Serum markers of oxidative stress and endometriosis.

PURPOSE OF INVESTIGATION: To assess the changes secondary to chronic inflammation in women with and without pelvic endometriosis by the determination of serum thiols and carbonyls. MATERIALS AND METHODS: Sixty-seven women with endometriosis consecutively submitted to laparoscopy and 41 women without endometriosis consecutively submitted to tubal ligation (control group) were selected. Serum levels of total thiols and carbonyls were determined in both groups. RESULTS: Patients with endometriosis had significantly lower thiol levels than controls (342.37 +/- 142.09 microM vs 559.60 +/- 294.05 microM) (p < 0.001), as well as significantly lower carbonyl levels (8.97 +/- 3.76 microM vs 16.40 +/- 9.26 microM) (p < 0.001). Other clinical characteristics were not associated with changes in marker levels. The cutoff point established by the ROC curve was 396.44 microM for the thiols, with 73.1% sensitivity and 80.5% specificity, and 14.9 microM for the carbonyls, with 94% sensitivity and 51.2% specificity. CONCLUSIONS: The serum thiol levels revealed an increase in oxidative stress related to the development of pelvic endometriosis.

Increased plasma and endothelial cell expression of chemokines and adhesion molecules in chronic kidney disease.

Chemokines and adhesion molecules are involved in early events of atherogenesis. In the present study, we investigated the effects of the uremic milieu on the expression of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), soluble vascular adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) and their relationship to cardiovascular status. Plasma samples were obtained from patients in different stages of chronic kidney disease (CKD). Cardiovascular status was evaluated by intima-media thickness and endothelial dysfunction by flow mediation dilatation and proteinuria. In vitro studies were performed using human umbilical endothelial cells exposed to uremic plasma or plasma from healthy subjects. MCP-1, IL-8, sVCAM-1 and sICAM-1 levels in plasma and in supernatant were analyzed by enzyme-linked immunosorbent assay. The population consisted of 73 (mean age 57 years; 48% males) CKD patients with glomerular filtration rate (GFR) of 37 +/- 2 ml/min. MCP-1 and sVCAM-1 plasma levels were negatively correlated with GFR (rho = -0.40, p < 0.0005 and rho = -0.42, p < 0.0005, respectively). Fibrinogen was positively correlated with MCP-1, sICAM-1 and sVCAM-1 (rho = 0.33, p < 0.005, rho = 0.32, p < 0.05 and rho = 0.25, p < 0.05, respectively) and ultra-high-sensitivity C-reactive protein was positively correlated with sICAM-1 (rho = 0.25, p < 0.0005). Plasma IL-8 had a significant positive correlation with proteinuria (rho = 0.31, p < 0.01). There was a time- and CKD-stage-dependent MCP-1, IL-8 and sVCAM-1 endothelial expression (p < 0.05). In summary, plasma levels of markers of endothelial cell activation (MCP-1 and sVCAM-1) are increased in more advanced CKD. Exposure of endothelial cells to uremic plasma results in a time- and CKD-stage-dependent increased expression of MCP-1, IL-8 and sVCAM-1, suggesting a link between vascular activation, systemic inflammation and uremic toxicity. Future studies are necessary to investigate whether these biomarkers add predictive value in comparison to the previously described ones. Also, endothelial response to uremic toxicity should be viewed as a potential target for intervention in order to reduce morbidity and mortality in CKD-related cardiovascular disease.

Toxic effects of DDT and methyl mercury on the hepatocytes from Hoplias malabaricus.

Here, we examined the impact of dichlorodiphenyltrichloroethane (DDT) and monomethyl mercury (MeHg) on the redox milieu and survival of hepatocytes from Hoplias malabaricus (traíra). After isolation and attachment of cells, we established one control and four treatments: DDT (50nM of DDT), MeHg I (0.25microM of MeHg), MeHg II (2.5microM of MeHg) and DDT * MeHg I (combination of 50nM of DDT and 0.25microM of MeHg). After four days the exposed hepatocytes presented significantly increased damage in lipids (all treatments), proteins (DDT * MeHg I and MeHg II) and reduced cell viability (all treatments). Also the antioxidant enzymes catalase, glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase and superoxide dismutase were affected. The current data showed that despite of some protective responses, the increased disturbs on membrane lipids and proteins, increased hydrogen peroxide levels, and decreased glutathione concentration and cell viability strongly indicate oxidative stress as the reason of hepatotoxicity due to DDT and MeHg exposure. In addition, DDT and MeHg together had greater effect than alone when G6PDH and glutathione-S-transferase activities and lipids damage were considered. These findings are indicative of hepatotoxicity occurring at realistic concentrations of DDT and MeHg found in Amazonian fish tissues.

Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue.

Mesenchymal stem cells (MSCs) have been investigated as promising candidates for use in new cell-based therapeutic strategies such as mesenchyme-derived tissue repair. MSCs are easily isolated from adult tissues and are not ethically restricted. MSC-related literature, however, is conflicting in relation to MSC differentiation potential and molecular markers. Here we compared MSCs isolated from bone marrow (BM), umbilical cord blood (UCB), and adipose tissue (AT). The isolation efficiency for both BM and AT was 100%, but that from UCB was only 30%. MSCs from these tissues are morphologically and immunophenotypically similar although their differentiation diverges. Differentiation to osteoblasts and chondroblasts was similar among MSCs from all sources, as analyzed by cytochemistry. Adipogenic differentiation showed that UCB-derived MSCs produced few and small lipid vacuoles in contrast to those of BM-derived MSCs and AT-derived stem cells (ADSCs) (arbitrary differentiation values of 245.57 +/- 943 and 243.89 +/- 145.52 mum(2) per nucleus, respectively). The mean area occupied by individual lipid droplets was 7.37 mum(2) for BM-derived MSCs and 2.36 mum(2) for ADSCs, a finding indicating more mature adipocytes in BM-derived MSCs than in treated cultures of ADSCs. We analyzed FAPB4, ALP, and type II collagen gene expression by quantitative polymerase chain reaction to confirm adipogenic, osteogenic, and chondrogenic differentiation, respectively. Results showed that all three sources presented a similar capacity for chondrogenic and osteogenic differentiation and they differed in their adipogenic potential. Therefore, it may be crucial to predetermine the most appropriate MSC source for future clinical applications.

High levels of active quiescin Q6 sulfhydryl oxidase (QSOX) are selectively present in fetal serum.

The participation of thiol-oxidoreductases such as thioredoxin during implantation, embryogenesis and fetal development has been extensively studied. Here, we analyzed the expression of the thioredoxin superfamily enzyme quiescin Q6/sulfhydryl oxidase (QSOX) during development. Results show that QSOX is present in fetal bovine serum (4 months' gestation), but its levels decrease with time after birth (from P1 to P60). We also demonstrate that a sulfhydryl oxidase activity correlates with QSOX expression in such sera, suggesting a putative role in the redox modulation of developmental programs.

Metabolism of acetaldehyde to methyl and acetyl radicals: in vitro and in vivo electron paramagnetic resonance spin-trapping studies.

Acetaldehyde oxidation by enzymes and cellular fractions has been previously shown to produce radicals that have been characterized as superoxide anion, hydroxyl, and acetyl radicals. Here, we report that acetaldehyde metabolism by xanthine oxidase, submitochondrial particles and whole rats produces both the acetyl and the methyl radical, although only the latter was unambiguously identified in vivo. Electron paramagnetic resonance (EPR) characterization of both radicals was possible by the use of two spin traps, 5,5-dimethyl 1-pyrroline N-oxide (DMPO) and alpha-(4-pyridyl 1-oxide)-N-t-butylnitrone (POBN), and of acetaldehyde labeled with (13)C. The POBN-acetyl radical adduct proved to be unstable, but POBN was employed to monitor acetaldehyde metabolism by Sprague-Dawley rats because previous studies have shown its usefulness for in vivo spin trapping. EPR analysis of the bile collected from treated and control rats showed the presence of the POBN-methyl and of an unidentified, biomolecule-derived, POBN adduct. Because decarbonylation of the acetyl radical is one of the routes for methyl radical formation from acetaldehyde, detection of the latter in bile provides strong evidence for the production of both radicals in vivo. The results may be relevant to understanding the toxic effects of acetaldehyde itself and of its more relevant biological precursor, ethanol.

Oxidation of acetaldehyde by peroxynitrite and hydrogen Peroxide/Iron(II). Production Of acetate, formate, and methyl radicals.

Production of free radicals from acetaldehyde oxidation by enzymes and cellular fractions is a well-known process. The toxic effects of acetaldehyde, however, are usually attributed to its reactions with biomolecules to produce adducts. Here, we demonstrate that hypothetical adducts produced from attack of acetaldehyde by two important biological oxidants, peroxynitrite and hydrogen peroxide, decompose to produce acetate, formate, and methyl radicals. Acetate, formate, nitrate, and nitrite were characterized and quantified by capillary electrophoresis. Radicals were detected and quantified by the EPR spectra produced in the presence of spin traps 3, 5-dibromo-4-nitrosobenzenesulfonic acid and 5,5-dimethyl-1-pyrroline N-oxide. Kinetic studies and product analysis were performed at different pHs. The results demonstrate that production of methyl radicals during oxidation of acetaldehyde by hydrogen peroxide was strictly dependent on the presence of iron(II) and occurred via two routes. One involved acetaldehyde attack by the hydroxyl radical to produce the acetyl radical that decomposes to methyl radical and carbon monoxide. The other route involved acetaldehyde attack by deprotonated hydrogen peroxide to produce a hypothetical intermediate that reductively cleaves via the action of present iron(II) to produce radicals. The latter mechanism predominates in the case of peroxynitrite, but radical formation does not require metal ions. Most of the hypothetical adduct produced from acetaldehyde and peroxynitrite (k = 680 M(-)(1) s(-)(1) at pH 7.4 and 37 degrees C) decays to nitrate and regenerates the aldehyde [Uppu, R. M., et al. (1997) Chem. Res. Toxicol. 10, 1331], but about 30% of it produces acetate, formate, and methyl radicals. Part of these oxidized products result from beta-scission and 1,2-shift reactions of the 1-hydroxyethoxyl radical which, together with nitrogen dioxide, freely diffuses from the adduct (20% yields). The results provide yet another example of the metal-independent free radical reactivity of peroxynitrite and may be relevant to the toxic effects associated with heavy drinking and diabetes.

Nucleic acid alkylation by free radical metabolites of ethanol. Formation of 8-(1-hydroxyethyl)guanine and 8-(2-hydroxyethyl)guanine adducts.

Alcohol consumption is associated with an increased risk of several types of malignancy by mechanisms that remain to be elucidated. Most of the ingested ethanol is converted to acetaldehyde but the formation of free radical metabolites such as the 1-hydroxyethyl radical has been also demonstrated to occur in vitro and in vivo. Here we tested the possibility of ethanol-derived free radicals alkylating nucleic acid and nucleic acid components. Ethanol oxidation by Fenton systems has been extensively used to mimic ethanol metabolism to free radical intermediates and it was also employed in our studies. Two adducts, 8-(1-hydroxyethyl)guanine and 8-(2-hydroxyethyl)guanine, were isolated in incubations containing guanine/ethanol/hydrogen peroxide/iron(II) at pH 1 under anaerobic conditions. The adducts were produced in comparable yields and were characterized by ultraviolet absorption, mass spectrometry, and proton nuclear magnetic resonance spectroscopy. Both adducts were also produced in incubations containing DNA and RNA at pH 4 and 7. Under these conditions, the obtained yields of 8-(1-hydroxyethyl)guanine were about 10 times higher than those of 8-(2-hydroxyethyl)guanine. Higher yields of both adducts were obtained at pH 4 than at pH 7 and with RNA as compared with DNA. As expected, nucleic acid oxidation products such as 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydroadenine were also produced under the employed experimental conditions. Their yields tended to increase in the presence of ethanol, particularly at pH 4, suggesting that ethanol can protect oxidized bases from further degradation. Parallel spin-trapping experiments with alpha-4-pyridyl-1-oxide N-tert-butylnitrone and 3,5-dibromo-4-nitrosobenzenesulfonic acid confirmed that ethanol was oxidized to both the 1-hydroxyethyl and 2-hydroxyethyl radicals by hydrogen peroxide/iron(II) at pH 4-7 in the presence and in the absence of nucleic acids. The results demonstrate that free radical metabolites of ethanol can alkylate nucleic acids in vitro. Both the 1-hydroxyethyl and 2-hydroxyethyl radicals may play a role in ethanol-mediated toxicity.