Effects of the antiproliferative cyclopentenone prostaglandin A1 on glutathione metabolism in human cancer cells in culture

Homeostatic mechanisms for the maintenance of glutathione (GSH) are fundamental in the provision of a cellular defense against electrophilic/oxidant challenges. Cyclopentenone prostaglandins (CP-PGs) are powerful antiproliferative endogenous substances that may act as electrophilic regulating compounds, by virtue of the presence of an alpha,beta-unsaturated carbonyl group in the cyclopentane ring. Nevertheless, differential resistance to CP-PG cytotoxic/cytostatic effect has been reported in different cell types. It is reported that the activity/expression of gamma-glutamylcysteine synthetase (gamma-GCS, the rate-limiting enzyme in GSH biosynthesis) can be inducibly activated by electrophiles, including CP-PGs. The response of the human cancer strains HEp-2 (larynx carcinoma) and HL-60 (promyelocytic leukemia) cells to treatment with the CP-PG PGA1 in culture was investigated by evaluating the time-course of GSH synthesis and activity of enzymes of GSH metabolism, other than gamma-GCS, after PGA1 addition. HEp-2 cells, being more resistant to PGA1 cytotoxic and cytostatic effects, have basal GSH levels that were 2.4-fold higher than that of HL-60 cells. The activities of GSH S-transferase (GST), glutathione reductase (GSRd) and glutathione peroxidase (GSPx) are constitutively higher in HL-60 cells than in HEp-2 cells (respectively, 17.0-, 28.5- and 12.3-fold). When challenged with PGA1, both cell types exhibited a dose-dependent rise in GSH content that was maximal 18 h after PGA1 addition and was preceded by a rise in GST and GSRd activities in both cell types (at 12 h). GSPx activity increased only in HEp-2 (PGA1 evoked a 93.4%-inhibition in HL-60 cells). Moreover only HEp-2 cells exhibited early capacity to enhance GSH content (1-2 h just after PGA1 addition). These results and earlier data showing that leukemia cells are sensitive to CP-PG treatment suggest that deficiencies in GSH metabolism may be strategically in therapeutic approaches to the treatment of human leukemias.     

A homology model for rat mu class glutathione S-transferase 4-4

Glutathione S-transferases (GSTs) are an important class of phase II (de)toxifying enzymes, catalyzing the conjugation of glutathione (GSH) to electrophilic species. Recently, a number of cytosolic GSTs was crystallized. In the present study, molecular modeling techniques have been used to derive a three-dimensional homology model for rat GST 4-4 based upon the crystal structure of rat GST 3-3, both members of the mu class. GST 3-3 and GST 4-4 isoenzymes share a sequence homology of 88%. GST 4-4 distinguishes itself from GST 3-3 in being much more efficient and stereoselective in the nucleophilic addition of GSH to epoxides and alpha,beta-unsaturated ketones. GST 3-3, however, is much more efficient in catalyzing nucleophilic aromatic substitution reactions. In this study, several known substrates of GST 4-4 were selected and their GSH conjugates docked into the active site of GST 4-4. GSH conjugates of phenanthrene 9(S),10(R)-oxide and 4,5-diazaphenanthrene 9(S),10(R)-oxide were docked into the active site of both GST 3-3 and GST 4-4. From these homology modeling and docking data, the difference in stereoselectivity between GST 3-3 and GST 4-4 for the R- and S-configured carbons of the oxirane moiety could be rationalized. The data acquired from a recently derived small molecule model for GST 4-4 substrates were compared with the results of the present protein homology model of GST 4-4. The energy optimized positions of the conjugates in the protein model agreed very well with the original relative positions of the substrates within the substrate model, confirming the usefulness of small molecule models in the absence of structural protein data. The protein homology model, together with the substrate model, will be useful to further rationalize the substrate selectivity of GST 4-4, and to identify new potential GST 4-4 substrates.     

Busulfan-glutathione conjugation catalyzed by human liver cytosolic glutathione S-transferases

We have examined the catalytic activity of glutathione S-transferases (GST) in the conjugation of busulfan with glutathione (GSH) in human liver cytosol, purified human liver GST, and cDNA-expressed GST-alpha 1-1. Human liver microsomes and cytosol were incubated with 40 microM busulfan and 1 mM GSH. Cytosol catalyzed the formation of the GSH-busulfan tetrahydrothiophenium ion (THT+) in a concentration-dependent manner, whereas microsomes lacked activity. The total and spontaneous rates of THT+ formation increased with pH (pH range, 6.50-7.75), with the maximum difference at pH 7.4. Due to the limited aqueous solubility of busulfan, a K(m) for busulfan was not determined. The intrinsic clearance (Vmax/K(m)) of busulfan conjugation was 0.167 microliter/min/mg with 50-1200 microM busulfan and 1 mM GSH. GSH Vmax and K(m) for busulfan conjugation were 30.6 pmol/min/mg and 312 microM, respectively. Ethacrynic acid (0.03-15 microM) inhibited cytosolic busulfan-conjugating activity with 40 microM busulfan and 1 mM GSH. Enzyme-mediated THT+ formation was decreased 97% by 15 microM ethacrynic acid with no effect on the spontaneous reaction. In incubations with affinity-purified liver GST and GST-alpha 1-1, the intrinsic clearance for busulfan conjugation was 0.87 and 2.92 microliters/min/mg, respectively. Busulfan is a GST substrate with a high K(m) relative to concentrations achieved clinically (1-8 microM).     

Human glutathione S-transferases

Human glutathione S-transferases (GSTs) are a functionally diverse family of soluble enzymes of detoxification that use reduced glutathione (GSH) in conjugation and reduction reactions. Toxic electrophiles, including a variety of carcinogens, are substrates for the GSTs and after conjugation or reduction they are more easily excreted into bile or urine. Many of the GSTs have been cloned, and the three-dimensional structures of GSTs from several species, including humans, have been determined. These data have provided significant insight into how the GSTs function as enzymes. Many GST substrates are inducers of GST gene expression; nonsubstrate inducers include H2O2 and other reactive oxygen species. The regulatory elements of several human GST genes have been partially characterized, and the regulation of the GSTs in humans appears to be very different from that in rodents. Several polymorphisms of GST expression occur commonly in humans and have been associated with an increased susceptibility to certain cancers, particularly when combined with other genetic and environmental factors such as smoking. The role of GSTs in protecting cells from injury by toxic electrophiles continues to be developed.     

Age and gender dependent levels of glutathione and glutathione S-transferases in human lymphocytes

Glutathione S-transferases (GSTs) are a family of enzymes involved in the detoxification of a wide range of chemicals including chemical carcinogens. Human cytosolic GSTs are divided into four major classes; alpha, mu, pi and theta. This study was performed to evaluate the influence of age and gender on the GST isoenzyme expression and glutathione (GSH) content in lymphocytes. Blood was collected from 124 healthy controls, which were divided into age groups of 20-40 years (21 females, 20 males), 40-60 years (20 females, 21 males) and 60-80 years (20 females, 22 males). Lymphocytes were isolated by density centrifugation on Histopaque-1077. After homogenization, cytosolic fractions were isolated. Herein, GST isoenzyme levels were determined by densitometrical analysis of western blots after immunodetection with monoclonal antibodies. Total GSH content was determined by high performance liquid chromatography after conjugation with monobromobimane. Spearman rank correlation and Wilcoxon rank sum tests were used for statistical evaluation. Lymphocytic GSTmu and pi levels were not correlated with age or gender. GSTalpha was not detectable in lymphocytes. GSH contents were not different in males and females, but decreased with age in both males and females. In age group 60-80, GSH content was significantly lower as compared with age groups 20-40 and 40-60 in both sexes. Since high GSH is an essential factor in the detoxification of many compounds, these data indicate that the detoxification potential of the GSH/GST system in lymphocytes may decrease with age in man.     

Glutathione-dependent biotransformation of the alkylating drug thiotepa and transport of its metabolite monoglutathionylthiotepa in human MCF-7 breast cancer cells

In this study, the role of glutathione S-transferase (GST) P1-1, the cellular reduced glutathione (GSH) status, and ATP-dependent efflux pumps in the cellular glutathione-dependent biotransformation of thiotepa and transport of the main metabolite monoglutathionylthiotepa in relation to cytotoxicity was studied in control and GST-P1-1-transfected MCF-7 cell lines. It was demonstrated that an enhanced cellular level of GST-P1-1 leads to an enhanced formation of monoglutathionylthiotepa, which is transported out of the cell into the medium. Monoglutathionylthiotepa was able to reversibly inhibit the activity of purified GST-P1-1, but only at nonphysiological concentrations, indicating that feedback inhibition of GST by its metabolites is not a relevant process in vivo. The GST activity, cellular GSH level, and/or ATP-dependent efflux of monoglutathionylthiotepa were modulated using ethacrynic acid, D,L-buthionine-S,R-sulfoximine, probenecid, and verapamil to understand the interplay between GSTs, glutathione conjugation, and efflux of glutathione conjugates in more detail. Inhibition of the GSH biosynthesis by D,L-buthionine-R,S-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced the glutathione conjugation of thiotepa and potentiated the cytotoxicity of thiotepa. Pretreatment of cells with ethacrynic acid resulted in decreased formation of monoglutathionylthiotepa as a result of inhibition of GST in the GST-P1-1 transfectant. In addition, the intracellular amount of monoglutathionylthiotepa increased in both of the cell lines on exposure to ethacrynic acid, indicating that transport of the glutathione conjugate was partially inhibited by the glutathione conjugate of ethacrynic acid. Transport activity of monoglutathionylthiotepa could also be inhibited by probenecid and verapamil, inhibitors of organic anion transport, without influencing the biotransformation capacity of the cells. It was demonstrated that inhibition of glutathione conjugate efflux by probenecid and verapamil leads to enhanced cytotoxicity, which indicates that besides thiotepa, monoglutathionylthiotepa is also cytotoxic for the cells. Only enhanced biotransformation and subsequent transport of the glutathione conjugate into the medium (which occurs with the GST-P1-1 transfectant) results in enhanced viability. Therefore, it was concluded that only enhanced biotransformation of thiotepa represents a real detoxification pathway when the resulting conjugate is transported out of the cells. Altogether, the results indicate that it is not the overexpression of GST per se but the interplay between GSH/GST and glutathione conjugate efflux pumps that results in increased resistance to alkylating anticancer drugs such as thiotepa.     

Effect of ifosfamide treatment on glutathione and glutathione conjugation activity in patients with advanced cancers

Several studies have suggested that the glutathione/glutathione S-transferase (GSH/GST) system is involved in resistance of tumors toward ifosfamide and other cytostatic agents. Besides, ifosfamide metabolites (in vitro) as well as ifosfamide treatment (in vivo) have been shown to decrease cellular GSH availability. In the present study, the in vivo effects of three different ifosfamide treatment schedules on the GSH/GST system were studied in patients with advanced cancers (n = 24): continuous i.v. infusions of 1300 mg/m2 daily for 10 days and 5000 mg/m2/day for 24 h, as well as a 4-h infusion of 3000 mg/m2 daily for 3 days. The GSH/GST system was characterized by administering bromisoval, a probe drug to assess GSH conjugation activity in vivo, as well as by daily monitoring of GSH concentrations in blood cells and plasma. Bromisoval pharmacokinetics was assessed before and at the end of the ifosfamide treatment. Blood cell GSH levels decreased significantly (P < 0.05) during the 3- and 10-day ifosfamide treatment schedules; the 24-h treatment had no effect. The ifosfamide treatment schedules had only minimal effects on bromisoval pharmacokinetics. Assuming that the kinetics of the probe drug provide an accurate reflection of enzyme activity, this suggests that GST activity remains unchanged. Because GSH conjugation of bromisoval enantiomers requires both GST activity and GSH availability, these results also indicate that, despite the 35% decrease in GSH in blood cells of two patient groups, the GSH availability of the cancer patients was not rate-limiting for GSH conjugation of bromisoval enantiomers. If GSH levels in blood cells reflect those in tumors/other tissues, the present results indicate that ifosfamide may be used clinically to decrease GSH levels. However, whether a 35% decrease is sufficient to increase tumor sensitivity toward (other) cytostatics remains uncertain.     

Biotransformation of the naturally occurring isothiocyanate sulforaphane in the rat: identification of phase I metabolites and glutathione conjugates

Sulforaphane (SFN) is a naturally occurring isothiocyanate present in cruciferous vegetables, such as broccoli, that has been identified as a potent inducer of glutathione S-transferase activities in laboratory animals. The present studies were carried out to elucidate the metabolic fate of SFN in the rat. Particular emphasis was placed on glutathione (GSH)-dependent pathways because conjugation with GSH is a major route by which many isothiocyanates are eliminated in mammals. Male Sprague-Dawley rats were administered a single dose of SFN (50 mg kg-1 ip), and bile and urine were collected over ascorbic acid. Analysis of biological fluids was carried out by ionspray LC-MS/MS using the neutral loss (129 Da) and precursor ion (m/z 164) scan modes to detect GSH and N-acetylcysteine (NAC) conjugates, respectively. In bile, five thiol conjugates (designated M1-M5) were detected. Metabolites M2 and M4 were identified as the GSH conjugates of SFN and erucin (ERN, the sulfide analog of SFN), respectively, by comparing their LC-MS/MS properties with those of standards obtained by synthesis. M1 was characterized as the GSH conjugate of a desaturated metabolite of SFN (tentatively assigned the structure of delta 1-SFN), suggesting that the parent compound also undergoes oxidative metabolism. Metabolites M3 and M5 were identified as the NAC conjugates of SFN and ERN, respectively, and together with the NAC conjugate of delta 1-SFN, these species also were detected in urine. Quantitative determination of the former two mercapturates in urine indicated that approximately 60% and approximately 12% of a single dose of SFN is eliminated in 24 h as the NAC conjugates of SFN and ERN, respectively. The corresponding figures in rats dosed with ERN were approximately 67% and approximately 29%. When the GSH conjugate of SFN was incubated with phosphate buffer (pH 7.4, 37 degrees C), < 1% of the conjugate dissociated to liberate free SFN. On the other hand, the conjugate underwent a facile thiol exchange reaction (> 70% conversion) when incubated in the presence of excess cysteine, thereby acting as an effective carbamoylating agent. It is concluded that SFN undergoes metabolism by S-oxide reduction and dehydrogenation and that GSH conjugation is the major pathway by which the parent compound and its phase I metabolites are eliminated in the rat.     

Inhibition of embryonic retinoic acid synthesis by aldehydes of lipid peroxidation and prevention of inhibition by reduced glutathione and glutathione S-transferases

Inhibition of conceptal biosynthesis of all-trans-retinoic acid (t-RA) by aldehydes generated from lipid peroxidation was investigated. Oxidative conversion of all-trans-retinal (t-RAL, 18 microM) to t-RA catalyzed by rat conceptal cytosol (RCC) was sensitive to inhibition by trans-2-nonenal (tNE), nonyl aldehyde (NA), 4-hydroxy-2-nonenal (4HNE), and hexanal. With an initial molar ratio of aldehyde/t-RAL of 2:1, tNE, NA, and 4HNE caused 70, 65, and 40% reductions of t-RA synthesis, respectively. Hexanal reduced generation of t-RA by approximately 50% as the ratio of aldehyde/t-RAL was raised to 20:1. tNE significantly increased the Km of the reaction and kinetic analyses indicated a mixed competitive/noncompetitive inhibition. By contrast, analogous reactions catalyzed by adult rat hepatic cytosol (ARHC) were highly resistant to inhibition by the same aldehydes. Significant inhibition (> 40% reduction of t-RA generation) by 4HNE, NA, and tNE were achieved at high molar ratios of aldehyde/t-RAL (> 175:1). Hexanal did not inhibit the reaction significantly even at very high ratios of aldehyde/t-RAL (> 2,000:1). Interestingly, when reduced glutathione (GSH, 10 mM) alone or GSH plus glutathione S-transferase (GST) were added to RCC-catalyzed reactions, additions of tNE or 4HNE showed either no significant inhibition or a partial lack of inhibition. Results suggested that GSH-dependent conjugation with 4HNE proceeded slowly compared to conjugation with tNE. To test the hypothesis that GST-catalyzed GSH conjugation can effectively prevent inhibition of t-RA synthesis by aldehydic products of lipid peroxidation, triethyltin bromide (TEB, a potent inhibitor of GST, 20 microM) was added to ARHC-catalyzed reactions when hexanal or tNE were present in the incubations. Eighty and 60% of hexanal and tNE inhibition, respectively, were observed. This was apparently due to TEB blockage of GST-catalyzed GSH conjugation reactions and thus strongly supported the stated hypothesis.     

Structures of herbicides in complex with their detoxifying enzyme glutathione S-transferase - explanations for the selectivity of the enzyme in plants

BACKGROUND: Glutathione S-transferases (GSTs) are detoxifying enzymes present in all aerobic organisms. These enzymes catalyse the conjugation of glutathione with a variety of electrophilic compounds. In plants, GSTs catalyse the first step in the degradation of several herbicides, such as triazines and acetamides, thus playing an important role in herbicide tolerance. RESULTS: We have solved the structures of GST-I from maize in complex with an atrazine-glutathione conjugate (at 2.8 A resolution) and GST from Arabidopsis thaliana (araGST) in complex with an FOE-4053-glutathione conjugate (at 2.6 A resolution). These ligands are products of the detoxifying reaction and are well defined in the electron density. The herbicide-binding site (H site) is different in the two structures. The architecture of the glutathione-binding site (G site) of araGST is different to that of the previously described structure of GST in complex with two S-hexylglutathione molecules, but is homologous to that of GST-I. CONCLUSIONS: Three features are responsible for the differences in the H site of the two GSTs described here: the exchange of hydrophobic residues of different degrees of bulkiness; a slight difference in the location of the H site; and a difference in the degree of flexibility of the upper side of the H site, which is built up by the loop between helices alpha4 and alpha5. Taking these two structures as a model, the different substrate specificities of other plant GSTs may be explained. The structures reported here provide a basis for the design of new, more selective herbicides.     

Thiol ester hydrolysis catalyzed by glutathione S-transferase A1-1

rGSTA1-1 has been shown to catalyze the hydrolysis of the thiol ester glutathionyl ethacrynate (E-SG). In contrast, neither the retro-Michael addition with the substrate EA-SG, to yield GSH and ethacrynic acid (EA), nor the conjugation reaction between GSH and EA to yield the thiol ester E-SG was catalyzed to any measurable extent under similar conditions. The steady state kcat and KM for hydrolysis of E-SG by wild type rGSTA1-1 were 0.11 +/- 0.009 min-1 and 15.7 +/- 1.6 mM, respectively. The site-directed mutant, Y9F, in which the catalytic Tyr-9 is substituted with Phe, was completely inactive in this reaction. To uncover a mechanistic signature that would distinguish between direct hydrolysis and covalent catalysis involving acylation of Tyr-9, solvent isotope exchange and mass spectrometry experiments were performed. No 18O incorporation into the starting thiol ester was detected with initial velocity solvent isotope exchange experiments. However, covalent adducts corresponding to acylated protein also were not observed by electrospray ionization mass spectrometry, even with an assay that minimized the experimental dead time and which allowed for detection of N-acetyltyrosine acylated with EA in a chemical model system. The kon and koff rate constants for association and dissociation of E-SG were determined, by stopped flow fluorescence, to be 5 x 10(5) s-1 M-1 and 6.7 s-1, respectively. Together with the isotope partitioning results, these rate constants were used to construct partial free energy profiles for the GST-catalyzed hydrolysis of E-SG, assuming that Tyr-9 acts as a general acid-base catalyst. The "one-way flux" of the thiol esterase reaction results directly from the thermodynamic stability of the products after rate-limiting attack of the thiol ester by H2O or Tyr-9, and is sufficient to drive the hydrolysis to completion, in contrast to GST-catalyzed breakdown of other GSH conjugates.     

Glutathione-doxorubicin conjugate expresses potent cytotoxicity by suppression of glutathione S-transferase activity: comparison between doxorubicin-sensitive and -resistant rat hepatoma cells

The cytotoxic mechanism of a conjugate of doxorubicin (DXR) and glutathione (GSH) via glutaraldehyde (GSH-DXR) was investigated using DXR-sensitive (AH66P) and -resistant (AH66DR) rat hepatoma cells. GSH-DXR accumulated in AH66DR cells as well as in AH66P cells without efflux by P-gp and exhibited the potent cytocidal activity against both cells compared with DXR. To examine whether thiol from GSH-DXR affected the expression of cytotoxicity, two conjugates of DXR, with modified peptides containing alanine or serine substituted for cysteine in GSH were prepared and their cytotoxicities determined. Substitution of these amino acids for cysteine resulted in an approximately two- to fourfold reduction in cytotoxic activity against both cell lines compared with the effect of GSH-DXR. Depletion of intracellular GSH by treatment of both cells with buthionine sulphoximine did not change the cytotoxic activity of DXR, BSA-DXR or GSH-DXR. By co-treating the cells with tributyltin acetate, an inhibitor of glutathione S-transferase (GST), and either DXR, BSA-DXR or GSH-DXR, the cytotoxicity was markedly increased. Interestingly, GSH-DXR showed non-competitive inhibition of GST activity and its IC50 value was 1.3 microM. These results suggested that the inhibition of GST activity by GSH-DXR must be an important contribution to the expression of potent cytotoxicity of the drug.     

Evidence of idiotypic modulation in the immune response to gp43, the major antigenic component of Paracoccidioides brasiliensis in both mice and humans

The kinetics of the glutathione (GSH) conjugation of (+)- and (-)-enantiomers of anti- as well as syn-3,4-dihydroxy-1,2-oxy-1,2,3, 4-tetrahydrobenzo[c]phenanthrene (B[c]PDE) catalyzed by murine GSH S-transferase (GST) isoenzymes has been investigated. Murine GSTs exhibited significant differences in their enantioselectivity toward B[c]PDE stereoisomers. For example, while pi class isoenzyme mGSTP1-1 was virtually inactive toward stereoisomers with 1S configuration [(-)-syn-and (+)-anti-B[c]PDE], these stereoisomers were good substrates for alpha class isoenzyme mGSTA1-2. When GST activity was measured as a function of varying B[c]PDE concentration (10-320 microM) at a fixed saturating concentration of GSH (2 mM), each isoenzyme examined obeyed Michaelis-Menten kinetics with all four B[c]PDE stereoisomers. Alpha class isoenzyme mGSTA4-4 exhibited negligible activity toward all four stereoisomers of B[c]PDE. The catalytic efficiency of mGSTA1-2 was approximately 1.5- to 15-fold higher than other murine GSTs in the GSH conjugation of (-)-anti-B[c]PDE, which among the four B[c]PDE stereoisomers is the most potent pulmonary carcinogen in the newborn mouse model and a potent skin tumor-initiator. While alpha class isoenzymes mGSTA3-3 and mGSTA1-2 were equally efficient in the GSH conjugation of (+)-anti-B[c]PDE, their catalytic efficiencies toward this stereoisomer were significantly higher than those of mGSTP1-1 and mGSTM1-1. Likewise, mGSTA1-2 was relatively more efficient than other GSTs in the GSH conjugation of both enantiomers of syn-B[c]PDE. In summary, our results indicate that (a) murine GSTs significantly differ in their enantioselectivity in the GSH conjugation of B[c]PDE stereoisomers, which may partially account for the observed differences in the carcinogenic potency of B[c]PDE stereoisomers, and (b) mGSTA1-2 and mGSTA3-3 play a major role in the detoxification of B[c]PDE.     

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 and isoelectric point, specificity towards model substrates, kinetic parameters, sensitivity to 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.     

Formation of catechol estrogen glutathione conjugates and gamma-glutamyl transpeptidase-dependent nephrotoxicity of 17beta-estradiol in the golden Syrian hamster

In an animal model of hormone-mediated carcinogenesis, male golden Syrian hamsters develop renal carcinoma following prolonged exposure to 17beta-estradiol. The basis for the species and tissue specificity is unclear. Detailed information on the disposition of 17beta-estradiol in this model is lacking. Because catechol estrogens have been implicated in this model of carcinogenesis, we investigated the metabolism and nephrotoxicity of 17beta-estradiol in golden Syrian hamsters, with emphasis on the formation of catechol estrogen thioethers. 17beta-Estradiol (50 micromol/kg, i.p.) is a mild nephrotoxicant, causing significant elevations in the urinary excretion of gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase, glutathione S-transferase (GST) and glucose. Increases in renal protein carbonyls and lipid hydroperoxides, which are markers of oxidative damage, also occur after administration of 17beta-estradiol (50 micromol/kg, i.p.). 17beta-Estradiol-mediated nephrotoxicity is reduced by treating animals with acivicin, an inhibitor of gamma-GT, implying that toxicity is mediated by metabolites requiring metabolism by this enzyme. Following administration of 17beta-[14C]estradiol (100 micromol/kg) to hamsters, 9.7% of the dose is recovered in bile after 5 h, the majority (7.9%) representing aqueous metabolites. Seven catechol estrogen GSH conjugates were identified, 2-hydroxy-1,4-bis-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-17beta-estradiol, 2-hydroxy-4-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-estrone, 2-hydroxy-1-(glutathion-S-yl)-estrone, 4-hydroxy-1-(glutathion-S-yl)-17beta-estradiol, and 2-hydroxy-1-(glutathion-S-yl)-17beta-estradiol. At 5.4 micromol/kg of 17beta-estradiol, a dose-reflective of daily exposure levels in the hamster model of nephrocarcinogenicity, 12% of the dose is recovered within 5 h as a combination of GSH conjugates of 2- and 4-hydroxy-17beta-estradiol and 2- and 4-hydroxyestrone. In summary, oxidation of catechol estrogens, followed by GSH conjugation, occurs in vivo and 17beta-estradiol is a mild nephrotoxicant in a manner dependent on the activity of gamma-GT.     

Susceptibility to esophageal cancer and genetic polymorphisms in glutathione S-transferases T1, P1, and M1 and cytochrome P450 2E1

Genetic polymorphisms in enzymes involved in carcinogen metabolism have been shown to influence susceptibility to cancer. Cytochrome P450 2E1 (CYP2E1) is primarily responsible for the bioactivation of many low molecular weight carcinogens, including certain nitrosamines, whereas glutathione S-transferases (GSTs) are involved in detoxifying many other carcinogenic electrophiles. Esophageal cancer, which is prevalent in China, is hypothesized to be related to environmental nitrosamine exposure. Thus, we conducted a pilot case-control study to examine the association between CYP2E1, GSTM1, GSTT1, and GSTP1 genetic polymorphisms and esophageal cancer susceptibility. DNA samples were isolated from surgically removed esophageal tissues or scraped esophageal epithelium from cases with cancer (n = 45), cases with severe epithelial hyperplasia (n = 45), and normal controls (n = 46) from a high-risk area, Linxian County, China. RFLPs in the CYP2E1 and the GSTP1 genes were determined by PCR amplification followed by digestion with RsaI or DraI and Alw26I, respectively. Deletion of the GSTM1 and GSTT1 genes was examined by a multiplex PCR. The CYP2E1 polymorphism detected by RsaI was significantly different between controls (56%) and cases with cancer (20%) or severe epithelial hyperplasia (17%; P < 0.001). Persons without the RsaI variant alleles had more than a 4-6-fold risk of developing severe epithelial hyperplasia (adjusted odds ratio, 6.0; 95% confidence interval, 2.3-16.0) and cancer (adjusted odds ratio, 4.8; 95% confidence interval, 1.8-12.4). Polymorphisms in the GSTs were not associated with increased esophageal cancer risk. These results indicate that CYP2E1 may be a genetic susceptibility factor involved in the early events leading to the development of esophageal cancer.     

Bioactivation of a toxic metabolite of valproic acid, (E)-2-propyl-2,4-pentadienoic acid, via glucuronidation. LC/MS/MS characterization of the GSH-glucuronide diconjugates

The hepatotoxicity of the anticonvulsant drug valproic acid may be associated with the formation of potentially reactive metabolites, one of which is (E)-2-propyl-2,4-pentadienoic acid ((E)-2,4-diene VPA). This report describes the characterization of new GSH-related conjugates of this diene. Bile samples collected from male Sprague-Dawley rats dosed ip with (E)-2,4-diene VPA (100 mg/kg) were analyzed by LC/MS/MS. Initial Q1 parent in scanning indicated that the daughter ions m/z 162 and 123 could be derived from the ions at m/z 624 and 480, respectively. Subsequent collision-induced dissociation (CID) of these parent ions revealed a common neutral loss of 176 Da which is diagnostic for glucuronides. A similar neutral loss of 176 Da was observed in daughter ion spectra of the biliary metabolites arising from [2H7]-4-ene VPA dosed ip to rats, where the ion fragments containing the VPA portion were 7 amu higher than those derived from the unlabeled drug. CID of the ion at m/z 624 also gave fragments characteristics for GSH conjugates such as the loss of glycine and glutamate moieties. Based on the MS data, the metabolites were assigned the diconjugate structures 1-O-(2-propyl-5-(glutathion-S-yl)-3-pentenoyl)-beta-D-glucur onide (5-GS-3-ene VPA-glucuronide I, MH+, 624) and the corresponding 5-NAC-3-ene VPA-glucuronide (MH+, 480). Further proof of structural identity was obtained from 1H NMR of HPLC-purified metabolites. The amount of biliary 5-GS-3-ene VPA-glucuronide I was 7-fold greater than the corresponding 5-GS-3-ene VPA, the sum of the two metabolites accounting for 6.6% of the dose. Incubation of 1-O-(2-propyl-2,4-pentadienoyl)-beta-D-glucuronide (2,4-diene VPA-glucuronide) with GSH in the presence or absence of GST enzyme led to the formation of 5-GS-3-ene VPA-glucuronide I which was readily detected by LC/MS/MS, suggesting that in vivo the diconjugate may arise from the reaction of GSH with 2,4-diene VPA-glucuronide. To our knowledge, this is the first recorded instance in which glucuronide formation activates a drug to further conjugate with GSH via a Michael addition reaction.     

Polymorphic expression of the glutathione S-transferase P1 gene and its susceptibility to Barrett's esophagus and esophageal carcinoma

Factors determining individual susceptibility to esophageal cancer or premalignant Barrett's epithelium are still largely unclear. An imbalance between phase I drug metabolism [e.g., cytochrome P450 (CYP)] and phase II detoxification [e.g., glutathione S-transferase (GST)] may contribute to the development of these diseases. Polymorphic variants in the CYP1A1 gene were described leading to increased levels of bioactive compounds, whereas polymorphisms in GST genes often resulted in impaired detoxification. We studied the frequencies of polymorphic variants in CYP1A1, GSTP1, GSTT1, and GSTM1 genes in 98 patients with Barrett's epithelium and 34 patients with esophageal cancer. The results were compared with those obtained from 247 healthy blood donors. DNA was extracted, and PCR-RFLP methods were used to detect genetic polymorphisms. Chi2 analysis, Spearman rank correlation, and Wilcoxon rank sum tests were used for statistical evaluation. Polymorphisms in CYP1A1, GSTM1, and GSTT1 occurred at an equal frequency in patients and controls. Occurrence of the polymorphic GSTP1b variant in the GSTP1 gene resulted in a significantly lower GST enzyme activity (P < 0.05), and GSTP1b was found significantly more often in patients with Barrett's epithelium (70%; P < 0.001) and patients with esophageal adenocarcinoma (76%; P = 0.005), as compared to healthy blood donors (41%). In conclusion, presence of the GSTP1b allele leads to lower GST enzyme activity levels and, consequently, impaired detoxification. This most important esophageal GST isoform may, therefore, contribute to the development of Barrett's epithelium and adenocarcinoma.     

Methionine and cysteine affect glutathione level, glutathione-related enzyme activities and the expression of glutathione S-transferase isozymes in rat hepatocytes

Methionine and cysteine are constituents of glutathione. To understand the effects of these two sulfur amino acids on the glutathione (GSH)-dependent detoxification defense system, intracellular GSH and GSH-related enzyme activities, including GSH peroxidase, GSH reductase, GSH S-transferase (GST) and gamma-glutamylcysteine synthetase, were determined. In addition, the expression of three GST isozymes and carbonic anhydrase III (CA III) was examined. Hepatocytes isolated from male Sprague-Dawley rats were cultured with 0.1, 0.3, 0.5 or 1.0 mmol/L each of L-methionine and L-cysteine, for up to 7 d. Cells incubated with 0.5 or 1.0 mmol/L methionine and cysteine had increased intracellular GSH. A twofold increase was observed on d 6 compared with freshly isolated hepatocytes (P < 0.05). However, intracellular GSH was lower in cells treated with 0.3 or 0.1 mmol/L each of methionine and cysteine than in cells tested with 0.5 or 1.0 mmol/L. Although the GSH level differed significantly between cells cultured with 0.3 or 1.0 mmol/L of methionine and cysteine, GSH-related enzymes did not differ at these two concentrations. The activity generally remained constant for the first 24 h, then increased up to d 4. Immunodetection analysis revealed no difference in the level of CA III and GST isoforms, Ya, Yb and Yp, with amino acids each at a concentration of at least 0.3 mmol/L. Yp expression steadily increased up to d 7. Most proteins decreased rapidly after 48 h when cultured with 0.1 mmol/L of methionine and cysteine; however, the Yp level increased up to d 6. In conclusion, results indicate that a twofold increase of intracellular GSH is reached by adding methionine and cysteine at a concentration >0.5 mmol/L to the culture medium. The concentrations of methionine and cysteine for maintaining hepatic GSH are higher than for GSH-related enzyme activity and for GST isoform expression.