Oral administration of Azadirachta indica (L.) seed kernel active principle protects rat liver hepatocytes and testis seminiferous tubules from phenobarbitol-induced damage.

The male albino rat testis and liver showed tissue modification upon exposure to phenobarbitol (PB), 24 mg/100 g of body weight, for about 3 weeks and upon staining of their sections with hematoxylin and eosin. In this procedure, the control liver showed normal hepatocytes with centrally placed nuclei, and the PB-treated hepatocyte showed degeneration of cytoplasm and nucleus, necrosis and fragmentation of nucleus, and pushing of nucleus to periphery. The control rat testis showed epithelial layer having broad seminiferous tubules, spermatids, mature spermatozoa, and lumen of seminiferous tubules, and the PB-treated rat testis showed degenerative and necrotic changes in seminiferous tubules and clumping of seminiferous tubules. These changes almost returned to normal conditions in rat liver and testis upon the oral administration of an antioxidant that is present in Azadirachta seed-kernel extract (ASKE, 100 mg/kg body weight). In the case of enzymes, glutathione transferase and glutathione peroxidase were induced upon PB or ASKE treatment and the combination of both the treatments. The lipid peroxides were reduced in all the three cases in both liver and testis. The histological studies and enzymatic analysis revealed that the potential role of ASKE in the protection of the testis and liver tissues from PB-induced damage.

Comparative study on glutathione transferases of rat brain and testis under the stress of phenobarbitol and beta-methylcholanthrene.

A comparative study was made on the tissue specific expression of glutathione transferases (GST) in brain and testis after exposure of rat to phenobarbitol (PB) and b-methylcholanthrene (MC). Glutathione transferases, a family of multifunctional proteins are involved in intracellular transport processes and in detoxication of electrophilic xenobiotics by catalyzing reactions such as conjugation, isomerization, reduction and thiolysis. On purification, the yield of GST proteins by affinity chromatography was 39% in testis and 32% in brain. The affinity purified testis GSTs were resolved by chromatofocusing into six anionic and four cationic isozymes, and in brain glutathione transferases were resolved into four anionic and three cationic isozymes, suggesting the presence of multiple isozymes with Yc, Yb, Ybeta and Ydelta in both of them. In testis and brain, these isozymes at identical pI values showed variable functions with a battery of substrates and the cationic isozymes of brain and testis showed identical properties in CHP (cumene hydroperoxide) at pH values of above 7.0. Substrate specificity studies and immunoblot analysis of testis and brain proteins revealed that they play a predominant role in the detoxication of phenobarbitol or beta-methylcholanthrene. Expression of the isozymes in testis and brain on exposure to PB and MC indicated elevated subunit variation. In both testis and brain, Ydelta of pi class was expressed on PB treatment and Yc of alpha class and Ybeta of mu class was expressed in MC treated testis and only Yc was predominantly expressed in MC treated brain. Thus these subunits expression is considered as markers for carcinogenesis and specific to chemical toxicity under phenobarbitol and beta-methylcholanthrene stress.

Effect of phenobarbital on the induction of glutathione S-transferases in rat testis.

Glutathione S-transferases are a family of multifunctional proteins involved in intracellular transport processes and drug detoxication. In rats, these enzymes are dimeric proteins, and exist as cytosolic and microsomal proteins. The affinity purified rat testicular glutathione S-transferases are comprised of four subunits, Yc of alpha class, Yb and Ybeta of mu class and Ydelta of pi class. On chromatofocusing, they were resolved into six anionic and four cationic isozymes. The cationic isozymes were found to be abundant. All these isozymes on electrophoresis were found to contain heteromers except for two isozymes. The expression of individual subunits and their activity was elevated on treatment with multiple doses of phenobarbital in rat testis. Among all of these, according to the immunological studies, Ydelta, a pi class glutathione S-transferase, was induced predominantly in response to phenobarbital.

Does glutathione S-transferase Pi (GST-Pi) a marker protein for cancer?

Glutathione S-transferases (GSTs, EC 2.5.1.18) are multifunctional and multigene products. They are versatile enzymes and participate in the nucleophilic attack of the sulphur atom of glutathione on the electrophilic centers of various endogenous and xenobiotic compounds. Out of the five, alpha, micro, pi, sigma and theta, major classes of GSTs, GST-pi has significance in the diagnosis of cancers as it is expressed abundantly in tumor cells. This protein is a single gene product, coded by seven exons, that is having 24 kDa mass and pI value of 7.0. Four upstream elements such as two enhancers, and one of each of AP-1 site and GC box regulate pi gene. During chemical carcinogenesis because of jun/fos oncogenes (AP-1) regulatory elements, specifically GST-pi is expressed in liver. Therefore this gene product could be used as marker protein for the detection of chemical toxicity and carcinogenesis.

A comparative study on the effect of phenobarbitol and beta-methylcholanthrene on glutathione S-transferases of rat testis.

Glutathione S-transferases (GSTs; EC, 2.5.1.18) ubiquitously distributed in all life forms, are a family of multigene and multifunctional dimeric proteins, which play a main role in drug detoxication. On purification and on electrophoresis, rat testicular glutathione S-transferases showed that it comprises of four subunits, Yc of alpha class, Yb and Ybeta of mu class and Ydelta of pi class. On chromatofocusing they were resolved into six anionic and four cationic isozymes. The substrate specificity studies and immunoblot analysis of testis proteins revealed that Ydelta of pi class GST was induced predominantly in response to phenobarbitol and Yc of alpha class and Ybeta of mu class were elevated specifically on treatment with methylcholanthrene (MC). These results show that structural variation between the two carcinogens induces different types of GST subunits. Therefore, these subunits may be used as marker proteins for specific chemical toxicity of rat testis.

Isolation of antioxidant principle from Azadirachta seed kernels: determination of its role on plant lipoxygenases.

An antioxidant principle was isolated from Azadirachta indica seed using high pressure liquid chromatography with a hydrophobic reverse-phase chromatography column. The eluted molecule had lambdamax at 224 and 272 nm and was a potent inhibitor of plant lipoxygenases. In in vivo studies of horsegram during germination, low levels of lipoxygenase activity and lipid peroxides were found upon treatment with the Azadirachta extract. The antioxidant property of Azadirachta indica has not been previously reported.

Isolation of 75Se binding protein from hepatic tissues of chick embryos.

Distribution study using 75Se shows that maximum accumulation was in liver tissues after 24h of 75Se administration. Induction of selenium binding protein (Se-P) in hepatic tissues of chick embryo was observed. Chick embryo hepatic Se-P was isolated after 24h of 75Se treatment using Sephadex G-75 column chromatography. Fractions of induced protein shows the presence of maximum concentration of 75Se. This induced protein was found to have an approximate molecular weight of 56 KD on molecular sieve. It also showed an absorbance maxima at 254 nm, which indicates the presence of high concentrations of sulphydryl groups.

Serum lipid peroxides and lipids in urban and rural Indian men.

Serum lipid peroxides, lipids, blood pressure, body mass index, and dietary intake in 190 urban men were compared with 190 age-matched rural men. Significantly higher levels of lipid peroxides, serum cholesterol, low-density lipoprotein cholesterol, and triglycerides were seen in urban men, compared with rural men. In rural men, serum lipid peroxides were related negatively to age; the same was observed in the 70 + y age group in the urban population. All the lipid constituents were related positively to age in both populations. There were statistically significant correlations between lipid peroxides and serum cholesterol and triglycerides in the urban men. The marked elevation of lipid peroxides and lipids in urban men may be the result of urbanization, including exposure to environmental pollutants.

Dietary supplementation of vitamin E protects heart tissue from exercise-induced oxidant stress.

Exhaustive endurance exercise in adult female albino rats (C-Ex) increased the generation of free radicals (R.) in the myocardium, probably through enhanced oxidative mechanisms. Free radical mediated lipid peroxidation measured in the form of tissue MDA content also increased in C-Ex animals, suggesting the exercise-induced oxidative stress in these animals. Dietary supplementation of Vit E, for a period of 60 days significantly increased Vit E incorporation into the serum and myocardium, more so in the myocardium. Vit E supplementation to exercising animals completely abolished the radical production. The protection of Vit E against oxidative stress appears to be not mediated through the improvement of antioxidant mechanisms by enzymes like SOD, catalase and Se-GSH Px. However the non Se-GSH Px, the enzyme involved in the reduction of endoperoxides increased significantly in control and Vit E fed animals in response to exercise. The protection of Vit E against exercise-induced oxidative stress was correlated with its multivarious activities like a) scavenger of free radicals; b) inhibition of lipoxygenases; and c) reduction of peroxides in association with lipoxygenases. These studies indicate that dietary supplementation of Vit E protects the animals from the possible oxidative damages of endurance exercise.

Glutathione-S-transferase, superoxide dismutase, xanthine oxidase, catalase, glutathione peroxidase and lipid peroxidation in the liver of exercised rats.

Glutathione-S-transferase (GST), superoxide dismutase (SOD), Xanthine oxidase, selenium-dependent glutathione peroxidase (GPxI), catalase activities and malondialdehyde (MDA) content were determined in liver of three groups of exercised rats (E) viz., one day (E1), 10 days (E10) and 60 days (E60). GST, SOD and xanthine oxidase activities increased significantly with the increase in exercise period. Lipid peroxidation, expressed in terms of MDA formation, also increased in the liver of all the three groups. But catalase activity decreased significantly during exercise. Further, GPxI did not show any significant change in its activity in response to exercise. Our findings indicate that: 1) The significant increase in GST activity suggests their induction aimed at counteracting the oxidant stress induced during exercise; 2) The significant increase in xanthine oxidase and SOD activities indicates the generation of more superoxide anion radicals and their removal, respectively. 3) The significant reduction in catalase activity denotes the decreased formation of hydrogenperoxides during exercise; and 4) The pattern of changes in the activity level of GPxI indicate its least participation during exercise. However, in another way it is giving a scope for the involvement of GPxII associated with GST in the reduction of organic hydroperoxides. Further more, the relative increase in MDA is considered as the indicator of the rate of lipid peroxidation in the wake of exhaustive exercise.

Elevation of rat liver mRNA for selenium-dependent glutathione peroxidase by selenium deficiency.

Selenium-dependent glutathione peroxidase (Se-GSH-Px, GSH-H2O2 oxidoreductase EC 1.11.1.9) is the best characterized selenoprotein in higher animals, but the mechanism whereby selenium becomes incorporated into the enzyme protein remains under investigation. To elucidate the mechanism of insertion of selenium into Ge-GSH-Px further, we have systematically analyzed and compared the results of Western blot, in vitro translation immunoprecipitation, and Northern blot experiments conducted with liver proteins and RNAs obtained from rats fed on selenium-deficient and selenium-supplemented diets. The anti-serum employed in this study was raised against an electrophoretically pure Se-GSH-Px preparation obtained from rat livers by a simplified purification procedure involving separation by high performance liquid chromatography on a hydrophobic interaction column. Different forms of Se-GSH-Px, including apo-protein, cross-reacted with this antiserum and Western blot analysis found no Se-GSH-Px protein present in livers from rats fed on selenium-deficient diets. By contrast, a distinct protein band corresponding to purified Se-GSH-Px was detected in livers from selenium-supplemented animals, a result consistent with the finding that the Se-GSH-Px activity was reduced to undetectable levels in livers of selenium-deficient rats. The in vitro translation experiments, however, indicated not only that mRNA for Se-GSH-Px was present during selenium deficiency but also that its translation products contained 2-3-fold as much immunoprecipitable protein as the products of poly(A) RNA from livers of selenium-supplemented rats. This result suggests that the Se-GSH-Px mRNA may be increased in the selenium-deficient state. Elevated levels of Se-GSH-Px mRNA were directly demonstrated in Northern blot experiments employing cDNA clone pGPX1211 as a probe. A similar increase in Se-GSH-Px mRNA was observed in such other tissues as kidney, testis, brain, and lung tissue, in selenium-deficient states. The present data support the co-translational mechanism for the incorporation of selenium into Se-GSH-Px in rat liver.

Expression of glutathione peroxidase I gene in selenium-deficient rats.

We have characterized a cDNA pGPX1211 encoding rat glutathione peroxidase I. The selenocysteine in the protein corresponded to a TGA codon in the coding region of the cDNA, similar to earlier findings in mouse and human genes, and a gene encoding the formate dehydrogenase from E. coli, another selenoenzyme. The rat GSH peroxidase I has a calculated subunit molecular weight of 22,155 daltons and shares 95% and 86% sequence homology with the mouse and human subunits, respectively. The 3'-noncoding sequence (greater than 930 bp) in pGPX1211 is much longer than that of the human sequences. We found that glutathione peroxidase I mRNA, but not the polypeptide, was expressed under nutritional stress of selenium deficiency where no glutathione peroxidase I activity can be detected. The failure of detecting any apoprotein for the glutathione peroxidase I under selenium deficiency and results published from other laboratories supports the proposal that selenium may be incorporated into the glutathione peroxidase I co-translationally.

Expression of glutathione S-transferases in rat brains.

The tissue-specific expression of glutathione S-transferases (GSTs) in rat brains has been studied by protein purification, in vitro translation of brain poly(A) RNAs, and RNA blot hybridization with cDNA clones of the Ya, Yb, and Yc subunit of rat liver GSTs. Four classes of GST subunits are expressed in rat brains at Mr 28,000 (Yc), Mr 27,000 (Yb), Mr 26,300, and Mr 25,000. The Mr 26,3000 species, or Y beta, has an electrophoretic mobility between that of Ya and Yb, similar to the liver Yn subunit(s) reported by Hayes (Hayes, J. D. (1984) Biochem. J. 224, 839-852). RNA blot hybridization of brain poly(A) RNAs with a liver Yb cDNA probe revealed two RNA species of approximately 1300 and approximately 1100 nucleotides. The band at approximately 1300 nucleotides was absent in liver poly(A) RNAs. The Mr 25,000 species, or Y delta, can be immunoprecipitated by antisera against rat heart and rat testis GSTs, but not by antiserum against rat liver GSTs. Therefore, the Y delta subunit may be related to the "Mr 22,000" subunit reported by Tu et al. (Tu, C.-P.D., Weiss, M.J., Li, N., and Reddy, C. C. (1983) J. Biol. Chem. 258, 4659-4662). The abundant liver GST subunits, Ya, are not expressed in rat brains as demonstrated by electrophoresis of purified brain GSTs and a lack of isomerase activity toward the Ya-specific substrate, delta 5-androstene-3,17-dione. This is apparently because of the absence of Ya mRNA expression prior to RNA processing. The data on the preferential expression of Yc subunits in rat brains, together with the differential phenobarbital inducibility of the Ya subunit(s) in rat liver reported by Pickett et al. (Pickett, C. B., Donohue, A. M., Lu, A. Y. H., and Hales, B. F. (1982) Arch. Biochem. Biophys. 215, 539-543), suggest that the Ya and Yc genes for rat GSTs are two functionally distinct gene families even though they share 68% DNA sequence homology. The expression of multiple GSTs in rat brains suggests that GSTs may be involved in physiological processes other than xenobiotics metabolism.