Analysis of genotoxic potentiality of stevioside by comet assay.

Stevioside is a natural non-caloric sweetener extracted from Stevia rebaudiana (Bertoni) leaves. It has been widely used in many countries, including Japan, Korea, China, Brazil and Paraguay, either as a substitute for sucrose in beverages and foods or as a household sweetener. The aim of this work was to study its genotoxic potentiality in eukaryotic cells. Wistar rats were treated with stevioside solution (4mg/mL) through oral administration (ad libitum) and the DNA-induced damage was evaluated using the single cell gel electrophoresis (comet assay). The results showed that treatment with stevioside generates lesions in peripheral blood, liver, brain and spleen cells in different levels, the largest effect being in liver. Therefore, these undesired effects must be better understood, once the data present here point to possible stevioside mutagenic properties.

Biological effects of stevioside on the survival of Escherichia coli strains and plasmid DNA.

Stevioside is widely used daily in many countries as a non-caloric sugar substitute. Its sweetening power is higher than that of sucrose by approximately 250-300 times, being extensively employed as a household sweetener, or added to beverages and food products. The purpose of this study was to ascertain stevioside genotoxic and cytotoxic potentiality in different biological systems, as its use continues to increase. Agarose gel electrophoresis and bacterial transformation were employed to observe the occurrence of DNA lesions. In addition to these assays, Escherichia coli strains were incubated with stevioside so that their survival fractions could be obtained. Results show absence of genotoxic activity through electrophoresis and bacterial transformation assays and drop of survival fraction of E. coli strains deficient in rec A and nth genes, suggesting that stevioside (i) is cytotoxic; (ii) could need metabolization to present deleterious effects on cells; (iii) is capable of generating lesions in DNA and pathways as base excision repair, recombination and SOS system would be important to recover these lesions.

[The cholesterol-lowering effect of black, carioquinha and red beans (Phaseolus vulgaris, L.) in hypercholesterolemic rats]. / Efeito dos feijões (Phaseolus vulgaris, L.) preto, carioquinha e vermelho na redução do colesterol sangüíneo de ratos hipercolesterolêmicos.

The propose of the present work was to investigate the cholesterol-lowering effect of black carioquinha and red beans (Phaseolus vulgaris, L.), widely consumed in Brazil, in hypercholesterolemic rats. Five groups of 8 male rats, Wistar strain, initial body weight of 200 g were kept at 25 degrees in a light-dark cycle of 12 h, for 28 days. The group Standard received a basal casein diet. Group Control was formulated by the addition of 1% cholesterol to the basal diet to produce hypercholesterolemia in the rats. The other groups received similar diets to the Control, substituted by 30% black (BB), carioquinha (CB) or red (RB) beans, on dry-weight. The addition of 1% cholesterol promoted an increase of 49% in the levels of total blood cholesterol on Control group, compared with the Standard. The bean diets reduced total blood cholesterol (non-significant): BB reduced 16%, RB 12% and CB 11%, in relation to the Control. The addition of cholesterol to the diets promoted lipid deposition in the rat livers, even in those fed the bean diets. It seems that the reduction of cholesterol in blood is followed by its retention in the rat livers.

Influence of parasite presence on the immunologic profile of peripheral blood mononuclear cells from chagasic patients after specific drug therapy.

The aim of this study is to evaluate the effects of parasite clearance on the immunological profile of peripheral blood mononuclear cells (PBMC) from chagasic patients submitted to specific drug therapy. PBMC were examined by flow cytometry and proliferative responsiveness to Trypanosoma cruzi-related stimuli. Three groups of patients were studied: not treated (NT), treated not cured (TNC) and cured (C). All data were compared to values from uninfected individuals (NI). NT displayed a lower percentage of CD3+ cells as compared to NI, while TNC and C had mean values that were between those from NI and NT. Infected patients had double the percent of CD3+ HLA-DR+ cells, independent of the efficacy of the treatment. Thus, absence of circulating parasites did not reduce T cell activation in Chagas' disease. NT displayed a higher percentage of CD5+ B cells as compared to NI, while TNC and C had mean values between those from NI and NT. In contrast to the phenotypic data, the in vitro mean proliferative responses to parasite-related stimuli of PBMC from C were reduced to the low mean levels observed in NI. These striking differences were statistically different from the high responses seen in NT and TNC. Our data suggest that proliferative responses of PBMC from C reflect immunological changes due elimination of parasite. However, successful treatment did not alter the levels of peripheral T cell activation.

Lytic antibody titre as a means of assessing cure after treatment of Chagas disease: a 10 years follow-up study.

A complement-mediated lysis test (CoML) using living trypomastigotes was compared with conventional serological methods and with haemoculture. Over a 10 years follow-up period evidence was obtained which supported the view that chagasic patients, treated with nitroheterocyclic drugs, in whom CoML had reverted to negative, might be considered cured despite conventional serology remaining positive.

Characteristics of oleate binding to liver plasma membranes and its uptake by isolated hepatocytes.

To clarify mechanisms of hepatic free fatty acid uptake, [3H]oleate uptake by isolated rat hepatocytes was studied, using solutions of 150 microM bovine serum albumin at oleate:albumin molar ratios of 0.033-6.7:1. Oleate partitioning between liver plasma membranes and albumin was also studied, and used to ascertain the membrane binding function for oleate. The experimental uptake curve was complex, but could be resolved by computer fitting into a sum of two components, one a saturable and the second a linear function of the unbound oleate concentration. The saturable component comprises > 90% of total oleate uptake when the oleate:albumin molar ratio is < 2.5, but < 50% when this ratio is > 5. Membrane binding also consisted of a sum of a saturable and a linear component. By comparison of the computer-fitted uptake and binding functions, separate rate constants for the transfer into the cell of the saturably and non-saturably bound oleate were estimated to be 0.7 s-1 and 0.05 s-1, respectively. The former is compatible with a specific, protein-mediated process. It is 15-times greater than the corresponding rate constant for transfer of non-saturably bound oleate into the cell, which in turn is similar to reported rates of non-specific 'flip-flop' of fatty acids across lipid bilayers. The observed kinetics are not consistent with models in which uptake occurs principally from the albumin-bound pool of oleate, or solely from the oleate which has partitioned passively into the lipid bilayer of the plasma membrane.

Effects of endotoxin on iron uptake by the hepatocyte.

Administration of endotoxins is often followed within 12-24 h by marked hypoferremia. Because the hepatocyte is the major site of both iron storage and transferrin synthesis, we have investigated the effects of an Escherichia coli endotoxin (lipopolysaccharide, LPS) on these parameters on isolated hepatocytes from normal Wistar rats (ND), rats previously treated intraperitoneally with 2.5 mg/kg (LD) or 25 mg/kg (HD) LPS, and control rats injected intraperitoneally with sterile saline (CD). No effects were observed on iron uptake from transferrin by ND cells incubated in vitro with up to 350 micrograms LPS/10(7) hepatocytes. There was also no significant difference in iron uptake between CD, HD, and LD hepatocytes 1 h after LPS injection. However, hepatocytes isolated 24 h after LPS administration took up iron significantly faster than controls. The uptake of non-transferrin-bound iron was also increased in HD and LD hepatocytes at 24 h but only in HD cells at 1 h. Transferrin binding was not altered in LPS-treated cells from ND rats but was depressed in cells from LPS-treated rats both at 1 h and at 24 h after injection. Transferrin receptor recycling was significantly increased at 24 h in cells from both LD and HD rats. Transferrin and total protein synthesis were also depressed at 1 h in LPS-treated rats, returning to normal values at 24 h. Direct preincubation of ND cells, however, failed to increase synthesis except at the highest concentrations of LPS. We conclude that LPS has an immediate (although indirect) effect on protein synthesis by the hepatocyte but not on iron uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Idiotype stimulation of T lymphocytes from Trypanosoma cruzi-infected patients.

Anti-Trypanosoma cruzi epimastigote antibodies (anti-epi) from pooled and individual sera from patients with chronic Chagas' disease were purified on immunoaffinity columns of epimastigotes antigens (epi) coupled to activated Sepharose 4B. SDS-PAGE analysis of purified anti-epi preparations showed only the presence of human IgG H and L chains. These antibodies preparations showed similar Western blotting profiles as the sera pools from which they originated. The main polypeptides recognized by anti-epi had apparent molecular masses 31, 46, 51, 75 and 85 kDa. No difference in these patterns were detected between anti-epi from pooled sera of cardiac (anti-epiC) and indeterminate (anti-epiI) clinical forms. Anti-epi preparations (20 to 60 micrograms/ml) of pooled and individual sera stimulated proliferation of homologous and autologous PBMN or T-lymphocyte-enriched population. The stimulatory ability was dependent upon the PBMN-anti-epi combinations. There is no direct correlation between the level of PBMN response to epi and anti-epi stimuli. Comparison of the stimulatory activities of anti-epiC vs anti-epiI on PBMN of either cardiac or indeterminate group of patients indicate that anti-epiC is significantly more active than anti-epiI (p less than 0.025). These data demonstrate the presence of auto-anti-idiotypic-T cells in chagasic patients and lead to the possibility that idiotype/anti-idiotype interactions may play a role in determining the pathogenesis of chagasic cardiopathy.

Depression of iron uptake from transferrin by isolated hepatocytes in the presence of ethanol is a pH-dependent consequence of ethanol metabolism.

Incubation of freshly isolated rat hepatocytes with highly purified radiolabeled rat transferrin in weakly buffered medium in the presence of 10 mM ethanol resulted in a marked diminution of iron uptake by these cells, associated with a greater pH depression than in ethanol-free control studies. This effect on iron uptake persisted, even when the cells were preincubated for 90 min with ethanol before the addition of transferrin. Increasing the buffering capacity of the system or the addition of a metabolic inhibitor of alcohol dehydrogenase (4-methylpyrazole) returned iron uptake to control values. Acetaldehyde, acetate, lactate (products of ethanol metabolism), and 3-butanol (an alcohol not metabolized by alcohol dehydrogenase) had no influence on iron uptake. Further investigation of iron uptake over the pH range 6-8.5 revealed a marked dependency of iron uptake on the extracellular pH. Leucine incorporation into cell protein was also found to be pH dependent. It is suggested that, in the light of current understanding of transferrin recycling by other cell types, the disturbances of iron homeostasis observed in alcoholics can be partially accounted for by alterations in their acid-base metabolism.

Cellular immune responses of chagasic patients to antigens derived from different Trypanosoma cruzi strains and clones.

The reactivity of peripheral blood mononuclear cells (PBMN) from 62 chagasic patients to antigens prepared with different Trypanosoma cruzi strains and clones belonging to different zymodemes was evaluated by the incorporation of 3H-thymidine into DNA. Standardization of experimental conditions was carried out by establishing the proper antigen concentration (15-20 micrograms protein), the adequate period of time (5-6 days) and the best cell concentration (300,000/well). Individual analysis of 62 patients showed 2 distinct patterns of cellular response. One group of patients (32%) had low cellular responses to all antigens tested while the remaining patients had high response to at least 1 of the antigens. No relationship of the immune responsiveness to the patients' clinical forms could be established. In addition, the PBMN response to different strain and clone antigens was not statistically significant. Thus, it appears that the cellular response induced by any particular clone or strain represents an expression of the stimulation of their common antigenic make-up.

Transferrin metabolism in alcoholic liver disease.

The metabolism of transferrin was studied using purified 125I-labeled transferrin in 11 alcoholic patients; six with fatty liver and five with cirrhosis. Six healthy subjects whose alcohol intake was les than 40 gm daily were studied as a control group. There were no significant differences in the mean fractional catabolic rate and plasma volume in the alcoholic groups when compared with control subjects. A significantly decreased mean serum transferrin concentration was found in the alcoholic cirrhotic patients (1.8 +/- 0.3 gm per liter vs. 2.9 +/- 0.2; p less than 0.01), resulting from diminished total body synthesis (0.9 +/- 0.2 mg per kg per hr vs. 1.8 +/- 0.2; p less than 0.01). In contrast, in the patients with alcoholic fatty liver, the mean total body transferrin synthesis (2.4 +/- 0.3 mg per kg per hr) was significantly increased when compared with controls (p less than 0.05). For all the alcoholic patients, the serum transferrin correlated with transferrin synthesis (r = + 0.70; p less than 0.01) but the serum iron did not. These results suggest that, in alcoholic cirrhosis, transferrin synthesis is decreased, probably reflecting diminished synthetic capacity by the liver. In contrast, in patients with alcoholic fatty liver, transferrin turnover is accelerated.