Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 338-354-Physiological Impacts of Endocrine Disrupting Chemicals
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-344
Helena Fedora Schteingart*1, Christian M Sobarzo2, Rosana Nogueira de Morais3, Livia Lustig2 and Berta Denduchis2
1Hospital de Niños R. Gutiérrez, Buenos Aires, Argentina, 2Facultad de Medicina, UBA, Buenos Aires, Argentina, 3Departamento de Fisiologia, Universidade Federal do Paraná, Curitiba-PR, Brazil
MEHP, an active metabolite of di-(2-ethylhexyl) phthalate (DEHP), is recognized as a reproductive toxicant, causing testicular atrophy and disruption of cellular redox state. Environmental toxicants trigger oxidative stress in the testis; increasing reactive oxygen species that led to lipid peroxidation (LPO) of cell membranes. We previously described (1) high levels of Glutathione (GSH) in Sertoli cell (SC), essential for cell protection against oxidative stress. Glutathione-S-Transferases (G-S-T), is present in the testis and catalyze conjugation of GSH with toxic substrates, such MEHP. We previously observed (2) that DEHP induces in vivo changes in the expression of specific junction proteins in rat testis. The aim of this study is to analyze MEHP effect on the integrity of intercellular junctions on SC primary cultures and to evaluate oxidative stress and GSH role.

Sertoli cells were isolated from 20-day-old male rats and cultured for 5 days. Cells were exposed to MEHP (200 µM) alone or MEHP plus N-Acetyl-cysteine (NAC - 1mM) during the last 24 h of culture. The localization and expression of adherens (N-cadherin, α, ß and p120-catenins),  tight (occludin, claudin-11, zonula occludens-1(ZO-1)), and gap (connexin 43 (Cx-43)) junction proteins were evaluated by immunofluorescence (IF) and Western blot (Wb) (2). Intracellular GSH level and G-S-T activity were determined by spectrophotometry (2).  Alternatively, SC were treated only the last hour, and LPO levels were quantified by the FOX2 method (3). Results were expressed as percentage of control value, ANOVA and Tukey’s test.

As an indicative of oxidative stress, LPO concentrations (161±17%, P<0.005 vs control) were higher in MEHP treated SC. Exposure to MEHP reduced GSH levels to 19±4% (P<0.001 vs control). NAC treatment increased GSH levels in comparison to control (185±12%, P<0.001). The addition of MEHP plus NAC reduced GSH levels to 150±12% (P<0.01 vs NAC). These results indicate that NAC helped to partially prevent MEHP effects. A concomitant increase in G-S-T activity was observed under MEHP treatment (162±26%, P<0.01 vs control), indicating that GSH could conjugate MEHP to detoxify the exposed SC. MEHP induced a delocalization of the IF signal from SC membrane to the cytoplasm for N-cadherin, α-catenin and ZO-1 proteins. By Wb analysis, MEHP significant increased N-cadherin, α and ß-catenin and ZO-1 and reduced Cx-43 expression levels. No changes in p-120 catenin, claudin-11 and occludin were found. Also, we observed a down regulation of N-cadherin and α-catenin expression in SC treated with MEHP plus NAC, similar to control SC. Our data suggest that disruption of intercellular junctions induced by MEHP in Sertoli cell may be mediated through oxidative stress signaling.

(1). Gualtieri, A.F., Iwachow,M.A., Venara,M.,Rey,R.A., & Schteingart,H.F. (2011) Bisphenol A effect on glutathione synthesis and recycling in testicular Sertoli cells. J Endocrinol Invest, 34, e102-e109. (2). Sobarzo,C.M., Lustig,L., Ponzio,R., & Denduchis,B. (2006) Effect of di-(2-ethylhexyl) phthalate on N-cadherin and catenin protein expression in rat testis. Reprod Toxicol., 22, 77-86. (3). Jiang,Z.Y., Woollard,A.C., & Wolff,S.P. (1991) Lipid hydroperoxide measurement by oxidation of Fe2+ in the presence of xylenol orange. Comparison with the TBA assay and an iodometric method. Lipids, 26, 853-856.

Nothing to Disclose: HFS, CMS, RN, LL, BD

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Sources of Research Support: Supported by CONICET and BID-PICT 2008- 0521 ANPCYT