Pre-clinical evaluation of an innovative oral nano-formulation of baicalein for modulation of radiation responses.

There is an unmet medical need for non-toxic and effective radiation countermeasures for prevention of radiation toxicity during planned exposures. We have earlier shown that intraperitoneal administration of baicalein (BCL) offers significant survival benefit in animal model. Safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of baicalein has been reported in pre-clinical model systems and also in healthy human volunteers. However, clinical translation of baicalein is hindered owing to poor bioavailability due to lipophilicity. In view of this, we fabricated and characterized in-situ solid lipid nanoparticles of baicalein (SLNB) with effective drug entrapment and release kinetics. SLNB offered significant protection to murine splenic lymphocytes against 4 Gy ionizing radiation (IR) induced apoptosis. Oral administration of SLNB exhibited ~70% protection to mice against whole body irradiation (WBI 7.5 Gy) induced mortality. Oral relative bioavailability of BCL was enhanced by over ~300% after entrapment in the SLNB as compared to BCL. Oral dosing of SLNB resulted in transient increase in neutrophil abundance in peripheral blood. Interestingly, we observed that treatment of human lung cancer cells (A549) with radioprotective dose of SLNB exhibited radio-sensitization as evinced by decrease in survival and clonogenic potential. Contrary to antioxidant nature of baicalein in normal cells, SLNB treatment induced significant increase in cellular ROS levels in A549 cells probably due to higher uptake and inhibition of TrxR. Thus, a pharmaceutically acceptable SLNB exhibited improved bioavailability, better radioprotection to normal cells and sensitized cancer cells to radiation induced killing as compared to BCL suggesting its possible utility as an adjuvant during cancer radiotherapy.

Free and nanoencapsulated vitamin D3 : effects on E-NTPDase and E-ADA activities in an animal model with induced arthritis.

UNLABELLED: The effect of vitamin D3 in oral solution (VD3 ) and vitamin D3 -loaded nanocapsules (NC-VD3 ) was analysed in animals with complete Freund's adjuvant (CFA) induced arthritis (AR). For this purpose, we evaluated scores for arthritis, thermal hyperalgesia and paw oedema, as well as histological analyses and measurements of the activity of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-adenosine deaminase (E-ADA) enzymes in rat lymphocytes. Haematological and biochemical parameters were also determined. The doses administered were 120 UI/day of VD3 and 15.84 UI/day of NC-VD3 . Fifteen days after the induction of AR, the groups were treated for 15 days with vitamin D3 . The results demonstrated that VD3 was able to reduce arthritis scores, thermal hyperalgesia and paw oedema in rats with CFA-induced arthritis. However, treatment with NC-VD3 did not reduce arthritis scores. The histological analyses showed that both formulations were able to reduce the inflammatory changes induced by CFA. The activity of E-NTPDase in rat lymphocytes was higher in the AR compared with the control group, while the activity of E-ADA was lower. This effect was reversed after the 15-day treatment. Data from this study indicates that both forms of vitamin D3 seem to contribute to decreasing the inflammatory process induced by CFA, possibly altering the activities of ectoenzymes. Copyright © 2016 John Wiley & Sons, Ltd. SIGNIFICANCE OF THE STUDY: The effects promoted by both formulations of vitamin D3 , either in oral solution or nanoencapsulated form, strongly suggests the softening of the inflammatory process induced by complete Freund's adjuvant (CFA), possibly altering the E-NTPDase and E-ADA activities. However, it is known that vitamin D has a beneficial effect on the modulation of the immune system components responsible for the inflammatory process. Moreover, the establishment of responses to treatment with vitamin D3 may provide an alternative for inhibiting the proinflammatory response, assisting in our understanding of the immunopathology of this disease and possibly improving the signs and symptoms that hinder the quality of life of patients with rheumatoid arthritis. HIGHLIGHTS: Evaluation of the effects on the E-NTPDase and E-ADA activities in an animal model of induced arthritis. Two formulations of vitamin D3 were used: form oral solution and nanoencapsulated. Vitamin D3 seems to contribute to the inflammatory process induced by CFA. Vitamin D3 possibly alters the E-NTPDase and E-ADA activities. Vitamin D3 may be an alternative supplementary treatment for chronic arthritis.

Hypercholesterolemia and Ecto-enzymes of Purinergic System: Effects of Paullinia cupana.

Hypercholesterolemia is a metabolic disorder characterized by high levels of low-density lipoprotein and blood cholesterol, causing inflammatory lesion. Purinergic signaling modulates the inflammatory and immune responses through adenine nucleotides and nucleoside. Guaraná has hypocholesterolemic and antiinflammatory properties. Considering that there are few studies demonstrating the effects of guaraná powder on the metabolism of adenine nucleotides, we investigated its effects on the activity of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-adenosine deaminase activity in lymphocytes of rats with diet-induced hypercholesterolemia. The rats were divided into hypercholesterolemic and normal diet groups. Each group was subdivided by treatment: saline, guaraná powder 12.5, 25, or 50 mg/kg/day and caffeine concentration equivalent to highest dose of guaraná, fed orally for 30 days. An increase in adenosine triphosphate hydrolysis was observed in the lymphocytes of rats with hypercholesterolemia and treated with 25 or 50 mg/kg/day when compared with the other groups. The hypercholesterolemic group treated with the highest concentration of guaraná powder showed decreased ecto-adenosine deaminase activity compared with the normal diet groups. Guaraná was able to reduce the total cholesterol and low-density lipoprotein cholesterol to basal levels in hypercholesterolemic rats. High concentrations of guaraná associated with a hypercholesterolemic diet are likely to have contributed to the reduction of the inflammatory process.

Effect of Uncaria tomentosa extract on purinergic enzyme activities in lymphocytes of rats submitted to experimental adjuvant arthritis model.

BACKGROUND: Considering that adjuvant arthritis is an experimental model of arthritis widely used for preclinical testing of numerous anti-arthritic agents, which were taken by a large number of patients worldwide, it is of great interest to investigate the therapeutic action of compounds with anti-inflammatory properties, such as Uncaria tomentosa extract. Moreover, there are no studies demonstrating the effect of U. tomentosa on the metabolism of adenine nucleotides published so far. Thus, the purpose of the present study is to investigate the effects of U. tomentosa extract on E-NTPDase and E-ADA activities in lymphocytes of Complete Freund's Adjuvant (CFA) arthritis induced rats. METHODS: To evaluate the effect of U. tomentosa extract on the activity of E-NTPDase and ADA in lymphocytes, the rats were submitted to an experimental adjuvant arthritis model. Peripheral lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Data were analyzed by a one- or two-way ANOVA. Post hoc analyses were carried out by the Student-Newman-Keuls (SNK) Multiple Comparison Test. RESULTS: E-NTPDase activity was increased in arthritic untreated. Arthritic rats which received U. tomentosa extract, presented similar results to the control group. However, results obtained for adenosine hydrolysis by E-ADA were not altered in arthritic rats. U. tomentosa extract did not alter E-NTPDase and E-ADA activity in healthy animals. CONCLUSIONS: The present investigation supports the hypothesis that the increased E-NTPDase activity verified in arthritic rats might be an attempt to maintain basal levels of ATP and ADP in the extracellular medium, since the arthritis induction causes tissue damage and, consequently, large amounts of ATP are released into this milieu. Also, it highlights the possibility to use U. tomentosa extract as an adjuvant to treat arthritis.

Effect of Scutia buxifolia Reissek in nucleotidase activities and inhibition of platelet aggregation.

This study aimed to determine the effect of lyophilized aqueous extracts of Scutia buxifolia on NTPDase and 5'-nucleotidase activity on platelets and lymphocytes as well as the profile of the platelet aggregation. In vitro tests were used to investigate the effect of the aqueous crude extract obtained from S. buxifolia leaves (SbL) and stem bark (SbS) on enzymatic activities and platelet aggregation. The platelets and lymphocytes were exposed to lyophilized aqueous extracts of S. buxifolia at concentrations of 1-200 µg/mL in the presence of ATP, ADP, AMP as substrates, during the enzymatic assay, as well as the platelet aggregation. The results showed that SbS and SbL potently inhibited NTPDase and 5'-nucleotidase in platelets and lymphocytes. ADP-induced aggregation was inhibited by the SbS (50, 100, and 200 µg/mL) and SbL (200 µg/mL). In addition, these results suggest that S. buxifolia have compounds, such as gallic acid, chlorogenic acid, caffeic acid, quercetin, rutin, and kaempferol, which cause a decrease the NTPDase and 5'-nucleotidase activity, resulting in alterations in adenine nucleotide levels and protection against ADP-induced platelet aggregation.

Antagonistic effects of Satureja hortensis essential oil against AFB1 on human lymphocytes in vitro.

Satureja hortensis L. (Lamiaceae) has been used as a folk remedy to treat various such as cramps, muscle pains, nausea, indigestion, diarrhea, and infectious diseases. In this study, the antagonistic effects of essential oil of S. hortensis (SHE) were studied against aflatoxin B1 (AFB1) in human lymphocytes in vitro. The analysis of the essential oil was performed by using Gas chromatography-mass spectrometry (GC-MS). Anti-genotoxic effects of the SHEs was evaluated using sister chromatid exchange (SCE), micronuclei (MN) tests against AFB1. Also level of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities used to determine the anti-oxidative effects of the SHEs. This result showed AFB1 (5 microM) increased the frequencies of SCE, MN and the level of MDA. AFB1 at the same concentration decreased the activities of SOD and GPx. However, different concentrations of SHE with AFB1 decreased the frequency of SCE and MN and level of MDA and also increased the activities of SOD and GPx significantly. Especially, the 1.0, 1.5, 2.0 microL dose of SHE are more effective than other doses. The results of this experiment have clearly shown that SHE has strong antioxidative and antigenotoxic effects, these biological activities of SHEs can be due to its component.

Antioxidants restore protoporphyrinogen oxidase in variegate porphyria patients.

BACKGROUND: Variegate porphyria (VP) is the result of decreased protoporphyrinogen oxidase (PPOX) activity and results in the accumulation of porphyrins and porphyrin precursors. Our aims were to analyse the basal antioxidant defences and oxidative damage markers and the effects of a diet supplementation with vitamins E and C on the oxidant/antioxidant status and PPOX gene expression in lymphocytes of variegate porphyria (VP) patients. MATERIALS AND METHODS: Twelve women affected by VP and 12 control women participated in a randomized and double-blind crossover study. Each participant took either 50 mg/day vitamin E and 150 mg/day vitamin C or a placebo for 6 months. RESULTS: Lymphocyte PPOX gene expression, together with catalase and glutathione peroxidase activities, was reduced in VP women. No differences were observed in the levels of malondialdehyde and protein carbonyl derivatives. Stimulated lymphocyte H2 O2 production was higher in porphyric women. Supplementation with antioxidant vitamins increased PPOX expression in VP patients. Glutathione reductase (GRd) and superoxide dismutase (SOD) activities were higher in the treatment groups. CONCLUSIONS: Lymphocytes from VP patients show reduced PPOX expression and present a greater susceptibility to producing H2 O2 and impaired H2 O2 detoxifying mechanisms. Supplementation with vitamins E and C restores PPOX expression in VP patients and enhances GRd and SOD activity, suggesting the potential benefits of a diet rich in vitamins E and C in these patients.

Genotoxic and cytotoxic effects of storax in vitro.

The aim of this study is to investigate the effects of the storax balsam, which is a kind of sweet gum obtained from the Liquidambar orientalis Mill trees, on cell viability, cytotoxicity and genotoxicity in human lymphocyte in vitro. We studied the genotoxic effects of the extract of storax balsam (SE) using sister chromatid exchange (SCE) test system. Also the cytotoxic and inhibitory effects on cell proliferation of SE were evaluated using lactate dehydrogenase (LDH) assay and cell proliferation (WST-1) assay. The SCE frequency was increased when the cells were treated with 1.6 and 4.0 µg/mL SE concentrations (p < 0.05). Moreover, treatment of the cells with the same concentrations significantly depleted the cell number at 24th and 48th hours and elevated the LDH levels (p < 0.05) at 48th hour. These results suggest that SE can be used as an alternative antibacterial and antipathogenic agent due to its cytotoxic and genotoxic effects.

Hematological indices and activity of NTPDase and cholinesterase enzymes in rats exposed to cadmium and treated with N-acetylcysteine.

The present study aimed to investigate the influence of N-acetylcysteine (NAC) on cadmium (Cd) poisoning by evaluating Cd concentration in tissues, hematological indices as well as the activity of NTPDase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes of rats exposed to Cd and co-treated with NAC. For this purpose, the rats received Cd (2 mg/kg) and NAC (150 mg/kg) by gavage every other day for 30 days. Animals were divided into four groups (n = 6-8): control/saline, NAC, Cd, and Cd/NAC. Cd exposure increased Cd concentration in plasma, spleen and thymus, and NAC co-treatment modulated this augment in both lymphoid organs. Cd exposure reduced red blood cell count, hemoglobin content and hematocrit value. Cd intoxication caused a decrease in total white blood cell count. NAC treatment per se caused an increase in lymphocyte and a decrease in neutrophil counts. On contrary, Cd exposure caused a decrease in lymphocyte and an increase in neutrophil and monocyte counts. NAC reversed or ameliorated the hematological impairments caused by Cd poisoning. There were no significant alterations in the NTPDase activity in lymphocytes of rats treated with Cd and/or NAC. Cd caused a decrease in the activities of lymphocyte AChE, whole blood AChE and serum BChE. However, NAC co-treatment was inefficient in counteracting the negative effect of Cd in the cholinesterase activities. The present investigation provides ex vivo evidence supporting the hypothesis that Cd induces immunotoxicity by interacting with the lymphoid organs, altering hematological parameters and inhibiting peripheral cholinesterase activity. Also, it highlights the possibility to use NAC as adjuvant against toxicological conditions.

Guanidinoacetate methyltransferase (GAMT) deficiency in two Tunisian siblings: clinical and biochemical features.

BACKGROUND: Guanidinoacetate methyltransferase (GAMT) deficiency is a recently described disorder and few cases have been reported to date. As it is a treatable pathology, we seek to contribute to its better understanding, particularly to further elucidate its biochemical diagnosis for early treatment. METHODS: The patients, two brothers aged 13 years (P1) and 11 years (P2), have been explored for signs and symptoms suggestive of inborn errors of metabolism. The quantification of creatine (Cr), guanidinoacetate (GAA), and GAMT activity was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The two brothers presented a similar clinical picture: developmental delay, epilepsy, axial hypotonia, spastic tetraparesis, severe mental and language delay, and autistic behaviour. GAA concentrations were markedly increased in plasma and in urine [2796 and 3342 micromol/mmol creatinine (control range: 4 - 220 micromol/mmol creatinine)/14 and 29 micromol/L (control range: 0.35 - 1.8 micromol/L), respectively] while plasma and urine creatine concentrations were at the lower normal range limit. Activity of GAMT in lymphoblasts was extremely reduced (< 0.01 nmol/mg protein/hour) compared to healthy subjects. GAMT activity was found to be intermediary in patients' parents. CONCLUSIONS: It appears that the clinical picture is heterogeneous but should be considered as potential signs of creatine metabolism disorders, however, the biochemical diagnosis is reliable as the enzyme activity is zero in most cases. To date, it is still too early to establish correlations between symptoms and biochemical profile. However, the identification of additional cases of GAMT deficiency should help elucidate such relationships and the progress of patients treated with creatine in conjunction with ornithine supplementation.

SNP and mRNA expression for glutathione peroxidase 4 in Kashin-Beck disease.

Kashin-Beck disease (KBD) is a chronic endemic osteoarthropathy, which mainly occurs in West and Northeast China. Epidemiological studies suggest that Se deficiency is an important environmental factor for the incidence of KBD. Glutathione peroxidase 4 (GPx4) belongs to the glutathione peroxidase family, which is crucial for optimal antioxidant defences. Our purpose is to investigate the putative association between GPx4 polymorphisms and the risk of KBD. Restriction fragment length polymorphism-PCR was used to detect two SNP (rs713041, rs4807542) in 219 cases and 194 controls in Han Chinese subjects, and quantitative analysis for the GPx4 mRNA level was performed by the real-time PCR method. The results revealed that linkage disequilibrium existed in the two SNP. A significant difference was observed in the haplotype A-T (P = 0·0066) of GPx4, which was obviously lower in the KBD cases (0·006 v. 0·032 %). Correlation analysis based on a single locus showed no association between each SNP and KBD risk. Furthermore, the GPx4 mRNA level was dramatically lower in the blood of KBD patients. Overall, our finding indicated GPx4 polymorphisms and decreased mRNA level may be related to the development of KBD in the Chinese population, suggesting GPx4 as a possible candidate susceptibility gene for KBD.

Characteristics of lymphocyte nuclear factor-κB signal transduction kinase expression in aging process and regulatory effect of epimedium flavonoids.

OBJECTIVE: To study the characteristics of lymphocyte nuclear factor kappa B (NF-κB) signal transduction kinase-related molecular mRNA differential expressions at various month age segments in aging process and the intervening effect of Epimedium flavonoids (EF) on it. METHODS: Sixty SD rats were divided into six groups, according to animals' age, i.e., the 3 days (d) group, the 4 months (m) group, the 10 m group, the 18 m group, the 27 m group, and the 27 m+EF group. RNA was extracted from separated splenic lymphocytes. Adopting NF-κB signal path functional genome oligonucleotide gene-chip (128 related genes), the integral characteristics and differences of NF-κB signal transduction kinase-related mRNA expressions were determined, and the intervening effect of EF was examined. RESULTS: The mean level of the NF-κB signal transduction kinase-related mRNA expressions in rats' splenic lymphocytes lowered with aging; the highest expression was presented at 3 d after birth, and then, it lowered gradually, with the lowest level at 18 m or 27 m. After EF intervention, the expression level was raised to the 10-18 m level in the aged rats. CONCLUSION: The changing rules of lymphocyte NF-κB-signal-transduction-kinase-related mRNA expressions in various stages of aging are helpful for selecting the well time for preventing and intervening aging, and will also give a hint to the molecular index for assessment of senility retarding researches.

Urinary excretion of 3-hydroxyisovaleryl carnitine is an early and sensitive indicator of marginal biotin deficiency in humans.

Mounting evidence indicates that marginal biotin deficiency is not rare, contrary to previous assumptions. Accordingly, robust indicators of biotin status would be useful. In a study of 10 healthy adults, we recently provided evidence that abnormally increased plasma concentration of 3-hydroxyisovaleryl carnitine (3HIA-carnitine) is a sensitive indicator of marginal biotin deficiency. We sought to determine whether urinary excretion of 3HIA-carnitine (expressed as the ratio to urinary creatinine) significantly increases in marginal biotin deficiency. Marginal, asymptomatic biotin deficiency was induced experimentally in the same 10 healthy adults (8 women) by feeding undenatured egg white with meals for 28 d. Biotin status was repleted by a mixed general diet plus biotin supplementation. Urinary excretion of 3HIA-carnitine was determined by liquid chromatography-tandem MS on d 0, 14, and 28 (depletion) and on d 35 and 50 (repletion). Mean urinary 3HIA-carnitine concentration increased with depletion (P < 0.0001; d 0 vs. 28) and decreased with repletion (P = 0.0002; d 28 vs. 50). Urinary 3HIA-carnitine excretion was greater than the upper limit of normal in 9 of 10 participants by d 14 and decreased to within normal limits by d 50 in all participants. This study provides evidence that urinary excretion of 3HIA-carnitine is an early and sensitive indicator of marginal biotin deficiency. The ease of collection of untimed urine samples and application of a new analytical method with simplified sample preparation suggest that urinary 3HIA-carnitine is likely to be a useful indicator for large population studies.

Soybean and green tea polyphenols improve immune function and redox status in very old ovariectomized mice.

In previous work we have observed that ovariectomy in rodents, a good model of mimicking human ovarian hormone loss, causes premature aging of the immune system. The prooxidative and inflammatory state that underlies the aging process is the base of that premature immunosenescence. It has been found that nutritional interventions with polyphenolic antioxidants constitute a good alternative to rejuvenate age-affected immune functions. In this study, we administered a diet supplemented with polyphenols (coming from soybean isoflavones and green tea) to sham-operated and ovariectomized mature mice for 15 weeks, until they reached a very old age. We have studied the effect of this supplementation on a broad range of parameters of immune function (in macrophages and lymphocytes) and oxidative stress (enzymatic and nonenzymatic antioxidant defences, oxidant compounds, and lipid peroxidation damage) in peritoneal leukocytes. The results showed that ovariectomy accelerates the age-related impairment of immune functions in very old mice as well as the oxidative and proinflammatory imbalance, and that the administration of soybean isoflavones and green tea improve the immune and redox state in these animals. Because the immune system is a good marker of health and a predictor of longevity, we suggest that an adequate nutritional treatment with polyphenols could be a highly recommended tool to fight against the detrimental effects of the lack of female sex hormones, through an improvement of the immune cell functions and redox state.

Curcumin protects DNA damage in a chronically arsenic-exposed population of West Bengal.

Groundwater arsenic contamination has been a health hazard for West Bengal, India. Oxidative stress to DNA is recognized as an underlying mechanism of arsenic carcinogenicity. A phytochemical, curcumin, from turmeric appears to be potent antioxidant and antimutagenic agent. DNA damage prevention with curcumin could be an effective strategy to combat arsenic toxicity. This field trial in Chakdah block of West Bengal evaluated the role of curcumin against the genotoxic effects of arsenic. DNA damage in human lymphocytes was assessed by comet assay and fluorescence-activated DNA unwinding assay. Curcumin was analyzed in blood by high performance liquid chromatography (HPLC). Arsenic induced oxidative stress and elucidation of the antagonistic role of curcumin was done by observation on reactive oxygen species (ROS) generation, lipid peroxidation and protein carbonyl. Antioxidant enzymes like catalase, superoxide dismutase, glutathione reductase, glutathioneS-transferase, glutathione peroxidase and non-enzymatic glutathione were also analyzed. The blood samples of the endemic regions showed severe DNA damage with increased levels of ROS and lipid peroxidation. The antioxidants were found with depleted activity. Three months curcumin intervention reduced the DNA damage, retarded ROS generation and lipid peroxidation and raised the level of antioxidant activity. Thus curcumin may have some protective role against the DNA damage caused by arsenic.

Molecular consequences of genetic variations in the glutathione peroxidase 1 selenoenzyme.

Accumulating data have implicated the selenium-containing cytosolic glutathione peroxidase, GPx-1, as a determinant of cancer risk and a mediator of the chemopreventive properties of selenium. Genetic variants of GPx-1 have been shown to be associated with cancer risk for several types of malignancies. To investigate the relationship between GPx-1 enzyme activity and genotype, we measured GPx-1 enzyme activity and protein levels in human lymphocytes as a function of the presence of two common variations: a leucine/proline polymorphism at codon 198 and a variable number of alanine-repeat codons. Differences in GPx activity among these cell lines, as well as in the response to the low-level supplementation of the media with selenium, indicated that factors other than just genotype are significant in determining activity. To restrict the study to genotypic effects, human MCF-7 cells were engineered to exclusively express allelic variants representing a combination of either a codon 198 leucine or proline and either 5 or 7 alanine-repeat codons following transfection of GPx-1 expression constructs. Transfectants were selected and analyzed for GPx-1 enzyme activity and protein levels. GPx-1 with 5 alanines and a leucine at codon 198 showed a significantly higher induction when cells were incubated with selenium and showed a distinct pattern of thermal denaturation as compared with GPx-1 encoded by the other examined alleles. The collective data obtained using both lymphocytes and MCF-7 indicate that both intrinsic and extrinsic factors cooperate to ultimately determine the levels of this enzyme available to protect cells against DNA damage and mutagenesis.

A soccer match's ability to enhance lymphocyte capability to produce ROS and induce oxidative damage.

Soccer-associated oxidative stress has barely been studied. The aims of this study were to establish the effect of a soccer training match and the effect of a diet supplementation with a multivitamin complex and coenzyme Q during 3 months of soccer training on the pro-oxidant and antioxidant status of lymphocytes. In a randomized, double-blind trial, 19 male preprofessional soccer players were treated with either an antioxidant nutrient cocktail or placebo for 90 days. After this period the athletes played a soccer match lasting 60 min. All determinations were made under basal conditions before and after the training period and after the match. Basal lymphocyte hydrogen peroxide (H2O2) production did not change after the 3 months of training. Catalase activity decreased (about 50%) after the 3 months, whereas glutathione reductase increased its activity (150-200%) both with placebo and in the supplemented group. Basal ascorbate levels were maintained during the training period, whereas a-tocopherol and MDA decreased (about 40%) in both groups. The match increased H2O2 production (180%) in both groups when the lymphocytes were stimulated with phorbol myristate acetate, and it also increased MDA levels (150%). Antioxidant enzyme activities and antioxidant vitamin levels were maintained before and after the match. Regular soccer training modifies the lymphocyte strategy to eliminate ROS and increases protection against oxidative damage. A friendly soccer match raises lymphocyte capacity to produce ROS and oxidative damage, but it is not enough to induce a defensive response, thus leading to a situation of postexercise oxidative stress. Supplementation with low doses of antioxidant vitamins and coenzyme Q does not modify the endogenous antioxidant response to training.

Lycopene supplementation (passata sauce) reduces apoptosis but does not affect oxidant-responsive heme oxygenase-1 in human lymphocytes.

OBJECTIVE: We tested the hypothesis that lycopene supplementation reduces the expression of oxidant-responsive heme oxygenase-1 (HO-1) in basal conditions and in response to an oxidant challenge and determined whether this is temporally associated with increased cell viability. METHODS: We determined basal and stimulated ex vivo expression of HO-1 and cell viability in lymphocytes from volunteers after lycopene supplementation. Twenty-four healthy young men on a low lycopene diet consumed 1) 170 g of passata sauce with butter or 2) butter alone for 3 wk in a randomized crossover design. RESULTS: Plasma lycopene concentrations at the end of the tomato and control trials were 0.54 +/- 0.20 versus 0.20 +/- 0.15 micromol/L, respectively (P < 0.05). There was a significant increase in the proportion of live cells (91 +/- 5% versus 87 +/- 9%) and a corresponding reduction in apoptosis (6 +/- 4% versus 11 +/- 9%) in untreated lymphocytes after supplementation (P < 0.05), with no effect on cell viability in response to hydrogen peroxide treatment. HO-1 protein expression in basal conditions and induction of HO-1 after hydrogen peroxide treatment was not different between trials. CONCLUSION: Lycopene supplementation did not affect basal oxidative stress or susceptibility to oxidant-induced stress as indicated by the expression of the oxidant-responsive protein HO-1 and cell viability in response to hydrogen peroxide treatment. However, lycopene supplementation significantly reduced apoptosis in freshly harvested untreated lymphocytes. We conclude that this was not through an oxidant-mediated mechanism because of the lack of an effect on oxidant-responsive HO-1.

Verbena officinalis essential oil and its component citral as apoptotic-inducing agent in chronic lymphocytic leukemia.

We evaluated the pro-apoptotic activity of Verbena officinalis essential oil and of its main component citral, on lymphocytes collected from normal blood donors and patients with chronic lymphocytic leukemia (CLL). The number of apoptotic cells was greater in CLL patients than in healthy subjects at all different times of incubation (4, 8 and 24 hours) for samples treated with Verbena officinalis essential oil (A) and citral (B) as well vs controls at different concentrations (0.1% and 0.01%). The greater pro-apoptotic ability was shown by both essential oil of Verbena officinalis and citral at lower concentrations (after 4 h A 0.1%: 17.8% vs 37.1%; A 0.01%: 15.8% vs 52%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%; after 8 h A 0.1%: 23% vs 38%; A 0.01%: 22.2% vs 55%; B 0.1%: 32% vs 42.2%; B 0.01%: 22% vs 54.3%; after 24 h A 0.1%: 5% vs 20.7%; A 0.01%: 25.8% vs 47.2%; B 0.1%: 18.4% vs 46.4%; B 0.01%: 15.8% vs 54.2%). Patients carrying deletion 17p13 (p53 mutation) showed a reduced ability to undergo apoptosis with respect to patients with other genomic aberrations or normal karyotype. The proapoptotic activity of Verbena officinalis essential oil and citral is thought to be due to a direct procaspase 3 activation. These data further support evidence that indicate natural compounds as a possible lead structure to develop new therapeutic agents.

[Effects of benzene and selenium on telomerase in mouse lymphocytes in vivo].

OBJECTIVE: To study the effects of benzene and selenium on telomerase in mouse lymphocytes in vivo and evaluate telomerase activity as an early marker of benzene effects on lymphocytes. METHODS: Male Kunming mice were divided randomly into 8 groups, including negative control group, reagent control group, 100 mg/kg benzene group, 200 mg/kg benzene group, 400 mg/kg benzene group, 200 mg/kg benzene + 0.75 mg/kg selenium group, 200 mg/kg benzene + 1.50 mg/kg selenium group and 200 mg/kg benzene + 3.00 mg/kg selenium group, 5 mice in each group. The mice in different groups were treated with different methods, once daily for 5 days. After 48 hours of the final exposure, lymphocytes were separated and the telomerase activities were detected with TRAPELISA. RESULTS: Compared with negative and reagent control groups, the telomerase activity was increased after treatment with different dose of benzene and at the dose of 100 mg/kg benzene group it was significantly increased (P < 0.01). At the dose of 200 mg/kg benzene + 0.75 mg/kg selenium group, it was significantly increased (P < 0.01). Compared with the counterpart treated with 200 mg/kg benzene group, the expression of telomerase was increased at the different concentrations after treatment with benzene combined with selenium and it was significantly increased at the dose of 200 mg/kg benzene + 0.75 mg/kg selenium group (P < 0.05). CONCLUSION: Increased telomerase activity in lymphocytes stimulated by benzene at different concentrations indicates activation and proliferation of these lymphocytes of mice in vivo. Telomerase activity is probably a sensitive early marker of lymphocyte proliferation by benzene. Selenium can upregulate the telomerase activity.