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217 p., Grapes and red wine are the main sources of resveratrol, a polyphenol widely known for its beneficial effects on various diseases. However, other parts of the vine (wood, canes and roots) are important and cheap sources of other bioactive stilbenes. The main objective of this thesis was 1) to carry out a preliminary screening of a series of novel stilbenes (including glucosides and oligomers of resveratrol) from Vitis vinifera vine for their cytotoxic and anti¿inflammatory potential, and 2) to select the most potent one to establish its mechanism of action. We tested cytotoxicity in human hepatocarcinoma HepG2 (wild-type p53) and Hep3B (p53-null), and inflammation in the murine macrophage RAW 264.7 cell line. Several glucosides, which had never before been extracted from the woody part of the vine, showed moderate cytotoxic activity. Piceatannol and the oligomers, ¿-viniferin, hopeaphenol and isohopeaphenol, inhibited the inflammatory response decreasing the levels of nitric oxide, reactive oxygen species, and cytokines. Of the oligomers tested, R2¿viniferin (tetramer) was the most potent in inducing cell death in HepG2, not affecting human non¿transformed hepatocyes. R2¿viniferin exerted its toxic action by a p53¿dependent mechanism, that involved DNA damage, increased H2O2, upregulation of mitochondrial SOD, and apoptosis. We conclude that Vitis vinifera cane is a source of R2¿viniferin, a promising natural compound in the development of chemotherapies against p53¿positive human hepatocellular carcinomas.

Increasing evidence suggests that the antioxidant paraoxonase proteins, PON1, PON2, and PON3, have a role in reproduction and may be synthesized by ovarian cells. The aim of this work was to investigate whether human ovarian granulosa cells (GC) express paraoxonases 1, 2, and 3 (PON1, PON2, and PON3) at both the transcriptional and protein levels. Cells were purified from follicle samples of women undergoing ovarian stimulation at oocyte retrieval. We analyzed mRNA by polymerase chain reaction using specific primers for the different variants and quantified the proteins by Western blot using commercially available human recombinant PON proteins as standards. The protein subcellular distribution was determined by immunofluorescence and confocal microscopy and the cell cycles by flow cytometry. Thymidine was used for cellular synchronization at G1/S. Human hepatoma HepG2 and immortalized granulosa COV434 cell lines were used to optimize methodologies. mRNAs from PON1, the two variants of PON2, and PON3 were detected in GC. The cells actively secreted PON1 and PON3, as evidenced by the protein detection in the incubation medium. PON1 and PON3 were mainly distributed in the cytoplasm and notably in the nucleus, while PON2 colocalized with mitochondria. Subcellular nucleo-cytoplasmic distribution of PON1 was associated with the cell cycle. This is the first evidence describing the presence of mRNAs and proteins of the three members of the PON family in human ovarian GC. This study provides the basis of further research to understand the role of these proteins in GC, which will contribute to a better understanding of the reproduction process.

217 p., Grapes and red wine are the main sources of resveratrol, a polyphenol widely known for its beneficial effects on various diseases. However, other parts of the vine (wood, canes and roots) are important and cheap sources of other bioactive stilbenes. The main objective of this thesis was 1) to carry out a preliminary screening of a series of novel stilbenes (including glucosides and oligomers of resveratrol) from Vitis vinifera vine for their cytotoxic and anti¿inflammatory potential, and 2) to select the most potent one to establish its mechanism of action. We tested cytotoxicity in human hepatocarcinoma HepG2 (wild-type p53) and Hep3B (p53-null), and inflammation in the murine macrophage RAW 264.7 cell line. Several glucosides, which had never before been extracted from the woody part of the vine, showed moderate cytotoxic activity. Piceatannol and the oligomers, ¿-viniferin, hopeaphenol and isohopeaphenol, inhibited the inflammatory response decreasing the levels of nitric oxide, reactive oxygen species, and cytokines. Of the oligomers tested, R2¿viniferin (tetramer) was the most potent in inducing cell death in HepG2, not affecting human non¿transformed hepatocyes. R2¿viniferin exerted its toxic action by a p53¿dependent mechanism, that involved DNA damage, increased H2O2, upregulation of mitochondrial SOD, and apoptosis. We conclude that Vitis vinifera cane is a source of R2¿viniferin, a promising natural compound in the development of chemotherapies against p53¿positive human hepatocellular carcinomas.

149 p., Los ciclos de hiperstimulación ovárica controlada son ampliamente utilizados en las técnicas de reproducción asistida debido al mayor número de óvulos que se obtienen por ciclo y al consecuente aumento de las probabilidades de éxito de los tratamientos. Sin embargo, la manipulación hormonal que conllevan estos ciclos puede producir efectos adversos tanto a nivel ovárico como gestacional, entre ellos alteraciones redox. En esta tesis doctoral se ha estudiado el sistema antioxidante del líquido folicular (microambiente del ovocito) de una misma mujer después de un ciclo de ovulación natural y después de un ciclo de hiperestimulación ovárica controlada. Para ello se han utilizado técnicas espectrofotométricas, espectrometría de masas, inmunotransferencia western, técnicas de cultivos celulares y microscopia confocal. Los resultados mostraron que los ciclos de hiperestimulación ovárica provocan una disminución de la protección antioxidante del líquido folicular y por tanto del ovocito, frente a los ciclos naturales en los que el ovocito se encuentra más protegido. Esto se vio reflejado, entre otras cosas, en una disminución de la actividad de los enzimas PON, los cuales hemos caracterizado por primera en las células de la granulosa humanas, lo que nos lleva a sugerir un posible papel en los procesos reproductivos.

PGC-1a controls, to a large extent, the capacity of cells to respond to changing nutritional requirements and energetic demands. The key role of metabolic reprogramming in tumor development has highlighted the potential role of PGC-1a in cancer. To investigate how loss of PGC-1a activity in primary cells impacts the oncogenic characteristics of spontaneously immortalized cells, and the mechanisms involved, we used the classic 3T3 protocol to generate spontaneously immortalized mouse embryonic fibroblasts (iMEFs) from wild-type (WT) and PGC-1a knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1a KO iMEFs formed larger and more proliferative primary tumors than WT counterparts, and fostered the formation of lung metastasis by B16 melanoma cells. These characteristics were associated with the reduced capacity of KO iMEFs to respond to cell contact inhibition, in addition to an increased ability to form colonies in soft agar, an enhanced migratory capacity, and a reduced growth factor dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear b-catenin and c-myc in KO iMEFs. Evaluation of the metabolic adaptations of the immortalized cell lines identified a decrease in oxidative metabolism and an increase in glycolytic flux in KO iMEFs, which were also more dependent on glutamine for their survival. Furthermore, glucose oxidation and tricarboxylic acid cycle forward flux were reduced in KO iMEF, resulting in the induction of compensatory anaplerotic pathways. Indeed, analysisi of aminoacid and lipid patterns supported the efficient use of tricarboxylic acid cycle intermediates to synthesize lipids and proteins to support elevated cell growth rates. All these characteristics have been observed in aggressive tumors and support a tumor suppressor role for PGC-1a, restraining metabolic adaptations in cancer.

Currently natural products derived from plants are receiving huge attention because of their antitumor activities. In previous work we reported that an aqueous leaf extract of Vismia baccifera induced toxicity in HepG2. The present study focuses on the mechanisms of the cytotoxic actions induced by the extract. Results showed that V. baccifera was innocuous in non-transformed human HH4 hepatocytes. In HepG2 it caused deregulation of antioxidant status (increasing superoxide dismutase expression and decreasing glutathione levels and glutathione peroxidase activity) and accumulation of reactive oxygen species, particularly hydrogen peroxide. The extract induced a) cell cycle arrest at G(2)/M phase, b) phosphorylation of ATM (protein kinase ataxia-telangiectasia mutated) and gamma H2AX (gamma-histone family 2A variant), c) caspase-3 activation, and e) deregulation of the Bax/Bcl family, increasing pro-apoptotic proteins. ATM did not seem to be involved in gamma H2AX activation. Co-incubation with catalase prevented the alterations elicited by V. baccifera in HepG2. Taking together, these results indicate that hydrogen peroxide mediates the HepG2 cytotoxic response and provide evidence for more in-depth studies of the signaling involved.

Oxidative stress is associated with obstetric complications during ovarian hyperstimulation in women undergoing in vitro fertilization. The follicular fluid contains high levels of proteins, which are the main targets of free radicals. The aim of this work was to determine specific biomarkers of non-enzymatic oxidative modifications of proteins from follicular fluid in vivo, and the effect of ovarian stimulation with gonadotropins on these biomarkers. For this purpose, 27 fertile women underwent both a natural and a stimulated cycle. The biomarkers, glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), N-epsilon-(carboxymethyl)lysine (CML), and N-epsilon-(carboxyethyl)lysine (CEL), were measured by gas-liquid chromatography coupled to mass spectrometry. Results showed that follicular fluid contained products of protein modifications by direct metal-catalyzed oxidation (GSA and AASA), glycoxidation (CML and CEL), and lipoxidation (CML). GSA was the most abundant biomarker (91.5%). The levels of CML amounted to 6% of the total lesions and were higher than AASA (1.3%) and CEL (1.2%). In the natural cycle, CEL was significantly lower (p < 0.05) than in the stimulated cycle, suggesting that natural cycles are more protected against protein glycoxidation. These findings are the basis for further research to elucidate the possible relevance of this follicular biomarker of advanced glycation end product in fertility programs.

Most of the in vitro studies using liver cell lines have been performed under atmospheric oxygen partial pressure (21% O-2). However, the oxygen concentrations in the liver and cancer cells are far from this value. In the present study, we have evaluated the influence of oxygen on 1) the tumor cell lines features (growth, steadystate ROS levels, GSH content, activities of antioxidant enzymes, p66 Shc and SOD expressions, metalloproteinases secretion, migration, invasion, and adhesion) of human hepatocellular carcinoma cell lines, and b) the response of the cells to an oxidant stimulus (aqueous leaf extract of the V. baccifera plant species). For this purpose, three hepatocarcinoma cell lines with different p53 status, HepG2 (wild-type), Huh7 (mutated), and Hep3B (deleted), were cultured (6-30 days) under atmospheric (21%) and more physiological (8%) pO(2). Results showed that after long-term culturing at 8% versus 21% O-2, the cellular proliferation rate and the steady-state levels of mitochondrial O-2-were unaffected. However, the intracellular basal ROS levels were higher independently of the characteristics of the cell line. Moreover, the lower pO(2) was associated with lower glutathione content, the induction of p66 Shc and Mn-SOD proteins, and increased SOD activity only in HepG2. This cell line also showed a higher migration rate, secretion of active metalloproteinases, and a faster invasion. HepG2 cells were more resistant to the oxidative stress induced by V. baccifera. Results suggest that the longterm culturing of human hepatoma cells at a low, more physiological pO(2) induces antioxidant adaptations that could be mediated by p53, and may alter the cellular response to a subsequent oxidant challenge. Data support the necessity of validating outcomes from studies performed with hepatoma cell cultures under ambient O-2.

195 p., Nowadays there is an increasing interest in finding bioactive compounds which can be used as chemopreventive agents against cancer. In this work, we have studied the antitumoral actions of aqueous extracts from leaves of the Colombian Amazonian Vismia baccifera plant on the human hepatocellular carcinoma HepG2 cell line. Our results showed that V. baccifera induced a cytotoxic response to HepG2, increasing the levels of mitochondrial O2- and intracellular ROS (particularly hydrogen peroxide), inducing depletion of GSH, cell cycle arrest at G2/M phase, endoplasmic reticulum stress, autophagy and apoptosis. Interestingly, the cytotoxic actions exerted by V. baccifera are exclusive of cancer cells. Hydrogen peroxide, whose intracellular accumulation was early induced by the extract, mediated the cytotoxic response. V. baccifera promoted deregulation of antioxidant enzymes, this effect being secondary to the accumulation of ROS. The intracellular GSH depletion was not the cause, but the consequence of V. baccifera-induced toxicity. The plant extract also affected HepG2 signaling by activating and inhibiting specific pathways. Moreover, we validated the experimental approach we have used to study the toxic response under conditions of 21% atmospheric pO2, ruling out any possible superimposed oxidative stress derived from the culture conditions. Our results highlight the importance of analyzing in depth the actions of this plant to generate knowledge that can lay the bases for coadjuvant or anti-cancer therapy.

There is an increasing interest to identify plant-derived natural products with antitumor activities. In this work, we have studied the effects of aqueous leaf extracts from Amazonian Vismia and Piper species on human hepatocarcinoma cell toxicity. Results showed that, depending on the cell type, the plants displayed differential effects; thus, Vismia baccifera induced the selective killing of HepG2, while increasing cell growth of PLC-PRF and SK-HEP-1. In contrast, these two last cell lines were sensitive to the toxicity by Piper krukoffii and Piper putumayoense, while the Piperaceae did not affect HepG2 growth. All the extracts induced cytotoxicity to rat hepatoma McA-RH7777, but were innocuous (V. baccifera at concentrations < 75 mu g/mL) or even protected cells from basal death (P. putumayoense) in primary cultures of rat hepatocytes. In every case, cytotoxicity was accompanied by an intracellular accumulation of reactive oxygen species (ROS). These results provide evidence for the anticancer activities of the studied plants on specific cell lines and suggest that cell killing could be mediated by ROS, thus involving mechanisms independent of the plants free radical scavenging activities. Results also support the use of these extracts of the Vismia and Piper genera with opposite effects as a model system to study the mechanisms of the antitumoral activity against different types of hepatocarcinoma.

Diverse plants of ethnobotanic interest in Amazonia are commonly used in traditional medicine. We determined the antioxidant potential against lipid peroxidation, the antimicrobial activity, and the polyphenol composition of several Amazonian plants (Brownea rosademonte, Piper glandulosissimum, Piper krukoffii, Piper putumayoense, Solanum grandiflorum, and Vismia baccifera). Extracts from the plant leaf, bark, and stem were prepared as aqueous infusions, as used in folk medicine, and added to rat liver microsomes exposed to iron. The polyphenolic composition was detected by reverse-phase HPLC coupled to diode-array detector and MS/MS analysis. The antimicrobial activity was tested by the spot-on-a-lawn method against several indicator microorganisms. All the extracts inhibited lipid oxidation, except the P. glandulosissimum stem. The plant extracts exhibiting high antioxidant potential (V. baccifera and B. rosademonte) contained high levels of flavanols (particularly, catechin and epicatechin). By contrast, S. grandiflorum leaf, which exhibited very low antioxidant activity, was rich in hydroxycinnamic acids. None of the extracts showed antimicrobial activity. This study demonstrates for the first time the presence of bioactive polyphenolic compounds in several Amazonian plants, and highlights the importance of flavanols as major phenolic contributors to antioxidant activity.

In spite of their high oxidisability. long-chain n-3 PUFA protect against CVD. Dietary fatty acids modulate the fatty acid composition of lipoproteins involved in atherosclerosis. We thought that if long-chain n-3 PUFA were able to increase NO production by the aorta, then by its antioxidant activity the NO will prevent lipid peroxidation. However, the beneficial effect of NO in vivo on VLDL + LDL oxidation would only be possible if NO could diffuse to their lipidic core. Rats were fed maize oil- or fish oil as menhaden oil- (MO) rich diets for 8 weeks, to study the effects of MO on aortic NO production. NO diffusion into VLDL + LDL. the extent of oxidation in native VLDL + LDL and their oxidisability ex vivo. Aortic NO production and its alpha-tocopherol content were increased and n-3 PUFA were incorporated into the VLDL + LDL. In spite of the higher per-oxidisability and the low alpha-tocopherol in native VLDL + LDL from rats fed MO. native VLDL + LDL from the two groups shared similar electrophoretic patterns. conjugated dienes. thiobarbituric acid-reactive substances, total antioxidant capacity, and NO diffusibility on VLDL + LDL indicative of an in vivo protection against oxidation. However, these results do not correlate with the ex vivo oxidisability of VLDL + LDL, as NO is lacking. Thus. the in vivo beneficial effects can be explained by increased alpha-tocopherol in aorta and by a compensatory effect of NO on VLDL divided by LDL against the low alpha-tocopherol levels, which may contribute to the anti-atherogenic properties of fish oil.

There is a relationship between the fatty acid profile in skeletal muscle phospholipids and peripheral resistance to insulin in adults, but similar data have not been reported in infancy and childhood. The objective of this study was to investigate the fatty acid composition of skeletal muscle and adipose tissue across the paediatric age range. The fatty acid profile of skeletal muscle phospholipids and adipose tissue triacylglycerols was analysed in ninety-three healthy Spanish infants and children distributed into four groups: group 1 (0 to < 2 years, n 10); group 2 (2 to < 5 years, n 41); group 3 (5 to < 10 years, n 24); group 4 (10 to 15 years, n 18). In skeletal muscle phospholipids, oleic acid (18: 1n-9cis) content decreased significantly whereas that of linoleic (18: 2n-6) acid increased significantly with age (P for trend < 0.01). In adipose tissue, the contents of triacylglycerol and linoleic acid increased significantly across the paediatric age range (P for trend < 0.01), whereas dihomo-gamma-linolenic (20: 3n-6) and arachidonic (20: 4n-6) showed significant differences between groups. The variations in fatty acid composition observed with age indicated an imbalance in dietary n-3/n-6 long-chain PUFA.