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

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

Interaction of Keap1 Modified by 2-<i>tert</i>-Butyl-1,4-benzoquinone with GSH: Evidence for <i>S</i>‑Transarylation

2-<i>tert</i>-Butyl-1,4-benzoquinone (TBQ), an electrophilic metabolite of butylated hydroxyanisole (BHA), causes activation of Nrf2 together with <i>S</i>-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 <i>S</i>-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 <i>S</i>-transarylation of the Keap1-TBQ adduct. Pretreatment with l-buthionine-(<i>S</i>,<i>R</i>)-sulfoximine and <i>N</i>-acetylcysteine prior to TBQ exposure of HepG2 cells suggested that the Keap1-TBQ adduct appears to undergo GSH-mediated <i>S</i>-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

Activation of the Kelch-like ECH-Associated Protein 1 (Keap1)/NF-E2-Related Factor 2 (Nrf2) Pathway through Covalent Modification of the 2‑Alkenal Group of Aliphatic Electrophiles in Coriandrum sativum L.

Phytochemicals able to activate the transcription factor NF-E2-related factor 2 (Nrf2) were isolated from an extract of Coriandrum sativum L. (<i>C. sativum</i>) 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 (<i>E</i>)-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, (<i>E</i>)-2-butenal caused Keap1 modification and Nrf2 activation, whereas butanal did not. These results suggest that (<i>E</i>)-2-alkenals with an α,β-unsaturated aldehyde moiety, which is a common substituent in phytochemicals isolated from <i>C. sativum</i> leaves, activate the Keap1/Nrf2 pathway associated with cellular protection

Glutathione Adduct of Methylmercury Activates the Keap1–Nrf2 Pathway in SH-SY5Y Cells

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

Sodium-Glucose Cotransporter 2 Inhibitor and a Low Carbohydrate Diet Affect Gluconeogenesis and Glycogen Content Differently in the Kidney and the Liver of Non-Diabetic Mice.

A low carbohydrate diet (LCHD) as well as sodium glucose cotransporter 2 inhibitors (SGLT2i) may reduce glucose utilization and improve metabolic disorders. However, it is not clear how different or similar the effects of LCHD and SGLT2i are on metabolic parameters such as insulin sensitivity, fat accumulation, and especially gluconeogenesis in the kidney and the liver. We conducted an 8-week study using non-diabetic mice, which were fed ad-libitum with LCHD or a normal carbohydrate diet (NCHD) and treated with/without the SGLT-2 inhibitor, ipragliflozin. We compared metabolic parameters, gene expression for transcripts related to glucose and fat metabolism, and glycogen content in the kidney and the liver among the groups. SGLT2i but not LCHD improved glucose excursion after an oral glucose load compared to NCHD, although all groups presented comparable non-fasted glycemia. Both the LCHD and SGLT2i treatments increased calorie-intake, whereas only the LCHD increased body weight compared to the NCHD, epididimal fat mass and developed insulin resistance. Gene expression of certain gluconeogenic enzymes was simultaneously upregulated in the kidney of SGLT2i treated group, as well as in the liver of the LCHD treated group. The SGLT2i treated groups showed markedly lower glycogen content in the liver, but induced glycogen accumulation in the kidney. We conclude that LCHD induces deleterious metabolic changes in the non-diabetic mice. Our results suggest that SGLT2i induced gluconeogenesis mainly in the kidney, whereas for LCHD it was predominantly in the liver

Involvement of Reactive Persulfides in Biological Bismethylmercury Sulfide Formation

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 <i>in vitro.</i> (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 <i>in vivo</i> 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 <i>S</i>-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