Toxicology 290 (2011) 334–341 Contents lists available at SciVerse ScienceDirect Toxicology journal homepage: www.elsevier.com/locate/toxicol 4-Nonylphenol triggers apoptosis and affects 17--Estradiol receptors in calvarial osteoblasts Maria Giovanna Sabbieti a,∗,1 , Dimitrios Agas a,1 , Francesco Palermo a , Gilberto Mosconi a , Giorgio Santoni b , Consuelo Amantini b , Valerio Farfariello b , Luigi Marchetti a a School of Biosciences and Biotechnology, University of Camerino, Camerino, MC, Italy b School of Pharmacy, University of Camerino, Camerino, MC, Italy article info Article history: Received 20 September 2011 Received in revised form 17 October 2011 Accepted 20 October 2011 Available online 25 October 2011 Keywords: Alkylphenols Osteoblasts Apoptosis abstract The present research examines the effects of 4-nonylphenol (4-NP) on mouse primary calvarial osteoblasts (COBs). Incubation of the cells with 4-NP at 10 -5 M and 10 -6 M striking decreased osteoblasts viability and phosphatidylserine (PS) exposure, measured by Annexin V, was greatly enhanced. In addi- tion, an up-regulation of Bax/Bcl2 ratio with a drop in m and an increase of cleaved caspase 9 and 3 was found, suggesting that the alkylphenol induced osteoblast death via the mitochondrial-dependent apoptotic pathway. Interestingly, treatment with 4-NP was also able to increase cleaved caspase 8 in parallel with the truncated active Bid (t-Bid) suggesting that 4-NP-mediated apoptosis depends on cross talk between the extrinsic and intrinsic pathways. It is of relevance, that the apoptotic effects of 4-NP overcame 17--Estradiol (17- E 2 ) induced-survival on osteoblasts. Also, the alkylphenol interfered with 17- E 2 regulated estrogen receptors expression. © 2011 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The formation of bone is a dynamic metabolic process and involves a complex series of events, such as the proliferation and differentiation of osteoprogenitor cells that eventually result in the formation of a mineralized extracellular matrix (Stein et al., 1990; Bresford et al., 1993; Inoue et al., 1996). Bone resorption and depo- sition are tightly coupled, and their balance defines both bone mass and quality. The resorption of pre-existing bone by mature osteo- clasts is followed by the formation of new bone by osteoblasts. Estrogens are important regulators of female bone development and play a critical role in maintaining bone volume (Riggs et al., 1998). The effects of estrogens are mediated by two nuclear recep- tors: estrogen receptor alpha (ER) and estrogen receptor beta (ER). ER and ER are both expressed in many cell types, but at lower levels than those found in reproductive tissues (Zallone, 2006). Estrogen and estrogen receptors (ERs) are important for bone formation in humans (Smith et al., 1994) and mice (Windahl et al., 2002). Estrogen has been shown to induce apoptosis in bone ∗ Corresponding author at: School of Biosciences and Biotechnology, University of Camerino, Via Gentile III da Varano, Camerino I-62032, MC, Italy. Tel.: +39 737 402715; fax: +39 737 402708. E-mail address: giovanna.sabbieti@unicam.it (M.G. Sabbieti). 1 The authors contribute equally to the paper. resorbing osteoclasts (Kameda et al., 1997; Kousteni et al., 2002) but it can also exert anti-apoptotic effects in osteoblasts, leading to an overall building of bone (Kousteni et al., 2002). In addition, estradiol enhanced BMP-induced Runx2 and osteocalcin expres- sion (Matsumoto et al., 2010). Moreover, several pieces of evidence indicate that estrogen has a bone-anabolic function that directly affects osteoblasts (Ernst et al., 1989; Bellido et al., 1993; Ikegami et al., 1993; Weitzmann and Pacifici, 2006). The endocrine disrupting chemicals (EDCs) are exogenous agents that interfere with the synthesis, secretion, transport, action and elimination of various endogenous hormones, which sustain development, reproduction and behavior (Kavlock et al., 1996; Fang et al., 2001; Markey et al., 2003). After ingestion and/or absorption, the EDCs tend to accumulate in the tissue (Clotfelter et al., 2004; Rhind et al., 2009, 2010) and alter endocrine functions through a variety of mechanisms including nuclear receptor binding and activation (Masuyama et al., 2000; Blume et al., 2000). Among these chemicals, alkylphenol ethoxylates show estrogen-like effects in various wildlife species. They are widely used in the manufacture of plastics, detergents, paints and pesti- cides (Nimrod and Benson, 1996) and 4-nonylphenol (4-NP) is the major degradation product of alkylphenol ethoxylates. The formation of bone is easily influenced by the exposure of osteoblasts and osteoclasts to chemical compounds (Yuhara et al., 1999; Yamagishi et al., 2001; Notoya et al., 2004, 2006). 4-NP induces Ca 2+ elevation and Ca 2+ -independent cell death in human osteosarcoma cells (Wang et al., 2005). However, at present, 0300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2011.10.014