VISIBLE CHEMILUMINESCENCE ASSOCIATED WITH THE REACTION BETWEEN METHEMOGLOBIN OR OXYHEMOGLOBIN WITH HYDROGEN PEROXIDE

Abstract Visible chemiluminescence is emitted in the irreversible deactivation of hemoglobin or methemoglobin with excess H₂O₂. The emission takes place in two phases. The most intense one lasts a few seconds and is followed by a second phase of lower intensity that remains for longer periods. This second phase presents chaotic or sustained oscillations. Free radicals are implicated in the luminescent process since the emission can be reduced by free radical scavengers such as 6-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid (Trolox) or ascorbic acid. These additives lead to a delay in reaching the maximum intensity, which can be related to their consumption, implying substantial recycling of the hemoprotein. Chemiluminescence is also observed in the oxidation of hemin by H₂O₂, suggesting a role for the heme group in the processes leading to the excited state production. The lower intensity observed in the presence of hemin can be related to the contribution of the globin chains.     

SINGLET OXYGEN INDUCED DNA DAMAGE AND MUTAGENICITY IN A SINGLE-STRANDED SV40-BASED SHUTTLE VECTOR

The effects of singlet oxygen (1O2), generated by the thermal decomposition of water soluble NDPO2 (endoperoxide of the disodium 3,3'-(1,4-naphthylidene) dipropionate), on a single-stranded shuttle vector were analysed. 1O2 induces a much higher level of breaks in the phosphodiester backbone of single-stranded than double-stranded DNA. This may be due to a higher accessibility of guanine residue, primarily damaged by 1O2. The damaged vector was transfected into monkey COS7 cells where single-stranded DNA was converted to the double-stranded replicative form DNA. After 3 days, extrachromosomal DNA was extracted and the plasmids rescued in E. coli to study mutagenesis. There is a significant increase in mutation frequency of damaged single-stranded DNA in comparison to untreated DNA. It is concluded that 1O2 induces breaks in the backbone of single-stranded DNA and that the 1O2-damaged molecules are mutated after passage through mammalian cells.     

Peroxidase Activity May Play a Role in the Cytotoxic Effect of Indole Acetic Acid*

Peroxidase activity in neutrophils is higher than in thioglycollate macrophages, while in lymphocytes this enzyme activity is very low. Indole-3-acetic acid is oxidized by peroxidase and the role of this enzyme in the cytotoxic effect of the compound was evaluated by measuring oxygen consumption, light emission and cell death in neutrophils, macrophages and lymphocytes. The increase in light emission, oxygen consumption and rate of cell death in cells cultured in the presence of indole-3-acetic acid presented a direct correlation with the peroxidase activity of the cells as follows: neutrophils > thioglycollate macrophages > resident macrophages > lymphocytes. Indeed, in lymphocytes that possess very low peroxidase activity, indole-3-acetic acid did not result in an increase in light emission or oxygen consumption and it was not cytotoxic.     

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 [O(2) ((1)Delta(g))]-derived oxidation products of tryptophan (W) were analyzed using collision-induced dissociation coupled with (18)O-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 (18)O-labeling studies. Isomeric WOH lost mainly H(2)O + CO, while WOOH showed preferential elimination of C(2)H(5)NO(3) 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, (18)O-labeling, and H(2)(18)O 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 O(2) ((1)Delta(g))-mediated oxidation of W provides a useful step toward the structural characterization of oxidized peptides and proteins.     

Direct evidence of singlet molecular oxygen [O2(1Deltag)] production in the reaction of linoleic acid hydroperoxide with peroxynitrite

Peroxynitrite (ONOO-), a biologically active species, can induce lipid peroxidation in biological membranes, thereby leading to the formation of various hydroperoxides. We report herein on the formation of singlet molecular oxygen [O(2) ((1)Delta(g))] in the reaction of peroxynitrite with linoleic acid hydroperoxide (LAOOH) or (18)O-labeled LAOOH. The formation of O(2) ((1)Delta(g)) was characterized by (i) dimol light emission in the red spectral region (lambda > 570 nm) using a red-sensitive photomultiplier; (ii) monomol light emission in the near-infrared region (lambda = 1270 nm) with a liquid nitrogen-cooled germanium diode or a photomultiplier coupled to a monochromator; (iii) the enhacing effect of deuterium oxide on chemiluminescence intensity, as well as the quenching effect of sodium azide; and (iv) chemical trapping of O(2) ((1)Delta(g)) or (18)O-labeled O(2) ((1)Delta(g)) with the 9,10-diphenylanthracene (DPA) and detection of the corresponding DPAO(2) or (18)O-labeled DPA endoperoxide by HPLC coupled to tandem mass spectrometry. Moreover, the presence of O(2) ((1)Delta(g)) was unequivocally demonstrated by a direct spectral characterization of the near-infrared light emission attributed to the transition of O(2) ((1)Delta(g)) to the triplet ground state. For the sake of comparison, O(2) ((1)Delta(g)) deriving from the thermolysis of the endoperoxide of 1,4-dimethylnaphthalene or from the H(2)O(2)/hypochlorite and H(2)O(2)/molybdate systems were also monitored. These novel observations identified the generation of O(2) ((1)Delta(g)) in the reaction of LAOOH with peroxynitrite, suggesting a potential O(2) ((1)Delta(g))-dependent mechanism that contributes to cytotoxicity mediated by lipid hydroperoxides and peroxynitrite reactions in biological systems.     

Generation of Singlet Molecular Oxygen by Lipid Hydroperoxides and Nitronium Ion†

Singlet molecular oxygen is a reactive species involved in biological oxidative processes. The major cellular targets of singlet molecular oxygen are unsaturated fatty acids in the membrane, as well as nucleic acids and proteins. The aim of this study was to investigate whether lipids and commercial hydroperoxides generate singlet molecular oxygen, in presence of nitronium and activated nitronium ion. For this purpose, monomol light emitted in the near-infrared region (λ = 1270 nm) was used to monitor singlet molecular oxygen decay in different solvents, with different hydroperoxides and in the presence of azide. Direct measurements of the singlet molecular oxygen spectrum at 1270 nm recorded during the reaction between lipids and commercial hydroperoxides and nitronium ions unequivocally demonstrated the formation of this excited species.     

Photochemically generated stable cation radical of phenothiazine aggregates in mildly acid buffered solutions

This work characterizes, for the first time, the photochemical behavior of the antipsychotic drugs thioridazine (TR), trifluoperazine (TFP), and fluphenazine (FP) influenced by the aggregation state of the molecules. Samples of monomeric and aggregated forms of phenothiazines were submitted to 20 min of irradiation at 254 nm for intervals of 1, 5, 10, 15, 20, or 25 days. In high phenothiazine concentrations, the irradiation led to the appearance of absorbance bands in the visible region peaking at 633 nm for TR and 509 nm for FP and TFP. In the dark, at room temperature and at 4 degrees C, these bands disappeared, after approximately 15 and approximately 60 min, respectively, but reappeared after a new irradiation session. These visible bands were assigned to stable cation radicals that were characterized by direct EPR measurements and by flash photolysis. Photogenerated stable cation radicals in the phenothiazine aggregates at room temperature are formed probably due to the stacking of the thiazine phenyl moieties. For the monomeric forms of phenothiazines, the spectral changes observed during the irradiation suggested the formation of sulfoxide and hydroxylated derivates. Oxidized derivates were detected by mass spectrometry of the aggregated forms of phenothiazines (>100 microM) only in the samples irradiated for more than 20 days. In contrast, monomeric phenothiazines were totally converted to the oxidized forms after 20 min of irradiation. Surface tension measurements of phenothiazines revealed that, in concentrations above 100 microM, the drugs formed aggregates. In the case of TR, small-angle X-ray scattering measurements indicated that this compound forms large lamellar-like aggregates in aqueous solutions.     

13C2]-Acetaldehyde promotes unequivocal formation of 1,N2-propano-2'-deoxyguanosine in human cells

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.