32P-postlabelling method for the detection of 7-alkylguanine adducts formed by the reaction of different 1,2-alkyl epoxides with DNA.
A 32P-postlabelling method is reported for the detection of 7-alkylguanines, the major adducts formed by the reaction of 1,2-alkylepoxides with DNA. Calf thymus DNA was reacted in vitro with different epoxides (ethylene oxide through octylene oxide) and digested with micrococcal nuclease and spleen phosphodiesterase to 3'-nucleotides. The adduct enrichment was carried out by an ion-exchange method and adducts were labelled with [gamma-32]ATP in a T4-polynucleotide kinase-mediated reaction. The labelled adducts, prior to resolution by two-dimensional TLC, were treated with nuclease P1. The recoveries of adducts formed by different epoxides ranged from 3 to 10%, tending to increase with the increase in the chain length of the substitutions. The 32P-postlabelled adducts were also analysed by HPLC coupled with a radioisotope detector. This method has been applied for the detection of 7-alkylguanine adducts in rats exposed to different alkenes. The method has potential for use in measuring human exposure to alkenes or their corresponding epoxides as well as the endogenously formed 7-alkylguanine adducts.
Kumar
R
R
Center for Nutrition and Toxicology, Karolinska Institute, NOVUM, Huddinge, Sweden.
Staffas
J
J
Försti
A
A
Hemminki
K
K
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry / Q000187:drug effects*; D018736:DNA Adducts; D004852:Epoxy Compounds / Q000633:toxicity*; D006147:Guanine / Q000032:analysis*; D010761:Phosphorus Radioisotopes; D011874:Radiometry; D051381:Rats
D018736:DNA Adducts; D004852:Epoxy Compounds; D010761:Phosphorus Radioisotopes; D006147:Guanine; D004247:DNA