Activity Of GSTs Research Articles

Analysis of Arabidopsis glutathione-transferases in yeast

The genome of Arabidopsis thaliana encodes 54 functional glutathione transferases (GSTs), classified in seven clades. Although plant GSTs have been implicated in the detoxification of xenobiotics, such as herbicides, extensive redundancy within this large gene family impedes a functional analysis in planta. In this study, a GST-deficient yeast strain was established as a system for analyzing plant GSTs that allows screening for GST substrates and identifying substrate preferences within the plant GST family. To this end, five yeast genes encoding GSTs and GST-related proteins were simultaneously disrupted. The resulting yeast quintuple mutant showed a strongly reduced conjugation of the GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl). Consistently, the quintuple mutant was hypersensitive to CDNB, and this phenotype was complemented by the inducible expression of Arabidopsis GSTs. The conjugating activity of the plant GSTs was assessed by in vitro enzymatic assays and via analysis of exposed yeast cells. The formation of glutathione adducts with dinitrobenzene was unequivocally verified by stable isotope labeling and subsequent accurate ultrahigh-resolution mass spectrometry (ICR-FTMS). Analysis of Arabidopsis GSTs encompassing six clades and 42 members demonstrated functional expression in yeast by using CDNB and NBD-Cl as model substrates. Subsequently, the established yeast system was explored for its potential to screen the Arabidopsis GST family for conjugation of the fungicide anilazine. Thirty Arabidopsis GSTs were identified that conferred increased levels of glutathionylated anilazine. Efficient anilazine conjugation was observed in the presence of the phi, tau, and theta clade GSTs including AtGSTF2, AtGSTF4, AtGSTF6, AtGSTF8, AtGSTF10, and AtGSTT2, none of which had previously been known to contribute to fungicide detoxification. ICR-FTMS analysis of yeast extracts allowed the simultaneous detection and semiquantification of anilazine conjugates as well as catabolites.

Effects of atrazine and chlorpyrifos on activity and transcription of glutathione S-transferase in common carp (Cyprinus carpio L.)

Glutathione S-transferase isoenzymes (GSTs) play a critical role in detoxification pathways. Here we report the tissue distribution of four antioxidant GSTs gene in common carp, and their expression profiles. We also investigated the GSTs activity in different tissues after exposure to the agricultural chemicals atrazine (ATR), chlorpyrifos (CPF), and their mixture. Relative changes in the mRNA abundance of the GST isoforms were examined by real time PCR in liver, brain, kidney and gill of common carp. After exposure and recovery, we observed a statistically significant decrease in the GSTs activity in animals exposed to high concentrations of ATR (428μg/L), CPF (116μg/L), and their mixture (113μg/L). At basal levels of tissue expression, four GSTs transcript were detected in liver, brain, kidney, and gill. High expression levels were found in all examined tissues. Transcription of some GST isoforms, GST kappa (GSTK), GST theta (GSTT) and GST rho (GSTR), decreased after exposure to CPF and ATR for the entire experimental period in both the kidney and gill. However, increased transcription of GST mu (GSTM) was observed in the kidney or gill 20d after exposure to ATR or CPF, respectively. Transcription of both GSTT and GSTR was inhibited for the entire experimental period in the brain, kidney and gill of animals exposed to the ATR/CPF mixture, but transcription of GSTM was induced in the liver after 40d of exposure. In summary, changes in the GSTs activity and their transcription varied within each organ and among organs of common carp after exposure to ATR, CPF, and their mixture.

Effect of Organophosphate Phoxim Exposure on Certain Oxidative Stress Biomarkers in the Silkworm

Organophosphorus (OP) insecticides are widely used in agriculture, which are toxic to insect pests and nontarget organisms. The current study mainly assessed the effect of the pesticide phoxim on oxidative stress by certain biomarkers in the fat body and midgut of the silkworm, Bombyx mori (L.), after exposure to 50% lethal concentration (LC50) of phoxim for 2 h. Malondialdehyde (MDA) content, activity of glutathione transferase (GST), and expression of GST at transcriptional level were assayed. LC50 value of phoxim was 2.5 mg/liter at 2-h exposure for the day 3 of the fifth-instar larvae. After exposure of phoxim, MDA content in the fat body significantly increased at 4-20 h posttreatment (p.t.),the highest increase was approximately 4.11-fold from 0.451 +/- 0.053 to 1.854 +/- 0.113 nmol/mg protein compared with corresponding control. In the midgut, significant increase in the MDA content (from 1.40- to 3.16-fold) was observed at 8-42 h p.t. The activity of GSTs increased to 1.48-2.00-fold at 24-42 h p.t. and 1.33-1.48-fold at 20-24 h p.t. in the fat body and midgut, respectively. The peroxidase activity of GSTs also was induced, which increased to 1.46-2.06-fold and 1.31-1.50-fold in the fat body and midgut, respectively. BmGSTe8 showed a late up-regulation of transcripts at 24-42 h after exposure to phoxim, which might contribute to the improved phoxim tolerance of silkworm larvae. These results indicated that phoxim could trigger oxidative stress and that MDA content and GST activity might be used as biomarkers of OP insecticide exposure. In addition, activity of GSTs were more inducible in the fat body than in midgut.

Insecticide resistance in Aedes aegypti populations from Ceará, Brazil

BackgroundOrganophosphates and pyrethroids are used widely in Brazil to control Aedes aegypti, the main vector of dengue viruses, under the auspices of the National Programme for Dengue Control. Resistance to these insecticides is widespread throughout Brazil. In Ceará the vector is present in 98% of districts and resistance to temephos has been reported previously. Here we measure resistance to temephos and the pyrethroid cypermethrin in three populations from Ceará and use biochemical and molecular assays to characterise resistance mechanisms.ResultsResistance to temephos varied widely across the three studied populations, with resistance ratios (RR95) of 7.2, 30 and 192.7 in Juazeiro do Norte, Barbalha and Crato respectively. The high levels of resistance detected in Barbalha and Crato (RR95 ≥ 30) imply a reduction of temephos efficacy, and indeed in simulated field tests reduced effectiveness was observed for the Barbalha population. Two populations (Crato and Barbalha) were also resistant to cypermethrin, whilst Juazeiro do Norte showed only an altered susceptibility. The Ile1011Met kdr mutation was detected in all three populations and Val1016Ile in Crato and Juazeiro do Norte. 1011Met was significantly associated with resistance to cypermethrin in the Crato population. Biochemical tests showed that only the activity of esterases and GSTs, among the tested detoxification enzymes, was altered in these populations when compared with the Rockefeller strain.ConclusionsOur results demonstrate that two A. aegypti populations from Ceará are under strong selection pressure by temephos, compromising the field effectiveness of this organophosphate. Our results also provide evidence that the process of reducing resistance to this larvicide in the field is difficult and slow and may require more than seven years for reversal. In addition, we show resistance to cypermethrin in two of the three populations studied, and for the first time the presence of the allele 1016Ile in mosquito populations from northeastern Brazil. A significant association between 1011Met and resistance was observed in one of the populations. Target-site mechanisms seem not to be implicated in temephos resistance, reinforcing the idea that for the studied populations, detoxification enzymes most likely play a major role in the resistance to this insecticide.

Resistance of Spanish codling moth (Cydia pomonella) populations to insecticides and activity of detoxifying enzymatic systems

Resistance of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) to insecticides has become a major problem in many apple and pear production areas. Our aim was to determine the level of insecticide resistance in Spanish field populations. Seven field populations collected from apple, Malus domestica Borkhausen (Rosaceae), orchards and three laboratory susceptible strains of codling moth were studied. Damage at harvest in all the conventional orchards from which codling moth populations were collected was higher than the economic threshold. The efficacy of eight insecticides, with five modes of action, was evaluated by topical application of the diagnostic concentrations on post-diapausing larvae. The enzymatic activity of mixed-function oxidases (MFOs), glutathione transferases (GSTs), and esterases (ESTs) was evaluated for each population. The susceptibility to insecticides and the biochemical activity of the three laboratory strains and one organic orchard population were not significantly different. Field populations were less susceptible to the tested insecticides than the susceptible strains, especially for azinphos-methyl, diflubenzuron, fenoxycarb, and phosalone. The efficacy of all insecticides was significantly dependent on the activity of MFOs. Only the toxicity of the three insecticides most used in Spain when the populations were collected (azinphos-methyl, fenoxycarb, and phosalone) was also dependent on the activity of ESTs and GSTs activity. According to our results, the control failures were due to the existence of populations resistant to the main insecticides used.

Functional characteristic of PC12 cells with reduced microsomal glutathione transferase 1.

Microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activities and is active in biotransformation of xenobiotics and in defense against oxidative stress. To assess MGST1 role in the development and functioning of PC12 cells, we constructed a cell line with reduced MGST1 (PC12_M). Real-time PCR and immunoblot assays showed MGST1 expression lowered to 60 % and immunocytochemical analyses demonstrated an altered concentration and distribution of the enzyme. PC12_M cells revealed a larger tendency to grow in clusters, weaker adhesion, irregular shape of bodies, short neurite outgrowth and higher percentage of necrotic cells (34 %). The total GSTs activity determined with non-specific substrate CDNB (1-chloro-2,4-dinitrobenzene) decreased by 15-20 %, whereas that with DCNB (2,4-dichloro-1-nitrobenzene), a substrate more specific for cytosolic GSTs, was similar to the one in control cells. This suggests that reduction of MGST1 cannot be compensated by other glutathione transferases. In PC12_M cells the total glutathione content was higher by 15-20 %, whereas the GSSG/GSH ratio was lower than in control cells. Moreover, the laminin-dependent migration rate was much faster in control cells than in PC12_M, suggesting some alterations in the metastatic potential of the line with suppressed MGST1. The amount of MAP kinases (p38, JNK, ERK1/2) was elevated in PC12_M cells but their phosphorylation level declined. Microarray analysis showed changed expression of several genes, which may be linked with differentiation and necrosis of PC12_M cells. Our data suggest that MGST1 could be an important regulator of PC12 cells development and might have significant effects on cell growth and proliferation, probably through altered expression of genes with different biological function.

Alternation between dietary protein depletion and normal feeding cause liver damage in mouse

The effect of frequent protein malnutrition on liver function has not been intensively examined. Thus, the effects of alternating 5days of a protein and amino acid-free diet followed by 5days of a complete diet repeated three times (3 PFD-CD) on female mouse liver were examined. The expression of carbonic anhydrase III (CAIII), fatty acid synthase (FAS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSTP1) in liver were assessed by proteomics, reverse transcriptase-polymerase chain reaction and Northern blotting. The activities of liver GSTs, glutathione reductase (GR) and catalase (CAT), as well as serum glutamic-oxaloacetic transaminase (SGOT) and glutamic-pyruvic transaminase (SGPT) were also tested. Additionally, oxidative damage was examined by measuring of protein carbonylation and lipid peroxidation. Liver histology was examined by light and electron microscopy. Compared with control mice, 3 PFD-CD increased the content of FAS protein (+90%) and FAS mRNA (+30%), while the levels of CAIII and CAIII mRNAs were decreased (-48% and -64%, respectively). In addition, 3 PFD-CD did not significantly change the content of GSTP1 but produced an increase in its mRNA level (+20%), while it decreased the activities of both CAT (-66%) and GSTs (-26%). After 3 PFD-CD, liver protein carbonylation and lipid peroxidation were increased by +55% and +95%, respectively. In serum, 3 PFD-CD increased the activities of both SGOT (+30%) and SGPT (+61%). In addition, 3 PFD-CD showed a histological pattern characteristic of hepatic damage. All together, these data suggest that frequent dietary amino acid deprivation causes hepatic metabolic and ultrastructural changes in a fashion similar to precancerous or cancerous conditions.

A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

BackgroundInsecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations.Methodology/Principal FindingsWe have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically.Conclusions/SignificanceThe novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control.

QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIP ANALYSIS OF CURCUMIN AND ITS DERIVATIVES AS GST INHIBITORS BASED ON COMPUTATIONAL CHEMISTRY CALCULATION

The Quantitative Structure-Activity Relationship (QSAR) study was established on curcumin and its derivatives as glutathione S-transferase(s) (GSTs) inhibitors using atomic net charges as the descriptors. The charges were resulted by semiempirical AM1 and PM3 quantum-chemical calculations using computational chemistry approach. The inhibition activity was expressed as the concentration that gave 50% inhibition of GSTs activity (IC50). The selection of the best QSAR equation models was determined by multiple linear regression analysis. This research was related to the nature of GSTs as multifunctional enzymes, which play an important role in the detoxification of electrophilic compounds, the process of inflammation and the effectivity of anticancer compounds. The result showed that AM1 semiempirical method gave better descriptor for the construction of QSAR equation model than PM3 did. The best QSAR equation model was described by : log 1/IC50 = -2,238 - 17,326 qC2' + 1,876 qC4' + 9,200 qC6' The equation was significant at 95% level with statistical parameters : n = 10, m = 3, r­ = 0,839, SE = 0,254, F = 4,764, F/Ftable = 1,001. Keywords: QSAR analysis, curcumin, glutathione S-transferase(s) (GSTs), atomic net charge

Effect of acrylamide on chick embryonic liver glutathione S-transferases

Glutathione S-transferases (GSTs, EC 2.5.1.18), as the detoxifying enzymes, play an important protective role in embryonic tissues. The GSTs of eleventh-day chick embryonic liver were purified to electrophoretic homogeneity with an overall yield of 31%. The SDS-PAGE analysis of chick embryonic liver GSTs resolved in to three bands, CL1, CL2 and CL3, with relative molecular weights of 27.0, 26.0 and 25.0 kDa respectively. On 72 hours of treatment with acrylamide (AC) (0.1, 0.2 and 0.3 mg) the specific activities of GSTs with CDNB, pNBC, EPNP, BSP, Δ55A and pNPA increased significantly. Total GPx levels were decreased with CHP in liver samples. Immunoblot analysis of AC-treated liver samples showed a dose-dependent increase in the induction of GSTs. CL1 of theta class, CL2 of Mu class and CL3 of alpha class were induced with AC treatment. The theta, mu and alpha classes induced by AC might inhibit the initiation of carcinogenesis.

Biological activity of the essential oil from the leaves of Piper sarmentosum Roxb. (Piperaceae) and its chemical constituents on Brontispa longissima (Gestro) (Coleoptera: Hispidae)

The results showed that the essential oil from Piper sarmentosum has strong antifeedant and toxicity effects on Brontispa longissima. The best antifeedant and contact toxicity effects were observed in the 1st–2nd instar larvae. The essential oil also displayed a notable fumigation effect on the eggs and pupae of B. longissima. It took the control-treated insects 43.34 d to complete one generation, while the insects treated with 2000 mg/L essential oil needed 73.58 d. The essential oil was analyzed by gas chromatography/mass spectrometry and 41 components were identified. Myristicin (65.22%) and trans-caryophyllene (13.89%) were the major components. Myristicin exhibited strong antifeedant and contact toxicity effects on both the 3rd instar larvae and the imagoes of B. longissima, when it showed a significant fumigation effect on the eggs and pupae of B. longissima. Another P. sarmentosum essential oil and myristicin all showed a strong inhibiting effect on the growth and development of B. longissima along with the activity of AChE, CarE, GSTs and Na +, K +-ATPase in B. longissima larvae.

Establishment of a multiplex ligation-dependent SNP genotyping method and its application in the detection of genes related to chemotherapeutic drugs in breast cancer

To establish a method for SNP genotyping of multi-genes by allele-specific oligonucleotide probe ligation mediated by a thermostable ligase, and to explore the genetic polymorphisms of drug-metabolizing enzymes in breast cancer patients and their association with chemotherapeutic responses. 10 SNP loci of enzyme genes related to chemotherapeutic drugs such as taxanes, anthracyclines and cyclophosphamide were selected, and were genotyped for blood samples from 126 breast cancer patients by the established method. Their correlations with therapeutic responses were retrospectively evaluated. The lower detection limit of genomic DNA by this developed method was 6.25 ng. The fluorescent peak locations of ligation products on ABI PRISM 377 DNA sequencer were accurate and consistent with prospective sizes in bases (Bias range 0.08 - 0.69 bp, x(-) = 0.31 bp, s = 0.18 bp). Same genotyping results were obtained for repeat tests of 8 random samples, which were further confirmed by sequencing analysis. The 10 SNP loci were polymorphic of different frequency in the breast cancer patients. The combinations with GSTP1 genotypes and GSTM1 genotypes were related to anthracycline-based chemotherapy efficacy (P = 0.037), and the low GSTs activity group (GSTP1 variant allele + GSTM1 null) showed the best effects (85.7%). GSTM1 genotypes and their combinations with GSTP1 and/or CYP3A5*3 genotypes were related to taxane-based therapy efficacy (P < 0.05 for all), and both the low GSTs activity group and the drug slow-metabolising group (low GSTs activity group + CYP3A5*3 wild allele) showed better effects (100%). The established method is reliable and applicable in multiplex SNPs genotyping of multi-genes. SNPs combination may have a better clinical application value for prediction of chemotherapeutic responses.

“Restoration” of Glutathione Transferase Activity By Single-site Mutation of The Yeast Prion Protein Ure2

The yeast prion Ure2p is composed of an N-terminal prion domain, and a C-terminal globular domain, which shows similarity to glutathione transferases (GSTs) in both sequence and structure. Ure2p protects Saccharomyces cerevisiae cells from heavy metal ion and oxidant toxicity. Ure2p shows glutathione-dependent peroxidase (GPx) activity, which is often an adjunct activity of GSTs, but wild-type Ure2p shows no detectable GST activity toward the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB). The structural basis for the substrate specificity of Ure2p enzymatic activity is an interesting problem that is fundamental to understanding the in vivo roles of Ure2p and its relationship to the GST structural family. The critical catalytic residue in the other known GSTs is Ser, Tyr or Cys. Here, we demonstrate that residue N124 is important for the GPx activity of Ure2p, and a wild-type level of activity is maintained in N124S, but not in N124Y/C. Interestingly, we found that the single-site mutations A122C and N124A/V (but not N124S/Y/C) ”restore” the GST activity of Ure2p toward CDNB, while causing a substantial reduction in GPx activity. The steady-state kinetics for the GST activity of A122C appears to follow a ping-pong mechanism. In contrast, the GST activity of 124-site mutants shows a sequential mechanism, as is observed for the native GPx activity of Ure2p, and typical GST enzymes. These findings shed light on the evolutionary relationship of Ure2p with other GST family members, and contribute to our understanding of catalytic promiscuity and divergent evolution.

MUDPIT ANALYSIS OF PSEUDOMONAS AERUGINOSA SECRETOME: CONSEQUENCES OF OXYGEN LIMITATION IN CLINICAL AND LABORATORY STRAINS

CF cell line IB3−1 presented with increased GSTT1 and GSTM1 mRNA and protein expression levels in comparison with the isogenic non-CF cell line C38. AZM exposure significantly decreased GSTT1 and GSTM1 mRNA and protein expression in IB3−1, restoring the levels observed in C38 cells. In another CF cell line, 2CFSMEo-, AZM produced 45% reductions in GSTT1 and GSTM1 mRNA levels. The macrolide JM, known to lack clinical anti-inflammatory properties, had no significant effects on GSTT1 and GSTM1 mRNA expression in all cell lines. GSTP1 was not differentially expressed in all CF and non-CF cells and its expression was unaffected by AZM. AZM reduced GST activity of about 25% and 40% in IB3−1 and 2CFSMEo− cells, respectively. Finally, GSTs activity and the expression of GSTT1 and GSTM1 proteins in CF cells, were reduced by interleukin 10 (IL-10), an anti-inflammatory cytokine, approximately to the same level detected after treatment with AZM, shedding light on a possible correlation between GSTs inhibition and anti-inflammatory properties of AZM. Downregulation of GSTT1 and GSTM1 expression may result in increased availability of intracellular GSH making CF cells less susceptible to oxidative stress induced by chronic inflammation. Thus these results suggest novel anti-oxidant properties for AZM. *CC and GB equally contributed to this work. This study is supported by Italian CF Research Foundation (FFC10/2005; 17/2006); Comitato di Vicenza-Associazione Veneta per la lotta contro la Fibrosi Cistica; Azienda Ospedaliera Verona, Italy.

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.

THE ROLES OF DETOXIFYING ENZYMES AND AChE INSENSITIVITY IN METHAMIDOPHOS RESISTANCE DEVELOPMENT AND DECLINE IN NILAPARVATA LUGENS

AbstractMethamidophos resistance of brown planthopper (Nilaparvata lugens Stål, BPH) was selected in laboratory. After successive selection for 9 generations, the selection was ceased by rearing BPH without contact with any insecticide for 9 generations. In the full course, the successive changes of esterase activity, MFO activity, GSTs activity and AChE insensitivity were analyzed. The results showed that the change of esterase activity was high correlated with that of methamidophos in the full course, which indicated that esterase played very important role both in the resistance development and in the resistance decline. However, the change of AChE insensitivity only significantly correlated with that of resistance in the development stage, and the change of MFO activity or GSTs activity only significantly correlated with that of the resistance in the decline stage, which indicated the changes of AChE insensitivity, MFO activity or GSTs activity only played some roles in different stages of the resistance change.

An enzyme based dechlorination of a polychlorinated biphenyl (PCB) mixture, Aroclor 1248, using glutathione S-transferases from the northern quahog Mercenaria mercenaria.

The glutathione S-transferases from the northern quahog (Mercenaria mercenaria) from control and contaminated sites were subjected to incubation with polychlorinated biphenyls in an Aroclor 1248 mixture. Subsequent solvent-solvent extraction and gas chromatographic analysis revealed that 2 of the 28 congeners present in the Aroclor 1248 mixture were affected by the presence of the GST. The first of the aforementioned congeners with a retention time of 36.7 min by gas chromatographic analysis decreased in total content. The largest decrease in the 36.7 min peak was the result of incubation with GSTs purified from quahogs taken from the Superfund site in New Bedford Harbor, New Bedford, MA. The second of the affected congeners with a retention time of 59 min showed an increase, which could result from the glutathione conjugation to the PCB congener. The Aroclor 1248 mixture (a limited number of its constituent congeners) also acts as a competitive inhibitor of the GST activity, which is indicative of a substance interacting with the free GST in solution. The ultimate result of the conjugation of the PCB congener to GSH would be the formation of a hydrophillic conjugate of an otherwise insoluble PCB. The PCB-GSH conjugating activity of the quahog GSTs may ultimately serve as a tool for PCB remediation.

Catalytic activities of human alpha class glutathione transferases toward carcinogenic dibenzo[a,l]pyrene diol epoxides.

In this study, human glutathione transferases (GSTs) of alpha class have been assayed with the ultimate carcinogenic (-)-anti- and (+)-syn-diol epoxides (DEs) derived from the nonplanar dibenzo[a,l]pyrene (DBPDE) and the (+)-anti-diol epoxide of the planar benzo[a]pyrene [(+)-anti-BPDE] in the presence of glutathione (GSH). In all DEs, the benzylic oxirane carbon reacting with GSH, possess R-absolute configuration. GSTA1-1 demonstrated activity with all DEs tested whereas A2-2 and A3-3 only were active with the DBPDE enantiomers. With GSTA4-4, no detectable activity was observed. GSTA1-1 was found to be the most efficient enzyme and demonstrated a catalytic efficiency (k(cat)/K(m)) of 464 mM(-)(1) s(-)(1) with (+)-syn-DBPDE. This activity was about 7-fold higher than that observed with (-)-anti-DBPDE and more than 65-fold higher than previously observed with less complex fjord-region DEs. GSTA3-3 also demonstrated high k(cat)/K(m) with the DEs of DBP and a high preference for the (+)-syn-DBPDE enantiomer [190 vs 16.2 mM(-)(1) s(-)(1) for (-)-anti-DBPDE]. Lowest k(cat)/K(m) value of the active enzymes was observed with GSTA2-2. In this case, 30.4 mM(-)(1) s(-)(1) was estimated for (+)-syn-DBPDE and 3.4 mM(-)(1) s(-)(1) with (-)-anti-DBPDE. Comparing the activity of the alpha class GSTs with (-)-anti-DBPDE and (+)-anti-BPDE revealed that GSTA1-1 was considerable more active with the former substrate (about 25-fold). Molecular modeling studies showed that the H-site of GSTA1-1 is deeper and wider than that of GSTA4-4. This is mainly due to the changes of Ser212-->Tyr212 and Ala216-->Val216, which cause a shallower active site, which cannot accommodate large substrates such as DBPDE. The higher activity of GSTA1-1 with (+)-syn-DBPDE relative to (-)-anti-DBPDE is explained by the formation of more favorable interactions between the substrate and the enzyme-GSH complex. The presence of GSTA1-1 in significant amounts in human lung, a primary target tissue for PAH carcinogenesis, may be an important factor for the protection against the harmful action of this type of potent carcinogenic intermediates.

Proposed reductive metabolism of artemisinin by glutathione transferases in vitro

Artemisinin is a sesquiterpene lactone containing an endoperoxide bridge. It is a promising new antimalarial and is particularly useful against the drug resistant strains of Plasmodium falciparum. It has unique antimalarial properties since it acts through the generation of free radicals that alkylate parasite proteins. Since the antimalarial action of the drug is antagonised by glutathione and ascorbate and has unusual pharmacokinetic properties in humans, we have investigated if the drug is broken down by a typical reductive reaction in the presence of glutathione transferases. Cytosolic glutathione transferases (GSTs) detoxify electrophilic xenobiotics by catalysing the formation of glutathione (GSH) conjugates and exhibit glutathione peroxidase activity towards hydroperoxides. Artemisinin was incubated with glutathione, NADPH and glutathione reductase and GSTs in a coupled assay system analogous to the standard assay scheme with cumene hydroperoxide as a substrate of GSTs. Artemisinin was shown to stimulate NADPH oxidation in cytosols from rat liver, kidney, intestines and in affinity purified preparations of GSTs from rat liver. Using human recombinant GSTs hetelorogously expressed in Escherichia coli, artemisinin was similarly shown to stimulate NADPH oxidation with the highest activity observed with GST M1-1. Using recombinant GSTs the activity of GSTs with artemisinin was at least two fold higher than the reaction with CDNB. Considering these results, it is possible that GSTs may contribute to the metabolism of artemisinin in the presence of NADPH and GSSG-reductase We propose a model, based on the known reactions of GSTs and sesquiterpenes, in which (1) artemisinin reacts with GSH resulting in oxidised glutathione; (2) the oxidised glutathione is then converted to reduced glutathione via glutathione reductase; and (3) the latter reaction may then result in the depletion of NADPH via GSSG-reductase. The ability of artemisinin to react with GSH in the presence of GST may be responsible for the NADPH utilisation observed in vitro and suggests that cytosolic GSTs are likely to be contributing to metabolism of artemisinin and related drugs in vivo.

Activation and detoxication of aflatoxin B 1

Aflatoxin B 1 (AFB 1) is a potent hepatocarcinogen in experimental animals and a hazard to human health in several parts of the world. Implementation of rational intervention plans requires understanding of aspects of the roles of individual chemical steps involved in its disposition. AFB 1 is activated to AFB 1 exo-8,0-epoxide primarily by cytochrome P450 (P450) enzymes, particularly P450 3A4. However, P450 3A4 and other P450s also oxidize AFB 1 to less dangerous products. The exo-epoxide is unstable in H 2O ( t 1 2 1 s at 25°C, k = 0.6 s −1) and the diol product undergoes base-catalyzed rearrangement to a dialdehyde that reacts with protein lysine residues. AFB 1 exo-8,9-epoxide reacts with DNA to give adducts in high yield (> 98%). This interaction is characterized by a K d of ∼ 1.4 mM, intercalation between base pairs, and rapid reaction with the guanyl N 7 atom ( k ∼ 40 s −1). A proton field on the periphery of DNA is postulated to catalyze hydrolysis and also conjugation. Rat and especially human epoxide hydrolase show very little rate acceleration of hydrolysis of AFB 1 exo- or endo-8,9-epoxide. However, glutathione transferases (GSTs) can catalyze AFB 1 exo-8,9-epoxide conjugation. Kinetic analysis indicates a range of ratios of k cat/ K d varying from 10 to 1700 s −1 M −1, with the polymorphic GST M1-1 having the highest activity of the human GSTs. Studies with human hepatocytes indicate a major role for GST M1-1 in AFB 1 conjugation and that the model chemoprotective agent oltipraz can act by both inducing GSTs and inhibiting P450s.