In vitro interaction of organic mercury compounds with soluble glutathione S-transferases from rat liver

The in vitro interaction of organic mercury compounds with rat liver glutathione S-transferases (GST) was studied, using reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The inhibition of the GST activity was dose dependent, but not linear. The different GST isoenzymes were inhibited to different degrees. Kinetic studies never revealed competitive inhibition, with CDNB or with GSH as the variable substrate. Titration of remaining GSH in appropriate incubation mixtures with organomercurials revealed no GST catalyzed conjugation of these compounds with GSH. These experiments showed a spontaneous conjugation of the mercury compounds with GSH, explaining the parabolic inhibition observed in the kinetic studies with GSH as the variable substrate. Both organic and inorganic mercury are spontaneously conjugated with GSH, but interact with GST by direct binding to these proteins. This binding could have a protective function against mercury. No qualitative differences between organic and inorganic mercury were detected.     

In vitro interaction of dithiocarb with rat liver glutathione S-transferases

The in vitro interaction of dithiocarb (DTC) with rat liver glutathione S-transferase was studied, using reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The inhibition of the GST activity by DTC was dose dependent, but not linear. The different GST isoenzymes were inhibited to different degrees. Kinetic studies revealed uncompetitive inhibition towards GSH for GST AA, and an intermediate kinetic pattern between uncompetitive and noncompetitive inhibition for the other GST isoenzymes. With respect to CDNB, mixed type inhibition was found for most GST isoenzymes, and nearly uncompetitive inhibition for GST AA and M. Titration of remaining GSH in appropriate incubation mixtures with DTC revealed no GST catalyzed conjugation of DTC with GSH. It is concluded that DTC interact with GST by direct binding to these proteins. This binding could have a protective function against DTC.     

Inhibition of rat liver glutathione S-transferases by glutathione conjugates and corresponding L-cysteines and mercapturic acids

Glutathione S-transferases from rat liver were partially purified by ion exchange chromatography. Active peaks, tentatively identified as containing the 1-2, 2-2, 3-3, 3-4, 4-4 and 5-5 isoenzymes were kept for study. The glutathione conjugates, S-hexyl-, S-benzyl- and S-(2,4-dinitrophenyl) L-glutathione were tested as inhibitors of the enzymes. The 1-2, 2-2, 3-3 and 3-4 fractions were inhibited to similar extents by these conjugates. For all enzymes the hexyl conjugate at 0.1 mM concentration was strongly inhibitory, the benzyl conjugate moderately so and the dinitrophenyl compound was only weakly inhibitory. In contrast, the epoxide conjugating activity in the 4-4 and 5-5 peak was barely affected by the substituted glutathiones at 0.1 mM concentrations. Studies on a purified ligandin (isoenzyme 1-2) from rat liver showed that further metabolism of the glutathione conjugates, to the corresponding cysteines or mercapturic acids, resulted in products with inhibitory properties approximately three orders of magnitude less potent than those of the parent S-substituted glutathiones.     

Inhibition by sulfasalazine of LTC synthetase and of rat liver glutathione S-transferases

Sulfasalazine inhibited the formation of sulfidopeptide leukotrienes in ionophore A23187-challenged rat basophil leukemia cells in a dose-dependent fashion (EC50 = 0.11 mM). This compound also inhibited the solubilized, particulate LTC synthetase of RBL cells (EC50 = approximately 0.4 mM in the presence of a standard substrate mixture). The inhibition of LTC synthetase was paralleled by the capacity of sulfasalazine to potently inhibit several subfractions of the cytosolic rat liver glutathione S-transferases. The kinetics of the inhibition of the glutathione S-transferases, with 2,4-dinitrochlorobenzene as the substrate, were consistent with competitive inhibition with respect to glutathione (Ki values 0.21 +/- 0.05 to 0.46 +/- 0.096 microM in three discrete fractions). Inhibition with respect to the chromophoric substrate was uncompetitive in two of the three fractions examined (K'i values 0.61 +/- 0.13 and 1.05 +/- 0.14 microM) and non-competitive in the third (Ki = 0.72 microM). The inhibition of the LTC synthetase of RBL cells was also competitive with respect to glutathione (Ki = 120 microM). Both 5-aminosalicyclic acid and N'-2-pyridylsulfanilamide inhibited the one glutathione S-transferase fraction which was examined, and N'-2-pyridylsulfanilamide also inhibited the LTC synthetase. However, the kinetics of the inhibition of the liver enzyme by these compounds were not consistent with a competitive mechanism relative to glutathione, and the Ki values were at least 100 times greater than the ones for sulfasalazine on the same enzyme.     

Glutathione and glutathione S-transferases in rat liver after inhalation of halothane and enflurane

Male Sprague-Dawley rats were exposed in inhalation chambers to halothane and enflurane in concentrations from 50 ppm-1000 ppm (0.0025-0.05 minimum alveolar concentration; MAC) 6 h a day for 3-9 days. Repeated subanaesthetic concentrations were used to avoid effects of general anaesthesia and to increase the metabolized fraction of the inhaled anaesthetics. Exposure to 0.05 MAC of halothane (500 ppm) and enflurane (1000 ppm) for 9 days reduced the activity of glutathione S-transferases. A decrease in liver concentration of reduced glutathione (GSH) was observed after inhalation of enflurane, probably caused by metabolic release of inorganic fluoride. The results indicate a decreased detoxifying capacity of rat liver under the given conditions. Inhalation of occupational related concentrations of the anaesthetics (50 ppm) did neither affect the activity of the transferases nor the concentration of GSH in rat liver.     

Structural, functional and hybridization studies of the glutathione S-transferases of rat liver

We have purified five forms of glutathione S-transferase from rat liver. One form was the glutathione S-transferase B (ligandin), which is composed of two non-identical subunits with molecular weights of 22,000 (Ya) and 25,000 (Yc). Two of the other transferases were Ya and Yc homodimers. The other two transferases were also homodimers, but their subunit, Yb, had a molecular weight of 24,000. The three proteins containing either Ya or Yc subunits had similar substrate specificities, and all three contained peroxidase activity. The greatest peroxidase activity was present in proteins containing the Yc subunit. Enzymes composed of Yb subunits had minimal peroxidase activity in addition to different substrate specificities. The Ya and Yc containing enzymes bound the ligands bilirubin, and indocyanine green with high affinity (KD less than 5 microM), although the KD values of the YcYc protein were consistently 4- to 12-fold greater than those of the other two transferases. Studies were performed to define the origins of the various isozymes. There was no evidence for conversion of Yc to either Ya or Yb during storage or under conditions favorable to proteolysis. Hybridization studies were performed under denaturing conditions (6 M guanidine-HCl), and a YaYc hybrid was formed from the YaYa and YcYc proteins. In addition, both YaYa and YcYc hybrids were formed from transferase B. The hybrids were functionally similar to the proteins isolated originally from the liver. Attempts to form a YaYb hybrid from the YbYb and YaYa transferases were unsuccessful. This result is consistent with the lack of this enzyme form in the liver. Glutathione S-transferase B and the Ya and Yc homodimers appeared to be hybrids of common subunits. These three transferases had very similar functional and structural characteristics and differed from the transferases that are composed of Yb subunits.     

Interaction of organotin compounds with three major glutathione S-transferases in zebrafish

Glutathione S-transferases (GSTs) play an important role in cellular detoxification as enzymatic mediators of glutathione (GSH) conjugation with a wide range of deleterious compounds, enabling their easier extrusion out of the organism. GSTs are shown to interact with organotin compounds (OTCs), known environmental pollutants, either as substrates, serving as electrophilic targets to the nucleophilic attack of GSH, or as noncompetitive inhibitors by binding to GST active sites and disrupting their enzymatic functions. There is a wide range of deleterious biological effects caused by OTCs in low concentration range. Their environmental concentrations, further potentiated by bioaccumulation in aquatic organisms, correspond with inhibitory constants reported for Gsts in zebrafish, which implies their environmental significance. Therefore, our main goal in this study was to analyze interactions of three major zebrafish Gsts – Gstp1, Gstr1, and Gstt1a – with a series of ten environmentally relevant organotin compounds. Using previously developed Gst inhibition assay with recombinant Gst proteins and fluorescent monochlorobimane as a model substrate, we determined Gst inhibitory constants for all tested OCTs. Furthermore, in order to elucidate nature of Gst interactions with OTCs, we determined type of interactions between tested Gsts and the strongest OTC inhibitors. Our results showed that OTCs can interact with zebrafish Gsts as competitive, noncompetitive, or mixed-type inhibitors. Determined types of interactions were additionally confirmed in silico by molecular docking studies of tested OTCs with newly developed Gst models. In silico models were further used to reveal structures of tested Gsts in more detail and identify crucial amino acid residues which interact with OTCs within Gst active sites. Our results revealed more extensive involvement of Gstr1 and Gstp1 in detoxification of numerous tested OTCs, with low inhibitory constants in nanomolar to low micromolar range and different types of inhibition, whereas Gstt1a noncompetitively interacted with only two tested OTCs with significantly higher inhibitory constants.     

Covalent binding of acrylonitrile to specific rat liver glutathione S-transferases in vivo.

Acrylonitrile (AN) is an industrial vinyl monomer that is acutely toxic. When administered to rats, AN covalently binds to tissue proteins in a dose-dependent but nonlinear manner [Benz, F. W., Nerland, D. E., Li, J., and Corbett, D. (1997) Fundam. Appl. Toxicol. 36, 149-156]. The nonlinearity in covalent binding stems from the fact that AN rapidly depletes liver glutathione after which the covalent binding to tissue proteins increases disproportionately. The identity of the tissue proteins to which AN covalently binds is unknown. The experiments described here were conducted to begin to answer this question. Male Sprague-Dawley rats were injected subcutaneously with 115 mg/kg (2.2 mmol/kg) [2,3-(14)C]AN. Two hours later, the livers were removed, homogenized, and fractionated into subcellular components, and the radioactively labeled proteins were separated on SDS-PAGE. One set of labeled proteins was found to be glutathione S-transferase (GST). Specific labeling of the mu over the alpha class was observed. Separation of the GST subunits by HPLC followed by scintillation counting showed that AN was selective for subunit rGSTM1. Mass spectral analysis of tryptic digests of the GST subunits indicated that the site of labeling was cysteine 86. The reason for the high reactivity of cysteine 86 in rGSTM1 was hypothesized to be due to its potential interaction with histidine 84, which is unique in this subunit.     

Differential regulation of male rat liver glutathione S-transferases: Effects of orchidectomy and hormone replacement

The effects of orchidectomy and hormone replacement on glutathione S-transferase activities in adult male rat liver were investigated. Due to the overlapping yet distinct substrate specificities of the hepatic glutathione S-transferases, we measured activity in cytosol toward four substrates: 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, trans-4-phenylbut-3-en-2-one and p-nitrobenzyl chloride. Orchidectomy resulted in a decrease in transferase activity toward 1-chloro-2,4-dinitrobenzene, trans-4-phenylbut-3-en-2-one and p-nitrobenzyl chloride to 76, 64 and 70% of control. In contrast, transferase activity toward 1,2-dichloro-4-nitrobenzene was increased to 137% of control. To determine the role of specific androgens in the hormonal dependence of the glutathione S-transferases, rats were subcutaneously implanted for 4 weeks with either blank or steroid-filled sustained-release capsules at the time of orchidectomy. Transferase activities toward 1-chloro-2,4-dinitrobenzene or p-nitrobenzyl chloride were increased to control levels by testosterone but not by any of its 5α-reduced metabolites. Transferase activity toward trans-4-phenylbut-3-en-2-one was increased to control level by either dihydrotestosterone or 5α-androstan-3-α, 17β-diol. Activity toward 1,2-dichloro-4-nitrobenzene was decreased to control level by all of the androgens studied, testosterone, dihydrotestosterone, 5α-androstan-3β,17β-diol or 5α-androstan-3α,17β-diol. Thus, the hepatic glutathione S-transferases are under separate control and are differentially regulated by testosterone and its 5α-reduced metabolites.     

Halothane: inhibition and activation of rat hepatic glutathione S-transferases

Multiple halothane anesthesias (1.25 MAC for 1 hr on 3 alternate days) of male Long-Evans rats initially decreased by up to 30% and subsequently increased to up to 185% liver cytosolic glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene, 3,4-dichloro-1-nitrobenzene and trans-4-phenyl-3-buten-2-one and glutathione peroxidase activity. Halothane rapidly and reversibly activated hepatic cytosolic glutathione S-transferases and purified isoenzyme 1-2 but not isoenzymes 1-1 and 3-3. At high concentrations of halothane (ca. 22 mM), maximal activation was ca. 25%. Halothane, enflurane, isoflurane and methoxyflurane, but not the halothane metabolite 1-chloro-2,2-difluoroethylene, inhibited a mixture of liver cytosolic glutathione S-transferases with time (ca. 30% inhibition/15 min). The inhibition exhibited pseudo-first order kinetics (kobs = 0.13 min-1) and an I50 for halothane of greater than or equal to 15 mM. Halothane inhibited glutathione S-transferases 3-3, 3-4, and 4-4 by 50-60%, but did not affect isoenzymes 1-1 and 1-2. The ability of halothane to diminish hepatic glutathione S-transferase activity in vivo may in part reflect the time-dependent inhibition of glutathione S-transferase isoenzymes containing the 3- and 4-subunits.     

Effect of streptozotocin on the glutathione S-transferases of mouse liver cytosol

Streptozotocin (STZ) increased the activity of mouse hepatic glutathione (GSH) S-transferases assayed with 1-chloro-2,4-dinitrobenzene. Nicotinamide administered prior to STZ prevented the hyperglycemia indicative of STZ-induced diabetes, but had no effect on the increase in GSH S-transferase activity caused by the drug. Another diabetogenic agent, alloxan, did not alter GSH S-transferase activity. Thus, streptozotocin may be increasing GSH S-transferase activity directly, and not as a result of the diabetic state the drug induces. Two transferases were characterized from mouse liver cytosol. One was a homodimer with a subunit molecular weight of about 28,000 and a pI of about 8.2. The other was also a homodimer with a subunit molecular weight of about 27,500 and a pI of about 9.2. The pI 8.2 GSH S-transferase was induced by STZ, while the pI 9.2 transferase was decreased by the drug. At least one other transferase appeared to be induced by STZ. Two other nitroso compounds, chlorozotocin and diethylnitrosamine, also increased GSH S-transferase activity, suggesting that this effect may be nitroso related.     

Methyl linoleate ozonide: a substrate for rat glutathione S-transferases

Glutathione S-transferases (GST) were shown to be capable of reducing the toxicity of the ozonide of methyl linoleate (MLO) by catalyzing its reaction with reduced glutathione (GSH). MLO was a substrate for both cytosolic and microsomal GST. Isoenzyme 2-2 demonstrated the highest specific activity. Oxidised glutathione and aldehydes were identified as products of the reaction, with unstable glutathione-conjugates being formed as intermediates only. It was concluded that the GST-activity toward MLO may be similar to the GST-peroxidase activity with lipid hydroperoxides as substrates.     

Interaction of acrylamide with glutathione in rat erythrocytes

Evidence is presented for an enzyme-catalyzed conjugation of acrylamide (ACR) in rat erythrocytes. Daily exposure of rats to ACR for a period of 7, 14 and 21 days resulted in a time-dependent decrease in glutathione content. In vitro incubation of ACR with rat erythrocytes suspension caused a concentration-dependent decrease in glutathione levels. Red blood cell (RBC) enzyme-catalyzed conjugation of ACR with glutathione increased with protein concentration and was dependent on pH and time of incubation. Glutathione-S-transferase (GST) activity using acrylamide and 1-chloro 2,4-dinitrobenzene (CDNB) as substrates followed the order: liver > kidney > brain > erythrocytes. Glutathione peroxidase activity of RBCs was inhibited by the in vitro addition of ACR to erythrocytes. These results suggest that rat erythrocytes are equipped with the mechanism which can inactivate toxic electrophilic chemicals, such as acrylamide.     

Effect of diabetes and starvation on the activity of rat liver epoxide hydrolases, glutathione S-transferases and peroxisomal beta-oxidation

The activities of peroxisomal beta-oxidation, cytosolic and microsomal epoxide hydrolase as well as soluble glutathione S-transferases have been determined in the livers of alloxan- and streptozotocin-diabetic male Fischer-344 rats. Five, seven and ten days after initiation of diabetes serum glucose levels were elevated 3.6-, 5.7- to 6.2- and 6-fold, while the activities of peroxisomal beta-oxidation and cytosolic epoxide hydrolase were elevated 1.5- and 2.5-fold, 1.4- and 2.7-fold and 1.3- and 2.0-fold, respectively. The activities of microsomal epoxide hydrolase and glutathione S-transferases were reduced to about 71% and 80% of controls. Application of 10 I.U./kg depot insulin twice a day for 10 consecutive days to alloxan-diabetic individuals approximately restored the initial glucose levels and enzyme activities except for peroxisomal beta-oxidation. Starvation of Fischer-344 rats for 48 hours and 5 days similarly resulted in a 1.3-fold to 2.1-fold and 1.2- to 1.6-fold increase in peroxisomal beta-oxidation and cytosolic epoxide hydrolase activity, respectively. Microsomal epoxide hydrolase was significantly decreased to 57% and 61% of control activity whereas glutathione S-transferase was only marginally reduced to 91% and 92%. Except for glutathione S-transferases initial enzyme activities were restored upon refeeding within 10 days. These results are similar to those obtained upon feeding of hypolipidemic compounds with peroxisome proliferating activity, and may indicate that high levels of free fatty acids or their metabolites which are known to accumulate in liver in both metabolic states may act as endogenous peroxisome proliferators.     

Induction of class alpha glutathione S-transferases by 4-methylthiazole in the rat liver: role of oxidative stress

The expression of glutathione S-transferase (GST) is a crucial factor in determining the sensitivity of cells and organs in response to a variety of toxicants. Expression of class alpha GST genes by methyl-substituted thiazoles was assessed in the rat liver. Northern blot analysis revealed that 4-methylthiazole (4-MT) elevated rGSTA2, A3, A5 and M1 mRNAs in the liver by 19-, 4-, 6- and 9-fold at 24 h after treatment, respectively, as compared to control. Consecutive 3-day treatment with 4-MT resulted in 4- to 7-fold increases in rGSTA and M1 mRNAs. Multiple treatments with 5-methylthiazole (5-MT) caused marginal increases in GST mRNAs in spite of the large increases in certain GST mRNAs at 24 h. Either 4, 5-dimethylthiazole (DT) or 2,4,5-trimethylthiazole (TT) minimally affected the rGSTA and rGSTM mRNA expression at 1-3 day(s). Western blot analysis showed that 4-MT induced rGSTA1/2, rGSTA3/5 and rGSTM1 proteins by 2.6-, 2.1- and 2.1-fold at 3 days, respectively, while other methylthiazoles failed to induce the GST subunits. Starving rats were treated with a lower dose of methylthiazoles to study the role of oxidative stress in the mRNA expression. The levels in rGSTA2/3/5 mRNAs were significantly enhanced by 4-MT in starving rats, whereas rGSTM1/2 mRNAs were not further increased. Other methylthiazoles were inactive in enhancing the mRNAs in starving animals. Pretreatment of starving rats with either cysteine or methionine completely prevented the increases in class alpha GST mRNAs by 4-MT. Data showed that 4-MT induces class alpha GSTs with the increases in the mRNAs, whereas 5-methyl-, dimethyl- and trimethyl-substituted thiazoles were minimally active. Increases in the class alpha GST mRNAs by 4-MT may be associated with the oxidative stress in hepatocytes, as supported by starvation and sulfur amino acid experiments.     

Interaction of nefopam and orphenadrine with the cytochrome P-450 and the glutathione system in rat liver

Nefopam, a cyclic analogue of orphenadrine, exhibits a type I (substrate) and a type II (ligand) interaction with ferri-cytochrome P-450 in control and phenobarbitone induced rat hepatic microsomes respectively. In-vitro metabolism of nefopam in phenobarbitone-induced microsomes leads to the production of a reactive metabolite which complexes with cytochrome P-450. In contrast to the known complexation of orphenadrine, complexation by nefopam can be inhibited by glutathione (GSH, 0.1-1.0 mM). However, in-vivo administration of nefopam to rats does not diminish the GSH content of liver cytosol nor increase oxidized glutathione levels nor alter the activities of GSH transferase and GSH peroxidase. In-vivo administration does not lead to cytochrome P-450 induction nor cytochrome P-450 complexation as has been shown for orphenadrine. Finally, nefopam inhibits the NADPH dependent endogenous H2O2 production in both control and phenobarbitone-induced microsomes.     

Interaction of doxorubicin with nuclei isolated from rat liver and kidney

The interaction of doxorubicin with nuclei isolated from rat liver and kidney was studied by fluorospectrometry . The nuclei had at least two different types of binding sites for the drug. Both Mg2+ and Ca2+ competitively inhibited the binding of doxorubicin to the nuclei, which showed a remarkable temperature dependency. No significant difference was observed between the numbers of binding sites (n = 6.70 X 10(-2) mol/mol of DNA for liver; 6.41 X 10(-2) mol/mol of DNA for kidney) or the affinity constants (Ka = 4.85 X 10(5) M-1 for liver; 5.41 X 10(5) M-1 for kidney) under quasi-physiological conditions. These results obtained from in vitro binding experiments support previous suggestions that the differences in the in vivo distribution of doxorubicin among tissues are not due to differences in the nuclear binding of the drug. The amount of nuclei per gram of tissue is the primary determinant of the characteristic tissue distribution of doxorubicin.