Detoxification Of Electrophiles Research Articles

Alkylcysteine Sulfoxide C-S Monooxygenase Uses a Flavin-Dependent Pummerer Rearrangement.

Flavoenzymes are highly versatile and participate in the catalysis of a wide range of reactions, including key reactions in the metabolism of sulfur-containing compounds. S-Alkyl cysteine is formed primarily by the degradation of S-alkyl glutathione generated during electrophile detoxification. A recently discovered S-alkyl cysteine salvage pathway uses two flavoenzymes (CmoO and CmoJ) to dealkylate this metabolite in soil bacteria. CmoO catalyzes a stereospecific sulfoxidation, and CmoJ catalyzes the cleavage of one of the sulfoxide C-S bonds in a new reaction of unknown mechanism. In this paper, we investigate the mechanism of CmoJ. We provide experimental evidence that eliminates carbanion and radical intermediates and conclude that the reaction proceeds via an unprecedented enzyme-mediated modified Pummerer rearrangement. The elucidation of the mechanism of CmoJ adds a new motif to the flavoenzymology of sulfur-containing natural products and demonstrates a new strategy for the enzyme-catalyzed cleavage of C-S bonds.

Abstract P046: Variable Effect Of Gstm1 Knockout In Mice

Glutathione S-transferase μ-1 (GSTM1) is an enzyme that has a role in the detoxification of electrophiles, including xenobiotics and products of oxidative stress. In humans, the GSTM1(0) null allele deletion variant is highly prevalent and is associated with both an increase in oxidative stress and an increased risk/severity of a variety of diseases, including cancers, chronic kidney disease, hypertension, and coronary artery disease.Recently, we generated a Gstm1 knockout ( Gstm1 -/- ) mouse line on a 129S6 background. Using these animals, we demonstrated that the loss of GSTM1 resulted in increased oxidative stress and greater kidney injury with either subtotal nephrectomy-induced chronic kidney disease or angiotensin II-induced hypertension, in accordance with clinical data.Following myocardial infarction, reperfusion of the heart results in additional tissue damage that is also mediated by acute oxidative stress. Here, we used an ex vivo Langendorff-perfused heart model of acute cardiac ischemia-reperfusion injury (IRI). Contrary to the expectation that hearts from Gstm1 -/- mice would be more susceptible to IRI, we found that the loss of the antioxidant enzyme GSTM1 was protective in males compared to age-matched wild-type controls. In contrast, the Gstm1 -/- genotype was deleterious in female hearts as expected.To explore the hypothesis that the loss of GSTM1 causes compensatory upregulation of other GSTs and that this effect varies based on both tissue and sex, we examined mRNA expression of α, θ, κ, μ, and π classes of Gst genes via qPCR and corresponding protein expression via HPLC and western blot. We found significant differences between male vs. female and heart vs. kidney in several GSTs both in their expression in wild-type mice and in the change in expression between wild-type and Gstm1 -/- mice. These results suggest that the severity of cardiac IRI may depend on the adaptive response mediated by genes encoding GST enzymes.

Diverse Physiological Functions of Cation Proton Antiporters across Bacteria and Plant Cells.

Membrane intrinsic transport systems play an important role in maintaining ion and pH homeostasis and forming the proton motive force in the cytoplasm and cell organelles. In most organisms, cation/proton antiporters (CPAs) mediate the exchange of K+, Na+ and Ca2+ for H+ across the membrane in response to a variety of environmental stimuli. The tertiary structure of the ion selective filter and the regulatory domains of Escherichia coli CPAs have been determined and a molecular mechanism of cation exchange has been proposed. Due to symbiogenesis, CPAs localized in mitochondria and chloroplasts of eukaryotic cells resemble prokaryotic CPAs. CPAs primarily contribute to keeping cytoplasmic Na+ concentrations low and controlling pH, which promotes the detoxification of electrophiles and formation of proton motive force across the membrane. CPAs in cyanobacteria and chloroplasts are regulators of photosynthesis and are essential for adaptation to high light or osmotic stress. CPAs in organellar membranes and in the plasma membrane also participate in various intracellular signal transduction pathways. This review discusses recent advances in our understanding of the role of CPAs in cyanobacteria and plant cells.

Nrf2 Activation and Its Coordination with the Protective Defense Systems in Response to Electrophilic Stress.

Molecular responses mediated by sensor proteins are important for biological defense against electrophilic stresses, such as xenobiotic electrophile exposure. NF-E2-related factor 2 (Nrf2) has an essential function as a master regulator of such cytoprotective molecular responses along with sensor protein Kelch-like ECH-associated protein 1. This review focuses on Nrf2 activation and its involvement with the protective defense systems under electrophilic stresses integrated with our recent findings that reactive sulfur species (RSS) mediate detoxification of electrophiles. The Nrf2 pathway does not function redundantly with the RSS-generating cystathionine γ-lyase pathway, and vice versa.

Environmental Electrophile-Mediated Toxicity in Mice Lacking Nrf2, CSE, or Both.

Background:Transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) plays a key role in detoxification of electrophiles via formation of glutathione (GSH) adducts and subsequent excretion into extracellular spaces. We found that reactive sulfur species (RSS), such as cysteine persulfides produced by cystathionine (CSE), capture environmental electrophiles through formation of sulfur adducts. However, contributions of Nrf2 and CSE to the blockage of environmental electrophile-mediated toxicity remain to be evaluated.Objectives:The aim of this study was to clarify roles that CSE and Nrf2 play in the protection against various environmental electrophiles. We also wished to clarify the molecular basis of the developmental window of toxicity through investigating expression levels of Nrf2, RSS-producing enzymes, and sulfur nucleophiles during developmental stages of mice.Methods:Wild-type (WT), CSE knockout (KO), Nrf2 KO, Nrf2/CSE double KO (DKO) mice, and their primary hepatocytes were analyzed in this study. Cadmium (Cd), methylmercury (MeHg), 1,4-naphthoquinone, crotonaldehyde, and acrylamide were used. We conducted Western blotting, real-time polymerase chain reaction (PCR), 3-(4,5-dimethylthiazol-2-yl)-2,5-triphenyl tetrazolium bromide (MTT) assays, liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) analysis, alanine transaminase (ALT) activity, histopathological analysis, and rotarod test.Results:Primary hepatocytes from DKO mice were significantly more sensitive to the environmental electrophiles than each single KO counterpart. Both Nrf2 and CSE single KO mice were highly susceptible to Cd and MeHg, and such sensitivity was further exacerbated in the DKO mice. Lower-level expressions of CSE and sulfur nucleophiles than those in adult mice were observed in a window of developmental stage.Conclusions:Our mouse model provided new insights into the response to environmental electrophiles; while Nrf2 is recognized as a key transcription factor for detoxification of environmental electrophiles, CSE is crucial factor to repress their toxicity in a parallel mode. In addition, the sensitivity of fetuses to MeHg appears to be, at least in part, associated with the restricted production of RSS due to low-level expression of CSE. https://doi.org/10.1289/EHP4949

酸化ストレスと活性イオウ分子の解毒代謝：新しいエネルギー代謝・イオウ呼吸の発見

酸化ストレスと活性イオウ分子の解毒代謝：新しいエネルギー代謝・イオウ呼吸の発見

6 - Differential Redox Changes in Liver Mitochondrial Proteomes from Phenotypically Distinct Mouse Models of Glutathione Deficiency

Intrahepatocyte generation of free radicals and depletion of glutathione (GSH) are believed to be key mediators involved in the cellular pathogenic events underlying fatty liver disease (FLD). As the most abundant intracellular non-protein thiol, GSH has an important biochemical role in electrophile detoxification and oxidant elimination. Additionally, changes in GSH/GSSG (its oxidized form) redox couple may modify protein functions via redox post-translational modification. We have developed two unique transgenic mouse models in which hepatic GSH synthesis is repressed by abolishing the expression of the catalytic (GCLC) or the modifier (GCLM) subunit of glutamate-cysteine ligase, the rate-limiting enzyme in GSH biosynthesis. Our previous studies show that these mice have GSH deficiencies at different levels but exhibit either resistance to steatosis (GCLM knockout mice) or develop spontaneous liver pathologies featuring all clinical stages of FLD (liver-specific GCLC knockout mice). Importantly our studies show that functional integrity of the mitochondria is a key determinant of the hepatic phenotype under conditions of chronic GSH deficiency. The purpose of the current study is to investigate the hypothesis that GSH plays an important regulatory role in mitochondrial redox signaling network. We performed redox proteomics analysis of liver mitochondrial proteomes derived from two phenotypically distinct mouse models. Cellular redox buffering couples (GSH-GSSG, NADH-NAD and NADPH-NADP) were measured in parallel. The proteomics analysis revealed phenotype-associated Cysteine redox profiles of mitochondrial proteins that are involved in oxidative phosphorylation, lipid metabolism, xenobiotic metabolism, and cellular stress response. Biochemically, decreased NAD/NADH ratio appears to correlate with a protective phenotype. In conclusion, our preliminary data suggest a redox-based role of GSH in modulating mitochondrial pathways that are mechanistically linked to the pathogenesis of FLD.

Review of Nrf2-regulated genes induced in response to antioxidants

Nuclear factor (erythroid-derived 2) -like 2 (Nrf2) is a transcription factor that plays an important role in the cellular protection against free radical damage and reduce the incidence of radical derived degenerative diseases such as cancer. Nrf2 is referred to as the "master regulator" of the antioxidant response due to the fact that it modulates the expression of several genes including phase-2 and antioxidant enzymes playing a crucial role in detoxification of electrophiles and reactive oxygen species (ROS), including glutathione-S-transferase (GST), gamma-glutamyl cysteine ligase (γ-GCL), glutathione-S-reductase (GSR), NAD(P)H:quinoneoxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), etc. Following dissociation from it obligatory partner Kelch like ECHassociated protein 1 (Keap1), Nrf2 translocates to the nucleus and transactivates the antioxidant response element (ARE) in the promoter region of several antioxidant genes. In this review, we discuss the role of the Nrf2 system, with particular focus on Nrf2-controlled target genes.

NemR Is a Bleach-sensing Transcription Factor

Little is known about how bacteria sense or respond to reactive chlorine species, such as bleach. NemR is a redox-regulated transcription factor which senses bleach. NemR controls expression of genes encoding electrophile detoxification enzymes, which increase bleach resistance. We demonstrate a bleach-sensing bacterial response system and a new mechanism contributing to bacterial bleach survival. Hypochlorous acid (HOCl), the active component of household bleach, also functions as a powerful antimicrobial during the innate immune response. Despite its widespread use, surprisingly little is known about how cells sense or respond to HOCl. We now demonstrate that Escherichia coli NemR is a redox-regulated transcriptional repressor, which uses the oxidation status of HOCl-sensitive cysteine residues to respond to bleach and related reactive chlorine species. NemR controls bleach-mediated expression of two enzymes required for detoxification of reactive electrophiles: glyoxalase I and N-ethylmaleimide reductase. Both enzymes contribute to bacterial bleach survival. These results provide evidence that bleach resistance relies on the capacity of organisms to specifically sense reactive chlorine species and respond with the up-regulation of enzymes dedicated to detoxification of methylglyoxal and other reactive electrophiles.

Cefadroxil potency as cancer co-therapy candidate by glutathione s-transferase mechanism

Background: Glutathione S-transferases (GSTs) have an important role in the detoxification of electrophiles, such as some anticancer drugs. Compounds with phenolic and/or α,b-unsaturated carbonyl group have been known as GSTs inhibitor in vitro. Cefadroxil in vitro decreased GST-Pi activity but not GSTs in rat kidney cytosol. GST inhibitor in a specific organ and of a specific class is needed for safety in cancer chemotherapy. The study aims to observe the effect of cefadroxil on GSTs in vivo in rat kidney cytosol and then compare it to those seen for liver, lung, and spleen in vivo.

Interactions of sulforaphane and dimethyl sulfoxide with methyl methanesulfonate, urethane, 4-nitroquinoline-1-oxide and hydrogen peroxide in the Drosophila melanogaster wing spot test

Sulforaphane (SF) is an isothiocyanate present in Brassicaceae, vegetables that induce the detoxification of electrophiles and reactive oxygen species. SF has been correlated with chemoprevention mechanisms against degenerative diseases. We tested if the SF had an effect against methyl methanesulfonate (MMS), urethane (URE), 4-NQO and H2O2. SF (>95% purity, 0.14, 0.28, 0.56mM) was diluted in a DMSO/Tw80/EtOH mixture (DTE) corresponding to 25, 50, 100% of lyophilized broccoli. The SF treatment (0.14mM) was positive for small spots in the ST cross and negative in the HB cross. In the HB cross, SF (0.28mM) was genotoxic. In the ST cross, the SF treatments showed a tendency to reduce the genotoxic damage caused by MMS, which could be explained by the radical scavenging action of the DTE mixture. In the ST cross, the frequency of small spots in the SF 0.14mM/URE treatment was similar to that of Water/URE, which can be explained by a DTE and SF scavenger action. In both crosses, the results for the direct oxidants, 4-NQO and H2O2, were different and must be related to differential modulation of CYPs expression and the SF and DTE scavenger properties.

Current research of effect of glutathione S-transferases on eye oxidative damage

Glutathione S-transferases (GSTs) are large super-gene family.It is traditionally thought to be a kind of electrophilic detoxification substance.However,recent research showed that it has a powerful antioxidant role,but its gene polymorphism is also a risk factor of variety diseases.Researches verified that GSTs are widely found in the epithelial tissue in human and eye tissues,such as iris,ciliary,trabecular meshwork,lens,macula and retina.GSTs play strong preventive and treating roles for many oxidative-induced eye diseases,including primary open-angle glaucoma ( POAG),age-related cataract and age-related macular degeneration ( AMD ).The antioxidative mechanism of GSTs to eye diseases is in study.The current researches about the element concept,distributions in eye tissues,gene polymorphism of GSTs and its relationship with eye oxidative damage were reviewed. Key words: Glutathione S-transferases; Oxidative damage/eye; Gene polymorphism

Optimization of Fluorescently Labeled Nrf2 Peptide Probes and the Development of a Fluorescence Polarization Assay for the Discovery of Inhibitors of Keap1-Nrf2 Interaction

Activation of the antioxidant response element (ARE) upregulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein Keap1 and the transcription factor Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small-molecule inhibitors of the Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance assay. The FP assay exhibits considerable tolerance toward DMSO and produced a Z′ factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high-throughput screening assay to discover small-molecule inhibitors of Keap1-Nrf2 interaction.

Genome-wide methylation analysis identifies involvement of TNF-α mediated cancer pathways in prostate cancer

Altered signaling pathways resulting from aberrant changes in epigenetic parameters may play a pivotal role in carcinogenesis. To identify biological pathways likely to be affected by methylation-mediated alterations in gene expression in prostate cancer, we performed a genome-wide methylation analysis of 27,578 CpG sites, corresponding to 14,495 genes on a pooled sample of 12 pairs of prostate tumor and adjacent normal tissues. In all, 972 CpG sites were significantly hypermethylated while 209 sites were hypomethylated in prostate tumor tissue (FDR adjusted p-value < 0.05; fold change ⩾ 2) corresponding to 1043 unique genes, which is consistent with genome-wide gene-specific hypermethylation patterns previously observed in multiple cancer models. Global hypomethylation in prostate tumor was also detected by measuring methylation changes in ALU repeat sequences. Pathway analysis of the genes with altered methylation patterns identifies the involvement of a cancer related network of genes whose activity may be heavily regulated by TNF-α in prostate tumorigenesis. Our results suggest that epigenetic dysregulation of cellular processes relevant to TNF-α-dependent apoptosis and electrophile detoxification may be intimately involved in prostate carcinogenesis. These findings may lend credence to the possibility of using tumor-specific alterations in methylation patterns as biomarkers in estimating prognosis and assessing treatment options for prostate cancer.

Mechanism of ligand-gated potassium efflux in bacterial pathogens

Gram negative pathogens are protected against toxic electrophilic compounds by glutathione-gated potassium efflux systems (Kef) that modulate cytoplasmic pH. We have elucidated the mechanism of gating through structural and functional analysis of Escherichia coli KefC. The revealed mechanism can explain how subtle chemical differences in glutathione derivatives can produce opposite effects on channel function. Kef channels are regulated by potassium transport and NAD-binding (KTN) domains that sense both reduced glutathione, which inhibits Kef activity, and glutathione adducts that form during electrophile detoxification and activate Kef. We find that reduced glutathione stabilizes an interdomain association between two KTN folds, whereas large adducts sterically disrupt this interaction. F441 is identified as the pivotal residue discriminating between reduced glutathione and its conjugates. We demonstrate a major structural change on the binding of an activating ligand to a KTN-domain protein. Analysis of the regulatory interactions suggests strategies to disrupt pathogen potassium and pH homeostasis.

Role of phase 2 drug-metabolizing enzymes modulated by extracts from 78 herbal medicines in detoxification of electrophiles and lung cancer chemotherapy

Role of phase 2 drug-metabolizing enzymes modulated by extracts from 78 herbal medicines in detoxification of electrophiles and lung cancer chemotherapy

Gain of Nrf2 Function in Non-Small-Cell Lung Cancer Cells Confers Radioresistance

Nuclear factor erythroid-2 related factor 2 (Nrf2), a redox-sensitive transcription factor, regulates the expression of antioxidant enzymes and several anti-apoptotic proteins, which confer cytoprotection against oxidative stress and apoptosis. Constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by upregulation of glutathione, thioredoxin, and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. In this study, we show that RNAi-mediated lowering of Nrf2 levels in non-small-cell lung cancer (NSCLC) cell lines (A549 and H460) led to a dramatic increase in endogenous reactive oxygen species (ROS) levels. Similarly, γ-irradiation-induced formation of protein carbonyls were significantly higher in Nrf2-depleted lung cancer cells, suggesting increased lethality of ionizing radiation in the absence of Nrf2. Radiation-induced protein oxidation in Nrf2shRNA cells correlated with reduced survival as measured by clonogenic assay. Radiation-induced cell death was abrogated by pretreatment with antioxidants such as N-acetyl-L-cysteine, glutathione, and vitamin-E, highlighting the importance of antioxidants in conferring protection against radiation injury. Using genetically-modified gain and loss of function models of Nrf2, in mouse embryonic fibroblasts, we establish that constitutive activation of Nrf2 protects against ionizing radiation toxicity and confers radioresistance. Thus, targeting Nrf2 activity in radioresistant tumors could be a promising strategy to circumvent radioresistance.

Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli

Bacillithiol (BSH), the alpha-anomeric glycoside of L-cysteinyl-D-glucosamine with L-malic acid, is a major low-molecular-weight thiol in Bacillus subtilis and related bacteria. Here, we identify genes required for BSH biosynthesis and provide evidence that the synthetic pathway has similarities to that established for the related thiol (mycothiol) in the Actinobacteria. Consistent with a key role for BSH in detoxification of electrophiles, the BshA glycosyltransferase and BshB1 deacetylase are encoded in an operon with methylglyoxal synthase. BshB1 is partially redundant in function with BshB2, a deacetylase of the LmbE family. Phylogenomic profiling identified a conserved unknown function protein (COG4365) as a candidate cysteine-adding enzyme (BshC) that co-occurs in genomes also encoding BshA, BshB1, and BshB2. Additional evolutionarily linked proteins include a thioredoxin reductase homolog and two thiol:disulfide oxidoreductases of the DUF1094 (CxC motif) family. Mutants lacking BshA, BshC, or both BshB1 and BshB2 are devoid of BSH. BSH is at least partially redundant in function with other low-molecular-weight thiols: redox proteomics indicates that protein thiols are largely reduced even in the absence of BSH. At the transcriptional level, the induction of genes controlled by two thiol-based regulators (OhrR, Spx) occurs normally. However, BSH null cells are significantly altered in acid and salt resistance, sporulation, and resistance to electrophiles and thiol reactive compounds. Moreover, cells lacking BSH are highly sensitive to fosfomycin, an epoxide-containing antibiotic detoxified by FosB, a prototype for bacillithiol-S-transferase enzymes.

Glutathione S-transferase polymorphisms and survival in African-American and White colorectal cancer patients

Background: Glutathione S-transferase (GST) enzymes are involved in electrophile detoxification. The authors investigated the association between GST genotype and survival in a racially diverse, population-based cohort of colorectal cancer (CRC) patients followed for a median of 9.6 years. Methods: Interviews were conducted with 315 African-American and White CRC patients in Connecticut, 1987–1991. Tumor tissue ( n = 197) was later retrieved from hospital of diagnosis and assayed using multiplex PCR (GSTM1 and GSTT1) and PCR and RFLP analysis (GSTP1). Cox proportional hazards models provided adjusted hazard ratios (HR) and 95% confidence intervals (CI). Results: Individuals with Ile/Val or Val/Val GSTP1 genotypes had a decreased risk of death (multivariate adjusted HR = 0.72, 95% CI: 0.48, 1.09) relative to those with wild type (Ile/Ile). Among those who received chemotherapy, this benefit was more pronounced (HR = 0.35, 95% CI: 0.16, 0.79); the interaction of reduced function GSTP1 genotype and chemotherapy was significant ( P = 0.05). GSTM1 and GSTT1 genotype were not associated with survival. GSTM1, GSTT1, and GSTP1 genotype did not vary by race and did not contribute significantly to the survival disadvantage observed in African-Americans. Conclusions: In summary, GSTP1 genotype may play a role in CRC survival in African-Americans and Whites, particularly among those who receive chemotherapy.

Catechol estrogens mediated activation of Nrf2 through covalent modification of its quinone metabolite to Keap1

The catechol metabolites (2-OHE and 4-OHE) of estrogen enter a redox cycle, thereby generating not only reactive oxygen species (ROS) but also electrophilic quinones. It is well recognized that chemicals causing oxidative stress or electrophiles activate a transcription factor Nrf2 that is negatively regulated by Keap1, leading to up-regulation of downstream proteins responsible for detoxification of electrophiles in cells. The purpose of the present study is to explore the roles of oxidative and electrophilic stress in Nrf2 activation caused by redox-active catechol estrogens. Exposure of RAW264.7 cells to 2- and 4-OHE activated Nrf2, resulting in induction of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC). Under these conditions, intracellular oxidants were generated; however, subsequent examinations revealed that quinoid metabolites derived from 2- and 4-OHE mainly participate in the Nrf2 activation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis revealed Keap1 undergoes modification by such quinoid species through multiple reactive thiol groups. These results suggest that Nrf2 activation during redox cycling of catechol estrogens is dominantly attributable to formation of their ortho-quinones that covalently bind to Keap1.