Some quantitative considerations about DNA adduct enrichment procedures for 32P-postlabelling.
The concentrations of 2'-deoxyribonucleoside-3'-monophosphates remaining in calf-thymus DNA digests after nuclease P1 digestion or extraction into 1-butanol, the most commonly used adduct enrichment procedures prior to the application of the 32P-postlabelling assay, were measured using HPLC and 32P-postlabelling methods. When 10 micrograms of DNA digested to mononucleotides was used, the total amount of nucleotides remaining in the samples were approximately 4 and approximately 14 pmol after nuclease P1 treatment or 1-butanol extraction respectively. The influence of various concentrations of normal nucleotides on the labelling efficiency of a 2'-deoxyguanosine-3'-monophosphate adduct of benzo[a]pyrene diol-epoxide was also studied and found to depend upon the ratio of normal nucleotides/adducted nucleotides present in the sample. Also, the ATP/normal nucleotides ratio in the phosphorylation reaction may affect the quantitation of the adducts and thus deserves due consideration.
Vaca
C E
CE
Molecular Epidemiology Unit, Center for Nutrition and Toxicology, NOVUM, Karolinska Institute, Huddinge, Sweden.
Löfgren
M
M
Hemminki
K
K
D020001:1-Butanol; D000818:Animals; D001564:Benzo(a)pyrene / Q000737:chemistry; D000440:Butanols; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018736:DNA Adducts; D004249:DNA Damage; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D009711:Nucleotides / Q000032:analysis / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D015719:Single-Strand Specific DNA and RNA Endonucleases
D000440:Butanols; D018736:DNA Adducts; D003838:Deoxyadenine Nucleotides; D009711:Nucleotides; D010761:Phosphorus Radioisotopes; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; C012873:2'-deoxy-5'-adenosine monophosphate; D020001:1-Butanol; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases