Liver Cytosolic Enzyme Research Articles

Novel insights into conjugation of antitumor-active unsymmetrical bisacridine C-2028 with glutathione: Characteristics of non-enzymatic and glutathione S-transferase-mediated reactions

Unsymmetrical bisacridines (UAs) are a novel potent class of antitumor-active therapeutics. A significant route of phase II drug metabolism is conjugation with glutathione (GSH), which can be non-enzymatic and/or catalyzed by GSH-dependent enzymes. The aim of this work was to investigate the GSH-mediated metabolic pathway of a representative UA, C-2028. GSH-supplemented incubations of C-2028 with rat, but not with human, liver cytosol led to the formation of a single GSH-related metabolite. Interestingly, it was also revealed with rat liver microsomes. Its formation was NADPH-independent and was not inhibited by co-incubation with the cytochrome P450 (CYP450) inhibitor 1-aminobenzotriazole. Therefore, the direct conjugation pathway occurred without the prior CYP450-catalyzed bioactivation of the substrate. In turn, incubations of C-2028 and GSH with human recombinant glutathione S-transferase (GST) P1-1 or with heat-/ethacrynic acid-inactivated liver cytosolic enzymes resulted in the presence or lack of GSH conjugated form, respectively. These findings proved the necessary participation of GST in the initial activation of the GSH thiol group to enable a nucleophilic attack on the substrate molecule. Another C-2028-GSH S-conjugate was also formed during non-enzymatic reaction. Both GSH S-conjugates were characterized by combined liquid chromatography/tandem mass spectrometry. Mechanisms for their formation were proposed. The ability of C-2028 to GST-mediated and/or direct GSH conjugation is suspected to be clinically important. This may affect the patient's drug clearance due to GST activity, loss of GSH, or the interactions with GSH-conjugated drugs. Moreover, GST-mediated depletion of cellular GSH may increase tumor cell exposure to reactive products of UA metabolic transformations.

Therapeutic potential of xanthones from Swertia chirata in breast cancer cells.

Background & objectives:Medicinal plants like Swertia chirata are rich sources of different xanthones. This study was aimed to assess the cytotoxic potential of four most abundant xanthones present in S. chirata both in vivo and in vitro in Ehrlich ascites carcinoma (EAC), a mouse transplantable breast carcinoma cell line and two human breast carcinoma cell lines (MCF-7 and MDA-MB-231).Methods:Four xanthones derived from S. chirata namely 1-hydroxy-3,7,8-trimethoxyxanthone (XA), 1,8-dihydroxy-3,5-dimethoxyxanthone (XB), 1-hydroxy-3,5,8-trimethoxyxanthone (XC) and 1,5,8-trihydroxy-3-methoxyxanthone (XD) were used for determination of sub-lethal dose on the cell lines EAC, MCF-7, MDA-MB-231 and verified toxicity of sub-lethal dose on normal murine fibroblast cells. Cytotoxicity was measured in vivo and survivability of mice was plotted accordingly. Therapeutic efficacy of XD was evaluated both in vivo and in vitro by determination of lipid peroxidation (LPO), reactive oxygen species (ROS) generation and by quantitating the enzyme status (GSH, catalase, superoxide dismutase) in treated and untreated samples. DNA damage was evaluated using comet and DNA fragmentation assays. Furthermore, apoptotic effect was analyzed by flow cytometry and validated by TUNEL assay and Western blotting.Results:Among all the xanthones tested XD showed IC50 at the lowest dose, and normal cells were unaffected at this dose. Survivability of mice increased significantly when treated with XD compared to other xanthones and cisplatin. Significantly increased ROS and LPO were found in cancer cells as a result of XD treatment which was unaltered in normal cell line. XD induced DNA damage and apoptosis in cancer cell lines.Interpretation & conclusions:Our experimental data indicate that XD may potentially act as a chemotherapeutic agent by enhancing ROS in breast cancer cells thereby leading to apoptosis.

Bromo-dragonfly, a psychoactive benzodifuran, is resistant to hepatic metabolism and potently inhibits monoamine oxidase A

Bromo-dragonfly is a benzodifuran derivative known as one of the most potent 5-HT2A-receptor agonists within this chemical class, with long-lasting effects of up to 2–3 days. In addition to hallucinogenic effects, the drug is a potent vasoconstrictor, resulting in severe adverse effects, such as necrosis of the limbs. In some cases, intoxication has had fatal outcomes. Little is known about the metabolism of bromo-dragonfly. The aims of this study were to investigate the pharmacokinetics of bromo-dragonfly, determine the plasma protein binding, examine the human hepatic metabolism in vitro, and compare with those of its close analogue, 2C-B-fly. Additionally, we assayed the inhibition potency of both compounds on the monoamine oxidase (MAO) A- and B-mediated oxidative deamination of serotonin (5-HT) and dopamine, respectively. Liquid chromatography high-resolution mass spectrometry was used for metabolism studies in pooled human liver microsomes (HLM), pooled human liver cytosol (HLC) and recombinant enzymes. Inhibition studies of the deamination of 5-HT and dopamine were carried out using LC–MS/MS. Bromo-dragonfly was not metabolised in the tested in vitro systems. On the other hand, 2C-B-fly was metabolised in HLM by CYP2D6 and in HLC to some extent, with the main biotransformations being monohydroxylation and N-acetylation. Furthermore, MAO-A metabolised 2C-B-fly, producing the aldehyde metabolite, which was trapped in vitro with methoxyamine. Inhibition experiments revealed that bromo-dragonfly is a competitive inhibitor of MAO-A with a Ki of 0.352 μM. The IC50 value for bromo-dragonfly indicated that the inhibition of MAO-A may be clinically relevant. However, more data are needed to estimate its impact on the increase of 5-HT in vivo.

New glutathione conjugate of pyrrolizidine alkaloids produced by human cytosolic enzyme-dependent reactions in vitro.

The toxic metabolites of pyrrolizidine alkaloids (PAs) are initially formed by cytochrome P450-mediated oxidation reactions and primarily eliminated as glutathione (GSH) conjugates. Although the reaction between the reactive metabolites and GSH can occur spontaneously, the role of the cytosolic enzymes in the process has not been studied. The toxic metabolites of selected PAs (retrorsine, monocrotaline, senecionine, lasiocarpine, heliotrine or senkirkine) were generated by incubating them in 100mM phosphate buffer (pH7.4) containing liver microsomes of human, pig, rat or sheep, NADPH and reduced GSH in the absence or presence of human, pig, rat or sheep liver cytosolic fraction. The supernatants were analyzed using liquid chromatography connected to Finnigan LTQ ion-trap, Agilent QTOF or Thermo Scientific Q Exactive Focus quadrupole-orbitrap mass spectrometers. Retrorsine, senecionine and lasiocarpine yielded three GSH conjugates producing [M-H]- ions at m/z 439 (7-GSH-DHP (CHO)), m/z 441 (7-GSH-DHP (OH)) and m/z 730 (7,9-diGSH-DHP) in the presence of human liver cytosolic fraction. 7-GSH-DHP (CHO) was a novel metabolite. Monocrotaline, heliotrine and senkirkine did not produce this novel 7-GSH-DHP (CHO) conjugate. 7-GSH-DHP (CHO) disappeared when incubated with hydroxylamine, and a new oxime derivative was formed. This metabolite was formed only by the human liver cytosolic enzymes but not in the presence of rat or sheep liver cytosolic fractions under otherwise identical reaction conditions. 7-GSH-DHP (CHO) has not been reported before, and thus it was considered as a novel metabolite of PAs. This may clarify the mechanisms involved in PA detoxification and widely observed but less understood species differences in response to PA exposure.

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes

The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes.DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CLint) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11.DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes.Apparent CLint values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CLint values for total metabolism (CYP and CES enzymes) per gram of adult human liver.Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

Ameliorative effect of Ficus dalhousiae Miq. (Moraceae) methanolic leaf extract on carbon tetrachloride induced hepatic and renal toxicity

<p>Developing traditional medicine in the field of hepatology and nephrology research is the key to pharmacology. This study demonstrates the mechanism of hepatoprotective and renal protective activity of <em>Ficus dalhousiae</em> Miq. (Moraceae) methanolic leaf extract on carbon tetrachloride induced hepatic and renal toxicity. Shade dried powder was subjected to shoxlet extraction with methanol and assessed for hepatoprotective and renal protective activities. Hepatotoxicity and renal toxicity were induced in rats by single oral dose of CCl<sub>4</sub> diluted with olive oil (1:1 v/v; ml/kg body weight) after pretreatment of methanolic extract for seven days. Sixteen hrs after CCl<sub>4</sub> administration, rats were sacrificed and biochemical markers like Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) estimated followed by the measurement of liver and kidney cytosolic antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and reactive oxygen species (ROS). The data were analysed by one way analysis of variance (ANOVA). The extract at the doses of 150 and mg/kg b.w. significantly reduces elevated levels of ALT, AST, ALP and LDH. The extracts also showed significant increase in the reduced level of SOD, CAT and GSH. The ROS activity also found down regulated. The activity of methanolic extracts were comparable with the standard Silymarin. These findings not only showed potential hepatoprotective and renal protective activities of <em>Ficus dalhousiae</em> but also manifested by restoring antioxidant enzymes. With this pilot study we can justify the medicinal importance of this plant.</p>

Human Tissue Distribution of Carbonyl Reductase 1 Using Proteomic Approach With Liquid Chromatography-Tandem Mass Spectrometry

Human tissue distribution of carbonyl reductase 1 (CBR1) is quite controversial in the literature. To understand the differences, CBR1 protein abundance in human intestine, liver, and kidney has been determined using a proteomic approach with liquid chromatography-tandem mass spectrometry. The results show that CBR1 distribution in the 3 tissues is relatively similar, within 2- to 3-folds of each other. Intestine has the highest CBR1 enzyme level (106 pmol/mg protein) followed by liver (76 pmol/mg protein) and kidney (39 pmol/mg protein). The high abundance of CBR1 in the intestine and kidney suggests the critical role of this enzyme in gut first-pass metabolism and extra-hepatic clearance. CBR1 is also detected, for the first time, in the microsomal fractions with 5- to 17-fold lower amounts than cytosols of the corresponding tissues. The average CBR1 protein amount is 14 pmol/mg protein in human hepatocytes. Individual variability of CBR1 is about 4-fold based on 13 lots of cryopreserved human hepatocytes. The relative expression factor between human liver cytosol and human recombinant CBR1 enzyme (hr-CBR1) is 0.0042. The relative activity factor between human liver cytosol and hr-CBR1 is 0.040, suggesting that significant amount of CBR1 protein in the hr-CBR1 has no catalytic activity.

Identification of a suitable and selective inhibitor towards aldehyde oxidase catalyzed reactions

1. Aldehyde oxidase (AO) is a liver cytosolic molybdoflavoprotein enzyme whose importance in drug metabolism is gaining in the recent. The objective of this work is to find a potent and selective inhibitor for AO activity using phthalazine oxidation as a marker reaction.2. Among organic solvents tested, it was identified that methanol was not a suitable choice for AO activity even at concentrations less than 0.2% v/v. Acetonitrile and DMSO did not show any effect till 0.5% v/v but thereafter activites tend to decrease.3. For selectivity, 23 compounds were selected and evaluated for their effects on AO and nine CYP450 enzymes. Among the tested compounds chlorpromazine, estradiol, hydralazine, quetiapine and raloxifene were selected based on their potency of inhibition towards AO activity.4. Raloxifene was found to be a non-specific inhibitor of all major tested CYP450 enzymes and was excluded as a selective inhibitor for AO. Quetiapine also showed a degree of inhibition towards the major CYP450 tested. Hydralazine used as a specific inhibitor during the past for AO activity demonstrated a stimulation of AO activity at high and low concentrations respectively and the inhibition noted to be time dependent while inhibiting other enzymes like monoamine oxidase.5. Estradiol showed no inhibition towards the tested CYP450 enzymes and thus proved to be a selective and specific inhibitor for AO activity with an uncompetitive mode of inhibition.

Antioxidant and Hepatoprotective Activities of Ethanolic Extracts of Leaves of Premna esculenta Roxb. against Carbon Tetrachloride-Induced Liver Damage in Rats

Premna esculenta Roxb. (family Verbenaceae) is a shrub used by the ethnic people of Chittagong Hill Tracts of Bangladesh for the treatment of hepatocellular jaundice. The present study was done to evaluate the hepatoprotective and the in vivo antioxidant activity of ethanolic extracts of leaves of the plant in carbon tetrachloride-induced liver damage in rats. Hepatotoxicity was induced in rats by i.p. injection of CCl4 diluted with olive oil (1:1 v/v; 1 mL/kg body weight) on alternate days for 7 days. After 7 days of pretreatment of test extracts, the biochemical markers such as Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Alkaline Phosphatase (ALP), total protein, and albumin were estimated followed by the measurement of liver cytosolic antioxidant enzymes such as superoxide dismutase, catalase, and peroxidase. The data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett's t-test. The extract both at the doses of 200 and 400 mg/kg p.o. significantly (P < 0.001) reduced the elevated levels of SGPT, SGOT, ALP and increased the reduced levels of total protein and albumin compared to the CCl4-treated animals. The extracts also showed a significant (P < 0.001) increase in the reduced levels of superoxide dismutase (SOD), catalase, and peroxidase. The effects of the extracts on these parameters were comparable with those of the standard, silymarin. The findings of the study indicate that the leaf extract of P. esculenta showed a potential hepatoprotective activity and the protective action might have manifested by restoring the hepatic SOD, catalase, and peroxidase levels. The results justify the traditional use of this plant in liver disorders.

Screening for reactive metabolites using electro-optical arrays featuring human liver cytosol and microsomal enzyme sources and DNA.

We demonstrate for the first time the combination of human liver cytosol and microsomal enzyme sources into an electro-optical array to screen for reactive metabolites produced in multi-enzyme metabolic processes.

Improvement of Diabetic Complication by Hydrangea Dulcis Folium in Streptozotocin-Induced Diabetic Rats

The improvement of diabetic complications such as lipid lowering and anti-oxidative potential of Hydrangea Dulcis Folium (HDF) was studied in streptozotocin-induced diabetic rats. Male Sprague-Dawley rats were divided into 3 groups after induction of streptozotocin (STZ)-diabetes: normal control; diabetic control; diabetic-HDF supplement (hot water extract 40 g/kg diet); and fed experimental diets for 3 weeks. Serum glucose and insulin concentrations, serum lipid profile, intraperitoneal glucose tolerance test, and liver cytosolic antioxidant enzyme levels were measured. The HDF supplement significantly decreased serum glucose concentration, increased insulin level, and improved glucose homeostasis in diabetic control rats. The total cholesterol and triglyceride concentrations in the serum and liver were markedly reduced by HDF treatment in STZ-diabetic rats. Moreover, low density lipoprotein (LDL)-, VLDL-, and high density lipoprotein (HDL)-cholesterol levels were ameliorated in HDF supplemented diabetic rats. Decreased fecal excretions of cholesterol, triglyceride, and bile acid in diabetic rats were significantly increased by HDF consumption. HDF supplement reversed the effects of the oxidative stress system of liver in diabetic rats. Lipid peroxidation of diabetic rats, assessed by thiobarbituric acid reactive substance (TBARS), as well as superoxide dismutase (SOD) activities were significantly increased, and glutathione contents were decreased in diabetic rats. HDF supplement reverted these parameters to near normal value. Our data suggest that HDF supplement could be used to improve the glucose and lipid metabolism as well as to reduce the imbalance between the generation of reactive oxygen species (ROS) and the scavenging enzyme activity in preventing diabetic complications.

Differential Enzymatic Reductions Governing the Differential Hypoxia-Selective Cytotoxicities of Phenazine 5,10-Dioxides

Some derivatives of phenazine 5,10-dioxide are selectively toxic to hypoxic cells commonly found in solid tumors. Previous studies of the phenazine 5,10-dioxide mechanism of action indicated that a bioreduction process could be involved in its selective toxicities, maybe as result of its potential H(*)-releasing capability in hypoxia. The major unresolved aspect of the mechanism of phenazine 5,10-dioxides is the identity of the reductase(s) in the cell responsible for activating the drug to its toxic form and metabolites. We have studied the metabolism in both hypoxia and oxia of some selected 2-amino and 2-hydroxyphenazine 5,10-dioxides, 1- 5, using rat liver microsomal and cytosol fractions. Differential hypoxic/oxic metabolism was found to be correlated to a compound's cytotoxic selectivity but, in general, without metabolic differences between liver microsomal or cytosolic enzymes. Dicoumarol and ketoconazole were found to inhibit the hypoxic metabolism of the most selective phenazine 5,10-dioxide, 1, inferring a role for DT-diaphorase and cytochrome P450. The least hypoxic selective agents, 4 and 5, possess different hypoxia-metabolic profiles as compared to derivative 1, explaining the differential cytotoxic biological behavior. The nonselective derivative, 2, suffered bioreduction in both conditions and, according to the inhibition studies with dicoumarol and ketoconazole, involves both DT-diaphorase and cytochrome P450. The nontoxic derivative, 3, showed poor bioreductive behavior.

Development of Assay Methods for Endogenous Inorganic Sulfur Compounds and Sulfurtransferases and Evaluation of the Physiological Functions of Bound Sulfur

Inorganic sulfur compounds, such as S(2-), SO(3)(2-) and S(2)O(3)(2-), are produced from sulfur- containing amino acids as intermediary metabolites in mammalian tissues through complex pathways and are ultimately incorporated into sulfate. Reduced sulfur is also produced via the desulfuration of cysteine by several sulfurtransferases present in mammalian tissues; these enzymes include gamma-cystathionase (gamma-CST), and 3-mercaptopyruvate sulfurtransferase (3-MST). This reduced sulfur is then incorporated into pools of active reduced sulfur (sulfane sulfur; polysulfides, polythionates, thiosulfate, thiosulfonates and elemental sulfur) that are involved in the detoxication of cyanide and in the biosynthesis of iron-sulfur cluster. Sulfane sulfur is labile and is reduced to H(2)S by reducing agents. The physiological function of these sulfur species is less clear. We have found that a reduced sulfur species is commonly present in mammalian sera and tissues as a high molecular weight material and as both a high and a low molecular weight material, respectively; we designated this sulfur species as "bound sulfur." Bound sulfur can be easily liberated as sulfide by reduction with DTT. This review describes sensitive and specific assay method for determining the presence of inorganic sulfur compounds as well as bound sulfur and related sulfurtransferases in biological samples. The physiological functions of bound sulfur in rat tissues were also evaluated using these assay methods. Bound sulfur was found to be located primarily in the rat liver cytosolic fraction in the form of high molecular weight components. The capacity of bound sulfur production was enriched in the cytosol fraction and depended on gamma-CST. Bound sulfur also affected redox regulation by modifying active thiol residues in some liver cytosol enzymes and effectively inhibited cytochrome P-450-dependent lipid peroxidation induced by CCl(4) and t-BuOOH.

Metabolism of the CysteineS-Conjugate of Busulfan Involves a β-Lyase Reaction

The present work documents the first example of an enzyme-catalyzed beta-elimination of a thioether from a sulfonium cysteine S-conjugate. beta-(S-Tetrahydrothiophenium)-L-alanine (THT-A) is the cysteine S-conjugate of busulfan. THT-A slowly undergoes a nonenzymatic beta-elimination reaction at pH 7.4 and 37 degrees C to yield tetrahydrothiophene, pyruvate, and ammonia. This reaction is accelerated by 1) rat liver, kidney, and brain homogenates, 2) isolated rat liver mitochondria, and 3) pyridoxal 5'-phosphate (PLP). A PLP-dependent enzyme in rat liver cytosol that catalyzes a beta-lyase reaction with THT-A was identified as cystathionine gamma-lyase. This unusual drug metabolism pathway represents an alternate route for intermediates in the mercapturate pathway.

MAK-4 and -5 supplemented diet inhibits liver carcinogenesis in mice

BackgroundMaharishi Amrit Kalash (MAK) is an herbal formulation composed of two herbal mixtures, MAK-4 and MAK-5. These preparations are part of a natural health care system from India, known as Maharishi Ayur-Veda. MAK-4 and MAK-5 are each composed of different herbs and are said to have maximum benefit when used in combination. This investigation evaluated the cancer inhibiting effects of MAK-4 and MAK-5, in vitro and in vivo.MethodsIn vitro assays: Aqueous extracts of MAK-4 and MAK-5 were tested for effects on ras induced cell transformation in the Rat 6 cell line assessed by focus formation assay. In vivo assays: Urethane-treated mice were put on a standard pellet diet or a diet supplemented with MAK-4, MAK-5 or both. At 36 weeks, livers were examined for tumors, sera for oxygen radical absorbance capacity (ORAC), and liver homogenates for enzyme activities of glutathione peroxidase (GPX), glutathione-S-transferase (GST), and NAD(P)H: quinone reductase (QR). Liver fragments of MAK-fed mice were analyzed for connexin (cx) protein expression.ResultsMAK-5 and a combination of MAK-5 plus MAK-4, inhibited ras-induced cell transformation. In MAK-4, MAK-5 and MAK4+5-treated mice we observed a 35%, 27% and 46% reduction in the development of urethane-induced liver nodules respectively. MAK-4 and MAK4+5-treated mice had a significantly higher ORAC value (P < 0.05) compared to controls (200.2 ± 33.7 and 191.6 ± 32.2 vs. 152.2 ± 15.7 ORAC units, respectively). The urethane-treated MAK-4, MAK-5 and MAK4+5-fed mice had significantly higher activities of liver cytosolic enzymes compared to the urethane-treated controls and to untreated mice: GPX(0.23 ± 0.08, 0.21 ± 0.05, 0.25 ± 0.04, 0.20 ± 0.05, 0.21 ± 0.03 U/mg protein, respectively), GST (2.0 ± 0.4, 2.0 ± 0.6, 2.1 ± 0.3, 1.7 ± 0.2, 1.7 ± 0.2 U/mg protein, respectively) and QR (0.13 ± 0.02, 0.12 ± 0.06, 0.15 ± 0.03, 0.1 ± 0.04, 0.11 ± 0.03 U/mg protein, respectively). Livers of MAK-treated mice showed a time-dependent increased expression of cx32.ConclusionOur results show that a MAK-supplemented diet inhibits liver carcinogenesis in urethane-treated mice. The prevention of excessive oxidative damage and the up-regulation of connexin expression are two of the possible effects of these products.

Antioxidative and anti-diabetic effects of amaranth (Amaranthus esculantus) in streptozotocin-induced diabetic rats

The anti-diabetic and antioxidative effect of amaranth grain (AG) and its oil fraction (AO) was studied in streptozotocin-induced diabetic rats. Male Sprague-Dawley rats were divided into four groups after induction of STZ-diabetes: normal control; diabetic control; diabetic-AG supplement (500 g kg(-1) diet); diabetic-AO supplement (100 g kg(-1) diet) and fed experimental diets for 3 weeks. Serum glucose, insulin, activities of serum marker enzymes of liver function and liver cytosolic antioxidant enzymes were measured. The AG and AO supplement significantly decreased the serum glucose and increased serum insulin level in diabetic rats. Serum concentration of liver function marker enzymes, GOT and GPT, were also normalized by AG and AO treatment in diabetic rats. Liver cytosolic SOD and GSH-reductase activities were significantly increased, and catalase, peroxidase and GSH-Px activities were decreased in diabetic rats. AG and AO supplement reverted the antioxidant enzyme activities to near normal values. Hepatic lipid peroxide product was significantly higher, and GSH content was decreased in diabetic rats. However, AG and AO supplement normalized these values. Our data suggest that AG and AO supplement, as an antioxidant therapy, may be beneficial for correcting hyperglycaemia and preventing diabetic complications.

Acute cadmium intoxication induces alpha-class glutathione S-transferase protein synthesis and enzyme activity in rat liver

Acute cadmium intoxication affects glutathione S-transferase (GST) in rat liver. It has been found that 24 h after i.p. cadmium administration to rats, at a dose of 2.5 mg CdCl 2 kg −1 body weight, the activity of this enzyme in liver cytosol increased by 40%. A less stimulatory effect persisted till 48 h and thereafter the enzyme activity normalized. Since, GST isoenzymes belong to different classes in mammalian tissues, we used quantitative immunoassays to verify which family of GST isoenzymes is influenced by this intoxication. Only alpha-class glutathione S-transferase (α-GST) proteins were detected in rat liver cytosol and their level increased by about 25%, 24 h after cadmium treatment. No pi-GST isoforms were found in liver cytosol from either normal or cadmium-treated rats. Co-administration of actinomycin D with cadmium normalized both the protein level and the activity of α-GST, suggesting that some effect occurs on enzyme transcription of these isoenzymes by this metal. On the other hand, it seems unlikely that the stimulatory effect is due to the high level of peroxides caused by lipid peroxidation, since Vitamin E administration strongly reduced the TBARS level, but did not cause any GST activity decrease.

Epoxidation of the methamphetamine pyrolysis product, trans-phenylpropene, to trans-phenylpropylene oxide by CYP enzymes and stereoselective glutathione adduct formation☆

Pyrolytic products of smoked methamphetamine hydrochloride are well established. Among the various degradation products formed, trans-phenylpropene (trans-beta-methylstyrene) is structurally similar to styrene analogues known to be bioactivated by CYP enzymes. In human liver microsomes, trans-phenylpropene was converted to the epoxide trans-phenylpropylene oxide (trans-2-methyl-3-phenyloxirane) and cinnamyl alcohol. Incubation of trans-phenylpropene with microsomes in the presence of enzyme-specific P450 enzyme inhibitors indicated the involvement of CYP2E1, CYP1A2, and CYP3A4 enzymes. Both (R,R)-phenylpropylene oxide and (S,S)-phenylpropylene oxide were formed in human liver microsomal preparations. Enantiomers of trans-phenylpropylene oxide were stereoselectively and regioselectively conjugated in a Phase II drug metabolism reaction catalyzed by human liver cytosolic enzymes consisting of conjugation with glutathione. The structure of the phenylpropylene oxide-glutathione adduct is consistent with nucleophilic ring-opening by attack at the benzylic carbon. Exposure of cultured C6 glial cells to (S,S)-phenylpropylene oxide produced a cytotoxic response in a concentration-dependent manner based on cell degeneration and death.

Rat liver glutathione S-transferase activity stimulation following acute cadmium or manganese intoxication.

The effect of cadmium or manganese administration on rat liver glutathione S-transferase (GST) has been investigated. The activity of this enzyme in liver cytosol, where almost all the cellular activity is present, had increased by more than 36% 24 h after a single i.p. injection of CdCl(2) (2.5 mg kg(-1) b.w.) or MnCl(2) (2.0 mg kg(-1) b.w.). After shorter and longer time intervals, a lower enzyme activity stimulation was observed in both cases. When liver cytosol was incubated for 10 min with 75 microM CdCl(2) or 40 microM MnCl(2), no effect was observed on enzyme activity. The increase in GST following cadmium or manganese administration was blocked by prior administration of actinomycin D, indicative of a possible transcription-dependent response. The liver soluble GST from both control and metal-treated rats was not at all affected by Vitamin E, in the range of 20-300 microM. By contrast, hematin was seen to be a competitive inhibitor of this liver enzyme from both types of rats by using CDNB as substrate and the K(i) value was equal to 0.22 microM. The possibility that under the conditions used class alpha GST isoenzymes are affected by cadmium or manganese is discussed.

Crystallization and preliminary X-ray diffraction analysis of recombinant hydrolase domain of 10-formyltetrahydrofolate dehydrogenase.

10-Formyltetrahydrofolate dehydrogenase (FDH) is an abundant enzyme in liver cytosol. It is important for the regulation of 10-formyltetrahydrofolate/tetrahydrofolate pools, for de novo purine biosynthesis and for the removal of formate in the form of CO(2). The enzyme is a natural fusion of two unrelated genes and consists of two functional catalytic domains. Here, the crystallization of the N-terminal domain of FDH is reported. This domain binds folate and functions as a 10-formyltetrahydrofolate hydrolase. The crystals grow as either spear-shaped needles or large plates, with the largest crystals reaching dimensions of 1.2 x 0.2 x 0.05 mm. Diffraction analysis revealed the space group to be P2(1)2(1)2, with unit-cell parameters a = 100.00, b = 64.63, c = 64.59 A. Based on the estimated solvent content, there is one 34 kDa molecule in the asymmetric unit. A native data set extending to 2.3 A resolution has been collected with good merging statistics.