Faculty Bibliography

Reaction of the rodent carcinogen acrylamide (AM) at pH 7.0 and 37 degrees C for 10 and 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), and thymidine (dThd) resulted in the formation of 2-formamidoethyl and 2-carboxyethyl adducts via Michael addition. The alkylated 2'-deoxynucleoside adducts isolated (% yield after 40 days) were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo) (8%), N6-CE-dAdo (21%) (via Dimroth rearrangement of 1-CE-dAdo), 1-CE-dGuo (4%), 7-(2-formamidoethyl)-Gua (7-FAE-Gua) (6%), 7, 9-bis-FAE-Gua (1%) (formed by reaction of AM with depurinated 7-FAE-Gua during the course of the reaction), and 3-FAE-dThd (4%). The products isolated following in vitro reaction of AM with calf thymus DNA at pH 7.0 and 37 degrees C for 40 days were (nmol/mg DNA) 1-CE-dAdo (5.5), N6-CE-dAdo (1.4), 3-CE-dCyd (2.8), 1-CE-dGuo (0.3), and 7-FAe-Gua (1.6). Compound 3-FAE-dThd was not detected. Structures were assigned on the basis of chemical properties, UV spectra, and electron impact, chemical ionization, desorption chemical ionization, Californium-252 fission fragment ionization, and fast atom bombardment mass spectra. A facile hydrolysis of the amide group to a carboxylic acid was observed when AM alkylated a ring nitrogen adjacent to an exocyclic nitrogen atom. In previous studies, we had observed an analogous phenomenon when studying the in vitro reactions of acrylonitrile with DNA, i.e., a facile hydrolysis of nitrile to carboxylic acid when acrylonitrile alkylated (via Michael addition) a ring nitrogen adjacent to an exocyclic nitrogen. Since the nitrile group hydrolyzes to a carboxylic acid via an amide intermediate, we had hypothesized in the present study that the same facile hydrolysis of amide to carboxylic acid would occur under identical stereochemical conditions as had occurred with the nitrile group. Thus, in vitro alkylation of calf thymus DNA by both acrylonitrile and, in the present study, AM, resulted in mixed adduct formation.

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 5.0 and/or pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), N6-methyl-2'-deoxyadenosine (N6-Me-dAdo) and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. Adducts were not detected after 4 h. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, 1-CE-N6-Me-dAdo and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. Evidence is presented which strongly suggests that N6-CE-dAdo was formed by Dimroth rearrangement of 1-CE-dAdo during the reaction between AN and dAdo. The carboxyethyl adducts resulted from initial cyanoethylation (by Michael addition) at a ring nitrogen adjacent to an exocyclic nitrogen atom followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is an autocatalyzed reaction resulting from the formation of a cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated were 1-CE-Ade (26%), N6-CE-Ade (8%), 3-CE-Cyt (1%), 7-CNE-Gua (26%), 7,9-bis-CNE-Gua (4%), imidazole ring-opened 7,9-bis-CNE-Gua (19%) and 3-CNE-Thy (16%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts. Three of the adducts (1-CE-Ade, N6-CE-Ade and 3-CE-Cyt) were identical to adducts previously reported by us to be formed following in vitro reaction of the carcinogen beta-propiolactone (BPL) and calf thymus DNA. The results demonstrate that AN can directly alkylate DNA in vitro at a physiological pH and temperature.

Chemical, infrared, and thermal energy analyses have provided evidence for the presence of the N-nitroso functional group in extracts of airborne particles. The total molar N-nitroso concentrations in New York City air are equivalent to the total concentrations of polycyclic aromatic hydrocarbons. Since 90 percent of the N-nitroso compounds that have been tested are carcinogens, the newly discovered but untested materials may represent a significant environmental hazard

The rodent carcinogens dimethylcarbamyl chloride (DMCC) and diethylcarbamyl chloride (DECC) react with dGuo (pH 7.0-7.5, 37 degrees C, 4 h) to form the O6-acyl derivatives 6-dimethylcarbamyloxy-2'-deoxyguanosine (6-DMC-dGuo) and 6-diethylcarbamyloxy-2'-deoxyguanosine (6-DEC-dGuo), respectively. Reaction of DMCC with dThd under identical conditions yielded 4-dimethylamino-thymidine (4-DMA-dThd). Compounds 6-DMC-dGuo and 6-DEC-dGuo undergo a nucleophilic aromatic substitution reaction with dimethylamine (DMA) to form 6-dimethylamino-2'-deoxyguanosine (6-DMA-dGuo) via displacement of the C-6 dialkylcarbamyloxy moiety. The substitution reaction did not take place when diethylamine or NH3 were substituted for DMA. The structures of the new compounds 6-DMC-dGuo, 6-DEC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were deduced from chemical analyses and syntheses, UV and nuclear magnetic resonance (NMR) spectra and electron impact, isobutane chemical ionization and source insertion isobutane chemical ionization mass spectra. It was postulated that 4-DMA-dThd was formed following reaction of the transient intermediate 4-DMC-dThd with DMA formed by hydrolysis of DMCC. Calf thymus DNA was reacted in vitro with DMCC (pH 7.0-7.5, 37 degrees C, 4 h) and the modified DNA hydrolyzed enzymatically to 2'-deoxynucleosides. Compounds 6-DMC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were identified in the hydrolysate by high-pressure liquid chromatography (HPLC). In an identical manner 6-DEC-dGuo was identified following in vitro reaction of DECC with calf thymus DNA. Compounds 6-DEC-dGuo and 6-DMC-dGuo possess novel structures with respect to the types of adducts known to be formed between carcinogens and bases in DNA. The implications of these findings with respect to chemical mutagenesis and carcinogenesis is discussed. The structural relationship between N4-dimethyl-5-methylcytosine (4-dimethylamino-Thy) formed in DNA following in vitro reaction with DMCC and 5-methylcytosine, the only modified base found in vertebrate DNA is noted