Spin Trap DMPO Research Articles

Photoactivity tests of TiO2 and ZnO sunscreen ingredients

ABSTRACTNanosized titanium dioxide (TiO2) and zinc oxide (ZnO) are widely used as inorganic sunscreen pigments. However, these metal oxide particles may also be photocatalytic and generate DNA-damaging reactive oxygen species. Therefore, we evaluated the photochemical properties of the whole sunscreen emulsions that contained nanoscale components and the inorganic particles derived from these sunscreens using several assays such as dichlorofluorescein fluorescence, the decolorization of Congo red dye and DMPO spin trap electron paramagnetic resonant spectroscopy. The results of these three tests showed that samples with ZnO nanoscale materials were more photoactive than the samples that contained TiO2 nanoparticles.

Use of EPR and FTIR to detect biological effects of ultrasound and microbubbles on a fibroblast cell line

Structural and functional effects of exposing murine fibroblasts (NIH 3T3) to therapeutic ultrasound at 1MHz frequency are described. These bioeffects can be attributed to the formation of free radical species by sonolysis of water. When cavitation occurs, dissociation of water vapor into H atoms and OH radicals is observed; these H atoms and OH radicals combine to form H(2), H(2)O(2), and HO(2). The radicals can chemically modify biomolecules, for example enzymes, DNA, and lipids. Generation of free radicals during exposure to ultrasound with or without encapsulated microbubbles (contrast agents) was studied by use of electron paramagnetic resonance with DMPO spin trapping. Recently the potential for possible use of these microbubbles in gene therapy has been investigated, because of the ability of the stabilized microbubbles to release their content when exposed to ultrasound. Structural changes were studied by Fourier-transform infrared spectroscopy, and induction of possible genotoxic damage by exposure of the cells to therapeutic ultrasound at 1MHz frequency with our experimental device was verified by use of the cytokinesis-block micronucleus assay.

Effect of microwave radiation on the (Raman) lattice phonons in selected titanium dioxide solid specimens

The effects of 2.45-GHz microwave radiation on the Raman-active lattice phonons of two selected (Wako) anatase and rutile specimens and on Degussa P-25 TiO 2 nanoparticles were probed by in situ microscopic Raman spectroscopy in the solid phase with samples subjected to microwave irradiation (MW) or in combination with UV illumination (UV/MW). Significant changes were seen in the Raman band intensities for the pure anatase E g mode at 143 cm −1 and for the rutile vibration at 446 cm −1, whereas only negligible changes in intensity were observed for the 144 cm −1 band of P-25 nanoparticles exposed to MW and UV/MW radiation. It is deduced that microwaves have a negligible impact on the lattice vibrational modes (phonons) of P-25 titania, even though this specimen was thermally heated by the microwaves. The photodegradation of phenol was re-visited to examine the photoactivity of the TiO 2 specimens selected for the in situ Raman study, as attested by the number of OH radicals produced (DMPO spin trap ESR) under UV versus UV/MW irradiations, and by the related enhanced dynamics under UV/MW irradiation relative to UV alone or UV/CH.

Photoinduced reactivity of Au–H intermediates in alcohol oxidation by gold nanoparticles supported on ceria

H-Au-np/CeO2 species, that are important intermediates in the alcohol oxidation by gold nanoparticles, show a photoinduced reactivity. UV-visible photoexcitation in deaerated conditions causes a hydride transfer from gold to the spin trap DMPO. The photoassisted breaking of the Au–H bond restores the catalyst in its active form. As a consequence, photochemical excitation has a positive effect on the catalytic oxidation of 2-pentanol under anaerobic conditions, demonstrating that light can be used in place of oxygen. This new photocatalytic activity of H-Au-np/CeO2 species is of general interest in the field of catalysis with gold nanoparticles, since the photoinduced hydride shift from gold surface to organic substrates opens up exciting possibilities to use alcohols for hydrogenation reactions.

Enhanced catalytic degradation of AO7 in the CeO2–H2O2 system with Fe3+ doping

Degradation of Acid Orange 7 (AO7) was employed to evaluate the catalytic activity of the Fe3+ doped CeO2 in the presence of H2O2. Fe3+ doping in the ceria improved the degradation of AO7 in the dark as well as under the visible light irradiation and showed the highest catalytic activity at Fe/Ce=1/100 (FC100). XPS, Raman and EPR studies showed that Fe3+ doping in the CeO2 greatly affected the concentration of Ce3+. FC100 was found to have the highest Ce3+ concentration. Raman and EPR studies confirmed the presence of surface peroxide-like species, which was derived from the complex of the surface Ce3+ and H2O2. DMPO spin trapping EPR studies indicated that peroxide-like species played a key role during the dark-reaction, while OH radical induced the degradation of AO7 under visible irradiation. The amounts of both active species were remarkably increased in the FC100/H2O2 system than those in the CeO2/H2O2 system.

Bisphenol A-glycidyl methacrylate induces a broad spectrum of DNA damage in human lymphocytes

Bisphenol A-glycidyl methacrylate (BisGMA) is monomer of dental filling composites, which can be released from these materials and cause adverse biologic effects in human cells. In the present work, we investigated genotoxic effect of BisGMA on human lymphocytes and human acute lymphoblastic leukemia cell line (CCRF-CEM) cells. Our results indicate that BisGMA is genotoxic for human lymphocytes. The compound induced DNA damage evaluated by the alkaline, neutral, and pH 12.1 version of the comet assay. This damage included oxidative modifications of the DNA bases, as checked by DNA repair enzymes EndoIII and Fpg, alkali-labile sites and DNA double-strand breaks. BisGMA induced DNA-strand breaks in the isolated plasmid. Lymphocytes incubated with BisGMA at 1 mM were able to remove about 50% of DNA damage during 120-min repair incubation. The monomer at 1 mM evoked a delay of the cell cycle in the S phase in CCRF-CEM cells. The experiment with spin trap—DMPO demonstrated that BisGMA induced reactive oxygen species, which were able to damage DNA. BisGMA is able to induce a broad spectrum of DNA damage including severe DNA double-strand breaks, which can be responsible for a delay of the cell cycle in the S phase.

Abstract 5209: Oxidative Stress Uncouples eNOS in a BH4-independent Manner via the Formation of Protein Thiol-radicals

Imbalance of nitric oxide (NO) and superoxide (·O 2 − ) production in the endothelium contributes to many cardiovascular diseases, including hypertension, atherosclerosis, and heart failure. Under conditions of oxidative stress, NO synthase (NOS) can switch from NO to ·O 2 − generation. We have demonstrated that endothelial NOS (eNOS) is reversibly S-glutathiolated in vivo and in vitro , in response to oxidative stress. This redox modification of eNOS leads to uncoupling, with decreased eNOS-derived NO and increased ·O 2 − . There are several proposed mechanisms that can lead to protein S-glutathiolation: thiol-disulfide exchange with GSSG; formation of thiyl radicals, sulphenic acid, or protein-S-nitrosothiols, which in turn react with GSH. Here we demonstrate the oxidant induced formation of an eNOS protein thiyl radical using immunoblotting, immunostaining, and mass spectrometry. BH 4 -free eNOS was used to self generate ·O 2 − , which in turn can modify sulfhydryl groups to form protein radicals. We trapped these short lived protein radicals with the spin trap DMPO. Immunoblotting using an anti-DMPO antibody, demonstrated the formation of eNOS protein radicals, which were abolished by SOD and L-NAME, and were Ca 2+ /CaM sensitive, indicating that protein radical formation is due to ·O 2 − generation from the eNOS heme domain. With BH 4 reconstituted eNOS, which will only generate NO, formation of the eNOS protein radical is completely inhibited. Furthermore, in endothelial cells treated with menadione to trigger cellular ·O 2 − generation, formation of eNOS protein radical as visualized with confocal microscopy was increased compared to control, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Using mass spectrometric analysis, we identified Cys908 as the only residue involved in protein radical formation. Of note, we have also demonstrated the oxidant-induced S-glutathiolation of Cys908. Thus, eNOS protein radical formation provides the groundwork for a mechanism of ·O 2 − -directed regulation of eNOS, involving S-glutathiolation; defining a unique pathway for the redox regulation of cardiovascular function. This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).

Hydrogen Peroxide Is a Second Messenger in Phase 2 Enzyme Induction by Cancer Chemopreventive Dithiolethiones

The ability of three dithiolethione cancer chemopreventives, oltipraz 1, anetholedithione (ADT) 2, 1,2-dithiole-3-thione (D3T) 3, and the major metabolite, 4, of 1, to induce the cytoprotective enzyme NQO1 in Hepa 1c1c7 cells and the inhibition of this induction by catalase are demonstrated. The ability of 1, 3, and 4 to form O(2)(*) has been reported, and it is here demonstrated that 2 decomposes in the presence of GSH to form, upon addition of the nitrone spin trap DMPO, the DMPO-OH adduct that is detectable by EPR. Decomposition of 2 in the presence of GSH elicits, upon the addition of hydroethidine and excitation at 510 nm, fluorescence at 580 nm that is diminished by the addition of superoxide dismutase. The compound 4, is a product of the reduction of 1, and it is demonstrated that 2 and 3 decompose in the presence of reductants such as thiolates and NaBH(4), followed by addition of CH(3)I, to form the dimethylated products of reductive cleavage of the S(1)-S(2) bond. The same products are isolated subsequent to lysis in buffer containing CH(3)I of Hepa 1c1c7 cells treated with 2 or 3. Reductive cleavage of 2 and 3 in aqueous ethanol by NaBH(4) in an argon atmosphere, followed by acidic destruction of remaining borohydride and neutralization and introduction of O(2) results in the reformation of 2 and 3 to the extent of 80 and 33%, respectively. The data in toto are consistent with a model in which dithiolethiones, generally, undergo reductive cleavage in Hepa 1c1c7 cells, thereby resulting in the generation of O(2)(*) that dismutates to H(2)O(2), that subsequently, by direct or indirect means, effects the nuclear translocation of transcription factor Nrf2, that upregulates phase 2 enzyme expression.

Oxidative stress-induced protein radical formation precedes follicular dendritic cell apoptosis in murine sepsis (92.3)

Abstract New studies indicate that most deaths from sepsis are actually the result of a substantially impaired immune response that is due to extensive death of immune effector cells including follicular dendritic cells (FDCs). However studies by Hotchkiss et al, and others have shown that FDC's undergo caspase 3-mediated apoptosis at 36 h post sepsis with profound depletion of these cells by 48 hours, but the underlying causes of apoptosis have been unknown. This study hypothesizes the role of oxidative stress-induced protein radical formation in FDCs as the principle cause of apoptosis in murine sepsis. This study utilizes immuno-spin trapping to detect DMPO (spin trap) nitrone adducts in FDCs isolated from Lipopolysaccharide (LPS)-primed mice after flow sorting. The isolated FDCs were also screened by LC/MS/MS analysis to detect possible proteins that contained DMPO nitrone adducts. Results indicated that mice administered LPS had DMPO nitrone adduct formation at 24 hours post sepsis as detected by western blot analysis and confocal microscopy. Mice administered allopurinol mice showed significant decrease in adduct formation, annexin V binding, thus indicating the role of xanthine oxidase in oxidative stress-induced protein radical formation and apoptosis. This clearly suggested a role of xanthine oxidase-mediated oxidative stress in FDC apoptosis in the germinal center of septic mice.

Antioxidant properties of plant extracts: an EPR and DFT comparative study of the reaction with DPPH, TEMPOL and spin trap DMPO

This work presents a comparative study of the antioxidant activity of extracts from nine plant species belonging to the Brazilian flora - Swartzia langsdorffii, Machaerium villosum, Pterogyne nitens, Pera glabrata, Aegiphyla sellowiana, Copaifera langsdorffii, Chrysophyllum inornatum, Iryanthera juruensis, Didymopanax venosum - acting on the free-radicals DPPH (2,2-diphenyl-1-picrylhydrazyl) and TEMPOL (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy-1-oxyl) by electron paramagnetic resonance (EPR), and acting on the hydroxyl radical (OH•) by the spin-trapping technique generated by a Fenton reaction. Results showed that the extracts of Iryanthera juruensis and Chrysophyllum inornatum display the strongest antioxidant activities. The results of scavenging tests were clarified by computational calculations - density functional theory (DFT), local density approximation (LDA) with SIESTA code - indicating that the energy released in the reduction reaction of TEMPOL is less than DPPH. Due to its availability and cost DPPH is more often used in these tests than TEMPOL, however TEMPOL should be considered when studying processes dealing with smaller energies.

Immuno-spin trapping of a post-translational carboxypeptidase B1 radical formed by a dual role of xanthine oxidase and endothelial nitric oxide synthase in acute septic mice

Post-translational modification of proteins due to exposure to radicals and other reactive species are markers of metabolic and inflammatory oxidative stress such as sepsis. This study uses the nitrone spin-trap DMPO and a combination of immuno-spin trapping and mass spectrometry to identify in vivo products of radical reactions in mice. We report the detection of dose-dependent production of DMPO-carboxypeptidase B1 (CPB1) adducts in the spleens of mice treated with lipopolysaccharide (LPS). Additionally, we report significant detection of DMPO-CPB1 adducts in mice experiencing normal physiological conditions. Treatments with inhibitors and experiments with knock-out mice indicate that xanthine oxidase and endothelial nitric oxide synthase are important sources of the reactive species that lead to CPB1 adduct formation. We also report a significant loss of CPB1 activity following LPS challenge in conjunction with an increase in CPB1 protein accumulation. This suggests the presence of a possible mechanism for CPB1 activity loss with compensatory protein production.

Mechanisms involved in A2E oxidation

A2E is one of the bis-retinoid pyridinium compounds that accumulate as lipofuscin pigments in retinal pigment epithelial (RPE) cells in association with aging and in some inherited forms of retinal degeneration. Here we observed that 430 nm irradiation of A2E in the presence of the spin trap DMPO, led to the appearance of a superoxide dismutase-inhibitable electron paramagnetic resonance (EPR) spectrum characteristic of DMPO-OH; this finding was indicative of hydroxyl radical (OH) formation following initial spin trapping of superoxide anion by DMPO. We also observed an increase in dihydroethidium (HEt) fluorescence and luminol-based chemiluminescence that on the basis of inhibition by superoxide dismutase, was indicative of superoxide anion generation when A2E was irradiated at 430 nm in cell-free systems. Nevertheless, while A2E was readily oxidized in the presence of a singlet oxygen generator, superoxide anion did not serve to oxidize A2E. Specifically, by HPLC quantitation and FAB-mass spectroscopy, there was no evidence of A2E oxidation when A2E was incubated with a superoxide anion generator (xanthine/xanthine oxidase) in a variety of solvents (100% PBS, 30% DMSO in PBS, 100% MeOH and CHCl 3) or in the presence of detergent. On the other hand, however, peroxy-A2E, an oxidized form of A2E with an endoperoxide moiety on the short-arm of the molecule, readily underwent further oxygen addition when incubated with xanthine/xanthine oxidase. Superoxide anion may be generated by irradiation of A2E but is not involved in the early events that oxidize A2E. Superoxide can contribute to the further oxidation of already-oxidized A2E.

Characterization of Superoxide Anion Scavenging Compounds in Roselle (Hibiscus sabdariffa L.) Extract by Electron Spin Resonance and LC/MS

Roselle (Hibiscus sabdariffa L.) is known to possess strong antioxidative capacity mainly due to its high content of anthocyanins and ascorbic acid. This study aimed to elucidate the ratio of contribution of these compounds in Roselle extract towards superoxide anion scavenging capabilities through analysis using electron spin resonance (ESR) coupled with the hypoxanthine/xanthine oxidase system and DMPO spin trapping technique. The ESR analysis indicated that the IC50 of Roselle extract against superoxide anion was 0.25 mg/ml. After depleting the ascorbate in Roselle extract (0.25 mg/ml) with ascorbate oxidase, the remaining superoxide anion scavenging capacity was determined to be 30.5%, which revealed that ascorbic acid substantially facilitated the scavenging process. By both HPLC and LC/MS analyses, the major active anthocyanins in Roselle crude pigments were identified as delphinidin-3-sambubioside (71.2% of total peak area), delphinidin-3-glucoside (8.7% of total peak area) and cyanidin-3-sambubioside (16.1% of total peak area). Approximately 80% of the superoxide anion scavenging capacities of these anthocyanins was from delphinidin-derivatives (delphinidin-3-sambubioside and delphinidin-3-glucoside); the other 20% was from cyanidin-derivative (cyanidin-3-sambubioside).

Chemical natures of 6-formylpterin nucleoside analogs

Pterin is an electron transfer compound in biological systems. Among the analogs, 6-formylpterin (6FP) has been demonstrated to have many marked physiological activities. In the present study, we have developed the synthetic procedure for nucleoside analogs of 6FP and prepared 1-(beta-D-ribofuranosyl)-2-(N,N-diethylaminomethyleneamino)-6-formylpteridin-4-one (RDEF) and 1-(beta-D-ribofuranosyl)-2-(piperidine-1-ylmethyleneamino)-6-formylpteridin-4-one (RPIF) in which the 1-position is glycosylated. By electron paramagnetic resonance analysis coupled with the DMPO spin trapping technique, it has been elucidated that O2 was converted to H2O2 by RDEF at pH 7.4 in the presence of NADH in the dark. This result indicates that marked reactive oxygen species (ROS) generation activities of 6FP occurring under light illumination, which gives rise to the particular physiological activities such as induction of apoptosis, can be reproduced in the cellular and living systems by such derivatives and gives suggestion for designing pharmaceutical compounds that generate appropriate and controllable amounts of ROS in vivo.

Electron Transfer between a Tyrosyl Radical and a Cysteine Residue in Hemoproteins: Spin Trapping Analysis

We investigated electron transfer between a tyrosyl radical and cysteine residue in two systems, oxyhemoglobin (oxyHb)/peroxynitrite/5,5-dimethyl-1-pyrroline N-oxide (DMPO) and myoglobin (Mb)/hydrogen peroxide/DMPO, using a combination of techniques including ESR, immuno-spin trapping (IST), and ESI/MS. These techniques show that the nitrone spin trap DMPO covalently binds to one or more amino acid radicals in the protein. Treating oxyHb with peroxynitrite and Mb with H2O2 in the presence of a low DMPO concentration yielded secondary Cys-DMPO radical adduct exclusively, whereas in the presence of high DMPO, more of the primary Tyr-DMPO radical adduct was detected. In both systems studied, we found that, at high DMPO concentrations, mainly tyrosyl radicals (Hb-Tyr42/Tyr24 and Mb-Tyr103) are trapped and the secondary electron-transfer reaction does not compete, whereas in the presence of low concentrations of DMPO, the secondary reaction predominates over tyrosyl trapping, and a thiyl radical is formed and then trapped (Hb-Cys93 or Mb-Cys110). With increasing concentrations of DMPO in the reaction medium, primary radicals have an increasing probability of being trapped. MS/MS was used to identify the specific Tyr and Cys residues forming radicals in the myoglobin system. All data obtained from this combination of approaches support the conclusion that the initial site of radical formation is a Tyr, which then abstracts an electron from a cysteine residue to produce a cysteinyl radical. This complex phenomenon of electron transfer from one radical to another has been investigated in proteins by IST, ESR, and MS.

Curcumin protects against cytotoxic and inflammatory effects of quartz particles but causes oxidative DNA damage in a rat lung epithelial cell line

Chronic inhalation of high concentrations of respirable quartz particles has been implicated in various lung diseases including lung fibrosis and cancer. Generation of reactive oxygen species (ROS) and oxidative stress is considered a major mechanism of quartz toxicity. Curcumin, a yellow pigment from Curcuma longa, has been considered as nutraceutical because of its strong anti-inflammatory, antitumour and antioxidant properties. The aim of our present study was to investigate whether curcumin can protect lung epithelial cells from the cytotoxic, genotoxic and inflammatory effects associated with quartz (DQ12) exposure. Electron paramagnetic resonance (EPR) measurements using the spin-trap DMPO demonstrated that curcumin reduces hydrogen peroxide-dependent hydroxyl-radical formation by quartz. Curcumin was also found to reduce quartz-induced cytotoxicity and cyclooxygenase 2 (COX-2) mRNA expression in RLE-6TN rat lung epithelial cells (RLE). Curcumin also inhibited the release of macrophage inflammatory protein-2 (MIP-2) from RLE cells as observed upon treatment with interleukin-1 beta (IL-1β) and tumour necrosis factor-alpha (TNFα). However, curcumin failed to protect the RLE cells from oxidative DNA damage induced by quartz, as shown by formamidopyrimidine glycosylase (FPG)-modified comet assay and by immunocytochemistry for 8-hydroxydeoxyguanosine. In contrast, curcumin was found to be a strong inducer of oxidative DNA damage itself at non-cytotoxic and anti-inflammatory concentrations. In line with this, curcumin also enhanced the mRNA expression of the oxidative stress response gene heme oxygenase-1 (ho-1). Curcumin also caused oxidative DNA damage in NR8383 rat alveolar macrophages and A549 human lung epithelial cells. Taken together, these observations indicate that one should be cautious in considering the potential use of curcumin in the prevention or treatment of lung diseases associated with quartz exposure.

Reaction of the carbonate radical with the spin-trap 5,5-dimethyl-1-pyrroline- N-oxide in chemical and cellular systems: Pulse radiolysis, electron paramagnetic resonance, and kinetic-competition studies

Carbonate radicals (CO 3 − ) can be formed biologically by the reaction of OH with bicarbonate, the decomposition of the peroxynitrite–carbon dioxide adduct (ONOOCO 2 −), and enzymatic activities, i.e., peroxidase activity of CuZnSOD and xanthine oxidase turnover in the presence of bicarbonate. It has been reported that the spin-trap DMPO reacts with CO 3 − to yield transient species to yield finally the DMPO–OH spin adduct. In this study, the kinetics of reaction of CO 3 − with DMPO were studied by pulse radiolysis, yielding a second-order rate constant of 2.5 × 10 6 M − 1 s − 1 . A Fenton system, composed of Fe II–DTPA plus H 2O 2, generated OH that was trapped by DMPO; the presence of 50–500 mM bicarbonate, expected to convert OH to CO 3 − , markedly inhibited DMPO–OH formation. This was demonstrated to be due mainly to a fast reaction of CO 3 − with Fe II–DTPA ( k = 6.1 × 10 8 M − 1 s − 1 ), supported by kinetic analysis. Generation of CO 3 − by the Fenton system was further proved by analysis of tyrosine oxidation products: the presence of bicarbonate caused a dose-dependent inhibition of 3,4-dihydroxiphenylalanine with a concomitant increase of 3,3′-dityrosine yields, and the presence of DMPO inhibited tyrosine oxidation, in agreement with the rate constants with OH or CO 3 − . Similarly, the formation of CO 3 − by CuZnSOD/H 2O 2/bicarbonate and peroxynitrite–carbon dioxide was supported by DMPO hydroxylation and kinetic competition data. Finally, the reaction of CO 3 − with DMPO to yield DMPO–OH was shown in peroxynitrite-forming macrophages. In conclusion, CO 3 − reacts quite rapidly with DMPO and may contribute to DMPO–OH yields in chemical and cellular systems; in turn, the extent of oxidation of other target molecules (such as tyrosine) by CO 3 − will be sensitive to the presence of DMPO.

Top-Down and Bottom-Up Mass Spectrometric Characterization of Human Myoglobin-Centered Free Radicals Induced by Oxidative Damage

In an effort to determine the utility of top-down mass spectrometric methodologies for the characterization of protein radical adducts, top-down approaches were investigated and compared to the traditional bottom-up approaches. Specifically, the nature of the radicals on human myoglobin induced by the addition of hydrogen peroxide and captured by the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was investigated. The most abundant ion observed in the electrospray mass spectrum of this reaction mixture corresponds in mass to the human myoglobin plus one DMPO molecule. In addition, a second ion of lower abundance is observed, which corresponds to a second DMPO molecule being trapped on myoglobin. Top-down analyses using Fourier transform ion cyclotron resonance mass spectrometry can be used to characterize proteins and, thus, were performed on several different charge-state ions of both the native and the mono-DMPO nitrone adduct of human myoglobin. Data produced from the top-down analyses are very complex yet information rich. In the case of DMPO-modified human myoglobin, the top-down data localized the DMPO spin trap to residues 97-110 of the myoglobin. The observation of the y43+5 fragment ion arising from C-terminal cleavage to the cysteine-110 residue in the MS/MS spectrum of DMPO-modified myoglobin and not in the unmodified myoglobin implicates a change to this residue, specifically, DMPO adduction. On the other hand, using the traditional bottom-up approach of peptide mapping and MS sequencing methodologies, two DMPO radical adducts on human myoglobin were identified, Cys-110 and Tyr-103. The bottom-up approach is more proven and robust than the top-down methodologies. Nonetheless, the bottom-up and top-down approaches to protein characterization are complementary rather than competitive approaches with each having its own utility.

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H 2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.

Influence of humic substances on the photolysis of aqueous pesticide residues

The present work investigated the direct and indirect photolysis of pesticide residues (atrazine, imazaquin, iprodione), in aqueous solutions and under UV–visible radiation (280–480 nm). Different kinds of humic substances (HS) were added to samples in order to evaluate their behaviour as possible photocatalysts and their effect on the photolysis of pesticides. The fulvic acids were purchased from the International Humic Substances Society, and they were added to samples in concentrations ranging from 1 to 150 mg l −1. Titanium dioxide was used as the photocatalyst, in concentration ranging from 10 to 150 mg l −1. Pesticides photolysis were measured by UV–visible absorption spectroscopy and differential pulse polarography with all used pesticides, reaching total degradation after 2 h of irradiation, thus indicating a fast direct photolysis. Photocatalysis by TiO 2 could increase the pesticides photolysis rate up to 40%. This effect, however, was not observed for imazaquin photolysis. Again, except for imazaquin, HS presence showed a positive effect in increasing pesticide degradation, but only within specific concentration ranges (below 10 mg l −1 for iprodione and about 30 mg l −1 for atrazine). Above these ranges HS induce a decrease in the pesticides photolysis rate. Spin-trapping measurements by electronic paramagnetic resonance spectroscopy, using the spin-trap DMPO, showed that HS are able to photogenerate hydroxyl radicals, increasing the pesticides molecule degradation. However, the HS also react with the photogenerated hydroxyl radical, influencing the pesticide photolysis, leading to a decrease in the photolysis rate and causing it to be strongly dependent on the nature and concentration of residues in the water to be treated.