Measurement of DNA breakage in specific germ-cell stages of male mice exposed to acrylamide, using an alkaline-elution procedure

Abstract

DNA breakage in spermiogenic stages of the mouse was studied after exposure to acrylamide (AA), using an alkaline-elution technique. At daily intervals over a 3-week period following i.p. injection of 100 mg AA/kg, mature spermatozoa were recovered from treated ([ 3H]dThd-labeled) and control ([ 14C]dThd-labeled) animals, and were lysed together on polycarbonate filters; the DNA was eluted with a high-pH (12.0) buffer. Elution of germ-cell DNA from AA-exposed animals increased (more DNA-strand breaks) in stages sensitive to the dominant-lethal effects of AA (late spermatids to early spermatozoa) (Shelby et al., 1986). The stage-related pattern of AA-induced DNA breakage also paralleled the pattern of sperm alkylation and protamine alkylation found to be produced by AA (Sega et al., 1989). While dominant-lethal damage from AA exposure is greatest in the spermatids and early spermatozoa, no such damage was observed in pachytene spermatocytes and early spermatids (Shelby et al., 1986). Therefore, AA-induced DNA breakage was also studied directly in pachytene spermatocytes and in early spermatids at short intervals (up to 4 days) after exposure. DNA breakage was clearly detected in these cell stages, with maximum breakage occurring at 1 day after treatment. At later times, the breakage gradually decreased, presumably as a result of DNA repair. By the time these cell stages gave rise to functional spermatozoa, DNA breaks that could have produced dominant-lethal events had apparently been reduced to a level where no genetic effect could be observed.