K. Elmendorff-dreikorn et al., Assessment of DNA damage and repair in human peripheral blood mononuclear cells using a novel DNA unwinding technique, CELL MOL B, 45(2), 1999, pp. 211-218
A newly developed, fast and sensitive microplate assay (Fast Micromethod(R)
) was used for the assessment of gamma-radiation-induced DNA damage in peri
pheral blood mononuclear cells (PBMC) from healthy donors of various ages a
nd from cancer patients undergoing radiotherapy. This assay detects the pre
sence 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 (>1
2.0); it requires only minimal amounts of material (similar to 3 x 10(3) ce
lls/well) and can be performed within 3 hrs. or less. EDTA blood samples we
re collected from patients not undergoing chemotherapy prior and immediatel
y after irradiation, or were collected from healthy donors and irradiated e
x 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 radiot
herapy treatment, markedly varied between different individuals. To examine
the effect of age on DNA damage, the basal levels of DNA damage in PBMC fr
om 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 fou
nd 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. Measuremen
ts of the level of induced DNA damage in PBMC isolated from blood after 2 G
y irradiation with Co-60 gamma-rays revealed no significant differences bet
ween 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 as
sessment of the individual radiosensitivity of patients prior to radiation
therapy.