Chemical and physical damage to DNA can cause mutations and ultimately cancer, cardiovascular disease, aging, and other diseases (1-4). This damage can result from endogenous agents or exogenous chemicals. Many types of damage are known, and the biological effects can vary considerably. One of the prominent types of damage is alkylation at the O-6 atom of guanine (5, 6). O 6 -AlkylG 4 lesions are some of the more mutagenic lesions formed from DNA-alkylating agents (5, 7). The ability of O 6 -alkylG adducts to cause mutations has been demonstrated directly in site-specific mutagenesis experiments with defined adducts (8 -12). Further support for the view that these are deleterious species derives from the existence of specific DNA repair systems for this type of damage in almost all species, ranging from most bacteria to humans (13,14).Different alkylating agents form O 6 -alkylG adducts, and structure-activity relationships are important for understanding the basic mechanisms of how DNA polymerases function as well as issues such as carcinogenesis. Ϫ and HIV-1 RT (16) and by others with several DNA polymerases, mostly bacterial (7,17,18). Some of the more significant conclusions with pol T7Ϫ and HIV-1 RT were that the bulk of the adduct at the O-6 atom had an inhibitory effect and that an inactive polymerase-oligonucleotide complex is in equilibrium with the functional form (16,19).Studies with pol T7 Ϫ and HIV-1 RT indicated that the effect of size at the N-2 atom is more severe than at the O-6 atom (16, * This work was supported in part by United States Public Health Service Grants R01 CA059887, R01 CA115309 (to L. A. P.), R01 ES010375, and P30 ES000267 (to F. P. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains a MALDI-TOF mass spectrum and capillary gel electrophoresis analysis of the O 6 -PobG 36-mer, an electrophoretic gel showing the lack of extension of an O 6 -PobG:G mispair by pol , and mass spectral analyses used to obtain the results shown in Fig. 7 4 The generic term "alkyl" is used to include both alkyl and aralkyl (Bz) groups for convenience. 5 The abbreviations used are: Bz, benzyl; CID, collision-induced dissociation; dCTP␣S, 2Ј-deoxycytidine 5Ј-O-(1-thiotriphosphate); DTT, dithiothreitol; ESI, electrospray ionization; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization/time-of-flight; MS, mass spectrometry; Pob, 4-oxo-4-(3-pyridyl)butyl; PCNA, proliferating cell nuclear antigen; pol, (DNA) polymerase; pol T7 Ϫ , bacteriophage pol T7 exonuclease-deficient; RT, reverse transcriptase; UDG, uracil DNA glycosylase; HIV-1, human immunodeficiency virus, type 1.

Furan is an environmental chemical that induces liver toxicity and tumor formation in rodents, leading to its classification as a probable human carcinogen. cis-2-Butene-1,4-dial, the metabolite considered responsible for furan's toxicological effects, is mutagenic in the Ames assay and reacts with 2'-deoxycytidine (dCyd), 2'-deoxyadenosine (dAdo), and 2'-deoxyguanosine (dGuo) to form previously characterized diastereomeric adducts. The initially formed dCyd adducts are stable to rearrangement, while the dAdo and dGuo adducts are unstable and rearrange to form secondary products. On the basis of UV absorbance, fluorescence, 1H NMR, and mass spectral data, the rearrangement product of the dAdo adduct was identified as the substituted etheno-dAdo adduct, 1''-[3-(2'-deoxy-beta-D-erythropentafuranosyl)-3H-imidazo[2,1-i]purin-8-yl]ethane-2''-al. The NMR characterization of the O-methyloxime derivative of the secondary dGuo adduct, along with mass spectral and UV data on the underivatized adduct, allowed for its structural assignment as the substituted etheno-dGuo compound, 3-(2'-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(ethane-2''-al)[1,2-alpha]purine-9-one. The characterization of the primary and secondary products formed in the reaction of cis-2-butene-1,4-dial with nucleosides is important for understanding the mechanism of furan-induced carcinogenesis. These secondary adducts retain a reactive aldehyde with the potential to form cross-links and are likely to contribute significantly to furan's toxic and carcinogenic effects.

Furan is a toxic and carcinogenic compound used in industry and commonly found in the environment. The mechanism of furan's carcinogenesis is not well-understood and may involve both genotoxic and nongenotoxic pathways. Furan undergoes oxidation by cytochrome P450 to cis-2-butene-1,4-dial, which is thought to mediate furan's toxic effects. Consistently, cis-2-butene-1,4-dial readily reacts with glutathione, amino acids, and nucleosides. To determine the importance of DNA alkylation in furan-induced carcinogenesis, we developed an assay for the detection of cis-2-butene-1,4-dial-derived DNA adducts. DNA samples were treated with O-benzyl-hydroxylamine, which reacts with the aldehyde functionality of the DNA adducts. Enzyme hydrolysates of these samples were then analyzed by capillary electrospray tandem mass spectrometry with selected reaction monitoring. The dCyd and dAdo adducts were detected in digests of DNA treated with nanomolar concentrations of cis-2-butene-1,4-dial. In addition, these adducts were present in DNA isolated from Ames assay strain TA104 treated with mutagenic concentrations of cis-2-butene-1,4-dial. These data support the hypothesis that cis-butene-1,4-dial is a genotoxic metabolite of furan. This method will allow us to explore the role of these adducts in furan-induced carcinogenesis.

ABSTRACT:Furan is a liver carcinogen and toxicant. Furan is oxidized to the reactive dialdehyde, cis-2-butene-1,4-dial, by microsomal enzymes. This reactive metabolite readily reacts with glutathione nonenzymatically to form conjugates. A high-performance liquid chromatography-electrochemical method for the detection of cis-2-butene-1,4-dial-glutathione (GSH) conjugates in microsomal preparations was developed to measure the extent of furan metabolism to cis-2-butene-1,4-dial in vitro. Previously unobserved mono-GSH reaction products of cis-2-butene-1,4-dial were detected in addition to the already characterized bis-GSH conjugates. Chemical characterization of these compounds indicated that the ␣-amino group of glutathione had reacted with cis-2-butene-1,4-dial to form a thiol-substituted pyrrole adduct. The analytical method was used to estimate the extent of furan oxidation in rat liver microsomes from untreated or acetone-pretreated F344 rats as well as in human P450 2E1 Supersomes. Our results confirm that cytochrome P450 2E1 can catalyze the oxidation of furan to cis-2-butene-1,4-dial. However, the data are also consistent with the involvement of other P450 enzymes in the oxidation of furan in untreated animals. This assay will be a valuable tool to explore tissue and species differences in rates of furan oxidation.