DNA Damage in Sperm Assayed by Alkaline Elution

Sega, Gary

Abstract

This project will use alkaline elution procedures to study DNA damage (strand breaks and cross-links) produced in meiotic and postmeiotic germ cell stages of male mice after multagen exposure. The test mutagens have been chosen for one or more of the following reasons: Differences in reaction mechanism; differences in germ cell stage sensitivities to the mutagens; ability to cross-link DNA; likely human exposure. The mutagens to be used are methyl methanesulfonate (MMS), methyl nitrosourea (MNU), ethyl nitrosourea (ENU). X-rays, triethylenemelamine (TEM), ethylene dibromide (EDB), and ethylene oxide (Eto).--Animals will be pre-labeled with with (3H) Delta Thd (treated group) or (14C) Delta Thd (control group). Sperm will be recovered from the vas of multagen-treated and control animals over a 3-4 week period following treatment and placed on a polycarbonate filter. After sperm lysis, DNA will be eluted through the filter using an alkaline buffer. The fraction of DNA eluted will be determined by measurement of 3H- and 14C radioactivities using a liquid scintillation counter. Differences between the fraction of treated- and control DN eluted will serve as a measure of DNA damage caused by mutagen treatment. Exposure-response curves will be established for each mutagen and the lowest exposure that produces a measurable change in DNA elution will be determined.--The objectives of the proposal are to: use germ cells of the male mouse as a model test system to validate the use of alkaline elution as a means to screen for DNA damage in human sperm; determine if DNA strand breaks are present in the mutagen-treated germ cells before fertilization; establish the relationship between DNA strand breaks or crosslinks in mouse germ cells and the extent of genetic damage; establish the relationship between DNA rapair in the germ cells and the removal of DNA strand breaks or cross-links; establish the relationship between mutagen binding to sperm protamine and the induction of DNA strand breaks; determine the sensitivity of alkaline elution for detecting DNA damage in mammalian germ cells following low level exposures to multagens.