. A concomitant increase in G-S-T activity was observed under MEHP treatment for 24h, indicating that GSH could conjugate to MEHP to detoxify the exposed SC. As an indicative of oxidative stress, LPO concentrations were higher in 1h-MEHP treated SC, than in controls.   Conclusion: Our data suggest that MEHP induces disruption on the integrity of intercellular junctions on Sertoli cell primary cultures and that the reduction of GSH levels to compensate the toxic triggered oxidative stress.