EXAMINATION OF TOXICITY OF CLOSTRIDIUM-PERFRINGENS EPSILON-TOXIN IN THE MDCK CELL-LINE

The epithelial Madin Darby canine kidney (MDCK) cell line was examined as a model to study the toxicity of Clostridium perfringens epsilon-toxin. The MDCK cell line was used because it is a monolayer cell line sensitive to epsilon-toxin. Using the neutral red (NR) retention assay (an indicator of lysosomal integrity), the concentration of toxin causing death in 50% of the cell population (LC(50)) was 900 pM, although this was found to vary between production batches. epsilon-Toxin was found to act rapidly but with a lag phase of 1 hr (NR assay). Pulsing the cultures with toxin (up to 4800 pM) indicated that the duration of exposure required to exert an effect was potentially very short (2.5 min). Increasing the duration of exposure beyond 3 hr did not decrease cell viability any further. Experiments with protease inhibitors failed to inactivate the toxin. Ethylenediaminetetraacetic acid (EDTA) was found to potentiate the lethality of the toxin by 90% This may be due to non-specific chaotropic effects such as membrane destabilization. By exposing cultures of MDCK cells to epsilon-toxin for a second time, resistance to the effects of the toxin was increased by 43%. The factor(s) controlling resistance to the toxin may have a heritable component.