**3. Effects of endocrine disruptors on the immune response**

In several species, the affinity for ER of several agonists, including natural estrogens like 17-estradiol, estrone or estriol, and estrogenic disruptor compounds, like 17αethynilestradiol or diethylstilbestrol, has been tested. The different types of ER show differential binding preferences for ligands and their expression patterns are tissuedependant (Iwanowicz & Ottinger, 2009). Taking into account that ER and AR are widely distributed in immune tissues, including the spleen, liver and anterior kidney (Lynn et al., 2008; Shved et al., 2009; Slater et al., 1995; Todo et al., 1999), the study of endocrine disruptor compounds as potential aquatic pollutants has taken on some importance for fish immunologists. Several anatomical and morphological changes were observed in lymphoid tissues following exposure to xenoestrogens and xenoandrogens. Spleno-somatic and hepato-somatic indices and thymus volume are affected by exposure to sex-steroids (androgens and estrogens) or to their related endocrine disruptor compounds (Grinwis et al., 2009; Kurtz et al., 2007; Tellez-Banuelos et al., 2009; van Ginneken et al., 2009). In the gilthead seabream the dietary intake of 17α-ethynilestradiol promotes the up-regulation of several genes related with leukocyte recruitment (e.g. E-selectin (sele), the CC chemokinelike 4 (CCL4), TNFα and IL-8). Moreover, the heavy chain of IgM and IgT genes has also been seen to be up-regulated (Cabas et al., 2011). An increase in the spleno-somatic index was also recorded.

Surprisingly, *in vitro* 17α-ethynilestradiol treatment of gilthead seabream endothelial cells dramatically reduces the expression of chemokines, adhesion molecules and MMPs in

vascular physiology (Ihionkhan et al., 2002; Straub, 2007). Gilthead seabream endothelial cells constitutively express ER and ER1 but not ER2 (Liarte et al., 2011c). However, few studies have been carried out into the effect of 17-estradiol on endothelial cell physiology in fish. In the Japanese eel, 17-estradiol stimulated the production of vascular endothelial cell growth factor in endothelial cells (Huang et al., 2006). In the gilthead seabream endothelial cell cultures, 17-estradiol induced the expression of genes coding for chemokines, adhesion molecules and MMPs, which agrees with previous studies that demonstrated that 17-estradiol promotes acidophilic granulocyte infiltration into the testis (Chaves-Pozo et al., 2007). These effects contrast with that which occurs in mammals, where 17-estradiol inhibits *in vivo* the migration of leukocytes into inflamed areas and exerts tissue-protective activities through the down-regulation of adhesion molecules and the proforms of MMPs (Straub, 2007). On the other hand, 17-estradiol did not affect the expression in endothelial cells of the genes encoding major pro-inflammatory cytokines, such as IL-1β, IL-6 and TNFα, which may prevent the detrimental effects of 17-estradiol-

Little is known about androgens and their receptors in fish endothelial cells. There are very recent studies that suggest that androgens influence fish endothelial cell physiology, although further effort is needed to really understand how androgens affect endothelial cells and their molecular pathways. Trout testicular endothelial cells possess AR, as located by immunocytochemistry (Galas et al., 2009). In the gilthead seabream, recent studies determined that testosterone up-regulated TNFα, cyclooxigenase 2 (Cox2) and IL-1β, and down-regulated TGFβ and aromatase (the enzyme that transforms testosterone into 17-

In several species, the affinity for ER of several agonists, including natural estrogens like 17-estradiol, estrone or estriol, and estrogenic disruptor compounds, like 17αethynilestradiol or diethylstilbestrol, has been tested. The different types of ER show differential binding preferences for ligands and their expression patterns are tissuedependant (Iwanowicz & Ottinger, 2009). Taking into account that ER and AR are widely distributed in immune tissues, including the spleen, liver and anterior kidney (Lynn et al., 2008; Shved et al., 2009; Slater et al., 1995; Todo et al., 1999), the study of endocrine disruptor compounds as potential aquatic pollutants has taken on some importance for fish immunologists. Several anatomical and morphological changes were observed in lymphoid tissues following exposure to xenoestrogens and xenoandrogens. Spleno-somatic and hepato-somatic indices and thymus volume are affected by exposure to sex-steroids (androgens and estrogens) or to their related endocrine disruptor compounds (Grinwis et al., 2009; Kurtz et al., 2007; Tellez-Banuelos et al., 2009; van Ginneken et al., 2009). In the gilthead seabream the dietary intake of 17α-ethynilestradiol promotes the up-regulation of several genes related with leukocyte recruitment (e.g. E-selectin (sele), the CC chemokinelike 4 (CCL4), TNFα and IL-8). Moreover, the heavy chain of IgM and IgT genes has also been seen to be up-regulated (Cabas et al., 2011). An increase in the spleno-somatic index

Surprisingly, *in vitro* 17α-ethynilestradiol treatment of gilthead seabream endothelial cells dramatically reduces the expression of chemokines, adhesion molecules and MMPs in

induced inflammation through leukocyte recruitment (Liarte et al., 2011c).

**3. Effects of endocrine disruptors on the immune response** 

estradiol) gene expression (Águila et al., 2011).

was also recorded.

VaDNA-activated endothelial cells unlike in 17β-estradiol-treated endothelial cells (see point 2.4). Although, the differential expression profile in stimulated 17-ethynilestradioltreated endothelial cells, compared with 17-estradiol-treated endothelial cells, indicates that this compound would be able to impair the recruitment and activation of fish leukocytes, other molecular pathways might promote an inflammatory process in the gonad *in vivo,* as described by Cabas et al. (2011). These data show the complex effect of endocrine disruptor compounds on immune functions and the need to deepen our knowledge of their molecular action mechanism. As also occurs in mammals, 17-estradiol, but not 17αethynilestradiol, significantly enhances nitric oxide production in gilthead seabream endothelial cells, indicating that some estrogens regulate nitric oxide production by endothelial cells from fish to mammals (Arnal et al., 1996; Liarte et al., 2011c; Nilsson, 2007). As far as we know, most studies on this topic have dealt with the effects of estrogenic and anti-androgenic disruptor compounds on reproductive functions. It is known that these disruptor compounds mainly affect several enzymes in the steroidogenic pathway, such as 20β-hydroxysteroid deshydrogenase, 17β- hydroxysteroid deshydrogenase and 11βhydroxysteroid deshydrogenase, aromatase and 5α-reductase (Rempel & Schlenk, 2008). Further studies are needed into androgenic disruptor compounds as well as into estrogenic, anti-androgenic and androgenic disruptor compound mixtures to better understand how chemically and pharmaceutically polluted water might affect the reproductive and immune function of fish. Future studies and analyses along these times are being undertaken in our laboratory.
