Trypanosoma cruzi Needs a Signal Provided by Reactive Oxygen Species to Infect Macrophages.

BACKGROUND: During Trypanosoma cruzi infection, macrophages produce reactive oxygen species (ROS) in a process called respiratory burst. Several works have aimed to elucidate the role of ROS during T. cruzi infection and the results obtained are sometimes contradictory. T. cruzi has a highly efficiently regulated antioxidant machinery to deal with the oxidative burst, but the parasite macromolecules, particularly DNA, may still suffer oxidative damage. Guanine (G) is the most vulnerable base and its oxidation results in formation of 8-oxoG, a cellular marker of oxidative stress. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the contribution of ROS in T. cruzi survival and infection, we utilized mice deficient in the gp91phox (Phox KO) subunit of NADPH oxidase and parasites that overexpress the enzyme EcMutT (from Escherichia coli) or TcMTH (from T. cruzi), which is responsible for removing 8-oxo-dGTP from the nucleotide pool. The modified parasites presented enhanced replication inside murine inflammatory macrophages from C57BL/6 WT mice when compared with control parasites. Interestingly, when Phox KO macrophages were infected with these parasites, we observed a decreased number of all parasites when compared with macrophages from C57BL/6 WT. Scavengers for ROS also decreased parasite growth in WT macrophages. In addition, treatment of macrophages or parasites with hydrogen peroxide increased parasite replication in Phox KO mice and in vivo. CONCLUSIONS: Our results indicate a paradoxical role for ROS since modified parasites multiply better inside macrophages, but proliferation is significantly reduced when ROS is removed from the host cell. Our findings suggest that ROS can work like a signaling molecule, contributing to T. cruzi growth inside the cells.

Strong Associations Exist among Oxidative Stress and Antioxidant Biomarkers in the Circulating, Cellular and Urinary Anatomical Compartments in Guatemalan Children from the Western Highlands.

BACKGROUND: A series of antioxidant enzymes and non-enzymatic compounds act to protect cells from uncontrolled propagation of free radicals. It is poorly understood, though, to what extent and how their interaction is harmonized. OBJECTIVES: To explore associative interactions among a battery of urinary and blood biomarkers of oxidative stress and enzymatic and non-enzymatic markers of the antioxidant defense system in children from low income households. METHODS: For this cross-sectional descriptive study, urine, red cells, and plasma were sampled in 82 preschool children attending three daycare centers in Quetzaltenango Guatemala. The urinary oxidative stress biomarkers studied were F2-isoprostanes and 8-hydroxy-deoxy-guanosine. Red cell enzyme activities measured were: catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase. Circulating non-enzymatic antioxidants selected were: retinol, tocopherols, ß-carotene and coenzymes Q9 and Q10. RESULTS: In a Spearman rank-order correlation hemi-matrix, of 55 paired combinations of the 11 biomarkers, 28 (51%) were significantly correlated among each other (p ≤ 0.05), with the strongest association being retinol and tocopherols (r = 0.697, p<0.001), and 4 associations (9%) showed a trend (p> 0.5 to ≤ 0.10). F2-isoprostanes showed the greatest number of cross-associations, having significant interactions with 8 of the 10 remaining biomarkers. Goodness-of-fit modeling improved or maintained the r value for 24 of the significant interactions and for one of the 5 borderline associations. Multiple regression backward stepwise analysis indicated that plasma retinol, ß-carotene and coenzyme Q10 were independent predictors of urinary F2-isoprostanes. CONCLUSION: Numerous significant associations resulted among biomarkers of oxidation and responders to oxidation. Interesting findings were the apparent patterns of harmonious interactions among the elements of the oxidation-antioxidation systems in this population.

Photosensitizing properties of biopterin and its photoproducts using 2'-deoxyguanosine 5'-monophosphate as an oxidizable target.

UV-A radiation (320-400 nm) induces damage to the DNA molecule and its components through photosensitized reactions. Biopterin (Bip) and its photoproducts 6-formylpterin (Fop) and 6-carboxypterin (Cap) accumulate in the skin of human beings suffering from vitiligo, a depigmentation disorder where the protection against UV radiation fails because of the lack of melanin. This study was aimed to evaluate the photosensitizing properties of oxidized pterins present in the skin and to elucidate the mechanisms involved in the photosensitized oxidation of purine nucleotides by pterins in vitro. For this purpose, steady-state and time-resolved experiments in acidic (pH 5.0-5.8) aqueous solution were performed using Bip, Fop and Cap as photosensitizers and the nucleotide 2'-deoxyguanosine 5'-monophosphate (dGMP) as an oxidizable target. The three pterin derivatives are able to photosensitize dGMP, being Fop the most efficient sensitizer. The reactions proceed through two competing pathways: (1) electron transfer from dGMP to triplet excited-state of pterins (type I mechanism) and (2) reaction of dGMP with (1)O(2) produced by pterins (type II mechanism). Kinetic analysis revealed that the electron transfer pathway is the main mechanism and the interaction of dGMP with the triplet excited-state of pterins and the formation of the corresponding dGMP radicals were demonstrated by laser flash photolysis experiments. The biological implications of the results obtained are also discussed.

The photosensitizing activity of lumazine using 2'-deoxyguanosine 5'-monophosphate and HeLa cells as targets.

Lumazines are an important family of heterocyclic compounds present in biological systems as biosynthetic precursors and/or products of metabolic degradation. Upon UV irradiation, the specific compound called lumazine (pteridine-2,4(1,3H)-dione) is able to generate singlet oxygen (1O2), which is one of the main chemical species responsible for photodynamic effects. To further assess the photosensitizing capability of lumazine (Lum) experiments were performed using the nucleotide 2'-deoxyguanosine 5'-monophosphate (dGMP) and, independently, cervical cancer cells (HeLa cell line) as targets. In the dGMP experiments, the data revealed that dGMP indeed undergoes oxidation/oxygenation photoinduced by Lum. Moreover, dGMP disappearance proceeds through two competing pathways: (1) electron transfer between dGMP and excited-state Lum (Type I process) and (2) reaction of dGMP with 1O2 produced by Lum (Type II process). The multistep processes involved are convoluted and susceptible to changes in experimental conditions. The independent studies with HeLa cells included fluorescence analysis of cell extracts and phototoxicity experiments performed at the single-cell level. Results showed that, upon Lum uptake and irradiation, photodynamic effects occur. In particular, the mitochondria and cell membrane were perturbed, both of which reflect key stages in cell death. The data reported herein illustrate how the irradiation of an endogenous biological compound can have various effects which, depending on the system, can be manifested in different ways.

Quenching of the fluorescence of aromatic pterins by deoxynucleotides.

Steady-state and time-resolved studies of the fluorescence of four aromatic unconjugated pterins (pterin (Ptr), 6-(hydroxymethyl)pterin (Hmp), 6-methylpterin (Mep), and 6,7-dimethylpterin (Dmp)) in aqueous solutions in the presence of different nucleotides (2'-deoxyguanosine 5'-monophosphate (dGMP), 2'-deoxyadenosine 5'-monophosphate (dAMP), and 2'-deoxycytosine 5'-monophosphate (dCMP)) have been performed using the single-photon counting technique. The singlet excited states of acid forms of pterins are deactivated by purine nucleotides (dGMP and dAMP) via a combination of dynamic and static processes. The efficiency of the dynamic quenching is high, independently of the nature of the purine base of the nucleotide and of the chemical structure of the substituents linked to the pterin moiety. Analysis of the static quenching indicates that ground-state association between pterins and purine nucleotides takes place, but the formation of the corresponding complexes is significant only at relatively high reactant concentrations. The quenching of the fluorescence of acid forms of pterin derivatives by dCMP, a pyrimidine nucleotide, is slightly less efficient than the quenching by purine nucleotides and is purely dynamic. In alkaline media, the fluorescence quenching is much less efficient than in acidic media, the deactivation by purine nucleotides being purely dynamic, whereas quenching by dCMP is negligible. Possible mechanisms for the quenching of fluorescence of pterin derivatives by the different nucleotides are discussed.

Oxidation of 2'-deoxyguanosine 5'-monophosphate photoinduced by pterin: type I versus type II mechanism.

UV-A radiation (320-400 nm) induces damage to the DNA molecule and its components through different photosensitized reactions. Among these processes, photosensitized oxidations may occur through electron transfer or hydrogen abstraction (type I) and/or the production of singlet molecular oxygen ((1)O2) (type II). Pterins, heterocyclic compounds widespread in biological systems, participate in relevant biological processes and are able to act as photosensitizers. We have investigated the photosensitized oxidation of 2'-deoxyguanosine 5'-monophosphate (dGMP) by pterin (PT) in aqueous solution under UV-A irrradiation. Kinetic analysis was employed to evaluate the participation of both types of mechanism under different pH conditions. The rate constant of (1)O2 total quenching (k(t)) by dGMP was determined by steady-state analysis of the (1)O2 NIR luminescence, whereas the rate constant of the chemical reaction between (1)O2 and dGMP (k(r)) was evaluated from kinetic analysis of concentration profiles obtained by HPLC. The results show that the oxidation of dGMP photosensitized by PT occurs through two competing mechanisms that contribute in different proportions depending on the pH. The dominant mechanism in alkaline media involves the reaction of dGMP with (1)O2 produced by energy transfer from the PT triplet state to molecular oxygen (type II). In contrast, under acidic pH conditions, where PT and the guanine moiety of dGMP are not ionized, the main pathway for dGMP oxidation involves an initial electron transfer between dGMP and the PT triplet state (type I mechanism). The biological implications of the results obtained are also discussed.

Applications of capillary electrophoresis with laser-induced fluorescence for analysis of dGMP-BPDE adduct.

DNA adducts are thought to be crucial to the initiation of mutational and carcinogenic processes. Polycyclic aromatic hydrocarbons (PAHs) have been identified as one major source of carcinogenic risk since they can bind to DNA thus forming an adduct. Quantification of this adduct is important because it may correlate to the risk for cancer development. In this study, the adduct formed between 2'-deoxyguanosine 5'-monophosphate and benzo[ a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) was analyzed by capillary electrophoresis. Both capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) modes with laser-induced fluorescence detection were used for the separation and analysis of DNA adducts. The exploration of capillary electrophoresis in several modes provided different separation mechanisms in which the stereochemical forms of the adduct could be separated. The best result obtained was using a coated fused-silica capillary in Tris-TAPS buffer, which provided high sensitivity with a detection limit of 2.5x10(-9) mol L(-1). MECC separation of the BPDE adduct, although less sensitive, provided an efficient enantioselective separation option.