Investigation of the positive response of ethyl acrylate in the mouse lymphoma genotoxicity assay

To develop a better understanding of the relationship between ethyl acrylat e (EA)-induced cytotoxicity and mutation frequency in the mouse lymphoma as say (MLA) we measured the effects of EA treatment to hit cells on: (1) nonp rotein sulfhydryl (NPS) levels; (2) mitochondrial rhodamine 123(Rh123) upta ke; (3) the DNA elution slope (single-strand breakage) and Y intercept of f itted curves (cytotoxicity and double-strand breakage) in the alkaline elut ion assay; (4) the appearance of apoptosis; and (5) the pulsed-field gel el ectrophoretic resolution of high-molecular-weight DNA. EA reduced NPS in bo th a time- and concentration-dependent manner. By 30 min, greater than or e qual to 20 mu g/ml EA reduced NPS by 50% or greater. By 4 h, greater than o r equal to 10 mu g/ml markedly decreased both MPS cell content (greater tha n or equal to 71.5% reduction) and mitochondrial Rh123 uptake (10-50 mu g/m l; 9-62%), the latter effect being further enhanced by washing and incubati on for an additional 2 h (12-85%). EA did not induce single-strand breaks i n the alkaline elution assay. Only highly cytotoxic EA concentrations (80-8 7% reduction in RCG at 40-50 mu g/ml) caused both increases in the elution slope and parallel. drops (Y intercept) in the elution curve in the alkalin e elution assay. Conventional agarose gel electrophoretic analysis of the D NA neutral fraction of these high dose preparations showed evidence for bot h apoptosis (180-bp oligonucleosomal DNA laddering effect) and random smear ing of DNA (necrosis). Pulsed-field gel electrophoresis of directly loaded high dose cell preparations revealed both high- and low-molecular-weight DN A double-strand breaks, but only at the highest concentrations. These obser vations indicated that the EA-induced mutagenic response correlated best wi th cellular cytotoxicity mediated by NPS depletion and mitochondrial membra ne impairment. (C) 1998 Society of Toxicology.