Measurement of DNA Double-Strand Breaks with Giant DNA and High Molecular-Weight DNA Fragments by Pulsed-Field Gel Electrophoresis

Reactive oxygen species (ROS) such as hydroxyl radicals (
*
OH), superoxide anions (O
2−
) and hydrogen peroxide (H
2
O
2
) have been shown to damage to chromosomal
DNA
and other cellular components, resulting in DNA degradation, protein denaturation, and lipid peroxidation (
1
,
2
). We know a little about the in vivo action mechanism of ROS produced by anticancer drugs and by X-ray irradiation on chromatin DNA in the nuclei of intact cells. DNA damage induced by ROS in vivo or in the cultured cell system is classified into singleand double-strand breaks and nucleotide base oxidative modifications (
2
-
4
). The application of gel electrophoresis to the measurement of DNA doublestrand breaks has been described by some workers for DNA irradiated in vitro (
5
-
7
). Double-strand breaks are generally thought to have a greater biological consequence than single-strand DNA breaks because they can lead directly to chromosomal aberrations, and more frequently to the loss of genetic information (
6
,
8
). Ionizing radiation such as X-ray and γ-ray are, in general, thought to produce
*
OH radicals from water molecules in or around the target sites in the DNA, and these in turn attack DNA and break it down (
1
,
3
). In addition, the involvement of such radicals in the induction of apoptosis has been suggested in several cell lines (
9
-
12
).