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.

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 RARß by 2.0-fold (quantitative real-time PCR). Our data show that RARß 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 RARß is epigenetically altered.

Evaluation of sperm DNA peroxidation in fertile and subfertile dogs.

Oxidative stress (OS) has been recognized as one of the most important causes of male infertility. The antioxidant activities of seminal plasma and epididymal fluid are not enough to prevent OS, which can damage sperm membranes and DNA, so antioxidant supplementation has been used as a treatment of male infertility. The aim of this experiment was to evaluate the DNA peroxidation before and after antioxidant supplementation with vitamin C and E in dogs with and without fertility problems. A total of eleven dogs were used and were divided in two groups: fertile group (G1), dogs with normal spermiogram (n = 5); subfertile group (G2): dogs with low sperm count (<20 × 10(6) sptz/ml) and/or more than 30% of total sperm pathology (n = 6). Both groups received 500 mg/day of vitamin C and 500 mg/day of vitamin E for 60 days. A semen sample was collected before (M1) and after (M2) oral supplementation. Samples were analysed for DNA peroxidation by measuring the 8-hydroxy-2'-deoxyguanosine concentration. No significant difference was observed between groups at either time. Oral supplementation with 500 mg/day of vitamin C and 500 mg/day of vitamin E did not change the DNA peroxidation in fertile and subfertile dogs.

The inflammatory stimulus of a natural latex biomembrane improves healing in mice

The aim of the present study was to compare healing obtained with biomembranes with the natural healing process (sham) using biochemical and immunohistological assays. C57BL/6 mice were divided into 4 groups of 15 mice each and received different subcutaneous implants: natural latex biomembrane (NLB), denatured latex (DL), expanded polytetrafluorethylene (ePTFE), or sham. On the 2nd, 7th, and 14th days post-treatment, 5 mice per group were sacrificed and biopsied for the following measurements: oxidative stress based on malondialdehyde (MDA), myeloperoxidase (MPO) and hydrogen peroxide by the method of ferrous oxidation-xylenol orange (FOX), as well as glutathione and total proteins; histological evaluation to enumerate inflammatory cells, fibroblasts, blood vessels, and collagen, and immunohistochemical staining for inducible nitric oxide synthase, interleukin-1β, vascular endothelial growth factor (VEGF), and transforming growth factor-β1 (TGF-β1). On day 2 post-treatment, NLB stimulated a dense inflammatory infiltrate mainly consisting of polymorphonuclear cells, as indicated by increased MPO (P < 0.05), but oxidative stress due to MDA was not observed until the 7th day (P < 0.05). The number of blood vessels was greater in NLB (P < 0.05) and DL (P < 0.05) mice compared to sham animals on day 14. NLB induced fibroplasia by day 14 (P < 0.05) with low expression of TGF-β1 and collagenesis. Thus, NLB significantly induced the inflammatory phase of healing mediated by oxidative stress, which appeared to influence the subsequent phases such as angiogenesis (with low expression of VEGF) and fibroplasia (independent of TGF-β1) without influencing collagenesis.

Water extracts of cabbage and kale inhibit ex vivo H2O2-induced DNA damage but not rat hepatocarcinogenesis

The chemopreventive potential of water extracts of the Brassica vegetables cabbage and kale was evaluated by administering their aqueous extracts in drinking water ad libitum to Wistar rats submitted to Ito’s hepatocarcinogenesis model (CB group and K group, respectively - 14 rats per group). Animals submitted to this same model and treated with water were used as controls (W group - 15 rats). Treatment with the vegetable extracts did not inhibit (P > 0.05) placental glutathione S-transferase-positive preneoplastic lesions (PNL). The number of apoptotic bodies did not differ (P > 0.05) among the experimental groups. Ex vivo hydrogen peroxide treatment of rat livers resulted in lower (P < 0.05) DNA strand breakage in cabbage- (107.6 ± 7.8 µm) and kale- (110.8 ± 10.0 µm) treated animals compared with control (120.9 ± 12.7 µm), as evaluated by the single cell gel (comet) assay. Treatment with cabbage (2 ± 0.3 µg/g) or kale (4 ± 0.2 µg/g) resulted in increased (P < 0.05) hepatic lutein concentration compared with control (0.5 ± 0.07 µg/g). Despite the absence of inhibitory effects of cabbage and kale aqueous extracts on PNL, these Brassica vegetables presented protection against DNA damage, an effect possibly related to increased hepatic lutein concentrations. However, it must be pointed out that the cause-effect relationship between lutein levels and protection is hypothetical and remains to be demonstrated.

Effect of beta-carotene supplementation on rats submitted to chronic ethanol ingestion.

Chronic alcohol consumption is directly related to the induction of liver damage. The continuous use of ethanol induces the isoenzyme cytochrome P450CYP2E1, which promotes the formation of free radicals, resulting in lipid peroxidation. Among the main antioxidants are vitamin E, reduced glutathione (GSH), vitamin C, and beta-carotene. beta-Carotene has antioxidant activity per se, with a probable protective effect against different types of cancer. However, some studies have shown an increased number of cancer cells when beta-carotene is administered in the presence of chronic ethanol ingestion. On this basis, the objective of the present study was to assess the effect of beta-carotene supplementation on rats chronically treated with a hydroalcoholic solution by determining the levels of vitamin E, beta-carotene, GSH, and thiobarbituric acid reactive species (TBARS). Both the plasma and liver concentrations of beta-carotene were higher in the supplemented groups. Plasma vitamin E levels were decreased in the control group and liver vitamin E levels were significantly decreased (p < 0.05) in all groups compared to basal levels. GSH levels were increased over basal values in the group supplemented with beta-carotene for 14 days. TBARS values were increased as much as four-fold in the control group at 14 days, and declined again at 28 days, whereas they were increased in the supplemented group, with the increase remaining until the end of the experiment. The study indicates that beta-carotene had no beneficial effect as an antioxidant on rats submitted to chronic alcohol intake, and could be act as prooxidant when administered with ethanol.

Vitamin A and all-trans and 9-cis retinoic acids inhibit cell proliferation during the progression phase of hepatocarcinogenesis in Wistar rats.

The effects of vitamin A and all-trans and 9-cis retinoic acids on the progression phase of hepatocarcinogenesis were evaluated in this study. For this purpose, male Wistar rats were first submitted to the resistant hepatocyte model of carcinogenesis (diethylnitrosamine for initiation and 2-acetylaminofluorene for selection/promotion). Ten months after initiation, the animals were distributed into four groups and treated by gavage, every other day and during eight weeks, with corn oil (control group), vitamin A (10 mg/kg of body wt), all-trans retinoic acid (10 mg/kg body wt), or 9-cis retinoic acid (10 mg/kg body wt). After this period, the animals were killed one hour after intraperitoneal administration of 5-bromo-2-deoxyuridine (BrdU, 100 mg/kg body wt). At the time of sacrifice, liver samples were collected for histopathological (hematoxylin-eosin) examination and immunohistochemical detection of glutathione S-transferase and BrdU, as well as for analysis of retinol and retinoic acid concentrations. Histopathological examination showed the lowest incidence of hepatocarcinomas in vitamin A-treated animals. Moreover, groups treated with retinoids demonstrated lower hepatic BrdU labeling indexes in the neoplastic lesions, as well as in their respective surrounding tissues, than controls. Thus vitamin A and all-trans and 9-cis retinoic acid strongly inhibited cell proliferation when administered during the progression phase of hepatocarcinogenesis. Therefore, the anticarcinogenic effects that have been attributed to these retinoids could be partially related to their capacity of inhibiting in vivo cell proliferation.

Lipid peroxidation in nicotinamide-deficient and nicotinamide-supplemented rats.

Supplementation or deficiency of nicotinamide in rats may interfere with the oxidative balance, with excess leading to greater lipid peroxidation, measured by TBARS, and deficiency causing a greater consumption of antioxidants such as vitamin E and glutathione. Urinary N-methylnicotinamide excretion was much more marked in the supplemented group, whereas the difference between deficient and control animals was nonsignificant.

Effect of different vitamin E levels on lipid peroxidation in streptozotocin-diabetic rats.

Administration of streptozotocin is used to induce diabetes in experimental models, causing a selective destruction of pancreatic beta islet cells associated with generation of free radicals. Supplementation with antioxidant vitamins such as vitamin E is a protective factor against free radicals. The objective of this study was to determine the effect of administration of a diet supplemented with, or deficient in vitamin E to streptozotocin diabetic rats, controlled or not with insulin, on plasma glucose, hepatic vitamin E and hepatic thiobarbituric acid reactive substance (TBARS) levels before streptozotocin and 24 hours and one and two weeks after drug administration. Deficiency of vitamin E alone increased TBARS levels, and streptozotocin elevated TBARS two times in deficient groups, regardless of insulin control. In rats supplemented with vitamin E, a reduction of plasma glucose and liver vitamin E was observed two weeks after streptozotocin administration (p < 0.05). In conclusion, vitamin E supplementation probably protected against lipoperoxidation and contributed to the absence of elevation of plasma glucose levels, and vitamin E deficiency produced an increase in hepatic TBARS levels in streptozotocin diabetic rats.

Methionine supplementation of soya products: effects on nitrogen balance parameters.

Soybean protein is one of the best quality foods available. Contribution of soy to human nutrition increases because of its overall positive nutritional profile, low cost, high protein and excellent functional properties. Addition of methionine to rat soybean diets improve biological value of soy protein. Few studies on methionine fortification of soya protein were carried in infants, but fortification of baby formulas with this amino acid is usually found. This study was carried out to demonstrate in malnourished children that the effect of methionine supplementation of soya milk and soy isolated protein, as well as to compare with their results to cows' milk. A total of 30 malnourished children, 1 to 3 years old, admitted to our metabolic unit and distributed in groups of 6 children were studied. They were fed experimental formulas with cows' milk, soya milk, soya milk plus methionine, soya isolated and soya isolated plus methionine. Nutrient compositions of formulas were calculated to be similar to mothers' milk. DL-methionine, 1.5 g per 100 g protein content was added to soya milk and soya isolated formulas. Two nitrogen balances, 3 days each, were carried out. Fecal and urinary nitrogen, serum proteins, creatinine and urea in serum and urine were followed during the study. Results showed differences of intake and retention of nitrogen between some of the groups, but there were no statistically significant differences on protein absorption in the groups. No differences were demonstrated in serum proteins, total nitrogen and other serum and urine parameters analyzed. Cows' milk fed children presented the highest nitrogen retention in both balance studies. The addition of methionine to the soya milk formula increased the nitrogen retention, not reaching the cows' milk levels and did not have the same effect when added to the isolate soy protein.

Absorption, by humans, of beta-carotene from fortified soybean oil added to rice: effect of heat treatment.

OBJECTIVE: This study was carried out to evaluate the absorption of beta-carotene in humans when rice is prepared with refined cooking soybean oil fortified with beta-carotene and to assess the effect of heat treatment on its bioavailability. METHODS: Sixteen healthy adults subjects participated in two experimental trials. Studies were carried out during two experimental periods of 11 days with a 12-day interval between them. Beta carotene was added to the soybean cooking oil and rice was cooked with it or it was added to the rice after cooking. Experimental diets included these two kinds of rice during the first day and fasting blood samples were collected on different days. All of the test diets were low in carotenoids. Plasma carotenoids were measured by HPLC method. beta-carotene absorption was calculated through postabsorptive peak rise in plasma beta-carotene and the total area under the absorption curve was determined by the trapezoidal method for the 11-day period. RESULTS: Absorption of carotene from heated or unheated fortified soybean oil were similar. Peak plasma carotene rise was different in men and women, p < 0.05 (0.66 +/- 0.097 vs. 1.04 +/- 0.117 mumol/l, respectively). Plasma alpha-carotene and retinol showed no variation. CONCLUSIONS: Results demonstrate that beta-carotene added to soybean oil used in the preparation of rice is absorbed, heated or not, and could be a practical source of provitamin A. Developing countries looking for strategies to increase vitamin A intake could use fortification of vegetable oils with synthetic beta-carotene as a simple method.

[Urinary excretion of vitamin A and thiobarbituric acid reactive substances in AIDS patients]. / Excreção urinária de vitamina A e substâncias reativas ao ácido tiobarbitúrico em pacientes com AIDS.

In the present investigation 28 serologically positive HIV-1 patients, 16 patients with AIDS (< 200/mm3 CD4+ T lymphocytes) and 12 with HIV-1 (200 to 500/mm3 CD4+ T lymphocytes) were studied. The Control Group consisted of 11 healthy individuals. The occurrence of alterations in the anthropometric parameters were higher in AIDS patients, compared to HIV-1 and controls patients, indicating a greater level of malnutrition. All individuals in present study showed normal plasma vitamin A levels. Contrasting, urinary excretion of vitamin A were higher in the AIDS Group (0.23 +/- 0.20 mumol/l) than in the HIV Group (0.19 +/- 0.12 mumol/l) and considerably higher than in the Control Group (0.06 +/- 0.05 mumol/l). Urinary excretion of TBARS also were higher in AIDS Group (3.34 +/- 2.65 mumol/l) compared to HIV (1.71 +/- 0.74 mumol/l) and Control Group (1.70 +/- 0.75 mumol/l). These results demonstrate a greater level of malnutrition and elevate excretion of vitamin A and SRATB in urine of AIDS patients. Therefore, monitoring of nutritional status, especially in relation to vitamin A is recommended for patients with HIV and AIDS.

Urinary excretion and plasma vitamin E levels in patients with AIDS.

Individuals with acquired immunodeficiency virus (HIV) and patients with acquired immunodeficiency syndrome (AIDS) present a variety of pathologic alterations that influence their nutritional status during various stages of the disease. Previous studies have reported a reduction in plasma vitamin E levels in these patients associated with a higher production of free radicals. Individuals with infection, fever, or acute diarrhea excrete considerable amounts of vitamin A in urine. This observation raised the hypothesis that this may also be the case for vitamin E and that its urinary excretion may play a significant role in the reduction of plasma vitamin E levels. In the present investigation, 28 serologically positive HIV-1 (HIV group) divided into a group of 16 patients with AIDS (< 200/mm3 CD4+ T lymphocytes) were studied. The control group consisted of 11 healthy individuals. Urinary and plasma vitamin E levels were determined by high-performance liquid chromatography. Patients with AIDS presented reduced plasma vitamin E levels (15.25 +/- 12.19 mumol/L) compared with the HIV (26.40 +/- 17.01 mumol/L) and control (40.03 +/- 31.80 mumol/L) groups. On the other hand, urinary excretion was higher in the AIDS group (0.86 +/- 0.99 mumol/24 h) than in the HIV group (0.62 +/- 0.46 mumol/24 h) and considerably higher than in the control group (0.05 +/- 0.13 mumol/24 h). These results indicate elevated vitamin E excretion in the urine of both patients with AIDS and patients with HIV-1, levels is recommended for patients with HIV and patients with AIDS and, if necessary, the combination of existing medical therapy with vitamin supplementation to maintain the nutritional status related to vitamin E.

Effect of heat treatment on the biological value of beta-carotene added to soybean cooking oil in rats.

Vitamin A deficiency is one of the major nutritional problems in the world, most common in developing countries. Food fortification is a recognised approach to supply vitamins and minerals to needed populations. Vegetable cooking oils were previously suggested by us as a carrier for vitamin A fortification. Fortification of cooking oil with beta-carotene could also be a strategy to prevent vitamin A deficiency. The objective of this article is to start studies on the use of cooking soya oil as a vehicle for synthetic carotene, to evaluate its stability to heat treatment, and to test its bioavailability and bioconversion to vitamin A in rats. Batches of carotene-fortified soybean oil were prepared, containing 2, 4 and 8 RE/g of diet. Some of them were heated to test its stability. At 100 degrees C there was no loss of carotene, at higher temperature carotene retention was 65%. The bioavailability and bioconversion of beta-carotene added to soybean oil was measured through feeding nursing rats and their pups method. Weight gain was good and plasma vitamin A increased significantly in all groups. Liver vitamin A values of rats fed diets with fortified soybean oil heated at 100 degrees C was similar to the 4 RE non-heated fortified oil group (0.72 +/- 0.06 and 0.64 +/- 0.08 mumol/g, respectively). Heated at 170 degrees C the liver total vitamin A value was reduced (0.45 +/- 0.04 mumol/g), but kept bioavailable vitamin A equivalent to 2 RE (0.47 +/- 0.09 mumol/g). Bioconversion of beta-carotene to vitamin A was validated by the plasma and liver findings. beta-carotene added to soybean oil showed good stability to heat and its bioconversion to vitamin A was shown in rat assays. beta-carotene mixed well with edible soybean oil and the fortified cooking oil showed potential as a carrier to be used for the prevention of vitamin A deficiency.

Effect of different dietary levels of vitamin E on lipid peroxidation in rats.

The regulation of normal oxidative balance include the maintenance of adequate levels of dietary antioxidants such as vitamin E. The objective of this investigation was to study the effect of three different dietary levels of vitamin E (normal, supplemented 20 times higher and deficient) on plasma and liver lipid peroxidation, assayed by determination of thiobarbituric acid reactive substances (TBARS) and vitamin E in plasma and liver and hepatic reduced glutathione. Administration of dietary vitamin E caused a dose-dependent increase in liver and plasma concentration of this vitamin to 42.11 micrograms/g liver and 29.52 mumol/l respectively, in the supplemented group, and a low concentration of TBARS, 0.67 nmol/mg protein, in liver. The group receiving the diet without vitamin E showed high values of hepatic TBARS, 2.95 nmol/mg protein, and low values of reduced glutathione and reduced concentration of hepatic and plasma vitamin E (1.75 micrograms/g liver and 3.67 mumol/l, respectively). In conclusion, the vitamin E deficiency alone induces the liver lipid peroxidation in rats, and maintenance of adequate or higher vitamin E levels acts as a protective factor against free radical generation.

A comparison of beta-carotene and vitamin A effects on a hepatocarcinogenesis model.

The effects of beta-carotene (beta C) or vitamin A (VA) administration for 8 consecutive weeks were compared in male Wistar rats submitted to the resistant hepatocyte model (RH model) of hepatocarcinogenesis. Animals treated with corn oil (CO), instead of carotenoid or retinoid, served as controls. At the end of the study, beta C treatment resulted in a substantial reduction in the hepatocyte nodule incidence, total number of nodules and in the nodule multiplicity, as well as in the number and size of hepatic gamma-glutamyltranspeptidase (gamma GT)-positive foci. In contrast, animals administered with VA presented a 100% nodule incidence and only a moderate decrease in the total number of hepatocyte nodules. These showed to be in the great majority larger than nodules observed after beta C treatment. Moreover, VA administration resulted in similar number and size of gamma GT-positive foci than controls. In addition, the hepatic concentrations of total VA increased in both, beta C and VA treated animals. However, as expected, increases in the hepatic carotenoid concentrations could be only observed after beta C application. Therefore, changes in the hepatic levels of beta C, and not of VA, resulted in appreciable inhibitory effects on preneoplastic lesions of the liver. The evidence implies that the chemopreventive property of beta C is unrelated to its provitamin A activity.