ASSESSMENT OF ASPECTS OF THE TOXICITY OF CLOSTRIDIUM-PERFRINGENS EPSILON-TOXIN USING THE MDCK CELL-LINE

1 The epithelial Madin Darby Canine Kidney (MDCK) cell line was used t o study the toxicity of epsilon-toxin from Clostridium perfringens. Th e epithelial MDCK cell line is known to be sensitive to epsilon-toxin of Clostridium perfringens and to investigate its mechanism of action, the neutral red assay has been used to determine the viability of cul tures of this cell line. 2 Comparison of the LC(50)s obtained at 34 de grees C and 0 degrees C showed that the lethality of epsilon-toxin was reduced by 18-fold at the lower temperature. The effect of temperatur e on epsilon-toxin lethality is unlikely to be due to reductions in me mbrane fluidity for the addition of Ca2+ or Mg2+ (2 mM) to buffer cont aining toxin was without effect. Varying the pH of the toxin-containin g buffer from 6.9 to 8.7 did not increase the lethality of the toxin, though the most acidic pH used (5.8) was found to potentiate its actio n on MDCK cells. 3 The effect of inhibiting endocytosis on the lethali ty of epsilon-toxin was also investigated by incubating cultures of MD CK cells with and without sodium azide over a range of concentrations of toxin. The co-administration of sodium azide did not reduce the tox icity of epsilon-toxin, suggesting that energy-dependent uptake proces ses such as endocytosis were unlikely to he involved in its mechanism of action. The results are, however, consistent with known receptor-ba sed mechanisms of uptake and with other mechanisms of internalisation across the plasma membrane. epsilon-toxin thus interacts with cell sur faces by a temperature sensitive mechanism potentiated by low pH.