THE CALORIMETRIC-RESPIROMETRIC RATIO - ITS POTENTIAL AS A CYTOTOXICITY TEST

The ratio between heat flux and oxygen flux, the calorimetric ratio, i s an enthalpy budget device used to identify anaerobic pathways in the presence of respiration. Ratios that are more exothermic (i.e. more n egative) than the average for catabolic substrates (-450kJ/mol O-2 +/- 5%; Thornton's rule), are usual for cells established in culture, inc luding suspension-adapted LS-L929 mouse fibroblasts. A common reason f or this is a high level of glycolysis, to produce lactate, simultaneou sly with aerobic pathways. To test the idea that the calorimetric-resp irometric (CR) ratio is a revealing cytotoxic endpoint, LS cells grown in serum-rich medium were insulted with known metabolic poisons. Malo nate, a, l-dinitrophenol (2,4-DNP) and a mixture of antimycin A and ro tenone increased the CR ratio to degrees largely explained by greater lactate flux, the CR700 values being 22 mu M malonate, 56 mu M2,4-DNP and, for the mixture, 2 mu M antimycin A and 5 mu M rotenone. Higher c oncentrations of 2,-4-DNP gave an ''exothermic gap'' for which there w as no explained pathway. Iodoacetate decreased the CR ratio while inhi biting glycolysis, a result which can be explained by the hypothesis t hat substrates available in the serum were degraded by mitochondrial p athways and thereby substituted for the normal input from the Krebs cy cle, which had been arrested by pyruvate starvation. In a balanced sal t solution containing only 5.5mM glucose, the metabolic rate slowed an d the CR ratio was more exothermic (CR700 = 6 mu M), giving a ''gap'' for which there was no explanation. Ten MEIC chemicals gave CR700 endp oints which closely corresponded to the order of toxicity for a batter y of tests using animal cells. The CR method thus provides a good basi s for investigating the mechanisms by which chemicals have toxic effec ts on cells.