C-Myc is a Nrf2-interacting protein that negatively regulates phase II genes through their electrophile responsive elements.

c-Myc is a transcription factor that is implicated in many cellular processes including proliferation, apoptosis and cancers. Recently, c-Myc was shown to be involved in regulation of glutamate cysteine ligase through E-box sequences. This investigation examined whether c-Myc also regulates phase II genes through interaction with the electrophile response element (EpRE). Experiments were conducted in human bronchial epithelial cells using si-RNA to knock down c-Myc. RT-PCR and reporter assays were used to measure transcription and promoter activity. c-Myc downregulated transcription and promoter activity of phase II genes. Chromatin immunoprecipitation verified binding of c-Myc to EpRE while coimmunoprecipitation demonstrated interaction of c-Myc with Nrf2. c-Myc also forms a ternary complex with Nrf2 and p-c-Jun. Finally, c-Myc decreased Nrf2 stability. Thus, our results suggest regulation of the EpRE/Nrf2 signaling pathway by c-Myc through both interaction with the EpRE binding complex and increased degradation of Nrf2.

The role of c-Jun phosphorylation in EpRE activation of phase II genes.

The transcription factors that bind to EpRE's play a key role in the regulation of phase II genes. In this study, we examined whether c-Jun, a partner of Nrf2 in binding to EpRE's, requires phosphorylation by JNK for binding and transcriptional activation. We used chromatin immunoprecipitation assays to measure the recruitment of transcription factors to EpRE sequences in NQO2, GCLC, and GCLM; Western analysis for phosphorylation of JNK; and EpRE-driven reporters along with a JNK-specific inhibitor peptide to determine the potential importance of c-Jun phosphorylation. Human bronchial epithelial (HBE1) and human hepatoma (HepG2) cells were exposed to 4-hydroxy-2-nonenal (HNE), and differences in the regulation of the same EpRE sequences were examined. We found that binding of c-Jun to EpRE sequences increased subsequent to HNE exposure in HepG2 cells; however, in HNE-exposed HBE1 cells, the binding of only phosphorylated c-Jun to the three EpRE sequences increased. Despite the increase in binding of phosphorylated c-Jun, reporter assays for EpRE's showed that inhibition of c-Jun phosphorylation had variable effects on basal and HNE-induced transcription of GCLC and GCLM in HBE1 cells. Thus, in terms of its role in mediating HNE induction of EpRE-mediated transcription, c-Jun seems to be a partner of Nrf2 and, whereas its phosphorylated form may predominate in one cell type versus another, the effects of phosphorylation of c-Jun on transcription can vary with the gene. This contrasts markedly with the well-established requirement for phosphorylation of c-Jun in the activation of AP-1/TRE-mediated transcription.

Synergistic cytotoxic effect of tetrachlorocatechol and sodium azide in Escherichia coli: toxicity, metabolism, and mechanistic aspects.

Pentachlorophenol (PCP) is used in industrial and domestic applications, including as a biocide and a wood preservative. Metabolism of PCP undergoes oxidative dechlorination, forming tetrachlorocatechol (TCC) and tetrachlorohydroquinone (TCHQ). Both sodium azide (NaN(3)) and TCC appear naturally in soil. None of them are cytotoxic by themselves or facilitate autooxidation. Here, we show that their combination leads to synergistic cytotoxicity (>6 log bacterial killing) to Escherichia coli. The rate of oxygen consumption in a cell-free system showed that NaN(3) increases TCC oxidation by 520-fold. The synergism coefficient to cells was calculated as 96 or greater, and we have shown the formation of a new compound. It is suggested that the intermediate species, o-tetrachlorosemiquinine, and an unknown, nitrogen-centered free radical, both visualized by electron-spin resonance, are harmful species responsible for the synergistic cytotoxicity of TCC/NaN(3), rather than the endproduct formed during the reaction. Desferrioxamine and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide offered nearly complete protection, but through radical scavenging rather than through chelating properties. The mechanism of damage for TCC compared to its analogue, TCHQ, were investigated, and whereas the cellular damage of TCHQ/NaN(3) is through a site-specific mechanism, in the case of TCC/NaN(3) it is through the accumulation of the component(s) in the bacterial cell membrane, eventually leading to dysfunction, as evidenced by electron microscopy.

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal.

During the past several years, major advances have been made in understanding how reactive oxygen species (ROS) and nitrogen species (RNS) participate in signal transduction. Identification of the specific targets and the chemical reactions involved still remains to be resolved with many of the signaling pathways in which the involvement of reactive species has been determined. Our understanding is that ROS and RNS have second messenger roles. While cysteine residues in the thiolate (ionized) form found in several classes of signaling proteins can be specific targets for reaction with H(2)O(2) and RNS, better understanding of the chemistry, particularly kinetics, suggests that for many signaling events in which ROS and RNS participate, enzymatic catalysis is more likely to be involved than non-enzymatic reaction. Due to increased interest in how oxidation products, particularly lipid peroxidation products, also are involved with signaling, a review of signaling by 4-hydroxy-2-nonenal (HNE) is included. This article focuses on the chemistry of signaling by ROS, RNS, and HNE and will describe reactions with selected target proteins as representatives of the mechanisms rather attempt to comprehensively review the many signaling pathways in which the reactive species are involved.

Synergism between the toxicity of chlorophenols and iron complexes.

Synergistic interactions could prove to be relevant when evaluating the toxicity of environmental pollutants in a complex mixture, especially when organic and inorganic substances co-occur at concentrations currently considered to be low-toxic or sublethal. Escherichia coli cells (SR-9 strain) were used as a model system for studying the cellular toxicity of environmental pollutants. Exposure of bacterial cells to a combination of pentachlorophenol (PCP) and a positively charged complex of iron or copper caused a dramatic inhibition of growth and an increase in cell death. Incubation of bacterial cells with PCP and either ferric-1,10-phenanthroline complex [Fe3+(OP)3]3+ (500 and 5 microM, respectively) or cupric-1,10-phenanthroline complex [Cu2+(OP)2]2+ (400 and 0.05 microM, respectively) showed two and four log units of cell death, respectively, in 30 min. In contrast, only minor amounts of cell death were observed with each component alone. Similar effects have been shown for other positively charged complexes of transition metals and for other biocides. The observed synergism was associated with the formation of novel noncharged and lipophilic ternary complexes, which contain PCP anions (or other polychlorinated anions) and the iron (or copper) complex. The ternary complexes demonstrated effective transport of their components into the cells.