GST Isoforms Research Articles

Human mitochondrial glutathione transferases: Kinetic parameters and accommodation of a mitochondria-targeting group in substrates

Glutathione-S-transferases are key to the cellular detoxification of xenobiotics and products of oxidative damage. GSTs catalyse the reaction of glutathione (GSH) with electrophiles to form stable thioether adducts. GSTK1-1 is the main GST isoform in the mitochondrial matrix, but the GSTA1-1 and GSTA4-4 isoforms are also thought to be in the mitochondria with their distribution altering in transformed cells, thus potentially providing a cancer specific target. A mitochondria-targeted version of the GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), MitoCDNB, has been used to manipulate the mitochondrial GSH pool. To finesse this approach to target particular GST isoforms in the context of cancer, here we have determined the kcat/Km for the human isoforms of GSTK1-1, GSTA1-1 and GSTA4-4 with respect to GSH and CDNB. We show how the rate of the GST-catalysed reaction between GSH and CDNB analogues can be modified by both the electron withdrawing substituents, and by the position of the mitochondria-targeting triphenylphosphonium on the chlorobenzene ring to tune the activity of mitochondria-targeted substrates. These findings can now be exploited to selectively disrupt the mitochondrial GSH pools of cancer cells expressing particular GST isoforms.

Association of Illegal Behavior with GST Isoforms Null Genotyping in Methamphetamine Abuse Cases

Association of Illegal Behavior with GST Isoforms Null Genotyping in Methamphetamine Abuse Cases

Molecular dynamics-derived pharmacophores of Schistosoma glutathione transferase in complex with bromosulfophthalein: Screening and analysis of potential inhibitors

Schistosoma glutathione transferases (GSTs) have been identified as attractive drug targets for the design of novel antischistosomals. Here, we used in silico methods to validate the discriminative inhibitory properties of bromosulfophthalein (BSP) against the 26-kDa GST from S. japonicum (Sj26GST), and the 28-kDa GST from S. haematobium (Sh28GST), versus human GST (hGST) isoforms alpha (hGSTA), mu (hGSTM) and pi (hGSTP). The use of BSP as an archetypal selective inhibitor was harnessed to produce molecular dynamics-derived pharmacophores of the two targets. Pharmacophore-based screening using a large dataset of experimental and approved drug compounds was performed to produce a shortlist of candidates. The top candidate for each target was prioritised via molecular docking, yielding guanosine-3′-monophosphate-5′-diphosphate (G3D) for Sj26GST, and quercetin-3′-O-phosphate (Q3P) for Sh28GST. Comparative molecular dynamics studies of both candidates compared to BSP showed similar characteristics of binding stability and strength, suggesting their potential to emulate the inhibitory effects of BSP.

Engineering a Pseudo-26-kDa Schistosoma Glutathione Transferase from bovis/haematobium for Structure, Kinetics, and Ligandin Studies.

Glutathione transferases (GSTs) are the main detoxification enzymes in schistosomes. These parasitic enzymes tend to be upregulated during drug treatment, with Schistosoma haematobium being one of the species that mainly affect humans. There is a lack of complete sequence information on the closely related bovis and haematobium 26-kDa GST isoforms in any database. Consequently, we engineered a pseudo-26-kDa S. bovis/haematobium GST (Sbh26GST) to understand structure–function relations and ligandin activity towards selected potential ligands. Sbh26GST was overexpressed in Escherichia coli as an MBP-fusion protein, purified to homogeneity and catalyzed 1-chloro-2,4-dinitrobenzene-glutathione (CDNB-GSH) conjugation activity, with a specific activity of 13 μmol/min/mg. This activity decreased by ~95% in the presence of bromosulfophthalein (BSP), which showed an IC50 of 27 µM. Additionally, enzyme kinetics revealed that BSP acts as a non-competitive inhibitor relative to GSH. Spectroscopic studies affirmed that Sbh26GST adopts the canonical GST structure, which is predominantly α-helical. Further extrinsic 8-anilino-1-naphthalenesulfonate (ANS) spectroscopy illustrated that BSP, praziquantel (PZQ), and artemisinin (ART) might preferentially bind at the dimer interface or in proximity to the hydrophobic substrate-binding site of the enzyme. The Sbh26GST-BSP interaction is both enthalpically and entropically driven, with a stoichiometry of one BSP molecule per Sbh26GST dimer. Enzyme stability appeared enhanced in the presence of BSP and GSH. Induced fit ligand docking affirmed the spectroscopic, thermodynamic, and molecular modelling results. In conclusion, BSP is a potent inhibitor of Sbh26GST and could potentially be rationalized as a treatment for schistosomiasis.

The soluble glutathione transferase superfamily: role of Mu class in triclabendazole sulphoxide challenge in Fasciola hepatica

Fasciola hepatica (liver fluke), a significant threat to food security, causes global economic loss for the livestock industry and is re-emerging as a foodborne disease of humans. In the absence of vaccines, treatment control is by anthelmintics; with only triclabendazole (TCBZ) currently effective against all stages of F. hepatica in livestock and humans. There is widespread resistance to TCBZ and its detoxification by flukes might contribute to the mechanism. However, there is limited phase I capacity in adult parasitic helminths with the phase II detoxification system dominated by the soluble glutathione transferase (GST) superfamily. Previous proteomic studies have demonstrated that the levels of Mu class GST from pooled F. hepatica parasites respond under TCBZ-sulphoxide (TCBZ-SO) challenge during in vitro culture ex-host. We have extended this finding by exploiting a sub-proteomic lead strategy to measure the change in the total soluble GST profile (GST-ome) of individual TCBZ-susceptible F. hepatica on TCBZ-SO-exposure in vitro culture. TCBZ-SO exposure demonstrated differential abundance of FhGST-Mu29 and FhGST-Mu26 following affinity purification using both GSH and S-hexyl GSH affinity. Furthermore, a low or weak affinity matrix interacting Mu class GST (FhGST-Mu5) has been identified and recombinantly expressed and represents a new low-affinity Mu class GST. Low-affinity GST isoforms within the GST-ome was not restricted to FhGST-Mu5 with a second likely low-affinity sigma class GST (FhGST-S2) uncovered. This study represents the most complete Fasciola GST-ome generated to date and has supported the potential of subproteomic analyses on individual adult flukes.

Deletion of Glutathione S-Transferase Omega 1 Activates Type I Interferon Genes and Downregulates Tissue Factor.

GST omega 1 (GSTO1) is an atypical GST isoform that is overexpressed in several cancers and has been implicated in drug resistance. Currently, no small-molecule drug targeting GSTO1 is under clinical development. Here we have validated GSTO1 as an impactful target in oncology. Transcriptional profiling coupled with proteomics uncovered novel pharmacodynamic markers and cellular pathways regulated by GSTO1. CRISPR/Cas9 GSTO1 knockout (KO) cell lines failed to form tumors or displayed growth delay in vivo; they also formed smaller 3D spheroids in vitro. Multiomics analysis in GSTO1 KO cells found a strong positive correlation with cell adhesion molecules and IFN response pathways and a strong negative correlation with Myc transcriptional signature. In addition, several clinically used drugs showed significant synthetic lethality with loss or inhibition of GSTO1. Transcription and protein expression of tissue factor (gene name, F3) were downregulated in response to GSTO1 KO. F3 is associated with poor patient survival and promotion of tumor progression in multiple cancers and is a known risk factor for metastasis. Transcription of F3 was regulated by IL1β, whose secretion decreased upon inhibition of GSTO1, suggesting that IL1β links GSTO1 expression and F3 transcription. In summary, our results implicate GSTO1 as a potential therapeutic target in cancer and offer new mechanistic insights into its significant role in cancer progression. SIGNIFICANCE: These findings validate GSTO1 as a therapeutic target in cancer and implicate GSTO1 in the modulation of tumor growth, immune responses, and expression of F3.

Effects of Gsta4 deficiency on age-related cochlear pathology and hearing loss in mice.

The glutathione transferase (GST) detoxification system converts exogenous and endogenous toxins into a less toxic form by conjugating the toxic compound to reduced glutathione (GSH) by a variety of GST enzymes. Of the ~20 GST isoforms, GSTA4 exhibits high catalytic efficiency toward 4-hydroxynonenal (4-HNE), one of the most abundant end products of lipid peroxidation that contributes to neurodegenerative diseases and age-related disorders. Conjugation to GSH by GSTA4 is thought to be a major route of 4-HNE elimination. In the current study, we investigated the effects of Gsta4 deficiency on age-related cochlear pathology and hearing loss using young (3-5months old) and old (24-25months old) Gsta4+/+ and Gsta4-/- mice that were backcrossed onto the CBA/CaJ mouse strain, a well-established model of age-related hearing loss (AHL). At 3-5months of age, loss of Gsta4 resulted in decreased total GSTA activity toward 4-HNE in the inner ears of young mice. However, there were no differences in the levels of 4-HNE in the inner ears between Gsta4+/+ and Gsta4-/- mice at 3-5 or 24-25months of age. No histological abnormalities were observed in the cochlea and no hearing impairments were observed in young Gsta4-/- mice. At 24-25months of age, both Gsta4+/+ and Gsta4-/- mice showed elevated ABR thresholds compared to 3-month-old mice, but there were no differences in ABR thresholds, cochlear spiral ganglion neuron densities, or stria vascularis thickness between Gsta4+/+ and Gsta4-/- mice. Together, these results suggest that under normal physiological conditions or during normal aging, GSTA4 is not essential for removal of 4-HNE in mouse inner ears.

Nuclear receptor AHR-mediated xenobiotic detoxification pathway involves in atrazine-induced nephrotoxicity in quail (Coturnix C. coturnix)

Atrazine (ATR), one of the most widely used pesticides in agricultural production, are gradually concerned due to potential ecosystem and health risks. Further, the induction of ATR nephrotoxicity and detoxification response is still unknown. To evaluate ATR-induced nephrotoxicity, quails were treated with 0, 50, 250 or 500 mg/kg ATR by gavage administration for 45 days. Histopathology indicated that ATR exposure caused renal tubular epithelial cell swelling and endoplasmic reticulum degeneration, suggesting that ATR exposure causes renal impairment even renal diseases. Notably, ATR interfered cytochrome P450 system (CYP450s) homeostasis by enhancing contents or activities of CYP450s (total CYP450, Cyt b5, AH, APND, NCR and ERND) and the expression of CYP450 isoforms (CYP1A, CYP1B, CYP2C and CYP3A). ATR triggered phase II detoxifying reaction, reflected by the elevated GSH level, GST activity and the up-regulation of GST isoforms (GSTa, GSTa3 and GSTt1) and GSH synthetase (GCLC). Moreover, ABC transporters were activated to expel ATR from the body by increasing expression of MRP1 and P-GP gene. Accompanying these alterations, the nuclear receptors (AHR, CAR and PXR) were activated by ATR in a dose-dependent manner. Analysis results of present study demonstrated that the induction of phase II detoxifying enzyme system and ABC transporters could be modulated by nuclear receptors response and CYP450s disturbance in low-dose ATR-treated quail. In conclusion, all data suggested that nuclear receptors AHR-mediated detoxification pathway was involved in ATR-induced nephrotoxicity. These results provided new evidence about the nephrotoxic effects of ATR on the response of biotransformation and detoxification system.

Response of the Chinese Soft-Shelled Turtle to Acute Heat Stress: Insights From the Systematic Antioxidant Defense.

Understanding the responses of animals to acute heat stress can help to reveal and predict the effect of more frequent extreme hot weather episodes on animal populations and ecosystems in the content of global climate change. Antioxidant defenses can help to protect animals against oxidative stress caused by intense temperature variation. In the present study, systematic antioxidant responses to acute heat stress (Δ15°C and maintained for 12 h) and subsequent recovery were assessed by evaluating gene transcript levels and relative enzyme activities in tissues of Pelodiscus sinensis, a subtropical freshwater turtle. Targets included nuclear factor erythroid 2-related factor 2 (Nrf2, the upstream transcription factor), antioxidant enzymes, and the glutathione (GSH) and ascorbic acid (AA) systems. Results showed three main patterns of expression change among antioxidant genes: (1) gene expression of Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase 4 (GPx 4), and catalase (CAT) increased in response to heat stress or recovery in the liver; (2) transcripts of most genes did not change in brain, liver, and kidney of P. sinensis; and (3) expression of several GST isoforms were affected by heat stress or recovery in brain and kidney. However, relative enzyme activities involved in antioxidant defense were little affected by acute heat stress and recovery, indicating a relatively conservative antioxidant response in P. sinensis. Furthermore, results for malondialdehyde (MDA) levels indicated that acute heat stress and recovery did not cause a net increase in oxidative damage in turtle tissues and, in particular, MDA levels in spleen decreased along with increased splenic ascorbic acid concentration. Overall, the present study revealed a conservative antioxidant response in P. sinensis, which may be indicative of a high basal stress tolerance and relate with adaptation to climate change in freshwater turtles.

Fasciola hepatica GST downregulates NF-\u03baB pathway effectors and inflammatory cytokines while promoting survival in a mouse septic shock model

Parasitic helminths and helminth-derived molecules have demonstrated to possess powerful anti-inflammatory properties and confirmed therapeutic effects on inflammatory diseases. The helminth Fasciola hepatica has been reported to suppress specific Th1 specific immune responses induced by concurrent bacterial infections, thus demonstrating its anti-inflammatory ability in vivo. In this study, we demonstrate that native F. hepatica glutathione S-transferase (nFhGST), a major parasite excretory-secretory antigen, majorly comprised of Mu-class GST isoforms, significantly suppresses the LPS-induced TNFα and IL1β of mouse bone-marrow derived macrophages in vitro and the pro-inflammatory cytokine/chemokine storm within C57BL/6 mice exposed to lethal doses of LPS increasing their survival rate by more than 85%. Using THP1-Blue CD14 cells, a human monocyte cell line, we also demonstrate that nFhGST suppresses NF-κB activation in response to multiple TLR-ligands, including whole bacteria clinical isolates and this suppression was found to be dose-dependent and independent of the timing of exposure. Moreover, the suppressive effect of nFhGST on NF-κB activation was shown to be independent of enzyme activity or secondary structure of protein. As part of its anti-inflammatory effect nFhGST target multiple proteins of the canonic and non-canonic NF-κB signaling pathway as well as also JAK/STAT pathway. Overall, our results demonstrate the potent anti-inflammatory properties of nFhGST and its therapeutic potential as an anti-inflammatory agent.

GST transcriptional changes induced by a toxic Microcystis aeruginosa strain in two bivalve species during exposure and recovery phases.

Previous studies have demonstrated the modulation of glutathione transferases (GSTs) induced by microcystin (MC) alone or in combination with other cyanobacterial secondary metabolites in bivalves. However, interspecies information about which and how GST isoforms are affected by these secondary metabolites is still scarce, especially considering the dynamic process involving their uptake and elimination routes. In this context, the role of GSTs gene expression changes in response to a toxic Microcystis aeruginosa extract were examined for Mytilus galloprovincialis and Ruditapes philippinarum during exposure and recovery phases. The expression levels of sigma 1, sigma 2, pi and mu-class GST genes were analyzed in the hepatopancreas of both bivalve species during cyanobacteria extract exposure (24 h) and post-exposure (24 and 72 h). Only a significant induction of sigma 1-class GST expression was observed for R. philippinarum upon 24-hour exposure of both bivalve species to Microcystis extract. During the recovery phase, GST transcriptional changes for M. galloprovincialis were characterized by an early induction (24 h) of sigma 1 and sigma 2 transcripts. On the other hand, GST transcriptional changes for R. philippinarum during post-exposure phase were characterized by an early induction (24 h) of sigma 1 and mu transcripts and a later induction (72 h) of the four analyzed GST transcripts. Such differences reflect variable GST response mechanisms to cope with MC-producing cyanobacterial blooms exposure between these two bivalve species, revealing a higher sensitivity of R. philippinarum to Microcystis-induced stress than M. galloprovincialis. The results also suggest a much higher level of activity of the GST detoxification system during the recovery phase compared to the period of the stress exposure for both bivalve species.

Acute Toxicity and Modulation of an Antioxidant Defence System in the Brackish Water Flea Diaphanosoma celebensis Exposed to Cadmium and Copper

Heavy metals are one of the greatest concerns for aquatic ecosystems, as land-derived contaminants continuously flow into estuaries and coastal regions. Thus, there is a need to develop robust model species for marine ecotoxicological research. Here, we investigated the acute toxic effect of cadmium (Cd) and copper (Cu) in a Cladoceran, the brackish water flea Diaphanosoma celebensis. Modulation of total glutathione level, GSH-related antioxidant (GPx, GST, and GR) activities, and transcription of six antioxidant enzyme-coding genes (Cu/Zn-SOD, Mn-SOD, GST-theta, GST-mu, GST-kappa, and CAT) were analyzed in response to Cd and Cu exposure for 24 h. Based on the acute test, the 24-h LC50 values were determined to be as follows; 65.33 mg/L (95% C.I. 55.36-75.33 mg/L) for Cd, and 5.68 mg/L (95% C.I. 4.89-6.52 mg/L) for Cu. No significant change was observed in the total glutathione level. Both GR and GST activities were significantly induced, whereas GPx activity showed varied responses to exposure to the two different metals. In terms of transcriptional profiles, Cu/Zn-SOD mRNA was upregulated at low concentration of Cu, whereas Mn-SOD was elevated at low and medium concentrations of Cd. Among the GST isoforms, GST-mu was greatly increased in a concentration - dependent manner in response to both Cu and Cd exposure. These findings suggest that Cu and Cd can modulate antioxidant systems with different modes of action, and that SODs and GST-mu may be sensitive molecular markers for monitoring heavy metal pollution in brackish water ecosystems. This study also suggests that D. celebensis may be a useful model species for ecotoxicological study.

High-performance liquid chromatography-based assay for glutathione transferase theta 2 activity: Application to characterize interindividual variability in human liver fractions

Human glutathione transferase T2-2 (GSTT2-2) is one of the enzymes considered to play a role in inactivation of toxicants and carcinogens. The expression level of this enzyme is determined by genetic and environmental factors, which may lead to differences in susceptibility. As a specific assay for GSTT2-2 so far a spectroscopical assay based on GSH-conjugation of menaphthyl sulfate (MSu) was used. This spectrophotometric assay, however, appeared too insensitive to accurately quantify the GSTT2-2 activities in a panel of 20 human liver samples. More recently, expression levels of GSTT2-2 in biological samples are quantified by measuring mRNA levels. Since mRNA-levels do not always correlate well with enzyme activity, a specific and sensitive assay is required. In the present study a highly sensitive high-performance liquid chromatography (HPLC)-based method was developed. By applying the new method, firstly, the specificity of GSTT2-2 among 15 recombinant human GST isoforms in catalyzing GSH-conjugation of MSu was confirmed. In addition, a 65-fold inter-individual variation of GSTT2-2 activity was found from the individual liver fractions. By applying the method to individual liver fractions, a 65-fold inter-individual variation of GSTT2-2 activity was found. As a second application, the role of GSTT2-2 in GSH-conjugation of the environmental carcinogen 1-methylpyrene sulfate (MPS) was studied by correlation analysis with GSTT2-2-catalyzed MSu conjugation. The relatively poor correlation suggested that other GSTs also contribute to MPS-conjugation, as confirmed by incubations with recombinant GSTs.

Modulation of hepatic glutathione transferases isoenzymes in three bivalve species exposed to purified microcystin-LR and Microcystis extracts

This study compares the role of hepatic cytosolic glutathione transferases (cGST) isoforms of three different bivalve species to a Microcystis aeruginosa extract and purified MC-LR exposure (both at 150 μg MC-LR L−1) for 24 h. Characterization and alterations of the cytosolic GST activities in Mytilus galloprovincialis, Ruditapes philippinarum and Corbicula fluminea were measured using four class-specific substrates and changes in individual GST isoforms expression were achieved by a subsequent two-dimensional electrophoresis analysis. Evaluation of cGST activity basal levels using the four class-specific substrates denoted quantitative differences between the three bivalves. Purified MC-LR did not induce any significant response from bivalves. On the other hand, cell extracts caused significant alterations according to bivalves and substrates. Among the three bivalves, only R. philippinarum showed a significant induction of cGST activity using generic 1-chloro-2,4-dinitrobenzene (CDNB) substrate. However, no significant alterations were detected in these clams by cell extracts using the other specific substrates. In contrast, C. fluminea revealed significant induction of cGST activity when using 3,4-dichloronitrobenzene (DCNB) and ethacrynic acid (EA). In M. galloprovincialis, cell extracts promoted a significant decrease of cGST activity when using EA substrate. Altered protein expression was quantitatively detected upon exposure to cell extracts for one spot in R. philippinarum and another for C. fluminea, both upregulated (2.0 and 8.5-fold, respectively) and identified as a sigma1-class GST in the case of the first. The results showed that the three bivalves presented specific adaptive biotransformation responses to MCs and other cyanobacteria compounds supported by the modulation of distinct cGST classes.

Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice

Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage.

Mechanistic evaluation and transcriptional signature of a glutathione S-transferase omega 1 inhibitor.

Glutathione S-transferase omega 1 (GSTO1) is an atypical GST isoform that is overexpressed in several cancers and has been implicated in drug resistance. Currently, no small-molecule drug targeting GSTO1 is under clinical development. Here we show that silencing of GSTO1 with siRNA significantly impairs cancer cell viability, validating GSTO1 as a potential new target in oncology. We report on the development and characterization of a series of chloroacetamide-containing potent GSTO1 inhibitors. Co-crystal structures of GSTO1 with our inhibitors demonstrate covalent binding to the active site cysteine. These potent GSTO1 inhibitors suppress cancer cell growth, enhance the cytotoxic effects of cisplatin and inhibit tumour growth in colon cancer models as single agent. Bru-seq-based transcription profiling unravelled novel roles for GSTO1 in cholesterol metabolism, oxidative and endoplasmic stress responses, cytoskeleton and cell migration. Our findings demonstrate the therapeutic utility of GSTO1 inhibitors as anticancer agents and identify the novel cellular pathways under GSTO1 regulation in colorectal cancer.

Developmental expression and oxidative stress induction of proteome of glutathione S-transferases in Aedes albopictus (Diptera: Culicidae)

The combination of proteomic approach and affinity chromatography (GSTrap™ HP) has been used to identify and investigate the differential expression of GSTs under toxic challenge and during the course of development in Aedes albopictus. There were multiple isoforms of glutathione transferases (GSTs) expressed in the three developmental stages of Aedes albopictus. On a 2D gel map, the results indicate that there were 19 spots with a molecular mass in the range of 24–30kDa. Three types of GSTs were identified from the spots; namely GSTS1, GSTD1, and GST Theta. Spots identified as GSTS1 and GSTD1 were further designated as GSTS1–1 to GSTS1–5 and GSTD1–1 to GSTD1–13 respectively. Isoforms GSTD1–10, GSTD1–11, GSTD1–12, GSTD1–13, and GSTT were absent in the adult life stage. Isoenzymes GSTS1–3, GSTD1–2, and GSTD1–6 appeared as the major expressed isoforms in all the life stages. The effect of free radical generators such as hydrogen peroxide and paraquat on the expression of the GST isoforms in larvae mosquitoes was also investigated. Isoforms GSTD1–1, GSTD1–4, GSTS1–4, and GSTD1–12 had >1.5 fold increase when the larvae were challenged with both chemicals. GSTS1–3, GSTS1–4, GSTD1–1, GSTD1–2, GSTD1–3, GSTD1–6, and GSTD1–12 were significantly expressed (p<0.05) with >1.5 fold change when the larvae were challenged with hydrogen peroxide. On the contrary, different GST isoforms were responsive towards paraquat challenge. Those isoforms were GSTS1–2, GSTS1–4, GSTD1–1, GSTD1–5, GSTD1–9, GSTD1–11, GSTD1–12, and GSTT (with >1.5 fold change). The purified larvae GSTs seemed to have limited substrate specificities with the ability to only conjugate 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB). The GSTs have however shown that they were capable of detoxifying lipid peroxidation products and possessing glutathione peroxidase activities.

Assembly of ligands interaction models for glutathione-S-transferases from Plasmodium falciparum, human and mouse using enzyme kinetics and molecular docking

BackgroundGlutathione-s-transferases (GSTs) are enzymes that principally catalyze the conjugation of electrophilic compounds to the endogenous nucleophilic glutathione substrate, besides, they have other non-catalytic functions. The Plasmodium falciparum genome encodes a single isoform of GST (PfGST) which is involved in buffering the toxic heme, thus considered a potential anti-malarial target. In mammals several classes of GSTs are available, each of various isoforms. The human (human GST Pi-1 or hGSTP1) and mouse (murine GST Mu-1 or mGSTM1) GST isoforms control cellular apoptosis by interaction with signaling proteins, thus considered as potential anti-cancer targets. In the course of GSTs inhibitors development, the models of ligands interactions with GSTs are used to guide rational molecular modification. In the absence of X-ray crystallographic data, enzyme kinetics and molecular docking experiments can aid in addressing ligands binding modes to the enzymes. MethodsKinetic studies were used to investigate the interactions between the three GSTs and each of glutathione, 1-chloro-2,4-dinitrobenzene, cibacron blue, ethacrynic acid, S-hexyl glutathione, hemin and protoporphyrin IX. Since hemin displacement is intended for PfGST inhibitors, the interactions between hemin and other ligands at PfGST binding sites were studied kinetically. Computationally determined binding modes and energies were interlinked with the kinetic results to resolve enzymes-ligands interaction models at atomic level. ResultsThe results showed that hemin and cibacron blue have different binding modes in the three GSTs. Hemin has two binding sites (A and B) with two binding modes at site-A depending on presence of GSH. None of the ligands were able to compete hemin binding to PfGST except ethacrynic acid. Besides bind differently in GSTs, the isolated anthraquinone moiety of cibacron blue is not maintaining sufficient interactions with GSTs to be used as a lead. Similarly, the ethacrynic acid uses water bridges to mediate interactions with GSTs and at least the conjugated form of EA is the true hemin inhibitor, thus EA may not be a suitable lead. ConclusionsGlutathione analogues with bulky substitution at thiol of cysteine moiety or at γ-amino group of γ-glutamine moiety may be the most suitable to provide GST inhibitors with hemin competition.

Glutathione transferase activity of vacuoles, plastids, and tissue extracts of red beetroot

Glutathione transferase (GST) activity revealed in vacuoles of red beetroot (Beta vulgaris L.) cells was investigated in comparison with the GST activity of plastids and extracts of tissues. The level of GST activity determined by spectrophotometric method proved fairly high in water extracts and membrane fractions of isolated vacuoles and plastids, as well as in water extracts of tissues. In the objects studied, pH dependence of the GST activity slightly differed. Optimal pH for the vacuolar GST activity was in the range 7.0–7.5, for the GST of plastids and tissue extracts it was 7.5. The GSTs differed in specificity to the substrates fluorodifen and ethacrynic acid. The activity of the vacuolar and tissue extract GSTs with fluorodifen was significantly higher than that of the GST from plastids. Ethacrynic acid, often used as a competitive inhibitor of GST, almost completely inhibited the GST activity assayed with 1-chloro-2,4-dinitrobenzene as a main substrate. However, ethacrynic acid was a substrate only for the GSTs of vacuoles and tissue extract, but not for the GST of plastids. Using zymography allowing estimation of the GST activity in a gel after electrophoresis of proteins, several zones of enzymatic activity were revealed in all objects that may correspond to different isozymes. It was found that the composition of the vacuolar GST isoforms and their substrate specificity may differ from the GSTs of other cellular structures. It is assumed that vacuole, having quite high activity of GST, should make a significant contribution to intracellular detoxification processes.

Bisphenol A-Induced Ovotoxicity Involves DNA Damage Induction to Which the Ovary Mounts a Protective Response Indicated by Increased Expression of Proteins Involved in DNA Repair and Xenobiotic Biotransformation.

Bisphenol A (BPA) is an endocrine disrupting chemical with ubiquitous human exposure. BPA causes primordial follicle loss and DNA damage in germ cells, thus we hypothesized that BPA induces ovarian DNA damage, thereby precipitating follicle loss. We also anticipated that the ovary activates DNA repair and xenobiotic biotransformation to minimize oocyte damage and/or, activate cell death signaling to deplete follicles. Postnatal day 4 F344 rat ovaries were cultured in medium containing vehicle control (1% dimethylsulfoxide [DMSO]) ± BPA (440 µM) for 2-8 days. BPA reduced (P < 0.05) small primary, large primary and secondary follicle numbers after 2 days, followed by a reduction (P < .05) in primordial follicle numbers after 4 days. Phosphorylated H2AX (γH2AX) and Ataxia-telangiectasia mutated (ATM), markers of DNA double-strand breaks, were increased (P < .05) in abundance prior to observed follicle loss. DNA repair genes (Atm, Prkdc, Xrcc6, Brca1, Mre11a, Rad50, and Smc1a) were increased (P < .05) after 1 day of BPA exposure. mRNA encoding Meh, Gstm, c-kit, Kitlg, and Akt were increased (P < .05), as was MEH, AKT, pAKT, Jun N-terminal kinase, and P53 protein abundance, while GST isoforms pi and Nuclear factor erythroid-related factor 2 proteins were decreased (P < .05) by BPA exposure. These data demonstrate the dynamic ovarian response to BPA exposure, which indicates that BPA, via biotransformation, may be converted to a DNA alkylating agent, causing ovarian DNA damage, to which the ovary mounts a protective response and further our knowledge on the biological impacts of BPA on the female germline.