Evidence of Paraoxonases 1, 2, and 3 Expression in Human Ovarian Granulosa Cells.

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.

Screening of Natural Stilbene Oligomers from Vitis vinifera for Anticancer Activity on Human Hepatocellular Carcinoma Cells.

The characterization of bioactive resveratrol oligomers extracted from Vitis vinifera canes has been recently reported. Here, we screened six of these compounds (ampelopsin A, trans-ε-viniferin, hopeaphenol, isohopeaphenol, R2-viniferin, and R-viniferin) for their cytotoxic activity to human hepatocellular carcinoma (HCC) cell lines p53 wild-type HepG2 and p53-null Hep3B. The cytotoxic efficacy depended on the cell line. R2-viniferin was the most toxic stilbene in HepG2, with inhibitory concentration 50 (IC50) of 9.7 ± 0.4 µM at 72 h, 3-fold lower than for resveratrol, while Hep3B was less sensitive (IC50 of 47.8 ± 2.8 µM). By contrast, hopeaphenol (IC50 of 13.1 ± 4.1 µM) and isohopeaphenol (IC50 of 26.0 ± 3.0 µM) were more toxic to Hep3B. Due to these results, and because it did not exert a large cytotoxicity in HH4 non-transformed hepatocytes, R2-viniferin was selected to investigate its mechanism of action in HepG2. The stilbene tended to arrest cell cycle at G2/M, and it also increased intracellular reactive oxygen species (ROS), caspase 3 activity, and the ratio of Bax/Bcl-2 proteins, indicative of apoptosis. The distinctive toxicity of R2-viniferin on HepG2 encourages research into the underlying mechanism to develop the oligostilbene as a therapeutic agent against HCC with a particular genetic background.

Metabolic adaptations in spontaneously immortalized PGC-1α knock-out mouse embryonic fibroblasts increase their oncogenic potential.

PGC-1α 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-1α in cancer. To investigate how loss of PGC-1α 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-1α knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1α 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 ß-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, analysis of amino acid 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-1α, restraining metabolic adaptations in cancer.

Enkephalinase activity is modified and correlates with fatty acids in frontal cortex depending on fish, olive or coconut oil used in the diet.

OBJECTIVE: Enkephalins are neuropeptides involved in functions such as pain modulation and/ or cognitive processes. It has been reported that dietary fat modifies enkephalins in the brain. Since enkephalins are hydrolyzed by enkephalinases, the study of the influence of dietary fats, differing in their degree of saturation, on brain fatty acids content and enkephalinase activity is important to understand its regulatory role on neuropeptides under different type of diets. METHODS: We analyzed enkephalinase activity, assayed with alanine-ß-naphthylamide as sub-strate, in frontal cortex of adult male rats fed diets supplemented with fish oil, olive oil or coconut oil, which markedly differed in the saturation of their fatty acids. RESULTS: Rats fed a diet enriched with coconut oil had lower soluble enkephalinase activity than the group fed olive oil (p<0.01) and fish oil (p<0.05) whereas rats fed a diet enriched with fish oil had lower membrane-bound enkephalinase activity than the group fed with olive (p<0.001) or coconut oil (p<0.05). Significant negative correlations were observed between certain fatty acids and enkephalinase activities in the groups fed with olive and coconut oils. No correlations were observed in the group fed with fish oil. CONCLUSIONS: Dietary fat modifies enkephalinase activity in the frontal cortex depending on the degree of saturation of the used oil. It is postulated that the functions, in which enkephalins are involved, such as pain modulation or cognitive functions, may also be affected according to the type of oil used in the diet.

Unraveling the in vitro antitumor activity of Vismia baccifera against HepG2: role of hydrogen peroxide.

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 G2/M phase, b) phosphorylation of ATM (protein kinase ataxia-telangiectasia mutated) and γH2AX (γ-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 γH2AX 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.

Influence of oxygen partial pressure on the characteristics of human hepatocarcinoma cells.

Most of the in vitro studies using liver cell lines have been performed under atmospheric oxygen partial pressure (21% O2). 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, steady-state 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%) pO2. Results showed that after long-term culturing at 8% versus 21% O2, the cellular proliferation rate and the steady-state levels of mitochondrial O2- were unaffected. However, the intracellular basal ROS levels were higher independently of the characteristics of the cell line. Moreover, the lower pO2 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 long-term culturing of human hepatoma cells at a low, more physiological pO2 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 O2.

Piper and Vismia species from Colombian Amazonia differentially affect cell proliferation of hepatocarcinoma cells.

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 µg/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.

Lipid oxidation inhibitory effects and phenolic composition of aqueous extracts from medicinal plants of Colombian Amazonia.

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.

Involvement of G-463A MPO gene polymorphism in the response of postmenopausal women to hormone therapy.

OBJECTIVE: The aims of this work were to determine (1) the effects of estrogen plus progestogen therapy (EPT) and raloxifene on oxidative stress and cardiovascular risk biomarkers in postmenopausal women and (2) the involvement of the functional G-463A polymorphism of the myeloperoxidase (MPO) gene in the therapy responses. METHODS: Postmenopausal women (45-55 y old) were assigned to three groups receiving (1) EPT (continuous 50 µg transdermal estradiol daily and 200 mg/d micronized progesterone orally the first 14 d of each month; n = 21), (2) raloxifene (60 mg daily; n = 17), and (3) no treatment (control; n = 21). Blood and urine samples were taken before and after 6 months of therapy. Measurements were serum lipid profile, C-reactive intercellular adhesion molecule 1 (ICAM-1), α-tocopherol, γ-tocopherol, uric acid, total antioxidant activity (TAA), malondialdehyde, and urinary 1,4-dihydroxynonane-mercapturic acid (the major urinary 4-hydroxynonenal metabolite). The G-463A MPO polymorphism was analyzed by polymerase chain reaction and restriction fragment length polymorphism. RESULTS: EPT significantly decreased TAA and the levels of ICAM-1, not modifying other cardiovascular risk or oxidative stress markers. The raloxifene and control groups experienced no modifications in oxidative stress or endothelial dysfunction markers. The MPO genotype specifically influenced the outcomes in the EPT group. Thus, TAA decreased significantly in GG (high-expression genotype) homozygotes, whereas ICAM-1 levels were reduced in A allele carriers. CONCLUSIONS: EPT exerted a negative action on the serum oxidant/antioxidant balance in the MPO GG homozygotes and a positive effect on the ICAM-1 endothelial dysfunction marker in carriers of the low-expression A allele. This observation provides evidence of the importance of this polymorphism in the response to EPT.

Doxorubicin induces ceramide and diacylglycerol accumulation in rat hepatocytes through independent routes.

Doxorubicin (DOX) is a potent anticancer drug, whose clinical use is limited due to its toxicity. This toxicity has been associated with free radicals generated during the drug metabolism. We previously found that DOX increased the intracellular diacylglycerol (DAG) levels at 1h in isolated rat hepatocytes, probably by mobilizing choline-enriched phospholipids. In this work, we studied the effects of DOX on oxidative stress markers, and the possible contribution of ceramide metabolism to DAG accumulation. Other possible routes of DAG production, such as impairment of triacylglycerol (TAG) synthesis, and their connection with oxidative stress were also investigated. Time-course experiments revealed that DOX decreased intracellular GSH at 2h, but did not affect cell viability, ATP or malondialdehyde (MDA) levels at any time. DOX did not modify the intracellular levels of [(3)H]-ceramide during the first 90 min of exposure, but increased it significantly at 2h. [(3)H]-Sphingomyelin remained unchanged during the whole period. These results indicate that ceramide metabolism is not involved in the early DAG response to DOX. The drug markedly increased the incorporation of [(3)H]-oleate into intracellular DAG from 60 min. In contrast, DOX reduced the incorporation of [(3)H]-oleate into intracellular phospholipids and TAG. DOX inhibited TAG synthesis at the DAG acyltransferase step. These results suggest that DOX increases the intracellular levels of the lipid messengers, ceramide and DAG, by independent mechanisms. Activation of the de novo synthesis of ceramide is probably involved in the sphingolipid accumulation, while inhibition of TAG synthesis contributes to DAG accumulation, this response being independent of oxidative damage.

Long-chain n-3 polyunsaturated fatty acid from fish oil modulates aortic nitric oxide and tocopherol status in the rat.

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 MOon 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 peroxidisability 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 a-tocopherol in aorta and by a compensatory effect of NO onVLDL + LDL against the low alpha-tocopherol levels, which may contribute to the anti-atherogenic properties of fish oil.

Detection of catechol-O-methyltransferase Val158Met polymorphism by a simple one-step tetra-primer amplification refractory mutation system-PCR.

The G-->A transition at nucleotide 21881 of the human catechol-O-methyltransferase (COMT) gene represents a functional genetic polymorphism (Val158Met), rendering an enzyme with reduced activity that has been associated with psychiatric disorders and estrogen-related cancers. A new method for the detection of this polymorphism is described, based on the tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), with a single PCR to discriminate both alleles. Two primers amplify a common amplicon independently of the allele considered. At the same time, two primers are used, differing in the 3' base. In the Val/Val or Met/Met conditions, amplification occurs both in the general amplicon and in the specific allele; in the Val/Met condition three different amplicons are produced. Direct DNA sequencing of a COMT region containing the G/A polymorphism demonstrates the validity of this tetra-primer ARMS-PCR method. Reevaluation by PCR-RFLP revealed 100% accordance for genotype adscription. Subjects carrying the COMT(HH) genotype in a Spanish population comprised 28%, and the COMT(LL) homozygotes amounted to 21%. The described method provides a fast and reliable approach for determining COMT polymorphism that can be useful in large clinical studies using minimal quantity of DNA, avoiding the timely and costly use of restriction enzymes.

No effect of menstrual cycle on LDL oxidizability and particle size.

OBJECTIVES: Premenopausal women have a lower incidence of cardiovascular disease than men, but this female advantage disappears after menopause, suggesting that female sex hormones exert some cardioprotective effects. One of the mechanisms proposed to explain this cardioprotection is the antioxidant properties of estrogens. The aim of this work was to assess whether fluctuations in ovarian hormones, particularly 17beta-estradiol (E(2)), during the menstrual cycle were associated with changes in the low-density lipoprotein (LDL) particle size, fatty acyl composition, alpha-tocopherol content and in vitro oxidizability. METHODS: Twenty-eight healthy premenopausal women (mean age: 32.2 years) participated in the study. Blood was drawn on days 3 (menstrual phase), 14 (follicular phase) and 22 (luteal phase) of the menstrual cycle for plasma determinations and LDL isolation. Plasma E(2), progesterone, follicle-stimulating hormone and luteinizing hormone were determined by immunoassay. LDL oxidation by Cu(2+)- and 2,2'-azobis (2-amidinopropane) was measured by the formation of conjugated dienes, LDL particle size by quasi-elastic light scattering, fatty acyl composition by gas chromatography, alpha-tocopherol by reversed phase HPLC. A within-subjects analysis of variance was performed to determine significant differences of the variables over the course of a subject's menstrual cycle. RESULTS: The LDL oxidizability indices (lag time before the onset of propagation and the maximal oxidation rate) did not change during the menstrual cycle. The LDL particle size (24.8+/-1.7 nm diameter), alpha-tocopherol (11.7+/-3.7 nmol/mg LDL protein) and fatty acyl composition also remained constant. CONCLUSIONS: The LDL physicochemical properties and oxidizability are not affected by menstrual cycle phase.

Superoxide anions are involved in doxorubicin-induced ERK activation in hepatocyte cultures.

Doxorubicin (DOX), an antineoplastic agent widely used for the treatment of cancer, belongs to the anthracycline family of antitumor antibiotics. DOX may undergo one-electron reduction to the corresponding semiquinone free radical by flavin-containing reductases. Under aerobic conditions, the semiquinone radical reacts rapidly with oxygen to generate superoxide anion, undergoing redox cycling. At moderate concentrations, reactive oxygen species (ROS) play an important role as regulatory mediators in signaling processes. We have shown that DOX increased phosphorylation of enzymes comprising mitogen-activated protein (MAP) kinase cascades in primary hepatocyte cultures, and that this action was independent of oxidant damage. In particular, extracellular signal-regulated kinase (ERK) was phosphorylated by the drug treatment. In this work, we have determined the possible involvement of particular free radicals in DOX-induced ERK phosphorylation in hepatocyte cultures by using specific free radical scavengers. The levels of ERK phosphorylation were measured by Western blot analysis with an anti-Thr202/Tyr204-phosphorylated p44/p42 MAPK antibody. Deferoxamine (DFO; iron chelator), catalase (hydrogen peroxide-removing enzyme), or alpha-tocopherol (peroxyl-radical scavenger) did not affect DOX-increased ERK phosphorylation levels. However, the cell-permeable superoxide dismutase mimetic MnTBAP and the flavin-containing enzyme inhibitor diphenyleneiodonium reverted DOX-induced effects. These results suggest that superoxide anions, probably generated by DOX metabolism, are involved in the effects of the anthracycline on the MAP kinase cascade activation.

Doxorubicin-induced MAPK activation in hepatocyte cultures is independent of oxidant damage.

Doxorubicin (DOX) is a potent anticancer drug, whose clinical use is limited on account of its toxicity. DOX cytotoxic effects have been associated with reactive oxygen species (ROS) generated during drug metabolism. ROS induce signaling cascades leading to changes in the phosphorylation status of target proteins, which are keys for cell survival or apoptosis. The mitogen-activated protein kinase (MAPK) cascades are routes activated in response to oxidative stress. In this work, the effects of DOX on cytotoxicity, indicators of oxidative stress (malondialdehyde -MDA- and GSH), and the phosphorylation status of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 kinases were analyzed in primary cultures of rat hepatocytes. DOX (1-50 microM) did not modify lactate dehydrogenase (LDH) release into the medium, the levels of MDA (determined by high-performance liquid chromatography [HPLC]) or the intracellular GSH during the incubation time up to 6 h. GSH levels from mitochondria extracted by Percoll gradient from cultured hepatocytes were not modified by DOX, thus excluding its depletion or any impaired mitochondrial uptake. Characterization of proteins by Western blot analysis revealed that DOX increased phosphorylation of p38 kinases and JNK1 and JNK2 in a dose- and time-dependent manner. DOX also increased ERK2 phosphorylation at latter time points. In conclusion, DOX triggers activation of ERK, JNK, and p38 kinases in primary cultures of rat hepatocytes independently of oxidant damage.