Diminished Resistance to Hyperoxia in Brains of Reproductively Senescent Female CBA/H Mice.

BACKGROUND We have explored sex differences in ability to maintain redox balance during acute oxidative stress in brains of mice. We aimed to determine if there were differences in oxidative/antioxidative status upon hyperoxia in brains of reproductively senescent CBA/H mice in order to elucidate some of the possible mechanisms of lifespan regulation. MATERIAL AND METHODS The brains of 12-month-old male and female CBA/H mice (n=9 per sex and treatment) subjected to 18-h hyperoxia were evaluated for lipid peroxidation (LPO), antioxidative enzyme expression and activity - superoxide dismutase 1 and 2 (Sod-1, Sod-2), catalase (Cat), glutathione peroxidase 1 (Gpx-1), heme-oxygenase 1 (Ho-1), nad NF-E2-related factor 2 (Nrf2), and for 2-deoxy-2-[18F] fluoro-D-glucose (18FDG) uptake. RESULTS No increase in LPO was observed after hyperoxia, regardless of sex. Expression of Nrf-2 showed significant downregulation in hyperoxia-treated males (p=0.001), and upregulation in hyperoxia-treated females (p=0.023). Also, in females hyperoxia upregulated Sod-1 (p=0.046), and Ho-1 (p=0.014) genes. SOD1 protein was upregulated in both sexes after hyperoxia (p=0.009 for males and p=0.011 for females). SOD2 protein was upregulated only in females (p=0.008) while CAT (p=0.026) and HO-1 (p=0.042) proteins were increased after hyperoxia only in males. Uptake of 18FDG was decreased after hyperoxia in the back brain of females. CONCLUSIONS We found that females at their reproductive senescence are more susceptible to hyperoxia, compared to males. We propose this model of hyperoxia as a useful tool to assess sex differences in adaptive response to acute stress conditions, which may be partially responsible for observed sex differences in longevity of CBA/H mice.

Antitumor effect of Croatian propolis as a consequence of diverse sex-related dihydropyrimidine dehydrogenase (DPD) protein expression.

The aim of this study was to detect the antitumor properties of Croatian propolis in BALB/c male and female mice injected with 4T1 mammary carcinoma. Furthermore, the gender-dependence of this effect and the possible involvement of combined effect of propolis and 5-Fluorouracil (5FU) on dihydropyrimidine dehydrogenase (DPD) transcriptional and translational level, were determined. In combination with 5FU propolis treatment induced gender-related effects. The results of the study revealed that pretreatment of mice with propolis combined with 5FU treatment prolonged the suppressive effect of 5FU on tumor growth and reduced the number of metastasis only in male mice. Only males pretreated with propolis prior to 5FU administration had decreased DPD protein level indicating higher sensitivity to 5FU. Thus, benefitial effects of propolis in male tumor-bearing mice treated with 5FU might be explained by increased sensitivity to 5FU as the result of translationally downregulated DPD.

Endomorphin-suppressed nitric oxide release from mice peritoneal macrophages.

Endomorphins are newly discovered mu-opioid receptor selective immunocompetent opioid peptides. Endomorphin 1 is predominantly distributed in brain, while endomorphin 2 is widely allocated in the spinal cord. Lately, endomorphins have been investigated as modulators of reactive oxygen and nitrogen species. Nitric oxide is short lived radical involved in various biological processes such as regulation of blood vessel contraction, inflammation, neurotransmission and apoptosis. The aim of this work was to investigate the in vivo effects of endomorphins on nitric oxide release and NOS 2 isoenzyme upregulation in mice peritoneal macrophages additionally challenged ex vivo with lipopolysaccharide. The results showed that endomorphin 1 or endomorphin 2 in vitro did not change NO release from peritoneal mouse macrophages during a 48 h incubation period. On the other hand in vivo endomorphins had suppressive effect on NO release as well as on NOS 2 and IL-1 protein concentration. The most of suppressive effect in vivo of both endomorphins was blocked with 30 min pretreatment with mu-receptor selective antagonist beta-FNA, which proved involvement of opioid receptor pathway in suppressive effects of endomorphins.

Improved antioxidant and anti-inflammatory potential in mice consuming sour cherry juice (Prunus Cerasus cv. Maraska).

The present investigation tested the in vivo antioxidant efficacy (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase; Gpx), lipid peroxidation (LPO) and anti-inflammatory properties (cyclooxygenase-2; COX-2) of sour cherry juices obtained from an autochthonous cultivar (Prunus cerasus cv. Maraska) that is grown in coastal parts of Croatia. Antioxidant potential was tested in mouse tissue (blood, liver, and brain), LPO (liver, brain) and anti-inflammatory properties in glycogen elicited macrophages. Additionally, the concentration of cyanidin-3-glucoside, cyanidin-3-rutinoside, pelargonidin-3-glucoside, pelargonidin-3-rutinoside and total anthocyanins present in Prunus cerasus cv. Maraska cherry juice was determined. Mice were randomly divided into a control group (fed with commercial food pellets) and 2 experimental groups (fed with commercial food pellets with 10% or 50% of cherry juice added). Among the anthocyanins, the cyanidin-3-glucoside was present in the highest concentration. These results show antioxidant action of cherry juice through increased SOD (liver, blood) and Gpx (liver) activity and decreased LPO concentration. The study highlights cherry juice as a potent COX-2 inhibitor and antioxidant in the liver and blood of mice, but not in the brain. Thus, according to our study, Prunus cerasus cv. Maraska cherry juice might potentially be used as an antioxidant and anti-inflammatory product with beneficial health-promoting properties.

Antioxidant effects of flavonoid from Croatian Cystus incanus L. rich bee pollen.

Oxidant/antioxidant status, estrogenic/anti-estrogenic activity and gene expression profile were studied in mice fed with Cystus incanus L. (Cistaceae) reach bee pollen from location in Central Croatia's Dalmatia coast and offshore islands. Seven phenolic compounds (out of 13 tested) in bee pollen sample were detected by high performance liquid chromatography (HPLC) analysis. Phenolics detected in C. incanus L. bee pollen belong to flavonol (pinocembrin), flavanols (quercetin, kaempferol, galangin, and isorhamnetin), flavones (chrysin) and phenylpropanoids (caffeic acid). Bee pollen as a food supplement (100mg/kgbw mixed with commercial food pellets) compared to control (commercial food pellets) modulated antioxidant enzymes (AOE) in the mice liver, brain and lysate of erythrocytes and reduced hepatic lipid peroxidation (LPO). Bee pollen induced 25% of anti-estrogenic properties while no estrogenic activity was found. Differential gene expression profile analyses after bee pollen enriched diet identify underexpressed gene Hspa9a, Tnfsf6 (liver) and down-regulated gene expression of Casp 1 and Cc121c (brain) which are important in the apoptosis pathway and chemotaxis. These results indicate that used bee pollen possess a noticable source of compounds with health protective potential and antioxidant activity.

Differential response to lipid peroxidation in male and female mice with age: correlation of antioxidant enzymes matters.

The aim of this study was to correlate the activity of superoxide dismutase, catalase and glutathione peroxidase in liver and brain of 1, 4 and 18 months old CBA mice of both sexes. In liver, decreased superoxide dismutase and increased glutathione peroxidase activities were observed during aging in male mice. In brain, the increase of catalase and glutathione peroxidase activity during aging was observed only in female mice. Regardless of tissue examined, different sex-related correlation pattern of antioxidant enzyme activity was demonstrated in young and old mice. The cooperation between antioxidant enzymes becomes more coherent with increased lipid peroxidation concentration in liver and brain of older female mice. On the contrary, in older male mice the link among three antioxidant enzymes becomes weaker, regardless of lipid peroxidation concentration which increased in liver and decreased in brain during aging. In older mice lower partial coefficient of correlation than pair correlation demonstrates the influence of the third party in the cooperation of two antioxidant enzymes. The results imply stronger correlative links in old female than male mice, which might explain why old females are better protected from oxidative stress than males.

Oxidant/antioxidant properties of Croatian native propolis.

Native propolis was defined as propolis powder collected from the continental part of Croatia and prepared according to a patented process that preserves all the propolis natural nutritional and organoleptic qualities. Nine phenolic compounds (out of thirteen tested) in propolis sample were detected by high performance liquid chromatography (HPLC) analysis. Among them chrysin was the most abundant (2478.5 microg/g propolis). Contrary to moderate antioxidant activity of propolis examined in vitro (ferric reduction antioxidant power; FRAP-assay), propolis as a food supplement modulated antioxidant enzymes (AOE) and significantly decreased lipid peroxidation processes (LPO) in plasma, liver, lungs, and brain of mice. The effect was dose- and tissue-dependent. The lower dose (100 mg/kg bw) protected plasma from oxidation, whereas the higher dose (300 mg/kg bw) was pro-oxidative. Hyperoxia (long-term normobaric 100% oxygen) increased LPO in all three organs tested. The highest vulnerability to oxidative stress was observed in lungs where hyperoxia was not associated with augmentation of AOE. Propolis protected lungs from hyperoxia by increased catalase (CAT) activity. This is of special importance for lungs since lungs of adult animals are highly vulnerable to oxidative stress because of their inability to augment AOE activity. Because of its strong antioxidant and scavenging abilities, native propolis might be used as a strong plant-based antioxidant effective not only in physiological conditions but also in cases that require prolonged high concentration of oxygen.

Methionine-enkephalin modulated regulation of oxidant/antioxidant status in liver of CBA mice.

Reactive oxygen species (ROS) are formed by all aerobic organisms, and are involved in the numerous physiological and pathophysiological processes. Opioid peptides belong to a class of bioactive compounds of great interest because of their opiate-like activity. We determined the influence of methionine-enkephalin (MENK) on age-associated oxidant/antioxidant status in liver of CBA mice. Lipid peroxidation (LPO), total superoxide dismutase (tSOD), catalase (CAT), and glutathione peroxidase (Gpx) activities of 1, 4, 10 and 18 months old male and female control and MENK treated (10 mg/kg bw) CBA mice were determined. MENK showed gender-related effect on both oxidant/antioxidant parameters. It stimulated LPO in males, but suppressed in females. CAT and Gpx activities were lowered upon MENK exposure in males, but in females the activities were modulated by MENK. The relative mRNA levels for the antioxidant enzymes CuZnSOD, MnSOD, CAT and Gpx-1 were determined by reverse transcriptase polymerase chain reaction (RT-PCR) in groups where differences in activities between control and treated samples were observed. Changes of mRNA level in MENK treated groups showed that transcriptional regulation is both gender- and age-related. Comparison of enzyme activities and mRNA levels in control and MENK treated groups showed that, in some cases parallel changes occurred, while in other cases nonparallel changes were found. These results suggest that transcriptional changes are in accordance with enzyme activities in some cases, while in other cases posttranscriptional regulation of antioxidant enzymes may exist.

Met-enkephalin modulates resistance to oxidative stress in mouse brain.

The purpose of this study was to investigate the effect of opioide peptide Met-enkephalin (MENK) on resistance to oxidative stress in the brain of 4, 10 and 18 months old CBA mice of both sexes. This was done by determination of oxidant status via lipid peroxidation (LPO) and antioxidant status by determination of total superoxide dismutase (tSOD), catalase (CAT) and glutathione peroxidase (Gpx). Results showed that brain of adult male mice is less resistant to oxidative stress than brain of adult females. The difference is mainly due to higher CAT activity and lower LPO activity in female brain. MENK decreased resistance to stress in the brain of both sexes but the effect appeared earlier in males (10 months of age) than in females (18 months of age). Also, MENK could pronounce its effect on resistance to oxidative stress in a gender-related manner: in female mice via regulation of antioxidant enzyme activities and in male mice via regulation of oxidant processes respectively.

Age and gender differences in antioxidant enzyme activity: potential relationship to liver carcinogenesis in male mice.

The present study was undertaken to determine whether age- and gender-related changes in lipid peroxidation (LPO) were attributable to differences in hepatic antioxidant defense mechanisms of aging 1-, 4-, 10-or 18-month-old male and female CBA mice. Specifically, total superoxide dismutase (tSOD), glutathione peroxidase (Gpx) and catalase (CAT) activities were examined. As an indicator of liver oxidative damage, we determined LPO, expressed in terms of thiobarbituric acid reactive substances (TBARS). LPO increased in both sexes with age. tSOD seems to be a relatively inert antioxidative enzyme in both sexes of mice. The main changes in antioxidant capacity of mice liver during aging were associated with sex-related CAT and Gpx increments observed in males but not in females. Surprisingly, more than 60% of 18-month-old males (but none of females) which started to appear at 10-months developed hepatic tumors. The results show that (1) the increased liver antioxidant capacity of CAT and Gpx in male mice might be a sign of oxidative stress; (2) the increase in CAT and Gpx activities in male mice is strongly correlated with incidence of hepatic tumors; (3) the significantly increased SOD activity in tumor-bearing mice might have induced damage with accumulated hydrogen peroxide H2O2.

Sex-dependent antioxidant enzyme activities and lipid peroxidation in ageing mouse brain.

We investigated whether oxidant status and antioxidant enzyme activities during ageing of mouse brain are regulated in sex-dependent manner. In the homogenate from the brain of 1, 4, 10 and 18 months old male and female CBA mice, lipid peroxidation (LPO), total superoxide dismutase (tSOD), catalase (CAT) and glutathione peroxidase (Gpx) were determined. LPO was age- and sex-related, favoring males over females throughout the lifespan with the peak in both sexes at 10 months of age. Throughout ageing, no difference in tSOD activity between male and female brains was observed, except in immature 1 month old mice. Gender-related difference in Gpx activity was observed, with higher level in females comparing to males, reaching statistical significance in senescent (18 months old) animals. CAT activity was drastically changed with ageing in both the male and female brain. We found different age associated trends in CAT activity in males and females: decreased with age in males and increased with age in females. Taken together, the present findings indicate that brains of female mice have lower oxidant and higher antioxidant capacity mostly related to CAT and to a lesser extent to Gpx activity.

Enkephalin degradating enzymes in pheochromocytoma patients.

Adrenal gland as a major source of enkephalins on the periphery can be affected by a rare adrenal gland tumor, adrenal pheochromocytoma. It has been demonstrated that this tumor might be associated with altered concentration of enkephalin-like peptides. The effect of these peptides can be either prolonged or abbreviated by two neutrophil membrane bound enzymes; aminopeptidase N (APN) and neutral endopeptidase (NEP). We assumed that altered enkephalin level in pheochromocytoma patients (but not in patients with non-functional adenomas or tumors of different origin) might result in differently regulated APN and/or NEP activity. We measured APN and NEP activity on surface of neutrophils, level of lipid peroxidation (LPO) in plasma and enkephalin concentration in plasma in patients with pheochromocytomas, non-functional adenomas, malignant renal tumors and healthy controls. Catheholamines and vanyllmandelic acid (VMA) were measured in 24-h urine of pheochromocytoma patients. NEP and APN activity on neutrophils from all pheochromocytoma patients was significantly increased as compared with healthy controls, non-functional adenomas and malignant renal tumors. In all pheochromocytoma patients NEP activity was reduced almost to the control level after surgery. At the same time APN activity was in some patients up- and in others down-regulated. In comparison, elevated levels of cateholamines and VMA were found after multiple determinations in 6 out of 10 pheochromocytoma patients. Although preliminary, this study has shown specifically and consistently up-regulated NEP activity on neutrophils from pheochromocytoma patients, and uniformly decreased NEP activity in these patients after adrenalectomy.

Age-associated alteration of lipid peroxidation and superoxide dismutase activity in CBA and AKR mice.

Oxidative modification of lipids, proteins and DNA by reactive oxygen species in the organism and imbalance between the concentrations of free radicals and the antioxidant defenses may be related to processes such as aging and diseases (cardiovascular, neurodegenerative, cancer, etc.). Although the relationship between oxidant status and antioxidant defence in aging of different species, organs or sexes has been investigated extensively, the studies have produced conflicting results. In order to determine the extent of age-associated alteration, oxidant production and antioxidant status were measured in tissues of CBA and AKR mice of both sexes. At the same time we will focus on lipid peroxidation (LPO) process and superoxide dismutase activity (SOD) of AKR mice related to ontogeny of thymic lymphoma in mice of different age and sex. Male and female CBA and AKR mice aged 3, 6, 12 or 18 months were used. Lipid-bound sialic acid (LSA) content was determined as a malignancy marker. LPO processes of CBA and AKR mice were monitored according to the presence of thiobarbituric acid reactive substances (TBARS) in liver and thymus, and antioxidant status as SOD activity in whole blood. TBARS concentration increased significantly with age in the liver of CBA and AKR mice of both sexes, but only in male thymuses of both strains. TBARS concentration in female thymuses of both strains was unchanged during aging. Thus, age-associated LPO processes of tumor-free mice of both strains were tissue-dependent. In the liver of tumor-bearing CBA and AKR mice as well as in thymuses of AKR mice, TBARS concentration significantly decreased and was neither sex nor tissue related. SOD activity was strain-dependent but independent of sex. However, SOD activity in mice with developed thymomas was drastically reduced in comparison to tumor-free mice. Our data indicate that age associated LPO processes in both strains are only tissue-dependent and SOD activity mainly strain-dependent in tumor-free mice. In tumor-bearing mice LPO processes and SOD activity were not tissue, sex or strain dependent.

Met-enkephalin modulates lipid peroxidation and total sialic acid level in CBA mice in age- and sex-dependent manners.

Age- and sex-associated differences in lipid peroxidation (LPO), and total sialic acid content (TSA) in response to abuse of drugs have been reported both in humans and experimental animals. However, no data on the influence of gender and age on these parameters have been reported for methionine-enkephalin (MENK). In this study we examined the influence of age and gender on MENK-induced LPO levels in the liver and TSA content in splenocytes of CBA mice. LPO production, which was age- and gender-associated was differentially regulated by MENK at a dose of 10 mg or 2.5 mg/kg body weight. At the higher dose, MENK stimulated LPO production in younger males and females but suppressed only in older male mice. At the lower dose, MENK induced strong suppression in males while being without any effect in females. In TSA levels, the age-associated increase was greater in males and much lower in females, with higher TSA levels in younger (2.5, 4.5 months) and decreased levels in older female mice (9 months) being observed. Contrary to the effect on LPO level, TSA level in MENK-treated mice was suppressed in both sexes but only in young 2.5-month-old mice. These data provide evidence that some immunomodulatory properties of MENK are age- and gender-associated which may be relevant to the potential use of MENK as adjuvant therapy in patients with immunocompromised status.