O nosso laboratório estuda os eventos primários e secundários causados pela geração de espécies reativas de oxigênio em estruturas celulares e biomoléculas. Atualmente, enfatizamos a ação genotóxica de produtos gerados durante a oxidação de lipídeos c onstitutintes das membranas biológicas.

O processo de lipoperoxidação tem sido associado a diversos mecanismos celulares que podem acarretar câncer, inflamação e envelhecimento. Até recentemente, assumia-se que as principais fontes de lesão em DNA humano eram provenientes de derivados ativa dos de substâncias exógenas. Entretanto, cada vez mais surgem evidências que substâncias endógenas, produzidas pelo metabolismo oxidativo e durante inflamações são prováveis fontes de danos ao DNA. Nossos interesses concentram-se na caracterização da estr utura química e nos mecanismos de formação de adutos de DNA, em sistemas in vitro, em células isoladas e in vivo. A proteção contra este tipo de dano, por antioxidantes conhecidos e os possíveis mecanismos de reparo são também objetos da nossa pesquisa.


Nossos Links:

• www.biophysics.mcw.edu/oxsoc/
  
• www.brunel.ac.uk/org/sfrr
  
• www.marisa5.iq.usp.br
  

• Carvalho, VM; Di Mascio, P. ; Arruda Campos, I.; Douki, T. ; Cadet, J.; Medeiros, MHG ; ( 1998 ),Formation of 1,N6-etheno-2'-deoxyadenosine adducts by trans,trans-2,4-decadienal,Chem. Res. Toxicol.- [11 ]: 1042- 1047
  
  

  Trans,trans-2,4-decadienal (DDE) is an important breakdown-product of lipid peroxidation. This aldehyde is cytotoxic to mammalian cells and is known to be implicated in DNA damage. Therefore, attempts were made in the present work to assess the reactivity of DDE with 2'-deoxyadenosine (dAdo). It was shown that DDE is able to bind to 2'-deoxyadenosine, yielding highly fluorescent products. Besides 1,N6-etheno-2'-deoxyadenosine (edAdo), two other related adducts were isolated by reverse phase high performance liquid chromatography and characterized on the basis of their UV, fluorescence, nuclear magnetic resonance and mass spectrometry features. The reaction mechanism for the formation of the DDE-2'-deoxyadenosine adducts involves 2,4-decadienal epoxidation and subsequent addition to the N2 amino group of 2'-deoxyadenosine, followed by cyclization at the N-1 site. Adducts differ by the length of carbon side chain and the number of hydroxyl groups. The present data indicate that DDE can be epoxidized by peroxides and the resulting products are able to form several adducts with 2'-deoxyadenosine and/or DNA. Endogenous DNA adduct formation can contribute to the already reported high cytotoxicity of DDE to mammalian cells.

  
  

• Douki T; Onuki J ; Medeiros MHG;Bechara EJH ; Cadet J; Di Mascio P ; ( 1998 ),Hydroxyl radicals are involved in the oxidation of isolated and cellular DNA bases by 4-aminolevulinic acid,FEBS Lett.- [428 ]: 93- 96
  
  

  5-Aminolevulinic acid (ALA) is a heme precursor, pathological accumulation of which is associated with liver cancer, We show that the reactive oxygen species produced upon ALA metal-catalyzed oxidation promote the formation of several radical-induced base degradation products in isolated DNA. The distribution of modified bases is similar to that obtained upon gamma irradiation. This observation strongly suggests the involvement of hydroxyl radicals in the ALA-mediated DNA damage. Increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 5-hydroxy-2'-deoxycytidine in organ DNA of rats chronically treated with ALA were observed. This is strongly suggestive of the implication of hydroxyl radicals in the ALA-induced degradation of cellular DNA.

  
  

• Douki T; Onuki J ; Medeiros MHG;Bechara EJH ; Cadet J, Di Mascio P ; ( 1998 ),DNA alkylation by 4,5-dioxovaleric acid, the final oxidation product of 5-aminolevulinic acid,Chem. Res. Toxicol.- [11 ]: 150- 157
  
  

  The heme precursor 6-aminolevulinic acid (ALA) accumulates under pathological conditions, namely, acute intermittent porphyria (AIP) and tyrosinosis, two diseases that are associated with increased liver cancer incidence. This has been previously linked to an enhanced production of reactive oxygen species generated by a metal-catalyzed ALA oxidation process, which was shown to cause DNA single-strand breaks and guanine oxidation within both isolated and cellular DNA. In the present work, we established that the final oxidation product of ALA, 4,5-dioxovaleric acid (DOVA), is an efficient alkylating agent of the guanine moieties within both nucleoside and isolated DNA. Adducts were produced through the formation of a Schiff base involving the N-2-amino group of 2'-deoxyguanosine (dGuo) and the ketone function of DOVA, respectively. The modified dGuo nucleosides were characterized, following reduction into stable secondary amines, by extensive NMR, infrared, and mass spectrometry analyses. A method, based on the use of HPLC with electrochemical detection, was then developed for the sensitive measurement of the DOVA-dGuo adducts. Using this assay, we showed that the guanine moieties of isolated DNA can undergo the same reaction as the free nucleoside. The present data provide additional information on the genotoxic potential of ALA and reinforce the hypothesis that AIP may be involved in the induction of primary liver cell carcinoma.

  
  

• DiMascio P; Briviba K ; Sasaki ST; Catalani LH ; Medeiros MHG; Bechara EJH ; Sies H ; ( 1977 ),The reaction of peroxynitrite with tert-butyl hydroperoxide produces singlet molecular oxygen,Biol. Chem. Hoppe-Seyler- [378 ]: 1071- 1074
  
  

  Peroxynitrite, a biological oxidant, can induce lipid peroxidation in biological membranes which leads to the formation of various hydroperoxides, Here, we report the formation of singlet oxygen (O-1(2)) in the reaction of peroxynitrite with tert-butyl hydroperoxide (t-BOOH) used as a model compound for organic hydroperoxides. The formation of singlet oxygen was observed by (i) dimol emission in the red spectral region, (ii) monomol emission in the infrared region at 1270 nm and by (iii) chemical trapping of singlet oxygen with anthracene-9,10-diyldiethyl disulfate (EAS), The emission signal was increased when H2O was replaced by deuterium oxide and was quenched by sodium azide. When singlet oxygen was generated in the reaction of peroxynitrite with t-BOOH, the O-1(2) quenching rate constant for sodium azide was estimated from a Stern-Volmer plot as 1.3 x 10(8) M-1 s(-1) which is in line with known values, The O-1(2) generation in the peroxynitrite/t-BOOH reaction was also detected by the formation of the endoperoxide of EAS, These data establish the generation of O-1(2) in the reaction of peroxynitrite with t-BOOH and suggest a potential involvement of O-1(2) in peroxynitrite-mediated reactions in biological systems.

  
  

• DiMascio P; Medeiros MHG ; Sies H; Bertolotti S ; Braslavsky SE; Veloso DP ; Sales BHLN; Magalhaes E ; Braz R;Bechara EJH ; ( 1997 ),Quenching of singlet molecular oxygen by natural furan diterpen,J. Photochem. Photobiol.B: Biol.- [38 ]: 169- 173
  
  

  Singlet molecular oxygen O-2((1) Delta(g)) is a reactive oxygen species capable of damaging biological molecules and triggering oxidative stress, The quenching of this species by naturally occurring furans was measured using O-2((1) Delta(g)) from two different sources: (1) the thermal decomposition of 1,4-dimethylnaphthalene endoperoxide (1,4-DMNO2); (2) tetraphenylporphyrin sulphonate photosensitization. Four furan diterpenes were isolated from alcoholic extracts of Sucupira branca (Pterodon sp,, Leguminosae) seeds, which are used for the treatment of rheumatic diseases, suggesting anti-inflammatory activity. Using time-resolved near-IR (NIR) emission after photosensitization, the overall (physical + chemical) O-2((1) Delta(g)) quenching rate constants by these compounds were found to be of the order of 10(8) M-1 s(-1), with the exception of the conjugated furan diterpenes 3,5,6-trimethoxy-7,8-furano-3',4'-methylenedioxy-2 '',3 ''-flavane and 3,4,5,6-tetramethoxy-7,8-furano-2 '',3 ''-flavane for which 20-fold lower values were observed, These lower values are attributed to the conjugation of the furan moiety with the aromatic ring, which prevents (2 + 4) cycloaddition of O-2((1) Delta(g)) to the furan ring, Although the non-conjugated furans exhibit a low scavenging effect on lipid peroxidation in microsomes, their high O-2((1) Delta(g)) quenching constant may minimize the extent of tissue damage associated with the inflammatory process.

  
  

• Di Mascio P; Briviba K ; Bechara EJH; Medeiros MHG ; Sies H ; ( 1996 ),Reaction of peroxynitrite and hydrogen peroxide to produce singlet molecular oxygen,Methods Enzymol.- [269 ]: 395- 400
  
  

  
  

• Onuki J; Ribas AV ; Medeiros MHG; Araki K ; Toma HE; Catalani L ; Di Mascio P ; ( 1996 ),Supramolecular cationic tetraruthenated porphyrin induces single-strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine formation in DNA in the presence of light,Photochem. Photobiol.- [63 ]: 272- 277
  
  

  The aim of this investigation is the evaluation of DNA interaction of with tetraruthenated porphyrin (TRP) and of DNA damage in the presence of light, Direct-fluorescence and electronic absorption measurements after incubation of DNA with TRP indicate strong binding between pBR322 DNA or calf thymus DNA with the modified porphyrin, Exposure of pBR322 DNA to TRP (up to 3 mu M) and light leads to single-strand break formation as determined by the conversion of the supercoiled form (form I) of the plasmid into the nicked circular form (form II). Oxidative DNA base damage was evaluated by the detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after irradiation of calf thymus DNA in the presence of the TRP. The data demonstrated a dose and time dependence with each type of DNA damage. These data indicate (1) a specificity of the binding mode and (2) type I and II photoinduced mechanisms leading to strand scission activity and 8-oxodGuo formation, Accordingly, singlet molecular oxygen formation, after TRP excitation, was confirmed by near-infrared emission. From these investigations a potential application of TRP in photodynamic therapy is proposed.

  
  

• Medeiros MHG; Di Mascio P ; Pinto AP; Vargas RR ; Bechara EJH ; ( 1996 ),Horseradish peroxidase-catalyzed conjugation of eugenol with basic amino acids,FREE RADIC. RES.- [25 ]: 5- 12
  
  

  L-Lysine is shown to yield an adduct with the quinone methide intermediate formed during the horseradish peroxidase (HRP)-catalyzed aerobic oxidation of eugenol (4-allyl-2-methoxyphenol). Adduct formation is evidenced by (i) lysine quenching of the characteristic quinone methide absorption band measured at 350 nm; arginine and gamma-aminobutyric acid, but not alanine or propionic acid showed similar behaviour (ii) lysine-promoted a 400 mV decrease of the eugenol oxidation voltammetric wave (1.00 V), concomitantly with an increase in current intensity and (iii) reverse phase HPLC isolation of the lysine eugenol adduct, followed by GCMS analysis. The MS spectrum is consistent with a 2:1 lysine:eugenol adduct (MW = 455). If operative in vivo, binding of lysine to eugenol might lead to protein inactivation and possibly be involved in eugenol toxicity.

  
  

• Di Mascio, P; Medeiros, MHG ; Menck, CF ; ( 1996 ),DNA damage by singulet molecular oxygen, protection by polyamines and biological effect,Journal de Chimie Physique- [93 ]: 64- 69
  
  

  DNA damage induced by singlet molecular oxygen (O-1(2)) and their biological consequences were investigated, using the thermodissociation of the water soluble endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2), which produces only O-1(2), with no reactive intermediates or secondary products. By following electrophoretic mobility of single and double stranded DNA on agarose gels, we found that O-1(2)-treatment induces single and double strand breaks formation. Single strand breaks are efficiently prevented with biomolecules, such as the polyamines, spermine and spermidine. The lethal and mutagenic consequences of the O-1(2)-induced DNA lesions were demonstrated after introduction of plasmid DNA into bacteria and mammalian cells. There is a clear increase on the mutation frequency, function of the NDPO2 concentration, when plasmids, single and double stranded, are introduced into monkey COS7 cells. This suggests that O-1(2)-damaged DNA is processed in mammalian cells by an error prone repair. The mutation spectrum reveals that 98% of the mutations involve G:C base pairs, thus, the high mutagenicity of O-1(2) are probably due to lesions on guanines. The data supports the idea that a direct bypass of guanine lesions is responsable for the O-1(2) mutagenicity.

  
  

• Medeiros, MHG; Carvalho, VM ; Farias, LP; Loureiro, APM ; ( 1995 ),DNA damage induced by secondary lipid oxidation products,Ciên. Cult.- [47 ]: 336- 339
  
  

  
  

• Bechara, EJH;Medeiros, MHG ; Costa, CA; Di Mascio, P. ; Catalani, LH; Soares, CHL ; Wendel, CMA; Onuki,J. ; Monteiro, HP; Abdalla, DSP ; Penatti, CAA. ; Cristoff, M.; Hermes-Lima, M ; Pereira, B.; Demasi, M. ; ( 1995 ),Enolizable Carbonyl and Imino Metabolites May Act as Endogenous Sources of Reactive Oxygen Species,Ciên. Cult.- [47 ]: 346- 357
  
  

  
  

• Medeiros, MHG; Baptista, RC ; Di Mascio, P. ; ( 1995 ),Quimiluminescência Fraca em Sistemas Biológicos,Rev. Ciênc. Farm.- [16 ]: 23- 35
  
  

  
  

• PEREIRA B; ROSA LFBC ; SAFI DA; MEDEIROS MHG ; CURI R; BECHARA EJH ; ( 1994 ),SUPEROXIDE-DISMUTASE, CATALASE, AND GLUTATHIONE-PEROXIDASE ACTIVITIES IN MUSCLE AND LYMPHOID ORGANS OF SEDENTARY AND EXERCISE-TRAINED RATS,Physiol. Behav.- [56 ]: 1095- 1099
  
  

  The effect of swimming-training upon the activities of the enzymes involved in the generation of reducing-equivalents (citrate synthase-mitochondria and glucose-6-phosphate dehydrogenase-cytosol) and of antioxidant enzymes (CuZn- and Mn-SOD, catalase and glutathione peroxidase) in the lymphoid organs (thymus, mesenteric lymph nodes and spleen) was examined. The skeletal muscles (soleus-red and gastrocnemius-white) were also studied. Although our data suggest an apparently random, organ-specific change in enzymatic activity, some interesting trends can be observed. Firstly, the increased citrate synthase and Mn-SOD activities observed in red, but not in white muscle, corroborate the well-known effect of endurance exercise-training on mitochondrial oxidative metabolism. Secondly, there was an inverse relationship between TBARs-monitored lipoperoxidation and glutathione peroxidase activity in all tissues studied, what is in accordance with the previous findings showing that such enzyme exerts the fine control of intracellular lipoperoxide concentration. Except in the case of the spleen, there was a trend for elevated glucose-6-phosphate dehydrogenase activity, coadjuvant of glutathione peroxidase in the antioxidant response to physical exercise in all tissues. Thirdly, Mn-SOD and catalase were conspicuously associated to oxidative stress in the thymus, while glutathione and catalase could be linked to this parameter in the spleen. Fourthly, the lymph nodes seem to be more dependent on the glucose-6-phosphate dehydrogenase/glutathione peroxidase pair for protection against damage promoted by physical exercise. Mn-SOD and catalase activities were lower in the lymph nodes after swimming training. Finally, the observed changes in antioxidant enzyme activities in the spleen, lymph nodes and thymus indicate that endurance training affects the aerobic metabolism of these immune-related organs. Whether these changes lead to the impaired immune function reported in exercise remains to be found.

  
  

• DIMASCIO P; BECHARA EJH ; MEDEIROS MHG; BRIVIBA K ; SIES H ; ( 1994 ),SINGLET MOLECULAR-OXYGEN PRODUCTION IN THE REACTION OF PEROXYNITRITE WITH HYDROGEN-PEROXIDE,FEBS LETTERS- [355 ]: 287- 289
  
  

  Peroxynitrite and hydrogen peroxide are mediators of cytotoxicity. This study shows that the peroxynitrite anion reacts with hydrogen peroxide to release oxygen accompanied by emission of chemiluminescence (CL). Direct characterization of this light emission attributes it to the transition of singlet molecular oxygen to the triplet ground state. Chemiluminescence was monitored: (i) by dimol light emission in the red spectral region (> 610 nm) using a red-sensitive photomultiplier; and (ii) by monomol light emission in the infrared (1270 Mn) with a liquid nitrogen-cooled germanium diode. These properties of photoemission and the enhancing effect of deuterium oxide on CL intensity as well as the quenching effect of sodium azide are diagnostic of molecular oxygen in the excited singlet state. For comparison, singlet molecular oxygen arising from the thermolysis of the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate or from the hypochlorite/H2O2 system was also monitored. These novel observations identify a potential singlet oxygen-dependent mechanism contributing to cytotoxicity mediated by peroxynitrite and hydrogen peroxide.

  
  

• ONUKI J; MEDEIROS MHG ; BECHARA EJH; DI MASCIO P ; ( 1994 ),5-AMINOLEVULINIC ACID INDUCES SINGLE-STRAND BREAKS IN PLASMID PBR322 DNA IN THE PRESENCE OF FE2+ IONS,Biochim. Biophys. Acta- [1225 ]: 259- 263
  
  

  5-Aminolevulinic acid (ALA), a heme precursor accumulated in chemical and inborn porphyrias, has been demonstrated to produce reactive oxygen species upon metal-catalyzed aerobic oxidation and to cause oxidative damage to proteins, liposomes and subcellular structures. Exposure of plasmid pBR322 DNA to ALA (0.01-3 mM) in the presence of 10 mu M Fe2+ ions causes DNA single-strand breaks (ssb), revealed by agarose gel electrophoresis as an increase in the proportion of the open circular form (75 +/- 7.5% at 3 mM ALA) at the expense of the supercoiled form. Addition of either anti-oxidant enzymes such as superoxide dismutase (10 mu g/ml) and catalase (20 mu g/ml), or a metal chelator (DTPA, 2.5 mM), or a HO. scavenger(mannitol, 100 mM) inhibited the damage (by 30, 45, 55, and 81%, respectively), evidencing the involvement of O-2(-)., H2O2 and HO. (by the Haber-Weiss reaction) in this process. Hydrogen peroxide (100 mu M) or Fe2+ (10 mu M) alone were of little effect on the extent of DNA ssb. The present data may shed light on the correlation reported between primary liver-cell carcinoma and intermittent acute porphyria.

  
  

• MEDEIROS MHG; DIMASCIO P ; GRUNDEL S; SOBOLL S ; SIES H; BECHARA EJH ; ( 1994 ),CATABOLISM OF 5-AMINOLEVULINIC ACID TO CO2 BY RAT-LIVER MITOCHONDRIA,ARCH. BIOCHEM. BIOPHYS.- [310 ]: 205- 209
  
  

  5-Aminolevulinic acid (ALA), the heme precursor accumulated in plasma and several organs of carriers of acute intermittent porphyria, hereditary tyrosinemia, and saturnism, was previously shown to yield reactive oxygen species upon metal-catalyzed aerobic oxidation and to cause the in vivo and in vitro impairment of rat liver mitochondrial functions. We have studied the uptake and catabolism of [5-C-14]ALA to CO2 by isolated rat liver mitochondria (RLM) with the aim of determining whether possible ALA-driven oxidative injury to mitochondria can also occur into the matrix. Using silicone oil centrifugation of [5-C-14]ALA-treated RLM, ALA was found to partition evenly into the intra- and extramatrix space of the mitochondrial preparations. The yield of evolved (CO2)-C-14 is very low (0.2%), responds to the concentration of added ADP, and is inhibited by malonate (75% at 2 mm), iproniazid (45% at 2 mm), beta-chloroalanine (36% at 1 mm), and aminooxyacetate (55% at 0.1 mm). With both iproniazid and aminooxyacetate, the percentage of inhibition is the same as that observed with the latter inhibitor alone. These data indicate that ALA decarboxylation by the Krebs cycle is a minor process and that it is initiated enzymically (transaminase) and not by metal-catalyzed ALA autoxidation.

  
  

• Bechara, EJH, Medeiros, MHG, ; Di Mascio, P. ; Monteiro, HP, Lima, MH, ; Pereira, B., Demasi, M., ; Costa, C.A., Abdalla, D., ; Onuki, J., Wendel, CMA, ; ( 1993 ),A Free Radical Hypothesis of Lead Poisoning and Inborn Porphyrias Associated with 5_aminolelulinic Acid. Overload,Química Nova- [16 ]: 385- 392
  
  

  
  

• KHAN AU, DI Mascio P, MEDEIROS MHG ; Wilson T ; ( 1992 ),SPERMINE AND SPERMIDINE PROTECTION OF PLASMID DNA AGAINST SINGLE-STRAND BREAKS INDUCED BY SINGLET OXYGEN,PROC. NATL. ACAD. SCI. USA- [89 ]: 11428- 11430
  
  

  Oxidative damage to DNA induced by singlet molecular oxygen (O-1(2)*) includes single-strand breaks, which the biologically occurring O-1(2)* quenchers spermine and spermidine are shown to prevent. These polyamines at a physiological concentration (10 mM) reduce the percentage of the open circular form of pBR322 plasmid DNA, which is generated at the expense of the native supercoiled form when the plasmids are incubated with a chemical source of O-1(2)*, the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene)dipropionate. Spermine and spermidine can be expected to protect DNA against other damaging effects of O-1(2)*.

  
  

• MEDEIROS MHG, SIES H ; ( 1989 ),CHEMILUMINESCENT OXIDATION OF RIBOSE CATALYZED BY HORSERADISH-PEROXIDASE IN PRESENCE OF HYDROGEN-PEROXIDE,FREE RADICAL BIOLOGY AND MEDICINE - [6 ]: 565- 571
  
  

  
  

• MEDEIROS MHG, WEFERS H, SIES H ; ( 1987 ),GENERATION OF EXCITED SPECIES CATALYZED BY HORSERADISH-PEROXIDASE OR HEMIN IN THE PRESENCE OF REDUCED GLUTATHIONE AND H2O2,FREE RADICAL BIOLOGY AND MEDICINE - [3 ]: 107- 110
  
  

  
  

• Medeiros, MHG; Bechara, EJH ; ( 1986 ),Chemiluminescent Aerobic Oxidation of Protein Adducts with Glycolaldehyde Catalyzed by Horseradish Peroxidase,Arch. Biochem. Biophys.- [248 ]: 435- 439
  
  

  
  

• Bechara, E.J.H., Medeiros, MHG ; ( 1984 ),Chemiluminescence of Schiff bases catalyzed by horseradish peroxidase,Oxygen Radicals in Chemistry and Biology (W. Bors, M. Saran, D. Tait, eds)- [ ]: 539- 542
  
  

  
  

• MEDEIROS MHG, BECHARA EJH, NAOUM PC, ; Mourão, CA ; ( 1983 ),OXYGEN-TOXICITY AND HEMOGLOBINEMIA IN SUBJECTS FROM A HIGHLY POLLUTED TOW,ARCHIVES OF ENVIRONMENTAL HEALTH - [38 ]: 11- 16
  
  

  
  

• Medeiros, M.H.G. ; Bechara, E.J.H. ; ( 1983 ),Estudos de Ativação de Oxigênio em Residentes de Vila Parisi,Química Nova- [6 ]: 131- 133
  
  

  
  

• MEDEIROS MHG ; MARCHIORI PE ; BECHARA EJH ; ( 1982 ),SUPEROXIDE-DISMUTASE, GLUTATHIONE-PEROXIDASE, AND CATALASE ACTIVITIES IN THE ERYTHROCYTES OF PATIENTS WITH INTERMITTENT ACUTE PORPHYRIA,CLINICAL CHEMISTRY- [28 ]: 242- 242