State of the liver antioxidant system and content of matrix metalloproteinase-2 of the large intestine under the effect of maleimide derivative in experimental colorectal carcinogenesis in rats

The maleimide derivative--1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrol-2.5-dione (MI-1) with cytostatic activity did not cause substantial changes of liver antioxidant system and level of matrix metalloproteinase-2 in intestinal mucosa after chronic treatment (for 20 weeks). MI-1 did not cause significant changes in the content of thiobarbituric-active products and plasma membrane protein carbonyl groups in the rat liver. However activities of superoxide dismutase, glutathione peroxidase, and content of reduced glutathione were decreased in both doses--0.027 and 2.7 mg/kg. The level of matrix metalloproteinase-2 in intestinal mucosa was decreased just in maximum dose--2.7 mg/kg. The contents of thiobarbituric-active products, protein carbonyl groups, reduced glutathione, matrix metalloproteinase-2, activities of glutathione peroxidase and glutathione-S-transferase in the liver cells have increased in 1.2-dimethylhydrazine-induced colon cancer in rats. The activities of enzymes of the first line of antioxidant defense--superoxide dismutase and catalase were decreased to 40%. The maleimide derivative prevents development of oxidation stress and partially reduce them to control level.     

Fatty acid-binding capacity of cytosolic proteins of various rat tissues: effect of postnatal development, starvation, sex, clofibrate feeding and light cycle

Fatty acid-binding capacity of dealbuminized, delipidated cytosolic proteins from rat tissues was studied with a radiochemical binding assay. Oleate-binding capacity ranges from 1.6 to 4.4 pmol/micrograms cytosolic protein in liver, heart, kidney, adrenal, brain, skeletal muscle and diaphragm. Differences in binding affinity indicate the presence of different fatty acid-binding proteins in these tissues. No change in fatty acid-binding protein content of heart and liver cytosol was observed during postnatal development up to 70 days. Starvation did not affect the fatty acid-binding capacity of heart cytosol, but increased the oleate-binding capacity in liver cytosol. Sex-related differences of binding by heart and liver cytosolic proteins were found with oleate, but not with palmitate. Fatty acid-binding capacity of liver and heart cytosol did not show marked diurnal variation. Clofibrate treatment had different effects on the oleate-binding capacity of cytosolic proteins: an increase in liver and kidney, no change in skeletal muscle and a decrease in heart. The results are discussed in relation to data concerning fatty acid oxidation.     

Time course effects of vanadium supplement on cytosolic reduced glutathione level and glutathione S-transferase activity

The influence of vanadium, an important dietary micronutrient, was evaluated on the cytosolic reduced glutathione (GSH) content and glutathione S-transferase (GST) activity in several rat target tissues. Supplementation of drinking water with vanadium at the level of 0.2 or 0.5 ppm for 4, 8, or 12 wk was found to increase the GSH level with a concomitant elevation in GST activity in the liver followed by small intestine mucosa, large intestine mucosa, and kidney. The results were almost dose-dependent and mostly pronounced with 0.5 ppm vanadium after 12 wk of its continuous supplementation. Neither the GSH level nor GST activity was significantly altered in forestomach and lung following vanadium supplementation throughout the study. The levels of vanadium that were found to increase the content of GSH and activity of GST in the liver, intestine, and kidney did not exert any toxic manifestation was evidenced from water and food consumption as well as the growth responses of the experimental animals. Moreover, these doses of vanadium did not impair either hepatic or renal functions as they did not alter the serum activities of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), sorbitol dehydrogenase (SDH), as well as serum urea and creatinine levels. All these results clearly indicate that vanadium under the doses employed in our study has a significant inducing role on GSH content with a concurrent elevation in GST activity in the liver and specific extrahepatic tissues without any apparent sign of cytotoxicity. This attribute of vanadium may have a greater importance in terms of biotransformation and detoxification of xenobiotics, including carcinogens. In addition, since the ability to afford an increment in the endogenous GSH-GST pool by anticarcinogenic natural substances has been found to correlate with their activity to inhibit neoplastic transformation, the trace element vanadium may be considered as a novel anticancer agent.     

Reduced glutathione inhibits the alkylation by N-nitrosodimethylamine of liver DNA in vivo and microsomal fraction in vitro

The transfer of radioactivity from N-nitroso-[14C]dimethylamine to trichloroacetic acid precipitable macromolecules in the microsomal fraction of rat liver was investigated. This transfer was found to depend on N-nitrosodimethylamine being metabolized. Cytosolic fraction and cytosol enriched with reduced glutathione inhibited the binding of radioactivity to acid insoluble proteins. Depletion of glutathione in rat liver with diethylmaleate prior to i.v. administration of 10 mg N-nitroso-[14C]dimethylamine/kg led to an increase in O6-methylguanine and N-7-methylguanine in DNA. If rats were fed disulfiram for 6 days (2 g/kg feed), glutathione and glutathione S-transferase were enhanced, and the degree of methylation of guanine by N-nitrosodimethylamine was greatly reduced, as was the metabolism of N-nitrosodimethylamine in the intact animal. Fasting rats for 24 h did not change the N-nitrosodimethylamine-demethylase activity in vitro but greatly enhanced the methylation of guanine in vivo, while the glutathione content and glutathione S-transferase activity were not changed compared to fed animals.     

Age-related changes in antioxidant enzyme activities in the small intestine and liver from Wistar rats.

The present study was designed to determine age-related changes in intestinal and hepatic antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and glutathione-S-transferase (GST), and lipid peroxidation in male Wistar rats (n = 8) aged 2 wk, 2.5 mon, 5 mon, 10 mon, and 23 mon. In the small intestine, cytosolic SOD, GSH-PX activities and lipid peroxidation were not affected by age, but intestinal GST activity was noticeably enhanced as age increased. In particular, intestinal GST activity in 23 mon old rats was 3 times as strong as that in 2 wk old rats. In the liver, the activity of hepatic cytosolic SOD was not affected by age, whereas GSH-PX and GST activities in rats aged 10 mon and 23 mon were much stronger than those in rats aged 2 wk, 2.5 mon, and 5 mon. The increased lipid peroxidation in 2.5 mon and 5 mon old rats was observed when compared with that of other groups. It is therefore concluded from the results presented here that age greatly increases GST activity in the small intestinal mucosae and increasing GSH-PX, GST activities and lipid peroxidation in the liver from male Wistar rats.     

Distribution of leucine 2,3-aminomutase activity in various organs of the rat and in subcellular organelles of rat liver

Leucine 2,3-aminomutase, the cobalamine-dependent enzyme involved in the conversion of β-leucine to leucine, has been shown to be widely distributed in rat organs. The activity is highest in skeletal and cardiac muscle, lower in brain, liver, and kidney, and very low in small intestine. In rat liver, the activity is nearly all in the cytosolic fraction.     

S-(1,2-dicarboxyethyl)glutathione and activity for its synthesis in rat tissues

The contents of S-(1,2-dicarboxyethyl)glutathione (DCE-GS) in several tissues of rat were determined by HPLC. The peptide was present at concentrations (nmol/g tissue) of 119 in lens, 71.6 in liver, and 27.4 in heart. It was, however, not detected in spleen, kidney, cerebrum, or cerebellum. In rat liver, DCE-GS was located primarily in the cytosolic fraction. The substrates for the enzymic synthesis of DCE-GS were GSH and L-malate. In rats, the DCE-GS-synthesizing activity was found to be highest in the liver and in the cytosol of rat liver subcellular fractions. The DCE-GS-synthesizing enzyme was partially purified from rat liver cytosolic fraction by ammonium sulfate fractionation, Phenyl Superose chromatography, hydroxyapatite chromatography, and gel filtration. The molecular mass of the enzyme was estimated to be 53 kDa by gel filtration and SDS-PAGE, showing it to be a monomeric protein. The Km values for GSH and L-malate were 2.3 and 4.0 mM at 37 degrees C, respectively. The enzyme did not utilize 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, p-nitrophenyl bromide, trans-4-phenyl-3-buten-2-one, or p-nitrobenzyl chloride, which were substrates for previously characterized glutathione S-transferases. The isolated enzyme preparation showed no fumarase activity, which supported the conclusion that the formation of DCE-GS was not the result of a nonenzymic reaction following the synthesis of fumarate from L-malate by the isolated enzyme. The N-terminal amino acid of this polypeptide was presumably blocked since no sequence was obtained by automatic sequencing after electro-blotting onto a siliconized-glass fiber (SGF) sheet.     

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.     

Possible pathways of lipid peroxide reduction in the liver and their intracellular localization

Lipid peroxidase activity in rat liver was studied. Rat liver cytosolic fraction was found to be capable of reducing lipid hydroperoxides. On the contrary, no lipid hydroperoxide reduction was observed in microsomes. It was found that at least two proteins in rat liver cytosol are capable of reducing phospholipid hydroperoxides. One of them is precipitated by 33-55% at (NH4)2SO4 saturation and requires reduced glutathione (GSH) as a hydrogen donor, while the other one is precipitated by 55-80% at (NH4)2SO4 saturation and reduces phospholipid hydroperoxides in the presence of a unidentified low molecular weight cytosolic factor, but not GSH or NADPH.     

Antioxidant Systems in Tissues of Senescence Accelerated Mice

Significant decrease in the level of lipid antioxidants (measured from the kinetics of the induced chemiluminescence in brain homogenate) and of the hydrophilic antioxidant carnosine as well was observed in the brain of 14-16-month-old mice of SAMP1 line, which is characterized by accelerated accumulation of senile features, in comparison with the control line SAMR1. In the brain of SAMP1 animals the activity of cytosolic Cu/Zn-containing superoxide dismutase (SOD) was reduced, while the activity of membrane-bound Mn-SOD was at an extremely low level. The activity of glutathione-dependent enzymes (glutathione peroxidase, glutathione reductase, and glutathione transferase) did not differ in the brain of SAMP1 and SAMR1 animals, and catalase activity was similarly low in both cases. At the same time, excess concentration of excitotoxic compounds, significantly exceeding that for the control line, was determined in the brain and blood of SAMP1 animals. The activity of glutathione enzymes in liver and heart as well as the activity of cytosolic Cu/Zn-SOD in liver did not differ in the two studied lines, while the activity of erythrocyte glutathione peroxidase was slightly increased, and the activity of liver catalase and erythrocyte Cu/Zn-SOD was significantly decreased for SAMP1 compared with SAMR1. The results demonstrate that the accelerated ageing of SAMP1 animals is connected to a significant extent with the decreased efficiency of the systems utilizing reactive oxygen species (ROS) in tissues.     

Effect of clofibrate treatment on aconitase activity in rat liver and other tissues

The activity of both mitochondrial and cytosolic aconitases was significantly increased in the livers of male rats following treatment with the hypolipidemic drug clofibrate. Cycloheximide or puromycin administration to rats inhibited the inducing effect of clofibrate on the enzyme activity. Aconitase activity in small intestine homogenate was also increased by clofibrate. The drug did not affect the enzyme activity in rat kidney, heart and brain.     

Effect of cigarette smoke on lipid peroxidation and antioxidant enzymes in albino rat

Effect of cigarette smoke on lipid peroxidation (LPX) and antioxidant enzymes like catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) in various organs like brain, heart, lung, liver and kidney of the albino rats exposed to cigarette smoke for 30 min/day for a period of 30 days were assayed. It was observed that the lipid peroxide levels in liver, lung and kidney were enhanced in case of animals exposed to cigarette smoke, whereas brain and heart did not show any change as compared to control animals. The activity of the antioxidant enzymes was also elevated in liver, lung and kidney of the test animals whereas, brain and heart did not show any change in the activities of all of these antioxidant enzymes except glutathione-s-transferase which was increased in brain also. The level of reduced glutathione (GSH) was lowered in liver, lung and kidney of the tested animals when compared with the control animals but there was no significant change in brain and heart. The results of our study suggest that cigarette smoke induces lipid peroxidation in liver, lung and kidney, and the antioxidant enzymes levels were enhanced in order to protect these tissues against the deleterious effect of the oxygen derived free radicals. The depletion of reduced glutathione in these organs could be due to it's utilization by the tissues to mop off the free radicals.     

The effects of dietary sulfur on selenium utilization by the rat

The antagonistic effects of S on Se utilization were studied by measurement of tissue Se concentrations and glutathione peroxidase (GSHpx, E.C.1.11.1.9) activities in rats offered Torula yeast-based diets supplemented with sodium sulfate to provide 1.5, 3, or 5 g S/kg diet. Increasing sulfur concentrations in the diet caused small but significant decreases in GSHpx activity of liver, spleen, heart, lung small intestine, and skeletal muscle. Also significantly decreased were Se concentrations in liver and hair. Dietary sulfur content had no significant effect on body weight gain of the rats. With an adequate supply of Se in the diet, there are probably no major deleterious effects of dietary S on Se metabolism in the rat.     

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.     

The effect of clofibrate feeding on the NADP-linked dehydrogenases activity in rat tissue

Administration of clofibrate to the rat increased several fold the activity of malic enzyme in the liver. Clofibrate treatment resulted also in an increased activity of the hepatic hexose monophosphate shunt dehydrogenases but was without effect on NADP-linked isocitrate dehydrogenase. The increased activity of malic enzyme in the liver resulting from the administration of clofibrate was inhibited by ethionine and puromycin, which suggests that de novo synthesis of the enzyme protein did occur as the result of the drug action. In contrast to the liver malic enzyme, the enzyme activity in kidney cortex increased only two-fold, whereas in the heart and skeletal muscle the activity was not affected by clofibrate administration.     

Effect of clofibrate on intracellular enzyme distribution in the rat liver

The effect of chronic administration of a hypolipaemic agent--clofibrate--on the subcellular distribution of liver enzymes in male rats was studied. Clofibrate produced an increase in the number of peroxisomes and also enhanced the activity of aconitase and histidine: glyoxylate aminotransferase (HGA) in liver homogenate. Differential centrifugation of homogenate revealed an elevation of the relative amounts of catalase, HGA and isocitrate dehydrogenase in the soluble cell fraction in clofibrate pretreated animals. Clofibrate induced peroxisomal HGA but failed to alter the amounts of catalase, urate oxidase and isocitrate dehydrogenase in the particles. In both the experimental and control groups the activity of aconitase, malate dehydrogenase (NAD+), creatine phosphokinase and glutathione reductase was observed in mitochondrial fractions and was not detected in purified peroxisomes.     

Effect of chronic ethanol treatment under partial catalase inhibition on the activity of enzymes related to peroxide metabolism in rat liver and heart

1. In order to test the hypothesis that the alcoholic cardiomyopathy under partial catalase inhibition is associated with the activation of lipid peroxidation in cardiomyocytes (Panchenko et al., Experientia 43, 580-581, 1987), the effects of ethanol and catalase inhibitor 3-amino-1,2,4-triazole (aminotriazole) on rat heart and liver content of reduced glutathione and on the activity of enzymes related to peroxide metabolism: catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and glucose-6-phosphate dehydrogenase were investigated. 2. In accordance with the data obtained by Kino (J. molec, cell. Cardiol. 13, 5-12, 1981), when ethanol (36% of dietary calories) and aminotriazole were simultaneously administered an alcoholic cardiomyopathy developed while in the liver moderate fatty degeneration was revealed. 3. Chronic combined or separate administration of ethanol and aminotriazole was shown to increase glutathione concentration and glutathione-S-transferase activity in rat liver. In the groups of animals which received isocaloric carbohydrates in the diet instead of ethanol the liver glucose-6-phosphate dehydrogenase was increased. 4. Acute and chronic aminotriazole injections led to catalase inactivation and in the latter case also to inhibition of the liver superoxide dismutase and glutathione peroxidase activities. 5. Ethanol and aminotriazole treatment did not alter the glutathione level and the activity of all enzymes tested (except catalase) in rat myocardium.     

Distribution of adenosine deaminase in some rat tissues. Inhibition by ethanol and dimethyl sulfoxide

The level of adenosine deaminase in various rat tissues has been tested. The enzyme activity of cytosolic fractions decreased in the following order: lung greater than spleen greater than small intestine greater than stomach greater than kidney greater than heart greater than liver greater than skeletal muscle greater than forebrain greater than cerebellum. The enzyme had identical patterns from tissue to tissue with respect to Km, V, and Ki values for ethanol and for dimethyl sulfoxide, with respect to electrophoretic behaviour and to inhibition by antibodies anti-rat brain adenosine deaminase.     

Effect of the ulcerogenic agent cysteamine on the content of glutathione and glutathione-dependent enzymes in the mucous membrane of the gastroduodenal region in the rat

A comparative study of reduced and oxidized glutathione forms and the activity of glutathione-dependent enzymes (glutathione peroxidase, glutathione-S-transferase, and glutathione reductase) has been performed in the rat mucous membranes of different gastroduodenal areas 24 hours after the injection of cysteamine--a specific ulcerogenic agent. It has been shown that cysteamine causes a decrease in the concentration of reduced and an increase in the concentration of oxidized glutathione forms in all gastroduodenal areas. The fall in reduced glutathione form concentration is the greatest in the duodenal mucosa. A considerable decrease in glutathione-dependent enzyme activity, especially glutathione-S-transferase, was observed in duodenal mucosa. It is concluded that glutathione and glutathione-dependent enzyme system may be directly related to pathogenetic mechanisms of gastroduodenal ulcer formation.     

Antioxidant and detoxifying enzymes in the liver and kidney of pheasants after intoxication by herbicides MCPA and ANITEN

The activity of antioxidant and detoxifying enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase, glutathione-S-transferase (GST), the contents of thiobarbituric acid reactive substances, and the superoxide dismutase and glutathione-S-transferase isoenzyme patterns, were determined in the liver and kidney of pheasants after acute intoxication by herbicides MCPA and ANITEN I. In the liver, the activity of antioxidant enzymes was significantly decreased in the group given ANITEN I. New superoxide dismutase isoforms (pI 6.30, 6.85, 7.00) and higher intensity of isoform with pI 6.60 were observed after isoelectrofocusing in all experimental groups. In the kidney, the activity of superoxide dismutase was significantly decreased, and a higher intensity of superoxide dismutase isoforms (pI 6.00 and 6.60) was observed in all experimental groups. The contents of thiobarbituric acid reactive substances were significantly increased in the group with ANITEN I. The glutathione-S-transferase isoenzyme pattern was studied by using subunit-specific substrates and by Western blotting. The activity of glutathione-S-transferase with ethacrynic acid and cross-reactivity with rat subunit 7 was lower in all experimental groups in the kidney and liver, except in the liver of the group given a higher dose of ANITEN I. In this group, we have found a 2.10-fold higher activity to ethacrynic acid and a strong induction of subunit 7. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 235–244, 1998