Intensity of peroxidation processes and activity of antioxidant enzymes in rat tissues at high chromium level in the diet

The data on the influence of chromium in different tissues of rats at its consumption with mixed fodder in the form of CrCl3 x 6H2O on the intensity of peroxidation processes and activity of antioxidant enzymes are presented. The degree of high chromium content in the studied tissues of rats at its addition to mixed fodder in the amount of 200 microg/kg during 30 days was established. Chromium content in the rat tissues decreased in the order: the spleen, heart, kidneys, lungs, brain, liver, skeletal muscle. In all tissues of rats fed with mixed fodder with chromium addition, except for skeletal muscles, content of lipid peroxidation products--hydroperoxide and TBARS-products decreased. The content of lipid peroxidation products decreased in the spleen, kidneys, liver and lungs. Also in all organs and tissues of rats the activity of glutathione peroxidase, glutathione reductase and catalase increased at the action of chromium. In the brain and kidneys the level of reduced glutathione increased. Superoxide dismutase activity was significantly higher not only in the heart and skeletal muscles of animals and is probably equal in the lungs and liver, and in other organs--the brain, kidneys and spleen in animals of the studied group the enzyme activity was lower as compared to animals of the control group. Obtained results demonstrate the regulatory influence of chromium on free radical process in the rat tissues.     

Oxidation and reduction of glutathione in rat organs during the administration of ethonium

An intramuscular administration of ethonium in a dose of 5 and 10 mg/kg 4-6 h later decreases the level of the reduced glutathione and increases that of oxidized glutathione and the glutathione peroxidase activity in the liver and kidneys of albino rats. The duration of the preparation effect depends on the dose and organ, changes in the glutathione reductase activity in the liver, kidneys, lungs, spleen and heart are not found. Participation of ethonium in formation of peroxides in the organism tissues is shown possible.     

Immunohistochemical detection of subunit A(M) of lactate dehydrogenase in tissues of the developing chick

Summary The distribution of A(M) subunits of lactate dehydrogenase (mainly LDH₅) in developing muscle, heart, liver, lung, kidney and cartilage tissue of chicken embryos was examined by the indirect fluorescent antibody technique. Antibodies against porcine LDH₅, purified by affinity chromatography, were used for this purpose. In special areas of newly formed myofibrils in somitic myoblasts fluorescence was already detected after 4 days of incubation, and located at the same place in muscle tissue of all advanced developmental stages examined. During the myotube stage of muscle development staining was also located in the peripheral thickened cytoplasma of the myotubes. The myocardium did not exhibit any fluorescent staining in the developmental stages examined. Endocardium, epicardium and pericardium, however, were fluorescent in young developmental stages. The liver showed fluorescence in 5- to 8-day embryos mainly in the endothelial cells of the blood sinusoids. In 9- to 12-day embryos the bile ducts became fluorescent. In lungs after 9- to 12-day development the epithelium and the surrounding tissues of bronchi exhibited strong immunofluorescence. The mesonephros exhibited faint granular fluorescence in tubule-forming cells and their membranes after 4–9 days of incubation. Advanced developmental stages only exhibited fluorescent blood cells. This latter staining is at least partly due to non-specific reactions of blood cell membranes with FITC-conjugated anti-rabbit IgG. Cartilage is characterized by non-specific fluorescence, but in embryos older than 8 days strong granular fluorescence of chondrocytes and staining of the perichondrium distinguished sections treated with anti-LDH₅ antibodies from control sections reacted only with FITC-conjugated anti-rabbit IgG. In addition, strong fluorescent staining was detectable in certain areas of the 5-day neural tube and faint staining in the mucosa of the intestine from embryos older than 10 days.     

Effect of bacterial lipopolysaccharide on the content of lipid peroxidation products in lungs and other organs of mice

The influence of lipopolysaccharide fromEscherichia coli (LPS, 17 mg/kg body weight) on the lipid peroxidation process in organs of mice was studied. The content of conjugated dienes (CD), lipid peroxides (LP), malondialdehyde (MDA) (all three lipid peroxidation by-products), peroxidase (PO) activity and wet-to-dry weight ratio in lungs, heart, spleen, kidneys and liver were determined 1.5 h after intravenous injection of LPS. Animals observed at this time-point had reduced activity and decreased body temperature by about 2°C, however, all analysed organs did not reveal any changes of wet-to-dry weight ratio comparing to organs from mice injected with sterile, pyrogen free 0,9% NaCl. Only extracts from heart and lungs showed significant increase in the tissue level of at least two lipid peroxidation products. The heart content of CD, MDA, and LP was about 1.5-, 1.3-, and 2.4-fold higher than in control group. In lungs CD and MDA increased 3.3- and 1.3-times but in spleen only content of LP was elevated. In these organs the suppression of PO activity was also observed. Liver and kidneys did not reveal any convincing enhancement of lipid peroxidation process and alterations of PO activity. Since free radical reactions are involved in lipid peroxidation process and inactivation of PO these results suggest that heart, lungs and spleen are the organs mostly exposed to oxidative stress during the first 1.5 h after single injection of LPS in mice.Abbreviations CD conjugated dienes - LP lipid peroxides - LPS lipopolysaccharide - MDA malondialdehyde - PMNL polymorphonuclear leukocytes - PO peroxidase - TBA thiobarbituric acid     

Immunohistochemical detection of human gastrointestinal glutathione peroxidase in normal tissues and cultured cells with novel mouse monoclonal antibodies.

This is the first report to describe the successful detection of human gastrointestinal glutathione peroxidase in normal tissues by Western blotting and immunohistochemical staining techniques. Four hybridoma clones producing monoclonal antibodies (MAbs) against the human gastrointestinal glutathione peroxidase were established from mice immunized with a gastrointestinal glutathione peroxidase-derived peptide. The MAbs did not crossreact with other members of the glutathione peroxidase family, be it cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, or extracellular glutathione peroxidase. Although the MAbs were found to react with a 24-kD protein in a Western blotting assay using gastric carcinoma cell extracts as antigen, they did not react with a B-lymphoblastoid cell extract. Immunohistochemical staining showed gastrointestinal glutathione peroxidase localized in the cytoplasm and in the nucleus of gastric carcinoma cells. Moreover, gastrointestinal glutathione peroxidase was detected in tissue extracts of human stomach, small intestine, large intestine, liver, and gallbladder by Western blotting, and its localization was immunohistochemically confirmed in the mucosal epithelia of the basal area of gastric pits and intestinal crypts.     

Effect of clofibrate treatment on glutathione content and the activity of the enzymes related to peroxide metabolism in rat liver and heart

Clofibrate treatment was shown to increase the content of reduced glutathione in rat liver and kidney, but did not alter the glutathione level in heart, brain, spleen and small intestine. Clofibrate did not affect the activity of superoxide dismutase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase in rat liver and heart. The drug decreased the activity of glutathione-S-transferase in the cytosolic fraction of liver homogenate. Glutathione-S-transferase activity in small intestine was also reduced. The administration of clofibrate decreased the content of polypeptides with mol. wt of 22,000 and 24,000 (possible monomers of glutathione-S-transferase) in the cytosolic fraction of liver cells.     

Ontogeny of the Neutral Amino Acid Transporter SNAT1 in the Developing Rat

Summary System A is a highly regulated, Na⁺-dependent transporter that accepts neutral amino acids containing short, polar side chains. System A plays an important role during rat development as decreased pup weights are observed in dams infused during gestation with a non-metabolizable System A substrate. Given the potential importance of SNAT1 during development in the rat brain, we examined whether SNAT1 would be present at an earlier gestation during organogenesis in multiple organs by immunohistochemistry and immunoblotting. SNAT1 protein was observed in the developing lungs, intestines, kidneys, heart, pancreas, and skeletal muscle of rats at prenatal days 14, 17, 19, 21, and postnatal day 2 rats. SNAT1 protein expression decreased in the liver and intestine shortly after birth and as the rat matured. SNAT1 expression was constant throughout development in the lungs and kidney and increased in the heart from prenatal day 19 to postnatal day 2. Highest levels of expression in older animals were seen in organs undergoing rapid cell division.     

Modulation of the expression of the Cip/Kip family of cyclin-dependent kinase inhibitors in foetal developing lungs of hamsters

We examine the cell proliferation activity and expression of cyclin-dependent kinase inhibitors of the Cip/Kip family, p21Cip1, p27Kip1 and p57Kip2, in foetal hamster lungs to determine the expression patterns of the cyclin-dependent kinase inhibitors and to clarify the relationship between expression of the cyclin-dependent kinase inhibitors and lung development. Foetal hamster lungs on gestational days 12.5-16 (the day of birth) and adult lungs were fixed in 4% paraformaldehyde. Frozen sections were immunostained for the cyclin-dependent kinase inhibitors, and examined by immunostaining for Ki-67 and bromodeoxyuridine to determine the proliferation activity of the foetal lungs. During the foetal period, cell proliferation activity, as analysed by Ki-67 or bromodeoxyuridine labelling, decreased with development of the lung. In contrast to the gradual decrease of cell proliferation activity, cells with p27Kip1 immunoreactivity increased with development. On the other hand, p21Cip1-positive cells were most prominent around gestational day 14.5, while after birth positive cells decreased markedly. A few p57Kip2-positive cells were detected in the bronchiolar epithelium on gestational day 14.5. Western blotting analyses confirmed these immunostaining patterns. Thus, the levels of the cyclin-dependent kinase inhibitors of the Cip/Kip family are modulated in the lungs during the foetal period, and each shows a unique expression pattern. The cyclin-dependent kinase inhibitors may play roles not only in regulating cell proliferation activity but also in regulating other functions such as differentiation in the lung during the foetal period.     

Glutathione and ascorbic acid metabolism and antioxidant enzyme activity in the tissues of vitamin E-deficient rats

In has been shown in the experiments on male rats that alimentary vitamin E deficit causes the decrease of reduced glutathione and ascorbic acid concentration in the liver and lungs and that of glutathione-S-transferase, glutathione reductase in the liver and lungs, catalase in the liver and glutathione peroxidase in the heart activity, but increases the amount of glutathione disulfide in the liver and lungs and superoxide dismutase and gamma-glutamyltransferase activity in the liver. The data obtained show the selective character of reaction participants of the antioxidant system of rats' organism to the deficit of one of the antioxidant factors--vitamin E and also testify to complex interrelation between separate members of this system.     

Influence of age on Cu/Zn-superoxide dismutase and indole 2,3-dioxygenase activities in rat tissues

The aim of this study was to investigate in vitro the variations with age of the activities of the two antioxidant enzymes Cu/Zn-superoxide dismutase (SOD) and indole 2,3-dioxygenase (IDO) in metabolically active tissues of rats of various ages. In rats aged one week and 2-3 months the highest Cu/Zn-SOD activity was found in the liver and the lowest in the small intestine. At 12 and 18 months of age, the activity was higher in the brain and kidneys, when compared to the small intestine, lungs and liver. Cu/Zn-SOD activity decreased significantly after 2-3 months of age with advancing age in all tissues examined. In newborn rats IDO activity was present only in the small intestine. In the group of rats aged 2-3 months, the highest specific activity was observed in the small intestine and the lowest in the lungs and kidneys, whereas at 12 months of age, the highest IDO activity was found in the brain, with kidneys presenting the lowest activity. At 18 months, IDO returned to be more elevated in the small intestine. At 12 months of age the values of IDO in the tissues varied slightly, while at 18 months similar activities were found between the lungs and brain and between the small intestine and kidneys. In relation to age, IDO specific activity declined in the small intestine, after 2-3 months of age. In the lungs, the activity remained unchanged; in the brain and in the kidneys activity decreased significantly from 2-3 to 18 months of age. In conclusion, this study demonstrates an age-related decline in Cu/Zn-SOD and IDO activities, the two enzymes responsible for scavenging O2*-.     

Immunolocalization of cellular glutathione peroxidase in adult rat lungs and quantitative analysis after postembedding immunogold labeling

To determine the distribution of cellular glutathione peroxidase in rat lungs, the tissues were stained immunohistochemically. Quantitative analysis was performed in certain cell types of alveolar linings, after the ultrathin sections were stained by a postembedding immunogold technique. Immunoblot analysis revealed that homogenates of rat liver, heart, and lungs all gave a single band. Under the light microscope, the following tissues were stained intensely: epithelial cells, smooth muscle cells and glands of bronchi and bronchioles, type II alveolar cells, and alveolar macrophages. Under immunoelectron microscopy, type II alveolar cells and macrophages were abundant in mitochondria. The mitochondria, nucleus, and cytoplasm of macrophages were labeled almost twice as densely as the respective compartments of type II alveolar cells. Within cell types, the mitochondria were labeled twice as densely as the nuclei. The other particles were less than half as densely labeled as the nuclei. The labeling was slightly less dense in the cytoplasm than in the nucleus. The present study revealed that glutathione peroxidase occurred predominantly in the epithelial linings and metabolically active sites in rat lungs. The tissues that were previously found to be rich in superoxide dismutases were also rich in glutathione peroxidase.     

Influence of silver,mercury, lead,cadmium, and selenium on glutathione peroxidase and transferase activities in rats

At the levels used in the experiments, mercury and silver significantly depressed the activity of glutathione peroxidase (assayed with either H₂O₂ or cumene-OOH) in rat tissues, whereas cadmium or lead had no effect on this activity. The most pronounced effects of mercury and silver on glutathione peroxidase were found in the liver and kidneys, with much less effect in the testes and erythrocytes. Similar trends for the effects of these metals were noted for tissue selenium levels. Silver and mercury significantly depressed the selenium concentrations, but cadmium and lead had no effect upon the selenium levels. Mercury and silver had no effect upon the activity of glutathione transferase in liver and testes, but mercury caused a significant initial increase of its activity in the kidneys. At no time did silver have any significant effect on its activity in this organ.     

Trypanosoma cruzi: isolation and characterization of two clones derived from neonatal mice

The Tulahuen strain of Trypanosoma cruzi was cloned in 15 C3H/Anf neonatal mice. Ten of these 15 neonates became parasitemic before the 12th day and died before the 19th day after the inoculation of a single bloodstream trypomastigote. Two clones were selected and maintained, while the other isolates which did not grow in a liquid metacyclic stage culture (LMC) medium were eventually discarded. The kinetics of in vitro growth and transformation from epimastigote to metacyclic trypomastigote of these two clones were characterized in LMC medium at 27 degrees C. Infectivities for vertebrate cells in vitro were retained by these two clones during the period of cultivation. The tropism for brain, heart, lungs, esophagus, stomach, large intestine, liver, pancreas, spleen, lymph nodes, kidneys, bladder, and skeletal muscles was also examined in the mice. The communication describes the establishment and characterization of T. cruzi clones. The utilization of these cloned parasites should produce some advantages in generating reproducible data in investigations.     

Effects of beta-carotene on oxidative stress in normal and diabetic rats

Increasing interest in the role of oxidative stress and beta-carotene in disease and prevention led us to examine the results of beta-carotene's administration in diabetic rats, a model for high-oxidative stress. In this experiment, amounts of lipid peroxidation, glutathione, and glutathione disulfide, and activity levels of catalase, glutathione peroxidase, glutathione reductase, superoxide dismutase, and gamma-glutamyl transpeptidase were measured in the liver, kidney, and heart of Sprague-Dawley rats with streptozotocin-induced diabetes, and after treatment with 10 mg/kg/day of beta-carotene for 14 days. Beta-carotene treatment resulted in the reversal of the diabetes-induced increase in hepatic and cardiac catalase activity, the decreased levels of glutathione disulfide in the heart, and the increased cardiac and renal levels of lipid peroxidation. Treatment with beta-carotene exacerbated the increased glutathione peroxidase activity in the heart and the decreased catalase activity in the kidneys. In contrast to reduced hepatic glutathione levels in untreated diabetic rats, beta-carotene treatment increased glutathione levels in diabetic rats. Increased hepatic gamma-glutamyl transpeptidase activity in diabetic rats was not reduced by treatment. Thus, beta-carotene therapy for 14 days prevented/reversed some, but not all, diabetes-induced changes in oxidative stress parameters.     

A Simultaneous Visualization of the Antioxidant Enzymes Glutathione Peroxidase and Catalase on Polyacrylamide Gels

A simple and sensitive method for the simultaneous visualization of glutathione peroxidase and catalase on polyacrylamide gels is described. The procedure included: (I) running samples on a 7. 5% polyacryla-mide gel, (2) soaking the gel in a certain concentration of reduced glutathione (0.25–2.0 mM). (3) soaking the gel in GSH plus H_zO_z or cumene hydroperoxide, (4) finally staining with a 1% ferric chloride I% potassium ferricyanide solution. The best concentration of glutathione for simultaneous visualization of glutathione peroxidase and catalase was 0.25rnM; I.5mM glutathione was the best concentration for visualization of glutathione peroxidase alone. The method is sensitive enough to detect catalase and glutathione peroxidase in mouse liver homogenates and also it is specific for glutathione peroxidase since other peroxidases such as lactoperoxidase, horseradish peroxidase and glutathione S-transferase cannot be visualized. Using this method, it was found that unlike catalase. glutathione peroxidase is heat resistant (68°C. 1min), but sensitive to 10mM sodium iodoacetate.     

Immunofluorescent study of DNA polymerase alpha in rat cells and tissues

An indirect immunofluorescent test based on globulin preparation from a highly specific antiserum against rat liver DNA polymerase alpha was used to direct the enzyme in sections of various tissues of the rat. The immunofluorescent staining was found in cells of the thymus and the wall of intestine crypt, in sparse cells of the intestinal muscular layer, and in cells of the embryo skin epithelium. In sections of liver the intensity of staining and the number of stained cells increased significantly during regeneration. The immunoglobulins did not interact with the cytoplasm and nuclei of skeletal muscle myotubes, with the epithelial cells at the top of intestinal villi, and with erythrocytes. The intracellular localization of the fluorescence observed was of two general types: 1) staining in the region of the nuclear envelope and/or in the cytoplasm; 2) an additional intranuclear staining. The staining of the first type is characteristic of the cells of intact liver and of leyomyocytes. It was also observed in the proliferating cells of thymus and crypt wall, and in cultured myogenic L6 cells. Cells of the embryo skin epithelium, the satellite cells in the skeletal muscle, and about one half of the regenerating liver cells appeared to have the second type of staining. These data serve an indication of possible histotypical differences in in the intracellular localization of DNA polymerase alpha in proliferating cells. It is proposed that the presence of DNA polymerase in resting cells is in association with their ability to respond to the mitogenic stimulus.     

Functional analysis of the receptor binding domain of SARS coronavirus S1 region and its monoclonal antibody

Severe acute respiratory syndrome (SARS) is caused by the SARS coronavirus (CoV). The spike protein of SARS-CoV consists of S1 and S2 domains, which are responsible for virus binding and fusion, respectively. The receptor-binding domain (RBD) positioned in S1 can specifically bind to angiotensin-converting enzyme 2 (ACE2) on target cells, and ACE2 regulates the balance between vasoconstrictors and vasodilators within the heart and kidneys. Here, a recombinant fusion protein containing 193-amino acid RBD (residues 318–510) and glutathione S-transferase were prepared for binding to target cells. Additionally, monoclonal RBD antibodies were prepared to confirm RBD binding to target cells through ACE2. We first confirmed that ACE2 was expressed in various mouse cells such as heart, lungs, spleen, liver, intestine, and kidneys using a commercial ACE2 polyclonal antibody. We also confirmed that the mouse fibroblast (NIH3T3) and human embryonic kidney cell lines (HEK293) expressed ACE2. We finally demonstrated that recombinant RBD bound to ACE2 on these cells using a cellular enzyme-linked immunosorbent assay and immunoassay. These results can be applied for future research to treat ACE2-related diseases and SARS.     

Structural and biochemical differentiation of the guinea-pig colon during foetal development

Summary We have studied some aspects of the morphological and biochemical differentiation of the foetal guinea-pig colonic epithelium. At day 40 the epithelium was organised in ridges and appeared pseudo-stratified. Folding of the epithelium, followed by villus formation, occurred between days 45 and 55, and by day 50 mucus-secreting goblet cells appeared at the bases of the colonic villi. By day 55 most epithelial cells, including goblet cells, possessed numerous microvilli which, by day 65, had become organised into well developed brush-borders. Between day 55 and term (day 65–68) mucosal depth increased markedly and the colon attained its final glandular morphology.Biochemical studies showed the specific activities of the microvillar hydrolases to be much lower in the washed colon than in either foetal meconium or small intestine at all times during development. Furthermore, a membrane fraction highly enriched in microvillus hydrolase activities was prepared from foetal colonic meconium using techniques originally devised to isolate the foetal small intestinal microvillus membrane. This meconial subfraction was almost identical in polypeptide composition to the highly-purified foetal small intestinal microvillus membrane. Identification of the colonic microvillus membrane was hampered by the absence of reliable membrane markers. Nevertheless, a fraction 14-fold enriched in aminopeptidase activity was prepared from day 40 foetal colon and its polypeptide composition compared by SDS-PAGE to that of the small intestinal microvillus membrane at the same age.     

Tissue Distribution of Selenium and Effect of Season and Age on Selenium Content in Roe Deer from Northwestern Poland

The aim of the study was to compare selenium concentrations in different organs of roe deer from northwestern Poland. Samples of liver, kidneys, heart and lungs, collected from 74 roe deer shot during the hunting seasons of 2008–2009 in northwestern Poland, were studied. Selenium concentration in the organs was determined spectrofluorimetrically. Mean selenium concentration was 0.06 μg/g w.w. in the liver, 0.41 μg/g w.w. in the kidneys and 0.05 μg/g w.w. in the heart and lungs. Season had a significant effect on selenium concentration in the liver, kidneys, lungs and heart. In all the organs, the highest selenium concentration was found in spring and the lowest in autumn and winter. All animals studied were deficient in selenium. The low selenium concentration in the liver or heart can disturb their function, and in the future, it may be a factor contributing to the population decline of roe deer in the northwestern part of Poland.     

The effect of Ketoconazole on experimental paracoccidioidomycosis in the Syrian hamster: immunological and histopathological study

Summary The effect of Ketoconazole (KTZ) on the hamster experimental intratesticular paracoccidioidomycosis was studied employing different treatment schedules. KTZ long course treatment beginning at an early stage of the infection was effective in preventing fungal proliferation, dissemination to lymph nodes, spleen and kidneys, and in maintaining low levels of humoral and cellular specific immune responses. KTZ short course treatment starting at an advanced stage of disease resulted in a more severe histopathological picture without significant changes in the immunological profile. The drug prolonged the life span of hamsters infected with Paracoccidioides brasiliensis, but did not prevent mortality. Toxic necrosis of the bone marrow occurred in normal animals receiving 120 mg/kg/day of KTZ but with lower doses no morphologic alterations were observed in heart, lungs, kidneys, adrenals, spleen, liver, intestine or bone marrow.