Glutathione transferase isoenzymes in cultured rat hepatocytes.
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Animals↗
Biomedical subjects
B Ketterer
Publications and source records attributed to B Ketterer.
Stereoselectivity of rat liver glutathione transferase isoenzymes for alpha-bromoisovaleric acid and alpha-bromoisovalerylurea enantiomers.
The stereoselectivity of purified rat GSH transferases towards alpha-bromoisovaleric acid (BI) and its amide derivative alpha-bromoisovalerylurea (BIU) was investigated. GSH transferase 2-2 was the only enzyme to catalyse the conjugation of BI and was selective for the (S)-enantiomer. The conjugation of (R)- and (S)-BIU was catalysed by the isoenzymes 2-2, 3-3 and 4-4. Transferase 1-1 was less active, and no catalytic activity was observed with transferase 7-7. Isoenzymes 1-1 and 2-2 of the Alpha multigene family preferentially catalysed the conjugation of the (S)-enantiomer of BIU (and BI), whereas isoenzymes 3-3 and 4-4 of the Mu multigene family preferred (R)-BIU. The opposite stereoselectivity of conjugation of BI and BIU previously observed in isolated rat hepatocytes and the summation of activities of enzymes known to be present in hepatocytes on the basis of present data are in accord.
Animals↗
The role of glutathione transferase in the detoxication and repair of lipid and DNA hydroperoxides.
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Animals↗
The spontaneous and enzymatic reaction of N-acetyl-p-benzoquinonimine with glutathione: a stopped-flow kinetic study.
The spontaneous and glutathione (GSH) transferase catalyzed reactions of GSH and N-acetyl-p-benzoquinonimine (NABQI) have been studied by stopped-flow kinetics. The spontaneous reaction was shown to be first order in NABQI, GSH and inversely proportional to the H+ concentration; e.g., at pH 7.0 and 25 degrees C the second-order rate constant was 3.2 X 10(4) M-1 s-1. Data for the enzymatic reaction gave values for Km of 27, 1.3, 7, and 7 microM and values for kappa cat of 90, 37, 5.1, and 165 s-1 for rat liver GSH transferases 1-1, 2-2, 3-3, and 7-7, respectively. Over a wide range of reactant concentrations and pH, the spontaneous reaction yields three products, namely a GSH conjugate, 3-(glutathion-S-yl)acetaminophen; a reduction product, acetaminophen; and an oxidation product, glutathione disulfide in the proportions 2:1:1. Analysis of products formed after enzymatic reaction showed that both GSH conjugation and the reduction of NABQI to acetaminophen were catalyzed to an extent characteristic of each isoenzyme. With respect to GSH conjugation, GSH transferase isoenzymes were effective in the order 7-7 greater than 2-2 greater than 1-1 greater than 3-3 greater than 4-4, and with respect to NABQI reduction these isoenzymes were effective in the order 1-1 greater than 2-2 greater than 7-7 the position of isoenzymes 3-3 and 4-4 being uncertain. Human GSH transferases delta, mu, and pi behave similarly to the homologous rat enzymes, i.e., toward conjugation in the order pi greater than delta greater than mu and the reduction delta greater than mu greater than pi (for nomenclature see W. B. Jakoby, B. Ketterer, and B. Mannervik, (1984) Biochem. Pharmacol. 33, 2539-2540). Possible mechanisms of the reaction and its effect on the toxicity of NABQI are discussed.
Acetaminophen↗
Protective role of glutathione and glutathione transferases in mutagenesis and carcinogenesis.
Glutathione (GSH) alone detoxifies electrophiles with an effectiveness which depends on the rate of the reaction and the concentration of GSH. If electrophiles are substrates for GSH transferase isoenzymes, the effectiveness of detoxication is much enhanced due to the increased rate of reaction and it is also independent of GSH concentration to low levels of GSH depletion, since the Km for GSH is approximately 0.1 mM. In this paper detoxication of electrophilic metabolites of the hepatocarcinogen N-methyl-4-aminoazobenzene which are not substrates for GSH transferases and the carcinogenic electrophile derived from the hepatocarcinogen aflatoxin B1 which is a poor substrate is compared with detoxication of electrophiles which are good substrates and which although bacterial mutagens are not carcinogenic in organs containing the appropriate GSH transferases. GSH transferases detoxify not only electrophiles derived from xenobiotics, but also endogenous electrophiles which are usually the consequence of free radical damage in the presence of oxygen to lipids and DNA and include lipid and DNA hydroperoxides and alkenals arising from the decomposition of lipid hydroperoxides. Studies in the rat and other mammals show the GSH transferases to be dimers in which the subunits are members of a gene super-family. There are three, perhaps four multigene families namely, alpha containing subunits 1, 2, 8 and 10; mu containing subunits 3, 4, 6 and 9; pi containing subunit 7 and subunits 5 and 5* which are so far unassigned. Subunit 5* is apparently restricted to the nucleus and is noteworthy for its activity towards DNA hydroperoxides. Studies in the human are not as advanced as in the rat but so far reveal close similarities. The ability of GSH transferases to detoxify electrophiles is important in carcinogenesis at a number of points. They may inhibit initiation and tumour proportion, but they may be advantageous to the developing tumour cell, and may be acquired in increased amounts during malignant progression. In many tumour cells the development of lines resistant to anticancer drugs is associated with an increased expression of GSH transferases, particularly GSH transferase pi in human cells.
Animals↗
The separation of glutathione transferase subunits by using reverse-phase high-pressure liquid chromatography.
A simple method is described for the separation and quantification of the subunits of GSH transferases present in rat tissue extracts. This method, involving GSH-agarose affinity chromatography followed by reverse-phase h.p.l.c., is rapid and sufficiently sensitive to measure 5 micrograms of each subunit in a mixture. Examples are given of its application to extracts of rat kidney, adrenal, testicular interstitial cells and seminiferous tubules. The analysis of seminiferous tubules indicates that the technique may be of value for the identification of novel subunits. Preliminary separations of subunits from human GSH transferases are also described.
Animals↗
The binding of an aminoazo dye carcinogen to a specific methionine residue in rat liver alcohol dehydrogenase in vivo.
On the administration of 3'-methyl-N,N-dimethyl-4-aminoazobenzene to rats pure aminoazo dye-bound alcohol dehydrogenase accounting for 45% of the total soluble protein bound aminoazo dye is isolated from the liver soluble supernatant. Tryptic digestion of that purified aminoazo dye-bound enzyme yields an aminoazo dye-bound nonapeptide which has a sequence identical to amino acids 301-309 in the known sequence of alcohol dehydrogenase (H. Jornvall and O. Markovic, Eur. J. Biochem., 29 (1972) 167-174) with the exception of methionine 306 which is replaced by an aminoazo dye modified amino acid. The nature of the aminoazo dye adduct was determined by studying the structure of the related tetrapeptide obtained by Pronase B digestion and shown by proton NMR spectroscopy and fast atom bombardment mass spectroscopy to have the structure 3-(Val. Asn. Pro. Homocystein-S-yl)-4-methylamino-3'-methylazobenzene. This carcinogen-protein adduct is assumed to arise from attack of the ultimate carcinogenic metabolite, N-sulphonyloxy-4-methylamino-3'-methylazobenzene (FF. Kadlubar, J.A. Miller and E.C. Miller, Cancer Res., 36 (1976) 2350-2359) at the sulphur of methionine 306 followed by spontaneous S-demethylation. This highly specific reaction of carcinogen with alcohol dehydrogenase lowers its Vmax and increases its Km with cyclohexanone thereby reducing its catalytic efficiency for this substrate. This highly specific reaction of the carcinogen with alcohol dehydrogenase may be regarded as a major detoxication reaction.
Alcohol Dehydrogenase↗
Molecular biology of glutathione transferases.
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Amino Acid Sequence↗
In vivo and in vitro formation of glutathione conjugates from the K-region epoxides of 1-nitropyrene.
4,5-Epoxy-4,5-dihydro-1-nitropyrene (1-nitropyrene 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-nitropyrene 9,10-oxide), which are electrophilic metabolites formed during the metabolism of the environmental pollutant, 1-nitropyrene, reacted slowly with glutathione. The rate of conjugation was greatly enhanced by the addition of purified rat liver glutathione (GSH) transferases, with transferases 3-3 and 4-4 exhibiting higher catalytic activities than transferases 1-1, 2-2 and 7-7. Two GSH conjugates were formed from each of the oxides: 1-nitropyrene 4,5-oxide gave a 1:1 mixture of 4-(glutathion-S-yl)-5-hydroxy-4,5-dihydro-1-nitropyrene and 5-(glutathion-S-yl)-4-hydroxy-4,5-dihydro-1-nitropyrene while 1-nitropyrene 9,10-oxide gave a 2:1 mixture of 9-(glutathion-S-yl)-10-hydroxy-9,10-dihydro-1-nitropyrene and 10-(glutathion-S-yl)-9-hydroxy-9,10-dihydro-1-nitropyrene. Both K-region oxides were converted to trans-dihydrodiols by hepatic microsomal epoxide hydrase, and faster rates were observed with 1-nitropyrene 4,5-oxide. In subsequent experiments [4,5,9,10-3H]1-nitropyrene was administered to Sprague-Dawley rats by intravenous and intraperitoneal injections. HPLC analysis of biliary metabolites indicated the presence of four GSH conjugates that were identical to those obtained from reactions of the K-region oxides with GSH. In addition, glucuronide conjugates were detected from trans-4,5-dihydroxy-4,5-dihydro-1-nitropyrene (1-nitropyrene trans-4,5-dihydrodiol) but not trans-9,10-dihydroxy-9,10-dihydro-1-nitropyrene (1-nitropyrene trans-9,10-dihydrodiol). These data combined with earlier studies indicate that 1-nitropyrene is oxidized preferentially to 1-nitropyrene 4,5-oxide and that, while the main detoxification pathway of 1-nitropyrene 9,10-oxide is GSH conjugation, 1-nitropyrene 4,5-oxide is excreted via both GSH conjugation and dihydrodiol formation followed by O-glucuronidation.
Animals↗
Lipid peroxidation in choline-methionine deficiency.
A deficiency of choline and methionine is hepatocarcinogenic and is associated with an apparent increase in lipid peroxidation. In this study the susceptibility of microsomes and nuclei to ferritin-dependent lipid peroxidation is examined together with the status of the peroxidation-protective systems. Choline-methionine deficiency caused an increase in Se-independent GSH peroxidases (GSH transferase subunit 2) and membrane vitamin E but a decrease in Se-dependent GSH peroxidase and microsomal GSH peroxidase activity. Choline-methionine deficient microsomes and nuclei were 4-fold more susceptible to lipid peroxidation induced in vitro by physiological concentrations of ferritin/ascorbate/ADP; and the peroxidation was less effectively inhibited by GSH and soluble GSH peroxidases than controls. The results indicate that a decreased level of Se-dependent and membrane GSH peroxidases is involved in the increase in lipid peroxidation observed in choline-methionine deficiency.
Choline Deficiency↗
Tissue distribution of rat glutathione transferase subunit 7, a hepatoma marker.
Polyadenylated RNA isolated from NN-dimethyl-4-aminoazobenzene-induced rat hepatoma was used to prepare a cDNA library in lambda gt10. Full-length clones complementary to mRNA coding for glutathione transferase subunit 7 were isolated and one of these clones (pGSTr7) was fully characterized. In Northern blot analysis, mRNA hybridizing to 32P-labelled pGSTr7 was found in poly(A)-containing RNA isolated from seven normal rat tissues but not from testis and liver. A similar hybridizing mRNA species was also detected in human placental mRNA. The same probe, used in a Southern blot analysis of genomic DNA, suggests the presence of a multigene family in the rat.
Animals↗
Thymine hydroperoxide, a substrate for rat Se-dependent glutathione peroxidase and glutathione transferase isoenzymes.
The thymine hydroperoxide, 5-hydroperoxymethyluracil, is a substrate for Se-dependent glutathione (GSH) peroxidase and the Se-independent GSH peroxidase activity associated with the GSH transferase fraction. These enzymes may contribute to repair mechanisms for damage caused by oxygen radicals. GSH transferases 1-1, 2-2, 3-3, 4-4, 6-6, and 7-7 [(1984) Biochem. Pharmacol. 33, 2539-2540] are shown to differ considerably in their ability to utilize this substrate. For example, high activity is found in GSH transferase 6-6 which is the major isoenzyme in spermatogenic tubules where DNA synthesis is so active and faithful DNA replication so important. The activity of the purified GSH transferase isoenzymes towards 5-hydroperoxymethyluracil is comparable with their activity towards other endogenous substrates related to cellular peroxidation such as linoleate hydroperoxide and 4-hydroxynon-2-enal or biologically important xenobiotic metabolites such as benzo(a)pyrene-7,8-diol-9,10-oxide.
Animals↗
Differential tissue expression of the glutathione transferase multigene family.
The content of GSH transferase mRNAs in poly(A)-containing RNA isolated from eight rat tissues was examined by immunoprecipitation of cell-free translation products and by Northern blotting. Considerable tissue-specific distribution and heterogeneity of immunoprecipitable GSH transferase subunits 1 and 2 synthesized in vitro was observed. These results were confirmed by Northern blotting using a 32P-labelled subunit 1 cDNA probe. The same probe, used in a Southern blot analysis of genomic DNA, provided confirmation that GSH transferase subunits 1 and 2 comprise a multigenic family in the rat. The results show that the selection of cDNA clones coding for chosen subunits can be made easier by making use of qualitative and quantitative tissue differences in GSH transferase mRNAs.
Animals↗
Glutathione transferases and carcinogenesis.
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Animals↗
Sulfamate formation is a major route for detoxification of 2-amino-3-methylimidazo[4,5-f]quinoline in the rat.
The major biliary metabolite of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in the rat was identified as the sulfamate derivative, N-[3-methylimidazo[4,5-f]quinolin-2-yl] sulfamic acid. Identification was accomplished primarily by u.v., 1H-n.m.r. and mass spectrometry of the material isolated from bile and confirmed by comparison with material synthesized by reaction of chlorosulfonic acid with IQ. The sulfamate was shown to be non-mutagenic in bacterial forward mutation assays. Greater than 20% of an administered dose of IQ could be recovered from feces (17%) and urine (5%) as the sulfamate. Very little unmetabolized IQ was recovered in bile, urine, or feces. Thus, the unusual process of N-sulfation is a major contributor to the detoxification and elimination of IQ in the rat.
Animals↗
Glutathione transferases in primary rat hepatomas: the isolation of a form with GSH peroxidase activity.
A previously uncharacterized glutathione (GSH) transferase which is not apparent in normal liver, accounts for at least 25% of the soluble GSH transferase content of primary hepatomas induced by feeding N,N-dimethyl-4-aminoazobenzene. This enzyme is readily isolated, has an isoelectric point of 6.8, is composed of two identical subunits of apparent Mr 26000 and has GSH transferase activity towards a number of substrates including benzo(a)pyrene-7,8-diol-9,10-oxide. It is unusual in that it has GSH peroxidase activity towards fatty acid hydroperoxides but not towards the model substrates, cumene hydroperoxide and t-butyl hydroperoxide. It has been shown by tryptic peptide analysis to be distinct from GSH transferases composed of subunits 1, 2, 3, 4 or 6 and has been designated GSH transferase 7-7.
Animals↗
Glutathione conjugation of the carcinogenic and mutagenic electrophile (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetra hydrobenzo[a]pyrene catalyzed by purified rat liver glutathione transferases.
The kinetics of the enzyme-catalyzed conjugation of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]-pyrene [(+/-)-anti-BPDE] with glutathione (GSH) by the following purified soluble rat liver GSH transferases: 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4 have been studied. When BPDE concentration was varied while GSH concentration remained constant (1 mM), linear Lineweaver-Burk plots were obtained: maximum rates of conjugation mediated by GSH transferases 1-1, 1-2, 2-2, 3-3, 3-4 and 4-4 were 105, 72, 83, 35, 179 and 357 nmol/min/mg protein, respectively. When GSH concentration was varied while BPDE concentration remained constant (40 microM), biphasic Lineweaver-Burk plots were obtained in each case with a break point of 0.2 mM GSH below which the affinity of these enzymes for GSH was apparently greater. These results are discussed with respect to the detoxication of benzo[a]pyrene (BP) in vivo.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Studies on the detoxication of microsomally-activated aflatoxin B1 by glutathione and glutathione transferases in vitro.
Aflatoxin B1 (AFB1)-8,9-oxide, the proposed ultimate carcinogen is conjugated enzymically with glutathione (GSH) to give 8-(S-glutathionyl)-9-hydroxy-8,9-dihydro AFB1 (AFB1-SG). The GSH conjugate isolated from rat bile was shown, on the basis of 1H n.m.r. to be identical to AFB1-SG. Of the seven soluble rat liver GSH transferases tested, namely GSH transferases 1-1, 1-2, 2-2, 3-3, 3-4, 4-4 and 5-5 (see reference 1 for the new system of nomenclature), only the first three were active with microsomally generated AFB1-8,9-oxide, their rates of conjugation being 1.1, 0.61 and 0.64 nmol/min/mg enzyme, respectively. AFB1-SG is a thioacetal, but it was not formed from the incubation of the hemiacetal, AFB1-8,9-dihydrodiol, with GSH or GSH plus GSH transferase 1-1 plus 1-2. The covalent binding of in vitro microsomally activated AFB1 to DNA and the formation of AFB1-SG were linearly related to AFB1 concentration in the range of 0.2-2 micrograms/ml. DNA binding was decreased by 38% by the competing formation of AFB1-SG throughout this range of concentrations. These results are in accord with the observation of Scott Appleton et al. (Cancer Res., 42, 3659-3662) that, in the rat in vivo, there is no evident threshold for the binding of AFB1 to DNA. These findings are also consistent with the further observation, reported in this paper that GSH and GSH transferases have no effect on the mutagenicity of microsomally activated AFB1 to Salmonella typhimurium TA 100.
Aflatoxin B1↗