vitamin-k-semiquinone-radical and plumbagin

The breakage/reunion reaction of DNA topoisomerase II (TOP2) can be interrupted by DNA intercalators (e.g., doxorubicin), enzyme binders (e.g., etoposide), or DNA lesions (e.g., abasic sites) to produce TOP2-mediated DNA damage. Here, we demonstrate that thiol alkylation of TOP2 can also produce TOP2-mediated DNA damage. This conclusion is supported by the following observations using purified TOP2: (1) Thiol-reactive quinones were shown to induce TOP2-mediated DNA cleavage. (2) Thiol-reactive compounds such as N-ethylmaleimide (NEM), disulfiram, and organic disulfides [e.g., 2,2'-dithiobis(5-nitropyridine)] were also shown to induce TOP2-mediated DNA cleavage with similar reaction characteristics as thiol-reactive quinones. (3) TOP2-mediated DNA cleavage induced by thiol-reactive quinones was completely abolished using mutant yeast TOP2 with all cysteine residues replaced with alanine (cysteineless TOP2). These results suggest the possibility that cellular DNA damage could occur indirectly through thiolation of a nuclear protein, TOP2. The implications of this reaction in carcinogenesis and apoptotic cell death are discussed.

Topics: Alanine; Alkylation; Animals; Cysteine; DNA Damage; DNA Topoisomerases, Type II; Drosophila; Humans; Intercalating Agents; KB Cells; Mutagenesis, Site-Directed; Naphthoquinones; Quinones; Sulfhydryl Compounds; Tumor Cells, Cultured; Vitamin K

MarR negatively regulates expression of the multiple antibiotic resistance operon (marRAB) in Escherichia coli. In this study, it was demonstrated that sodium salicylate, plumbagin, 2, 4-dinitrophenol, and menadione-inducers of the marRAB operon in whole cells-all interfered with the repressor activity of MarR in vitro. It is proposed that these compounds can interact directly with MarR to affect its repressor activity.

Topics: 2,4-Dinitrophenol; Antineoplastic Agents, Phytogenic; Bacterial Proteins; Drug Resistance, Microbial; Escherichia coli; Escherichia coli Proteins; Naphthoquinones; Operon; Plasmids; Repressor Proteins; Restriction Mapping; Sodium Salicylate; Vitamin K

We have identified an RNA polymerase sigma factor, sigmaR, that is part of a system that senses and responds to thiol oxidation in the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2). Deletion of the gene (sigR) encoding sigmaR caused sensitivity to the thiol-specific oxidant diamide and to the redox cycling compounds menadione and plumbagin. This correlated with reduced levels of disulfide reductase activity and an inability to induce this activity on exposure to diamide. The trxBA operon, encoding thioredoxin reductase and thioredoxin, was found to be under the direct control of sigmaR. trxBA is transcribed from two promoters, trxBp1 and trxBp2, separated by 5-6 bp. trxBp1 is transiently induced at least 50-fold in response to diamide treatment in a sigR-dependent manner. Purified sigmaR directed transcription from trxBp1 in vitro, indicating that trxBp1 is a target for sigmaR. Transcription of sigR itself initiates at two promoters, sigRp1 and sigRp2, which are separated by 173 bp. The sigRp2 transcript was undetectable in a sigR-null mutant, and purified sigmaR could direct transcription from sigRp2 in vitro, indicating that sigR is positively autoregulated. Transcription from sigRp2 was also transiently induced (70-fold) following treatment with diamide. We propose a model in which sigmaR induces expression of the thioredoxin system in response to cytoplasmic disulfide bond formation. Upon reestablishment of normal thiol levels, sigmaR activity is switched off, resulting in down-regulation of trxBA and sigR. We present evidence that the sigmaR system also functions in the actinomycete pathogen Mycobacterium tuberculosis.

Topics: Amino Acid Sequence; Base Sequence; Cloning, Molecular; Diamide; Gene Expression Regulation, Bacterial; Genes, Bacterial; Homeostasis; Models, Genetic; Molecular Sequence Data; Mutation; Naphthoquinones; Oxidative Stress; Promoter Regions, Genetic; Sigma Factor; Signal Transduction; Streptomyces; Sulfhydryl Compounds; Thioredoxin-Disulfide Reductase; Thioredoxins; Transcription, Genetic; Vitamin K

Addition of hydrogen peroxide, menadione, or plumbagin to growing cultures of the fission yeast Schizosaccharomyces pombe increased trehalase activity. The effect was inhibited only slightly in the presence of cycloheximide, indicating that the stimulation of trehalase triggered by oxidative stress is mostly due to posttranscriptional activation. Northern blot analysis of trehalase mRNA level revealed that oxidative stress also induces a moderate rise in transcription of trehalase. Mutants disrupted in genes encoding elements of the mitogen-activated protein kinase (MAPK) cascade showed a reduced increase in trehalase activity upon oxidative challenge, which was coincident with a block in transcription of trehalase. Taken together, the results support the idea that the enhancement of trehalase by oxidative stress is due to enzyme activation (via the Pka1/Sck1 phosphorylation pathway) and induction of trehalase mRNA (via the MAPK signaling pathway). In spite of the trehalase increase, a net accumulation of trehalose was noticed during the oxidative stress.

Topics: Cycloheximide; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Hydrogen Peroxide; Naphthoquinones; Oxidative Stress; RNA Processing, Post-Transcriptional; RNA, Messenger; Schizosaccharomyces; Transcription, Genetic; Trehalase; Vitamin K

We isolated a promoter that is inducible by paraquat, a superoxide-generating agent, from Escherichia coli using the promoter-probe plasmid pRS415. Sequence analysis revealed that the promoter derives from the ribA gene encoding GTP cyclohydrolase II, which is the first enzyme in the biosynthetic pathway of riboflavin. We fused the lacZ gene with the ribA promoter to monitor the expression of the gene in the single-copy state. LacZ expression from the ribA promoter was induced about eight-fold by 200 microM paraquat. Other known superoxide generators, menadione and plumbagin, also induced the expression of beta-galactosidase in the fusion strain. On the other hand, no significant induction was observed following treatment with hydrogen peroxide, ethanol or heat shock. Induction of beta-galactosidase was significantly reduced by the introduction of a delta sox-8::cat or soxS3::Tn10 mutation into the fusion strain, indicating that the ribA gene is a member of the soxRS regulon. The transcriptional start site was determined by primer extension analysis and putative binding sites for SoxS in both orientations were identified. GTP cyclohydrolase II activity in soluble extracts of E. coli increased more than three-fold on treatment with paraquat. This increase was dependent on the soxRS locus, and reflects the increase in transcript levels. However, flavin pools did not change significantly. A possible role for ribA induction during superoxide stress is discussed.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; beta-Galactosidase; Binding Sites; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; GTP Cyclohydrolase; Molecular Sequence Data; Naphthoquinones; Oxidative Stress; Paraquat; Promoter Regions, Genetic; Recombinant Fusion Proteins; Regulon; RNA, Bacterial; RNA, Messenger; Trans-Activators; Transcription Factors; Vitamin K

The purpose of this study was to examine the possibility of using Artemia salina as a test organism in the search for compounds having the ability to protect against superoxide-mediated toxicity. The basic procedure for the assay using Artemia salina was performed as described in previous literature, with minor modifications. We found that Artemia salina are extremely sensitive to menadione bisulfite, a compound whose toxicity is probably mediated by intracellular superoxide generation. Desferrioxamine (desferal), a compound with known protective effects, was shown to display dramatic protective activity in our system. We also observed that an inhibitor of endogenous superoxide dismutase (SOD) activity increased the toxicity of menadione toward Artemia salina. In conclusion, this simple, inexpensive, and convenient assay could be a valuable addition to a screening effort in the search for compounds that will be protective against damage by superoxide or other active oxygen species.

Topics: Animals; Artemia; Biological Assay; Deferoxamine; Dimethyl Sulfoxide; Drug Evaluation, Preclinical; Drug Interactions; Hydrogen Peroxide; Naphthoquinones; Paraquat; Potassium Cyanide; Reactive Oxygen Species; Sensitivity and Specificity; Superoxide Dismutase; Superoxides; Ubiquinone; Vitamin K; Vitamin K 3

Exponentially growing Saccharomyces cerevisiae cells are more sensitive to oxidants such as hydrogen peroxide and superoxides than stationary phase cells. Using disruption mutations in the genes encoding the two S. cerevisiae superoxide dismutases, we show that the principal mechanism of toxicity of redox-cycling compounds, such as menadione and plumbagin, is via the production of superoxide anions. Using two-dimensional polyacrylamide gel electrophoresis we have compared the pattern of protein expression in cells labelled with L-[35S]methionine and stressed with either H2O2 or menadione. Three groups of proteins were evident: those whose levels are elevated by both H2O2 and menadione, and those specifically induced by either H2O2 or menadione. Experiments with promoter fusions demonstrated that one of the heat inducible forms of HSP70 (SSA1) was inducible with H2O2. Furthermore, induction of the yeast H2O2-responsive TRX2 promoter by menadione required the metabolism of menadione.

Topics: Base Sequence; Cell Cycle; DNA Primers; DNA, Fungal; Gene Expression Regulation, Fungal; Genes, Fungal; Hydrogen Peroxide; Molecular Sequence Data; Mutagenesis; Naphthoquinones; Oxidative Stress; Saccharomyces cerevisiae; Superoxide Dismutase; Superoxides; Vitamin K

Redox-cycling agents catalyze the flow of reducing equivalents to oxygen; this process generates superoxide ion and other reduced oxygen species. Measurements of redox-cycling activity have been performed previously by studying cyanide-resistant oxygen consumption (respiration) of Escherichia coli cells. E. coli strain GK100, lacking both terminal oxidases, has almost no measurable respiration. We show that the use of this strain eliminates the requirement for cyanide in measurements of redox-cycling activity. The addition of either menadione sodium bisulfite or plumbagin, well-known redox-cycling agents, to GK100 cells resulted in high levels of oxygen consumption. The rate of menadione bisulfite-induced oxygen consumption in this respiration-deficient strain, in the absence of cyanide, was comparable to the cyanide-resistant respiration of isogenic respiration-proficient E. coli strains. In GK100 cells, cyanide increased menadione bisulfite-induced oxygen consumption but had no effect on plumbagin-induced oxygen consumption.

Topics: Cyanides; Cytochrome b Group; Cytochrome d Group; Cytochromes; Electron Transport; Escherichia coli; Escherichia coli Proteins; Mutation; Naphthoquinones; Oxidation-Reduction; Oxygen Consumption; Sulfites; Vitamin K

Several naphthoquinones, except 2-hydroxy-1,4-naphthoquinone, were found to inhibit microsomal cytochrome P-450-linked monooxygenase activities in rabbit liver and human placenta. In particular, 5-hydroxy-1,4-naphthoquinone inhibited placental estrogen biosynthesis more effectively than it did hepatic drug oxidation reactions. There was little contribution by superoxide radicals to these enzyme inhibitions by naphthoquinones. Spectrophotometric studies revealed that naphthoquinones bind to the cytochrome P-450 component of the monooxygenase complex in both microsomal systems, suggesting that the inhibition is caused by direct interaction of these compounds with the heme.

Topics: Androstenedione; Animals; Cytochrome c Group; Cytochrome P-450 Enzyme System; Humans; Microsomes; Microsomes, Liver; Naphthoquinones; Oxygenases; Placenta; Rabbits; Spectrophotometry; Vitamin K

Topics: Animals; Antiprotozoal Agents; Chlorpromazine; Clofazimine; Electron Transport; Leishmania donovani; Leishmania mexicana; Leishmaniasis; Leishmaniasis, Visceral; Methylene Blue; Methylphenazonium Methosulfate; Mice; Mice, Inbred BALB C; Naphthoquinones; Nifurtimox; Vitamin K