Hydroxyl-specific fluorescence labeling of ABP-deoxyguanosine, PhIP-deoxyguanosine, and AFB1-formamidopyrimidine with BODIPY-FL.
Detection and analysis of DNA adducts resulting from endogenous or exogenous exposures to carcinogens are essential not only for quantifying biologically effective doses but also for establishing relationships between exposure and cancer risk. We have developed and validated a procedure of high sensitivity and specificity based on fluorescence labeling of DNA adducts combined with high-performance liquid chromatography-laser-induced fluorescence detection. The fluorescent dye 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) was used to label the deoxynucleoside adducts N-(2'-deoxyguanosine-8-yl)-4-aminobiphenyl and N-(2'-deoxyguanosine-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and the base adduct aflatoxin B(1)-formamidopyrimidine by acylation. The labeling reaction was carried out on adducts at 1pmol to 30nmol concentrations at 25 degrees C for 4h in dichloromethane with 200- to 5000-fold excess of BODIPY FL. BODIPY FL and its activating agents 1,3-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were used at a molar ratio of 1:2:2. Under these conditions, all of the above adducts were quantitatively converted to bis-labeled products, as confirmed by mass spectrometry. Sites of derivatization of adduct deoxynucleosides were established primarily by nuclear magnetic resonance and by collision-induced dissociation mass spectrometric analysis, which indicated that the bis-BODIPY groups were located predominantely on the 3'- and 5'-hydroxyl groups of the deoxyribose ring.
Jang
Hyun Gyung
HG
Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Park
Misun
M
Wishnok
John S
JS
Tannenbaum
Steven R
SR
Wogan
Gerald N
GN
D016604:Aflatoxin B1 / Q000031:analogs & derivatives / Q000737:chemistry / Q000302:isolation & purification*; D000611:Aminobiphenyl Compounds / Q000737:chemistry; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D001896:Boron Compounds / Q000737:chemistry*; D002273:Carcinogens / Q000737:chemistry / Q000302:isolation & purification; D018736:DNA Adducts / Q000737:chemistry / Q000302:isolation & purification*; D003849:Deoxyguanosine / Q000031:analogs & derivatives*; D004781:Environmental Exposure / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007093:Imidazoles; D007202:Indicators and Reagents; D009153:Mutagens / Q000737:chemistry / Q000302:isolation & purification; D011743:Pyrimidines / Q000302:isolation & purification; D012680:Sensitivity and Specificity
C502239:2-amino-1-methyl-6-phenolimidazo(4,5-b)pyridine-DNA adduct; C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000611:Aminobiphenyl Compounds; D015415:Biomarkers; D001896:Boron Compounds; D002273:Carcinogens; D018736:DNA Adducts; D005456:Fluorescent Dyes; D007093:Imidazoles; D007202:Indicators and Reagents; D009153:Mutagens; D011743:Pyrimidines; C072611:aflatoxin B1-DNA adduct; C061392:aflatoxin B1-formamidopyrimidine; D016604:Aflatoxin B1; D003849:Deoxyguanosine