Green tea reduces body fat via upregulation of neprilysin.

BACKGROUND/OBJECTIVE: Consumption of green tea has become increasingly popular, particularly because of claimed reduction in body weight. We recently reported that animals with pharmacological inhibition (by candoxatril) or genetic absence of the endopeptidase neprilysin (NEP) develop an obese phenotype. We now investigated the effect of green tea extract (in drinking water) on body weight and body composition and the mediating role of NEP. SUBJECTS/METHODS: To elucidate the role of NEP in mediating the beneficial effects of green tea extract, 'Berlin fat mice' or NEP-deficient mice and their age- and gender-matched wild-type controls received the extract in two different doses (300 or 600 mg kg-1 body weight per day) in the drinking water. RESULTS: In 'Berlin fat mice', 51 days of green tea treatment did not only prevent fat accumulation (control: day 0: 30.5% fat, day 51: 33.1%; NS) but also reduced significant body fat (green tea: day 0: 27.8%, day 51: 20.9%, P<0.01) and body weight below the initial levels. Green tea reduced food intake. This was paralleled by a selective increase in peripheral (in kidney 17%, in intestine 92%), but not central NEP expression and activity, leading to downregulation of orexigens (like galanin and neuropeptide Y (NPY)) known to be physiological substrates of NEP. Consequently, in NEP-knockout mice, green tea extract failed to reduce body fat/weight. CONCLUSIONS: Our data generate experimental proof for the assumed effects of green tea on body weight and the key role for NEP in such process, and thus open a new avenue for the treatment of obesity.

Accurate and reproducible ion mobility measurements for chemical standard evaluation.

Chemical standards are used to calibrate ion mobility spectrometers (IMS) for accurate and precise identification of target compounds. Research over the past 30 years has identified several positive and negative mode compounds that have been used as IMS standards. However, the IMS research community has not come to a consensus on any chemical compound(s) for use as a reference standard. Also, the reported K(0) values for the same compound analyzed on several IMS systems can be inconsistent. In many cases, mobility has not been correlated with a mass identification of an ion. The primary goal of this work was to provide mass-identified mobility (K(0)) values for standards. The results of this work were mass-identified K(0) values for positive and negative mode IMS chemical standards. The negative mode results of this study showed that TNT is a viable negative mode reference standard. New temperature-dependent K(0) values were found by characterizing drift gas temperature and water content; several examples were found of temperature-dependent changes for the ion species of several standards. The overall recommendation of this study is that proposed IMS standards should have temperature-dependent K(0) values quoted in the literature instead of using a single K(0) value for a compound.

Chemistry and biochemistry of lipid peroxidation products.

Oxidative stress and resulting lipid peroxidation is involved in various and numerous pathological states including inflammation, atherosclerosis, neurodegenerative diseases and cancer. This review is focused on recent advances concerning the formation, metabolism and reactivity towards macromolecules of lipid peroxidation breakdown products, some of which being considered as 'second messengers' of oxidative stress. This review relates also new advances regarding apoptosis induction, survival/proliferation processes and autophagy regulated by 4-hydroxynonenal, a major product of omega-6 fatty acid peroxidation, in relationship with detoxication mechanisms. The use of these lipid peroxidation products as oxidative stress/lipid peroxidation biomarkers is also addressed.

4-hydroxynonenal: a membrane lipid oxidation product of medicinal interest.

A comprehensive focus on 4-hydroxynonenal (HNE) as candidate molecule in a variety of pathophysiological conditions occurring in humans is here provided. Despite an active, now well characterized, metabolism in most cells and tissues, HNE can be easily detected and quantified by means of several methods, although with different sensitivity. Measurements of HNE and/or stable metabolites in biological fluids are already applied as lipid peroxidation/oxidative stress markers in a huge number of human disease processes, often sustained by inflammatory reactions. A primary involvement of this aldehydic product of membrane lipid oxidation in inflammation-related events, as well as in regulation of cell proliferation and growth, in necrotic or apoptotic cell death, appears supported by its marked ability to modulate several major pathways of cell signaling and, consequently, gene expression. The actual knowledge of HNE reactivity, metabolism, signaling and modulatory effect in the various human organs should provide a solid background to the investigation of the aldehyde's contribution to the pathogenesis of human major chronic diseases and would likely promote advanced and oriented applications not only in diagnosis and prevention but also in molecular treatment of human diseases.

Plasticity and impact of the central renin-angiotensin system during development of ethanol dependence.

Pharmacological and genetic interference with the renin-angiotensin system (RAS) seems to alter voluntary ethanol consumption. However, understanding the influence of the RAS on ethanol dependence and its treatment requires modeling the neuroadaptations that occur with prolonged exposure to ethanol. Increased ethanol consumption was induced in rats through repeated cycles of intoxication and withdrawal. Expression of angiotensinogen, angiotensin-converting enzyme, and the angiotensin II receptor, AT1a, was examined by quantitative reverse transcription polymerase chain reaction. Increased ethanol consumption after a history of dependence was associated with increased angiotensinogen expression in medial prefrontal cortex but not in nucleus accumbens or amygdala. Increased angiotensinogen expression also demonstrates that the astroglia is an integral part of the plasticity underlying the development of dependence. The effects of low central RAS activity on increased ethanol consumption were investigated using either spirapril, a blood-brain barrier-penetrating inhibitor of angiotensin-converting enzyme, or transgenic rats (TGR(ASrAOGEN)680) with reduced central angiotensinogen expression. Spirapril reduced ethanol intake in dependent rats compared to controls. After induction of dependence, TGR(ASrAOGEN)680 rats had increased ethanol consumption but to a lesser degree than Wistar rats with the same history of dependence. These data suggest that the central RAS is sensitized in its modulatory control of ethanol consumption in the dependent state, but pharmacological or genetic blockade of the system appears to be insufficient to halt the progression of dependence.

alpha-Tocopherol increases caspase-3 up-regulation and apoptosis by beta-carotene cleavage products in human neutrophils.

It has been found that beta-carotene cleavage products (CarCP), besides having mutagenic and toxic effects on mitochondria due to their prooxidative properties, also initiate spontaneous apoptosis of human neutrophils. Therefore, it was expected that antioxidants such as alpha-tocopherol would inhibit the stimulation of apoptosis and caspase-3 activity by CarCP. However, we found that alpha-tocopherol increases caspase-3 up-regulation and stimulation of apoptosis of human neutrophils by CarCP. Ascorbic acid does not alter this caspase-3 up-regulating and proapoptotic effect exerted by alpha-tocopherol. Both alpha-tocopherol and ascorbic acid, in the absence of CarCP, decrease intracellular caspase-3 activity and spontaneous apoptosis of neutrophils. Uric acid alone or in combination with CarCP does not exert apparent effects on caspase-3 activity and apoptosis. Up-regulating effect of alpha-tocopherol is not observed in the presence of retinol that markedly stimulates apoptosis by itself, whereas increase of caspase-3 activity is induced by concomitant addition of alpha-tocopherol and beta-ionone, a cyclohexenyl degradation product of beta-carotene with shorter aliphatic chain.

Beta-carotene breakdown products enhance genotoxic effects of oxidative stress in primary rat hepatocytes.

Since it has to be expected that individuals exposed to oxidative stress who take supplements of beta-carotene are simultaneously exposed to both beta-carotene cleavage products (CPs) and oxidative stress, and both exposures have been demonstrated to cause genotoxic effects in primary rat hepatocytes, cyto- and genotoxic effects on primary rat hepatocytes after supplementation of the medium with increasing concentrations of a CP mixture during exposure to oxidative stress by treatment with either DMNQ (2,3-dimethoxy-1,4-naphthoquinone) or hypoxia/reoxygenation (Hy/Reox) was investigated. The cytological endpoints analysed were the mitotic indices, the percentages of apoptotic and necrotic cells, the percentages of micronucleated (MN) cells and the number of chromosomal aberrations (CAs) and sister chromatid exchanges (SCE). The results obtained clearly demonstrate that the CP mixture enhances the genotoxic effects of oxidative stress exposure, whereas it had no effect at all on the endpoints of cytotoxicity studied. These results further support the hypothesis that CP might be responsible for the reported carcinogenic response in the beta-CArotene and Retinol Efficacy Trial (CARET) and Alpha-Tocopherol Beta-carotene Cancer prevention (ATBC) chemoprevention trials.

Inhibition of defective adenylosuccinate lyase by HNE: a neurological disease that may be affected by oxidative stress.

Adenylosuccinate lyase is an enzyme of fumarase superfamily that participates in the purine biosynthetic pathway, catalysing the nonhydrolytic cleavage of succinyl groups from SAICA ribotide and adenylosuccinate. Enzyme defects are associated with a human inherited disease, which arises from single point mutations to the gene and results in mild to severe psychomotor retardation, epilepsy, muscle wasting, and autistic features. Adenylosuccinate lyase activity is lost to a different extent in the patients. Diminished levels of enzyme have been attributed to loss of catalytic activity, protein instability, or environmental factors. P100A/D422Y mutation represents a feasible model for studying the effect of cell milieu on the activity of the impaired enzyme. The defective enzyme is inhibited by micromolar concentrations of trans-4-hydroxy-2-nonenal (HNE), a major product of membrane peroxidation that has been found to accumulate in brain tissues of patients with neurodegenerative disorders. It is suggested that inactivation of defective adenylosuccinate lyase by HNE and other membrane peroxidation products may account, at least in part, for the impairment of neurological functions and recurrent worsening of the symptoms.

Inhibition of ion transport ATPases by HNE.

4-Hydroxy-2,3-trans-nonenal (HNE), a major lipid peroxidation product, has been shown to react with specific amino acid residues of proteins and alter their function. In vitro exposure of erythrocyte ghosts and neutrophil membranes to HNE results in the inhibition of ion transport ATPases. Neutrophil membrane Ca2+-ATPase is strongly inhibited by micromolar concentrations of HNE, while HNE is considerably less effective against neutrophil Mg2+-ATPase and the erythrocyte ghost enzymes.

Cyto- and genotoxic potential of beta-carotene and cleavage products under oxidative stress.

Free radical attack on beta-carotene results in the formation of high amounts of cleavage products with prooxidant activities towards subcellular organelles such as mitochondria, a finding which could provide an explanation for the contradictory results obtained with beta-carotene in clinical efficacy and cancer prevention trials. Since primary hepatocytes proved to be very sensitive indicators for the genotoxic action of suspect mutagens/carcinogens we therefore investigated a beta-carotene cleavage products mixture (CP), apo-8'-beta-carotenal (apo-8') and beta-carotene in the primary rat hepatocyte assay in the presence and absence of oxidative stress provided by hypoxia/reoxygenation (Hy/re). The endpoints tested were: the mitotic indices, the percentages of necrotic and apoptotic cells, micronucleated cells (MN), chromosomal aberrations (CA) and sister chromatid exchanges (SCE). The results obtained indicate a genotoxic potential of both CP and apo-8' already in the concentration range of 100 nM and 1 microM, i.e. at physiologically relevant levels of beta-carotene and beta-carotene breakdown products. In contrast, no significant cytotoxic effects of these substances were observed, nor did beta-carotene induce significant cytotoxic or genotoxic effects at concentrations ranging from 0.01 up to 10 microM. However, when beta-carotene is supplemented during oxidative stress induced by hypoxia/reoxygenation, a dose-dependent increase of CP is observed accompanied by increasing genotoxicity. Furthermore, when beta-carotene cleavage products were supplied during oxidative stress significant additional increases of genotoxic effects were observed, the additional increases indicating an additive effect of both exposures. Summarizing, these results provide strong evidence that beta-carotene breakdown products are responsible for the occurrence of carcinogenic effects found in the Alpha-Tocopherol Beta-carotene-Cancer prevention (ATBC) study and the beta-CArotene and RETinol Efficacy (CARET) Trial.

Effect of carotenoid oxidation products on neutrophil viability and function.

Human neutrophils are short-lived cells that play important roles in host defense and acute inflammation by releasing hydrolytic and cytotoxic proteins and reactive oxygen derivatives. Apoptosis, a physiological mechanism for cell death, regulates both production and survival of neutrophils, representing a basic biological mechanism for this type of cells. Carotenoids may react with toxic oxygen metabolites released by neutrophils to form a multitude of carotenoid cleavage products that exert, in turn, relevant prooxidative biological effects. Recent data suggest that carotenoid oxidation products may affect neutrophil viability and function by exerting proapoptotic activity and interfering with superoxide production by activated cells. The prooxidant and proapoptotic activities of carotenoid oxidation products could account, at least in some cases, for the procancerogenic properties of carotenoid rich diet.

Cytotoxic and genotoxic effects of beta-carotene breakdown products on primary rat hepatocytes.

According to Siems and colleagues, free radical attack on beta-carotene results in the formation of high amounts of cleavage products with prooxidant activities towards subcellular organelles such as mitochondria. This finding may be an explanation for the contradictory results obtained with beta-carotene in clinical efficacy and cancer prevention trials. Since primary hepatocytes proved to be very sensitive indicators of the genotoxic action of suspect mutagens/carcinogens we therefore investigated a beta-carotene cleavage products mixture (CP), apo8'- carotenal (apo8') and beta-carotene utilizing primary cultures of rat hepatocytes. The end-points tested were: the mitotic index, the percentage of necrotic and apoptotic cells, micronucleated cells, chromosomal aberrations and sister chromatid exchanges (SCE). Our results indicate a genotoxic potential of both CP and apo8' already at the concentrations 100 nM and 1 microM, i.e. at pathophysiologically relevant levels of beta-carotene and beta-carotene breakdown products. A 3 h treatment with CP induced statistically significant levels of micronuclei at concentrations of 0.1, 1 and 10 microM and chromosomal aberrations at concentrations of 1, 5 and 10 microM. Apo8' induced statistically significant levels of micronuclei at concentrations of 0.1, 1 and 5 microM and chromosomal aberrations at concentrations of 0.1, 1 and 10 microM. Statistically significant increases in SCE induction were only observed at a concentration of 10 microM CP and apo8'. In contrast, no significant cytotoxic effects of these substances were observed. Since beta-carotene induced neither significant cytotoxic nor genotoxic effects at concentrations ranging from 0.01 up to 10 microM, these observations indicate that most likely beta-carotene breakdown products are responsible for the occurrence of carcinogenic effects found in the Alpha-Tocopherol Beta-Carotene Cancer Prevention (ATBC) Study and the Beta-CArotene and RETinol Efficacy Trial (CARET).

High sensitivity of plasma membrane ion transport ATPases from human neutrophils towards 4-hydroxy-2,3-trans-nonenal.

Lipid peroxidation results in release of 4-hydroxy-2,3-trans-nonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. The effect of HNE on the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase, and calmodulin-stimulated Ca(2+)-ATPase has been studied both in erythrocyte ghosts and in neutrophil membrane preparations. Neutrophil Ca(2+)-ATPase was strongly inhibited by micromolar concentrations of HNE (IC(50) = 12 microM), that means in the range of pathophysiologically relevant HNE levels. The IC(50) value for neutrophil Na(+)/K(+)-ATPase was about 40 microM. HNE was considerably less effective against neutrophil Mg(2+)-ATPase and the erythrocyte ghost enzymes (IC(50) values range from 91 to 240 microM). The data suggest that HNE may play a specific role in the regulation of neutrophil calcium homeostasis in response to oxidative stress.

Oxidative stress in cardio renal anemia syndrome: correlations and therapeutic possibilities.

Cardiovascular injury has been shown to be the most critical factor affecting quality of life and mortality in patients suffering from chronic renal failure. Oxidative stress has been thought to be an important risk factor for cardiovascular disorders. As oxidative stress parameters with high cardiovascular risk factor 4-hydroxynonenal and other aldehydic lipid peroxidation products, F2-isoprostanes, homocysteine, and cholesterol oxidation products were measured in chronic renal failure patients. 4-Hydroxynonenal and some cholesterol oxidation products correlated well with the degree of renal anemia. F2-isoprostane levels were related to inflammation, whereas homocysteine was increased due to malnutrition. Further, cholesterol oxidation products correlated well with the consumption of lipophilic antioxidants such as alpha-tocopherol. There was an almost linear correlation between the left ventricular mass index and 4-hydroxynonenal. Both parameters furthermore showed an inverse relationship to hemoglobin concentration. The correction of renal anemia by means of erythropoietin therapy led to an efficient strengthening of the antioxidative defence system. The improvement of the antioxidative capacity is of complex nature comprising both enzymatic pathways and low molecular antioxidants. The correction of renal anemia with its well documented reduction of the cardiovascular risk can be regarded as an antioxidative therapy, demonstrating the clinical efficiency of antioxidative protection in patients with chronic renal failure.

Effects of kinins on mammalian spermatozoa and the impact of peptidolytic enzymes.

Effects of kinins, mainly bradykinin (Bk), and other components of the kallikrein-kinin system on sperm motility and further fertility-related functions have been described repeatedly. However, reported data are in part controversial and the mechanism of kinin effects on sperm motility is not yet understood. In the present report we describe a significant promoting effect of Bk on sperm motility at subnanomolar concentrations. This effect was stabilized and even increased by suppression of Bk hydrolysis in semen samples. As sperm membrane-bound angiotensin-converting enzyme and neutral metalloendopeptidase are mainly involved in Bk hydrolysis, an effective cocktail of enzyme inhibitors promoting the sperm motility consists of phosphoramidon and lisinopril (both at 10-7 m). The effects of Bk on sperm cells are not mediated by the B2 Bk receptor. Using several biochemical, molecular and genetic methods we could not detect any Bk receptor on spermatozoa.

Oxidative stress in chronic lymphoedema.

BACKGROUND: Chronic lymphoedema is one of the most frequent and debilitating complications after surgical and radiological tumour treatment. Prevention and therapy of lymphoedema is therefore an important problem of the rehabilitation of those patients. AIM: To investigate whether chronic lymphoedema results in increased oxidative stress. DESIGN: Prospective case-control study. METHODS: We obtained venous blood samples from patients (n=38) with chronic lymphoedema and determined biomarkers of prooxidative reactions and of antioxidative defense system in the erythrocytes or blood plasma: reduced and oxidized glutathione (GSH and GSSG), and lipid peroxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE). Healthy volunteers (n=90) and patients who had undergone surgical and/or radiotherapeutic treatment of tumours without consequent lymphoedema (n=20) acted as controls. RESULTS: The blood of patients with chronic lymphoedema contained lower concentrations of GSH and higher levels of GSSG and of MDA and HNE, compared with the control group. MDA was increased by about three-fold in the serum of the lymphoedema patients. Accelerated free radical formation and lipid peroxidation processes were further demonstrated by the liberation of MDA and HNE into the blood serum after manual lymph drainage. DISCUSSION: Our data demonstrate enhanced formation of reactive oxygen species (ROS) and accelerated lipid peroxidation processes in chronic lymphoedematous tissue. The strengthening of antioxidative defense mechanisms could be useful in the therapy of chronic lymphoedema.

Oxidative stress in chronic renal failure as a cardiovascular risk factor.

Myocardial injury has been shown to be the most critical factor influencing quality of life and mortality in patients with chronic renal failure. Oxidative stress has been postulated to be an important risk factor for cardiovascular disorders. One reason for oxidative stress in patients with renal failure is the underlying disease itself. Renal toxicity, ischemia/reperfusion and immunological disorders of the kidney result in an elevated formation of reactive oxygen species active in the pathogenesis of kidney disease. However, treatment procedures were also shown to induce oxidative stress. Increased formation of free radicals leads to an accelerated lipid peroxidation (LPO). Furthermore, secondary aldehydic LPO products, e.g. malondialdehyde (MDA) and 4-hydroxynonenal (HNE), are formed which were shown to deplete antioxidants, inhibit protein syntheses, mitochondrial respiration, and enzyme functions. F2-isoprostanes, also metabolites of polyunsaturated fatty acids, represent an additional in vivo marker of oxidative stress. Both isoprostanes and aldehydic LPO products can be removed by hemodialysis, however, this suggests only in part their binding to other molecules which cause tissue damage. Protein carbonyls are end-products of such interventions. Oxysterols, another form of free-radical initiated oxidation products, were shown to initiate atherosclerosis and plaque formation increasing dramatically the risk of coronary heart disease. Today there is no doubt that the correction of the oxidant/antioxidant imbalance in patients with chronic renal failure is an important approach for the reduction of the risk of those patients to develop cardiovascular disorders. The complete correction of renal anemia represents an effective means of strengthening antioxidant capacity and, therefore, of reducting cardiovascular risk potential.

Elevated serum concentration of cardiotoxic lipid peroxidation products in chronic renal failure in relation to severity of renal anemia.

Patients with end-stage renal disease undergoing hemodialysis (HD) are exposed to oxidative stress. Increased levels of malondialdehyde (MDA) and 4-hydroxylnonenal (HNE) were found in plasma of uremic patients indicating accelerated lipid peroxidation (LPO) as a consequence of multiple pathogenetic factors. The catabolism and action of those products was already intensively studied. As highly reactive metabolites they are able to bind to proteins, nucleic acids, and other molecules. Doing so, they exert molecular signal effects in cells and are able to exacerbate tissue and organ damage, e.g. cardiotoxic effects. Since renal anemia was shown to promote oxidative stress as well, the aim of our investigation was to examine its role in HD patients. Therefore, two groups of HD patients were investigated (group I Hb < 10 g/dl, group II Hb > 10 g/dl) and serum concentrations of MDA, HNE, and of protein carbonyls, a marker for protein oxidation, were determined. All HD patients had significantly higher levels of the LPO products MDA and HNE compared with controls. However, group I patients showed higher MDA and HNE concentrations compared to group II patients. The same result could be seen for protein carbonyls. During HD concentration of both LPO products decreased. However, this was not the case for protein carbonyls. These results lead to the conclusion that optimized correction of the renal anemia may result in a significant reduction of oxidative stress and therefore in the reduction of organ tissue damage. In this way correction of renal anemia will reduce the cardiovascular risk and comorbidity of HD patients improving their prognosis.

Homocysteine in chronic renal failure in relation to renal anemia and to oxidative stress parameters 4-hydroxynonenal and malondialdehyde.

Homocysteine serum levels were measured in patients with end-stage renal disease in relation to severity of renal anemia and oxidative stress parameters such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA). The predialytic homocysteine serum levels of the patients are five times as high as in healthy controls. It was found that homocysteine does not correlate to hemoglobin concentration and to oxidative stress, but rather to parameters of nutrition status such as albumine concentration and protein catabolic rate. The homocysteine accumulation represents a cardiovascular risk factor which is statistically independent of oxidative stress, but dependent on nutrition or energy status in patients with chronic renal failure.