Environmental pollutants and the immune response.

Environmental pollution is one of the most serious challenges to health in the modern world. Pollutants alter immune responses and can provoke immunotoxicity. In this Review, we summarize the major environmental pollutants that are attracting wide-ranging concern and the molecular basis underlying their effects on the immune system. Xenobiotic receptors, including the aryl hydrocarbon receptor (AHR), sense and respond to a subset of environmental pollutants by activating the expression of detoxification enzymes to protect the body. However, chronic activation of the AHR leads to immunotoxicity. KEAP1-NRF2 is another important system that protects the body against environmental pollutants. KEAP1 is a sensor protein that detects environmental pollutants, leading to activation of the transcription factor NRF2. NRF2 protects the body from immunotoxicity by inducing the expression of genes involved in detoxification, antioxidant and anti-inflammatory activities. Intervening in these sensor-response systems could protect the body from the devastating immunotoxicity that can be induced by environmental pollutants.

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.

Capture of Electrophilic Quinones in the Extracellular Space: Evidence for a Phase Zero Reaction.

Electrophilic quinones are produced during the combustion of gasoline in the atmosphere. Although these reactive species covalently bind to protein-based nucleophiles in cells, resulting in the formation of protein adducts involved in the modulation of redox signaling pathways and cytotoxicity, the extracellular regulation of quinones is not understood. In this study, incubation of 1,2-naphthoquinone (1,2-NQ) with the low-molecular-weight fraction of mouse plasma resulted in the consumption of cysteine (CysSH) in the plasma in a concentration-dependent manner. Covalent modification of albumin was markedly repressed by the addition of either the low-molecular-weight fraction of mouse plasma or CysSH, suggesting that CysSH protects by forming a conjugate with 1,2-NQ. Similar phenomena also occurred for other atmospheric quinones 1,4-NQ and 1,4-benzoquinone (1,4-BQ). The addition of cystine to a culture medium without amino acids enhanced the release of CysSH from A431 cells and blocked 1,2-NQ-mediated arylation of intracellular proteins, suggesting that 1,2-NQ interacts with extracellular CysSH. Liquid chromatography-tandem mass spectrometry analysis revealed that 1,2-NQ and 1,4-BQ undergoes nucleophilic attack by CysSH, yielding a 1,2-NQH2-SCys adduct and 1,4-BQH2-SCys adduct, respectively. Unlike 1,2-NQ and 1,4-BQ, the authentic 1,2-NQH2-SCys adduct and 1,4-BQH2-SCys adduct had little effect on the covalent modification of cellular proteins and viability of A431 cells. These results suggest that electrophilic quinones are readily trapped by CysSH released from A431 cells, forming less-toxic CysSH adducts and thereby repressing covalent modification of cellular proteins. These findings provide evidence for the existence of a "phase zero" reaction of electrophiles prior to their uptake by cells.

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.

Cystathionine γ-Lyase Self-Inactivates by Polysulfidation during Cystine Metabolism.

Cystathionine γ-lyase (CSE) is an enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. It also has ß-lyase activity toward cystine, generating cysteine persulfide (Cys-SSH). The chemical reactivity of Cys-SSH is thought to be involved in the catalytic activity of particular proteins via protein polysulfidation, the formation of -S-(S)n-H on their reactive cysteine residues. The Cys136/171 residues of CSE have been proposed to be redox-sensitive residues. Herein, we investigated whether CSE polysulfidation occurs at Cys136/171 during cystine metabolism. Transfection of wild-type CSE into COS-7 cells resulted in increased intracellular Cys-SSH production, which was significantly increased when Cys136Val or Cys136/171Val CSE mutants were transfected, instead of the wild-type enzyme. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CSE polysulfidation occurs at Cys136 during cystine metabolism. In vitro incubation of CSE with CSE-enzymatically synthesized Cys-SSH resulted in the inhibition of Cys-SSH production. In contrast, the mutant CSEs (Cys136Val and Cys136/171Val) proved resistant to inhibition. The Cys-SSH-producing CSE activity of Cys136/171Val CSE was higher than that of the wild-type enzyme. Meanwhile, the cysteine-producing CSE activity of this mutant was equivalent to that of the wild-type enzyme. It is assumed that Cys-SSH-producing CSE activity could be auto-inactivated via the polysulfidation of the enzyme during cystine metabolism. Thus, the polysulfidation of CSE at the Cys136 residue may be an integral feature of cystine metabolism, which functions to down-regulate Cys-SSH synthesis by the enzyme.

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.

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.

Biotransformation of Morphinone and Its Glutathione Adduct Derived from Morphine by Anaerobic Gut Microbes in Guinea Pigs.

Morphinone (MO) and its glutathione adduct (MO-GSH) are excreted into bile of guinea pigs after subcutaneous administration of morphine (M). In the present study, we examined metabolites of M in guinea pig feces. Surprisingly, minimal amounts of MO and MO-GSH were excreted into the feces, whereas dihydromorphine (DHM) and dihydromorphinone (DHMO), which are not found in bile of guinea pigs administered M, were detected in the feces. Incubation of MO and MO-GSH with the contents of the large intestine under anaerobic conditions resulted in their conversion into DHMO. These results suggest that MO-GSH undergoes C-S cleavage by gut microbes to form MO, which is anaerobically reduced to DHMO excreted into feces.

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.

Alterations in UPR Signaling by Methylmercury Trigger Neuronal Cell Death in the Mouse Brain.

Methylmercury (MeHg), an environmental toxicant, induces neuronal cell death and injures specific areas of the brain. MeHg is known to induce oxidative and endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) pathway has a dual nature in that it regulates and protects cells from an overload of improperly folded proteins in the ER, whereas excessively stressed cells are eliminated by apoptosis. Oxidative stress/ER stress induced by methylmercury exposure may tilt the UPR toward apoptosis, but there is little in vivo evidence of a direct link to actual neuronal cell death. Here, by using the ER stress-activated indicator (ERAI) system, we investigated the time course signaling alterations of UPR in vivo in the most affected areas, the somatosensory cortex and striatum. In the ERAI-Venus transgenic mice exposed to MeHg (30 or 50 ppm in drinking water), the ERAI signal, which indicates the activation of the cytoprotective pathway of the UPR, was only transiently enhanced, whereas the apoptotic pathway of the UPR was persistently enhanced. Furthermore, detailed analysis following the time course showed that MeHg-induced apoptosis is strongly associated with alterations in UPR signaling. Our results suggest that UPR modulation could be a therapeutic target for treating neuropathy.

Inducible Systemic Gcn1 Deletion in Mice Leads to Transient Body Weight Loss upon Tamoxifen Treatment Associated with Decrease of Fat and Liver Glycogen Storage.

GCN1 is an evolutionarily-conserved ribosome-binding protein that mediates the amino acid starvation response as well as the ribotoxic stress response. We previously demonstrated that Gcn1 mutant mice lacking the GCN2-binding domain suffer from growth retardation and postnatal lethality via GCN2-independent mechanisms, while Gcn1-null mice die early in embryonic development. In this study, we explored the role of GCN1 in adult mice by generating tamoxifen-inducible conditional knockout (CKO) mice. Unexpectedly, the Gcn1 CKO mice showed body weight loss during tamoxifen treatment, which gradually recovered following its cessation. They also showed decreases in liver weight, hepatic glycogen and lipid contents, blood glucose and non-esterified fatty acids, and visceral white adipose tissue weight with no changes in food intake and viability. A decrease of serum VLDL suggested that hepatic lipid supply to the peripheral tissues was primarily impaired. Liver proteomic analysis revealed the downregulation of mitochondrial ß-oxidation that accompanied increases of peroxisomal ß-oxidation and aerobic glucose catabolism that maintain ATP levels. These findings show the involvement of GCN1 in hepatic lipid metabolism during tamoxifen treatment in adult mice.

Glycoprotein non-metastatic melanoma protein B functions with growth factor signaling to induce tumorigenesis through its serine phosphorylation.

Breast cancer is the most common cancer among women. Glycoprotein non-metastatic melanoma protein B (GPNMB), a type I transmembrane protein that is highly expressed in many cancers, including breast cancer, has been shown to be a prognostic factor. We previously reported that GPNMB overexpression confers tumorigenic potential, as evidenced by invasive tumor growth in vivo, sphere formation, and cellular migration and invasion to non-tumorigenic mammary epithelial cells. In this study, we focused on the serine (S) residue in the intracellular domain of GPNMB (S530 in human isoform b and S546 in mouse), which is predicted to be a phosphorylation site. To investigate the roles of this serine residue, we made an antibody specific for S530-phosphorylated human GPNMB and a point mutant in which S530 is replaced by an alanine (A) residue, GPNMB(SA). Established GPNMB(SA) overexpressing cells showed a significant reduction in sphere formation in vitro and tumor growth in vivo as a result of decreased stemness-related gene expression compared to that in GPNMB(WT)-expressing cells. In addition, GPNMB(SA) impaired GPNMB-mediated cellular migration. Furthermore, we found that tyrosine kinase receptor signaling triggered by epidermal growth factor or fibroblast growth factor 2 induces the serine phosphorylation of GPNMB through activation of downstream oncoproteins RAS and RAF.

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.

Spatiotemporal analysis of the UPR transition induced by methylmercury in the mouse brain.

Methylmercury (MeHg), an environmental toxicant, induces neuronal cell death and injures a specific area of the brain. MeHg-mediated neurotoxicity is believed to be caused by oxidative stress and endoplasmic reticulum (ER) stress but the mechanism by which those stresses lead to neuronal loss is unclear. Here, by utilizing the ER stress-activated indicator (ERAI) system, we investigated the signaling alterations in the unfolded protein response (UPR) prior to neuronal apoptosis in the mouse brain. In ERAI transgenic mice exposed to MeHg (25 mg/kg, S.C.), the ERAI signal, which indicates activation of the cytoprotective pathway of the UPR, was detected in the brain. Interestingly, detailed ex vivo analysis showed that the ERAI signal was localized predominantly in neurons. Time course analysis of MeHg exposure (30 ppm in drinking water) showed that whereas the ERAI signal was gradually attenuated at the late phase after increasing at the early phase, activation of the apoptotic pathway of the UPR was enhanced in proportion to the exposure time. These results suggest that MeHg induces not only ER stress but also neuronal cell death via a UPR shift. UPR modulation could be a therapeutic target for treating neuropathy caused by electrophiles similar to MeHg.

Synthesis of Reactive Sulfur Species in Cultured Vascular Endothelial Cells after Exposure to TGF-ß1: Induction of Cystathionine γ-Lyase and Cystathionine ß-Synthase Expression Mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 Pathways.

Transforming growth factor-ß1 (TGF-ß1) occurs at high levels at damage sites of vascular endothelial cell layers and regulates the functions of vascular endothelial cells. Reactive sulfur species (RSS), such as cysteine persulfide, glutathione persulfide, and hydrogen persulfide, are cytoprotective factors against electrophiles such as reactive oxygen species and heavy metals. Previously, we reported that sodium trisulfide, a sulfane sulfur donor, promotes vascular endothelial cell proliferation. The objective of the present study was to clarify the regulation and significance of RSS synthesis in vascular endothelial cells after exposure to TGF-ß1. Bovine aortic endothelial cells in a culture system were treated with TGF-ß1 to assess the expression of intracellular RSS, the effect of RSS on cell proliferation in the presence of TGF-ß1, induction of RSS-producing enzymes by TGF-ß1, and intracellular signal pathways that mediate this induction. The results suggest that TGF-ß1 increased intracellular RSS levels to modulate its inhibitory effect on proliferation. The increased production of RSS, probably high-molecular-mass RSS, was due to the induction of cystathionine γ-lyase and cystathionine ß-synthase, which are RSS-producing enzymes, and the induction was mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 pathways in vascular endothelial cells. TGF-ß1 regulates vascular endothelial cell functions such as proliferation and fibrinolytic activity; intracellular high-molecular-mass RSS, which are increased by TGF-ß1, may modulate the regulation activity in vascular endothelial cells.

Cysteine Trisulfide Protects E. coli from Electrophile-Induced Death through the Generation of Cysteine Hydropersulfide.

Hydropersulfide and polysulfide species have recently been shown to elicit a wide variety of biological and physiological responses. In this study, we examine the effects of cysteine trisulfide (Cys-SSS-Cys; also known as thiocystine) treatment on E. coli. Previous studies in mammalian cells have shown that Cys-SSS-Cys treatment results in protection from the electrophiles. Here, we show that the protective effect of Cys-SSS-Cys treatment against electrophile-induced cell death is conserved in E. coli. This protection correlates with the rapid generation of cysteine hydropersulfide (Cys-SSH) in the culture media. We go on to demonstrate that an exogenous phosphatase expressed in E. coli, containing only a single catalytic cysteine, is protected from electrophile-induced inactivation in the presence of hydropersulfides. These data together demonstrate that E. coli can utilize Cys-SSS-Cys to generate Cys-SSH and that the Cys-SSH can protect cellular thiols from reactivity with the electrophiles.

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

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

Transcriptional Induction of Cystathionine γ-Lyase, a Reactive Sulfur-Producing Enzyme, by Copper Diethyldithiocarbamate in Cultured Vascular Endothelial Cells.

As toxic substances can enter the circulating blood and cross endothelial monolayers to reach parenchymal cells in organs, vascular endothelial cells are an important target compartment for such substances. Reactive sulfur species protect cells against oxidative stress and toxic substances, including heavy metals. Reactive sulfur species are produced by enzymes, such as cystathionine γ-lyase (CSE), cystathionine ß-synthase, 3-mercaptopyruvate sulfurtransferase, and cysteinyl-tRNA synthetase. However, little is known about the regulatory mechanisms underlying the expression of these enzymes in vascular endothelial cells. Bio-organometallics is a research field that analyzes biological systems using organic-inorganic hybrid molecules (organometallic compounds and metal coordinating compounds) as molecular probes. In the present study, we analyzed intracellular signaling pathways that mediate the expression of reactive sulfur species-producing enzymes in cultured bovine aortic endothelial cells, using copper diethyldithiocarbamate (Cu10). Cu10 selectively upregulated CSE gene expression in vascular endothelial cells independent of cell density. This transcriptional induction of endothelial CSE required both the diethyldithiocarbamate scaffold and the coordinated copper ion. Additionally, the present study revealed that ERK1/2, p38 MAPK, and hypoxia-inducible factor (HIF)-1α/HIF-1ß pathways mediate transcriptional induction of endothelial CSE by Cu10. The transcription factors NF-κB, Sp1, and ATF4 were suggested to act in constitutive CSE expression, although the possibility that they are involved in the CSE induction by Cu10 cannot be excluded. The present study used a copper complex as a molecular probe to reveal that the transcription of CSE is regulated by multiple pathways in vascular endothelial cells, including ERK1/2, p38 MAPK, and HIF-1α/HIF-1ß. Bio-organometallics appears to be an effective strategy for analyzing the functions of intracellular signaling pathways in vascular endothelial cells.

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.