Immunoaffinity clean-up combined with 32P-postlabelling analysis of 1,N6-ethenoadenine and 3,N4-ethenocytosine in DNA.
Immunoaffinity gels able to bind 1,N6-ethenodeoxyadenosine 3'-monophosphate (3'-epsilon dAMP) and 3,N4-ethenodeoxycytidine 3'-monophosphate (3'-epsilon dCMP) were prepared. These gels retained their specific binding capacity for 3'-epsilon dAMP or 3'-epsilon dCMP in the presence of a large excess (> 2 mg per column) of unmodified 3'-deoxynucleotide monophosphates. 3'-epsilon dAMP and 3'-epsilon dCMP were 32P-postlabelled in the presence of [gamma-32P]ATP and T4 polynucleotide kinase to give either 3',5'-[5'-32P]-bisphosphates or 5'-[32P]monophosphates. 3',5'-[5'-32P]Bisphosphates were recovered from the labelling mixture by immunoprecipitation and quantitated by Cerenkov counting (method A). The detection limit of this procedure was 1 fmol with an efficiency of 80% for both ethenonucleotides. Alternatively, 5'-[32P]epsilon dAMP and 5'-[32P]epsilon dCMP were analysed by two-dimensional TLC on PEI cellulose and autoradiography (method B). The detection limit of method B was 50 amol of ethenonucleotide. Methods A and B are complementary and can quantify the formation and repair of 3'-epsilon dAMP and 3'-epsilon dCMP in DNA from rats exposed to a low level of vinyl chloride.
Guichard
Y
Y
International Agency for Research on Cancer, Lyon, France.
Nair
J
J
Barbin
A
A
Bartsch
H
H
D000225:Adenine / Q000031:analogs & derivatives* / Q000032:analysis; D000818:Animals; D001345:Autoradiography; D002273:Carcinogens / Q000633:toxicity; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D002855:Chromatography, Thin Layer; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D004249:DNA Damage; D005069:Evaluation Studies as Topic; D066298:In Vitro Techniques; D010761:Phosphorus Radioisotopes; D011233:Precipitin Tests; D014752:Vinyl Chloride / Q000633:toxicity
C018289:3,N(4)-ethenocytosine; D002273:Carcinogens; D010761:Phosphorus Radioisotopes; C004981:1,N(6)-ethenoadenine; D003596:Cytosine; D004247:DNA; D000225:Adenine; D014752:Vinyl Chloride