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.

The repair protein O 6 -alkylguanine-DNA alkyltransferase (AGT) protects cells from the mutagenic and carcinogenic effects of alkylating agents by removing O 6 -alkylguanine adducts from DNA. Recently, we established that AGT protects against the mutagenic effects of pyridyloxobutylation resulting from the metabolic activation of the tobacco-specific nitrosamines (TSNA) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosonornicotine by repairing O 6 -[4-oxo-4-(3-pyridyl)butyl]guanine (O 6 -pobG). There have been several epidemiologic studies examining the association between the I143V/K178R AGT genotype and lung cancer risk. Two studies have found positive associations, suggesting that AGT proteins differ in their repair of DNA damage caused by TSNA. However, it is not known how this genotype alters the biochemical activity of AGT. We proposed that AGT proteins may differ in their ability to remove large O 6 -alkylguanine adducts, such as O 6 -pobG, from DNA. Therefore, we examined the repair of O 6 -pobG by wild-type (WT) human, I143V/K178R, and L84F AGT proteins when contained in multiple sequence contexts, including the twelfth codon of H-ras, a mutational hotspot within this oncogene. The AGT-mediated repair of O 6 -pobG was more profoundly influenced by sequence context than that of O 6 -methylguanine. These differences are not the result of secondary structure (hairpin) formation in DNA. In addition, the I143V/K178R variant seems less sensitive to the effects of sequence context than the WT or L84F proteins. These studies indicate that the sequence dependence of O 6 -pobG repair by human AGT (hAGT) varies with subtle changes in protein structure. These data establish a novel functional difference between the I143V/K178R protein and other hAGTs in the repair of a toxicologically relevant substrate, O 6 -pobG.

The hepatocarcinogen and toxicant, furan, requires metabolic activation to elicit its toxic effects. The available experimental evidence indicate that the overall metabolism of furan is initiated via cytochrome P450 catalyzed oxidation to cis-2-butene-1,4-dial. This α,β-unsaturated dialdehyde reacts in vitro with protein and DNA nucleophiles. To determine if this compound is an in vivo intermediate in the metabolism of furan, rats were treated with either [ 12 C 4 ]furan or [ 13 C 4 ]furan and urine was collected for 24 h. Capillary LC/MS/MS analysis of the urine indicated that one of the metabolites was a mono-glutathione conjugate of cis-2-butene-1,4-dial. These results indicate that glutathione conjugation of the reactive metabolite of furan occurs in vivo. This metabolite may serve as a useful marker for furan exposure and metabolism in risk assessment studies.

The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are potent carcinogens in animal models and likely human carcinogens. Both NNK and NNN can be activated to a pyridyloxobutylating agent. This alkylating agent contributes to the carcinogenic effects of NNK and NNN via the formation of miscoding DNA adducts. One of these adducts, O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG) has been characterized as a mutagenic adduct which is a substrate for the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). Repair of O6-alkylguanine adducts by AGT protects cells from the mutagenic and carcinogenic effects of alkylating agents and is likely to play a similar role in shielding cells from the adverse effects of pyridyloxobutylating agents. Therefore, we examined the mutagenicity of the model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc), in Salmonella typhimurium YG7108 expressing hAGT. Expression of hAGT protected cells from NNKOAc-induced mutagenicity. Interestingly, hAGT did not shield cells from the toxicity of this agent. To confirm that the repair of O6-pobG was increased in the bacteria expressing hAGT, we measured levels of this adduct in NNKOAc-treated cultures. The levels of O6-pobG were lower in DNA from bacteria expressing hAGT. This work establishes an important role for O6-pobG in mediating the mutagenic, and possibly carcinogenic, effects of pyridyloxobutylating compounds.

Oxidation of deoxyribose in DNA produces a variety of electrophilic residues that are capable of reacting with nucleobases to form adducts such as M 1 dG, the pyrimidopurinone adduct of dG. We now report that deoxyribose oxidation in DNA leads to the formation of oxadiazabicyclo(3.3.0) octaimine adducts of dC and dA. We previously demonstrated that these adducts arise in reactions of nucleosides and DNA with trans-1,4-dioxo-2-butene, the β-elimination product of the 2-phosphoryl-1,4-dioxobutane residue arising from 5′-oxidation of deoxyribose in DNA and with cis-1,4-dioxo-2-butene, a metabolite of furan. Treatment of DNA with enediyne antibiotics capable of oxidizing the 5′-position of deoxyribose (calicheamicin and neocarzinostatin) led to a concentration-dependent formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA, while the antibiotic bleomycin, which is capable of performing only 4-oxidation of deoxyribose, did not give rise to the adducts. The non-specific DNA oxidant, γ-radiation, also produced the adducts that represented ~0.1% of the 2-phosphoryl-1,4-dioxobutane residues formed during the irradiation. These results suggest that the oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA could represent endogenous DNA lesions arising from oxidative stresses that also give rise to other DNA adducts.