Sex Differences in Estrogen Regulation of Renal 11β-Hydroxysteroid Dehydrogenases
Alteration of placental type 2 11β-hydroxysteroid dehydrogenase expression and activity by lead
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and in this way mercury out of cells. The resulting loss of GSH however must be compensated by regeneration or de novo synthesis. Thus, the interplay of GSH synthesizing, metabolizing and conjugating enzymes is required to confer that mercury can be rapidly bound and removed from cells. Aim: To clarify the role of GSH system enzymes in placental mercury toxicokinetics, we (1) established conditions to detect the proteins in human placenta in situ, (2) analyzed various cell lines for endogenous protein expression, and (3) conducted gene knockdown to confirm that proteins are involved in mercury toxicokinetics. Methods: Tissue collected from term healthy human placentas was either immediately frozen or HOPE-fixed and paraffin-embedded. Protein locali- zation was studied by immunofluorescence microscopy on 2-4 mm tissue sections. Protein expression in human placenta, BeWo, Q1, n-tera and HeLa cells was analyzed by using commercial antibodies on total protein lysates. siRNA-mediated silencing of candidate genes was achieved by applying lipid- based forward transfection. Mercury accumulation was analysed by CV-AFS in transfected cells versus control cells (treated with nontargeting siRNAs). Results and conclusion: All tested cell lines express GSH system enzymes. GSTA1 is exclusively expressed in placenta cells. The proteins are localized mainly in the cytosolic fraction of placenta cells. Preliminary data from gene knockdown experiments confirm involvement of GSH system en- zymes in cellular mercury toxicokinetics. Supported by LifeScience2010, NFB P2.163-N. ALTERATION OF PLACENTAL TYPE 2 11b-HYDROXYSTEROID DEHYDROGENASE EXPRESSION AND ACTIVITY BY LEAD Joey St-Pierre, Marc Fraser, Cathy Vaillancourt INRS-Institut Armand Frappier and BioMed Research Center, Laval, Quebec, Canada The placenta protects the foetus from the transfer of maternal stress hormone, cortisol. The placenta regulates the transfer of maternal stress by the type 2 11b-hydroxysteroid dehydrogenase (11b-HSD2), which converts maternal cortisol to inactive cortisone. Prenatal maternal stress may in- fluence the expression and activity of 11b-HSD2 and in turn affect fetal development. Lead (Pb), an environmental stressor, can increase the risk of obstetrical complications and alter fetal brain development. Unfortunately, the effect of Pb on placental 11b-HSD2 has never been studied. Objective: The objective of this study is to determine the effect of increasing concentrations of Pb on the expression and activity of 11b-HSD2 in the BeWo placental cell line, a model of villous trophoblast, the func- tional unit of the placenta expressing the 11b-HSD2. BeWo cells were exposed 24h to different Pb concentrations with or without forskolin (20 mM, a potent trophoblast differentiation agent). Pb concentrations were established from literature and a cohort where environmental contami- nants were previously measured. Gene expression was analysed by RT- qPCR and protein expression by Western blot. 11b- HSD2 activity was assessed by radioenzymatic assay (conversion of cortisol to cortisone). Results: Western blot results demonstrate a reduction of protein expression by 45% at 0.01 nM, 55% at 0.1 nM and 45% at 1000 nM Pb compared to untreated cells, while no effect on the expression of 11b-HSD2 mRNA (RT-qPCR) was observed. Radioenzymatic assays show a significant reduction in conversion rate of cortisol to cortisone at 0.01, 1, 10, 100 and 1000 nMPb. None of the Pb concentrations analyzed affects viability parameters of villous trophoblast (no effect on cell viability, cell proliferation or production of b-hCG). Conclusion: This study shows that Pb alters the activity of placental 11b-HSD2 and suggests that current acceptable level of Pb exposure should be revised. P2.164-N. SELECTIVE SEROTONIN-REUPTAKE INHIBITORS (SSRIS) INDUCE THE ESTROGEN BIOSYNTHETIC ENZYME AROMATASE (CYP19) IN TROPHOBLAST-LIKE BEWO CHORIOCARCINOMA CELLS Andr ee-Anne Hudon-Thibeault, Laetitia Laurent, Thomas Sanderson, Cathy Vaillancourt INRS-Institut Armand Frappier and BioMed research Center, Laval, Quebec, Canada Objective: Depression occurs in up to 25% of pregnancies and selective serotonin-reuptake inhibitors (SSRIs) are the main antidepressants pre- scribed. Depression and/or SSRIs are associated with obstetrical compli- cations and alterations of fetal development. Since estrogens are involved in the etiology of depression, it is crucial to study the relationship between depression, antidepressant treatment and estrogens in the placenta. We hypothesize that SSRIs increase serotonin levels, which stimulates placental estrogen production through up-regulation of the enzyme aro- matase (CYP19). The objective of this study was to evaluate if SSRIs affect the activity and expression of placental CYP19. Methods: BeWo human choriocarcinoma cells (12,500 cells/well in 96-well plate), which we used as a model of the villous trophoblast, were exposed to SSRIs (fluoxetine and its metabolite norfluoxetine, citalopram, sertraline, paroxetine and venlafaxine) within a physiologically relevant concentra- tion range (0.03 - 3 mM). DMSO was used as vehicle control. CYP19 activity was determined by tritiated-water release assay using 1b-[ 3 H]-andro- stenedione as substrate. CYP19 gene expression levels were determined by RT-qPCR and normalized using SDHA, 18SRNA and HPRT1 reference genes. Results: Fluoxetine and paroxetine induced CYP19 activity by 188% and 168%, respectively, at 1 mM and 281% and 185%, respectively, at 3 mM. Sertraline induced CYP19 significantly (270%) at 1 mM only. Co-treatment of fluoxetine with PKC inhibitors GF109203X or G € O6976 (100 nM) partially inhibited fluoxetine-mediated CYP19 induction. CYP19 mRNA expression was induced 1.5 fold by fluoxetine (at 3 mM). Conclusion: We conclude that the estrogen biosynthetic enzyme CYP19 is induced by several SSRIs in BeWo cells at physiologically relevant con- centrations. Our results will contribute to understand better the in- teractions between serotonin and estrogens as well as the influence of depression and SSRI treatment on these interactions during pregnancy. Ultimately, our study will allow for an improved risk-benefit analysis of SSRI treatment for depression during pregnancy. P2.165. BENZO(A)PYRENE EXPOSURE DURING PREGNANCY: ACCUMULATION AND EFFECTS ON PRIMARY CULTURES OF HUMAN TROPHOBLASTS Guillaume Pidoux a , Nicolas Auzeil b , Pascale Gerbaud a , Anne Regazzetti b , Christelle Simasotchi a , Delphine Darg ere b , Fatima Ferreira a , Maud Brossard a , Olivier Lapr evote b , Dani ele Evain- Brion a , Sophie Gil a a UMR-S 1139, INSERM, University of Paris Descartes, Paris, France; b UMR 8638 CNRS, Universit e Paris Descartes, Paris, France Objectives: The exposure to environmental contaminants is responsible for the development of human pathologies. Polycyclic aromatic hydrocarbons (PAHs) are notably found in diesel exhaust particles, foods and cigarette smoke. Bathing in the maternal blood, the syncytiotrophoblast is a main target of these environmental contaminants, which might affect its essential functions during pregnancy. The aim of this study was to evaluate 1) the placental uptake of Benzo(a)pyrene (BaP), the prototypical PAH, by using the ex vivo human perfused cotyledon model 2) its effects, in vitro on the dif- ferentiation of human primary cytotrophoblasts in syncytiotrophoblast. Methods: Placentas from uncomplicated full-term pregnancies were collected immediately after delivery and cotyledons were perfused with or without BaP. Placental uptake was calculated by determining endogenous accumulation during pregnancy or just after a perfusion experiment. BaP concentrations were achieved by mass spectrometry. The effect of BaP on trophoblast differentiation was characterized in vitro by the exposition of human cytotrophoblasts to this polluant. Cell fusion process and the for- mation of a functional multinucleated syncytiotrophoblast were studied. Finally, various approaches were used to determine intra-cellular cell signaling induced by BaP. Results: The mean mass of endogenous BaP in the cotyledon is the ng/g of tissue while after exposure, accumulation of BaP reached the mg/g of tissue. Concentrations of BaP above 1 mM were cytotoxic on trophoblastic cells. Fusion assays indicated a decreased in trophoblast fusion by 40% while concomitantly it was an increased in hCG secretion into media for cyto- trophoblasts 24h-exposed to 1 mM BaP. Co-treatment by the aryl Abstracts / Placenta 35 (2014) A1eA112 A111
Transcript of Alteration of placental type 2 11β-hydroxysteroid dehydrogenase expression and activity by lead
Abstracts / Placenta 35 (2014) A1eA112 A111
and in this waymercury out of cells. The resulting loss of GSH howevermustbe compensated by regeneration or de novo synthesis. Thus, the interplay ofGSH synthesizing, metabolizing and conjugating enzymes is required toconfer that mercury can be rapidly bound and removed from cells.
Aim: To clarify the role of GSH system enzymes in placental mercurytoxicokinetics, we (1) established conditions to detect the proteins inhuman placenta in situ, (2) analyzed various cell lines for endogenousprotein expression, and (3) conducted gene knockdown to confirm thatproteins are involved in mercury toxicokinetics.Methods: Tissue collected from term healthy human placentas was eitherimmediately frozen or HOPE-fixed and paraffin-embedded. Protein locali-zation was studied by immunofluorescence microscopy on 2-4 mm tissuesections. Protein expression in human placenta, BeWo, Q1, n-tera and HeLacells was analyzed by using commercial antibodies on total protein lysates.siRNA-mediated silencingof candidate geneswasachievedbyapplying lipid-based forward transfection. Mercury accumulation was analysed by CV-AFSin transfected cells versus control cells (treated with nontargeting siRNAs).Results and conclusion: All tested cell lines express GSH system enzymes.GSTA1 is exclusively expressed in placenta cells. The proteins are localizedmainly in the cytosolic fraction of placenta cells. Preliminary data fromgene knockdown experiments confirm involvement of GSH system en-zymes in cellular mercury toxicokinetics.Supported by LifeScience2010, NFB
P2.163-N.ALTERATION OF PLACENTAL TYPE 2 11b-HYDROXYSTEROIDDEHYDROGENASE EXPRESSION AND ACTIVITY BY LEAD
Joey St-Pierre, Marc Fraser, Cathy Vaillancourt INRS-Institut ArmandFrappier and BioMed Research Center, Laval, Quebec, Canada
The placenta protects the foetus from the transfer of maternal stresshormone, cortisol. The placenta regulates the transfer of maternal stress bythe type 2 11b-hydroxysteroid dehydrogenase (11b-HSD2), which convertsmaternal cortisol to inactive cortisone. Prenatal maternal stress may in-fluence the expression and activity of 11b-HSD2 and in turn affect fetaldevelopment. Lead (Pb), an environmental stressor, can increase the risk ofobstetrical complications and alter fetal brain development. Unfortunately,the effect of Pb on placental 11b-HSD2 has never been studied.
Objective: The objective of this study is to determine the effect ofincreasing concentrations of Pb on the expression and activity of 11b-HSD2in the BeWo placental cell line, a model of villous trophoblast, the func-tional unit of the placenta expressing the 11b-HSD2. BeWo cells wereexposed 24h to different Pb concentrations with or without forskolin (20mM, a potent trophoblast differentiation agent). Pb concentrations wereestablished from literature and a cohort where environmental contami-nants were previously measured. Gene expression was analysed by RT-qPCR and protein expression by Western blot. 11b- HSD2 activity wasassessed by radioenzymatic assay (conversion of cortisol to cortisone).Results:Westernblot resultsdemonstratea reductionofproteinexpressionby45% at 0.01 nM, 55% at 0.1 nM and 45% at 1000 nMPb compared to untreatedcells, while no effect on the expression of 11b-HSD2 mRNA (RT-qPCR) wasobserved. Radioenzymatic assays show a significant reduction in conversionrate of cortisol to cortisone at 0.01, 1, 10, 100 and 1000 nMPb. None of the Pbconcentrations analyzed affects viability parameters of villous trophoblast (noeffect on cell viability, cell proliferation or production of b-hCG).Conclusion:This studyshows thatPbalters theactivityofplacental 11b-HSD2and suggests that current acceptable level of Pb exposure should be revised.
P2.164-N.SELECTIVE SEROTONIN-REUPTAKE INHIBITORS (SSRIS) INDUCE THEESTROGEN BIOSYNTHETIC ENZYME AROMATASE (CYP19) INTROPHOBLAST-LIKE BEWO CHORIOCARCINOMA CELLS
Andr�ee-Anne Hudon-Thibeault, Laetitia Laurent, ThomasSanderson, Cathy Vaillancourt INRS-Institut Armand Frappier and BioMedresearch Center, Laval, Quebec, Canada
Objective: Depression occurs in up to 25% of pregnancies and selectiveserotonin-reuptake inhibitors (SSRIs) are the main antidepressants pre-scribed. Depression and/or SSRIs are associated with obstetrical compli-cations and alterations of fetal development. Since estrogens are involvedin the etiology of depression, it is crucial to study the relationship betweendepression, antidepressant treatment and estrogens in the placenta. Wehypothesize that SSRIs increase serotonin levels, which stimulatesplacental estrogen production through up-regulation of the enzyme aro-matase (CYP19). The objective of this study was to evaluate if SSRIs affectthe activity and expression of placental CYP19.
Methods: BeWohuman choriocarcinoma cells (12,500 cells/well in 96-wellplate),whichweused as amodel of the villous trophoblast,were exposed toSSRIs (fluoxetine and its metabolite norfluoxetine, citalopram, sertraline,paroxetine and venlafaxine) within a physiologically relevant concentra-tion range (0.03 - 3 mM). DMSO was used as vehicle control. CYP19 activitywas determined by tritiated-water release assay using 1b-[3H]-andro-stenedione as substrate. CYP19 gene expression levels were determined byRT-qPCR and normalized using SDHA, 18SRNA and HPRT1 reference genes.Results: Fluoxetine and paroxetine induced CYP19 activity by 188% and168%, respectively, at 1 mM and 281% and 185%, respectively, at 3 mM.Sertraline induced CYP19 significantly (270%) at 1 mM only. Co-treatmentof fluoxetine with PKC inhibitors GF109203X or G€O6976 (100 nM) partiallyinhibited fluoxetine-mediated CYP19 induction. CYP19 mRNA expressionwas induced 1.5 fold by fluoxetine (at 3 mM).Conclusion: We conclude that the estrogen biosynthetic enzyme CYP19 isinduced by several SSRIs in BeWo cells at physiologically relevant con-centrations. Our results will contribute to understand better the in-teractions between serotonin and estrogens as well as the influence ofdepression and SSRI treatment on these interactions during pregnancy.Ultimately, our study will allow for an improved risk-benefit analysis ofSSRI treatment for depression during pregnancy.
P2.165.BENZO(A)PYRENE EXPOSURE DURING PREGNANCY: ACCUMULATIONAND EFFECTS ON PRIMARY CULTURES OF HUMAN TROPHOBLASTS
Guillaume Pidoux a, Nicolas Auzeil b, Pascale Gerbaud a, AnneRegazzetti b, Christelle Simasotchi a, Delphine Darg�ere b, FatimaFerreira a, Maud Brossard a, Olivier Lapr�evote b, Dani�ele Evain-Brion a, Sophie Gil a aUMR-S 1139, INSERM, University of Paris Descartes,Paris, France; bUMR 8638 CNRS, Universit�e Paris Descartes, Paris, France
Objectives: The exposure to environmental contaminants is responsible forthe development of human pathologies. Polycyclic aromatic hydrocarbons(PAHs) are notably found in diesel exhaust particles, foods and cigarettesmoke. Bathing in the maternal blood, the syncytiotrophoblast is a maintarget of these environmental contaminants,whichmight affect its essentialfunctions during pregnancy. The aim of this study was to evaluate 1) theplacental uptake of Benzo(a)pyrene (BaP), the prototypical PAH, by using theex vivo human perfused cotyledon model 2) its effects, in vitro on the dif-ferentiation of human primary cytotrophoblasts in syncytiotrophoblast.
Methods: Placentas from uncomplicated full-term pregnancies werecollected immediately after delivery and cotyledons were perfused with orwithout BaP. Placental uptake was calculated by determining endogenousaccumulation during pregnancy or just after a perfusion experiment. BaPconcentrations were achieved by mass spectrometry. The effect of BaP ontrophoblast differentiation was characterized in vitro by the exposition ofhuman cytotrophoblasts to this polluant. Cell fusion process and the for-mation of a functional multinucleated syncytiotrophoblast were studied.Finally, various approaches were used to determine intra-cellular cellsignaling induced by BaP.Results: The mean mass of endogenous BaP in the cotyledon is the ng/g oftissue while after exposure, accumulation of BaP reached the mg/g of tissue.Concentrations of BaP above 1 mM were cytotoxic on trophoblastic cells.Fusion assays indicated a decreased in trophoblast fusion by 40% whileconcomitantly it was an increased in hCG secretion into media for cyto-trophoblasts 24h-exposed to 1 mM BaP. Co-treatment by the aryl
