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Amphotericin B: Relationship between nephrotoxicity and inhibition of PKA and p38 MAPK signaling pathways
S140
Abstracts / Toxicology Letters 221S (2013) S59–S256
P12-08 Amphotericin B: Relationship between nephrotoxicity and inhibition of PKA and p38 MAPK signaling pathways F.D. Franc¸a 1,∗ , C.A. Tagliati 2 , M.M. Chaves 1 1 Laboratory of Biochemistry, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 2 Laboratory of Toxicology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
The CULTEX® Long-Term Cultivation (LTC) system was specifically designed for the computer-controlled cultivation of cells grown on commercial cell culture inserts under ALI conditions. The system allows a continuous as well as intermittent medium supply and the medium level is controlled by an ultrasonic sensor. The results demonstrate that the CULTEX® LTC module enables the long-term cultivation of epithelial cells under stable and reproducible conditions and can thus highly enhance the reliability of toxicological studies. http://dx.doi.org/10.1016/j.toxlet.2013.05.276
Amphotericin B is one of the most effective antifungal agents; however, its use is often limited due to nephrotoxicity. The purpose of this study was to evaluate the change in cell viability in LLC-PK1 and MDCK cell lines, as well as the effect of inhibiting the PKA and p38 MAPK signaling pathways on nephrotoxicity caused by Amphotericin B. Amphotericin B proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity (MTT) assay, and caused DNA fragmentation, determined by flow cytometry using the Propidium iodide (PI) dye. In MDCK cells, the inhibition of PKA signaling pathways (using H89 inhibitor) and p38 MAPK (using PD169316 inhibitor) caused a significant reduction in DNA fragmentation. Both cell lines were able to recover from the toxic effect caused by Amphotericin B and, in both cases, the inhibition of PKA and P38 MAPK was unable to hinder the capacity of cell recovery, as assessed by Neutral Red assay. Therefore, considering the present study’s results as a whole, it can be concluded that the inhibition of PKA and p38 MAPK signaling pathways can aid in preventing nephrotoxicity caused by Amphotericin B without altering the capacity of cellular recovery at the molecular level. Acknowledgments: FAPEMIG (APQ-00596-08); CNPq (473125/ 2012-9). http://dx.doi.org/10.1016/j.toxlet.2013.05.275
P12-09 An automated system for a stable and reproducible long-term cultivation of epithelial cells at the air–liquid interface: The CULTEX® LTC module Michaela Aufderheide ∗ , Jessica Rach, Niklas Möhle Cultex Laboratories GmbH, Feodor-Lynen-Str. 21, 30625 Hannover, Germany In vitro approaches, especially cell culture techniques, have gained more and more importance in toxicology during the last decade, due to the ongoing progress in the development of novel technologies and the increasing call for alternatives to animal testing. One major requirement for the reliability of toxicological data is their reproducibility. For that purpose, identical cell cultures are required for repeatable experiments. However, the handling of cell cultures is laborious and vulnerable to person-specific variations as well as pipetting errors. Thus, automated cell cultivation systems are highly desirable for in vitro toxicology. There are various automated systems that are specifically designed to fulfill particular requirements, ranging from different bioreactors up to sophisticated modular working stations. Today, cell cultures that reflect the in vivo conditions in the best possible manner are often used for toxicological studies. The lung, skin and intestinal epithelium are target organs of many drugs and pollutants and are thus the subject of most toxicological studies. The differentiation of epithelial cells in vitro is achieved by cultivating them at the air–liquid interface (ALI).
P12-10 An in vitro model of the renal proximal tubule composed of small intestinal submucosa (SIS) and human kidney-derived cells (hKDCs) Anke Hoppensack 1,∗ , Johanna Schanz 1 , Christian Kazanecki 2 , David Colter 2 , Heike Walles 1 1
Fraunhofer IGB, Stuttgart, Germany, 2 Advanced Technologies and Regenerative Medicine, LLC, Somerville, USA Introduction: Epithelial cells of the renal proximal tubulus (RPT) are targeted by toxic substances due to their transport functions and metabolic activity. Simulating the RPT in vitro has been hindered by the lack of a cell–scaffold combination that allows for epithelial monolayer formation. By using natural matrices, an in vivo-mimicking environment can be created to promote epithelial morphogenesis. Accordingly, we cultured hKDCs on the SIS, a well-known natural scaffold, to develop a model of the RPT. Methods: SIS was prepared by mechanical removal of the mucosa from porcine jejunum and decellularization with sodium desoxycholate (3.4%). hKDCs were cultured on the SIS and, for comparison, on collagen type I-coated PET membrane inserts. Furthermore, growth of the renal cell line HK-2 on the SIS was investigated. For co-culture experiments, human dermal endothelial cells (HDMECs) were used. Cultures were analysed by (immune)histology and scanning electron microscopy. Results: hKDCs formed a monolayer with cuboidal to high-prismatic morphology on the SIS. In contrast, hKDCs on PET-membranes and HK-2 cells on the SIS grew in multiple layers without developing physiological morphology. hKDCs cultured on the SIS showed basement membrane and brush border formation as well as the predominant expression of markers of the RPT epithelium whereas markers of later tubule segments were weakly or not expressed. Furthermore, the co-culture with HDMECs was successfully established. Conclusion: Our results demonstrate that the SIS highly promotes the formation of a polarized, epithelial monolayer by hKDCs. Thus, this cell-scaffold combination represents a promising approach for in vitro toxicology assays. http://dx.doi.org/10.1016/j.toxlet.2013.05.277
P12-11 An investigation into the toxicity and genotoxicity of brominated flame retardants in SHSY-5Y cells Jelena Sostare ∗ , Francesco Michelangeli, Nikolas Hodges University of Birmingham, UK Brominated flame-retardants (BFRs) are widespread environmental pollutants and ubiquitous contaminants in humans.
Abstracts / Toxicology Letters 221S (2013) S59–S256
P12-08 Amphotericin B: Relationship between nephrotoxicity and inhibition of PKA and p38 MAPK signaling pathways F.D. Franc¸a 1,∗ , C.A. Tagliati 2 , M.M. Chaves 1 1 Laboratory of Biochemistry, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 2 Laboratory of Toxicology, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
The CULTEX® Long-Term Cultivation (LTC) system was specifically designed for the computer-controlled cultivation of cells grown on commercial cell culture inserts under ALI conditions. The system allows a continuous as well as intermittent medium supply and the medium level is controlled by an ultrasonic sensor. The results demonstrate that the CULTEX® LTC module enables the long-term cultivation of epithelial cells under stable and reproducible conditions and can thus highly enhance the reliability of toxicological studies. http://dx.doi.org/10.1016/j.toxlet.2013.05.276
Amphotericin B is one of the most effective antifungal agents; however, its use is often limited due to nephrotoxicity. The purpose of this study was to evaluate the change in cell viability in LLC-PK1 and MDCK cell lines, as well as the effect of inhibiting the PKA and p38 MAPK signaling pathways on nephrotoxicity caused by Amphotericin B. Amphotericin B proved to be cytotoxic for both cell lines, as assessed by the mitochondrial enzyme activity (MTT) assay, and caused DNA fragmentation, determined by flow cytometry using the Propidium iodide (PI) dye. In MDCK cells, the inhibition of PKA signaling pathways (using H89 inhibitor) and p38 MAPK (using PD169316 inhibitor) caused a significant reduction in DNA fragmentation. Both cell lines were able to recover from the toxic effect caused by Amphotericin B and, in both cases, the inhibition of PKA and P38 MAPK was unable to hinder the capacity of cell recovery, as assessed by Neutral Red assay. Therefore, considering the present study’s results as a whole, it can be concluded that the inhibition of PKA and p38 MAPK signaling pathways can aid in preventing nephrotoxicity caused by Amphotericin B without altering the capacity of cellular recovery at the molecular level. Acknowledgments: FAPEMIG (APQ-00596-08); CNPq (473125/ 2012-9). http://dx.doi.org/10.1016/j.toxlet.2013.05.275
P12-09 An automated system for a stable and reproducible long-term cultivation of epithelial cells at the air–liquid interface: The CULTEX® LTC module Michaela Aufderheide ∗ , Jessica Rach, Niklas Möhle Cultex Laboratories GmbH, Feodor-Lynen-Str. 21, 30625 Hannover, Germany In vitro approaches, especially cell culture techniques, have gained more and more importance in toxicology during the last decade, due to the ongoing progress in the development of novel technologies and the increasing call for alternatives to animal testing. One major requirement for the reliability of toxicological data is their reproducibility. For that purpose, identical cell cultures are required for repeatable experiments. However, the handling of cell cultures is laborious and vulnerable to person-specific variations as well as pipetting errors. Thus, automated cell cultivation systems are highly desirable for in vitro toxicology. There are various automated systems that are specifically designed to fulfill particular requirements, ranging from different bioreactors up to sophisticated modular working stations. Today, cell cultures that reflect the in vivo conditions in the best possible manner are often used for toxicological studies. The lung, skin and intestinal epithelium are target organs of many drugs and pollutants and are thus the subject of most toxicological studies. The differentiation of epithelial cells in vitro is achieved by cultivating them at the air–liquid interface (ALI).
P12-10 An in vitro model of the renal proximal tubule composed of small intestinal submucosa (SIS) and human kidney-derived cells (hKDCs) Anke Hoppensack 1,∗ , Johanna Schanz 1 , Christian Kazanecki 2 , David Colter 2 , Heike Walles 1 1
Fraunhofer IGB, Stuttgart, Germany, 2 Advanced Technologies and Regenerative Medicine, LLC, Somerville, USA Introduction: Epithelial cells of the renal proximal tubulus (RPT) are targeted by toxic substances due to their transport functions and metabolic activity. Simulating the RPT in vitro has been hindered by the lack of a cell–scaffold combination that allows for epithelial monolayer formation. By using natural matrices, an in vivo-mimicking environment can be created to promote epithelial morphogenesis. Accordingly, we cultured hKDCs on the SIS, a well-known natural scaffold, to develop a model of the RPT. Methods: SIS was prepared by mechanical removal of the mucosa from porcine jejunum and decellularization with sodium desoxycholate (3.4%). hKDCs were cultured on the SIS and, for comparison, on collagen type I-coated PET membrane inserts. Furthermore, growth of the renal cell line HK-2 on the SIS was investigated. For co-culture experiments, human dermal endothelial cells (HDMECs) were used. Cultures were analysed by (immune)histology and scanning electron microscopy. Results: hKDCs formed a monolayer with cuboidal to high-prismatic morphology on the SIS. In contrast, hKDCs on PET-membranes and HK-2 cells on the SIS grew in multiple layers without developing physiological morphology. hKDCs cultured on the SIS showed basement membrane and brush border formation as well as the predominant expression of markers of the RPT epithelium whereas markers of later tubule segments were weakly or not expressed. Furthermore, the co-culture with HDMECs was successfully established. Conclusion: Our results demonstrate that the SIS highly promotes the formation of a polarized, epithelial monolayer by hKDCs. Thus, this cell-scaffold combination represents a promising approach for in vitro toxicology assays. http://dx.doi.org/10.1016/j.toxlet.2013.05.277
P12-11 An investigation into the toxicity and genotoxicity of brominated flame retardants in SHSY-5Y cells Jelena Sostare ∗ , Francesco Michelangeli, Nikolas Hodges University of Birmingham, UK Brominated flame-retardants (BFRs) are widespread environmental pollutants and ubiquitous contaminants in humans.