High doses of riboflavin and the elimination of dietary red meat promote the recovery of some motor functions in Parkinson's disease patients

Abnormal riboflavin status in the absence of a dietary deficiency was detected in 31 consecutive outpatients with Parkinson's disease (PD), while the classical determinants of homocysteine levels (B6, folic acid, and B12) were usually within normal limits. In contrast, only 3 of 10 consecutive outpatients with dementia without previous stroke had abnormal riboflavin status. The data for 12 patients who did not complete 6 months of therapy or did not comply with the proposed treatment paradigm were excluded from analysis. Nineteen PD patients (8 males and 11 females, mean age ± SD = 66.2 ± 8.6 years; 3, 3, 2, 5, and 6 patients in Hoehn and Yahr stages I to V) received riboflavin orally (30 mg every 8 h) plus their usual symptomatic medications and all red meat was eliminated from their diet. After 1 month the riboflavin status of the patients was normalized from 106.4 ± 34.9 to 179.2 ± 23 ng/ml (N = 9). Motor capacity was measured by a modification of the scoring system of Hoehn and Yahr, which reports motor capacity as percent. All 19 patients who completed 6 months of treatment showed improved motor capacity during the first three months and most reached a plateau while 5/19 continued to improve in the 3- to 6-month interval. Their average motor capacity increased from 44 to 71% after 6 months, increasing significantly every month compared with their own pretreatment status (P < 0.001, Wilcoxon signed rank test). Discontinuation of riboflavin for several days did not impair motor capacity and yellowish urine was the only side effect observed. The data show that the proposed treatment improves the clinical condition of PD patients. Riboflavin-sensitive mechanisms involved in PD may include glutathione depletion, cumulative mitochondrial DNA mutations, disturbed mitochondrial protein complexes, and abnormal iron metabolism. More studies are required to identify the mechanisms involved.

Effect of dietary fat saturation on lipid metabolism, arachidonic acid turnover and peritoneal macrophage oxidative stress in mice

We investigated the effects of a saturated fat diet on lipid metabolism and arachidonic acid (AA) turnover in mouse resident peritoneal macrophages. The pro-oxidative effect of this diet was also studied. Female C57BL/6 mice were weaned at 21 days of age and assigned to either the experimental diet containing coconut oil (COCO diet), or the control diet containing soybean oil as fat source (10 mice per group). The fat content of each diet was 15% (w/w). Mice were fed for 6 weeks and then sacrificed. The concentration of total lipids, triglycerides, (LDL + VLDL)-cholesterol, thiobarbituric acid-reactive substances (TBARS) and reduced glutathione were increased in the plasma of mice fed the COCO diet, without changes in phospholipid or total cholesterol concentrations compared to control. The concentrations of total cholesterol, free and esterified cholesterol, triglycerides, and TBARS were increased in the macrophages of COCO-fed mice, while the content of total phospholipids did not change. The phospholipid composition showed an increase of phosphatidylcholine and a decrease of phosphatidylethanolamine. The [³H]-AA distribution in the phospholipid classes showed an increase in phosphatidylcholine and phosphatidylethanolamine. Incorporation of [³H]-cholesterol into the macrophages of COCO-fed mice and into the cholesterol ester fraction was increased. The COCO diet did not affect [³H]-AA uptake but induced an increase in [³H]-AA release. The COCO diet also enhanced AA mobilization induced by lipopolysaccharide. These results indicate that the COCO diet, high in saturated fatty acids, alters the lipid metabolism and AA turnover of peritoneal macrophages in female mice and also produces a significant degree of oxidative stress.

Cytotoxicity and genotoxicity of low doses of mercury chloride and methylmercury chloride on human lymphocytes in vitro

Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well-known and humans are exposed by consumption of contaminated fish, shellfish and algae. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes. Short-term human leukocyte cultures from 10 healthy donors (5 females and 5 males) were set-up by adding drops of whole blood in complete medium. Cultures were separately and simultaneously treated with low doses (0.1 to 1000 µg/l) of HgCl2 and CH3HgCl and incubated at 37ºC for 48 h. Genotoxicity was assessed by chromosome aberrations and polyploid cells. Mitotic index was used as a measure of cytotoxicity. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl when compared to control, whether alone or in an evident sinergistic combination with HgCl2. The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 µg/l alone, and at 1, 10, 100, and 1000 µg/l when combined with HgCl2, showing a synergistic cytotoxic effect. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic. Such effects may indicate early cellular changes with possible biological consequences and should be considered in the preliminary evaluation of the risks of populations exposed in vivo to low doses of mercury.

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus

To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 µg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 µg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 ± 393 cells/mm²) compared to control (1886 ± 892 cells/mm²; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 ± 56 cells/mm² (2 µg/g) and 845 ± 82 cells/mm² (6 µg/g), also lower than control (1312 ± 31 cells/mm²; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 µg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

Methylmercury intoxication activates nitric oxide synthase in chick retinal cell culture

The visual system is a potential target for methylmercury (MeHg) intoxication. Nevertheless, there are few studies about the cellular mechanisms of toxicity induced by MeHg in retinal cells. Various reports have indicated a critical role for nitric oxide synthase (NOS) activation in modulating MeHg neurotoxicity in cerebellar and cortical regions. The aim of the present study is to describe the effects of MeHg on cell viability and NOS activation in chick retinal cell cultures. For this purpose, primary cultures were prepared from 7-day-old chick embryos: retinas were aseptically dissected and dissociated and cells were grown at 37ºC for 7-8 days. Cultures were exposed to MeHg (10 µM, 100 µM, and 1 mM) for 2, 4, and 6 h. Cell viability was measured by MTT method and NOS activity by monitoring the conversion of L-[H³]-arginine to L-[H³]-citrulline. The incubation of cultured retina cells with 10 and 100 µM MeHg promoted an increase of NOS activity compared to control (P < 0.05). Maximum values (P < 0.05) were reached after 4 h of MeHg incubation: increases of 81.6 ± 5.3 and 91.3 ± 3.7%, respectively (data are reported as mean ± SEM for 4 replicates). MeHg also promoted a concentration- and time-dependent decrease in cell viability, with the highest toxicity (a reduction of about 80% in cell viability) being observed at the concentration of 1 mM and after 4-6 h of incubation. The present study demonstrates for the first time the modulation of MeHg neurotoxicity in retinal cells by the nitrergic system.

Immunoglobulin production is impaired in protein-deprived mice and can be restored by dietary protein supplementation

Most contacts with food protein and microbiota antigens occur at the level of the gut mucosa. In animal models where this natural stimulation is absent, such as germ-free and antigen-free mice, the gut-associated lymphoid tissue (GALT) and systemic immunological activities are underdeveloped. We have shown that food proteins play a critical role in the full development of the immune system. C57BL/6 mice weaned to a diet in which intact proteins are replaced by equivalent amounts of amino acids (Aa diet) have a poorly developed GALT as well as low levels of serum immunoglobulins (total Ig, IgG, and IgA, but not IgM). In the present study, we evaluated whether the introduction of a protein-containing diet in 10 adult Aa-fed C57BL/6 mice could restore their immunoglobulin levels and whether this recovery was dependent on the amount of dietary protein. After the introduction of a casein-containing diet, Aa-fed mice presented a fast recovery (after 7 days) of secretory IgA (from 0.33 to 0.75 mg/mL, while in casein-fed mice this value was 0.81 mg/mL) and serum immunoglobulin levels (from 5.39 to 10.25 mg/mL of total Ig). Five percent dietary casein was enough to promote the restoration of secretory IgA and serum immunoglobulin levels to a normal range after 30 days feeding casein diet (as in casein-fed mice - 15% by weight of diet). These data suggest that the defect detected in the immunoglobulin levels was a reversible result of the absence of food proteins as an antigenic stimulus. They also indicate that the deleterious consequences of malnutrition at an early age for some immune functions may be restored by therapeutic intervention later in life.

Involvement of glutamate and reactive oxygen species in methylmercury neurotoxicity

This review addresses the mechanisms of methylmercury (MeHg)-induced neurotoxicity, specifically examining the role of oxidative stress in mediating neuronal damage. A number of critical findings point to a central role for astrocytes in mediating MeHg-induced neurotoxicity as evidenced by the following observations: a) MeHg preferentially accumulates in astrocytes; b) MeHg specifically inhibits glutamate uptake in astrocytes; c) neuronal dysfunction is secondary to disturbances in astrocytes. The generation of reactive oxygen species (ROS) by MeHg has been observed in various experimental paradigms. For example, MeHg enhances ROS formation both in vivo (rodent cerebellum) and in vitro (isolated rat brain synaptosomes), as well as in neuronal and mixed reaggregating cell cultures. Antioxidants, including selenocompounds, can rescue astrocytes from MeHg-induced cytotoxicity by reducing ROS formation. We emphasize that oxidative stress plays a significant role in mediating MeHg-induced neurotoxic damage with active involvement of the mitochondria in this process. Furthermore, we provide a mechanistic overview on oxidative stress induced by MeHg that is triggered by a series of molecular events such as activation of various kinases, stress proteins and other immediate early genes culminating in cell damage.

Opposite lipemic response of Wistar rats and C57BL/6 mice to dietary glucose or fructose supplementation

The metabolic effects of carbohydrate supplementation in mice have not been extensively studied. In rats, glucose- and fructose-rich diets induce hypertriacylglycerolemia. In the present study, we compared the metabolic responses to two monosaccharide supplementations in two murine models. Adult male Wistar rats (N = 80) and C57BL/6 mice (N = 60), after 3 weeks on a standardized diet, were submitted to dietary supplementation by gavage with glucose (G) or fructose (F) solutions (500 g/L), 8 g/kg body weight for 21 days. Glycemia was significantly higher in rats after fructose treatment (F: 7.9 vs 9.3 mM) and in mice (G: 6.5 vs 10 and F: 6.6 vs 8.9 mM) after both carbohydrate treatments. Triacylglycerolemia increased significantly 1.5 times in rats after G or F supplementation. Total cholesterol did not change with G treatment in rats, but did decrease after F supplementation (1.5 vs 1.4 mM, P < 0.05). Both supplementations in rats induced insulin resistance, as suggested by the higher Homeostasis Model Assessment Index. In contrast, mice showed significant decreases in triacylglycerol (G: 1.8 vs 1.4 and F: 1.9 vs 1.4 mM, P < 0.01) and total cholesterol levels (G and F: 2.7 vs 2.5 mM, P < 0.05) after both monosaccharide supplementations. Wistar rats and C57BL/6 mice, although belonging to the same family (Muridae), presented opposite responses to glucose and fructose supplementation regarding serum triacylglycerol, free fatty acids, and insulin levels after monosaccharide treatment. Thus, while Wistar rats developed features of plurimetabolic syndrome, C57BL/6 mice presented changes in serum biochemical profile considered to be healthier for the cardiovascular system.

Association of CYP7A1 -278A>C polymorphism and the response of plasma triglyceride after dietary intervention in dyslipidemic patients

We investigated the effect of the -278A>C polymorphism in the CYP7A1 gene on the response of plasma lipids to a reduced-fat diet for 6 to 8 weeks in a group of 82 dyslipidemic males with a mean age of 46.0 ± 11.7 years. Individuals who presented at least one high alteration in total cholesterol, low-density lipoprotein cholesterol or triglyceride values were considered to be dyslipidemic. Exclusion criteria were secondary dyslipidemia due to diabetes mellitus, renal, liver, or thyroid disease. None of the subjects were using lipid-lowering medication. Baseline and follow-up lipid concentrations were measured. The genotypes were determined by the digestion of PCR products with the BsaI restriction endonuclease. There were statistically significant reductions in plasma total cholesterol, low-density lipoprotein cholesterol and triglyceride concentrations after dietary intervention. The minor allele C has a frequency of 43%. Carriers of the C allele had significantly lower triglyceride concentrations (P = 0.02) than AA homozygotes. After adjustment of covariates, subjects with the AC and CC genotypes showed a greater reduction in triglyceride concentrations compared to subjects with the AA genotype. Multiple linear regression analyses showed that the AC and CC CYP7A1 genotypes accounted for 5.2 and 6.2% of triglyceride concentration during follow-up and adjusted percent of change of triglyceride concentration, respectively. The present study provides evidence that -278A>C polymorphism in the CYP7A1 gene can modify triglyceride concentrations in response to a reduced fat diet in a dyslipidemic male population. This gene represents a potential locus for a nutrigenetic directed approach.

Protective effects of organoselenium compounds against methylmercury-induced oxidative stress in mouse brain mitochondrial-enriched fractions

We evaluated the potential neuroprotective effect of 1-100 µM of four organoselenium compounds: diphenyl diselenide, 3’3-ditri-fluoromethyldiphenyl diselenide, p-methoxy-diphenyl diselenide, and p-chloro-diphenyl diselenide, against methylmercury-induced mitochondrial dysfunction and oxidative stress in mitochondrial-enriched fractions from adult Swiss mouse brain. Methylmercury (10-100 µM) significantly decreased mitochondrial activity, assessed by MTT reduction assay, in a dose-dependent manner, which occurred in parallel with increased glutathione oxidation, hydroperoxide formation (xylenol orange assay) and lipid peroxidation end-products (thiobarbituric acid reactive substances, TBARS). The co-incubation with diphenyl diselenide (100 µM) completely prevented the disruption of mitochondrial activity as well as the increase in TBARS levels caused by methylmercury. The compound 3’3-ditrifluoromethyldiphenyl diselenide provided a partial but significant protection against methylmercury-induced mitochondrial dysfunction (45.4 ± 5.8% inhibition of the methylmercury effect). Diphenyl diselenide showed a higher thiol peroxidase activity compared to the other three compounds. Catalase blocked methylmercury-induced TBARS, pointing to hydrogen peroxide as a vector during methylmercury toxicity in this model. This result also suggests that thiol peroxidase activity of organoselenium compounds accounts for their protective actions against methylmercury-induced oxidative stress. Our results show that diphenyl diselenide and potentially other organoselenium compounds may represent important molecules in the search for an improved therapy against the deleterious effects of methylmercury as well as other mercury compounds.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARβ by 2.0-fold (quantitative real-time PCR). Our data show that RARβ may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARβ is epigenetically altered.

Methylmercury inhibits prolactin release in a cell line of pituitary origin

Heavy metals, such as methylmercury, are key environmental pollutants that easily reach human beings by bioaccumulation through the food chain. Several reports have demonstrated that endocrine organs, and especially the pituitary gland, are potential targets for mercury accumulation; however, the effects on the regulation of hormonal release are unclear. It has been suggested that serum prolactin could represent a biomarker of heavy metal exposure. The aim of this study was to evaluate the effect of methylmercury on prolactin release and the role of the nitrergic system using prolactin secretory cells (the mammosomatotroph cell line, GH3B6). Exposure to methylmercury (0-100 μM) was cytotoxic in a time- and concentration-dependent manner, with an LC50 higher than described for cells of neuronal origin, suggesting GH3B6 cells have a relative resistance. Methylmercury (at exposures as low as 1 μM for 2 h) also decreased prolactin release. Interestingly, inhibition of nitric oxide synthase by N-nitro-L-arginine completely prevented the decrease in prolactin release without acute neurotoxic effects of methylmercury. These data indicate that the decrease in prolactin production occurs via activation of the nitrergic system and is an early effect of methylmercury in cells of pituitary origin.

Drying of sicilian lemon residue: influence of process variables on the evaluation of the dietary fiber produced

In this study, the effect of the process variables of the air-drying of Sicilian lemon residues on some technological properties of the fibers produced was studied. The determination and modeling of desorption isotherms were used to establish the equilibrium moisture content at 60, 75, and 90 °C using the static method with 8 saturated salt solutions. The best fit was obtained with BET and GAB models. The drying process was conducted in a vertical tray dryer and delineated according to a central composite experimental design (2²) using the following as factors: air velocity (0.5, 0.75 and 1 m/s) and temperature (60, 75, and 90 °C), and it presented a good fit to the exponential model (R² > 99.9%). The experimental design responses evaluated were the technological properties of the fibers: water-holding, oil-holding, and swelling capacity. Since these properties were present in high levels, the lemon residues could be used to increase content of fibers in foods resulting in the addition of nutritional benefits for the consumers.