Submitted to: American Society of Animal Science
Publication Type: Abstract Only
Publication Acceptance Date: January 1, 2002
Publication Date: January 1, 2002
Citation: Shappell, N.W. 2002. Toxicity of ergovaline on Caco2 cells as assessed by MTT, alarmarBlue, and DNA analysis. [abstract]. Journal of Animal Science. 80(Suppl 1):30. Technical Abstract: The exact mechanism of fescue toxicity has yet to be established, but it has been associated with an inability to thrive. Ergovaline has been identified as the major ergopeptine alkaloid associated with fungal infections of tall fescue. The gastrointestinal (G.I.) toxicity of ergovaline was evaluated in a Caco2 cell system which mimics the G.I. epithelium. Cells were plated at 1 X 10e3 cells per well (96 well plates) in DMEM, with 9.1% fetal bovine serum. Ergovaline in methanol was tested at 1 X 10 e-4 to -11M beginning on da 1, 8, and 18 in culture. Acute and chronic toxicity was assessed after 24 and 72h of exposure to ergovaline. Treatment periods were chosen to study undifferentiated, semi-differentiated, and completely differentiated cells. Cell toxicity was assessed by MTT (thiazolyl blue) reduction (mitochondrial succinate dehyrdogenase activity), alamarBlue assay (cytochrome oxidase activity), and total DNA. Undifferentiated cells were sensitive to 0.1 mM ergovaline after acute exposure(74% 56% and 53% of control values for MTT, alamarBlue, and DNA respectively, P less than or equal to 0.0001) or chronic exposure (6% 13% and 0.3% as indicated above). By day 11 in culture, cell toxicity to ergovaline had decreased, and after 24h of exposure an ~12% increase in MTT was seen (1 nM, 10 nM, and 0.1 mM - P less than or equal to 0.02, 0.01, and 0.0002, respectively). After 72h of exposure to 0.1 mM ergovaline, all three parameters were reduced ~20 to 30% (MTT 26% P less than or equal to 0.0001, alamarBlue 31% P less than or equal to 0.0001, and DNA 16% P less than or equal to 0.006). Fully differentiated cells exhibited increased MTT activity (~ 20% after 24h exposure at all concentrations except 0.1 nM, while alamarBlue activity was decreased at all concentrations (~15%). An ~15% elevation in MTT was found after 72h exposure from 1 nM to 10 mM ergovaline, while both MTT and alamarBlue activity decreased ~13% with 0.1 mM ergovaline. No change in DNA was found until 72h of exposure, when DNA was reduced ~12% over most concentrations. These findings indicate variable sensitivity of G.I. cells to ergovaline, dependent on the state of differentiation. Ergovaline (0.1 mM) is toxic to undifferentiated cells, while differentiated cells are much more resistant to its toxic effects.