Submitted to: In Vitro Cellular And Developmental Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/26/2003
Publication Date: 7/8/2003
Citation: Shappell, N.W. 2003. Ergovaline toxicity on Caco-2 cells as assessed by MTT, alamarBlue, and DNA assay. In Vitro Cellular and Developmental Biology-Animal. 39:329-335. Interpretive Summary: Fescue toxicosis is an illness that affects animals that consume fesuce pasture that is contaminated with mold producing ergovaline (a toxin). It is unclear exactly how ergovaline makes animals ill. It has been reported that ergovaline is rapidly eliminated from the blood, but total elimination cannot be accounted for by the sum of urinary and fecal elimination. The ultimate goal is to study ergovaline absorption/transport in gut cells. Before this could be done, non-toxic conditions (concentrations of ergovaline and exposure times) had to be established. If conditions were toxic to the cells, true absorption would not be measured, but instead would include leakage of ergovaline through cells. Caco-2 cells, established from a human colon tumor, exist in two differentiate states: undifferentiated or dividing, and differentiated or nondividing - having enzymes and structures that are present in only mature cells. Membranes of the normal gut are composed of both of these cells states. Ergovaline toxicity was found to be dependent on both cell state and exposure period. At the highest concentrations tested, dividing cells were found to be highly sensitive (cells died) to ergovaline. As the exposure period increased to 72 hr, greater toxicity was found. Non-dividing cells were relatively resistant to ergovaline. These findings may help explain why animals with fescue toxicosis thrive, as the gut requires constant replacement of cells in the membrane with new cells. If the dividing replacement cells are killed by ergovaline, the membrane may become unhealthy and "holey". Results from one assay(MTT)were sometimes contradictory to the other two assays used to quantify toxicity and raise issues with MTT's use in toxicity studies.
Technical Abstract: The exact mechanism of fescue toxicity in animals has yet to be established, but it has been associated with an inability to thrive. Ergovaline is the major ergopeptine alkaloid associated with fungal infections of tall fescue. Therefore, toxicity of ergovaline to gastrointestinal(G.I.) cells was evaluated in Caco2 cells (derived from a colon adenocarcinoma) at a concentration of 1 X 10-4 to -11M ergovaline beginning on day 1, 8, and 18 of culture. Toxicity was evaluated under acute and chronic exposure conditions(24 and 72h). Treatment periods were chosen to study undifferentiated, semi-differentiated, and completely differentiated cells. Cell loss/metabolic activity was assessed by thiazolyl blue reduction (MTT, mitochondrial succinate dehydrogenase activity), alamarBlue assay (cytochrome oxidase activity), and DNA quantitation. Undifferentiated cells were sensitive to 1 X 10-4 M ergovaline after acute exposure (from 52-74% of control values depending on assay). After 72h of exposure to 1 X 10-4 M ergovaline, in all three assays treatment means were reduced to ~70-80% of controls means. By day 11 in culture, ergovaline toxicity had decreased. With 24h exposure an apparent paradoxical increase in MTT was seen at some concentrations. This increase in MTT was also found in fully differentiated cells (day 21), while alamarBlue activity decreased. No change in DNA was found until 72h of exposure, when DNA was reduced ~12% over most concentrations. These findings indicate differentiation state dependent-sensitivity of Caco2 cells to ergovaline; potential problems of the MTT assay as an indicator of cellular toxicity, and usefulness of alamarBlue assay over DNA assay for toxicity assessment