The anti-inflammatory and anti-oxidative effect of a classical hypnotic bromovalerylurea mediated by the activation of NRF2.

The Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (KEAP1-NRF2) system plays a central role in redox homeostasis and inflammation control. Oxidative stress or electrophilic compounds promote NRF2 stabilization and transcriptional activity by negatively regulating its inhibitor, KEAP1. We have previously reported that bromovalerylurea (BU), originally developed as a hypnotic, exerts anti-inflammatory effects in various inflammatory disease models. However, the molecular mechanism underlying its effect remains uncertain. Herein, we found that by real-time multicolor luciferase assay using stable luciferase red3 (SLR3) and green-emitting emerald luciferase (ELuc), BU potentiates NRF2-dependent transcription in the human hepatoblastoma cell line HepG2 cells, which lasted for more than 60 h. Further analysis revealed that BU promotes NRF2 accumulation and the transcription of its downstream cytoprotective genes in the HepG2 and the murine microglial cell line BV2. Keap1 knockdown did not further enhance NRF2 activity, suggesting that BU upregulates NRF2 by targeting KEAP1. Knockdown of Nfe2l2 in BV2 cells diminished the suppressive effects of BU on the production of pro-inflammatory mediators, like nitric oxide (NO) and its synthase NOS2, indicating the involvement of NRF2 in the anti-inflammatory effects of BU. These data collectively suggest that BU could be repurposed as a novel NRF2 activator to control inflammation and oxidative stress.

Comparison of the detrimental features of microglia and infiltrated macrophages in traumatic brain injury: A study using a hypnotic bromovalerylurea.

Microglia and blood-borne macrophages in injured or diseased brains are difficult to distinguish because they share many common characteristics. However, the identification of microglia-specific markers and the use of flow cytometry have recently made it easy to discriminate these types of cells. In this study, we analyzed the features of blood-borne macrophages, and activated and resting microglia in a rat traumatic brain injury (TBI) model. Oxidative injury was indicated in macrophages and neurons in TBI lesions by the presence of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Generation of mitochondrial reactive oxygen species (ROS) was markedly observed in granulocytes and macrophages, but not in activated or resting microglia. Dihydroethidium staining supported microglia not being the major source of ROS in TBI lesions. Furthermore, macrophages expressed NADPH oxidase 2, interleukin-1ß (IL-1ß), and CD68 at higher levels than microglia. In contrast, microglia expressed transforming growth factor ß1 (TGFß1), interleukin-6 (IL-6), and tumor necrosis factor α at higher levels than macrophages. A hypnotic, bromovalerylurea (BU), which has anti-inflammatory effects, reduced both glycolysis and mitochondrial oxygen consumption. BU administration inhibited chemokine CCL2 expression, accumulation of monocytes/macrophages, 8-OHdG generation, mitochondrial ROS generation, and proinflammatory cytokine expression, and markedly ameliorated the outcome of the TBI model. Yet, BU did not inhibit microglial activation or expression of TGFß1 and insulin-like growth factor 1 (IGF-1). These results indicate that macrophages are the major aggravating cell type in TBI lesions, in particular during the acute phase. Activated microglia may even play favorable roles. Reduction of cellular energy metabolism in macrophages and suppression of CCL2 expression in injured tissue may lead to amelioration of TBI.

Effects of hypnotic bromovalerylurea on microglial BV2 cells.

An old sedative and hypnotic bromovalerylurea (BU) has anti-inflammatory effects. BU suppressed nitric oxide (NO) release and proinflammatory cytokine expression by lipopolysaccharide (LPS)-treated BV2 cells, a murine microglial cell line. However, BU did not inhibit LPS-induced nuclear translocation of nuclear factor-κB and subsequent transcription. BU suppressed LPS-induced phosphorylation of signal transducer and activator of transcription 1 (STAT1) and expression of interferon regulatory factor 1 (IRF1). The Janus kinase 1 (JAK1) inhibitor filgotinib suppressed the NO release much more weakly than that of BU, although filgotinib almost completely prevented LPS-induced STAT1 phosphorylation. Knockdown of JAK1, STAT1, or IRF1 did not affect the suppressive effects of BU on LPS-induced NO release by BV2 cells. A combination of BU and filgotinib synergistically suppressed the NO release. The mitochondrial complex I inhibitor rotenone, which did not prevent STAT1 phosphorylation or IRF1 expression, suppressed proinflammatory mediator expression less significantly than BU. BU and rotenone reduced intracellular ATP (iATP) levels to a similar extent. A combination of rotenone and filgotinib suppressed NO release by LPS-treated BV2 cells as strongly as BU. These results suggest that anti-inflammatory actions of BU may be attributable to the synergism of inhibition of JAK1/STAT1-dependent pathways and reduction in iATP level.

Isoenzyme-selective inhibition of glutathione conjugation in vivo: selective inhibition of the conjugation of S-2-Bromoisovalerylurea in the rat.

Glutathione S-transferases (GSTs) play a major role in the (de-)toxification of many endogenous and xenobiotic substrates. To assess their contribution in (de-)toxification, specific in vivo inhibitors that ideally are selective for a single isoenzyme of GST are required. In the present study, selective inhibition of the alpha class GST by the glutathione analog (R)-5-ethyloxycarbonyl-2-gamma-(S)-glutamylamino-N-2-hept ylpentamide (Et-R-Hep) was studied. In rat liver cytosol and in isolated rat hepatocytes, only the conjugation of the (S)-enantiomer of (RS)-2-bromoisovalerylurea (BIU), which is conjugated mainly by alpha class GST 2-2 (Te Koppele et al., Biochem. J. 252:137-142, 1988), was inhibited by Et-R-hep. The conjugation of (R)-BIU, which is mainly catalyzed by mu class GSTs 3-3 and 4-4, was unaffected. In anesthetized rats to which an infusion of (RS)-BIU was administered, the biliary excretion of the glutathione conjugate of (S)-BIU was inhibited by up to 67% after administration of Et-R-hep (an i.v. bolus dose of 200 mu mol/kg followed by an infusion of 6.7 mu mol/min/kg for 30 min). The extent of inhibition decreased gradually to reach 40% at the end of the experiment (4 hr after administration of the inhibitor). The conjugation of (R)-BIU was unaffected. Thus, the inhibitor Et-R-Hep shows preferential inhibition of the alpha-GST substrate (S)-BIU. Although Et-R-Hep is not specific for alpha class GST, it may be used to assess the role of this class of GST in (de)-toxification and conjugation in vivo.

Stereospecific glutathione conjugation of (R)- and (S)-2-bromoisovalerylurea in freshly isolated rat kidney proximal tubular cells.

The glutathione conjugation of 2-bromoisovalerylurea (BIU) was studied in isolated proximal tubular kidney cells of the rat. Racemic (R,S)-BIU was incubated with the cell suspension, and the incubation medium was analysed for the diastereomeric glutathione (GSH) conjugates, cysteine conjugates and mercapturates that can be formed from (R)- and (S)-BIU. Only the mercapturate formed from (R)-BIU was found, as well as its cysteine precursor. No GSH conjugates were detected. These results indicate that these cells conjugate only the (R)-BIU enantiomer, and that the GSH conjugate is immediately further metabolized to its cysteine conjugate and mercapturate.

Extracellular calcium alleviates cell toxicity due to hepatotoxins that induce lipid peroxidation, but has no effect on toxins that do not cause lipid peroxidation. A study in isolated rat hepatocytes.

The effect of extracellular calcium on cell death, induced by hepatotoxins that induce lipid peroxidation [diethyl maleate (DEM), allyl alcohol (AA) and bromoisovalerylurea (BIU)] and hepatotoxins that do not induce lipid peroxidation [disulfiram (DSF), N-hydroxy-2-acetyl-aminofluorene (N-OH-AAF) and tetrahydroaminoacridine (THA)] was studied in freshly isolated rat hepatocytes. Extracellular calcium strongly delayed the onset of toxicity of DEM, AA and BIU as detected by lipid peroxidation, depletion of free protein thiol groups and cell death. This protective effect of calcium was decreased at higher concentrations of the toxic compounds. In contrast, no effect of calcium was observed on toxicity induced in the absence of lipid peroxidation by DSF, N-OH-AAF and THA. Addition of calcium was also without effect on the protein thiol depletion. These results indicate that calcium only alleviates cytotoxicity which is induced by thiol depletion resulting from lipid peroxidation. Cytotoxicity as a result of protein thiol depletion through disulfide formation is not affected by extracellular calcium.

Lipid peroxidation-dependent and -independent protein thiol modifications in isolated rat hepatocytes: differential effects of vitamin E and disulfiram.

Exposure of isolated rat hepatocytes to allyl alcohol (AA), diethyl maleate (DEM) and bromoisovalerylurea (BIU) induced lipid peroxidation, depletion of free protein thiols to about 50% of the control value and cell death. Vitamin E completely prevented lipid peroxidation, protein thiol depletion and cell death. A low concentration (0.1 mM) of the lipophylic disulfide, disulfiram (DSF), also prevented the induction of lipid peroxidation by the hepatotoxins; however, in the presence of DSF, protein thiol depletion and cell death occurred more rapidly. Incubation of cells with a high concentration (10 mM) of DSF alone led to 100% depletion of protein thiols and cell death, which could not be prevented by vitamin E. The level of free protein thiols in cells, decreased to 50% by exposure to AA, DEM and BIU, could be reversed to 75% of the initial level by dithiothreitol (DTT) treatment, indicating that the protein thiols were partially modified into disulfides and partially into other, stable thiol adducts. The 100% depletion of protein thiols by DSF was completely reversed by DTT treatment. The involvement of lipid peroxidation in protein thiol depletion was studied by measuring the effect of a lipid peroxidation product, 4-hydroxynonenal (4-HNE), on protein thiols in a cell free liver fraction. 4-HNE did not induce lipid peroxidation in this system, but protein thiols were depleted to 30% of the initial value, irrespective of the presence of vitamin E. DTT treatment could reverse this for only 25%. Similar, DSF-induced protein thiol depletion could be reversed completely by DTT. We conclude that (at least) two types of protein thiol modifications can occur after exposure of hepatocytes to toxic compounds: one due to interaction of endogeneously generated lipid peroxidation products with protein thiols, which is not reversible by the action of DTT, and one due to a disulfide interchange between disulfides like DSF and protein thiols, which can be reversed by the action of DTT.