The response of gram-negative,thermophilic bacteria to oxygen

Nine gram-negative, thermophilic bacteria (LEH-1, CC-6, BI-1, NR-9, K-2,Thermus aquaticus, T. thermophilus, T. sp. X-1, andThermomicrobium roseum) tolerated increased oxygen tensions (0.6–1.0 atm). All of the strains demonstrated catalase, peroxidase, and superoxide dismutase activity. Except forT. roseum, the specific activity of one or more of these enzymes was higher under aerated culture, as compared to static conditions. Polyacrylamide gel electrophoresis (PAGE) indicated that each organism possessed only one superoxide dismutase, and each was tentatively identified as having a manganese prosthetic group. PAGE also demonstrated that the number of isozymes of catalase varied from one to three, depending on the organism. Finally, the characterization of these enzymes supported previous tentative groupings of these thermophiles.     

Expression of antioxidant enzymes in rat kidney during ischemia-reperfusion injury

The effect of ischemia-reperfusion on activity, protein and m-RNA levels of catalase, copper-zinc and manganese containing superoxide dismutases and glutathione peroxidase, the enzymes that are involved in free radical detoxification was studied in rat kidney. Ischemia alone did not alter either the activities or protein levels of superoxide dismutase and glutathione peroxidase. However, catalase activity was found to be inhibited to 82% of control. The inhibition of catalase was due to the inactivation of the enzyme as there was no significant change in enzyme protein level. Reperfusion following ischemia, however, led to a significant decrease in both the activities as well as the protein levels of all the antioxidant enzymes. The observed overall decrease in total superoxide dismutase activity was the net effect of a decrease in copper-zinc superoxide dismutase while manganese superoxide dismutase activity was found to be increased following reperfusion. This observed increased manganese superoxide dismutase activity was the result of its increased protein level. The mRNA levels for catalase, superoxide dismutases, and glutathione peroxidase were observed to be increased (100–145% of controls) following ischemia; reperfusion of ischemic kidneys, however, resulted in a significant decrease in the levels of mRNAs coding for all the enzymes except manganese superoxide dismutase which remained high. These results suggest that in tissue, the down regulation of the antioxidant enzyme system could be responsible for the pathophysiology of ischemia-reperfusion injury.     

Mutations in Escherichia coli that effect sensitivity to oxygen

Fifteen oxygen-sensitive (Oxys) mutants of Escherichia coli were isolated after exposure to UV light. The mutants did not form macroscopic colonies when plated aerobically. They did form macroscopic colonies anaerobically. Oxygen, introduced during log phase, inhibited the growth of liquid cultures. The degree of inhibition was used to separate the mutants into three classes. Class I mutants did not grow after exposure to oxygen. Class II mutants were able to grow, but at a reduced rate and to a reduced final titer, when compared with the wild-type parent. Class III mutants formed filaments in response to oxygen. Genetic experiments indicated that the mutations map to six different chromosomal regions. The results of enzymatic assays indicated that 7 of the 10 class I mutants have low levels of catalase, peroxidase, superoxide dismutase, and respiratory enzymes when compared with the wild-type parent. Mutations in five of the seven class I mutants which have the low enzyme activities mapped within the region 8 to 13.5 min. P1 transduction data indicated that mutations in three of these five mutants, Oxys-6, Oxys-14, and Oxys-17, mapped to 8.4 min. The correlation of low enzyme levels and mapping data suggests that a single gene may regulate several enzymes in response to oxygen. The remaining three class I mutants had wild-type levels of catalase, peroxidase, and superoxide dismutase, but decreased respiratory activity. The class II and III mutants had enzyme activities similar to those of the wild-type parent. Our results demonstrate that mutations in at least six genes can be expressed as oxygen sensitivity. Some of these genes may be involved in respiration or cell division or may regulate the expression of several enzymes.     

Delayed ultraviolet light-induced cessation of respiration by inadequate aeration of Escherichia coli.

Inadequately aerated Escherichia coli B/r cultures did not shut their respiration off 60 min after ultraviolet light (52 M/m2 at 254 nm) as they did when well supplied with oxygen. Since cessation of respiaration is associated with cell death, the result suggested that oxygen toxicity by superoxide radicals generated by cell metabolism might be responsible for cell death. The specific activity of superoxide dismutase, which scavenges O2- radicals, increased twofold after 90 min of adequate aeration, but the specific activity of catalase remained constant. Respiration and viability of irradiated cells were affected not at all by the presence of superoxide dismutase and only slightly by the presence of catalase. Metal ions such as Mn2+ and Fe2+ inducers of superoxide dismutase, had no effect on respiration and viability. When irradiated cells were incubated under N2 for 90 min, the respiration, growth, and viability time-course responses were the same as for the cells not exposed to anareobiosis. We conclude that superoxide anions generated at the time of irradiation play no part in cessation delays the ultraviolet light-induced synthesis of proteins responsible for the irreversible cessation of respiration.     

Superoxide, hydrogen peroxide, and oxygen toxicity in two free-living nematode species

Two species of free-living nematodes, Turbatrix aceti and Caenorhabditis elegans, exhibited a marked sensitivity to 3 atm of 100% O2. Environmental changes in pH and temperature, which altered nematode respiration, resulted in alterations in the survival of these organisms under high pO2. Levels of defensive enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and dianisidine peroxidase were measured in the two species. No changes in the level of superoxide dismutase or catalase activity were induced by exposure of the nematodes to high pO2. Manipulation of these two enzymes was however achieved using the inhibitors 3-amino-1,2,4-triazole and diethyldithiocarbamate. 3-Amino-1,2,4-triazole (20 mM) eliminated greater than or equal to 80% of the catalase activity in vivo and diethyldithiocarbamate (5 mM) decreased the level of CuZn superoxide dismutase by greater than or equal to 70%. Both of these compounds increased the sensitivity of C. elegans to high pO2 toxicity. Compounds capable of intracellular redox-cycling with O2- -production, such as plumbagin, increased CN- -resistant respiration in the nematodes and imposed an O2-dependent toxicity. These experiments demonstrate the toxicity of intracellular O2- and H2O2 in nematodes and the importance of superoxide dismutase and catalase in providing a defense against these toxic molecules in vivo.     

Effect of flooding on the activities of some enzymes of activated oxygen metabolism,the levels of antioxidants,and lipid peroxidation in senescing tobacco leaves

Effects of flooding on the activities of some enzymes of activated oxygen metabolism, the levels of antioxidants, and lipid peroxidation in senescing leaves of tobacco were investigated. As judged by the decrease in chlorophyll and protein levels, flooding accelerated the senescence of tobacco leaves. Total peroxide and the lipid peroxidation product, malondialdehyde, increased in both control and flooding-treated leaves with increasing duration of the experiment. Throughout the duration of the experiment, flooded leaves had higher levels of total peroxide and malondialdehyde than did control leaves. Flooding resulted in an increase in peroxidase and ascorbate peroxidase activities and a reduction of superoxide dismutase activity in the senescing leaves. Glycolate oxidase, catalase, and glutathione reductase activities were not affected by flooding. Flooding increased the levels of total ascorbate and dehydroascorbate. Total glutathione, reduced form glutathione, or oxidized glutathione levels in flooded leaves were lower than in control leaves during the first two days of the experiment, but were higher than in control leaves at the later stage of the experiment. Our work suggests that senescence of tobacco induced by flooding may be a consequence of lipid peroxidation possibly controlled by superoxide dismutase activity. Our results also suggest that increased rates of hydrogen peroxide in leaves of flooded plants could lead to increased capacities of the scavenging system of hydrogen peroxide.Abbreviations GSH reduced form glutathione - GSSG oxidized form glutathione - GSSG reductase glutathione reductase - MDA malondialdehyde - SOD superoxide dismutase     

The response to oxidative stress of Fusobacterium nucleatum grown in continuous culture

Fusobacterium nucleatum ATCC 10953 was grown in continuous culture and the atmosphere changed stepwise from nitrogen (anaerobiosis) to a mixture of air: oxygen (40:60). No significant differences in biomass were observed and the baseline low level of superoxide dismutase increased only slightly; catalase and peroxidase activities were never detected but the level of NADH oxidase increased more than three-fold when oxygen was introduced into the system. In relation to acidic end-products, the relative proportion of acetate increased while that of butyrate decreased. Due mainly, it would seem, to NADH oxidase activity, the culture maintained a low redox potential (E(h)=-274 mV) even under an atmosphere of 40% oxygen in air and dissolved oxygen was not detected. This may, in part, explain the protective role of F. nucleatum for anaerobes in both biofilm and planktonic phases of aerated, mixed cultures of oral bacteria.     

Catalase, superoxide dismutase, and the production of O2-sensitive mutants of Bacillus coagulans

A number of facultatively anaerobic members of the genus Bacillus were screened for their catalase, diaminobenzidine peroxidase, and superoxide dismutase activities. A strain of Bacillus coagulans (7050) lacking peroxidatic activity and containing single catalatic and superoxide dismutase activities was selected. Responses of the superoxide dismutase activity and catalase level to the partial pressure of oxygen, and Fe and Mn levels, as well as to aerobic and fermentative metabolism, were determined. There appeared to be a relationship between high endogenous catalase levels and the high H2O2 evolution and KCN insensitivity of B. coagulans respiration. Bacillus coagulans 7050 was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and screened for the expression of oxygen intolerance. All of the 38 stable oxygen sensitive mutants obtained had very low or completely absent catalatic activity and catalase protein. No mutant lacked superoxide dismutase, although five showed significantly lowered levels of the enzyme. Exogenous bovine liver catalase restored aerotolerance and reduced cell pleomorphism in the mutants.     

Enzymatic defenses against the toxicity of oxygen and of streptonigrin in Escherichia coli.

Anaerobically grown Escherichia coli K-12 contain only one superoxide dismutase and that is the iron-containing isozyme found in the periplasmic space. Exposure to oxygen caused the induction of a manganese-containing superoxide dismutase and of another, previously undescribed, superoxide dismutase, as well as of catalase and peroxidase. These inductions differed in their responsiveness towards oxygen. Thus the very low levels of oxygen present in deep, static, aerobic cultures were enough for nearly maximal induction of the manganese-superoxide dismutase. In contrast, induction of the new superoxide dismutase, catalase, and peroxidase required the much higher levels of oxygen achieved in vigorously agitated aerobic cultures. Anaerobically grown cells showed a much greater oxygen enhancement of the lethality of streptonigrin than did aerobically grown cells, in accord with the proposal that streptonigrin can serve as an intracellular source of superoxide. Anaerobically grown cells in which enzyme inductions were prevented by puromycin were damaged by exposure to air. This damage was evidenced both as a decline in viable cell count and as structural abnormalities evident under an electron microscope.     

Factors Related to the Oxygen Tolerance of Anaerobic Bacteria

The effect of atmospheric oxygen on the viability of 13 strains of anaerobic bacteria, two strains of facultative bacteria, and one aerobic organism was examined. There were great variations in oxygen tolerance among the bacteria. All facultative bacteria survived more than 72 h of exposure to atmospheric oxygen. The survival time for anaerobes ranged from less than 45 min for Peptostreptococcus anaerobius to more than 72 h for two Clostridium perfringens strains. An effort was made to relate the degree of oxygen tolerance to the activities of superoxide dismutase, catalase, and peroxidases in cell-free extracts of the bacteria. All facultative bacteria and a number of anaerobic bacteria possessed superoxide dismutase. There was a correlation between superoxide dismutase activity and oxygen tolerance, but there were notable exceptions. Polyacrylamide gel electropherograms stained for superoxide dismutase indicated that many of the anaerobic bacteria contained at least two electrophoretically distinct enzymes with superoxide dismutase activity. All facultative bacteria contained peroxidase, whereas none of the anaerobic bacteria possessed measurable amounts of this enzyme. Catalase activity was variable among the bacteria and showed no relationship to oxygen tolerance. The ability of the bacteria to reduce oxygen was also examined and related to enzyme content and oxygen tolerance. In general, organisms that survived for relatively long periods of time in the presence of oxygen but demonstrated little superoxide dismutase activity reduced little oxygen. The effects of medium composition and conditions of growth were examined for their influence on the level of the three enzymes. Bacteria grown on the surface of an enriched blood agar medium generally had more enzyme activity than bacteria grown in a liquid medium. The data indicate that superoxide dismutase activity and oxygen reduction rates are important determinants related to the tolerance of anaerobic bacteria to oxygen.     

Effects of combined quercetin and coenzyme Q(10) treatment on oxidative stress in normal and diabetic rats

Reactive oxygen species may be actively involved in the genesis of various pathological states such as ischemia-reperfusion injury, cancer, and diabetes. Our objective was to determine if subacute treatment with combined antioxidants quercetin and coenzyme Q(10) (10 mg/kg/day ip for 14 days) affects the activities of antioxidant enzymes in normal and 30-day streptozotocin-induced diabetic Sprague-Dawley rats. Quercetin treatment raised blood glucose concentrations in normal and diabetic rats, whereas treatment with coenzyme Q(10) did not. Liver, kidney, heart, and brain tissues were excised and the activities of catalase, glutathione reductase, glutathione peroxidase, superoxide dismutase, and concentrations of oxidized and reduced glutathione were determined. In the liver of diabetic rats, superoxide dismutase, glutathione peroxidase, and levels of both oxidized and reduced glutathione were significantly decreased from the nondiabetic control, and these effects were not reversed when antioxidants were administered. In kidney, glutathione peroxidase activity was significantly elevated in the diabetic rats as compared to nondiabetic rats, and antioxidant treatment did not return the enzyme activity to nondiabetic levels. In heart, catalase activity was increased in diabetic animals and restored to normal levels after combined treatment with quercetin and coenzyme Q(10). Cardiac superoxide dismutase was lower than normal in quercetin- and quercetin + coenzyme Q(10)-treated diabetic rats. There were no adverse effects on oxidative stress markers after treatment with quercetin or coenzyme Q(10) singly or in combination. In spite of the elevation of glucose, quercetin may be effective in reversing some effects of diabetes, but the combination of quercetin + coenzyme Q(10) did not increase effectiveness in reversing effects of diabetes.     

Hepatoprotective and antioxidant property of Andrographis paniculata (Nees) in BHC induced liver damage in mice

Andrographis paniculata (AP) treatment prevents BHC induced increase in the activities of enzymes y-Glutamyl transpeptidase, glutathione-S-transferase and lipid peroxidation. The activities of antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase and the levels of glutathione were decreased following BHC effect. Administration of AP showed protective effects in the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase as well the level of glutathione. The activity of lipid peroxidase was also decreased. The result indicate antioxidant and hepatoprotective action of A. paniculata.     

Superoxide radicals and antiradical defence of propionic acid bacteria

Superoxide dismutase activity was demonstrated for 6 strains of 3 propionibacteria species. Rather high level of superoxide dismutase activity found in propionibacteria was in accordance with high level of catalase activity reported for propionibacteria previously. Both these activities were shown to have cytozolic localization. For the first time peroxidase activity was detected in gel-fractionated crude cell extracts of propionibacteria. The ability to produce superoxide radicals in NADH-dependent oxidation system was revealed for three strains of the bacteria. The level of superoxide production by the membrane particles of the propionic acid bacteria correlated with the levels of superoxide dismutase and catalase activities and was the lowest for Propionibacterium shermanii. The ability to perform monovalent oxygen reduction during succinate oxidation was not revealed. The intact cells of P. globosum, P. vannielii, P. shermanii apparently did not excrete superoxide radicals into culture fluid during respiration.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Superoxide dismutase and peroxidase are coordinately regulated in differentiated and transformed tissues of Nicotiana tabacum

We used a series of normal and Agrobacterium-transformed, bacteria-free tobacco tissue cultures which differ in their levels of histodifferentiation to test the relationship of superoxide dismutase, peroxidase, and catalase to oncogenic transformation and differentiation. When compared with normal callus, tumor callus contained reduced levels of both superoxide dismutase and peroxidase, and a reduced number of isozymes of both enzymes. Teratomas characterized by limited but abnormal differentiation showed increases in superoxide-dismutase activity and isozymes, but levels of peroxidase activity lower than those found in normal callus despite an increase in the number of peroxidase isozymes. A similar disparity between low peroxidase activity and high isozyme number in the shoot suggests that there are increased levels of peroxidase inhibitors or of molecules which interfere with the spectrophotometric assay for peroxidase in more differentiated tissues. As judged by the number of isozymes of both superoxide dismutase and peroxidase in each tissue, the following conclusions are warranted: first, tobacco copper/zinc superoxide dismutases and peroxidases are encoded in several duplicated loci which are regulated independently. Second, transformation is associated with a decrease in both the specific activity and isozyme number of superoxide dismutase. Third, the partial release from the total inhibition of expression of differentiated function exhibited by teratoma is associated with an increase in both the activity and isozyme number of superoxide dismutase. Finally, the expression of superoxide dismutase and peroxidase isozymes appears to be coordinated during differentiation in a manner that is consistent with their role in an integrated mechanism for the removal of reduced oxygen species.     

Biosynthesis of oxygen-detoxifying enzymes in Bdellovibrio stolpii.

Axenically grown Bdellovibrio stolpii (i.e., grown independently of the host) was examined for superoxide dismutase, catalase, and peroxidase activities. Kinetics of enzyme synthesis were determined for aerobically grown cultures and for cultures exposed to 100% oxygen. Enzymatic activities varied with the age of the culture. Normally grown cultures exhibited maximum activity during the first 10 h of growth and again as the stationary phase was approached, beginning at about 48 h. Polyacrylamide gel electropherograms of cell-free extracts revealed that B. stolpii contained one major band (1) and two minor bands (II, III) of superoxide dismutase activity. Each of these enzymes was inactivated by H2O2, indicating that they were iron-containing enzymes. Manganese-containing superoxide dismutase was not detected in B. stolpii. Increased oxygenation did not appreciably stimulate enzyme synthesis, for only superoxide dismutase was induced, reaching maximum activity at 10 h and then rapidly falling to normal levels. Superoxide dismutase appears to be the main enzymatic defense against oxygen toxicity in B. stolpii. Induction of superoxide dismutase with 100% oxygen was manifested as an increase in the intensities of the two minor bands of activity, suggesting that isozyme I is constitutive, whereas isozymes II and III are inducible. The induction of isozymes II and III by 100% oxygen was prevented by an inhibitor of protein biosynthesis, chloramphenicol.     

Leishmanial superoxide dismutase: A possible target for chemotherapy

Leishmania tropica promastigotes stimulate macrophages to produce activated oxygen as measured by luminol-enhanced chemiluminescence. Exogenous superoxide dismutase and catalase inhibit this by 95%, implying that both superoxide and hydrogen peroxide are generated. Whereas leishmania have undetectable levels of catalase, and very little glutathione peroxidase, they have relatively high amcunts of superoxide dismutase (23 units/mg protein). The leishmanial superoxide dismutase is cyanide-insensitive but azide- and peroxide-sensitive, suggesting that the enzyme may be iron-containing. Furthermore, the leishmanial superoxide dismutase is insensitive to diethyldithiocarbamate, which inhibits vertebrate enzymes. Thus, leishmania may contain a superoxide dismutase which is different from its host's enzyme. A specific inhibitor of this enzyme might serve as an antileishmanial agent.     

Catalase, glutathione peroxidase, and superoxide dismutase in the rete mirabile and gas gland epithelium of six species of marine fishes

Rete mirabile and gas gland epithelium from the swim bladders of six species of marine fishes were assayed for catalase, glutathione peroxidase, and superoxide dismutase activity. Correlation of the results of these assays with measurements of the concentration of oxygen in the lumen of the normal steady state swim bladders revealed that swim bladders in species containing higher levels of oxygen also exhibited higher levels of superoxide dismutase activity in the rete mirabile/gas gland epithelium region. There appeared to be no correlation between oxygen concentration and the level of catalase or glutathione peroxidase activity. Induction of the inflatory reflex in Opsanus tau by a single deflation of the swim bladder resulted in an increase in the percent of oxygen in the swim bladder lumen 18 to 24 hours later, but this was not accompanied by any significant increases in antioxidant enzyme activity. Swim bladders that were deflated three times at 24-hour intervals showed further increases in oxygen concentration at the end of the 72-hour period but no alteration in superoxide dismutase activity.     

Metabolic responses of Lemna minor to lead ions II. Induction of antioxidant enzymes in roots

Duckweed Lemna minor L. was grown on Wang culture medium supplemented with lead ions for 24 hours. Metal was tested at 1.5, 3 and 6 mg·dm⁻³ concentrations. The response of antioxidant enzymes, such as superoxide dismutase, catalase and peroxidase in lead-treated roots of duckweed was investigated. Lead ions had no effect on the spectrum of catalase and peroxidase isoenzymes while a new isoform of superoxide dismutase appeared on the Pb treated roots. A lead-depended increase in activities of superoxide dismutase and peroxidase was observed, whereas catalase activity was maintained at relatively constant values at lower lead concentrations and then decreased markedly below control level.