Intracellular Catalase Activity Research Articles

Irr regulates brucebactin and 2,3-dihydroxybenzoic acid biosynthesis, and is implicated in the oxidative stress resistance and intracellular survival of Brucella abortus

Brucella abortus faces iron deprivation in both nature and the host. To overcome this limitation, Brucella secretes the siderophores 2,3-dihydroxybenzoic acid and brucebactin. A Fur-like protein named Irr has previously been characterized in B. abortus; this protein is present in the alpha-2 group of Proteobacteria only, where it negatively regulates haem biosynthesis when iron is scarce. Additional evidence that Irr also regulates the synthesis of both siderophores is presented here. Transcriptional lacZ fusion and chemical determinations revealed that Irr induced the transcription of the operon involved in the synthesis of the catecholic siderophores, which were consequently secreted under conditions of iron limitation. Irr was able to bind the upstream region of the operon, as shown by electrophoretic mobility shift assay. A B. abortus irr mutant showed higher intracellular haem content, catalase activity and resistance to hydrogen peroxide than the wild-type strain. The mutation also improved the replication and survival of iron-depleted bacteria within cultured mammalian cells. Although the pathogenesis of Brucella correlates with its ability to replicate intracellularly, pathogenicity was not attenuated when assayed in a murine model.

Hydrogen peroxide-induced astaxanthin biosynthesis and catalase activity in Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) in shake-flask cultures was exposed to 10-20 mmol/L H(2)O(2) at various culture stages, and the astaxanthin production was significantly increased by H(2)O(2) fed at 0 or 24 h (exponential phase), but only slightly at 48 h (near stationary phase). The astaxanthin production was enhanced most significantly with double feeding of 10 mmol/L H(2)O(2) at 0 and 24 h, reaching a cellular content of 1.30 mg/g cell and a volumetric yield of 10.4 mg/L, which were 83 and 65% higher, respectively, than those of the control (0.71 mg/g cell and 6.3 mg/L). The intracellular catalase (CAT) activity was also increased after H(2)O(2) treatment. The increases in CAT and astaxanthin of cells could be detected within 4 h of H(2)O(2) treatment. The increase in the astaxanthin content of cells was concomitant with a notable decrease in the beta-carotene content. The older yeast cells at late culture stage (120 h), due perhaps in part to their higher astaxanthin contents, were more tolerant to H(2)O(2) toxicity than the younger cells (24 h). No enhancement of the astaxanthin biosynthesis was attained when H(2)O(2) was added to the yeast culture together with a sufficient amount of exogenous CAT. The results suggest that astaxanthin biosynthesis in X. dendrorhous can be stimulated by H(2)O(2) as an antioxidative response.

Effect of abiotic stress on photosynthesis and respiration in Chlamydomonas reinhardtii: Induction of oxidative stress

Our results indicate that 200 mM NaCl, 0.2 μM methyl viologen, 4 mM arsenite or 300 μM of cadmium in the culture medium are highly toxic for Chlamydomonas reinhardtii productivity. All of them inhibit the photosynthetic-electron flow, while cyanide-sensitive respiration remains high in the alga. The addition of NaCl, 200 mM to the culture medium immediately blocks the photosynthetic activity of the alga which is partially recovered, after 1 h of treatment, remaining high during the following 24 h. At short time (4 h) methyl viologen (MV) inhibits the photosynthetic activity of the alga, while respiration inhibition was only evident at 0.5 μM of MV. After 24 h treatment, 0.2 μM MV inhibits drastically the photosynthetic activity of C. reinhardtii, while photosystem I was still functional. At short time, cadmium and arsenite inhibit the alga photosynthetic activity, while only arsenite 4 mM inhibits both photosynthesis and respiration in 24 h-treated C. reinhardtii cells. Catalase activity of C. reinhardtii was stimulated by abiotic stress, being NaCl the most efficient inductor of the enzyme. After 24 h treatment with NaCl 200 mM, the intracellular catalase activity of the alga reaches 337.5 ± 2.8 μmol O 2 mg −1 Chl min −1, which means a 20-fold higher level than in the control cells. The degree of catalase stimulation correlates the NaCl concentration in the culture medium. These data indicate that abiotic stress induces in C. reinhardtii an increase of intracellular peroxide, which parallel a significant inhibition of the photosynthetic-electron flow.

Resistance of Penicillium piceum F-648 to hydrogen peroxide under short term and prolonged oxidative stress

Resistance of Penicillium piceum F-648 to hydrogen peroxide under short-term and prolonged oxidative stress was studied. An increase in the activity of intracellular catalase in fungal cells after short-term exposure to hydrogen peroxide was shown. Activation of fungal cells induced by H2O2 depends on H2O2 concentration, time of exposure, and the growth phase of the fungus. Variants of P. piceum F-648 that produced two forms of extracellular catalase with different catalytic properties were obtained due to prolonged adaptation to H2O2. Catalase with low affinity for substrate was produced predominantly by the parent culture and variant 3; however, a high substrate affinity of catalase was observed in variant 5. Variant 5 of P. peniceum F-648 displayed a high catalytic activity and operational stability of catalase in the presence of phosphate ions and the concentration of substrate less than 30 mM at pH more than 7.

Unique presence of a manganese catalase in a hyperthermophilic archaeon, Pyrobaculum calidifontis VA1.

We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1. Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity. The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70 degrees C. The enzyme exhibited a K(m) value of 170 mM toward H(2)O(2) and a k(cat) value of 2.9 x 10(4) s(-1).subunit(-1) at 25 degrees C. Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da. The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes. In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide. Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese. Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea. Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese. Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (kat(Pc)). The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp. YS 8-13. Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene. Moreover, a homology search with the sequence of kat(Pc) revealed that no orthologue genes were present on the archaeal genomes, including those from the "aerobic" (hyper)thermophilic archaea Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii. Therefore, Kat(Pc) can be considered a rare example of a manganese catalase from archaea.

Intracellular superoxide dismutase, catalase, and glutathione peroxidase activities and membrane lipid peroxide levels in Fusarium acuminatum upon environmental changes in a defined medium

The variations of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities and lipid peroxide (LPO) levels in Fusarium acuminatum, an aerobic filamentous fungus, were investigated depending on the carbon and nitrogen sources during the incubation period. Fungus was cultivated in growing medium containing either maltose or saccharose in 5–25 g/L concentration range as a carbon source and either glycine or peptone in 5–35 g/L concentration range as a nitrogen source at 28 °C and 100 rpm. The observed highest SOD, CAT, and GSH-Px activities were 31.2±0.655, 62.5±5.23, and 1.52±0.0122 IU/mg in the presence of 20 g/L maltose and 73.96±1.48, 74.46±2.94, 3.48±0.083 IU/mg in the 15 g/L glycine-containing medium at 16 days, respectively. At the same time, the minimum LPO level was observed at 20 g/L maltose and 15 g/L glycine compared with the other carbon and nitrogen sources. The results showed a negative correlation between antioxidant enzyme activities and membrane LPO levels in F. acuminatum cells.

Effects of Fusaric Acid on Reactive Oxygen Species and Antioxidants in Tomato Cell Cultures

Generation of O2– and H2O2 as well as the activities of superoxide dismutase, catalase, ascorbate peroxidase, guaiacol peroxidase, dehydroascorbate reductase and ascorbate content were studied in tomato cell cultures in response to fusaric acid – a nonspecific toxin of phytopathogenic Fusarium species. Toxin treatment resulted in decreased cell viability which was preceded by culture medium alkalinization up to 0.65 pH unit and enhanced extracellular O2– production. The H2O2 level was not significantly affected. In toxin‐treated cultures, a transient, significant increase occurred in intracellular superoxide dismutase, catalase, guaiacol peroxidase and ascorbate peroxidase activities. Fusaric acid‐induced ascorbate turnover modulation led to up to a twofold increase in dehydroascorbic acid accumulation, and a decrease in the associated ascorbate redox ratio. It was concomitant with a significant decrease in dehydroascorbate reductase activity. These results support previous observations that the pro‐ and anti‐oxidant systems are involved in response to fusaric acid treatment although differential response of H2O2 and its metabolism‐related enzymes between the whole leaf and cell culture assays was found.

Chronic Ethanol Consumption Alters the Glutathione/Glutathione Peroxidase-1 System and Protein Oxidation Status in Rat Liver

Background: Alcohol-induced liver damage is associated with oxidative stress, which might be linked to disturbances in liver antioxidant defense mechanisms. The effect of chronic ethanol consumption on the mitochondrial and cytosolic glutathione/glutathione peroxidase-1 (GSHPx-1) system and oxidative modification of proteins was therefore studied in the rat. Methods: Male Sprague-Dawley® rats were fed liquid diets that provided 36% total calories as ethanol for at least 31 days. Pair-fed controls received isocaloric diets with ethanol calories substituted with maltose-dextrins. Mitochondrial and cytosolic fractions were prepared from livers and assayed for GSHPx-1 and glutathione reductase activities and total and oxidized concentrations of glutathione. Catalase activity was measured in the postmitochondrial supernatant. Levels of GSHPx-1, lactate dehydrogenase, and the beta subunit of the F1 portion of the ATP synthase protein were determined by western blot analysis. Concentrations of mitochondrial and cytosolic protein carbonyls were measured to assess ethanol-induced oxidation of proteins. Results: Chronic ethanol consumption significantly decreased cytosolic and mitochondrial GSHPx-1 activities by 40% and 30%, respectively. Levels of GSHPx-1 protein in cytosol were unaffected by ethanol feeding, whereas there was a small decrease in GSHPx-1 protein levels in mitochondria isolated from ethanol-fed rats. Glutathione reductase activities were increased in both intracellular compartments and catalase activity was increased as a consequence of ethanol exposure. Cytosolic total glutathione was mildly decreased, whereas ethanol feeding increased mitochondrial levels of total glutathione. Chronic ethanol feeding significantly increased both cytosolic and mitochondrial concentrations of protein carbonyls by 30% and 60%, respectively. Conclusions: This study demonstrates that chronic ethanol-induced alterations in the glutathione/GSHPx-1 antioxidant system might promote oxidative modification of liver proteins, namely those of the mitochondrion, which could contribute to the adverse effects of ethanol on the liver.

Chronic Ethanol Consumption Alters the Glutathione/Glutathione Peroxidase‐1 System and Protein Oxidation Status in Rat Liver

Alcohol-induced liver damage is associated with oxidative stress, which might be linked to disturbances in liver antioxidant defense mechanisms. The effect of chronic ethanol consumption on the mitochondrial and cytosolic glutathione/glutathione peroxidase-1 (GSHPx-1) system and oxidative modification of proteins was therefore studied in the rat. Male Sprague-Dawley rats were fed liquid diets that provided 36% total calories as ethanol for at least 31 days. Pair-fed controls received isocaloric diets with ethanol calories substituted with maltose-dextrins. Mitochondrial and cytosolic fractions were prepared from livers and assayed for GSHPx-1 and glutathione reductase activities and total and oxidized concentrations of glutathione. Catalase activity was measured in the postmitochondrial supernatant. Levels of GSHPx-1, lactate dehydrogenase, and the beta subunit of the F1 portion of the ATP synthase protein were determined by western blot analysis. Concentrations of mitochondrial and cytosolic protein carbonyls were measured to assess ethanol-induced oxidation of proteins. Chronic ethanol consumption significantly decreased cytosolic and mitochondrial GSHPx-1 activities by 40% and 30%, respectively. Levels of GSHPx-1 protein in cytosol were unaffected by ethanol feeding, whereas there was a small decrease in GSHPx-1 protein levels in mitochondria isolated from ethanol-fed rats. Glutathione reductase activities were increased in both intracellular compartments and catalase activity was increased as a consequence of ethanol exposure. Cytosolic total glutathione was mildly decreased, whereas ethanol feeding increased mitochondrial levels of total glutathione. Chronic ethanol feeding significantly increased both cytosolic and mitochondrial concentrations of protein carbonyls by 30% and 60%, respectively. This study demonstrates that chronic ethanol-induced alterations in the glutathione/GSHPx-1 antioxidant system might promote oxidative modification of liver proteins, namely those of the mitochondrion, which could contribute to the adverse effects of ethanol on the liver.

Free radical activity and antioxidant defense mechanisms in patients with hyperthyroidism due to Graves’ disease during therapy

Free radical-mediated oxidative stress has been implicated in the etiopathogenesis of several autoimmune disorders. We investigated the prooxidant–antioxidant status in order to evaluate the possible deleterious role of oxidative phenomena in patients with Graves’ disease. Thirty patients with hyperthyroidism due to Graves’ disease were investigated. Thirty age-matched healthy subjects were studied as a control group. Free radical activity indices, antioxidant defense systems, and thyroid and pituitary hormone levels were measured in fasting blood samples. Blood samples were taken before initiation of therapy and after attainment of euthyroid state. A significant increase in lipid peroxidation activity indices, i.e., conjugated dienes and thiobarbituric acid-reacting substances, was found in blood serum of the patients with untreated Graves’ disease. These changes were accompanied by a decrease in plasma thiol and erythrocyte lysate thiol groups concentrations. Hyperthyroidism resulted in a marked increase in intracellular antioxidant enzymes, i.e., superoxide dismutase, catalase and glutathione peroxidase activities as compared to the controls. Extracellular anti-free radical scavenging systems potential, measured by glutathione reductase activity and total antioxidant status level, was found to be significantly decreased in untreated Graves’ patients. Treatment with thiamazole resulted in normalization of the free radical and antioxidant activity indices. The obtained results indicate an enhanced generation of reactive oxygen species and impairment of cellular and extracellular antioxidant systems potential in patients with Graves’ disease. The attainment of euthyroid state led to an improvement in oxidative stress indices and antioxidant potential parameters.

Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture.

A novel method for increasing dissolved oxygen concentration in culture media has been developed. It involves adding hydrogen peroxide (H2O2) to the medium which is then decomposed to oxygen and water by catalase. Some factors affecting oxygenation of culture were investigated. Maximal oxygen concentration occurred in 50 ml of the medium containing 0-2 g wet mycelium and 0.2% glucose at pH 5.0. A new apparatus for automated addition of H2O2 to the bioreactor to keep the dissolved oxygen concentration constant over the range 1-100% +/- 2% was tested. A significant increase (over sixfold) of intracellular catalase activity was obtained while the dissolved oxygen concentration remained stable (30% +/- 2%).

Decreased Intracellular Superoxide Levels Activate Sindbis Virus-induced Apoptosis

Infection of many cultured cell types with Sindbis virus (SV), an alphavirus, triggers apoptosis through a commonly utilized caspase activation pathway. However, the upstream signals by which SV activates downstream apoptotic effectors, including caspases, remain unclear. Here we report that in AT-3 prostate carcinoma cells, SV infection decreases superoxide (O-2) levels within minutes of infection as monitored by an aconitase activity assay. This SV-induced decrease in O-2 levels appears to activate or modulate cell death, as a recombinant SV expressing the O-2 scavenging enzyme, copper/zinc superoxide dismutase (SOD), potentiates SV-induced apoptosis. A recombinant SV expressing a mutant form of SOD, which has reduced SOD activity, has no effect. The potentiation of SV-induced apoptosis by wild type SOD is because of its ability to scavenge intracellular O-2 rather than its ability to promote the generation of hydrogen peroxide. Pyruvate, a peroxide scavenger, does not affect the ability of wild type SOD to potentiate cell death; and increasing the intracellular catalase activity via a recombinant SV vector has no effect on SV-induced apoptosis. Moreover, increasing intracellular O-2 by treatment of 3T3 cells with paraquat protects them from SV-induced death. Altogether, our results suggest that SV may activate apoptosis by reducing intracellular superoxide levels and define a novel redox signaling pathway by which viruses can trigger cell death.

Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy.

Recent evidence suggests that oxidative mechanisms may be involved in vascular smooth muscle cell (VSMC) hypertrophy. We previously showed that angiotensin II (Ang II) increases superoxide production by activating an NADH/NADPH oxidase, which contributes to hypertrophy. In this study, we determined whether Ang II stimulation of this oxidase results in H2O2 production by studying the effects of Ang II on intracellular H2O2 generation, intracellular superoxide dismutase and catalase activity, and hypertrophy. Ang II (100 nmol/L) significantly increased intracellular H2O2 levels at 4 hours. Neither superoxide dismutase activity nor catalase activity was affected by Ang II; the SOD present in VSMCs is sufficient to metabolize Ang II-stimulated superoxide to H2O2, which accumulates more rapidly than it is degraded by catalase. This increase in H2O2 was inhibited by extracellular catalase, diphenylene iodonium, an inhibitor of the NADH/NADPH oxidase, and the AT1 receptor blocker losartan. In VSMCs stably transfected with antisense p22phox, a critical component of the NADH/NADPH oxidase in which oxidase activity was markedly reduced, Ang II-induced production of H2O2 was almost completely inhibited, confirming that the source of Ang II-induced H2O2 was the NADH/NADPH oxidase. Using a novel cell line that stably overexpresses catalase, we showed that this increased H2O2 is a critical step in VSMC hypertrophy, a hallmark of many vascular diseases. Inhibition of intracellular superoxide dismutase by diethylthiocarbamate (1 mmol/L) also resulted in attenuation of Ang II-induced hypertrophy (62+/-2% inhibition). These data indicate that AT1 receptor-mediated production of superoxide generated by the NADH/NADPH oxidase is followed by an increase in intracellular H2O2, suggesting a specific role for these oxygen species and scavenging systems in modifying the intracellular redox state in vascular growth.

Selection of biochemical mutants of Aspergillus niger with enhanced catalase production.

The production of extracellular catalase in a submerged culture by a number of biochemical mutants has been evaluated. Eight of these mutants showed increased extracellular catalase, the level of which ranged widely in individual cases from 44% to over 94% in comparison with the parental strain. Studies of the relationship between a criterion of selection and the frequency of mutation showed that the highest frequency of positive mutations (15.8% and 24.2%) was obtained with respect to mutants resistant to ethidium bromide (1 mmol/l) and sodium gluconate (45%) respectively. The time course of growth and enzyme production by the most active mutant, AM-20, showed extra- and intracellular catalase activities increasing about 2- and 2.6-fold respectively, compared with the parental strain.

Optimization of catalase biosynthesis in submerged cultures of Aspergillus niger mutant.

The effect of some medium components, viscous substances and metabolic inhibitors, on catalase production by mutant Aspergillus niger has been studied in shake culture. Altering the composition of the basal medium, particularly substituting NaNO3 for KNO3, and peptone for yeast extract brought an increase in extra- and intracellular catalase activity by 1.5- and 3-fold, respectively. The addition of 2.0-6.0 mg sodium alginate or pectin/ml as viscous additive to the medium, containing glucose as carbon source, increased the medium viscosity and catalase production in shake culture by about 2.8- to 3.0-fold. The highest yield of extracellular catalase activity of A. niger was obtained in the presence of sodium orthovanadate and Triton X-100, which improved the activity of this enzyme by about 1.5-2.2-fold. A significant increase in intracellular catalase activity was observed in the presence of hematin, Tween 80 and sodium orthovanadate (1.7-, 1.6- and 1.4-fold respectively). The time course of growth and enzyme production by A. niger in the optimized medium is also reported.

Adaptation of Lung Antioxidants to Cigarette Smoking in Humans

We investigated the effect of free radical scavengers, micronutrient antioxidants, on antioxidant enzyme activities in cigarette smokers. We measured the intracellular superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and vitamin E and beta-carotene levels in the bronchoalveolar cells of 14 smokers before and after 6 weeks of supplementation with vitamins E and C and beta-carotene. Eight nonsmokers served as control subjects. CAT and GPx activities were higher in BAL cells from smokers compared with nonsmokers (20.5 +/- 2.3 vs 9.6 +/- 1.3 U/10(6) cells; p = 0.027; 0.90 +/- 0.10 vs 0.46 +/- 0.12 U/10(6) cells; p = 0.049, respectively), while there was no difference in SOD activity between the two groups. Likewise, vitamin E and beta-carotene concentrations were markedly higher in smokers' lung lavage cells (403.3 +/- 81.0 in smokers vs 16.6 +/- 5.3 ng/10(6) cells in nonsmokers, and 1.23 +/- 0.21 in smokers vs 0.15 +/- 0.04 ng/10(6) cells in nonsmokers, respectively). The serum levels of vitamin E and C and beta-carotene increased by 2.0-, 1.6-, and 8.9-fold in smokers after supplementation, which were similar to nonsmokers. Similarly, BAL cell vitamin E increased from 403.3 +/- 81.0 to 477.4 +/- 97.7 ng/10(6) cells and beta-carotene increased from 1.23 +/- 0.21 to 4.32 +/- 0.45 ng/10(6) cells (p < 0.05). Despite increased concentrations of vitamins in serum as well as beta-carotene levels in BAL cells, there was no significant down regulation in SOD, CAT, or GPx activities in the lung lavage cells. These data suggest that augmentation of micronutrient antioxidants in smokers and nonsmokers does not appear to have an effect on antioxidant enzyme activities, suggesting a differential regulation of these defenses.

An Investigation of the response of lymphoblastoid cells to hydrogen peroxide-derived oxidative stress

The synthesis of heat shock proteins (HSPs) may play a significant role in the mechanism of cellular protection against oxidative injury [ I , 21. We have shown that T-lymphoid (Molt-3) cells exhibit enhanced susceptibility to H202-induced cytogenetic damage and that these cells also have low levels of intracellular catalase activity [3]. The aim of this study was to investigate the response of human T-lymphoid cells (Molt-3) to H202-derived oxidative stress. The endpoints we analysed were HSP synthesis and cellular antioxidant enzyme activities. Molt-3 cells were maintained in RPMI 1640 containing 10 % (v/v) FCS, 100 IU/ml streptomycin/penicillin and incubated at 37'C in 9 5 5 % air:CO2 humidified athmosphere. Previous experiments had shown that a dose of 10 pM H202 was non-toxic to Molt-3 cells and a dose of 100 pM H202 was cytotoxic. In the light of these findings, the following treatment groups were set up; control cells where no H202 was added; cells pretreated with a single dose of 10 pM H202, incubated for 18 h at 37 'C and then challenged with a 30 min exposure to 100 pM H202. cells were washed free of H202 and were then further incubated for 72h. Other treatment groups consisted of cells receiving pretreatment with 10 pM H202 only or receiving 100 pM H202 challenge only. Cell viability was determined by the Trypan-Blue exclusion test. Control and H202 treated cells were harvested at 72 h post challenge treatment and total cell lysates prepared by sonication. Cellular antioxidant assays were then performed for superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT), according to the methods described 14-61 respectively. To assess HSP synthesis treated cells were cultured in RPMI 1640 supplemented with 20 pCi/ml L-[35S]-methionine (specific activity > lo00 Ci/mmol), harvested and washed twice in PBS. After sonication total cell lysates were solubilized in 2% (w/v) SDS. 10% (v/v) glycerol, 62 mM Tris-HCI. pH 6.8. 5% (v/v) mercaptoethanol. 0.0025% (wh.) bromophenol blue. Samples were boiled and subjected to I-D SDS-PAGE. HSPs were identified by probing PVDF membranes with anti-Hsc/Hsp 70 (C92F3A-5) recognising both constitutive and inducible forms, anti Hsp-70 (C92F3A-5) inducible form only and anti-heme-oxygenase (HO1) (1:lOOO dilution, v/v), and detection was achieved with a reporter alkaline phosphatase (AP)-linked reporter antibody (1:3O,o00 dilution, v/v) and a AP staining kit. Table 1 compares the cellular antioxidant enzyme status of Molt-3 cells pretreated with 10 pM H202 with control groups. Following pretreatment there was a marked increase intracellular GPx activity i.e., 62 f 10.00 mU/mg protein (P< 0.05) compared to 38 f 8.8 mU mg protein for untreated cells. There was no significant alteration in the activities of SOD and CAT in pretreated and challenged groups. These results show that GPx activity increases in Molt-3 cells in response to a non-toxic H202 exposure. Table 1. lntracellular antioxidant enzyme activities in Molt-3 cells in culture following exposure to H202. Treatment SOD GPx . CAT Group (U/mg) (mU/mg) (Uhng)

A positive correlation between catalase activity and ascorbate uptake in the tissues of guinea pigs and cultured cells of mammals.

We recently reported that the concentration of supplemental ascorbate which inhibits cell growth is positively related to intracellular catalase activity. It is assumed that the cells with high catalase activity are resistant to high concentrations of ascorbate since catalase can decompose hydrogen peroxide (H2O2) induced by the auto-oxidation of ascorbate in cultured medium. In this study, we investigated whether intracellular catalase activity affects the uptake of ascorbate into animal tissue and cultured cells. Ascorbate concentrations in the tissues of guinea pigs and various cultured cells, with and without supplementation of ascorbate, were determined to evaluate the efficiency of ascorbate uptake. We found a positive correlation between the efficiency of ascorbate uptake and catalase activity in various tissues of guinea pigs (r = 0.767, p < 0.05). Furthermore, a positive correlation between the two was also found in various species of cultured cells. This study indicates that tissues and cells with higher efficiency of ascorbate uptake are required for higher catalase activity, presumably for the decomposition of H2O2 from ascorbate.

Catalase and hydrogen peroxide cytotoxicity in cultured cardiac myocytes

We examined the role of intracellular catalase activity in modulating hydrogen peroxide (H 2O 2)-induced cytotoxicity in cultured chick embryo cardiac myocytes. Injury was quantitated by release of lactate dehydrogenase (LDH). Application of 1.5 m m H 2O 2 to myocytes caused LDH release beginning at 2 h. Inactivation or inhibition of catalase with aminotriazole or sodium azide increased LDH release but did not cause earlier release. Free catalase which entered or became associated with myocytes, but not catalase bound to agarose beads, which did not enter or become associated with myocytes, was protective. Separate experiments demonstrated that myocyte catalase activity decreased by 27% between 1 and 4 h of H 2O 2 exposure. Treatment with aprotinin, a protease inhibitor, prevented the H 2O 2-induced fall in catalase activity at 4 h but treatment with deferoxamine, an iron chelator, had no effect on catalase activity. Thus, with exposure of cardiac myocytes to H 2O 2, the magnitude of the cytotoxicity is modulated by endogenous or cell associated exogenous catalase. It is proposed that in addition to excessive accumulation of H 2O 2, a reduction in intracellular catalase activity may be required before substantial cell injury occurs during H 2O 2 exposure. Activation of proteases may cause the reduction in catalase activity in this setting.

Autocrine production of extracellular catalase prevents apoptosis of the human CEM T-cell line in serum-free medium.

CCRF-CEM is a human T-cell line originally isolated from a child with acute lymphoblastic leukemia. At cell densities > 2 x 10(5) cells per ml, CEM cells grow in serum-free medium, but at lower cell densities the cultures rapidly undergo apoptosis, or programmed cell death. The viability of low-density CEM cells could be preserved by supplementing the serum-free medium with "conditioned" medium from high-density CEM cultures, but a variety of known growth factors and lymphokines were ineffective. Fractionation of conditioned medium by sequential chromatography on DEAE-cellulose, propyl agarose, chromatofocusing, and hydrophobic-interaction HPLC resulted in the isolation of a 60-kDa protein capable of sustaining CEM growth in the absence of serum. The active protein was identified as human catalase based on its amino acid sequence and composition and was subsequently shown to exhibit catalase activity and to be replaceable by human erythrocyte catalase or bovine liver catalase. Comparison of the level of intracellular catalase activity with the amount released into the culture medium demonstrated that the latter accounted for < 3% of the total catalase activity present in the cell culture. These findings show that, despite its low amount, the catalase released by CEM cells, and perhaps by T cells in general, provides a critical first line of defense against hydrogen peroxide (H2O2) present in the extracellular milieu.