Reproductive Toxicology 27 (2009) 133–139 Contents lists available at ScienceDirect Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox Estrogenicity of food-associated estrogenic compounds in the fetuses of female transgenic mice upon oral and IP maternal exposure  Marcel G.R. ter Veld a,1 , E. Zawadzka a,2 , Ivonne M.C.M. Rietjens a , Albertinka J. Murk a,b,∗ a Toxicology Section, Wageningen University, Tuinlaan 5, 6703 HE, Wageningen, The Netherlands b Wageningen Imares, Postbox 68, 1970 AB IJmuiden, The Netherlands article info Article history: Received 5 June 2008 Received in revised form 27 January 2009 Accepted 30 January 2009 Available online 11 February 2009 Keywords: Estrogen receptor Fetus Placental transfer Reporter gene mice Endocrine disruption Food packaging Plasticizer Pseudo-estrogen abstract The present study investigated to what extent seven food-associated in vitro estrogenic compounds can induce estrogenic effects in the fetuses of pregnant female mice with an estrogen receptor (ER)- mediated luciferase (luc) reporter gene system. The luc-induction was determined either 8h after maternal dosing with a single intraperitoneal (IP) dose or 24 h after the last of a series of 8 daily oral dosages. Three known estrogens, 17-estradiol (E 2 ), 17-ethynylestradiol (EE) and 17-estradiol 3,17-dipropionate (EP) were used as positive controls at 1mg/kgbw and DMSO as solvent control. The food-associated estrogenic compounds tested were: bisphenol A (BPA), nonylphenol (NP) both at 50 mg/kg bw, dichlorodiphenyldichloroethylene (p,p ′ -DDE) at 50 mg/kg bw, quercetin at 16.6 mg/kg bw, and di-isoheptyl phthalate (DIHP), di-(2-ethylhexyl) phthalate (DEHP) and di-(2-ethylhexyl) adipate (DEHA) all at 100 mg/kg bw. Exposure to E 2 , EE and EP resulted in significant luc inductions upon both oral and/or IP dosing in a variety of tissues including liver, tibia and femurs, and upon IP dosing also in fetuses. BPA, NP, DEHA, DEHP, DIHP, DDE and quercetin were unable to significantly induce luc activity in fetuses. However, after maternal oral exposure during gestation to NP, BPA and DIHP placental luc activity was significantly lowered. The results indicate that at the current levels of exposure to food-associated estrogenic compounds, estrogenic effects to the fetus are not expected. The significant luc reduction in the placenta, should be further studied for its significance for fetal development and relevance for the human situation. © 2009 Published by Elsevier Inc. 1. Introduction Estrogenic compounds are ubiquitously present in the food chain. Well-known phyto-estrogens are genistein and quercetin, present in fruits and vegetables [1,2]. These phyto-estrogens were recently proven to be estrogenic in vitro in a human osteoblastic transgenic cell line containing either one of the two estrogen recep- Abbreviations: DDE, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene; EEF, Estra- diol Equivalence Factor; EEQ, Estradiol Equivalents; ER, estrogen receptor; ER-luc, estrogen receptor reporter gene (luciferase); GD, gestation day; IF, induction factor; IP, intraperitoneal; PND, postnatal day; RLU, relative light units.  Funding: This study was financially supported by Netherlands Society for Research (NWO-Aspasia) grant no. 015.001.130. ∗ Corresponding author at: Toxicology Section, Wageningen University, Tuinlaan 5, 6703 HE, Wageningen, The Netherlands. Tel.: +31 317483233; fax: +31 317484931. E-mail address: tinka.murk@wur.nl (A.J. Murk). 1 Current address: Department of Toxicology and Drug Disposition, Schering- Plough, Post Box 20, 5340 BH, Oss, The Netherlands. 2 Current address: Department of Andrology and Reproductive Endocrinology, Medical University of Lodz, 91-425 Lodz, Sterlinga 5, Poland. tors (ERs); ER or ER [3]. Next to phyto-estrogens present in food, pseudo-estrogens are present in food packaging, such as plastic wraps and tin coatings [4–6]. Well-known examples of estrogenic compounds in food packaging are bisphenol A (BPA), nonylphenol (NP) and several phthalates. In our laboratory several other food- associated compounds have been shown to be estrogenic in vitro as well [6]. BPA has been shown to be estrogenic in vivo as well in estrogen receptor luciferase reporter gene (ER-luc) female mice 8 h after intraperitoneal (IP)-exposure but this effect was no longer observed 24 h after IP-exposure [7,8]. In this previous study the IP- dosed diethylstilbestrol (DES) and BPA induced a time-dependent luc activity in the fetuses of the pregnant female mice. This demon- strates the usefulness of the model to detect transfer of estrogens to fetuses of IP-exposed pregnant ER-reporter gene female mice already 8 h after exposure [7,8]. The effects of DES still were measur- able after 24 h as well. The study was performed using IP-exposure which leads to high dosage peaks in the blood plasma, and not using the more realistic oral exposure that has been shown to result in much lower responses in Luc-male mice before [9]. Very early (fetal) life stages are generally considered to be more susceptible to exposure to toxicants because of the relatively low levels of defence 0890-6238/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.reprotox.2009.01.010