In vitro genotoxicity assessment of functional ingredients: DHA, rutin and α-tocopherol.

The in vitro genotoxicity of three compounds widely used as functional ingredients, docosahexaenoic acid (DHA), rutin and α-tocopherol, was assessed. A miniaturized version of the Ames test in Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains (following the principles of OECD 471), and the in vitro micronucleus test in TK6 cells (OECD 487) were performed. This strategy is recommended by the European Food Safety Authority for the in vitro genotoxicity assessment of food and feed. In addition, this approach was complemented with the in vitro standard and enzyme-modified comet assay (S9-/S9+) using hOGG1, EndoIII and hAAG in order to assess potential premutagenic lesions in TK6 cells. Rutin showed an equivocal response in the in vitro micronucleus test and also was a potent Salmonella typhimurium revertant inductor in the Ames test. DHA showed equivocal results in the in vitro micronucleus test. In this regard, DHA and rutin seemed to interact with the DNA at a chromosomal level, but rutin is also capable of producing frameshift mutations. No genotoxicity was observed in cells treated with α-tocopherol. This article complements the evidence already available about the genotoxicity of these compounds. However, more studies are needed in order to elucidate the consequences of their use as functional ingredients in human health.

α-Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile for gene delivery: Design, synthesis and transfection.

Natural antioxidants and vitamins have potential to protect biological systems from peroxidative damage induced by peroxyl radicals, α-tocopherol (Vitamin E, lipid soluble) and ascorbic acid (vitamin C, water soluble), well known natural antioxidant molecules. In the present study we described the synthesis and biological evaluation of hybrid of these two natural antioxidants with each other via ammonium di-ethylether linker, Toc-As in gene delivery. Two control cationic lipids N14-As and Toc-NOH are designed in such a way that one is with ascorbic acid moiety and no tocopherol moiety; another is with tocopherol moiety and no ascorbic acid moiety respectively. All the three cationic lipids can form self-assembled aggregates. The antioxidant efficiencies of the three lipids were compared with free ascorbic acid. The cationic lipids (Toc-As, N14-As and Toc-NOH) were formulated individually with a well-known fusogenic co-lipid DOPE and characterization studies such as DNA binding, heparin displacement, size, charge, circular dichroism were performed. The biological characterization studies such as cell viability assay and in vitro transfection studies were carried out with the above formulations in HepG2, Neuro-2a, CHO andHEK-293T cell lines. The three formulations showed their transfection efficiencies with highest in Toc-As, moderate inN14-As and least in Toc-NOH. Interestingly, the transfection efficiency observed with the antioxidant based conjugated lipid Toc-As is found to be approximately two and half fold higher than the commercially available lipofectamine 2000 at 4:1 charge ratio in Hep G2 cell lines. In the other cell lines studied the efficiency of Toc-As is found to be either higher or similarly active compared to lipofectamine 2000. The physicochemical characterization results show that Toc-As lipid is showing maximum antioxidant potency, strong binding with pDNA, least size and optimal zeta potential. It is also found to be least toxic in all the cell lines studied especially in Neuro-2a cell lines when compared to other two lipids. In summary, the designed antioxidant lipid can be exploited as a delivering system for treating ROS related diseases such as malignancy, brain stroke, etc.

A Vitamin E-Enriched Antioxidant Diet Interferes with the Acute Adaptation of the Liver to Physical Exercise in Mice.

Physical exercise is beneficial for general health and is an effective treatment for metabolic disorders. Vitamin E is widely used as dietary supplement and is considered to improve non-alcoholic fatty liver disease by reducing inflammation and dyslipidemia. However, increased vitamin E intake may interfere with adaptation to exercise training. Here, we explored how vitamin E alters the acute exercise response of the liver, an organ that plays an essential metabolic role during physical activity. Mice fed a control or an α-tocopherol-enriched diet were subjected to a non-exhaustive treadmill run. We assessed the acute transcriptional response of the liver as well as glucocorticoid signalling and plasma free fatty acids (FFA) and performed indirect calorimetry. Vitamin E interfered with the exercise-induced increase in FFA and upregulation of hepatic metabolic regulators, and it shifted the transcriptional profile of exercised mice towards lipid and cholesterol synthesis while reducing inflammation. Energy utilization, as well as corticosterone levels and signalling were similar, arguing against acute differences in substrate oxidation or glucocorticoid action. Our results show that high-dose vitamin E alters the metabolic and inflammatory response of the liver to physical exercise. The interference with these processes may suggest a cautious use of vitamin E as dietary supplement.

UV-vis degradation of α-tocopherol in a model system and in a cosmetic emulsion-Structural elucidation of photoproducts and toxicological consequences.

The UV-vis photodegradation of α-tocopherol was investigated in a model system and in a cosmetic emulsion. Both gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) and high performance liquid chromatography coupled with ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (LC-UHR-MS) were used for photoproducts structural identification. Nine photoproduct families were detected and identified based on their mass spectra and additional experiments with α-tocopherol-d9; phototransformation mechanisms were postulated to rationalize their formation under irradiation. In silico QSAR (Quantitative Structure Activity Relationship) toxicity predictions were conducted with the Toxicity Estimation Software Tool (T.E.S.T.). Low oral rat LD50 values of 466.78mgkg-1 and 467.9mgkg-1 were predicted for some photoproducts, indicating a potential toxicity more than 10 times greater that of α-tocopherol (5742.54mgkg-1). In vitro assays on Vibrio fischeri bacteria showed that the global ecotoxicity of the α-tocopherol solution significantly increases with irradiation time. One identified product should contribute to this ecotoxicity enhancement since in silico estimations for D. magna provide a LC50 value 4 times lower than that of the parent molecule.

Formulation and physiochemical study of α-tocopherol based oil in water nanoemulsion stabilized with non toxic, biodegradable surfactant: Sodium stearoyl lactate.

The unique properties such as high optical clarity, stability and enhanced bioavailability of nanoemulsion make them useful for food, cosmetic and pharmaceutical industries. In this work, sodium stearoyl lactate and Tween 80 surfactants were collectively used to fabricate alpha tocopherol based oil in water nanoemulsion using high energy ultrasonication method. The spherical nature of pure and drug loaded nanoemulsion has been confirmed with transmission electron microscopy (TEM). The influence of pH, dilution, surfactant concentration and ionic strength on average particle size of pure and nutraceutical (benzylisothiocyanate and curcumin) encapsulated emulsion was examined. The prepared emulsion exhibited good stability up to 90days in salt solution (50-200mM) and different pH conditions. The cumulative release % of benzylisothiocyanate and curcumin was found to be 50.29% in 36h and 89.15% in 150h respectively. The antioxidant activity of pure, benzylisothiocyanate, curcumin and cocktail (benzylisothiocyanate and curcumin) nanoemulsion was calculated with 2,2-diphenyl-1-picrylhydrazyl radical. The IC50 value of different antioxidant showed that benzylisothiocyanate nanoemulsion acted as better antioxidant as compared to pure and curcumin encapsulated nanoemulsion. Also the cell viability of pure nanoemulsion was found to be 24% on hep G2 cell. The effect of UV light irradiation on curcumin and benzylisothiocyanate stability was carried out in different solvent conditions (water/ethanol and nanoemulsion). The degradation of curcumin by the impact of UV light was successfully controlled by trapping in NEm.

THE SENSITIVITY OF CELLS WITH THE VARIOUS LEVEL OF NAD(P)H:QUINONE OXIDOREDUCTASE 1 TO CYTOTOXIC ACTION OF QUINONIMINES AND α-TOCOPHEROL SYNTHETIC DERIVATIVES.

The effects of α-tocopherol with shortened to 6 carbon atoms side chain (α-Toc-C6), α-tocopherol succinate (α-TS) and quinonimine 2,6-dichlorophenolindophenol (DCPIP) on DT-diaphorase activity and viability of rat thymocytes, splenocytes and hepatocytes were investigated. It was shown that the lowest basal activity of the enzyme is inherent in splenocytes. In comparison to splenocytes, DT-diaphorase activity was 1.4 and 5 times higher in thymocytes and hepatocytes, respectively. It was found that the sensitivity of cells to the cytotoxic effect of DCPIP was inversely proportional to the basal level of DT-diaphorase activity and accompanied by its activation with subsequent inhibition at non-toxic and toxic concentrations, respectively. Hepatocytes were least sensitive to the cytotoxic effect of α-Toc-C6. In thymocytes and splenocytes α-Toc-C6 exerts inhibitory effects on DT-diaphorase, whereas in hepatocytes an increased activity of the enzyme was observed, which probably caused their high survival rate. Simultaneous induction of cytochrome P450 enzyme expression by α-Toc-C6 in hepatocytes is also possible. Cytotoxic effect of α-TS does not depend on the basal level of DT-diaphorase activity in cells, is not accompanied by its induction and it is most likely determined by the non-specific esterase activity.

High-Dose α-Tocopherol Supplementation Does Not Induce Bone Loss in Normal Rats.

Oxidative stress affects bone turnover. Preventative effects of antioxidants such as vitamin E on reduced bone mineral density and fractures associated with aging, osteoporosis, and smoking have been examined in animals and humans. The effects of vitamin E (α-tocopherol; αT) on bone health have yielded conflicting and inconclusive results from animal studies. In this study, to determine the bone effects of αT, we investigated the in vivo effects of αT on the bone mineral density, bone mass, bone microstructure, bone resorption, and osteogenesis through peripheral quantitative computed tomography (pQCT) measurements, micro-computed tomography (micro-CT) analyses, and bone histomorphometry of lumbar vertebrae and femurs in normal female Wistar rats fed diets containing αT in different quantities (0, 30, 120, or 600 mg/kg diet) for 8 weeks. To validate our hypotheses regarding bone changes, we examined ovariectomized rats as an osteoporosis model and control sham-operated rats in parallel. As expected, ovariectomized rats had reduced bone mineral density in lumbar vertebrae and the distal metaphyses of their femurs, reduced bone mass and deteriorated microstructure of cancellous bones in the vertebral body and distal femur metaphyses, and reduced bone mass due to resorption-dominant enhanced bone turnover in secondary cancellous bones in these sites. In comparison, αT administered to normal rats, even at the highest dose, did not induce reduced bone mineral density of lumbar vertebrae and femurs or a reduced bone mass or fragile microstructure of cancellous bones of the vertebral body and distal femur metaphyses. Instead, αT-fed rats showed a tendency for an osteogenesis-dominant bone mass increase in secondary cancellous bones in the vertebral body, in which active bone remodeling occurs. Thus, αT consumption may have beneficial effects on bone health.

Non-clinical safety and biodistribution of AS03-adjuvanted inactivated pandemic influenza vaccines.

Pandemic-influenza vaccines containing split-inactivated-virus antigen have been formulated with the immunostimulatory Adjuvant System AS03 to enhance the antigen immunogenicity and reduce antigen content per dose. AS03 is an oil-in-water emulsion containing α-tocopherol, squalene and polysorbate 80. To support the clinical development of AS03-adjuvanted pandemic-influenza vaccines, the local and systemic toxicity of test articles containing split-influenza A(H5N1) and/or AS03 were evaluated after 3-4 intramuscular (i.m.) injections in rabbits. Treatment-related effects were restricted to mild inflammatory responses and were induced primarily by the test articles containing AS03. The injection-site inflammation was mild at 3 days, and minimal at 4 weeks after the last injection; and was reflected by signs of activation in the draining lymph nodes and by systemic effects in the blood including a transient increase of neutrophils. In addition, a study in mice explored the biodistribution of A(H5N1) vaccines or AS03 through radiolabelling the antigen or constituents of AS03 prior to injection. In this evaluation, 57-73% of AS03's principal constituents had cleared from the injection site 3 days after injection, and their different clearance kinetics were suggestive of AS03's dissociation. All these AS03 constituents entered into the draining lymph nodes within 30 min after injection. In conclusion, the administration of repeated doses of the H5N1/AS03 vaccine was well tolerated in the rabbit, and was primarily associated with transient mild inflammation at the injection site and draining lymph nodes. The biodistribution kinetics of AS03 constituents in the mouse were consistent with AS03 inducing this pattern of inflammation.

Excessive α-tocopherol exacerbates microglial activation and brain injury caused by acute ischemic stroke.

The vitamin E family includes both tocopherols and tocotrienols, where α-tocopherol (αTOC) is the most bioavailable form. Clinical trials testing the therapeutic efficacy of high-dose αTOC against stroke have largely failed or reported negative outcomes when a "more is better" approach to supplementation (>400 IU/d) was used. This work addresses mechanisms by which supraphysiologic αTOC may contribute to stroke-induced brain injury. Ischemic stroke injury and the neuroinflammatory response were studied in tocopherol transfer protein-deficient mice maintained on a diet containing αTOC vitamin E at the equivalent human dose of 1680 IU/d. Ischemic stroke-induced brain injury was exacerbated in the presence of supraphysiologic brain αTOC levels. At 48 h after stroke, S100B and RAGE expression was increased in stroke-affected cortex of mice with elevated brain αTOC levels. Such increases were concomitant with aggravated microglial activation and neuroinflammatory signaling. A poststroke increase in markers of oxidative injury and neurodegeneration in the presence of elevated brain αTOC establish that at supraphysiologic levels, αTOC potentiates neuroinflammatory responses to acute ischemic stroke. Exacerbation of microglial activation by excessive αTOC likely depends on its unique cell signaling regulatory properties independent of antioxidant function. Against the background of clinical failure for high-dose αTOC, outcomes of this work identify risk for exacerbating stroke-induced brain injury as a result of supplementing diet with excessive levels of αTOC.

Design and characterization of an ocular topical liposomal preparation to replenish the lipids of the tear film.

PURPOSE: Dry eye (DE) includes a group of diseases related to tear film disorders. Current trends for DE therapy focus on providing lipid components to replace the damaged lipid layer. Formulations that contain aqueous and mucin-like compounds may have additional therapeutic benefits for DE patients. The aim of this work was to design and evaluate novel formulations having the potential to become topical treatment for DE. METHODS: Unpreserved liposomal formulations composed of phosphatidylcholine (PC), cholesterol, and α-tocopherol (vit E) were prepared by the thin-film hydration technique. Formulations were characterized in terms of liposome size, pH, surface tension, osmolarity, and viscosity. In vitro tolerance assays were performed on macrophage, human corneal, and conjunctival cell lines at short- and long-term exposures. In vivo ocular tolerance was studied after instillation of the formulation. RESULTS: The mean liposome size was less than 1 µm and surface tension < 30 mN/m for all formulations. The final liposomal formulation (PC-cholesterol-vit E in a ratio of 8:1:0.8) had physiological values of pH (6.45 ± 0.09), osmolarity (289.43 ± 3.28 mOsm), and viscosity (1.82 ± 0.02 mPa · s). Cell viability was greater than 80% in the corneal and conjunctival cells. This formulation was well tolerated by experimental animals. CONCLUSIONS: The unpreserved liposomal formulation has suitable properties to be administered by a topical ophthalmic route. The liposome-based artificial tear had good in vitro and in vivo tolerance responses. This formulation, composed of a combination of liposomes and bioadhesive polymers, may be used successfully as a tear film substitute in DE therapy.

Safety and pharmacokinetic studies of liposomal antioxidant formulations containing N-acetylcysteine, α-tocopherol or γ-tocopherol in beagle dogs.

The safety and pharmacokinetic profile of liposomal formulations containing combinations of the antioxidants α-tocopherol, γ-tocopherol or N-acetylcysteine in beagle dogs was examined. Each group consisted of beagle dogs of both genders with a control group receiving empty dipalmitoylphosphatidylcholine (DPPC) liposomes (330 mg/kg DPPC, EL), and test groups receiving liposomes prepared from DPPC lipids with (i) N-acetylcysteine (NAC) (60 mg/kg NAC [L-NAC]); (ii) NAC and α-tocopherol (αT) (60 mg/kg NAC and 25 mg/kg α-tocopherol [L-αT-NAC]) and (iii) NAC and γ-tocopherol (60 mg/kg NAC and 25 mg/kg γ-tocopherol (γT) [L-γT-NAC]). The dogs in the control group (EL) and three test groups exhibited no signs of toxicity during the dosing period or day 15 post treatment. Weight gain, feed consumption and clinical pathology findings (hematology, coagulation, clinical chemistry, urinalysis) were unremarkable in all dogs and in all groups. Results from the pharmacokinetic study revealed that the inclusion of tocopherols in the liposomal formulation significantly increased the area under the curve (AUC) and ß-half life for NAC; the tocopherols had greater impact on the clearance of NAC, where reductions of central compartment clearance (CL) ranged from 56% to 60% and reductions of tissue clearance (CL2) ranged from 73% to 77%. In conclusion, there was no treatment-related toxicity in dogs at the maximum feasible dose level by a single bolus intravenous administration while the addition of tocopherols to the liposomal formulation prolonged the circulation of NAC in plasma largely due to a decreased clearance of NAC.

Iodinated α-tocopherol nano-emulsions as non-toxic contrast agents for preclinical X-ray imaging.

Micro-computed tomography (micro-CT) is an emerging imaging modality, due to the low cost of the imagers as well as their efficiency in establishing high-resolution (1-100 µm) three-dimensional images of small laboratory animals and facilitating rapid, structural and functional in vivo visualization. However use of a contrast agent is absolutely necessary when imaging soft tissues. The main limitation of micro-CT is the low efficiency and toxicity of the commercially available blood pool contrast agents. This study proposes new, efficient and non-toxic contrast agents for micro-CT imaging. This formulation consists of iodinated vitamin E (α-tocopheryl 2,3,5-triiodobenzoate) as an oily phase, formulated as liquid nano-emulsion droplets (by low-energy nano-emulsification), surrounded by a hairy PEG layer to confer stealth properties. The originality and strength of these new contrast agents lie not only in their outstanding contrasting properties, biocompatibility and low toxicity, but also in the simplicity of their fabrication: one-step synthesis of highly iodinated oil (iodine constitutes 41.7% of the oil molecule weight) and its spontaneous emulsification. After i.v. administration in mice (8.5% of blood volume), the product shows stealth properties towards the immune system and thus acts as an efficient blood pool contrast agent (t(1/2) = 9.0 h), exhibiting blood clearance following mono-exponential decay. A gradual accumulation predominantly due to hepatocyte uptake is observed and measured in the liver, establishing a strong hepatic contrast, persistent for more than four months. To summarize, in the current range of available or developed contrast agents for preclinical X-ray imaging, this agent appears to be one of the most efficient.

Acute toxicity study of liposomal antioxidant formulations containing N-acetylcysteine, α-tocopherol, and γ-tocopherol in rats.

Liposomes have been used for the delivery of antioxidants to different tissues and organs for the treatment of oxidative stress-induced injuries. In this study, the acute toxicity of a single dose of intravenously (i.v.) administered liposomal antioxidant formulation, containing N-acetylcysteine (NAC) with or without α-tocopherol (α-T) or γ-tocopherol (γ-T), in rats was examined. Each group consisted of 5 male and 5 female Sprague-Dawley rats, with a control group receiving empty dipalmitoylphosphatidylcholine (DPPC) liposomes (660 mg/kg) and test groups receiving DPPC liposomes (660 mg/kg) entrapped with 1) NAC (200 mg/kg), 2) NAC (200 mg/kg) and α-T (83.3 mg/kg), and 3) NAC (200 mg/kg) and γ-T (71.4 mg/kg). These dose levels were determined from the dose-range-finding study and were considered to be the maximum feasible dose (MFD) levels, based on the volume of 10 mL/kg and physical properties and viscosity of the test articles that could be safely administered to rats by an i.v. injection. Two weeks after treatment (day 15), rats in the control group and three test groups exhibited no clinical signs of toxicity during the dosing period or during the 14-day post-treatment period. Weight gain and food consumption in all animals was appropriate for the age and sex of animals. Clinical pathology findings (e.g., hematology, coagulation, clinical chemistry, and urinalysis) were unremarkable in all rats and in all groups. In conclusion, the results of this study showed no treatment-related toxicity in rats at the MFD level by a single bolus i.v. administration.

Inhibition of NF-κB nuclear translocation via HO-1 activation underlies α-tocopheryl succinate toxicity.

α-Tocopheryl succinate (α-TOS) inhibits oxidative phosphorylation at the level of mitochondrial complex I and II, thus promoting cancer cell death through mitochondrial reactive oxygen species (ROS) generation. Redox imbalance activates NF-E2 p45-related factor 2 (Nrf2), a transcription factor involved in cell protection and detoxification responses. Here we examined the involvement of heme oxygenase-1 (HO-1) in the regulation of nuclear factor κB (NF-κB) signaling by short exposure to α-TOS in prostate cancer cells. A short-term (4 h) exposure to α-TOS causes a significant reduction in cell viability (76%±9%) and a moderate rise in ROS production (113%±8%). α-TOS alters glutathione (GSH) homeostasis by inducing a biphasic effect, i.e., an early (1 h) decrease in intracellular GSH content (56%±20%) followed by a threefold rise at 4 h. α-TOS increases nuclear translocation and electrophile-responsive/antioxidant-responsive elements binding activity of Nrf2, resulting in up-regulation of downstream genes cystine-glutamic acid exchange transporter and HO-1, while decreasing NF-κB nuclear translocation. This effect is suppressed by the pharmacological inhibition of HO-1 and mimicked by the end-products of HO activity, i.e., bilirubin and carbon monoxide. Results suggest a little understood mechanism for α-TOS-induced inhibition of NF-κB nuclear translocation due to HO-1 up-regulation.

Pharmacological dose of {alpha}-tocopherol induces cardiotoxicity in Wistar rats determined by echocardiography and histology.

The effect of pharmacological dose of α-tocopherol on heart health was determined in Wistar rats. Animals were randomly assigned to either C (control, n = 11) or E (α-tocopherol, n = 11) group. Animals received corn oil (C) or α-tocopherol dissolved in corn oil (250 mg α-tocopherol/[kg body wt/day]) (E) by gavage for a 7-week period. Rats underwent echocardiogram and were analyzed for cardiomyocyte histology and cardiac α-tocopherol absorption at the end of the study period. As compared to the C group, α-tocopherol-supplemented group showed significantly (p < 0.05) lower body weight (E, 412.8 g vs C, 480.3 g) and total cardiac weight (E, 0.94 g vs C, 1.08 g); cardiomyocyte histological impairment; smaller left ventricle (LV) (LV end-diastolic diameter (E, 7.22 mm vs C, 7.37 mm), lower LV systolic [left ventricle fractional shortening (E, 47.6% vs C, 53.6%) and ejection fraction ratio (E, 85.4 vs C, 89.9)] and diastolic [early peak velocities of diastolic transmitral flow (E, 64.6 cm/sec vs C, 75.1 cm/sec)] function. The α-tocopherol uptake in target tissue was confirmed by determination of α-tocopherol concentration medians in cardiac tissue (E, 109.91 nmol/kg vs C, 52.09 nmol/kg). The current study indicates that pharmacological dose of α-tocopherol supplementation can induce cardiotoxicity in healthy rats.

Maternal dietary loads of α-tocopherol depress protein kinase C signaling and synaptic plasticity in rat postnatal developing hippocampus and promote permanent deficits in adult offspring.

Vitamin E (α-tocopherol) supplementation has been tested as prophylaxis against gestational disorders associated with oxidative damage. However, recent evidence showing that high maternal α-tocopherol intake can adversely affect offspring development raises concerns on the safety of vitamin E extradosages during pregnancy. Besides acting as an antioxidant, α-tocopherol depresses cell proliferation and modulates cell signaling through inhibiting protein kinase C (PKC), a kinase that is deeply involved in neural maturation and plasticity. Possible effects of α-tocopherol loads in the maturing brain, where PKC dysregulation is associated to developmental dysfunctions, are poorly known. Here, supranutritional doses of α-tocopherol were fed to pregnant and lactating dams to evaluate the effects on PKC signaling and morphofunctional maturation in offspring hippocampus. Results showed that maternal supplementation potentiates hippocampal α-tocopherol incorporation in offspring and leads to marked decrease of PKC phosphorylation throughout postnatal maturation, accompanied by reduced phosphorylation of growth-associated protein-43 and myristoylated alanine-rich C kinase substrate, two PKC substrates involved in neural development and plasticity. Although processes of neuronal maturation, synapse formation and targeting appeared unaffected, offspring of supplemented mothers displayed a marked reduction of long-term synaptic plasticity in juvenile hippocampus. Interestingly, this impairment persisted in adulthood, when a deficit in hippocampus-dependent, long-lasting spatial memory was also revealed. In conclusion, maternal supplementation with elevated doses of α-tocopherol can influence cell signaling and synaptic plasticity in developing hippocampus and promotes permanent adverse effects in adult offspring. The present results emphasize the need to evaluate the safety of supranutritional maternal intake of α-tocopherol in humans.

Antioxidant therapy attenuates oxidative stress in the blood of subjects exposed to occupational airborne contamination from coal mining extraction and incineration of hospital residues.

Coal mining and incineration of solid residues of health services (SRHS) generate several contaminants that are delivered into the environment, such as heavy metals and dioxins. These xenobiotics can lead to oxidative stress overgeneration in organisms and cause different kinds of pathologies, including cancer. In the present study the concentrations of heavy metals such as lead, copper, iron, manganese and zinc in the urine, as well as several enzymatic and non-enzymatic biomarkers of oxidative stress in the blood (contents of lipoperoxidation = TBARS, protein carbonyls = PC, protein thiols = PT, α-tocopherol = AT, reduced glutathione = GSH, and the activities of glutathione S-transferase = GST, glutathione reductase = GR, glutathione peroxidase = GPx, catalase = CAT and superoxide dismutase = SOD), in the blood of six different groups (n = 20 each) of subjects exposed to airborne contamination related to coal mining as well as incineration of solid residues of health services (SRHS) after vitamin E (800 mg/day) and vitamin C (500 mg/day) supplementation during 6 months, which were compared to the situation before the antioxidant intervention (Ávila et al., Ecotoxicology 18:1150-1157, 2009; Possamai et al., Ecotoxicology 18:1158-1164, 2009). Except for the decreased manganese contents, heavy metal concentrations were elevated in all groups exposed to both sources of airborne contamination when compared to controls. TBARS and PC concentrations, which were elevated before the antioxidant intervention decreased after the antioxidant supplementation. Similarly, the contents of PC, AT and GSH, which were decreased before the antioxidant intervention, reached values near those found in controls, GPx activity was reestablished in underground miners, and SOD, CAT and GST activities were reestablished in all groups. The results showed that the oxidative stress condition detected previously to the antioxidant supplementation in both directly and indirectly subjects exposed to the airborne contamination from coal dusts and SRHS incineration, was attenuated after the antioxidant intervention.

alpha-Tocopheryl succinate causes mitochondrial permeabilization by preferential formation of Bak channels.

Mitocans are drugs selectively killing cancer cells by destabilizing mitochondria and many induce apoptosis via generation of reactive oxygen species (ROS). However, the molecular events by which ROS production leads to apoptosis has not been clearly defined. In this study with the mitocan alpha-tocopheryl succinate (alpha-TOS) the role of the Bcl-2 family proteins in the mechanism of malignant cell apoptosis has been determined. Exposure of several different cancer cell lines to alpha-TOS increased expression of the Noxa protein, but none of the other proteins of the Bcl-2 family, an event that was independent of the cellular p53 status. alpha-TOS caused a profound conformational change in the pro-apoptotic protein, Bak, involving oligomerization in all cell types, and this also applied to the Bax protein, but only in non-small cell lung cancer cells. Immunoprecipitation studies indicated that alpha-TOS activates the two BH1-3 proteins, Bak or Bax, to form high molecular weight complexes in the mitochondria. RNAi knockdown revealed that Noxa and Bak are required for alpha-TOS-induced apoptosis, and the role of Bak was confirmed using Bak- and/or Bax-deficient cells. We conclude that the major events induced by alpha-TOS in cancer cells downstream of ROS production leading to mitochondrial apoptosis involve the Noxa-Bak axis. It is proposed that this represents a common mechanism for mitochondrial destabilization activated by a variety of mitocans that induce accumulation of ROS in the early phases of apoptosis.

Possible adverse effect of high delta-alpha-tocopherol intake on hepatic iron overload: enhanced production of vitamin C and the genotoxin, 8-hydroxy-2'- deoxyguanosine.

Excess hepatic iron generates reactive oxygen species that result in oxidative stress and oxidative damage to the liver. Vitamins have hitherto been considered to be a possible remedy. The aim of this study was to determine if high doses of delta-alpha-tocopherol supplementation in iron overload would ameliorate the oxidative stress. Four groups of 20 male Wistar albino rats were studied: group 1 (control) was fed normal diet, group 2 (Fe) 0.75% Ferrocene iron, group 3 (FV gp) 0.75% Ferrocene/delta-alpha-tocopherol (10x RDA), group 4 (V gp) normal diet/delta-alpha-tocopherol. After 12 months, serum iron, reduced glutathione, catalase, vitamin C, Oxygen Radical Absorbance Capacity, lipid peroxidation, 8-hydroxy-2'-deoxyguanosine (8-OHdG), aspartate transaminase (AST), and alanine transaminase (ALT) were measured. Vitamin C levels were: F gp = 5.04 +/- 0.09; FV gp = 5.85 +/- 0.13 (micromol/l) (p < 0.05). 8-hydroxy-2'-deoxyguanosine levels were: F gp = 143.6 +/- 6.4; FV gp = 179.2 +/- 18.2 (ng/ml) (p < 0.05). Oxidative liver damage, as determined by serum AST and ALT levels, was not attenuated by alpha-tocopherol. A positive correlation existed between vitamin C and 8-OHdG, suggesting possible delta-alpha-tocopherol toxicity.

Reduction of cryoprotectant toxicity in cells in suspension by use of a sodium-free vehicle solution.

Propane-1,2-diol (PD) possesses physico-chemical properties that make it a useful biological vitrifying agent but, although of relatively low toxicity, it still has substantial damaging effects on cells. This study aimed to identify possible toxic mechanisms using primary cell cultures from vascular tissue: these were exposed to the cryoprotectant at room temperature to avoid any possibility of hypothermic injury. Toxicity was evaluated by measuring the ability of the cells to divide in culture after exposure to the cryoprotectant. A variety of interventions, which addressed either possible consequences of PD exposure, or known mediators of other types of cell injury, were utilized in an attempt to inhibit PD toxicity. Some comparative studies with dimethyl sulphoxide (Me2SO) exposure were also made. Replacing sodium in the vehicle solution with choline was the only intervention that reduced PD toxicity. It did so both in smooth muscle cells, where the loss of functional capacity was reduced from 56% to 13%, and in endothelial cells. where the reduction was from 40% to 18%. Similar observations were also made in smooth muscle cells exposed to Me2SO. We failed to find evidence for a role of pH regulation, for oxidative injury and/or an involvement of redox-active iron as a mediator of the injury. The results strongly suggest that the influx of sodium into the cell provides one mechanism whereby both PD and Me2SO exert their toxic effects. We suggest that the use of choline-based vehicle solutions in cryopreservation would be beneficial.