Structure of DNA adduct formed with aminophenylnorharman, being responsible for the comutagenic action of norharman with aniline

Chem Res Toxicol. 2002 Oct;15(10):1288-94. doi: 10.1021/tx020007p.


A mutagenic heterocyclic amine (HCA), 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), is produced in the presence of S9 mix by the reaction of norharman and aniline, both of which are nonmutagenic and abundantly present in our environment. It has been previously reported that APNH-DNA adducts were detected in DNA of Salmonella typhimurium strain incubated with APNH and S9 mix. In the present study, we examined the structures of APNH-DNA adducts using the (32)P-postlabeling method and various spectrometry techniques. When the reaction mixture of N-acetoxy-APNH and 2'-deoxyguanosine 3'-monophosphate (3'-dGp) was analyzed, three adduct spots (two major and one minor) were observed by (32)P-postlabeling under modified-standard conditions. No adduct formation was observed for reaction mixtures of N-acetoxy-APNH with 3'-dAp, 3'-dTp, or 3'-dCp. The two major adduct spots (spots 1 and 2) detected by TLC were extracted and subjected to HPLC along with the standards 3',5'-pdGp-C8-APNH and 5'-pdG-C8-APNH, which were independently chemically synthesized. On the basis of the results of co-chromatography, spots 1 and 2 were identified to be 5'-monophosphate and 3',5'-diphosphate forms of dG-C8-APNH. When the extract of spot 2 (3',5'-pdGp-C8-APNH) was further digested with nuclease P1 and phosphodiesterase I, a spot corresponding to spot 1 (5'-pdG-C8-APNH) was newly observed on TLC. From these observations, both of the two major spots were concluded to be dG-C8-APNH. A similar DNA adduct pattern to that apparent in vitro was observed in various organs of F344 rats fed 40 ppm of APNH for 4 weeks. The levels of APNH-DNA adducts were highest in the liver and colon, with RAL values of 1.31 +/- 0.26 and 1.32 +/- 0.11 adducts/10(7)nucleotides, respectively. Thus, APNH was demonstrated to form DNA adducts primarily at the C-8 position of guanine residues in vitro and in vivo, like other mutagenic and carcinogenic HCAs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aniline Compounds / toxicity*
  • Animals
  • Carbolines
  • Colon
  • DNA Adducts*
  • Drug Interactions
  • Harmine / analogs & derivatives*
  • Harmine / toxicity*
  • Indoles / analysis
  • Indoles / chemistry*
  • Liver
  • Male
  • Mutagens / toxicity*
  • Pyridines / analysis
  • Pyridines / chemistry*
  • Rats
  • Rats, Inbred F344
  • Tissue Distribution


  • Aniline Compounds
  • Carbolines
  • DNA Adducts
  • Indoles
  • Mutagens
  • Pyridines
  • Harmine
  • norharman
  • 9-(4'-aminophenyl)-9H-pyrido(3,4-b)indole
  • aniline