Leukotriene C4 formation catalyzed by three distinct forms of human cytosolic glutathione transferase

The ability of three distinct types of human cytosolic glutathione transferase to catalyze the formation of leukotriene C4 from glutathione and leukotriene A4 has been demonstrated. The near-neutral transferase (mu) was the most efficient enzyme with Vmax= 180 nmol X min-1 X mg-1 and Km= 160 microM. The Vmax and Km values for the basic (alpha-epsilon) and the acidic (pi) transferases were 66 and 24 nmol X min-1 X mg-1 and 130 and 190 microM, respectively. The synthetic methyl ester derivative of leukotriene A4 was somewhat more active as a substrate for all the three forms of the enzyme.     

Liver and biliary levels of glutathione and thiobarbituric acid reactants after acute lindane intoxication

The i.p. administration of 60 mg kg-1 body weight of lindane, the gamma-isomer of hexachlorocyclohexane, to fed rats led to an enhancement of hepatic lipid peroxidation after 24 h of treatment. This was evidenced by significant increases in the hepatic production and biliary release of thiobarbituric acid reactive substances, and in the biliary release of glutathione disulphide. Under these conditions, the content of cytochrome P450 was enhanced concomitantly with increases in the total microsomal oxygen uptake, superoxide radical generation and (+)-catechin (cyanid-3-ol) sensitive respiration. The glutathione status of hepatocytes was altered by lindane as the content and biliary release of glutathione disulphide was drastically augmented, leading to a decrease in the cellular and biliary GSH/GSSG ratios. It is suggested that lindane treatment leads to an induced oxidative capacity, which, in turn, alters the glutathione status of the liver tissue.     

Modulation of benthiocarb in vitro inhibited neonate rat (Wistar strain) brain Na+K+-ATPase by norepinephrine

Effect in vitro of benthiocarb, an organocarbamate herbicide on neonate rat (3 day old) brain was studied to understand the interaction of benthiocarb with Na+K+-ATPase. Na+K+-ATPase of the developing rat brain was selected as an index enzyme since alterations in the Na+K+-ATPase activity leads to neuronal dysfunction. The assay of Na+K+-ATPase in the presence of 1-8 mu moles of benthiocarb showed decreased activity and a concentration dependent inhibition of Na+K+-ATPase was noticed upto 7 mu moles of benthiocarb. Based on IC50 values (median concentration), 50% inhibition of the enzyme was observed with 5 mu moles of benthiocarb. Norepinephrine (NE) was selected to study the modulation of benthiocarb inhibited enzyme. Maximum increase (76.7%) of Na+K+-ATPase was noticed with 35 mu moles of NE and effective concentration (EC50) of NE which produced 50% activation of the enzyme was found to be 20 mu moles. This study suggests that NE acts as a protective agent in reversing the benthiocarb in vitro inhibited neonate rat brain Na+K+-ATPase.     

Sulphate uptake and metabolism in the chrysomonad, monochrysis lutheri.

The intracellular concentration of inorganic 35SO4 in Monochrysis lutheri cells exposed to 0.513 mM Na235SO4 for up to 6-hr remained constant at about 0.038 mM. The exchange rate of this 35SO4 with the external unlabelled sulphate was negligible compared to the rate of influx across the plasmalemma (0.032 mu moles/g cells/hr). The flux of free 35SO4 to organic 35S was 0.029 mu moles/g cells/hr. Assuming an internal electrical potential in the cells of -70 mV, this intracellular concentration of inorganic 35SO4 was well in excess of that obtainable by passive diffusion as calculated from the Nernst equation. These results indicate that sulphate is accumulated by an active mechanism rather than by facilitated diffusion. Sulphate uptake appears to occur via a carrier-mediated membrane transport system which conforms to Michaelis-Menten type saturation kinetics with a Km of 3.2 X 10(-5) M and Vmax of 7.9 X 10(-5) mu moles sulphate/hr/10(5) cells. Uptake was dependent on a source of energy since the metabolic inhibitor CCCP almost completely inhibited uptake under both light and dark conditions and DCMU caused a 50% decrease in uptake under light conditions. Under dark conditions, uptake remained at about 80% of that observed under light conditions and was little affected by DCMU, indicating that the energy for uptake could be supplied by either photosynthesis or respiration. A charge and size recognition site in the cell is implied by the finding that sulphate uptake was inhibited by chromate and selenate but not by tungstate, molybdate, nitrate or phosphate. Chromate did not inhibit photosynthesis. Cysteine and methionine added to the culture medium were apparently capable of exerting inhibition of sulphate uptake in both unstarved and sulphate-starved cells. Cycloheximide slightly inhibited sulphate uptake over an 8-hr period indicating, either a slow rate of entry of the inhibitor into the cells or a slow turnover of the protein(s) associated with sulphate transport.     

Alleviation of lindane induced toxicity in testis of Swiss mice (Mus musculus) by combined treatment with vitamin C, vitamin E and alpha-lipoic acid

Mitigation of lindane induced toxicity in testis of Swiss mice by combined treatment with vitamin C, vitamin E and alpha-lipoic acid has been evaluated. Male healthy mice (40), 8-10 weeks old were randomly selected and divided into 4 groups, control (C); lindane (L); antioxidant (A) and antioxidant plus lindane (A+L). Group C animals were administered only the vehicle (olive oil); in group L lindane was administered orally at a dose of 40 mg/kg body wt.; in group A combination of antioxidants at a dose of 125 mg/kg body wt.(vitamin C: 50 mg/kg body wt., vitamin E: 50 mg/kg body wt. and alpha-lipoic acid: 25 mg/kg body wt.) was administered orally; in group A+L both antioxidants (125 mg/kg body wt.) and lindane (40 mg/kg body wt.) were administered at their respective doses. In group A+L antioxidants were administered 1 h prior to lindane administration. All treatments were continuously given for 60 days. Histopathological changes due to lindane intoxication indicated shrunken and distorted seminiferous tubules, sparse Leydig cells and blood vessels and atrophy in the tissue. The testis weight also decreased significantly. Lindane treated group showed increased lipid peroxidation, whereas glutathione, glutathione peroxidase, superoxide dismutase, catalase and protein were significantly decreased compared to control. Lindane induced damage was minimized by administration of antioxidants. Results suggest that combined pretreatment with antioxidants can alleviate the damage caused to testis by lindane.     

Effects of L-thyroxine and L-triiodothyronine on protein and nucleic acid contents of liver of 6N-2-propylthiouracil treated hypothyroid singi fish, Heteropneustes fossilis bloch

Three consecutive injections of 12.5 X 10(-10) and 25 X 10(-10) moles/g of L-thyroxine (T4) or a single injection of L-triiodothyronine (T3) at 7.5 X 10(-10) moles/g to Singi fish caused an increase in liver protein and RNA contents, whereas similar injections of 50 X 10(-10) moles/g of T4 or 75 X 10(-10) moles/g of T3 caused a fall in these cellular constituents in liver. Treatments of Singi fish with thiourea (1 mg/ml) for 30 days caused a fall in the protein and RNA contents in liver which were restored to the euthyroid control level by a single injection of 7.5 X 10(-10) moles/g of T3 or three consecutive injections of T4 at 12.5 X 10(-10) moles/g dose. Administration of T4 (12.5 X 10(-10) moles/g, three consecutive injections) along with 6N-2-propylthiouracil (PTU) at 20 micrograms/g of b. w. in six consecutive injections to the thiourea treated (hypothyroid) fish failed to cause any change in hepatic protein and RNA contents in comparison to only PTU-treated hypothyroid fish, but a single injection of 7.5 X 10(-10) moles/g of T3 to the PTU-treated hypothyroid fish increased these cellular constituents of liver. A dose-dependent biphasic nature of thyroid hormone action, a higher potency of T3 than T4 and the probable 'prohormone' nature of T4 have been documented in case of Singi fish in the present experiments.     

Inhibition of lindane-induced toxicity using alpha-lipoic acid and vitamin E in the brain of Mus musculus

In the present investigation, we have used adenosine triphosphatase (ATPase) activity as biochemical test of toxic action of lindane that was explained by lipid peroxidation model. Study was also undertaken to ascertain the potential protective role of alpha-lipoic acid (ALA) and vitamin E on the same parameters. Highly acute dose of lindane, i.e., 40 mg/kg bw for 18 h exposure, was used for creating lesions in brain. Lipid peroxidation was measured in terms of glutathione peroxidase and thio barbituric acid-reacting substances (TBARS). Various brain regions under investigation were cerebellum and pons-medulla oblongata. Healthy, male, Swiss mice (7–8 weeks old) were allocated into four groups. First group was control, second group was treated with lindane, third group was treated purely with antioxidants, and fourth group received both antioxidants and lindane treatment. Results revealed the significant difference (at 1% and 5% in all groups) in all studied parameters from control. Increased TBARS level in second group suggests that lindane enhances the production of free radicals in studied brain regions. Antioxidants under test are efficient remedy for neurotoxicity caused by lindane. We conclude that lindane manifests toxic effects on brain ATPase and enhances lipid peroxidation. ALA and vitamin E in combination may provide protection against lindane-induced acute toxicity.     

Assay of free-radical toxicity and antioxidant effect on the Hep 3B cell line: a test survey using lindane

The content of reduced glutathione and of glutathione disulfide as well as the activities of glutathione reductase, glutathione peroxidase, glutathione S-transferases, catalase and superoxide dismutases were determined in human hepatoma Hep 3B cells in relation to free-radical toxicity in order to appreciate the defense capacities of these cells compared to data on normal hepatocytes. When Hep 3B cells were exposed to lindane, a known inducer of free-radical production, superoxide dismutase activity appeared as the best-adapted cellular parameter for early detection of the resulting free-radical toxicity.Abbreviations AAS atomic absorption spectrometry - CDNB 1-chloro-2,4-dinitrobenzene - DMEM Dulbecco's modified Eagle medium - GPx glutathione peroxidase - G.Red glutathione reductase - GSH reduced glutathione - GSSG glutathione disulfide - GST glutathione S-transferases - Prot proteins - SOD superoxide dismutase     

Energy transfer from tryptophan residues to pyridoxal 5'-phosphate at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase

Forster's mechanism of radiationless energy transfer has been used to estimate average distance between tryptophan residues and pyridoxal 5'-phosphate bound at the active site of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase. This distance was found to depend on the activity of the enzyme and was 29 A for a freshly purified enzyme (activity 1.7 mu moles CO2 fixed/min/mg protein) and 37 A for a 6 week old enzyme stored at 4 degrees C (activity 0.07 mu moles CO2 fixed/min/mg protein).     

Mechanism of Copper-Catalyzed Oxidation of Glutathione

The mechanism of copper-catalyzed glutathione oxidation was investigated using oxygen consumption, thiol depletion, spectroscopy and hydroxyl radical detection. The mechanism of oxidation has kinetics which appear biphasic. During the first reaction phase a stoichiometric amount of oxygen is consumed (1 mole oxygen per 4 moles thiol) with minimal *OH production. In the second reaction phase, additional (excess) oxygen is consumed at an increased rate and with significant hydrogen peroxide and *OH production. The kinetic and spectroscopic data suggest that copper forms a catalytic complex with glutathione (1 mole copper per 2 moles glutathione). Our proposed reaction mechanism assumes two parallel processes (superoxide-dependent and peroxide-dependent) for the first reaction phase and superoxide-independent for the second phase. Our current results indicate that glutathione, usually considered as an antioxidant, can act as prooxidant at physiological conditions and therefore can participate in cellular radical damage.     

Effect of treatment of cow’s urine “Gomutra” and antioxidants in alleviating the lindane-induced oxidative stress in kidney of Swiss mice (Mus musculus)

The study aimed to evaluate the effect of cow urine and combination of antioxidants against lindane induced oxidative stress in Swiss mice. Male healthy mice, 8–10 weeks old, weighing 30 ± 5 g were randomly selected and divided into eight groups, namely, control (C); lindane (L); antioxidant (A), antioxidant+lindane (A+L), cow urine (U), cow urine+lindane (U+L), cow urine+antioxidants (U+A) and cow urine+antioxidants+lindane (U+A+L). Group C animals were administered only the vehicle (olive oil); doses selected for other treatments were: lindane: 40 mg/kg b.w.; antioxidants: 125 mg/kg b.w. (vitamin C: 50 mg/kg b.w., vitamin E: 50 mg/kg b.w., α-lipoic acid: 25 mg/kg b.w.) and cow urine: 0.25 ml/kg b.w. In group A+L and U+L antioxidants and cow urine were administered 1 h prior to lindane administration and in group U+A and U+A+L cow urine was administered 10 min before antioxidants. All treatments were administered orally continuously for 60 days. Lindane treated group showed increased lipid peroxidation, whereas glutathione, glutathione peroxidase, superoxide dismutase, catalase, protein and endogenous levels of vitamin C and E were significantly decreased compared to control. Administration of cow urine and antioxidants alleviated the levels of these biochemical parameters.     

Coccidian parasites (Apicomplexa: Eimeriidae) from insectivores. VII. Six new species from the hairy-tailed mole, Parascalops breweri

Sixteen hairy-tailed moles, Parascalops breweri, collected from the northeastern U.S.A. were examined for coccidian oocysts; all were infected with multiple species of coccidia and 3 genera were represented. Two cyclosporans, 2 eimerians, and 2 isosporans are described as new species. Sporulated oocysts of Cyclospora ashtabulensis n. sp. are subspheroid to ellipsoid, 18 X 14 (14-23 X 11-19) microns, and sporocysts are ovoid, 12 X 7 (8-14 X 5-9) microns; C. ashtabulensis was found in 7 of 16 (44%) moles. Sporulated oocysts of Cyclospora parascalopi n. sp. are spheroid, 17 X 14 (13-20 X 11-20) microns, and sporocysts are ovoid, 11 X 7 (8-14 X 5-8) microns; C. parascalopi was found in 8 of 16 (50%) moles. Sporulated oocysts of Eimeria aethiospora n. sp. are subspheroid to ellipsoid, 19 X 13 (15-24 X 10-16) microns, and sporocysts are ovoid, 11 X 6 (8-13 X 4-7) microns; E. aethiospora was found in 4 of 16 (25%) moles. Sporulated oocysts of Eimeria titthus n. sp. are subspheroid, 16 X 14 (13-19 X 11-17) microns, and sporocysts are ellipsoid, 11 X 6 (9-13 X 4-7) microns; E. titthus was found in 4 of 16 (25%) moles. Sporulated oocysts of Isospora ashtabulensis n. sp. are ellipsoid, 20 X 14 (16-24 X 10-18) microns, and sporocysts are ovoid, 10 X 7 (7-14 X 5-10) microns; I. ashtabulensis was found in 5 of 16 (31%) moles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of glutathione S-transferase in the freshwater bivalve Sphaerium corneum as a biomarker for short-term toxicity tests?

A study was carried out to examine the use of glutathione S-transferase (GST) in freshwater bivalves as a short-term biochemical marker for organic contaminants. In contrast with earlier reports, induction of GST activity towards 1-Chloro-2,4-dinitrobenzene (CDNB) was not found in any of the experiments performed. These included 10–12 days exposures to sediments spiked with 2,3′,4,4′,5-pentachlorobiphenyl, benzo[a]pyrene or lindane and 7–8 days exposures to dieldrin, lindane or benzo[a]pyrene in a water-only system. A nonsignificant decrease in GST activity (ca. − 20%) was found at the highest sediment concentration of lindane (10,000 μg/kg) and at the highest water concentrations of dieldrin (4.9 and 29 μg/l). Between experiments differences in GST activity were found that were ascribed to seasonal influences and maintenance in the laboratory. It is concluded that under the applied conditions glutathione S-transferase activity of Sphaerium corneum towards CDNB is not a suitable short-term biomarker with respect to the toxicants tested.     

Optimal production of glutathione by controlling the specific growth rate of yeast in fed-batch culture

The optimal of the specific growth rate was obtained with simple mathematical model in a yeast fed-batch cultures. The model was based on the mass balance around the fed-batch system and the relationship between the specific growth rate, mu, and the specific production rate of glutathione, rho(G). The optimal profile of mu was calculated as a bang-bang type, That is mu, should start from the maximum value, mu(max) and should be kept at mu(max); then mu should be switched to mu(c), which gives a maximum value of rho(G). It was proven from the maximum principle that switching was needed only once, with the switching time from mu(max) to mu(c) depending on the final required glutathione content. Finally, this ideal profile of mu for the maximum production of glutathione was realized by manipulating the substrates feed rate in the fed-batch culture. Using the extended Kalman filter and a programmed-controller/feedback-compensator (PF) system, mu could be controlled at the optimal profile obtained. As a result, the maximum production of glutathione was accomplished fairly successfully. However, further improvement in the controller performance for mu is desired. The control strategy employed here can be applied to other batch reaction processes.     

Degradation of lindane by cell-free preparations of Clostridium sphenoides.

Cell-free preparation of Clostridium sphenoides degraded the insecticide lindane, the gamma-isomer of 1,2,3,4,5,6-hexachlorocyclohexane, to the gamma-isomer of 3,4,5,6-tetrachloro-1-cyclohexene. The activity appeared to be associated with the membrane fraction and required reduced glutathione. The tetrachlorocy-clohexene intermediate was further metabolized by the membrane fraction to unknown substances.     

Degradation of lindane by cell-free preparations of Clostridium sphenoides.

Cell-free preparation of Clostridium sphenoides degraded the insecticide lindane, the gamma-isomer of 1,2,3,4,5,6-hexachlorocyclohexane, to the gamma-isomer of 3,4,5,6-tetrachloro-1-cyclohexene. The activity appeared to be associated with the membrane fraction and required reduced glutathione. The tetrachlorocy-clohexene intermediate was further metabolized by the membrane fraction to unknown substances.     

Cleavage of DNA by model complexes of cytochrome P-450

Thiolate-hemin complexes as chemical models for cytochrome P-450 have been shown to cause cleavage of DNA. The cleavage of DNA to open-circular and linear forms depended on the structure of thiol ligand and the thiol ligand:hemin ratio at pH 7.8. Complete cleavage of DNA was observed by complexes containing thioglycolate ethylester and mercaptoethanol at 400-600 moles excess of thiol ligand to hemin, those containing cysteine, cysteine methylester and cysteine ethylester at 50-200 moles excess, and those containing mercaptopropionylglycine, glutathione, glutathione dimethylester, penta- and nonapeptides at 5-100 moles excess. Inhibition experiments suggested the involvement of active oxygen species in the cleavage of DNA.     

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV.

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.     

Structure-activity relations of the dark-colour-inducing neurohormone of locusts

The dark-colour-inducing effect of several peptides in comparison to that of the dark-colour-inducing neurohormone (DCIN, [His(7)]-corazonin) of locusts was investigated by a bioassay based on nymphs of a DCIN-deficient albino mutant of Locusta migratoria. The study was aimed at elucidating the active part of the DCIN and to explore the contribution of its amino acids to the activity. Graded doses of all peptides were injected in oil. [Arg(7)]-corazonin and DCIN were equally effective. Certain arthropod neuropeptides having the -SXGW- partial sequence (a part of the DCIN and of [Arg(7)]-corazonin; X=His and X=Arg, respectively) yielded the following findings: Scg-AKH-II (adipokinetic hormone II of Schistocerca gregaria X=Thr), Grb-AKH ( adipokinetic hormone of Gryllus bimaculatus X=Thr) and RPCH (red pigment concentrating hormone of crustaceans X=Pro) evoked a moderate darkening response, but Lom-AKH-II (adipokinetic hormone II of L. migratoria X=Ala) was ineffective. Step by step shortening of the sequence of the DCIN at the N-terminal, from pGlu-3-11DCIN to pGlu-9-11DCIN, resulted in a decreasing activity, but even pGlu-9-11DCIN induced a weak response with high doses. Shortening of the DCIN from the C-terminal revealed a moderate activity of 1-7DCIN-NH(2) and a weak activity of 1-5DCIN-NH(2). An octadecapeptide which induces dark colour in moth larvae, having the pentamer FTPRL-NH(2) at its C-terminal, evoked no darkening in the albino locusts. We conclude that although the -SXGW- partial sequence has some role in induction of darkening, for obtaining maximal effect the whole sequence of the DCIN (or of [Arg(7)]-corazonin) is necessary.     

Acute lindane intoxication: A study on lindane tissue concentration and oxidative stress-related parameters in liver and erythrocytes

Treatment of rats with daily dosis of 20 mg of lindane/kg for 3 consecutive days led to the accumulation of the insecticide in several tissues, including erythrocytes and liver. Lindane did not alter the hematocrit and hemoglobin concentration but reduced methemogiobin levels by 17%. Red blood cells from controls and lindane-treated rats, exposed to t-butyl hydroperoxide, exhibited comparable rates of oxygen uptake and visible chemiluminescence, whereas the induction period that precedes oxygen uptake was significantly enhanced in the latter group. Lindane treatment did not modify the activity of erythrocyte glutathione peroxidase, glucose-6-phosphate dehydrogenase, catalase, and methemoglobin reductase, being the total content of glutathione and superoxide dismutase activity significantly increased. The liver from lindane-treated rats showed an enhanced microsomal pro-oxidant activity, evidenced by higher cytochrome P450 content and NADPH-cytochrome c reductase and NADPH oxidase activities. The higher enzyme activities led to an increased superoxide anion generation (adrenochrome formation) and lipid peroxidation (measured either by the production of thiobarbituric acid reactants and spontaneous visible chemiluminescence). Concomitantly, liver glutathione content and the activity of glutathione peroxidase-glutathione reductase couple were augmented by lindane treatment, without any change in superoxide dismutase activity, together with a reduction in that of catalase. Results suggest that lindane does not alter the prooxidant/antioxidant status of the erythrocyte in conditions of a significant cellular accumulation of the insecticide, which might exert direct action on enzymatic systems leading to enhanced superoxide dismutase activity and glutathione content. In the liver, lindane-induced pro-oxidant condition was not accompanied by cell injury, probably due to the adaptative increase in some antioxidant mechanisms of the hepatocyte.