Development of a (32)P-postlabeling/HPLC method for detection of dehydroretronecine-derived DNA adducts in vivo and in vitro.
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.
Yang
Y
Y
National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
Yan
J
J
Churchwell
M
M
Beger
R
R
Chan
P
P
Doerge
D R
DR
Fu
P P
PP
Chou
M W
MW
D000818:Animals; D002273:Carcinogens / Q000378:metabolism* / Q000633:toxicity; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts / Q000032:analysis* / Q000138:chemical synthesis / Q000302:isolation & purification; D003838:Deoxyadenine Nucleotides / Q000032:analysis / Q000737:chemistry; D003848:Deoxyguanine Nucleotides / Q000032:analysis / Q000737:chemistry; D005092:Exonucleases / Q000737:chemistry / Q000378:metabolism; D005260:Female; D007553:Isotope Labeling / Q000379:methods*; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000378:metabolism; D016686:Monocrotaline / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D010761:Phosphorus Radioisotopes / Q000737:chemistry; D011763:Pyrrolizidine Alkaloids / Q000138:chemical synthesis; D051381:Rats; D011916:Rats, Inbred F344; D015203:Reproducibility of Results; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D002273:Carcinogens; D018736:DNA Adducts; D003838:Deoxyadenine Nucleotides; D003848:Deoxyguanine Nucleotides; D010761:Phosphorus Radioisotopes; D011763:Pyrrolizidine Alkaloids; C077233:2'-deoxyguanylic acid; C014795:retronecine; C012873:2'-deoxy-5'-adenosine monophosphate; D016686:Monocrotaline; D004247:DNA; D005092:Exonucleases; C022650:spleen exonuclease; D015719:Single-Strand Specific DNA and RNA Endonucleases; D008836:Micrococcal Nuclease; C007241:dehydroretronecine