Regulation of microsomal and cytosolic glutathione S-transferase activities by S-nitrosylation

There is increasing evidence that S-nitrosylation is a mechanism for the regulation of protein function via the modification of critical sulfhydryl groups. The activity of rat liver microsomal glutathione S-transferase (GST) is increased after treatment with N-ethylmaleimide (NEM), a sulfhydryl alkylating reagent, and is also increased under conditions of oxidative stress. In the present study, preincubation of purified rat liver microsomal GST with S-nitrosoglutathione (GSNO) or the nitric oxide (NO) donor, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO), resulted in a 2-fold increase in enzyme activity. This increase in activity was reversed by dithiothreitol. The initial treatment of microsomal GST with either GSNO or DEA/NO was associated with an 85% loss of free sulfhydryl groups. After removal of the nitrosylating agents over a 6-hr period, approximately 50% of the enzyme was still nitrosylated, as determined by redox chemiluminescence. Furthermore, preincubation of either purified enzyme or hepatic microsomes with GSNO or DEA/NO prevented further enzyme activation by NEM, suggesting that NEM and the NO donors interact with a common population of sulfhydryl groups in the enzyme. In contrast, both NEM and NO donors partially inhibited the activity of cytosolic GST isoforms. The inhibitory activity of NEM and NO donors was much more evident when the GST pi isoform was used instead of a mixture of GST isoforms. These data suggest that there may be differential regulation of microsomal and cytosolic GST activities under conditions of nitrosative stress.     

Inactivation of hepatic enzymes by inhalant nitrite—In vivo and in vitro studies

We examined the effects of acute isobutyl nitrite (ISBN) exposure on the activity of several hepatic enzymes. Two strains of adult male mice (Balb/c and C57BL/6) were exposed to 900 ppm ISBN or ambient air for 45 minutes. The enzyme activity of hepatic cytochrome P450 (CYP)-mediated deethylation, glutathione S-transferase (GST), and carboxylesterase (CBE) was monitored through the substrates 3-cyano-7-ethoxycoumarin (CEC), 1-chloro-2,4-dinitrobenzene, and p-nitrophenyl acetate, respectively. Acute ISBN exposure led to a significant reduction in hepatic CYP-mediated CEC deethylation, GST, and CBE activity in Balb/c mice (of 81.5%, 74.7%, and 25.2%, respectively, vs control mice, each at P < .05) when livers were harvested immediately after inhalant exposure. The corresponding decreases in C57BL/6 mice were smaller (with reductions of 21.8%, 18.8%, and 13.3%, respectively, each at P < .05). This enzyme activity, tested in C57BL/6 mice only, returned to control values after a 24-hour period of nonexposure. Follow-up mechanistic investigations using rat liver GST indicated that ISBN-mediated enzyme inactivation was not caused by its metabolites: inorganic nitrite ion (NO2-) or nitric oxide. This inactivation could be prevented, but not reversed, by added glutathione, suggesting irreversible protein oxidation. Using different NO donors as comparative agents, we found that GST inactivation by ISBN was not associated with protein S-nitrosylation or disulfide formation, but with tyrosine nitration. Inhalant nitrite exposure, therefore, led to a significant reduction in hepatic enzyme activity in mice, possibly through tyrosine nitration of hepatic proteins. This effect raises the possibility of drug-drug metabolic interactions from inhalant nitrite abuse. However, determining the applicability of these findings to humans will require further study.     

Tyrosine nitration in blood vessels occurs with increasing nitric oxide concentration.

1. Experiments were designed to explore the effects of nitric oxide (NO) donors on generation of superoxide (O2.-) and peroxynitrite (ONOO-) in rabbit aortic rings. 2. Following inhibition of endogenous superoxide dismutase (SOD), significant basal release of O2.- was revealed (0.9 +/- 0.01 x 10(-12) mol min-1 mg-1 tissue). Generation of O2.- increased in a concentration-dependent manner in response to NADH or NADPH (EC50 = 2.34 +/- 1.18 x 10(-4) and 6.21 +/- 1.79 x 10(-3) M respectively, n = 4). NADH-stimulated O2.- chemiluminescence was reduced by approximately 85% in the presence of exogenous SOD (15 x 10(3) U ml-1). 3. Incubation of aortic rings with S-nitrosoglutathione (GSNO; 1 x 10(-5)-3 x 10(-3) M) or sodium nitroprusside (SNP; 1 x 10(-8)-1 x 10(-3) M), resulted in a concentration-dependent quenching of O2.- chemiluminescence which was proportional to NO release. 4. ONOO- formation was assessed indirectly by determining protein tyrosine nitration in rabbit aorta using a specific antibody against nitrotyrosine. Basally and in the presence of NADH, a single band was detected. Incubation of aortic rings with either GSNO (1 x 10(-3) M) alone or GSNO with NADH resulted in the appearance of additional nitrotyrosine bands. Incubation of serum albumin with GSNO alone did not cause nitrotyrosine formation. In contrast, incubation with 3-morpholinosydonomine (SIN-1; 1 x 10(-3) M, 10 min), resulted in marked nitration of albumin which was reduced by oxyhaemoglobin or SOD. Incubation of albumin with GSNO and pyrogallol, a O2.- generator, also resulted in protein nitration. 5. Addition of exogenous NO results in nitrotyrosine formation in rabbit aortic rings. Nitrotyrosine formation is likely to result from the reaction of exogenous NO and basal endogenous O2.- resulting in the formation of ONOO-. Formation of ONOO- and nitration of tyrosine residues potentially could lead to vascular damage and might represent unexpected adverse effects of long-term nitrate therapy.     

Possible regulation mechanism of microsomal glutathione S-transferase activity in rat liver

After rats were injected with the reduced glutathione (GSH) depletor phorone (diisopropylidene acetone, 250 mg/kg, i.p.), there was a significant increase in microsomal glutathione S-transferase activity in the liver. The maximum activity was observed 24 hr after injection and was about 2-fold that of the control activity. Diethylmaleate (500 mg/kg, i.p.) had the same effect. Twenty-four hours after phorone injection (250 mg/kg, i.p.), the concentrations of GSH and oxidized glutathione (GSSG) in the liver were increased about 2-fold. Under the same conditions, the level of mixed disulfides with microsomal proteins (GSS-protein) was also increased. Further, the activity of microsomal glutathione S-transferases was increased by the in vitro addition of disulfide compounds such as GSSG, cystine and homocystine, and the activity increased by GSSG was reduced to control levels by incubating with the corresponding sulfhydryl compounds such as GSH, cysteine and homocysteine respectively. Thus, microsomal glutathione S-transferase activity appears to be regulated by the formation and/or cleavage of a mixed disulfide bond between the sulfhydryl group present in the enzyme and GSSG. Therefore, the increase of microsomal glutathione S-transferase activity after phorone injection may be due to the formation of a mixed disulfide bond between the sulfhydryl group in the enzyme and GSSG.     

Selective peroxynitrite scavenging activity of 3-methyl-1,2-cyclopentanedione from coffee extract

It has been known that reactive oxygen and nitrogen species such as nitric oxide (NO), superoxide radical (*O2-) and their byproduct peroxynitrite (ONOO-) induce cellular and tissue injury, ultimately resulting in several human diseases. In this study, we examined scavenging effects of 3-methyl-1,2-cyclopentanedione (MCP) from coffee extract on the reactivity of those toxic molecules. MCP significantly inhibited both the oxidation of 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) by reactive oxygen species (ROS) (mainly *O2-) from kidney homogenate (41% at 100 microM) and the generation of fluorescent 4,5-diaminofluorescein (DAF-2) by NO from sodium nitroprusside (IC50 (concn producing 50% inhibition), 63.8 microM). More potently, however, MCP suppressed the oxidation of dihydrorhodamine 123 (DHR 123) to fluorescent rhodamine 123 mediated by authentic ONOO- with an IC50 value of 3.3 microM. The neutralizing effect of the reactivity of ONOO- by MCP was due to electron donation, not nitration of the compound. Additionally, MCP also decreased ONOO- formation of nitrotyrosine adducts of glutathione (GSH) reductase, and consequently protected the enzyme activity of GSH reductase against decreasing by ONOO-, indicating that MCP may prevent ONOO- -induced damage of GSH reductase. Furthermore, MCP only weakly suppressed NO production, which is one of the upstream sources of ONOO- in-vivo, suggesting that NO production may be not a pharmacological target for MCP. Taken together, our results suggest that MCP may be regarded as a selective regulator of ONOO- -mediated diseases via direct scavenging activity of ONOO-.     

Peroxynitrite scavenging mode of alaternin isolated from Cassia tora

Peroxynitrite (ONOO-), formed from the reaction of superoxide (.O2-) and nitric oxide (NO), is a potent oxidant that contributes to the oxidation of various cellular constituents, including lipids, amino acids, sulfhydryls and nucleotides. It can cause cellular injury, such as DNA fragmentation and apoptotic cell death. ONOO- toxicity is also reported to be involved in inflammatory and neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and atherosclerosis. Moreover, the necessity for a strong ONOO- scavenger is important because of the lack of endogenous enzymes that protect against the damage caused by ONOO-. The aim of this study was to evaluate the ability of natural products to scavenge ONOO-. We tested various plant extracts for their ONOO- scavenging activity. Among them, extract from Cassia tora, which is well known as an oriental herb in traditional medicine, showed potent ONOO- scavenging activity. Further analysis identified the phenolic active components, alaternin and nor-rubrofusarin glucose, as potent ONOO- scavengers. Spectrophotometric analysis demonstrated that alaternin and nor-rubrofusarin glucose led to a decrease in the ONOO- -mediated nitration of tyrosine through electron donation. In bovine serum albumin, alaternin, but not nor-rubrofusarin glucose, showed significant inhibition of ONOO- -mediated nitration in a dose-dependent manner. We believe alaternin can be developed as an effective ONOO- scavenger for the prevention of ONOO- -associated diseases.     

Effects of angiotensin II on vascular endothelial cells: formation of receptor-mediated reactive nitrogen species

Angiotensin II (ANG II) participates in many cardiovascular disease states, but the mechanisms involved are not completely defined. Doses of ANG II that do not affect blood pressure significantly can still cause early changes in vascular endothelial performance and cell-specific protein 3-nitrotyrosine formation (protein-3NT, marker of peroxynitrite formation) in vivo. Here, we have tested the hypothesis that ANG II induces endothelial cell peroxynitrite (ONOO-) formation in vitro, and investigated the mechanisms involved. Endothelial cells were incubated with ANG II (1nM-250 microM), and protein nitration was assessed by immunoblotting. ANG II caused concentration-dependent increases in protein-3NT above detectable basal control levels, at concentrations greater than 100nM. This response was inhibited significantly by co-incubation with losartan or diphenyleneiodonium chloride. Endothelial cell lysates incubated with nitrated protein standards demonstrated significant protein-3NT modification activity only in the presence of serum. However, endothelial cell lysates did not modify the free amino acid form of 3NT (free-3NT) in identical experimental conditions, assessed by capillary electrophoresis. Finally, free-3NT was cytotoxic to cultured endothelial cells (fitted LC(50)=98 microM). These data demonstrate that stimulation of angiotensin receptor subtype 1 by ANG II can cause increased endothelial cell protein nitration in vitro in the absence of other cell types or stimuli, at concentrations that are pathophysiologically relevant. Furthermore, endothelial cells selectively modified nitrated protein tyrosine residues only in the presence of a cofactor(s), and did not modify the free modified amino acid. Protein nitration may be a regulated endothelial signaling process, while free-3NT may be toxic to endothelial cells.     

Peroxynitrite scavenging activity of lithospermate B from Salvia miltiorrhiza

Peroxynitrite (ONOO-) is produced by the reaction of superoxide (O2-) with nitric oxide. ONOO- damages proteins through nitration or oxidation. For protection from ONOO- induced protein modifications, ONOO- scavengers should be supplemented. Evidence was obtained that lithospermate B extracted from Salvia miltiorrhiza showed the strongest scavenging activity among its constituents. Its ONOO- scavenging activity is via an electron donation mechanism. A dihydroxyl group and a double bond seem to be essential structure requirements. The data from the experiments further confirmed a protective effect of lithospermate B on bovine serum albumin and low-density lipoprotein against ONOO-. This study demonstrated that lithospermate B with hydroxyl groups and double bonds exerts an anti-nitration effect by scavenging ONOO-.     

Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl-1,4-naphthoquinone)

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) resulted in a dose-dependent depletion of intracellular reduced glutathione (GSH), most of which was oxidized to glutathione disulfide (GSSG). Menadione metabolism was also associated with a dose- and time-dependent inhibition of glutathione reductase, impairing the regeneration of GSH from GSSG produced during menadione-induced oxidative stress. Inhibition of glutathione reductase by pretreatment of hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) greatly potentiated both GSH depletion and GSSG formation during the metabolism of low concentrations of menadione. Concomitant with GSH oxidation, mixed disulfides between glutathione and protein thiols were formed. The amount of mixed disulfides produced and the kinetics of their formation were dependent on both the intracellular GSH/GSSG ratio and the activity of glutathione reductase. The mixed disulfides were mainly recovered in the cytosolic fraction and, to a lesser extent, in the microsomal and mitochondrial fractions. The removal of glutathione from protein mixed disulfides formed in hepatocytes exposed to oxidative stress was dependent on GSH and/or cysteine and appeared to occur predominantly via a thiol-disulfide exchange mechanism. However, incubation of the microsomal fraction from menadione-treated hepatocytes with purified glutathione reductase in the presence of NADPH also resulted in the reduction of a significant portion of the glutathione-protein mixed disulfides present in this fraction. Our results suggest that the formation of glutathione-protein mixed disulfides occurs as a result of increased GSSG formation and inhibition of glutathione reductase activity during menadione metabolism in hepatocytes.     

活性氮对肝脏微粒体谷胱甘肽转移酶1修饰激活的研究进展

活性氮导致肝脏微粒体谷胱甘肽转移酶1(MGST1)修饰激活是近年来研究热点之一,由于其半胱氨酸(Cys49)可感受亲电子剂等而被修饰,担任"感受器"角色,行使对机体的保护作用。以过氧亚硝酸盐和S-亚硝基谷胱甘肽为代表的活性氮,可引起MGST1的酪氨酸(Tyr92)和Cys49发生若干种修饰,包括硝基化、S-亚硝酰化、谷胱甘肽化、巯基氧化等,均可介导酶激活,其中Tyr92的硝基化修饰可望揭示MGST1的"第二个感受器",与机体对硝基化应激的保护机制相关。     

A novel superoxide dismutase-based trap for peroxynitrite used to detect entry of peroxynitrite into erythrocyte ghosts

Peroxynitrite (ONOO-) is a relatively stable oxidant produced by activated macrophages and neutrophils. To detect peroxynitrite, a novel human superoxide dismutase (SOD) trap was developed by substituting a tyrosine near the copper in the active site. The copper can catalyze nitration of this tyrosine by peroxynitrite. The nitrated tyrosine can serve as a reporter for peroxynitrite by measuring the extent of nitration with Western blots developed with a nitrotyrosine antibody. The new SOD mutant differs from bovine SOD whose sole tyrosine is far removed from the active site. Nitration of bovine SOD was second-order with respect to SOD concentration, whereas nitration of the new mutant SODs followed first-order kinetics with respect to peroxynitrite. The tyrosine SODs were used to assess whether peroxynitrite crosses erythrocyte membranes through the band 3 anion exchange protein. Tyrosine-containing SOD entrapped within normal human erythrocyte ghosts became nitrated in proportion to peroxynitrite concentration. The band 3 anion exchange protein inhibitors, phenyl isothiocyanate (PITC) and 4, 4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), inhibited up to 90% of the nitration. The erythrocyte membrane proteins, spectrin, band 3 anion exchange protein, and proteins 4.1 and 4.2, were also nitrated. Nitration of erythrocyte membrane proteins was also inhibited by PITC and DIDS. These data suggest that the band 3 anion exchange protein is the major route for the entry of peroxynitrite into erythrocytes. The ability of peroxynitrite to cross cell membranes can contribute to its toxicity by allowing access to intracellular target molecules.     

N-acetyl-p-benzoquinone imine-induced protein thiol modification in isolated rat hepatocytes.

Incubation of isolated rat hepatocytes with N-acetyl-p-benzoquinone imine (NAPQI) or 3,5-dimethyl-N-acetyl-p-benzoquinone imine (3,5-Me2-NAPQI) resulted in a concentration-dependent decrease in the protein thiol content of the mitochondrial, cytosolic and microsomal fractions. On a concentration basis, 3,5-Me2-NAPQI induced a more marked depletion of protein thiols than did NAPQI. Sodium dodecyl sulphate-polyacrylamide gel electrophoretic separation of the proteins of each fraction showed that different proteins had different susceptibilities to modification of their cysteine residues by the quinone imines. A few protein bands showed a decreased protein thiol content following incubation with non-toxic concentrations of quinone imines, whereas other proteins were affected by higher concentrations. Concentrations of quinone imines that were highly cytotoxic induced a general loss of protein thiols. NAPQI-induced protein thiol depletion occurred within 5 min and remained essentially unchanged for at least 30 min. In contrast, protein thiol depletion induced by 3,5-Me2-NAPQI increased over the 30-min time course of the experiment. Toxic concentrations of 3,5-Me2-NAPQI caused the formation of high molecular mass aggregates in all three subcellular fractions after 30 min of incubation. The observed crosslinking was not due to protein disulfide formation. However, no aggregate formation was observed after exposure of hepatocytes to NAPQI. One of the major target proteins of quinone imine-induced protein thiol depletion was a 17 kDa microsomal protein that was identified as the microsomal glutathione S-transferase. Exposure of hepatocytes and isolated liver microsomes to the quinone imines resulted in an up to four-fold increase in the specific activity of the microsomal glutathione S-transferase. In conclusion, our results are consistent with the suggestion of a critical role of protein thiol depletion in quinone imine-induced cytotoxicity.     

Metabolism of tridiphane (2-(3,5-dichlorophenyl)-2(2,2,2-trichloroethyl)oxirane) by hepatic epoxide hydrolases and glutathione S-transferases in mouse

The transformation of the herbicide tridiphane (Tandem, Dowco 356, 2-(3,5-dichlorophenyl)-2(2,2,2-trichloroethyl)oxirane by the epoxide-metabolizing enzymes, epoxide hydrolases (EH) and glutathione S-transferases (GST), was investigated in mouse liver microsomes and cytosol. The microsomal EH catalyzed the formation of tridiphane diol. The production of this metabolite was prevented by cyclohexene oxide at 1 mM, a known inhibitor of microsomal EH. The structure of the diol was verified by comparison of retention time or Rf of the compound with those of an authentic standard using gas-liquid chromatography or thin-layer chromatography techniques. The diol formed a diester with 1-butane boronic acid or an aldehyde with lead tetraacetate. Mass spectral analysis supported the structural assignment. After optimization of the assay conditions, kinetic constants for the hydration of tridiphane by the microsomal EH were determined (Km = 65 microM and Vmax = 0.9 nmol/min/mg protein). Dietary exposure of mice to the hypolipidemic drug clofibrate at a dose of 0.5% (w/w) for 2 weeks increased by 173% the metabolism of tridiphane to tridiphane diol by the microsomal fraction. No diol could be detected following incubation of tridiphane with the cytosolic EH, even after induction by clofibrate. Tridiphane was also a substrate for GST, but administration of clofibrate did not change the specific activity for the formation of the glutathione conjugate. The herbicide was a rather weak inhibitor of the microsomal EH and the cytosolic GST activities measured with cis-stilbene oxide and trans-stilbene oxide as substrates with I50's of 3.0 x 10(-5) and 1.8 x 10(-4)M, respectively. Tridiphane diol was a poor inhibitor of the enzymes studied, and the glutathione conjugate of tridiphane caused marked inhibition of only the GST activity (I50, 2.0 x 10(-5)M). By contrast the activity of cytosolic EH (trans-stilbene oxide) was relatively insensitive to the addition of tridiphane or of tridiphane metabolites.     

Glutathione S-transferase-mediated chlorothalonil metabolism in liver and gill subcellular fractions of channel catfish

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile) is a broad spectrum fungicide that is a potent acute toxicant to fish. Therefore, the metabolism of chlorothalonil was investigated in liver and gill cytosolic and microsomal fractions from channel catfish (Ictalurus punctatus) using HPLC. All fractions catalyzed the metabolism of chlorothalonil to polar metabolites. Chlorothalonil metabolism by cytosolic fractions was reduced markedly when glutathione (GSH) was omitted from the reaction mixtures. The lack of microsomal metabolism in the presence of either NADPH or an NADPH-regenerating system indicated direct glutathione S-transferase (GST)-catalyzed conjugation with GSH without prior oxidation by cytochrome P450. Cytosolic and microsomal GSTs from both tissues were also active toward 1-chloro-2,4-dinitrobenzene (CDNB), a commonly employed reference substrate. In summary, channel catfish detoxified chlorothalonil in vitro by GST-catalyzed GSH conjugation in the liver and gill. The present report is the first to confirm microsomal GST activity toward CDNB in gill and toward chlorothalonil in liver, and also of gill cytosolic GST activity towards chlorothalonil, in an aquatic species.     

Mutation of tyrosine 190 to alanine eliminates the inactivation of cytochrome P450 2B1 by peroxynitrite

We have previously reported that cytochrome P450 2B1 was inactivated by peroxynitrite and that the decrease in the catalytic activity correlated with an increase in the nitration of tyrosine. Digestion of the peroxynitrite-treated P450 2B1 with Lys C followed by amino acid sequencing of the major nitrotyrosine-containing peptide demonstrated that it spanned residues 160-225. This peptide contains two tyrosine residues at positions 190 and 203. In this study, we mutated Tyr 190 to Ala (Y190A) and Tyr 203 to Ala (Y203A) in wild-type recombinant P450 2B1 (WT) in order to identify the specific residue(s) that is nitrated and to determine whether nitrotyrosine formation is reponsible for the peroxynitrite-mediated inactivation of P450 2B1. All three P450s were expressed in Escherichia coli, purified to homogeneity, and characterized. The catalytic activities for four different substrates of P450 2B1 increased approximately 2-fold for the Y203A mutant, but decreased by about 60% for the Y190A mutant when compared to WT. The addition of peroxynitrite to the P450s resulted in concentration-dependent decreases in the catalytic activities of WT and Y203A, but no loss of the catalytic activities of Y190A. The extent of tyrosine nitration of Y190A by peroxynitrite decreased by approximately 75% as compared with WT or the Y203A protein. Following digestion of the peroxynitrite-modified proteins with Lys C, a major nitrotyrosine-containing peptide was detected from WT and Y203A, but not from Y190A. Collectively, these results indicate that Tyr 190 is the target residue for peroxynitrite-mediated nitration and that nitration of this tyrosine is a responsible for the inactivation of P450 2B1. Modeling studies suggest that Tyr 190 may play a structural role in maintaining the integrity of the protein for maximal activity through hydrogen bonding with Glu 149.     

Mouse strain differences in glutathione S-transferase activity and aflatoxin B1 biotransformation

Previous studies have suggested that mice are resistant to the carcinogenic effects of aflatoxin B1 (AFB1) and that this resistance is largely the result of expression of an isoenzyme of glutathione S-transferase (GST) with high activity toward AFB1-8,9-epoxide. Significant interstrain differences in cytosolic GST activities toward a variety of substrates have been reported in mice. If such differences exist for the conjugation of AFB1-8,9-epoxide, then there may be significant mouse strain differences in susceptibility to AFB1-induced hepatocarcinogenicity. The hepatic microsomal and cytosolic biotransformation of AFB1 was studied in 8 different strains of mice fed a purified diet. GST-mediated conjugation of AFB1-8,9-epoxide with glutathione and GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA) and cumene hydroperoxide (CHP) were determined with cytosolic fractions from 8-10 pooled livers. Specific activities of cytochrome-P-450-mediated oxidation of AFB1 to aflatoxin Q1 (AFQ1), aflatoxin M1 (AFM1), and aflatoxin P1 (AFP1), as well as the reactive intermediate AFB1-8,9-epoxide, were determined with hepatic microsomal fractions from each mouse strain. No striking differences in specific activity between mouse strains were observed for any of the P-450- or GST-mediated enzymatic pathways measured, although some statistically significant differences were found. GST specific activities toward AFB1-8,9-epoxide, CDNB, DCNB, ECA and CHP ranged from 1.5-2.1, 2,830-5,370, 81-144, 38-69 and 32-73 nmol/mg protein/min, respectively. The rate of formation of AFB1-8,9-epoxide ranged from 208 to 465 pmol/mg protein/min. The specific activities of AFQ1,AFM1, and AFP1 formation by microsomes ranged from 36-70, 161-326, and 252-426 pmol/mg protein/min, respectively. Mice fed a standard rodent chow diet showed evidence of microsomal and cytosolic enzyme induction when compared to mice fed a purified diet. The lack of substantial differences in enzyme specific activities between mouse strains suggests that interstrain variations in the hepatocarcinogenic effects of AFB1 in mice should not be large.     

Studies on the activation of rat liver microsomal glutathione transferase in isolated hepatocytes.

The mechanism of activation of microsomal glutathione transferase in isolated liver cells by diisapropylidene acetone (phorone) was investigated. Phorone (1 mM) causes a time-dependent increase (up to 2.6-fold) in the glutathione transferase activity of microsomes isolated from treated hepatocytes. Since phorone reacts with sulfhydryl groups, the possibility that this compound activated microsomal glutathione transferase directly was studied. It was found that neither the activity of the purified enzyme nor that in isolated microsomes is affected by phorone. It has been suggested [Masukawa T and Iwata H, Biochem Pharmacol 35: 435-438, 1986] that activation of microsomal glutathione transferase by phorone in vivo is mediated through thiol-disulfide interchange involving oxidized glutathione (GSSG). It is shown here that the glutathione transferase activity of isolated microsomes, which was increased by the addition of 10 mM GSSG, can be decreased to the basal level with 0.1 M dithioerythritol. Dithioerythritol, on the other hand, only marginally decreases the glutathione transferase activity in microsomes isolated from phorone-treated hepatocytes. This finding argues against a role for thiol-disulfide interchange in the activation of the enzyme by phorone. Furthermore, the glutathione depletion caused by phorone does not seem to be responsible for activation per se, since other thiol depletors [e.g. diethylmaleate (DEM)] do not affect the activity of the enzyme. Immunoblot analysis of microsomes isolated from phorone-treated hepatocytes did not reveal any partial proteolysis which might have accounted for the activation. It is suggested that activation of microsomal glutathione transferase by phorone proceeds through a mechanism which might reflect an in vivo regulation of this enzyme. Additional compounds which have been shown to activate the microsomal glutathione transferase in vivo were also tested and significant activation was obtained with 1,2-dibromoethane (1.4-fold) but not with DEM or carbon tetrachloride. Activation was also obtained with 1-chloro-2,4-dinitrobenzene (CDNB) (1.6-fold) and to a small extent with t-butyl hydroperoxide (1.2-fold). The activation by 1,2-dibromoethane and CDNB is probably mediated through covalent binding, considering the known alkylating properties of these compounds. CDNB is the first substrate shown to activate the microsomal glutathione transferase implying that electrophilic compounds which are substrates can increase the rate of their own elimination by reacting with this enzyme. In addition, activation by t-butyl hydroperoxide indicates that oxidative stress can activate microsomal glutathione transferase.     

苯巴比妥诱导下大鼠肝微粒体药酶活性与膜流动性变化的相关性

本文用ANS和DPH为荧光探剂,研究苯巴比妥(PB)诱导下大鼠肝微粒体膜脂区流动性与膜药酶活性变化的相关性。结果表明,经PB诱导后在增加肝微粒体蛋白质含量,P-450含量及NADPH-细胞色素C还原酶等酶活性的同时,肝微粒体膜流动性明显增大,且膜深层流动性的增大与膜氨基比林N-脱甲基酶、细胞色素C还原酶活性增加有明显的直线正相关。膜胆固醇/碑脂比值明显降低。此结果提示,肝微粒体膜流动性的适当增大与PB增加单胺氧化酶系统活性之间可能存在着某种联系。     

Incorporation of the hydrophobic probe N-t-BOC-L-tyrosine tert-butyl ester to red blood cell membranes to study peroxynitrite-dependent reactions

We have previously demonstrated that red blood cells (RBC) are an important sink of intravascularly generated peroxynitrite even in the presence of physiological concentrations of CO2 or other plasmatic biotargets. Once inside erythrocytes, peroxynitrite reacts fast with oxyhemoglobin (oxyHb; k2=2 x 10(4) M(-1) s(-1) at 37 degrees C and pH 7.4) and isomerizes to nitrate. Herein, we investigated whether, in spite of the fast diffusion and consumption of extracellularly added peroxynitrite by intraerythrocytic oxyHb, peroxynitrite-dependent radical processes could occur at the RBC membrane, focusing on tyrosine nitration. For this purpose, the hydrophobic tyrosine analogue N-t-BOC-L-tyrosine tert-butyl ester (BTBE) was successfully incorporated for the first time to a biological membrane, that is, RBC membrane, with incorporation yields approximately 1-3 x 10(7) molecules per RBC. The membrane integrity of BTBE-containing RBC was not significantly altered after BTBE incorporation as demonstrated by permeability studies. The probe was then used to study peroxynitrite-dependent reactions. The addition of peroxynitrite to BTBE-containing RBC suspensions resulted in BTBE nitration and dimerization to 3-nitro-BTBE and 3,3'-di-BTBE, respectively, indicative of peroxynitrite-derived radicals reactions in the membrane. Peroxynitrite addition to RBC also caused tyrosine nitration of membrane-associated proteins. The free radical nature of the process was also shown by the detection of protein-derived radicals by DMPO-immunospin trapping. While the presence of extracellular CO2 was potently inhibitory of intracellular oxyHb oxidation, membrane protein and BTBE nitration by peroxynitrite at 相似文献   

On the role of thiol groups in the inhibition of liver microsomal Ca2+ sequestration by toxic agents

ATP-dependent Ca2+ sequestration by rat liver microsomes was assayed using three different methods, and characterized with regard to the effect of various inhibitors. When glucose and hexokinase were added in combination to deplete ATP in the incubation, Ca2+ uptake was followed by rapid release of Ca2+ from the microsomes. Ca2+ sequestration was inhibited by reagents that cause alkylation (e.g. p-chloromercuribenzoate) or oxidation (e.g. diamide) of protein sulfhydryl groups. Moreover, pretreatment of the microsomes with cystamine, which causes formation of mixed disulfides with protein thiols, also resulted in the inhibition of Ca2+ sequestration. It is concluded that microsomal Ca2+ sequestration is critically dependent on protein sulfhydryl groups, and that modification of protein thiols may be an important mechanism for the inhibition of microsomal Ca2+ sequestration by a variety of toxic agents.