Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: In vitro and in vivo studies
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.
References (58)
- et al.
Steroid estrogens in ocean sediments
Chemosphere
(2005) - et al.
Signaling pathways involved in the physiological response of mussel hemocytes to bacterial challenge: the role of stress-activated p38 MAPK
Dev. Comp. Immunol.
(2002) - et al.
Effects of PCB congeners on the immune function of Mytilus hemocytes: alterations of tyrosine kinase-mediated cell signaling
Aquat. Toxicol.
(2003) - et al.
Rapid Effect of 17beta-estradiol on cell signaling and function of Mytilus hemocytes
Gen. Comp. Endocrinol.
(2004) - et al.
Environmental estrogens can affect the function of mussel hemocytes through rapid modulation of kinase pathways
Gen. Comp. Endocrinol.
(2004) - et al.
‘In vivo’ effects of Bisphenol A in Mytilus hemocytes: modulation of kinase-mediated signalling pathways
Aquat. Toxicol.
(2005) - et al.
Effects of the brominated flame retardant Tetrabromobisphenol-A (TBBPA) on cell signaling and function of Mytilus hemocytes: involvement of MAP kinases and protein kinase C
Aquat. Toxicol.
(2005) - et al.
Effect of environmental factors and parasitism on hemolymph lysozyme and protein of American oysters (Crassostrea virginica)
J. Invert. Pathol.
(1989) - et al.
Effects of municipal effluents on serotonin and dopamine levels in the freshwater mussel Elliptio complanata
Comp. Biochem. Physiol.
(2003) - et al.
Endocrine disruption and health effects of caged mussels, Elliptio complanata, placed downstream from a primary-treated municipal effluent plume for 1 year
Comp. Biochem. Physiol.
(2004)
Evaluation of estrogenic effects of municipal effluents to the freshwater mussel Elliptio complanata
Comp. Biochem. Physiol.
Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens, and xenoestrogens
Toxicology
Effects of 17β-estradiol exposure in the mussel Mytilus galloprovincialis: a possible regulating role for steroid acyltransferases
Aquat. Toxicol.
Tissue preferential expression of estorgen receptor gene in the marine snail, Thais clavigera
Gen. Comp Endocrinol.
Short-term bioaccumulation, circulation and metabolism of estradiol-17beta in the oyster Crassostrea gigas
J. Exp. Mar. Biol., Ecol.
Estrogen synthesis in relaton to gonadal development of Japanese scallop Patinopecten yessoensis: gonadal profile and immunolocalization of P450 aromatase and estrogen
Comp. Biochem. Physiol.
Endocrine disruptors in marine organisms: Approaches and perspectives
Comp. Biochem. Physiol.
Laboratory exposure to 17β-estradiol fails to induce vitellogenin and estrogen receptor gene expression in the marine invertebrate Mytilus edulis
Aquat. Toxicol.
The endocrine disrupting effect of municipal effluent on the zebra mussel (Dreissena polymorpha)
Aquat. Toxicol.
Studies on endogenous steroids from the marine mollusc Mytilus edulis L. by gas chromatography and mass spectrometry
Comp. Biochem. Physiol.
Vitellogenin levels in mussel hemolymph—a suitable marker for the exposure for estrogens?
Comp. Biochem. Physiol.
Estradiol and endocrine disrupting compounds adversely affect development of sea urchin embryos at environmentally relevant concentrations
Aquat. Toxicol.
Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals
Environ. Health Perspect.
The immune system as a potential target for environmental estrogens (endocrine disrupters): a new emerging field
Toxicology
Estrogens from sewage in coastal marine environments
Environ. Health Perspect.
Effects of tumor necrosis factor alpha (TNFalpha) on Mytilus hemocytes: role of stress-activated MAP kinases
Biol. Cell.
Determination of vitellogenin-like properties in Mya arenaria hemolymph (Saguenay Fijord, Canada): a potential biomarker for endocrine disruption
Environ. Toxicol.
Immunocompetence of bivalve hemocytes as evaluated by a miniaturised phagocytosis assay
Environ. Toxicol.
Accurate prediction of the response of freshwater fish to a mixture of estrogenic chemicals
Environ. Health Perspect.
Cited by (106)
Motility of Mytilus galloprovincialis hemocytes: Sensitivity to paracetamol in vitro exposure
2023, Aquatic ToxicologyRecycling and ecotoxicity of flax/PLA composites: Influence of seawater aging
2023, Composites Part C: Open AccessComparative immune responses of blue mussel and zebra mussel haemocytes to simultaneous chemical and bacterial exposure
2023, Fish and Shellfish Immunology