Comparison between the DNA precipitation and alkali unwinding assays for detecting DNA strand breaks and cross-links.
A new DNA precipitation assay used together with the alkali unwinding assay may provide a rapid means of detecting DNA damage in addition to strand breaks based on the relative amount of damage measured by the two assays. X-rays, Adriamycin, 4-nitroquinoline-N-oxide, N-methyl-N'-nitrosoguanidine, bleomycin, RSU 1172, and five other drugs produced the same relative amount of strand breakage by using the DNA precipitation and alkali unwinding assays. However, strand breaks produced by the bifunctional alkylating agents bis(2-chloroethyl)nitrosourea, RSU 1069, and RSU 1131 were detected with greater efficiency by the DNA precipitation assay, while the unwinding assay measured more strand breaks than the precipitation assay after damage by the topoisomerase inhibitors VP-16 and VM-26 and the DNA-condensing agents acridine orange and pyronin Y. Based on the reported mechanisms of action of these drugs, and studies with known DNA cross-linking agents, it appears that in addition to DNA strand breaks, the alkali unwinding assay is more sensitive to interstrand than to DNA-protein cross-links, while the DNA precipitation assay can be used to detect both types of cross-links. While quantification of specific lesions is not possible with this approach, the concomitant use of these two assays may provide a rapid and simple method for screening genotoxic drugs for DNA damage, and may also help to differentiate between DNA lesions which include strand breaks, interstrand and protein cross-links, DNA-phosphate adducts, and DNA-drug precipitates.
Olive
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British Columbia Cancer Research Center, Vancouver, Canada.
Chan
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D000818:Animals; D002330:Carmustine / Q000494:pharmacology; D002478:Cells, Cultured; D011232:Chemical Precipitation; D002945:Cisplatin / Q000494:pharmacology; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D011506:Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D011506:Proteins; D004247:DNA; D002945:Cisplatin; D002330:Carmustine