Irreversible inhibition of human glutathione S-transferase isoenzymes by tetrachloro-1,4-benzoquinone and its glutathione conjugate

The quinones tetrachloro-1,4-benzoquinone (1,4-TCBQ) and its glutathione conjugate (GS-1,4-TCBQ) are potent irreversible inhibitors of most human glutathione S-transferase (GST) isoenzymes. Human pi, psi, and mu are almost completely inhibited at a molar ratio 1,4-TCBQ/GST = 2/1. The isoenzyme B1B1 was inhibited up to 75%, and higher concentrations (1,4-TCBQ/GST = 6/1) were needed to reach this maximum effect. For these isoenzymes 75-85% of the maximal amount of inhibition was already reached on incubation of equimolar ratios of 1,4-TCBQ and subunit GST, while approximately 1 nmol (0.82-0.95) 1,4-[U-14C]TCBQ per nmol subunit GST could be covalently bound. These results suggest that these GST isoenzymes possess only one cysteine in or near the active site of GST, which is completely responsible for the inhibition. In agreement, human isoenzyme B2B2 which possesses no cysteine, was not inhibited and no 1,4-TCBQ was bound to it. The rate of inhibition was studied at 0 degrees: 1,4-TCBQ, trichloro-1,4-benzoquinone and GS-1,4-TCBQ all inhibit GST very fast. Especially for B1B1, the inhibition by the glutathione conjugate is significantly faster than inhibition by 1,4-TCBQ: the glutathione moiety seems to target the quinone to the enzyme. For the other isoenzymes only minor differences are observed between 1,4-TCBQ and its glutathione conjugate under the conditions used.     

Differential induction of rat hepatic glutathione S-transferase isoenzymes by hexachlorobenzene and benzyl isothiocyanate. Comparison with induction by phenobarbital and 3-methylcholanthrene

Male Wistar rats were treated with hexachlorobenzene, benzyl isothiocyanate, phenobarbital or 3-methylcholanthrene. Hepatic cytosolic glutathione S-transferase (GST) activity was determined with the substrates 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, ethacrynic acid and trans-4-phenyl-3-buten-2-one. Cytosolic glutathione peroxidase activity was measured with cumene hydroperoxide. GST activity toward 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene and ethacrynic acid was enhanced by all compounds, hexachlorobenzene and 3-methylcholanthrene causing the largest and the smallest increase respectively. Trans-4-phenyl-3-buten-2-one-conjugating activity exhibited only small changes, while peroxidase activity with cumeme hydroperoxide was not changed by any of the inducing agents. GST isoenzymes were purified on S-hexylglutathione Sepharose 6B and separated by means of FPLC-chromatofocusing, to evaluate effects on the GST isoenzyme pattern. Hexachlorobenzene and phenobarbital both caused an increase in the relative amounts of subunits 1 and 3 when compared with subunits 2 and 4 respectively. For 3-methylcholanthrene only induction of subunit 1 was observed, possibly due to the relatively low induction levels of total GST activity. In benzyl isothiocyanate-treated animals, an induction of subunit 3 was found as well as an increase in the relative amount of subunit 2. Thus, benzyl isothiocyanate behaves differently from hexachlorobenzene, phenobarbital and 3-methylcholanthrene as an inducing agent of rat hepatic glutathione S-transferases.     

Inhibition of glutathione S-transferases by thonningianin A, isolated from the African medicinal herb, Thonningia sanguinea, in vitro.

There is evidence that increased expression of glutathione S-transferase (EC: 2.5.1.18, GST) is involved in resistance of tumor cells against chemotherapeutic agents. In this study we investigated the inhibitory effects of thonningianin A (Th A), a novel antioxidant isolated from the medicinal herb, Thonningia sanguinea on uncharacterized rat liver GST and human GST P1-1. Using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, rat liver cytosolic GST activity was inhibited by Th A in a concentration dependent manner with 50% inhibition concentration (IC50) of 1.1 microM. When Th A was compared with known potent GST inhibitors the order of inhibition was tannic acid>cibacron blue>hematin>Th A>ethacrynic acid with CDNB as substrate. Th A also exhibited non-competitive inhibition towards both CDNB and glutathione. Furthermore, using 1,2-dichloro-4-nitrobenzene, ethacrynic acid and 1,2-epoxy-3-(p-nitrophenoxy) propane as substrates Th A at 1.0 microM inhibited cytosolic GST by 2%, 12% and 36% respectively. Human GST P1-1 was also inhibited by Th A with an IC50 of 3.6 microM. While Th A showed competitive inhibition towards CDNB it exhibited non-competitive inhibition towards GSH of the human GST P1-1. These results suggest that Th A represents a new potent GST in vitro inhibitor.     

Interaction of benzo- and naphthoquinones with soluble glutathione S-transferases from rat liver

The in vitro interaction of 1,4-benzoquinone, 1,2- and 1,4-naphthoquinone, and 2-methyl-1,4-naphthoquinone with rat liver glutathione S-transferases (GST) was studied, using reduced glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) as substrates. The inhibition of the GST activity by quinones in crude extracts was dose dependent. While most of the dihydroxynaphtalenes investigated also inhibited the GST activity, dihydroxybenzenes and catecholamines did not. The quinones inhibited all the GST isoenzymes, albeit at different degrees. Kinetic studies revealed mixed type function inhibition towards glutathione and competitive inhibition towards CDNB, implicating that quinones are GST substrates. This was further confirmed by titration of remaining glutathione in appropriate incubation mixtures. These results indicate that GST could have a protective function against quinones, and that catecholamines are conjugated with glutathione via a reactive quinone intermediate.     

Inhibition of human hepatic glutathione S-transferase isozymes by ethacrynic acid and its metabolites.

The comparative inhibition of ethacrynic acid (EA) and its known metabolites against glutathione S-transferase (GST) was investigated using human livers procured from kidney donors. EA and all three metabolites of EA had an inhibitory effect against conjugation between 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH). The GSH adduct of EA (EA-GSH) was the most potent inhibitor of GSTs; EA-GSH was approximately one order of magnitude more potent than the parent EA, while L-cysteine conjugate of EA (EA-cysteine) and N-acetyl-L-cysteine conjugate of EA (EA-mercapturate) were approximately two orders of magnitude less potent than the parent EA. Further metabolism of EA-GSH conjugate is suggested to be a detoxification process in terms of GST activities.     

Inhibition of human glutathione S-transferases by bile acids

Glutathione S-transferase (GST) isoenzymes isolated from various human tissues are differentially inhibited by bile acids. Trihydroxy bile acid (lithocholate) was found to be more inhibitory to all the human GST isoenzymes tested in this study, as compared to the monohydroxy (cholate) and dihydroxy (chenodeoxycholate) bile acids. Among the three major classes of GST, mu class isoenzymes are generally inhibited to a greater extent than the alpha and pi class isoenzymes. The results of this study also indicate that differential inhibition of GST by various bile acids may be used to distinguish closely related GST isoenzymes within the mu class of GST isoenzyme. Likewise, the pi class or the anionic isoenzymes of human kidney, placenta, and erythrocytes can be distinguished using bile acid inhibition studies. These studies also provide further support for tissue-specific expression of GST isoenzymes in humans.     

Selective expression of the three classes of glutathione S-transferase isoenzymes in mouse tissues

The suitability of mouse as an animal model for studying the glutathione S-transferase (GST)-mediated detoxification mechanisms has been studied by analyzing the expression of the alpha, mu, and pi classes of glutathione S-transferase isoenzymes in mouse brain, heart, kidney, spleen, liver, and muscle. Individual isoenzymes from each of these tissues have been purified, characterized, and classified into the three known classes of GST. These studies demonstrate that GST isoenzymes are variably expressed in different mouse tissues, suggesting that their expression is tissue specific. A major isoenzyme, belonging to the pi class, with a pI value in the range of 8.6-9.1 and an approximate subunit Mr value of 22,500 was detected in each tissue investigated in this study. A variable number of mu class isoenzymes with subunit Mr values of 26,500 were expressed in all mouse tissues studied, except spleen and muscle. Only liver and kidney showed the expression of an alpha class isoenzyme, each having a basic pI value and subunit Mr of approximately 25,000. Another minor acidic alpha class isoenzyme, also with a subunit Mr value of 25,000, was detected in liver, kidney, and brain. While multiple GST isoenzymes were detected in all other tissues studied, only spleen showed the presence of a single isoenzyme, which belonged to the pi class. These results reveal considerable differences in the GST isoenzyme composition of mouse tissues as compared to rat and human tissues. However, several apparent similarities in mouse and human tissues exist, suggesting that the mouse model can be used to analyze the GST-mediated detoxification mechanisms in humans.     

Purification and characterization of hepatic glutathione transferases from an insectivorous marsupial,the brown antechinus (Antechinus stuartii)

1. Five unique glutathione transferase isoenzymes were purified from the hepatic cytosol of an insectivorous marsupial, the brown antechinus. The purified GSTs were characterized by structural and catalytic properties including apparent molecular weight andisoelectricpoint,specificity towards modelsubstrates,kineticparameters,sensitivityto inhibitors and cross-reactivity with antisera raised against human GSTs. 2. An alpha class GST, Antechinus GST 1-1, predominated in the hepatic cytosol, representing 71% of the total GST purified. The substrate specificity of Antechinus GST 1-1 was similar to that of other alpha class GSTs, particularly with respect to its high activity with cumene hydroperoxide. The mu class was represented by three GST isoenzymes, Antechinus GST 3-3, GST 3-4 and GST 4-4. These isoenzymes represented 8, 2 and 10% of the total GST purified respectively. A single GST, Antechinus GST 22, belonged to the pi class of GSTs and represented 12% of the total GST purified. The hepatic GST isoenzyme ratio (by class) observed in the brown antechinus was more similar to that observed in the human than in rat. 3. A previous study investigating a herbivorous marsupial, the brushtail possum (Trichosurus vulpecula) also identified a predominant hepatic GST belonging to the alpha class and displaying peroxidase activity. The evolutionary conservation of a similar predominant GST isoenzyme in these marsupials suggests that they play an important role in the detoxication metabolism of these unique mammals.     

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.     

Interaction of chlorophenoxyalkyl acid herbicides with rat-liver glutathione S-transferases

The in vitro interaction of four chlorophenoxyalkyl (CPA) acid herbicides with rat-liver glutathione S-transferase (GST) was studied using reduced glutathione and 1-chloro-2,4-dinitrobenzene as substrates. Inhibition of GST activity by the CPA acids in crude extracts was dose dependent. Ring substitution and side-chain length were shown to be of importance in determining the extent of GST inhibition. While GST AA, an isoenzyme of GST, was stimulated by two CPA acids, each of the other GST isoenzymes (A, B, C, E and M) was inhibited, to different degrees. Kinetic studies revealed a mixed type inhibition of the isoenzymes. Conjugates of CPA acids with glutathione were not formed. These results indicate that CPA acids interact with GST by binding directly to these proteins, possibly at a different locus from that of the substrate. The binding of CPA acids to GST may, therefore, have a protective function against these herbicides.     

Inhibition of hepatic glutathione-S-transferases by fatty acids and fatty acid esters

Micromolar concentrations of polyunsaturated fatty acids and ascorbate esters of saturated fatty acids were found to cause a marked inhibition of rat and mouse hepatic glutathione-S-transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene. Arachidonic acid was approximately 25 times more potent in inhibiting rat GST than palmitic acid which was the least effective. Both linoleic and arachidonic acids did not inhibit rat liver GST when ethacrynic acid was used as substrate while the reverse was true with 1,2-dichoro-4-nitrobenzene. In contrast, all the chemicals tested inhibited rat liver GST activity towards 4-nitropyridine N-oxide, indicating isozyme specificity.     

Inhibition of various glutathione S-transferase isoenzymes by RRR-alpha-tocopherol.

The activity of human cytosolic glutathione S-transferases (GSTs) can positively or negatively be changed by various compounds. It is for instance known that RRR-alpha-tocopherol inhibits GST P1-1 [Haaften van R.I.M. et al. (2001) Alpha-tocopherol inhibits human glutathione S-transferase pi. BBRC 280, 631-633]. The effect of RRR-alpha-tocopherol on the other isoenzymes of GST in purified forms of the isoenzymes and in human liver cytosol (GST M and GST A) and lysate of human erythrocytes (GST P) is studied. It is found that all isoenzymes (purified enzymes and enzymes present in homogenates) are inhibited, in a concentration-dependent way, by RRR-alpha-tocopherol. GST P is in both cases inhibited with the highest potency compared to the other isoenzymes. It also appeared that the purified GST P1-1 isoenzyme is non-competitively inhibited by RRR-alpha-tocopherol. The IC(50) values of RRR-alpha-tocopherol for the purified isoenzymes of GST are much lower compared to the IC(50) values for human lysate and human liver cytosol. This is probably due to binding of RRR-alpha-tocopherol to proteins, e.g. albumin and hemoglobin, with higher affinity than to GST; so more RRR-alpha-tocopherol is needed to inhibit the enzyme. However, the inhibition of GSTs by RRR-alpha-tocopherol can still be of physiological relevance, because due to dermal application of cosmetic products very high concentrations vitamin E can be reached in the skin, where GST P1-1 is present. RRR-alpha-tocopherol might also be a good lead compound for the development of a new class of inhibitors of GST that can be used as adjuvant in cancer therapy.     

Inhibition of soluble glutathione S-transferase by diuretic drugs

Glutathione transferases are believed to play an important protective role in the various tissues of animals and man by catalysing the glutathione conjugation of electrophilic drugs and electrophilic drug metabolites. Many of these compounds have the potential to react with vital cellular macromolecules in the absence of this enzyme system. We have investigated the interaction of a number of high ceiling diuretics with the glutathione transferases contained in the cytosolic fraction of the rat liver. Of bumetanide, ethacrynic acid, furosemide, indacrynic acid and tienilic acid, only ethacrynic acid was conjugated with glutathione. Further experiments revealed that ethacrynic, indacrynic and tienilic acids are all potent inhibitors of glutathione S- aryltransferase . Glutathione S- alkyltransferase and glutathione S-epoxide transferase were also inhibited by the diuretics, but to a lesser extent than glutathione S- aryltransferase . The diuretics giving the greatest inhibition of these reactions were chemically related to ethacrynic acid. The concept where inhibition of glutathione-S-transferase by a drug may enhance its own toxicity is considered. This mechanism has also the potential of enhancing the toxicity of other concurrently administered drugs which normally require glutathione S-transferase for detoxication.     

Protective activity of different hepatic cytosolic glutathione S-transferases against DNA-binding metabolites of aflatoxin B1

To evaluate the role of glutathione S-transferase (GST) isoenzymes in induced resistance of hepatocytes to aflatoxin B1 (AFB1), we compared DNA protective activities of different hepatic cytosol preparations and purified GSTs from normal rats, rats exposed to different polychlorinated biphenyls (PCBs), and rats with carcinogen-induced hepatocellular neoplasms, with cytosols or purified GSTs from mouse, rainbow trout, and human livers. These comparisons were performed in an in vitro assay for [3H]AFB1-DNA binding after activation by rat liver microsomes. Cytosol and S-hexylglutathione-affinity-purified GST preparations from livers of mice consistently had strong protective activity against AFB1-DNA binding. The majority of this activity was dependent on the presence of reduced glutathione (GSH) but some GSH-independent protection was observed in mouse hepatic cytosol, but not in purified GST preparations. We found that all of the GSH-dependent DNA-protective activity in mouse liver eluted as a single GST isoenzyme by hydroxyapatite chromatography. Preparations of cytosol and purified GSTs from normal rat liver, rainbow trout liver, and human liver had much less AFB1-specific DNA protective activity than GSTs found in mouse liver preparations. Cytosol from rats with carcinogen-generated liver neoplasms and livers induced with 3,3',4,4'-tetrachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl had more GST activity toward CDNB than cytosol from normal rat liver. When equivalent units of GST activity (CDNB) were compared, there was little difference observed between the DNA-protective activities of PCB-induced and normal rat liver cytosols, yet cytosol from rat liver neoplasms was more protective. Purified GST-P (7-7), the GST isoenzyme most induced in carcinogen-generated rat liver neoplasms, was not protective when added at protein concentrations found to be protective for total GSTs isolated from these neoplasms. These studies demonstrate that the resistance of mouse liver to AFB1 can be explained primarily by a single constitutive GST isoenzyme (YaYa or 4-4) with a relatively high activity toward DNA-binding metabolites of AFB1. GST isoenzymes with such high specific DNA protective activity against AFB1 metabolites were not evident in human, rat, or rainbow trout liver or in PCB-induced or neoplastic rat liver preparations.     

Mouse liver glutathione S-transferase isoenzyme activity toward aflatoxin B1-8,9-epoxide and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide

As part of the studies of the biochemical basis for species differences in biotransformation of the carcinogen aflatoxin B1 (AFB1) and its modulation by phenolic antioxidants, we have investigated the role of mouse liver glutathione S-transferase (GST) isoenzymes in the conjugation of AFB1-8,9-epoxide. Isoenzymes of GST were purified to electrophoretic homogeneity from Swiss-Webster mouse liver cytosol by affinity chromatography and chromatofocusing. The isoenzyme fractions were characterized in terms of activity toward surrogate substrates and immunologic cross-reactivity with antisera to rat GSTs. The major isoenzymes were identified as SW 4-4, SW 3-3, and SW 1-1. The specific activity of SW 4-4 toward AFB1-8,9-epoxide was at least 50- and 150-fold greater than that of SW 3-3 and SW 1-1, respectively. Relatively high activity toward another epoxide carcinogen, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, was observed with both SW 4-4 and SW 3-3. SW 1-1 had the highest activity toward 1-chloro-2,4-dinitrobenzene (CDNB) whereas SW 4-4 had relatively low CDNB activity. Following pretreatment with 0.75% butylated hydroxyanisole in the diet, the fraction of total GST contributed by SW 1-1 appeared to increase dramatically, whereas in control mice SW 3-3 constituted the predominant isoenzyme. The high GST activity of mouse liver cytosol toward AFB1-8,9-epoxide is apparently due to an isoenzyme that contributes little to the overall cytosolic CDNB activity.     

Glutathione S-transferases and glutathione peroxidases in doxorubicin-resistant murine leukemic P388 cells

Energy-dependent rapid drug efflux is believed to be a major factor in cellular resistance to doxorubicin (DOX). However, several recent studies have demonstrated that cellular DOX retention alone does not always correlate with its cytotoxicity and suggest that mechanisms other than rapid drug efflux may also be important. In the present study, we have compared glutathione (GSH) S-transferase (GST), selenium-dependent GSH peroxidase and selenium-independent GSH peroxidase II activities in DOX-sensitive (P388/S) and resistant (P388/R) mouse leukemic cells. The GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (EA) was markedly higher in P388/R cells compared to P388/S cells. Purification of GST by GSH-affinity chromatography from an equal number of P388/S and P388/R cells revealed an increased amount of GST protein in P388/R cells. Immunological studies indicated that alpha and pi type GST isoenzymes were 1.27- and 2.2-fold higher, respectively, in P388/R cells compared to P388/S cells. Selenium-dependent GSH peroxidase activity was similar in both the cell lines, whereas selenium-independent GSH peroxidase II activity was approximately 1.36-fold higher in P388/R cells compared to P388/S cells. These results suggest that increased GSH peroxidase II activity in P388/R cells may contribute to cellular DOX resistance by enhancing free radical detoxification in this cell line.     

Characterization of hepatic glutathione S-transferases in coho salmon (Oncorhynchus kisutch)

The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon (Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24-26kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS-MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.     

Glutathione conjugation of styrene 7,8-oxide enantiomers by major glutathione transferase isoenzymes isolated from rat livers

Male Sprague-Dawley rat liver cytosol mediated regioselective conjugation of styrene 7,8-oxide (STO) enantiomers with glutathione in completely trans-ring-opening manner to afford (1S)-S-(1-phenyl-2-hydroxyethyl)glutathione and (2R)-S-(2-phenyl-2-hydroxyethyl)glutathione in the ratio 22:1 for (R)-STO and also to afford (1R)-S-(1-phenyl-2-hydroxyethyl)glutathione and (2S)-S-(2-phenyl-2-hydroxyethyl)glutathione in the ratio 12:1 for (S)-STO. In the above cytosolic reactions, (R)-STO was conjugated 1.8 times faster than (S)-STO, while the (R)- to (S)-ratio in rate of the conjugation was 2.7 when racemic STO was used as a substrate. A kinetic study, carried out by using six major glutathione transferase (GST) isoenzymes isolated from the cytosol, indicated that GSTs 3-3, 3-4 and 4-4 (class mu enzymes) had much higher Kcat/Km values towards both STO enantiomers than the other three major isoenzymes, GSTs 1-1, 1-2 and 2-2 (class alpha enzymes). All the class mu enzymes mediated preferential glutathione conjugation of (R)-STO to (S)-STO. On the contrary, the class alpha enzymes catalysed the conjugation of (S)-STO preferentially to (R)-STO. The kinetic study strongly suggested that GSTs determining the higher enantioselectivity towards (R)-STO in the rat liver cytosol were the class mu enzymes, especially GST 3-3, which had the highest Kcat/Km value towards (R)-STO as well as the highest (R) to (S) ratio in the enantioselectivity among the six isoenzymes examined. GST 7-7, isolated as a major enzyme from the liver cytosol of the animals bearing hepatic hyperplastic nodules which were induced by chemical carcinogens, catalysed preferential GSH conjugation of (S)-STO to (R)-STO.     

Phosphono analogs of glutathione: inhibition of glutathione transferases, metabolic stability, and uptake by cancer cells

Glutathione transferases (GSTs) have been shown to play an important role in multiple drug resistance in cancer chemotherapy. The inactivation of GST isoforms could lead to an enhanced activity of cytotoxic drugs. Thus, we have developed glutathione phosphono analogs [(S)-gamma-glutamyl-(2RS)-(+/-)-2-amino-(dialkoxyphosphinyl)-ac etylgl ycines], which were previously shown to be inhibitors of GSTP1-1. In the present study, the inhibition characteristics of these analogs, including isoenzyme specificities, type of inhibition, and determination of K(i) values, were determined. The inhibition of class alpha GSTs was competitive towards GSH. A mixed-type, non-competitive inhibition of class mu and pi GSTs was observed. The K(i) values varied between 880 +/- 210 and 0.45 +/- 0.1 microM. The inhibitors were most effective towards class mu GSTs. In order to investigate the potential use of these GST inhibitors in intact cellular systems, two additional approaches were examined. Firstly, the metabolic stability was tested with purified gamma-glutamyl transpeptidase and cell homogenates as well as during incubation of cell lines. No appreciable degradation was observed in any of the tested systems. Secondly, to facilitate cellular uptake, three derivatives were synthesized in which the glycine carboxylic group was esterified. Uptake and a possible intracellular cleavage to the corresponding free acids were monitored by HPLC analysis. The esters were effectively transported into HT29 (colon cancer) and EPG85-257P (gastric cancer) cells, respectively, and readily converted into the more active free acids. In conclusion, the tested inhibitors may be regarded as model compounds for the development of modulating agents in cancer chemotherapy.