Sandra Sobočanec

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 Croatias 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.

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.

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. Suprisingly, 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.

Cyp4a14 overexpression induced by hyperoxia in female CBA mice as a possible contributor of increased resistance to oxidative stress

Abstract The beneficial effects of hyperoxia have been noted in treatment of several diseases and pathological states. However, the excessive production of ROS under hyperoxic conditions can directly damage cellular macromolecules if the imbalance in antioxidant status exists. Cytochrome P450 (Cyp) 4a14 has an important role in the metabolism of lipids and as a source of ROS in oxidative stress. This study investigated the oxidant/antioxidant status as a response to hyperoxia treatment in liver of young CBA/Hr mice of both sexes and whether the observed response is mediated by Cyp4a14 via PPAR isoforms in a sex-dependent manner. The overexpression of Cyp4a14, lack of both LPO and of 4-hydroxynonenal(HNE)-protein adducts revealed by immunohistochemical analysis in hyperoxia-treated females indicates their greater resistance to hyperoxia compared to males, which is parallelled to changes in PPARβ/δ and PPARγ expression. These results suggest the presence of sex-dependent changes in all investigated parameters, which points out sex-related susceptibility towards oxidative stress and hyperoxia treatment of various pathological conditions and diseases.

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.

Neutrophil neutral endopeptidase variation and its regulation by opioid peptides

The opioid peptide methionine-enkephalin (MENK) has significant immunomodulatory ability in addition to its neurotransmitter function. Since neutral endopeptidase (NEP, CD10, enkephalinase EC 3.4.24.11) cleaves opioid peptides, the presence and activity of NEP in neutrophils from different persons might be responsible for the diverse, neuropeptide-induced, responses of neutrophils from different donors [Ann. N. Y. Acad. Sci. 650 (1992) 146]. The results obtained showed statistically significant differences in NEP activity among donors (high, medium and low). A 10-fold higher NEP activity in neutrophils (160-280 nmol/10(6) cells/h) and in their corresponding membrane preparations (550 nmol/mg protein/min) in our study, as compared to literature data, was a result of high specificity and affinity of Suc-Ala-Ala-Phe-pNA as substrate. In control nontreated neutrophils, the number of CD10 positive cells were not correlated with NEP activity. However, in neutrophils treated with a physiological (10(-10) M) concentration of MENK, two main events occurred; not only did the number of CD10 positive cells correlate with NEP activity, but contrary to control samples, MENK upregulated the expression of CD10 marker as demonstrated by an increase of mean florescence intensity (F-mean) in donors with low NEP activity. Taken together, these data add some clarity to the diverse activity of enkephalins in association with enzyme cleavage of those molecules.

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 1 activates nitric oxide synthase 2 activity and downregulates nitric oxide synthase 2 mRNA expression

Endomorphins 1 and 2 are newly discovered opioid tetrapeptides whose structure is more resistant to enzymatic degradation than that of other opioid peptides. Endomorphins 1 and 2 are considered as endogenous ligands with a high affinity for mu receptors. A number of studies have shown that opioid peptides per se can induce release of nitric oxide from rodent and human immune cells. Endomorphins seemed to be involved in the process of vasodilatation by stimulating release of nitric oxide. In our study we stimulated in vitro J774 macrophages with different concentrations of endomorphin 1 or 2 for measuring nitric oxide release and nitric oxide synthase 2 (NOS 2) mRNA expression. Results showed that 48 h incubation did not enhance nitric oxide release when measured with the Griess method. On the other hand, using real-time amperometric detection of nitric oxide release shortly after challenge with endomorphins, we showed that only 10(-6) M endomorphin 1 was able to stimulate nitric oxide release from a J774 macrophage cell line by activation of NOS 2 isoenzyme. The peak release was 1000-1500 s after stimulation and was in the range of nitric oxide release stimulated with 10 microg/ml lipopolysaccharide. In contrast to this, endomorphin 2 failed to induce nitric oxide release in all tested concentrations. Using a specific inhibitor of nitric oxide synthase 2 (N-(3-[aminomethyl]benzyl)acetamidine, 1400W) we eliminated the stimulatory effect of endomorphin 1 on nitric oxide release. The expression of mRNA for NOS 2 in J774 macrophages, after 30 min incubation with either lipopolysaccharide or 10(-6) M endomorphin 1 was not upregulated. As expected, lipopolysaccharide induced de novo NOS 2 transcription within 4 h. At the same time, in contrast to lipopolysaccharide, mRNA expression of cells treated with endomorphin 1 was downregulated. Since a mu-opioid receptor specific antagonist beta-funaltrexamine hydrochloride inhibited nitric oxide release from endomorphin 1-treated cells, the effect seemed to be mu-opioid receptor mediated.

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.