Determination of Phosphofructokinase and Enolase Activities in Cultured Mouse Neuroblastoma Cells: Application to the In Vitro Detection of Neurotoxic Effects

Abstract

Simple methods for the determination of phosphofructokinase (PFK) and total enolase (END activities in cultured mouse neuroblastoma cells have been developed. The influences of changes in glucose metabolism, induced by chlorpromazine, cycloheximide, 2,4-dinitrophenol and iodoacetic acid, on PFK and ENL activities in vitro were compared as a practical application of the methods. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed for 24 hours and cytotoxic effects were evaluated. All the determinations were carried out in the same 96-well tissue culture plates in which exposure took place. Chlorpromazine and cycloheximide produced opposite effects on both PFK and ENL activities. While chlorpromazine increased PFK activity by 50% and decreased ENL activity by 30%, cycloheximide inhibited PFK activity by 50% and increased ENL activity by 45%. The response to dinitrophenol was quite different; both PFK and ENL activities were increased, by 45% and 35% respectively. After exposure of the cells to iodoacetic acid, neither PFK nor ENL activities showed statistically significant differences from the levels in control cells. The different responses elicited by the four toxicants suggest that the two enzymes selected are useful for differentiating among diverse types of mechanism of action.