Elsevier

Aquatic Toxicology

Volume 81, Issue 1, 15 February 2007, Pages 36-44
Aquatic Toxicology

Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: In vitro and in vivo studies

https://doi.org/10.1016/j.aquatox.2006.10.010Get rights and content

Abstract

Endocrine disrupting compounds (EDCs) are almost ubiquitous in the aquatic environment. In the marine bivalve Mytilus the natural estrogen 17β-estradiol (E2) and different EDCs have been recently demonstrated to affect the function of the immune cells, the hemocytes. The effects were Tamoxifen-sensitive and were mediated by rapid modulation of kinase-mediated transduction pathways. In this work we compared the in vitro effects of individual estrogenic chemicals (E2, EE: 17α-ethynyl estradiol; MES: mestranol; NP: nonylphenol; NP1EC: nonylphenol monoethoxylate carboxylate; BPA: bisphenol A; BP: benzophenone) on hemocyte parameters: lysosomal membrane stability (LMS), phagocytosis, lysozyme release. LMS was the most sensitive effect parameter, showing a decreasing trend at increasing concentrations of estrogens. EC50 values obtained from LMS data were utilized to calculate the estradiol equivalency factor (EEF) for each compound; these EEFs allowed for an estimation of the estrogenic potential of a synthetic mixture with a composition very similar to that previously found in waters of the Venice lagoon. Concentrated mixtures significantly affected hemocyte parameters in vitro and the effects were prevented by Tamoxifen. Significant effects of the mixture were also observed in vivo, at longer exposure times and at concentrations comparable with environmental exposure levels. The results indicate that Mytilus immune parameters can be suitably utilized to evaluate the estrogenic potential of environmental samples.

Introduction

Endocrine disrupting compounds (EDCs) include both natural and synthetic steroid estrogens, as well as a variety of estrogen-mimicking chemicals, such as alkylphenols and alkylphenol ethoxylates, PCBs, dioxins, various pesticides and herbicides. EDCs have been found in freshwater, estuarine and marine environments (Atkinson et al., 2003, Peck et al., 2004, Braga et al., 2005), thus representing a potential hazard for aquatic species (Rotchell and Ostrander, 2003, Sumpter and Johnson, 2005). Most studies on the effects and mechanisms of action of estrogenic chemicals have been focused on vertebrates: many EDCs have been shown to bind both mammalian and fish estrogen receptors (ERs) (McLachlan, 2001, Rotchell and Ostrander, 2003). The estrogenic potency of EDCs is commonly related to that of 17β-estradiol (E2), the most potent natural estrogen, by using different assays; these are based on the measurement of responses mediated by the common ‘genomic’ mechanism of action of estrogens in vertebrate cells, that is binding to intracellular ERs that act as ligand-inducible transcription factors thus modulating the expression of estrogen responsive genes (Andersen et al., 1999, Gutendorf and Westendorf, 2001, Brian et al., 2005).

Although invertebrates represent approximately 95% of animal species, and a significant component of aquatic ecosystems, information on the effects and mechanisms of action of EDCs in these organisms is scarce compared to that available for vertebrates (Jobling et al., 2003, Verslycke et al., 2004, Roepke et al., 2005, Ohelmann et al., 2006). The evaluation of the effects of EDCs on invertebrates represents a complex task, because of the limited knowledge on the endocrine systems, their differences in a large number of species, and on the mechanisms of action of natural hormones (Ohelmann and Schulte-Ohelmann, 2003, Porte et al., 2006). Among aquatic invertebrates, in molluscs endogenous estrogens have been identified, and evidence for ER-like receptors has been obtained in different species (Reis-Henriques et al., 1990, Gagnè et al., 2001, Di Cosmo et al., 2002, Canesi et al., 2004a, Osada et al., 2003, Osada et al., 2004). ER-like receptors have been recently characterized in gastropods (Thornton et al., 2003, Kajiwara et al., 2006) and cephalopods (Keay et al., 2006). These studies showed that molluscan ERs, although phylogenetically clustered with other steroid receptors, appear to be functionally different: their transcriptional activity is constitutive, and not activated by estrogens.

Bivalves are sessile, filter-feeding molluscs that can represent a primary target for estrogenic chemicals, due to their high bioaccumulation and low biotransformation potential for contaminants (Ortiz-Zarragoitia and Cajaraville, 2006). Recent progress has been made on the role of estrogens on bivalve reproduction (Osada et al., 2003, Osada et al., 2004, Gauthier-Clerc et al., 2005), steroid biosynthesis and metabolism (Janer et al., 2005a, Janer et al., 2005b) and, in analogy with vertebrate studies, on the induction of Vitellogenin (Vtg)-like proteins in males by EDCs, although the relationship between estrogen exposure and Vtg induction has not been fully clarified (Blaise et al., 1999, Gagnè et al., 2001, Riffeser and Hock, 2002, Ortiz-Zarragoitia and Cajaraville, 2006). In the marine mussel Mytilus, exposure to E2 does not affect the expression of ER- and Vtg-like sequences (Puinean et al., 2006). Effects of municipal effluents containing EDC mixtures have been demonstrated in non-reproductive tissues (Gagnè et al., 2001, Gagnè et al., 2004, Quinn et al., 2004); a role for EDCs in neuroendocrine disruption of reproduction has been suggested (Gagnè and Blaise, 2003).

In Mytilus hemocytes, the cells responsible for innate immunity (Canesi et al., 2002a), E2 was shown to modulate a number of functional parameters, including lysosomal membrane stability (LMS), extracellular lysozyme release, phagocytosis, oxyradical production (Canesi et al., 2004a, Canesi et al., 2006). Low nM concentrations stimulated the immune function, whereas higher concentrations were inhibitory. The effects of E2 were prevented by the antiestrogen Tamoxifen and were mediated by rapid, ‘non-genomic’ mechanisms of action similar to those identified in mammalian cells (Lösel et al., 2003), involving activation of cytosolic kinases such as MAPKs (mitogen activated protein kinases) and PKC (protein kinase C), and phosphorylation of transcription factors such as STATs (signal transducers and activators of transcription) and CREB (cAMP responsive element binding protein) (Canesi et al., 2004a, Canesi et al., 2006), that play a key role in the hemocyte immune response (Canesi et al., 2005a). Different EDCs were also shown to rapidly affect hemocyte lysosomal function and signalling through Tamoxifen-sensitive modulation of kinase pathways, although at higher concentrations, and with distinct effects depending on the compound (Canesi et al., 2003, Canesi et al., 2004b). The effects were confirmed in vivo, in mussels injected with bisphenol A, at concentrations similar to those utilised for E2 (Canesi et al., 2005b).

In this work, we extended our studies on the in vitro effects of EDCs on the hemocyte function in terms of number and type of individual compounds, concentration range, and effect parameters. The effects of different concentrations of a set of widely distributed estrogenic compounds on LMS were evaluated and the results compared to those obtained with the natural estrogen E2. Specific immune parameters, such as phagocytosis and release of hydrolytic enzymes, were also evaluated. The same effect endpoints were measured in hemocytes exposed to a synthetic mixture containing E2 and the examined estrogens in proportions similar to those previously found in environmental samples from the Venice Lagoon (Pojana et al., 2004). Finally, the effects of the synthetic mixture on immune parameters were evaluated by an in vivo experiment, conducted at longer exposure times, in the hemocytes of mussels injected with the mixtures at environmental concentrations.

Section snippets

Chemicals

All reagents were of analytical grade. 17β-Estradiol (E2) and 17α-ethinylestradiol (EE) were from Sigma (St. Louis, MO); bisphenol A (BPA) and 4-n-nonylphenol (NP) were from Riedel-de Haen (Germany); mestranol (MES) and benzophenone (BP) were obtained from Fluka (Büchs, Switzerland); nonylphenol monoethoxylate carboxylate (NP1EC) (purity ∼ 90%) was purchased by Ciba Speciality Chemicals (Basel, Switzerland) and further purified by semipreparative HPLC up to a final >99% purity. Stock solutions

Effects of individual compounds on hemocyte parameters

Lysosomal membrane stability (LMS): the effects of different concentrations of individual estrogenic chemicals on lysosomal membrane stability of Mytilus hemocytes were first evaluated and compared to those of E2. The mean annual values of NRR time in control hemocytes, evaluated over a 18-month period, were 122 ± 12 min (mean ± S.D.) and no significant seasonal-related differences were observed. However, in order to minimize the chance of seasonal changes in LMS sensitivity to EDC treatment,

Discussion

The results demonstrate that in mussel hemocytes short-term exposure to individual estrogenic compounds induced rapid changes in hemocyte parameters in vitro. A complete dose–response curve was obtained for LMS, clearly showing that all the EDC tested were effective at concentrations higher (nM–μM) than those of E2 (low nM). The results confirm that in the hemocytes the lysosomal vacuolar system, that plays a key role in different aspects of the immune function, represents a sensitive target

Acknowledgment

The Authors thank Prof. John Sumpter, of the Brunel University (London, U.K.) for his critical reading of the manuscript.

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