Neuroendocrine effects of the endocrine disruptors Vinclozolin and Equol in the adult male rat

Abstract

Numerous anthropogenic chemicals and natural plant-derived products can mimic or disrupt the normal function of endogenous sex steroid hormones by direct interaction with their specific nuclear receptors. These compounds were classified as ‘Endocrine Disruptors’ (EDs). However, there is increasing evidence that at least some EDs are rather promiscuous with regard to the sex steroid receptor selectivity, i.e. they bind to more than one nuclear receptor. An example of such promiscuous EDs is fungicide Vinclozolin (VZ) which can exert androgen receptor (AR) antagonistic as well as estrogen receptors (ERs) agonistic actions. Likewise, Equol (EQ), a metabolite of the isoflavonoid daidzein, interacts with ERs, however, as suggested recently, EQ may also exert antiandrogenic activity. Despite the proven adverse effects of EDs on development and reproductive function, however, little efforts have been made to investigate the potential pharmacological actions of VZ and EQ on a variety of brain functions, particulary the neuroendocrine regulation of the HPG (hypothalamo-pituitary-gonadal) axis function when male individuals are exposed to these EDs. Therefore, the present study is designed to investigate whether and how VZ and EQ affect the brain and neuroendocrine function in adult male rats upon 5-days oral treatment. Examined parameters were potential effects of VZ and EQ on gene expression in the brain (medial preoptic area/anterior hypothalamus (MPOA/AH), mediobasal hypothalamus/median eminence (MBH/ME), striatum, hippocampus and amygdala), pituitary, prostate, seminal vesicles, and epididymis. In addition, the impact of equol on male mating behavior was examined. As reference compound, the pure antiandrogenic drug flutamide (FLUT) was employed. At the end of treatment interval or after completion with mating test, animals were sacrificed. The trunk blood was collected and brains and pituitaries were removed. Prostates, seminal vesicles, epididymides, testes, and livers were dissected and weighed. Changes in levels of serum hormones (LH, T, DHT, TSH, T4, T3) and mRNA expressions of relevant genes (sex steroid receptors, sex steroid-regulated genes) were measured by RIA and qRT-PCR, respectively. Serum concentrations of test substances were analyzed by HPLC-UV detection. The present study revealed that VZ decreased weight of epididymides and increased serum levels of LH and T. In the hypothalamic brain areas, VZ affected the expression of GnRH and both ERs subtypes ER"Α" and ER"Β". In the extrahypothalamic brain areas, VZ altered expression of both AR and ERs. In the pituitary, VZ up-regulated expression of GnRH receptor, LH"Β", "Α"-subunit, and TERP-1/-2. In the prostate, VZ increased and decreased levels of AR and ER mRNA, respectively. In the seminal vesicles, VZ increased levels of AR and ER"Α" mRNA expression. In the epididymis, VZ up-regulated AR and ER"Β" mRNA levels. Upon the pituitary-thyroid axis, VZ exerted direct effects on the pituitary thyrotropes resulting in decreased serum TSH and T4 levels. While FLUT displayed the typical pattern of an AR antagonist, such as decreased weights of ventral prostate, seminal vesicles, and epididymides, and increased levels of serum LH, T and DHT without effects on serum PRL levels, EQ exerted opposite effects. The hypothalamic effects of EQ and FLUT comprised changes of GnRH and GnRH receptor expression, while in the extrahypothalamic areas both compounds altered ER"Α" and AR expression in a similar manner. In contrast, effects of EQ and FLUT in the pituitary were different due to the opposite changes in ER"Α"-, TERP-1/-2-, GnRH receptor-, LH"Β"-, and "Α"-subunit expression. In the prostate, EQ and FLUT both affected ER and AR expression. EQ did not modulate the expression of male mating behavior, whereas FLUT completely inhibited it. The similar reductions in serum T4 and T3 levels caused by EQ and FLUT treatments was primary due to the direct action on the thyrotropes, and secondary to the hepatic clearance of thyroid hormones following liver enzyme induction as assessed by the increased liver weight. In summary, the present study provided the first in vivo data demonstrating that: 1. VZ is not a ‘pure’ antiandrogen, since it exerts mixed AR antagonistic/ERs agonistic actions observed at the levels of mRNA expression of selected AR- and ERs-regulated genes in the brain, pituitary, and male accessory sex organs; 2. EQ displays a clear-cut endocrine activity within the HPG axis which is attributed to be estrogenic;3. EQ does not exert any antiandrogenic effects on brain, pituitary and prostate functions, and on male mating behavior; 4. FLUT exerts its potent antiandrogenic actions in the brain and its neuroendocrine regulation of the HPG axis, and control of androgen-induced male mating behavior; 5. As demonstrated for FLUT, a short-term (5 days) in vivo oral administration utilizing intact adult male rats should be a powerful tool to characterize putative antiandrogenic EDs with regard to neuroendocrine and male sexual behavioral aspects.