title: Evaluation of different toxicity assays applied to proliferating cells and to stratified epithelium in relation to permeability enhancement with glycocholate.
abstract: The purpose of the present study was to evaluate different toxicity assays for use on proliferating buccal TR146 cells and on stratified TR146 epithelium and to compare these results to the permeability enhancing effect of glycocholate (GC). Both the proliferating cells and the epithelium were exposed to different GC concentrations for 4 h. The MTS/PMS assay and neutral red (NR) retention were performed along with quantitation of ATP, lactate dehydrogenase (LDH) and extracellular protein. The toxicity was calculated as the IC50 value relative to the control. Increase in 3H-mannitol permeability across the epithelium concurrent with a decrease in the transepithelial electrical resistance (TEER) was also determined. The robustness of the epithelium was significantly higher than that of the proliferating cells (P <0.01). The ATP assay was the most sensitive assay with IC50 values of 6.4 and 11.5 mM for proliferating cells and epithelium, respectively. Intracellular LDH quantitation was the least sensitive method and extracellular LDH could not be used as a measure of toxicity partly due to interaction between LDH and GC. The effect on permeability and TEER could be correlated to the IC50 values obtained for the epithelium. The present study clearly demonstrates that for a correlation between toxicity and permeability enhancement, both studies should be performed on the epithelium.
authors.affiliation: Department of Pharmaceutics, The Danish University of Pharmaceutical Sciences, 2 Universitetsparken, DK-2100 Copenhagen, Denmark.
authors.forename: Heidi Ugelstad
authors.initials: HU
authors.lastname: Eirheim
authors.forename: Christoffer
authors.initials: C
authors.lastname: Bundgaard
authors.forename: Hanne Mørck
authors.initials: HM
authors.lastname: Nielsen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects* / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects*; D003902:Detergents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D006000:Glycocholic Acid / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D008353:Mannitol / Q000378:metabolism; D008773:Methylphenazonium Methosulfate / Q000378:metabolism; D009061:Mouth Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009499:Neutral Red / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D018675:Toxicity Tests
chemical_list: D003902:Detergents; D011506:Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; D009499:Neutral Red; D008773:Methylphenazonium Methosulfate; D008353:Mannitol; D000255:Adenosine Triphosphate; D006000:Glycocholic Acid