Submitted to: In Vitro Cellular and Developmental Biology - Animals
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 21, 2005
Publication Date: June 21, 2005
Citation: Shappell, N.W., Smith, D.J. 2005. Ergovaline movement across CACO-2 cells. In Vitro Cellular and Developmental Biology - Animals 41:245-251. Interpretive Summary: Ergovaline is a compound produced by fungi that grow in the seed head of some pasture grass (tall fescue). While the fungal infection provides drought tolerance and pest resistance to the plant, animals that graze on the infected fescue become sick (fescue toxicosis). This condition is associated with failure to thrive, inability to withstand heat-stress, hoof rot, and miscarriage. Because of the expense and limited availability of purified ergovaline, its role as a direct cause of fescue toxicosis has not been verified. For a direct effect of ergovaline to be established, first dietary absorption must be proven. Therefore, ergovaline movement across human intestinal cells was assessed using Caco-2 cells established from a cancerous human intestinal tumor. These cells were grown on a semi-permeable membrane attached to a cylindrical rigid support forming a well-like chamber that rests inside another well. The upper chamber with the semi-permeable membrane mimics a bait bucket and the cells grow together to create a barrier/layer equivalent to the intestinal tissue. In an aqueous solution, ergovaline can change to another chemical form, ergovalinine (called an isomer). Therefore a nutrient solution containing ergovaline was allowed to pre-equilibrate to form a constant ratio of ergovaline/inine. This solution was added to the upper chamber (top side of the cell layer). Ergovaline/inine disappearance from the upper chamber, presence in the cells, and appearance in the lower chamber was measured over 12 hours. Rates of movement were not different for the isomers. In the absence of cells, ergovaline/inine was transferred to the basal chamber in a linear manner for three hours regardless of the concentration used, after which no more compound accumulated (concentration plateaued). When cells were present, the rate of ergovaline/inine movement slowed to about 25% the rate of movement in the absence of cells. The rate of transfer determined in our laboratory using human intestinal cells were similar to those reported for ergovaline transfer across tissue slices from the third stomach of sheep (7.5 versus 7nanograms/cm2/minute). After six h in the presence of cells, ~25% of the lower and 40% of the higher ergovaline/inine dose added to the upper chamber had accumulated in the lower chamber. The isomers readily crossed the intestinal cells intact and at similar rates. Only 1-2 % of the compound was retained by the cells, and there was no indication that the cells changed or metabolized ergovaline/inine to other compounds. Because both isomers were found to pass through the intestinal cell layer, either or both could be involved in causing symptoms of fescue toxicosis at other sites of the body.
Technical Abstract: Ergovaline’s role in the direct causation of fescue toxicosis first requires establishment of its dietary absorption. Therefore, ergovaline movement across human intestinal cells was assessed using Caco-2 cells derived from human colon carcinoma. Pre-equilibrated mixtures of ergovaline/ergovalinine (6.6 microM and 22 microM; 60:40 mixture of isomers) were added to the apical compartment and isomer movements were assessed by HPLC of extracted media. Mathematical models for were ergovaline movement were developed. Rates of movement were not different for the isomers. In the absence of cells, basal accumulation of isomers was essentially linear for three h regardless of loading concentration, after which basal accumulation of ergovaline/inine plateaued. Rates of ergovaline/inine movement in the presence of cells slowed to about 25% the rate of movement in the absence of cells (22 microM kt = 0.0133 no cells, 0.0035 with cells, p<0.05). Mass transfer rate was 7.5 ng/cm2/min and was similar to that reported for ergovaline using a parabiotic chamber with sheep omasum. After six h in the presence of cells, ~25% and 40% of the total ergovaline/inine administered had accumulated in the basal compartment for 6.6 and 22 microM treatments, respectively. Ergovaline and its naturally occurring isomer, ergovalinine, readily crossed intestinal cells intact and at similar rates. Either isomer, or a combination of both, could be involved in the pathogenesis of fescue toxicosis at sites distal to the intestine.