Michel Clément

Age-related changes in antioxidant enzymes and lipid peroxidation in brains of control and transgenic mice overexpressing copper-zinc superoxide dismutase

The aim of our study was first to obtain a comprehensive profile of the brain antioxidant defense potential and peroxidative damage during aging. We investigated copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), seleno-dependent glutathione peroxidase (GSH-PX), glutathione reductase (GSSG-R) activities, endogenous and in vitro stimulated lipid peroxidation in 40 brains of control mice divided into 3 age groups: 2 months (young), 12 months (middle-aged) and 28 months (old). We found a positive correlation between age and activities of CuZnSOD (r = 0.47; P < 0.01) and GSH-PX (r = 0.72; P < 0.0001). CuZnSOD and GSH-PX activities are independently regulated during brain aging since temporal changes of these two enzymes do not correlate. No modification in MnSOD activity and basal lipid peroxidation was observed as a function of age. Nevertheless, stimulated lipid peroxidation was significantly higher at 12 months (6.53 +/- 0.71 mumole MDA/g tissue) than at 2 months (5.69 +/- 0.90) and significantly lower at 28 months (5.13 +/- 0.33) than at 12 months. Second, we used genetic manipulations to construct transgenic mice that specifically overexpress CuZnSOD to understand the role of CuZnSOD in neuronal aging. The human CuZnSOD transgene expression was stable during aging. The increased CuZnSOD activity in the brain (1.9-fold) of transgenic mice resulted in an enhanced rate of basal lipid peroxidation and in increased MnSOD activity in the 3 age groups. Other antioxidant enzymes did not exhibit modifications indicating the independence of the regulation between CuZnSOD and glutathione-related enzymes probably due to their different cellular localization in the brain.

Docosahexaenoic acid-rich phospholipid supplementation: effect on behavior, learning ability, and retinal function in control and n-3 polyunsaturated fatty acid deficient old mice.

This study investigated the effects of docosahexaenoic acid (DHA)-rich phospholipid supplementation on behavior, electroretinogram and phospholipid fatty acid (PUFA) composition in selected brain regions and retina in old mice. Two groups of mice were fed a semisynthetic balanced diet or a diet deficient in α-linolenic acid. At the age of 8 months, half of each diet group was supplemented with DHA. In the open field, no differences in motor or exploratory activities were observed between the four diet groups. In the light/dark test of anxiety, the time spent in the light compartment was significantly higher in both supplemented groups than in control and deficient groups. Learning performance in the Morris water maze was significantly impaired in deficient old mice, but was completely restored by the phospholipid supplementation. The electroretinogram showed a significant alteration of a-and b-wave amplitudes in control compared to deficient mice. Phospholipid supplementation induced a significant increase of b-wave amplitude in both control and deficient groups and restored normal fatty acid composition in brain regions and retina in deficient mice. DHA-rich phospholipids may improve learning ability, visual function and reverse biochemical modifications in old mice fed an n-3 polyunsaturated fatty acid-deficient diet; they also may improve visual function in old mice fed a balanced diet.

Graded dietary levels of RRR-γ-tocopherol induce a marked increase in the concentrations of α- and γ-tocopherol in nervous tissues, heart, liver and muscle of vitamin-E-deficient rats

Abstract The effect of dietary RRR- γ -tocopherol supplementation on serum and tissue α - and γ -tocopherol concentrations was studied in vitamin-E-deficient rats fed diets containing adequate levels of RRR- α -tocopherol and graded levels of RRR- γ -tocopherol over a 60 day period. Feeding rats with a RRR- α -tocopherol-supplemented diet induced in forebrain, sciatic endoneurium, skeletal muscle, heart and liver a marked increase in α -tocopherol concentration. In contrast, feeding rats with a diet containing the same level of RRR- γ -tocopherol induced a small increase in γ -tocopherol concentrations in brain, sciatic endoneurium, skeletal, muscle, heart and liver and a slight but significant decrease in α -tocopherol concentration in all tissues examined. In rats fed diets containing a constant level of RRR- α -tocopherol and graded levels of RRR- γ -tocopherol, the concentrations of α -tocopherol in all tissues were much higher than those in rats fed a control diet containing RRR- α -tocopherol alone. The higher the γ / α ratio, the more the α -tocopherol concentrations increased. Significant positive linear regressions were found between the γ / α ratio and the α - and γ -tocopherol concentrations in most of the tissues examined. These results indicate that when γ -tocopherol was supplied continuously in the diet γ -tocopherol accumulated significantly in the tissues but to a much smaller extent than when rats were fed with RRR- α -tocopherol. These experiments also indicate that γ -tocopherol did not depress the serum and tissue α -tocopherol concentrations. On the contrary, γ -tocopherol supplements induced a marked increase in α -tocopherol concentrations in the serum and tissues. These results suggest that there is a relationship between α - and γ -tocopherol levels in vivo and that the biopotency of α -tocopherol should be reevaluated especially when high levels of γ -tocopherol were present in the diet.

Effect of dietary α-linolenic acid deficiency on habituation

Abstract Three weeks before mating, two groups of SWISS OF 1 mice were fed a diet that was similar but contained either peanut oil poor in alpha-linolenic acid [18: 3 (n-3)] (n − 3 deficient = deficient mice = (n-3)−) or peanut + rapeseed oil rich in alpha-linolenic acid (n − 3 nondeficient = controls = (n − 3)+). Pups, fed the same diet as their dams, aged 45 to 62 days were used for brain lipid analysis and for behavioral experiments, aimed at determining whether there is a relation between the dietary intake of alpha-linolenate and a simple form of learning: habituation. The behavior of mice was compared using four models: exploration recorded in a photocell actimeter, activity in an open-field, duration of immobility in the forced swimming test and number of escape attempts from a small closed space. Habituation was measured by testing the mice in the same situation after some time had elapsed since the first test. Exploration in the photocell actimeter was significantly reduced between day 1 and 4 in nondeficient mice, but, not in deficient mice. The number of square crossings in the open-field was significantly reduced on the second test neither in the control nor in the deficient mice. In the forced swimming test, the habituation (increase in duration of immobility) was significantly greater (255%) in nondeficient than in deficient mice (163%) In the escape attempt experiment, the habituation showed a trend to be greater in controls than in deficient mice (p = 0.061) and was significantly greater in females than in males (p = 0.028) These results suggest that a simple form of learning, habituation, occurs more slowly in mice fed a diet deficient in alpha-linolenic acid.

Influence of a dietary α-linolenic acid deficiency on learning in the Morris water maze and on the effects of morphine

Female OF1 mice were fed on a diet deficient in alpha-linolenic acid or on a control diet 3 weeks before mating and throughout pregnancy and lactation. Pups fed on the same diet as their mothers were used for experiments. The effects of dietary alpha-linolenic acid deficiency were studied in a model of learning, the Morris water maze, and on the following effects of morphine: increase in locomotor activity, modifications of rectal temperature and analgesia. In the place and in the cue versions of the Morris water maze, learning occurred at the same speed in the two diet groups; however, in the place version of the test, the level of the performance was significantly lower in the deficient mice. The probe trial and the extinction procedure did not show any difference between the two diet groups. The morphine-induced increase in locomotor activity occurred significantly earlier and was greater in the deficient diet group. Morphine induced an early hypothermia followed by a late hyperthermia; the hypothermia was significantly greater and the hyperthermia significantly smaller in the deficient mice. The pain thresholds and the morphine-induced analgesia were unmodified by the dietary deficiency. The plasma levels of morphine were similar in the two diet groups.

Hepatic lipid peroxidation and mitochondrial susceptibility to peroxidative attacks during ethanol inhalation and withdrawal

Male Sprague-Dawley rats were exposed to increasing concentrations (15-22 mg/l) of ethanol vapor over a 4-day period. The hepatic lipid peroxide level as well as the sensitivity of mitochondria and microsomes to peroxidative attacks were studied during the early stage of alcohol intoxication, at the end of the inhalation period and, finally, during withdrawal. The level of hepatic lipid peroxide started to increase significantly after the first day of ethanol inhalation, whereas the in vitro mitochondrial sensitivity to peroxidation induced by ADP X Fe3+ in the presence of an O(2)-generating system was still unaltered after a 2-day inhalation period. Both the hepatic peroxide level and the mitochondrial sensitivity to peroxidation were significantly enhanced at the end of the 4-day inhalation period. Such an enhancement was still apparent 24 h after withdrawal, a time at which no more ethanol was present in the blood. Lipid peroxidation returned to normal values only 48 h after withdrawal. Microsomes were less affected than mitochondria by the ethanol treatment. It is suggested that the alterations of lipid peroxidation are related to the presence and/or the metabolism of ethanol at an early stage of inhalation, whereas changes in the membrane structure would be responsible for the maintenance of enhanced lipid peroxidation 24 h after ethanol withdrawal.

Learning deficits in first generation OF1 mice deficient in (n-3) polyunsaturated fatty acids do not result from visual alteration

The effects of (n-3) polyunsaturated fatty acids (PUFA) diet deficiency on learning, electroretinogram and retinal fatty acid composition were assessed for the first time in OF1 mice. Pups fed the same diets (deficient in alpha-linolenic acid or a control) as their dams were used aged 7 weeks for passive avoidance test and fatty acid analysis of retinal phospholipids. Visual function was measured by electroretinography in 4- and 7-week-old mice. The (n-3) PUFA-deficient diet significantly decreased learning performance and retinal docosahexaenoic acid level in adult mice. The electroretinogram showed a significant alteration of b-wave amplitude in deficient mice at 4 weeks but not at 7 weeks. These results show that learning deficits in mice fed a diet deficient in (n-3) PUFA were not due to visual alteration.

Uptake of dietary RRR-α- and RRR-γ-tocopherol by nervous tissues, liver and muscle in vitamin-E-deficient rats

The time course of RRR-alpha-tocopherol and RRR-gamma-tocopherol uptake by liver, muscle and selected nervous tissues was studied in vitamin-E-deficient rats fed diets containing either RRR-alpha-tocopherol or RRR-gamma-tocopherol over a 60 day period. Feeding rats with a RRR-alpha-tocopherol-supplemented diet induced in brain, cerebellum, sciatic endoneurium and muscle a marked and regular increase in alpha-tocopherol concentration. In addition, the tocopherol concentration in liver reached a plateau very rapidly. In contrast, feeding rats with a diet containing the same level of RRR-gamma-tocopherol induced a very small increase in gamma-tocopherol concentration in brain, cerebellum, sciatic endoneurium and muscle, no change in alpha-tocopherol concentration of brain and muscle and a slight but significant decrease in alpha-tocopherol concentration in sciatic endoneurium and cerebellum. These results indicate that when gamma-tocopherol was supplied continuously in the diet gamma-tocopherol accumulated significantly in the tissues but to a much lesser extent than when rats were fed with RRR-alpha-tocopherol. These results also show that in the tocopherol-deficient rat, gamma-tocopherol does not significantly affect the residual alpha-tocopherol concentrations in brain or cerebellum, except poorly in sciatic endoneurium.

Alteration of Δ-6 desaturase by vitamin E in rat brain and liver

Abstract Δ-6 Desaturase, measured at substrate saturation using linoleic acid, was found to be increased by more than two-fold when the content of vitamin E in brain microsomal membrane suspension was increased (up to 7.5 μg/mg membrane protein, i.e. 100 μg/g tissue from which microsomes were prepared). In contrast, this activity was reduced by 25% in the liver. This raises the question of the multiple role of vitamin E in membranes, the control of membrane polyunsaturated fatty acids through synthesis, and their protection against peroxidation.

Unaltered brain membranes after prolonged intake of highly oxidizable long-chain fatty acids of the (n-3) series.

Feeding rats a diet enriched in n-3 polyunsaturated fatty acids (Menhaden oil) increased the content in eicosapentaenoic acid 20:5 n-3 of brain phospholipids. Conversely 22:4 n-6 was reduced. These changes were not associated with alterations in either vitamin E concentration or glutathione peroxidase and catalase activities in cerebrum and cerebellum. No increase in peroxidative damage was found. Interestingly the major very-long-chain fatty acids (22:6 n-3 and 22:5 n-3) were not affected.