Soft Electrophiles Research Articles

Cyanide Trapping of Iminium Ion Reactive Metabolites: Implications for Clinical Hepatotoxicity.

Reactive metabolite formation is a major mechanism of hepatotoxicity. Although reactive electrophiles can be soft or hard in nature, screening strategies have generally focused on the use of glutathione trapping assays to screen for soft electrophiles, with many data sets available to support their use. The use of a similar assay for hard electrophiles using cyanide as the trapping agent is far less common, and there is a lack of studies with sufficient supporting data. Using a set of 260 compounds with a defined hepatotoxicity status by the FDA, a comprehensive literature search yielded cyanide trapping data on an unbalanced set of 20 compounds that were all clinically hepatotoxic. Thus, a further set of 19 compounds was selected to generate cyanide trapping data, resulting in a more balanced data set of 39 compounds. Analysis of the data demonstrated that the cyanide trapping assay had high specificity (92%) and a positive predictive value (83%) such that hepatotoxic compounds would be confidently flagged. Structural analysis of the adducts formed revealed artifactual methylated cyanide adducts to also occur, highlighting the importance of full structural identification to confirm the nature of the adduct formed. The assay was demonstrated to add the most value for compounds containing typical structural alerts for hard electrophile formation: half of the severe hepatotoxins with these structural alerts formed cyanide adducts, while none of the severe hepatotoxins with no relevant structural alerts formed adducts. The assay conditions used included cytosolic enzymes (e.g., aldehyde oxidase) and an optimized cyanide concentration to minimize the inhibition of cytochrome P450 enzymes by cyanide. Based on the demonstrated added value of this assay, it is to be initiated for use at GSK as part of the integrated hepatotoxicity strategy, with its performance being reviewed periodically as more data is generated.

Role of Oxidative Stress and Reactive Metabolites in Cytotoxicity & Mitotoxicity of Clozapine, Diclofenac and Nifedipine in CHO-K1 Cells In Vitro.

To provide in vitro data on toxicity mechanisms of clozapine, diclofenac and nifedipine. CHO-K1 cells were used as in vitro model to explore mechanisms of cytotoxicity of the test drugs. Cytotoxic mechanisms of clozapine (CLZ), diclofenac (DIC) and nifedipine (NIF) were studied in CHO-K1 cells in vitro. All three drugs induce adverse reactions in some patients with partially unknown mechanisms. Following the determination of time- and dose-dependency of cytotoxicity by the MTT test, cytoplasmic membrane integrity was explored by the LDH leakage test. Both end-points were further examined in the presence of soft and hard nucleophilic agents, glutathione (GSH) and potassium cyanide (KCN), respectively, and either individual or general cytochrome P450 (CYP) inhibitors, whether CYP-catalysed formation of electrophilic metabolites play a role in the observed cytotoxicity and membrane damage. The generation of reactive metabolites during the incubations was also explored. Formation of malondialdehyde (MDA) and oxidation of dihydrofluorescein (DCFH) were monitored whether peroxidative membrane damage and oxidative stress take place in cytotoxicity. Incubations were also conducted in the presence of chelating agents of EDTA or DTPA to explore any possible role of metals in cytotoxicity by facilitating electron transfer in redox reactions. Finally, mitochondrial membrane oxidative degradation and permeability transition pore (mPTP) induction by the drugs were tested as markers of mitochondrial damage. The presence of an individual or combined nucleophilic agents significantly diminished CLZ- and NIF-induced cytotoxicities, while the presence of both agents paradoxically increased DIC-induced cytotoxicity by a factor of three with the reason remaining unknown. The presence of GSH significantly increased DIC-induced membrane damage too. Prevention of membrane damage by the hard nucleophile KCN suggests the generation of a hard electrophile upon DIC and GSH interaction. The presence of CYP2C9 inhibitor sulfaphenazol significantly diminished DIC-induced cytotoxicity, probably by preventing the formation of 4-hydroxylated metabolite of DIC, which further converts to an electrophilic reactive intermediate. Among the chelating agents, EDTA caused a marginal decrease in CLZ-induced cytotoxicity, while DIC-induced cytotoxicity was amplified by a factor of five. Both reactive and stable metabolites of CLZ could be detected in the incubation medium of CLZ with CHO-K1 cells, which are known to have low metabolic capacity. All three drugs caused a significant increase in cytoplasmic oxidative stress by means of DCFH oxidation, which was confirmed by increased MDA from cytoplasmic as well as mitochondrial membranes. The addition of GSH paradoxically and significantly increased DIC-induced MDA formation, in parallel with the increase in membrane damage when DIC and GSH combined. Our results suggested that the soft electrophilic nitrenium ion of CLZ is not responsible for the observed in vitro toxicities, and this may originate from a relatively low amount of the metabolite due to the low metabolic capacity of CHO-K1. A hard electrophilic intermediate may contribute to cellular membrane damage incubated with DIC, while a soft electrophilic intermediate seems to exacerbate cell death by a mechanism other than membrane damage. A significant decrease in cytotoxicity of NIF by GSH and KCN suggested that both soft and hard electrophiles contribute to NIF-induced cytotoxicity. All three drugs induced peroxidative cytoplasmic membrane damage, while only DIC and NIF induced peroxidative mitochondrial membrane damage, which suggested mitochondrial processes may contribute to adverse effects of these drugs in vivo.

Machine Learning Enables Accurate Prediction of Quinone Formation during Drug Metabolism.

Metabolism helps in the elimination of drugs from the human body by making them more hydrophilic. Sometimes, drugs can be bioactivated to highly reactive metabolites or intermediates during metabolism. These reactive metabolites are often responsible for the toxicities associated with the drugs. Identification of reactive metabolites of drug candidates can be very helpful in the initial stages of drug discovery. Quinones are soft electrophiles that are generated as reactive intermediates during metabolism. Quinones make up more than 40% of the reactive metabolites. In this work, a reliable data set of 510 molecules was used to develop machine learning and deep learning-based predictive models to predict the formation of quinone-type metabolites. For representing molecules, two-dimensional (2D) descriptors, PubChem fingerprints, electro-topological state (E-state) fingerprints, and metabolic reactivity-based descriptors were used. Developed models were compared to the existing Xenosite web server using the untouched test set of 102 molecules. The best model achieved an accuracy of 86.27%, while the Xenosite server could achieve an accuracy of only 52.94% on the test set. Descriptor analysis revealed that the presence of greater numbers of polar moieties in a molecule can prevent the formation of quinone-type metabolites. In addition, the presence of a nitrogen atom in an aromatic ring and the presence of metabolophores V51, V52, and V53 (SMARTCyp descriptors) decrease the probability of quinone formation. Finally, a tool based on the best machine learning models was developed, which is accessible at http://14.139.57.41/quinonepred/.

Concomitant selenoenzyme inhibitor exposures as etiologic contributors to disease: Implications for preventative medicine

The physiological activities of selenium (Se) occur through enzymes that incorporate selenocysteine (Sec), a rare but important amino acid. The human genome includes 25 genes coding for Sec that employ it to catalyze challenging reactions. Selenoenzymes control thyroid hormones, calcium activities, immune responses, and perform other vital roles, but most are devoted to preventing and reversing oxidative damage. As the most potent intracellular nucleophile (pKa 5.2), Sec is vulnerable to binding by metallic and organic soft electrophiles (E*). These electron poor reactants initially form covalent bonds with nucleophiles such as cysteine (Cys) whose thiol (pKa 8.3) forms adducts which function as suicide substrates for selenoenzymes. These adducts orient E* to interact with Sec and since Se has a higher affinity for E* than sulfur, the E* transfers to Sec and irreversibly inhibits the enzyme's activity. Organic electrophiles have lower Se-binding affinities than metallic E*, but exposure sources are more abundant. Individuals with poor Se status are more vulnerable to the toxic effects of high E* exposures. The relative E*:Se stoichiometries remain undefined, but the aggregate effects of multiple E* exposures are predicted to be additive and possibly synergistic under certain conditions. The potential for the combined Se-binding effects of common pharmaceutical, dietary, or environmental E* require study, but even temporary loss of selenoenzyme activities would accentuate oxidative damage to tissues. As various degenerative diseases are associated with accumulating DNA damage, defining the effects of complementary E* exposures on selenoenzyme activities may enhance the ability of preventative medicine to support healthy aging.

Acrylamide Induces Neurotoxicity in SH-SY5Y Cells via NLRP3-mediated Pyroptosis.

Acrylamide (ACR), a soft electrophile, is a typical environmental and food contaminant that presents potential health hazards and, consequently, is attracting increasing attention in the quest for its control. ACR neurotoxicity has been widely reported in experimental animals and attributed to neuroinflammation; however, the mechanisms involved therein require clarification. In this study, we used a neuron cell model to investigate the mechanisms of ACR-induced neuroinflammation and pyroptosis. The results showed that ACR treatment induced lytic cell death morphologically under both the canonical pyroptotic pathway (NOD-like receptor protein 3 (NLRP3)-apoptosis-associated speck-like protein containing CARD (ASC)-cysteinyl aspartate specific proteinase 1 (caspase-1)-gasdermin D (GSDMD)-interleukin-1β (IL-1β)/interleukin-18 (IL-18)) and an alternative pyroptotic pathway (cysteinyl aspartate specific proteinase 3 (caspase-3)-gasdermin E (GSDME)-IL-1β/IL-18) in SH-SY5Y cells. Moreover, the lactate dehydrogenase (LDH) production, cytokines release, and lytic cell death induced by ACR were diminished by caspase-1 and -3 inhibitors. Furthermore, the knockdown of caspase-1 by small interfering RNA attenuated ACR-induced lytic cell death, suggesting that canonical pyroptosis (the NLRP3-caspase 1-GSDMD-IL-1β signaling axis) played a primary role in the ACR-induced pyroptosis. Of the two pyroptotic-related pathways, the NLRP3 inflammasome cascade was activated first within the 6-h period of ACR exposure, while the activation of the alternative pyroptotic pathway was delayed. Collectively, these results indicate that ACR mainly induces NLRP3-related neuroinflammation and pyroptosis in SH-SY5Y cells, which is, thus, suggestive of an alternative mechanism for ACR-induced neurotoxicity.

Convenient Synthesis of N-Alkyl-2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetamides and Methyl-2-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)acetylamino]alkanoates.

A series of 27 new quinoxaline derivatives (N-alkyl-[2-(3-phenyl-quinoxalin-2-ylsulfanyl)]acetamides, methyl-2-[2-(3-phenylquinoxalin-2-ylsulfanyl)-acetylamino]alkanoates, and their corresponding dipeptides) were prepared from 3-phenylquinoxaline-2(1H)-thione based on the chemoselective reaction with soft electrophiles. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to study the efficacy of 27 compounds on cancer cell viability and proliferation. A total of 13 compounds (4a–c, 5, 6, 8c, 9c, 9f, 10a, 10b, 11c, 12b, and 12c) showed inhibitory action on HCT-116 cancer cells and 15 compounds (4a–c, 5, 6, 8c, 9a, 9c, 9f, 9h, 10b, 11c, 12a, 12b, and 12c) showed activity on MCF-7 cancer cells, with compound 10b exhibiting the highest inhibitory action (IC50 1.52 and 2 μg/mL, respectively) on both cell lines. The molecular modeling studies on the human thymidylate synthase (hTS) homodimer interface showed that these compounds are good binders and could selectively inhibit the enzyme by stabilizing its inactive conformation. The study also identified key residues for homodimer binding, which could be used for further optimization and development.

Mapping the dynamics of Nrf2 antioxidant and NFκB inflammatory responses by soft electrophilic chemicals in human liver cells defines the transition from adaptive to adverse responses

A comprehensive understanding of the dynamic activation and crosstalk between different cellular stress response pathways that drive cell adversity is crucial in chemical safety assessment. Various chemicals have electrophilic properties that drive cell injury responses in particular oxidative stress signaling and inflammatory signaling. Here we used bacterial artificial chromosome-based GFP cellular stress reporters with live cell confocal imaging, to systematically monitor the differential modulation of the dynamics of stress pathway activation by six different soft electrophiles: sulforaphane, andrographolide, diethyl maleate, CDDO-Me, ethacrynic acid and tert-butyl hydroquinone. The various soft electrophiles showed differential potency and dynamics of Nrf2 activation and nuclear translocation. These differences in Nrf2 dynamics correlated with distinct activation pattern of Nrf2 downstream targets SRNX1 and HMOX1. All soft electrophiles caused a strong dose dependent suppression of a cytokine-induced NFĸB response represented by suppression of NFĸB nuclear oscillation and inhibition of the downstream target gene activation A20 and ICAM1, which followed the potency of Nrf2 modulation but occurred at higher concentration close to saturation of Nrf2 activation. RNAi-based depletion of RelA resulted in a prolonged presence of Nrf2 in the nucleus after soft electrophile treatment; depletion of Nrf2 caused the induction of NFĸB signaling and activation of its downstream targets A20 and ICAM1. A systematic transcriptome analysis confirmed these effects by soft electrophiles on Nrf2 and NFκB signaling crosstalk in human induced-pluripotent stem cell-derived hepatocyte-like cells. Altogether our data indicate that modulation of Nrf2 by soft electrophiles may have consequences for efficient inflammatory signaling.

Docking-based virtual screening studies aiming at the covalent inhibition of SARS-CoV-2 MPro by targeting the cysteine 145

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 which has infected millions of people worldwide. The main protease of SARS-CoV-2 (MPro) has been recognized as a key target for the development of antiviral compounds. Taking advantage of the X-ray crystal complex with reversible covalent inhibitors interacting with the catalytic cysteine 145 (Cys145), we explored flexible docking studies to select alternative compounds able to target this residue as covalent inhibitors. First, docking studies of three known electrophilic compounds led to results consistent with co-crystallized data validating the method for SARS-CoV-2 MPro covalent inhibition. Then, libraries of soft electrophiles (overall 41 757 compounds) were submitted to docking-based virtual screening resulting in the identification of 17 molecules having their electrophilic group close to the Cys145 residue. We also investigated flexible docking studies of a focused approved covalent drugs library including 32 compounds with various electrophilic functional groups. Among them, the calculations resulted in the identification of four compounds, namely dimethylfumarate, fosfomycin, ibrutinib and saxagliptin, able first, to bind to the active site of the protein and second, to form a covalent bond with the catalytic cysteine.

Exposure to acrylamide decreases noradrenergic axons in rat brain

PurposeAcrylamide is known to induce disorders in the central nervous system in humans and experimental animals. The present study investigated effects of exposure to acrylamide on adult neurogenesis, noradrenergic axons and the level of norepinephrine in the brain of male rats. MethodFour groups of 12 male Wistar rats each were exposed to acrylamide at 0, 0.2, 2 and 20 mg/kg body weight by gavage for 5 weeks. Six rats of each groups were injected with 5-bromo-2′-deoxy-uridine (BrdU) after five-week exposure to acrylamide to examine proliferative cells in the dentate gyrus using immunostaining. Density of noradrenergic and serotonergic axons in the prefrontal cortex, hippocampus and cortex behind the bregma was quantified. Remaining 6 rats were decapitated after the last exposure and brains were dissected out to measure monoamine level in the hippocampus and prefrontal cortex using high performance liquid chromatography. ResultExposure to acrylamide dose-dependently decreased the density of noradrenergic axons in the prefrontal cortex with a significant change at 20 mg/kg. Norepinephrine level decreased in the hippocampus at 20 mg/kg. Exposure to acrylamide at 20 mg/kg or less did not change the number of BrdU positive cells, but the result should be considered preliminary. ConclusionThe results show that oral exposure to acrylamide induces decrease in noradrenergic axons and norepinephrine level in the brain of rats. Given the similar effects are observed in 1-bromopropane-exposed rats, there may be the common mechanism in the toxicity of soft electrophiles to the central nervous system.

Therapeutic potential of N-acetylcysteine in acrylamide acute neurotoxicity in adult zebrafish

Two essential key events in acrylamide (ACR) acute neurotoxicity are the formation of adducts with nucleophilic sulfhydryl groups on cysteine residues of selected proteins in the synaptic terminals and the depletion of the glutathione (GSx) stores in neural tissue. The use of N-acetylcysteine (NAC) has been recently proposed as a potential antidote against ACR neurotoxicity, as this chemical is not only a well-known precursor of the reduced form of glutathione (GSH), but also is an scavenger of soft electrophiles such as ACR. In this study, the suitability of 0.3 and 0.75 mM NAC to protect against the neurotoxic effect of 0.75 mM ACR has been tested in vivo in adult zebrafish. NAC provided only a mild to negligible protection against the changes induced by ACR in the motor function, behavior, transcriptome and proteome. The permeability of NAC to cross blood-brain barrier (BBB) was assessed, as well as the ACR-scavenging activity and the gamma-glutamyl-cysteine ligase (γ-GCL) and acylase I activities. The results show that ACR not only depletes GSx levels but also inhibits it synthesis from NAC/cysteine, having a dramatic effect over the glutathione system. Moreover, results indicate a very low NAC uptake to the brain, probably by a combination of low BBB permeability and high deacylation of NAC during the intestinal absorption. These results strongly suggest that the use of NAC is not indicated in ACR acute neurotoxicity treatment.

Proteomic analysis of hippocampal proteins in acrylamide-exposed Wistar rats.

Acrylamide has been used industrially and also found in certain foods cooked at high temperatures. Previous reports described acrylamide-related human intoxication who presented with ataxia, memory impairment, and/or illusion. The aim of this study was to characterize the molecular mechanisms of neurotoxicity of acrylamide by analyzing the expression levels of various proteins in the hippocampus of rats exposed to acrylamide. Male Wistar rats were administered acrylamide by gavage at 0, 2, and 20mg/kg for 1week or 0, 0.2, 2, and 20mg/kg for 5weeks. At the end of the experiment, the hippocampus was dissected out and proteins were extracted for two-dimensional difference gel electrophoresis combined with matrix-assisted laser-desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF/MS). MALDI-TOF/TOF/MS identified significant changes in two proteins in the 1-week and 22 proteins in the 5-week exposure groups. These changes were up-regulation in 9 and down-regulation in 13 proteins in the hippocampus of rats exposed to acrylamide at 20mg/kg for 5weeks. PANTHER overrepresentation test based on the GO of biological process showed significant overrepresentation in proteins annotated to nicotinamide nucleotide metabolic process, coenzyme biosynthetic process, pyruvate metabolic process, and carbohydrate metabolic process. The test also showed significant overrepresentation in proteins annotated to creatinine kinase activity for the GO of molecular function as well as myelin sheath, cytoplasmic part, and cell body for the GO of cellular component. Comparison with a previous proteomic study on hippocampal proteins in rats exposed to 1-bromopropane identified triosephosphate isomerase, mitochondrial creatine kinase U-type, creatine kinase β-type and proteasome subunit α type-1 as proteins affected by exposure to acrylamide and 1-bromopropane, suggesting a common mechanism of neurotoxicity for soft electrophiles.

Role of microglial activation and neuroinflammation in neurotoxicity of acrylamide in vivo and in vitro.

Acrylamide, a soft electrophile, is widely used in the industry and laboratories, and also contaminates certain foods. Neurotoxicity and neurodegenerative effects of acrylamide have been reported in humans and experimental animals, although the underlying mechanism remains obscure. Activation of microglia and neuroinflammation has been demonstrated in various neurodegenerative diseases as well as other pathologies of the brain. The present study aimed to investigate the role of microglial activation and neuroinflammation in acrylamide neurotoxicity. Male 10-week-old Wistar rats were exposed to acrylamide by gavage at 0, 0.2, 2, or 20mg/kg BW, once per day for 5weeks. The results showed that 5-week exposure to acrylamide induced inflammatory responses in the cerebral cortex, evident by upregulated mRNA and protein expression of pro-inflammatory cytokines IL-1β, IL-6, and IL-18. Acrylamide also induced activation of microglia, indicated by increased expression of microglial markers, CD11b and CD40, and increased CD11b/c-positive microglial area and microglial process length. In vitro studies using BV-2 microglial cells confirmed microglial inflammatory response, as evident by time- (0-36h; 50μM) and dose- (0-500μM; 24h) dependent increase in mRNA expression of IL-1β and IL-18, as well as the inflammatory marker iNOS. Furthermore, acrylamide-induced upregulation of pro-inflammatory cytokines was mediated through the NLRP3 inflammasome pathway, as evident by increased expression of NLRP3, caspase 1, and ASC in the rat cerebral cortex, and by the inhibitory effects of NLRP3 inflammasome inhibitor on the acrylamide-induced upregulation of NLRP3, caspase 1, IL-1β, and IL-18 in BV-2 microglia.

Electrophilic fatty acids impair RAD51 function and potentiate the effects of DNA-damaging agents on growth of triple-negative breast cells

Homologous recombination (HR)-directed DNA double-strand break (DSB) repair enables template-directed DNA repair to maintain genomic stability. RAD51 recombinase (RAD51) is a critical component of HR and facilitates DNA strand exchange in DSB repair. We report here that treating triple-negative breast cancer (TNBC) cells with the fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO2) in combination with the antineoplastic DNA-damaging agents doxorubicin, cisplatin, olaparib, and γ-irradiation (IR) enhances the antiproliferative effects of these agents. OA-NO2 inhibited IR-induced RAD51 foci formation and enhanced H2A histone family member X (H2AX) phosphorylation in TNBC cells. Analyses of fluorescent DSB reporter activity with both static-flow cytometry and kinetic live-cell studies enabling temporal resolution of recombination revealed that OA-NO2 inhibits HR and not nonhomologous end joining (NHEJ). OA-NO2 alkylated Cys-319 in RAD51, and this alkylation depended on the Michael acceptor properties of OA-NO2 because nonnitrated and saturated nonelectrophilic analogs of OA-NO2, octadecanoic acid and 10-nitro-octadecanoic acid, did not react with Cys-319. Of note, OA-NO2 alkylation of RAD51 inhibited its binding to ssDNA. RAD51 Cys-319 resides within the SH3-binding site of ABL proto-oncogene 1, nonreceptor tyrosine kinase (ABL1), so we investigated the effect of OA-NO2-mediated Cys-319 alkylation on ABL1 binding and found that OA-NO2 inhibits RAD51-ABL1 complex formation both in vitro and in cell-based immunoprecipitation assays. The inhibition of the RAD51-ABL1 complex also suppressed downstream RAD51 Tyr-315 phosphorylation. In conclusion, RAD51 Cys-319 is a functionally significant site for adduction of soft electrophiles such as OA-NO2 and suggests further investigation of lipid electrophile-based combinational therapies for TNBC.

Oxidative Stress in Methylmercury-Induced Cell Toxicity

Methylmercury (MeHg) is a hazardous environmental pollutant, which elicits significant toxicity in humans. The accumulation of MeHg through the daily consumption of large predatory fish poses potential health risks, and the central nervous system (CNS) is the primary target of toxicity. Despite well-described neurobehavioral effects (i.e., motor impairment), the mechanisms of MeHg-induced toxicity are not completely understood. However, several lines of evidence point out the oxidative stress as an important molecular mechanism in MeHg-induced intoxication. Indeed, MeHg is a soft electrophile that preferentially interacts with nucleophilic groups (mainly thiols and selenols) from proteins and low-molecular-weight molecules. Such interaction contributes to the occurrence of oxidative stress, which can produce damage by several interacting mechanisms, impairing the function of various molecules (i.e., proteins, lipids, and nucleic acids), potentially resulting in modulation of different cellular signal transduction pathways. This review summarizes the general aspects regarding the interaction between MeHg with regulators of the antioxidant response system that are rich in thiol and selenol groups such as glutathione (GSH), and the selenoenzymes thioredoxin reductase (TrxR) and glutathione peroxidase (Gpx). A particular attention is directed towards the role of the PI3K/Akt signaling pathway and the nuclear transcription factor NF-E2-related factor 2 (Nrf2) in MeHg-induced redox imbalance.

The Chemical Reactivity of Anthocyanins and Its Consequences in Food Science and Nutrition.

Owing to their specific pyrylium nucleus (C-ring), anthocyanins express a much richer chemical reactivity than the other flavonoid classes. For instance, anthocyanins are weak diacids, hard and soft electrophiles, nucleophiles, prone to developing π-stacking interactions, and bind hard metal ions. They also display the usual chemical properties of polyphenols, such as electron donation and affinity for proteins. In this review, these properties are revisited through a variety of examples and discussed in relation to their consequences in food and in nutrition with an emphasis on the transformations occurring upon storage or thermal treatment and on the catabolism of anthocyanins in humans, which is of critical importance for interpreting their effects on health.

Redox Regulation of RAD51 and Homologous Recombination by Peroxiredoxin 1 and Electrophilic Nitro-Fatty Acids

Peroxiredoxin 1 (PRDX1) senses and reduces peroxides and coordinates the signaling actions of its binding partners. We show that PRDX1 protects RAD51 from oxidation-induced repression of homologous recombination (HR) repair following ɣ-irradiation. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. The oxidation of RAD51 Cys319, a thiol located in the SH3 domain targeted by ABL, is prevented by PRDX1. Alkylation of RAD51 Cys319 by the electrophilic fatty acid nitroalkene 10- nitro-octadec-9-enoic acid (OA-NO2) inhibited RAD51-ABL protein complex formation and downstream Y315 phosphorylation. In breast cancer cells, this reactivity of OA-NO2 was manifested by diminished RAD51 foci formation and sensitization of cells to irradiation. These data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR by PRDX1 and soft electrophiles such as OA-NO2.

138 - Redox Regulation of RAD51 and Homologous Recombination by Peroxiredoxin 1 and Electrophilic Nitro-fatty Acids

Peroxiredoxin 1 (PRDX1) senses and reduces peroxides and coordinates the signaling actions of its binding partners. Loss of Prdx1 function in mice has been shown to enhance susceptibility to various cancers, including breast cancer. Homologous recombination (HR) is a critical process that enables template directed DNA repair to maintain genomic stability. RAD51 is a critical component of HR, which facilitates strand-exchange to repair DNA double-strand breaks (DSB). The role of PRDX1 in DNA damage and RAD51 directed repair was investigated. We show that PRDX1 protects RAD51 from oxidation-induced repression of homologous recombination (HR) repair following γ-irradiation. PRDX1-deficient human breast cancer cells and mouse embryonic fibroblasts display disrupted RAD51 foci formation and decreased HR, resulting in increased DNA damage and sensitization of cells to irradiation. PRDX1 was found to directly bind RAD51 with increasing irradiation and loss of PRDX1 enhanced sulfenylation of RAD51. Site-directed mutagenesis revealed RAD51 Cys319, a thiol located in the SH3 domain targeted by ABL, as a primary target of oxidation. Pharmacological treatment with the thiol reactive electrophilic fatty acid nitroalkene 10-nitro-octadec-9-enoic acid (OA-NO2) targeted the C319 residue of RAD51. Alkylation of RAD51 Cys319 OA-NO2 inhibited RAD51-ABL protein complex formation and downstream Y315 phosphorylation. In breast cancer cells, this reactivity of OA-NO2 was manifested by diminished RAD51 foci formation, enhanced H2AX phosphorylation and sensitization of cells to irradiation. These data establish RAD51 Cys319 as a functionally-significant site for the redox regulation of HR and cellular responses to IR by PRDX1 and soft electrophiles such as OA-NO2.

Machine learning-based models to predict modes of toxic action of phenols to Tetrahymena pyriformis

The phenols are structurally heterogeneous pollutants and they present a variety of modes of toxic action (MOA), including polar narcotics, weak acid respiratory uncouplers, pro-electrophiles, and soft electrophiles. Because it is often difficult to determine correctly the mechanism of action of a compound, quantitative structure-activity relationship (QSAR) methods, which have proved their interest in toxicity prediction, can be used. In this work, several QSAR models for the prediction of MOA of 221 phenols to the ciliated protozoan Tetrahymena pyriformis, using Chemistry Development Kit descriptors, are reported. Four machine learning techniques (ML), k-nearest neighbours, support vector machine, classification trees, and artificial neural networks, have been used to develop several models with higher accuracies and predictive capabilities for distinguishing between four MOAs. They showed global accuracy values between 95.9% and 97.7% and area under Receiver Operator Curve values between 0.978 and 0.998; additionally, false alarm rate values were below 8.2% for training set. In order to validate our models, cross-validation (10-folds-out) and external test-set were performed with good behaviour in all cases. These models, obtained with ML techniques, were compared with others previously reported by other researchers, and the improvement was significant.

From Borane to Borylene without Reduction: Ambiphilic Behavior of a Monovalent Silylisonitrile Boron Species.

Deprotonation of [(cAAC)BH2 (CN)] provided clean access to the stable boryl anion, [(cAAC)BH(CN)]- . Whereas the addition of soft electrophiles occurred at the nucleophilic boron center, harder silyl electrophiles added to the harder terminal cyano nitrogen. The resulting [(cAAC)BH(CNSiPh3 )] species behaved like a silylium boryl nucleophile as well as a neutral silylisonitrile borylene.

Glutathione Adduct Patterns of Michael-Acceptor Carbonyls.

Glutathione (GSH) has so far been considered to facilitate detoxification of soft organic electrophiles through covalent binding at its cysteine (Cys) thiol group, followed by stepwise catalyzed degradation and eventual elimination along the mercapturic acid pathway. Here we show that in contrast to expectation from HSAB theory, Michael-acceptor ketones, aldehydes and esters may form also single, double and triple adducts with GSH involving β-carbon attack at the much harder N-terminus of the γ-glutamyl (Glu) unit of GSH. In particular, formation of the GSH-N single adduct contradicts the traditional view that S alkylation always forms the initial reaction of GSH with Michael-acceptor carbonyls. To this end, chemoassay analyses of the adduct formation of GSH with nine α,β-unsaturated carbonyls employing high performance liquid chromatography and tandem mass spectrometry have been performed. Besides enriching the GSH adductome and potential biomarker applications, electrophilic N-terminus functionalization is likely to impair GSH homeostasis substantially through blocking the γ-glutamyl transferase catalysis of the first breakdown step of modified GSH, and thus its timely reconstitution. The discussion includes a comparison with cyclic adducts of GSH and furan metabolites as reported in literature, and quantum chemically calculated thermodynamics of hard-hard, hard-soft, and soft-soft adducts.