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To develop a better understanding of the relationship between ethyl acrylate (EA)-induced cytotoxicity and mutation frequency in the mouse lymphoma assay (MLA) we measured the effects of EA treatment to ML cells on: (1) nonprotein sulfhydryl (NPS) levels; (2) mitochondrial rhodamine 123 (Rh123) uptake; (3) the DNA elution slope (single-strand breakage) and Y intercept of fitted curves (cytotoxicity and double-strand breakage) in the alkaline elution assay; (4) the appearance of apoptosis; and (5) the pulsed-field gel electrophoretic resolution of high-molecular-weight DNA. EA reduced NPS in both a time- and concentration-dependent manner. By 30 min, &gt; or = 20 micrograms/ml EA reduced NPS by 50% or greater. By 4 h, &gt; or = 10 micrograms/ml markedly decreased both NPS cell content (&gt; or = 71.5% reduction) and mitochondrial Rh123 uptake (10-50 micrograms/ml; 9-62%), the latter effect being further enhanced by washing and incubation for an additional 2 h (12-85%). EA did not induce single-strand breaks in the alkaline elution assay. Only highly cytotoxic EA concentrations (80-87% reduction in RCG at 40-50 micrograms/ml) caused both increases in the elution slope and parallel drops (Y intercept) in the elution curve in the alkaline elution assay. Conventional agarose gel electrophoretic analysis of the DNA neutral fraction of these high dose preparations showed evidence for both apoptosis (180-bp oligonucleosomal DNA laddering effect) and random smearing of DNA (necrosis). Pulsed-field gel electrophoresis of directly loaded high dose cell preparations revealed both high- and low-molecular-weight DNA double-strand breaks, but only at the highest concentrations. These observations indicated that the EA-induced mutagenic response correlated best with cellular cytotoxicity mediated by NPS depletion and mitochondrial membrane impairment.

Investigation of the Positive Response of Ethyl Acrylate in the Mouse Lymphoma Genotoxicity Assay