Participation of active oxygen species in the induction of chromosomal aberrations by cadmium chloride in cultured Chinese hamster cells

The effect of various scavengers of active oxygen species on the induction of chromosomal aberrations by cadmium chloride (CdCl2) was investigated in cultured Chinese hamster V79 cells. Incidences of chromosomal aberrations by CdCl2 were partially or fully reduced by the presence of catalase, mannitol (a scavenger of hydroxyl radicals) and butylated hydroxytoluene (BHT, an antioxidant). These findings may indicate participation of the active oxygen species such as hydrogen peroxide (H2O2) or hydroxyl radicals in the clastogenicity of cadmium. In contrast, superoxide dismutase (SOD) and dimethylfuran (a scavenger of singlet oxygen) did not influence incidences of chromosomal aberrations by CdCl2. These results suggest that superoxide anion and singlet oxygen are not directly involved in the clastogenicity of the metal. The presence of aminotriazole (an inhibitor of catalase) increased incidences of chromosomal aberrations by CdCl2. This emphasizes participation of H2O2 in the clastogenicity of cadmium.     

Induction of chromosomal aberrations in active oxygen-generating systems. II. A study with hydrogen peroxide-resistant cells in culture

Cells hyper-resistant to hydrogen peroxide (H2O2) were obtained from a Chinese hamster cell line (CHL) by repeated treatments with H2O2 at stepwise increasing concentrations. A clonal line (R-8) was approximately 10 times more resistant to H2O2 than the parental cells, and retained its resistance for about 2 months in normal medium. However, with further passages after the completion of the present study, the elevated resistance gradually decreased. Although the concentration of H2O2 required to induce chromosomal aberrations in 50% of treated cells was about 10 times higher in R-8 than in the parental cells, there were no distinct differences between the cells in the induction of chromosomal aberrations by 3 alkylating agents (N-methyl-N'-nitro-N-nitrosoguanidine, N-ethyl-N-nitrosourea and mitomycin C). The catalase activity of R-8 was 10-fold in comparison with the parental cells, but no obvious differences were seen in the activities of superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase. Therefore, the elevated H2O2-resistance seemed to be associated with the enhanced catalase activity. The induction of chromosomal aberrations in two O2- generating systems--xanthine oxidase plus hypoxanthine (XO + HX), and paraquat--was compared between R-8 cells and the ordinary CHL cells. XO + HX produced chromosomal aberrations in the parental cells but not in the R-8, while paraquat induced almost the same level of aberrations in both cell lines. This finding suggests that different active oxygens are responsible for the induction of aberrations in these two O2- generating systems, i.e., H2O2 in XO + HX and O2- in paraquat.     

Difference in superoxide toxicity between 4,7-dicyanobenzofurazan and paraquat.

The O2-. production by aerobically cultured Escherichia coli in the presence of benzofurazan (1), 4,7-dimethylbenzofurazan (2), 4,7-dibromobenzofurazan (3), 4-bromo-6-cyanobenzofurazan (4), and 4,7-dicyanobenzofurazan (5) was examined by using the cytochrome c reduction method in order to elucidate the mechanism of cytotoxicity of benzofurazans. Adding compound 5 to E. coli cell suspension caused cytochrome c reduction, which was completely inhibited by superoxide dismutase. The rate of cytochrome c reduction was in the order of 1 = 2 = 3 less than 4 less than 5, which correlates well with that of the reduction potentials of these benzofurazans. Adding glucose to the E. coli cell suspension-compound 5-cytochrome c system accelerated the rate of cytochrome c reduction. The formation of 4,7-dicyanobenzofurazan anion radical in the cell suspension-compound 5-glucose system in the absence of O2 was followed by ESR spectroscopy. The ESR signal of the anion radical disappeared when O2 was added. Compound 5 was shown to have an approximately 10-fold greater increasing effect on the flux of O2-. by E. coli than paraquat (PQ) by the cytochrome c reduction method. The results were confirmed by the electrochemical method with an oxygen electrode. However, compound 5 had a bacteriostatic, but not lethal, effect, while PQ had both effects. The effect of compound 5 and PQ on lethality of E. coli showed a dramatic difference when E. coli was exposed to these two compounds and washed prior to testing the effects of that exposure. This difference probably arose because compound 5 readily leaked from the cells during dilution and plating. Also, the reduced form of compound 5 exits from the cells more readily than the reduced form of PQ and then generates O2-. in the medium by autoxidation. This suggests the importance of the intracellular production of O2-., rather than the extracellular production of O2-., for lethal effect.     

Induction of chromosomal aberrations in cultured Chinese hamster cells in a superoxide-generating system

The induction of chromosomal aberrations in a superoxide-generating system using xanthine oxidase and hypoxanthine was investigated in cultured Chinese hamster cells. The production of chromosomal aberations in this system was inhibited by the addition of cytochrome C. This finding indicates that the generation of superoxide was the primary requirement for induction of chromosomal aberrations. On the other hand, superoxide dismutase showed no effect on the frequency of chromosomal aberrations, whereas catalase was effective in preventing the aberrations. It is conceivable, therefore, that the induction of chromosomal aberrations in the superoxide-generating system may be directly or indirectly due to hydrogen peroxide formed in the cultured medium as a result of the spontaneous dismutation reaction of superoxide.     

Involvement of Oxidative Stress in Paraquat-Induced Metallothionein Synthesis Under Glutathione Depletion

The inhibition of glutathione (GSH) synthesis by -buthionine-SR-sulfoximine (BSO) causes aggravation of hepatotoxicity of paraquat (PQ), an oxidative-stress inducing substance, in mice. On the other hand, synthesis of metallothionein (MT), a cysteine-rich protein having radical scavenging activity, is induced by PQ, and the induction by PQ is significantly enhanced by pretreatment of mice with BSO. The purpose of present study is to examine whether generation of reactive oxygens is involved in the induction of MT synthesis by PQ under inhibition of GSH synthesis. Administration of PQ to BSO-pretreated mice increased hepatic lipid peroxidation and frequency of DNA single strand breakage followed by manifestation of the liver injury and induction of MT synthesis. Both vitamin E and deferoxamine prevented MT induction as well as lipid peroxidation in the liver of mice caused by administration of BSO and PQ. In cultured colon 26 cells, both cytotoxicity and the increase in MT mRNA level caused by PQ were significantly enhanced by pretreatment with BSO. Facilitation of PQ-induced reactive oxygen generation was also observed by BSO treatment. These results suggest that reactive oxygens generated by PQ under inhibition of GSH synthesis may stimulate MT synthesis. GSH depletion markedly increased reactive oxygen generation induced by PQ, probably due to the reduced cellular capability to remove the radical species produced.     

Bacteria form intracellular free radicals in response to paraquat and streptonigrin. Demonstration of the potency of hydroxyl radical

The generation of oxygen reduction products by Neisseria gonorrhoeae FA1090 upon exposure to streptonigrin (SNG) and paraquat (PQ2+) and their toxicity was examined. N. gonorrhoeae exhibited maximal cyanide-insensitive respiration, which was employed as an indicator of superoxide (O2-) formation, in the presence of 0.064 mM streptonigrin and 90 mM PQ2+, respectively. Using the concentrations of SNG and PQ2+ described above, complete lethality (greater than 10(8) cells/ml) was observed among cells exposed to SNG, whereas PQ2+ reduced viability by only 3 logs. In an attempt to determine the oxygen radical species generated by gonococci when exposed to SNG, dimethyl sulfoxide, Fe3+, KCN, and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we were able to detect .OH manifested as the methyl adduct (DMPO-CH3). The production of the latter species was not inhibited by catalase, suggesting intracellular .OH generation. When PQ2+ was substituted for SNG, only low levels of DMPO-CH3 were observed, the production of which ceased within 8 min. SNG and PQ2+, added to a O2(-)- generating system in the presence of Fe3+, promoted increased .OH generation. The iron chelator diethyl-enetriaminepentaacetic acid enhanced the generation of spin-trapped .OH and O2- in the presence of PQ2+. The addition of catalase to this system, however, eliminated the DMPO-CH3 signal, showing that the .OH in this system was extracellular. PQ2+-mediated generation of extracellular .OH in the presence of Fe3+-diethylenetriaminepentaacetic acid EDTA did not enhance the killing of gonococci by PQ2+. These data show that the lethality of SNG relative to PQ2+ is due to the inherent ability of SNG to catalyze the formation of critical levels of intracellular .OH, detectable through the use of spin trapping techniques.     

Induction of superoxide dismutase, chromosomal aberrations and sister-chromatid exchanges by paraquat in Chinese hamster fibroblasts

We have recently shown that Bloom syndrome fibroblasts have elevated levels of superoxide dismutase activity compared to those of normal fibroblasts. Based on this observation we decided to test whether an increased rate of superoxide radical production could be responsible for the induction of superoxide dismutase and of chromosomal aberrations and sister-chromatid exchanges characteristic of Bloom syndrome. Utilizing the superoxide-generating herbicide paraquat in Chinese hamster fibroblasts, we assayed the cells for dismutase activity, chromosomal aberrations and sister-chromatid exchanges. All 3 parameters investigated demonstrated a dose-dependent increase with paraquat and, consequently, with the superoxide produced. Since the induction of the enzyme is mediated by its substrate, the superoxide anion radical, we concluded that the increased dismutase activity (in Bloom syndrome and paraquat-treated cells) may be a secondary manifestation of an overall imbalance in oxygen metabolism and that this elevated enzymatic activity is insufficient to detoxify the high superoxide levels, which results in elevated levels of chromosomal damage.     

5,5-dimethyl-1-pyrroline-N-oxide alone enhances the spontaneous superoxide generation by primaquine.

Primaquine (PQ), a well-known antimalarial drug, has been reported to generate superoxide (O2-) in the presence of reducing agents such as NADPH. In the present study, chemiluminescence was detected by adding only PQ to aqueous 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo-[1,2-alpha] pyrazin-3-one (MCLA), which is a specific chemiluminescent probe for O2-, and was quenched by superoxide dismutase (SOD), indicating that PQ alone can generate O2- in aerobic conditions. Furthermore, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) enhanced the O2- generation by PQ. Superoxide spin adduct, DMPO-OOH, was also detected by ESR both in aqueous solutions and in dimethyl sulfoxide with DMPO. The level of O2- generation showed a linear correlation with the DMPO concentration, and SOD competitively inhibited the DMPO-OOH formation. The results suggested that in aerobic conditions PQ is autoxidized to 5-hydroxy-PQ, which generates O2-, and DMPO accelerates the autoxidation process by trapping O2-. DMPO or M4PO alone enhances the spontaneous O2- generation by PQ, therefore cautious evaluation is necessary in all studies using the ESR/spin trapping technique to elucidate the mechanism of PQ-related radical generation.     

9,10-Phenanthraquinone in diesel exhaust particles downregulates Cu,Zn-SOD and HO-1 in human pulmonary epithelial cells: intracellular iron scavenger 1,10-phenanthroline affords protection against apoptosis

9,10-Phenanthraquinone (PQ), a major quinone contained in diesel exhaust particles and atmospheric PM(2.5), undergoes one-electron reduction by flavin enzymes such as NADPH-cytochrome P450 reductase, leading to production of reactive oxygen species in vitro. We have detected an ESR signal for superoxide (O(2)(-)) and hydroxyl radicals ((.)OH) by the spin trap method when PQ was mixed with P450 reductase, NADPH, and iron(III). When we examined the effects of PQ on A549 human pulmonary epithelial cells, PQ induced apoptosis with a LC(50) of approximately 7 microM. Formation of protein carbonyls was also detected in cells after treatment with PQ, suggesting that PQ induces oxidative damage. Iron chelators such as 1,10-phenanthroline (OP), desferrioxamine mesylate, and deferiprone respectively afforded protection against the toxic effects of PQ. Furthermore, treatment of A549 cells with 10-20 microM PQ for 12 h specifically down-regulated protein levels of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and heme oxygenase-1 (HO-1) by more than 50%. Pretreatment of cells with OP (10 microM) markedly reduced the down-regulation of Cu,Zn-SOD and HO-1 and protein carbonyl formation in response to PQ. The inhibitor of Cu,Zn-SOD, diethyldithiocarbamate, enhanced the toxic effects of 5 microM PQ. The present findings suggest that PQ causes iron-mediated oxidative damage that is exacerbated by the concomitant down-regulation of Cu,Zn-SOD.     

Superoxide dismutase protects against paraquat-mediated dioxygen toxicity and mutagenicity: studies in Salmonella typhimurium

Paraquat is univalently reduced to the relatively stable, but oxygen-sensitive, paraquat radical (PQ.+). This PQ.+ can react with dioxygen to generate the superoxide radical, which can further generate other more deleterious species of oxygen free radicals (i.e., hydroxyl radical, OH.). These oxygen free radicals are known to cause chromosomal breaks; therefore, it was logical to postulate that paraquat is a mutagen. This proved to be the case when tested in a modified Ames test using a liquid incubation assay. Salmonella typhimurium strains TA98 and TA100 were grown in the presence of various concentrations of PQ, as well as in the presence of known mutagenic compounds: mitomycin C, azide, and proflavine. Paraquat was much more toxic and mutagenic in a simple nutritionally restricted medium than in a rich complex medium and these toxic and mutagenic effects were oxygen dependent. Furthermore, cells containing high levels of superoxide dismutase were more resistant to the toxic and mutagenic effects of paraquat than were cells containing a normal level of this enzyme.     

Nickel compound-induced DNA single-strand breaks in chromosomal and nuclear chromatin in human blood lymphocytes in vitro: role of oxidative stress and intracellular calcium

The effects of nickel sulfate, and soluble forms of nickel carbonate hydroxide (NiCH), nickel subsulfide, and nickel oxide on delayed induction of DNA single-strand breaks (DNA SSBs) in chromosomal and nuclear chromatin of human blood lymphocytes in culture were studied. After 46 h of initial culture in supplemented RPMI-1640 media at 37 degrees C, human whole blood lymphocytes in culture were exposed to low concentrations (0-15 microM) of different nickel (Ni) compounds for 2 h, whereas only RPMI-1640 medium served as control. Immediately after 2 h of such exposure, both control and Ni-treated cells were washed with the same medium and incubated further in fresh complete RPMI-1640 culture medium for another 24h. After a total 70 h of incubation, cells were then arrested at metaphase. Two hours later, the induction of DNA SSBs involving both metaphase chromosomal and interphase nuclear chromatin was measured using the method of electron microscopy in situ end-labeling. The metaphase chromosomal chromatin showed significantly higher DNA SSBs (as measured by an increase in immunogold particles per microm2 chromatin) due to 15 microM NiCH and NiO when compared to the corresponding control value. Both NiCH and nickel oxide produced significantly higher induction of DNA SSBs than those of nickel subsulfide and nickel sulfate in chromosomal chromatin. The DNA SSBs in chromosomal chromatin were found to be significantly higher than those in nuclear chromatin due to different Ni compounds. Overall, the genotoxic potency seems to be decreased as follows: NiCH>nickel oxide>or=nickel subsulfide>nickel sulfate. Pretreatment of human blood lymphocytes with either catalase (a H2O2 scavenger), or superoxide dismutase (a scavenger of O2- radical) or dimethylthiourea (a hydroxyl radical scavenger), or N-acetylcysteine (GSH precursor) significantly reduced DNA SSBs in both chromosomal and nuclear chromatin induced by NiCH, suggesting the involvement of different types of oxidative stress in such genotoxicity. Deferoxamine (a highly specific iron chelator) pretreatment prevented NiCH-induced DNA SSBs in both chromosomal and nuclear chromatin suggesting a role of iron-mediated oxidative stress generating hydroxyl radical in such genotoxicity. Simultaneous treatment with either verapamil (an inhibitor of Ca 2+ through plasma membranes), or dantrolene (an inhibitor of mobilization of [Ca2+]i from endoplasmic reticulum), or BAPTA (a Ca2+ chelator) significantly reduced Ni compound-induced DNA SSBs in both chromosomal and nuclear chromatin, suggesting that Ni compound-induced destabilization of calcium homeostasis may also involved in the induction of such DNA SSBs.     

The biological activity of hydrogen peroxide. IV. Enhancement of its clastogenic actions by coadministration of L-histidine

An enhancing effect of L-histidine (L-His) was detected on the induction by hydrogen peroxide (H2O2) of chromosomal aberrations of both the chromosome type and the chromatid type, in human embryonic fibroblasts. The maximum efficiency of induction was about 8-fold higher in the presence of L-His than in the presence of H2O2 alone, at a concentration of L-His of 50 microM. D-His and DL-His showed lower enhancing effects than L-His, with approximately 2-fold and 5-fold enhancement of induction of chromosomal aberrations, respectively. L-Histidinol and L-His-methyl ester, among various derivatives of L-His tested, also enhanced this process. However, the effects of these derivatives were smaller than those of L-His in a range of concentrations equivalent to that of the most effective dose of L-His (50 microM), while they produced greater enhancement than L-His at concentrations higher than 200 microM. Other derivatives of L-His, such as L-carnosine, urocanic acid, imidazolepyruvic acid, 1-methyl-L-His, imidazolelactic acid, imidazoleacetic acid and histamine and imidazole itself did not enhance the frequency of chromosomal aberrations induced by H2O2. These results indicate that at least both the imidazole ring and the amino group are essential components of the chemical structure of L-His required for the enhancing effect. Moreover, in order to cause such an enhancing effect, L-His had to be applied together with H2O2 to cells, because the enhancing effect of L-His was not observed with cells which were washed after pretreatment with L-His. The preliminary study suggested that this enhancing effect depends on the His-peroxide adduct derived from L-His and H2O2. None of the amino acids tested other than His produced any enhancing effect on the induction of chromosomal aberrations by H2O2.     

Effects of iron chelators and glutathione depletion on the induction and repair of chromosomal aberrations by tert-butyl hydroperoxide in cultured Chinese hamster cells

The effects of iron chelators and glutathione (GSH) depletion on the induction of chromosomal aberrations by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. t-BuOOH in a concentration range of 0.1-1.0 mM induced chromosomal structural aberrations, consisting mainly of chromatid gaps and breaks, in a dose-dependent fashion. The divalent iron chelator o-phenanthroline almost completely suppressed the formation of chromosomal aberrations while the trivalent chelator desferrioxamine was less effective. GSH depletion did not affect the formation of chromosomal aberrations and DNA single-strand breaks (ssb) by t-BuOOH. DNA ssb by 0.5 mM t-BuOOH were repaired within 60 min of treatment in both GSH-depleted (GSH-) and non-depleted (GSH+) cells. In contrast, chromosomal aberrations increased a little during the 60 min after treatment in both GSH- and GSH+ cells. The aberrations were then repaired in GSH+ cells but those in GSH- cells were maintained to a great extent during 20 h of post-treatment incubation. These results indicate that divalent iron mediates the induction of chromosomal aberrations by t-BuOOH. That t-BuOOH-induced chromosomal aberrations remain even after DNA ssb were repaired suggests involvement of other lesions than DNA ssb in the formation of chromosomal aberrations by the hydroperoxide.     

Inducibility of chromosomal aberrations by metal compounds in cultured mammalian cells.

Metal compounds were tested for their ability to induce chromosomal aberrations in cultured mammalian cells. Chromosomal aberrations were induced by the application of some Cr, Mn and Ni compounds. Among 6-valent Cr compounds, K2Cr2O7 and CrO3 induced high levels of aberrations, at rates which were similar for Cr-equivalent doses. The perchromate compounds were more efficient in producing chromosomal aberrations than was a chromate compound, K2CrO4. A 3-valent Cr compound, Cr2(SO4)3, was less toxic and failed to induce a demonstrable increase in chromosomal aberrations. KMnO4 induced aberrations, but at a low rate. As to Ni compounds, NiCl2 and (CH3COO)2Ni induced few aberrations. Administration of K2Ni(CN)4 induced only gaps. NiS induced a low but definite increase in chromosomal aberrations. The rate of these aberrations increased with an increase in treatment time from 24 to 48 h, indicating a time-dependent increase in the hereditable toxicity of metal compounds. CdCl2 and HgCl2 were somewhat toxic, but failed to induce chromosomal aberrations in the present study.     

Role of superoxide anion in the fungicidal activity of murine peritoneal exudate macrophages against Penicillium marneffei.

Penicillium marneffei is an important opportunistic fungal pathogen. The mechanisms of host defense against P. marneffei are not fully understood. In the present study, we, for the first time, investigated the role of superoxide anion (O2-) in the killing of two forms of P. marneffei, yeast cells and conidia, and the role of this killing mediator in the fungicidal activity of IFN-gamma-stimulated murine peritoneal macrophages. P. marneffei yeast cells were susceptible to the killing effect of activated macrophages and chemically generated O2, while conidia were not. These results suggested that O2- played some role in the fungicidal activity of macrophages. However, an oxygen radical scavenger, superoxide dismutase (SOD), did not suppress, but rather enhanced the fungicidal activity of IFN-gamma-stimulated macrophages against P. marneffei yeast cells. This inconsistency was explained by the release of insufficient concentrations of O2- by activated macrophages as compared with the amount of O2- necessary for the killing of yeast cells, which was predicted in a chemical generating system. On the other hand, SOD enhanced the production of nitric oxide (NO) by IFN-gamma-activated macrophages, and their increased fungicidal activity was significantly inhibited by N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase. Our results suggested that O2- does not function as the killing mediator of macrophages against P. marneffei, but rather plays an important role in the regulation of the NO-mediated killing system by suppressing NO production.     

The response of Azotobacter chroococcum to oxygen: superoxide-mediated effects.

Nitrogenase in Azotobacter chroococcum whole cells was inhibited by enzymically generated superoxide anion (O2-), hydrogen peroxide, and ethyl hydrogen peroxide. The degree of inhibition produced by O2- was related to the quantity of oxygen supplied to the organisms in continuous cultures. O2- also inhibited oxygen uptake by whole cells. These O2- mediated inhibitions were prevented by bovine superoxide dismutase. The quantities of superoxide dismutase (SOD), and catalase associated with cells grown under varying oxygen concentrations were determined. The role of hydrogen peroxide, and of the hydroxyl radical (.OH) in nitrogenase inhibition was examined. The response of Azotobacter chroococum to oxygen was evaluated with respect to the observed effects of O2- on the organism, and some explanation is given to account for nitrogenase sensitivity to oxygen.     

Transplacental action of sodium nitrite on embryonic cells of Syrian golden hamster

Hamster embryos were treated with various doses of NaNO2 in utero, by its oral administration to the mothers, and then the embryonic cells were examined for micronucleus formation, chromosomal aberrations, morphological or malignant transformation and drug-resistant mutations. For induction of resistant mutations, the cells were cultured in normal medium for 72 h, and then selected in media containing 8-azaguanine (10 or 20 microgram/ml) or 1 mM ouabain. This treatment with NaNO2 caused marked dose-dependent induction of 8-azaguanine- and ouabain-resistant mutations. Cultured embryonic fibroblasts in the resting state also showed a marked dose-dependent increase in micronucleus formation but not an increase in chromosomal aberrations. This treatment also caused morphological and neoplastic transformation of the cells. Transplacental oral treatment with DMN, as a positive control, caused changes of similar extent in biological effects of embryonic fibroblasts, and in addition it caused chromosomal aberrations in metaphase plates. On the contrary, transplacental oral application of NaNO2 did not induce any biological change in cultured embryonic fibroblasts.     

The mechanism of action of lymphokines. IX. The enzymatic basis of hydrogen peroxide production by lymphokine-activated macrophages

The purpose of this study was to elucidate the biochemical basis of the enhanced hydrogen peroxide (H2O2) production by guinea pig peritoneal macrophages (MP) cultured in lymphokine (LK)-containing medium. The markedly augmented H2O2 generation by these cells, demonstrable by the horseradish peroxidase (HRP)-catalyzed oxidation of phenol red, is distinguished by its lack of dependence on a second stimulus. We demonstrate that H2O2 production is truly spontaneous and is not caused by a stimulant present among the H2O2 assay reagents. The principal candidate for such a role was HRP type II (a mixture of five isoenzymes) that was reported to be capable of eliciting an oxidative burst in MP. Four distinct HRP isoenzymes that were found incapable of provoking an oxidative response were nevertheless adequate for demonstrating H2O2 production by LK-activated MP. Blocking the MP receptor for mannose by the addition of mannan to the assay system resulted in enhanced detection of H2O2 by low concentrations of HRP type II and by three out of four HRP isoenzymes. Treatment of MP with LK-containing medium for 72 hr did not result in a significant change in the activity of cellular superoxide dismutase (SOD) compared with MP cultured for the same length of time in control medium. By using the specific inhibitor of copper, zinc-containing SOD, sodium diethyldithiocarbamate (DDC), and the universal SOD inhibitor, sodium nitroprusside, we found that the predominant enzyme in guinea pig peritoneal MP is probably manganese-containing SOD. Incubation of LK-activated MP with nitroprusside resulted in almost total inhibition of H2O2 production and a simultaneous switch to superoxide (O2-) liberation. Similar exposure to DDC had no effect. These data indicate that H2O2 produced by LK-activated MP is derived exclusively by enzymatic dismutation of O2- mediated by a manganese-containing SOD. The increase in spontaneous H2O2 production induced by LK is therefore secondary to augmented O2- production that occurs at a cellular location where O2- is accessible to SOD. The enzymatic basis of the enhanced oxygen radical production was investigated by determining the kinetic parameters of the O2- -forming NADPH oxidase of resting LK-treated MP in a cellfree system in which O-2 production was induced by sodium dodecyl sulfate. The Km for NADPH and the Vmax of the enzyme of LK-treated MP were not different from those of the enzyme of MP incubated in control medium. We conclude that LK treatment of MP does not modulate the NADPH oxidase itself but, most likely, a process related to activation of the enzyme.     

The activation of the rat copper/zinc superoxide dismutase gene by hydrogen peroxide through the hydrogen peroxide-responsive element and by paraquat and heat shock through the same heat shock element.

Copper/zinc superoxide dismutase (SOD1) protects cells against oxidative hazards by the dismutation of superoxide radicals. The promoter activity of the SOD1 gene was increased 3-5-fold by hydrogen peroxide, paraquat (PQ) and heat shock. Functional analyses of the regulatory region of the SOD1 gene by deletions, mutations, and heterologous promoter systems confirmed the induction of the SOD1 gene by H(2)O(2) through the hydrogen peroxide-responsive element (HRE) (between nucleotides -533 and -520). Gel mobility shift assays showed that the existence of an H(2)O(2)-inducible protein bound to the oligonucleotide of the HRE. Similar analyses showed that the heat shock activated the SOD1 promoter through the heat shock element (HSE) (between nucleotides -185 and -171). A strong specific far-shifted complex with the oligonucleotide of the HSE was observed by the treatment of heat shock. When cells were treated with PQ, a strong far-shifted complex with the HSE was observed and was competed out by the cold HSE probe, indicating that PQ also activated the SOD1 promoter through the same HSE site. It is very interesting to note that chemical and physical stresses, such as PQ and heat shock, respectively, activated the SOD1 promoter through the same cis-element HSE. These results indicate that the SOD1 was inducible by H(2)O(2) through the HRE and by PQ and heat shock through the same HSE to protect cells from oxidative hazards.     

Enhancement of Amphotericin B Activity Against Candida albicans by Superoxide Radical

This study aimed to examine the involvement of oxidative damage in amphotericin B (AmB) activity against Candida albicans using the superoxide (O2-) generator paraquat (PQ). The effects of PQ on AmB activities against growth, viability, membrane permeability and respiration were examined in a wild-type parent strain (K) and a respiration-deficient mutant (KRD-19) since PQ-induced superoxide generation depends on respiration. In the parent strain, the minimal inhibitory concentration (MIC) of AmB, 0.25 microg/ml, tested with a liquid culture was lowered to 0.025 microg/ml by 1 mM PQ. Such a PQ-induced decrease in the MIC value of AmB was minimal in the mutant. Similar PQ-induced enhancement of AmB activity toward the parent strain was also observed with growth on an agar medium. In viability tests, when candidal cells were exposed to AmB (0.1 microg/ml) for I h, the lethality of AmB was enhanced by 1 mM PQ only in the parent strain. Exogenous superoxide dismutase and catalase failed to diminish the enhancing effect of PQ on the growth inhibitory activity of AmB in the parent strain, suggesting an interaction between superoxide and AmB in candidal cells. The enhancement of AmB activity by PQ, observed preferentially in the wild-type strain, can be explained by extensive superoxide generation depending on respiration. These results suggest that oxidative damage induced by superoxide is involved in AmB activity against C. albicans.