Dosimetry of the chemical mutagen ethyl methanesulfonate in spermatozoan DNA from Drosophila melanogaster

Abstract

Summary A method for determining the chemical dose of ethyl methanesulfonate (EMS) to the DNA of Drosophila melanogaster sperm cells has been developed. Males fed as larvae on media containing [ Me - 3 H]thymidine were fed0.025 M , 0.0125 M and 0.008 M concentrationd of [I- 14 C]ethyl methanesulfonate ([ 14 ]EMS) as adults. Our feeding procedures and concentrations were chosen so as to duplicate as nearly as possible the treatments other workers have used. The amount of 3 H per sperm cell was determined by microscopically counting sperm cells, washing them with cold trichloroacetic acid (TCA) onto a MIllipore filter, and counting radioactivity with a liquid scintillation spectrometer. 14 C and 3 H counts were determined for oligodeoxynucleotides released from labeled sperm DNA by electrophoretically purified deoxyribonuclease. From these two counts, the specific activity of [ 14 C]EMS and the amount of 3 H per cell, the number of ethylations per sperm cell DNA was found to range from 1.10 7 to 3.10 8 . Estimates for the number of nucleotides per sperm cell DNA of Drosophila are found from the literature to range from 1.6· 10 8 to 4.5 · 10 8 . It has, therefore, been concluded that the concentrations of [ 14 C]EMS used in these experiments were able to ethylate between 3% and 100% of the nucleotides in the sperm cell DNA of Drosophila, assuming one ethylation per nucleotide. The results of our chemical dosimetry experiments are also discussed in relation to comparative mutagenesis studies with other genetically well studied organisms, such as the mouse. On the basis of the extensive DNA ethylation found in Drosophila sperm combined with previously published reports on sex-linked recessive lethal frequencies and production of mosaics following EMS treatment of Drosophila, it is hypothesized that ethylation of a rare site or combination of sites in the sperm DNA is responsible for the mutagenic action of EMS. Previous models models suggesting the production of mutations by incorrect base pairing of 7-ethylguanine following treatment by EMS cannot explain mutagenesis in Drosophila.