Assessment of DNA damage and repair in human peripheral blood mononuclear cells using a novel DNA unwinding technique

Abstract

A newly developed, fast and sensitive microplate assay (Fast Micromethod) was used for the assessment of gamma-radiation-induced DNA damage in peripheral blood mononuclear cells (PBMC) from healthy donors of various ages and from cancer patients undergoing radiotherapy. This assay detects the presence of DNA single-strand breaks and alkali-labile sites by monitoring the rate of DNA unwinding under alkaline conditions using the fluorescent dye PicoGreen, which preferentially binds to double-stranded DNA at high pH (>12.0); it requires only minimal amounts of material (approximately 3 x 10(3) cells/well) and can be performed within 3 hrs. or less. EDTA blood samples were collected from patients not undergoing chemotherapy prior and immediately after irradiation, or were collected from healthy donors and irradiated ex vivo. The results revealed that the amount of DNA strand breaks in PBMC, induced by application of a single dose to patients in the course of radiotherapy treatment, markedly varied between different individuals. To examine the effect of age on DNA damage, the basal levels of DNA damage in PBMC from a total of 30 healthy donors were determined: 10 were 20 to 30 years of age, 10 were 40 to 60 years of age and 10 were >70 years of age. It was found that the mean basal level of DNA damage from donors in the >70-year age group was significantly higher (by 97%) than that of the 20- to 30-year age group and 27% higher than that of the 40- to 60-year age group. Measurements of the level of induced DNA damage in PBMC isolated from blood after 2 Gy irradiation with 60Co gamma-rays revealed no significant differences between donors aged 20-30 and 40-60. However, there was a strong increase (by 2.3- to 2.9-fold) in radiosensitivity in the age group >70. The microplate assay described may be used as a pretherapeutic sensitivity test for the assessment of the individual radiosensitivity of patients prior to radiation therapy.