Detection and quantification of depurinated benzol[alpha] pyrene-adducted DNA bases in the urine of cigarette smokers and women exposed to household coal smoke

Polycyclic aromatic hydrocarbons (PAM) are metabolized to electrophiles tha t can bind to DNA bases and destabilize the N-glycosyl bond, causing rapid depurination of the adducted bases. Recent studies support depurination of DNA as a mechanism central to the genesis of H-ras mutations in PAM-treated mouse skin. Depurinating adducts account for 71.% of all DNA adducts forme d in mouse skin treated with benzo[a]pyrene (BP). This study analyzed urine of cigarette smokers, coal smoke-exposed women, and nonexposed controls fo r the presence and quantities of the depurinated BP-adducted DNA bases, 7-( benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) and 7-(benzo[a]pyren-6-yl)adenine ( BP-6-N7Ade). Since these adducted bases originate from reaction of the BP r adical cation with double-stranded DNA and not with RNA or denatured DNA, t heir presence in urine is indicative of DNA damage. Urine samples were frac tionated by a combination of SepPak extraction and reverse-phase HPLC, and then analyzed by tandem mass spectrometry and capillary electrophoresis wit h laser-induced fluorescence. BP-adducted bases were detected in the urine from three of seven cigarette smokers and three of seven women exposed to c oal smoke, but were not detected in urine from the 13 control subjects. Con centrations were estimated to be 60-340 and 0.1-0.6 fmol/mg of creatinine e quivalent of urine for coal smoke-exposed women (maximum possible BP intake of ca. 23 000 ng/day) and cigarette smokers (BP intake of ca. 800 ng/day), respectively, exhibiting a sensitive response to BP exposures. BP-6-N7Gua was present at ca. 20-300 times the concentration of BP-6-N7Ade in the urin e of coal smoke-exposed women, but was not detected in the urine of cigaret te smokers. This difference may be due to the remarkably different BP expos ures experienced by the two groups of PAM-exposed individuals. These result s justify more extensive studies of depurinated BP-adducted DNA bases as po tential biomarkers of PAM-associated cancer risk.