329 In Vitro Cell. Dev. Biol.—Animal 39:329–335, July/August 2003  2003 Society for In Vitro Biology 1071-2690/03 $18.00+0.00 ERGOVALINE TOXICITY ON CACO-2 CELLS AS ASSESSED BY MTT, ALAMARBLUE, AND DNA ASSAYS NANCY W. SHAPPELL 1 USDA ARS Biosciences Research Laboratory, 1605 Albrecht Boulevard, Fargo, North Dakota 58105 (Received 30 June 2003; accepted 26 August 2003) SUMMARY The exact mechanisms of fescue toxicity in animals have 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. Gastro- intestinal (GI) toxicity of ergovaline (10 -11 to 10 -4 M) was evaluated in Caco-2 cells (mimicking the GI epithelium) beginning on days 1, 8, and 18 of culture. Acute and chronic toxicity was assessed after 24 and 72 h of exposure. Treatment periods were chosen to study undifferentiated, semidifferentiated, and completely differentiated cells. Cell loss and metabolic activity were assessed by thiazolyl blue reduction (3-(4,5-dimethylthiozole-2-yl)-2,5,-biphenyl tetrazolium bromide [MTT], mitochondrial succinate dehyrdogenase activity), alamarBlue assay (cytochrome oxidase activity), and deoxyribonucleic acid (DNA) quantitation. Undifferentiated cells were sensitive to 1  10 -4 M ergovaline after acute exposure (from 52 to 74% of control values depending on assay). After 72 h of exposure to 1  10 -4 M ergovaline, in all three assays, treatment means were reduced to 10% of the control means. By day 11 in culture, ergovaline toxicity to cells had decreased. With 24 h 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), whereas alamarBlue activity decreased. No change in DNA was found until 72 h of exposure, when DNA was reduced 12% over most concentrations. These findings indicate differentiation state–dependent sensitivity of Caco-2 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. Key words: ergovaline; ergopeptine; fescue toxicosis; gastrointestinal cells. INTRODUCTION Ergovaline is one of a class of compounds (ergopeptine alkaloids) that is produced by endophytes (fungi) that infect forages such as fescue (Fig. 1). The association of ergot poisoning with consumption of infected cereals was made centuries ago and the condition was described as ‘‘St. Anthony’s fire.’’ The symptoms include the vas- cular effects of the poison, including numb, cold, or blue limbs, and absence of peripheral arterial pulses (Yater and Cahill, 1936). One of these compounds, ergotamine, has been used clinically in the treatment of migraines. (Physicians’ Desk Reference, 1995). Consumption of endophyte-contaminated fescue by livestock can result in a syndrome referred to as ‘‘fescue toxicosis.’’ The economic losses to the cattle industry due to contaminated fescue have been estimated to be as great as $609 million annually (Hoveland, 1993). The symptoms include inappetence, failure to thrive, inability to regulate body temperature (heat-stress susceptibility), reproductive and lactational failure, and hoof rot (Williams et al., 1975; Rhodes et al., 1991; Porter and Thompson, 1992; Paterson et al., 1995; Blaney et al., 2000; Sharma et al., 2002). Although these symptoms fail to act as conclusive diagnostic indicators, depressed serum pro- lactin concentrations are reliably indicative of fescue toxicosis (Pat- erson et al., 1995). The mechanism(s) of toxicity has(ve) yet to be 1 To whom correspondence should be addressed at E-mail: shappeln@ fargo.ars.usda.gov fully understood. Dopamine, serotonin, and norepinephrine ago- nists–antagonists have been evaluated for their ability to exacerbate or alleviate ergot alkaloid effects because ergot alkaloids are known to cause contraction of smooth muscles and central nervous system symptoms (review by Berde and Schild, 1978; Boling et al., 1989; Samford-Grigsby et al., 1997; Bennett-Wimbush and Loch, 1998). Studies of ergot alkaloids have indicated a short half-life of cir- culating ergot peptide alkaloids, with serum half-lives of 1h (Nimmerfall and Ronsethaler, 1976). Plasma ergovaline was found to have a half-life of less than 1 h in both sheep and goats after intravenous administration (Jaussaud et al., 1998; Durix et al., 1999). Although tissue concentrations were not reported, others have assumed the presence of ergot alkaloid biological activity in tissues to be reflective of ergot alkaloid concentrations. Rothlin (1946/1947) reported tissue biological activity from highest to low- est to be liver  kidney  lung  spleen  skeletal muscles. Oral administration of 14 C-ergotamine (an ergot peptide alkaloid) to mon- keys resulted in 70% fecal, 7% urinary, and 24% biliary elimi- nation, with a presumed absorption of 31% (Meszaros et al., 1975). These values reflect radioactivity and not necessarily parent compound. Although some work has been published using ruminant gastric tissues to quantitate GI transport (Hill et al., 2001), research efforts have been hindered by the limited availability and expense of purified ergot alkaloids. It is the goal of this laboratory to study the absorption of ergovaline using an established gastrointestinal