Mouse strain variations in the magnitude of induction of liver DT-diaphorase and hereditary transmission of the trait

1. Strain variations among mice in terms of cytosolic DT-diaphorase activity were studied in liver, kidney, stomach and heart tissues with or without the administration of 3-tert-butyl-4-hydroxyanisole (BHA). 2. BHA induced DT-diaphorase activity in all strains examined, and the magnitude of induction varied depending on the strain and tissue. Among the 10 inbred strains tested, BALB/c and C57BL mice showed relatively large magnitudes of induction for liver DT-diaphorase, whereas C3H and CBA mice showed relatively small magnitudes. 3. Results of examinations of BALB/c-C3H-F1, -F2 and C57BL-CBA-F1 mice revealed that smaller magnitudes of induction of liver DT-diaphorase were inherited essentially as a dominant trait. The hereditary trait could be adequately explained by postulating two gene loci that regulate the magnitude of induction. 4. The possible significance of DT-diaphorase activity in chemical carcinogenesis was discussed.     

Reduction of Brain Antioxidant Defense Upon Treatment with Butylated Hydroxyanisole (BHA) and Sudan III in Syrian Golden Hamster

Treatment with the antioxidant butylated hydroxyanisole (BHA) or the azo dye Sudan III during two weeks led to changes in the brain enzymatic antioxidant defense of Syrian golden hamsters. BHA was able to induce liver superoxide dismutase (SOD) 2-fold but had no effect on the brain SOD activity, whereas SOD activity was reduced to 50% in brain and remained unchanged in liver with Sudan III. These two substances are known inducers of DT-diaphorase and in fact this enzymatic activity was induced 4- and 6-fold in liver with BHA and Sudan III, respectively. However, BHA promoted a significant 40% reduction, whereas no change was observed with Sudan III in brain DT-diaphorase activity. Glutathione(GSH)-related enzymatic activities were also assayed in brain and liver. No induction was observed with BHA or Sudan III for any of the activities tested in hamster brain: GSH S-transferase (GST), GSH peroxidase (GSH-Px) and glutathione disulfide (GSSG) reductase (GR). Only 1.3- and 1. 4-fold increases of GST and GR activities were observed in liver and no change in any of these enzymatic activities in brain with BHA; a partial limitation of permeability to BHA of the blood-brain barrier may explain this results. Furthermore, Sudan III promoted reductions in all these GSH-related enzymatic activities in brain and liver. The possible explanations for these results are discussed.Deceased 4th November 1998     

Hepatic levels of cytosolic, microsomal and 'mitochondrial' epoxide hydrolases and other drug-metabolizing enzymes after treatment of mice with various xenobiotics and endogenous compounds

This study was performed in order to study the response of epoxide hydrolases in different subcellular compartments of mouse liver to treatment with various compounds. Male C57BL/6 mice were treated with 31 different compounds--including traditional inducers of xenobiotic-metabolizing systems, liver carcinogens, stilbene derivatives, endogenous compounds and various other drugs and xenobiotics. The effects on liver somatic index; protein contents in 'mitochondria', microsomes and cytosol prepared from the liver; epoxide hydrolase activity towards trans- or cis-stilbene oxide in these three fractions; microsomal cytochrome P-450 content; cytosolic and 'mitochondrial' glutathione transferase activity and cytosolic DT-diaphorase activity were then determined. Cytosolic epoxide hydrolase activity was induced by chlorinated paraffins, di(2-ethylhexyl)phthalate and clofibrate and depressed by alpha-naphthylisothiocyanate, 3-methylcholanthrene, benzil and quercitin. Radial immunodiffusion revealed similar changes in the amount of enzyme protein present, except for two cases, where the increase in amount was larger; and the enzyme seems to be inhibited by benzil. Microsomal epoxide hydrolase activity was induced by these same compounds and several others as well, including dibenzoylmethane, butylated hydroxyanisole and polychlorinated biphenyls. 'Mitochondrial' epoxide hydrolase activity towards trans-stilbene oxide was not affected by those compounds which induced the cytosolic enzyme, but increased about two-fold after treatment with 2-acetylaminofluorene, DL-ethionine, aflatoxin B1 and phenobarbital. There does not seem to be any co-regulation of different forms of epoxide hydrolase in mouse liver. In general small effects were observed on liver weight and protein contents in the different subcellular fractions. Polychlorinated biphenyls were the most potent of the 8 compounds which induced cytochrome P-450, while butylated hydroxyanisole induced cytosolic glutathione transferase activity to the highest extent. 'Mitochondrial' glutathione transferase activity was most induced by certain of the stilbene derivatives. The most potent inducers of DT-diaphorase activity were 3-methylcholanthrene, polychlorinated biphenyls and dinitrotoluene.     

Mechanism of coumarin action: sensitivity of vitamin K metabolizing enzymes of normal and warfarin-resistant rat liver

The in vitro effects of two coumarin anticoagulants, warfarin and difenacoum, on rat liver microsomal vitamin K dependent carboxylase, vitamin K epoxidase, vitamin K epoxide reductase, and cytosolic vitamin K reductase (DT-diaphorase) from the livers of normal and a warfarin-resistant strain of rats have been determined. Millimolar concentrations of both coumarins are required to inhibit the carboxylase and epoxidase activities in both strains of rats. Sensitivity of DT-diaphorase to coumarin inhibition differs when a soluble or liposomal-associated substrate is used, but the diaphorases isolated from both strains of rats have comparable sensitivity. The anticoagulant difenacoum is an effective rodenticide in the warfarin-resistant strain of rats, and the only enzyme studied from warfarin-resistant rat liver that demonstrated a significant differential inhibition by the two coumarins used was the vitamin K epoxide reductase. This enzyme also showed the greatest sensitivity to coumarin inhibition among the enzymes studied. These results support the hypothesis that the physiologically important site of action of coumarin anticoagulants is the vitamin K epoxide reductase.     

Biospecific adsorption of hepatic DT-diaphorase on immobilized dicoumarol. II. Purification of mitochondrial and microsomal DT-diaphorase from 3-methylcholanthrene-treated rats

Liver mitochondrial and microsomal DT-diaphorase have been purified from 3-methylcholanthrene-treated rats. A 1150-fold and 3500-fold purification of mitochondrial and microsomal DT-diaphorase, respectively, is achieved after solubilization of the membranes with deoxycholate followed by affinity chromatography on azodicoumarol Sepharose 6B and subsequent gel filtration on Sephadex G-100. From this purification procedure, 65–70% of mitochondrial DT-diaphorase is recovered and the purified enzyme has a specific activity comparable to that of cytosolic DT-diaphorase; i.e., 50.4 kat/kg protein. Microsomal DT-diaphorase is obtained with a yield of 45% and a specific activity of 15.5 kat/kg protein.Purified mitochondrial DT-diaphorase exhibits an absorption spectrum characteristic of a flavoprotein and very similar to that of the cytosolic enzyme. Purification of both mitochondrial and microsomal DT-diaphorase results in fractions enriched in a polypeptide with a molecular weight of 28,000 which comigrates with purified cytosolic DT-diaphorase on SDS-polyacrylamide gel electrophoresis. Employing antiserum raised against cytosolic DT-diaphorase, immunological identity between DT-diaphorase isolated from the three cell fractions is observed with both the Ouchterlony immunodiffusion technique and fused rocket immunoelectrophoresis. The latter method also reveals that DT-diaphorase isolated from mitochondria and microsomes contains several antigenic forms identical to those observed in purified cytosolic DT-diaphorase. Furthermore, this antiserum inhibits DT-diaphorase to about the same extent whether the enzyme is isolated from mitochondria, microsomes, or cytosol. In addition, this antiserum efficiently inhibits membrane-bound microsomal DT-diaphorase.     

Effects of fructose 6-phosphate and adenylates on the activities of rabbit liver fructose bisphosphatase and phosphofructokinase.

The kinetics of induction of cytosolic DT-diaphorase (NAD(P)H dehydrogenase-quinone, EC 1.6.99.2) by benzo(a)pyrene (BP) in the liver of the 8-day-old rat has been studied. After a lag phase of 8 h, DT-diaphorase reaches its maximum activity in three waves, with plateau levels of activity between 15–18, 26–36, and 40 h after administration of BP, at 4, 15, and 26 times the basal activity, respectively. A lower degree of induction of DT-diaphorase could be observed in the kidney cortex of the young rat and in the liver of the adult rat. No induction was observed in the fetal liver and in the adult kidney cortex. Lead acetate treatment of the adult rat resulted in induction of DT-diaphorase by BP in the liver and in the kidney cortex. Induction could not be observed in the regenerating liver of the adult rat. Experiments with picolinic acid (PA)—as a G₁ inhibitor—administered simultaneously or at different time intervals after BP administration resulted in an inhibition of induction, depending on the time of administration of picolinic acid. It is concluded that a mitotic cell cycle is necessary for DT-diaphorase induction by BP. Evidence is presented that BP acts in late G₁. The kinetics of induction of aryl hydrocarbon hydroxylase (AHH) by BP in liver microsomes of the 8-day-old rat has been compared with the induction of cytosolic DT-diaphorase. The effect of PA on the induction of AHH has also been studied. In view of the differences in kinetics of induction and in the effects of PA, it is concluded that the induction of AHH and that of DT-diaphorase are dissociated. AHH induction may take place in all hepatocytes, in contrast to DT-diaphorase induction.     

Effect of inducers of DT-diaphorase on the toxicity of 2-methyl- and 2-hydroxy-1,4-naphthoquinone to rats

It has previously been shown that rats pre-treated with butylated hydroxyanisole (BHA), a well-known inducer of the enzyme DT-diaphorase, are protected against the toxic effects of 2-methyl-1,4-naphthoquinone but are made more susceptible to the harmful action of 2-hydroxy-1,4-naphthoquinone. In the present experiments, the effects of BHA have been compared with those of other inducers of DT-diaphorase. Rats were dosed with BHA, butylated hydroxytoluene (BHT), ethoxyquin (EQ), dimethyl fumarate (DMF) or disulfiram (DIS) and then challenged with a toxic dose of the naphthoquinones. All the inducers protected against the haemolytic anaemia induced by 2-methyl-1,4-naphthoquinone in rats, with BHA, BHT and EQ being somewhat more effective than DMF and DIS. A similar order of activity was recorded in the relative ability of these substances to increase hepatic activities of DT-diaphorase, consistent with a role for this enzyme in facilitating conjugation and excretion of this naphthoquinone. In contrast, all the compounds increased the haemolytic activity of 2-hydroxy-1,4-naphthoquinone. DMF and DIS were significantly more effective in this regard than BHA, BHT and EQ. DMF and DIS also caused a much greater increase in levels of DT-diaphorase in the intestine, suggesting that 2-hydroxy-1,4-naphthoquinone is activated by this enzyme in the gut. BHA, BHT and EQ had no effect on the nephrotoxicity of 2-hydroxy-1,4-naphthoquinone, but the severity of the renal lesions was decreased in rats pre-treated with DMF and DIS. The results of the present experiments show that modulation of tissue levels of DT-diaphorase may not only alter the severity of naphthoquinone toxicity in vivo, but may also change the relative toxicity of these substances to different target organs.     

Effects of modulation of tissue activities of DT-diaphorase on the toxicity of 2,3-dimethyl-1,4-naphthoquinone to rats

The enzyme DT-diaphorase mediates the two-electron reduction of quinones to hydroquinones. It has previously been shown that the toxicity of 2-methyl-1,4-naphthoquinone to rats is decreased by pre-treatment of the animals with compounds that increase tissue levels of this enzyme. In contrast, the severity of the haemolytic anaemia induced in rats by 2-hydroxy-1,4-naphthoquinone was increased in animals with high levels of DT-diaphorase. In the present experiments, the effect of alterations in tissue diaphorase activities on the toxicity of a third naphthoquinone derivative, 2,3-dimethyl-1,4-naphthoquinone, has been investigated. This compound induced severe haemolysis and slight renal tubular necrosis in control rats. Pre-treatment of the animals with BHA, a potent inducer of DT-diaphorase, diminished the severity of the haemolysis induced by this compound and abolished its nephrotoxicity. Pre-treatment with dicoumarol, an inhibitor of this enzyme, caused only a slight increase in the haemolysis induced by 2,3-dimethyl-1,4-naphthoquinone, but provoked a massive increase in its nephrotoxicity. Modulation of DT-diaphorase activity in animals may therefore not only alter the severity of naphthoquinone toxicity, but also cause pronounced changes in the site of toxic action of these substances. The factors that may control whether induction of DT-diaphorase in animals will decrease or increase naphthoquinone toxicity are discussed.     

Effect of butylated hydroxyanisole on the toxicity of 2-hydroxy-1,4-naphthoquinone to rats

It has previously been shown that rats pre-treated with butylated hydroxyanisole (BHA), a well-known inducer of the enzyme DT-diaphorase, are protected against the harmful effects of 2-methyl-1,4-naphthoquinone. This is consistent with a role for diaphorase in the detoxification of this quinone, but it is not known if increased tissue levels of this enzyme give protection against other naphthoquinone derivatives. In the present study, rats were dosed with BHA and then challenged with a toxic dose of 2-hydroxy-1,4-naphthoquinone, a substance that causes haemolytic anaemia and renal damage in vivo. Pre-treatment with BHA had no effect upon the nephrotoxicity of 2-hydroxy-1,4-naphthoquinone, but the severity of the haemolysis induced by this compound was increased in the animals given BHA. DT-Diaphorase is known to promote the redox cycling of 2-hydroxy-1,4-naphthoquinone in vitro, with concomitant formation of 'active oxygen' species. The results of the present experiment suggest that activation of 2-hydroxy-1,4-naphthoquinone by DT-diaphorase may also occur in vivo and show that increased tissue levels of DT-diaphorase are not always associated with naphthoquinone detoxification.     

Strain differences of the ability to hydroxylate methotrexate in rats

Converting activity of methotrexate (MTX) to 7-hydroxymethotrexate (7-OH-MTX) was examined using eight strains of rats. Marked variability of the activity was found in liver cytosols from the rats. The highest activity was observed with Sea:SD rats, followed by LEW/Sea and Jcl:Wistar rats. The lowest activity was observed with WKA/Sea rats. The difference in the activity between Sea:SD and WKA/Sea strains was 104-fold. The variation was correlated to the strain difference of benzaldehyde oxidase activity in the rats. The cytosolic 7-hydroxylase activities in other tissues of Sea:SD rats were much higher than those of WKA/Sea, similarly to the case in liver. The liver microsomes of Sea:SD rats exhibited no 7-hydroxylase activity toward MTX even in the presence of NADPH. The cytosolic 7-hydroxylating activity of the livers of Sea:SD rats was inhibited by menadione, β-estradiol, chlorpromazine and disulfiram, inhibitors of aldehyde oxidase, but not oxypurinol, an inhibitor of xanthine oxidase. The purified aldehyde oxidase from the livers of Sea:SD rats exhibited a significant 7-hydroxylating activity toward MTX. However, xanthine oxidase had no ability to hydroxylate MTX. These facts suggest that MTX hydroxylating activity in rats is predominantly due to aldehyde oxidase, and the strain differences are due to the variations of the flavoenzyme level.     

Triacylglycerol metabolism in the phenobarbital-treated rat

1. Various aspects of triacylglycerol metabolism were compared in rats given phenobarbital at a dose of 100mg/kg body wt. per day by intraperitoneal injection; controls were injected with an equal volume of 0.15m-NaCl by the same route. Animals were killed after 5 days of treatment. 2. Rats injected with phenobarbital demonstrated increased liver weight, and increased microsomal protein per g of liver. Other evidence of microsomal enzyme induction was provided by increased activity of aminopyrine N-demethylase and cytochrome P-450 content. Increased hepatic activity of γ-glutamyltransferase (EC 2.3.2.2) occurred in male rats, but not in females, and was not accompanied by any detectable change in the activity of this enzyme in serum. 3. Phenobarbital treatment increased the hepatic content of triacylglycerol after 5 days in starved male and female rats, as well as in non-starved male rats; non-starved females were not tested in this regard. At 5 days after withdrawal of the drug, there was no difference in hepatic triacylglycerol content or in hepatic functions of microsomal enzyme induction between the treated and control rats. 4. After 5 days, phenobarbital increased the synthesis in vitro of glycerolipids in cell-free liver fractions fortified with optimal concentrations of substrates and co-substrates when results were expressed per whole liver. The drug caused a significant increment in the activity of hepatic diacylglycerol acyltransferase (EC 2.3.1.20), but did not affect the activity per liver of phosphatidate phosphohydrolase (EC 3.1.3.4) in cytosolic or washed microsomal fractions. A remarkable sex-dependent difference was observed for this latter enzyme. In female rats, the activity of the microsomal enzyme per liver was 10-fold greater than that of the cytosolic enzyme, whereas in males, the activities of phosphohydrolases per liver from both subcellular fractions were similar. 5. The phenobarbital-mediated increase in hepatic triacylglycerol content could not be explained by a decrease in the hepatic triacylglycerol secretion rate as measured by the Triton WR1339 technique. Since the hepatic triacylglycerol showed significant correlation with microsomal enzyme induction functions, with hepatic glycerolipid synthesis in vitro and with diacylglycerol acyltransferase activity, it is likely to be due to enhanced triacylglycerol synthesis consequent on hepatic microsomal enzyme induction. 6. In contrast with rabbits and guinea pigs, rats injected with phenobarbital showed a decrease in serum triacylglycerol concentration in the starved state; this decrease persisted for up to 5 days after drug administration stopped, and did not occur in non-starved animals. It seems to be independent of the microsomal enzyme-inducing properties of the drug, and may be due to the action of phenobarbital at an extrahepatic site.     

Strain differences in the induction of mono-oxygenase activity in mouse skin by topical clobetasol propionate: evidence of a role for the hr locus

The effect of the topical application of clobetasol propionate on cutaneous ethoxycoumarin O'dealkylation (EOD) has been studied in various strains of mice. Clobetasol propionate markedly increased cutaneous EOD activity in adult hairless mice only. Similar treatment of adult haired C57BL/6J mice, or adult haired DBA/2J mice had no significant effect on cutaneous EOD activity. In contrast 3 methylcholanthrene induced cutaneous EOD activity in both hairless and C57 strains to a far greater extent than in the DBA strain. EOD activity in hairless mice non-responsive to polycyclic hydrocarbons, derived by selective breeding of hairless and DBA strains was induced by clobetasol propionate to a similar extent to that observed in responsive hairless strains. Hepatic EOD activity was not induced by clobetasol propionate in any of the strains tested. Strain differences in the induction of EOD by clobetasol propionate were not related to differences in either the concentration of cytosolic glucocorticoid receptor in the skin, the dissociation constant of the cytosolic receptor, or differences in percutaneous absorption. Polycyclic hydrocarbons did not compete with triaminolone acetonide for binding to the cytosolic glucocorticoid receptor. Strain differences in the induction of EOD activity by clobetasol propionate appear therefore not to be related to strain differences in either the Ah receptor of the glucocorticoid receptor, but to be regulated by the hr locus.     

Expression of mammalian DT-diaphorase in Escherichia coli: purification and characterization of the expressed protein.

A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase [NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)] mRNA was expressed in Escherichia coli. The pKK-DTD4 cDNA was obtained by extending the 5'-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH2-terminal codons using polymerase chain reaction (PCR). Restriction sites for EcoRI and HindIII were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by the PCR reaction. The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites. E. coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promotor. The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10(-5)M. After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase. The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis. It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase. The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm. Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme. Edman protein sequencing of the first 20 amino acid residues at the NH2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH2 terminus and has an alanine as the first amino acid. The remaining 19 amino acid residues at the NH2 terminus were identical with those of the DT-diaphorase purified from rat liver cytosol.     

Enzyme induction by daunorubicin in neonatal heart cells in culture

The effect of the antineoplastic agent daunorubicin on beating heart cells from neonatal rats was investigated with respect to cell damage and induction of enzymes possibly involved in drug metabolism. Of the enzymes assayed DT-diaphorase and glutathione-S-transferase showed a two-to-four fold increase in activity: higher concentrations of daunorubicin inactivated glutathione-S-transferase. Daunorubicin toxicity increased in the presence of dicoumarol, a specific inhibitor of DT-diaphorase. These results indicate that both DT-diaphorase and glutathione-S-transferase may be involved in the metabolism of daunorubicin.     

Age-related changes of lipid fractions and total fatty acids in liver lipids and heart lipids of female and male rats aged 37-1200 days (liver) and 331-1200 days (heart)

1. Total lipids and the lipid fractions cholesterol ester, triacylglycerol, free cholesterol, free fatty acids and phospholipids, as well as the fatty acid patterns of total lipids, were measured in liver homogenates of female and male rats (Wistar SPF, strain Hannover) aged 37-1213 days. 2. The same parameters were measured in the apex of the heart in female and male rats aged 331-1213 days. 3. All parameters were monitored every 49th day. Five female and five male animals were used in each experiment. 4. The lipid fractions in liver showed a positive linear regression vs age, whereas all lipids in rat heart showed a negative regression vs age in both sexes. 5. The significance of regression vs age of fatty acids was much less than that in the lipid fractions of liver and heart of these animals.     

Purification and characterization of a cytosolic transglutaminase from a cultured human tumour-cell line.

The cytosolic glutathione transferases (GSTs) with basic pI values have been studied in mouse liver after treatment with 2,3-t-butylhydroxyanisole (BHA), cafestol palmitate (CAF), phenobarbital (PB), 3-methylcholanthrene (3-MC) and trans-stilbene oxide (t-SBO). The cytosolic GST activity was induced by all compounds except for 3-MC. Three forms of GST were isolated by means of affinity chromatography and f.p.l.c. The examination of protein profiles and enzymic activities with specific substrates showed that the three GSTs correspond to those found in control animals, i.e. GSTs MI, MII and MIII. The class Mu GST MIII accounted for the major effect of induction, whereas the class Alpha GST MI and the class Pi GST MII were unchanged or somewhat down-regulated. The greatest induction was obtained with BHA, PB and CAF. The activities of other glutathione-dependent enzymes were also studied. An increase in glutathione reductase and thioltransferase activities was observed after BHA, PB or CAF treatment; glyoxalase I and Se-dependent glutathione peroxidase were depressed in comparison with the control group in all cases studied.     

Decreased activity and impaired induction of nitric oxide synthase by lipopolysaccharides in streptozotocin-induced diabetic rats

The effect of diabetes was determined on nitric oxide synthase (NOS) activity in rat heart and liver. The diabetes was induced by streptozotocin (STZ) and NOS activity was determined after 1 or 12 weeks post-STZ injection. In both tissues, the majority of NOS activity was associated with endothelial constitutive calcium-sensitive NOS (ecNOS) isoform and found in the particulate (100,000xg pellet) fraction in young rats. The diabetes as well as age reduced this activity significantly in heart, whereas only the age caused a decrease in ecNOS activity in liver tissue. Lipopolysaccharides (LPS) induced calcium-insensitive iNOS activity in both young and old rats. The induction was significantly higher (up to 10-fold) in liver as compared to heart. Although the maximum induction of iNOS in young rats was almost similar in diabetic tissues as compared to control animals, there was a lag period for induction of iNOS in diabetic tissues. In old diabetic rats, the induction by LPS was almost completely abolished. These results suggest that diabetes causes either no change or a decrease in ecNOS activity and impairment in the induction of iNOS by LPS in rat heart and liver.     

Modulatory influence of Adhatoda vesica (Justicia adhatoda) leaf extract on the enzymes of xenobiotic metabolism,antioxidant status and lipid peroxidation in mice

The effect of two different doses (50 and 100 mg/kg body wt/day for 14 days) of 80% ethanolic extract of the leaves of Adhatoda vesica were examined on drug metabolizing phase I and phase II enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase and lipid peroxidation in the liver of 8 weeks old Swiss albino mice. The modulatory effect of the extract was also examined on extra-hepatic organs viz. lung, kidney and forestomach for the activities of glutathione S-transferase, DT-diaphorase, superoxide dismutase and catalase. Significant increase in the activities of acid soluble sulfhydryl (-SH) content, cytochrome P450, NADPH-cytochrome P450 reductase, cytochrome b₅, NADH-cytochrome b₅ reductase, glutathione S-transferase (GST), DT-diaphorase (DTD), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were observed in the liver at both dose levels of treatments. Adhatoda vesica acted as bifunctional inducer since it induced both phase I and phase II enzyme systems. Both the treated groups showed significant decrease in malondialdehyde (MDA) formation in liver, suggesting its role in protection against prooxidant induced membrane damage. The cytosolic protein was significantly inhibited at both the dose levels of treatment indicating the possibility of its involvement in the inhibition of protein synthesis. BHA has significantly induced the activities of GR and GSH in the present study. The extract was effective in inducing GST and DTD in lung and forestomach, and SOD and CAT in kidney. Thus, besides liver, other organs viz., lung, kidney and forestomach were also stimulated by Adhatoda, to increase the potential of the machinery associated with the detoxification of xenobiotic compounds. But, liver and lung showed a more consistent induction. Since the study of induction of the phase I and phase II enzymes is considered to be a reliable marker for evaluating the chemopreventive efficacy of a particular compound, these findings are suggestive of the possible chemopreventive role played by Adhatoda leaf extract.     

Polychlorinated biphenyls-induced lipid peroxidation as measured by thiobarbituric acid-reactive substances in liver subcellular fractions of rats

Rats were given a 0.05% polychlorinated biphenyls (PCB) diet supplemented with adequate nutrients for 10 days and not only PCB-induced lipid peroxidation as measured by thiobarbituric acid (TBA)-reactive substances but also variations of lipid peroxides scavengers in liver and its subcellular fractions (nuclei and cell debris, mitochondrial, microsomal and cytosolic fractions) were investigated. The lipid peroxidation in liver and subcellular fractions in the PCB-treated group increased significantly except in the nuclei and cell debris fraction. The increase in lipid peroxidation in the microsomal fraction appeared to be associated in part with the decrease in vitamin E (alpha-tocopherol) content and induction of drug-metabolizing enzymes. In the cytosolic fraction, the total lipid content increased, glutathione peroxidase (GSHPx) activity decreased and the quantity of free radical-reactive substances suppressing lipid peroxidation was low as measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) value. From these results, the increase in lipid peroxidation in the cytosolic fraction in the PCB-treated group was ascribed to the abundance and availability of oxidizable substrate attended with fatty liver, to the decline in GSHPx activity, and to the insufficiency in antioxygenic activity as observed by the decrease in the DPPH value.     

Lysosomal beta-glucosidase of rat liver

Studies were undertaken to characterize the beta-glucosidase activity in freshly homogenized liver from Sprague-Dawley rats. About 95% of the total beta-glucosidase activity was associated with the particulate fraction, whereas only about 3-7% was found in the cytosol. Storage of fresh liver at room temperature for several hours or repeated freezing and thawing of fresh rat liver prior to homogenization, solubilized 20-30% of the total hepatic beta-glucosidase activity. An additional 30% could be solubilized by extracting the particulate sediments with water or Triton X-100. The enzymatic activity in both the particulate and solubilized fractions optimally hydrolyzed 4-methylumbelliferyl-beta-D-glucoside as well as the glycolipid substrate, glucosylceramide, at an acidic pH. The rates of hydrolysis of either substrate by all subcellular fractions were stimulated by addition of sodium taurocholate or phosphatidylserine. The particulate, cytosolic and solubilized enzymes bound to concanavalin A, were inhibited by conduritol B epoxide and migrated more electronegatively on cellulose acetate than the cytosolic acid beta-glucosidase from human liver or spleen. These data indicated that the liver of Sprague-Dawley rats contained primarily the lysosomal acid beta-glucosidase ('glucocerebrosidase') and little, if any, 'nonspecific' beta-glucosidase. This, and the fact that about 60% of the rat hepatic beta-glucosidase could be solubilized by autolysis, freezing and rethawing or extraction with water, contrasts with the beta-glucosidases in human liver since about 80% of the total beta-glucosidase activity is cytosolic and does not hydrolyze glucosylceramide.(ABSTRACT TRUNCATED AT 250 WORDS)