32P-postlabelling method for the detection of 7-alkylguanine adducts formed by the reaction of different 1,2-alkyl epoxides with DNA

Carcinogenesis. 1995 Mar;16(3):483-9. doi: 10.1093/carcin/16.3.483.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid
  • Chromatography, Thin Layer
  • DNA / chemistry
  • DNA / drug effects*
  • DNA Adducts*
  • Epoxy Compounds / toxicity*
  • Guanine / analysis*
  • Phosphorus Radioisotopes
  • Radiometry
  • Rats

Substances

  • DNA Adducts
  • Epoxy Compounds
  • Phosphorus Radioisotopes
  • Guanine
  • DNA