Determination of DNA superhelicity and extremely low levels of DNA strand breaks in low numbers of nonradiolabeled cells by DNA-4′,6-diamidino-2-phenylindole fluorescence in nucleoid gradients

Abstract

Nonradiolabeled cells derived from animals or cell cultures can be examined in nucleoid gradients labeled with 4′,6-diamidino-2-phenylindole to quantitate extremely low levels of DNA strand breaks and DNA superhelicity. In vitro: 4′,6-Diamidino-2-phenylindole detection of DNA in nucleoid gradients requires only 15,000 nonradiolabeled human dermal fibroblasts per gradient. Quantization of increased DNA strand breaks, relative to controls, occurring at frequencies as low as one break per 2.2 × 10 10 daltons DNA is possible. This technique was employed to examine low-passage (“young”) and high-passage (“old”) normal human dermal fibroblasts. DNA from old cells contains approximately one more strand break per 5 × 10 9 daltons that does the DNA from young cells, and this difference is unobservable using conventional radiolabeled alkaline-sucrose gradients. Decreased DNA negative superhelicity in the old cells was also observed. In vivo: This technique was used to examine nucleoids derived from female Fisher rat livers treated with aflatoxin B 1. Treated cells exhibit decreased DNA negative superhelicity. Since this technique cannot quantitate DNA strand breaks occurring at the higher levels required by alkaline techniques, it is proposed that this technique is a necessary complement to alkaline techniques when low levels of DNA strand breaks must be quantitated.