M E BAKER and G HARDIMAN Transcriptional analysis of 52:3 R241–R256 Review endocrine disruption Transcriptional analysis of endocrine disruption using zebrafish and massively parallel sequencing Michael E Baker and Gary Hardiman1,2 Correspondence Department of Medicine, University of California San Diego, 9500 Gilman Drive 0605, La Jolla, should be addressed California 92093-0605, USA to M E Baker or G Hardiman 1CSRC and BIMRC, San Diego State University, 5500 Campanile Drive, San Diego, California 92182-7720, USA Emails 2Department of Medicine, Medical University of South Carolina, 135 Cannon Street, Suite 303 MSC 835, [email protected] or Charleston, South Carolina 29425, USA [email protected] Abstract Endocrine-disrupting chemicals (EDCs), including plasticizers, pesticides, detergents, and Key Words pharmaceuticals, affect a variety of hormone-regulated physiological pathways in humans " endocrine disruptors and wildlife. Many EDCs are lipophilic molecules and bind to hydrophobic pockets in steroid " steroid receptors receptors, such as the estrogen receptor and androgen receptor, which are important in " zebrafish vertebrate reproduction and development. Indeed, health effects attributed to EDCs include " massive parallel sequencing reproductive dysfunction (e.g. reduced fertility, reproductive tract abnormalities, and skewed male:female sex ratios in fish), early puberty, various cancers, and obesity. A major concern is the effects of exposure to low concentrations of endocrine disruptors in utero and post partum, which may increase the incidence of cancer and diabetes in adults. EDCs affect transcription of hundreds and even thousands of genes, which has created the need for new Journal of Molecular Endocrinology tools to monitor the global effects of EDCs. The emergence of massive parallel sequencing for investigating gene transcription provides a sensitive tool for monitoring the effects of EDCs on humans and other vertebrates, as well as elucidating the mechanism of action of EDCs. Zebrafish conserve many developmental pathways found in humans, which makes zebrafish a valuable model system for studying EDCs, especially on early organ development because their embryos are translucent. In this article, we review recent advances in massive parallel sequencing approaches with a focus on zebrafish. We make the case that zebrafish exposed to EDCs at different stages of development can provide important insights on EDC Journal of Molecular effects on human health. Endocrinology (2014) 52, R241–R256 Endocrine disruptors these chemicals are used as plasticizers, pesticides, deter- gents, and pharmaceuticals, providing important benefits There is increasing concern about the effects on the health for modern industrial societies. However, some of these of humans and wildlife of organic and inorganic contaminants that have been introduced into the environ- chemicals disrupt a variety of hormone-regulated physio- ment by manufacturing industries (Colborn et al.1993, logical pathways, including reproductive responses Calafat et al. 2008, Diamanti-Kandarakis et al.2009, mediated by the estrogen receptor (ER) and androgen Heindel & vom Saal 2009, Rubin 2011, Baker & receptor (AR) in humans (Kelce et al.1998, Sonnenschein & Chandsawangbhuwana 2012, Baker et al.2012). Many of Soto 1998, Calafat et al.2008, Soto et al.2008, 2009, http://jme.endocrinology-journals.org Ñ 2014 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-13-0219 Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 02:02:25AM via free access Review M E BAKER and G HARDIMAN Transcriptional analysis of 52:3 R242 endocrine disruption Swan 2008, Diamanti-Kandarakis et al.2009, Rubin 2011) E2, can disrupt physiological responses mediated by the ER and wildlife (Oehlmann et al.2008, 2009, Lange et al.2009). (Sonnenschein & Soto 1998, Diamanti-Kandarakis et al. In humans, exposure to endocrine-disrupting chemicals 2009, Rubin 2011, Baker & Chandsawangbhuwana 2012). (EDCs) may lead to premature puberty in females, and The ER, AR, and other steroid receptors are nuclear decreased reproductive ability in men. Of major concern is receptors, a large and diverse family of transcription transient exposure to EDCs in utero and in newborns, which factors (Escriva et al. 2000, Bertrand et al. 2004, Baker can have toxic effects on reproduction and development, as 2005, Markov et al. 2009, Bertrand et al. 2011, Krasowski well as causing some endocrine-related cancers and heart et al. 2011, Sladek 2011) that are activated by a variety of disease later in life (Grun & Blumberg 2006, Henley & lipophilic molecules (Chawla et al. 2001, Ingraham & Korach 2006, Soto et al.2008, Swan 2008). Redinbo 2005, Markov et al. 2009, Huang et al. 2010, Estrogens, androgens, other steroids (Fig. 1), and Baker 2011, Sladek 2011). Nuclear receptors regulate many EDCs (Fig. 2) are small molecules, lipophilic in differentiation, development, homeostasis, and responses nature, which are thought to explain binding of EDCs to stress in humans and other vertebrates. Thus, to the ER, AR, and other steroid receptors. For example, disruption of nuclear receptor signaling by EDCs poses a chemicals, such as 4-nonylphenol, which contain a threat to many physiological responses in humans, fish, phenolic group that mimics the A ring on estradiol (E2), and other animals (Grun & Blumberg 2006, Swan 2008, and bisphenol A (BPA), which mimic the A and D rings on Diamanti-Kandarakis et al.2009, Heindel & vom Saal 2009, OH OH CH OH CH 3 CH3 3 17 D CH C CH3 3 AB HO 3 O O H Estradiol Testosterone Dihydrotestosterone Female reproduction Male reproduction Male reproduction OH Journal of Molecular Endocrinology CH OH CH3 2 CH2OH OH O O O CH O CH3 CH3 OH OH CH 3 CH3 CH3 O O O Progesterone Cortisol Aldosterone Female reproduction Response to stress Electrolyte balance Figure 1 Adrenal and sex steroids. Estradiol (E2) is the canonical female sex steroid. tissues that are not directly involved in reproduction. Cortisol is involved in However, E2 is more than a reproductive steroid: E2 has important actions in response to stress, metabolism of carbohydrates and lipids, bone turnover, heart, brain, liver, bone in females and males (Heldring et al. 2007, lung maturation, and homeostasis of the immune, cardiovascular, and CNSs Sugiyama et al. 2010, Gao & Dahlman-Wright 2011, Baker 2013). Moreover, (Sapolsky et al. 2000, Tomlinson et al. 2004, Zhou & Cidlowski 2005, E2 has an important role in prostate physiology (Weihua et al. 2002, Prins Odermatt & Gumy 2008, McEwen 2012). Although these actions are et al. 2011). Thus, EDC binding to the ER can affect reproductive physiology mediated primarily by the glucocorticoid receptor (GR); in some cells, in males as well as non-reproductive physiology in males and females. cortisol is a transcriptional activator of the mineralocorticoid receptor Testosterone and 5a-dihydrotestosterone (DHT) are two male androgens (MR). Aldosterone regulates electrolyte homeostasis by controlling (Sharifi & Auchus 2012), which also are important hormones for females. In transport of sodium and potassium in kidney and gut through transcrip- humans, progesterone is important for successful implantation of the tional activation of the MR (Hawkins et al. 2012, Martinerie et al. 2013). fertilized egg (Graham & Clarke 1997, Smith 2007). Progesterone However, MR also is important in other organs such as heart and brain, in antagonists, such as RU486, can interfere with implantation and prevent which the MR may be activated by aldosterone or cortisol (Tomlinson et al. pregnancy. However, as with E2, progesterone has actions in males and in 2004, Funder 2009). http://jme.endocrinology-journals.org Ñ 2014 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JME-13-0219 Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 02:02:25AM via free access Review M E BAKER and G HARDIMAN Transcriptional analysis of 52:3 R243 endocrine disruption AB CH OH 3 OH CH3 C D HO OH F CH3 FF O CI O A B H CH3 N H HO HO H C OH O H 3 H N H C C Estradiol Diethylstilbestrol [DES] 4-Nonylphenol 3 N H HO H H O H CI H Cl H CH O H CI CI 3 CH CH CH 5α-DHT Hydroxy-flutamide Vinclozolin H3C 3 2 3 CI Active androgen Anti-androgen Fungicide CC C H 2 CH3 HO OH HO OH CI Cl Cl Bisphenol A MBP o,p′-DDT CH CI 3 O O S O HO P O CI O CH Cl Cl 3 N CI CI N N CH3 N O H CH3 CI O CI Cl CH3 O HO CI p,p′-DDE Linuron Fenitrothion HO DDT metabolite Herbicide Insecticide OCH CI H3CO 3 O Methoxychlor 4-Hydroxyl-trichloro-biphenyl Zearalenone Figure 2 Structures of xenoestrogens and xenoandrogens. (A) Xenoestrogens substituents also can disrupt estrogen physiology. Examples are DDT (Kuiper et al. 1998, Sonnenschein & Soto 1998, Diamanti-Kandarakis et al. (Sonnenschein & Soto 1998), a pesticide, and polychlorinated hydroxy- 2009). A key property of xenoestrogens is the presence of one or more biphenyls (PCBs), which are used in transformers and capacitors (Korach phenolic groups in a structure that mimics either A or the A and D rings et al. 1988, Kuiper et al. 1998). (B) Xenoandrogens (Kelce et al. 1998, of E2. Examples are diethylstilbestrol, (DES), a synthetic estrogen, Sonnenschein & Soto 1998, Diamanti-Kandarakis et al. 2009, Luccio-Camelo 4-nonylphenol (NP) and bisphenol A (BPA), which are used in plastics, & Prins 2011). A variety of structures, which do not have much in common 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene (MBP), a metabolite of BPA with either DHT or T can bind the AR. Vinclozolin, a fungicide, is an (Yamaguchi et al. 2005, Baker & Chandsawangbhuwana 2012), which antiadrogen, as are p,p0-DDE, a metabolite of p,p0-DDT, the insecticide has over 100-fold higher affinity for the ER than BPA and zearalenone, fenitrothion (Tamura et al. 2001) and the herbicide linuron (Lambright which is a toxic estrogenic metabolite synthesized by some fungi et al.