Cell-permeable superoxide dismutase and glutathione peroxidase mimetics afford superior protection against doxorubicin-induced cardiotoxicity: the role of reactive oxygen and nitrogen intermediates.

The use of the potent antitumor antibiotic doxorubicin (DOX) is hampered because of its severe cardiac toxicity that leads to the development of cardiomyopathy and heart failure. In this study, we have developed a cell culture model for DOX-induced myocardial injury using primary adult rat cardiomyocytes that were cultured in serum-free medium and exposed to 1 to 40 microM DOX. DOX caused a dose-dependent release of sarcosolic enzyme lactate dehydrogenase (LDH) from cultured myocytes. The release of LDH was prevented by the cell-permeable superoxide dismutase (SOD) mimetic (MnTBAP), but was unaffected by either cell-impermeable SOD enzyme, or manganese (II) sulfate. Ebselen, a glutathione peroxidase (GPx) mimetic, enhanced the protection of cardiomyocytes afforded by MnTBAP. DOX caused the increased formation of oxidants in cardiomyocytes, and MnTBAP lowered the amount of intracellular oxidants induced by DOX. In addition, DOX selectively inactivated aconitase in cardiomyocytes, and MnTBAP partially reversed this inactivation. Ebselen further amplified the protective effect of MnTBAP on aconitase activity. These results suggest that the SOD mimetic MnTBAP prevents DOX-induced damage to cardiomyocytes and that the GPx mimetic ebselen synergistically enhanced the cardioprotection afforded by MnTBAP. Relevance of these findings to minimizing cardiotoxicity in cancer treatment is discussed.     

Comparison of lignin peroxidase, horseradish peroxidase and laccase in the oxidation of methoxybenzenes.

Lignin peroxidase oxidizes non-phenolic substrates by one electron to give aryl-cation-radical intermediates, which react further to give a variety of products. The present study investigated the possibility that other peroxidative and oxidative enzymes known to catalyse one-electron oxidations may also oxidize non-phenolics to cation-radical intermediates and that this ability is related to the redox potential of the substrate. Lignin peroxidase from the fungus Phanerochaete chrysosporium, horseradish peroxidase (HRP) and laccase from the fungus Trametes versicolor were chosen for investigation with methoxybenzenes as a homologous series of substrates. The twelve methoxybenzene congeners have known half-wave potentials that differ by as much as approximately 1 V. Lignin peroxidase oxidized the ten with the lowest half-wave potentials, whereas HRP oxidized the four lowest and laccase oxidized only 1,2,4,5-tetramethoxybenzene, the lowest. E.s.r. spectroscopy showed that this congener is oxidized to its cation radical by all three enzymes. Oxidation in each case gave the same products: 2,5-dimethoxy-p-benzoquinone and 4,5-dimethoxy-o-benzoquinone, in a 4:1 ratio, plus 2 mol of methanol for each 1 mol of substrate. Using HRP-catalysed oxidation, we showed that the quinone oxygen atoms are derived from water. We conclude that the three enzymes affect their substrates similarly, and that whether an aromatic compound is a substrate depends in large part on its redox potential. Furthermore, oxidized lignin peroxidase is clearly a stronger oxidant than oxidized HRP or laccase. Determination of the enzyme kinetic parameters for the methoxybenzene oxidations demonstrated further differences among the enzymes.     

Ligninase-mediated phenoxy radical formation and polymerization unaffected by cellobiose:quinone oxidoreductase

Phanerochete chrysosporium ligninase (+ H2O2) oxidized the lignin substructure-related compound acetosyringone to a phenoxy radical which was identified by ESR spectroscopy. Cellobiose:quinone oxidoreductase (CBQase) + cellobiose, previously suggested to be a phenoxy radical reducing system, was without effect on the radical. Ligninase polymerized guaiacol and it increased the molecular size of a synthetic lignin. These polymerizations, reflecting phenoxy radical coupling reactions, were also unaffected by the CBQase system. We conclude that ligninase catalyzes phenol polymerization via phenoxy radicals, which CBQase does not affect. The CBQase system also did not produce H2O2, and its physiological role remains obscure. Glucose oxidase + glucose did produce H2O2 as expected, but, like CBQase, it did not reduce the phenoxy radical of acetosyringone. Because intact cultures of P. chrysosporium depolymerize lignins, it is likely that phenol polymerization by ligninase is prevented or reversed in vivo by an as yet undescribed system.     

Human immunodeficiency virus type 1 envelope glycoproteins gp120 and gp160 induce interleukin-6 production in CD4+ T-cell clones.

Polyclonal B-cell activation is a characteristic feature of AIDS and of the AIDS-related complex. Since the immunoregulatory cytokine interleukin-6 (IL-6) plays a major role in inducing B-cell differentiation, we examined the effects of native human immunodeficiency virus type 1 envelope glycoproteins gp120 and gp160 on IL-6 induction. In this study, we have demonstrated that both gp120 and gp160 have the ability to induce IL-6 mRNA and biologically active IL-6 protein secretion in peripheral blood mononuclear cells in vitro. The envelope protein preparations had no detectable endotoxin as tested by the Limulus amebocyte lysate assay, and hence we can rule out the effect of contaminating endotoxin, which is a potent inducer of IL-6 in monocyte/macrophage cell cultures. In addition, we have shown that the envelope glycoproteins act directly on CD4(+)-cloned T cells to induce IL-6 production in the absence of monocytes. These findings indicate that monocytes and T cells both contribute to the secretion of IL-6, which plays an important role in the pathogenesis of B-cell activation in human immunodeficiency virus infection.     

Oxygen activation during the interaction between MPTP metabolites and synthetic neuromelanin--an ESR-spin trapping, optical, and oxidase electrode study

Oxidase electrode measurements as well as optical and electron spin resonance spectroscopic data have shown that synthetic neuromelanin oxidizes the neurotoxin metabolite 1-methyl-4-phenyl-2,3-dihydropyridinium in a dose-dependent manner forming 1-methyl-4-phenylpyridinium and hydrogen peroxide. Hydroxyl radicals are formed in this reaction which is promoted by iron chelates. In contrast, neither 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine nor 1-methyl-4-phenylpyridinium reacts with synthetic neuromelanin in a similar fashion. The mechanism of selective toxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in pigmented neuronal cells is discussed in the light of these findings.     

Homologous recombination between human immunodeficiency viral DNAs in cultured human cells: analysis of the factors influencing recombination

Recombination between HIV DNAs was analyzed using DNA transfection in cell cultures and the optimal conditions for efficient recombination were determined. Recombinant plasmid DNA substrates were constructed from HIV proviral DNAs and the success of recombination was measured by the production of viable hybrid virus. The process of recombination between HIV DNAs was shown to be i) dependent on homology between the truncated HIV DNAs and ii) maximum with concentrations of the truncated DNAs 3ug and above. HIV isolates with heterogeneity in their primary sequence, thus offer an ideal system for the analysis of the requirement of homologous recombination. In addition, recombination methodology would be useful for generating hybrid HIVs for the analysis of specific viral gene functions.     

Ligninase of Phanerochaete chrysosporium. Mechanism of its degradation of the non-phenolic arylglycerol beta-aryl ether substructure of lignin.

This study examined the ligninase-catalysed degradation of lignin model compounds representing the arylglycerol beta-aryl ether substructure, which is the dominant one in the lignin polymer. Three dimeric model compounds were used, all methoxylated in the 3- and 4-positions of the arylglycerol ring (ring A) and having various substituents in the beta-ether-linked aromatic ring (ring B), so that competing reactions involving both rings could be compared. Studies of the products formed and the time courses of their formation showed that these model compounds are oxidized by ligninase (+ H2O2 + O2) in both ring A and ring B. The major consequence with all three model compounds is oxidation of ring A, leading primarily to cleavage between C(alpha) and C(beta) (C(alpha) being proximal to ring A), and to a lesser extent to the oxidation of the C(alpha)-hydroxy group to a carbonyl group. Such C(alpha)-oxidation deactivates ring A, leaving only ring B for attack. Studies with C(alpha)-carbonyl model compounds corresponding to the three basic model compounds revealed that oxidation of ring B leads in part to dealkoxylations (i.e. to cleavage of the glycerol beta-aryl ether bond and to demethoxylations), but that these are minor reactions in the model compounds most closely related to lignin. Evidence is also given that another consequence of oxidation of ring B in the C(alpha)-carbonyl model compounds is formation of unstable cyclohexadienone ketals, which can decompose with elimination of the beta-ether-linked aromatic ring. The mechanisms proposed for the observed reactions involve initial formation of aryl cation radicals in either ring A or ring B. The cation radical intermediate from one of the C(alpha)-carbonyl model compounds was identified by e.s.r. spectroscopy. The mechanisms are based on earlier studies showing that ligninase acts by oxidizing appropriately substituted aromatic nuclei to aryl cation radicals [Kersten, Tien, Kalyanaraman & Kirk (1985) J. Biol. Chem. 260, 2609-2612; Hammel, Tien, Kalyanaraman & Kirk (1985) J. Biol. Chem. 260, 8348-8353].     

Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates.     

An electron spin resonance study of the reduction of peroxides by anthracycline semiquinones

Direct and spin-trapping electron spin resonance methods have been used to study the reactivity of semiquinone radicals from the anthracycline antibiotics daunorubicin and adriamycin towards peroxides (hydrogen peroxide, t-butyl hydroperoxide and cumene hydroperoxide). Semiquinone radicals were generated by one-electron reduction of anthracyclines, using xanthine/xanthine oxidase. It is shown that the semiquinones are effective reducing agents for all the peroxides. From spin-trapping experiments it is inferred that the radical product is either OH (from H2O2) or an alkoxyl radical (from the hydroperoxides) which undergoes beta-scission to give the methyl radical. The rate constant for reaction of semiquinone with H2O2 is estimated to be approx. 10(4)-10(5) M-1 X s-1. The reduction does not appear to require catalysis by metal ions.