Antioxygenic Enzymes Research Articles

Spermidine alleviates the growth of saline-stressed ginseng seedlings through antioxidative defense system

Protective effects of exogenous spermidine (Spd), activity of antioxygenic enzymes, and levels of free radicals in a well-known medicinal plant, Panax ginseng was examined. Seedlings grown in salinized nutrient solution (150mM NaCl) for 7d exhibited reduced relative water content, plant growth, increased free radicals, and showing elevated lipid peroxidation. Application of Spd (0.01, 0.1, and 1mM) to the salinized nutrient solution showed increased plant growth by preventing chlorophyll degradation and increasing PA levels, as well as antioxidant enzymes such as CAT, APX, and GPX activity in the seedlings of ginseng. During salinity stress, Spd was effective for lowering the accumulation of putrescine (Put), with a significant increase in the spermidine (Spd) and spermine (Spm) levels in the ginseng seedlings. A decline in the Put level ran parallel to the higher accumulation of proline (Pro), and exogenous Spd also resulted in the alleviation of Pro content under salinity. Hydrogen peroxide (H2O2) and superoxide (O2−) production rates were also reduced in stressed plants after Spd treatment. Furthermore, the combined effect of Spd and salt led to a significant increase in diamine oxidase (DAO), and subsequent decline in polyamine oxidase (PAO). These positive effects were observed in 0.1 and 1mM Spd concentrations, but a lower concentration (0.01mM) had a very limited effect. In summary, application of exogenous Spd could enhance salt tolerance of P. ginseng by enhancing the activities of enzyme scavenging system, which influence the intensity of oxidative stress.

Alleviation of oxidative stress induced by spider mite invasion through application of elicitors in bean plants

Spider mite invasion induces oxidative stress on bean plants and increased soluble sugars, phenole, proline and peroxidase activity, but decreased catalase activity and ascorbic acid and carotenoid concentration. Application of elicitors significantly enhanced spider mite tolerance by decreasing hydrogen peroxide, malondialdehyde, electrolyte leakage, and increasing peroxidase activity and antioxidant compounds reported previously, leading to increasing ion percentage in plant shoot. This finding suggests that elicitors might be activating antioxygenic enzymes and elevating antioxidants there by controlling free radical generation, hence preventing membrane peroxidation and denaturation of biomolecules resulting into improving plant growth. Keywords: Tetranychus urticae, Phaseolus vulgaris , salicylate, methyl jasmonate, antioxidants.

Putrescine alleviation of growth in salt stressed Brassica juncea by inducing antioxidative defense system

Seedlings of Indian mustard (Brassica juncea L.cv. RH-30) grown in controlled condition (irradiance 75 Wm(-2), RH 60-70% and temp. 25 +/- 2 degrees C) for 7d and watered with Hoagland's solution containing different level of NaCL (50-250 mmol/L NaCl) with or without putrescine (PUT, 0.1 mmol/L) were examined for PUT amelioration of NaCl induced inhibition in seedling growth by altering activity of antioxygenic enzymes and level of free radicals in the leaves. Salinity caused reduction in seedling growth and biomass accumulation was parallel to increased superoxide (*O2-), hydrogen peroxide (H2O2) levels, lipid peroxidation (MDA content) and electrolyte leakage in leaf tissues which were reversed significantly by PUT. The antioxygenic enzymes viz superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX) and glutathione reductase (GR) were differentially altered, depending on salt level. PUT induction of enzyme was in the following order APX>GR>CAT>SOD>POD in leaf tissues of salt stressed seedlings. PUT increased the level of glutathione and carotenoids in leaf tissues. This finding suggests that PUT might be activating antioxygenic enzymes and elevating antioxidants there by controlling free radical generation, hence preventing membrane peroxidation and denaturation of biomolecules resulting into improved seedling growth under salinity.

Effect of methionine on longevity, antioxygenic enzymes and lipid peroxides in Callosobruchus maculatus F.

The effect of methionine on longevity, antioxygenic enzymes and lipid peroxides in Callosobruchus maculatus was investigated using C. maculatus maintained on Vigna radiata seeds soaked in seven levels of methionine, 0, 1, 3, 5, 7, 9 and 12 mg/ml water. The life span and activities of catalase and peroxidase were higher in Callosobruchus maculatus infesting Vigna radiata seeds soaked in methionine as compared to their control cohorts. However, the lipid peroxide (malondialdehyde; MDA) levels generally remained unaltered. The present observation on the increased longevity in C. maculatus with methionine level is an extension of earlier findings on its anti-ageing effect in higher animals.

Effect of BHA on longevity, antioxygenic enzymes and peroxides in Callosobruchus maculatus

Life span and antioxygenic enzyme activities of insects reared on optimal BHA concentration (1 mM) soaked seeds were higher than their respective controls. However, the reproductive potential, peroxides and the level of free radicals declined. The increased longevity with BHA could be attributed to its free radical quenching effect, thereby leading to the decreased peroxide levels and the increased antioxygenic enzyme activities of the insects.

Effect of ascorbic acid on longevity and biochemical alterations in Callosobruchus maculatus F. (Coleoptera: Bruchidae)

Optimal ascorbic acid concentration (1 mM) increased the median (LT 50) and maximum (LT 100) life spans, decreased age-independent susceptibility to death (a 0), reproductive period, number of eggs laid/female but prolonged the post-reproductive period in Callosobruchus maculatus. The activities of respiratory enzymes and the levels of metabolic end-products declined while the activities of antioxygenic enzymes increased. The increased longevity of insects reared on ascorbic acid soaked seeds may be interpreted in terms of conservation of energy by way of decreased reproductive potentiality and the maintenance of a homeostatic balance between pro-oxidant generation and antioxidant defences.

Effect of propyl gallate feeding on antioxygenic and respiratory enzymes in liver of mice

Antioxygenic enzymes provide defense to the cell against free radicals produced during respiration. This study deals with the effect of propyl gallate feeding on antioxygenic and respiratory enzymes in liver of male mice of different age groups. Antioxidant increased the activities of catalase, glutathione reductase, ATPase and NADH-dehydrogenase significantly in 8-month-old mice, whereas the increase in activity in early and late growth stages was observed only at selective concentrations. The increased activities of antioxygenic enzymes on propyl gallate feeding may be attributed to an increase in the activities of respiratory enzymes.

Effect of ascorbic acid on longevity, catalase and lipid peroxidation in Callosobruchus maculatus F.

The natural defense against peroxidative damages inflicted by oxygen derived free radicals is provided by antioxygenic enzymes. Feeding of exogenous antioxidants increases the life span of insects by decreasing age-independent susceptibility to death. The present study describes the effect of L-ascorbic acid on the life span, catalase activity, and lipid peroxidation in Callosobruchus maculatus, a non-feeding insect. Life span studies were carried out on insects emerging from unsoaked, water soaked and ascorbic acid (0.1, 0.5, 1.0, 50, 100 and 250 mM) soaked seeds. The increased life span at 1 mM ascorbic acid concentration was accompanied by declined mortality rate, age-independent susceptibility to death, lipid peroxidation and increased catalase activity in both sexes of the insect. The differential distribution of catalase activity with respect to age and sex in whole body homogenates was observed in the insects. The increased longevity of C. maculatus on ascorbic acid treatment could be attributed to a decline in lipid peroxidation and increase in catalase activity of the insects.

Lipid peroxidation and activities of antioxygenic enzymes in vitro in mercuric chloride treated human erythrocytes.

Mercury can exist in the environment as metal, as monovalent and divalent salts and as organomercurials, the most important of which is methyl mercury. There are reports which suggest that heavy metals like mercury, lead and cadmium have prooxidant catalytic activity and can initiate lipid peroxidation by generating free radicals and thereby interfering with the antioxidant system of the cell. Higher concentrations of mercury have been used earlier to assess the extent of lipid peroxidation in bovine erythrocytes. However, the interaction between mercury and the red blood cell is not properly understood. Therefore, in the present study the in vitro toxicity by lower concentrations of mercuric chloride on human erthrocytes in relation to their effect on lipid peroxidation and some enzymes which have a protective role in such a condition are reported.

Aluminum effect on the activity of superoxide dismutase and of other antioxygenic enzymes in vitro

The effect of Al on superoxide dismutase (SOD) and on other antioxygenic enzymes: horseradish peroxidase, catalase, and glutathione peroxidase, has been investigated in vitro. In the case of SOD, the effect of metal chelators (EDTA and deferoxamine) and a possible synergistic effect with iron salts have also been tested using the pyrogallol assay. There is no significant inhibitory effect of Al on the activity of any of the above-mentioned enzymes. Noticeable increases in SOD activity were observed when metal chelators were added to the medium, but not when high concentrations of Al were present too, in the case of deferoxamine (DFO). The former fact seems to be a consequence of the chelation of transition metal ions that catalyze pyrogallol autoxidation by a mechanism not inhibitable by SOD, interfering in its action, which may account for part of the DFO antioxidant effect observed in vivo. The latter phenomenon could be owing to a saturation of the chelating capacity of DFO by an excess of Al present in the medium, which should bring the system back to the interfering conditions explained above. It can be concluded that Al, either in the presence or in the absence of iron salts, does not inhibit SOD activity in vitro. Moreover, no significant binding of Al to SOD was demonstrated, and the amounts of its metal constituents, Cu and Zn, were not affected by preincubation of the enzyme with Al. The effect of the different compounds tested on the rate of autoxidation of the indicating scavenger, pyrogallol, and a suitable hypothesis on their role in the oxidation process are also discussed.

Characterization of an oxygen-tolerant cell line derived from Chinese hamster ovary

To study the cellular defense mechanism against oxygen toxicity, an oxygen-tolerant cell line from Chinese hamster ovary (CHO) was obtained by multistep adaptation to increased O2 levels. The hyperoxia-adapted (HA) cells were able to proliferate under an atmosphere of 99% O2/1% CO2, an O2 tension lethal to the parental (control) cells. When grown under normoxic conditions (20% O2/1% CO2/79% N2) the cells remained tolerant for at least 8 weeks, suggesting a genetic basis for the oxygen tolerance. Compared to the parental cells, the HA cells were irregularly shaped, had larger mitochondria, contained more lipid droplets and showed a reduced growth rate. Ultrastructural morphometry revealed a 1.8-fold (p less than 0.001) increase of the mitochondrial volume fraction in the HA cells, resulting from an increase in both number and average volume of the mitochondria. The volume fraction of peroxisomes was increased over two-fold in the HA cells, as appeared from a approximately 1.9-fold (p less than 0.001) increase in number and a 1.2-fold (p less than 0.025) increase in size. There was no evidence for ultrastructural damage in the HA cells. Specific activities of antioxygenic enzymes were considerably higher in the HA cells compared to controls: CuZn-superoxide dismutase, X 2.5; Mn-superoxide dismutase, X 2.1; catalase, X 4.0; glutathione peroxidase, X 1.9. Oxygen tolerance in CHO cells is therefore associated with increased levels of antioxygenic enzymes, confirming the proposed important role of these enzymes in the defense against oxygen toxicity.

Genetic control of cellular longevity in Neurospora crassa: A relationship between cyclic nucleotides, antioxidants, and antioxygenic enzymes

Short-lived conidial longevity mutants of Neurospora were studied. Mutations at a number of closely-linked and probably functionally-redundant genes lead to deficiency of five anti-oxygenic enzymes, including superoxide dismutase.

Dominance and complementation relationships of conidial longevity mutants of Neurospora crassa

Previously we reported the occurrence of 16 linked conidial longevity determinant genes in Neurospora. Mutations of those genes are characterized by a reduction of longevity, a pleiotropic morphological defect, and deficiency of five antioxygenic enzymes. On the basis of the linkage and biochemical data, it was proposed that the genes are spatially and functionally redundant. The results of the present investigation support the hypothesis of functional redundancy. All of the mutants examined were dominant to wild type and failed to complement in heterokaryons. These results are discussed in terms of two molecular models of gene function.

Genetically determined conidial longevity is positively correlated with superoxide dismutase, catalase, glutathione peroxidase, cytochrome c peroxidase, and ascorbate free radical reductase activities in Neurospora crassa

Aging of post-mitotic cells, the conidia, of Neurospora crassa is defined as the time-dependent loss of viability under a constant laboratory environment which probably resembles the organism's tropical habitat; namely, at 30°C, 85–100% relative humidity under white light. Median lifespan is defined as the age at which survival of a conidial population has declined to 50% of that of a fully viable population at birth. A collection of short ( age −) and long-lived ( age +) mutants were previously selected from the wild-type whose median lifespan is 22 days. Thus, five groups of strains with distinct lifespans of 7, 22, 36, 50 and 60 days were defined. The purposes of the present investigation were to determine if the activities of anti-oxygenic enzymes are correlated with lifespan and to elucidate the function of the cellular longevity determinant genes. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) were highly-correlated with lifespan; whereas glutathione reductase and non-specific peroxidase activities were not correlated. The short-lived mutants were also deficient in cytochrome c peroxidase (CPX) and ascorbate free radical reductase (AFR), but not deficient in dehydroascorbate reductase. (These latter three enzymes were not examined in age + mutants.) By isoelectric focusing analysis, the deficiencies of SOD, CAT, and GPX activities of age − mutants were defined in terms of specific isozymes. The mutants were specifically deficient in a cyanide-resistant mitochondrial isozyme of SOD. Sixteen age − gens, called the age-1 complex, were previously mapped on one arm of the seven chromosomes. On the basis of mapping and complementation data, it was inferred that the genes are spatially and functionally redundant. The hypothesis of functional redundancy is also supported by the enzyme data. Of seven mutants examined, representing seven of the age - genes, all were deficient in SOD, CAT and CPX, and six were deficient in AFR . Of four mutants examined, representing four of the genes, all were deficient in GPX. The results indicate a molecular basis for the previously observed photosentivity of the mutants. Deficiency of the antioxygenic enzymes may render the cells more susceptible to lethal macromolecular damage by photochemically generated free radicals and peroxides. A model is proposed in which age - and age + mutations occur at genes whose function is the regulation of the synthesis of the five antioxygenic enzymes. Thus, age - and age +mutations, respectively, repress and depress the amount of synthesis relative to the constitutive level of wild-type age °. Other observations pertaining to the biochemical and developmental regulation of these in Neurospora are discussed. We speculate that the large number of genes in the age-1 complex may provide a genetic mechanism for the amplication of regulatory signals (proteins?) in response to an increase in flux of free radicals and peroxides.

Ageing of Neurospora crassa. II. Organic hydroperoxide toxicity and the protective role of antioxidant and the antioxygenic enzymes

Cumene hydroperoxide and tert.-butyl hydroperoxide at sublethal concentrations initially prevent growth of mycelia of wild-type Neurospora crassa, but after a time the cells grow at a subnormal steady-state rate. The antioxidant nordihydroguaiaretic acid protects unadapted cells from hydroperoxide inhibition, leading to a decrease in the time before growth begins, an increase in steady-state growth rate and an increase in biomass production. The results of growth transfer experiments and enzyme measurements indicated that the acquired resistance to the hydroperoxides is physiological and most likely involves the induction of the synthesis of the antioxygenic enzymes superoxide dismutase, glutathione peroxidase and glutathione reductase. Nordihydroguaiaretic acid normalizes the levels of activities of glutathione peroxidase and glutathione reductase during culture with hydroperoxide. Molecular-induced homolysis of the hydroperoxides, a process that is induced by unsaturated fatty acids of membrane lipids, leads to lipid autoxidation in a chain reaction which produces lipid hydroperoxides, which in turn decomposes to form more free radicals. Nordihydroguaiaretic acid, a well-known free radical scavenger, probably serves to minimize hydroperoxide decomposition, lipid autoxidation and molecular damage from free radicals, whereas the coupled enzyme system glutathione peroxidase and glutathione reductase minimizes these processes by decomposing the hydroperoxides to harmless alcohols. We suggest that either free radicals derived from these processes or some consequent non-radical products may serve as the inducers of this enzyme system, rather than the hydroperoxide substrates.

Ageing of Neurospora crassa. IV. Induction of senescence in wild type by dietary amino acid analogs and reversal by antioxidants and membrane stabilizers

The extensional growth rate of wild-type 74 A8 N. crassa in the presence of various concentrations of 19 amino acid analogs was measured. Seven analogs were not inhibitory at concentrations in the range of one to 10mM. Of the remaining 12 analogs, nine inhibited growth in a novel way. The kinetics of growth in the presence of these analogs at 30° were characterized by seven sequential phases: (1) lag; (2) acceleration of growth rate; (3) steady-state growth rate; (4) exponential rate of decline of growthrate; (5) no growth or growth rate ⩽ 0.1 mm h −1; (6) acceleration of growth rate; and (7) steady state. At 33°, phases 6 and 7 did not occur and irreparable death of the clones occurred. The mechanism by which the clones acquired resistance at 30° appeared to involve a combination of physiological adaptation and cellular selection. Dietary application of either free radical scavengers or surface-active membrane ‘stabilizers’ alleviated or prevented the inhibition and deterioration of growth rate which occurred in the presence of the nine amino acid analogs. Culture with either 4-fluorophenylalanine or ethionine led to an increase of the activities of antioxygenic enzymes glutathione peroxidase, glutathione reductase and superoxide dismutase. The amino acid analogs that cause senescence and death of growing cells are known to be incorporated into proteins and such proteins are generally abnormal. Because a substantial fraction of cellular protein occurs in membranes and the proteins synthesized by mitochondria are exclusively intrinsic membrane proteins, we suggest that a primary consequence of errors in protein synthesis is the production of faulty membranes. The deterioration of such membranes with associated lipid autoxidation and free radical production proceeding as a chain reaction at an exponential rate may in itself contribute to the exponential rate of cellular deterioration which is characteristic of the ageing process. According to this hypothesis, dietary membrane stabilizers, free radical scavengers and antioxygenic enzymes protect cells from error catastrophy arising from the chain of events leading from membrane deterioration.

Ageing of Neurospora crassa. I. Evidence for the free radical theory of ageing from studies of a natural-death mutant

A recessive mutant of Neurospora crassa, called natural-death, is characterized by a decreasing clonal growth potential under all nutritional conditions and the irreversible cessation of growth. The primary molecular defect of this mutant is not known. Evidence presented here, based upon measurements of the activities and thermolabilities of several enzymes, suggests that faulty protein synthesis is probably not a cause of the senescence and death of the mutant, as suggested by Lewis and Holliday ( Nature, 228 (1970) 877). Three lines of evidence indicate that lipid autoxidation and associated free radical reactions contribute to the senescence and death of this mutant: (1) The relative times before the onset of senescence and death of mutant clones in the last 40% of their chronological life-span were prolonged 2 to 3-fold by either dietary antioxidants or selenite and the total life-span was increased by 40% to 80%. These compounds also alleviated the senescent morphology and enhanced biomass production; (2) Senescing clones accumulated a green fluorescent pigment in situ, but dietary antioxidant nordihydroguaiaretic acid prevented this accumulation. The fluorescent pigment exhibited the spectral properties of lipofuscin, an end product of lipid autoxidation; (3) Relative to wild type, mycelial extracts of the mutant exhibited a 2 to 4-fold excess of activities of the antioxygenic enzymes superoxide dismutase, glutathione peroxidase and glutatione reductase. We briefly review: (1) the roles of antioxygenic enzymes and antioxidants in their protection against cellular damage from lipid autoxidation and free radical reactions; and (2) the major lines of evidence which appear to support a form of the free radical theory of ageing, encompassing the interrelated processes of membrane deterioration, lipid autoxidation and deleterious free radical reactions as the major causes of cellular deterioration.