Inhibition Of Glutathione S-transferase Research Articles

BENZYLIDENE CYCLOPENTANONE DERIVATIVES AS INHIBITORS OF RAT LIVER GLUTATHIONE S-TRANSFERASE ACTIVITIES

The effect of the curcumin analogues, 2,6-bis-(4-hydroxy-3-methoxy benzylidene) cyclopentanone (B1) and two of its derivatives on m class glutathione S-transferases (GSTs) from phenobarbital-induced and uninduced rat liver cytosol has been studied to elucidate their anti-inflammatory activity. GST activity was monitored spectrophotometrically using 1,2-dichloro-4-nitrobenzene. B1 was the most potent inhibitor of GSTs, both in uninduced and in phenobarbital-induced rat liver cytosol. These inhibitory properties might be explained as part of the anti-inflammatory activity of benzylidene cyclopentanone derivatives (B1 and B12). Keywords: curcumin; benzylidene cyclopentanone; inhibitory potency; glutathione S-transferases mesoporous

ACTIVITY OF PROPYL p-BENZOYLOXYBENZOATE AS A GLUTATHIONE S-TRANSFERASE(S) INHIBITOR: THE COMPARISON BETWEEN COMPUTATIONAL CHEMISTRY APPROACH AND EMPIRICAL OBSERVATION

The activity of propyl p-benzoyloxybenzoate as a glutathione S-transferase(s) (GSTs) inhibitor has been examined through computational chemistry based theoretical approach and laboratory experiment. 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 effectivities of anticancer compounds. Quantitative Structure-Activity Relationship (QSAR) study, which was established on curcumin and its derivatives using computational chemistry approach, was used to examine the theoretical activity of p-benzoyloxybenzoate as a GSTs inhibitor. Empirical observation on GSTs inhibition was examined using formation reaction model of GS-CNB conjugate through conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) with GSTs (prepared from rat's liver) as catalysts. The result showed that the difference between the activities of propyl p-benzoyloxybenzoate as a GSTs inhibitor obtained from the computational chemistry approach and the empirical observation were not statistically significant at 95% level of confidence. Keywords: Propyl p-benzoyloxybenzoate, inhibitor, glutathione S-transferase (GSTs), QSAR

Biomarkers as a tool to assess effects of chromium (VI): Comparison of responses in zebrafish early life stages and adults

The present work aims to compare the sensitivity of embryos and adult zebrafish to chromium (VI) (as potassium dichromate) focusing on biomarkers (cholinesterase, glutathione S-transferase and lactate dehydrogenase) as endpoints. Zebrafish eggs showed less sensitivity to Cr (VI) (96 h-LC 50 = 145.7 mg/L) than adults (96 h-LC 50 = 39.4 mg/L) probably due to the protective action of the chorion. However, biomarkers were much more responsive in larvae than in adults and gave clear indications about Cr (VI) mode of action: it seems to be neurotoxic (inhibited cholinesterase), to inhibit glutathione S-transferase activity and to interfere with cellular metabolic activity (changes in lactate dehydrogenase activity) in larvae. In adults, only glutathione S-transferase was responsive, showing a clear inhibition. The responsiveness of the analyzed biomarkers in larvae reinforces the idea of the usefulness of early life stage assays in the assessment of chemicals effects. Moreover, early life stage assays also contributed with relevant information regarding anomalies in larvae development and behavior. Further research should focus on the use of biomarkers to assess long term effects which are ecologically more relevant.

Sulforaphane induces glutathione S‐transferase isozymes which detoxify aflatoxin B1‐8,9‐epoxide in AML 12 cells

The aflatoxin B(1)-8,9-epoxide (AFBO) is hepatocarcinogenic intermediate of aflatoxin B(1) (AFB(1)) and is detoxified by glutathione S-transferases (GSTs). In this study, we investigated whether sulforaphane (SFN) could increase the rate of conjugation between AFBO and glutathione (GSH) as well as which of the GST isozymes were involved in the conjugation reaction. The conjugation potential was inhibited dose dependently with curcumin, an inhibitor of GSTs. SFN induced the expression of GST A3, GST A4, GST M1, GST P1, and GST T1 in alpha mouse line (AML) 12 cells. The cells treated with SFN (10 microM) for 12 h showed a 35-fold increase in conjugation potential of AFBO with GSH compared with the vehicle-treated cell. The conjugation potential was blocked partially by transfection of cells with siRNAs against each of the GST isozymes. The activity of GST A3 had the strongest effect on the conjugation potential. SFN treatment also increased total GST activity detected with 1-chloro-2,4-dinitrobenzene (CDNB) up to 4.3-fold. The induction fold was much lower than that detected with AFBO. These results suggest that the chemopreventive effect of SFN on the decomposition of AFBO is related to the upregulation of several GST isozymes genes. The increase of GST activity by SFN was extremely specific toward the conjugation reaction of AFBO compared with CDNB. Therefore, this system for detecting GST activity seems to be an excellent method for screening chemopreventive compounds toward AFB(1) toxicity.

Advantages of Human Hepatocyte-Derived Transformants Expressing a Series of Human Cytochrome P450 Isoforms for Genotoxicity Examination

Metabolites of chemicals can often be ultimate genotoxic species; thus, in vitro routine testing requires the use of rat liver S9. However, there is a question as to whether this represents an appropriate surrogate for human metabolism. We have previously demonstrated the usefulness of HepG2 transformants expressing major human cytochrome P450 (CYP) isoforms to assess the genotoxicity of metabolites. We further assessed the advantages of these transformants from the following three aspects. First, the sensitivity of these transformants was confirmed with micronucleus (MN) induction by 7,12-dimethylbenz[a]anthracene or ifosfamide in transformants expressing the corresponding CYP1A1 or CYP2B6 and CYP2C9, respectively. Second, by using these transformants, beta-endosulfan, a chemical for which the CYP isoforms contributing to its genotoxicity are unknown, was found to induce MN through the CYP3A4-mediated pathway. This result was confirmed by the facts that the decreased CYP3A4 activity using a inhibitor or short interfering RNA (siRNA) repressed MN induction by beta-endosulfan and that endosulfan sulfate, one of the metabolites produced by CYP3A4, induced MN in the transformants harboring an empty vector. Third, the interaction between phase I and II drug-metabolizing enzymes was demonstrated by MN induction with inhibitors of uridine diphosphate (UDP)-glucuronosyltransferases in tamoxifen-treated transformants harboring the corresponding CYP3A4 or with inhibitors of glutathione S-transferase in safrole-treated transformants harboring the corresponding CYP2D6, whereas neither tamoxifen nor safrole alone induced MN in any transformant. These advantages provide the benefits of newly established transformants for in vitro genotoxicity testing that reflects comprehensive metabolic pathways including not only human CYP isoforms but also the phase II enzymes.

In Pursuit of Bioactive Constituents from Sri Lankan Medicinal Plants

In search for bioactive natural products in Sri Lankan medicinal plants, Terminalia arjuna (Roxb.) Wight & Arnot. (Combretaceae), Nelumbo nucifera (Nymphaeaceae), Nymphaea stellata (Nymphaeaceae), Barleria prionitis Linn. (Acanthaceae), Caesalpinia bonduc L. (Fabaceae) and Artocarpus nobilis (Artocarpaceae) were identified to exhibit anti-microbial, free radical scavenging, anti-tyrosinase and acetylcholinesterase (AchE) and glutathione s-transferase (GST) inhibitory activities. Bio-assay guided fractionation of extracts of these plants by chromatography afforded active constituents and spectroscopic techniques including multi-dimensional NMR techniques were utilized to elucidate the structures of compounds. Two antibacterial compounds, olean-1α, 3β, 22β-triol-12-en-28-oic acid and olean-1α, 3β, 22β-triol-12-en-28-oic acid-3β-D-glucopyranoside were isolated from ethyl acetate extract of the stem bark of T. arjuna. Stamen extracts of N. nucifera and N. stellata afforded free radical scavenging compounds, Kaemperferol and Cyanadin 3-O-2((Prime))-O-galloyl-β-galactopyranoside). Extracts of B. prionitis, a plant extensively used in Ayurveda, exhibited GST and AchE inhibitory, antibacterial and free radical scavenging activities and the ethanolic extracts afforded iridoid glycosides as potential GST and AchE inhibitors. Chemical studies of C. bonduc, which is used in indigeneous system of medicine, afforded GST inhibiting 13,14-seco-steroids. Extracts of A. nobilis exhibited AchE and GST inhibitory and antibacterial activities and afforded cycloartane-type triterpenoids, artocarpuate A and B and flavonoids, artoblidxanthone and artoninse exhibiting GST and AchE inhibitory activities. Acknowledgements: Research students, Gunathilaka S, Abeysekera K, Gayani P G and Kosmulalage K S who involved in various aspects of this research are greatly acknowledged.

Inhibition by Cu2+ and Cd2+ of a mu‐class glutathione S‐transferase from shrimp Litopenaeus vannamei

Glutathione S-transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of xenobiotics and aiding its excretion from the cell. The present work presents the inhibition of a mu-class GST of the marine shrimp Litopenaeus vannamei by copper (Cu2+) and cadmium (Cd2+). The protein was overexpressed in bacteria and its enzymatic activity measured using 1-chloro-2,4-dinitrobenzene. The mean inhibitory concentration (IC(50)) for shrimp GST against Cu2+) was 4.77 microM and for Cd2+ was 0.39 microM. A molecular model of the protein based on the crystal structure of a maize GST bound to cadmium showed that the metal binds in the GSH-binding site by coordination with Asp and Gln residues. These results are consistent with the experimental data and suggest that sublethal concentration of metals may affect the capacity of the organism to detoxify pesticides or xenobiotics.

Biochemical warfare on the reef: the role of glutathione transferases in consumer tolerance of dietary prostaglandins.

BackgroundDespite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla.Principal Findings Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity.SignificanceThe high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may operate as ‘all-purpose’ detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.

Increased transcription of Glutathione S-transferases in acaricide exposed scabies mites.

BackgroundRecent evidence suggests that Sarcoptes scabiei var. hominis mites collected from scabies endemic communities in northern Australia show increasing tolerance to 5% permethrin and oral ivermectin. Previous findings have implicated detoxification pathways in developing resistance to these acaricides. We investigated the contribution of Glutathione S-transferase (GST) enzymes to permethrin and ivermectin tolerance in scabies mites using biochemical and molecular approaches.ResultsIncreased in vitro survival following permethrin exposure was observed in S. scabiei var. hominis compared to acaricide naïve mites (p < 0.0001). The addition of the GST inhibitor diethyl maleate restored in vitro permethrin susceptibility, confirming GST involvement in permethrin detoxification. Assay of GST enzymatic activity in mites demonstrated that S. scabiei var. hominis mites showed a two-fold increase in activity compared to naïve mites (p < 0.0001). Increased transcription of three different GST molecules was observed in permethrin resistant S. scabiei var. canis- mu 1 (p < 0.0001), delta 1 (p < 0.001), and delta 3 (p < 0.0001). mRNA levels of GST mu 1, delta 3 and P-glycoprotein also significantly increased in S. scabiei var. hominis mites collected from a recurrent crusted scabies patient over the course of ivermectin treatment.ConclusionsThese findings provide further support for the hypothesis that increased drug metabolism and efflux mediate permethrin and ivermectin resistance in scabies mites and highlight the threat of emerging acaricide resistance to the treatment of scabies worldwide. This is one of the first attempts to define specific genes involved in GST mediated acaricide resistance at the transcriptional level, and the first application of such studies to S. scabiei, a historically challenging ectoparasite.

Inhibition of glutathione-S-transferase as a treatment strategy for multidrug resistance in childhood rhabdomyosarcoma

Multidrug resistance (MDR) is a common problem in the treatment of childhood rhabdomyosarcoma (RMS). A complete reversal of MDR is currently not possible. The aim of this study was to investigate the role of glutathione-S-transferase (GST) as mechanism of MDR in childhood RMS and to analyze possible reversal strategies. Female athymic mice underwent xenotransplantation with embryonal or alveolar RMS cells and were treated with vincristine. Gene expression analysis using Affymetrix HU-Gene 1.0 arrays revealed 2314 differentially expressed genes between the groups in alveolar RMS and 1387 in embryonal RMS. Ingenuity pathway analysis revealed a cluster of 5 overexpressed genes of the GST family in animals treated with vincristine, putative mediating the development of MDR. In order to analyze possible GST activity after chemotherapy with other commonly used drugs (doxorubicin, topotecan), cell culture experiments with alveolar and embryonal RMS cells were carried out. Specific GST activity was quantified using the clorodinitrobenzol conjugation with glutathione. Increased GST activity was found after incubation with cytotoxic agents in all cell lines. Highest induction of GST activity was found in embryonal RMS (up to 12-fold). After incubation with the GST inhibitors, tumor cell viability was decreased depending on the type of tumor cell and inhibitor used. We detected a novel mechanism for MDR in childhood RMS mediated via genes and proteins of the GST family. Reversal of these effects may be achieved by GST inhibitors in part. The GST family represents a promising target for further treatment strategies in childhood RMS.

Inhibition of the Initiation Stage of Carcinogenesis by Salvia disermas Constituents

Phytochemical studies of an ethanolic extract of the aerial parts of Salvia disermas resulted in the isolation of seven known compounds, rosmarinic (1) and caffeic (2) acids, salvigenin (3), luteolin (4), luteolin 7-O-beta-arabinoside (5), luteolin 7-O-beta-glucoside (6), and ocotillol II (7). The initiation stage of carcinogenesis is triggered by activation of procarcinogens by phase I enzymes, such as cytochrome P-450 1A, and oxidative stress that leads to DNA damage. The initiation stage is countered by phase II detoxification enzymes such as glutathione S-transferases (GST), quinine reductase (QR), epoxide hydrolase (mEH) besides conjugation with thiols. We aimed to investigate the cancer chemopreventive and tumour anti-initiating activity of the ethanolic extract of the aerial parts of Salvia disermas and its constituents. The S. disermas extract was a promising inhibitor of CYP1A activity, inducer of GST, QR, and mEH activities, enhancer of thiol content, radical scavenger, and inhibitor of DNA damage. On the other hand, 3 was an enhancer of thiol content and QR activity, while 4 was an inhibitor of CYP1A activity, inducer of QR activity, and radical scavenger of ROO*, and 5 was an inducer of GST activity and inhibitor of DNA damage. The present study indicated that the ethanolic extract of S. disermas and 4 are promising anti-initiating and multipotent blocking agents.

The effect of sixteen medicinal plants used in the Brazilian pharmacopoeia on the expression and activity of glutathione S-transferase in hepatocytes and leukemia cells

Glutathione S-transferase (GST) is a family of enzymes involved in the detoxification of electrophilic compounds. Different classes of GST are expressed in various organs, such as liver, lungs, stomach and others. Expression of GST can be modulated by diet components and plant-derived compounds. The importance of controlling GST expression is twofold: increasing levels of GST are beneficial to prevent deleterious effects of toxic and carcinogenic compounds, while inhibition of GST in tumor cells may help overcoming tumor resistance to chemotherapy. A screening of 16 plants used in the Brazilian pharmacopoeia tested their effects on GST expression in hepatocytes and Jurkat (leukemia) T-cells. The methanol extracts of five plants inhibited GST expression in hepatocytes. Three plants significantly inhibited and four others induced GST expression in Jurkat cells. Among these, the extracts of Bauhinia forficata Link. (Leguminosae) and Cecropia pachystachya Tréc. (Urticaceae) inhibited GST expression at relatively low concentrations. With the exception of B. forficata, all plants were cytotoxic when administered to Jurkat cells at high doses (1 mg/mL) and some extracts were considerably cytotoxic even at lower concentrations.

Inhibition of glutathione S-transferases (GSTs) activity from cowpea storage bruchid, Callosobrochus maculatus Frabiricius by some plant extracts

Crude ethanolic extracts of Tithonia diversifolia, Cyperus rotundus and Hyptis suavolens have insecticidal activity against Callosobrochus maculatus Frabiricius. The ethanol extracts of the plants have positive results for alkaloids, saponin, tannins and flavonoids. Antioxidant and reducing properties were also determined in the crude ethanol extracts. Cowpea storage bruchid, C. maculatus glutathione S-transferases was a potential target for the plants extracts. Gluathione S-transferase from cowpea storage bruchid was purified by affinity gel chromatography of glutathione sepharose. Inhibition effect of the plant extracts on the GST was studied by spectrophotometric method. The binding of the extract was competitive by the Dixon plot with Ki of 84, 132 and 180 ug/mL for T. diversifolia , C. rotundus and H. suavolens, respectively. We suggest that reported efficacy of the extract is due to the antioxidant properties and competitve binding inhibition on GST may contribute to the pharmacological basis of the efficacy against cowpea storage bruchid, C. maculatus Frabiricius and its attendant managements.

Comparing the role of glutathione-S-transferase and mitochondrial aldehyde dehydrogenase in nitroglycerin biotransformation and the correlation with calcitonin gene-related peptide

Both glutathione-S-transferase (GST) and mitochondrial aldehyde dehydrogenase (ALDH-2) have been reported to participate in the biotransformation of nitroglycerin. In this study, we explored which is the major player in nitroglycerin biotransformation. In vivo, rats were treated with nitroglycerin, the blood pressure and plasma calcitonin gene-related peptide (CGRP) were measured. The inhibitor of GST (ethacrynic acid) or ALDH-2 (cyanamide) was given before nitroglycerin treatment; In vitro, the isolated aorta rings were incubated with nitroglycerin to obtain the concentration–response curve. Ethacrynic acid or cyanamide was pre-incubated with the rings before nitroglycerin treatment. The release of CGRP from the aorta rings was determined. Both ethacrynic acid and cyanamide were able to reverse the depressant action of nitroglycerin while the inhibitory effect of cyanamide was more profound. However, combined administration of both inhibitors did not produce an additive effect. The change of plasma CGRP level positively correlated with the change of nitroglycerin-induced hypotensive effects. In the isolated aorta rings, vasodilator responses to nitroglycerin were reduced in the presence of ethacrynic acid or cyanamide while the inhibitory effect of cyanamide was more profound. However, combined administration of both inhibitors did not produce an additive effect. The change of CGRP release from the rings positively correlated with the nitroglycerin-induced vasodilator responses. The present results suggest that both GST and ALDH-2 are involved in nitroglycerin action while ALDH-2 plays a major role, and the change of CGRP contents closely correlates with the biotransformation of nitroglycerin.

In vitro antifilarial activity of glutathione S-transferase inhibitors

Female adult bovine filarial worms Setaria digitata were extracted with phosphate-buffered saline (pH 7.4) and glutathione S-transferase (GST) activity and protein content were determined. The protein content, GST enzyme activity, and specific activity were 10.61 +/- 3.41 mg ml(-1), 0.09 +/- 0.019 micromol min(-1) ml(-1), and 0.009 +/- 0.002 micromol min(-1) mg(-1) protein, respectively. The GST inhibition studies were performed with and without the inhibitors resulted from earlier molecular docking studies viz., ethacrynic acid, plumbagin, and curcumin for which the IC(50) values were 19.42, 51.41, and 114.86 microM, respectively. The in vitro macrofilaricidal activity of these molecules was studied by worm motility and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay at 24- and 48-h incubation. Plumbagin and ethacrynic acid showed 100% inhibition in worm motility at lower concentrations of 3.19 and 6.6 microM, respectively, at 48-h incubation while curcumin was effective at 54.29 microM. In MTT reduction assay, the ED(50) values (50% inhibition in formazan formation) for plumbagin, ethacrynic acid, and curcumin at 48-h incubation were 1.20, 2.48, and 19.86 microM, respectively. MTT reduction assay showed that plumbagin was the most effective in killing the adult S. digitata worms followed by ethacrynic acid and curcumin. In conclusion, all the three molecules selected by molecular modeling and docking studies inhibited the GST enzyme isolated from S. digitata and exhibited macrofilaricidal activity in vitro.

Prolactin confers resistance against cisplatin in breast cancer cells by activating glutathione-S-transferase

Resistance to chemotherapy is a major obstacle for successful treatment of breast cancer patients. Given that prolactin (PRL) acts as an anti-apoptotic/survival factor in the breast, we postulated that it antagonizes cytotoxicity by chemotherapeutic drugs. Treatment of breast cancer cells with PRL caused variable resistance to taxol, vinblastine, doxorubicin and cisplatin. PRL prevented cisplatin-induced G(2)/M cell cycle arrest and apoptosis. In the presence of PRL, significantly less cisplatin was bound to DNA, as determined by mass spectroscopy, and little DNA damage was seen by gamma-H2AX staining. PRL dramatically increased the activity of glutathione-S-transferase (GST), which sequesters cisplatin in the cytoplasm; this increase was abrogated by Jak and mitogen-activated protein kinase inhibitors. PRL upregulated the expression of the GSTmu, but not the pi, isozyme. A GST inhibitor abrogated antagonism of cisplatin cytotoxicity by PRL. In conclusion, PRL confers resistance against cisplatin by activating a detoxification enzyme, thereby reducing drug entry into the nucleus. These data provide a rational explanation for the ineffectiveness of cisplatin in breast cancer, which is characterized by high expression of both PRL and its receptor. Suppression of PRL production or blockade of its actions should benefit patients undergoing chemotherapy by allowing for lower drug doses and expanded drug options.

Multiple mechanisms responsible for differential susceptibilities ofSitobion avenae(Fabricius) andRhopalosiphum padi(Linnaeus) to pirimicarb

Both Sitobion avenae (Fabricius) and Rhopalosiphum padi (Linnaeus) are the most important pests of wheat in China and usually coexist on the late period of wheat growth. Pirimicarb was introduced into China for wheat aphid control in early 1990s, and differential susceptibilities of Sitobion avenae (Fabricius) and Rhopalosiphum padi (Linnaeus) to pirimicarb have been observed. A bioassay exhibited that Rhopalosiphum padi possessed significantly higher susceptibility to pirimicarb than Sitobion avenae. The addition of synergists DEF, an esterase inhibitor, PBO, a cytochrome P450 monooxygenase inhibitor, and DEM, a glutathione S-transferase inhibitor, resulted in apparent reductions in the differential susceptibilities, suggesting the involvement of the above three detoxification enzymes in the differential susceptibility to pirimicarb between Sitobion avenae and Rhopalosiphum padi. A biochemical analysis showed that the activities of carboxylesterases and glutathione S-transferases were significantly higher in Sitobion avenae than in Rhopalosiphum padi, consistent with the results of synergism. Acetylcholinesterase is the target enzyme of pirimicarb and the sensitivity of acetylcholinesterase to pirimicarb was significantly higher in Rhopalosiphum padi than in Sitobion avenae. The combined results suggest that multiple mechanisms are likely to be responsible for differential susceptibilities to pirimicarb between Sitobion avenae and Rhopalosiphum padi. The results obtained from this study should be helpful in the rational applications of insecticides.

Analysis of glutathione adducts of patulin by means of liquid chromatography (HPLC) with biochemical detection (BCD) and electrospray ionization tandem mass spectrometry (ESI-MS/MS)

A novel method for the identification of glutathione/electrophile adducts that are inhibiting glutathione-S-transferase (GST) activity was developed and applied for the analysis of the mycotoxin patulin. The method is based on high-performance liquid chromatography (HPLC) coupled to a continuous-flow enzyme reactor serving as biochemical detector (BCD) in parallel to electrospray mass spectrometric detection (ESI-MS). This HPLC-BCD technique combines a separation step and the detection of the inhibition and is therefore ideally suited for the analysis of the activity of single patulin/glutathione adducts within a complex mixture of adducts. Two out of at least 15 detected patulin-glutathione adducts showed strong GST inhibition. In ESI-MS, the inhibitory active adducts were characterized by [M + H]+ ions with m/z 462.1138 and m/z 741.2011, respectively. They could be identified as a dihydropyranone adduct containing one molecule glutathione and a ketohexanoic acid bearing two glutathione molecules.

Moonlighting protein in Starkeyomyces koorchalomoides: Characterization of dihydrolipoamide dehydrogenase as a protein acetyltransferase utilizing acetoxycoumarin as the acetyl group donor

In this report we have identified for the first time a transacetylase (TAase) in a mesophilic fungi Starkeyomyces koorchalomoides catalyzing the transfer of acetyl group from polyphenolic acetate (PA) to a receptor protein glutathione S-transferase (GST). An elegant assay procedure was established for TAase based on its ability to mediate inhibition of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model PA. Utilizing this assay procedure, S. koorchalomoides TAase was purified to homogeneity. TAase was found to have MW of 50 kDa. The purified enzyme exhibited maximum activity at 45 °C at pH 6.8. The N-terminal sequence of purified fungal TAase ( ANDASTVED) showed identity with corresponding N-terminal sequence of dihydrolipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme and an E3 component of pyruvate dehydrogenase complex (PDHC). TAase was found to have all the properties of LADH and avidly interacted with the anti-LADH antibody. TAase catalyzed acetylation of GST by DAMC was identified by LC–MS/MS and a single lysine residue (Lys-113) was found to be acetylated. Further, recombinant LADH from Streptococcus pneumoniae lacking lipoyl domain was found to exhibit little TAase activity, suggesting the role of lipoyl domain in the TAase activity of LADH. These observations bear evidence for the protein acetyltransferase activity of LADH. Such an activity of LADH can be attributed as a moonlighting function of the enzyme.

Chalcone Derivatives Inhibit Glutathione S‐Transferase P1‐1 Activity: Insights into the Interaction Mode of α, β‐Unsaturated Carbonyl Compounds

Resistance to chemotherapeutic drugs has long been a considerable barrier to successful treatment of many cancers and over-expression of glutathione S-transferase P1-1 is correlated to carcinogenesis and resistance of cancer cells against chemotherapeutic agents. This study throws light on the role of chalcone derivatives, a new class of glutathione S-transferase P1-1 inhibitors potentially to overcome glutathione S-transferase P1-1-mediated chemotherapy resistance. Nineteen alpha-substituted chalcone derivatives were synthesized and their in vitro inhibitory effects on glutathione S-transferase P1-1 were determined. We interestingly found that most of these compounds showed inhibitory effect on glutathione S-transferase P1-1 activity. In addition, molecular field-based similarity analysis provides the necessary three-dimensional molecular field properties of alpha, beta-unsaturated carbonyl derivatives to inhibit glutathione S-transferase P1-1 activity. Thus, these compounds have great potential to be developed into novel chemotherapeutic sensitizers.