Effect of beta-lapachone on superoxide anion and hydrogen peroxide production in Trypanosoma cruzi.

Addition of beta-lapachone, an o-naphthoquinone endowed with trypanocidal properties to respiring Trypanosoma cruzi epimastigotes induced the release of O2- and H2O2 from the whole cells to the suspending medium. The same beta-lapachone concentration (4 micron) that released H2O2 at maximal rate completely inhibited T. cruzi growth in a liquid medium. The position isomer, alpha-lapachone, did not stimulate O2- and H2O2 release, and did not inhibit epimastigote growth. beta-Lapachone was able to stimulate H2O2 production by the epimastigote homogenate in the presence of NADH as reductant. The same effect was observed with the mitochondrial fraction supplemented with NADH, where beta-lapachone enhanced the generation of O2- and H2O2 4.5- and 2.5-fold respectively. beta-Lapachone also increased O2- and H2O2 production (2.5 and 2-fold respectively) by the microsomal fraction with NADPH as reductant. Cyanide-insensitive NADH and NADPH oxidation by the mitochondrial and microsomal fractions (quinone reductase activity) was stimulated to about the same extent by beta-lapachone. alpha-Lapachone was unable to increase O2- and H2O2 production and quinone reductase activity of the mitochondrial and microsomal fractions.     

Effects of beta-lapachone, a peroxide-generating quinone, on macromolecule synthesis and degradation in Trypanosoma cruzi

Incubation of Trypanosoma cruzi epimastigotes with beta-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), a lipophilic o-quinone, produced inhibition of [3H]thymidine, [3H]uridine, and L-[3H]leucine incorporation into DNA, RNA, and protein, respectively. With 1.6 microM beta-lapachone, DNA synthesis was preferentially inhibited. The inhibition was irreversible, and time and concentration dependent. Other effects of beta-lapachone were (a) inhibition of 3H precursor uptake into epimastigotes, (b) exaggerated degradation of DNA, RNA, and protein, (c) increased unscheduled synthesis of DNA, and (d) increased number of strand breaks in nuclear and kinetoplast DNA. DNA damage by 1.6 microM beta-lapachone was repaired by reincubating the drug-treated epimastigotes in fresh medium for 24 h, but with 7.8 microM beta-lapachone DNA damage was irreversible. The p-quinone isomer alpha-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione), was less effective than beta-lapachone, especially on DNA and RNA synthesis, and did not stimulate unscheduled DNA synthesis. Since beta-lapachone redox cycling in T. cruzi generates oxygen radicals while alpha-lapachone does not (A. Boveris, R. Docampo, J. F. Turrens, and A. O. M. Stoppani (1978) Biochem. J. 175, 431-439), the summarized results support the hypothesis that oxygen radicals contribute to beta-lapachone toxicity in T. cruzi.     

Mild thermotolerance induced at 40°C protects HeLa cells against activation of death receptor-mediated apoptosis by hydrogen peroxide

Preexposure to mild temperatures such as 40°C induces thermotolerance, whereby cells resist subsequent exposure to a toxic insult. This study investigates the protective effect of mild thermotolerance (3h, 40°C) against activation of death receptor-mediated apoptosis by H(2)O(2) in HeLa cells. H(2)O(2) (5-50μM) caused rapid activation (1-3h) of the Fas death receptor pathway of apoptosis, which was evident by up-regulation of the death ligand FasL and recruitment of the adaptor protein Fas-associated death domain to the plasma membrane. This resulted in activation of caspase-8 and caspase-2, which led to activation of the cross-talk pathway involving Bid cleavage, t-Bid translocation to mitochondria, and caspase-9 activation. These changes were all diminished in thermotolerant cells. Mild thermotolerance also protected cells against cytotoxicity from H(2)O(2) as well as execution-phase events of apoptosis such as caspase-3 activation and chromatin condensation. The antioxidant polyethylene glycol-catalase abolished FasL induction and caspase-8 activation due to H(2)O(2). FasL up-regulation; activation of caspases-8, -2, -9, and -3; and chromatin condensation were decreased by the p53 inhibitor pifithrin-α, implicating p53 as an upstream factor in the activation of death receptor-mediated apoptosis by H(2)O(2). This study advances knowledge about the protective effect of adaptive responses induced by mild stresses, such as fever temperatures, against induction of apoptosis by oxidative stress.     

Hydrogen peroxide induces rapid digestion of oligodendrocyte chromatin into high molecular weight fragments

High molecular weight (HMW) fragmentation of nuclear chromatin was studied in cultured rat oligodendrocytes (OL) exposed to hydrogen peroxide (H2O2). Intact genomic DNA was isolated by agarose embedding, and analyzed by field inversion gel electrophoresis, with and without S1 endonuclease digestion to detect and discriminate between single and double stranded fragmentations, respectively. The exposure of OL to H2O2 resulted in a very rapid degradation of chromosomal DNA into HMW fragments that reflect native chromatin structure. Hence, within 10 min after the addition of 1 mM H2O2, a discrete pool representing approximately 45% of the nuclear chromatin underwent single strand digestion into >400 kb fragments likely at AT-rich matrix attachment regions. Subsequent accumulation of single stand breaks at these regions led to bifilar scission. Ultimately, chromatin within this susceptible pool was cleaved at remaining matrix attachment regions into 50-200 kb fragments. Chromatin digestion could be elicited with H2O2 concentrations as low as 50 microM. After the removal of H2O2, most >400 kb fragments were religated within 2 h; however, digestion into 50-200 kb fragments was irreversible. The DNA digestion was not accompanied by the degradation of nuclear proteins, i.e., lamins A/C and poly (ADP-ribose) polymerase indicating that chromatin fragmentation is unlikely to be mediated by proteolysis. In conclusion, H2O2 at pathologically relevant concentrations induces a very rapid and extensive digestion of OL chromatin into HMW fragments. Because the chromatin fragmentation is only partly reversible, it may be a decisive factor in committing oxidatively stressed OL to degeneration and/or death.     

Hydrogen peroxide mediates higher order chromatin degradation

Although a large body of evidence supports a causative link between oxidative stress and neurodegeneration, the mechanisms are still elusive. We have recently demonstrated that hydrogen peroxide (H(2)O(2)), the major mediator of oxidative stress triggers higher order chromatin degradation (HOCD), i.e. excision of chromatin loops at the matrix attachment regions (MARs). The present study was designed to determine the specificity of H(2)O(2) in respect to HOCD induction. Rat glioma C6 cells were exposed to H(2)O(2) and other oxidants, and the fragmentation of genomic DNA was assessed by field inversion gel electrophoresis (FIGE). S1 digestion before FIGE was used to detect single strand fragmentation. The exposure of C6 cells to H(2)O(2) induced a rapid and extensive HOCD. Thus, within 30 min, total chromatin was single strandedly digested into 50 kb fragments. Evident HOCD was elicited by H(2)O(2) at concentrations as low as 5 micro M. HOCD was mostly reversible during 4-8h following the removal of H(2)O(2) from the medium indicating an efficient relegation of the chromatin fragments. No HOCD was induced by H(2)O(2) in isolated nuclei indicating that HOCD-endonuclease is activated indirectly by cytoplasmic signal pathways triggered by H(2)O(2). The exposure of cells to a synthetic peroxide, i.e. tert-butyrylhydroperoxide (tBH) also induced HOCD, but to a lesser extent than H(2)O(2). Contrary to the peroxides, the exposure of cells to equitoxic concentration of hypochlorite and spermine NONOate, a nitric oxide generator, failed to induce rapid HOCD. These results indicate that rapid HOCD is not a result of oxidative stress per se, but is rather triggered by signaling cascades initiated specifically by H(2)O(2). Furthermore, the rapid and extensive HOCD was observed in several rat and human cell lines challenged with H(2)O(2), indicating that the process is not restricted to glial cells, but rather represents a general response of cells to H(2)O(2).     

Levels of histone H4 diacetylation decrease dramatically during sea urchin embryonic development and correlate with cell doubling rate

Basic proteins in nuclei and nucleosomes at different stages of development in Arbacia punctulata sea urchins were analyzed directly by in situ protamine release of chromosomal proteins into Triton/acid/urea-polyacrylamide gels. The predominant protein band in the H4 region of 2-cell through 64-cell stage embryos migrates with the mobility expected for diacetylated histone H4 (i.e. H4aa), whereas after blastulation (approximately 300 cells) the predominant H4 species is the unmodified form, H4O. In early embryos this H4aa band is highly labeled in vivo with [3H]acetic acid. The ratio of H4aa:H4O is more than 20-fold greater at the rapidly dividing 2-cell stage than at pluteus stage. This is true for both newly synthesized H4 labeled with [3H]lysine and total H4 (stained). Enhanced acetylation is also found in nucleosomes. The relative amount of this acetylated H4 species correlates roughly with the rate of cell doubling during early embryogenesis, and decreases as the average nucleosomal repeat increases. The results are indicative of a dynamically changing chromatin structure through development, as well as an intimate role of diacetylated histone H4 in the maturation of newly replicated chromatin.     

Use of histone antibodies for studying chromatin topography and the phosphorylation of chromatin subunits

Polyclonal and monoclonal antibodies specific for histones as well as sera directed against synthetic peptides of histones were used to probe the topography of chromatin subunits. In native chromatin, the regions corresponding to residues 130-135 of H3 and 6-18 of H2B were found to be exposed and able to interact with antibodies whereas the regions 26-35 and 36-43 of H2B and 80-89 and 85-102 of H4 were not. In vitro phosphorylation of H3 and H5 in native chromatin or of H3 in H1/H5-depleted chromatin led to a marked drop in the binding of antibodies specific for residues 130-135 of H3 and 6-18 of H2B. Phosphorylation of H1/H5-depleted chromatin also altered the degree of exposure of certain H2A epitopes but it did not affect the surface accessibility of residues 1-11 of H2B.     

NAD(P)H:Quinone oxidoreductase activity is the principal determinant of beta-lapachone cytotoxicity

beta-Lapachone activates a novel apoptotic response in a number of cell lines. We demonstrate that the enzyme NAD(P)H:quinone oxidoreductase (NQO1) substantially enhances the toxicity of beta-lapachone. NQO1 expression directly correlated with sensitivity to a 4-h pulse of beta-lapachone in a panel of breast cancer cell lines, and the NQO1 inhibitor, dicoumarol, significantly protected NQO1-expressing cells from all aspects of beta-lapachone toxicity. Stable transfection of the NQO1-deficient cell line, MDA-MB-468, with an NQO1 expression plasmid increased apoptotic responses and lethality after beta-lapachone exposure. Dicoumarol blocked both the apoptotic responses and lethality. Biochemical studies suggest that reduction of beta-lapachone by NQO1 leads to a futile cycling between the quinone and hydroquinone forms, with a concomitant loss of reduced NAD(P)H. In addition, the activation of a cysteine protease, which has characteristics consistent with the neutral calcium-dependent protease, calpain, is observed after beta-lapachone treatment. This is the first definitive elucidation of an intracellular target for beta-lapachone in tumor cells. NQO1 could be exploited for gene therapy, radiotherapy, and/or chemopreventive interventions, since the enzyme is elevated in a number of tumor types (i.e. breast and lung) and during neoplastic transformation.     

DNA-protein cross-linking between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells.

Formation of DNA-protein cross-links between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells is reported. Chromatin was isolated from cells, and subsequently hydrolyzed and derivatized. Analysis of derivatized hydrolysates by gas chromatography/mass spectrometry with selected-ion monitoring showed that 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]-L-tyrosine (Thy-Tyr cross-link) was formed. The presence of this DNA-protein cross-link in control cells was also observed at a level of approximately 7 molecules per 10(6) DNA nucleotides. Exposure of cells to ionizing radiation at doses between 8.7 and 82 Gy (J.kg-1) increased the amount of the Thy-Tyr cross-link linearly up to approximately fourfold over the background level. At doses higher than 82 Gy, the yield approached a plateau. Treatment of cells with H2O2 (0.5 to 10 mM) also increased the amount of the Thy-Tyr cross-link in a concentration-dependent manner. Addition of dimethyl sulfoxide and o-phenanthroline in the culture medium afforded partial inhibition of cross-link formation. Addition of catalase inhibitor KCN prior to H2O2 treatment increased the yield of cross-linking over the level observed with H2O2 treatment alone. Pretreatment of cells with ascorbic acid for 24 h without H2O2 caused formation of the Thy-Tyr cross-link. This DNA-protein cross-link in chromatin of cells is proposed to be formed by mechanisms involving a radical addition reaction and/or a radical-radical combination involving thymine and tyrosine radicals. Hydroxyl radical mediated by chromatin-bound metal ions is proposed to cause the formation of the Thy-Tyr cross-link in H2O2-treated cells.     

beta-lapachone induces cell cycle arrest and apoptosis in human colon cancer cells

BACKGROUND: Human colon cancers have a high frequency of p53 mutations, and cancer cells expressing mutant p53 tend to be resistant to current chemo- and radiation therapy. It is thus important to find therapeutic agents that can inhibit colon cancer cells with altered p53 status. beta-Lapachone, a novel topoisomerase inhibitor, has been shown to induce cell death in human promyelocytic leukemia and prostate cancer cells through a p53-independent pathway. Here we examined the effects of beta-lapachone on human colon cancer cells. MATERIALS AND METHODS: Several human colon cancer cell lines, SW480, SW620, and DLD1, with mutant or defective p53, were used. The antiproliferative effects of beta-lapachone were assessed by colony formation assays, cell cycle analysis, and apoptosis analysis, including annexin V staining and DNA laddering analysis. The effects on cell cycle and apoptosis regulatory proteins were examined by immunoblotting. RESULTS: All three cell lines, SW480, SW620, and DLD1, were sensitive to beta-lapachone, with an IC(50) of 2 to 3 microM in colony formation assays, a finding similar to that previously reported for prostate cancer cells. However, these cells were arrested in different stages of S phase. At 24 hr post-treatment, beta-lapachone induced S-, late S/G2-, and early S-phase arrest in SW480, SW620, and DLD1 cells, respectively. The cell cycle alterations induced by beta-lapachone were congruous with changes in cell cycle regulatory proteins such as cyclin A, cyclin B1, cdc2, and cyclin D1. Moreover, beta-lapachone induced apoptosis, as demonstrated by annexin V staining, flow cytometric analysis of DNA content, and DNA laddering analysis. Furthermore, down-regulation of mutant p53 and induction of p27 in SW480 cells, and induction of pro-apoptotic protein Bax in DLD1 cells may be pertinent to the anti-proliferative and apoptotic effects of beta-lapachone on these cells. CONCLUSIONS: beta-Lapachone induced cell cycle arrest and apoptosis in human colon cancer cells through a p53-independent pathway. For human colon cancers, which often contain p53 mutations, beta-lapachone may prove to be a promising anticancer agent that can target cancer cells, especially those with mutant p53.     

Chemical nature of DNA-protein cross-links produced in mammalian chromatin by hydrogen peroxide in the presence of iron or copper ions

We report on the elucidation of DNA-protein cross-links formed in isolated mammalian chromatin upon treatment with H2O2 in the presence of iron or copper ions. Analysis of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization showed the presence of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine (thymine-tyrosine cross-link) on the basis of the gas chromatographic and mass spectrometric characteristics of the trimethylsilylated authentic compound. Other DNA-protein cross-links involving thymine and the aliphatic amino acids and cytosine and tyrosine, which were known to occur in nucleohistone gamma-irradiated under anoxic conditions, were not observed. This was due to inhibition by oxygen as clearly shown by experiments that were carried out using ionizing radiation under both oxic and anoxic conditions instead of using H2O2 and metal ions. However, oxygen did not inhibit formation of the thymine-tyrosine cross-link in gamma-irradiated chromatin or in chromatin treated with H2O2 and metal ions. The yield of the thymine-tyrosine cross-link was higher upon treatment with H2O2/chelated Fe3+ ions than with H2O2/unchelated Fe3+ ions. By contrast, H2O2/unchelated Cu2+ ions produced a higher yield than H2O2/chelated Cu2+ ions. Almost complete inhibition of cross-link formation was provided by the hydroxyl radical scavengers mannitol and dimethyl sulfoxide when H2O2/chelated metal ions were used. On the other hand, scavengers only partially inhibited formation of cross-links when H2O2/unchelated metal ions were used, possibly indicating the site-specific nature of cross-linking. Superoxide dismutase afforded partial inhibition only when chelated ions were used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective release of HMG nonhistone proteins during DNase digestion of Tetrahymena chromatin at different stages of the cell cycle.

The possible role of LG-1, a Tetrahymena specific HMG protein found in the macronuclear chromatin (Hamana, K. and Iwai, K. (1979) J. Biochem. 86, 789-794), was examined in relation to the chromatin structure. The chromatin isolated from cells synchronized at different stages of the cell cycle contained about one molecule of LG-1 per nucleosome. Limited digestion of the chromatin with DNase I or micrococcal nuclease selectively released LG-1 with the nucleosomal core histones and H1 remained insoluble, bound to the resistant DNA. Depending on the cell stages several types of chromatin structure were distinguished by their nuclease sensitivity. However, the chromatin at different stages exhibited the similar behavior of the LG-1 release with the nucleases as a function of the degree of chromatin solubilization. The results suggest that LG-1 proteins play a role in the chromatin organization which is rather independent of the cell stages.     

Dexamethasone-mediated regulation of death and differentiation of muscle cells. Is hydrogen peroxide involved in the process?

The hypothetical involvement of H2O2 in dexamethasone-mediated regulation of muscle cell differentiation and elimination was studied. Rat L6 myoblasts and mouse C2C12 satellite cells were chosen for acute (24 h) and chronic (5 or 10 day) experiments. Mitogenicity and anabolism were both affected by H2O2. Micromolar concentrations of H2O2 inhibited DNA while stimulating protein synthesis. At the millimolar level, H2O2 led to cell death by apoptosis.Synthetic glucocorticoi - dexamethasone (Dex) was shown to effect muscle cell fate similarly to H2O2. Chronic treatment with H2O2 or Dex dose-dependently accelerated either the formation of myotubes or cell elimination. Dex-induced cell death slightly differed from classical apoptosis and was featured by the symptoms of cell senescence such as extensive cytoplasm vacuolisation, accumulation of inclusion-bodies and lack of low molecular weight oligonucleosomal DNA fragmentation but chromatin condensation. Antioxidants (sodium ascorbate, N-acetyl-L-cysteine, catalase) abrogated Dex-dependent cell death. We conclude that H2O2 directly influences myogenesis and muscle cell elimination. Moreover, H2O2 can be considered as the potent mediator of glucocorticoid-dependent effects on muscle cells.     

In vivo formation of single-strand breaks in DNA by hydrogen peroxide is mediated by the Haber-Weiss reaction

Phenanthroline and bipyridine, strong chelators of iron, protect DNA from single-strand break formation by H2O2 in human fibroblasts. This fact strongly supports the concept that these DNA single-strand breaks are produced by hydroxyl radicals generated by a Fenton-like reaction between intracellular Fe2+ and H2O2: H2O2 + Fe2+----Fe3+ + OH- + OH: Corroborating this idea is the fact that thiourea, an effective OH radical scavenger, prevents the formation of DNA single-strand breaks by H2O2 in nuclei from human fibroblasts. The copper chelator diethyldithiocarbamate, a strong inhibitor of superoxide dismutase, greatly enhances the in vivo production of DNA single-strand breaks by H2O in fibroblasts. This supports the idea that Fe3+ is reduced to Fe2+ by superoxide ion: O divided by 2 + Fe3+----O2 + Fe2+; and therefore that the sum of this reaction and the Fenton reaction, namely the so-called Haber-Weiss reaction, H2O2 + O divided by 2----O2 + OH- + OH; represents the mode whereby OH radical is produced from H2O2 in the cell. EDTA completely protects DNA from single-strand break formation in nuclei. The chelator therefore removes iron from the chromatin, and although the Fe-EDTA complex formed is capable of reacting with H2O2, the OH radical generated under these conditions is not close enough to hit DNA. Therefore iron complexed to chromatin functions as catalyst for the Haber-Weiss reaction in vivo, similarly to the role played by Fe-chelates in vitro.     

H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced higher order chromatin degradation: A novel mechanism of oxidative genotoxicity

The genotoxicity of reactive oxygen species (ROS) is well established. The underlying mechanism involves oxidation of DNA by ROS. However, we have recently shown that hydrogen peroxide (H2O2), the major mediator of oxidative stress, can also cause genomic damage indirectly. Thus, H2O2 at pathologically relevant concentrations rapidly induces higher order chromatin degradation (HOCD), i.e. enzymatic excision of chromatin loops and their oligomers at matrix-attachment regions. The activation of endonuclease that catalyzes HOCD is a signalling event triggered specifically by H2O2. The activation is not mediated by an influx of calcium ions, but resting concentrations of intracellular calcium ions are required for the maintenance of the endonuclease in an active form. Although H2O2-induced HOCD can efficiently dismantle the genome leading to cell death, under sublethal oxidative stress conditions H2O2-induced HOCD may be the major source of somatic mutations.     

Antimalarial activity of phenazines from lapachol, beta-lapachone and its derivatives against Plasmodium falciparum in vitro and Plasmodium berghei in vivo

The antimalarial activity of benzo[a]phenazines synthesized from 1,2-naphthoquinone, lapachol, beta-lapachone and several derivatives have been tested against Plasmodium falciparum in vitro using isolates of parasites with various susceptibilities to chloroquine and/or mefloquine. Parasite growth in the presence of the test drugs was measured by incorporation of [(3)H]-hipoxanthine in comparison to controls with no drugs, always testing in parallel chloroquine, a standard antimalarial. Among seven benzophenazines tested, four had significant in vitro activities; important, the parasites resistant to chloroquine were more susceptible to the active phenazines in vitro. The doses of phenazines causing 50% inhibition of parasite growth varied from 1.67 to 9.44 microM. The two most active ones were also tested in vivo against Plasmodium berghei in mice, in parallel with lapachol and beta-lapachone. The 3-sulfonic acid-beta-lapachone-derived phenazine was the most active causing up to 98% inhibition of parasitaemia in long term treatment (7 doses) subcutaneously, whereas the phenazine from 3-bromo-beta-lapachone was inactive. Thus, these simple phenazines, containing polar (-Br,-I) and ionizable (-SO(3)H, -OH) groups, easily synthesized from cheap, natural or synthetic precursors (lapachol and beta-lapachone), at rather low cost, provide prototypes for development of new antimalarials aiming the chloroquine resistant parasites.