Oxygen supply to bacterial suspensions of high cell densities by hydrogen peroxide

The supply of heterotrophically growing suspensions of Alcaligenes eutrophus PHB^?4 with oxygen formed by the continuous addition of H₂O₂ in the presence of bovine liver catalase was found to be restricted to well-defined conditions. The catalase-H₂O₂ system proved to be suitable during the growth at low cell densities equivalent to 2 g dry weight/liter. When under these conditions the oxygen concentration was held constant at 1.8 mg O₂/liter, the cells grew for 6–8 hr at a rate almost identical to that observed with conventional aeration. However, aeration with H₂O₂ for longer durations (10–20 hr) and at higher cell densities (5?20 g dry weight/liter) led invariably to cell damage and retardation of growth. The impairment of growth observed during the oxygen supply by the catalase?H₂O₂ system was traced back to the formation of gradually increasing steady-state concentrations of H₂O₂ in the medium. Possible sites of cell damage by H₂O₂ such as membrane function, excretion and function of siderophores, and synthesis of cell polymers have been studied, and the cytotoxic mechanism of low concentrations of H₂O₂ was discussed.     

Efficiency of bovine liver catalase as a catalyst to cleave H2O2 added continually to buffer solutions

Empirical estimations of H₂O₂ concentration in a system containing bovine liver catalase and continually supplied with H₂O₂ were done to evaluate the efficiency of the enzyme to cleave H₂O₂. It was found that the continuous addition of H₂O₂ leads to the formation of steady-state concentrations of H₂O₂ in the medium. At a constant catalase concentration both the level and the duration of the steady state are dependent on the flow rate of H₂O₂. The increase of the catalase concentration in the medium does not change the steady-state level, it merely leads to the maintenance of the steady state for longer durations. At higher flow rates of H₂O₂, no steady state could be maintained, even when catalase was present in high excess. The incomplete cleavage of H₂O₂ by catalase under these conditions is due to the low affinity of catalase toward H₂O₂ (high K_m value, apparent K_m = 0.1M H₂O₂) and to the rapid inactivation of the enzyme during the continuous addition of H₂O₂.     

Factors determining the degree of anaerobiosis of Bifidobacterium strains

Summary The degree of sensitivity of twelve Bifidobacterium (Lactobacillus bifidus) strains to O₂ was determined by measuring the size of the inhibition zones obtained when the bacteria were grown in deep agar cultures under air, and by measuring growth in aerated cultures. The size of the inhibition zones varied from 1 to 23 mm. Growth in aerated cultures differed markedly for the strains investigated. No strain grew on agar plates under aerobic conditions.The small inhibition zone of three Bifidobacterium strains might be explained by the presence of a weak catalase activity, which removes traces of H₂O₂ possibly formed. It is also possible that the NADH oxidase of these strains does not form H₂O₂ at all. Most probably, the lack of growth on an agar medium results from the fact that these strains only grow below a certain oxidation-reduction potential.One strain, which was rather insensitive to O₂, formed a small amount of H₂O₂ from NADH oxidation. The absence of H₂O₂ in aerated liquid cultures and cell suspensions of this strain, which lacked catalase and NAD peroxidase activity, must be explained by removal of the traces of H₂O₂ formed, by an unknown peroxidase system or by a chemical reaction with pyruvate formed during glucose fermentation.For two strains, which were moderately sensitive to O₂, accumulation of H₂O₂ seems to be the principal reason for anaerobiosis. H₂O₂ turned out to inactivate specifically fructose-6-phosphate phosphoketolase, a key enzyme of the fermentation pathway of bifidobacteria.In the culture medium of two strains, which were extremely sensitive to O₂, no H₂O₂ could be detected after aeration. During anaerobic growth of these strains, the oxidation-reduction potential of the culture decreased so much that neutral red was decolourized. Cell suspensions of these strains only fermented glucose when cysteine was added. It was concluded that these strains required a low oxidation-reduction potential for growth and fermentation.     

Clonal growth of lymphoid cells in serum-free media requires elimination of H2O2 toxicity

We have recently described the development of a serum-free medium that contains casein, insulin, testosterone, transferrin, and linoleic acid and that supports the long-term growth of a wide variety of lymphoid cells. A problem of culturing cells in this medium is the difficulty of cloning cells or growing cells at low density. We now describe the formulation of a chemically defined medium that supports the clonal growth of the murine S49 T lymphoma cell line. This medium contains catalase, insulin, transferrin, testosterone, Na₂SeO₃, and dilinoleoyl phosphatidylcholine and contains less than 50 μg/ml total protein. The two novel additions in this medium are catalase, which replaces casein and dilinoleoyl phosphatidylcholine, which substitutes for linoleic acid in this defined medium. In addition to S49 cells, the medium described above supports the long-term growth of other lymphoid cells, including human and murine hybridomas. We propose that catalase functions to degrade H₂O₂ that is present in the cultures and that casein, bovine serum albumin, and other proteins commonly included in media for cultured cells may also scavenge H₂O₂. Na₂SeO₃ also partially protects against the death of cells at clonal density and this protection may, like catalase, be due to removal of H₂O₂. Our results suggest that H₂O₂ is an important cytotoxic agent that prevents growth of lymphoid cells during culture in serum-free media and perhaps in serum-containing media as well.     

Transposon-Induced Catalase-Deficient Agrobacterium tumefaciens

Agrobacterium tumefaciens MKR, a nonpathogenic strain, has three catalase isozymes and one superoxide dismutase but no detectable peroxidase activity. A large number (8400) of transconjugants were obtained with pSUP1011::Tn5 suicide vector. The transposition frequencies were found to be greater in biparental mating than in triparental mating with helper plasmid. Mutants MLA31, MLA32, MLA41, and MLA41(a), generated by transposon mutagenesis, all lacked one of the catalase isozymes. Mutants were more susceptible to cell death than the wild type upon direct exposure to 10.0 mmol L⁻¹ H₂O₂. The specific activity of the enzyme catalase was found to be higher in nitrogen-rich growth medium than carbon-rich growth medium. Received: 28 January 1997 / Accepted: 25 February 1997     

Hydrogen Peroxide Metabolism in Yeasts

A catalase-negative mutant of the yeast Hansenula polymorpha consumed methanol in the presence of glucose when the organism was grown in carbon-limited chemostat cultures. The organism was apparently able to decompose the H₂O₂ generated in the oxidation of methanol by alcohol oxidase. Not only H₂O₂ generated intracellularly but also H₂O₂ added extracellularly was effectively destroyed by the catalase-negative mutant. From the rate of H₂O₂ consumption during growth in chemostat cultures on mixtures of glucose and H₂O₂, it appeared that the mutant was capable of decomposing H₂O₂ at a rate as high as 8 mmol · g of cells⁻¹ · h⁻¹. Glutathione peroxidase (EC 1.11.1.9) was absent under all growth conditions. However, cytochrome c peroxidase (CCP; EC 1.11.1.5) increased to very high levels in cells which decomposed H₂O₂. When wild-type H. polymorpha was grown on mixtures of glucose and methanol, the CCP level was independent of the rate of methanol utilization, whereas the level of catalase increased with increasing amounts of methanol in the substrate feed. Also, the wild type decomposed H₂O₂ at a high rate when cells were grown on mixtures of glucose and H₂O₂. In this case, an increase of both CCP and catalase was observed. When Saccharomyces cerevisiae was grown on mixtures of glucose and H₂O₂, the level of catalase remained low, but CCP increased with increasing rates of H₂O₂ utilization. From these observations and an analysis of cell yields under the various conditions, two conclusions can be drawn. (i) CCP is a key enzyme of H₂O₂ detoxification in yeasts. (ii) Catalase can effectively compete with mitochondrial CCP for hydrogen peroxide only if hydrogen peroxide is generated at the site where catalase is located, namely in the peroxisomes.     

Establishment and cellular characteristics of a hepatocyte cell line (oums-31) derived from an acatalasemic mouse

Summary Liver cell lines with very low catalase activity were established from an acatalasemic mouse. Hepatocytes isolated by a collagenase-liver-perfusion technique were cultured in Williams’ E medium supplemented with 10% fetal bovine serum. The acatalasemic liver cell line showed approximately 20% of the catalase activity of a normal mouse liver cell line, whereas its glutathione peroxidase activity was approximately equal to that of the normal liver cell line. DNA sequence analysis of this cell line showed the same mutation in the catalase gene as is seen in the acatalasemic mouse. Our observation of intracellular content of hydrogen peroxide (H₂O₂) radical and increased susceptibility of the cells to H₂O₂ were compatible with the existence of low catalase activity in the acatalasemic mouse. This hepatocyte cell line should be useful for studying effects of oxidative radical stress at the cellular level.     

Mechanisms of cell death induction by L-amino acid oxidase, a major component of ophidian venom

L-amino acid oxidase (LAAO) from the Malayan pit viper induces both necrosis and apoptosis in Jurkat cells. Cell death by necrosis is attributed to H₂O₂ produced by oxidation of α-amino acids. In the presence of catalase that effectively scavenges H₂O_2, a switch to apoptosis is observed. The major factors contributing to apoptosis are proposed to be: (i) generation of toxic intermediates from fetal calf serum (ii) binding and internalization of LAAO. The latter process appears to be mediated by the glycan moiety of the enzyme as desialylation reduces cytotoxicity. D-amino acid oxidase (DAAO), which catalyzes the same reaction as LAAO but lacks glycosylation, triggers necrosis as a consequence of H₂O₂ production but not apoptosis in the presence of catalase. Thus induction of cell death by LAAO appears to involve both the generation of H₂O₂ and the molecular interaction of the glycan moiety of the enzyme with structures at the cell surface. S. R. Ande, P. R. Kommoju contributed equally to this work.     

A Mechanism of Resistance to Hydrogen Peroxide in Vibrio rumoiensis S-1

A possible mechanism of resistance to hydrogen peroxide (H₂O₂) in Vibrio rumoiensis, isolated from the H₂O₂-rich drain pool of a fish processing plant, was examined. When V. rumoiensis cells were inoculated into medium containing either 5 mM or no H₂O₂, they grew in similar manners. A spontaneous mutant strain, S-4, derived from V. rumoiensis and lacking catalase activity did not grow at all in the presence of 5 mM H₂O₂. These results suggest that catalase is inevitably involved in the resistance and survival of V. rumoiensis in the presence of H₂O₂. Catalase activity was constitutively present in V. rumoiensis cells grown in the absence of H₂O₂, and its occurrence was dependent on the age of the cells, a characteristic which is observed for the HP II-type catalase of Escherichia coli. The presence of the HP II-type catalase in V. rumoiensis cells was evidenced by partial sequencing of the gene encoding the HP II-type catalase from this organism. A notable difference between V. rumoiensis and E. coli is that catalase is accumulated at very high levels (~2% of the total soluble proteins) in V. rumoiensis, in contrast to the case for E. coli. When V. rumoiensis cells which had been exposed to 5 mM H₂O₂ were centrifuged, most intracellular proteins, including catalase, were recovered in the medium. On the other hand, when V. rumoiensis cells were grown on plates containing various concentrations of H₂O₂, individual cells had a colony-forming ability inferior to those of E. coli, Bacillus subtilis, and Vibrio parahaemolyticus. Thus, it is suggested that when V. rumoiensis cells are exposed to high concentrations of H₂O₂, most cells will immediately be broken by H₂O₂. In addition, the cells which have had little or no damage will start to grow in a medium where almost all H₂O₂ has been decomposed by the catalase released from broken cells.     

Growth-Dependent Catalase Localization in Exiguobacterium oxidotolerans T-2-2T Reflected by Catalase Activity of Cells

A psychrotolerant and H₂O₂-resistant bacterium, Exiguobacterium oxidotolerans T-2-2^T, exhibits extraordinary H₂O₂ resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state.     

Response of the Microaerophilic Bifidobacterium Species, B. boum and B. thermophilum, to Oxygen

We investigated the effects of O₂ on Bifidobacterium species using liquid shaking cultures under various O₂ concentrations. Although most of the Bifidobacterium species we selected showed O₂ sensitivity, two species, B. boum and B. thermophilum, demonstrated microaerophilic profiles. The growth of B. bifidum and B. longum was inhibited under high-O₂ conditions accompanied by the accumulation of H₂O₂ in the medium, and growth was restored by adding catalase to the medium. B. boum and B. thermophilum grew well even under 20% O₂ conditions without H₂O₂ accumulation, and growth was stimulated compared to anoxic growth. H₂O-forming NADH oxidase activities were detected dominantly in cell extracts of B. boum and B. thermophilum under acidic reaction conditions (pH 5.0 to 6.0).     

Identification of a soluble protein stimulator of plasmalogen biosynthesis and stearoyl-coenzyme a desaturase

Stimulation of the desaturation of 1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine (GPE), which forms ethanolamine plasmalogens, by a component of the 105,000g supernatant has been previously reported. We have isolated the stimulatory protein and identified it as catalase. Purified rat liver catalase or commercial bovine liver catalase is as effective in stimulating microsomal 1-alkyl-2-acyl-GPE desaturation as the soluble proteins. The stimulatory effect of these proteins is eliminated by catalase inhibitors. It appears that catalase stimulates the desaturation of 1-alkyl-2-acyl-GPE by preventing inactivation of the enzyme system by H₂O₂ or a decomposition product of H₂O₂. The cytochrome b₅ content and NADH oxidation are depressed in Fischer R-3259 sarcoma microsomes by H₂O₂; this effect is eliminated by catalase. However, since measurable inhibition of 1-alkyl-2-acyl-GPE desaturase by H₂O₂ still occurred in the presence of catalase, the inhibition by H₂O₂ cannot be explained solely on the basis of cytochrome b₅ inactivation. The desaturation of stearoyl-coenzyme A, a reaction analogous in many respects to 1-alkyl-2-acyl-GPE desaturation, was also found to be stimulated by catalase.     

Relationship between changes in ploidy and stable cellular resistance to hydrogen peroxide

Stable hydrogen peroxide (H₂O₂)-resistant variants of the Chinese hamster ovary HA-1 line have been isolated by culturing cells in progressively increasing concentrations of H₂O₂ (>200 days, in 50–800 μM H₂O₂). Increases in catalase activity in these variant cell lines were shown to correlate with increased H₂O₂ resistance. Stable (>240 days) H₂O₂-resistant cell lines, seven quasidiploid (21–22 chromosomes/cell) and six quasitetraploid (40–44 chromosomes/cell) were clonally isolated from the 800 μM adapted H₂O₂-resistant variants which were heterogeneous with respect to ploidy. The H₂O₂ dose-modifying factors (DMFs) were 3, 5, 8, 13, 15, 26, and 27 for the seven quasidiploid cell lines, and 21, 32, 38, 40, 42, and 49 for the six quasitetraploid cell lines. The mean DMF was 14±10 for the former and 37±10 for the latter. Our data show that on the average the quasitetraploid cell lines were significantly more resistant to H₂O₂-mediated cell killing than the quasidiploid cell lines derived from the same mixed population of 800 μM H₂O₂-adapted cells. When catalase activities (k units/cell) of the HA-1 cells and three of the clonally derived cell lines (two quasidiploid and one quasitetraploid) were determined and plotted vs. H₂O₂–DMF, a positive linear correlation was obtained (correlation coefficient = 0.99). This result was further confirmed when immunoreactive catalase protein/cell was detected by Western blots. Our data show that chronic exposure of cells to H₂O₂ stress (800 μM) was accompanied by increases in quasitetraploid cells within the population. Quasitetraploid cell lines derived from this population demonstrated increased stable H₂O₂-resistance which may be related to stable increases in the expression of catalase.     

Selective cytochemical localization of peroxidase,cytochrome oxidase and catalase in rat liver with 3,3′-diaminobenzidine

Summary In rat liver, three different enzymes with peroxidatic activity are demonstrated with modifications of the DAB-technique: peroxidase in the endoplasmic reticulum of Kupffer cells, catalase in peroxisomes and cytochrome oxidase in mitochondria. The major problem of the DAB-methods is their limited specifity so that often in tissues incubated for one enzyme the other two proteins are also stained simultaneously. We have studied the conditions for selective staining of each of these three enzymes in rat liver fixed either by perfusion with glutaraldehyde or by immersion in a modified Karnovsky's glutaraldehyde-formaldehyde fixative. The observations indicate that in perfusion fixed material selective staining can be obtained by reduction of the incubation time (5 min) and the use of optimal conditions for each enzyme. In livers fixed by immersion the distribution of the staining is patchy and irregular and usually longer incubation times (15–30 min) are required. Selective staining of peroxidase in Kupffer cells was obtained by brief incubation at room temperature in a medium containing 2.5 mM DAB in cacodylate buffer pH 6.5 and 0.02% H₂O₂. The exclusive staining for cytochrome oxidase in cristae of mitochondria was achieved after short incubation in 2.5 mM DAB in phosphate buffer pH 7.2 containing 0.05% cytochrome c. For selective demonstration of catalase in peroxisomes the tissue was incubated in 5 mM DAB in Teorell-Stenhagen (or glycine-NaOH) butffer at pH 10.5 and 0.15% H₂O₂. The prolongation of the incubation time in peroxidase medium caused marked staining of both mitochondria and peroxisomes. In the cytochrome oxidase medium longer incubations led to slight staining of peroxisomes. The catalase medium was quite selective for this enzyme so that even after incubation for 120 min only peroxisomes stained.     

The effects of disruption of genes for peroxiredoxin-2, glutathione peroxidase-1, and catalase on erythrocyte oxidative metabolism

Peroxiredoxin-2 (Prdx2), a potent peroxide reductant, is the third most abundant protein in the erythrocyte and might be expected to play a major role in the cell's oxidative defenses. However, in this study, experiments with erythrocytes from mice with a disrupted Prdx2 gene found that the cells were not more sensitive to exogenous H₂O₂ or organic peroxides than wild type. Intraerythrocytic H₂O₂ was increased, however, indicating an important role for Prdx2 in detoxifying endogenously generated H₂O₂. These results are consistent with proposals that red cell Prdx2 acts stoichiometrically, not catalytically, in reducing peroxides. Additional experiments with mice with disrupted catalase or glutathione peroxidase (Gpx1) genes showed that Gpx1 is the only erythrocyte enzyme that reduces organic peroxides. Catalase^?/? cells were readily oxidized by exogenous H₂O₂. Cells lacking both catalase and Gpx1 were more sensitive to exogenous H₂O₂ than cells lacking only catalase. A kinetic model proposed earlier to rationalize results with Gpx1^?/? erythrocytes also fits the data with Prdx2^?/? cells and indicates that although Gpx1 and Prdx2 both participate in removing endogenous H₂O₂, Prdx2 plays a larger role. Although the rate of H₂O₂ production in the red cell is quite low, Prdx2-deficient mice are anemic, suggesting an important role in erythropoiesis.     

Identification of hydrogen peroxide as a photoproduct toxic to human cells in tissue-culture medium irradiated with “daylight” fluorescent light

Summary Hydrogen peroxide, lethal for human cells, is produced in Dulbecco's modified Eagle's tissue culture medium when exposed to “daylight” fluorescent light. Addition of pure H₂O₂ and use of the enzyme catalase demonstrate that about 40% of the toxicity in irradiated medium is due to generated peroxide. Riboflavin and tryptophan, or riboflavin and tyrosine, are the components necessary for formation of lethal levels of H₂O₂ during light exposure. Supported by an American Cancer Society Research Grant and a Public Health Service Research Career Development Award to Richard J. Wang.     

Protection of Bacillus larvae from Oxygen Toxicity with Emphasis on the Role of Catalase

Sporulation of Bacillus larvae NRRL B-3650 occurred only at aeration rates lower than those used for cultivation of most Bacillus species. One possible explanation for the requirement for a low level of aeration in B. larvae is that toxic forms of oxygen such as H₂O₂ and superoxide are involved. The superoxide dismutase levels of strain B-3650 were similar to those of Bacillus subtilis 168 during sporulation, and no NADH peroxidase was present. Catalase activity was absent during exponential growth and first appeared near the start of the stationary phase. The catalase activity was 2,700 times less than that in B. subtilis 168 at the same stage of development. Therefore, the relative deficiency of catalase (and NADH peroxidase) might be the cause of the apparent O₂ toxicity. It was postulated that B. larvae might accumulate H₂O₂ in the medium and exhibit more than normal sensitivity to H₂O₂. Experimental results did not verify either postulate, but the possibilities of intracellular accumulation of H₂O₂ and unusual sensitivity to endogenous H₂O₂ were not excluded. The catalase present in early-stationary-phase cells was soluble, heat labile, and inhibited by cyanide, azide, and hydroxylamine. An increase in catalase activity also occurred at the time of appearance of refractile spores in both B. larvae NRRL B-3650 and B. subtilis 168. The level of catalase activity in strain B-3650 was 5,400 times less than that in B. subtilis 168 at this stage. In B. larvae, this second increase occurred primarily within the developing endospore. The activity in spore extracts was particulate, heat stable, and inhibited by hydroxylamine but not by azide or cyanide. Synthesis of catalase in B. larvae was unaffected by H₂O₂, O₂, or glucose.     

The relationship of hydrogen peroxide to the inhibition of the glyoxalase activity of intact erythrocytes by x-radiation

The x-irradiation of a dilute suspension of erythrocytes results in a decrease in the glyoxalase activity of the cells as a result of a fall in the reduced glutathione level. The present paper deals with the possible role of H₂O₂ in this reaction. The addition of intact erythrocytes to physiological saline previously irradiated with 150,000 r or 225,000 r results in a fall in the glyoxalase activity of the cells. The inhibition is prevented by the preincubation of the irradiated saline with catalase and is reversed by the addition of plasma, glucose, adenosine, and inosine to the cell suspension. An inhibition of the glyoxalase activity is also produced by the addition of H₂O₂ to the suspension of erythrocytes. The inhibitory effect of H₂O₂ can be prevented and largely reversed by plasma, glucose, adenosine, and inosine. Methylglyoxal is also protective under these conditions. Hydrogen peroxide formed continuously and in low concentrations by enzyme systems appears to be more effective than added H₂O₂ in inhibiting the glyoxalase system. The inhibition by H₂O₂-producing enzyme systems is minimized by the addition of catalase, plasma, glucose, methylglyoxal, and to a lesser extent, by adenosine and inosine, and is accentuated by the addition of sodium azide. The results are discussed in relation to the role of H₂O₂ and catalase in the toxicity of ionizing radiations.