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The in vitro estrogenic activity of fluoxetine and norfluoxetine
S300
Abstracts / Toxicology Letters 238S (2015) S56–S383
but it significantly reduced the mitomycin C (MMC) induced-MN frequencies at the concentrations of 50–2000 M. However at the highest concentration of SA (5000 M), the MN frequencies increased significantly when compared to the samples treated with MMC. In conclusion, SA seems to have anticlastogenic effects at the non-cytotoxic concentrations in V79 cells. http://dx.doi.org/10.1016/j.toxlet.2015.08.859
P13-081 Mitochondrial complex I inhibitors: Mechanistic investigations in the context of safety evaluation S. Heinz ∗ , H. Ellinger-Ziegelbauer, L. Schladt, B. Lawrenz, A. Freyberger, G. Schmuck Bayer Pharma AG, Wuppertal, Germany
P13-080 The in vitro estrogenic activity of fluoxetine and norfluoxetine D. Lupu ∗ , A. Pop, J. Cherfan, B. Kiss, F. Loghin “Iuliu Hatieganu” University of Medicine and Pharmacy, Faculty of Pharmacy, Toxicology, Cluj-Napoca, Romania Question: Fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI), has been linked to reproductive and endocrine toxicity in aquatic organisms, rodents and humans. Recent studies have shown that FLX can induce estrogenic effects in vitro and in vivo and can act as an anti-reproductive neuroendocrine disruptor. This compound is metabolized in vivo to norfluoxetine, an active molecule for which there are no published data on its endocrine effects. This study aimed to assess the capacity of norfluoxetine (NFLX) to elicit estrogenic or antiestrogenic effects in vitro, individually and in binary mixtures with FLX, as these compounds are present together during in vivo exposure. Methods: The in vitro (anti)estrogenic activity of NFLX and FLXNFLX equimolar mixtures was assessed using a firefly luciferase reporter construct in the T47D-Kbluc breast cancer cell line. These cells express nuclear estrogen receptors (ERs) that can activate the transcription of the luciferase reporter gene upon binding of ER agonists. Light emission was monitored for NFLX, FLX and mixtures of NFLX-E2, FLX-E2, NFLX-FLX, NFLX-FLX-E2. Cytotoxicity was assessed using a resazurin-based assay. Results: During individual testing, both compounds were able to induce a significant increase in luciferase activity compared to control. In binary mixtures with E2 (30 pM constant concentration) a significant increase in luminescence was observed at low micromolar FLX and NFLX concentrations compared to E2 alone. Equimolar mixtures of fluoxetine and norfluoxetine also elicited an increased response in the presence of E2 compared to the response of E2 alone. Conclusion: Fluoxetine and norfluoxetine can induce estrogenic effects in vitro at micromolar concentrations. In mixtures with E2 these compounds potentiate the activity of E2. However, further studies are needed to clarify if the luminescence increase induced by FLX and NFLX is due to interactions with the reporter system and if the observed estrogenic effects may have detrimental consequences for human exposure. http://dx.doi.org/10.1016/j.toxlet.2015.08.860
Complex I is the first enzyme complex of the mitochondrial respiratory chain responsible for oxidative phosphorylation. Inhibitors of this complex such as metformin are suggested to exert antitumor activity of those tumors relying on oxidative metabolism, and are therefore of great interest in oncology research. In this context the aim of our studies is to identify biomarkers and enhance mechanistic insight for improved screening during drug development, with the primary focus on compounds which have a tolerable risk-benefit balance. Correspondingly, rats were treated for 1, 3 or 14 days with several compounds known to be associated with complex I inhibition, including rotenone, metformin and phenformin using published MTD doses and routes of application. At necropsy, a core spectrum of organs including possible targets was collected for histopathological examinations. Selected organs with a high energy demand or with induced histopathological lesions were subjected to microarray analyses to monitor the effect of complex I inhibitors on gene expression at a global level. In line with the pharmacological action of these compounds, expression levels of genes associated with mitochondrial energy metabolism were decreased. Potentially as a response to reduced mitochondrial energy supply, genes associated with lipid metabolism were increased. In addition, we observed decreased expression of genes associated with cell adhesion, migration and proliferation. Following rotenone and phenformin treatment histopathological investigations identified acute degenerative liver and bile duct lesions, and bone marrow depletion increasing over time. Interestingly, treatment with metformin increased glycogen storage in the liver, whereas with the other compounds glycogen stores were depleted. Moreover, with each of the complex I inhibitors, the adrenal glands developed cortical vacuolation, which might indicate either higher stress levels or an influence on steroid metabolism. Due to the complexity of findings, various mechanisms have to be taken into account which could explain the results of this investigation. http://dx.doi.org/10.1016/j.toxlet.2015.08.861
P13-082 Mitochondrial and lysosomal permeabilization and reactive oxygen species mediated Patulin and Sterigmatocystin cytotoxicity on CHO-K1 Z. Nidhal 1,∗ , F. Hanen 1 , M. Beatriz 2 , M.J. Ruiz Leal 2 , H. Bacha 1 , S. Abid-Essefi 1 1 2
Faculty of Dentistry, Biochemistry, Monastir, Tunisia Faculty of Pharmacy, Toxicology, Valencia, Spain
Ingestion of contaminated food is a main route of exposure to different industrial and environmental contaminants. Mycotoxins constitute an example of naturally occurring contaminants; they are hazardous secondary fungal metabolites which are found in feed and other food materials that occur simultaneously in food or raw materials. The objectives of this study were to evaluate the inhibition of cell viability, induction of the reactive oxygen species (ROS) production and damage to sub-cellular organelles (mito-
Abstracts / Toxicology Letters 238S (2015) S56–S383
but it significantly reduced the mitomycin C (MMC) induced-MN frequencies at the concentrations of 50–2000 M. However at the highest concentration of SA (5000 M), the MN frequencies increased significantly when compared to the samples treated with MMC. In conclusion, SA seems to have anticlastogenic effects at the non-cytotoxic concentrations in V79 cells. http://dx.doi.org/10.1016/j.toxlet.2015.08.859
P13-081 Mitochondrial complex I inhibitors: Mechanistic investigations in the context of safety evaluation S. Heinz ∗ , H. Ellinger-Ziegelbauer, L. Schladt, B. Lawrenz, A. Freyberger, G. Schmuck Bayer Pharma AG, Wuppertal, Germany
P13-080 The in vitro estrogenic activity of fluoxetine and norfluoxetine D. Lupu ∗ , A. Pop, J. Cherfan, B. Kiss, F. Loghin “Iuliu Hatieganu” University of Medicine and Pharmacy, Faculty of Pharmacy, Toxicology, Cluj-Napoca, Romania Question: Fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI), has been linked to reproductive and endocrine toxicity in aquatic organisms, rodents and humans. Recent studies have shown that FLX can induce estrogenic effects in vitro and in vivo and can act as an anti-reproductive neuroendocrine disruptor. This compound is metabolized in vivo to norfluoxetine, an active molecule for which there are no published data on its endocrine effects. This study aimed to assess the capacity of norfluoxetine (NFLX) to elicit estrogenic or antiestrogenic effects in vitro, individually and in binary mixtures with FLX, as these compounds are present together during in vivo exposure. Methods: The in vitro (anti)estrogenic activity of NFLX and FLXNFLX equimolar mixtures was assessed using a firefly luciferase reporter construct in the T47D-Kbluc breast cancer cell line. These cells express nuclear estrogen receptors (ERs) that can activate the transcription of the luciferase reporter gene upon binding of ER agonists. Light emission was monitored for NFLX, FLX and mixtures of NFLX-E2, FLX-E2, NFLX-FLX, NFLX-FLX-E2. Cytotoxicity was assessed using a resazurin-based assay. Results: During individual testing, both compounds were able to induce a significant increase in luciferase activity compared to control. In binary mixtures with E2 (30 pM constant concentration) a significant increase in luminescence was observed at low micromolar FLX and NFLX concentrations compared to E2 alone. Equimolar mixtures of fluoxetine and norfluoxetine also elicited an increased response in the presence of E2 compared to the response of E2 alone. Conclusion: Fluoxetine and norfluoxetine can induce estrogenic effects in vitro at micromolar concentrations. In mixtures with E2 these compounds potentiate the activity of E2. However, further studies are needed to clarify if the luminescence increase induced by FLX and NFLX is due to interactions with the reporter system and if the observed estrogenic effects may have detrimental consequences for human exposure. http://dx.doi.org/10.1016/j.toxlet.2015.08.860
Complex I is the first enzyme complex of the mitochondrial respiratory chain responsible for oxidative phosphorylation. Inhibitors of this complex such as metformin are suggested to exert antitumor activity of those tumors relying on oxidative metabolism, and are therefore of great interest in oncology research. In this context the aim of our studies is to identify biomarkers and enhance mechanistic insight for improved screening during drug development, with the primary focus on compounds which have a tolerable risk-benefit balance. Correspondingly, rats were treated for 1, 3 or 14 days with several compounds known to be associated with complex I inhibition, including rotenone, metformin and phenformin using published MTD doses and routes of application. At necropsy, a core spectrum of organs including possible targets was collected for histopathological examinations. Selected organs with a high energy demand or with induced histopathological lesions were subjected to microarray analyses to monitor the effect of complex I inhibitors on gene expression at a global level. In line with the pharmacological action of these compounds, expression levels of genes associated with mitochondrial energy metabolism were decreased. Potentially as a response to reduced mitochondrial energy supply, genes associated with lipid metabolism were increased. In addition, we observed decreased expression of genes associated with cell adhesion, migration and proliferation. Following rotenone and phenformin treatment histopathological investigations identified acute degenerative liver and bile duct lesions, and bone marrow depletion increasing over time. Interestingly, treatment with metformin increased glycogen storage in the liver, whereas with the other compounds glycogen stores were depleted. Moreover, with each of the complex I inhibitors, the adrenal glands developed cortical vacuolation, which might indicate either higher stress levels or an influence on steroid metabolism. Due to the complexity of findings, various mechanisms have to be taken into account which could explain the results of this investigation. http://dx.doi.org/10.1016/j.toxlet.2015.08.861
P13-082 Mitochondrial and lysosomal permeabilization and reactive oxygen species mediated Patulin and Sterigmatocystin cytotoxicity on CHO-K1 Z. Nidhal 1,∗ , F. Hanen 1 , M. Beatriz 2 , M.J. Ruiz Leal 2 , H. Bacha 1 , S. Abid-Essefi 1 1 2
Faculty of Dentistry, Biochemistry, Monastir, Tunisia Faculty of Pharmacy, Toxicology, Valencia, Spain
Ingestion of contaminated food is a main route of exposure to different industrial and environmental contaminants. Mycotoxins constitute an example of naturally occurring contaminants; they are hazardous secondary fungal metabolites which are found in feed and other food materials that occur simultaneously in food or raw materials. The objectives of this study were to evaluate the inhibition of cell viability, induction of the reactive oxygen species (ROS) production and damage to sub-cellular organelles (mito-