Application of the $^{32}$P-Postlabelling Technique : A Study on Detection of DNA Adduct Induced by Azo Dyes rind Flavonoid Compounds

$^{32}$P-Postlabelling 방법의 응용 : Azo색소 및 Flavonoid화합물에 의해 유도되는 DNA Adduct의 겸출에 관한 연구

  • 김재현 (국립보건안전연구원 독성부) ;
  • 박창원 (국립보건안전연구원 독성부) ;
  • 박정식 (국립보건안전연구원 독성부) ;
  • 홍연탁 (국립보건안전연구원 독성부) ;
  • 김효정 (국립보건안전연구원 독성부) ;
  • 이주한 (국립보건안전연구원 독성부) ;
  • 이헌수 (국립보건안전연구원 독성부) ;
  • 이동권 (성균관대학교 약학대학)
  • Published : 1993.04.01


DNA addicts induced by putative chemical related to carcinogenesis were detected and determined by $^{32}$P-Postlabelling assay after exposure of 4 compounds comprising two auto dyes (amaranth, new coccine) and two flavonoid compounds (rutin, quercetin) to ICR mouse. DNA was isolated from mouse liver and digested enzymatically to deoxyribonucleoside 3'-monophosphate. The postincubation of DNA digests with nuclease Pl before $^{32}$P-labelling enhanced the technique's sensitivity. Nuclease Pl cleaves deoxyribonucleoside 3'-mono-phosphates of normal nucleotides to deoxyrihonucleosides which do not serve as substrates for polynucleotide kinase, while most of addicts were found to be totally or partially resistant to the 3'-dephosphorylating action of nuclease Pl. The adducted deoxyribonucleoside 3'-monophosphate was converted to 5'-$^{32}$P-labelled deoxynucleoside 3',5'-bisphosphate by T4 polynucleotide kinase. The nucleotides were separated by anion-exchange thin layer chromatography(TLC) on polyethyleneimine cellulose by 4-dimensional or 2-dimensional TLC then detected by autoradiography. The results show that DNA addicts were detected in liver DNA of ICR mouse after administration of amaranth and quercetin by 2-dimensional and/or 4-dimensional TLC.