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Acetaldehyde and crotonaldehyde are genotoxic aldehydes present in tobacco smoke and vehicle exhaust. The reaction of these aldehydes with 2'-deoxyguanosine in DNA produces α-methyl-γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo). Online HPLC-tandem mass spectrometry was utilized to accurately quantify 1,N(2)-propanodGuo in human urinary samples from 47 residents of São Paulo City (SP) and 35 residents of the rural municipality of São João da Boa Vista (SJBV) in the state of São Paulo. Significantly higher 1,N(2)-propanodGuo levels were found in the samples from SP donors than in samples from SJBV donors. Our results provide the first evidence that elevated levels of 1,N(2)-propanodGuo in urinary samples may be correlated with urban air pollution.

Acetaldehyde is an environmentally widespread genotoxic aldehyde present in tobacco smoke, vehicle exhaust and several food products. Endogenously, acetaldehyde is produced by the metabolic oxidation of ethanol by hepatic NAD-dependent alcohol dehydrogenase and during threonine catabolism. The formation of DNA adducts has been regarded as a critical factor in the mechanisms of acetaldehyde mutagenicity and carcinogenesis. Acetaldehyde reacts with 2'-deoxyguanosine in DNA to form primarily N(2)-ethylidene-2'-deoxyguanosine. The subsequent reaction of N(2)-ethylidenedGuo with another molecule of acetaldehyde gives rise to 1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo), an adduct also found as a product of the crotonaldehyde reaction with dGuo. However, adducts resulting from the reaction of more than one molecule of acetaldehyde in vivo are still controversial. In this study, the unequivocal formation of 1,N(2)-propanodGuo by acetaldehyde was assessed in human cells via treatment with [(13)C(2)]-acetaldehyde. Detection of labeled 1,N(2)-propanodGuo was performed by HPLC/MS/MS. Upon acetaldehyde exposure (703 μM), increased levels of both 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-εdGuo), which is produced from α,β-unsaturated aldehydes formed during the lipid peroxidation process, and 1,N(2)-propanodGuo were observed. The unequivocal formation of 1,N(2)-propanodGuo in cells exposed to this aldehyde can be used to elucidate the mechanisms associated with acetaldehyde exposure and cancer risk.

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after the incubation of 2'-deoxyguanosine (dGuo) with ChOOH and Cu(2+). In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) and 1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo) in DNA. This methodology permits direct online quantification of 2'-deoxyguanosine and ca. 500 amol of adducts in 100 microg of hydrolyzed DNA in the same analysis. Using the newly developed technique, accurate determinations of 1,N(2)-etheno-2'-deoxyguanosine and 1,N(2)-propano-2'-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 +/- 0.32 1,N(2)-epsilondGuo/10(8) dGuo and 3.43 +/- 0.33 1,N(2)-propanodGuo/10(8) dGuo) and rat tissue (liver, 2.47 +/- 0.61 1,N(2)-epsilondGuo/10(8) dGuo and 4.61 +/- 0.69 1,N(2)-propanodGuo/10(8) dGuo; brain, 2.96 +/- 1.43 1,N(2)-epsilondGuo/10(8) dGuo and 5.66 +/- 3.70 1,N(2)-propanodGuo/10(8) dGuo; and lung, 0.87 +/- 0.34 1,N(2)-epsilondGuo/10(8) dGuo and 2.25 +/- 1.72 1,N(2)-propanodGuo/10(8) dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental animals with pathological conditions and after air pollution exposure.

A ruthenium oxide hexacyanoferrate (RuOHCF) modified electrode was developed. Hydrodynamic voltammetry was employed to demonstrate the remarkable electrocatalytic activity toward the oxidation of 2'-deoxyguanosine. The RuOHCF modified electrode was used as amperometric detector for 2'-deoxyguanosine determination in a FIA apparatus. The influence of various experimental conditions was explored for optimum analytical performance, and at these experimental conditions, the method exhibited a linear response range to 2'-deoxyguanosine extending from 3.8 to 252 micromol L(-1) with detection limit of 94 nmol L(-1). Applications in DNA samples were examined, and the results for determination of 2'-deoxyguanosine were in good agreement with those obtained by HPLC analysis. Studies on the kinetics of the in vitro consumption of 2'-deoxyguanosine by acetaldehyde were also performed.

S(IV) (SO(2),HSO(3)(-)andSO(3)(2-)) autoxidation catalyzed by Cu(II)/tetraglycine complexes in the presence of DNA or 2'-deoxyguanosine (dGuo) resulted in DNA strand breaks and formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. Ni(II), Co(II) or Mn(II) (1.0x10(-4)M) complexes had much smaller effects. Cu(II)/tetraglycine (1.0x10(-4)M) in the presence of Ni(II) or Mn(II) (10(-7)-10(-6)M) and S(IV) showed remarkable synergistic effect with these metal ions producing a higher yield of 8-oxodGuo. Oxidation of dGuo and DNA damage were attributed to oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by transition metal ions. SO*(3)(-) and HO* radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline-N-oxide).

A recent study showed that tetrahydrofuran (THF), a widely used solvent, is carcinogenic in experimental animals. Despite its carcinogenic activity, there is a paucity of information regarding cellular toxicity, biomolecular damage, and genotoxicity induced by THF. We describe here the structural characterization of adducts produced by the reaction of oxidized THF with 2'-deoxyguanosine (dGuo-THF 1 and dGuo-THF 2), 2'-deoxyadenosine (dAdo-THF), and 2'-deoxycytidine (dCyd-THF). Adducts were isolated from in vitro reactions by reverse-phase HPLC and fully characterized on the basis of spectroscopic measurements. The stable derivatives obtained by the reduction of adducts with NaBH(4) (the case of dGuo-THF 1, dCyd-THF, and dAdo-THF) and the stable adduct dGuo-THF 2 were used as standards for optimization of chromatographic separations for adduct detection in DNA through HPLC/ESI/MS-MS. Using this methodology, we successfully detected the four adducts in calf thymus DNA reacted with oxidized THF. The present study also provides evidence that rat liver microsomal monooxigenases oxidize THF to the reactive electrophilic compounds that are able to damage the DNA molecule, as indicated by a significant increase in adduct dGuo-THF 1 level when NADPH was added to the THF/microsomes/dGuo incubation mixtures. Our data point to DNA-THF adducts as possible contributing factors to the toxicological effects of THF exposure.

It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides.

The reaction of 2'-deoxyguanosine with the alpha,beta-unsaturated aldehydes trans-2-octenal, trans-2-nonenal, trans-2-decenal, trans,trans-2,4-nonadienal, and trans,trans-2,4-decadienal in THF gives rise to three novel adducts: 3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-7-[3-hydroxy-1-(3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-3,5-dihydro-imidazo[1,2-a]purin-9-one-7-yl)-propyl]-3,5-dihydro-imidazo[1,2-a]purin-9-one (A7) and 3-(2'-deoxy-beta-D-erythro-pentafuranosyl)-7-(tetrahydrofuran-2-yl)-3,5-dihydro-imidazo[1,2-a]purin-9-one (A8 and A9), which are not observed in the absence of THF. These adducts were isolated from in vitro reactions by reversed-phase HPLC and fully characterized on the basis of spectroscopic measurements. Adduct A7 consists of two 1,N2-etheno-2'-deoxyguanosine (1,N2-epsilon dGuo) residues linked to a hydroxy-carbon side chain; adducts A8 and A9 are interconvertible 1,N2-epsilon dGuo derivatives bearing a THF moiety. The proposed reaction mechanism involves the electrophilic attack on 1,N2-epsilon dGuo by the carbonyl of 4-hydroxy-butanal, generated via ring opening of alpha-hydroxy-THF (THF-OH), yielding adducts A8 and A9. A further combination of these adducts with another 1,N2-epsilon dGuo produces the double adduct A7. These findings demonstrate that reactions of unsaturated aldehydes in the presence of THF produce novel condensation 1,N2-epsilon dGuo-THF adducts. Further studies would indicate the relevance of these adducts in THF toxicity.

The present paper focussed on the mechanistic aspect of the reaction of singlet oxygen (1O2), in its lowest excited state (1 Delta g), toward DNA constituents. It is well known that 1O2 is able to react with the guanine moiety of DNA to produce almost specifically 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). However, when 2'-deoxyguanosine (dGuo), free in solution, is the substrate, additional modified nucleosides are detected upon 1O2-mediated oxidation. The combined use of the thermolysis of a water-soluble naphthalene endoperoxide as a generator of 18O-singlet oxygen and the sensitivity of electrospray ionization-tandem mass spectrometry has allowed us to better understand the reactivity of 1O2 toward dGuo and 8-oxodGuo. As a striking observation, the secondary oxidation reaction of 8-oxodGuo, a main 1O2 oxidation product of dGuo, may explain, at least partly, the observed mutagenicity of 1O2.

Epidemiological studies testing the effect of beta-carotene in humans have found a relative risk for lung cancer in smokers supplemented with beta-carotene. We investigated the reactions of retinal and beta-apo-8'-carotenal, two beta-carotene oxidation products, with 2'-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N(2)-etheno-2'-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with beta-carotene or beta-carotene oxidation products, significantly increased levels of 1,N(2)-etheno-2'-deoxyguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of beta-carotene oxidation products.

A method involving on-line reversed-phase high-performance liquid chromatography with electrospray tandem mass spectrometry detection has been developed for the analysis of 1,N(2)-etheno-2'-deoxyguanosine in DNA. This methodology permits direct quantification of 20 fmol (7.4 adducts/10(8) dGuo) of the etheno adduct from approximately 350 microg of crude DNA hydrolysate. Using the newly developed technique, basal levels of 1,N(2)-etheno-2'-deoxyguanosine were determined in commercial calf thymus DNA (1.70 +/- 0.09 adducts/10(7) dGuo), in cultured mammalian cells (CV1-P) DNA (4.5 +/- 0.4 adducts/10(7) dGuo), and in untreated female rat liver DNA (5.22 +/- 1.37 adduct/10(7) dGuo). The mutagenicity of 1,N(2)-etheno-2'-deoxyguanosine had already been demonstrated by in vitro and in vivo systems. The method described here provides the first evidence of the occurrence of 1,N(2)-etheno-2'-deoxyguanosine as a basal endogenous lesion and may be usefully employed to assess the biological consequences of etheno DNA damage under normal and pathological conditions.

5-Aminolevulinic acid (ALA) is a heme precursor that accumulates in acute intermittent porphyria (AIP) due to enzymatic deficiencies in the heme biosynthetic pathway Its accumulation has been associated with several symptoms, such as abdominal pain attacks, neuromuscular weaknesses, neuropsychiatric alterations and increased hepatocellular carcinoma (HCC) incidence. The use of exogenous ALA to elevate porphyrin levels in tumor photodynamic therapy, adds further significance to ALA toxicology. Under ferritin mediated and metal catalyzed oxidation, ALA produces reactive oxygen species that can damage plasmid and isolated DNA in vitro, and increases the steady-state level of 8-oxo-7,8-dihydro-2'-deoxyguanosine in liver, spleen and kidney DNA and 5-hydroxy-2'-deoxycytidine in liver DNA of ALA-treated rats. The in vitro DNA damage could be partially inhibited by SOD, catalase, DTPA, mannitol and melatonin. ALA also promotes the formation of radical-induced base degradation products in isolated DNA. 4,5-Dioxovaleric acid, the final oxidation product of ALA, alkylates guanine moieties within both nucleoside and isolated DNA, producing two diastereoisomeric adducts. Dihydropyrazine derivatives of ALA generated by its dimerization, promote DNA strand-breaks and 8-oxodGuo formation in the presence of Cu2+. Together these results reinforce the hypothesis that the DNA damage induced by ALA may be associated with the development of HCC in individuals suffering from AIP.