The toxin BjussuLAAO-II induces oxidative stress and DNA damage, upregulates the inflammatory cytokine genes TNF and IL6, and downregulates the apoptotic-related genes BAX, BCL2 and RELA in human Caco-2 cells.

Colorectal carcinoma is one of the most common cancers in adults. As chemotherapy, the first-choice treatment for colorectal carcinoma, is often infeasible due to acquired tumor resistance and several adverse effects, it is important to discover and explore new molecules with better therapeutic action. Snake venom toxins have shown promising results with high cytotoxicity against tumor cells, but their mechanisms of action remain unclear. Here we examined how BjussuLAAO-II, an L-amino acid oxidase isolated from Bothrops jararacussu snake venom, exerts cytotoxicity towards colorectal adenocarcinoma human cells (Caco-2) and human umbilical vein endothelial cell line (HUVEC). A 24-h treatment with BjussuLAAO-II at 0.25 - 5.00 µg/mL diminished cell viability by decreasing (i) mitochondrial activity, assessed by reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and resazurin; (ii) the activity of acid phosphatases; and (iii) lysosomal function, assessed by neutral red uptake. BjussuLAAO-II also increased intracellular levels of reactive oxygen species and DNA damage, as assessed by fluorescence and the comet assay, respectively. BjussuLAAO-II altered the expression of cell proliferation-related genes, as determined by RT-qPCR: it elevated the expression of the inflammatory cytokine genes TNF and IL6, and lowered the expression of the apoptotic-related genes BAX, BCL2, and RELA. Therefore, BjussuLAAO-II induces Caco-2 cells death by acting on multiple intracellular targets, providing important data for further studies to assess whether these effects are seen in both tumor and normal cells, with the aim of selecting this drug for possible therapeutic purposes in the future.

Protective effects of the exopolysaccharide Lasiodiplodan against DNA damage and inflammation induced by doxorubicin in rats: Cytogenetic and gene expression assays.

The lasiodiplodan (LS) is a ß-(1→6)-d-glucan produced by the fungus Lasiodiplodia theobromae and some of the biological activities of LS were reported as hypoglycemic, anticoagulant, anti-proliferative and anticancer action; however, its effects on DNA instability and modulation of gene expression are still unclear. Aims of study were investigate the genotoxic effects of lasiodiplodan, and its protective activity against DNA damage induced by doxorubicin (DXR) and its impact on the expression of genes associated with DNA damage and inflammatory response pathways. Therefore, Wistar rats were treated (15 days) orally with LS (5.0; 10 and 20mg/kg bw) alone and in combination with DXR (15mg/kg bw; administrated intraperitoneally on 14th day) as well as their respective controls: distilled water and DXR. Monitoring of DNA damage was assessed by comet and micronucleus (MN) assays and gene expression was evaluated by PCR-Arrays. Treatments with LS alone did not induce disturbances on DNA; when LS was given in combination with DXR, comet and MN formations were reduced to those found in the respective controls. Moreover, LS was able to reduce the disturbances on gene expressions induced by DXR treatment, since the animals that receive LS associated with DXR showed no alteration in the expression of genes related to DNA damage response. Also, DXR induced several up- and down-regulation of several genes associated to inflammatory process, while the animals that received LS+DXR had their gene expression patterns similar to those found in the control group. In conclusion, our results showed that LS did not induce disturbances on DNA stability and significantly reduce the DNA damage and inflammation caused by DXR exposure. In addition, we give further information concerning the molecular mechanisms associated to LS protective effects which seems to be a promising nutraceutical with chemopreventive potential.

Effects of lutein and chlorophyll b on GSH depletion and DNA damage induced by cisplatin in vivo.

Recent studies have proposed the use of low concentrations of phytochemicals and combinations of phytochemicals in chemoprevention to reduce cytotoxicity and simulate normal ingestion through diet. The purpose of the present study was to evaluate whether the DNA damage, chromosome instability, and oxidative stress induced by cisplatin (cDDP) are modulated by a combination of the natural pigments lutein (LT) and chlorophyll b (CLb). The protective effects observed for synergism between phytochemicals have not been completely investigated. The comet assay and micronucleus test were performed and the catalase activities and glutathione (GSH) concentrations were measured in the peripheral blood, bone marrow, liver, and kidney cells of mice. The comet assay and micronucleus test results revealed that the pigments LT and CLb were not genotoxic or mutagenic and that the pigments presented antigenotoxic and antimutagenic effects in the different cell types evaluated. This protective effect is likely related to antioxidant properties in peripheral blood cells through the prevention of cDDP-induced GSH depletion. Altogether our results show that the combination of LT and CLb, which are both usually present in the same foods, such as leafy green vegetables, can be used safely.

Cisplatin induces mitochondrial oxidative stress with resultant energetic metabolism impairment, membrane rigidification and apoptosis in rat liver.

Cisplatin is a potent and widely used chemotherapeutic agent. Nephrotoxicity induced by this drug has been well documented. However, very little information is available on cisplatin-induced hepatotoxicity and its underlying mechanism remains unclear. High doses of cisplatin have been known to produce hepatotoxicity. Additionally, elevated expression of CYP 2E1 has been associated with enhanced cisplatin-induced hepatotoxicity. Several studies suggest that cisplatin toxicity occurs by the increased generation of reactive oxygen species (ROS) in mitochondria. Therefore, the present study examined, in vivo, the cisplatin-induced effects on hepatic mitochondrial structure and function as well as the occurrence of hepatocellular death by apoptosis. Adult male Wistar rats (200-220 g) were divided into two groups (n=8) treated as follows: (1) control group (saline solution, 1 ml 100 g(-1) body weight, i.p.) and (2) cisplatin group (10 mg kg(-1) body weight, i.p.). The animals were killed 72 h after the treatment. Hepatotoxicity was evidenced in the cisplatin group by the increased serum levels of alanine (ALT) and aspartate (AST) aminotransferases. The mechanism of cisplatin-induced hepatotoxicity was found to involve membrane rigidification; decreased GSH/GSSG ratio, ATP, GSH and NADPH levels; lipid peroxidation; oxidative damage of cardiolipin and protein sulfhydryl groups. Moreover, cell death by apoptosis was also demonstrated and the findings strongly suggest the participation of the mitochondrial signaling pathway in this process. Therefore, the results show the key role of mitochondria in the hepatotoxicity induced by cisplatin and delineate several mitochondrial processes that could be targeted in future cytoprotective therapy approaches.

Hydroxyl radical scavenger ameliorates cisplatin-induced nephrotoxicity by preventing oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria.

Nephrotoxicity is the major dose-limiting factor of cisplatin chemotherapy. Reactive oxygen species generated in mitochondria are thought to be the main cause of cellular damage in such injury. The present study examined, in vivo, the protective potential of the hydroxyl radical scavenger dimethylthiourea (DMTU) against cisplatin-induced effects on renal mitochondrial bioenergetics, redox state and oxidative stress. Adult male Wistar rats (200 to 220 g) were divided into four groups of eight animals each. The control group was treated only with an intraperitoneal (i.p.) injection of saline solution (1 ml/100 g body weight). The second group was given only DMTU (500 mg/kg body weight, i.p, followed by 125 mg/Kg, i.p., twice a day until they were killed). The third group was given a single injection of cisplatin (10 mg/kg body weight, i.p.). The fourth group was given DMTU (500 mg/kg body weight, i.p.), just before the cisplatin injection (10 mg/kg body weight, i.p.), followed by injections of DMTU (125 mg/kg body weight, i.p.) twice a day until they were killed. Animals were killed 72 h after the treatment. Besides not presenting any direct effect on mitochondria, DMTU substantially inhibited cisplatin-induced mitochondrial injury and cellular death by apoptosis, suppressing the occurrence of acute renal failure. All the following cisplatin-induced effects were prevented by DMTU: (1) increased plasmatic levels of creatinine and blood urea nitrogen (BUN); (2) decreased ATP content, calcium uptake and electrochemical potential; (3) oxidation of lipids, including cardiolipin; and oxidation of proteins, including sulfhydryl, and aconitase enzyme, as well as accumulation of carbonyl proteins; (4) depletion of the antioxidant defense (NADPH and GSH) and (5) increased activity of the apoptosis executioner caspase-3. Our findings show the important role played by mitochondria and hydroxyl radicals in cisplatin-induced nephrotoxicity, as well as the effectiveness of DMTU in preventing the renal mitochondrial damage caused by cisplatin. These results strongly suggest that protection of mitochondria by hydroxyl radical scavengers may be an interesting approach to prevent the kidney tissue damage caused by cisplatin-chemotherapy.

Cisplatin-induced nephrotoxicity is associated with oxidative stress, redox state unbalance, impairment of energetic metabolism and apoptosis in rat kidney mitochondria.

The clinical use of cisplatin (cis-diamminedichloroplatinum II) is highly limited by its nephrotoxicity. The precise mechanisms involved in cisplatin-induced mitochondrial dysfunction in kidney have not been completely clarified. Therefore, we investigated in vivo the effects of cisplatin on mitochondrial bioenergetics, redox state, and oxidative stress as well as the occurrence of cell death by apoptosis in cisplatin-treated rat kidney. Adult male Wistar rats weighing 200-220 g were divided into two groups. The control group (n = 8) was treated only with an intraperitoneal (i.p.) injection of saline solution (1 ml per 100 g body weight), and the cisplatin group (n = 8) was given a single injection of cisplatin (10 mg/kg body weight, i.p.). Animals were sacrificed 72 h after the treatment. The cisplatin group presented acute renal failure characterized by increased plasmatic creatinine and urea levels. Mitochondrial dysfunction was evidenced by the decline in membrane electrochemical potential and the substantial decrease in mitochondrial calcium uptake. The mitochondrial antioxidant defense system was depleted, as shown by decreased GSH and NADPH levels, GSH/GSSG ratio, and increased GSSG level. Moreover, cisplatin induced oxidative damage to mitochondrial lipids, including cardiolipin, and oxidation of mitochondrial proteins, as demonstrated by the significant decrease of sulfhydryl protein concentrations and increased levels of carbonylated proteins. Additionally, aconitase activity, which is essential for mitochondrial function, was also found to be lower in the cisplatin group. Renal cell death via apoptosis was evidenced by the increased caspase-3 activity. Results show the central role of mitochondria and the intensification of apoptosis in cisplatin-induced acute renal failure, highlighting a number of steps that might be targeted to minimize cisplatin-induced nephrotoxicity.

Antioxidant action of bixin against cisplatin-induced chromosome aberrations and lipid peroxidation in rats.

Cisplatin is one of the most active cytotoxic agents in the treatment of cancer, but has serious side effects, inducing nephrotoxicity and chromosome aberrations. In this study we evaluated the role of the carotenoid bixin on cisplatin-induced oxidative stress in Wistar rats through three markers of oxidative damage: chromosome aberrations, glutathione depletion and lipid peroxidation. The animals were divided into six treatment groups with six rats in each (n= 6). The dose of cisplatin (5.0 mg kg(-1)body wt.) was injected i.p. and bixin (2.5 or 5.0 mg kg(-1)body wt.) was given by gavage at 48, 24 h and 10 min before the cisplatin injection. The treatment with the highest dose of bixin resulted in a statistically significant reduction, by about 33%, in cisplatin-induced abnormal metaphases (P< 0.05). A single dose of cisplatin enhanced the formation of lipid peroxides in 29% and resulted in a 29% depletion in renal glutathione 24 h after cisplatin administration (P< 0.05). The pretreatment with bixin reduced the total number of chromosome aberrations, inhibited the increase in lipid peroxidation, and inhibited renal glutathione depletion induced by cisplatin. Since the pretreatment with bixin alone was safe, under the present experimental conditions, the results suggest that bixin may have future clinical application after further studies.

Oral administration of sodium selenite minimizes cisplatin toxicity on proximal tubules of rats.

Cisplatin (c-DDP) is a widely used antineoplastic drug whose main side effect is nephrotoxicity. Selenium, administered intravenously or intraperitoneally, has been shown to provided protection against c-DDP-induced nephrotoxicity in rats. In the present study, the protective effect of orally administered sodium selenite on c-DDP toxicity was further examined. Animals treated with c-DDP alone showed increased urinary volume, decreased creatinine clearance (GFR), and a rise in urinary N-acetyl-(beta-D-glucosaminidase) (NAG) isoenzyme B activity. When sodium selenite was given prior to c-DDP, rats showed less GFR decline, delayed urinary volume increases, and no urinary NAG isoenzyme B activity increment. It is suggested that a single oral dose of sodium selenite given prior to c-DDP administration, although not preventing deterioration of renal function, partially protects rats from early proximal tubular injury.

[Iron status, malnutrition and acute phase response in HIV-positive patients]. / Resposta de fase aguda, subnutrição e estado nutricional do ferro em adultos com AIDS.

Nutritional status and some iron metabolism parameters of acute phase response (APR) positive and APR-negative AIDS patients were studied. Twenty-nine AIDS patients were submitted to 24h food intake recall, anthropometry, and albumin, C-reactive protein (CRP), hemoglobin, ferritin, and total iron binding capacity (TIBC) measurements. Infection plus serum CRP > 7 mg/dl were criteria for APR presence. Protein-energy malnutrition (PEM) was ascertained by body mass index (BMI) lower than 18.5 kg/m2 and height-creatinine index (HCI < 70%). PEM (77.8 vs 40%) and pulmonary tuberculosis (44. 4 vs 9.5%) were more frequent in APR-positive patients, which also had lower serum albumin (3.7 +/- 0.9 vs 4.3 +/- 0.9 g/dl), TIBC (165. 8 +/- 110.7 vs 265.9 +/- 74.6 mg/dl) and blood hemoglobin (10.5 +/- 1. 8 vs 12.6 +/- 2.3g/dl). Iron intake was similar between groups; however, serum ferritin levels (median, range) were higher among APR-positive (568, 45.3-1814 vs 246, 18.4-1577 ng/ml) patients. HIV-positive adults with systemic response to invading pathogens showed worse nutritional status than those APR-negative. In APR-positive AIDS patients, anemia appears to be unrelated to recent iron intake.

Nutritional assessment of vitamin E in malnourished patients with AIDS.

Malnourished patients with acquired immunodeficiency syndrome (AIDS) may have low serum levels and reduced intake of alpha-tocopherol, mainly in the presence of acute-phase response. The aims of this study were to compare intake and serum levels of alpha-tocopherol between malnourished (MN) and non-malnourished (NMN) AIDS patients and to correlate alpha-tocopherol intake and serum levels. Undernutrition was defined as having a body mass index lower than 18. 5 kg/m(2) or a height-creatinine index lower than 70%. A semiquantitative food frequency questionnaire assessed alpha-tocopherol intake. High-performance liquid chromatography determined vitamin serum levels. The patients were divided into MN (n = 14) and NMN (n = 15) groups. There were no statistical differences in relation to clinical findings between MN and NMN, respectively, including moniliasis (7/14 versus 4/15), neurocryptoccocosis and neurotoxoplasmosis (6/14 versus 6/15), pulmonary tuberculosis (4/14 versus 2/15), and fever (1/14 versus 3/15). MN and NMN groups had similar peripheral blood CD(4) levels (111.4+/-87.1 versus 124.4+/-90.9 cells/mm(3)), and both groups had similar and adequate alpha-tocopherol intake (MN = 50.0+/-11.0 versus NMN = 47.2+/-16.5 mg) and serum levels (MN = 17.8+/-7.2 versus NMN = 19.8+/-6.3 micromol/L). Vitamin E intake and serum levels did not show a significant correlation (r = -0.22, P 0.05). Protein-energy nutrition status and acute-phase response were not factors determining vitamin status among AIDS patients.

Effects of the antioxidants curcumin and vitamin C on cisplatin-induced clastogenesis in Wistar rat bone marrow cells.

The use of dietary antioxidants to prevent antitumor agent-induced chromosomal damage in nontumor cells is currently eliciting considerable interest. Curcumin (CMN) is a dietary antioxidant that has been reported to protect against clastogenesis in in vivo and in vitro assays. This study was undertaken to investigate the modulatory effects of CMN on cisplatin-induced chromosomal aberrations in Wistar rat bone marrow cells and whether there is any potentiation of these effects with the combination between CMN and vitamin C (VC), which has been reported to reduce the clastogenic effect of many antitumor agents in in vivo assays. Animals treated with CMN plus a single dose of cisplatin, at 18, 24 or 72 h following treatment, presented a statistically significant reduction in the total amount of chromosomal damage and in the number of abnormal metaphases. The results also indicate that the combination between antioxidants would not be effective in protecting against cisplatin-induced chromosomal damage in animals sacrificed 24 h after cisplatin treatment. Under the present experimental conditions, CMN could prevent cisplatin-induced clastogenesis by acting as a free radical scavenger.

Dietary intake of selenium and its concentration in breast milk.

Selenium concentration was measured in the breast milk of 30 mothers at different stages of lactation and various body mass indices (BMI). For a maternal mean selenium intake meeting 100% of the Recommended Daily Allowance, mean milk selenium concentration was 14.06 ng/mL (range: 10.0-24.7 ng/mL). No significant correlation was found between the concentration of milk selenium with the stage of lactation, BMI, or dietary selenium intake.

Disaccharidase levels in normal epithelium of the small intestine of rats with iron-deficiency anemia.

Iron-deficiency anemia is the nutritional deficiency most frequently occurring throughout the world, which manifests as a complex systemic disease involving all cells, affecting enzyme activities and modifying protein synthesis. In view of these considerations, the objective of the present study was to determine the effects of iron-deficiency anemia on disaccharidases and on the epithelial morphokinetics of the jejunal mucosa. Newly weaned male Wistar rats were divided into 4 groups of 10 animals each: C6w received a standard ration containing 36 mg elemental iron per kg ration for 6 weeks; E6w received an iron-poor ration (5-8 mg/kg ration) for 6 weeks; C10w received an iron-rich ration (36 mg/kg ration) for 10 weeks; E10w received an iron-poor ration for 6 weeks and then an iron-rich ration (36 mg/kg) for an additional 4 weeks. Jejunal fragments were used to measure disaccharidase content and to study cell proliferation. The following results were obtained: 1) a significant reduction (P < 0.001) of animal weight, hemoglobin (Hb), serum iron and total iron-binding capacity (TIBC) in group E6w as compared to C6w; reversal of the alterations in Hb, serum iron and TIBC with iron repletion (E10w = C10w); animal weights continued to be significantly different in groups E10w and C10w. 2) Sucrase and maltase levels were unchanged; total and specific lactase levels were significantly lower in group E6w and this reduction was reversed by iron repletion (E10w = C10w). 3) The cell proliferation parameters did not differ between groups. On the basis of these results, we conclude that lactase production was influenced by iron deficiency and that this fact was not related to changes in cell population and proliferation in the intestinal mucosa.

Disaccharidase levels in normal epithelium of the small intestine of rats with iron-deficiency anemia

Iron-deficiency anemia is the nutritional deficiency most frequently occurring throughout the world, which manifests as a complex systemic disease involving all cells, affecting enzyme activities and modifying protein synthesis. In view of these considerations, the objective of the present study was to determine the effects of iron-deficiency anemia on disaccharidase and on the epithelial morphokinetics of the jejunal mucosa. Newly weaned male Wistar rats were divided into 4 groups of 10 animals each: C6w received a standard ration containing 36 mg elemental iron per Kg ration for 6 weeks; E6w received and iron-poor ration (5-8 mg/kg ration) for 6 weeks; C10w received an iron-rich ration (36 mg/kg ration) for 10 weeks; E10w received an iron-poor ration for 6 weeks and then an iron-rick ration (36 mg/kg) for an additional 4 weeks. Jejunal fragments were used to measure disaccharidase content and to study cell proliferation. The following results were obtained: 1) a significant reduction (P<0.001) of animal weight, hemoglobin (Hb), serum iron and total iron-binding capacity (TIBC) in groups E6w as compared to C6w; reversal of the alterations in Hb, serum iron and TIBC with iron repletion (E10w = C10w); animal weights continued to be significanly different in group E10w and C10w. 2) Sucrase and maltase levels were unchanged; total and specific lactase levels were significantly lower in group E6w and this reduction was reversed by iron repletion (E10w = C10w). 3) The cell proliferation parameters did not differ between groups. On the basis of these results, we conclude that lactase production was influenced by iron deficiency and that fact was not related to changes in cell population and proliferation in the intestinal mucosa.

[Dietary fiber intake in women with constipation]. / Ingestão de fibras alimentares em mulheres com constipação intestinal.

The objective of this study was to investigate the food pattern of women with constipation. It was carried out a 24 h recall and a food frequency survey in 31 constipated women and compared the results with a control of 31 women without constipation. Their mean daily crude fiber intake were no statistical different (2.33 +/- 1.4 vs 2.17 +/- 0.9 g/day), but the usual intake of fruits and vegetables of the constipated ones were irregular. These results call the attention for the influence of the kind and schedule of the food rich fiber intake on the intestinal habit.

[The use of drinking water as a vehicle of nutrients: experimental studies with iron]. / Utilização da água potável como veiculo de nutrientes: estudos experimentais com ferro.

This investigation is part of a large project, the objective of which is to study the possibility of utilizing drinking water as a carrier of nutrients, such as iron. Various iron salts and in different concentrations, were added to water and tested for their effect on color and turbidity. The bioavailability of various forms of iron salts added to drinking water was tested using the rat bioassay technique. The results of this study indicated that ferric ammonium citrate changed very little the color and turbidity of the water solutions. It was considered the best suitable form of iron to be added to drinking chloride water. Ferric ammonium citrate, ferrous sulfate and ferrous gluconate were all found effective in preventing rat anemia when added to their drinking water.

Acute effects of calcitriol and phosphate salts on mineral metabolism in children with hypophosphatemic rickets.

We investigated the acute effects of oral administration of 1,25-dihydroxyvitamin D (1,25-(OH)2D) and phosphate on the major mineral metabolism indexes in six children with vitamin D-resistant rickets treated with a long-term regimen of phosphate and calcitriol. Two acute tests were performed in which plasma calcium, phosphate, immunoreactive parathyroid hormone (iPTH) (intact molecule), 25-hydroxyvitamin D (25-OHD), and 1,25-(OH)2D levels were measured: the first after an oral phosphate load (20 mg/kg) was administered after calcitriol had been discontinued for 10 days, and the second after a calcitriol load (0.03 microgram/kg) plus the same phosphate load but with the children receiving the usual combination treatment. There were no significant differences in basal levels of calcium, phosphate, iPTH, 25-OHD, or 1,25-(OH)2D between the two tests, nor were delta percent calcium and 25-OHD values significantly different. The delta percent plasma phosphate concentration at 60 minutes was significantly higher during test 2 than during test 1 (p less than 0.01) and delta percent iPTH concentration at 60 minutes was significantly higher during test 1 than during test 2 (p less than 0.01). In test 2 the iPTH level returned to baseline at 180 minutes. Higher delta percent 1,25-(OH)2D values at 60 minutes were observed in test 2 than in test 1 (p less than 0.01). Furthermore, the delta percent 1,25-(OH)2D levels were still higher at 180 minutes in test 2 than during test 1 (p less than 0.01). Our study indicates that oral calcitriol has an inhibitory effect on iPTH secretion in the hours immediately after oral phosphate administration in children with vitamin D-resistant rickets.