C. P. Reddy Avula

Modulation of Antioxidant Enzymes and Apoptosis in Mice by Dietary Lipids and Treadmill Exercise

The current experiments were designed to study the effect of dietary n-6 and n-3 polyunsaturated fatty acids on antioxidant enzyme activity and dexamethasone (DEX)-induced apoptosis in spleen cells of sedentary (Sed) and treadmill-exercised (Ex) ICR male mice. Two-month-old mice maintained on AIN 76 formula diet, supplemented with either 5% corn oil (CO) or 5% fish oil (FO) diets, were trained on a treadmill to run from 45 to 50 min 1 km/day, 6 days a week for 12 weeks. After 12 weeks of exercise, both Sed and Ex groups were sacrificed. Blood and various tissues, including spleen, were collected asceptically. Increased serum and spleen homogenate peroxide [malondialdehyde (MDA)] levels were observed in mice fed FO (n-3 PUFA) diets, compared to mice fed CO (n-6 PUFA). However, exercise did not alter MDA levels in either CO- or FO-fed mice. Feeding n-3 PUFA significantly increased superoxide dismutase (SOD), catalase, and glutathione peroxidase activity of spleen homogenates. Exercise also significantly increased SOD and peroxidase in CO-fed animals, whereas catalase, glutathione peroxidase, and glutathione transferase were higher in FO-fed mice, compared to the Sed group. Apoptosis and necrosis were quantitated in splenocytes incubated with or without 1 μM Dex in RPMI medium for 8 and 24 hr. Cells were stained with Annexin V and propidium iodide (PI) for apoptotic and necrotic cells. FO-fed mice showed higher apoptosis (64 vs 50%) and necrosis (40 vs 22%) in spleen cells than CO-fed mice. Cells from FO-fed mice, incubated in medium alone, showed increased apoptosis (112%) 24 hr after incubation, and necrosis (37 and 70%) at 8 and 24 hr of incubation, compared to CO-fed mice. In Ex group, apoptosis was increased in both CO- and FO-fed mice only at 24 hr after incubation. In summary, these results indicate that FO (n-3 PUFA-enriched) diets increase apoptosis and antioxidant enzyme activity in spleen cells, probably due to elevated lipid peroxides.

Induction of apoptosis and apoptotic mediators in balb/C splenic lymphocytes by dietary n−3 and n−6 fatty acids

The present study was designed to investigate the effect of diatery n−6 and n−3 polyunsaturated fatty acids (PUFA) on anti-CD3 and anti-Fas antibody-induced apoptosis and its mediators in mouse spleen cells. Nutritionally adequate semipurified diets containing either 5% w/w corn oil (n−6 PUFA) or fish oil (n−3 PUFA) were fed to weanling female Balb/C mice, and 24 wk later mice were sacrificed. In n−3 PUFA-fed mice, serum and splenocyte lipid peroxides were increased by 20 and 28.3% respectively, compared to n−6 PUFA-fed mice. Further, serum vitamin F levels were decreased by 50% in the n−3 PUFA-fed group, whereas higher anti0Fas- and anti-CD3-induced apoptosis (65 and 66%) and necrosis (17 and 25%), compared to the n−6 PUFA-fed group, were found when measured with Annexin V and propidium iodide staining, respectively. In addition, decreased Bcl-2 and increased Fas-ligand (Fas-L) also were observed in the n−3 PUFA-fed group compared to the n−6 PUFA-fed group. No difference in the ratio of splenocyte subsets nor their Fas expression was observed between the n−3 PUFA-fed and n−6 PUFA-fed groups, whereas decreased proliferation of splenocytes was found in n−3 PUFA-fed mice compared to n−6 PUFA-fed mice. In conclusion, our results indicate that dietary n−3 PUFA induces higher apoptosis by increasing the generation of lipid peroxides and elevating Fas-L expression along with decreasing Bcl-2 expression. A reduced proliferative response of immune cells also was observed in n−3 PUFA-fed mice.

Modulation of antioxidant enzymes and lipid peroxidation in salivary gland and other tissues in mice by moderate treadmill exercise

The current experiments were designed to study the effect of moderate treadmill training exercise on lipid peroxides and antioxidant enzyme activity in various tissues of ICR mice. Three-month-old female mice were trained on a treadmill to run daily from 45 to 50 minutes, at 1 km per hour, 6 days a week for a total of 8 weeks. At the end of the 8-week endurance-training period, both sedentary control (SC) and exercise-trained (ET) mice were sacrificed, and various tissues were collected to measure antioxidant enzyme activity. The results showed weight gain and serum lipid peroxides significantly decreased in ET mice compared to SC mice. Also, although lipid peroxide levels in kidney and salivary glands were found to be significantly decreased in ET mice, these mice showed higher lipid peroxide levels in the liver compared to SC mice. No change was observed in heart and calf muscle tissue of the ET mice. Exercise was also noted to increase superoxide dismutase (SOD) activity in kidney, heart, and calf muscle homogenates. Increases in catalase activity were present in liver, heart, calf muscle, and salivary gland homogenates of ET mice compared to their SC counterparts. Exercise was also shown to increase glutathione peroxidase activity in liver, kidney, and heart homogenates, as well as glutathione transferase activity in liver and salivary gland homogenates. In addition, exercise training was found to increase reduced and total glutathione levels in heart, calf muscle, and salivary gland. These results indicate that moderate exercise is beneficial to the lowering of lipid peroxides and the increasing of antioxidant enzyme activity specifically in the salivary gland, and also in various organs. However, its beneficial effect on elevation of antioxidant enzymes and suppression of lipid peroxide varies from organ to organ.

Calorie restriction increases Fas/Fas-ligand expression and apoptosis in murine splenic lymphocytes

One‐month‐old male ICR mice were fed a nutritionally adequate, semipurified diet, either ad libitum (AL) or calorie restricted (CR) (40% less food) for 6 months and were killed to obtain spleens. Flow cytometric analysis revealed increased proportions of both CD4+ and CD8+ T cells in CR‐fed mice compared to AL‐fed mice. The T cell subsets of CR‐fed mice were also found to have higher levels of plasma membrane Fas receptor expression. Similarly, Fas‐ligand (Fas‐L) expression was higher in anti‐CD3‐stimulated CD4+ and CD8+ T cells. CR‐fed mice also had increased numbers of annexin V‐positive CD4+ and CD8+ T cells in stimulated splenic lymphocytes suggesting an increased potential for apoptosis. Fas and Fas‐L gene expression in splenic lymphocytes, which correlated closely with the observed increased rate of apoptosis, was significantly increased in CR‐fed mice compared to AL‐fed mice. In conclusion, these results indicate that CR increases the expression of Fas and Fas‐L which may contribute to the known beneficial effects of CR such as prolongation of life span by activating chronic physiologically mediated apoptosis.

Inhibition of Intracellular Peroxides and Apoptosis of Lymphocytes in Lupus-Prone B/W Mice by Dietary n-6 and n-3 Lipids with Calorie Restriction

Our earlier studies have shown that calorie restriction and n-3 fatty acids inhibit autoimmune disease and prolong life span. Experiments were designed to study the alteration of apoptosis and its mediators in B/W mice fed either n-6 fatty acids [5% corn oil (CO)] or n-3 fatty acids [5% fish oil (FO)] and either allowed access to the diet ad libitum (AL) or restricted in caloric intake by 40% (CR), from 4 weeks of age. At 4 months (young) and 9 months (old) mice were killed, splenic lymphocytes were isolated, and apoptosis was measured with Annexin V and PI staining. Apoptosis was decreased in splenic lymphocytes from both young and old CR mice compared to their respective AL-fed control groups regardless of fat source. Increasing apoptosis with age was observed in CO/AL, CO/CR, and FO/AL mice which correlated closely with significantly higher cellular peroxides measured by flow cytometry using dichlorofluourescein diacetate (DCFH-DA), whereas in both CO/CR and FO/CR peroxide levels remained low in old mice. Furthermore, CR increased the proliferative response of splenic lymphocytes and decreased the Fas (CD95) and Fas-L protein expression in CD4+ lymphocytes from old mice. Higher levels of Fas and Fas-L expression were observed in old mice compared to young mice. Bcl-2 levels were elevated in both young and old CR groups compared to the respective AL groups. Calorie restriction prevented the loss of CD8 cells in old mice fed both the CO and the FO diet. In summary, CR resulted in decreased apoptosis accompanied by alterations in Fas, Fas-L, and Bcl-2 expression in old mice, increased life span, and delayed onset of kidney disease.

Effect of dietary vitamin E on apoptosis and proliferation of murine splenocytes

Abstract Vitamin E (Vit E) is one of the most potent natural lipophilic antioxidants shown to inhibit proliferation of cancer cells and influence apoptosis in in-vitro systems. In the present investigation, we studied the effect of dietary Vit E on splenocyte proliferation, apoptosis, and necrosis. Two-month old male ICR mice were fed an AIN tm 76 formula diet for three months with 10% (W/W) corn oil, supplemented with Vit E at normal (75 IU/kg) or higher (500 IU/kg) levels, or without Vit E as zero Vit E group. In vivo analysis revealed both serum lipid peroxides (MDA) and splenocyte peroxides (dichlorofluorescein staining) were found to be reduced with increased dietary Vit E levels in a dose-dependent manner; whereas, serum Vit E levels were found to be augmented with increased levels of dietary Vit E. Also, apoptosis and necrosis were measured in splenocytes incubated with and without 1 μM dexamethasone (DEX) in RPMI media for eight and 21 hours. Cells were stained with Annexin V and Propidium Iodide (PI), and both apoptotic and necrotic cells were analyzed simultaneously by a FACScan desktop flow cytometer using the Cell Quest Program. High and normal levels of Vit E increased necrosis by 29 and 18% in the cells placed in media, and 67 and 34% in cells incubated with DEX for eight hours, respectively, in comparison to that in the zero Vit E-fed group. However, in splenocytes from the high Vit E group apoptosis was decreased by 32 and 49.5% in cells incubated for eight hours in media alone or with DEX, respectively. Furthermore, in the high Vit E group necrosis was increased by 25% and apoptosis was decreased by 17% in cells incubated for 21 hr in media alone, compared to that of the zero Vit E group. In addition, proliferation of splenocytes, either stimulated or unstimulated with mitogens, was also found to be decreased with increasing levels of dietary Vit E. In conclusion, our results indicate that dietary Vit E appears to alter both cell proliferation and programmed cell death (apoptosis vs. necrosis), by lowering lipid peroxides.