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Effects of the organophosphate pesticide Chlorpyriphos on the responses of Mytilus digestive gland to the natural estrogen 17β-Estradiol
Abstracts / Comparative Biochemistry and Physiology, Part A 151 (2008) S2–S10
The uptake of dyes into cells of midgut gland and brain tissue of crustacean from the North Sea was investigated to reveal the existence and specificity of multixenobiotic or multidrug resistance transporters. The lobster Homarus gammarus, the brown shrimp Crangon crangon and the isopod Idotea emarginata were used for this experiment to cover different groups of Crustacea. The multidrug resistance transporter expels small molecules from the cytosol by consumption of ATP. The ABC transporters (ATP binding cassette) are divided by structural means and in part by specificity into several major classes. The MDR1 p-glycoprotein is the best known and widely distributed enzyme. The multidrug resistance associated protein (MRP) was found to be present in many mammalian tissues and surprisingly in marine diatoms by the use of the drug MK571, which is considered to be a selective blocker of MRP transporter. MRP transports organic anions, glutathione and glucuronate of the phase II metabolism. The three crustaceans seem to express this transporter in the brain, whereas in the midgut gland it is present in the lobster H. gammarus. The inhibition of dye extrusion leads to a fast and increased fluorescence of dyes in the cytosol which was measured in this experimental approach. doi:10.1016/j.cbpa.2008.05.045
3. Modulation of the MXR response and cyclic AMP pathway in the Mediterranean mussel exposed to contaminant mixtures S. Franzellitti, E. Fabbri (CIRSA, University of Bologna, Bologna, Italy); A. Capuzzo (Department of Biology and Evolution, University of Ferrara, Ferrara, Italy); A. Viarengo (Deparment of Environmental and Life Sciences, University of Eastern Piedmont, Alessandria, Italy) Marine mussels display a high capability of adaptation to heavily contaminated environments. Stress tolerance in these organisms involves the activation of different response pathways aiming at the protection of cellular constituents. The Multi Xenobiotic Resistance (MXR) system is a broadly distributed physiological mechanism in aquatic organisms acting as a first-tier defense against contaminants. These transporters pump out xenobiotics, thus preventing their accumulation and toxic effects. Mussels were exposed for 6 day s to a contaminant mixture composed by the heavy metal Ni2+ and the pesticide chlorpyriphos (0.3 mg/L and 0.6 mg/L, respectively) or to the single contaminants. We have previously shown that in these experimental conditions Ni2+ and chlorpyriphos alone or in combination affected several parameters of mussel physiology. In the present study, a semi-quantitative RT-PCR approach was used to evaluate the expression profiles of three MXR-related gene products, Pgp Mrp2, and Mvp, in gills of Mediterranean mussels (Mytilus galloprovincialis). Primers were designed on MXR gene sequences previously detected in our laboratory. Tissue levels of the second messenger cAMP and activity of the cAMPdependent protein kinase (PKA) were also assessed to elucidate putative effects on the signal transduction system. Our results indicated that the mixture synergistically impaired the mussel MXR response, as Ni2+ and chlorpyriphos alone did not affect Pgp and Mrp2 expression, while their combination induced the overexpression of both transcripts. Similar response patterns were also detected for cAMP levels and the PKA activity, indicating a possible relationship between the effects of pollutants on cell signaling and the transcriptional regulation of the MXR response. doi:10.1016/j.cbpa.2008.05.046
S3
4. Effects of the organophosphate pesticide Chlorpyriphos on the responses of Mytilus digestive gland to the natural estrogen 17β-Estradiol L. Canesi, C. Borghi, G. Gallo (Università di Genova, Italy); F. Caprì, A. Viarengo, F. Dondero (Università del Piemonte Orientale ‘Amedeo Avogadro’, Italy)
Pesticides comprise very different chemical substances that can act as endocrine disrupters through different modes of action. Although the potencies of pesticides as estrogen agonists or antagonists and antiandrogens in vitro are low compared with those of the natural ligands, their ability to act via more than one mechanism might enhance the biological effect. Among the organophosphates, the insecticide chlorpyrifos (CPF) was shown to be weakly estrogenic and to increase transcription of ERβ in mammalian cells. In this work, the possible effects of sublethal concentrations of CPF (4.5 mg/l, 72 h) on the responses to 24 h post-injection with the natural estrogen 17β-estradiol (E2) in the digestive gland of mussels (Mytilus galloprovincialis) were evaluated. Pre-exposure to CPF increased responses to E2 (6.75 ng/g dw) in terms of lysosomal destabilisation and of transcription of selected genes (the Mytilus Estrogen Receptor MER2, GST-π, catalase), as evaluated by RT-Q-PCR. Moreover, data obtained by the cDNA microarray of M. galloprovincialis indicate that CPF, E2 or CPF/E2, induced significant upregulation of 61, 29, and 55 and downregulation of 22, 15, and 13 sequences, respectively. 24 genes were shared between CPF and CPF/E2 conditions, only 3 genes between E2 and the mix. Moreover, 38 genes were uniquely modulated by CPF/E2, this indicating that pollutant mixtures may give rise to unexpected effects at the molecular level, and that exposure to organophosphates can affect the physiological responses to natural estrogens. Gene ontology annotation was used to elucidate the putative mechanisms involved in the response to pollutanthormone perturbation and toxicity. doi:10.1016/j.cbpa.2008.05.047
5. Mixture activity of three estrogenic UV-filters in fish (Pimephales promelas) P.Y. Kunz (University of Applied Sciences Northwestern Switzerland, Switzerland / University of California Davis, USA); K. Fent (University of Applied Sciences Northwestern, Switzerland CH) UV-absorbing chemicals (UV-filters) are widely used in sunscreens and cosmetics, but also for the protection of numerous materials against UV-radiation. Depending on the desired protection range and factor, single compounds or combinations are employed. UV-filter residues are widespread in the environment, as residues were detected in natural waters, wastewater effluents and fish. We found that many UV-filter compounds elicit multiple hormonal activities in vitro, and estrogenic activity in fish. Thus far, only very little is known about the estrogenic activities of UV-filter mixtures. In this study, we analysed 3 commonly used UV-filters, for their estrogenic mixture activity. 3-Benzylidene camphor (3BC), benzophenone-1 (BP1) and benzophenone-2 (BP2) act as pure or partial hERα agonists in vitro and elicit estrogenic activity in fish. During a 14-day exposure of juvenile fathead minnow equieffective mixtures of 3BC, BP1 and BP2 were assessed for their vitellogenin induction at 6 effect levels (ECNOEC, EC1, EC5, EC10, EC20, and EC30). The joint effects of these
The uptake of dyes into cells of midgut gland and brain tissue of crustacean from the North Sea was investigated to reveal the existence and specificity of multixenobiotic or multidrug resistance transporters. The lobster Homarus gammarus, the brown shrimp Crangon crangon and the isopod Idotea emarginata were used for this experiment to cover different groups of Crustacea. The multidrug resistance transporter expels small molecules from the cytosol by consumption of ATP. The ABC transporters (ATP binding cassette) are divided by structural means and in part by specificity into several major classes. The MDR1 p-glycoprotein is the best known and widely distributed enzyme. The multidrug resistance associated protein (MRP) was found to be present in many mammalian tissues and surprisingly in marine diatoms by the use of the drug MK571, which is considered to be a selective blocker of MRP transporter. MRP transports organic anions, glutathione and glucuronate of the phase II metabolism. The three crustaceans seem to express this transporter in the brain, whereas in the midgut gland it is present in the lobster H. gammarus. The inhibition of dye extrusion leads to a fast and increased fluorescence of dyes in the cytosol which was measured in this experimental approach. doi:10.1016/j.cbpa.2008.05.045
3. Modulation of the MXR response and cyclic AMP pathway in the Mediterranean mussel exposed to contaminant mixtures S. Franzellitti, E. Fabbri (CIRSA, University of Bologna, Bologna, Italy); A. Capuzzo (Department of Biology and Evolution, University of Ferrara, Ferrara, Italy); A. Viarengo (Deparment of Environmental and Life Sciences, University of Eastern Piedmont, Alessandria, Italy) Marine mussels display a high capability of adaptation to heavily contaminated environments. Stress tolerance in these organisms involves the activation of different response pathways aiming at the protection of cellular constituents. The Multi Xenobiotic Resistance (MXR) system is a broadly distributed physiological mechanism in aquatic organisms acting as a first-tier defense against contaminants. These transporters pump out xenobiotics, thus preventing their accumulation and toxic effects. Mussels were exposed for 6 day s to a contaminant mixture composed by the heavy metal Ni2+ and the pesticide chlorpyriphos (0.3 mg/L and 0.6 mg/L, respectively) or to the single contaminants. We have previously shown that in these experimental conditions Ni2+ and chlorpyriphos alone or in combination affected several parameters of mussel physiology. In the present study, a semi-quantitative RT-PCR approach was used to evaluate the expression profiles of three MXR-related gene products, Pgp Mrp2, and Mvp, in gills of Mediterranean mussels (Mytilus galloprovincialis). Primers were designed on MXR gene sequences previously detected in our laboratory. Tissue levels of the second messenger cAMP and activity of the cAMPdependent protein kinase (PKA) were also assessed to elucidate putative effects on the signal transduction system. Our results indicated that the mixture synergistically impaired the mussel MXR response, as Ni2+ and chlorpyriphos alone did not affect Pgp and Mrp2 expression, while their combination induced the overexpression of both transcripts. Similar response patterns were also detected for cAMP levels and the PKA activity, indicating a possible relationship between the effects of pollutants on cell signaling and the transcriptional regulation of the MXR response. doi:10.1016/j.cbpa.2008.05.046
S3
4. Effects of the organophosphate pesticide Chlorpyriphos on the responses of Mytilus digestive gland to the natural estrogen 17β-Estradiol L. Canesi, C. Borghi, G. Gallo (Università di Genova, Italy); F. Caprì, A. Viarengo, F. Dondero (Università del Piemonte Orientale ‘Amedeo Avogadro’, Italy)
Pesticides comprise very different chemical substances that can act as endocrine disrupters through different modes of action. Although the potencies of pesticides as estrogen agonists or antagonists and antiandrogens in vitro are low compared with those of the natural ligands, their ability to act via more than one mechanism might enhance the biological effect. Among the organophosphates, the insecticide chlorpyrifos (CPF) was shown to be weakly estrogenic and to increase transcription of ERβ in mammalian cells. In this work, the possible effects of sublethal concentrations of CPF (4.5 mg/l, 72 h) on the responses to 24 h post-injection with the natural estrogen 17β-estradiol (E2) in the digestive gland of mussels (Mytilus galloprovincialis) were evaluated. Pre-exposure to CPF increased responses to E2 (6.75 ng/g dw) in terms of lysosomal destabilisation and of transcription of selected genes (the Mytilus Estrogen Receptor MER2, GST-π, catalase), as evaluated by RT-Q-PCR. Moreover, data obtained by the cDNA microarray of M. galloprovincialis indicate that CPF, E2 or CPF/E2, induced significant upregulation of 61, 29, and 55 and downregulation of 22, 15, and 13 sequences, respectively. 24 genes were shared between CPF and CPF/E2 conditions, only 3 genes between E2 and the mix. Moreover, 38 genes were uniquely modulated by CPF/E2, this indicating that pollutant mixtures may give rise to unexpected effects at the molecular level, and that exposure to organophosphates can affect the physiological responses to natural estrogens. Gene ontology annotation was used to elucidate the putative mechanisms involved in the response to pollutanthormone perturbation and toxicity. doi:10.1016/j.cbpa.2008.05.047
5. Mixture activity of three estrogenic UV-filters in fish (Pimephales promelas) P.Y. Kunz (University of Applied Sciences Northwestern Switzerland, Switzerland / University of California Davis, USA); K. Fent (University of Applied Sciences Northwestern, Switzerland CH) UV-absorbing chemicals (UV-filters) are widely used in sunscreens and cosmetics, but also for the protection of numerous materials against UV-radiation. Depending on the desired protection range and factor, single compounds or combinations are employed. UV-filter residues are widespread in the environment, as residues were detected in natural waters, wastewater effluents and fish. We found that many UV-filter compounds elicit multiple hormonal activities in vitro, and estrogenic activity in fish. Thus far, only very little is known about the estrogenic activities of UV-filter mixtures. In this study, we analysed 3 commonly used UV-filters, for their estrogenic mixture activity. 3-Benzylidene camphor (3BC), benzophenone-1 (BP1) and benzophenone-2 (BP2) act as pure or partial hERα agonists in vitro and elicit estrogenic activity in fish. During a 14-day exposure of juvenile fathead minnow equieffective mixtures of 3BC, BP1 and BP2 were assessed for their vitellogenin induction at 6 effect levels (ECNOEC, EC1, EC5, EC10, EC20, and EC30). The joint effects of these