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François Chauvigné,^1* Arnaud Menuet,^1* Laurianne Lesné,¹ Marie-Christine Chagnon,² Cécile Chevrier,¹ Jean-François Regnier,³ Jürgen Angerer,⁴ and Bernard Jégou¹

¹INSERM (Institut National de la Santé et de la Recherche Médicale), U625, GERHM, Université Rennes I, Campus de Beaulieu, Rennes, France; ²UMR FLAVIC, ENSBANA (Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l’Alimentation), Dijon, France; ³ARKEMA France, Département Toxicologie et Environnement, Colombes, France; ⁴Institut und Poliklinik für Arbeits-, Sozial- und Umweltmedizin, Erlangen, Germany

Abstract
Background: Endocrine-disrupting effects of phthalates are understood primarily from in utero exposures within the fetal rat testis. Nevertheless, their path of action, dose–response character, and cellular target(s) within the fetal testis are not known.

Objectives: In this study we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP) , mono-(2-ethylhexyl) phthalate (MEHP) , and several of their metabolites on the development of organocultured testes from rat fetus.

Methods: We removed testes from 14.5-day-old rat fetuses and cultured them for 1–3 days with or without DEHP, MEHP, and the metabolites.

Results: DEHP (10^–5 M) produced a proandrogenic effect after 3 days of culture, whereas MEHP disrupted testis morphology and function. Leydig cells were the first affected by MEHP, with a number of them being inappropriately located within some seminiferous tubules. Additionally, we found a time- and dose-dependent reduction of testosterone. By 48 hr, gonocyte proliferation had decreased, whereas apoptosis increased. Sertoli cell number was unaffected, although some cells appeared vacuolated, and production of anti-Müllerian hormone decreased in a time- and dose-dependent manner. The derived metabolite mono-(2-ethyl-5-hydroxyhexyl) phthalate was the only one to cause deleterious effects to the rat fetal testis in vitro.

Conclusion: We hope that this in vitro method will facilitate the study of different phthalate esters and other endocrine disruptors for direct testicular effects.

Key words: androgens, anti-Müllerian hormone, endocrine disruption, explant culture, fetal testis, gonocytes, phthalates. Environ Health Perspect 117:515–521 (2009) . doi:10.1289/ehp.11870 available via http://dx.doi.org/ [Online 1 December 2008]

Address correspondence to B. Jégou, INSERM, U625, GERHM, Campus de Beaulieu, Université Rennes I, Rennes, Bretagne F-35042 France. Telephone: 33-0-2-23-23-69-11. Fax: 33-0-2-23-23-50-55. E-mail: Bernard.jegou@inserm.fr

*These authors contributed equally to this work.

We thank S. Plummer for his advice with di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate (MEHP) , and H.M. Koch for his advice with the metabolites of MEHP. We also thank J.P. Cravedi for providing us with the ¹⁴C-MEHP.

This work was funded by INSERM (Institut National de la Santé et de la Recherche Médicale) , Ministère de l’Enseignement Supérieur et de la Recherche, and grants QLK-CT-200-00603 [EDEN (Exploring Novel Endpoints, Exposure, Low Dose- and Mixture-Effects in Humans, Aquatic Wildlife and Laboratory Animals) ] and 212844 [DEER (Developmental Effects of Environment on Reproductive Health) ] from the European Union, by grants from the European Council for Plasticisers and Intermediates, and by ANR-06-PNRA-008-05 (PLAST-IMPACT) .

J.F.R. is employed by ARKEMA France, which has three business segments: vinyl products (i.e. chlorine, polyvinyl chloride, and vinyl compounds) , industrial chemicals (i.e., acrylics and fluorochemicals) , and performance products (i.e., functional additives and technical polymers) . The remaining authors declare they have no competing financial interests.

Received 30 June 2008 ; accepted 1 December 2008.