Thiamine deficiency and recovery: impact of recurrent episodes and beneficial effect of treatment with Trolox and dimethyl sulfoxide.

At present, thiamine deficiency (TD) is managed with administration of high doses of thiamine. Even so, severe and permanent neurological disorders can occur in recurrent episodes of TD. In this study, we used a murine model to assess the efficacy of TD recovery treatments using thiamine with or without additional administration of the antioxidant Trolox or the anti-inflammatory dimethyl sulfoxide (DMSO) after a single or recurrent episode of TD. TD was induced for 9 days with deficient chow and pyrithiamine, and the recovery period was 7 days with standard amounts of chow and thiamine, Trolox, and/or DMSO. After these periods, we evaluated behavior, histopathology, and ERK1/2 modulation in the brain. Deficient animals showed reductions in locomotor activity, motor coordination, and spatial memory. Morphologically, after a single episode of TD and recovery, deficient mice showed neuronal vacuolization in the dorsal thalamus and, after two episodes, a reduction in neuronal cell number. These effects were attenuated or reversed by the recovery treatments, mainly in the treatments with thiamine associated with Trolox or DMSO. Deficient animals showed a strong increase in ERK1/2 phosphorylation in the thalamus, hippocampus, and cerebral cortex after one deficiency episode and recovery. Interestingly, after recurrent TD and recovery, ERK1/2 phosphorylation remained high only in the deficient mice treated with thiamine and/or Trolox or thiamine with DMSO. Our data suggest that a protocol for TD treatment with thiamine in conjunction with Trolox or DMSO enhances the recovery of animals and possibly minimizes the late neurological sequelae.

Protective Effect of the Intracellular Content from Potential Probiotic Bacteria against Oxidative Damage Induced by Acrylamide in Human Erythrocytes.

The aim of this study was to assess the protective effect of the intracellular content obtained from potential probiotic bacteria against acrylamide-induced oxidative damage in human erythrocytes. First, the antioxidant properties of 12 potential probiotic strains was evaluated. Two commercial probiotic bacteria were included as reference strains, namely, Lactobacillus casei Shirota and Lactobacillus paracasei 431. Data showed that the intracellular content from four strains, i.e., Lactobacillus fermentum J10, Lactobacillus pentosus J24 and J26, and Lactobacillus pentosus J27, showed higher (P < 0.05) antioxidant capacity in most methods used. Thereafter, the intracellular content of such pre-selected strains was able to prevent the disturbance of the antioxidant system of human erythrocytes exposed to acrylamide, thereby reducing cell disruption and eryptosis development (P < 0.05). Additionally, the degree of oxidative stress in erythrocytes exposed to acrylamide was significantly (P < 0.05) reduced to levels similar to the basal conditions when the intracellular content of Lact. fermentum J10, Lact. pentosus J27, and Lact. paracasei 431 were employed. Hence, our findings suggest that the intracellular contents of specific Lactobacillus strains represent a potential source of metabolites with antioxidant properties that may help reduce the oxidative stress induced by acrylamide in human erythrocytes.

Thiamine Deficiency Modulates p38MAPK and Heme Oxygenase-1 in Mouse Brain: Association with Early Tissue and Behavioral Changes.

Thiamine deficiency (TD) produces severe neurodegenerative lesions. Studies have suggested that primary neurodegenerative events are associated with both oxidative stress and inflammation. Very little is known about the downstream effects on intracellular signaling pathways involved in neuronal death. The primary aim of this work was to evaluate the modulation of p38MAPK and the expression of heme oxygenase 1 (HO-1) in the central nervous system (CNS). Behavioral, metabolic, and morphological parameters were assessed. Mice were separated into six groups: control (Cont), TD with pyrithiamine (Ptd), TD with pyrithiamine and Trolox (Ptd + Tr), TD with pyrithiamine and dimethyl sulfoxide (Ptd + Dmso), Trolox (Tr) and DMSO (Dmso) control groups and treated for 9 days. Control groups received standard feed (AIN-93M), while TD groups received thiamine deficient feed (AIN-93DT). All the groups were subjected to behavioral tests, and CNS samples were collected for cell viability, histopathology and western blot analyses. The Ptd group showed a reduction in weight gain and feed intake, as well as a reduction in locomotor, grooming, and motor coordination activities. Also, Ptd group showed a robust increase in p38MAPK phosphorylation and mild HO-1 expression in the cerebral cortex and thalamus. The Ptd group showed a decreased cell viability, hemorrhage, spongiosis, and astrocytic swelling in the thalamus. Groups treated with Trolox and DMSO displayed diminished p38MAPK phosphorylation in both the structures, as well as attenuated thalamic lesions and behavioral activities. These data suggest that p38MAPK and HO-1 are involved in the TD-induced neurodegeneration in vivo, possibly modulated by oxidative stress and neuroinflammation.

Enhanced Survival in Mice Exposed to Ionizing Radiation by Combination of Gamma-Tocotrienol and Simvastatin.

Ionizing radiation exposure is a major concern for active military service members, as well as civilian population. Considering that the exposure is not predictable, it is imperative that strategies to counteract radiation damage must be discovered. Recent in vitro studies performed in our laboratory demonstrated that the vitamin E analog gamma-tocotrienol (GT3) in combination with cholesterol-lowering drugs (Statins), synergistically induced endothelial thrombomodulin, an anticoagulant with radio-protective efficacy. It was hypothesized that the combination of treatment with both GT3 along with Statins would provide better radiation protection in vivo than each drug individually. CD2F1 mice were injected subcutaneously with either vehicle or single dose of GT3 (200 mg/kg body weight) 24 hours before irradiation followed by oral or subcutaneous administration of various doses of simvastatin (25, 50, and 100 mg/kg body weight) before exposure to lethal doses (11.5 and 12 Gy) of Cobalt-60 (60Co) gamma-irradiation. The combined treatment group exhibited enhanced radiation lethality protection substantially, accelerated white blood cell recovery, and augmented restoration of bone marrow cellularity when compared to the animals treated with either drug exclusively. This information clearly suggests that combined treatment could be used as a safeguard for military personnel from exposure to harmful ionizing radiation.

The effects of Trolox on the quality of sperm from captive squirrel monkey during liquefaction in the extender ACP-118™.

SummaryThe aim of this study was to evaluate the effect of incubating semen for different periods (90, 270 or 450 min) with or without Trolox® (100 or 150 µM) on the quality of sperm from Saimiri collinsi. Sperm motility, vigour, and plasma membrane integrity (PMI) were evaluated in both fresh semen and semen incubated for different time periods, i.e. 90, 270 or 450 min of incubation. Supplementation of semen extender with Trolox® 100 µM improved sperm motility, vigour and PMI for up to 270 min of incubation.

Oxidative Imbalance, Nitrative Stress, and Inflammation in C6 Glial Cells Exposed to Hexacosanoic Acid: Protective Effect of N-acetyl-L-cysteine, Trolox, and Rosuvastatin.

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurometabolic disorder caused by disfunction of the ABCD1 gene, which encodes a peroxisomal protein responsible for the transport of the very long-chain fatty acids from the cytosol into the peroxisome, to undergo ß-oxidation. The mainly accumulated saturated fatty acids are hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0) in tissues and body fluids. This peroxisomal disorder occurs in at least 1 out of 20,000 births. Considering that pathophysiology of this disease is not well characterized yet, and glial cells are widely used in studies of protective mechanisms against neuronal oxidative stress, we investigated oxidative damages and inflammatory effects of vesicles containing lecithin and C26:0, as well as the protection conferred by N-acetyl-L-cysteine (NAC), trolox (TRO), and rosuvastatin (RSV) was assessed. It was verified that glial cells exposed to C26:0 presented oxidative DNA damage (measured by comet assay and endonuclease III repair enzyme), enzymatic oxidative imbalance (high catalase activity), nitrative stress [increased nitric oxide (NO) levels], inflammation [high Interleukin-1beta (IL-1ß) levels], and induced lipid peroxidation (increased isoprostane levels) compared to native glial cells without C26:0 exposure. Furthermore, NAC, TRO, and RSV were capable to mitigate some damages caused by the C26:0 in glial cells. The present work yields experimental evidence that inflammation, oxidative, and nitrative stress may be induced by hexacosanoic acid, the main accumulated metabolite in X-ALD, and that antioxidants might be considered as an adjuvant therapy for this severe neurometabolic disease.

Electrophilic nitroalkene-tocopherol derivatives: synthesis, physicochemical characterization and evaluation of anti-inflammatory signaling responses.

Inflammation plays a major role in the onset and development of chronic non-communicable diseases like obesity, cardiovascular diseases and cancer. Combined, these diseases represent the most common causes of death worldwide, thus development of novel pharmacological approaches is crucial. Electrophilic nitroalkenes derived from fatty acids are formed endogenously and exert anti-inflammatory actions by the modification of proteins involved in inflammation signaling cascades. We have developed novel nitroalkenes derived from α-tocopherol aiming to increase its salutary actions by adding anti-inflammatory properties to a well-known nutraceutical. We synthesized and characterized an α-tocopherol-nitroalkene (NATOH) and two hydrosoluble analogues derived from Trolox (NATxME and NATx0). We analyzed the kinetics of the Michael addition reaction of these compounds with thiols in micellar systems aiming to understand the effect of hydrophobic partition on the reactivity of nitroalkenes. We studied NATxME in vitro showing it exerts non-conventional anti-inflammatory responses by inducing Nrf2-Keap1-dependent gene expression and inhibiting the secretion of NF-κB dependent pro-inflammatory cytokines. NATxME was also effective in vivo, inhibiting neutrophil recruitment in a zebrafish model of inflammation. This work lays the foundation for the rational design of a new therapeutic strategy for the prevention and treatment of metabolic and inflammation-related diseases.

Evaluation of Trypanocidal and Antioxidant Activities of a Selected Series of 3-amidocoumarins.

BACKGROUND: Neglected diseases are becoming more prevalent due to globalization. This has inspired active research in the development of new drugs for the treatment of parasitic diseases such as Chagas disease. OBJECTIVES: With the aim of finding new trypanocidal agents, we report the in vitro evaluation of a new series of 3-amidocoumarins with or without hydroxyl substituents at position 4 of the coumarin ring. METHODS: Electrochemical and biological assays were performed in order to assess the antioxidant and trypanocidal potential of these compounds and to better understand the mechanisms involved in their activity. RESULTS: Most of the studied compounds showed high trypanocidal activity against both epimastigote and trypomastigote forms, with IC50 values in the low micromolar range. Some of them have greater activity and selectivity than the reference compound, nifurtimox. CONCLUSION: Compound 2 is the most active of this series, being also non-cytotoxic against murine RAW 264.7 macrophages. Electrochemical and radical scavenging experiments were carried out, providing new information about the profile of the best derivatives, and the potential therapeutic application of the new 3-amidocoumarins.

Hydrazone Derivatives Enhance Antileishmanial Activity of Thiochroman-4-ones.

Cutaneous leishmaniasis (CL) is a neglected tropical disease, which causes severe skin lesions. Due to the lack of effective vaccines, and toxicity or reduced effectiveness of available drugs in addition to complex and prolonged treatments, there is an urgent need to develop alternatives for the treatment for CL with different mechanisms of action. In our effort to search for new promising hits against Leishmania parasites we prepared 18 acyl hydrazone derivatives of thiochroman-4-ones. Compounds were evaluated for their in vitro antileishmanial activity against the intracellular amastigote form of Leishmania panamensis and cytotoxic activity against human monocytes (U-937 ATCC CRL-1593.2). Our results show that derivatization of the thiochroman-4-ones with acyl hydrazones significantly enhances the antileishmanial activity. Among the compounds tested semicarbazone and thiosemicarbazone derivatives of thioflavanone 19 and 20 displayed the highest antileishmanial activities, with EC50 values of 5.4 and 5.1 µM and low cytotoxicities (100.2 and 50.1 µM respectively), resulting in higher indexes of selectivity (IS).

Phenolic Melatonin-Related Compounds: Their Role as Chemical Protectors against Oxidative Stress.

There is currently no doubt about the serious threat that oxidative stress (OS) poses to human health. Therefore, a crucial strategy to maintain a good health status is to identify molecules capable of offering protection against OS through chemical routes. Based on the known efficiency of the phenolic and melatonin (MLT) families of compounds as antioxidants, it is logical to assume that phenolic MLT-related compounds should be (at least) equally efficient. Unfortunately, they have been less investigated than phenols, MLT and its non-phenolic metabolites in this context. The evidence reviewed here strongly suggests that MLT phenolic derivatives can act as both primary and secondary antioxidants, exerting their protection through diverse chemical routes. They all seem to be better free radical scavengers than MLT and Trolox, while some of them also surpass ascorbic acid and resveratrol. However, there are still many aspects that deserve further investigations for this kind of compounds.

Insight into the messenger role of reactive oxygen intermediates in immunostimulated hemocytes from the scallop Argopecten purpuratus.

Reactive oxygen intermediates (ROI) are metabolites produced by aerobic cells which have been linked to oxidative stress. Evidence reported in vertebrates indicates that ROI can also act as messengers in a variety of cellular signaling pathways, including those involved in innate immunity. In a recent study, an inhibitor of NF-kB transcription factors was identified in the scallop Argopecten purpuratus, and its functional characterization suggested that it may regulate the expression of the big defensin antimicrobial peptide ApBD1. In order to give new insights into the messenger role of ROI in the immune response of bivalve mollusks, the effect of ROI production on gene transcription of ApBD1 was assessed in A. purpuratus. The results showed that 48 h-cultured hemocytes were able to display phagocytic activity and ROI production in response to the ß-glucan zymosan. The immune stimulation also induced the transcription of ApBD1, which was upregulated in cultured hemocytes. After neutralizing the ROI produced by the stimulated hemocytes with the antioxidant trolox, the transcription of ApBD1 was reduced near to base levels. The results suggest a potential messenger role of intracellular ROI on the regulation of ApBD1 transcription during the immune response of scallops.

Chemical constituents and their antibacterial activity from the tropical endophytic fungus Diaporthe sp. F2934.

AIMS: To isolate, characterize and determine the antibacterial activities of compounds produced by the endophytic fungus Diaporthe sp. F2934, cultivated on malt extract agar. METHODS AND RESULTS: The fungus was cultivated aseptically in Petri dishes containing malt extract agar at 25°C for 15 days. Crude extract was obtained from mycelium using ethyl acetate and sonication, and was fractioned using classic chromatography and HPLC. The structures of phomosines and chromanones were established by NMR experiments including HMQC, HMBC and COSY. Their molecular formulas were determined by ESI-TOFMS. We obtained six compounds: (1) 4H-1-benzopyra-4-one-2,3-dihydro-5-hydroxy-2,8-dimetyl, (2) 4H-1-benzopyran-4-one-2,3-dihydro-5-hydroxy-8-(hydroxylmethyl)-2-methyl, (3) 4H-1-benzopyra-4-one-2,3-dihydro-5-methoxyl-2,8-dimetyl, (4) phomosine A, (5) phomosine D and (6) phomosine C. Isolated compounds 1, 2 and 5 were inactive against 15 micro-organisms, but phomosines A and C were active against diverse Gram-negative and Gram-positive bacteria. CONCLUSIONS: A group of new chromanones and known phomosines have been isolated from the genus Diaporthe (Diaporthe sp. F2934). The results obtained confirm the wide chemical diversity produced by endophytic fungi, specifically the genus Diaporthe. In addition, phomosines A and C may be considered as antimicrobial agents that can be used to guide the development of new antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: Our phylogenetic analysis places Diaporthe sp. F2934 as sister to the Diaporthe cynaroidis clade. Three chromanones were isolated and identified, for the first time, using crude extract obtained from Diaporthe F2934. From this extract phomosines A, C and D were also purified. Regarding Staphylococcus aureus, the inhibition zone diameter (IZD) for phomosine A was 20% higher than the standard drug, vancomycin. When cultivated as described here, Diaporthe sp. F2934 produced new and antimicrobial compounds.

Ultrastructure evaluation of goat spermatozoa after freezing in a skim milk-based extender with Trolox supplementation.

The aim of this work was to evaluate the in vitro effect of adding Trolox in freezing extender for goat semen. Ejaculates from five bucks were evaluated, and when approved, the samples were pooled, diluted according to experimental groups [Trolox 0 (control), 30, 60 and 120 nmol ml(-1) ] and frozen in an automated system. Thawed samples (37 °C/30 s) were evaluated for plasma membrane (PMi) and acrosome integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics by CASA system. Spermatozoa ultrastructure was evaluated in fresh and post-thawed semen. No significant difference (P > 0.05) was observed among control and Trolox groups in the analyses of PMi, Aci, MMP and CASA in goat spermatozoa after thawing. Samples of 60 and 120 nmol ml(-1) Trolox groups had a higher percentage of cells that had intact plasma membranes in spermatozoa head than in the other groups, although they did not differ (P > 0.05) before being frozen. A higher percentage (P < 0.05) of spermatozoa with intact mitochondria was observed in fresh semen, control and Trolox 60 nmol ml(-1) groups than in the other groups. Addition of Trolox to skim milk extender at 60 nmol ml(-1) ultrastructurally preserves the plasma membrane and mitochondrial sheath integrity in goat spermatozoa after cryopreservation.

Effect of N-acetylarginine, a metabolite accumulated in hyperargininemia, on parameters of oxidative stress in rats: protective role of vitamins and L-NAME.

In the present investigation, we initially evaluated the in vitro effect of N-acetylarginine on thiobarbituric acid-reactive substances (TBA-RS), total sulfhydryl content and on the activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the blood, kidney and liver of rats. Results showed that N-acetylarginine, at a concentration of 5.0 µM, decreased the activity of CAT in erythrocytes, enhanced TBA-RS in the renal cortex, decreased CAT and SOD activities in the renal medulla and decreased CAT and increased SOD and GSH-Px activities in the liver of 60-day-old rats. Furthermore, we tested the influence of the antioxidants, trolox and ascorbic acid, as well as of the N(ω) -nitro-L-arginine methyl ester (L-NAME) on the effects elicited by N-acetylarginine on the parameters tested. Antioxidants and L-NAME prevented most of the alterations caused by N-acetylarginine on the oxidative stress parameters evaluated. Data indicate that oxidative stress induction is probably mediated by the generation of NO and/or ONOO(-) and other free radicals because L-NAME and antioxidants prevented the effects caused by N-acetylarginine in the blood, renal tissues and liver of rats. Our findings lend support to a potential therapeutic strategy for this condition, which may include the use of appropriate antioxidants for ameliorating the damage caused by N-acetylarginine.

Cell proliferation arrest and redox state status as part of different stages during senescence establishment in mouse fibroblasts.

Senescence phenotype can be achieved by multiple pathways. Most of them involve the activation of negative cell cycle regulators as well as a shift to an oxidative status. However, the exact participation of these events in senescence establishment and maintenance is not completely understood. In this study we investigated the content of three final cell cycle regulators, as well as the redox state in some critical points during the pre-senescent and the full-senescent states. Our results highlight the existence of a critical pre-phase in senescent phenotype establishment, in which cell proliferation stops with the participation of the cell cycle inhibitors, and a second maintenance stage where the exacerbated pro-oxidant state inside the cell induces the physiological decline characteristic in senescent cells.

Vitamin E-analog Trolox prevents endoplasmic reticulum stress in frozen-thawed ovarian tissue of capuchin monkey (Sapajus apella).

Ovarian fragments were exposed to 0.5 M sucrose and 1 M ethylene glycol (freezing solution; FS) with or without selenium or Trolox. Histological and ultrastructural analyses showed that the percentages of normal follicles in control tissue and in tissue after exposure to FS + 50 µM Trolox were similar. Trolox prevented endoplasmic reticulum (ER)-related vacuolization, which is commonly observed in oocytes and stromal tissue after exposure to FS. From the evaluated stress markers, superoxide dismutase 1 (SOD1) was up-regulated in ovarian tissue exposed to FS + 10 ng/ml selenium. Ovarian fragments were subsequently frozen-thawed in the presence of FS with or without 50 µM Trolox, followed by in vitro culture (IVC). Antioxidant capacity in ovarian fragments decreased after freeze-thawing in Trolox-free FS compared with FS + 50 µM Trolox. Although freezing itself minimized the percentage of viable follicles in each solution, Trolox supplementation resulted in higher rates of viable follicles (67 %), even after IVC (61 %). Furthermore, stress markers SOD1 and ERp29 were up-regulated in ovarian tissue frozen-thawed in Trolox-free medium. Relative mRNA expression of growth factors markers was evaluated after freeze-thawing followed by IVC. BMP4, BMP5, CTGF, GDF9 and KL were down-regulated independently of the presence of Trolox in FS but down-regulation was less pronounced in the presence of Trolox. Thus, medium supplementation with 50 µM Trolox prevents ER stress and, consequently, protects ovarian tissue from ER-derived cytoplasmic vacuolization. ERp29 but not ERp60, appears to be a key marker linking stress caused by freezing-thawing and cell vacuolization.

Manganese-exposed developing rats display motor deficits and striatal oxidative stress that are reversed by Trolox.

While manganese (Mn) is essential for proper central nervous system (CNS) development, excessive Mn exposure may lead to neurotoxicity. Mn preferentially accumulates in the basal ganglia, and in adults it may cause Parkinson's disease-like disorder. Compared to adults, younger individuals accumulate greater Mn levels in the CNS and are more vulnerable to its toxicity. Moreover, the mechanisms mediating developmental Mn-induced neurotoxicity are not completely understood. The present study investigated the developmental neurotoxicity elicited by Mn exposure (5, 10 and 20 mg/kg; i.p.) from postnatal day 8 to PN27 in rats. Neurochemical analyses were carried out on PN29, with a particular focus on striatal alterations in intracellular signaling pathways (MAPKs, Akt and DARPP-32), oxidative stress generation and cell death. Motor alterations were evaluated later in life at 3, 4 or 5 weeks of age. Mn exposure (20 mg/kg) increased p38(MAPK) and Akt phosphorylation, but decreased DARPP-32-Thr-34 phosphorylation. Mn (10 and 20 mg/kg) increased caspase activity and F2-isoprostane production (a biological marker of lipid peroxidation). Paralleling the changes in striatal biochemical parameters, Mn (20 mg/kg) also caused motor impairment, evidenced by increased falling latency in the rotarod test, decreased distance traveled and motor speed in the open-field test. Notably, the antioxidant Trolox™ reversed the Mn (20 mg/kg)-dependent augmentation in p38(MAPK) phosphorylation and reduced the Mn (20 mg/kg)-induced caspase activity and F2-isoprostane production. Trolox™ also reversed the Mn-induced motor coordination deficits. These findings are the first to show that long-term exposure to Mn during a critical period of neurodevelopment causes motor coordination dysfunction with parallel increment in oxidative stress markers, p38(MAPK) phosphorylation and caspase activity in the striatum. Moreover, we establish Trolox™ as a potential neuroprotective agent given its efficacy in reversing the Mn-induced neurodevelopmental effects.

In vitro stimulation of oxidative stress by hypoxanthine in blood of rats: prevention by vitamins e plus C and allopurinol.

We herein investigated the in vitro effect of hypoxanthine on the activities of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) in erythrocytes, as well as on thiobarbituric acid-reactive substances (TBA-RS), in the plasma of rats. Results showed that hypoxanthine, when added to the incubation medium, enhanced CAT (10.0 µM), GSH-Px and SOD (8.5 µM and 10.0 µM) activities in erythrocytes of 15-day-old rats, reduced CAT activity (10.0 µM) and enhanced GSH-Px activity (10.0 µM) in erythrocytes of 30-day-old rats, reduced CAT activity (10.0 µM) and enhanced GSH-Px activity (8.5 µM and 10.0 µM) in erythrocytes of 60-day-old rats, as compared to controls. In addition, hypoxanthine (10.0 µM) enhanced TBA-RS levels in the plasma of 30- and 60-day old rats. Furthermore, we also tested the influence of allopurinol, trolox, and ascorbic acid on the effects elicited by hypoxanthine on the antioxidant enzymes and TBA-RS. Allopurinol and/or administration of antioxidants prevented most alterations caused by hypoxanthine in the oxidative stress parameters evaluated. Findings suggest that hypoxanthine alters antioxidant defenses and induces lipid peroxidation in the blood of rats; however, in the presence of allopurinol and antioxidants, some of these alterations in oxidative stress caused are prevented. Data indicate that, in humans, antioxidant administration might serve as a potential adjuvant therapy for ameliorating the damage caused by hypoxanthine.

Phytanic acid disturbs mitochondrial homeostasis in heart of young rats: a possible pathomechanism of cardiomyopathy in Refsum disease.

Phytanic acid (Phyt) accumulates in tissues and biological fluids of patients affected by Refsum disease. Although cardiomyopathy is an important clinical manifestation of this disorder, the mechanisms of heart damage are poorly known. In the present study, we investigated the in vitro effects of Phyt on important parameters of oxidative stress in heart of young rats. Phyt significantly increased thiobarbituric acid-reactive substances levels (P < 0.001) and carbonyl formation (P < 0.01), indicating that this fatty acid induces lipid and protein oxidative damage, respectively. In contrast, Phyt did not alter sulfhydryl oxidation. Phyt also decreased glutathione (GSH) concentrations (P < 0.05), an important non-enzymatic antioxidant defense. Moreover, Phyt increased 2',7'-dichlorofluorescin oxidation (DCFH) (P < 0.01), reflecting increased reactive species generation. We also found that the induced lipid and protein oxidative damage, as well as the decreased GSH levels and increased DCFH oxidation provoked by this fatty acid were prevented or attenuated by the reactive oxygen species scavengers melatonin, trolox, and GSH, but not by the nitric oxide inhibitor N: (ω)-nitro-L: -arginine methyl ester, suggesting that reactive oxygen species were involved in these effects. Next, we verified that Phyt strongly inhibited NADH-cytochrome c oxidoreductase (complex I-III) activity (P < 0.001) in heart supernatants, and decreased membrane potential and the NAD(P)H pool in heart mitochondria, indicating that Phyt acts as a metabolic inhibitor and as an uncoupler of the electron transport chain. Therefore, it can be presumed that disturbance of cellular energy and redox homeostasis induced by Phyt may possibly contribute to the cardiomyopathy found in patients affected by Refsum disease.

Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts.

OBJECTIVE: Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of γ-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes. METHODS: Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer. RESULTS: The cell cycle was arrested in the G(0)/G(1) phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G(0)/G(1) phase and increased cell populations in the G(2)/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts. CONCLUSION: γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.