Mauricio Cesar Bof de Oliveira

Biological hydroperoxides and singlet molecular oxygen generation

The decomposition of lipid hydroperoxides (LOOH) into peroxyl radicals is a potential source of singlet molecular oxygen (1O2) in biological systems. Recently, we have clearly demonstrated the generation of 1O2 in the reaction of lipid hydroperoxides with biologically important oxidants such as metal ions, peroxynitrite and hypochlorous acid. The approach used to unequivocally demonstrate the generation of 1O2 in these reactions was the use of an isotopic labeled hydroperoxide, the 18O‐labeled linoleic acid hydroperoxide, the detection of labeled compounds by HPLC coupled to tandem mass spectrometry (HPLC‐MS/MS) and the direct spectroscopic detection and characterization of 1O2 light emission. Using this approach we have observed the formation of 18O‐labeled 1O2 by chemical trapping of 1O2 with anthracene derivatives and detection of the corresponding labeled endoperoxide by HPLC‐MS/MS. The generation of 1O2 was also demonstrated by direct spectral characterization of 1O2 monomol light emission in the near‐infrared region (λ = 1270 nm). In summary, our studies demonstrated that LOOH can originate 1O2. The experimental evidences indicate that 1O2 is generated at a yield close to 10% by the Russell mechanism, where a linear tetraoxide intermediate is formed in the combination of two peroxyl radicals. In addition to LOOH, other biological hydroperoxides, including hydroperoxides formed in proteins and nucleic acids, may also participate in reactions leading to the generation 1O2. This hypothesis is currently being investigated in our laboratory.

Tryptophan Oxidation by Singlet Molecular Oxygen [O2 (1Δg)]: Mechanistic Studies Using 18O-Labeled Hydroperoxides, Mass Spectrometry, and Light Emission Measurements

Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen [O2(1Deltag)]. In this study, two cis- and trans-tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. These photoproducts underwent thermal decay into the corresponding alcohols. Additionally, WOOHs were shown to decompose under heating or basification, leading to the formation of N-formylkynurenine (FMK). Using 18O-labeled hydroperoxides (W18O18OH), it was possible to confirm the formation of two oxygen-labeled FMK molecules derived from W18O18OH decomposition. This result demonstrates that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species.

Characterization of O2 (1Δg)-derived oxidation products of tryptophan: A combination of tandem mass spectrometry analyses and isotopic labeling studies

The fragmentation mechanisms of singlet oxygen [O2 (1Δg)]-derived oxidation products of tryptophan (W) were analyzed using collision-induced dissociation coupled with 18O-isotopic labeling experiments and accurate mass measurements. The five identified oxidized products, namely two isomeric alcohols (trans and cis WOH), two isomeric hydroperoxides (trans and cis WOOH), and N-formylkynurenine (FMK), were shown to share some common fragment ions and losses of small neutral molecules. Conversely, each oxidation product has its own fragmentation mechanism and intermediates, which were confirmed by 18O-labeling studies. Isomeric WOH lost mainly H2O + CO, while WOOH showed preferential elimination of C2H5NO3 by two distinct mechanisms. Differences in the spatial arrangement of the two isomeric WOHs led to differences in the intensities of the fragment ions. The same behavior was also found for trans and cis WOOH. FMK was shown to dissociate by a diverse range of mechanisms, with the loss of ammonia the most favored route. MS/MS analyses, 18O-labeling, and H218O experiments demonstrated the ability of FMK to exchange its oxygen atoms with water. Moreover, this approach also revealed that the carbonyl group has more pronounced oxygen exchange ability compared with the formyl group. The understanding of fragmentation mechanisms involved in O2 (1Δg)-mediated oxidation of W provides a useful step toward the structural characterization of oxidized peptides and proteins.

Mechanism of dioxindolylalanine formation by singlet molecular oxygen-mediated oxidation of tryptophan residues

The singlet molecular oxygen-oxidation of tryptophan generates diastereoisomeric dioxindolylalanine (diOia) along with hydroperoxides, alcohols and carbonyl compounds. Mechanistic investigations based on isotopic labeling and MS/MS analyses support diOia formation through a dioxetane intermediate.

Detection and characterization of cholesterol-oxidized products using HPLC coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry.

Oxidation of cholesterol (Ch) by a variety of reactive oxygen species gives rise mainly to hydroperoxides and aldehydes. Despite the growing interest in Ch-oxidized products, the detection and characterization of these products is still a matter of concern. In this work, the main Ch-oxidized products, namely, 3beta-hydroxycholest-5-ene-7alpha-hydroperoxide (7alpha-OOH), 3beta-5alpha-cholest-6-ene-5-hydroperoxide (5alpha-OOH), 3beta-hydroxycholest-4-ene-6alpha-hydroperoxide (6alpha-OOH), 3beta-hydroxycholest-4-ene-6beta-hydroperoxide (6beta-OOH), and 3beta-hydroxy-5beta-hydroxy-B-norcholestane-6beta-carboxaldehyde (ChAld), were detected in the same analysis using high-performance liquid chromatography (HPLC) coupled to dopant assisted atmospheric pressure photoionization tandem mass spectrometry. The use of selected reaction monitoring mode (SRM) allowed a sensitive detection of each oxidized product, while the enhanced product ion mode (EPI) helped to improve the confidence of the analyses. Isotopic labeling experiments enabled one to elucidate mechanistic features during fragmentation processes. The characteristic fragmentation pattern of Ch-oxidized products is the consecutive loss of H(2)O molecules, yielding cationic fragments at m/z 401, 383, and 365. Homolytic scissions of the peroxide bond are also seen. With (18)O-labeling approach, it was possible to establish a fragmentation order for each isomer. The SRM transitions ratio along with EPI and (18)O-labeled experiments give detailed information about differences for water elimination, allowing a proper discrimination between the isomers. This is of special interest considering the emerging role of Ch-oxidized products in the development of diseases.

DNA strand breaks and base modifications induced by cholesterol hydroperoxides.

Abstract 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 Cu2+. 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.