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Effect of P-Glycoprotein inducers on its expression and activity in Caco-2 cells
S116
Abstracts / Toxicology Letters 180S (2008) S32–S246
(at least in our case) and a care is to be taken to avoid false positive or negative results when measuring toxicity and proliferation with help of these test methods. Acknowledgements: Supported by GACR (grant 301/06/P047). doi:10.1016/j.toxlet.2008.06.459 V69 ES-cell derived ready-to-use cardiomyocytes: An efficient and predictive tool to detect cardiac specific cytotoxicity Silke Schwengberg ∗ , Ralf Kettenhofen, Heribert Bohlen Axiogenesis AG, Cologne, Germany Standardised in vitro cardiac cell and tissue models to determine cardiac-specific cytotoxicity are in short supply. Most scientists have to use primary preparations of cardiomyocytes. Freshly isolated primary cardiomyocytes, although showing good physiological properties, are time- and cost intensive to produce, difficult to standardise, and not suitable for long-term storage. Here we present data that murine embryonic stem cell-derived cardiomyocytes (Cor.At® ) provide a standardized, homogenous and reproducible cell system for the in vitro classification of a compound’s cardio-cytotoxic potential. After incubation with known cardiotoxic compounds, effects which directly affect the viability and integrity of cardiac cells can be determined using various endpoints: morphological changes, cell viability, and release of Troponin. Troponin levels in cell culture supernatant of Cor.At® cells treated with 1 M Emetine for 48 h were elevated 5 times compared to solvent controls. Comparison of viability of Cor.At® cells with mouse embryonic fibroblasts after treatment with known cardiotoxic compounds prove the specificity and clinical relevance of the assay. Cor.At® cells are available ready-to-use and can be stored frozen in different formats (vials, 96 well plates, coverslips), offering a time and cost effective tool for determination of cardiac specific cytotoxicity even for medium to high throughput screenings at early stages of drug development. The use of Cor.At® cells with a disease phenotype (i.e. hypertrophic cardiomyocytes) in the above described systems will complete the picture of predicted adverse cardiotoxic effects of a compound even in patients with cardiac risk factors, thus increasing safety- and function profiles already in pre-clinical stages of development. doi:10.1016/j.toxlet.2008.06.460 V70 Evaluation of cellular toxicity for cisplatin, arsenic and acetaminophen in the cancer and normal cell line Mohammad Shokrzadeh ∗ , Seyyed Soheil Saeedi Saravi Mazandaran University of Medical Sciences, Sari, Mazandaran, Islamic Republic of Iran Keywords: Cell toxicity; Clonogenic assay; Arsenic; Cisplatin; Acetaminophen Purposes: Cell culture is a process in which, the cells were isolated from original tissue are dispersed in the liquid media and then, placed in culture plate and the cells adhere together and propagate. Today this manner is used for assessment of cell toxicity and its mechanisms and different compounds on intracellular organs.
Methods: Clonogenic assay was used for assessment of cell toxicity and amount of cell death after a specific time that the cell were exposed to different compounds. Thus, IC50 in caner cell lines (HePG2, SKOV3 and A549) and normal cell (LLCPK1, CHO and HGF1) was assessed after exposure to cisplatin, acetaminophen and Arsenic. Results: Results showed that, Acetaminophen has maximum resistance and minimum sensitivity in CHO line with IC50 = 16.7 ± 1.06 and regarding HePG2 with IC50 = 18.6 ± 1.29. On the other hand, cisplatin showed minimum resistance and maximum Sensitivity in HePG2 with IC50 = 0.87 ± 0.07 and HGF1 with IC50 = 1.6 ± 0.21 and at last Arsenic showed minimum resistance and maximum sensitivity in A549 with IC50 = 4.59 ± 0.29 and LLCPK1 with IC50 = 1 ± 0.37. Conclusion: According to the evaluated IC50 , it showed differences between results of sensitivity of cell lines which exposed to the three examinated drugs (P < 0.05). Entirely, resistance in cancer cell lines is lower than normal cells, therefore, the results showed the importance of cell defensive mechanisms encounting different substances, such as glutathione. doi:10.1016/j.toxlet.2008.06.461 V71 Effect of P-Glycoprotein inducers on its expression and activity in Caco-2 cells Renata Silva 1,∗ , Anabela Cordeiro-da-Silva 2 , Sofia A.C. Lima 3 , Félix Carvalho 1 , Maria Lourdes Bastos 1 , Helena Carmo 1 , Fernando Remião 1 1
REQUIMTE, Toxicology Department, Faculty of Pharmacy, University of Porto, Porto, Portugal, 2 Biochemistry Department, Faculty of Pharmacy, University of Porto, Porto, Portugal, 3 IBMC, Institute of Molecular and Cellular Biology, University of Porto, Porto, Portugal P-glycoprotein (P-gp) is a membrane-bound glycoprotein belonging to the ATP-binding cassette transporter superfamily expressed in several organs including, liver, brain, kidney and small intestine. P-gp acts as a defence mechanism by “effluxing” drugs and toxic compounds from the cell membrane or cytoplasm to the extracellular space, thus affecting the absorption, disposition, and elimination of different xenobiotics. Caco-2 cells have been widely accepted as a reliable in vitro model for the rapid screening of the intestinal drug absorption and excretion mediated by P-gp. In fact, when grown on permeable supports Caco-2 cells from epithelial monolayers express many features characteristic of the normal human intestine such as spontaneous morphological and biochemical enterocytic differentiation. Thus, the main objective of the present work was to characterize the P-gp expression levels in caco-2 cells during their differentiation processes. To evaluate potential changes in those expression levels, as well as changes in P-gp activity, when exposed to several Pgp inducers, such as dexamethasone, vinblastine and rifampin were the other objectives. Alkaline phosphatase activity was measured to assess the cell differentiation. P-gp expression levels were determined by flow cytometry, using a CD243-PE conjugated antibody. The protein transport activity was evaluated using daunorubicin as a fluorescent transported P-gp subtract. Caco-2 cell P-gp ATPase activity was also evaluated by colorimetric assay. The observed results are important to characterize these cells in order to study the induction mechanism to protect cells from toxic compounds. doi:10.1016/j.toxlet.2008.06.462
Abstracts / Toxicology Letters 180S (2008) S32–S246
(at least in our case) and a care is to be taken to avoid false positive or negative results when measuring toxicity and proliferation with help of these test methods. Acknowledgements: Supported by GACR (grant 301/06/P047). doi:10.1016/j.toxlet.2008.06.459 V69 ES-cell derived ready-to-use cardiomyocytes: An efficient and predictive tool to detect cardiac specific cytotoxicity Silke Schwengberg ∗ , Ralf Kettenhofen, Heribert Bohlen Axiogenesis AG, Cologne, Germany Standardised in vitro cardiac cell and tissue models to determine cardiac-specific cytotoxicity are in short supply. Most scientists have to use primary preparations of cardiomyocytes. Freshly isolated primary cardiomyocytes, although showing good physiological properties, are time- and cost intensive to produce, difficult to standardise, and not suitable for long-term storage. Here we present data that murine embryonic stem cell-derived cardiomyocytes (Cor.At® ) provide a standardized, homogenous and reproducible cell system for the in vitro classification of a compound’s cardio-cytotoxic potential. After incubation with known cardiotoxic compounds, effects which directly affect the viability and integrity of cardiac cells can be determined using various endpoints: morphological changes, cell viability, and release of Troponin. Troponin levels in cell culture supernatant of Cor.At® cells treated with 1 M Emetine for 48 h were elevated 5 times compared to solvent controls. Comparison of viability of Cor.At® cells with mouse embryonic fibroblasts after treatment with known cardiotoxic compounds prove the specificity and clinical relevance of the assay. Cor.At® cells are available ready-to-use and can be stored frozen in different formats (vials, 96 well plates, coverslips), offering a time and cost effective tool for determination of cardiac specific cytotoxicity even for medium to high throughput screenings at early stages of drug development. The use of Cor.At® cells with a disease phenotype (i.e. hypertrophic cardiomyocytes) in the above described systems will complete the picture of predicted adverse cardiotoxic effects of a compound even in patients with cardiac risk factors, thus increasing safety- and function profiles already in pre-clinical stages of development. doi:10.1016/j.toxlet.2008.06.460 V70 Evaluation of cellular toxicity for cisplatin, arsenic and acetaminophen in the cancer and normal cell line Mohammad Shokrzadeh ∗ , Seyyed Soheil Saeedi Saravi Mazandaran University of Medical Sciences, Sari, Mazandaran, Islamic Republic of Iran Keywords: Cell toxicity; Clonogenic assay; Arsenic; Cisplatin; Acetaminophen Purposes: Cell culture is a process in which, the cells were isolated from original tissue are dispersed in the liquid media and then, placed in culture plate and the cells adhere together and propagate. Today this manner is used for assessment of cell toxicity and its mechanisms and different compounds on intracellular organs.
Methods: Clonogenic assay was used for assessment of cell toxicity and amount of cell death after a specific time that the cell were exposed to different compounds. Thus, IC50 in caner cell lines (HePG2, SKOV3 and A549) and normal cell (LLCPK1, CHO and HGF1) was assessed after exposure to cisplatin, acetaminophen and Arsenic. Results: Results showed that, Acetaminophen has maximum resistance and minimum sensitivity in CHO line with IC50 = 16.7 ± 1.06 and regarding HePG2 with IC50 = 18.6 ± 1.29. On the other hand, cisplatin showed minimum resistance and maximum Sensitivity in HePG2 with IC50 = 0.87 ± 0.07 and HGF1 with IC50 = 1.6 ± 0.21 and at last Arsenic showed minimum resistance and maximum sensitivity in A549 with IC50 = 4.59 ± 0.29 and LLCPK1 with IC50 = 1 ± 0.37. Conclusion: According to the evaluated IC50 , it showed differences between results of sensitivity of cell lines which exposed to the three examinated drugs (P < 0.05). Entirely, resistance in cancer cell lines is lower than normal cells, therefore, the results showed the importance of cell defensive mechanisms encounting different substances, such as glutathione. doi:10.1016/j.toxlet.2008.06.461 V71 Effect of P-Glycoprotein inducers on its expression and activity in Caco-2 cells Renata Silva 1,∗ , Anabela Cordeiro-da-Silva 2 , Sofia A.C. Lima 3 , Félix Carvalho 1 , Maria Lourdes Bastos 1 , Helena Carmo 1 , Fernando Remião 1 1
REQUIMTE, Toxicology Department, Faculty of Pharmacy, University of Porto, Porto, Portugal, 2 Biochemistry Department, Faculty of Pharmacy, University of Porto, Porto, Portugal, 3 IBMC, Institute of Molecular and Cellular Biology, University of Porto, Porto, Portugal P-glycoprotein (P-gp) is a membrane-bound glycoprotein belonging to the ATP-binding cassette transporter superfamily expressed in several organs including, liver, brain, kidney and small intestine. P-gp acts as a defence mechanism by “effluxing” drugs and toxic compounds from the cell membrane or cytoplasm to the extracellular space, thus affecting the absorption, disposition, and elimination of different xenobiotics. Caco-2 cells have been widely accepted as a reliable in vitro model for the rapid screening of the intestinal drug absorption and excretion mediated by P-gp. In fact, when grown on permeable supports Caco-2 cells from epithelial monolayers express many features characteristic of the normal human intestine such as spontaneous morphological and biochemical enterocytic differentiation. Thus, the main objective of the present work was to characterize the P-gp expression levels in caco-2 cells during their differentiation processes. To evaluate potential changes in those expression levels, as well as changes in P-gp activity, when exposed to several Pgp inducers, such as dexamethasone, vinblastine and rifampin were the other objectives. Alkaline phosphatase activity was measured to assess the cell differentiation. P-gp expression levels were determined by flow cytometry, using a CD243-PE conjugated antibody. The protein transport activity was evaluated using daunorubicin as a fluorescent transported P-gp subtract. Caco-2 cell P-gp ATPase activity was also evaluated by colorimetric assay. The observed results are important to characterize these cells in order to study the induction mechanism to protect cells from toxic compounds. doi:10.1016/j.toxlet.2008.06.462