Hydroxylase Activity Comparisons with Total Bulky Adducts and Aryl Hydrocarbon )pyrene Diol-Epoxide-DNA Adducts in Smokers' Lung: a An Improved Fluorometric Assay for Dosimetry of Benzo(

An improved high-performance liquid chromatography/fluorometric assay has been established to quantitate the benzo(«)pyrene (BP) tetrols released after acid hydrolysis of lung DN A from lung cancer patients, so that the formation of benzo(a)pyrene diol-epoxide-DNA adducts can be measured. The r-7,c-10,f-8,f-9-tetrahydroxy-7,8,9,10-tetrahydro-BP isolated by high-performance liquid chromatography was determined by chromatography in two different solvent systems and fluorescence spectroscopy. This assay has a detection limit of 2 pg of r-7,c-10,r-8,r-9tetrahydroxy-7,8,9,10-tetrahydro-BP, requires 100-500 ng of DNA, and can measure 1 adduct/10" unmodified nucleotides. As this assay does not use immunoaffinity chromatography or solvent extraction, it allows a >90°/orecovery of benzo(a)pyrene diol-epoxide-DNA adducts. This procedure has been tested on 13 DNA samples prepared from nontumorous lung parenchyma taken from lung cancer patients at sur gery and revealed the presence of DNA adducts of the anri-benzo(a)pyrene diol-epoxide in 9 of 11 samples from smokers and in 2 of 2 exsmokers. In only two samples from smokers the formation of adducts derived from .v)-n-bi'ii/i>(«)p\riiiu' diol-epoxide was detected. A 15-fold variation in DNA adduci level was found in 11 of 13 DNA samples, with a range of 0.6-9.9 adducts of benzo(a)pyrene diol-epoxide/10" nucle otides. In samples containing both amiand î>'n-benzo(a)pyrene diolepoxide-DNA adducts, the anti/syn adduci ratio is 2:1. A highly signif icant correlation was found between pulmonary microsomal aryl hydrocarbon hydroxylase activity and the level of benzo(a)pyrene diolepoxide-DNA adduci (r = 0.91; P < 0.001; n = 13). A crude linear correlation between the amounts of these adducts and those of bulky DNA adducts determined by -"P-postlabeling assay was observed in the same samples (r = 0.78; P < 0.02; n = 13). Thus this highly sensitive and specific procedure is suitable for measuring benzo(a)pyrene diolepoxide-DNA adducts in human tissues from environmentally exposed subjects and could be adapted to measure polycyclic aromatic hydrocar bons other than BP.