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Mitotically active, growth-arrested cells and proliferatively senescent cultures of human fetal lung fibroblasts (WI-38) were exposed to six different oxygen tensions for various lengths of time and then analyzed to determine the responses of their antioxidant defense system. Glutathione (GSH) concentration increased as a function of ambient oxygen tension in early passage cultures; the effect was larger in exponentially growing cultures than in those in a state of contact-inhibited growth arrest, but was absent in senescent cells. Conversely, the activity of glutathione disulfide reductase was greater in growth-arrested cultures than in mitotically active cells irrespective of oxygen tension. Glucose-6-phosphate dehydrogenase was lowest in log-phase cells exposed to different oxygen tensions for 24 h and in senescent cells. Both hypoxia and hyperoxia depressed selenium-dependent glutathione peroxidase activity in early passage cultures, while the activity of the enzyme progressively declined with increasing oxygen in senescent cells. The GSH S-transferase activity was unresponsive to changes in ambient oxygen tension in either young or senescent cultures. Manganese-containing superoxide dismutase (MnSOD) activity was unaffected by oxygen tension, but was elevated in young confluent cultures as compared with cultures in log-phase growth. MnSOD activity was significantly higher in senescent cultures than in early passage cultures and was also responsive to increased oxygen tension in senescent cultures. Copper–zinc-containing superoxide dismutases activity was not affected by oxygen tension or the passage of time, but it declined in senescent cultures.

Basal antioxidant defense levels are often aberrant in tumor cells; however, less attention has been given to differences in the way that normal and transformed cells respond to changes in oxidative stress. This study evaluated differences in the responses of various normal and transformed cell lines to different oxygen tensions. Exposure to hyperoxia generally failed to induce either the activity of GSH peroxidase (GPx) or the manganese-containing form of superoxide dismutase (MnSOD) after 48 h, although at 605 mm Hg oxygen, small inductions of MnSOD activity were observed in adult lung fibroblasts and amelanotic melanoma. Exposure to 605 mm Hg O2 for 48 h was inhibitory to GPx activity. MnSOD activity was strongly induced in virally transformed WI-38 cells by treatment with the herbicide paraquat or inhibition of GSH synthesis with BSO. In normal cells GSH concentration was proportional to ambient oxygen tension. Tumor cells exhibited greater GSH concentrations at low oxygen tensions than normal cells but were unable to increase GSH in response to elevation of oxygen tension. These results reveal differences in tumor and normal cell responses to changes in ambient oxygen tension and show that MnSOD activity is inducible when an appropriate stimulus is applied.

Diethylstilbestrol induces proliferation of Syrian hamster renal proximal tubular cells. By counting the number of cells in culture, we showed that liposomes containing superoxide dismutase or catalase suppressed diethylstilbestrol-induced proliferation, whereas empty liposomes or liposomes containing inactivated superoxide dismutase did not. Liposomes containing antioxidant enzymes did not suppress proliferation of cells in control media or of cells treated with ethinyl estradiol. In the absence of liposomes, exogenous superoxide dismutase did not suppress diethylstilbestrol-induced proliferation. The decrease in cell number when diethylstilbestrol-treated cells were treated with antioxidant enzyme-containing liposomes was not due to decreased cell viability. Results were confirmed by measuring a correlate of cell proliferation immunohistochemically, using an antibody to proliferating cell nuclear antigen. A larger proportion of diethylstilbestrol-treated cells than of control cell showed nuclear immunostaining with this antibody. The number of cells immunostained in diethylstilbestrol-treated cultures was sharply decreased by the addition of superoxide dismutase- or catalase-containing liposomes. Our studies suggest a role for active oxygen species in diethylstilbestrol-induced proliferation of cultured proximal tubular cells.

Oxygen toxicity is believed to arise from changes in the rates at which cells generate reactive oxygen species (ROS). Sensitivity to hyperoxia has been postulated to depend on levels of antioxidant defense. Human cells obtained from fetal tissues have lower antioxidant defenses than those obtained from adult tissue. The present study was performed to determine whether the differences in fetal and adult antioxidant defense levels modulated their responses to changes in the ambient oxygen concentration. Our results demonstrate that oxygen modulates the proliferation of human fetal and adult skin fibroblasts in a similar fashion. In general, skin fibroblasts grew better at approximately 31 mm Hg, regardless of donor age. Manganese superoxide dismutase, catalase, and glutathione peroxidase activities were lower in fetal cells than in adult fibroblasts. Copper/zinc superoxide dismutase and glucose-6-phosphate dehydrogenase were similar in fetal and postnatal tissues and were unaltered appreciably by hyperoxic exposure. Glutathione concentration increased at higher oxygen tensions; however, the increase was much greater in fetal cells than in cultures derived from adult skin. These observations demonstrate that the capacity of fetal and adult cells to cope with oxidative stress, while similar, result from distinct mechanisms.

The activities and mRNA abundances of enzymes that regulate the rate of electron flow through the electron transport chain (ETC), including NADH dehydrogenase, succinate dehydrogenase, and cytochrome c oxidase, were examined in young and senescent fetal lung fibroblasts (WI-38). We also determined the activities and mRNA abundances of antioxidant defenses including superoxide dismutase, catalase, and glutathione peroxidase. We confirmed our previous report of a senescence-related increase in the abundance of ND4, a mitochondrially encoded subunit of NADH dehydrogenase. The activities of cytochrome c oxidase and NADH dehydrogenase were also elevated in senescent cultures. No differences were observed in the mRNA abundances of COX-1, a mitochondrially encoded subunit of cytochrome c oxidase or of nuclearly encoded subunits of various electron transport components (SD, COX-4, and ND 51). Lucigenin-detected chemiluminescence and H2O2 generation were both elevated in senescent cells. Catalase activity was also elevated in senescent fibroblasts. However, no differences in catalase mRNA abundance were observed. A small decrease in GSH peroxidase (GPx) mRNA abundance was observed in senescent cells. No other changes in the activities or mRNA abundances of any of the antioxidant defenses were observed in early and late passage cultures. The relationships between oxidant generation, mitochondrial enzyme activities, and antioxidant defense observed during proliferative senescence are dissimilar to those detected between fetal and postnatal fibroblasts as well as those found between fibroblast lines obtained from young and old individuals. The relevance of the differences between these models is discussed.

We have determined the activities, protein, and mRNA abundances as well as the level of transcriptional activation of two intracellular forms of the free radical metabolizing enzyme superoxide dismutase in 29 human skin fibroblast lines established from donors of different ages. SOD-1 (a copper and zinc containing form of SOD) and SOD-2 (a manganese containing form of the enzyme) activities were both observed to be significantly lower in cell lines derived from fetal skin than in lines established from postnatal skin (ages 17-94 years). The percent of total activity contributed by SOD-1 decreased in an age-associated manner from approximately 50% in the fetal lines to less than 20% in lines established from old tissue donors. All of the cell lines were screened to exclude the possibility that they contained a polymorphism known to influence SOD-2 activity. Northern blot analysis revealed three SOD-1 mRNA transcripts that were 0.5, 0.7, and 1.9 kb in length. Although SOD-1 protein abundance was lower in fetal lines than in lines derived from postnatal donors, SOD-1 mRNA abundance did not differ between fetal cells and cell lines derived from young donors. SOD-2 protein abundance and mRNA abundance were both significantly lower in fetal lines than in postnatal lines. No postnatal age-dependent differences were observed in any of the SOD-2 parameters examined. Nuclear run-on analysis revealed that fetal cell lines exhibited a lower level of transcriptional initiation for SOD-1 than postnatal lines. The transcription of SOD-2 was readily detected in postnatal lines, but undetectable in fetal lines. These results are consistent with multiple levels of control for SOD-1 expression and with a strong transcriptional influence on SOD-2 expression.

Metabolic gradients are known to influence cellular differentiation at critical junctures of development. The existence of these gradients will stimulate variations in the rate of generation of active oxygen metabolites by metabolic pathways. Recent evidence suggests that cellular oxidation can induce changes in gene expression during normal development. Conversely, antioxidants such as ascorbate, alpha-tocopherol and beta-carotene are inhibitory to differentiation in many types of cells. One basis of oxidative influence on gene expression stems from changes in cellular calcium distribution.

The activities of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione S-transferases, GSSG reductase, thiol transferases, gamma glutamyl-cysteine synthetase, and glucose-6-phosphate dehydrogenase, and the concentrations of H2O2 and reduced and oxidized glutathione were determined in the various developmental stages of houseflies. Housefly development was correlated with a progressive increase of cellular oxidizing equivalents and a loss of cellular reducing capacity. The loss of reducing equivalents appeared to result from a decrease in the activity of enzymes involved in glutathione and NADPH synthesis and a concomitant increase in glutathione-oxidizing enzymes. Relatively little change was observed in SOD activity during housefly development; however, the electrophoretic pattern of MnSOD varied in a manner specific to developmental stage. A striking increase in H2O2 concentration occurred prior to pupation possibly due to changes in substrate catabolism. These results support the hypothesis that the cellular environment becomes progressively more oxidizing during development.

Metabolic gradients are established during early phases of development and their existence influences subsequent developmental events. Variations in oxygen supply and oxygen metabolism associated with the gradation of metabolic rate in embryos appear to form one basis for the influence of metabolic gradients on development. The rate of oxygen metabolism affects the rate of oxidant generation by various cellular biochemical pathways. Cells contain antioxidant defenses that respond to variations in cellular oxidant production. Large changes in the activity of the antioxidant enzyme superoxide dismutase and changes in cellular redox state occur during the differentiation of many types of cells. These changes correspond to an increased rate of oxidant production; the cellular environment becomes more prooxidizing during differentiation. Evidence is presented that implicates oxidants as a factor that can stimulate alterations in gene expression. Possible mechanisms by which oxidants influence gene expression are also discussed.

We have previously reported that calcium is required for the starvation-induced differentiation of the slime mold, Physarum polycephalum. With the exception of calcium, each component of the complex starvation medium may be withheld and the organism will still differentiate into spherules. The results of the present study reveal that spherulation will proceed normally when the microplasmoidal cells are transferred from nutrient medium to a citrate buffer containing only 8 mM Ca CI2. Electron microscopy and X-ray microprobe analysis reveal that there is an initial increase in the population of calcium-containing mitochondrial granules when the microplasmodia are induced to differentiate. However, as differentiation proceeds, these granules decrease in number and are virtually absent from the mitochondria of mature spherules. The accumulation and depletion of calcium-containing granules is not observed in a nondifferentiating strain of Physarum cultured under standard conditions, but is observed when this strain is first treated with a calcium-enriched nutrient medium that conditions it for spherulation. Changes in the cellular concentrations of NADH and lipid peroxides, and in the activity of superoxide dismutase, correspond temporally to the pattern of increase and depletion of the calcium-containing inclusions. The oxidative stress associated with starvation-induced spherulation may be a consequence of the active accumulation of calcium; the mobilization of this calcium may then be the event that initiates differentiation.

The effects of total inhibition of catalase, induced by 3-amino-1,2,4-triazole, on the adult housefly (Musca domestica) were examined. The lack of catalase activity had no effect on the longevity of the houseflies. Inorganic-peroxide concentration was elevated at younger ages, but declined in older flies. The rate of oxygen consumption by the flies was greatly decreased and the levels of oxidized as well as reduced glutathione were augmented. Superoxide dismutase activity showed a slight increase. This study suggests that loss of catalase activity does not affect survival of houseflies due to adaptive responses.

Confluent cultures of human skin fibroblast lines established from fetal and postnatal donors were exposed to a broad range of oxygen tensions (10-600 mmHg) for 1 wk; superoxide dismutase (SOD) activity was subsequently determined. Hyperoxia increased SOD activity slightly in postnatal lines but not in fetal lines. The magnitude of the increase in postnatal lines was not significant. Fetal lines exhibit only about one-fifth the SOD activity observed in postnatal lines. The results indicate that, while development-associated changes in SOD do occur in human cells, these alterations do not result from variations in ambient oxygen tension.

Microplasmodia of Physarum polycephalum differentiate into spherules when the CaCI2 concentration of their nutrient medium is increased to 54mM (high- calcium). The salts starvation medium routinely used to induce differentiation contains 8mM CaCI2. This medium will not induce spherulation in the ab- sence of a calcium salt; no other metal is essential. High-calcium also induces the spherulation of a strain of Physarum that had not been previously ob- served to spherulate. The striking increase in superoxide dismutase activity (SOD) and the decrease in glutathione concentration (GSH) that are charac- teristic of salts-induced spherulation do not occur in salts media containing high-calcium. In the absence of calcium, no significant change in SOD is observed and very little change in GSH occurs. The immediate effect of the oxidative stress associated with spherulation may be the release of calcium stores into the cytosol. The parameters modulating this stress are, in turn, sensitive to exogenous calcium concentrations. Cellular differentiation is ultimately due to qualita- tive and quantitative alterations in gene expression. At present the nature of the mechanisms governing devel- opmental changes in gene expression remains obscure. Cytoplasmic fields, gradients, and morphogenetic deter- minants are widely believed to modulate the transition of embryonic tissues into differentiated cell types (Na- menwirth, 1974; Summerbell, 1979; Summerbell et al., 1973; Wolpert, 1978). Among the factors which appear to play a role in development are the metabolic and chemical gradients observed in developing organisms; these arise from regional variations in surfaceholume ratio and from differential vascularization of the embry- onic tissues (Boveri, 1901; Browder, 1980; Caplan and Ordahl, 1978; Child, 1915). The effects of metabolic gra- dients on development may be partly due to differential nutrient supply; however, there is also evidence that variations in molecular oxygen concentration play a role in the regulation of developmental processes (Jargiello and Caplan, 1983; Caplan and Koutroupas, 1973). The mechanism by which oxygen and its metabolites affect gene expression is presently unknown.

The authors outline the progression of thought on the mechanism of the aging process, giving emphasis to environmental factors that influence genetic events. Discussion is limited to those theories that explain fundamental causes of aging and have a firm thermodynamic basis. The authors propose that the cumulative result of continual oxidative stress and other thermodynamic processes (such as amino-acid racemization and nonenzymatic glycosylation), resulting in altered function and increasing the net entropy of living systems, governs the rate of the aging process.

Metabolic gradients exist in developing organisms and are believed to influence development. It has been postulated that the effects of these gradients on development result from differential oxygen supplies to tissues. Oxygen has been found to influence the course of development. Cells and tissues in various stages of differentiation exhibit discrete changes in their antioxidant defenses and in parameters of oxidation. Metabolically generated oxidants have been implicated as one factor that directs the initiation of certain developmental events. Also implicated as factors that modulate developmental processes are the cellular distribution of ions and the cytoskeleton both of which can be influenced by oxidants. The interaction of oxidants with ion balance and cytoskeleton is discussed.

Evidence is presented that supports a role for the enzyme superoxide dismutase (SOD) in the differentiation of the slime mold, Physarum polycephalum. SOD activity increases 46-fold during differentiation. A strain of Physarum that does not differentiate exhibits no change in SOD activity. Addition of SOD, via liposomes, to the nondifferentiating strain induces differentiation; this effect is enhanced by an inhibitor of glutathione synthesis. Other antioxidants selected for study failed to induce differentiation. Conversely, oxidative treatments including introduction of d -amino acid oxidase, via liposomes, induced differentiation. Cellular oxidation is the probable cause of the SOD effect.