Rat Hepatic Glutathione S-transferase Research Articles

Specially processed cereals diet increases plasma levels of active antisecretory factor and up-regulates rat hepatic glutathione S-transferase mu

Objective Antisecretory factor (AF) inhibits pathologic fluid secretion and inflammation. AF is expressed in most tissues and is secreted into the blood. Challenge with bacterial enterotoxins increases AF activity. The plasma level of active AF is also increased after intake of certain food constituents, such as specially processed cereals, SPC. The exact molecular events that mediate these responses have remained obscure. The objective of this study was to investigate changes in protein expression in liver after SPC diet. Methods Rats were fed SPC or standard rodent diet for 18 d. The induction of AF in plasma was tested by ELISA. Changes in the liver proteome were analyzed by using 2D DIGE and LC-MS/MS. Further characterizations were done with Western blot and immunohistochemistry studies. Results The AF activity was increased after intake of SPC. Equivalent to recombinant AF, 6.6 ± 1.09 ng/well could be detected in control plasma compared to 26 ± 5.73 ng/well in plasma after SPC treatment. We found that the protein level of glutathione S-transferase mu (GST mu) was significantly up-regulated 1.2-fold in rat liver after stimulation with SPC (wheat). The result was further confirmed by Western blot analysis. Immunohistochemistry showed staining for GST mu1 and AF preferentially in the central parts of the liver lobuli. Conclusion Given the known role of GST mu1 in inducing defense, our results suggest that SPC-induced GST mu1 up-regulation can contribute to the positive clinical effects seen by SPC treatment.

Chemical synthesis of N-acetylcysteine conjugates of bile acids and in vivo formation in cholestatic rats as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

N-Acetylcysteine (NAC) conjugates of the five major bile acids occurring in man were synthesized in order to investigate the possible formation in vivo of these conjugates. Upon collision-induced dissociation, structurally informative daughter ions were observed. The transformation of cholyl-adenylate and cholyl-CoA thioester into a N-acetyl- S-(cholyl)cysteine by rat hepatic glutathione S-transferase was confirmed by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry (LC/ESI-MS 2). Lithocholic acid was administered orally to bile duct-ligated rats that also received NAC intraperitoneally. The NAC conjugate of lithocholic acid was identified in urine by means of LC/ESI-MS 2. Rapid hydrolysis of the BA-NAC conjugates by rabbit liver carboxylesterase was found, demonstrating the possible labile nature of the NAC conjugates formed in the liver.

Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization–linear ion trap mass spectrometry

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to undergo transacylation-type reactions with the thiol group of glutathione (GSH), leading to the formation of thioester-linked GSH conjugates. In the current study, we examined the transformation of cholyl-adenylate (CA–AMP) and cholyl-coenzyme A thioester (CA–CoA) into a cholyl- S-acyl GSH (CA–GSH) conjugate by rat hepatic glutathione S-transferase (GST). The reaction product was analyzed by liquid chromatography (LC)/electrospray ionization (ESI)–linear ion trap mass spectrometry (MS). The GST-catalyzed formation of CA–GSH occurred with both CA–AMP and CA–CoA. Ursodeoxycholic acid, lithocholic acid, and 2,2,4,4- 2H 4-labeled lithocholic acid were administered orally to biliary fistula rats, and their corresponding GSH conjugates were identified in bile by LC/ESI–MS 2. These in vitro and in vivo studies confirm a new mode of BA conjugation in which BAs are transformed into their GSH conjugates via their acyl-linked intermediary metabolites by the catalytic action of GST in the liver, and the GSH conjugates are then excreted into the bile.

Effect of naturally occurring phenolic acids on the expression of glutathione S-transferase isozymes in the rat

Naturally occurring plant phenols, protocatechuic and tannic acids, have been reported to be inhibitors of chemical mutagenesis and carcinogenesis in experimental models. Our previous studies, have shown that these compounds modulate the activity of phases 1 and 2 enzymes in rodents. The aim of the present study was to investigate whether these compounds affect protein levels of rat hepatic and renal glutathione S-transferase (GST) isozymes. Male Wistar rats were treated intraperitoneally with protocatechuic or tannic acid at 50 mg/kg body weight five times during 14 days. 3-Methylcholanthrene (MC) was administered at 20 mg/kg body weight on day 13 (the last treatment with phenolic compounds) and on day 14. Tissues were obtained from rats terminated 24 h after the last treatment. Western blot analysis with specific antibodies showed significant differences in the effect of the phenolic compounds in the liver and kidney. In the liver, protocatechuic acid significantly increased the constitutive GSTμ, while tannic acid reduced the GSTα protein level by 60%. Both plant phenols decreased all classes of constitutive GST isozymes in the kidney including GSTπ, and also the MC-induced GSTα and/or π protein levels. These results, as well as our previous reports, suggest that protocatechuic and tannic acids interfere with the pathways related to xenobiotic toxicities and carcinogenesis. This effect may be important for chemoprotective activity of these plant phenols.

Lipopolysaccharide inhibition of rat hepatic microsomal epoxide hydrolase and glutathione S-transferase gene expression irrespective of nuclear factor-κB activation

Lipopolysaccharide (LPS) is an endotoxin involved in septic shock syndrome and potentiates toxicant-induced liver injury. The effects of LPS on the constitutive and inducible expression of hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) genes were studied in rats. Northern blot analysis showed that treatment of rats with LPS caused suppression in mEH and GST gene expression. The mEH mRNA level was decreased in a time-dependent manner following a single dose of LPS (1 mg/kg, i.v.), resulting in levels of 52%, 22%, 17%, and 94% of those in untreated animals at 2, 6, 12, and 24 hr, respectively. The levels of rGSTA2 and rGSTA3 mRNA were suppressed in response to an LPS injection to the similar extents as observed in mEH mRNA, whereas rGSTM1 and rGSTM2 mRNA levels were less affected. LPS inhibited mEH gene expression at the doses of 1 μg or greater. Whereas treatment of rats with allyl disulfide (ADS), oltipraz (OZ) or pyrazine (PZ) at the dose of 50 mg/kg caused increases in the mEH mRNA level at 12 hr, a concomitant LPS injection (1 mg/kg) resulted in 80%–95% suppression of the inducible gene expression. The inducible rGSTA2, rGSTA3, rGSTM1, and rGSTM2 mRNA levels were also 50%–90% decreased at 12 hr after LPS treatment, with the relative change in rGSTA being greater than that in rGSTM. Three consecutive daily treatments with LPS (10 μg/kg/day) resulted in significant decreases of the constitutive and PZ (50 mg/kg/day, i.p. for 3 days)-inducible mEH and GST mRNA levels, which were consistent with those in the protein levels. Gel shift retardation analysis showed that LPS substantially activated the hepatic nuclear p65/p50 nuclear factor-κB (NF-κB) complex with the maximal effect observed at 1 hr at the doses of 1 μg/kg or greater. LPS-induced activation of nuclear NF-κB (1 μg/kg, i.v.) failed to be inhibited by concomitant treatment with the mEH and GST inducers, including ADS (300 mg/kg, p.o.), OZ (300 mg/kg, p.o.), and PZ (300 mg/kg, i.p.), indicating that NF-κB activation was not required for suppression of the gene expression by LPS. In contrast, GdCl 3, an inhibitor of mEH and GST expression, inhibited LPS-induced activation of the p65/p50 NF-κB. These gel shift analyses provided evidence that LPS-induced activation of the NF-κB was not responsible for alterations in the gene expression. In summary, the results of this research demonstrate that LPS effectively inhibits constitutive and inducible mEH and GST expression with decreases in their mRNA levels, and that LPS suppression in the expression of the detoxifying enzymes is not mediated with its activation of NF-κB.

Effect of oltipraz, alpha-tocopherol, beta-carotene and phenethylisothiocyanate on rat oesophageal, gastric, colonic and hepatic glutathione, glutathione S-transferase and peroxidase.

Four anticarcinogens (oltipraz, alpha-tocopherol, beta-carotene and phenethylisothiocyanate [PEITC]) were studied with respect to their effects on oesophageal, gastric, colonic and hepatic (i) glutathione (GSH) content, (ii) glutathione S-transferase (GST) enzyme activity, (iii) GST isoenzyme levels, and (iv) glutathione peroxidase (GPx) enzyme activity in male Wistar rats. GST enzyme activity was significantly increased in oesophagus (1.9X) and colon (1.2X) by PEITC and in liver (1.4X) by oltipraz. GST Alpha was doubled in the liver by oltipraz, alpha-tocopherol and PEITC. GST Mu levels were increased by beta-carotene and PEITC in stomach and liver, by oltipraz in liver and by alpha-tocopherol in stomach. PEITC induced colonic GST Pi levels (1.3X). GSH content was induced in liver by oltipraz (1.4X) and alpha-tocopherol (1.2X) and in colon by PEITC (1.6X). Each of the anticarcinogens tested increased GPx activity at one or more sites: Se-dependent and total GPx activities were induced in 31.3% and 37.5% of all possibilities, respectively. Major induction in total GPx was found in stomach by alpha-tocopherol (1.8X). In conclusion our data demonstrate that dietary administration of oltipraz, PEITC, alpha-tocopherol and beta-carotene, may exert chemopreventive effects in the digestive tract of the rat by enhancing GST, GPx, and, to a lesser extent, GSH levels.

Mechanistic study of the inhibition of aflatoxin b1-induced hepatotoxicity by dimethyl 4,4'-dimethoxy-5,6,5',6'-dimethylenedioxy biphenyl-2, 2'-dicarboxylate.

The mechanism of DDB (dimethyl 4,4'-dimethoxy-5,6,5',6'-dimethylenedioxy biphenyl-2,2'-dicarboxylate) prevention of aflatoxin B1 (AFB1)-induced hepatotoxicity in rats has been investigated. Pretreatment of DDB (200 mg/kg) daily for 4 days significantly suppressed (P < 0.05) the AFB1-induced hepatic damage as evidenced by the increase of serum marker enzymes. DDB induced rat hepatic cytochrome P450IA1, IIB1 and glutathione S-transferase activities. The hepatic microsomes derived from DDB treated rats increased the mutation frequency of AFB1 and enhanced the binding of AFB1 to DNA. However, the hepatic S9 fraction from DDB treated rats showed a protective effect against AFB1-induced damage. It is concluded that the protective effect of DDB against AFB1-induced damage might be mediated by the induced glutathione S-transferase activity and not from the accelerated hepatic cytochrome P450 detoxification pathway of AFB1 which was previously believed.

Enhancement of rat hepatic and gastrointestinal glutathione and glutathione S-transferases by alpha-angelicalactone and flavone.

The naturally occurring anticarcinogens flavone and alpha-angelicalactone incorporated separately and simultaneously in the diet at 0.5, 0.1, 0.05 and 0.01% w/w, were studied with respect to their effects on oesophageal, gastric, intestinal, colonic and hepatic (i) glutathione S-transferase (GST) enzyme activity, (ii) GST isozyme levels and (iii) glutathione (GSH) content in male Wistar rats. GST enzyme activity was significantly increased in the three treatment groups at one or more sites. The most substantial inductions were seen in oesophagus and stomach by 0.5% alpha-angelicalactone (1.9- and 2.3-fold respectively); and in small intestine, colon and liver by 0.5% combination diet (2.5-, 1.4- and 4.0-fold respectively). The inducing capacities declined with decreasing anticarcinogen concentrations. GST enzyme activity was induced in liver and to a lesser extent in small intestine and stomach. In general, in combination groups similar effects were seen as after treatment with alpha-angelicalactone or flavone separately. However, colonic GST enzyme activity was increased in the 0.5% combination group (1.4-fold), whereas in the corresponding flavone or alpha-angelicalactone groups no induction was observed. Concomitant changes in GST isozyme levels occurred. The involvement was the highest for GST-alpha (75%), followed by GST-mu (58%) and GST-pi (33%). Increased GSH levels were obtained in stomach and liver in all three treatment groups at various concentrations. These data demonstrate that dietary administration of flavone or alpha-angelicalactone, even at relatively low concentrations, may exert chemopreventive effects in stomach, small intestine, liver and to a lesser extent in oesophagus by enhancing the GST detoxification system, mainly by induction of GST-alpha and GST-mu isozymes. In addition, simultaneous administration of flavone and alpha-angelicalactone may result in anticarcinogenic effects in the colon by the same principle.

Ammonium 4-Chloro-7-sulfobenzofurazan: A Fluorescent Substrate Highly Specific for Rat Glutathione S-Transferase Subunit 3

Ammonium 4-chloro-7-sulfobenzofurazan (Sbf-Cl) is a water-soluble fluorescent reagent which is highly specific for thiol groups. We describe here a simple and sensitive fluorescence assay which is highly specific for subunit 3 of the rat glutathione S-transferases. Specific activities of isoenzymes 3-3 and 3-4 were an order of magnitude greater than those of isoenzymes 1-1, 1-2, 2-2, and 4-4, with glutathione and Sbf-Cl concentrations of 1 and 2 mM, respectively. The catalytic specificity constant, kcat/Km was in the range of 104-106 M−1 s−1, indicating that Sbf-Cl is a very good substrate for rat hepatic glutathione S-transferases. The specificity constants measured for the heterodimers 1-2 and 3-4 were greater than the values predicted when dimeric independence is assumed. This suggests that binding of Sbf-Cl to the glutathione S-transferases may result in steric alterations and subsequent elevation in enzymatic activity. Sbf-Cl was shown to be at least 10-fold more sensitive for the detection of rat GST isoenzyme 3-3 than DCNB. Consequently, Sbf-Cl will have an important future role in the investigation of the active site topology of glutathione S-transferases, in addition to its obvious potential as a substrate for the detection and quantitation of rat glutathione S-transferase subunit 3.

INDUCTION OF RAT HEPATIC AND INTESTINAL GLUTATHIONE S-TRANSFERASES AND GLUTATHIONE BY DIETARY NATURALLY-OCCURRING ANTICARCINOGENS

Effects of dietary naturally occurring anticarcinogens; quercetin, flavone, ellagic acid, ferulic acid, tannic acid, curcumin, coumarin, alpha-angelicalactone, fumaric acid and Brussels sprouts on male Wistar rat hepatic and intestinal (i) glutathione S-transferases (GST) enzyme activity, (ii) GST isozyme levels and (iii) glutathione (GSH) content were investigated. GST enzyme activity was significantly increased by all anticarcinogens tested, except fumaric acid, at least at one of the five sites investigated: proximal, middle, distal small intestine, large intestine and liver. Only alpha-angelicalactone gave an enhanced GST enzyme activity at all five sites. Large intestinal GST enzyme activity was increased only by quercetin (175%) and alpha-angelicalactone (138%). Concomitant changes in GST isozyme levels occurred. Class alpha GSTs were induced in 50% of the cases, especially in liver and upper parts of the intestine by quercetin, flavone, coumarin and alpha-angelicalactone. GST class pi levels were enhanced only at one site by quercetin, coumarin and alpha-angelicalactone. GST class mu changed in 14% of the cases, most profoundly in proximal and middle small intestine by flavone, coumarin and alpha-angelicalactone. Tannic acid and fumaric acid gave a significant raise in class alpha GSTs at almost all sites, whereas overall GST enzyme activity hardly changed. GSH was increased at various sites in 14% of the cases by Brussels sprouts, quercetin, flavone and alpha-angelicalactone. These data demonstrate that most anticarcinogens, in particular flavone, coumarin and alpha-angelicalactone, enhance GST activity in liver and intestine, mainly by induction of class alpha and mu isozymes.

Combined effects of cadmium and nickel on hepatic glutathione S-transferases in raTS

1. The acute combined effects of cadmium (Cd) and nickel (Ni) on rat hepatic glutathione S-transferase (GST) activities toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB), l,2-dichloro-4nitrobenzene (DCNB) and ethacrynic acid (EAA) were determined and compared to those of Cd or Ni alone. 2. Male adult rats (225–275 g) were administered either a single dose of Cd (3.58 mg CdCl 2 · H 2O kg , i.p.) 72 hr prior to sacrifice or a single dose of Ni (59.5 mg NiCl 2 · 6H 2O kg , s.c.) 16 hr prior to sacrifice. For the combined treatment, animals received the single dose of Ni 56 hr after the single dose of Cd and they were killed 16hr later. 3. Cd treatment alone did not produce any changes in the hepatic GST activities toward the substrates studied. 4. Ni treatment alone, however, significantly increased hepatic GST activity toward EAA whereas it was ineffective on GST activities for CDNB and DCNB. 5. Combined treatment of metals did not alter hepatic GST activities toward the substrates CDNB and DCNB. Hepatic GST activity for EAA, however, was significantly increased by the combined treatment. Nevertheless, the combined treatment did not augment the increase in GST activity for EAA noted by Ni treatment alone.

Induction of rat hepatic and intestinal glutathione S-transferases by dietary butylated hydroxyanisole

To obtain insight into the protection mechanism of butylated hydroxyanisole (BHA), a widely used food preservative with anticarcinogenic properties, we investigated the effects of dietary BHA on rat hepatic and intestinal glutathione S-transferase (GST) enzyme activity, and GST isozyme levels. In the proximal small intestine and liver, BHA supplementation significantly increased GST enzyme activity as compared with controls (2.3- and 1.7-fold, respectively, P < 0.05). GST class α and μ contents were significantly higher only in the small intestine (1.6–2.1-fold and 1.3–1.5-fold, respectively, P < 0.05), whereas GST class π was significantly induced in liver (4.6-fold, P < 0.05).

Activation of hepatic microsomal glutathione S-transferase of rats by a glutathione depletor, diethylmaleate.

The effect of glutathione depletor diethylmaleate on rat hepatic glutathione S-transferase and glutathione peroxidase was studied in vivo and in vitro. When diethylmaleate (600 mg/kg) was given i.p. to rats, liver glutathione was depleted within 2 h and recovered to the control level 5 h after diethylmaleate treatment. Both glutathione S-transferase and peroxidase activities in microsomes, not in cytosol, were markedly increased during glutathione depletion and only glutathione S-transferase activity remained at high levels after recovery of the glutathione content. The increase in microsomal glutathione S-transferase and peroxidase activities with concomitant exhaustion of glutathione was also observed by perfusion of the isolated liver with diethylmaleate (10 mM). When liver microsomes were incubated with diethylmaleate in vitro at 37 degrees C, glutathione S-transferase, but not peroxidase, activity was increased; the increase was not reversed by dithiothreitol. These results indicate that diethylmaleate activates microsomal glutathione S-transferase by direct reaction to the enzyme during glutathione depletion and suggest that glutathione S-transferase activity and glutathione peroxidase activity in the microsomal enzyme may be differently regulated.

Differential inhibition of rat hepatic glutathione S-transferase isoenzymes in the course of fascioliasis

The effects of a subclinical fascioliasis at various stages of its development (at week 3, 6 and 9 after infection by oral administration of 20 metacercariae of Fasciola hepatica) in rats were determined on the activity of enzymes involved in liver metabolism of glutathione and on the subunit pattern of cytosolic glutathione S-transferase. The parasitic pathology was ascertained by clinical observation of the rats and at autopsy. Hepatic microsomal cytochrome P-450 content was significantly decreased in infected rats by week 3 and 6 post-infection. Not correlatively, the catalytic activities of glutathione S-transferase towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene were significantly lowered in last stages of the experimental fascioliasis (by week 6 and 9 post-infection). These decreases were correlated to that of subunit 1 as determined by means of high-performance liquid chromatography of cytosolic proteins whereas subunit 6 could also be decreased. Fasciolasis did not alter cytosolic glutathione, glutathione reductase and glutathione peroxidase activities or plasma glutathione S-transferase activity accepting 1-chloro-2,4-dinitrobenzene as the substrate.

Hybrid formation of rabbit and rat hepatic glutathione S-transferases

1. 1. A reconstitution experiment resulted in the formation of new proteins between limited combinations of rat and rabbit hepatic glutathione S-transferases: AA(subunit composition: YcYc) and R3b(Y3Y3), Lig(YaYa) and R3b(Y3Y3), and A(Yb1Yb1) and R2(Y2Y2). 2. 2. It was demonstrated that the new protein formed between R2 and A had the subunit composition of Y2Yb1, suggesting a hybrid of rabbit (R2) and rat isozyme (A). 3. 3. This hybrid protein showed intermediate spec. acts between those of R2 and A when either 1-chloro-2,4-dinitrobenzene (CDNB) or 1,2-dichloro-4-nitrobenzene (DCNB) was employed as the substrate.

Inhibition of rat hepatic glutathione S-transferase placental form positive foci development by concomitant administration of antioxidants to carcinogen-treated rats

Inhibition potential of concomitant butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), catechol or sodium ascorbate (Na-AsA) administration on development of diethylnitrosamine (DEN) initiated glutathione S-transferase placental form (GST-P) positive foci in rat liver under the influence of 2-acetylaminofluorene (2-AAF) or 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB) plus partial hepatectomy (PH) was investigated. Whereas BHA, BHT and catechol exerted marked inhibitory effects, Na-AsA lacked any modifying potential. The compounds that demonstrated inhibition also induced GST-P in the hepatic periportal areas, suggesting that development of GST-P positive foci is negatively influenced by extra-focal increase in this enzyme form observed with BHA, BHT or catechol.

Reversible inhibition of rat hepatic glutathione s-transferase 1–2 by bilirubin

The inhibition of rat hepatic glutathione (GSH) S-transferase 1–2 by bilirubin exhibited pseudo first-order kinetics with k obs values of 0.0214 ± 0.0005 and 0.040 ± 0.008sec −1 at 4 and 8 μM bilirubin, when followed to 72 and 84% completion respectively. These correspond to calculated secondorder rate constants of 5.3 ± 0.1 × 10 3 and 5.0 ± 1.0 × 10 3/M·sec. The extent of inhibition of the transferase increased with bilirubin concentration, with half-maximal inhibition at 4 μM bilirubin. Inhibition was reversed by 10-fold dilution of bilirubin or by increasing the pH from 6.0 to 7.4. Premixing 0.2 to 0.5 μM albumin, hemoglobin or aldolase with bilirubin prevented inhibition of GSH S-transferase 1–2. Protection by these proteins occurred at a selected high concentration (0.2 to 0.4 μM) at which they reduced free bilirubin to concentrations (< 0.5 μM) that did not inhibit isoenzyme 1–2 significantly. No protection was afforded by a selected low protein concentration (0.001 to 0.01 μM) which did not strikingly reduce bilirubin levels in solution. We conclude that bilirubin inhibition of GSH S-transferase 1–2 appears to be a second-order process; the reaction is clearly first-order with respect to GSH S-transferase and appears also to be first-order with respect to bilirubin. It is proposed that (a) inhibition of GSH S-transferase 1–2 results from slow, reversible bilirubin binding, and (b) added proteins appear to prevent GSH S-transferase inhibition by binding high molar ratios of bilirubin.

Studies on the mechanism of haloacetonitriles toxicity: Inhibition of rat hepatic glutathione S-transferases in vitro

Acetonitrile (AN) and seven of its halogenated derivatives known to be water disinfectant by-products were evaluated for their action on hepatic cytosolic glutathione S-transferase (GST) activity using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. Increasing concentrations of acetonitrile, monofluoroacetonitrile (MFAN), monochloroacetonitrile (MCAN), and monobromoacetonitrile (MBAN) up to 10 m m failed to produce 50% inhibition of the activity of GST enzyme. However, dichloroacetonitrile (DCAN), trichloroacetonitrile (TCAN), dibromoacetonitrile (DBAN), and monoiodoacetonitrile (MIAN) were potent inhibitors with I50 values of 2.49, 0.34, 0.82, and 4.44 m m, respectively. At concentrations equivalent to their I50, MIAN, DCAN, and DBAN decrease both apparent K m and V max of the enzyme activity toward glutathione (GSH) to 20–50% of control. TCAN significantly increases both apparent K m and V max for GSH to 650 and 120% of control values, respectively. The inhibitory effect of haloacetonitriles (HAN) on hepatic GST activity toward CDNB was found to be of a mixed type. The inhibitory effect of DCAN, DBAN, and TCAN on the hepatic GST activity was found to be reversible and the activity was completely recovered after dialysis of the inhibited enzyme. MIAN, however, inhibited GST activity in an irreversible manner. Haloacetonitriles' induced inhibition of hepatic GST activity in vitro is consistent with that observed in vivo. The data presented in this study show that haloacetonitriles induced reversible inhibition of hepatic GST activities, and this effect may lead to decreased detoxification of other electrophilic chemicals.

Methyl linoleate ozonide as a substrate for rat glutathione S-transferases: Reaction pathway and isoenzyme selectivity

The 9,10-mono-ozonide of methyl linoleate was shown to be a substrate for rat hepatic cytosolic, rat lung cytosolic and rat hepatic microsomal glutathione S-transferases (GST). The activities of lung cytosol and liver microsomes with methyl linoleate ozonide (MLO) were found to be high relative to the activity demonstrated by liver cytosol, as compared with their respective activities towards 1-chloro-2,4-dinitrobenzene (CDNB). Only a slight catalytic activity towards the ozonide was noticed for rat lung microsomes. Isoenzyme 2-2 exhibited the highest specific activity (208 nmol/min/mg) when isoenzymes 1-1, 1-2, 2-2, 3-3, 3-4, 4-4 and 7-7 were compared. This isoenzyme accounts for approx. 25% of cytosolic GST protein in rat lung, while in rat liver it represents approx. 9%. This may partly explain the high activity towards the ozonide noticed for rat lung cytosol. No stable conjugates were formed as products of the reaction of MLO with glutathione; although two glutathione-conjugates were noticed on TLC, they were only formed as intermediate compounds. Coupling of an aldehyde dehydrogenase assay or a glutathione reductase assay to the GST-catalyzed conjugation, demonstrated that oxidized glutathione and aldehydes are formed as the major products in the reaction. To further confirm the formation of aldehydes, the products of the GST-catalyzed reaction were incubated with 2,4-dinitrophenylhydrazine, which resulted in hydrazone formation. In conclusion, the activity of the GST towards the ozonide of methyl linoleate is similar to their peroxidase activity with lipid hydroperoxides as substrates.

Irreversible inhibition of rat hepatic glutathione S-transferase isoenzymes by a series of structurally related quinones

The effect of several structurally related 1,4-benzoquinones (BQ) and 1,4-naphthoquinones (NQ) on the activity of rat hepatic glutathione S-transferases (GST) was studied. For the 1,4-benzoquinones, the extent of inhibition increased with an increasing number of halogen substituents. Neither the type of halogen nor the position of chlorine-atoms was of major importance. Similarly, 2,3-dichloro-NQ demonstrated a considerably higher inhibitory activity than 5-hydroxy-NQ. 2-Methyl derivatives of NQ did not inhibit GST activity at all. The irreversible nature of the inhibition was shown both by the time-course of the inhibition as well as by the fact that removal of the inhibitor by ultrafiltration did not restore the enzymatic activity. Incubation of quinones and enzyme in the presence of the competitive inhibitor S-hexylglutathione, slowed the inhibition considerably, indicating an involvement of the active site. Isoenzyme 3-3 was found to be most sensitive towards the whole series of inhibitors, whereas the activity of isoenzyme 2-2 was least affected in all cases. The inhibition by quinones is probably mainly due to covalent modification of a specific cysteine residue in or near the active site. The differential sensitivities of individual isoenzymes indicates that this residue is more accessible and/or easier modified in isoenzyme 3-3 than in any of the other isoenzymes tested. The findings suggest that quinones form a class of compounds from which a selective in vivo inhibitor of the GST might be developed.