Mu Class Research Articles

Role of Glutathione Transferases in the Mechanism of Brostallicin Activation

Brostallicin is a novel and unique glutathione transferase-activated pro-drug with promising anticancer activity, currently in phase I and II clinical evaluation. In this work, we show that, in comparison with the parental cell line showing low GST levels, the cytotoxic activity of brostallicin is significantly enhanced in the human breast carcinoma MCF-7 cell line, transfected with either human GST-pi or GST-mu. Moreover, we describe in detail the interaction of brostallicin with GSH in the presence of GSTP1-1 and GSTM2-2, the predominant GST isoenzymes found within tumor cells. The experiments reported here indicate that brostallicin binds reversibly to both isoenzymes with K(d) values in the micromolar range (the affinity being higher for GSTM2-2). Direct evidence that both GSTP1-1 and GSTM2-2 isoenzymes catalyze the Michael addition reaction of GSH to brostallicin has been obtained both by an HPLC-MS technique and by a new fluorometric assay. We also saw the rapid formation of an intermediate reactive species, which is slowly converted into the final products. This intermediate, identified as the alpha-chloroamido derivative of the GSH-brostallicin adduct, is able to alkylate DNA in a sequence-specific manner and appears to be the active form of the drug. The kinetic behavior of the reaction between brostallicin and GSH, catalyzed by GSTP1-1, has been studied in detail, and a minimum kinetic scheme that suitably describes the experimental data is provided. Overall, these data fully support and extend the findings that brostallicin could be indicated for the treatment of tumor overexpressing the pi or mu class GST.

Molecular Complex of Three Testis-Specific Isozymes Associated with the Mouse Sperm Fibrous Sheath: Hexokinase 1, Phosphofructokinase M, and Glutathione S-Transferase mu class 51

Mammalian sperm require ATP for motility, and most of it is generated by glycolysis. The glycolytic enzymes segregate to the principal piece region of the flagellum, where some are bound tightly to a novel cytoskeletal structure defining this region, the fibrous sheath (FS), and others are easily extracted with detergents. One of the latter is the spermatogenic cell-specific variant isozyme of hexokinase type 1 (HK1S), characterized by an N-terminal 24-amino acid spermatogenic cell-specific region (SSR). Yeast two-hybrid screens carried out using the SSR as bait determined that HK1S is tethered to muscle-type phosphofructokinase (PFKM) in the principal piece region. This led to the identification of four testis-specific Pfkm splice variants, one that overlapped a variant reported previously (Pfkm_v1) and three that were novel (Pfkm_v2, Pfkm_v3, and Pfkm_v4). They differ from Pfkm transcripts found in somatic cells by encoding a novel 67-amino acid N-terminal extension, the testis-specific region (TSR), producing a spermatogenic cell-specific PFKM variant isozyme (PFKMS). An antiserum generated to the TSR demonstrated that PFKMS is present in the principal piece and is insoluble in 1% Triton X-100 detergent. In subsequent yeast two-hybrid screens, the TSR was found to interact with glutathione S-transferase mu class 5 (GSTM5), identified previously as a spermatogenic cell-specific component of the FS. These results demonstrated that HK1S is tethered in the principal piece region by PFKMS, which in turn is bound tightly to GSTM5 in the FS.

Genetic variants in GSTM3 gene within GSTM4-GSTM2-GSTM1-GSTM5-GSTM3 cluster influence breast cancer susceptibility depending on GSTM1

Mu class of Glutathione-S-transferase (GSTM) genes arrange in a tandem on chromosome 1p13.3. The relationship between genetic variants in the GSTM1-5 gene cluster and breast cancer is still ambiguous. In the present study, 17 tagging single-nucleotide polymorphisms (SNPs) covering the GSTMs cluster were originally selected and 11 validated SNPs were used for genotyping 921 cases and 711 controls. The association analyses were performed according to the absence or presence of GSTM1. In the GSTM1-/- group, the allele frequency of one SNP in GSTM3 was significantly different between cases and controls (P = 2.0 x 10(-4), corrected P = 0.001), with odds ratio of 1.75 (95% confidence interval, 1.26-2.44). The observed association in the GSTM1-/- group was successfully replicated in an independent population set (familial/early-onset breast cancer cases, n = 267; community-based controls, n = 667). The combined P values were robust (10(-6)) and the false positive report probability (FPRP) values were low. In contrast, no susceptibility allele/haplotype was identified when the GSTM1 gene was present. Based on epidemiological observations, we further identified two genetic variants in the GSTM3 locus accounting for differential expression of GSTM3 in normal breast tissues by such means as altering binding of RNA-pol-II. Protective genotypes were correlated with higher GSTM3 expression levels. In conclusion, SNPs/haplotypes in the GSTM3 gene within the GSTMs gene cluster are likely to contribute to breast cancer risk when the GSTM1 is absent. We infer that GSTM3 catalyzing ability in normal breast tissue might protect against breast carcinogenesis.

Structures of yeast glutathione‐S‐transferase Gtt2 reveal a new catalytic type of GST family

Glutathione-S-transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand-bound forms, at 2.23 A, 2.20 A and 2.10 A, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues--tyrosine, serine and cysteine--distinguishes it from all other cytosolic GSTs of known structure. Site-directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino-terminus of helix-alpha1 because of stereo-hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.

Structural Basis for the Binding of the Anticancer Compound 6-(7-Nitro-2,1,3-Benzoxadiazol-4-Ylthio)Hexanol to Human Glutathione S-Transferases

Glutathione S-transferases (GST) constitute a superfamily of enzymes with diversified functions including detoxification from xenobiotics. In many human cancers, Pi class GST (GSTP1-1) is overexpressed and contributes to multidrug resistance by conjugating chemotherapeutics. In addition, GSTP1-1 displays antiapoptotic activity by interacting with c-Jun NH(2)-terminal kinase, a key regulator of apoptosis. Therefore, GSTP1-1 is considered a promising target for pharmaceutical treatment. Recently, a potent inhibitor of GSTs, 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), was identified and tested on several tumor cell lines demonstrating high antiproliferative activity. To establish the structural basis of NBDHEX activity, we determined the crystal structure of NBDHEX bound to either GSTP1-1 or GSTM2-2 (mu class). NBDHEX in both cases binds to the H-site but occupies different positions. Furthermore, the compound is covalently attached to the GSH sulfur in the GSTM2-2 crystal, forming a sigma-complex, although it is bound but not conjugated in the GSTP1-1 crystal. Several differences in the H-sites of the two isozymes determine the higher affinity of NBDHEX for GSTM2-2 with respect to GSTP1-1. One such difference is the presence of Ile(104) in GSTP1-1 close to the bound NBDHEX, whereas the corresponding position is occupied by an alanine in GSTM2-2. Mutation of Ile(104) into valine is a frequent GSTP1-1 polymorphism and we show here that the Ile(104)Val and Ile(104)Ala variants display a 4-fold higher affinity for the compound. Remarkably, the GSTP1-1/Ile(104)Ala structure in complex with NBDHEX shows a considerable shift of the compound inside the H-site. These data might be useful for the development of new anticancer compounds.

Monomer−Dimer Equilibrium in Glutathione Transferases: A Critical Re-Examination

Glutathione transferases (GSTs) are dimeric enzymes involved in cell detoxification versus many endogenous toxic compounds and xenobiotics. In addition, single monomers of GSTs appear to be involved in particular protein-protein interactions as in the case of the pi class GST that regulates the apoptotic process by means of a GST-c-Jun N-terminal kinase complex. Thus, the dimer-monomer transition of GSTs may have important physiological relevance, but many studies reached contrasting conclusions both about the modality and extension of this event and about the catalytic competence of a single subunit. This paper re-examines the monomer-dimer question in light of novel experiments and old observations. Recent papers claimed the existence of a predominant monomeric and active species among pi, alpha, and mu class GSTs at 20-40 nM dilution levels, reporting dissociation constants (K(d)) for dimeric GST of 5.1, 0.34, and 0.16 microM, respectively. However, we demonstrate here that only traces of monomers could be found at these concentrations since all these enzymes display K(d) values of <<1 nM, values thousands of times lower than those reported previously. Time-resolved and steady-state fluorescence anisotropy experiments, two-photon fluorescence correlation spectroscopy, kinetic studies, and docking simulations have been used to reach such conclusions. Our results also indicate that there is no clear evidence of the existence of a fully active monomer. Conversely, many data strongly support the idea that the monomeric form is scarcely active or fully inactive.

Glutathione S-transferase genetic polymorphisms (GSTM1, GSTT1 and GSTO2) in three Iranian populations

Genetic polymorphisms in genes encoding glutathione S-transferases M1 (GSTM1; a member of class mu), T1 (GSTT1; a member of class theta) and O2 (GSTO2; a member of class omega) have been defined previously. Studies have revealed that there were significant differences between populations for allelic frequencies of GSTT1, GSTM1 and GSTO2 N412D polymorphisms. To get more insight into the genetic structure of Iranian populations the present study was done on Iranian Georgians living in Frydoonshahr (Isfahan province) and two Persian populations who living in Shiraz (Fars province) and Frydoonshahr. Study subjects consisted of 401 unrelated healthy individuals. From these 121 were Georgians. The remaining subjects were Persians from either Frydoonshahr (n = 34) or Shiraz (n = 246). The genetic polymorphism of GSTT1, GSTM1 and GSTO2 N412D was detected by PCR-based method. The frequency of GSTT1 null genotype in Georgian and Persians of Frydoonshahr and Shiraz was 15.7, 35.2 and 24.8%, respectively. There was significant difference between these populations for the distributions of the GSTT1 genotypes (chi(2) = 7.00, df = 2, P = 0.030). No significant difference was observed between these populations for polymorphisms of GSTM1 (chi(2) = 1.682, df = 2, P = 0.431) and GSTO N142D (chi(2) = 4.622, df = 4, P = 0.328). The prevalence of GSTT1 null genotype in Iranian Georgians showed significant difference with Persians and other Asian countries, but it seems to be similar with the frequency which was reported from European populations.

Structural determinants in domain II of human glutathione transferase M2-2 govern the characteristic activities with aminochrome, 2-cyano-1, 3-dimethyl-1-nitrosoguanidine, and 1, 2-dichloro-4-nitrobenzene

Two human Mu class glutathione transferases, hGST M1-1 and hGST M2-2, with high sequence identity (84%) exhibit a 100-fold difference in activities with the substrates aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidine (cyanoDMNG), and 1,2-dichloro-4-nitrobenzene (DCNB), hGST M2-2 being more efficient. A sequence alignment with the rat Mu class GST M3-3, an enzyme also showing high activities with aminochrome and DCNB, demonstrated an identical structural cluster of residues 164-168 in the alpha6-helices of rGST M3-3 and hGST M2-2, a motif unique among known sequences of human, rat, and mouse Mu class GSTs. A putative electrostatic network Arg107-Asp161-Arg165-Glu164(-Gln167) was identified based on the published three-dimensional structure of hGST M2-2. Corresponding variant residues of hGSTM1-1 (Leu165, Asp164, and Arg167) as well as the active site residue Ser209 were targeted for point mutations, introducing hGST M2-2 residues to the framework of hGST M1-1, to improve the activities with substrates characteristic of hGST M2-2. In addition, chimeric enzymes composed of hGST M1-1 and hGST M2-2 sequences were analyzed. The activity with 1-chloro-2,4-dinitrobenzene (CDNB) was retained in all mutant enzymes, proving that they were catalytically competent, but none of the point mutations improved the activities with hGST M2-2 characteristic substrates. The chimeric enzymes showed that the structural determinants of these activities reside in domain II and that residue Arg165 in hGST M2-2 appears to be important for the reactions with cyanoDMNG and DCNB. A mutant, which contained all the hGST M2-2 residues of the putative electrostatic network, was still lacking one order of magnitude of the activities with the characteristic substrates of wild-type hGST M2-2. It was concluded that a limited set of point mutations is not sufficient, but that indirect secondary structural affects also contribute to the hGST M2-2 characteristic activities with aminochrome, cyanoDMNG, and DCNB.

Characterization of the binding of 8-anilinonaphthalene sulfonate to rat class Mu GST M1-1

Molecular docking and ANS-displacement experiments indicated that 8-anilinonaphthalene sulfonate (ANS) binds the hydrophobic site (H-site) in the active site of dimeric class Mu rGST M1-1. The naphthalene moiety provides most of the van der Waals contacts at the ANS-binding interface while the anilino group is able to sample different rotamers. The energetics of ANS binding were studied by isothermal titration calorimetry (ITC) over the temperature range of 5–30 °C. Binding is both enthalpically and entropically driven and displays a stoichiometry of one ANS molecule per subunit (or H-site). ANS binding is linked to the uptake of 0.5 protons at pH 6.5. Enthalpy of binding depends linearly upon temperature yielding a Δ C p of − 80 ± 4 cal K − 1 mol − 1 indicating the burial of solvent-exposed nonpolar surface area upon ANS-protein complex formation. While ion-pair interactions between the sulfonate moiety of ANS and protein cationic groups may be significant for other ANS-binding proteins, the binding of ANS to rGST M1-1 is primarily hydrophobic in origin. The binding properties are compared with those of other GSTs and ANS-binding proteins.

Cooperative kinetics of the recombinant glutathione transferase of Taenia solium and characterization of the enzyme

Glutathione transferases (GSTs) are essential enzymes in many organisms due their diverse functions and, in helminths they are the main detoxification system. For Taenia solium, two cytosolic GSTs with molecular masses of 25.5 and 26.5 kDa (Ts26GST) have been found. Ts26GST was cloned to be studied in its recombinant form (recTs26GST). Although the primary structure is related to the mu class, the kinetic parameters for CDNB ( V max = 51.5 μmol min −1 mg −1; K m = 1.06 mM; k cat = 22.2 s −1) are related with some alpha GSTs. The substrate and inhibitor class markers reaffirmed these bimodal characteristics. Inhibition studies with anthelminthics indicate that recTs26GST is sensitive to mebendazole, displaying a non competitive inhibition pattern suggesting that at least two molecules are binding to recTs26GST. On the other hand, the kinetic curves for CDNB and GSH showed a positive cooperativity that was corroborated using fluorometric assays. Those assays indicate that CDNB binding is highly influenced by GSH, probably by modulation of the Ts26GST conformational ensamble.

Psoroptes ovis: Identification of vaccine candidates by immunoscreening

Serum from successful vaccine trials against the sheep scab mite, Psoroptes ovis, was used to immunoscreen a cDNA library constructed from mixed-stage and gender P. ovis to identify potential recombinant vaccine candidates. Immunodominant recombinant proteins recognised by IgG in these sera were selected for further analysis. Two candidates were identified in this way; a catchin-like protein (CLP) and a novel mu class glutathione S-transferase (GST). Both candidates were expressed in bacteria as recombinant proteins, the GST as an active enzyme, and combined with four other recombinant allergens in a multi-component recombinant vaccine. Strong serum IgG responses were induced in sheep against each of the components of the recombinant vaccine, however, the protective efficacy of the vaccine could not be determined because of variability in the establishment of a challenge infection.

Molecular characterization of mu class glutathione- S-transferase from disk abalone ( Haliotis discus discus), a potential biomarker of endocrine-disrupting chemicals

The present study has reported the cloning, expression, and characterization of a mu class glutathione S-transferase (GST) gene, HdGSTM1, identified from disk abalone ( Haliotis dicus discus) cDNA library. HdGSTM1 encodes a polypeptide of 215 amino acids with a calculated molecular mass of 25 kDa. The recombinant HdGSTM1 exhibited a relatively low activity of 0.172 ± 0.01 μmol/min/mg protein toward 1-chloro-2, 4-dinitrobenzenel (CDNB), and 0.114 ± 0.03 μmol/min/mg protein toward ethacrynic acid (ECA). Kinetic analysis with respect to glutathione and CDNB gave a K m value of 0.734 ± 0.053 mM and 2.721 ± 0.236 mM, respectively. HdGSTM1 had an optimum temperature of 35 °C and an optimum pH of 8.0. It also showed stability in a wide range of temperatures and pH. Modeling structure analysis revealed that the low catalytic activity of HdGSTM1 was caused by the improper residues in key active sites. The transcripts of HdGSTM1 were detected in all five examined organs, with the highest levels in gills and gonads. After 48 h waterborne exposure of three model endocrine-disrupting chemicals (PAH, PCB, and TBT), the expression of HdGSTM1 was significantly induced in both gill and digestive tract tissues through semi-quantitative RT-PCR examination, suggesting good biomarker potentials.

Unique Resistance Mechanism to Dexamethasone by Glutathione S-Transferase M1 Involving p38 MAPK and NF-κB Pathways, Possible Prognostic Role for Childhood ALL.

Glutathione S-transferases (GSTs) are known as detoxification enzymes that catalyse the conjugation of glutathione to anticancer drugs. In addition to this activity, GSTM1, an isotype of the Mu class GSTs, has been suggested to act as a negative regulator of mitogen-activated protein kinase (MAPK) activation. About 40–60% of the population has a deficit in GSTM1 enzyme activity, and the patients possessing GSTM1 have been reported to have a greater risk of relapse in childhood acute lymphoblastic leukemia (ALL). However, the reasons for poor prognosis remain unclear. To establish our hypothesis that GSTM1-induced drug resistance was involved, GSTM1 was transfected into GSTM1-negative CCRF-CEM cell lines (hereafter, the transfectant is denoted as CEM/M1) and the drug sensitivity was compared with that of control cells (CEM/mock). CEM/M1 showed decreased sensitivity to melphalan and carmustine (relative resistance vs. CEM/mock: 1.6 and 2.1, respectively; p<0.05), but not to daunorubicin, vincristine or etoposide. Notably, sensitivity to dexamethasone (DEX) decreased to a degree of up to 8-fold (IC50:1.76 μM in CEM/M1 and 0.22 μM in CEM/mock; p<0.01). Glutathione depletion by buthionine sulfoximine abrogated the resistance of CEM/M1 to alkylating agents, but not to DEX. These results suggest that a different mechanism is involved in the resistance to DEX. To clarify the GSTM1-related DEX resistance, proapoptotic pathway via p38 MAPK was evaluated. In CEM/M1, DEX-induced apoptosis was significantly inhibited (annexin V-positive at 72hr: 27% in CEM/M1 and 57% in CEM/mock; p<0.01). Silencing of GSTM1 by siRNA restored the DEX resistance, indicating that the anti-apoptosis was caused by the expression of GSTM1. After the treatment with DEX, phosphorylation of p38 was upregulated in CEM/mock, but not in CEM/M1; and the level of Bim mRNA was higher in CEM/mock than it was in CEM/M1. These findings indicate that the overexpression of GSTM1 down-regulates the proapoptotic pathway via p38 MAPK-Bim. However, treatment with a p38 MAPK-inhibitor (SB202194) decreased the DEX-induced apoptosis in both CEM/mock and CEM/M1. The involvement in DEX-resistance seems to be partial. To investigate other mechanisms, NF-κB pathway was evaluated. The basal activity of NF-κB p50 was significantly higher in CEM/M1 than it was in CEM/mock. Inhibition of IκBα phosphorylation by BAY11-7082 increased DEX sensitivity in either cells. Of considerable importance, however, is that the increase by BAY11-7082 was prominent in CEM/M1 and the relative resistance decreased from 8 to 2-fold. It appears that the anti-apoptotic NF-κB pathway is also involved in a resistance to DEX. In conclusion, the expression of GSTM1 in CCRF-CEM caused the resistance to alkylating agents due to its catalytic activity and to DEX by its anti-apoptotic function. The latter is the first report demonstrating the relationship between the expression of GSTM1 and DEX resistance. The determination of GSTM1 expression could be a useful tool for the individualization of optimize therapy for childhood ALL.(Supported by JSPS grant C19591102 and JRFCP grant)

Hepatic and extrahepatic expression of glutathione S-transferase isozymes in mice and its modulation by naturally occurring phenolic acids

A simple plant phenolic acid, protocatechuic acid and a polyphenol, tannic acid are potential chemopreventive agents which inhibited the chemically induced carcinogenesis in many experimental models. We previously demonstrated that those compounds modulate the activity of xenobiotic detoxifying enzymes, including GST in mouse liver, kidney and epidermis. Intraperitoneal (i.p.) treatment with protocatechuic acid in the dose of 80 mg/kg for three consecutive days increased the GST activity in liver and kidney. In case of tannic acid the same effect was observed in kidney after i.p. administration of the single dose of 80 mg/kg. Topical application of phenolic acids resulted in enhancement of epidermal GST activity. The focus of this study was to further investigate the effects of these phenolic acids on the protein levels of GST isozymes in the same tissues using the treatment protocols used in our previous studies. The results confirmed the expression of GST alfa, mu, pi and theta in mouse liver, kidney and epidermis. Treatment with protocatechuic acid resulted in an increase of the expression of GST class mu in liver, but did not affect this isoform in skin and kidney. This compound inhibited the level of kidney GST pi by 35%. Tannic acid decreased the expression of GST mu, alpha and theta in liver. Application of the equimolar doses of both phenolic acids on mouse skin resulted in reduced level of the GST alpha protein. The results of our study indicate that, although moderate, the effect of protocatechuic acid and tannic acid on GST subunits in mice may play certain role in biological activity of these compounds. Of special importance could be the increased expression of GST mu in liver which is involved in detoxification of many carcinogens including polycyclic aromatic hydrocarbons.

Molecular cloning and enzymatic characterization of a class mu glutathione S-transferase of Paragonimus westermani

Glutathione S-transferase (GST) is a component of a second line of defense against bioreactive radicals derived from host immune attack. Paragonimus westermani causes acute or chronic lung diseases in mammals. A cDNA clone, PwGST#11, of adult P. westermani produced in the present study was 748 bp long and encoded an open reading frame of 217 amino acids with a starting methionine. The molecular mass of this putative polypeptide, Pw26GST, was estimated to be 25.1 kDa with an isoelectric point of 5.7. Pw26GST was homologous with the 26-kDa GSTs of trematodes and vertebrates. Nine of the ten amino acid residues lining the glutathione-binding pocket were conserved. Putative Pw26GST polypeptide was clustered with 26-kDa GSTs of trematodes belonging to the class mu. Recombinant Pw26GST protein generated bacterially, revealed GST enzyme activity toward an universal and class mu-specific substrates. Mouse antisera to recombinant Pw26GST protein recognized native 26-kDa GST of P. westermani but not the GSTs of any other trematodes. Collectively, Pw26GST was found to be a member of class mu GSTs of P. westermani.

GLUTATHIONE-S-TRANSFERASE EXPRESSION IN THE MURINE OVARY

Glutathione-S-Transferases (GST's) are a large family of enzymes which play a role in detoxification of xenobiotics by catalyzing the formation of glutathione conjugates. In addition, a number of studies have implicated a role for the GST family as regulators of signal transduction pathways, in particular mitogen-activated protein kinase (MAPK) pathways. 4-Vinylcyclohexane diepoxide (VCD) is an occupational chemical that destroys small pre-antral follicles in rodents. Previously, it has been shown that ovarian exposure to VCD resulted in increased activities of JNK and p38 MAPK. This study was designed to investigate whether one or more of the major GST classes might play a role in the VCD-induced ovotoxicity in mice. A post-natal day 4 (PND4) mouse whole ovary culture system (enriched in target small pre-antral follicles) was employed to examine expression of the major GST classes, alpha, mu, omega, pi, and theta, using Real-Time PCR. Comparison between the classes showed that GST alpha was expressed at the lowest level. Relative to GST alpha, GST omega, theta, mu and pi showed 12.9, 154, 201 and 352-fold greater expression, respectively. In addition, the effect of treatment with 15μM VCD for 15d in culture was examined. Expression of both GST pi and mu classes were increased (P<0.05) by 38% and 81%, respectively compared to control treatment. These findings demonstrate differential expression of the major GST classes in the ovary in response to xenobiotic exposure and suggest a potential role for GST pi and mu in ovarian biotransformation of VCD (Supported by ES09246 and Center grant 06694). (poster)

Polymorphisms of cytochrome P450 1A1, glutathione S-transferase class mu, and tumour protein p53 genes and the risk of developing gallbladder cancer in Japanese

Objectives: To examine the relationship between genetic polymorphisms of cytochrome P450 1A1 ( CYP1A1), glutathione S-transferase class mu ( GSTM1), and tumour protein p53 ( TP53) genes, and gallbladder cancer (GBC) risk, a case–control study was conducted. Design and methods: Genotypes of CYP1A1 T3801C, CYP1A1 Ile462Val, GSTM1, and TP53 Arg72Pro were determined in 54 cases of GBC and 178 controls. Results: The age-adjusted odds ratios (ORs) for the Ile/Val genotype of CYP1A1 Ile462Val polymorphism in women and the Arg/Pro genotype of TP53 Arg72Pro polymorphism in men were observed to be 2.70 (95% CI: 1.14–6.40) and 4.32 (95% CI: 1.08–17.2), respectively. No significant differences in the genotypic frequencies of CYP1A1 T3801C and GSTM1 polymorphisms were observed between controls and cases in both men and women. Conclusion: These results suggest that the Val allele of CYP1A1 Ile462Val polymorphism and the Pro allele of TP53 Arg72Pro polymorphism contribute to an increased risk of GBC among Japanese women and men, respectively.

Importance of a hypervariable active-site residue in human Mu class glutathione transferases catalyzing the bioactivation of chemotherapeutic thiopurine prodrugs

Glutathione transferases (GSTs) catalyze the bioactivation of the thiopurine prodrugs azathioprine, cis-6-(2-acetylvinylthio)purine (cAVTP) and trans-6-(2-acetylvinylthio)guanine (tAVTG), thereby releasing the antimetabolites 6-mercaptopurine and 6-thioguanine. In the GST Mu class, GST M1-1 has the highest catalytic efficiency, whereas GST M2-2 and other enzymes are less active. In the evolution of Mu class GSTs, residue 210 appears hypervariable and has particular functional significance. We demonstrate that the catalytic activity of GST M1-1 with cAVTP or tAVTG is successively diminished when wild-type Ser-210 is mutated into Ala followed by Thr. Conversely, mutating wild-type Thr-210 in GST M2-2 into Ala and Ser enhanced the corresponding activities. Comparisons were also made with GST M2-2 distinguished by Gly or Pro in position 210, as well as wild-type GSTs M4-4 and M5-5. The results suggest that the hydroxyl group of Ser in position 210 stabilizes the transition state of the GST-catalyzed reaction. The low activity of GSTs containing Thr in position 210 is probably due to steric hindrance caused by the β-methyl group of the side chain. The ratios of the different catalytic efficiencies were translated into differences in the Gibbs free energies of transition state stabilization. The effects of the mutations were qualitatively parallel for the alternative substrates, but vary significantly in magnitude. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.

Hepatocytes isolated from preneoplastic rat livers are resistant to ethacrynic acid cytotoxicity

Glutathione S-transferases (GSTs) are involved in the detoxification of xenobiotics, such as several cytostatic drugs, through conjugation with glutathione (GSH). Pi class GST (GST P) liver expression is associated with preneoplastic and neoplastic development and contributes with the drug-resistance phenotype. Ethacrynic acid (EA) is an inhibitor of rat and human GSTs. In addition, causes lipid peroxidation in isolated rat hepatocytes. Therefore, we decided to evaluate the role of the GST/GSH system in isolated hepatocytes from preneoplastic rat livers (IP) in the presence of EA and determine the cytotoxicity of the drug. Our results showed a resistance to the toxic effects of EA since viability and cellular integrity values were significantly higher than control. Initial levels of thiobarbituric acid reactive substances (TBARS) in IP hepatocytes were significantly higher than control and the presence of EA did not change TBARS levels. A diminution in intracellular total GSH was observed by treating with EA isolated hepatocytes from both groups. However, the initial total GSH levels were higher in IP hepatocytes than in control. Immunoblotting analysis showed the presence of GST P in IP animals only. Although alpha and mu class isoenzymes levels were decreased in IP hepatocytes, total GST activity was 1.5-fold higher than in control. In addition, multidrug-resistance protein 2 (Mrp2) showed fivefold decreased levels in IP hepatocytes. In conclusion, increased total GSH, decreased Mrp2 levels and the presence of GST P could be critical factors involved in the resistance of IP hepatocytes to the toxicity of EA.

Differences in the subunit interface residues of alternatively spliced glutathione transferases affects catalytic and structural functions

GSTs (glutathione transferases) are multifunctional widespread enzymes. Currently there are 13 identified classes within this family. Previously most structural characterization has been reported for mammalian Alpha, Mu and Pi class GSTs. In the present study we characterize two enzymes from the insect-specific Delta class, adGSTD3-3 and adGSTD4-4. These two proteins are alternatively spliced products from the same gene and have very similar tertiary structures. Several major contributions to the dimer interface area can be separated into three regions: conserved electrostatic interactions in region 1, hydrophobic interactions in region 2 and an ionic network in region 3. The four amino acid side chains studied in region 1 interact with each other as a planar rectangle. These interactions are highly conserved among the GST classes, Delta, Sigma and Theta. The hydrophobic residues in region 2 are not only subunit interface residues but also active site residues. Overall these three regions provide important contributions to stabilization and folding of the protein. In addition, decreases in yield as well as catalytic activity changes, suggest that the mutations in these regions can disrupt the active site conformation which decreases binding affinity, alters kinetic constants and alters substrate specificity. Several of these residues have only a slight effect on the initial folding of each subunit but have more influence on the dimerization process as well as impacting upon appropriate active site conformation. The results also suggest that even splicing products from the same gene may have specific features in the subunit interface area that would preclude heterodimerization.