Role of aldehydes in the toxic and mutagenic effects of nitrosamines.

α-Hydroxynitrosamine metabolites of nitrosamines decompose to a reactive diazohydroxide and an aldehyde. To test the hypothesis that the aldehydes contribute to the harmful effects of nitrosamines, the toxic and mutagenic activities of three model methylating agents were compared in Chinese hamster ovary cells expressing or not expressing human O6-alkylguanine DNA alkyltransferase (AGT). N-Nitrosomethylurethane (NMUr), acetoxymethylmethylnitrosamine (AMMN), and 4-(methylnitrosamino)-4-acetoxy-1-(3-pyridyl)-1-butanone (NNK-4-OAc) are all activated by ester hydrolysis to methanediazohydroxide. NMUr does not form an aldehyde, whereas AMMN generates formaldehyde, and NNK-4-OAc produces 4-oxo-1-(3-pyridyl)-1-butanone (OPB). Since these compounds were likely to alkylate DNA to different extents, the toxic and mutagenic activities of these compounds were normalized to the levels of the most cytotoxic and mutagenic DNA adduct, O6-mG, to assess if the aldehydes contributed to the toxicological properties of these methylating agents. Levels of 7-mG indicated that the differences in cytotoxic and mutagenic effects of these compounds resulted from differences in their ability to methylate DNA. When normalized against the levels of O6-mG, there was no difference between these three compounds in cells that lacked AGT. However, AMMN and NNK-4-OAc were more toxic than NMUr in cells expressing AGT when normalized against O6-mG levels. In addition, AMMN was more mutagenic than NNK-4-OAc and MNUr in these cells. These findings demonstrate that the aldehyde decomposition products of nitrosamines can contribute to the cytotoxic and/or mutagenic activity of methylating nitrosamines.

Translesion synthesis across O6-alkylguanine DNA adducts by recombinant human DNA polymerases.

Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O(6)-methyl and -benzyl (Bz) G. Recombinant human polymerase (pol) delta also copied past these two lesions but was totally blocked by O(6)-[4-oxo-4-(3-pyridyl)butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. The human translesion pols iota and kappa produced mainly only 1-base incorporation opposite O(6)-MeG and O(6)-BzG and had very low activity in copying O(6)-PobG. Human pol eta copied past all three adducts. Steady-state kinetic analysis showed similar efficiencies of insertion opposite the O(6)-alkylG adducts for dCTP and dTTP with pol eta and kappa; pol iota showed a strong preference for dTTP. pol eta, iota, and kappa showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O(6)-MeG but little, if any, for O(6)-BzG or O(6)-PobG. Analysis of the pol eta O(6)-PobG products indicated that the insertion of G was opposite the base (C) 5' of the adduct, but this product was not extended. Mass spectrometry analysis of all of the pol eta primer extension products indicated multiple components, mainly with C or T inserted opposite O(6)-alkylG but with no deletions in the cases of O(6)-MeG and O(6)-PobG. With pol eta and O(6)-BzG, products were also obtained with -1 and -2 deletions and also with A inserted (opposite O(6)-BzG). The results with pol eta may be relevant to some mutations previously reported with O(6)-alkylG adducts in mammalian cells.

Identification of a cis-2-butene-1,4-dial-derived glutathione conjugate in the urine of furan-treated rats.

The hepatocarcinogen and toxicant furan requires metabolic activation to elicit its toxic effects. The available experimental evidence indicates that the overall metabolism of furan is initiated via cytochrome P450 catalyzed oxidation to cis-2-butene-1,4-dial. This alpha,beta-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 monoglutathione 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.

Formation of 1,4-dioxo-2-butene-derived adducts of 2'-deoxyadenosine and 2'-deoxycytidine in oxidized DNA.

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 beta-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 nonspecific DNA oxidant, gamma-radiation, also produced the adducts that represented approximately 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.

DNA sequence context affects repair of the tobacco-specific adduct O(6)-[4-Oxo-4-(3-pyridyl)butyl]guanine by human O(6)-alkylguanine-DNA alkyltransferases.

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.

Detection of DNA adducts derived from the reactive metabolite of furan, cis-2-butene-1,4-dial.

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.

Glutathione trapping to measure microsomal oxidation of furan to cis-2-butene-1,4-dial.

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 alpha-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.

Synthesis of a 2'-deoxyguanosine adduct of cis-2-butene-1,4-dial, a reactive metabolite of furan.

Furan is a liver and kidney toxicant and a hepatocarcinogen in rodents. Its reactive metabolite, cis-2-butene-1,4-dial, reacts with nucleosides to form adducts in vitro. The reaction with 2'-deoxyguanosine generates 3-(2'-deoxy-beta-D-erythropentafuranosyl)-3,5,6,7-tetrahydro-6-hydroxy-7-(ethane-2"-al)-9H-imidazo[1,2-alpha]purine-9-one as the major reaction product. A synthetic approach to this adduct is presented in this report. The key step in this synthesis is the preparation of 2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-1-(1,2,5,6-tetrahydroxyhexan-3-yl)guanosine. Treatment of this intermediate with sodium periodate gave three reaction products: a one-substituted adduct, 2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-1-(2,5-dihydroxy-tetrahydrofuran-3-yl)guanosine; a 1,N(2)-cyclic adduct, 3-[2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-beta-D-erythropentafuranosyl]-6-hydroxy-8-formyl-5,6,7,8-tetrahydropyrimidino[1,2-alpha]purin-10(3H)-one; and the 1,N(2)-bicyclic adduct, 3-[2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-beta-D-erythropentafuranosyl]-3,5,6,7-tetrahydro-6-hydroxy-7-(ethane-2"-al)-9H-imidazo[1,2-alpha]purine-9-one. The one-substituted and 1,N(2)-cyclic reaction products were unstable and rearranged over time to yield the 1,N(2)-bicyclic 2'-deoxyguanosine adducts. The desired reaction product was obtained as a mixture of four diastereomers by removing the tert-butyldimethylsilyl groups with hydrogen fluoride. This synthetic approach to the cis-2-butene-1,4-dial-derived dGuo adducts confirms our previous structural characterization of the in vitro cis-2-butene-1,4-dial-dGuo reaction product. These studies demonstrate that the observed 1,N(2) bicyclic structure is the thermodynamically stable isomer, supporting our previous observations that this adduct is the major product formed in vitro. Finally, these studies provide the necessary groundwork for the preparation of oligonucleotides with site specifically incorporated cis-2-butene-1,4-dial-derived adducts.

O6-pyridyloxobutylguanine adducts contribute to the mutagenic properties of pyridyloxobutylating agents.

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.

The formation of substituted 1,N6-etheno-2'-deoxyadenosine and 1,N2-etheno-2'-deoxyguanosine adducts by cis-2-butene-1,4-dial, a reactive metabolite of furan.

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.

Solution structure of an O6-[4-oxo-4-(3-pyridyl)butyl]guanine adduct in an 11 mer DNA duplex: evidence for formation of a base triplex.

The pyridyloxobutylating agents derived from metabolically activated tobacco-specific nitrosamines can covalently modify guanine bases in DNA at the O(6) position. The adduct formed, O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine ([POB]dG), results in mutations that can lead to tumor formation, posing a significant cancer risk to humans exposed to tobacco smoke. A combined NMR-molecular mechanics computational approach was used to determine the solution structure of the [POB]dG adduct within an 11mer duplex sequence d(CCATAT-[POB]G-GCCC).d(GGGCCATATGG). In agreement with the NMR results, the POB ligand is located in the major groove, centered between the flanking 5'-side dT.dA and the 3'-side dG.dC base pairs and thus in the plane of the modified [POB]dG.dC base pair, which is displaced slightly into the minor groove. The modified base pair in the structure adopts wobble base pairing (hydrogen bonds between [POB]dG(N1) and dC(NH4) amino proton and between [POB]dG(NH2) amino proton and dC(N3)). A hydrogen bond appears to occur between the POB carbonyl oxygen and the partner dC's second amino proton. The modified guanine purine base, partner cytosine pyrimidine base, and POB pyridyl ring form a triplex via this unusual hydrogen-bonding pattern. The phosphodiester backbone twists at the lesion site, accounting for the unusual phosphorus chemical shift differences relative to those for the control DNA duplex. The helical distortions and wobble base pairing induced by the covalent binding of POB to the O(6)-position of dG help explain the significant decrease of 17.6 degrees C in melting temperature of the modified duplex relative to the unmodified control.