Conjugation of isoprene monoepoxides with glutathione, catalyzed by alpha, mu, pi and theta-class glutathione S-transferases of rat and man

In the present study, the enzymatic conjugation of the isoprene monoepoxides 3,4 epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II) with glutathione was investigated, using purified glutathione S-transferases (GSTs) of the alpha, mu, pi and theta-class of rat and man. HPLC analysis of incubations of EPOX-I and EPOX-II with [35S]glutathione (GSH) showed the formation of two radioactive fractions for each isoprene monoepoxide. The structures of the EPOX-I and EPOX-II GSH conjugates were elucidated with 1H-NMR analysis. As expected, two sites of conjugation were found for both isoprene epoxides. EPOX-II was conjugated more efficiently than EPOX-I. In addition, the mu and theta class glutathione S-transferases were much more efficient than the alpha and pi class glutathione S-transferases, both for rat and man. Because the mu- and theta-class glutathione S-transferases are expressed in about 50 and 40-90% of the human population, respectively, this may have significant consequences for the detoxification of isoprene monoepoxides in individuals who lack these enzymes. Rat glutathione S-transferases were more efficient than human glu tathione S-transferases: rat GST T1-1 showed about 2.1-6.5-fold higher activities than human GST T1-1 for the conjugation of both EPOX-I and EPOX-II, while rat GST M1-1 and GST M2-2 showed about 5.2-14-fold higher activities than human GST M1a-1a. Most of the glutathione S-transferases showed first order kinetics at the concentration range used (50-2000 microM). In addition to differences in activities between GST-classes, differences between sites of conjugation were found. EPOX-I was almost exclusively conjugated with glutathione at the C4-position by all glutathione S-transferases, with exception of rat GST M1-1, which also showed significant conjugation at the C3-position. This selectivity was not observed for the conjugation of EPOX-II. Incubations with EPOX-I and EPOX-II and hepatic S9 fractions of mouse, rat and man, showed similar rates of GSH conjugation for mouse and rat. Compared to mouse and rat, human liver S9 showed a 25-50-fold lower rate of GSH conjugation.     

Hepatic and branchial glutathione S-transferases of two fish species: Substrate specificity and biotransformation of microcystin-LR

Liver and gills of roach (Rutilus rutilus) and silver carp (Hypophthalmichthys molitrix) were examined for glutathione S-transferases (GSTs) contents and their substrate specificity and capacity to biotransform microcystin-LR (MC-LR). GSTs and other glutathione (GSH) affine proteins were purified using a GSH-agarose matrix and separated by anionic chromatography (AEC). Substrate specificities were determined photometrical for 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrobenzyl chloride (pNBC) and ethacrynic acid (ETHA). Biotransformation rate of MC-LR was determined by high performance liquid chromatography (HPLC). Roach exhibited different hepatic and branchial GST activities for used substrates (DNB, pNBC and DCNB) compared to silver carp but not for ethacrynic acid. It suggests that, both fish species have similar amount of pi and/or alpha class, which were the dominant GST classes in liver and gills. Gills of both fish species contained a higher number of GST isoenzymes, but with lower activities and ability of MC-LR biotransformation than livers. GST isoenzymes from roach had higher activity to biotransform MC-LR (conversion rate ranging up to 268 ng MC-LR min^? 1 mL^? 1 hepatic enzyme) than that isolated from silver carp. Without any prior contact to MC-LR or another GST inducer, roach seems to be better equipped for microcystin biotransformation than silver carp.     

Effects of benzo[a]pyrene on tissue activities of metabolizing enzymes and antioxidant system in normal and protein-malnourished rats

The effects of benzo[a]pyrene (B[a]P) on some drug-metabolizing and antioxidant systems in liver, lung, and stomach were investigated in normal and protein malnutrition (PM) rats. PM significantly inhibited tissue glutathione (GSH) content and increased hepatic lipid peroxidation. Cytochrome P450 isoform CYP1A1 was significantly increased in various tissues (42-73%). Also, lung glutathione S-transferase (GST) activity was significantly decreased (19%) in PM rats. On the other hand, B[a]P significantly induced tissue GSH of control and PM rats. Also, hepatic lipid peroxidation were significantly increased in control rats treated with B[a]P. Superoxide dismutase (SOD) activity was decreased by B[a]P treatment in PM rat stomach. B[a]P significantly induced both quinone reductase (QR) (in all tissues) and hepatic GST of control and PM rats. GST activity in PM rat liver was significantly higher than that of control rat liver after B[a]P treatment. Also, B[a]P induced hepatic CYP1A1 by 32-fold and 27-fold (P < or = 0.05) in control and PM rats, respectively. Stomach and hepatic UDP-glucuronosyltransferase activities were significantly decreased (34%) and increased (74%), respectively by B[a]P in PM rats. The results suggest that PM status has a modifying effect on the response of some antioxidant and metabolizing systems to a well-known carcinogen risk.     

Biochemical Characterization and Distribution of Glutathione S-Transferases in Leaping Mullet (Liza saliens)

In this study, feral leaping mullet (Liza saliens) liver cytosolic glutathione S-transferases (GSTs) were investigated and characterized using 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) as substrates. The average GST activities towards CDNB and EA were found to be 1365 +/- 41 and 140 +/- 20 nmol/min per mg protein, respectively. The effects of cytosolic protein amount and temperature ranging from 4 to 70 degrees C on enzyme activities were examined. While both activities towards CDNB and EA showed similar dependence on protein amount, temperature optima were found as 37 and 42 degrees C, respectively. In addition, the effects of pH on GST-CDNB and -EA activities were studied and different pH activity profiles were observed. For both substrates, GST activities were found to obey Michaelis-Menten kinetics with apparent V(max) and K(m) values of 1661 nmol/min per mg protein and 0.24 mM and 157 nmol/min per mg protein and 0.056 mM for CDNB and EA, respectively. Distribution of GST in Liza saliens tissues was investigated and compared with other fish species. Very high GST activities were measured in tissues from Liza saliens such as liver, kidney, testis, proximal intestine, and gills. Moreover, our results suggested that GST activities from Liza saliens would be a valuable biomarker for aquatic pollution.     

Sex difference in subunit composition of hepatic glutathione S-transferase in rats

The activities of hepatic cytosolic glutathione S-transferases (GSTs) towards 1,2-dichloro-4-nitrobenzene in male rats were higher than those in females, however, the enzyme activities towards 1-chloro-2,4-dinitrobenzene were not significantly different between the two sexes. SDS-PAGE analysis of GSTs purified from male and female rat hepatic cytosols by affinity column chromatography showed that there was a significant difference in the subunit composition between the two sexes. With regard to the several isozymes of GSTs in male and female rats, isozymes with basic and neutral/acidic isoelectric points were separated into seven molecular species by chromatofocusing. These sex differences in the quantitative proportions of GST isozymes were also confirmed by immunotitration using anti-GST-BL and -AC antibodies. On the other hand, glutathione peroxidase (GSH-Px) activities in rat hepatic cytosol towards hydrogen peroxide and cumene hydroperoxide were markedly higher in females than in males. Of the two types of GSH-Px, selenoenzyme (Se-GSH-Px) and the Se-independent enzyme (non-Se-GSH-Px), the former was found to be mainly responsible for the sex difference in the enzyme activities. Moreover, the GSH-Px activity of GSTs, non-Se-GSH-Px, was also higher in females than that in males. Since GST isozymes of the BL type are known to possess GSH-Px activity towards cumene hydroperoxide, the increased activities of non-Se-GSH-Px in the female hepatic cytosol seemed to be mainly due to the increased transferase activities of the isozymes, GST-L2 and -BL.     

Transient decrease of liver cytosolic glutathione S-transferase activities in rats given 1,2-dibromoethane or CCl4

In vivo treatment of fasted male rats with 1,2-dibromoethane (DBE) (0.4 mmol/kg) or carbon tetrachloride (CCl4) (4 mmol/kg) was found to rapidly alter the activities of liver cytosolic and microsomal glutathione S-transferases. Microsomal activities towards chloro-2,4-dinitrobenzene (CDNB) were increased 2 h after either treatment. Cytosolic activities towards CDNB and 3,4-dichloronitrobenzene (DCNB), but not 1,2-epoxy-3-(p-nitrophenoxy)-propane (ENPP), were selectively and transiently decreased after either treatment. Time course studies in DBE animals indicated that the decrease in cytosolic activity was not evident until 2 h although liver glutathione (GSH) concentrations were diminished within 15 min. In contrast, in CCl4 animals the decrease in cytosolic activity was evident within 15 min and was not accompanied by diminished GSH concentrations. By 4 h, cytosolic activities had rebounded to control levels in both DBE and CCl4-treated animals. Kinetic studies of the enzyme in liver cytosol from animals 2 h after treatment with DBE or CCl4 indicated that both treatments decreased the apparent Vmax while neither treatment altered the apparent Km. This pattern of change allows exclusion of a simple competitive mechanism of enzyme inhibition, but cannot distinguish between reversible non-competitive inhibition and irreversible inhibition. It is possible that the observed decreases in the activities of the abundant cytosal enzyme are due to 'sacrificial' covalent linkages between the enzyme and reactive metabolites of DBE or CCl4.     

Serum glutathione S-transferases: Perinatal development,sex difference,and effect of carbon tetrachloride administration on enzyme activity in the rat

Glutathione $\text{S}$-transferase activity was determined in rat, rabbit, and guinea pig serum using styrene 7,8-oxide (SO) and benzo (a) pyrene 4,5-oxide (4,5-BPO) as substrates. Of the species tested, rat had the highest transferase activity (62.5 and 3.2 nmol/min/ml serum for SO and 4,5-BPO, respectively) and rabbit had the lowest activity. Glutathione $\text{S}$-transferase activity was 60% higher in serum from male rats than in female rats. In rats, serum enzyme specific activities (nmol/min/mg protein) were less than 1% of hepatic enzyme activities with SO, 4,5-BPO, 1,2-dichloro-4-nitrobenzene (DCNB), and 1-chloro-2,4-dinitrobenzene (DNCB). Glutathione $\text{S}$-transferase activity was also determined in rat serum during perinatal development. Serum from rats at 18 days of gestation or from 1- and 4-day-old animals had barely detectable transferase activity. Activity increased with age and reached a maximum in 140-day-old animals. The intraperitoneal administration of diethyl maleate (DEM) (0.8 ml/kg) or L-methionine-DL-sulfoximine (MS) (200 mg/kg) to male rats had no effect on serum or hepatic glutathione $\text{S}$-transferase activities 2 or 26 hr after dosing. Treatment with carbon tetrachloride (CCl₄) (1 m1/kg) caused an 11-fold increase in serum transferase activity and a 40% decrease in liver specific activities 24 hr after administration.     

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

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

Dehydroepiandrosterone improves hepatic antioxidant reserve and stimulates Akt signaling in young and old rats

This study examined, in the liver of young and old (3- and 24-month-old, respectively) healthy Wistar rats, the in vivo effect of dehydroepiandrosterone (DHEA) (10mg/kg body weight) administered subcutaneously for 5 weeks. Reduced (GSH) and oxidized (GSSG) glutathione levels, glucose-6-phosphate dehydrogenase (G6PDH), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) activities, hydrogen peroxide concentration, GST and p-Akt/Akt immunocontent ratio were assessed in hepatic tissue. DHEA treatment significantly increased total glutathione content (17%) and GSH (22%) in 3- and 24-month-old treated groups when compared to control groups. The aging factor increased G6PDH (51%) and GPx (22%) activities as well as the hydrogen peroxide concentration (33%), independently of treatment. DHEA treatment increased p-Akt (54%) and p-Akt/Akt ratio (36%) immunocontents in both treated groups. Increased serum levels of alanine aminotransferase (ALT) in aged rats were reduced by DHEA treatment (34%).     

Combined effects of cadmium and nickel on testicular xenobiotic metabolizing enzymes in rats

When male rats were given a single dose of cadmium (Cd) (3.58 mg CdCl₂·H₂O/kg, ip) 72 hr prior to sacrifice, the testicular 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (EAA), 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP), and cumene hydroperoxide (CHPx) decreased significantly as compared to controls. Cd also inhibited reduced glutathione (GSH) level while increasing the lipid peroxidation (LP) level significantly. When the animals were given a single dose of nickel (Ni) (59.5 mg NiCl₂·6H₂O/kg, ip) 16 hr prior to sacrifice, significant decreases were observed in EROD and GST activities toward CDNB, EAA, EPNP, and CHPx, and GSH level. No significant alterations were noted in DCNB GST activity and LP level by Ni. For the combined treatment, rats received the single dose of Ni 56 hr after the single dose of Cd and were killed 16 hr later. In these animals, lesser depressions were observed on EROD activity and LP level than those of Cd alone. The combination of metals significantly inhibited GST activities and GSH level but not to a greater degree than noted by Cd or Ni alone. Plasma testosterone levels of Cd-, Ni-, and combination-treated rats decreased significantly compared to controls. The strongest depression was achieved by Cd alone. Cd, both alone and in combination with Ni, increased the tissue Ni uptake significantly. Ni, however, did not produce such an effect on the tissue uptake of Cd in either case. Cd treatment caused interstitial edema and coagulation necrosis in seminiferous tubules and also caused fibrinoidal necrosis in vascular endothelium. Ni treatment did not produce any pathological testicular alterations compared to controls. Combined treatment produced fewer pathological alterations (i.e., only interstitial edema) than that of Cd treatment. These results reveal that the combination of Cd and Ni does not have a synergistic effect on testicular xenobiotic metabolizing enzymes, and in contrast, Ni has an ameliorating effect on pathological disturbances caused by Cd alone in the rat testis.     

Rat glutathione S-transferase. Cloning of double-stranded cDNA and induction of its mRNA

Messenger RNA extracted from the livers of normal, phenobarbital-treated, and trans-stilbene oxide-treated rats was translated in a mRNA-dependent protein-synthesizing system. Immunoprecipitation of the translation products by antibodies against the Ya and Yc subunits of glutathione S-transferase detected two polypeptides of molecular weights 23,500 and 25,000. Subsequently, a clone containing glutathione S-transferase sequences was identified from a rat liver double-stranded cDNA library that had been prepared by homopolymeric tailing and cloning into the Pst I site of pBR322. Confirmation of the identity of the clone was obtained by recloning the 550-bp insert DNA into the phage vector M13 and utilizing the single strand recombinant phage DNA in specific hybrid selection of mRNA followed by translation and immunoprecipitation with antibodies to the Ya and Yc subunits. This recombinant phage, M13GST94, was also utilized in a new technique to synthesize 32P-labeled cDNA specific to the glutathione S-transferase insert DNA that was used subsequently in RNA excess solution hybridization to determine the relative concentration of glutathione S-transferase mRNA. Phenobarbital treatment resulted in a 3.2-fold increase in glutathione S-transferase mRNA over levels found in control rats, while trans-stilbene oxide increased glutathione S-transferase mRNA levels 5.7-fold. The DNA sequence of the clone was determined and utilized to propose a partial amino acid sequence.     

Induction of glutathione S-transferases in genetically inbred male mice by dietary ethoxyquin hydrochloride

1. Constitutive and ethoxyquin hydrochloride (EQ-HCl)-induced hepatic glutathione (GSH) S-transferase, GSH reductase, and GSH peroxidase activities were determined in 5 strains of 8-10 week old inbred male mice. 2. The constitutive GSH S-transferase (GST) activity varied from 2.9 (SJL/JCR) to 8.9 (C57BL/6NCR) mumol product formed/min/mg protein and the corresponding values for the EQ-HCl-treated mice were in the range of 15.3-25.3 mumol product formed/min/mg protein. 3. EQ-HCl induced GST activity in all the strains examined and this contrasted to the induction activity of Aroclor 1254 which was strain-dependent. GST activity was induced 2.9-fold in Aroclor 1254-responsive (C57BL/6) and 2.8-fold in non-responsive (DBA/2) mice, respectively.     

Time-dependent oxidative stress caused by benznidazole

Benznidazole (BZN) is a nitroimidazole derivative which has a notable trypanocide activity, and it is the only drug used in Brazil and Argentina for the treatment of Chagas' disease. The drug in current use is thought to act, at least in part, by inducing oxidative stress within the parasite. Imidazolic compounds are involved in the production of reactive oxygen species (ROS). In order to evaluate the effect of BZN on ROS production and on the antioxidant status of the host, male rats were treated for different periods of time (2, 4, 6, 10 and 30 days) with 40 mg BZN/kg body weight. After treatment, biomarkers of oxidative stress such as the activities of catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST) and glutathione reductase (GR), and also thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH), total glutathione (TG) and oxidized glutathione (GSSG) concentrations, were measured in crude hepatic homogenates. Our results revealed that BZN is able to cause tissue damage as shown by increased TBARS content, inhibition of some antioxidants and induction of other antioxidants in a concentration- and time-dependent manner. The tissue damage measured as TBARS increased up to the 10th day of treatment. GST activity was inhibited during the BZN treatment. On the other hand, CAT and GR showed similar increased activities at the beginning, followed by decreased activities at the end of the treatment. After 30 days of treatment, GR activity remained low while CAT activity was high, compared to controls. The SOD activities remained unchanged throughout the experimental period. GSH showed lower values at the beginning of BZN treatment but the hepatic concentrations were enhanced at the end of the experimental period. Total glutathione showed a similar profile, and oxidized glutathione showed higher values in rats treated with BZN. In conclusion, these results indicate that, at therapeutic doses, BZN treatment elicits an oxidative stress in rat hepatocytes.     

Structural determinants in domain II of human glutathione transferase M2-2 govern the characteristic activities with aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidine, and 1,2-dichloro-4-nitrobenzene

Two human Mu class glutathione transferases, hGST M1-1 and hGST M2-2, with high sequence identity (84%) exhibit a 100-fold difference in activities with the substrates aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidine (cyanoDMNG), and 1,2-dichloro-4-nitrobenzene (DCNB), hGST M2-2 being more efficient. A sequence alignment with the rat Mu class GST M3-3, an enzyme also showing high activities with aminochrome and DCNB, demonstrated an identical structural cluster of residues 164-168 in the alpha6-helices of rGST M3-3 and hGST M2-2, a motif unique among known sequences of human, rat, and mouse Mu class GSTs. A putative electrostatic network Arg107-Asp161-Arg165-Glu164(-Gln167) was identified based on the published three-dimensional structure of hGST M2-2. Corresponding variant residues of hGSTM1-1 (Leu165, Asp164, and Arg167) as well as the active site residue Ser209 were targeted for point mutations, introducing hGST M2-2 residues to the framework of hGST M1-1, to improve the activities with substrates characteristic of hGST M2-2. In addition, chimeric enzymes composed of hGST M1-1 and hGST M2-2 sequences were analyzed. The activity with 1-chloro-2,4-dinitrobenzene (CDNB) was retained in all mutant enzymes, proving that they were catalytically competent, but none of the point mutations improved the activities with hGST M2-2 characteristic substrates. The chimeric enzymes showed that the structural determinants of these activities reside in domain II and that residue Arg165 in hGST M2-2 appears to be important for the reactions with cyanoDMNG and DCNB. A mutant, which contained all the hGST M2-2 residues of the putative electrostatic network, was still lacking one order of magnitude of the activities with the characteristic substrates of wild-type hGST M2-2. It was concluded that a limited set of point mutations is not sufficient, but that indirect secondary structural affects also contribute to the hGST M2-2 characteristic activities with aminochrome, cyanoDMNG, and DCNB.     

Modulation of drug-metabolizing enzymes by extracts of a herbal medicine Evodia rutaecarpa in C57BL/6J mice

Evodia rutaecarpa is a traditional Chinese medicine used for the treatment of gastrointestinal disorders and headache. To assess the possible drug interactions, effects of methanol and aqueous extracts of E. rutaecarpa on drug-metabolizing enzymes, cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. Treatment of mice with methanol extract by gastrogavage caused a dose-dependent increase of liver microsomal 7-ethoxyresorufin O-deethylation (EROD) activity. In liver, methanol extract at 2 g/kg caused 47%, 7-, 8-, 4-fold, 81% and 26% increases of benzo(a)pyrene hydroxylation (AHH), EROD, 7-methoxyresorufin O-demethylation (MROD), 7-ethoxycoumarin O-deethylation (ECOD), benzphetamine N-demethylation, and N-nitrosodimethylamine N-demethylation activities, respectively. Aqueous extract at 2 g/kg caused 68%, 2-fold, and 83% increases of EROD, MROD, and ECOD activities, respectively. For conjugation activities, methanol extract elevated UGT and GST activities. Aqueous extract elevated UGT activity without affecting GST activity. Immunoblot analyses showed that methanol extract increased the levels of CYP1A1, CYP1A2, CYP2B-, and GSTYb-immunoreactive proteins. Aqueous extract increased CYP1A2 protein level. In kidney, both extracts had no effects on AHH, ECOD, UGT, and GST activities. Three major bioactive alkaloids rutaecarpine, evodiamine, and dehydroevodiamine were present in both extracts. These alkaloids at 25 mg/kg increased hepatic EROD activity. These results demonstrated that E. rutaecarpa methanol and aqueous extracts could affect drug-metabolizing enzyme activities. Rutaecarpine, evodiamine, and dehydroevodiamine contributed at least in part to the increase of hepatic EROD activity by extracts of E. rutaecarpa. Thus, caution should be paid to the possible drug interactions of E. rutaecarpa and CYP substrates.     

The effect of vitamin A deficiency on hepatic, renal and pulmonary glutathione S-transferase activities in the rat.

Feeding male weanling rats on a vitamin A-deficient diet for 6 weeks resulted in significant increases (44-57%) in glutathione S-aryl-, S-aralkyl- S-alkyl- and S-epoxidetransferase activities in the liver cytosol. Only the S-aralkyl- (27%) and S-alkyltransferase (14%) activities were significantly increased in the kidney as a result of deficiency. There was no effect on any of the pulmonary glutathione S-transferase activities. The increases in hepatic transferase activities were due primarily to increases (25-96%) in the apparent Vmax. There were no changes in the apparant Km of any of the four drug substrates employed. With 3,4-dichloronitrobenzene as the second substrate, the apparent Km for glutathione was increased by over 2-fold in vitamin A-deficient livers as compared with controls. The relationship between these results and enhanced susceptibility to chemical carcinogens in vitamin A deficiency is briefly discussed, and comparison is made between the effects of this nutritional state and pretreatment with drug inducers on the glutathione S-transferases.     

Protective effects of silymarin, a milk thistle (Silybium marianum) derivative on ethanol-induced oxidative stress in liver

The production of reactive oxygen species (ROS) is considered to be a major factor in oxidative cell injury. The antioxidant activity or the inhibition of the generation of free radicals is important in providing protection against such hepatic damage. Silymarin, derived from the milk thistle plant, Silybium marianum, has been used in traditional medicine as a remedy for diseases of the liver and biliary tract. In the present study, the effect of hepatoprotective drug silymarin on body weight and biochemical parameters, particularly, antioxidant status of ethanol-exposed rats was studied and its efficacy was compared with the potent antioxidant, ascorbic acid as well as capacity of hepatic regeneration during abstention. Ethanol, at a dose of 1.6 g/kg body wt/day for 4 wks affected body weight in 16-18 week-old male albino rats (Wistar strain weighing 200-220 g). Thiobarbituric acid reactive substance (TBARS) level, superoxide dismutase (SOD), and glutathione-s-transferase (GST) activities were significantly increased, whereas GSH content, and catalase, glutathione reductase (GR) and GPx (glutathione peroxidase) activities significantly reduced, on ethanol exposure. These changes were reversed by silybin and ascorbic acid treatment. It was also observed that abstinence from ethanol might help in hepatic regeneration. Silybin showed a significant hepatoprotective activity, but activity was less than that of ascorbic acid. Furthermore, preventive measures were more effective than curative treatment.     

Some effects of the fungicide propiconazole on cytochrome P450 and glutathione S-transferase in brown trout (Salmo trutta)

The fungicide propiconazole (1-(2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl)-1H-1,2,4-triazole) induced the hepatic cytochrome P4501A (CYP1A) activity towards ethoxyresorufin-O-deethylase (EROD), the content of CYP1A protein as quantified by enzyme-linked immunosorbent assay (ELISA) and the glutathione S-transferase (GST) activity towards the three commonly used substrates CDNB(1-chloro-2,4-dinitrobenzene), cumene hydroperoxide (CU) and ethachrynic acid (EA) in brown trout (Salmo trutta) depending on dose and body weight. An exponential dose–response relationship existed between propiconazole exposure and CYP1A activity. A 2. order polynomial regression of the propiconazole concentration (square root transformed) on the data for CDNB, EU and CU revealed a bell-shaped pattern of the GST induction. Reverse-phase HPLC of the GSH-affinity chromatography purified GST isozymes in trout exposed to respectively 8.3, 23, 93, 313 and 606 μg l⁻¹ propiconazole in the water indicated that the propiconazole treatment may lead to changes in the composition of the subunits compared to the controls. Thus, propiconazole exposure through the water changed the properties of the brown trout hepatic CYP1A and GST, and these changes may be used as a bioindicator on the molecular level of exposure and effect of propiconazole in controlled experiments. The use in monitoring of propiconazole exposure under natural field conditions is possible, however needs further investigation.     

Activation of rat glutathione transferases in class mu by active oxygen species

The activities of rat glutathione transferases (GSTs) 3-3, 3-4, 4-4 in Class mu towards 1-chloro-2,4-dinitrobenzene (CDNB) but not 1,2-dichloro-4-nitrobenzene were increased up to 5-fold during preincubation with 0.4 mM xanthine and xanthine oxidase in 50 mM potassium phosphate, pH 7.8, containing 0.1 mM EDTA. The activated GST 3-4, purified by S-hexylglutathione affinity chromatography after the treatment, had a higher specific activity (130 units/mg) than that of the nontreated (35 units/mg), the Km and Vmax values for glutathione or CDNB also were increased. Other rat GSTs in Class alpha and pi were inactivated by the same treatment. In the presence of superoxide dismutase, the activation of GST 3-4 did not occur.     

Effect of lithium on hepatic drug-metabolizing enzymes of protein-deficient rats

Protein deficiency was produced by feeding synthetic 8%-protein diet. Lithium carbonate at the dose level of 1.1g/kg diet was administered to normal and protein-deficient rats for a period of one mo. A significant inhibition in the levels of cytochrome (cyt) P₄₅₀, cyt b₅, glutathione (GSH), glutathione S-transferase (GST) and glutathione peroxidase (GPx), but an increase in γ-glutamyl transpeptidase (γ-GT), was observed in low-protein LP-fed rats. Lithium treatment to normal rats caused no significant change in the activities of cyt P₄₅₀, cyt b₅, GST, and GSH levels, whereas there was elevation in the activities of γ-GT and GPx and suppression in glutathione reductase (GRd) activity. Lithium administration to LP-fed rats resulted in significant increases in the hepatic γ-GT and GPx activities.