Elsevier

Hormones and Behavior

Volume 55, Issue 2, February 2009, Pages 319-328
Hormones and Behavior

Systemic administration of diarylpropionitrile (DPN) or phytoestrogens does not affect anxiety-related behaviors in gonadally intact male rats

https://doi.org/10.1016/j.yhbeh.2008.11.004Get rights and content

Abstract

The development of highly selective agonists for the two major subforms of the estrogen receptor (ERα and ERβ) has produced new experimental methodologies for delineating the distinct functional role each plays in neurobehavioral biology. It has also been suggested that these compounds might have the potential to treat estrogen influenced behavioral disorders, such as anxiety and depression. Prior work has established that the ERβ agonist, diarylpropionitrile (DPN) is anxiolytic in gonadectomized animals of both sexes, but whether or not this effect persists in gonadally intact individuals is unknown. Isoflavone phytoestrogens, also potent but less selective ERβ agonists, have also been shown to influence anxiety in multiple species and are becoming more readily available to humans as health supplements. Here we determined the effects of 0.5, 1 or 2 mg/kg DPN, 1 mg/kg of the ERα agonist propyl-pyrazole-triol (PPT), 3 or 20 mg/kg of the isoflavone equol (EQ) and 3 or 20 mg/kg of the isoflavone polyphenol resveratrol (RES) on anxiety behavior in the gonadally intact male rat using the light/dark box and the elevated plus maze. We first determined that DPN can be successfully administered either orally or by subcutaneous injection, although plasma DPN levels are significantly lower if given orally. Once injected, plasma levels peak rapidly and then decline to baseline levels within 3 h of administration. For the behavioral studies, all compounds were injected and the animals were tested within 3 h of treatment. None of the compounds, at any of the doses, significantly altered anxiety-related behavior. Plasma testosterone levels were also not significantly altered suggesting that these compounds do not interfere with endogenous androgen levels. The results suggest that the efficacy of ERβ agonists may depend on gonadal status. Therefore the therapeutic potential of ERβ selective agonists to treat mood disorders may be limited.

Introduction

To date, two major subforms of the estrogen receptor (ERα and ERβ) have been identified in mammals (Greene et al., 1986, Kuiper et al., 1996). Although the DNA binding domains of the two are nearly identical in structure, the ligand binding domain of ERβ is only partially homologous to that of ERα and has previously been shown to bind a spectrum of ligands including the androgen metabolite 17beta-diol (3beta-diol) (Handa et al., 2007, Lund et al., 2006), phytoestrogens, and a number of endocrine disrupting compounds (Kuiper et al., 1996, 1998). ERα and ERβ are differentially distributed in both adult (Shughrue et al., 1998, Zhang et al., 2002) and immature brains (Fried et al., 2004, Lemmen et al., 1999, Perez et al., 2003) of rodents and humans suggesting that the two ER subtypes may regulate different aspects of behavior and neuroendocrine function across the lifespan. The development of ligands specific for either ERα or ERβ has proven to be a powerful tool to help delineate the relative roles each ER subtype plays in the organization and activation of steroid sensitive physiology and behavior. It has also been proposed that these compounds may have therapeutic potential (Harris, 2007, Imamov et al., 2007, Osterlund et al., 2005). However little has been published regarding the basic pharmacokinetics of these compounds or how they alter hormone dependent physiology and behavior in gonadally intact animals. This information is essential in order to evaluate the therapeutic potential of these compounds.

The present study largely focused on the most commonly used ERβ specific ligand, diarylpropionitrile (DPN), a compound with a 70-fold higher binding affinity for ERβ than ERα (Katzenellenbogen et al., 2000). This compound has been used by a number of different investigators from a broad spectrum of fields to evaluate the relative contribution ERβ plays in the organization and activation of estrogen dependent physiology and behavior (Choleris et al., 2008, Harris, 2007). These prior experiments have employed a wide range of DPN doses, spanning 10 μg to 1.5 mg, but circulating levels of DPN were not determined making it difficult to discern which, if any, of these doses produce plasma levels that approximate circulating steroid hormone levels (Frasor et al., 2003, Lund et al., 2005, Meyers et al., 2001, Rhodes and Frye, 2006, Sanchez-Criado et al., 2004, Walf et al., 2004). In addition, all of these studies have administered DPN by subcutaneous (sc) injection. For some experimental designs oral administration may be advantageous because it is less stressful to the animal and would more closely replicate the route of administration for humans. It is currently unclear from the present literature how long DPN remains in plasma after it is administered or if it would survive oral administration. Therefore, the first goal of the present study was to compare plasma DPN levels following oral or sc injection at two different doses. The second goal was to determine how long DPN remains in plasma following a single sc injection or oral dose. We hypothesized that plasma DPN levels would be present but lower following oral administration than when given by sc injection and that, regardless of the route of administration, would rapidly clear from plasma.

We next examined how agonism of ERβ by DPN affects ERβ-dependent behavior in gonadally intact male rats. Agonism of ERβ by DPN has recently been shown to have significant anxiolytic effects in gonadectomized male and female rats using a variety of behavioral paradigms including the forced swim test, the elevated plus-maze and the open field test (Lund et al., 2005, Walf and Frye, 2005, Walf et al., 2004). In contrast, the ERα specific agonist propyl-pyrazole-triol (PPT) has an opposite or no effect. These studies strongly indicate a role for ERβ in the modulation of anxiety related behaviors but because all of these studies used gonadectomized animals, it remains unclear whether or not the anxiolytic effect of DPN would persist in a gonadally intact animal. This is a key consideration when evaluating the therapeutic potential of ER selective agonists like DPN because in this case the target subject would be a gonadally intact individual. At the very least, administration of DPN to a gonadally intact animal may modify blood steroid hormone levels, an effect which may result in altered physiology or behavior.

To examine these questions we first compared the effects of two doses of DPN (0.5 mg/kg and 2 mg/kg) on anxiety related behavior using the elevated plus maze and the light dark box. Next, we compared the effect of DPN (1 mg/kg) with the ERα agonist PPT (1 mg/kg) and two phytoestrogens on anxiety related behavior and plasma androgen levels in gonadally intact male rats. Phytoestrogens, steroidal chemicals produced by plants, have been found to mimic or interact with hormone signaling in animals and generally have a higher relative binding affinity (RBA) for ERβ than ERα (Kuiper et al., 1998, Morito et al., 2001) suggesting that they may be particularly potent modulators of ERβ-dependent physiology and behavior. They are also becoming more widely available in supplement form and are routinely advertised to “improve mood.” We have previously shown that isoflavone-rich diets can be anxiogenic when fed to gonadally intact male rats (Patisaul et al., 2005). Diets containing soy isoflavones have also been shown to alter anxious and aggressive behaviors in gonadally intact males of other species including non-human primates (Hartley et al., 2003, Moore et al., 2004, Simon et al., 2004). These data suggest that agonism of ERβ in gonadally intact males may produce distinct and opposite effects on anxiety than in castrated males.

The phytoestrogens chosen for the present study were equol (EQ) and resveratrol (RES). The two most prevalent phytoestrogen isoflavones obtained from soy based foods are genistein and daidzein but, in mammals, equol (7-hydroxy-3[4′hydroxyphenyl]-chroman) is the major metabolite of daidzein and is produced exclusively by microflora in the gut (Setchell et al., 2003b). EQ production varies greatly across species, but rats consuming soy rich diets generate especially high levels of EQ (Axelson et al., 1982, Lephart et al., 2000, Setchell et al., 2002). It has also been suggested that humans that can generate EQ are more likely to experience the hypothesized benefits of soy consumption (Setchell et al., 2002, Speroff, 2005). Therefore we hypothesized that the previously observed anxiogenic effect of an isoflavone rich diet on gonadally intact male rats (Patisaul et al., 2005) might largely be attributed to the activity of EQ. The bioflavonoid isoflavone polyphenol RES (trans-3,5,4′-trihydroxystilbene) is a major constituent of red wine and is now available in health food supplements. Interest in RES intensified following a widely publicized report that obese mice, maintained on a diet containing a relatively high dose of RES (approximately 22 mg/kg body weight per day), showed improved overall health, decreased organ pathology, and longer lifespan compared to obese control mice (Baur et al., 2006). RES has also been shown increase lifespan in other species including fish and C. elegans (Howitz et al., 2003, Valenzano et al., 2006). Because RES binds both ER subtypes with equal affinity (Bowers et al., 2000, Kuiper et al., 1997, Levenson et al., 2003, Schreihofer, 2005) and therefore may not be as potent as EQ and DPN at modulating ERβ-dependent physiology and behavior, we hypothesized that RES would have a lower potential to alter anxiety-related behavior than EQ or DPN. By comparing the effects of these phytoestrogens with the ERβ agonist DPN on anxiety-related behavior in gonadally intact male rats, we aimed to determine if any of these compounds might have therapeutic potential for mood disorders in humans.

Because we used gonadally intact animals for these experiments, behavioral change following exposure to any of the ERβ agonists could result from altered plasma testosterone (T) levels rather than as a direct effect of the administered compound. Several studies have demonstrated an anxiolytic effect of T administration in castrated male rats (Bitran et al., 1993, Fernandez-Guasti and Martinez-Mota, 2005, Frye and Seliga, 2001). Additionally, EQ has the unique ability to specifically bind 5 alpha-dihydro-testosterone (DHT), and thus the potential to inhibit the action of this potent androgen. Therefore we quantified plasma T levels following the conclusion of the behavioral experiments to determine if any of the observed changes in behavior were associated with modified plasma T levels.

Section snippets

Animal care

All animals (n = 86 Long Evans males, 18 Wistar males, 3–5 months of age) were maintained on a reverse light cycle (lights off at 10:00 h and on at 22:00 h) at 23 °C and 50% humidity and housed individually. Because standard lab chows are soy-based and thus contain significant amounts of phytoestrogens (Boettger-Tong et al., 1998, Thigpen et al., 1999) all animals were fed a semi-purified, phytoestrogen-free diet (5 K96, Purina Test Diets, Richmond, IN). Animal care, maintenance, and behavioral

Experiment 1: Plasma DPN levels were significantly higher following sc injection versus oral administration

For both doses, plasma DPN levels were higher following sc injection rather than oral administration (Table 1). An oral dose of 3 mg/kg bw produced plasma DPN levels approximately equivalent to an injected dose of 1 mg/kg bw indicating that although oral administration is possible, higher doses are needed to achieve the same plasma levels of DPN. Plasma DPN levels from the animals fed 1 mg/kg bw were close to the method detection limit.

Experiment 2: Plasma DPN levels are detectable within the first 2 h after administration

Plasma DPN levels following a single, oral 3 mg/kg bw dose

Discussion

The results from the present study demonstrate that DPN can be successfully administered either orally or by sc injection. Once administered, plasma DPN levels peak rapidly and decline to near the limit of detection within 3–4 h. Plasma testosterone levels are not significantly affected by DPN administration or phytoestrogen exposure relative to control animals. The anxiolytic effect of DPN, previously reported in gonadectomized animals (Koehler et al., 2005, Lund et al., 2005, Walf and Frye,

Acknowledgments

The authors are grateful to Mike Adams of Wake Forest University for supplying the equol and Gail Mahnken for conducting the HPLC analysis. We also thank BJ Welker and Linda Hester for their assistance with animal husbandry as well as the students and staff members of the College of Agricultural Engineering at NCSU for constructing the light/dark boxes. This work was supported by NIEHS grant R01 ES016001 to H.B. Patisaul and the College of Agriculture and Life Sciences at NCSU. Work on this

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