Cholesterol induces fetal rat enterocyte death in culture

The effect of cholesterol on fetal rat enterocytes and IEC-6 cells (line originated from normal rat small intestine) was examined. Both cells were cultured in the presence of 20 to 80 µM cholesterol for up to 72 h. Apoptosis was determined by flow cytometric analysis and fluorescence microscopy. The expression of HMG-CoA reductase and peroxisome proliferator-activated receptor gamma (PPARgamma) was measured by RT-PCR. The addition of 20 µM cholesterol reduced enterocyte proliferation as early as 6 h of culture. Reduction of enterocyte proliferation by 28 and 41% was observed after 24 h of culture in the presence and absence of 10% fetal calf serum, respectively, with the effect lasting up to 72 h. Treatment of IEC-6 cells with cholesterol for 24 h raised the proportion of cells with fragmented DNA by 9.7% at 40 µM and by 20.8% at 80 µM. When the culture period was extended to 48 h, the effect of cholesterol was still more pronounced, with the percent of cells with fragmented DNA reaching 53.5% for 40 µM and 84.3% for 80 µM. Chromatin condensation of IEC-6 cells was observed after treatment with cholesterol even at 20 µM. Cholesterol did not affect HMG-CoA reductase expression. A dose-dependent increase in PPARgamma expression in fetal rat enterocytes was observed. The expression of PPAR-gamma was raised by 7- and 40-fold, in the presence and absence of fetal calf serum, respectively, with cholesterol at 80 mM. The apoptotic effect of cholesterol on enterocytes was possibly due to an increase in PPARgamma expression.

Screening the mutagenic activities of coomonly used antiparasite drugs by the Simultest, a simplified Salmonella/microsome plate incorporation assay

The mutagenic activities of 16 anti-parasite drugs were screened by the Simultest in both qualitative (spot test) and quantitative (plate incorporation) assays with a Salmonella typhimurium pool composed by the indicator strains TA97, TA 98, TA100 and TA102. Four anti Chagas' disease drugs (nifurtimox, benznidazole, CL 64,855, and MK 436) and two anti-amebae drugs (metronidazole and tinidazole) gave positive results in qualitative tests and incorporation of rat liver microsomes did not alter the results. Comparative dose response curves of the mutagenic activities of CL 64,855, metronidazole and benznidazole obtained by the simultest and by individual Salmonella indicator strains demonstrated that both approachs have similar sensitivities. The results corroborate the validity of the Simultest, as a simplified, fast and economic version of the Ames test in preliminary screening of potential mutagenic drugs.

An attemp at reversibility and increase of the virulence of axenic strains of Entamoeba histolytica

In this study we have tried to verify whether the interaction "in vitro" with bacteria or small pieces of normal hamster liver would modify the pathogenic behavior of axenic strains of E. histolytica: avirulent ones (ICB-32 and ICB-RPS), of attenuated virulence (ICB-CSP and HM1) and of mean virulence (ICB-462). Every attempt to render virulent, recover or increase the virulence of axenic strains of E. histolytica has failed

Distribuição da xantina oxidase no fígado e no sôro de rato

The localization of the xanthine oxidase (X.O.) and xanthine dehydrogenase (X.D.) activities in rat liver have been studied using separation of cytoplasmic particles into fractions by differential centrifugation. The results clearly demonstrate that practically all the enzymic activity is present in the supernatant fluid corresponding to the cell sap containing the soluble proteins of the cell. No activity could be detected for the nuclear, mitocondrial and microsomal fractions. The enzymatic activity of the mixture of the four factions was 102 per cent of that of the original homogenate. The distribution of the xanthine dehydrogenase in the protein fractions of the rat serum was accomplished in preliminary experiments by means of 50% ammonium sulphate precipitation and subsequent dialysis against water. All enzymatic activity was confined to the globulin fractions of the serum. Paper electrophoresis was performed and the protein and lipoprotein fractions determined. A method for the localization of the X.D. activity in the protein fractions separated by paper electrophoresis was developed. The results obtained suggest that xanthine dehydrogenase is localized in the globulin fractions possessing mobilities of [alpha 1], [beta] and [gamma] globulins and are probably bound to the lipoproteins.

CL 64, 855, a potent do anti-Trypanosoma cruzi drug, is also mutagenic in the salmonella/microsome assay

The nitroimidazole-tiadiazole derivative CL 64,855 (2-amino-5-(1-methyl-5-nitro-2-imidazolyl)-1,3,4-thiadiazole, a potent anti-trypanosomal drug, was assayed in a short-term bacterial mutagenicity test with Salmonella typhimurium strains TA 98, TA 100 and TA 102. Results indicate that CL 64,855 is a potent frameshift mutagen detected by strains TA 98 and TA 102. CL 64,855 was able to revert the indicators strains at concentrations as low as 0.1 µg/plate. Metabolic activation experiments with rat liver microsomal fractions did not increase the mutagenic action of Cl 64,855.

Clinical versus ultrasound examination in the evaluation of hepatosplenic schistosomiasis mansoni in endemic areas

The best way to appraise the size of abdominal organs remains undefined. Herein we compare the size of liver and spleen in hepatosplenic schistosomiasis using clinical and ultrasound (US) examination, and the size of the organs measured by US with their visualization below the costal margin ("palpable by US"). For this study, 411 individuals from an endemic area for schistosomiasis mansoni in Brazil have been selected. We found that palpable spleens and left liver lobes are larger than non palpable ones. Also, 23% of normal spleens measured by US were palpable on clinical examination, and 22% of spleens increased in size on US were non palpable. A total of 21% of normal spleens were "palpable by US". We also found 54% of normal sized right liver lobes palpable on clinical examination, whilst 54% of the increased livers, measured by US, were non palpable. About 76% of normal right liver lobes were "palpable by US". We conclude that the association of clinical, ultrasound and magnetic resonance imaging (MRI) examinations, in the near future, should give the investigators the necessary tools to perform a more accurate clinical diagnosis of hepatosplenic schistosomiasis mansoni.

Chronic lactose intake modifies the gastric emptying of monosaccharides but not of disaccharides in weanling rats

Ninety-six weanling male Wistar rats were fed for four weeks one of two different chows: a normal rat chow containing 55.5% (w/w) starch (control group, N = 48) or a rat chow in which starch was partially replaced by lactose, in such a way that the experimental group (N = 48) received 35.5% (w/w) starch and 20% (w/w) lactose. The gastric emptying of fluid was then studied by measuring the gastric retention of four test meals containing lactose (5% or 10%, w/v) or glucose + galactose (5% or 10%, w/v). Homogenates of the small intestine were assayed for lactase activity. The gastric retention values were obtained 15 min after orogastric infusion of the liquid meals. The median values for gastric retention of the 5% lactose solutions were 37.7% for the control group and 37.0% for the experimental group (P>0.02). For the 10% lactose solution the median values were 51.2% and 47.9% (P>0.02) for the control and experimental groups, respectively. However, for the 2.5% glucose + 2.5% galactose meal the median gastric retention was lower (P<0.02) in the group fed a lactose-enriched chow (38.5%) than in the control group (41.6%). For the 5% glucose + 5% galactose solution the median values were not statistically different between groups, 65.0% for the control group and 58.8% for the experimental group. The median values of the specific lactase activity in the small intestine homogenate was 0.74 U/g in the control group and 0.91 U/g in the experimental group. These values were not statistically different (P>0.05). These results suggest that the prolonged ingestion of lactose by young adult rats changes the gastric emptying of a solution containing 5% monosaccharides. This adaptation may reflect the desensitization of intestinal nutrient receptors, possibly by an osmotic effect of lactose present in the chow.

Ca2+ dependence of gluconeogenesis stimulation by glucagon at different cytosolic NAD+-NADH redox potentials

The influence of Ca2+ on hepatic gluconeogenesis was measured in the isolated perfused rat liver at different cytosolic NAD+-NADH potentials. Lactate and pyruvate were the gluconeogenic substrates and the cytosolic NAD+-NADH potentials were changed by varying the lactate to pyruvate ratios from 0.01 to 100. The following results were obtained: a) gluconeogenesis from lactate plus pyruvate was not affected by Ca2+-free perfusion (no Ca2+ in the perfusion fluid combined with previous depletion of the intracellular pools); gluconeogenesis was also poorly dependent on the lactate to pyruvate ratios in the range of 0.1 to 100; only for a ratio equal to 0.01 was a significantly smaller gluconeogenic activity observed in comparison to the other ratios. b) In the presence of Ca2+, the increase in oxygen uptake caused by the infusion of lactate plus pyruvate at a ratio equal to 10 was the most pronounced one; in Ca2+-free perfusion the increase in oxygen uptake caused by lactate plus pyruvate infusion tended to be higher for all lactate to pyruvate ratios; the most pronounced difference was observed for a lactate/pyruvate ratio equal to 1. c) In the presence of Ca2+ the effects of glucagon on gluconeogenesis showed a positive correlation with the lactate to pyruvate ratios; for a ratio equal to 0.01 no stimulation occurred, but in the 0.1 to 100 range stimulation increased progressively, producing a clear parabolic dependence between the effects of glucagon and the lactate to pyruvate ratio. d) In the absence of Ca2+ the relationship between the changes caused by glucagon in gluconeogenesis and the lactate to pyruvate ratio was substantially changed; the dependence curve was no longer parabolic but sigmoidal in shape with a plateau beginning at a lactate/pyruvate ratio equal to 1; there was inhibition at the lactate to pyruvate ratios of 0.01 and 0.1 and a constant stimulation starting with a ratio equal to 1; for the lactate to pyruvate ratios of 10 and 100, stimulation caused by glucagon was much smaller than that found when Ca2+ was present. e) The effects of glucagon on oxygen uptake in the presence of Ca2+ showed a parabolic relationship with the lactate to pyruvate ratios which was closely similar to that found in the case of gluconeogenesis; the only difference was that inhibition rather than stimulation of oxygen uptake was observed for a lactate to pyruvate ratio equal to 0.01; progressive stimulation was observed in the 0.1 to 100 range. f) In the absence of Ca2+ the effects of glucagon on oxygen uptake were different; the dependence curve was sigmoidal at the onset, with a well-defined maximum at a lactate to pyruvate ratio equal to 1; this maximum was followed by a steady decline at higher ratios; at the ratios of 0.01 and 0.1 inhibition took place; oxygen uptake stimulation caused by glucagon was generally lower in the absence of Ca2+ except when the lactate to pyruvate ratio was equal to 1. The results of the present study demonstrate that stimulation of gluconeogenesis by glucagon depends on Ca2+. However, Ca2+ is only effective in helping gluconeogenesis stimulation by glucagon at highly negative redox potentials of the cytosolic NAD+-NADH system. The triple interdependence of glucagon-Ca2+-NAD+-NADH redox potential reveals highly complex interrelations that can only be partially understood at the present stage of knowledge

Antioxidant and free radical scavenging effects in extracts of the medicinal herb Achyrocline satureioides (Lam.) DC. ("marcela")

Achyrocline satureioides (Lam.) DC. (Compositae) is a medicinal herb used in Argentina, Uruguay, Brazil and Paraguay for its choleretic, antispasmodic and hepatoprotective properties. The presence of the flavonoid quercetin and its derivatives, and of different phenolic acids such as caffeic, chlorogenic and isochlorogenic acids in the aerial parts of this plant has led us to study the antioxidant activity of its extracts using different bioassays. The inhibition of luminol-enhanced chemiluminescence by the aqueous and methanolic extracts was used to show that their total reactive antioxidant potential index (TRAP; in µM Trolox equivalents) was 91.0 ± 15.4 and 128.1 ± 20.1 µM, respectively, while the total antioxidant reactivity index (TAR) was calculated to be 1537 ± 148 and 1910 ± 171 µM. Only the methanolic extract was capable of reducing iron (II)-dependent DNA damage. Lipid peroxidation was assessed by two different methods. The aqueous extract reduced hydroperoxide-initiated chemiluminescence in rat liver homogenates at all concentrations in a dose-dependent manner, with a calculated IC50 = 225 µg/ml, while the methanolic extract was only effective at higher concentrations (100 and 1000 µg/ml). Both aqueous and methanolic extracts were capable of reducing the production of thiobarbituric acid reactive substances (TBARS) in rat liver homogenates, with an IC50 >1000 µg/ml. The results obtained suggest that the extracts of A. satureioides possess significant free radical scavenging and antioxidant activity in vitro, a fact that should encourage future in vivo studies.

Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins

Intercellular communication may be regulated by the differential expression of subunit gap junction proteins (connexins) which form channels with differing gating and permeability properties. Endothelial cells express three different connexins (connexin37, connexin40, and connexin43) in vivo. To study the differential regulation of expression and synthesis of connexin37 and connexin43, we used cultured bovine aortic endothelial cells which contain these two connexins in vitro. RNA blots demonstrated discordant expression of these two connexins during growth to confluency. RNA blots and immunoblots showed that levels of these connexins were modulated by treatment of cultures with transforming growth factor-ß1. To examine the potential ability of these connexins to form heteromeric channels (containing different connexins within the same hemi-channel), we stably transfected connexin43-containing normal rat kidney (NRK) cells with connexin37 or connexin40. In the transfected cells, both connexin proteins were abundantly produced and localized in identical distributions as detected by immunofluorescence. Double whole-cell patch-clamp studies showed that co-expressing cells exhibited unitary channel conductances and gating characteristics that could not be explained by hemi-channels formed of either connexin alone. These observations suggest that these connexins can readily mix with connexin43 to form heteromeric channels and that the intercellular communication between cells is determined not only by the properties of individual connexins, but also by the interactions of those connexins to form heteromeric channels with novel properties. Furthermore, modulation of levels of the co-expressed connexins during cell proliferation or by cytokines may alter the relative abundance of different heteromeric combinations.

Protection against cephalosporin-induced lipid peroxidation and nephrotoxicity by (+)-cyanidanol-3 and vitamin E

The ability of the clinically used cephalosporins: cephalothin, cefotaxime and cefotiam to induce lipid peroxidation (LPO) and renal damage was compared to that of nephrotoxic cephaloridine under in vivo conditions. Glutathione was measured in rat liver or in renal cortex as non-protein sulfhydryls. LPO was measured in plasma, renal cortex and liver by the generation of malondialdehyde or as the increase in renal cortical concentration of conjugated dienes. Impairment of renal function was measured as the decrease in renal cortical accumulation of the organic anion p-aminohippurate (PAH). Administration of cephalosporins to rats as a single dose (2000 mg/kg, ip) induced a significant glutathione-depletion in the renal cortex with cephaloridine, and in the liver with cephaloridine, cephalothin and cefotiam. Treatment of rats with cephaloridine, cephalothin and cefotiam (200, 500, or 1000 mg kg-1 day-1, ip) for 5 days resulted in a dose-dependent increase of LPO in the renal cortex. While cephaloridine induced the highest concentration of conjugated diene, cefotaxime had no effect. Measurements of PAH accumulation in renal cortical slices from cephalosporin-treated rats showed a dose-dependent decrease in the renal cortical accumulation of PAH. Pretreatment with the antioxidants vitamin E or cyanidanol (400 mg kg-1 day-1, ip) 1 h before treatment with cephaloridine, cephalothin or cefotiam (1000 mg kg-1 day-1, ip) for 3 days inhibited cephalosporin-induced LPO and significantly reduced the impairment of renal cortical accumulation of PAH. The potential of different cephalosporins for inducing LPO and reducing PAH accumulation was ranked as follows: cephaloridine > cephalothin > cefotiam > cefotaxime.

The mutagenic and antimutagenic effects of the traditional phytoestrogen-rich herbs, Pueraria mirifica and Pueraria lobata

Pueraria mirifica is a Thai phytoestrogen-rich herb traditionally used for the treatment of menopausal symptoms. Pueraria lobata is also a phytoestrogen-rich herb traditionally used in Japan, Korea and China for the treatment of hypertension and alcoholism. We evaluated the mutagenic and antimutagenic activity of the two plant extracts using the Ames test preincubation method plus or minus the rat liver mixture S9 for metabolic activation using Salmonella typhimurium strains TA98 and TA100 as indicator strains. The cytotoxicity of the two extracts to the two S. typhimurium indicators was evaluated before the mutagenic and antimutagenic tests. Both extracts at a final concentration of 2.5, 5, 10, or 20 mg/plate exhibited only mild cytotoxic effects. The plant extracts at the concentrations of 2.5, 5 and 10 mg/plate in the presence and absence of the S9 mixture were negative in the mutagenic Ames test. In contrast, both extracts were positive in the antimutagenic Ames test towards either one or both of the tested mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide and benzo(a)pyrene. The absence of mutagenic and the presence of anti-mutagenic activities of the two plant extracts were confirmed in rec-assays and further supported by a micronucleus test where both plant extracts at doses up to 300 mg/kg body weight (equivalent to 16 g/kg body weight plant tuberous powder) failed to exhibit significant micronucleus formation in rats. The tests confirmed the non-mutagenic but reasonably antimutagenic activities of the two plant extracts, supporting their current use as safe dietary supplements and cosmetics.

Differential binding with ERα and ERβ of the phytoestrogen-rich plant Pueraria mirifica

Variations in the estrogenic activity of the phytoestrogen-rich plant, Pueraria mirifica, were determined with yeast estrogen screen (YES) consisting of human estrogen receptors (hER) hERα and hERβ and human transcriptional intermediary factor 2 (hTIF2) or human steroid receptor coactivator 1 (hSRC1), respectively, together with the β-galactosidase expression cassette. Relative estrogenic potency was expressed by determining the β-galactosidase activity (EC50) of the tuber extracts in relation to 17β-estradiol. Twenty-four and 22 of the plant tuber ethanolic extracts interacted with hERα and hERβ, respectively, with a higher relative estrogenic potency with hERβ than with hERα. Antiestrogenic activity of the plant extracts was also determined by incubation of plant extracts with 17β-estradiol prior to YES assay. The plant extracts tested exhibited antiestrogenic activity. Both the estrogenic and the antiestrogenic activity of the tuber extracts were metabolically activated with the rat liver S9-fraction prior to the assay indicating the positive influence of liver enzymes. Correlation analysis between estrogenic potency and the five major isoflavonoid contents within the previously HPLC-analyzed tuberous samples namely puerarin, daidzin, genistin, daidzein, and genistein revealed a negative result.

VEGF receptor expression decreases during lung development in congenital diaphragmatic hernia induced by nitrofen

Changes in vascular endothelial growth factor (VEGF) in pulmonary vessels have been described in congenital diaphragmatic hernia (CDH) and may contribute to the development of pulmonary hypoplasia and hypertension; however, how the expression of VEGF receptors changes during fetal lung development in CDH is not understood. The aim of this study was to compare morphological evolution with expression of VEGF receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1), in pseudoglandular, canalicular, and saccular stages of lung development in normal rat fetuses and in fetuses with CDH. Pregnant rats were divided into four groups (n=20 fetuses each) of four different gestational days (GD) 18.5, 19.5, 20.5, 21.5: external control (EC), exposed to olive oil (OO), exposed to 100 mg nitrofen, by gavage, without CDH (N-), and exposed to nitrofen with CDH (CDH) on GD 9.5 (term=22 days). The morphological variables studied were: body weight (BW), total lung weight (TLW), left lung weight, TLW/BW ratio, total lung volume, and left lung volume. The histometric variables studied were: left lung parenchymal area density and left lung parenchymal volume. VEGFR1 and VEGFR2 expression were determined by Western blotting. The data were analyzed using analysis of variance with the Tukey-Kramer post hoc test. CDH frequency was 37% (80/216). All the morphological and histometric variables were reduced in the N- and CDH groups compared with the controls, and reductions were more pronounced in the CDH group (P<0.05) and more evident on GD 20.5 and GD 21.5. Similar results were observed for VEGFR1 and VEGFR2 expression. We conclude that N- and CDH fetuses showed primary pulmonary hypoplasia, with a decrease in VEGFR1 and VEGFR2 expression.

Effects of estradiol benzoate on 5'-iodothyronine deiodinase activities in female rat anterior pituitary gland, liver and thyroid gland

There is little information on the possible effects of estrogen on the activity of 5'-deiodinase (5'-ID), an enzyme responsible for the generation of T3, the biologically active thyroid hormone. In the present study, anterior pituitary sonicates or hepatic and thyroid microsomes from ovariectomized (OVX) rats treated or not with estradiol benzoate (EB, 0.7 or 14 µg/100 g body weight, sc, for 10 days) were assayed for type I 5'-ID (5'-ID-I) and type II 5'-ID (5'-ID-II, only in pituitary) activities. The 5'-ID activity was evaluated by the release of 125I from deiodinated 125I rT3, using specific assay conditions for type I or type II. Serum TSH and free T3 and free T4 were measured by radioimmunoassay. OVX alone induced a reduction in pituitary 5'-ID-I (control = 723.7 ± 67.9 vs OVX = 413.9 ± 26.9; P<0.05), while the EB-treated OVX group showed activity similar to that of the normal group. Thyroid 5'-ID-I showed the same pattern of changes, but these changes were not statistically significant. Pituitary and hepatic 5'-ID-II did not show major alterations. The treatment with the higher EB dose (14 µg), contrary to the results obtained with the lower dose, had no effect on the reduced pituitary 5'-ID-I of OVX rats. However, it induced an important increment of 5'-ID-I in the thyroid gland (0.8 times higher than that of the normal group: control = 131.9 ± 23.7 vs ovx + EB 14 µg = 248.0 ± 31.2; P<0.05), which is associated with increased serum TSH (0.6-fold vs OVX, P<0.05) but normal serum free T3 and free T4. The data suggest that estrogen is a physiological stimulator of anterior pituitary 5'-ID-I and a potent stimulator of the thyroid enzyme when employed at high doses