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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

Redox-active quinones generate reactive oxygen species (ROS) through their redox cycling with electron donors. Hydrogen peroxide (H

Graphical abstract Abstract Intake of a variety of vegetables and fruit is found to be effective in promoting health; one of the reasons for this is activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a basic leucine zipper protein. Nrf2 is activated by chemical modification of Kelch-like ECH-associated protein 1 (Keap1), a negative regulator for this transcription factor, and specific phosphorylation of Nrf2, resulting in upregulation of its downstream gene products for antioxidant proteins, phase-II xenobiotic-metabolizing enzymes, and phase-III transporters. This type of activation plays a role in adaptive response and protection against oxidative and electrophilic stress. Multiple phytochemicals, such as curcumin, sulforaphane, and (E)-2-alkenals, have been identified as Nrf2 activators and may reduce the adverse health effects of oxidants and electrophiles. In this review, we introduce plant components that are known as Nrf2 activators and associated phytochemical-mediated reduction of risk from chemicals which cause oxidative and electrophilic stress. We also discuss the capture and inactivation of methylmercury, an electrophile, by sulfane sulfur atoms contained in garlic.

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 (B

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.

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 β-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 β-(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)

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.

A previous study of ours 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 extracellular transformation of MeHg to (MeHg)2S in the distal colon, and accordingly, the sulfur adduct MeHg was detected in the intestinal content and feces of mice given MeHg, suggesting that (MeHg)2S is formed from reactions of MeHg with RSS in the gut. In a cell-free system, we found that (MeHg)2S spontaneously undergoes degradation in a time-dependent fashion, resulting in formation of mercury sulfide, as determined by X-ray diffraction, and dimethylmercury (DMeHg), as evaluated by gas chromatography/mass spectrometry. We also identified DMeHg in expiration after intraperitoneal administration of (MeHg)2S to mice. Our present study therefore identifies a new fate of MeHg through (MeHg)2S as an intermediate, which leads to conversion of volatile DMeHg in the body.

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.

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.

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.

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.

Thioredoxin system–mediated persulfidation of Cys residues controls protein function and protects them from oxidative stress., Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO3H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness.

Background/Aims: In this study, we evaluated the functional impact of facilitatory presynaptic adenosine A2A and muscarinic M1 receptors in the recovery of neuromuscular tetanic depression caused by the blockage of high-affinity choline transporter (HChT) by hemicholinium-3 (HC-3), a condition that mimics a myasthenia-like condition. Methods: Rat diaphragm preparations were indirectly stimulated via the phrenic nerve trunk with 50-Hz frequency trains, each consisting of 500–750 supramaximal intensity pulses. The tension at the beginning (A) and at the end (B) of the tetanus was recorded and the ratio (R) B/A calculated. Results: Activation of A2A and M1 receptors with CGS21680 (CGS; 2 nmol/L) and McN-A-343c (McN; 3 μmol/L) increased R values. Similar facilitatory effects were obtained with forskolin (FSK; 3 μmol/L) and phorbol 12-myristate 13-acetate (PMA; 10 μmol/L), which activate adenylate cyclase and protein kinase C respectively. HC-3 (4 μmol/L) decreased transmitter exocytosis measured by real-time videomicroscopy with the FM4-64 fluorescent dye and prevented the facilitation of neuromuscular transmission caused by CGS, McN, and FSK, with a minor effect on PMA. The acetylcholinesterase inhibitor, neostigmine (NEO; 0.5 μmol/L), also decreased transmitter exocytosis. The paradoxical neuromuscular tetanic fade caused by NEO (0.5 μmol/L) was also prevented by HC-3 (4 μmol/L) and might result from the rundown of the positive feedback mechanism operated by neuronal nicotinic receptors (blocked by hexamethonium, 120 μmol/L). Conclusion: Data suggest that the recovery of tetanic neuromuscular facilitation by adenosine A2A and M1 receptors is highly dependent on HChT activity and may be weakened in myasthenic patients when HChT is inoperative.

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.

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 Na

Arsenic, a metalloid abundant in the earth’s crust, is ingested mainly through contaminated drinking water. Arsenite [As(III)], which is known to be toxic to humans, is detoxified through conjugation with glutathione (GSH), followed by excretion of the As(III)-GSH adducts via phase III transporters such as the multidrug resistance-associated protein. The Keap1-Nrf2 system regulates phase II xenobiotic-metabolizing enzymes such as glutamate-cysteine ligase and glutathione S-transferase, which are required to produce GSH and to conjugate chemicals and phase III transporters. Activation of the Keap1-Nrf2 system to upregulate downstream targets protects cells from As(III) toxicity. Multiple phytochemicals have been identified as Nrf2 activators and may reduce the adverse health effects of arsenic. These phytochemicals are briefly introduced in this review.

Air pollutants such as diesel exhaust particles (DEP) are thought to cause pulmonary diseases such as asthma as a result of oxidative stress. While DEP contain a large number of polycyclic aromatic hydrocarbons, we have focused on 9,10-phenanthrenequinone (9,10-PQ) and 1,2-naphthoquinone (1,2-NQ) because of their chemical properties based on their oxidative and chemical modification capabilities. We have found that 9,10-PQ interacts with electron donors such as NADPH (in the presence of enzymes) and dithiols, resulting in generation of excess reactive oxygen species (ROS) through redox cycling. We have also shown that 1,2-NQ is able to modify protein thiols, leading to protein adducts associated with activation of redox signal transduction pathways at lower concentrations and toxicity at higher concentrations. In this review, we briefly introduce our findings from the last two decades.

While it has long been believed that benzenes and naphthalenes are unable to activate the aryl hydrocarbon receptor (AhR) because they are poor ligands, we recently reported that these quinoid metabolites upregulated cytochrome P450 1A1 (CYP1A1) in Hepa1c1c7 cells (Abiko et al., 2015). In the current study, AhR activation, measured with a bioluminescence-based cell free assay, was induced by 1,2-naphthoquinone (1,2-NQ), a metabolite of naphthalene. Consistent with this, 1,4-benzoquinone (1,4-BQ), tert-butyl-1,4-BQ, and 1,4-NQ, as well as 1,2-NQ, all electrophilic mono- and bi-cyclic quinones, upregulated CYP1A1 mRNA and protein in HepG2 cells, whereas their parent aromatic hydrocarbons had little effect. Furthermore, immunofluorescence analysis confirmed that these quinones enhanced translocation of AhR to the nucleus.

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., Graphical abstract Image 1, Highlights • Environmental chemicals cause redox toxicity via oxidative stress. • NRF2 acts as a sensor of oxidative stress and a positive regulator of antioxidants. • NRF2 responds to metals, pesticides and atmospheric pollutants. • NRF2 and related pathways can be epigenetically regulated.

While N-acetyl-p-benzoquinoneimine (NAPQI), an electrophilic metabolite of acetaminophen (APAP), has been found to undergo GSH conjugation associated with its detoxification, interaction of NAPQI with nucleophilic per- and polysulfides produced by cystathionine γ-lyase (CSE), cystathionine β-synthase, and/or other enzymes is not known. In the present study, we found that sulfur adducts such as the NAPQIH2-SSSCys adduct and the NAPQIH2-SSG adduct are produced in biological samples of mice upon APAP exposure. Our in vitro experiments indicated that the formation of these novel APAP metabolites is, at least in part, attributable to the interaction of CysSSnSH produced by CSE and GSH persulfide with APAP-derived NAPQI.

Bismethylmercury sulfide (MeHg)2S has been found to be a detoxified metabolite of methylmercury (MeHg) that is produced by SH-SY5Y cells and in livers of rats exposed to MeHg. (MeHg)2S could be formed through the interactions between MeHg and sulfur species such as hydrogen sulfide (H2S or HS(-)), but the origin of its sulfur has not been fully identified. We herein examined the formation of (MeHg)2S through interactions between MeHg and persulfides, polysulfides, and protein preparations. Investigations using HPLC/atomic absorption spectrophotometry and EI-MS revealed that NaHS and Na2S4 react readily with MeHg to give (MeHg)2S, and similar results were found using GSH persulfide (GSSH) formed endogenously or generated enzymatically in vitro. (MeHg)2S was also formed by incubation of MeHg with liver and heart cytosolic fractions prepared from wild-type mice but not with those from mice lacking cystathionine γ-lyase (CSE) that catalyzes the formation of cysteine persulfide. Consistent with this, (MeHg)2S was detected in a variety of tissues taken from wild-type mice intraperitoneally injected with MeHg in vivo but not in those from MeHg-injected CSE knockout mice. By separating liver fractions by column chromatography, we found numerous proteins that contain persulfides: one of the proteins was identified as being glutathione S-transferase pi 1. These results indicate that the formation of (MeHg)2S can be attributed to interactions between MeHg and endogenous free persulfide species, as well as protein-bound cysteine persulfide.

Highly reactive quinone species produced by photooxidation and/or metabolic activation of mono- or bi-aromatic hydrocarbons modulate cellular homeostasis and electrophilic signal transduction pathways through the covalent modification of proteins. Polycyclic aromatic hydrocarbons, but not mono- or bi-aromatic hydrocarbons, are well recognized as ligands for the aryl hydrocarbon receptor (AhR). However, quinone species produced from mono- and bi-aromatic hydrocarbons could potentially cause AhR activation. To clarify the AhR response to mono- and bi-aromatic hydrocarbon quinones, we studied Cyp1a1 (cytochrome P450 1A1) induction and AhR activation by these quinones. We detected Cyp1a1 induction during treatment with quinones in Hepa1c1c7 cells, but not their parent compounds. Nine of the twelve quinones with covalent binding capability for proteins induced Cyp1a1. Cyp1a1 induction mediated by 1,2-naphthoquinone (1,2-NQ), 1,4-NQ, 1,4-benzoquinone (1,4-BQ) and tert-butyl-1,4-BQ was suppressed by a specific AhR inhibitor and was not observed in c35 cells, which do not have a functional AhR. These quinones stimulated AhR nuclear translocation and interaction with the AhR nuclear translocator. Interestingly, 1,2-NQ covalently modified AhR, which was detected by an immunoprecipitation assay using a specific antibody against 1,2-NQ, resulting in enhancement of xenobiotic responsive element (XRE)-derived luciferase activity and binding of AhR to the Cyp1a1 promoter region. While mono- and bi-aromatic hydrocarbons are generally believed to be poor ligands for AhR and hence unable to induce Cyp1a1, our study suggests that the quinones of these molecules are able to modify AhR and activate the AhR/XRE pathway, thereby inducing Cyp1a1. Since we previously reported that 1,2-NQ and tert-butyl-1,4-BQ also activate NF-E2-related factor 2, it seems likely that some of quinones are bi-functional inducers for phase-I and phase-II reaction of xenobiotics.

The key mechanism for acetaminophen hepatotoxicity is cytochrome P450 (CYP)-dependent formation of N-acetyl-p-benzoquinone imine, a potent electrophile that forms protein adducts. Previous studies revealed the fundamental role of glutathione, which binds to and detoxifies N-acetyl-p-benzoquinone imine. Glutathione is synthesized from cysteine in the liver, and N-acetylcysteine is used as a sole antidote for acetaminophen poisoning. Here, we evaluated the potential roles of transsulfuration enzymes essential for cysteine biosynthesis, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH), in acetaminophen hepatotoxicity using hemizygous (Cbs{sup +/−} or Cth{sup +/−}) and homozygous (Cth{sup −/−}) knockout mice. At 4 h after intraperitoneal acetaminophen injection, serum alanine aminotransferase levels were highly elevated in Cth{sup −/−} mice at 150 mg/kg dose, and also in Cbs{sup +/−} or Cth{sup +/−} mice at 250 mg/kg dose, which was associated with characteristic centrilobular hepatocyte oncosis. Hepatic glutathione was depleted while serum malondialdehyde accumulated in acetaminophen-injected Cth{sup −/−} mice but not wild-type mice, although glutamate–cysteine ligase (composed of catalytic [GCLC] and modifier [GCLM] subunits) became more activated in the livers of Cth{sup −/−} mice with lower K{sub m} values for Cys and Glu. Proteome analysis using fluorescent two-dimensional difference gel electrophoresis revealed 47 differentially expressed proteins after injection of 150 mg acetaminophen/kgmore » into Cth{sup −/−} mice; the profiles were similar to 1000 mg acetaminophen/kg-treated wild-type mice. The prevalence of Cbs or Cth hemizygosity is estimated to be 1:200–300 population; therefore, the deletion or polymorphism of either transsulfuration gene may underlie idiosyncratic acetaminophen vulnerability along with the differences in Cyp, Gclc, and Gclm gene activities. - Highlights: • Cbs{sup +/−}, Cth{sup +/−}, and especially Cth{sup −/−} mice were susceptible to APAP hepatic injury. • Hepatic glutathione became rapidly depleted upon APAP injection in Cth{sup −/−} mice. • Hepatic glutamate–cysteine ligase was activated by APAP injection and CTH deletion. • 2D DIGE identified 47 differentially expressed hepatic proteins by APAP injection. • Both transsulfuration enzymes are essential for protection against APAP injury.« less

Recently, we established a biotin-PEAC5-maleimide (BPM)-labeling assay, which can be used to determine the modification of electrophilic metals to proteins (Toyama et al., J. Toxicol. Sci., 38, 477-484, 2013). In the present study, we applied a BPM-labeling assay to detect protein S-modification by environmental organic electrophiles. After exposing A431 cells to 1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ), there was an inverse correlation between Western blot analysis with specific antibody against these electrophiles and that with BPM on the blot intensity to detect protein modification. Similar results were also observed using enzyme-linked immunosorbent assay (ELISA) with BPM. Modification of proteins in mouse liver cytosol by 5-hydroxy-1,4-NQ, 5,8-dihydroxy-1,4-NQ, 1,4-benzoquinone (1,4-BQ), tert-butyl-1,4-BQ, and N-acetyl-p-benzoquinoneimine, an electrophilic metabolite of acetaminophen, was detected using ELISA, but not non-electrophilic quinones or hydroquinone. We also tested whether ELISA could be used to detect electrophiles contained in the vapor phase of ambient air samples collected in the midtown area of Los Angeles. Taken together, the results suggested that the ELISA, developed in this study, can detect the existence of electrophilic quinones that covalently modify cellular proteins, resulting in modulation of redox-signal transduction pathways or cell damage.

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.

Methylmercury (MeHg) reacts readily with GSH, leading to the formation of a MeHg-SG adduct that is excreted into extracellular space through multidrug-resistance-associated protein (MRP), which is regulated by the transcription factor Nrf2. We previously reported that MeHg covalently modifies Keap1 and activates Nrf2 in human neuroblastoma SH-SY5Y cells. In the study presented here, we examined whether the MeHg-SG adduct could also modulate the Keap1-Nrf2 pathway because the formation of the Hg-S bond is believed to be reversible in the presence of a nucleophile. SH-SY5Y cells exposed to the synthetic ethyl monoester of the MeHg-SG adduct (which is hydrolyzed by cellular esterase(s) to give the MeHg-SG adduct) exhibited a concentration-dependent cellular toxicity that was enhanced by pretreatment with a specific MRP inhibitor. As expected, the MeHg-SG adduct was able to modify cellular proteins in the SH-SY5Y cells and purified Keap1. We also found that this prodrug, as well as MeHg, causes the cellular Keap1 in the cells to be modified, resulting in Nrf2 activation and, thereby, the upregulation of the downstream genes. These results suggest that the MeHg-SG adduct is not electrophilic but that it modifies protein thiols (including Keap1) through S-transmercuration and that rapid Nrf2-dependent excretion of the MeHg-SG adduct is essential in decreasing the cytotoxicity of MeHg.

2-tert-Butyl-1,4-benzoquinone (TBQ), an electrophilic metabolite of butylated hydroxyanisole (BHA), causes activation of Nrf2 together with S-arylation of its negative regulator Keap1 in RAW264.7 cells. In a previous study, we found that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) covalently modified with 1,2-naphthoquinone (1,2-NQ) undergoes S-transarylation by GSH, resulting in a decline of the GAPDH-1,2-NQ adduct and formation of a 1,2-NQ-SG adduct ( Miura , T. et al. ( 2011 ) Chem. Res. Toxicol. 24 , 1836 -1844 ). In the present study, we explored the possibility of GSH-dependent S-transarylation of the Keap1-TBQ adduct. Pretreatment with l-buthionine-(S,R)-sulfoximine and N-acetylcysteine prior to TBQ exposure of HepG2 cells suggested that the Keap1-TBQ adduct appears to undergo GSH-mediated S-transarylation because the resulting alterations in the intracellular GSH concentration affected Nrf2 activation caused by TBQ. In support of this hypothesis, a cell-free study demonstrated that incubation of the Keap1-TBQ adduct with GSH results in the removal of TBQ from Keap1 with the production of mono- and di-GSH adducts of TB(H)Q. These results suggest that GSH plays a role in reversible covalent modification of TBQ derived from BHA to Keap1 through the formation of a C-S bond.

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, Na 2 S 2 and Na 2 S 4 , blocked 1,4-NQ-dependent protein modification and HSF1 activation in A431 cells, the knockdown of Cys persulfide producing enzymes cystathionine β-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.

Methylmercury (MeHg) modifies cellular proteins via their thiol groups in a process referred to as “S-mercuration”, potentially resulting in modulation of the cellular signal transduction pathway. We examined whether low-dose MeHg could affect Akt signaling involved in cell survival. Exposure of human neuroblastoma SH-SY5Y cells of up to 2 μM MeHg phosphorylated Akt and its downstream signal molecule CREB, presumably due to inactivation of PTEN through S-mercuration. As a result, the anti-apoptotic protein Bcl-2 was up-regulated by MeHg. The activation of Akt/CREB/Bcl-2 signaling mediated by MeHg was, at least in part, linked to cellular defence because either pretreatment with wortmannin to block PI3K/Akt signaling or knockdown of Bcl-2 enhanced MeHg-mediated cytotoxicity. In contrast, increasing concentrations of MeHg disrupted Akt/CREB/Bcl-2 signaling. This phenomenon was attributed to S-mercuration of CREB through Cys286 rather than Akt. These results suggest that although MeHg is an apoptosis-inducing toxicant, this environmental electrophile is able to activate the cell survival signal transduction pathway at lower concentrations prior to apoptotic cell death.

The key mechanism for hepatotoxicity resulting from acetaminophen (APAP) overdose is cytochrome P450-dependent formation of N-acetyl-p-benzoquinone imine (NAPQI), a potent electrophilic metabolite that forms protein adducts. The fundamental roles of glutathione in the effective conjugation/clearance of NAPQI have been established, giving a molecular basis for the clinical use of N-acetylcysteine as a sole antidote. Recent evidence from in vitro experiments suggested that sulfide anions (S(2-)) to yield hydrogen sulfide anions (HS(-)) under physiological pH could effectively react with NAPQI. This study evaluated the protective roles of HS(-) against APAP-induced hepatotoxicity in mice. We utilized cystathionine γ-lyase-deficient (Cth(-/-)) mice that are highly sensitive to acetaminophen toxicity. Intraperitoneal injection of acetaminophen (150 mg/kg) into Cth(-/-) mice resulted in highly elevated levels of serum alanine/aspartate aminotransferases and lactate dehydrogenase associated with marked increases in oncotic hepatocytes; all of which were significantly inhibited by intraperitoneal preadministration of sodium hydrosulfide (NaHS). NaHS preadministration significantly suppressed APAP-induced serum malondialdehyde level increases without abrogating APAP-induced rapid depletion of hepatic glutathione. These results suggest that exogenous HS(-) protects hepatocytes by directly scavenging reactive NAPQI rather than by increasing cystine uptake and thereby elevating intracellular glutathione levels, which provides a novel therapeutic approach against acute APAP poisoning.

Methylmercury (MeHg) is an environmental electrophile that covalently modifies cellular proteins. In this study, we identified proteins that undergo S-mercuration by MeHg. By combining two-dimensional SDS-PAGE, atomic absorption spectrometry and ultra performance liquid chromatography mass spectrometry (UPLC/MS/MS), we revealed that ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a target for S-mercuration in human neuroblastoma SH-SY5Y cells exposed to MeHg (1 µM, 9 hr). The modification site of UCH-L1 by MeHg was Cys152, as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. MeHg was shown to inhibit the catalytic activity of recombinant human UCH-L1 in a concentration-dependent manner. Knockdown of UCH-L1 indicated that this enzyme plays a critical role in regulating mono-ubiquitin (monoUb) levels in SH-SY5Y cells and exposure of SH-SY5Y cells to MeHg caused a reduction in the level of monoUb in these cells. These observations suggest that UCH-L1 readily undergoes S-mercuration by MeHg through Cys152 and this covalent modification inhibits UCH-L1, leading to the potential disruption of the maintenance of cellular monoUb levels.

Recently, we established a biotin-PEAC5-maleimide (BPM)-labeling assay, which can be used to determine the modification of electrophilic metals to proteins (Toyama et al., J. Toxicol. Sci., 38, 477-484, 2013). In the present study, we applied a BPM-labeling assay to detect protein S-modification by environmental organic electrophiles. After exposing A431 cells to 1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ), there was an inverse correlation between Western blot analysis with specific antibody against these electrophiles and that with BPM on the blot intensity to detect protein modification. Similar results were also observed using enzyme-linked immunosorbent assay (ELISA) with BPM. Modification of proteins in mouse liver cytosol by 5-hydroxy-1,4-NQ, 5,8-dihydroxy-1,4-NQ, 1,4-benzoquinone (1,4-BQ), tert-butyl-1,4-BQ, and N-acetyl-p-benzoquinoneimine, an electrophilic metabolite of acetaminophen, was detected using ELISA, but not non-electrophilic quinones or hydroquinone. We also tested whether ELISA could be used to detect electrophiles contained in the vapor phase of ambient air samples collected in the midtown area of Los Angeles. Taken together, the results suggested that the ELISA, developed in this study, can detect the existence of electrophilic quinones that covalently modify cellular proteins, resulting in modulation of redox-signal transduction pathways or cell damage.

Sulfhydration by a hydrogen sulfide anion and electrophile thiolation by reactive sulfur species (RSS) such as persulfides/polysulfides (e.g., R-S-SH/R-S-Sn-H(R)) are unique reactions in electrophilic signaling. Using 1,2-dihydroxynaphthalene-4-thioacetate (1,2-NQH2-SAc) as a precursor to 1,2-dihydroxynaphthalene-4-thiol (1,2-NQH2-SH) and a generator of reactive oxygen species (ROS), we demonstrate that protein thiols can be modified by a reactive sulfenic acid to form disulfide adducts that undergo rapid cleavage in the presence of glutathione (GSH). As expected, 1,2-NQH2-SAc is rapidly hydrolyzed and partially oxidized to yield 1,2-NQ-SH, resulting in a redox cycling reaction that produces ROS through a chemical disproportionation reaction. The sulfenic acid forms of 1,2-NQ-SH and 1,2-NQH2-SH were detected by derivatization experiments with dimedone. 1,2-NQH2-SOH modified Keap1 at Cys171 to produce a Keap1-S-S-1,2-NQH2 adduct. Subsequent exposure of A431 cells to 1,2-NQ or 1,2-NQH2-SAc caused an extensive chemical modification of cellular proteins in both cases. Protein adduction by 1,2-NQ through a thio ether (C-S-C) bond slowly declined through a GSH-dependent S-transarylation reaction, whereas that originating from 1,2-NQH2-SAc through a disulfide (C-S-S-C) bond was rapidly restored to the free protein thiol in the cells. Under these conditions, 1,2-NQH2-SAc activated Nrf2 and upregulated its target genes, which were enhanced by pretreatment with buthionine sulfoximine (BSO), to deplete cellular GSH. Pretreatment of catalase conjugated with poly(ethylene glycol) suppressed Nrf2 activation by 1,2-NQH2-SAc. These results suggest that RSS-mediated reversible electrophilic signaling takes place through sulfenic acids formation under oxidative stress.

A highly enantioselective cyano-phosphorylation of aldehydes catalyzed by a YLi 3 tris(binaphthoxide) complex YLB 1 is described. The slow addition of diethyl cyanophosphonate 4 to aldehydes 5 in the presence of YLB 1 (10 mol %), H 2 O (30 mol %), tris(2,6-dimethoxyphenyl)phosphine oxide 3a (10 mol %), and BuLi (10 mol %) afforded cyanohydrin O -phosphates 6 in up to 98% yield and 97% ee. Mechanistic studies revealed that the addition of cyanide to aldehydes is irreversible and determines the enantioselectivity. The reaction mechanism is also discussed in detail.

Phytochemicals able to activate the transcription factor NF-E2-related factor 2 (Nrf2) were isolated from an extract of Coriandrum sativum L. (C. sativum) leaves by preparative octadecyl silica column chromatography. Ultraperformance liquid chromatography and liquid chromatography-tandem mass spectrometry analysis of the isolated components after derivatization with 2-diphenylacetyl-1,3-inandione-1-hydrazone and experiments with HepG2 cells revealed that (E)-2-alkenals with different carbon numbers play a role in Nrf2 activation in these cells. Such Nrf2 activation appears to be attributable to S-alkylation of Kelch-like ECH-associated protein 1 (Keap1), the negative regulator for Nrf2, as determined by a biotin-PEAC5-maleimide assay. Interestingly, (E)-2-butenal caused Keap1 modification and Nrf2 activation, whereas butanal did not. These results suggest that (E)-2-alkenals with an α,β-unsaturated aldehyde moiety, which is a common substituent in phytochemicals isolated from C. sativum leaves, activate the Keap1/Nrf2 pathway associated with cellular protection.

Covalent coupling between unsaturated bonds in fatty acids and thiol groups in peptides and proteins can occur through a free radical (thiol-ene) and Michael addition reactions. Recently, we have observed that fatty acid induced-SOD1 aggregation in vitro depends on protein free thiol groups and the presence of least one unsaturated bond in the fatty acid structure. Docosahexaenoic acid (DHA; C22:6n-3) is highly enriched in the brain and its oxidation generates products bearing hydroperoxides, hydroxides and ketone moieties. The later, is known to form covalent adducts with thiol groups via Michael addition while unsaturated fatty acid can undergo a thiol-ene addition reaction. To better understand how DHA and its oxidation products could lead to thiol modifications we have investigated their reaction with thiols in a tri-peptide (glutathione, GSH) and a protein (Keap1) in vitro by LC-MS/MS and biotin-PEAC5-maleimide (BPM) assay. A new covalent adduct between DHA and glutathione (GS-DHA adduct, m/z 634.3168) was detected by LC-MS/MS. However, this adduct was only detected when incubated with AAPH, a free radical-generating azo compound, which suggeststhat GS-DHA is generated in presence of thiylradical. On the other hand, DHA derived hydroperoxides and ketones reacted with GSH generating a GS-oxo-DHA adduct (m/z 648.2960) while the hydroxides did not. To evaluate the reactivity of DHA and its oxidation products under more physiological conditions we evaluated their reactivity towards a thiol-rich protein, Keap1, the negative regulator of Nrf2. Incubations of DHA with Keap1 showed protein thiolate consumption as well as aggregation. Studies is now being conducted to characterize the presence of covalent DHA-thiol adducts in the protein and also to compare the reactivity between DHA and its oxidized derivatives. Overall, our data show that both non-oxidized and oxidized DHA derivatives can modify thiol groups in peptides and proteins, which may potentially affect redox signaling pathways.

A highly enantioselective cyano-phosphorylation of aldehydes catalyzed by YLi3tris(binaphthoxide) complex (YLB, 1) is described. Slow addition of diethyl cyanophosphonate (4) to aldehydes 5 in the presence of 1 (10 mol%),H 2 O (30 mol%), tris(2,6-dimethoxyphenyl)phosphine oxide (10 mol%, 3a), and BuLi (10 mol%) afforded cyanohydrin O-phosphates 6 in up to 98% yield and 97% ee.