CYTOXICITY AS MEASURED BY TRYPAN BLUE AS A POTENTIALLY CONFOUNDING VARIABLE IN THE INVITRO ALKALINE ELUTION RAT HEPATOCYTE ASSAY

Rat hepatocytes treated in vitro with A2RA, an angiotensin II receptor antagonist, displayed an increased level of DNA-strand breaks as dete rmined by alkaline elution, without an appreciable increase in cytotox icity as determined by a trypan blue dye exclusion assay at harvest. T he alkaline elution profile appeared to have two components: a rapidly eluting component detected in the first fraction collected (often ass ociated with DNA from dead or dying cells), followed by a more slowly eluting component detected in the subsequent fractions. Further analys is of hepatocytes treated with A2RA by pulsed-field gel electrophoresi s and neutral elution revealed significant levels of DNA double-strand breaks. Electron microscopy (EM) showed pronounced damage to mitochon dria; although cell blebbing was seen using both EM and light microsco py, the plasma and nuclear membranes appeared intact when examined by EM. Cellular ATP levels decreased precipitously with increasing doses of A2RA, falling to less than 10% of control values at a dose of 0.213 mM A2RA, a concentration showing 100% relative viability by trypan bl ue at harvest. Thus, whereas in our experience trypan blue dye exclusi on accurately reflects cytotoxicity induced by the majority of test ag ents, in this rather unusual case, trypan blue did not accurately refl ect compound-induced cytotoxicity at harvest since there was no concur rent loss of membrane integrity. However, when hepatocytes treated wit h A2RA were incubated for either 3 h or 20 h in the absence of compoun d, a sharp, dose-dependent decline in viability was observed using try pan blue dye exclusion. Together with the initial, dose-dependent drop in the alkaline elution curve, these data suggest that the observed D NA double-strand breaks arose as a consequence of endonucleolytic DNA degradation associated with cytotoxicity, rather than by a direct comp ound-DNA interaction. Since DNA double-strand breaks behave under alka line denaturing conditions as two single-strand breaks and can therefo re produce increases in the alkaline-elution slope values, a necessary criteria for a valid positive result in this assay is that cytotoxici ty by trypan blue dye exclusion will not be greater than 30%. Our data , however, indicate that interpretation of the elution assay as a test for genotoxicity can still be confounded by the failure of the trypan blue dye exclusion assay to reflect cytotoxicity in the unusual insta nce when there is no concurrent, immediate loss of membrane integrity.