DETECTION OF DNA AND GLOBIN ADDUCTS OF POLYNUCLEAR AROMATIC HYDROCARBON DIOL EPOXIDES BY GAS-CHROMATOGRAPHY MASS-SPECTROMETRY AND [H-3] CH3I POSTLABELING OF RELEASED TETRAOLS

Gas chromatography-negative ion chemical ionization mass spectrometry- selected ion monitoring (GC-NICI-MS-SIM) was employed to detect tetram ethyl ether derivatives of tetraols formed upon hydrolysis of DNA and globin adducts derived from diol epoxides of benzo[a]pyrene (BP) and o ther polynuclear aromatic hydrocarbons (PAH). The tetramethyl ether de rivatives could also be detected by [H-3]CH3I postlabeling. The method ology involves the following steps: (1) isolation of DNA or globin; (2 ) mild acid hydrolysis under vacuum; (3) isolation of the resulting te traols and derivatization to the corresponding tetramethyl ethers usin g methyl sulfinyl carbanion and unlabeled or H-3-labeled CH3I; (4) ana lysis by GC-NICI-MS-SIM or HPLC with radioflow detection. The optimum conditions for hydrolysis of adducts and derivatization of the resulti ng tetraols as well as the feasibility of this approach for detecting PAH adducts in mice and humans were explored. Using the set of four BP -tetraols that can be formed upon hydrolysis of adducts formed from ox y-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) or oxy-c- 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BPDE) as models, the stability of the tetraols under the hydrolysis conditions was investi gated. Adducts derived from anti-BPDE yield predominantly the stable - 10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (trans-anti-BP-tetra ol), while adducts derived from syn-BPDE released cis-syn-BP-tetraol a s a major hydrolysis product. Hydrolysis under vacuum significantly in creased the recovery of tetraols. Conditions for derivatization of the BP-tetraols as well as tetraols derived from several other PAH anti-d iol epoxides were investigated. Tetramethyl ethers proved to be superi or derivatives that were stable, easy to prepare in high yields, and d etectable with high sensitivity by GC-NICI-MS-SIM (1-50 fmol per injec tion). Alternatively, these derivatives could be detected by HPLC with radioflow detection if [H-3]CH3I were employed for derivatization. Th e methodology was tested by comparing levels of DNA and globin adducts in mice treated with either unlabeled or H-3-labeled BP. Good agreeme nt was obtained among the GC-NICI-MS-SIM, [H-3]CH3I postlabeling, and conventional radiometric methods. Moreover, analysis of human hemoglob in by GC-NICI-MS-SIM resulted in detection of adducts derived from ant i-BPDE and -dihydroxy-t-3,4-epoxy-1,2,3,4-tetrahydrochrysene. The resu lts of this study demonstrate that GC-NICI-MS-SIM of tetramethyl ether s of tetraols formed by hydrolysis of PAH diol epoxide DNA and globin adducts is a promising approach for detection and quantitation of addu cts derived from a broad range of PAH.