Development of a (32)P-postlabeling/HPLC method for detection of dehydroretronecine-derived DNA adducts in vivo and in vitro

Chem Res Toxicol. 2001 Jan;14(1):91-100. doi: 10.1021/tx000149o.

Abstract

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a (32)P-postlabeling/HPLC method for detection of (i) two DHR-3'-dGMP and four DHR-3'-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR-3'-dGMP, DHR-3'-dAMP, and DHR-3',5'-dG-bisphosphate standards and characterization of their structures by mass and (1)H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and (32)P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR-3',5'-dG-bisphosphate adducts both in vitro and in vivo.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Carcinogens / metabolism*
  • Carcinogens / toxicity
  • Cattle
  • Chromatography, High Pressure Liquid / methods*
  • DNA / chemistry*
  • DNA / drug effects
  • DNA / metabolism
  • DNA Adducts / analysis*
  • DNA Adducts / chemical synthesis
  • DNA Adducts / isolation & purification
  • Deoxyadenine Nucleotides / analysis
  • Deoxyadenine Nucleotides / chemistry
  • Deoxyguanine Nucleotides / analysis
  • Deoxyguanine Nucleotides / chemistry
  • Exonucleases / chemistry
  • Exonucleases / metabolism
  • Female
  • Isotope Labeling / methods*
  • Micrococcal Nuclease / chemistry
  • Micrococcal Nuclease / metabolism
  • Monocrotaline / analogs & derivatives*
  • Monocrotaline / chemical synthesis
  • Monocrotaline / chemistry*
  • Monocrotaline / metabolism
  • Monocrotaline / toxicity
  • Phosphorus Radioisotopes / chemistry
  • Pyrrolizidine Alkaloids / chemical synthesis
  • Rats
  • Rats, Inbred F344
  • Reproducibility of Results
  • Single-Strand Specific DNA and RNA Endonucleases / chemistry
  • Single-Strand Specific DNA and RNA Endonucleases / metabolism
  • Spectrometry, Mass, Electrospray Ionization

Substances

  • Carcinogens
  • DNA Adducts
  • Deoxyadenine Nucleotides
  • Deoxyguanine Nucleotides
  • Phosphorus Radioisotopes
  • Pyrrolizidine Alkaloids
  • 2'-deoxyguanylic acid
  • retronecine
  • 2'-deoxy-5'-adenosine monophosphate
  • Monocrotaline
  • DNA
  • Exonucleases
  • spleen exonuclease
  • Single-Strand Specific DNA and RNA Endonucleases
  • Micrococcal Nuclease
  • dehydroretronecine