Grape pomace improves antioxidant capacity and faecal microflora of lambs.

Grape pomace is a by-product of winemaking process and rich in bioactive compounds such as plant polyphenols having antioxidant and antimicrobial activities. As known, oxidative stress may cause a number of pathological conditions in farm animals and thus affecting animal welfare and production. Moreover, pathogenic bacteria affect animals' health status. Thus, the objective of this study was to investigate whether lambs' feed supplemented with grape pomace enhances the antioxidant mechanisms and reduces the growth of pathogenic bacteria. For this purpose, redox status was assessed in blood and tissues using oxidative stress markers in lambs fed with diet supplemented with grape pomace. Moreover, the effect on bacterial growth was assessed by examining microbial populations in lambs' faecal microflora. The following oxidative stress markers were assessed: reduced glutathione (GSH), catalase activity (CAT), total antioxidant capacity (TAC), thiobarbituric acid-reactive substances (TBARS) and protein carbonyls (CARB). Twenty-four lambs were assigned to two experimental groups receiving standard or experimental diet for 55 days. Blood and tissues (liver, quadriceps muscle, spleen, heart and brain) were collected at two different time-points, 42 and 70 days post-birth, after ration administration. The results showed that lambs fed with experimental diet had significantly increased antioxidants mechanisms in blood and tissues as shown by increases in CAT and GSH compared to control. Moreover, lambs fed with the experimental diet exhibited decreased oxidative stress-induced damage to lipids and proteins as shown by decreases in TBARS and CARB respectively. In addition, the experimental diet enhanced the growth of facultative probiotic bacteria and inhibited the growth of pathogen populations such as Enterobacteriacae and E. coli. This is the first study investigating the effect of diet supplemented with grape pomace on the redox status and microbiota of lambs.

The effects of a water polo game on the blood redox status of male water polo players.

AIM: The purpose of this study was to examine the effects of a real water polo game on the oxidative stress and antioxidants markers of male water polo players. The importance of this study is that it is the first to examine the oxidative stress and antioxidants markers after a water polo game, considering the specific and high demands of the latter. METHODS: Twelve players (25.8±3.7 years) were assessed from two teams. The oxidative stress and antioxidants markers which were measured were TBARS, Protein Carbonyls, GSH and GSSG, Total Antioxidant Capacity (TAC) and Catalase. RESULTS: An increase was found after the game for TBARS, for catalase, for GSH, and GSSG. Protein Carbonyls, TAC and the GSH/GSSG ratio did not change significantly. CONCLUSION: The water polo game was found to be a strong stimulus for oxidative stress, while alterations seem to appear for the antioxidant capability, as indicated by increase of catalase and GSH. The increase of the latter is controversial to the usual effect post exercise. A possible explanation for this effect could be the activation of GSH synthesis in order to meet the increased needs for antioxidant protection. Also, it should be considered that blood samples were taken 10-20 min. after the game, so it is possible that in well-trained subjects, this time period is enough for the conversion of GSSG back to GSH. Yet, it can be stated that, despite the enhanced antioxidant defense of the players, oxidative stress could not be prevented.

Plasma GSH levels and Alzheimer's disease. A prospective approach.: Results from the HELIAD study.

BACKGROUND: Potential links between oxidative stress and the pathophysiology of Alzheimer's disease (AD) have been reported in the existing literature. Biological markers of oxidative stress, such as the reduced form of glutathione (GSH), may have a potential role as predictive biomarkers for AD development. The aim of the present study was to explore the longitudinal associations between plasma GSH and the risk of developing AD or cognitive decline, in a sample of community-dwelling, non-demented older adults. METHODS: Participants from the Hellenic Longitudinal Investigation of Aging and Diet (HELIAD) were included in the present prospective study. The sample used in the analyses consisted of 391 non-demented individuals over the age of 64 (mean age = 73.85 years; SD = 5.06), with available baseline GSH measurements and longitudinal follow-up. Plasma GSH was treated both as a continuous variable and as tertiles in our analyses. Cox proportional hazards models were used to evaluate the hazard ratio (HR) for AD incidence as a function of baseline plasma GSH. Generalized estimating equations (GEE) models were deployed to explore the associations between baseline plasma GSH and the rate of change of performance scores on individual cognitive domains over time. Models were adjusted for age, years of education and sex. Supplementary exploratory models were also adjusted for mild cognitive impairment (MCI) at baseline, risk for malnutrition, physical activity and adherence to the Mediterranean dietary pattern. RESULTS: A total of 24 incident AD cases occurred during a mean (SD) of 2.99 (0.92) years of follow-up. Individuals in the highest GSH tertile group (highest baseline plasma GSH values) had a 70.1% lower risk for development of AD, compared to those in the lowest one [HR = 0.299 (0.093-0.959); p = 0.042], and also demonstrated a slower rate of decline of their executive functioning over time (5.2% of a standard deviation less decline in the executive composite score for each additional year of follow-up; p = 0.028). The test for trend was also significant suggesting a potential dose-response relationship. CONCLUSION: In the present study, higher baseline plasma GSH levels were associated with a decreased risk of developing AD and with a better preservation of executive functioning longitudinally.

Grape pomace improves performance, antioxidant status, fecal microbiota and meat quality of piglets.

In the present study, grape pomace (GP) was used as feed additive in the diet of weaned piglets in order to develop innovative feedstuffs and to investigate their potential beneficial effects on welfare, productivity and meat quality. For examining the antioxidant capacity of the experimental feeds, 24 piglets of 20 days old were assigned to two experimental groups receiving standard or experimental diet for 30 days. Blood and tissues collections were performed at four different time-points, 2, 20, 35 and 50 days post birth. The collected tissues were brain, heart, kidney, liver, lung, quadriceps muscle, pancreas, spleen and stomach. The following oxidative stress markers were assessed: reduced glutathione (GSH), catalase activity, total antioxidant capacity (TAC), thiobarbituric acid reactive substances (TBARS), protein carbonyls (CARB) and H2O2 decomposition activity. The effect on bacterial growth was assessed by examining microbial populations in piglets' fecal microbiota. Furthermore, the average daily gain (ADG) was calculated and the fatty acid profile of quadriceps muscle was assessed. The results showed that piglets fed with the diet supplemented with GP, had significantly increased antioxidants mechanisms in almost all the tissues as shown by increases in GSH, H2O2 decomposition activity and TAC compared with control group. Piglets fed with the experimental diet exhibited decreased oxidative stress-induced damage to lipids and proteins as shown by decreases in TBARS and CARB in GP group compared with control. In addition, the experimental diet increased significantly ADG (by 23.65%) (P<0.05) and enhanced the growth of facultative probiotic bacteria (by up to 1.2 log colony forming units (CFU)/g) (P<0.05) and lactic acid bacteria (by up to 2.0 log CFU/g) (P<0.05) in GP group compared with the control group. GP supplementation inhibited the growth of pathogen populations such as Enterobacteriacae (by up to 1.8 log CFU/g) (P<0.05) and Campylobacter jejuni (by up to 1.0 log CFU/g) (P<0.05). Regarding fatty acid composition of meat, GP inclusion in piglets' diet increased significantly n-3 fatty acids (EPA; C20 : 5n-3, DHA; C22 : 6n-3, α-linolenic acid; C18 : 3n-3) and decreased significantly n-6/n-3 ratio compared with control (P<0.05). The results suggested that dietary GP supplementation may have a beneficial impact on piglets' welfare and may improve productivity as well as meat quality.

Assessment of antioxidant activity of extracts from unique Greek varieties of Leguminosae plants using in vitro assays.

BACKGROUND: It is believed that legumes are a very good source of micronutrients and phytochemicals that present chemopreventive activity against diseases such as diabetes, coronary heart disease and colon cancer. Methanolic and aqueous extracts from 11 unique varieties of Leguminosae family plants cultured in Greece were tested using three different in vitro assays in order to investigate the mechanisms by which phytochemicals present in these legumes exert their chemoprevention. MATERIALS AND METHODS: The extracts were tested by the 1, -diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, the hydroxyl radical- and the peroxyl radical-induced DNA strand scission assays. Hydroxyl (OH*) and peroxyl (ROO*) radicals were generated from ultraviolet (UV) photolysis of hydrogen peroxide (H2O2) and thermal decomposition of 2,2'-azobis-(2-amidinopropane hydrochloride) (AAPH) respectively. RESULTS: In the DPPH assay, all the tested extracts displayed potent radical scavenging efficiency. Furthermore, most of the Leguminosae family plant extracts exerted significant protective activity against DNA damage induced by both reactive oxygen species, although they were more effective in inhibiting ROO*-induced rather than OH*-induced DNA strand scission. CONCLUSION: The results suggest that the free radical scavenging activity of Leguminosae plants may be one of the mechanisms accounting for their chemoprevention.

Short duration exhaustive aerobic exercise induces oxidative stress: a novel play-oriented volitional fatigue test.

AIM: Exercise is associated with the generation of reactive oxygen and nitrogen species. This study examined the oxidative stress in response to a novel volitional fatigue test. METHODS: Eleven male college students performed a volitional fatigue test consisting of shuttle runs with a tennis racquet in the hand towards the left and right sidelines within the tennis singles court in an attempt to hit tennis balls until exhaustion. A tennis ball serving machine was adjusted to alternate feeds to the forehand and backhand sides of the subjects, standing at the baseline, at a frequency of 20 balls per minute. RESULTS: Mean time to volitional fatigue was 5.9+/-1.3 min and mean heart rate at volitional fatigue was 189+/-8.1 beats x min(-1). The volitional fatigue test resulted in significant increases in blood thiobarbituric acid-reactive substances (22%), protein carbonyls (58%), catalase activity (143%), total antioxidant capacity (34%) and oxidized glutathione (GSSG, 81%) concentration, as well as significant decreases in reduced glutathione (GSH, 15%) concentration and GSH/GSSG ratio (56%) immediately postexercise, as compared to the pre-exercise concentration. CONCLUSION: The data provide evidence that acute short duration exhaustive aerobic exercise in the form of a novel volitional fatigue test is capable of inducing oxidative stress. This novel test could serve as an alternative exercise modality to study oxidative stress.

Simulating real-life exposures to uncover possible risks to human health: A proposed consensus for a novel methodological approach.

In real life, consumers are exposed to complex mixtures of chemicals via food, water and commercial products consumption. Since risk assessment usually focuses on individual compounds, the current regulatory approach doesn't assess the overall risk of chemicals present in a mixture. This study will evaluate the cumulative toxicity of mixtures of different classes of pesticides and mixtures of different classes of pesticides together with food additives (FAs) and common consumer product chemicals using realistic doses after long-term exposure. Groups of Sprague Dawley (CD-SD) rats (20 males and 20 females) will be treated with mixtures of pesticides or mixtures of pesticides together with FAs and common consumer product chemicals in 0.0, 0.25 × acceptable daily intake (ADI)/tolerable daily intake (TDI), ADI/TDI and 5 × ADI/TDI doses for 104 weeks. All animals will be examined every day for signs of morbidity and mortality. Clinical chemistry hematological parameters, serum hormone levels, biomarkers of oxidative stress, cardiotoxicity, genotoxicity, urinalysis and echocardiographic tests will be assessed periodically at 6 month intervals. At 3-month intervals, ophthalmological examination, test for sensory reactivity to different types of stimuli, together with assessment of learning abilities and memory performance of the adult and ageing animals will be conducted. After 24 months, animals will be necropsied, and internal organs will be histopathologically examined. If the hypothesis of an increased risk or a new hazard not currently identified from cumulative exposure to multiple chemicals was observed, this will provide further information to public authorities and research communities supporting the need of replacing current single-compound risk assessment by a more robust cumulative risk assessment paradigm.

Comparison of the blood redox status between long-distance and short-distance runners.

Exercise increases the production of reactive oxygen species, which may damage a number of cell constituents. Organisms have developed a sophisticated antioxidant system for protection against reactive oxygen species. Our aim was to compare the adaptive responses of antioxidant mechanisms and the blood redox status of two groups of athletes, long-distance and short-distance runners. Thiobarbituric acid reactive substances, catalase activity and total antioxidant capacity was measured in the serum, while reduced and oxidized glutathione as well as their ratio were determined in blood hemolysates. Serum catalase activity (P<0.001) was found to be three times higher in long-distance compared to short-distance runners (25.4 vs. 8.9 micromol x min(-3) x ml(-1)), whereas the two groups did not differ in the other markers. Catalase activity also correlated significantly with maximal oxygen consumption in long-distance runners. In conclusion, we report here that long-distance and short-distance runners exhibit similar blood redox status judged by several oxidative stress indices, except for the much higher activity of catalase in long-distance runners. This different effect of the two training modules on catalase activity of long-distance runners might be partly due to the high oxygen load imposed during their repeated prolonged exercise bouts.

CDK-inhibitor olomoucine inhibits cell death after exposure of cell lines to cytosine-arabinoside.

Signal transduction for apoptosis or programmed cell death, after DNA damage in mammalian cells, is believed to involve activation of cyclin-dependent kinases (CDKs), especially CDK-1 (cdc2) and CDK-2. We used CDK-inhibitor olomoucine, a purine analogue to evaluate the role CDK inhibition on cytosine-arabinoside (Ara-C)-induced cell death. The two drugs showed an antagonistic effect, suggesting that apoptosis after exposure to Ara-C is inhibited by olomoucine. DNA-electrophoresis showed a clear inhibition of the apoptotic pattern when olomoucine was added to Ara-C. We conclude that CDK-inhibitor olomoucine inhibits cell death induced by Ara-C.

Chemopreventive activity of very low dose dietary tannic acid administration in hepatoma bearing C3H male mice.

Tannins are plant polyphenols comprising a heterogeneous group of compounds. Tannic acid is a common tannin found in tea, coffee, immature fruits, etc. and it has also been used as a food additive. An increasing body of experimental evidence supports the hypothesis that tannins exert anticarcinogenic activity in chemically induced cancers in animal models. In the present study, tannic acid was administered in very low doses in the drinking water of C3H male mice divided into three groups (75 mg/l, 150 mg/l and 300 mg/l). These animals carry a genetic defect and show a high incidence of spontaneous liver tumors (> 50%) at an age older than 12 months. The results showed a decrease in the overall incidence of hepatic neoplasms (adenomas plus carcinomas): 53.3% of animals in the control group developed hepatic neoplasms versus 33.3% in the group given a low dose of tannic acid, 26.6% in the group given a medium dose and 13.3% in the high dosage group. The difference was more pronounced in the animals with carcinomas: 4.44% of mice who received tannic acid developed carcinomas versus 33.3% of those in the control group. Tannic acid administration did not affect the PCNA labeling index of normal hepatocytes. It is concluded that tannic acid dietary intake in low doses can exert a strong dose-dependent chemoprotective activity against spontaneous hepatic neoplasm development in C3H male mice, most probably through antipromoting mechanisms.

Characterization of markers flanking the human SP-B locus.

We have previously identified a SP-B length polymorphism that appears with higher frequency in the RDS population (Biochem. J., 305, 1995, p583). This polymorphism encompasses a fairly large region, thus it is difficult to distinguish between variants with small size differences. Because of the importance of SP-B in normal lung function and the association of this SP-B polymorphism with RDS, we wished to identify and characterize polymorphic markers linked to the SP-B locus that would allow better resolution of SP-B alleles. In this report we a) characterized a novel (AAGG)n linked SP-B microsatellite marker; b) determined linkage of published markers with the SP-B locus and also determined the distance of each marker from the SP-B locus using medium and high resolution radiation hybrid panels; c) determined heterozygosity index and PIC values of the novel and known markers in various populations; and d) determined haplotypes using CEPH families. The availability of these SP-B linked markers/haplotypes will facilitate population and family based association studies. We are hopeful that the information gained will help to unravel the genetic complexity of RDS and respiratory diseases with regards to the SP-B locus.

Characterization of the protein protecting RNA from Acid precipitation and its evaluation as tumor-marker in gastrointestinal and gynecological cancer.

In a previous study, we observed increased protective activity against acid precipitation of poly (U) in the serum of tumor bearing mice and hepatoma patients. In the present study we purified and partially characterized the protein responsible for this phenomenon and measured the activity in the serum of patients with various cancers. Evidence is presented that this protein forms a complex with serum ribonucleases which results in the inactivation of enzymes and protection of RNA from acid precipitation. The serum protective activity against acid precipitation of poly (U) was measured in 37 patients with gastrointestinal cancer and 79 with gynecological cancer, along with 15 male subjects with nonmalignant diseases of the gastrointestinal system, 15 with uterine leiomyoma, 19 healthy men and 30 healthy women from the hospital personnel. The mean values found in patients with various tumors were about three times higher than those found in healthy individuals, while no significant overlapping of values was observed between cancer patients and controls. On the other hand, the activity was not increased in patients with nonmalignant diseases of gastrointestinal system and uterus.

Two-dimensional gel electrophoretic profile of rat basophilic leukemia (RBL) and mast cells.

RBL cells are not differentiated, but resemble mucosal mast cells (MMC). Two-dimensional (2-D) gel electrophoresis following isoelectric focusing (IEF) was performed using purified rat peritoneal mast cells and RBL cells. Certain similarities were identified with silver staining between mast cells and stationary phase (72 hr) RBL cells. RBL cells were also labelled with [35S]-cysteine in order to study the specific expression of proteins during logarithmic or stationary growth phases. Only stationary phase RBL cells appeared to specifically express three proteins of 42, 55 and 93 kD and were still capable of secreting histamine in response to immunoglobulin E (IgE) and specific antigen. These results suggest that specific RBL cell proteins may be used as markers for further analysis of their maturation/differentiation.

Increased protective activity against acid precipitation of poly(U) in the serum of tumor-bearing mice.

Transplantation of leukemia L1210 cells into DBA/2 mice and of Ehrlich ascites tumor cells into BALB/C mice resulted in a significant increase of protective activity against acid precipitation of poly (U) in the serum. The increase was observed as early as one day after the tumor transplantation and seems to be connected with cancer growth, since inoculation of L1210 cells into BALB/C mice did not affect the protective activity, evidently as a result of their well established inability to cause cancer in this strain. Furthermore, no increase of activity was observed when bacteria were inoculated into mice, or when the latter were partially hepatectomized. The results suggest that the protective activity against acid precipitation of poly (U) could prove to be a tumor marker for the early detection of cancer growth.

Enzymatic conversion of dihydrotestosterone from 3-keto to 3-enol form in the rat prostate.

Dihydrotestosterone (DHT) is the active androgen, as well as a strong tumor promoter in the prostate, where several enzymes are essential for the regulation of its activity. We localized four enzymes promoting the enolization of the 3-keto group of DHT in rat prostate. The enzymes were purified by ion-exchange chromatography, acetone fractionation and gel filtration to homogeneity, and found to have molecular sizes of 19.5, 22.0, 44.5 and 21.5 kDa. A partial characterization of the four enzymes revealed that their structure consisted of a common chain of 14.5 kDa with various subunits which differentiate the four enzymes from each other. All the enzymes exerted their activity only on 5-dihydro 3-keto steroids. The total enzymatic activity was strongly influenced by animal age, being very low before sexual maturation, as well as after castration. In the latter case the level of total activity fell to about 8% control animals. Activity was also estimated in human, pork, ram and bovine prostate and it was found that all these species have 20-25 times lower enzyme levels than rat. These results, in combination with the practically exclusive localization of the enzymes in the prostate, suggest a role relating to the bioavailability of DHT in this gland.

Albumin possesses intrinsic enolase activity towards dihydrotestosterone which can differentiate benign from malignant breast tumors.

Serum albumin was found to possess enolase activity towards the dihydrotestesterone (DHT) molecule, converting it from its 3-keto to 3-enol form. This activity was accompanied by albumin during all stages of purification, as well as following various treatments, a fact indicating that the enzymatic activity was an intrinsic property of albumin molecule and did not represent an impurity of the preparation. Enolase activity was decreased in parallel with the quantity of intact albumin molecules when proteolytic enzymes were used for their degradation. The activity was strongly inhibited by Ni (II) and Cu (II) ions, which bind to 3-histidine of the albumin molecule, as well as by oleic acid and cholesterol. It was also inhibited, in a reversible manner by surface-active agents. Enolase activity was found in all mammalian species studied, the specific activity however was very low in the serum of dogs. The administration of DHT to mice did not influence the albumin or enolase levels in their serum. The optimum pH of enolase was at 9.2, with a carbonate buffer solution. In addition to the serum enolase activity was found to be a feature of intracellular albumin. The two albumins exhibited the same specific activity and the same Km for DHT. The study of cytosolic albumin, obtained from human mammary gland tissue, revealed that benign and malignant tumors of this gland differed substantially with respect to their percentage of albumin. Significant differences were also observed in enolase activity, a consequence of the existence of a fraction of albumin in the malignant tissue in a polymeric form. This form exhibited a decreased enzymatic activity, compared to its monomeric form, exclusively encountered in benign breast specimens. The last observation, along with the quantitative differences of albumin in the two tissues, offers a possibility of reliable differentiation between benign and malignant breast tumors.

Value of serum protective activity against acid precipitation of poly (U) in the differentiation between cirrhotic patients with and without hepatocellular carcinoma.

The serum protective activity against acid precipitation of poly (U) and a-fetoprotein levels were compared in 39 cirrhotic patients with hepatocellular carcinoma (HCC) and in 33 patients with chronic liver disease (CLD) alone, in order to differentiate malignant and nonmalignant chronic liver disease. All but one (97.4%) patients with HCC were found to have serum protective activity levels of greater than or equal to 21 micrograms/ml, whereas all but one (97%) patients with CLD had serum protective activity levels of less than or equal to 20 micrograms/ml. Mean serum protective activity levels were significantly higher in the HCC group than in those with CLD (p less than 0.0001). Serum a-fetoprotein concentrations of over 500 ng/ml, suggesting malignancy, were observed in 54% of patients with HCC and in 15% of patients with CLD. Application of the best discriminating values for protective activity (greater than 21 micrograms/ml) and for a-fetoprotein (greater than 500 ng/ml) to 72 patients with or without HCC revealed an efficiency of 97.2% for protective activity and only 68.1% for a-fetoprotein.

Determination of xylazine and its metabolites by GC-MS in equine urine for doping analysis.

Xylazine and its main metabolites were detected in equine urine after a single-dose intravenous administration of 0.98 and 1.01 mg/kg body weight xylazine, respectively, in two horses, in order to be used for equine doping control routine analysis. The urine levels of the parent drug and its metabolites were determined using gas chromatography-mass spectrometry (GC-MS). Xylazine is metabolised rapidly, down to a concentration level of about 1.0 microg/ml after 1-3h administration. Seven metabolites were identified in urine. 4-Hydroxy-xylazine, the major metabolite, could be traced for 25 h and it is regarded as the long-term metabolite of xylazine in horse. 2,6-Dimethylaniline was, for the first time, reported as metabolite in equine.

Can traditional epidemiology detect cancer risks caused by occupational exposure to pesticides?

In order to investigate the possible relationship between cancer and occupational exposure to pesticides, we reviewed the latest literature of the epidemiological studies in this area coming to the conclusion that, while several studies indicate a link between certain pesticides and certain tumors, this information is still insufficient, and further research on the health consequences of exposure to pesticides is needed. Moreover, provided there is a risk, it is often too limited to be detected by available epidemiological techniques. Therefore, in addition to the epidemiological studies, the development of new biology, gene technology and medical biotechnology methods may significantly enhance the specificity of the epidemiological studies. Thus, the fusion of molecular biology and epidemiology into molecular epidemiology may provide more specific methods for monitoring the occupational dependent carcinogenic risk of individuals and groups.

Lipid peroxidation and tissue damage.

In recent years it has become apparent that the oxidation of lipids, or lipid peroxidation, is a crucial step in the pathogenesis of several disease states in adult and infant patients. Lipid peroxidation is a process generated naturally in small amounts in the body, mainly by the effect of several reactive oxygen species (hydroxyl radical, hydrogen peroxide etc.). It can also be generated by the action of several phagocytes. These reactive oxygen species readily attack the polyunsaturated fatty acids of the fatty acid membrane, initiating a self-propagating chain reaction. The destruction of membrane lipids and the end-products of such lipid peroxidation reactions are especially dangerous for the viability of cells, even tissues. Enzymatic (catalase, superoxide dismutasse) and nonenzymatic (vitamins A and E) natural antioxidant defence mechanisms exist; however, these mechanisms may be overcome, causing lipid peroxidation to take place. Since lipid peroxidation is a self-propagating chain-reaction, the initial oxidation of only a few lipid molecules can result in significant tissue damage. Despite extensive research in the field of lipid peroxidation it has not yet been precisely determined if it is the cause or an effect of several pathological conditions. Lipid peroxidation has been implicated in disease states such as atherosclerosis, IBD, ROP, BPD, asthma, Parkinson's disease, kidney damage, preeclampsia and others.