Inhibition of human glutathione S-transferases by curcumin and analogues.

Glutathione S-transferases (GSTs) are important phase II drug-metabolizing enzymes that play a major role in protecting cells from the toxic insults of electrophilic compounds. Curcumin, a promising chemotherapeutic agent, inhibits human GSTA1-1, GSTM1-1, and GSTP1-1 isoenzymes. In the present study, the effect of three series of curcumin analogues, 2,6-dibenzylidenecyclohexanone (A series), 2,5-dibenzylidenecyclopentanone (B series), and 1,4-pentadiene-3-one (C series) substituted analogues (n = 34), on these three human GST isoenzymes, and on human and rat liver cytosolic GSTs, was investigated using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Most of the 34 curcumin analogues showed less potent inhibitory activities towards GSTA1-1, GSTM1-1, and GSTP1-1 than the parent curcumin. Compounds B14 and C10 were the most potent inhibitors of GSTA1-1 and human liver cytosolic GSTs, with IC(50) values of 0.2-0.6 microM. The most potent inhibitors of GSTM1-1 were C1, C3 and C10, with IC(50) values of 0.2-0.7 microM. Similarly, GSTP1-1 was predominantly strongly inhibited by compounds of the C series C0, C1, C2 C10 and A0, with IC(50) values of 0.4-4.6 microM. Compounds in the B series showed no significant inhibition of GSTP1-1. Molecular Operating Environment (MOE) program-based quantitative structure-activity relationship (QSAR) analyses have also suggested the relevance of Van der Waals surface area and compound lipophilicity factors for the inhibition of GSTA1-1 and GSTM1-1 and partial charge factors for GSTP1-1. These results may be useful in the design and synthesis of curcumin analogues with either more or less potency for GST inhibition.

The novel antifungal agent PLD-118 is neither metabolized by liver microsomes nor inhibits cytochrome P450 in vitro.

PLD-118 is a novel, oral antifungal drug, under development for the treatment of Candida infections. Possible metabolism of PLD-118 by rat, dog and human S9 liver homogenates and inhibition of human cytochrome P450 (CYP) enzymes were investigated. PLD-118 (10 and 100 microM) incubated for 0-60 min with S9 fractions and NADPH was determined by HPLC, using the Waters AccQ.Tag method after derivatization of amino acids to stable, fluorescent derivatives. CYP assays were performed using pooled human liver microsomes with substrates, selective towards human CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, incubated at concentrations around the Km. Incubation mixtures were preincubated with PLD-118 (0.1-100 microM) or control inhibitor for 5 min. No metabolism of PLD-118 was detected with rat and dog S9 fractions. A small (8%) decrease in PLD-118 at 100 microM (not detected at 10 microM) with human microsomes was considered to be biologically irrelevant. PLD-118 did not inhibit any of the tested CYPs. PLD-118, at concentrations up to 100 microM, is not metabolized by rat, dog or human liver S9 homogenates and does not inhibit human CYPs in vitro, suggesting little likelihood for interaction of PLD-118 with drugs metabolized by these enzymes.

Prediction of isoprene diepoxide levels in vivo in mouse, rat and man using enzyme kinetic data in vitro and physiologically-based pharmacokinetic modelling.

The present study was designed to explain the differences in isoprene toxicity between mouse and rat based on the liver concentrations of the assumed toxic metabolite isoprene diepoxide. In addition, extrapolation to the human situation was attempted. For this purpose, enzyme kinetic parameters K(m) and V(max) were determined in vitro in mouse, rat and human liver microsomes/cytosol for the cytochrome P450-mediated formation of isoprene mono- and diepoxides, epoxide hydrolase mediated hydrolysis of isoprene mono- and diepoxides, and the glutathione S-transferases mediated conjugation of isoprene monoepoxides. Subsequently, the kinetic parameters were incorporated into a physiologically-based pharmacokinetic model, and species differences regarding isoprene diepoxide levels were forecasted. Almost similar isoprene diepoxide liver and lung concentrations were predicted in mouse and rat, while predicted levels in humans were about 20-fold lower. However, when interindividual variation in enzyme activity was introduced in the human model, the levels of isoprene diepoxide changed considerably. It was forecasted that in individuals having both an extensive oxidation by cytochrome P450 and a low detoxification by epoxide hydrolase, isoprene diepoxide concentrations in the liver increased to similar concentrations as predicted for the mouse. However, the interpretation of the latter finding for human risk assessment is ambiguous since species differences between mouse and rat regarding isoprene toxicity could not be explained by the predicted isoprene diepoxide concentrations. We assume that other metabolites than isoprene diepoxide or different carcinogenic response might play a key role in determining the extent of isoprene toxicity. In order to confirm this, in vivo experiments are required in which isoprene epoxide concentrations will be established in rats and mice.

Prediction of interindividual variation in drug plasma levels in vivo from individual enzyme kinetic data and physiologically based pharmacokinetic modeling.

A strategy is presented to predict interindividual variation in drug plasma levels in vivo by the use of physiologically based pharmacokinetic modeling and human in vitro metabolic parameters, obtained through the combined use of microsomes containing single cytochrome P450 enzymes and a human liver microsome bank. The strategy, applied to the pharmaceutical compound (N-[2-(7-methoxy-1-naphtyl)-ethyl]acetamide), consists of the following steps: (1) estimation of enzyme kinetic parameters K(m) and V(max) for the key cytochrome P450 enzymes using microsomes containing individual P450 enzymes; (2) scaling-up of the V(max) values for each individual cytochrome P450 involved using the ratio between marker substrate activities obtained from the same microsomes containing single P450 enzymes and a human liver microsome bank; (3) incorporation into a physiologically based pharmacokinetic model. For validation, predicted blood plasma levels and pharmacokinetic parameters were compared to those found in human volunteers: both the absolute plasma levels as well as the range in plasma levels were well predicted. Therefore, the presented strategy appears to be promising with respect to the integration of interindividual differences in metabolism and prediction of the possible impact on plasma and tissue concentrations of drugs in humans.

Determining the best animal model for human cytochrome P450 activities: a comparison of mouse, rat, rabbit, dog, micropig, monkey and man.

1. In the present study, nine cytochrome P450 enzyme activities in seven species were characterized to allow a practical means of comparing this important metabolic step between various test animals and man. 2. Enzyme activities and kinetic parameters were first determined towards marker substrates for human cytochrome P450 enzymes. Inhibition profiles were then determined with both antibodies directed against various cytochrome P450 enzymes and with chemical inhibitors. 3. Both the enzyme kinetic parameters/enzyme activities, and the inhibition profiles obtained for the animal species were compared with those obtained for human liver microsomes in order to postulate the animal species most similar to man with regard to each individual cytochrome P450 enzyme activity. 4. It was found that, as expected, none of the tested species was similar to man for all the measured P450 enzyme activities, but that in each species only some of the P450 enzyme activities could be considered as similar to man. 5. When it is known which human cytochrome P450 enzymes are involved in the metabolism of a compound, the comparative data presented here can be used for selecting the most suitable species for in vitro and in it no experiments.

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

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

Stereoselective conjugation of prostaglandin A2 and prostaglandin J2 with glutathione, catalyzed by the human glutathione S-transferases A1-1, A2-2, M1a-1a, and P1-1.

Prostaglandins containing an alpha,beta-unsaturated keto group, such as prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2), inhibit cell proliferation. These cyclopentenone prostaglandins may be conjugated with GSH chemically or enzymatically via glutathione S-transferases, and this has been suggested to result in inhibition of the antiproliferative mode of action. In the present study, the role of the major human GSTs in the conjugation of PGA2 and PGJ2 with GSH was investigated with purified enzymes, i.e., the Alpha-class enzymes GST A1-1 and GST A2-2, the Mu-class enzyme GST M1a-1a, and the Pi-class enzyme GST P1-1. The GSH conjugates were separated from the parent compound by HPLC and identified by fast atom bombardment mass spectrometry and 1H-NMR. Two GSH conjugates were found for both PGA2 and PGJ2, the R- and S-GSH conjugates of both prostaglandins. Incubation experiments with PGA2 and PGJ2 (70-600 microM) clearly showed the role of individual GSTs in the conjugation of PGA2 and PGJ2. Compared to the chemical reaction, enzyme activities towards PGA2 were up to 5.4 times as high (GSTA1-1) at the lowest concentration (70 microM), while at the highest concentration (600 microM) enzyme activities were up to 3.0 times as high (GST P1-1). For PGJ2, enzyme activities were up to 4.3 (GSTM1a-1a, 70 microM) and up to 3.1 (GSTM1a-1a, 600 microM) times as high. As expected, similar amounts of the R- and S-conjugates of both prostaglandins were found in the chemical reaction. Striking stereoselectivities in conjugating activities were observed for GST A1-1 and GST P1-1. GST A1-1 favors the formation of the R-GSH conjugates of both prostaglandins. GST P1-1 showed a clear selectivity with regard to the formation of the S-GSH conjugate of PGA2. However, this selectivity was not found for the formation of the S-GSH conjugate of PGJ2. GSTM1a-1a showed no stereoselectivity with regard to the GSH conjugation of both PGA2 and PGJ2. GSTA2-2 only showed some minor formation of the R-GSH conjugate of PGJ2. The possible implications of the observed stereoselectivity on the effects of PGA2 and PGJ2 are discussed.

The biotransformation of isoprene and the two isoprene monoepoxides by human cytochrome P450 enzymes, compared to mouse and rat liver microsomes.

The metabolism of isoprene was investigated with microsomes derived from cell lines expressing human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, or CYP3A4. The formation of epoxide metabolites was determined by gas chromatographic analysis. CYP2E1 showed the highest rates of formation of the isoprene monoepoxides 3,4-epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II), followed by CYP2B6. CYP2E1 was the only enzyme showing detectable formation of the diepoxide of isoprene, 2-methyl-1,2:3,4-diepoxybutane. Both isoprene monoepoxides were oxidized by CYP2E1 to the diepoxide at similar enzymatic rates. In order to determine the relative role of CYP2E1 in hepatic metabolism, isoprene as well as the two monoepoxides were also incubated with a series of ten human liver microsomal preparations in the presence of the epoxide hydrolase inhibitor cyclohexene oxide. The obtained activities were correlated with activities towards specific substrates for CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1 and CYP3A. The results were supportive for those obtained with single human P450 enzymes. Isoprene (monoepoxide) metabolism sowed a significant correlation with CYP2E1 activity, determined as chlorzoxazone 6-hydroxylation. CYP2E1 is therefore the major enzyme involved in hepatic metabolism of isoprene and the isoprene monoepoxides in vitro. To investigae species differences with regard to the role of epoxide hydrolase in the metabolism of isoprene monoepoxides, the epoxidation of isoprene by human liver microsomes was compared to that of mouse and rate liver microsomes. The amounts of monoepoxides formed as a balance between epoxidation and hydrolysis, was measured in incubations with and without the epoxide hydrolase inhibitor cyclohexene oxide. Inhibition of epoxide hydrolase resulted in similar rates of monoepoxide formation in mouse, rat and man. Without inhibitor, however, the total amount of monoepoxides present at the end of the incubation period was twice as high for mouse liver microsomes than for rat and even 15 times as high as for human liver microsomes. Thus, differences in epoxide hydrolase activity between species may be of crucial importance for the toxicity of isoprene in the various species.

Effect of a vegan diet on biomarkers of chemoprevention in females.

1. In order to study the potential beneficial effects of a vegan diet, a cross-sectional study was performed and several biomarkers of chemoprevention were measured in a population of female 'living food' eaters ('vegans'; n = 20) vs matched omnivorous controls (n = 20). 2. White blood cells obtained from fresh blood samples were subjected to the single-cell gel-electrophoresis assay. There was no statistically significant difference between the vegans and controls in the parameters 'tail length' and 'tail moment'. However, the 'tail moment' was significantly lower in a subset of the vegans (i.e.in those who did not use any vitamin and/or mineral supplements). 3. Fresh blood samples were exposed in vitro to the mutagen mitomycin C just prior to culturing. After culturing the number of binucleated lymphocytes with micronuclei was scored. There was no difference between the controls and vegans in the incidence of baseline micronuclei, nor in the number of mitomycin C-induced micronuclei. However, a significant correlation (r = -0.64, P < 0.01) between the number of mitomycin C-induced micronuclei and the activity of erythrocyte superoxide dismutase was found in the vegans. The number of baseline micronuclei increased with age in both groups. These findings may be of biological relevance. 4. The content of glutathione-S-transferase-alpha in plasma was not different between the vegans (n = 12) and controls (n = 12). 5. The present data indicate a few differences in biomarkers of chemopreventive potential in strict vegans vs matched omnivorous controls. The significance of these changes as biologically relevant indicators of beneficial effects of vegan diets in humans needs to be determined in studies with a larger number of subjects.

Effect of short-term dietary administration of eugenol in humans.

1. In order to study the antigenotoxic potential of eugenol in humans, ten healthy non-smoking males ingested a daily amount of 150 mg eugenol or the placebo for seven consecutive days. After a washout period of one week, groups ingesting eugenol or the placebo were crossed and received the other treatment for seven consecutive days. 2. On days 8 and 22 blood samples were taken for the assessment of standard clinical biochemical parameters. To study the possible antigenotoxic effect of eugenol, on day 8 and 22 blood samples were collected and exposed in vitro to the established genotoxic agents mitomycin C and vinblastine. After exposure the percentage of cells with chromosome aberrations and micronuclei was determined in cultured white blood cells. On days 8 and 22 paracetamol (500 mg p.o.) was administered as test substance to measure phase-II biotransformation capacity. Glutathione-S-transferase (GST) activities were determined in erythrocytes and blood plasma. 3. No significant differences in the clinical biochemical parameters were detected between the eugenol-period and the placebo-period, indicating that daily administration of 150 mg eugenol for 7 days has no toxic affects. 4. No significant differences on the cytogenetic parameters were found after ingestion of eugenol. Thus, there are no indications for an antigenotoxic potential of eugenol in humans, consuming daily 150 mg eugenol for 7 days. 5. A significant reduction in alpha-class GSTs in plasma (P < 0.05), but not in the other measured biotransformation parameters, was found in volunteers during the eugenol-periods as compared to the placebo-period. This may either reflect GST-inhibition by eugenol or protection against background damage of liver cells by eugenol.

Human cytochrome P450 enzyme selectivities in the oxidation of chlorinated benzenes.

1,2-Dichlorobenzene, 1,4-dichlorobenzene, 1,2,4-trichlorobenzene, 1,2,3,5-tetrachlorobenzene, and pentachlorobenzene were incubated with microsomes derived from cell lines expressing human CYP1A1, CYP1A2, CYP3A4, CYP2E1, or CYP2D6. The formation of phenolic metabolites as determined by gas chromatographic analysis revealed that CYP2E1 possessed the highest activity toward all chlorinated benzenes. Furthermore, CYP1A1 and CYP1A2 showed relatively high enzymatic activities toward the lower chlorinated benzenes (1,2-dichlorobenzene, 1,4-dichlorobenzene, 1,2,4-trichlorobenzene) and CYP3A4 toward the higher chlorinated benzenes (1,2,3,5-tetrachlorobenzene, pentachlorobenzene). CYP2D6 only showed low or nondetectable activity toward the investigated chlorobenzenes. The ratio between the activities of CYP2E1 and CYP3A4 with respect to the oxidation of chlorinated benzenes decreased from 150 (1,2-dichlorobenzene) to 1.8 (pentachlorobenzene). In order to estimate the relative contribution of CYP2E1 in hepatic metabolism of 1,2-dichlorobenzene and 1,2,4-trichlorobenzene in vitro, the rate of oxidation of these compounds by microsomal preparations from 22 human livers was correlated with activities toward specific substrates for CYP2E1, CYP3A, and CYP1A. The results were supportive for the results obtained with single human P450 enzymes. CYP2E1 is the major, if not the only, enzyme involved in the formation of 2,3-dichlorophenol, 3,4-dichlorophenol, 2,3,5-trichlorophenol, and 2,3,4-trichlorophenol, while CYP3A4 is responsible for the formation of 2,3,6-trichlorophenol. The formation of the major metabolite from 1,2,4-trichlorobenzene (2,4,5-trichlorophenol) was correlated with both CYP2E1 and CYP3A activity. Because of the decreasing ratio in activity between CYP2E1 and CYP3A4 with respect to the oxidation of chlorinated benzenes, it is concluded that the role of CYP2E1 toward chlorinated benzenes decreases with increasing number of chlorine atoms. The relative amount of CYP3A4 present then becomes an important determinant for metabolism.

Consumption of Brussels sprouts results in elevated alpha-class glutathione S-transferase levels in human blood plasma.

The effect of consumption of glucosinolate-containing Brussels sprouts on alpha-class glutathione S-transferase levels in human blood plasma was investigated in 10 healthy, male, non-smoking volunteers. Following a 3-week run-in period, five volunteers continued on a glucosinolate-free diet during a subsequent 3-week intervention period (control group), while the other five (sprouts group) consumed 300 g of cooked Brussels sprouts per day, at the expense of 300 g of a glucosinolate-free vegetable. alpha-Class glutathione S-transferases were measured by radioimmunoassay. In the control group, similar alpha-class glutathione S-transferase levels were observed in both periods (P = 0.814), while in the sprouts group the alpha-class glutathione S-transferase levels were elevated by a factor of 1.4 (P = 0.002). We hypothesize that the elevated alpha-class GST levels in plasma reflect GST-alpha induction in tissues such as liver and small intestine under non-toxic conditions. The present findings indicate that alpha-class GST levels in plasma may be used as a biomarker for alpha-class GST levels in tissues. In addition, they support the results of epidemiologic studies that consumption of cruciferous vegetables may result in a decreased cancer risk.

Ethacrynic acid and its glutathione conjugate as inhibitors of glutathione S-transferases.

1. The diuretic drug ethacrynic acid (EA) is a potent reversible inhibitor of rat and human glutathione S-transferases (GST), with I50-values (microM) of 4.6-6.0, 0.3-1.9 and 3.3-4.8 for alpha, mu and pi-class, respectively. 2. The reversible inhibition by the glutathione conjugate of EA is even stronger for alpha and mu-class, with I50-values (microM) of 0.8-2.8 and < 0.1-1.2, respectively, while the I50 for the pi-class is 11. 3. Inhibition of rat and human pi-class GST also occurs by covalent binding of ethacrynic acid. 14C-ethacrynic acid, 0.8 nmol EA per nmol pi-class GST could be incorporated, resulting in 65-93% inhibition of the catalytic activity. 4. Owing to the chemical nature of the covalent binding (Michael addition), this reaction should be reversible. Indeed, full restoration of the catalytic activity of GST P1-1 inactivated by covalently-bound EA was reached in about 125 h by incubation with an excess of glutathione. 5. EA has been used to inhibit GST in biological systems. The reversible covalent binding may very well play a role in the observed inhibition of GST by EA in vivo.

Interindividual differences in the in vitro conjugation of methylene chloride with glutathione by cytosolic glutathione S-transferase in 22 human liver samples.

The interindividual variation in the in vitro conjugation of methylene chloride with glutathione by cytosolic glutathione S-transferase (GST) was investigated with 22 human liver samples. In three of the samples no activity towards methylene chloride was observed. Eleven samples showed an activity ranging from 0.20 to 0.41 (0.31 +/- 0.08) nmol/min/mg protein, and eight samples an activity of 0.82-1.23 (1.03 +/- 0.14) nmol/min/mg protein. The activities towards 1-chloro-2,4-dinitrobenzene (CDNB) of these three groups were 1.17 +/- 0.25, 1.12 +/- 0.35 and 1.20 +/- 0.53 mumol/min/mg protein, respectively. In nine of the liver samples, the alpha-, mu- and pi-class GST subunits were quantified. In two of these samples, no activity was observed towards methylene chloride, while alpha-, mu- and pi-class subunits were expressed in these human liver cytosolic samples. As the highest activity towards methylene chloride was still 1.4 times lower than the activity in rat cytosol, the existence of the three populations seems to be of little importance for human risk assessment.

Isoenzyme selective irreversible inhibition of rat and human glutathione S-transferases by ethacrynic acid and two brominated derivatives.

In the present study it has been shown that ethacrynic acid can inhibit glutathione S-transferase (GST) of the pi-class irreversibly. [14C]Ethacrynic acid, 0.8 nmol/nmol human P1-1 and 0.8 nmol/nmol rat GST 7-7 could be incorporated, resulting in 65-93% inhibition of the activity towards 1-chloro-2,4-dinitrobenzene (CDNB). Isoenzymes of the alpha- and mu-class also bound [14C]ethacrynic acid, however without loss of catalytic activity. Incorporation ranged from 0.3 to 0.6 and 0.2 nmol/nmol enzyme for the mu- and alpha-class GST isoenzymes, respectively. For all isoenzymes, incorporation of [14C]ethacrynic acid could be prevented by preincubation with tetrachloro-1,4-benzoquinone, suggesting, that a cysteine residue is the target site. Protection of GST P1-1 against inhibition by ethacrynic acid by the substrate analog S-hexylglutathione, indicates an active site-directed modification. The monobromo and dibromo dihydro derivatives of ethacrynic acid were synthesized in an effort to produce more reactive compounds. The monobromo derivative did not exhibit enhanced irreversible inhibitory capacity. However, the dibromo dihydro derivative inhibited both human and rat GST isoenzymes of the pi-class very efficiently, resulting in 90-96% inhibition of the activity towards CDNB. Interestingly, this compound is also a powerful irreversible inhibitor of the mu-class GST isoenzymes, resulting in 52-70% inhibition. The two bromine atoms only marginally affect the strong (reversible) competitive inhibitory capacity of ethacrynic acid, with IC50 (microM) of 0.4-0.6 and 4.6-10 for the mu- and pi-class GST isoenzymes, respectively.

Purification and characterization of eight glutathione S-transferase isoenzymes of hamster. Comparison of subunit composition of enzymes from liver, kidney, testis, pancreas and trachea.

Eight dimeric isoenzymes of glutathione S-transferase (GST) were purified from liver, kidney and testis of the Syrian golden hamster, using S-hexylglutathione affinity chromatography and chromatofocusing. The isoenzymes were characterized according to their substrate selectivity, physical properties and amino acid sequence analysis. Thus a classification into Alpha, Mu and Pi classes was made in analogy with GSTs of other species. Two Alpha-class GSTs were purified, termed A1A1 (pI 8.9) and A1A2 (pI 8.6). Four Mu-class subunits were detected (M1-M4), all forming homodimers, with M2 and M3 also forming a heterodimer. The isoelectric points ranged from 5.9 to 8.6. One Pi-class isoenzyme was purified and termed P1P1 (pI 6.8). Using h.p.l.c. analysis, the subunit composition was determined in a number of organs. The major subunits in liver were A1 and M1. Subunit A1 was also the major subunit in the kidney. Subunit M1 was not detected in kidney, while subunit P1 was not found in the liver. Pancreas and trachea contained predominantly the Pi-class subunit, P1. GST in the testis was mostly of the Mu class. The major subunit was M4, and subunits M2 and M3 were exclusively detected in the testis.

Irreversible inhibition of rat glutathione S-transferase 1-1 by quinones and their glutathione conjugates. Structure-activity relationship and mechanism.

The irreversible inhibition of the rat glutathione S-transferase (GST) isoenzyme 1-1 by a series of halogenated 1,4-benzoquinones and their GSH conjugates was studied quantitatively by analysing the time course of enzyme inactivation. With increasing numbers of chlorine substituents, the rate of inhibition greatly increased. Incorporation of a GSH moiety in all cases increased the rate of inactivation compared with the non-substituted compound, and this was due to the increased affinity of the inhibitor for the active site. The ratio between the rates of inhibition for a given quinone with and without GSH substituent was largest for the three dichlorobenzoquinones, with the 2,6-isomer showing a 41-fold increase in rate of inhibition upon conjugation with GSH. The time courses of inhibition could be fitted either to a bi-exponential function (for the GSH conjugates and the higher chlorinated quinones) or to a mono-exponential function (all other quinones). It is concluded that the second component describes the affinity part of the reaction. GST 1-1 possesses two cysteine residues, with modification of one of these, probably located in the vicinity of the active site, having a major impact on the enzyme activity. Compounds with affinity towards the active site preferentially react with this residue. Non-specific quinones react equally with both cysteine residues. This was confirmed by the observation that complete inactivation of GST 1-1 by 2,5-dichlorobenzoquinone was achieved only after modification of two residues, whereas the corresponding GSH conjugate already completely inhibited the enzyme after modification of one residue.