N-acetylcysteine improves the quality of red blood cells stored for transfusion.

Storage inflicts a series of changes on red blood cells (RBC) that compromise the cell survival and functionality; largely these alterations (storage lesions) are due to oxidative modifications. The possibility of improving the quality of packed RBC stored for transfusion including N-acetylcysteine (NAC) in the preservation solution was explored. Relatively high concentrations of NAC (20-25 mM) were necessary to prevent the progressive leakage of hemoglobin, while lower concentrations (≥2.5 mM) were enough to prevent the loss of reduced glutathione during the first 21 days of storage. Peroxiredoxin-2 was also affected during storage, with a progressive accumulation of disulfide-linked dimers and hetero-protein complexes in the cytosol and also in the membrane of stored RBC. Although the presence of NAC in the storage solution was unable to avoid the formation of thiol-mediated protein complexes, it partially restored the capacity of the cell to metabolize H2O2, indicating the potential use of NAC as an additive in the preservation solution to improve RBC performance after transfusion.

Arylnitroalkenes as scavengers of macrophage-generated oxidants.

Oxygen and nitrogen derived molecules mediated oxidation and nitration have been involved in several pathological conditions. Conversely, nitric oxide and hydrogen peroxide are important signalization intermediates, whose concentrations are tightly regulated by specialized enzyme repertoires and should remain undisturbed by the addition of exogenous antioxidant molecules, as already demonstrated by intervention studies with antioxidant vitamins. Our goal was to develop specific antioxidants able to scavenge peroxynitrite anion, as well the radicals derived from the homolytic decomposition of its conjugated acid, nitrogen dioxide and hydroxyl radical. Fourteen substituted nitroalkenes, seven 4-substituted 1-(2-nitro-1Z-ethenyl)benzene, and seven 4-substituted (2-nitro-1Z-propenyl)benzene, with different stereochemical and electronic characteristics were synthesized and tested. Compounds with the electron donor group N,N-dimethylamino showed the highest reaction rates against peroxynitrite, and also reacted with its homolytic decomposition products, OH and NO2. While 1,1-dimethylamino-4-(2-nitro-1Z-ethenyl)benzene came up as a lead for future developments without the risk of interfering with signalization pathways, since it was highly specific for peroxynitrite and peroxynitrite derived radicals, its methylated analogous 1,1-dimethylamino-4-(2-nitro-1Z-propenyl)benzene was less specific and also reacted with NO and O2(-), the biological precursor of H2O2.

Analysis of two methods to evaluate antioxidants.

This exercise is intended to introduce undergraduate biochemistry students to the analysis of antioxidants as a biotechnological tool. In addition, some statistical resources will also be used and discussed. Antioxidants play an important metabolic role, preventing oxidative stress-mediated cell and tissue injury. Knowing the antioxidant content of nutritional components can help make informed decisions about diet design, and increase the commercial value of antioxidant-rich natural products. As a reliable and convenient technique to evaluate the whole spectrum of antioxidants present in biological samples is lacking, the general consensus is to use more than one technique. We have chosen two widely used and inexpensive methods, Trolox-equivalent antioxidant capacity and the ferric reducing antioxidant power assays, to evaluate the antioxidant content of several fruits, and to compare and analyze the correlation between both assays.

Antioxidant activity of uruguayan propolis. In vitro and cellular assays.

The antioxidant capacity of propolis from the southern region of Uruguay was evaluated using in vitro as well as cellular assays. Free radical scavenging capacity was assessed by ORAC, obtaining values significantly higher than those of other natural products (8000 µmol Trolox equiv/g propolis). ORAC values correlated well with total polyphenol content (determined by Folin-Ciocalteu method) and UV absorption. Total polyphenol content (150 mg gallic acid equiv/g propolis) and flavonoids (45 mg quercetin equiv/g propolis) were similar to values reported for southern Brazilian (group 3) and Argentinean propolis. Flavonoid composition determined by RP-HPLC indicates a strong poplar-tree origin. Samples high in polyphenols efficiently inhibit low-density lipoprotein lipoperoxidation and tyrosine nitration. In addition, Uruguayan propolis was found to induce the expression of endothelial nitric oxide synthase and inhibit endothelial NADPH oxidase, suggesting a potential cardiovascular benefit by increasing nitric oxide bioavailability in the endothelium.

Identification of chalcones as in vivo liver monofunctional phase II enzymes inducers.

Cancer preventive agents (CPA) are drugs able to suppress the carcinogen metabolic activation or block the formation of ultimate carcinogens. CPA could act through various molecular mechanisms, for example by interfering with the action of procarcinogen. This could be attained by increasing the phase II enzymes levels of quinone reductase (QR) and glutathione S-transferase (GST). New flavonoids, especially chalcones, have been identified as in vivo monofunctional phase II enzymes inducers. Oral administration of chalcone, 4, and both p-methoxy-substituted chalcones, 6 and 14, increased hepatic QR activity with concomitant decrease in CYP1A1 activity, a member of the most important group of phase I enzymes cytochrome P450. Among them, 4 also increased GST activity. While p-bromo-substituted chalcone 8 was the best inducer of QR it decreased hepatic GST expression and cytochrome P450, being the most effective decreasing cytochrome P450-expression. Thienyl-chalcone 20 being the bioisostere of chalcone 4 did not display the same in vivo profile in the phase I level modification. As chalcone 4 its bioisostere, chalcone 20, displayed low DNA strand breakage and absence of mutagenicity. Also, in our preliminary in vivo tumourigenesis/chemopreventive and acute-toxicity studies, chalcones 4, 6 and 8 showed the best behaviours as CPA justifying additional studies that are ongoing.

Kinetics of peroxiredoxins and their role in the decomposition of peroxynitrite.

Methodologies and results of studies on the kinetics of peroxiredoxins (Prx) are reviewed. Peroxiredoxins are broad-spectrum peroxidases that catalyze the reduction of H2O2, organic hydroperoxides and peroxynitrite by thiols. Their catalytic cycle starts with the oxidation of a particularly reactive cysteine residue (C(P)) to a sulfenic acid derivative by the peroxide substrate, the sulfenic acid then reacts with a thiol to form a disulfide, and the cycle is completed by thiol/disulfide exchange reactions that regenerate the ground-state enzyme. Depending on the subtype of peroxiredoxin, the thiol reacting with the primary oxidation product (E-SOH) may be a cysteine residue of a second subunit (typical 2-Cys Prx), a cysteine residue of the same subunit (atypical 2-Cys Prx) or reducing substrate (1-Cys Prx and at least one example of an atypical 2-Cys Prx). In a typical 2-Cys Prx the intra-subunit disulfide formation with the second "resolving" cysteine (C(R)) is mandatory for the reduction by the specific substrate, which is a protein characterized by a CXXC motif such as thioredoxin, tryparedoxin or AhpF. These consecutive redox reactions define the catalysis as an enzyme substitution mechanism, which is corroborated by a ping-pong pattern that is commonly observed in steady-state analyses, chemical identification of catalytic intermediates and stopped-flow analyses of partial reactions. More complex kinetic patterns are discussed in terms of cooperativity between the subunits of the oligomeric enzymes, generation of different oxidized intermediates or partial over-oxidation of C(P) to a sulfinic acid. Saturation kinetics is often not observed indicating that a typical complex between reduced enzyme and hydroperoxide is not formed and that, in these cases, formation of the complex between the oxidized enzyme and its reducing substrate is slower than the reaction within this complex. Working with sulphur catalysis, Prxs are usually less efficient than the heme- or selenium-containing peroxidases, but in some cases the k(+1) values (bimolecular rate constant for oxidation of reduced E by ROOH) are comparable, the overall range being 2 x 10(3)-4 x 10(7) M(-1)s(-1) depending on the hydroperoxide and the individual Prx. For the reduction of peroxynitrite k(+1) values of 1 x 10(6) up to 7 x 10(7) M(-1)s(-1) have been measured. The net forward rate constants k'(+2) for the reductive part of the cycle range between 2 x 10(4)-1 x 10(7) M(-1)s(-1). These kinetic characteristics qualify the peroxiredoxins as moderately efficient devices to detoxify hydroperoxides, which is pivotal to organisms devoid of more efficient peroxidases, and as most relevant to the detoxification of peroxynitrite. In higher organisms, their specific role is seen in the regulation of signalling cascades that are modulated by H2O2, lipid hydroperoxides or peroxynitrite.

Mutational analysis of DJ-1 in Drosophila implicates functional inactivation by oxidative damage and aging.

Inherited mutations in PARK7, the gene encoding DJ-1, are associated with loss of protein function and early-onset parkinsonism. Like human DJ-1 (hDJ-1), Drosophila DJ-1b protects against oxidative insult and is modified with oxidation. We demonstrate that hDJ-1 rescues flies mutant for DJ-1b, and that a conserved cysteine residue in the fly protein (C104, analogous to C106 in hDJ-1) is critical for biological antioxidant function in vivo. Targeted mutagenesis suggests that modification of DJ-1b at this residue inactivates the protective activity of the protein against oxidative stress. Further studies show that DJ-1 modification increases dramatically with age in flies, mice, and humans, with aged flies showing strikingly increased susceptibility to oxidative stress and markedly enhanced DJ-1b modification upon oxidative challenge. Overoxidation of DJ-1 with age and exposure to oxidative toxins may lead to inactivation of DJ-1 function, suggesting a role in susceptibility to sporadic Parkinson's disease.

Apolipoprotein A-I is a selective target for myeloperoxidase-catalyzed oxidation and functional impairment in subjects with cardiovascular disease.

In recent studies we demonstrated that systemic levels of protein-bound nitrotyrosine (NO(2)Tyr) and myeloperoxidase (MPO), a protein that catalyzes generation of nitrating oxidants, serve as independent predictors of atherosclerotic risk, burden, and incident cardiac events. We now show both that apolipoprotein A-I (apoA-I), the primary protein constituent of HDL, is a selective target for MPO-catalyzed nitration and chlorination in vivo and that MPO-catalyzed oxidation of HDL and apoA-I results in selective inhibition in ABCA1-dependent cholesterol efflux from macrophages. Dramatic selective enrichment in NO(2)Tyr and chlorotyrosine (ClTyr) content within apoA-I recovered from serum and human atherosclerotic lesions is noted, and analysis of serum from sequential subjects demonstrates that the NO(2)Tyr and ClTyr contents of apoA-I are markedly higher in individuals with cardiovascular disease (CVD). Analysis of circulating HDL further reveals that higher NO(2)Tyr and ClTyr contents of the lipoprotein are each significantly associated with diminished ABCA1-dependent cholesterol efflux capacity of the lipoprotein. MPO as a likely mechanism for oxidative modification of apoA-I in vivo is apparently facilitated by MPO binding to apoA-I, as revealed by cross-immunoprecipitation studies in plasma, recovery of MPO within HDL-like particles isolated from human atheroma, and identification of a probable contact site between the apoA-I moiety of HDL and MPO. To our knowledge, the present results provide the first direct evidence for apoA-I as a selective target for MPO-catalyzed oxidative modification in human atheroma. They also suggest a potential mechanism for MPO-dependent generation of a proatherogenic dysfunctional form of HDL in vivo.

The trypanothione-thiol system in Trypanosoma cruzi as a key antioxidant mechanism against peroxynitrite-mediated cytotoxicity.

Peroxynitrite, the reaction product between superoxide (O(*2)) and nitric oxide (*NO), is a powerful oxidizing species that contributes to macrophage competence against pathogens. In this context, peroxynitrite appears to play an important role in controlling infection by Trypanosoma cruzi, the unicellular parasite responsible for Chagas disease. T. cruzi contains various enzyme systems for the decomposition of hydroperoxides, all of which involve the participation of the low-molecular-weight dithiol trypanothione (N(1),N(8)-bis(glutathionyl)spermidine) as a critical redox partner. A large fraction of the trypanothione-dependent antioxidant capacity of T. cruzi is linked to the tryparedoxin-tryparedoxin peroxidase system which has critical protein thiol groups. In this report we demonstrate that dihydrotrypanothione is readily consumed during peroxynitrite challenge to cells to yield the corresponding trypanothione disulfide. On the other hand, glutathione, which is present in T. cruzi at lower concentrations than trypanothione, is consumed to a much lesser extent and mainly evolves to glutathione-protein mixed disulfides. The inhibition of glutathione biosynthesis by buthionine sulfoximine, which decreases glutathione concentration to 10% of control after 20 h, neither affects the concentration of dihydrotrypanothione nor sensitizes T. cruzi to peroxynitrite-mediated cytotoxicity. On the other hand, pretreatment of T. cruzi with diamide, which leads to a significant depletion (>70%) of dihydrotrypanothione, largely increases the extent of cellular nitration and inhibition of cell growth caused by peroxynitrite. Altogether, our findings support a key protective role for dihydrotrypanothione and the trypanothione-dependent antioxidant system in T. cruzi against peroxynitrite, which may facilitate the survival of trypanosomes within the oxidative environment of activated macrophages.