Improved thin-layer chromatographic separation of 32P-postlabeled DNA adducts.
DNA adducts represent the putative initiating event in the chemical carcinogenesis process. 32P-Postlabeling is one of several assays which have been developed for the sensitive detection of DNA adducts. An integral part of the 32P-postlabeling assay is the separation of adducted nucleotides by multidirectional, multisolvent, anion-exchange polyethyleneimine-cellulose thin-layer chromatography. Standard since the introduction of this assay has been the use of high-salt, high-urea solvents for the resolution of adducts during the D3 and D4 phases of the chromatography. Urea solvents are able to separate adducts resulting from a number of chemicals, however, they are time-consuming, retain a lot of background noise, may push adducts into inadequately resolved diagonal radioactive zones, and may not separate adducts of similar structure. In this study we introduce the use of a dilute ammonium hydroxide solvent for D4 chromatography and compare it to other standard solvents such as lithium chloride-Tris.HCl-urea, sodium phosphate-Tris.HCl-urea, and isopropanol-4 M ammonium hydroxide for adduct separation, resolution, recovery, retention of background noise, and chromatography development time. We found that 0.2 M ammonium hydroxide worked well for the recovery, separation, and resolution of a wide array of adducts derived from highly lipophilic polycyclic aromatic hydrocarbons and aromatic amines. In addition, this solvent required much less time (< 1/4) as compared to the other solvents and more importantly allowed the separation of adducts which otherwise comigrated and were not visible when using the other three D4 solvents.(ABSTRACT TRUNCATED AT 250 WORDS)
Spencer
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GG
Graduate Center for Toxicology, University of Kentucky, Lexington 40506-0054.
Beach
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AC
Gupta
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RC
D064753:Ammonium Hydroxide; D000818:Animals; D002273:Carcinogens / Q000302:isolation & purification*; D002645:Chickens; D002855:Chromatography, Thin Layer; D004247:DNA / Q000302:isolation & purification*; D006878:Hydroxides; D010761:Phosphorus Radioisotopes; D051381:Rats; D012997:Solvents
D002273:Carcinogens; D006878:Hydroxides; D010761:Phosphorus Radioisotopes; D012997:Solvents; D064753:Ammonium Hydroxide; D004247:DNA