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A theoretical model was developed taking into account the production and destruction of oxygen-derived free radicals. The steady state of the system was derived by using the rate equations of these reactions, and the stability of the system was tested. In the simplified model, only one stable steady state was found. However, we know that glutathione peroxidase can be inhibited by hydroperoxides, and, when incorporated into the model, this effect led to a complex situation with the presence of some stable and some unstable domains according to the concentration of either the enzyme or the hydroperoxide. This qualitative description of the system was compared with experimental data on the protection given by three antioxidant enzymes, and concordance of data was found which allows some quantification of the system. A general view of the efficiency of the three antioxidant enzymes and of the stability of the system according to their concentrations could be produced.

Ischemia is a situation occurring in several diseases including myocardial infarction and organ transplantation in which oxygenated blood supply is impaired. Ischemia leads to many cellular and tissue modifications, the most important one being cell death. Several explanations have been proposed to account for these modifications and cell death; among them is calcium overload. However, the influence of calcium concentration on the alteration of endothelial cell functions or viability during ischemia are still unknown. We developed here an in vitro model where human endothelial cell monolayers were submitted to hypoxia with or without reoxygenation and variation in calcium concentration was followed using a specific intracellular probe Fura 2. We observed a significant increase of [Ca2+]i during 2 h hypoxia reaching values similar to those observed during agonist stimulation of endothelial cells but far lower than values toxic for the cells. This increase was constant during the hypoxic incubation and was due mainly to an influx of extracellular calcium. Viability was also followed during hypoxia and using calcium channel blockers, we could show that there was no correlation between viability and the rise in calcium concentration. During the reoxygenation period, [Ca2+]i decreased to reach the normal value of resting cells after 45 min, suggesting that cells were still able to recover their calcium homeostasis. The use of a ketone body (beta-hydroxybutyrate) indicated that an energy deficiency was responsible for the hypoxia-induced increase in [Ca2+]i. We actually observed a 43% decrease in ATP concentration after 2 h hypoxia. This decrease was already significant after 30 min which thus precedes the changes in [Ca2+]i. These results show that during hypoxia, energy deficiency led to an increase in [Ca2+]i which is, however, too low to account for the loss of viability but which is within the range of concentrations observed during stimulation of endothelial cells. We propose that such increased intracellular calcium concentrations could play a role in the synthesis of mediators leading to the development of local inflammation.

Colorectal carcinoma is very common in western countries. It is in the front line of malignant pathology in France. The estimated number of new cases is almost 26,000 per year. The role played by diet in the onset of colorectal carcinomas is well established. The majority of studies indicate the protective role played by green vegetables. The role of other factors remains controversial: protective role of fibres, calcium and vitamins and favorizing role of fats, proteins, red meat, alcohol and calorie intake. The role of bile salts in increasing the size of adenomas and their malignant change is accepted. A number of therapeutic trials have involved modifications in bile salt concentrations. These should enable the proposal of a primary prevention strategy. The early detection of carcinomas and the secondary prevention of colorectal carcinoma (by detection of the adenomas which precede a high proportion of carcinomas) are areas of great interest. Detection of occult blood in stools as a mass screening test is currently being evaluated. Methods used to obtain a high participation rate among the population vary from one country to another, but are now well-defined. It will be necessary to wait for 2 years before being able to determine the effect on mortality and an even longer period to evaluate the effect on the incidence of colorectal carcinoma.

Venous stasis is a situation encountered commonly in varicose disorders. The potential implications of this decrease in oxygen levels in terms of the status of the cells of the vein were assessed. When endothelial cells are subjected to hypoxia, there is stimulation of the cells which shows itself as increased synthesis of prostaglandins and of PAF (Platelet Activating Factor). The synthesis of these typical mediators of inflammation results from activation by the calcium of phospholipase A2 which releases the arachidonic acid of phospholipids and this increase in intracellular calcium results itself from a fall in efficacy of calcium pumps due to the fall in ATP caused by hypoxia. Thus the fall in oxygen leads to the production of mediators of inflammation which activate leucocytes and result in local micro-inflammation which can be very rapidly eliminated if the circulation is restored but which can also cause irreversible damage to the vein by changes in venous tissue due to activated leucocytes which release proteases and free radicals after having penetrated the intima of the vein. These processes offer an explanation for the histological changes seen in varicose veins and the onset of localised pain during the development of such disease.

Aerobic cells contain various amounts of the three main antioxidant enzymes: superoxide dismutase (SOD), catalase and GSH peroxidase. These three enzymes are necessary for cell survival since inhibition of their activity leads to the arrest of cell mitosis and to cell death. Amongst them, GSH peroxidase was shown to be more efficient than catalase and much more than SOD. This result was obtained by comparing the cell protection against oxidative stress after their microinjection in the cytoplasm. With age, the level of these antioxidant enzymes does not change in several experimental models, so that it is not possible to explain the aging process by a lack of protection due to a decrease in the activity of these three enzymes. However, tissues and cells are more susceptible to free radical attacks with age. In order to understand the importance of free radicals in this process, we have to distinguish between their respective effects on cell mitosis, cell death and cell aging. The effects on mitosis and cell death are well described, and the results clearly show a threshold of response which is determined by the antioxidant content of the cell. There is now evidence that short free radical stresses can also speed up the aging of in vitro cultured human fibroblasts. However, such effects are not typical of free radicals but are also obtained with many other deleterious substances so that free radicals have to be considered as one amongst other factors responsible for influencing the evolution of a cell to an older stage or to cell death. The lowering of the general metabolism and of the free energy in old cells are probably the main factors responsible for the increased susceptibility of these cells to stresses such as oxidative stresses.

Numerous reported findings indicate that the etiology of venous diseases is multifactorial. One of the chief factors is certainly stasis of blood in the veins of the lower limbs during long periods spent standing up. This stasis causes tissue hypoxia which first affects the venous wall. Because of their location at the interface between blood and vein wall and because of their fragility, endothelial cells are the first to suffer from the lack of oxygen. With the aim of understanding these events, the authors have developed a model of endothelial cells in culture subjected to hypoxia in vitro which mimics the conditions encountered clinically. This model has enabled us to test various drugs commonly used in venous disease. These included GbE, the active ingredient of GINKOR FORT, diosmin and procyanidol oligomers. It was thus shown that only GbE was not toxic to endothelial cells. GbE was also found to be capable of effectively protecting cells exposed to hypoxia. Protection under the influence of diosmin was obtained only at concentrations very close to toxic doses. In contrast, procyanidol oligomers offered no protection. The protective effect of GbE is believed to be due to the action of terpenes on the energy metabolism of the cell.

Many findings indicate that the etiology of venous disease is multifactorial. One of the chief factors is certainly the stasis of blood in the lower limbs during long periods of standing. This stasis causes tissue hypoxia which first affects the venous wall. Because of their site at the blood/vein wall interface as well as their fragility, endothelial cells are the first to suffer from lack of oxygen. In order to understand these events, the authors have developed a model of endothelial cells in culture subjected to hypoxia in vitro which imitates conditions encountered clinically. This model was used to test different drugs commonly used in venous pathology. These included GbE, the active ingredient of GINKOR FORT, diosmine and procyanidolic oligomers. It was thus shown that only GbE was not toxic to endothelial cells. Furthermore, GbE was capable of effectively protecting cells subjected to hypoxia. Protection with diosmine was obtained only at concentrations very close to toxic doses. In contrast, procyanidolic oligomers afforded no protection. The protective effect of GbE is believed to be due to the action of terpenes on cellular energy metabolism.

Lipid peroxidation occurs during oxidative stress and leads to the formation of various active compounds. However, controversy remains about its importance in the events leading to cell death. One approach to estimate their role in cell death would be to test the toxicity of oxidative products generated during the stress. In this work, three of these products were incubated with human fibroblasts and their toxicities were compared. The three compounds tested are: linoleic acid peroxide (LOOH), malondialdehyde (MDA) and 4-hydroxynonenal (HNE). Three cellular parameters were assayed: viability, DNA synthesis estimated by thymidine incorporation and protein synthesis measured by leucine incorporation. Protection against cellular damages was also tested adding alpha-tocopherol in the culture medium. The results showed that the peroxide was more toxic than HNE and much more than MDA. The possibility of initiation and propagation of the free radical chain reaction could explain this highest toxicity. The fibroblasts seem to be protected by alpha-tocopherol against LOOH. These effects emphasize the crucial role of this lipophilic antioxidant to protect cells against peroxidation damages.

The protection of human diploid fibroblasts against high oxygen tension was investigated using various combinations of the three major antioxidant enzymes: superoxide dismutase, catalase and glutathione peroxidase. alpha-Tocopherol, a well-known hydrophobic antioxidant, was also tested in combination with the different enzymes. Microinjection of solutions containing different combinations of the three enzymes was compared with the injection of each single enzyme. We observed that the protections given by catalase or superoxide dismutase on the one hand, and by glutathione peroxidase on the other hand, were additive. Surprisingly, the combinations of catalase and superoxide dismutase were less effective than catalase alone and was even toxic at low SOD concentrations. Addition of alpha-tocopherol following the injection of any of the three enzymes was highly beneficial, but the strongest synergistic effect was obtained with glutathione peroxidase. These results stress the importance of membrane protection by alpha-tocopherol and indirectly by glutathione peroxidase. They also showed that any injection leading to the decrease in the O2.- or H2O2 concentration combined with one of these two protectors is very beneficial for the cells probably by decreasing the OH concentration. This is also proven by the very good protective effect obtained with desferrioxamine.

The resistance of human pulmonary fibroblasts (WI-38) and human umbilical vein endothelial cells to oxygen toxicity (1 atm O2) was compared. Endothelial cells were more sensitive than fibroblasts. They contained also less antioxidant enzymes except for SOD: respectively 132%, 96%, 70%, 59%, and 21% of the SOD, GSH peroxidase, GSH reductase, catalase, and G6PD content of fibroblasts. However, they contained 1.81-fold more GSH than fibroblasts. Their lower content of antioxidant enzymes can explain their higher sensitivity to oxygen. The efficiency of natural antioxidant molecules and enzymes in the protection of cells incubated 3 days under 1 atm O2 was studied. alpha-tocopherol added in the culture medium led to a significant protection, contrary to the result for ascorbic acid. Microinjection of catalase, SOD, and GSH peroxidase directly into the cells was also tested: the protection was concentration dependent for both types of cells but SOD did not protect the endothelial cells. Lower activities of the other enzymes were needed to achieve protection of the endothelial cells, compared to fibroblasts. Since endothelial cells were also shown to display lower antioxidant enzyme activities, it can be hypothesized that their content is optimized for survival in physiological conditions.

Recent discoveries have shown that venous diseases have a multifactorial etiology. One of the factors which is definitely involved in this pathologic process is the change in the concentration of oxygen. An increase in the concentration of oxygen, hyperoxia, or reoxygenation following hypoxia, damages the tissues by stepping up the production of free radicals. In addition, a reduction in oxygen concentration, or hypoxia, is also damaging, probably through a reduction in ATP synthesis. From a therapeutic standpoint, the veins, and more particularly the endothelium, must be protected against the impact on the tissue of these changes in oxygen concentration. In this study, the effects of Ginkor Fort were tested on cultured endothelial cells subjected to varying oxygen pressures. The results show that Ginkor Fort can provide good protection of endothelial cells against hyperoxia and hypoxia-reoxygenation. These beneficial effects are probably due to the presence of flavonoids in the Ginko biloba extract; these flavonoids have an anti-oxidant effect. In addition, this substance also protects the cells against hypoxia, possibly by increasing the availability of oxygen for ATP synthesis. This dual protective effect, which is produced by two different mechanisms, may account for the wide spectrum of Ginkor Fort in its use in venous diseases.

Glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase are the most important enzymes of the cell antioxidant defense system. However, these molecules are themselves susceptible to oxidation. The aim of this work was to estimate to what extent this system could be inactivated by its own substrates. We tested the effect of hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and hydroxyl and superoxide radicals on GPX, SOD and catalase. For GPX, a 50% inactivation was observed at 10(-1) M (30 min, 37 degrees C) for hydrogen peroxide, 3 x 10(-4) M (15 min, 37 degrees C) for cumene hydroperoxide and 5 x 10(-5) M (11 min, 37 degrees C) for t-butyl hydroperoxide. Unlike the hydroxyl radicals, superoxide anions did not inactivate this enzyme. Catalase was inactivated by hydroxyl radicals and by superoxide anions but organic peroxides had no effect. SOD was inactivated by 50% by hydrogen peroxide at 4 x 10(-4) M (20 min, 37 degrees C), but organic peroxides and hydroxyl radicals were ineffective on this enzyme. Since the three enzymes of the antioxidant system are susceptible to at least one of the oxidative reactive molecules, in the case of high oxidative stresses such an inhibition could take place, leading to an irreversible autocatalytical process in which the production rate of the oxidants will continuously increase, leading to cell death.

Respiratory activity of isolated rat liver mitochondria was assayed following in vitro exposure to oxygen radicals. Our results show that mitochondrial respiration is more sensitive to O2.(-) than to H2O2. However, ferrous ions drastically enhance the toxicity of the enzymatic system generating H2O2 because of the production of the hydroxyl radicals. A protection against those oxygen species could be given by SOD in the xanthine/xanthine oxidase system and by catalase with the glucose/glucose oxidase system. The most damaging system was the combination of Fe2+ with H2O2. In this case, OH. is formed in a Fenton-like reaction. The fact that the OH. is the most damaging molecule accounts for the finding that catalase and desferrioxamine were efficient protectors in this system. Threshold levels of O2.(-) and H2O2 able to inhibit the mitochondrial respiration have been estimated. It is concluded that under normal respiration such thresholds are not reached in vivo and that the impairment of the mitochondrial respiratory activity does not seem to originate only from the natural free radical production in those organelles. However, if the production of free radicals is such to exceed the defense capability, like under oxidative stress, then the critical threshold can be surpassed and the respiration impaired leading to irreversible damages.

Antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) have been injected into human fibroblasts exposed to 2 atm O2 in order to test if the threshold of oxidative damage versus antioxidant defenses could be modulated and if the damage remains reversible beyond the threshold. Cell damage was estimated by thymidine incorporation and cell survival curves. The proportion of dividing cells, measured by thymidine incorporation, rapidly decreased after O2 incubation: no cells could divide after 15 h of hyperoxia. However, cells incubated for a short time and injected with a high concentration of any of the three enzymes divided like non-oxygen-incubated cells: the enzymes could protect the cells against their loss of division potential. However, when cells were incubated for a longer period and/or when the injected enzyme concentration was lower, cells were either less or not protected and could no longer divide. These results suggest the presence of a threshold for the oxidative damage which cannot be totally repaired and which impairs the cell division; this threshold can, however, be modulated by supplementation of antioxidant enzymes, glutathione peroxidase being the most efficient.

Human WI-38 diploid fibroblasts have been cultivated under high toxic O2 pressure, and their survival curves are reported. Superoxide dismutase, catalase, or glutathione peroxidase provided some protection when injected in the cells exposed to O2. This protective effect, recorded after 3 or 4 days of incubation, was the most pronounced when cells were injected just before oxygen exposure. Quantitative injection assays have been performed for the three enzymes. Surprisingly, glutathione peroxidase was found to be much more effective than both catalase and superoxide dismutase, the latter being particularly inefficient.

The toxicity of nitrofurantoin was studied on human WI-38 fibroblasts: this chemical was lethal when added at concentrations higher than 5.10(-5) M in the culture medium. The protection afforded by antioxidants was then tested: alpha-tocopherol gave at 10(-4) M a light protection in contrast to ascorbic acid which even became toxic at high concentrations. We also tested catalase, superoxide dismutase and glutathione peroxidase introduced intracellularly by the microinjection technique. On a molecular basis, glutathione peroxidase was 23-times more efficient than catalase and 3000-times more than superoxide dismutase. The results also showed that a similar range of enzyme concentrations was found for the protection against high oxygen pressure. This suggests that, in the case of both oxygen and nitrofurantoin toxicity, the peroxide derivatives are the most toxic intermediates of the free radical attacks.

Chemical inhibitors of the different antioxidant enzymes were systematically testet either on purified enzymes of after incubation with human fibroblasts in culture. Inhibition values were obtained for catalase with aminotriazole, for superoxide dismutase with diethyldithiocarbamate, for glutathione peroxidase with mercaptosuccinate, for glutathione reductase with bischloroethylnitrosourea and for glutathione synthesis with buthionine sulfoximine. Viability of cells incubated with these inhibitors was then tested under normal conditions and under high oxygen pressure; the data were correlated with the above-mentioned inhibitory values. Cell viability was particularly affected when the glutathione-related enzymes, especially glutathione peroxidase, were inhibited.

An experimental model was developed in order to study the protective effect of antioxidant molecules. Human diploid WI-38 fibroblasts were cultivated under 2 atm of 95% O2. Antioxidants like alpha-tocopherol or superoxide dismutase (SOD) were added respectively in the culture medium or directly inside the cell through a microinjection technique. With both antioxidant molecules a protection was observed. In the control experiment, cells died within 6 or 8 days depending on the confluency and the malonaldehyde content increased sharply. This model represents a new tool in order to test other antioxidant systems towards an oxidative stress.

Antibodies were prepared against glutathione peroxidase, superoxide dismutase, and catalase. Inhibition of the enzyme activity was obtained with anti-Gpx and anti-SOD antibodies but not with anti-CAT antibodies. The antibodies were then injected into human fibroblasts and bovine chondrocytes in culture either under normal conditions or under 1 atm of oxygen. The injected anti-Gpx and anti-SOD antibodies increased the mortality rate of the fibroblasts incubated under 1 atm of oxygen. However, when cells were incubated under normal atmosphere, anti-Gpx antibodies inhibited the division while anti-SOD antibodies increased this capacity. Anti-Gpx antibodies injected into chondrocytes decreased their viability. Injection of control antiserum had no effect. These data stress the primary importance of Gpx as antioxidant under all conditions and the relative efficiency of SOD according to the balance between the radical production and the activity of the other antioxidant systems.