diethyl-maleate and ebselen

The Keap1-Nrf2 system plays a critical role in cellular defense against electrophiles and reactive oxygen species. Keap1 possesses a number of cysteine residues, some of which are highly reactive and serves as sensors for these insults. Indeed, point mutation of Cys151 abrogates the response to certain electrophiles. However, this mutation does not affect the other set of electrophiles, suggesting that multiple sensor systems reside within the cysteine residues of Keap1. The precise contribution of each reactive cysteine to the sensor function of Keap1 remains to be clarified. To elucidate the contribution of Cys151 in vivo, in this study we adopted transgenic complementation rescue assays. Embryonic fibroblasts and primary peritoneal macrophages were prepared from mice expressing the Keap1-C151S mutant. These cells were challenged with various Nrf2 inducers. We found that some of the inducers triggered only marginal responses in Keap1-C151S-expressing cells, while others evoked responses in a comparable magnitude to those observed in the wild-type cells. We found that tert-butyl hydroquinone, diethylmaleate, sulforaphane, and dimethylfumarate were Cys151 preferable, whereas 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PG-J(2)), 2-cyano-3,12 dioxooleana-1,9 diene-28-imidazolide (CDDO-Im), ebselen, nitro-oleic acid, and cadmium chloride were Cys151 independent. Experiments with embryonic fibroblasts and primary macrophages yielded consistent results. Experiments testing protective effects against the cytotoxicity of 1-chloro-2,4-dinitrobenzene of sulforaphane and 15d-PG-J(2) in Keap1-C151S-expressing macrophages revealed that the former inducer was effective, while the latter was not. These results thus indicate that there exists distinct utilization of Keap1 cysteine residues by different chemicals that trigger the response of the Keap1-Nrf2 system, and further substantiate the notion that there are multiple sensing mechanisms within Keap1 cysteine residues.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Substitution; Animals; Antioxidants; Azoles; Cytoskeletal Proteins; Dimethyl Fumarate; Fumarates; Gene Expression; Gene Expression Regulation; Glutamate-Cysteine Ligase; HEK293 Cells; Humans; Hydroquinones; Imidazoles; Isoindoles; Isothiocyanates; Kelch-Like ECH-Associated Protein 1; Macrophages, Peritoneal; Maleates; Mice; Mice, Transgenic; NAD(P)H Dehydrogenase (Quinone); NF-E2-Related Factor 2; Oleanolic Acid; Organoselenium Compounds; Oxidants; Oxidative Stress; Sulfoxides; Transcriptional Activation

Heme is a co-factor required for the stimulation of soluble guanylate cyclase (sGC) by nitric oxide (NO) and carbon monoxide, and sGC activation by these agents is inhibited by superoxide. Because heme promotes oxidant generation, we examined the influence of rat pulmonary microvascular endothelial cells (PMECs) with a stable human heme oxygenase-1 (HO-1) transfection and heme on oxidant generation and cGMP. Culture of PMEC with low serum heme decreased cGMP and the detection of peroxide with 10 microM 2',7'-dichlorofluorescin diacetate and increased HO-1 further decreased cGMP without altering the peroxide detection under these conditions. Under conditions where heme (30 microM) has been shown to stimulate cGMP production in PMECsby mechanisms involving NO and CO, heme increased the detection of peroxide in a PMEC-dependent manner and HO-1 transfection did not markedly alter the effects heme on peroxide detection. The addition of 1 microM catalase markedly inhibited the effects of heme on peroxide detection whereas increasing (0.1 mM ebselen) or decreasing (depleting glutathione with 7 mM diethylmaleate) rates of intracellular peroxide metabolism or inhibiting the biosynthesis of oxidants (with 10 microM diphenyliodonium or 0.1 mM nitro-L-arginine) had only modest effects. The detection of superoxide by 10 microM dihydroethidium from PMECs was not increased by exposure to heme. These actions of oxidant probes suggest that intracellular oxidants have a minimal influence on the response to heme. Thus, exposure of PMECs to heme causes a complex response involving an extracellular generation of peroxide-derived oxidant species, which do not appear to originate from increases in intracellular superoxide or peroxide. This enables heme and HO to regulate sGC through mechanisms involving NO and CO, which are normally inhibited by superoxide.

Topics: Animals; Antioxidants; Azoles; Catalase; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Enzyme Inhibitors; Heme; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Isoindoles; Lung; Maleates; Membrane Proteins; Nitric Oxide Synthase; Nitroarginine; Organoselenium Compounds; Oxidants; Rats; Reactive Oxygen Species; Transfection

We investigated the role of the glutathione redox cycle in endothelial cell injury induced by 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), an arachidonate lipoxygenase product. Pretreatment of endothelial monolayers with reduced glutathione (GSH) markedly suppressed 15-HPETE-induced cellular injury, which was determined by the 51Cr-release assay. 15-HPETE-induced cytotoxicity was modified by several GSH-modulating agents such as buthionine sulfoximine and 2-oxothiazolidine-4-carboxylate, indicating that this cyto-protective action of GSH was correlated with the intracellular GSH level. These GSH-modulating agents also modified the conversion of 15-HPETE to 15(S)-hydroxyeicosatetraenoic acid by endothelial cells. On the other hand, the exposure of endothelial cell monolayers to 15-HPETE did not deplete intracellular GSH levels but decreased GSH peroxidase activity. In addition, sodium selenite and ebselen, a stimulator and mimic of GSH peroxidase activity, respectively, displayed remarkable protective effects against 15-HPETE-induced cytotoxicity. These results suggest that intracellular GSH plays a pivotal role in the protection against 15-HPETE-induced endothelial cell injury, and that the decreased activity of GSH peroxidase activity is involved in 15-HPETE-induced cytotoxicity.

Topics: Animals; Antioxidants; Azoles; Buthionine Sulfoximine; Carotid Arteries; Cattle; Cells, Cultured; Endothelium, Vascular; Glutathione; Glutathione Peroxidase; Isoindoles; Kinetics; Leukotrienes; Lipid Peroxides; Maleates; Methionine Sulfoximine; Organoselenium Compounds; Oxidation-Reduction; Pyrrolidonecarboxylic Acid; Selenium; Sodium Selenite; Thiazoles; Thiazolidines