Analysis of chromate-induced DNA-protein crosslinks with the comet assay

Modifications of the comet assay have been introduced to measure crosslinks by determining the reduction of induced DNA migration. Our previous result s indicated that the modified protocol of the alkaline comet assay is a sen sitive tool for the detection of formaldehyde-induced DNA-protein crosslink s. But results for mitomycin C and cisplatin suggested that the modified pr otocol is not well suited for the evaluation of DNA-DNA crosslinkers. We no w used the comet assay to investigate in V79 cells the effect of potassium chromate (K2CrO4), another DNA-protein crosslinker, to see whether the resu lts obtained for formaldehyde can be generalized. However, chromate did not reduce spontaneous or radiation-induced DNA migration in the alkaline (pH 13) comet assay but led to a small but significant induction of DNA migrati on. A crosslinking effect of chromate could also not be detected with the a lkaline comet assay after postincubation of cells in normal medium after ch romate treatment to enable repair of other (migration-inducing) lesions tha t might mask the crosslinking effect. Exposure of slides to proteinase K fu rther increased DNA migration of chromate-treated cells, thus indicating th e presence of DNA-protein crosslinks. In contrast to the alkaline comet ass ay, a "neutral" version at pH 9 was suited to demonstrate reduced induction of DNA migration after gamma -irradiation of chromate-treated cells. The c rosslinking effect was seen immediately at the end of the chromate treatmen t as well as after a 3 h postincubation period. Using the "neutral" protoco l in combination with proteinase K, we were able to demonstrate the presenc e of DNA-protein crosslinks as the probable cause for the migration-reducin g effect. Further investigations will have to show whether this protocol ca n be recommended as a universal approach for the detection of DNA-protein c rosslinks and also of DNA-DNA crosslinks with the comet assay. (C) 2000 Els evier Science B.V. All rights reserved.