Analysis of polycyclic aromatic hydrocarbon-DNA adducts by postlabelling with the weak beta-emitters 35S-phosphorothioate and 33P-phosphate, immobilized boronate chromatography and high-performance liquid chromatography.
The wide range of carcinogenic activities of optical isomers of bay-region diol-epoxides of polycyclic aromatic hydrocarbons (PAHs) demands analytical techniques capable of distinguishing both the diastereoisomer and enantiomers of the PAH diol-epoxide (PAH-DE) responsible for DNA adduct formation. Our laboratory recently developed postlabelling procedures using the radioisotope 35S for HPLC analysis of benzo[a]pyrene (BaP)-DNA adducts formed in BaP-treated hamster cell cultures. To allow identification and complete separation of anti-PAH-DE-DNA adducts from syn-PAH-DE-DNA adducts, an immobilized boronate chromatography procedure was developed using a high-capacity boronate column. Analysis of 7,12-dimethyl-benz[a]anthracene (DMBA)-DNA adducts formed in treated hamster embryo cells by this boronate chromatography procedure and 35S-postlabelling demonstrated that both anti- and syn-DMBA-DE were bound to DNA. These analytical techniques also provided evidence that the potent carcinogen and environmental PAH dibenzo[a,l]pyrene was metabolized in cells to a DNA-binding intermediate with structural characteristics of an anti-PAH-DE. Conditions were also developed for using 33P-labelled ATP in the postlabelling assay. The use of [33P] instead of [35S]ATP offers the advantages of ease of labelling and greater sensitivity while still using a weak beta-emitter. The development of PAH-DNA adduct analysis techniques with improved sensitivity allows more detailed studies of how PAHs interact with DNA and how this leads to cancer induction.
Baird
W M
WM
Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907.
Lau
H H
HH
Schmerold
I
I
Coffing
S L
SL
Brozich
S L
SL
Lee
H
H
Harvey
R G
RG
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000032:analysis / Q000633:toxicity; D000818:Animals; D001564:Benzo(a)pyrene / Q000032:analysis; D002273:Carcinogens / Q000032:analysis / Q000633:toxicity; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D004247:DNA / Q000032:analysis* / Q000187:drug effects; D018736:DNA Adducts; D004249:DNA Damage; D010761:Phosphorus Radioisotopes; D011083:Polycyclic Compounds / Q000032:analysis* / Q000633:toxicity; D013237:Stereoisomerism; D013462:Sulfur Radioisotopes; D013873:Thionucleotides
D002273:Carcinogens; D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D011083:Polycyclic Compounds; D013462:Sulfur Radioisotopes; D013873:Thionucleotides; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; D015127:9,10-Dimethyl-1,2-benzanthracene; D004247:DNA