Methyl vinyl ketone and its analogs covalently modify PI3K and alter physiological functions by inhibiting PI3K signaling.

Reactive carbonyl species (RCS), which are abundant in the environment and are produced in vivo under stress, covalently bind to nucleophilic residues such as Cys in proteins. Disruption of protein function by RCS exposure is predicted to play a role in the development of various diseases such as cancer and metabolic disorders, but most studies on RCS have been limited to simple cytotoxicity validation, leaving their target proteins and resulting physiological changes unknown. In this study, we focused on methyl vinyl ketone (MVK), which is one of the main RCS found in cigarette smoke and exhaust gas. We found that MVK suppressed PI3K-Akt signaling, which regulates processes involved in cellular homeostasis, including cell proliferation, autophagy, and glucose metabolism. Interestingly, MVK inhibits the interaction between the epidermal growth factor receptor and PI3K. Cys656 in the SH2 domain of the PI3K p85 subunit, which is the covalently binding site of MVK, is important for this interaction. Suppression of PI3K-Akt signaling by MVK reversed epidermal growth factor-induced negative regulation of autophagy and attenuated glucose uptake. Furthermore, we analyzed the effects of the 23 RCS compounds with structures similar to MVK and showed that their analogs also suppressed PI3K-Akt signaling in a manner that correlated with their similarities to MVK. Our study demonstrates the mechanism of MVK and its analogs in suppressing PI3K-Akt signaling and modulating physiological functions, providing a model for future studies analyzing environmental reactive species.

Activation of HSP90/HSF1 Signaling as an Adaptive Response to an Electrophilic Metabolite of Morphine.

Morphinone (MO) is an electrophilic metabolite of morphine that covalently binds to protein thiols, resulting in toxicity in vitro and in vivo. We have previously identified a variety of redox signaling pathways that are activated during electrophilic stress. However, the role of MO in such activation remains unknown. In this study, we examined whether MO could activate heat shock protein (HSP) 90/heat shock factor (HSF) 1 signaling in HepG2 cells. MO exposure caused S-modification of HSP90 (determined using biotin-PEAC5-maleimide labeling) and nuclear translocation of transcription factor HSF1, thereby up-regulating its downstream genes encoding B-cell lymphoma 2-associated anthanogene 3 and heat shock 70 kDa protein 1. However, dihydromorphinone, a non-electrophilic metabolite of morphine, had little effect on HSF1 activation or upregulation of these genes, suggesting that covalent modification plays a role in this process and that the HSP90/HSF1 pathway is a redox-signaled adaptive response to morphine metabolism.

Activation of the Keap1/Nrf2 Pathway as an Adaptive Response to an Electrophilic Metabolite of Morphine.

Morphinone (MO) is an electrophilic metabolite of morphine that covalently binds to protein thiols via its α,ß-unsaturated carbonyl group, resulting in toxicity in vitro and in vivo. Our previous studies identified a variety of redox signaling pathways that are activated during electrophilic stress. Here, we examined in vitro activation of a signaling pathway involving Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2) in response to MO. Exposure of HepG2 cells to MO caused covalent modification of Keap1 thiols (evaluated using biotin-PEAC5-maleimide labeling) and nuclear translocation of Nrf2, thereby up-regulating downstream genes encoding ATP binding cassette subfamily C member 2, solute carrier family 7 member 11, glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, glutathione S-transferase alpha 1, and heme oxygenase 1. However, dihydromorphinone, a metabolite of morphine lacking the reactive C7-C8 double bond, had little effect on Nrf2 activation. These results suggest that covalent modification is crucial in the Keap1/Nrf2 pathway activation and that this pathway is a redox signaling-associated adaptive response to MO metabolism.

Identification and removal of aflatoxin coprecipitates derived from plant samples on immunoaffinity chromatographic purification.

The non-polar compounds that coprecipitate with aflatoxins and interfere aflatoxin analysis using an immunoaffinity column (IAC) were identified and an effective pretreatment method was developed in combination with IAC. The proanthocyanidins fractionated from cinnamon coprecipitated with four major aflatoxins (B1, G1, B2 and G2) and were effectively removed using zirconia-coated silica gel. A pretreatment method which combined zirconia-coated silica gel and an IAC was developed for LC-MS/MS analysis of aflatoxins and the combined method substantially improved the recovery of the analytes. The method validation for the quantification of aflatoxins in four types of spiked samples (bark, dried fruits, seeds and rhizomes) and a certified reference material showed favorable accuracy. Furthermore, the developed method was applied to the real samples which encouraged mold growth, and aflatoxins B1 and G1 were successfully detected in some of the samples on which mold grew. This is the first study revealing the causative agent of aflatoxin coprecipitation and developing a new technique to remove the matrix from plant samples. Thus, the method has the potential to become a standard analytical method for aflatoxins in food and medicinal plant samples.

Redox Homeostasis is Disturbed by Redox Cycling between Reactive Cysteines of Thioredoxin 1 and 9,10-Phenanthrenequinone, an Atmospheric Electron Acceptor.

9,10-Phenanthrenequinone (9,10-PQ) is a toxicant in diesel exhaust particles and airborne particulate matter ≤2.5 µm in diameter. It is an efficient electron acceptor that readily reacts with dithiol compounds in vitro, resulting in the oxidation of thiol groups and concomitant generation of reactive oxygen species (ROS). However, it remains to be elucidated whether 9,10-PQ interacts with proximal protein dithiols. In the present study, we used thioredoxin 1 (Trx1) as a model of proteins with reactive proximal cysteines and examined whether it reacts with 9,10-PQ in cells and tissues, thereby affecting its catalytic activity and thiol status. Intratracheal injection of 9,10-PQ into mice resulted in protein oxidation and diminished Trx activity in the lungs. Using recombinant wild-type and C32S/C35S Trx1, we found that Cys32 and Cys35 selectively serve as electron donor sites for redox reactions with 9,10-PQ that lead to substantial inhibition of Trx activity. Addition of dithiothreitol restored the Trx activity inhibited by 9,10-PQ. Exposure of cultured cells to 9,10-PQ caused intracellular reactive oxygen species generation that led to protein oxidation, Trx1 dimerization, p38 phosphorylation, and apoptotic cell death. Overexpression of Trx1 blocked these 9,10-PQ-mediated events. These results suggest that the interaction of the reactive cysteines of Trx1 with 9,10-PQ causes oxidative stress, leading to disruption of redox homeostasis.

A Convenient Assay to Detect Protein Oxidation Caused by Redox-Active Quinones.

Redox-active quinones generate reactive oxygen species (ROS) through their redox cycling with electron donors. Hydrogen peroxide (H2O2) causes S-oxidation of proteins and is associated with activation of the redox signaling pathway and/or toxicity (Chem. Res. Toxicol., 30, 2017, Kumagai et al.). In the present study, we developed a convenient assay based on a combination of an enzyme-linked immunosorbent assay and a biotin-PEAC5-maleimide assay and used it to determine protein S-oxidation by ROS during redox cycling of 9,10-phenanthrenequinone (9,10-PQ) and pyrroloquinoline quinone (PQQ). S-Oxidation of proteins in a mouse liver supernatant was detected during reaction of 9,10-PQ or PQQ with electron donors such as dithiothreitol or reduced nicotinamide adenine dinucleotide phosphate (NADPH), whereas cellular protein oxidation was not observed in the absence of electron donors. These results suggest that the developed assay is useful for the detection of S-oxidation of proteins.

Concentrations of nucleophilic sulfur species in small Indian mongoose (Herpestes auropunctatus) in Okinawa, Japan.

Reactive sulfur species (RSS), such as hydrogen per (poly)sulfide, cysteine per (poly)sulfide, glutathione per (poly)sulfide, and protein-bound per (poly)sulfides, can easily react with environmental electrophiles such as methylmercury (MeHg), because of their high nucleophilicity. These RSS are produced by enzymes such as cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) and are found in mammalian organs. Organs of wildlife have not been analyzed for hydrogen sulfide, cysteine, glutathione, and RSS. In this study, low molecular weight nucleophilic sulfur substances, including RSS, were quantified by stable isotope dilution assay-based liquid chromatography-mass spectrometry using ß-(4-hydroxyphenyl)ethyl iodoacetamide to capture the target chemicals in the small Indian mongoose which species possesses high mercury content as same as some marine mammals. Western blotting revealed that the mongoose organs (liver, kidney, cerebrum, and cerebellum) contained proteins that cross-reacted with anti-CBS and CSE antibodies. The expression patterns of these enzymes were similar to those in mice, indicating that mongoose organs contain CBS and CSE. Moreover, bis-methylmercury sulfide (MeHg)2S, which is a low toxic compound in comparison to MeHg, was found in the liver of this species. These results suggest that the small Indian mongoose produces RSS and monothiols associated with detoxification of electrophilic organomercury. The animals which have high mercury content in their bodies may have function of mercury detoxification involved not only Se but also RSS interactions.

The fate of methylmercury through the formation of bismethylmercury sulfide as an intermediate in mice.

A previous study by our group indicated that methylmercury (MeHg) is biotransformed to bismethylmercury sulfide [(MeHg)2S)] by interaction with reactive sulfur species (RSS) produced in the body. In the present study, we explored the transformation of MeHg to (MeHg)2S in the gut and the subsequent fate of (MeHg)2S in vitro and in vivo. An ex vivo experiment suggested the possibility of the extracellular transformation of MeHg to (MeHg)2S in the distal colon, and accordingly, the MeHg sulfur adduct was detected in the intestinal contents and feces of mice administered MeHg, suggesting that (MeHg)2S is formed through reactions between MeHg and RSS in the gut. In a cell-free system, we found that (MeHg)2S undergoes degradation in a time-dependent manner, resulting in the formation of mercury sulfide and dimethylmercury (DMeHg), as determined by X-ray diffraction and gas chromatography/mass spectrometry, respectively. We also identified DMeHg in the expiration after the intraperitoneal administration of (MeHg)2S to mice. Thus, our present study identified a new fate of MeHg through (MeHg)2S as an intermediate, which leads to conversion of volatile DMeHg in the body.

Activation of PTP1B/EGFR signaling and cytotoxicity during combined exposure to ambient electrophiles in A431 cells.

Chemical modification of the thiol group on protein tyrosine phosphatase (PTP) 1B triggers an activation of epidermal growth factor receptor (EGFR) signaling that is mimicked by environmental electrophiles through S-modification of PTP1B. While activation of PTP1B/EGFR by a single exposure to an electrophile has been established, the effects of combined exposure to electrophiles are unknown. Here, we examined the activation of EGFR signaling by combined exposure to ambient electrophiles in human epithelial carcinoma A431 cells. Simultaneous exposure to 1,2- and 1,4-naphthoquinone (NQ) augmented the S-modification of endogenous and recombinant human PTP1B (hPTP1B). Combined exposure of hPTP1B or A431 cells to 1,2- and 1,4-NQ escalated the inactivation of PTP compared with individual exposure. Phosphorylation of EGFR and its downstream kinase extracellular signal-regulated kinase (ERK) 1/2 by 1,2-NQ exposure was facilitated by simultaneous exposure to 1,2-NQ with 10 µM 1,4-NQ. An EGFR inhibitor diminished the phosphorylation of ERK1/2, indicating that ERK was phosphorylated following EGFR activation by the NQ cocktail. The combined exposure to NQs also accelerated cell death in A431 cells compared with each NQ alone. While no EGFR/ERK activation was seen following 1,4-benzoquinone (BQ) treatment, exposure to 1,4-NQ in the presence of 1,4-BQ increased 1,4-NQ-mediated activation of EGFR. This suggests that the enhancement of 1,4-NQ-dependent EGFR activation by 1,4-BQ is caused by a different mechanism than 1,2-NQ with 1,4-NQ. These results suggest that combined exposure to ambient electrophiles, even at low concentrations, can induce stronger activation of redox signaling than individual exposure. Our findings indicate that combining different electrophiles may produce unexpected effects.

Covalent N-arylation by the pollutant 1,2-naphthoquinone activates the EGF receptor.

The epidermal growth factor receptor (EGFR) is the most intensively investigated receptor tyrosine kinase. Several EGFR mutations and modifications have been shown to lead to abnormal self-activation, which plays a critical role in carcinogenesis. Environmental air pollutants, which are associated with cancer and respiratory diseases, can also activate EGFR. Specifically, the environmental electrophile 1,2-naphthoquinone (1,2-NQ), a component of diesel exhaust particles and particulate matter more generally, has previously been shown to impact EGFR signaling. However, the detailed mechanism of 1,2-NQ function is unknown. Here, we demonstrate that 1,2-NQ is a novel chemical activator of EGFR but not other EGFR family proteins. We found that 1,2-NQ forms a covalent bond, in a reaction referred to as N-arylation, with Lys80, which is in the ligand-binding domain. This modification activates the EGFR-Akt signaling pathway, which inhibits serum deprivation-induced cell death in a human lung adenocarcinoma cell line. Our study reveals a novel mode of EGFR pathway activation and suggests a link between abnormal EGFR activation and environmental pollutant-associated diseases such as cancer.

Lipophilic compounds in garlic decrease the toxicity of methylmercury by forming sulfur adducts.

Garlic (Allium sativum L.) contains numerous sulfur compounds. We have previously found that reactive sulfur species such as glutathione persulfide, glutathione polysulfide, protein-bound persulfides, and hydrogen sulfide can bind to methylmercury to give bismethylmercury sulfide, which is less toxic than methylmercury. It was not clear, however, whether such reactive sulfur species are present in garlic. The aim of the study presented here was to determine whether garlic contains reactive sulfur species that can bind to methylmercury. We extracted garlic with organic solvents and then performed silica gel column chromatography to separate constituents that could cause bismethylmercury sulfide to form. We found numerous garlic constituents could bind to methylmercury to form bismethylmercury sulfide. A hexane extract of garlic decreased methylmercury cytotoxicity in vitro and body weight loss in mice. The results suggest that ingesting garlic may decrease methylmercury toxicity by causing the formation of sulfur adducts that inhibit adverse reactions.

Effect of combined exposure to environmental aliphatic electrophiles from plants on Keap1/Nrf2 activation and cytotoxicity in HepG2 cells: A model of an electrophile exposome.

Electrophiles, ubiquitously found in the environment, modify thiol groups of sensor proteins, leading to activation of redox signaling pathways such as the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor E2 related factor 2 (Nrf2) pathway. Nrf2 activation by exposure to single electrophiles has been established. However, the effect of exposure to a combination of electrophiles on Nrf2 activation has not been well evaluated. The current study examined whether combined exposure to electrophiles enhances the modification of thiol groups and Keap1/Nrf2 activation in HepG2 cells. Six electrophiles [1,2-naphthoquinone (1,2-NQ), 1,4-NQ, 1,4-benzoquinone, (E)-2-hexenal (hexenal), (E)-2-decenal, and (E)-2-butenal] were tested for S-modification of albumin in vitro and for cytotoxicity to HepG2 cells. Interestingly, a mixture of the electrophiles enhanced S-modification of albumin and cytotoxicity compared with exposure to each electrophile separately. Herein, we focused on 1,2-NQ, 1,4-NQ, and hexenal to clarify the combined effect of electrophiles on Keap1/Nrf2 activation in HepG2 cells. A concentration addition model revealed that 1,2-NQ and/or 1,4-NQ additively enhanced hexenal-mediated S-modification of GSH in vitro, whereas the cytotoxicity of hexenal was synergistically increased by simultaneous exposure of HepG2 cells to the NQs. Furthermore, an NQ cocktail (2.5 µM each) that does not activate Nrf2 enhanced hexenal-mediated Nrf2 activation. These results suggest that combined exposure to electrophiles at low concentrations induces stronger activation of redox signaling compared with exposure to each electrophile alone and worsens their cytotoxicity.

Redox cycling of 9,10-phenanthrenequinone activates epidermal growth factor receptor signaling through S-oxidation of protein tyrosine phosphatase 1B.

[Page 358, Figure 5C] The label of Western blot data in Figure 5C in the paper was erroneously placed.

A strategy for repression of arsenic toxicity through nuclear factor E2 related factor 2 activation mediated by the (E)-2-alkenals in Coriandrum sativum L. leaf extract.

Activation of the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor E2 related factor 2 (Nrf2) system plays a role in repression of xenobiotic toxicity. The Coriandrum sativum L. leaf extract (CSLE) contains various aliphatic electrophiles such as (E)-2-decenal and (E)-2-dodecenal. In the present study, we examined the activation of Nrf2 coupled to chemical modification of Keap1 mediated by (E)-2-alkenals in CSLE, and the protective role of CSLE and (E)-2-alkenals against inorganic arsenite (iAsIII) cytotoxicity. Ultra-performance liquid chromatography-elevated collision energy mass spectrometry analysis revealed that (E)-2-decenal modified recombinant Keap1 at Cys241, Cys249, Cys257 and His274. Exposure of HepG2 cells to CSLE, (E)-2-decenal, or (E)-2-dodecenal upregulated Nrf2-related downstream signaling such as expression of phase-II xenobiotic-metabolizing enzymes and phase-III transporters involved in cytoprotection against iAsIII. Pretreatment with CSLE or (E)-2-butenal, a prototype of (E)-2-alkenal, prior to iAsIII exposure suppressed accumulation of iAsIII significantly and reduced iAsIII-induced cytotoxicity in cells. Oral administration of CSLE to C57BL/6 mice upregulated downstream proteins of Nrf2 and reduced accumulation of arsenic in liver tissue. The present study indicates that CSLE containing (E)-2-alkenals activates Nrf2, leading to a reduction in arsenic accumulation in vivo.

Redox cycling of 9,10-phenanthrenequinone activates epidermal growth factor receptor signaling through S-oxidation of protein tyrosine phosphatase 1B.

9,10-Phenanthrenequinone (9,10-PQ) is a polycyclic aromatic hydrocarbon quinone contaminated in diesel exhaust particles and particulate matter 2.5. It is an efficient electron acceptor that induces redox cycling with electron donors, resulting in excessive reactive oxygen species and oxidized protein production in cells. The current study examined whether 9,10-PQ could activate epidermal growth factor receptor (EGFR) signaling in A431 cells through S-oxidation of its negative regulators such as protein tyrosine phosphatase (PTP) 1B. 9,10-PQ oxidized recombinant human PTP1B at Cys215 and inhibited its catalytic activity, an effect that was blocked by catalase (CAT), whereas cis-9,10-dihydroxy-9,10-dihydrophenanthrene (DDP), which lacks redox cycling activity, had no effect on PTP1B activity. Exposure of A431 cells to 9,10-PQ, but not DDP, activated signaling through EGFR and its downstream extracellular signal-regulated kinase 1/2 (ERK1/2), coupled with a decrease of cellular PTP activity. Immunoprecipitation and UPLC-MSE revealed that PTP1B easily undergoes oxidation during exposure of A431 cells to 9,10-PQ. Pretreatment with polyethylene glycol conjugated with CAT (PEG-CAT) abolished 9,10-PQ-generated H2O2 production and significantly blocked the activation of EGFR-ERK1/2 signaling by 9,10-PQ, indicating the involvement of H2O2 in the activation because scavenging agents for hydroxyl radicals had no effect on the redox signal activation. These results suggest that such an air pollutant producing H2O2, activates EGFR-ERK1/2 signaling, presumably through the S-oxidation of PTPs such as PTP1B, and activation of the signal cascade may contribute, at least in part, to cellular responses in A431 cells.

Redox toxicology of environmental chemicals causing oxidative stress.

Living organisms are surrounded with heavy metals such as methylmercury, manganese, cobalt, cadmium, arsenic, as well as pesticides such as deltamethrin and paraquat, or atmospheric pollutants such as quinone. Extensive studies have demonstrated a strong link between environmental pollutants and human health. Redox toxicity is proposed as one of the main mechanisms of chemical-induced pathology in humans. Acting as both a sensor of oxidative stress and a positive regulator of antioxidants, the nuclear factor erythroid 2-related factor 2 (NRF2) has attracted recent attention. However, the role NRF2 plays in environmental pollutant-induced toxicity has not been systematically addressed. Here, we characterize NRF2 function in response to various pollutants, such as metals, pesticides and atmospheric quinones. NRF2 related signaling pathways and epigenetic regulations are also reviewed.

Interaction of Quinone-Related Electron Acceptors with Hydropersulfide Na2S2: Evidence for One-Electron Reduction Reaction.

We previously reported that 9,10-phenanthraquinone (9,10-PQ), an atmospheric electron acceptor, undergoes redox cycling with dithiols as electron donors, resulting in the formation of semiquinone radicals and monothiyl radicals; however, monothiols have little reactivity. Because persulfide and polysulfide species are highly reducing, we speculate that 9,10-PQ might undergo one-electron reduction with these reactive sulfides. In the present study, we explored the redox cycling capability of a variety of quinone-related electron acceptors, including 9,10-PQ, during interactions with the hydropersulfide Na2S2 and its related polysulfides. No reaction occurred when 9,10-PQ was incubated with Na2S; however, when 5 µM 9,10-PQ was incubated with either 250 µM Na2S2 or Na2S4, we detected extensive consumption of dissolved oxygen (84 µM). Under these conditions, both the semiquinone radicals of 9,10-PQ and their thiyl radical species were also detected using ESR, suggesting that a redox cycle reaction occurred utilizing one-electron reduction processes. Notably, the perthiyl radicals remained stable even under aerobic conditions. Similar phenomenon has also been observed with other electron acceptors, such as pyrroloquinoline quinone, vitamin K3, and coenzyme Q10. Our experiments with N-methoxycarbonyl penicillamine persulfide (MCPSSH), a precursor for endogenous cysteine persulfide, suggested the possibility of a redox coupling reaction with 9,10-PQ inside cells. Our study indicates that hydropersulfide and its related polysulfides are efficient electron donors that interact with quinones. Redox coupling reactions between quinoid electron acceptors and such highly reactive thiols might occur in biological systems.

Polysulfide Na2S4 regulates the activation of PTEN/Akt/CREB signaling and cytotoxicity mediated by 1,4-naphthoquinone through formation of sulfur adducts.

Electrophiles can activate redox signal transduction pathways, through actions of effector molecules (e.g., kinases and transcription factors) and sensor proteins with low pKa thiols that are covalently modified. In this study, we investigated whether 1,4-naphthoquinone (1,4-NQ) could affect the phosphatase and tensin homolog (PTEN)-Akt signaling pathway and persulfides/polysulfides could modulate this adaptive response. Simultaneous exposure of primary mouse hepatocytes to Na2S4 and 1,4-NQ markedly decreased 1,4-NQ-mediated cell death and S-arylation of cellular proteins. Modification of cellular PTEN during exposure to 1,4-NQ was also blocked in the presence of Na2S4. 1,4-NQ, at up to 10 µM, increased phosphorylation of Akt and cAMP response element binding protein (CREB). However, at higher concentrations, 1,4-NQ inhibited phosphorylation of both proteins. These bell-shaped dose curves for Akt and CREB activation were right-shifted in cells treated with both 1,4-NQ and Na2S4. Incubation of 1,4-NQ with Na2S4 resulted in formation of 1,4-NQ-S-1,4-NQ-OH. Unlike 1,4-NQ, authentic 1,4-NQ-S-1,4-NQ-OH adduct had no cytotoxicity, covalent binding capability nor ability to activate PTEN-Akt signaling in cells. Our results suggested that polysulfides, such as Na2S4, can increase the threshold of 1,4-NQ for activating PTEN-Akt signaling and cytotoxicity by capturing this electrophile to form its sulfur adducts.

1,4-Naphthoquinone activates the HSP90/HSF1 pathway through the S-arylation of HSP90 in A431 cells: Negative regulation of the redox signal transduction pathway by persulfides/polysulfides.

The current consensus is that environmental electrophiles activate redox signal transduction pathways through covalent modification of sensor proteins with reactive thiol groups at low concentrations, while they cause cell damage at higher concentrations. We previously exposed human carcinoma A431 cells to the atmospheric electrophile 1,4-naphthoquinone (1,4-NQ) and found that heat shock protein 90 (HSP90), a negative regulator of heat shock factor 1 (HSF1), was a target of 1,4-NQ. In the study presented here, we determined whether 1,4-NQ activates HSF1. We also examined whether such redox signaling could be regulated by nucleophilic sulfur species. Exposure of A431 cells to 1,4-NQ covalently modified cellular HSP90, resulting in repression of the association between HSF1 with HSP90, thereby enhancing HSF1 translocation into the nuclei. Liquid chromatography-tandem mass spectrometry analysis with recombinant HSP90 revealed that the modifications site were Cys412 and Cys564. We found that HSF1 activation mediated by 1,4-NQ upregulated downstream genes, such as HSPA6. HSF1 knockdown accelerated 1,4-NQ-mediated cytotoxicity in the cells. While simultaneous treatment with reactive persulfide and polysulfide, Na2S2 and Na2S4, blocked 1,4-NQ-dependent protein modification and HSF1 activation in A431 cells, the knockdown of Cys persulfide producing enzymes cystathionine ß-synthase (CBS) and/or cystathionine γ-lyase (CSE) enhanced these phenomena. 1,4-NQ-thiol adduct and 1,4-NQ-S-1,4-NQ adduct were produced during the enzymatic reaction of recombinant CSE in the presence of 1,4-NQ. The results suggest that activation of the HSP90-HSF1 signal transduction pathway mediated by 1,4-NQ protects cells against 1,4-NQ and that per/polysulfides can diminish the reactivity of 1,4-NQ by forming sulfur adducts.

Environmental Electrophiles: Protein Adducts, Modulation of Redox Signaling, and Interaction with Persulfides/Polysulfides.

Included among the many environmental electrophiles are aromatic hydrocarbon quinones formed during combustion of gasoline, crotonaldehyde in tobacco smoke, methylmercury accumulated in fish, cadmium contaminated in rice, and acrylamide in baked foods. These electrophiles can modify nucleophilic functions such as cysteine residues in proteins forming adducts and in the process activate cellular redox signal transduction pathways such as kinases and transcription factors. However, higher concentrations of electrophiles disrupt such signaling by nonselective covalent modification of cellular proteins. Persulfide/polysulfides produced by various enzymes appear to capture environmental electrophiles because of the formation of their sulfur adducts without electrophilicity. We therefore speculate that persulfide/polysulfides are candidates for the regulation of redox signal transduction pathways (e.g., cell survival, cell proliferation, and adaptive response) and toxicity during exposure to environmental electrophiles.