- Email: [email protected]
Human neurospheres as predictive in vitro test for developmental neurotoxicants
Abstracts / Toxicology Letters 196S (2010) S37–S351
the chondrocytes show signs of cell death by increasing the age of cells. So the feasibility of cell articular replacement therapy is reduced by aging. In this study, we investigated whether the bone morphogenetic protein-2 (BMP-2) could modulate the chondrocyte proliferation index and apoptosis, as well as the morphological changes. Methods: This study was performed in Anatomy department’s Cell Culture Lab. of Ahvaz Jundishapur University. Chondrocytes harvested from normal articular cartilage (AC) of four individuals (average 67–87 years, mean age 77 years) with no history of joint disease. The chondrocytes were isolated by mechanical and enzymatic treatment of AC and cultured in monolayer in the presence of 100 ng/ml BMP-2 and %1 FBS as a test group and %1 FBS alone as a control group. Then, the chondrocytes were harvested and assessed for morphology with invert microscopy, proliferation by using MTT-assay and apoptosis with caspase-3 immunocytochemistry. Results: The chondrocytes showed round and polygonal appearance with chondrocyte-like morphology in BMP-2 treated groups after 6 days. The MTT proliferation test did not show significant difference between test and control groups (P < 0.05). Positive immunoreactivity of caspase-3 was %1 and %20 in test and control groups, respectively. Conclusions: It appears that BMP-2 involves in suppression of dedifferentiation and apoptosis processes of cultured human articular chondrocytes. These findings may be beneficial for chondrocyte-based cartilage repair procedures. doi:10.1016/j.toxlet.2010.03.469
P201-015 An exposure system for in vitro cell culture investigations of cigarette smoke toxicity at the air–liquid interface J. Adamson, D. Azzopardi, M. Gac¸a British American Tobacco, United Kingdom We have developed a novel exposure system to investigate the biological and toxicological effects of in vitro cell cultures exposed to mainstream cigarette smoke at the air-liquid interface (ALI). Cigarette smoke, generated using a Borgwaldt RM20S smoking machine, is delivered to clear Perspex exposure chambers (patent publication number WO 03100417 A1), housing cells cultured on Transwell inserts. Particle dosimetry has been characterised within the system and delivery at biologically relevant doses has been demonstrated. In this study we have characterised an expanded whole smoke system designed to expose ALI cultures to eight different concurrent dilutions of whole smoke (previously only four dilutions could be generated) by assessing accuracy of syringe delivery and cytotoxicity within the whole smoke chamber. Syringes were tested using a methane gas standard and hydrocarbon analysis to assess how accurately the target dilution was met. Results indicated no significant difference in syringe output across all eight syringes at two methane targets, 200 ppm and 520 ppm (p = 0.051) and a repeatability error of 1.88% was achieved. The neutral red cytotoxicity assay was used to assess delivery of smoke to H292 lung epithelial cells on Transwells, exposed to the same dilution of smoke within a single chamber. Delivery of a 1:60 smoke to air dilution ratio, equivalent to ∼5 g/cm2 , indicated no significant difference in cell viability of ALI cultures in six different TranswellsTM within a single smoke chamber (p = 0.61). These results suggest our exposure system is a reliable and repeatable method for generating and exposing in vitro ALI cultures to whole smoke. Future studies will use this exposure system
S135
to investigate whole smoke verses smoke gas phase and single aerosol or individual gas phase components. This exposure system is adaptable for various cigarette formats and for other test aerosols, including environmental, industrial, pharmaceutical and cosmetic aerosols. doi:10.1016/j.toxlet.2010.03.470
P201-016 Human neurospheres as predictive in vitro test for developmental neurotoxicants K. Gassmann, T.D. Rockel, J. Abel, E. Fritsche Institut fuer umweltmedizinische Forschung at the Heinrich-Heine-University Duesseldorf, Germany Current developmental neurotoxicity (DNT) testing guidelines propose investigations in rodents, which require huge amount of animals. With regards to the 3Rs and the European Regulation of Chemicals (REACH), alternative testing strategies are needed, which refine and reduce animal experiments by allowing faster and cheaper screening. We have established a 3D test system for DNT screening based on primary human fetal neural progenitor cells which is embedded in the BMBF joint project: Development of predictive in vitro test for developmental neurotoxicity testing. Within this project, different cell models are compared with regard to their DNT predictability employing a battery of test compounds. In our system first results indicate that the well known developmental neurotoxicant methylmercury effects proliferation, migration and differentiation of neurospheres in a nanomolar range, while a negative test substance, the liver toxicant paracetamol, showed interference with these processes in millimolar concentrations. Furthermore, the DNT compounds MAM, valproic acid and lead also affect these endpoints, while glutamate, which is not developmentally neurotoxic, is well distinguishable. At the shorter timepoints, specific effects on those DNT endpoints are observed at concentrations which do not cause cytotoxicity. Taken together, we have established the human neurosphere model as a system-based in vitro test method for elucidating the potential of chemicals to disturb human brain development. Testing more chemicals will give us an answer on the predictability of our test system. doi:10.1016/j.toxlet.2010.03.471
P201-017 A co-culture approach to new in vitro skin sensitization assays combining Episkin and U937 or THP-1 A. Staropoli 1 , M. Ishikawa 2 , J.M. Ovigne 1 , T. Ashikaga 2 , J. Robert 1 , H. Itagaki 2 , Y. Kohno 2 , J.R. Meunier 1 1
L’Oréal, 2 Shiseido
L’Oreal and Shiseido are developing new in vitro skin sensitization assays based on a co-culture approach: the principle is to adapt respectively MUSST or h-CLAT cellular assays by integrating an Episkin in order to take into account some of the first steps of the sensitization reaction: penetration and metabolization of tested chemicals by the skin. Thus, U937 (L’Oréal) or THP-1 (Shiseido) cells are cultured beneath Episkin inserts. Chemicals are topically applied onto the Episkin. After 18 h or 2 h incubation respectively, the Episkin inserts were removed and the cells were cultured sep-
the chondrocytes show signs of cell death by increasing the age of cells. So the feasibility of cell articular replacement therapy is reduced by aging. In this study, we investigated whether the bone morphogenetic protein-2 (BMP-2) could modulate the chondrocyte proliferation index and apoptosis, as well as the morphological changes. Methods: This study was performed in Anatomy department’s Cell Culture Lab. of Ahvaz Jundishapur University. Chondrocytes harvested from normal articular cartilage (AC) of four individuals (average 67–87 years, mean age 77 years) with no history of joint disease. The chondrocytes were isolated by mechanical and enzymatic treatment of AC and cultured in monolayer in the presence of 100 ng/ml BMP-2 and %1 FBS as a test group and %1 FBS alone as a control group. Then, the chondrocytes were harvested and assessed for morphology with invert microscopy, proliferation by using MTT-assay and apoptosis with caspase-3 immunocytochemistry. Results: The chondrocytes showed round and polygonal appearance with chondrocyte-like morphology in BMP-2 treated groups after 6 days. The MTT proliferation test did not show significant difference between test and control groups (P < 0.05). Positive immunoreactivity of caspase-3 was %1 and %20 in test and control groups, respectively. Conclusions: It appears that BMP-2 involves in suppression of dedifferentiation and apoptosis processes of cultured human articular chondrocytes. These findings may be beneficial for chondrocyte-based cartilage repair procedures. doi:10.1016/j.toxlet.2010.03.469
P201-015 An exposure system for in vitro cell culture investigations of cigarette smoke toxicity at the air–liquid interface J. Adamson, D. Azzopardi, M. Gac¸a British American Tobacco, United Kingdom We have developed a novel exposure system to investigate the biological and toxicological effects of in vitro cell cultures exposed to mainstream cigarette smoke at the air-liquid interface (ALI). Cigarette smoke, generated using a Borgwaldt RM20S smoking machine, is delivered to clear Perspex exposure chambers (patent publication number WO 03100417 A1), housing cells cultured on Transwell inserts. Particle dosimetry has been characterised within the system and delivery at biologically relevant doses has been demonstrated. In this study we have characterised an expanded whole smoke system designed to expose ALI cultures to eight different concurrent dilutions of whole smoke (previously only four dilutions could be generated) by assessing accuracy of syringe delivery and cytotoxicity within the whole smoke chamber. Syringes were tested using a methane gas standard and hydrocarbon analysis to assess how accurately the target dilution was met. Results indicated no significant difference in syringe output across all eight syringes at two methane targets, 200 ppm and 520 ppm (p = 0.051) and a repeatability error of 1.88% was achieved. The neutral red cytotoxicity assay was used to assess delivery of smoke to H292 lung epithelial cells on Transwells, exposed to the same dilution of smoke within a single chamber. Delivery of a 1:60 smoke to air dilution ratio, equivalent to ∼5 g/cm2 , indicated no significant difference in cell viability of ALI cultures in six different TranswellsTM within a single smoke chamber (p = 0.61). These results suggest our exposure system is a reliable and repeatable method for generating and exposing in vitro ALI cultures to whole smoke. Future studies will use this exposure system
S135
to investigate whole smoke verses smoke gas phase and single aerosol or individual gas phase components. This exposure system is adaptable for various cigarette formats and for other test aerosols, including environmental, industrial, pharmaceutical and cosmetic aerosols. doi:10.1016/j.toxlet.2010.03.470
P201-016 Human neurospheres as predictive in vitro test for developmental neurotoxicants K. Gassmann, T.D. Rockel, J. Abel, E. Fritsche Institut fuer umweltmedizinische Forschung at the Heinrich-Heine-University Duesseldorf, Germany Current developmental neurotoxicity (DNT) testing guidelines propose investigations in rodents, which require huge amount of animals. With regards to the 3Rs and the European Regulation of Chemicals (REACH), alternative testing strategies are needed, which refine and reduce animal experiments by allowing faster and cheaper screening. We have established a 3D test system for DNT screening based on primary human fetal neural progenitor cells which is embedded in the BMBF joint project: Development of predictive in vitro test for developmental neurotoxicity testing. Within this project, different cell models are compared with regard to their DNT predictability employing a battery of test compounds. In our system first results indicate that the well known developmental neurotoxicant methylmercury effects proliferation, migration and differentiation of neurospheres in a nanomolar range, while a negative test substance, the liver toxicant paracetamol, showed interference with these processes in millimolar concentrations. Furthermore, the DNT compounds MAM, valproic acid and lead also affect these endpoints, while glutamate, which is not developmentally neurotoxic, is well distinguishable. At the shorter timepoints, specific effects on those DNT endpoints are observed at concentrations which do not cause cytotoxicity. Taken together, we have established the human neurosphere model as a system-based in vitro test method for elucidating the potential of chemicals to disturb human brain development. Testing more chemicals will give us an answer on the predictability of our test system. doi:10.1016/j.toxlet.2010.03.471
P201-017 A co-culture approach to new in vitro skin sensitization assays combining Episkin and U937 or THP-1 A. Staropoli 1 , M. Ishikawa 2 , J.M. Ovigne 1 , T. Ashikaga 2 , J. Robert 1 , H. Itagaki 2 , Y. Kohno 2 , J.R. Meunier 1 1
L’Oréal, 2 Shiseido
L’Oreal and Shiseido are developing new in vitro skin sensitization assays based on a co-culture approach: the principle is to adapt respectively MUSST or h-CLAT cellular assays by integrating an Episkin in order to take into account some of the first steps of the sensitization reaction: penetration and metabolization of tested chemicals by the skin. Thus, U937 (L’Oréal) or THP-1 (Shiseido) cells are cultured beneath Episkin inserts. Chemicals are topically applied onto the Episkin. After 18 h or 2 h incubation respectively, the Episkin inserts were removed and the cells were cultured sep-