Lifestyle and Sense of Coherence: A comparative analysis among university students in different areas of knowledge.

BACKGROUND: The concept of health has undergone profound changes. Lifestyle Medicine consists of therapeutic approaches that focus on the prevention and treatment of diseases. It follows that the quality of life of university students directly affects their health and educational progress. EXPERIMENTAL METHODOLOGY: Socioeconomic, lifestyle (LS), and Salutogenesis Theory/sense of coherence (SOC) questionnaires were administered to college students from three different areas. The results were analyzed for normality and homogeneity, followed by ANOVA variance analysis and Dunn and Tukey post hoc test for multiple comparisons. Spearman's correlation coefficient evaluated the correlation between lifestyle and sense of coherence; p values < 0.05 were considered statistically significant. RESULTS: The correlation between LS and SOC was higher among males and higher among Medical and Human sciences students compared to Exact sciences. Medical students' scores were higher than Applied sciences and Human sciences students on the LS questionnaire. Exact science students' scores on the SOC questionnaire were higher than Human sciences students. In the LS areas related to alcohol intake, sleeping quality, and behavior, there were no differences between the areas. However, women scored better in the nutrition domain and alcohol intake. The SOC was also higher in men compared to women. CONCLUSION: The results obtained demonstrate in an unprecedented way in the literature that the correlation between the LS and SOC of college students varies according to gender and areas of knowledge, reflecting the importance of actions on improving students' quality of life and enabling better academic performance.

High prevalence of gastroschisis in Brazilian triple side border: A socioenvironmental spatial analysis.

This research investigated the spatial association between socioenvironmental factors and gastroschisis in Brazilian triple side border. A geographic analysis for gastroschisis prevalence was performed considering census sector units using Global Moran Index, Local Indicator of Spatial Association Analysis and Getis Ord statistics. Sociodemographic factors included rate of adolescent and parturients over 35 years; population with no income and above 5 minimum wages; rate of late prenatal; and proximity to power transmission lines. Logistic regression models were applied to verify the association between socio-environmental factors and prevalence of gastroschisis. No global spatial correlation was observed in the distribution of gastroschisis (Moran´s I = 0.006; p = 0.319). However, multiple logistic regression showed census sectors with positive cases had higher probability to power transmission lines proximity (OR 3,47; CI 95% 1,11-10,79; p = 0,031). Yet, spatial scan statistic showed low risk for gastroschisis in southern city region (OR = 0; p = 0.035) in opposite to power transmission lines location. The study design does not allow us to attest the causality between power transmission lines and gastroschisis but these findings support the potential exposure risk of pregnant to electromagnetic fields.

Changes in Peripheral Blood Biomarkers with Aging and Neurodegenerative Disorders.

BACKGROUND: Aging is associated with most neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. Determination of peripheral blood biomarkers represents a low invasive approach for tracking early changes in body metabolism during aging. OBJECTIVE: This study addresses a cross-sectional analysis to identify changes in lipid, minerals, and antioxidant capacity as potential biomarkers to the onset of neurodegenerative diseases during aging. METHODS: A retrospective analysis was performed to determine age-related biomarkers from a clinical sample database. Next, one hundred volunteers between 20-59 (adult) and over 60 years (elderly) were selected for the motor and cognitive tests according to Unified Parkinson's Disease Rating Scale (UPDRS) and Mini-Mental State Examination (MMSE), respectively. Peripheral blood samples were also collected to determine circulating lipids, minerals, and antioxidant capacity. RESULTS: Lipid profile revealed an increase in triglycerides, total, and VLDL Cholesterol. Among the elderlies, HDL was lower than the adult group, particularly in volunteers with severe cognitive decline. Minerals involved in antioxidant defense such as iron, selenium, and manganese were lower in elderly people compared to adults. Catalase activity was also reduced among elderlies with mild cognitive impairment. CONCLUSION: Here, we show changes in key serum biomarkers correlate with aging and clinical cognitive decline among elderly people. These findings may contribute to the understanding of how biomarkers can be useful for early diagnosis and treatment of aging-related diseases.

Major birth defects in the Brazilian side of the triple border: a population-based cross-sectional study.

BACKGROUND: Major birth defects increase the risk of fetal death and pediatric hospitalization, which also impact on healthcare costs. Sociodemographic factors can drastically affect reproductive health and be used to discriminate the exposure to hidden risk factors. Foz do Iguassu is a Brazilian city located in the triple-border region of Brazil / Paraguay / Argentina with high rates of birth defects. However no study aimed to verify factors associated with this incidence or preventive care is reported. The current work investigated the prevalence of major birth defects and its association with maternal sociodemographic factors in Foz do Iguassu. METHODS: In this population-based cross-sectional study we used data of all live births occurred in Foz do Iguassu from 2012 to 2017. The associated sociodemographic variables such as maternal age, maternal education, maternal race, country of residence, maternal parity and onset of prenatal care were analyzed. Each major birth defect was described according to absolute and relative frequencies, Kruskal-Wallis and logistic regression models were used to evaluate variables associated with selected birth defects. RESULTS: The most prevalent major birth defects were Cleft Lip and/or Palate (9.5/10,000), gastroschisis (6.93/10,000), spina bifida (5.53/10,000), hydrocephalus (5.53/10,000), hypospadias (4.55/10,000), Down syndrome (4.23/10,000), anencephaly (2.93/10,000), anorectal atresia / stenosis (1.95/10,000), undetermined sex (1.95/10,000), esophageal atresia / stenosis with or without fistula (1.63/10,000) and limb reduction defects (1.30/10,000). Maternal age was associated with gastroschisis and Down syndrome. Only maternal education up to 7 years was statistically associated with major birth defects considering all other sociodemographic variables. CONCLUSION: Cleft Lip and/or Palate and Gastroschisis prevalence were higher than those found in the literature. This findings may suggest a distinct epidemiological behavior regarding major birth defects in the region. The work opens new perspectives for birth defects risk factors in the triple-border.

Treatment with sodium nitroprusside improves the endothelial function in aortic rings with endothelial dysfunction.

PURPOSE: Verify if sodium nitroprusside (SNP) is able to improve endothelial function and if this effect is independent of nitric oxide (NO) release of the compound. METHODS: Normotensive (2K) and hypertensive (2K-1C) wistar rats were used. Intact endothelium aortas were placed in a myograph and incubated with SNP: 0.1nM; 1nM or 10nM during 30min. Cumulative concentration-effect curves for acetylcholine (Ach) were realized to measure the relaxing capacity. Intracellular NO were measured (by DAF-2DA probe) in HUVEC treated with SNP 0.1nM or DETA/NO 0.1µM. The detection of intracellular superoxide radical (O2•-) was obtained by using DHE probe. RESULTS: Treatment of 2K-1C aortic rings with SNP (0.1; 1.0 and 10nM) improved endothelium dependent relaxation induced by acetylcholine. This improvement induced by SNP was verified at the concentration of 0.1nM, which does not release NO, suggesting that this effect was not induced due to NO release by SNP compound. Besides, we show that the cell treatment with 0.1nM of SNP decreased the fluorescence intensity to DHE in cells stimulated with angiotensin II. These results indicate that SNP decreases the concentration of O2•- in HUVEC cells. CONCLUSIONS: The SNP at a concentration that does not release NO inside the cells is able to attenuate endothelial dysfunction. DRUGS AND CHEMICALS: Acetylcholine (Ach) (PubChem CID:6060); angiotensin II human (Ang II) (PubChem CID: 16211177); diethylenetriamine/nitric oxide (DETA-NO) (PubChem CID 4518); dihydroethidium (DHE) (PubChem CID: 128682); phenylephrine (Phe) (PubChem CID: 5284443); sodium nitroprusside (SNP) (PubChem CID: 11963579); Thiazolyl Blue Tetrazolium Bromide (MTT) (PubChem CID: 64965); 4,5-diaminofluorescein diacetate (DAF-2DA); 4-hidroxy-Tempo (Tempol) (PubChem CID: 137994), were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Metabolic profiling of human endothelial cells during autophagy assessed in a biomimetic microfluidic device model.

AIMS: Autophagy is critical to endothelial function. We explored the effects of autophagy induced by serum deprivation on Human Umbilical Vascular Endothelial Cells (HUVEC) metabolome profile and its inhibition by the antimalarial drug chloroquine (CLQ) using a microfluidic biomimetic model. MAIN METHODS: The metabolites secreted by HUVEC into the circulating microfluidics were determined by liquid chromatography mass spectrometry (LC-MS) and further analyzed using Metaboanalyst 3.0 multivariate and pathway analysis tools. KEY FINDINGS: Principal component analysis showed the discrimination of metabolites between treated and control groups. The results also identified alterations in metabolites relevant to endothelial function such as arginine, glutamate and energy metabolism pathways. Interestingly, CLQ mostly reversed the changes induced by serum deprivation. SIGNIFICANCE: The knowledge of endothelial metabolic profile during autophagy may contribute to the identification of clinical biomarkers and potential therapeutic approaches based on the regulation of autophagy.

In vitro Treatment with cis-[Ru(H-dcbpy-)2(Cl)(NO)] Improves the Endothelial Function in Aortic Rings with Endothelial Dysfunction.

PURPOSE: The ruthenium complex cis-[Ru(H-dcbpy-)2(Cl)(NO)] (DCBPY) is a nitric oxide (NO) donor and studies suggested that the ruthenium compounds can inactivate O2-. The aim of this study is to test if DCBPY can revert and/or prevent the endothelial dysfunction. METHODS: Normotensive (2K) and hypertensive (2K-1C) wistar rats were used. To vascular reactivity study, thoracic aortas were isolated, rings with intact endothelium were incubated with: DCBPY: 0.1; 1 and 10µM, DCBPY plus hydroxocobalin (NO scavenger) or tempol during 30 minutes, and concentration effect curves to acetylcholine were performed. The potency values (pD2) and maximum effect (ME) were analyzed. The O2- was generated using hypoxantine xantine oxidase and the reduction of cytochrome c, NO consumption by O2- and the effect in avoid NO consumption was measured. RESULTS: In 2K-1C DCBPY at 0.1; 1 or 10µM improved the relaxation endothelium dependent induced by acetylcholine in aortic rings compared to control 2K-1C, and also improved ME. In rings from 2K incubation with DCBPY (0.1; 1.0 and 10 µM) did not change pD2 or ME. Incubation with 0.1 µM of DCBPY plus hydroxocobalamin did not modify the potency and ME in 2K-1C compared to DCBPY (0.1 µM). DCBPY and SOD inhibits the reduction of cytochrome c and inhibited the NO consumption by O2-, showing that O2- has been removed from the solution. CONCLUSION: Our results suggest that DCBPY at a lower concentration (0.1 µM) is not an NO generator, but can inactivate superoxide and improves the endothelial function.

Inhibition of autophagy by chloroquine stimulates nitric oxide production and protects endothelial function during serum deprivation.

BACKGROUND/AIMS: Autophagy plays a fundamental role in cell survival under stress conditions such as nutrient deprivation. Decreased nitric oxide (NO) production, which may contribute to vascular dysfunction, is one of the consequences of autophagy in endothelial cells. The antimalarial drug chloroquine (CLQ) inhibits autophagy by blocking autophagosome formation and has been proposed as adjuvant chemotherapy in other diseases. METHODS: Autophagy was induced by serum deprivation in Human Umbilical Vascular Endothelial Cells (HUVEC) as demonstrated by formation of Acidic Vesicular Organelles (AVOs), conversion of Microtubule-associated protein 1 light chain (LC3), and Sequestosome-1 (SQTM1/p62) degradation. Using endothelium-dependent vasorelaxation assays, intracellular NO production in an ex vivo rat aortic ring model pre-constricted with phenylephrine was estimated along with DAF-2 DA cell membrane-permeable NO sensitive fluorescent dye. RESULTS: The inhibition of autophagy by CLQ restored NO levels, protected against superoxide generation and preserved morphology as well as proliferation of HUVEC under serum deprivation. Interestingly, the incubation of rat aortic rings with CLQ resulted in endothelium-dependent relaxation mediated by the increase of NO. CONCLUSION: These findings emphasize the importance of autophagy in endothelial function and demonstrate the potential use of autophagy inhibitors to protect vascular function during nutrient deprivation.

Testosterone induces apoptosis in vascular smooth muscle cells via extrinsic apoptotic pathway with mitochondria-generated reactive oxygen species involvement.

Testosterone exerts both beneficial and harmful effects on the cardiovascular system. Considering that testosterone induces reactive oxygen species (ROS) generation and ROS activate cell death signaling pathways, we tested the hypothesis that testosterone induces apoptosis in vascular smooth muscle cells (VSMCs) via mitochondria-dependent ROS generation. Potential mechanisms were addressed. Cultured VSMCs were stimulated with testosterone (10(-7) mol/l) or vehicle (2-12 h) in the presence of flutamide (10(-5) mol/l), CCCP (10(-6) mol/l), mimetic manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP; 3 × 10(-5) mol/l), Z-Ile-Glu(O-ME)-Thr-Asp(O-Me) fluoromethyl ketone (Z-IETD-FMK; 10(-5) mol/l), or vehicle. ROS were determined with lucigenin and dichlorodihydrofluorescein; apoptosis, with annexin V and calcein; O2 consumption, with a Clark-type electrode, and procaspases, caspases, cytochrome c, Bax, and Bcl-2 levels by immunoblotting. Testosterone induced ROS generation (relative light units/mg protein, 2 h; 162.6 ± 16 vs. 100) and procaspase-3 activation [arbitrary units, (AU), 6 h; 166.2 ± 19 vs. 100]. CCCP, MnTMPyP, and flutamide abolished these effects. Testosterone increased annexin-V fluorescence (AU, 197.6 ± 21.5 vs. 100) and decreased calcein fluorescence (AU, 34.4 ± 6.4 vs. 100), and O2 consumption (nmol O2/min, 18.6 ± 2.0 vs. 34.4 ± 3.9). Testosterone also reduced Bax-to-Bcl-2 ratio but not cytochrome-c release from mitochondria. Moreover, testosterone (6 h) induced cleavage of procaspase 8 (AU, 161.1 ± 13.5 vs. 100) and increased gene expression of Fas ligand (2(ΔΔCt), 3.6 ± 1.2 vs. 0.7 ± 0.5), and TNF-α (1.7 ± 0.4 vs. 0.3 ± 0.1). CCCP, MnTMPyP, and flutamide abolished these effects. These data indicate that testosterone induces apoptosis in VSMCs via the extrinsic apoptotic pathway with the involvement of androgen receptor activation and mitochondria-generated ROS.

Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia.

BACKGROUND: We have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoK(ATP) protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoK(ATP) activity and evaluate the influence of this trait on cell redox state and viability. METHODS: Oxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells. RESULTS: HTG mice mononuclear cells displayed increased mitoK(ATP) activity, as evidenced by higher resting respiration rates that were sensitive to mitoK(ATP) antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoK(ATP) further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice. CONCLUSIONS: These results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoK(ATP) channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoK(ATP) channels. Thus, mitoK(ATP) opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.

SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates.

Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase 2 (ALDH2) and glutathione S-transferase pi 1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatin A increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G(0) /G(1) and S in HEK293 cells, whereas HEK293/SET showed G(2) /M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.

SET protein accumulates in HNSCC and contributes to cell survival: antioxidant defense, Akt phosphorylation and AVOs acidification.

OBJECTIVES: Determination of the SET protein levels in head and neck squamous cell carcinoma (HNSCC) tissue samples and the SET role in cell survival and response to oxidative stress in HNSCC cell lineages. MATERIALS AND METHODS: SET protein was analyzed in 372 HNSCC tissue samples by immunohistochemistry using tissue microarray and HNSCC cell lineages. Oxidative stress was induced with the pro-oxidant tert-butylhydroperoxide (50 and 250µM) in the HNSCC HN13 cell lineage either with (siSET) or without (siNC) SET knockdown. Cell viability was evaluated by trypan blue exclusion and annexin V/propidium iodide assays. It was assessed caspase-3 and -9, PARP-1, DNA fragmentation, NM23-H1, SET, Akt and phosphorylated Akt (p-Akt) status. Acidic vesicular organelles (AVOs) were assessed by the acridine orange assay. Glutathione levels and transcripts of antioxidant genes were assayed by fluorometry and real time PCR, respectively. RESULTS: SET levels were up-regulated in 97% tumor tissue samples and in HNSCC cell lineages. SiSET in HN13 cells (i) promoted cell death but did not induced caspases, PARP-1 cleavage or DNA fragmentation, and (ii) decreased resistance to death induced by oxidative stress, indicating SET involvement through caspase-independent mechanism. The red fluorescence induced by siSET in HN13 cells in the acridine orange assay suggests SET-dependent prevention of AVOs acidification. NM23-H1 protein was restricted to the cytoplasm of siSET/siNC HN13 cells under oxidative stress, in association with decrease of cleaved SET levels. In the presence of oxidative stress, siNC HN13 cells showed lower GSH antioxidant defense (GSH/GSSG ratio) but higher expression of the antioxidant genes PRDX6, SOD2 and TXN compared to siSET HN13 cells. Still under oxidative stress, p-Akt levels were increased in siNC HN13 cells but not in siSET HN13, indicating its involvement in HN13 cell survival. Similar results for the main SET effects were observed in HN12 and CAL 27 cell lineages, except that HN13 cells were more resistant to death. CONCLUSION: SET is potential (i) marker for HNSCC associated with cancer cell resistance and (ii) new target in cancer therapy.

Release of NO from a nitrosyl ruthenium complex through oxidation of mitochondrial NADH and effects on mitochondria.

Nitrosyl ruthenium complexes are promising NO donor agents with numerous advantages for the biologic applications of NO. We have characterized the NO release from the nitrosyl ruthenium complex [Ru(NO(2))(bpy)(2)(4-pic)](+) (I) and the reactive oxygen/nitrogen species (ROS/RNS)-mediated NO actions on isolated rat liver mitochondria. The results indicated that oxidation of mitochondrial NADH promotes NO release from (I) in a manner mediated by NO(2) formation (at neutral pH) as in mammalian cells, followed by an oxygen atom transfer mechanism (OAT). The NO released from (I) uncoupled mitochondria at low concentrations/incubation times and inhibited the respiratory chain at high concentrations/incubation times. In the presence of ROS generated by mitochondria NO gave rise to peroxynitrite, which, in turn, inhibited the respiratory chain and oxidized membrane protein-thiols to elicit a Ca(2+)-independent mitochondrial permeability transition; this process was only partially inhibited by cyclosporine-A, almost fully inhibited by the thiol reagent N-ethylmaleimide (NEM) and fully inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). These actions correlated with the release of cytochrome c from isolated mitochondria as detected by Western blotting analysis. These events, typically involved in cell necrosis and/or apoptosis denote a potential specific action of (I) and analogs against tumor cells via mitochondria-mediated processes.

Accumulation of the SET protein in HEK293T cells and mild oxidative stress: cell survival or death signaling.

SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 µM tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.

The human Suv3 helicase interacts with replication protein A and flap endonuclease 1 in the nucleus.

The hSuv3 (human Suv3) helicase has been shown to be a major player in mitochondrial RNA surveillance and decay, but its physiological role might go beyond this functional niche. hSuv3 has been found to interact with BLM (Bloom's syndrome protein) and WRN (Werner's syndrome protein), members of the RecQ helicase family involved in multiple DNA metabolic processes, and in protection and stabilization of the genome. In the present study, we have addressed the possible role of hSuv3 in genome maintenance by examining its potential association with key interaction partners of the RecQ helicases. By analysis of hSuv3 co-IP (co-immunoprecipitation) complexes, we identify two new interaction partners of hSuv3: the RPA (replication protein A) and FEN1 (flap endonuclease 1). Utilizing an in vitro biochemical assay we find that low amounts of RPA inhibit helicase activity of hSuv3 on a forked substrate. Another single-strand-binding protein, mtSSB (mitochondrial single-strand-binding protein), fails to affect hSuv3 activity, indicating that the functional interaction is specific for hSuv3 and RPA. Further in vitro studies demonstrate that the flap endonuclease activity of FEN1 is stimulated by hSuv3 independently of flap length. hSuv3 is generally thought to be a mitochondrial helicase, but the physical and functional interactions between hSuv3 and known RecQ helicase-associated proteins strengthen the hypothesis that hSuv3 may play a significant role in nuclear DNA metabolism as well.

Characterization of the stimulus for reactive oxygen species generation in calcium-overloaded mitochondria.

We have used two different probes with distinct detection properties, dichlorodihydrofluorescein diacetate and Amplex Red/horseradish peroxidase, as well as different respiratory substrates and electron transport chain inhibitors, to characterize the reactive oxygen species (ROS) generation by the respiratory chain in calcium-overloaded mitochondria. Regardless of the respiratory substrate, calcium stimulated the mitochondrial generation of ROS, which were released at both the mitochondrial-matrix side and the extra-mitochondrial space, in a way insensitive to the mitochondrial permeability transition pores inhibitor cyclosporine A. In glutamate/malate-energized mitochondria, inhibition at complex I or complex III (ubiquinone cycle) similarly modulated ROS generation at either mitochondrial-matrix side or extra-mitochondrial space; this also occurred when the backflow of electrons to complex I in succinate-energized mitochondria was inhibited. On the other hand, in succinate-energized mitochondria the modulation of ROS generation at mitochondrial-matrix side or extra-mitochondrial space depends on the site of complex III which was inhibited. These results allow a straight comparison between the effects of different respiratory substrates and electron transport chain inhibitors on ROS generation at either mitochondrial-matrix side or extra-mitochondrial space in calcium-overloaded mitochondria.

The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: ensuing energetic and oxidative stress implications.

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 µM) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca²âº efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP+ transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds.

Divergent role of heme oxygenase inhibition in the pathogenesis of sepsis.

The reduction of neutrophil migration to an infectious focus is associated with a high mortality in severe sepsis. Previously, we showed that heme oxygenase (HO) products downregulate neutrophil recruitment in a noninfectious inflammatory model. The present study was designed to determine the role of HO in sepsis induced by cecal ligation and puncture (CLP) model. We demonstrated that pretreatment, but not the combination of pretreatment plus posttreatment with zinc protoporphyrin IX (ZnPP IX), an HO inhibitor, prevented the reduction of CXCR2 on circulating neutrophils and the failure of intraperitoneal neutrophil migration to the site of infection. Consequently, bacterial dissemination, systemic inflammatory response, and organ injury were prevented. In addition, pretreatment with the HO inhibitor avoided hypotension and consequently increased survival. Moreover, in mice subjected to severe CLP, the pretreatment, but not the combination of pretreatment plus posttreatment with ZnPP IX, prevented the increase of plasmatic free heme observed in nontreated severe CLP. The administration of exogenous hemin to mice subjected to moderate sepsis consistently increased the mortality rate. Furthermore, hemin resulted in a reduction of neutrophil migration both in vivo and in vitro. Altogether, our results demonstrated that pretreatment with the HO inhibitor prevents the pathological findings in severe CLP. However, the combination of pretreatment plus posttreatment with ZnPP IX enhances sepsis severity because of an increase in circulating levels of heme, which is deleterious to the host tissues and also inhibits neutrophil migration.

The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler.

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 µM) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells.

Impact of adenosine nucleotide translocase (ANT) proline isomerization on Ca2+-induced cysteine relative mobility/mitochondrial permeability transition pore.

Mitochondrial membrane carriers containing proline and cysteine, such as adenine nucleotide translocase (ANT), are potential targets of cyclophilin D (CyP-D) and potential Ca(2+)-induced permeability transition pore (PTP) components or regulators; CyP-D, a mitochondrial peptidyl-prolyl cis-trans isomerase, is the probable target of the PTP inhibitor cyclosporine A (CsA). In the present study, the impact of proline isomerization (from trans to cis) on the mitochondrial membrane carriers containing proline and cysteine was addressed using ANT as model. For this purpose, two different approaches were used: (i) Molecular dynamic (MD) analysis of ANT-Cys(56) relative mobility and (ii) light scattering techniques employing rat liver isolated mitochondria to assess both Ca(2+)-induced ANT conformational change and mitochondrial swelling. ANT-Pro(61) isomerization increased ANT-Cys(56) relative mobility and, moreover, desensitized ANT to the prevention of this effect by ADP. In addition, Ca(2+) induced ANT "c" conformation and opened PTP; while the first effect was fully inhibited, the second was only attenuated by CsA or ADP. Atractyloside (ATR), in turn, stabilized Ca(2+)-induced ANT "c" conformation, rendering the ANT conformational change and PTP opening less sensitive to the inhibition by CsA or ADP. These results suggest that Ca(2+) induces the ANT "c" conformation, apparently associated with PTP opening, but requires the CyP-D peptidyl-prolyl cis-trans isomerase activity for sustaining both effects.