The influence of season on oxidant-antioxidant status in trained and sedentary subjects.

The association between oxidative stress and cardiovascular diseases is a widely accepted fact today. Generally, men have a higher risk of cardiovascular incidents and mortality from acute myocardial infarction and strokes. We have examined sport-associated circannual rhythms of oxidant and antioxidant processes by measuring plasma LPO, erythrocyte SOD, CAT, Gpx activity and plasma hormonal status in both sedentary and long-term trained men and women. We have shown seasonal variations in both oxidant and antioxidant status in all examined groups. The largest difference was observed in the oxidant status between sedentary men and women during autumn and winter, which is considered a period of high coronary risk for men. Sport decreased LPO in trained men in autumn, while the same effect in trained women was shifted towards summer. These data state that regular, long-term physical exercise training induces adaptive responses that confer protection against oxidative stress, as well as the beneficial effect of exercise with regard to season, particularly in men during a period of high coronary risk (autumn and winter, respectively) and in women during summer.

Met-enkephalin modulation of age-related changes in red cell antioxidant status.

Opioid peptides have been recognized as modulators of reactive oxygen species (ROS) in mouse macrophages and human neutrophils. Since the effect cannot be ascribed to its direct scavenger properties, in this study, we tested the hypothesis that methionine-enkephalin (MENK) modulates ROS by alteration of antioxidant enzyme activity (AOE). For this purpose superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) are measured in red blood cells of 1, 4, 10, and 18-month-old CBA mice of both sexes injected with 10 mg/kg MENK. The results indicate that MENK-affected antioxidant enzyme activity of red blood cells is age- but not sex-related. The most abundant effects were observed at the reproductive stage. Increased sensitivity to oxidative stress by opioid peptides was in both sexes mainly due to increased SOD activity followed by GPX decrease. Thus, the damage ascribed to opioid peptides might be, at least partly, ascribed to deleterious effects of accumulated hydrogen peroxide (H2O2).

Immunostimulatory effect of natural clinoptilolite as a possible mechanism of its antimetastatic ability.

PURPOSE: Many biochemical processes are closely related to ion exchange, adsorption, and catalysis. Zeolites reversibly bind small molecules such as oxygen or nitric oxide; they possess size and shape selectivity, the possibility of metalloenzyme mimicry, and immunomodulatory activity. These properties make them interesting for pharmaceutical industry and medicine. METHODS: The experiments were performed on mice. Different biochemical and molecular methods were used. RESULTS: Micronized zeolite (MZ) administered by gastric intubation to mice injected with melanoma cells significantly reduced the number of melanoma metastases. In mice fed MZ for 28 days, concentration of lipid-bound sialic acid (LSA) in serum increased, but lipid peroxidation in liver decreased. The lymphocytes from lymph nodes of these mice provoked a significantly higher alogeneic graft-versus-host (GVH) reaction than cells of control mice. After i.p. application of MZ, the number of peritoneal macrophages, as well as their production of superoxide anion, increased. However, NO generation was totally abolished. At the same time, translocation of p65 (NFkappaB subunit) to the nucleus of splenic cells was observed. CONCLUSION: Here we report antimetastatic and immunostimulatory effect of MZ and we propose a possible mechanism of its action.

The effect of methionine-enkephalin on nitric oxide release in mice is age and gender related.

Gender- and age-related differences in nitric oxide (NO) release and in response to drugs of abuse has been reported in both humans and experimental animals. So far, we have demonstrated in vivo methionine-enkephalin- (MENK-) modulated NO release in mice. However, no data on the influence of age and gender on this immunomodulatory effect of MENK have been reported. In this study we examined the influence of age (2, 4, 8 month old mice) and gender (male and female mice) on MENK-induced NO release of mouse peritoneal macrophages (PEMs) of the CBA strain of mice. NO release was not age but was gender related in that males generally produced more NO than females. The effect of MENK on NO release was age (demonstrated only in mature 4 and 8 month old mice) and gender related in that it could be observed only in female mice. Apoptotic cells that paralleled the increase of NO in MENK-treated female mice were, however, observed also in male mice although MENK was in males without effect. These data provide evidence that some immunomodulatory properties of MENK are age and gender related which may be relevant to the potential use of MENK in adjuvant therapy for immunocompromised status.

Effect of cisplatin and 6-bromo-6-deoxy-L-ascorbic acid on some biochemical and functional parameters in mice.

The results of the present study demonstrate that 6-bromo-6-deoxy-L-ascorbic acid (6-BrAA), an antioxidative derivative of ascorbic acid, is capable of lowering the toxicity of cisplatin, cis-diaminedichloroplatinum (cis-DDP), an anticancerogenic drug. The biological aspects and pharmacological significance of a combined treatment of these two substances were investigated in a mouse model. The results indicate that the effectiveness of 6-BrAA on biological response(s) is strongly dependent on the dose of cis-DDP. Injection of 10 mg/kg body weight (bw) of cis-DDP following pretreatment with 6-BrAA (480 mg/kg bw) enhances the tissue-protecting effect of 6-BrAA and reduces, to some extent, the ensuing nephro-, liver and spleen toxicity. On the other hand, 6-BrAA in animals treated with a higher dose of cis-DDP (15 mg/kg bw) leads to exacerbation of the toxic cis-DDP effect and concurrent loss of the protective potential of 6-BrAA with respect to tissue damage. The exact mechanism(s) of 6-BrAA protection and exacerbation of the toxic cis-DDP effect is unclear, although scavenging or generating of free radicals may play an important role. The results obtained may be of importance in planning the rational use of cis-DDP and 6-BrAA administration in the potential treatment of cancer.

In vitro inhibition of superoxide anion production and superoxide dismutase activity by zinc in human spermatozoa.

The in vitro effect of zinc on superoxide anion (O2-) generation and on SOD-like activity in spermatozoa of infertile men was investigated. The formation of superoxide anion was stimulated by NADPH and the level of superoxide anion was measured by the reduction of ferricytochrome c. Both Percoll-isolated (n = 14) and washed spermatozoa (n = 14) exposed to 1 mmol/L zinc (60 min, 37 degrees C), released less (p < 0.002 and p < 0.04, respectively) superoxide anions than did zinc-untreated spermatozoa. These results implicate a possible role for zinc as a scavenger of excessive superoxide anions produced by defective spermatozoa in semen after ejaculation. Additionally, zinc was found to dose-dependently inhibit superoxide dismutase (SOD)-like activity of spermatozoa in vitro. The inhibition of SOD-like activity by an equal concentration of zinc (1 mmol/L) was less pronounced in oligospermic (p < 0.002; n = 16) and asthenozoospermic (p < 0.0005; n = 20) than in normozoospermic samples (p < 0.0001; n = 20). This differential ability of zinc to inhibit SOD-like activity may be relevant to the physiological function of spermatozoa in fertilization. The evidence that zinc may elicit an inhibition of both superoxide anion production and SOD-like activity in human spermatozoa, indicate the existence of novel, zinc-related mechanism(s) involved in the oxidative events occurring after ejaculation, with a possible modulatory effect on germ cell function.

Influence of methionine-enkephalin on stress-induced parameters.

This study examines the influence of methionine-enkephalin (MENK) on stress-induced oxidative damage (lipid peroxidation; LPO) in mice liver homogenate, plasma corticosterone concentration (CS) and phagocytic activity of mouse splenocytes. The LPO value increased in the mice subjected to restraint stress and had no correlation to stress duration, while MENK had no effect. The CS concentration was enhanced after 6 h of stress and 6 h after injection of a low (2.5 mg/kg bw) dose of MENK. However, MENK and stress are adjunct modulators of LPO and corticosterone in vivo. LPO was additionally elevated when MENK (10 mg/kg bw) proceeded for 2 h after the onset of stress. However, corticosterone concentration seems to be regulated differently by the same dose of MENK depending on the duration of stress i.e. elevated in cases involving short periods of stress (2 h) and decreased in cases involving prolonged periods of stress (6 h). This modulation of LPO and corticosterone by 10 mg/kg bw of MENK and 2 h of restraint stress was paralleled with elevated phagocytosis.

Superoxide anion production and some sperm-specific enzyme activities in infertile men.

The relationship between superoxide anion production and the activities of sperm specific NADH-diaphorase and isozyme LDH-C4 has been investigated in 25 normozoospermic and 17 oligozoospermic semen samples. The samples were separated into high and low density sperm populations on discontinuous, two step (40%:80%) Percoll gradients prior to analysis. Superoxide anion generation was significantly higher (P < 0.001) and diaphorase activity significantly increased (P < 0.01) in both low and high density sperm fractions from oligozoospermic patients compared to the values found in normozoospermic men. A significant positive correlation (P < 0.01) between diaphorase activity and superoxide anion production was established in the oligozoospermic samples of low density sperm fraction. A tendency of statistically insignificant increase in LDH-C4 isozyme activity was observed in the oligozoospermic samples. The activity of this isozyme appears to have no association with superoxide anion generation or diaphorase activity. These findings seem to suggest that excessive superoxide anion production along with increased diaphorase activity point to biochemical abnormalities which may affect the functional status of sperms. Hence, determination of these parameters may provide useful data in male infertility of unknown etiology.

Side-effects of peptidoglycan monomer (PGM) treatment in mice.

The influence of single (4 mg/mouse) and multiple (1 mg/mouse per day for 5 consecutive days) injections of PGM on some hepatic enzymes, lipid peroxide generation in serum and liver, sialic acid concentration in serum and spleen and hepatic lysosomal membrane permeability was investigated. The studies performed showed that a single injection of PGM in vivo changed temporarily the permeability of lysosomal membranes, lipid peroxidation products and sialic acid concentration, and when administered in vitro modulated superoxide anion production and did not affect the activity of lysosomal membrane enzymes. Multiple injections of PGM did not cause significant changes in the examined parameters. Although the metabolic changes were time-limited and from the toxicological point of view, provoked transient effects, the results obtained may be of importance when using PGM in combined chemo-immunotherapy.

Effect of sialic acid on glycation-induced fluorescence of albumin.

The aims of this study were to determine whether in vitro nonenzymatic glycation of proteins by sialic acid (sialylation) induces the generation of fluorescence, and whether the presence of this keto sugar may affect the generation of fluorescence induced by other sugars. Incubation of bovine serum albumin (BSA; 1.5 mM) with sugars (50 mM in 0.2 M phosphate buffer, pH 7.4, at 37 degrees C) resulted in a time-dependent increase of formaldehyde release (moles/moles of protein). On the 17th day of incubation, the value was 0.53 +/- 0.06, 0.78 +/- 0.15 and 1.23 +/- 0.18 for sialic acid, fructose and glucose respectively, compared with 0.37 +/- 0.05 for BSA. The fluorescence intensity (arbitrary units/mg protein) was higher after 17 days of incubation with fructose (16.9 +/- 1.8) than with glucose (12.7 +/- 1.3), while no significant increase was noted with sialic acid compared with BSA (3.8 +/- 0.4). Fluorescence intensity increase by incubation with glucose (50 mM) was significantly reduced by sialic acid (20 mM) after both 10 (P < 0.001) and 14 (P < 0.001) days of incubation, while inhibition was weaker after 14 (P < 0.05) than after 10 (P < 0.001) days when fructose (50 mM) was used as the glycating agent. This indicates that sialic acid can be potentially used to limit the damage from adverse glycation-induced processes.

The effect of Met-enkephalin on mice liver lysosomes.

The unsedimentable activities of acid phosphatase (AP) and beta-glucosidase (BG), from mice liver lysosomes significantly increased 6 h after a single i/p injection of Met-enkephalin (MENK). The activity of AP in the serum at the same time remained unchanged. Multiple injections of MENK (8 x 10 mg/kg) induced a significant decrease in AP activity in the serum and no change in the unsedimentable activities of AP or BG. MENK did not elicit any significant extracellular release of lactate dehydrogenase (LDH) either, indicating that, under the experimental conditions described, the cells remained intact. Other parameters, such as the activities of AP and BG in the liver and total sialic acid content in the serum and spleen remained unaltered. Moreover, MENK in concentrations of 10(-12) M, 10(-8) M, 10(-6) M or 10(-4) M did not change the activities of the lysosomal enzyme markers AP or BG in vitro. These data indicate far less pronounced transient effects of MENK on lysosomal membranes and enzymes compared to Leu-enkephalin which may be relevant for the use of MENK in combined chemo-immunotherapy.

Modulation of superoxide anion release from human polymorphonuclear cells by Met- and Leu-enkephalin.

The present work describes the ability of Met- and Leu-enkephalin to modulate the superoxide anion (O2-) release from unstimulated human polymorphonuclear cells (PMN) and from PMN stimulated with phorbol myristate acetate (PMA). The direction (stimulation or suppression) and the magnitude of change were dependent upon the baseline reactivity of the donor's PMN. Both opioid peptides stimulated O2- release by PMN from donors with low baseline reactivity in a concentration-dependent manner. PMNs collected from donors with medium baseline reactivity incubated with Leu-enkephalin regardless of concentration released less O2- than control, nontreated PMNs. Met-enkephalin stimulated O2- release but only at 2 X 10(-15) M concentration. Superoxide anion release from PMNs of individuals with high baseline reactivity was concentration dependent and suppressed by Met- and Leu-enkephalin. Leu-enkephalin induced baseline reactivity was dependent upon progressive increase in the magnitude of change on O2- release (i.e., the higher the baseline the higher the magnitude of change in O2- generation). Met-enkephalin data show this also, but to a lesser extent. In cells stimulated with PMA, Met-enkephalin caused additional O2- release, while Leu-enkephalin was ineffective in triggering already stimulated cells. The modulating effect of both opioid peptides on superoxide anion release by human PMN is a short phenomenon that lasts up to 10 min after the addition of the peptide.

Immune response of stressed rats treated with drugs affecting serotoninergic and adrenergic transmission.

Stressful conditions interfere with immune response. One of the principal mechanisms is activation of hypothalamo-pituitary-suprarenal axis by central serotoninergic and adrenergic pathways. Alternative mechanisms bypassing the axis also take part in stress-induced immunomodulation. Immunosuppression caused by repeated restraints or over-crowding was usually accompanied by increased metabolism of serotonin in the brain (as indicated by increased level of its metabolite, 5-HIAA) and by increased levels of corticosterone in plasma. Changes in lymphatic tissues of stressed animals that result in suppression of immune response apparently "outlive" fluctuating changes in neurotransmitter and corticosterone levels. Drugs that alter serotoninergic or adrenergic transmission interfere with immunosuppressive effect of stress either synergistically (augmenting suppression) or antagonistically (preventing it). Since immunocompetent cells possess serotoninergic and adrenergic receptors, such drugs may exert their effect either via central neuroendocrine mechanisms, or by direct effects on immunocompetent cells.

Leu-enkephalin induced alteration of enzymes and sialic acid levels in vivo.

The influence in mice of 10 mg/kg Leu-enkephalin (LENK) on the activity of hepatic enzymes in serum and liver and sialic acid concentration in serum and spleen is described. The activity of acid and alkaline phosphatase and lactate dehydrogenase in serum was significantly enhanced 6 h after a single (10 mg/kg) LENK injection. This effect was not dose-dependent. The increase of acid phosphatase in serum was accompanied by a simultaneous decrease in the liver. While the level of serum sialic acid remained unchanged, it decreased in the spleen 6 h after a single LENK injection. The effect of LENK in the spleen and liver was dose-dependent; ie, the higher the dose the greater the effect. Multiple injections of Leu-enkephalin (6 x 10 mg/kg) induced the same degree of increase but an earlier rise in serum alkaline, acid phosphatase and lactate dehydrogenase levels than with a single injection. This was not accompanied by a change of activity in liver and spleen. These data may be relevant for use of LENK in combined chemo-immunotherapy, since liver enzyme changes may alter drug metabolism, and since sialic acid plays a regulatory role in immune processes.

Suppression of immune response in rats by stress and drugs interfering with metabolism of serotonin.

Rats immunized with sheep erythrocytes were stressed by repeated restraint and/or treated with a precursor of serotonin (5-hydroxytryptophan, 5-HTP) or with an inhibitor of serotonin synthesis (parachlorophenylalanine, PCPA). As expected, repeated stress reduced the plaque-forming cell (PFC) response. Treatment with 5-HTP also reduced the PFC response, and potentiated the immunosuppressive effect of stress. This was accompanied by increased metabolism of serotonin in the brain, as indicated by increased concentration of its metabolite, 5-hydroxyindoleacetic acid (5-HIAA) in cerebral tissue. Treatment with PCPA also suppressed the PFC response, but this suppression was accompanied by decreased levels of brain serotonin and of 5-HIAA. Plasma corticosterone levels were elevated, reaching statistical significance, in rats treated with PCPA. Both drugs suppressed the in vitro PFC response of peripheral blood lymphocytes. Thus, although 5-HTP and PCPA altered serotonin metabolism in the brain in a diametrically opposite manner, their effects on the immune response were the same. Putative central effects of the drugs on serotoninergic regulation of the immune response were apparently obscured by their effects on corticosterone secretion as well as by their direct effects on immunocompetent cells.

Influence of the thymus extract on the immunological function of animals with experimental diabetes.

After immunization with SRBC, the number of plaque-forming cells (PFC) in the spleen of alloxan-diabetic mice, in nondiabetic TIR mice and in alloxan-diabetic TIR mice was significantly decreased as compared with control non-diabetic donors. The ability of lymphocytes from alloxan-diabetic mice to adoptively restore the suppressed immune response of TIR mice, was reduced in comparison with the effect of lymphocytes from normal, nondiabetic donors. Local GVH reaction in nondiabetic rat recipients provoked by lymphocytes from control healthy mice was 5.6 +/- 0.7 mm. Significantly lower rate of local GVH reaction after injection of lymphocytes from diabetic donors was found in diabetic as in nondiabetic recipients as well. Treatment of alloxan-diabetic mice with thymus extract or with insulin, partly restored depressed function of the humoral and cellular system. Treatment of diabetic mice with both thymus extract and insulin, was even more effective in restoring of their immune reactivity. Diabetic condition strongly influenced the function of the immune system. This could be attributed to depletion of T-lymphocytes, changed relations between the lymphocyte subpopulations in diabetic donors, and disturbance of lymphocyte metabolism.