Simultaneous determination of DNA double strand breaks and DNA fragment size in cultured mammalian cells exposed to hydrogen peroxide/histidine or etoposide with CHEF electrophoresis.
A CHEF (contour clamped, homogenous electric field) assay allowing the measurement of chemically-generated DNA DSBs (double strand breaks), and the simultaneous estimation of the size of the resulting double stranded DNA fragments, in a single gel run, has been developed. This method combines a very high sensitivity for detecting DNA DSBs with a very good resolution over a broad range of megabase--sized DNA. This information can be obtained in a 68 h gel run, a time which is slightly elevated as compared to the CHEF DSB assay (approximately 20 h), but dramatically reduced as compared to other CHEF protocols utilized for resolving DNA fragments of 0.2-5.7 Mb (5-14 days). Treatment with 5-10 microM etoposide or 50-100 microM hydrogen peroxide/300 microM histidine produced DNA fragments with a mean size of 7.7 x 10(5) bp (from < or = -0.2 Mb) or 4.6 x 10(6) bp (from > or = 5.7-2.2 Mb), respectively.
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Istituto di Farmacologia e Farmacognosia, Università di Urbino, Italy.
Cattabeni
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Cantoni
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D000818:Animals; D016466:CHO Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D004247:DNA / Q000032:analysis* / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage; D004586:Electrophoresis / Q000379:methods; D005047:Etoposide / Q000633:toxicity*; D006639:Histidine / Q000633:toxicity*; D006861:Hydrogen Peroxide / Q000633:toxicity*
D006639:Histidine; D005047:Etoposide; D004247:DNA; D006861:Hydrogen Peroxide