Steroid hormone related effects of marine persistent organic pollutants in human H295R adrenocortical carcinoma cells Myrthe W. van den Dungen a,b,⇑ , Jeroen C.W. Rijk c , Ellen Kampman b , Wilma T. Steegenga b , Albertinka J. Murk a a Sub-department of Environmental Technology, Wageningen University, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands b Division of Human Nutrition, Wageningen University, Bomenweg 4, 6703 HD Wageningen, The Netherlands c RIKILT – Institute of Food Safety, Wageningen UR, Akkermaalsbos 2, 6708 WB Wageningen, The Netherlands article info Article history: Received 25 June 2014 Accepted 1 March 2015 Available online 9 March 2015 Keywords: Persistent organic pollutants (POPs) Gene expression Steroid hormones Adrenal cortex cell line H295R Endocrine disruption abstract Persistent organic pollutants (POPs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychloro- biphenyl (PCB) 126 and 153, perfluorooctanesulfonic acid (PFOS), hexabromocyclododecane (HBCD), 2,2 0 ,4,4 0 -tetrabromodiphenyl ether (BDE-47), tributyltin (TBT), and methylmercury (MeHg) can be accu- mulated in seafood and then form a main source for human exposure. Some POPs have been associated with changes in steroid hormone levels in both humans and animals. This study describes the in vitro effects of these POPs and mixtures thereof in H295R adrenocortical carcinoma cells. Relative responses for 13 steroid hormones and 7 genes involved in the steroidogenic pathway, and CYP1A1, were analyzed. PFOS induced the most pronounced effects on steroid hormone levels by significantly affecting 9 out of 13 hormone levels measured, with the largest increases found for 17b-estradiol, corticosterone, and cortisol. Furthermore, TCDD, both PCBs, and TBT significantly altered steroidogenesis. Increased steroid hormone levels were accompanied by related increased gene expression levels. The differently expressed genes were MC2R, CYP11B1, CYP11B2, and CYP19A1 and changes in gene expression levels were more sensitive than changes in hormone levels. The POP mixtures tested showed mostly additive effects, especially for DHEA and 17b-estradiol levels. This study shows that some seafood POPs are capable of altering steroido- genesis in H295R cells at concentrations that mixtures might reach in human blood, suggesting that adverse health effects cannot be excluded. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Environmental persistent organic pollutants (POPs), either his- torical or currently in use, accumulate in fatty tissues causing sea- food to be a main source for human exposure (Bilau et al., 2008; Liem et al., 2000; Montano et al., 2013). This is of major concern, because many of these marine POPs have been related to negative health effects such as reduced cognitive development, immune toxicity, neurological disorders, cancer, and endocrine disruption (Li et al., 2006; Yu et al., 2010). In addition to the well-known mechanism for dioxin-like compounds via the aryl hydrocarbon receptor (AhR) (Landers and Bunce, 1991; Okey et al., 1994), other mechanisms of toxic action have been suggested to cause adverse health effects, including disruption of thyroid (Brouwer et al., 1998) and steroid hormone systems (WHO, 2012). Such mechan- isms can be inappropriate activation or antagonism of the nuclear steroid receptors, modulating nuclear receptor coactivators, or interference with key enzymes involved in steroid hormone syn- thesis and metabolism (Sanderson, 2006). Several POPs have been shown to affect hormone levels in humans and animals (Steinberg et al., 2008; Turyk et al., 2008; Zimmer et al., 2009). To date, most studies focused on the effects of single POPs on levels of only a few different steroid hormones (Ding et al., 2007; Kraugerud et al., 2010, 2011), or extracted mixtures of only partially identified POPs were tested (Montano et al., 2011; Zimmer et al., 2011). http://dx.doi.org/10.1016/j.tiv.2015.03.002 0887-2333/Ó 2015 Elsevier Ltd. All rights reserved. Abbreviations: AhR, aryl hydrocarbon receptor; BDE-47, 2.2 0 ,4,4 0 -tetrabro- modiphenyl ether; CV, coefficient of variation; DHEA, dehydroepiandrosterone; EIA, enzyme immunoassay; HBCD, hexabromocyclododecane; HPA, hypothalamic- pituitary-adrenal; LOQ, limit of quantification; MC2R, melanocortin 2 receptor; MeHg, methylmercury; OECD, organization for economic co-operation and devel- opment; PCB, polychlorobiphenyl; PFOS, perfluorooctanesulfonic acid; POPs, persistent organic pollutants; SC, solvent control; S/N, signal-to-noise ratio; SPE, solid-phase extraction; TBT, tributyl chlorotin; TCDD, 2,3,7,8-tetrachlorodibenzo- p-dioxin. ⇑ Corresponding author at: Wageningen University, Tuinlaan 5, 6703 HE Wageningen, The Netherlands. Tel.: +31 317 485271; fax: +31 317 484931. E-mail addresses: myrthe.vandendungen@wur.nl (M.W. van den Dungen), rijk. jeroen@gmail.com (J.C.W. Rijk), ellen.kampman@wur.nl (E. Kampman), wilma. steegenga@wur.nl (W.T. Steegenga), tinka.murk@wur.nl (A.J. Murk). Toxicology in Vitro 29 (2015) 769–778 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit