Analysis of the action of the restriction endonuclease AluI using three different comet assay protocols.
OBJECTIVE
The comet assay offers the opportunity to measure the amount of DNA damage and the effectiveness of DNA repair in single cells. In a first part, experiments are presented comparing three different protocols of the comet assay technique with respect to the analysis of the induction of DNA damage after X-irradiation in isolated human lymphocytes and CHO cells. In a second part, the restriction enzyme AluI, an agent producing DNA double-strand breaks exclusively, was introduced into CHO cells by electroporation and the effects were analyzed using the different comet assay protocols. The experiments were carried out in order to test the assertion that comet assay techniques can measure different types of DNA damages at different pH conditions of lysis and electrophoresis.


METHODS
Three different comet assay protocols were used for the analysis of DNA damage in lymphocytes and CHO cells.


RESULTS
The results clearly indicate that among the three protocols the modified comet assay technique used by the authors showed the highest sensitivity in the radiotherapy-relevant dose range between 0 and 2 Gy. All three protocols were capable of detecting an effect by AluI. This effect, however, was clearly different from radiation effects. Whereas after radiation exposure all cell nuclei show a dose-dependent increase in DNA content in the comet tail, most of the cell nuclei were unaffected by an AluI uptake. Nevertheless, there was an effect by AluI that could be detected in all three assay versions: between 5% and 15% of the nuclei showed clearly abnormal comet morphologies.


CONCLUSIONS
Neither the strictly alkaline nor the strictly neutral comet assay is applicable in the radiation dose range of about 2 Gy. The restriction enzyme results show that other factors than just DNA strand breaks contribute to DNA migration into the tail of the comets.
Müller
Wolfgang-Ulrich
WU
Institute for Medical Radiobiology, University Hospital Essen, Essen, Germany. wolfgang-ulrich.mueller@uni-essen.de
Ciborovius
Jens
J
Bauch
Thomas
T
Johannes
Christian
C
Schunck
Christian
C
Mallek
Ulrike
U
Böcker
Wilfried
W
Obe
Günter
G
Streffer
Christian
C
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D020552:Comet Assay / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000187:drug effects* / Q000528:radiation effects*; D004249:DNA Damage; D004262:DNA Restriction Enzymes / Q000494:pharmacology; D015252:Deoxyribonucleases, Type II Site-Specific / Q000008:administration & dosage*; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D005260:Female; D006801:Humans; D008214:Lymphocytes / Q000187:drug effects* / Q000528:radiation effects*; D008297:Male; D008875:Middle Aged; D011829:Radiation Dosage; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014965:X-Rays
D004247:DNA; D004262:DNA Restriction Enzymes; C024669:endodeoxyribonuclease AluI; D015252:Deoxyribonucleases, Type II Site-Specific