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Stilbenes, of which resveratrol is the most studied, were described as antioxidant and anti-inflammatory molecules. Presumably, other stilbenes have similar biological properties, but the evidence ...

The paraoxonases (PONs) are antioxidant enzymes associated with beneficial effects against several diseases and some exposures. Little is known, however, about the role of PONs in human reproduction. This work was conducted to investigate whether any association existed between the activities of the PON enzymes (1, 2, and 3) with the follicular size and fertility parameters in assisted reproduction. The study included 100 subfertile women (patients) and 55 proven fertile women (oocyte donors), all undergoing an ovarian stimulation cycle. Follicular fluid from small (diameter12 mm) and large (diameter ≥18 mm) follicles was collected from each woman. The PONs were quantified in follicular fluid by immunoblotting. PON1 arylesterase and paraoxonase, PON2 methyl paraoxonase and PON3 simvastatinase activities from both donors and patients were significantly higher (P0.001) in follicular fluid from large follicles compared with small ones. In large follicles, PON3 activity was significantly higher (P0.01) in donors compared with patients. Follicular fluid PON1 arylesterase and paraoxonase activity was positively correlated with the number of retrieved oocytes in donors. This study shows an increase in the activities of PONs with follicle size, thus providing indirect evidence for the role of PONs in follicle maturation.

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 β-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., This work was funded by grants from the Spanish “Ministerio de Ciencia, Innovación y Universidades” (MICINN) and ERDF/FEDER funds, SAF2012-37693, SAF2015-63904-R, SAF2015-71521-REDC, RTI2018-093864-B-I00 to M.M., SAF2017-83043-R and B2017/BMD-3724 to S·C., PI15/00107 to A.M.R, the University of the Basque Country UPV/EHU grant GIU16/62) to J.l.R.S. and M.B.R.L., and the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement 721236-TREATMENT to M.M.

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-β-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 (pmembrane-bound enkephalinase activity than the group fed with olive (p 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.

International audience; Aim: Stilbenes are well-known phytoalexins present in vine and wine. Stilbene glucosides have been identified in wine; however, other than piceid, these compounds have never been reported to be present in the woody parts of grapevine. The aims of this study were to investigate the presence of stilbene glucosides in the woody parts of the vine and to evaluate their cytotoxic activity, in comparison with that of resveratrol, in human hepatoma HepG2 cells. Methods and results: Stilbene glucosides were isolated from a Vitis vinifera root extract. The extract was partitioned with ethyl acetate and fractionated by polyamide gel column chromatography. Pure compounds were obtained by semipreparative high-performance liquid chromatography. These were then identified by mass spectrometry and nuclear magnetic resonance (NMR) analyses, including analysis of two-dimensional NMR spectra. In addition to resveratrol, five stilbene glucosides were found: resveratroloside, resveratrol rutinoside, trans-epsilon-viniferin diglucoside, cis-epsilon-viniferin diglucoside and piceid. Of these, the first four showed cytotoxic effects against HepG2 cells when the crystal violet assay was used to determine cell viability. Conclusion: In addition to resveratrol and piceid, this is the first report of the presence of four glucosylated derivatives of resveratrol (resveratroloside, resveratrol rutinoside, and trans- and cis-epsilon-viniferin diglucosides) in the woody parts of vine. These compounds showed significant cytotoxicity against HepG2 cells. Significance and impact of the study: Stilbenes are well-known biological compounds. Grapevine is one of the main sources of this family of polyphenols. Other than piceid, stilbene glucosides have been identified in wine but never in the woody parts of vine. This is the first study in which five glucosylated derivatives of resveratrol were isolated from woody parts of vine. They were also shown to exert antiproliferative effects in human hepatoma HepG2 cells.

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. This work was supported by the Basque Government (ref. IT687-13), and University of the Basque Country UPV/EHU (CLUMBER UFI11/20, and pre-doctoral and post-doctoral grants to J.T.).

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., Graphical abstract fx1, Highlights • The influence of pO2 on human hepatocellular carcinoma cell features is analyzed. • Low oxygen tension (8% O2) induces antioxidant adaptations in HepG2. • Low O2 increases the migration and invasion rates of HepG2. • Antioxidant adaptations could be p53-dependent. • Validating results from in vitro studies using cells cultured at 21% O2 are required.

Free radicals are necessary in female reproduction, but at high concentrations they are associated with reproductive diseases and infertility. To maintain these species in non-harmful stationary concentrations, antioxidants act coordinately in reproductive tissues. Paraoxonases (PONs) are hydrolytic enzymes with a wide range of substrates capable of protecting against lipid peroxidation. Recently, PON1, PON2, and PON3 have been localized in follicular fluid (FF), a fluid that is constituted by components of the blood that freely diffuse through the hematofollicular barrier and by products of the local synthesis of ovarian cells. In this work, we describe for the first time the expression of PON1, PON2, and PON3 proteins at the transcriptional and protein levels in human granulosa cells. The proteins were localized intracellularly by confocal microscopy and quantified by western blot using commercial human recombinant PON proteins as standards. The expression of PONs in these cells indicate that their presence in FF is not exclusively due to simple diffusion from the blood and that these proteins have a role in reproduction.

Controlled ovarian hyperstimulation (COH) is used in standard protocols in in vitro fertilization (IVF). However, its use is associated with clinical complications such as ovarian hyperstimulation syndrome. The evidence indicates that the natural cycle (NC) has a lower risk and is more physiological. In this study, we have determined the antioxidant status of follicular fluid at the time of oocyte retrieval following NC or COH. Forty-one fertile women (oocyte donors) followed both cycles. In NC no exogenous gonadotrophins were used, and only hCG was given to induce ovulation. We determined TAA, ORAC, - and tocopherol, nitric oxide, and the activities of SOD, catalase, total and Se-dependent glutathione peroxidases, and paraoxonases. Results showed that TAA, -tocopherol, and the paraoxonase, arylesterase, and simvastatinase activities were significantly higher in NC, compared with COH. Our results indicate that NC is associated with higher follicle antioxidant protection and provide important insights into prevention of hyperstimulation side effects.

The total phenol and flavonoid contents of 19 Amazonian plants and their related antioxidant activities were determined. The extracts from the plant leaf, bark, root, fruit and/or stem were prepared as infusions, as are traditionally used in popular medicine. Total phenols ranged from 0.8 to 22.2 mg gallic acid equivalents/g and flavonoids from 0.0 to 10.2 mg catechin equivalents/g, by using Folin–Ciocalteau and aluminium chloride colourimetric methods. Differences were observed in phenol and flavonoid contents at the organic level, the leaf presenting greater values than the stem. All the extracts showed different degrees of antioxidant activity with TEAC, 1.1 up to 117.4 and ORAC, 7.8 up to 359.1 μmol Trolox equivalents/g. These values correlated with total phenol content ( r 2 = 0.90) and flavonoid content ( r 2 = 0.70 for TEAC; r 2 = 0.76 for ORAC). Piper putumayoense , Piper glandulosissimum , Piper krukoffii and Senna reticulata leaves and Brownea rosademonte bark showed elevated antioxidant activities, thus representing promising plant-sources of medicine.

In our previous works, we have demonstrated that terfenadine (TEF) induces DNA damage and apoptosis in human melanoma cell lines. In this present work, we have studied the effect of histamine on viability of A375 human melanoma cells and the cell-signalling pathways through which TEF may induce its apoptotic effect. We have found that exogenous histamine stimulates A375 melanoma cell proliferation in a dose- and time-dependent manner. Moreover, TEF-induced apoptosis seems to occur via other cellular pathways independent of the histamine-signalling system since co-treatment of histamine with TEF did not protect melanoma cells from the cytotoxic effect of TEF, and alpha fluoromethylhistidine did not induce the same cytotoxic effect of TEF. In addition, we have observed that knocking down the H1 histamine receptor (HRH1) by small interference RNA approach protects melanoma cells only slightly from TEF-induced apoptosis. To explore the molecular mechanisms responsible for histamine and TEF effect on the cell growth, we analysed intracellular cyclic nucleotides and Ca(2+) levels. TEF did not modify intracellular levels of cyclic adenosine 3',5'-monophosphate and cyclic guanine 3',5'-monophosphate; however, TEF induced a very sharp and sustained increase in cytosolic Ca(2+) levels in A375 melanoma cells. On the contrary, histamine did not modulate intracellular Ca(2+). TEF-induced Ca(2+) rise and apoptosis appear to be phospholipase C (PLC) dependent since neomycin and U73122, two inhibitors of PLC, abolished cytosolic Ca(2+) increase and protected the cells completely from cell death. Furthermore, inhibition of tyrosine kinase activity by genistein blocked cytosolic Ca(2+) rise and TEF-induced apoptosis. These results suggest that TEF modulates Ca(2+) homeostasis and induces apoptosis through other cellular pathways involving tyrosine kinase activity, independently of HRH1.

Ferrylmyoglobin (ferrylMb) may play a major role in vivo under certain pathological conditions. Preliminary experiments showed that ferrylmyoglobin induced a mild oxidative stress in rat hepatocytes, mainly reflected by early lipid peroxidation. One of the major functions of hepatocytes is the synthesis, secretion and distribution of lipids to other cells. The aim of this work was to examine whether ferrylMb affected the synthesis and secretion of triacylglycerols (TAG), and the possible involvement of lipid peroxidation on these effects. The heme protein completely impaired VLDL secretion, affecting both the lipid and apoB components of the lipoprotein particle. The incorporation of [3H]-oleate into newly synthesized diacylglycerol and TAG was not altered by ferrylMb. The co-treatment of cells with α-tocopherol prevented lipid peroxidation and concomitantly reverted VLDL TAG secretion to control values. Importantly, although ferrylMb dramatically blocked prelabeled TAG secretion, newly synthesized TAG secretion was not impaired. These data indicate that lipid peroxidation elicited by ferrylMb modulates the VLDL TAG secretion process, specifically affecting the stored intracellular TAG mobilization, rather than de novo synthesis. Apart from its potential role in vivo, ferrylmyoglobin constitutes a useful model for studying the interactions between lipid peroxidation and the specific TAG pool dependence for VLDL secretion.

Reactive oxygen species (ROS) and antioxidants are involved in the regulation of reproductive processes. Previous studies in infertile women undergoing an ovarian stimulation cycle have suggested a possible role for ROS in the occurrence of conception in In Vitro Fertilization. In this context the control of the redox balance of follicular fluid becomes essential for reproduction, so that the presence of enzymes with antioxidant activities, such as the paraoxonase (PON) system, would play a role in maintaining this balance. The objective of this work was a) to characterize the paraoxonase system in follicular fluid of women undergoing a controlled ovarian stimulation cycle, analysing the associated PON1, PON2, and PON3 activities, and b) to study the possible involvement of the PON system in follicular maturation. The enzyme activities were quantified in follicular fluid from large and small follicles from women undergoing an ovarian stimulation cycle in the IVI-Bilbao clinic. PON activities were quantified using spectrophotometric and HPLC techniques. Statistical comparisons were performed using the Student's t-test for paired data. Results indicate that follicular fluid presents paraoxonase activities which are detectable by the methods developed in this study. PON activities were associated with follicular maturation, suggesting that the PON system plays a role in oocyte maturation. This work was supported by research grants from the Ministry of Health and Consumption (FIS/FEDER PI11/02559), the Basque Country Government (Dep. Education, Universities and Research ref. IT687-13, and DCIT ref. S-PE13UN063), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01).

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.

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 &lt, 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.

Hemoglobin is a complex protein formed by various subunits interacting with each other. These non-covalent interactions, quaternary structure, are responsible for hemoglobin functioning as an excellent oxygen transporter, loading up with oxygen in the lungs and delivering it to tissues, where the oxygen pressure is lower. The communications between subunits make hemoglobin bind oxygen in a cooperative way, displaying sigmoidal saturation curves. In addition to oxygen, hemoglobin also transports H+, CO2, and 2,3-bisphosphoglycerate, and these molecules regulate the transport of oxygen. The quaternary structure of hemoglobin accounts for the different behavior with respect to another oxygen-binding protein, myoglobin, a monomeric protein, thus lacking quaternary structure, whose function is mainly to store oxygen. A simple question, “What consequences would the loss of the hemoglobin quaternary structure have on oxygenation?” helps the student to think about the behavior of hemoglobin and to integrate and relate all the elements affecting its binding with oxygen.

Phenols are universal components of plant material. Flavonoids are polyphenolic constituents of the diet with antioxidant properties by virtue of the structural arrangements and hydrogen-donating potential of their phenolic groups. The ability of specific phenolics to chelate transition metal ions is also of significance. In vitro , the antioxidant activity of the aglycone forms of these polyphenols is, in the main, greater than that of vitamin E and vitamin C. In the diet they are present principally as glycosylated, esterified or polymerized derivatives. Many factors, such as the extent of their gastrointestinal absorption, metabolism (possibly into derivatives with enhanced activity) and interaction with target cells, affect the bioavailability and activity of dietary polyphenols. The balance between beneficial and detrimental effects after their consumption is thus somewhat unpredictable. The possible sites at which these compounds might act as significant dietary micronutrient antioxidants are: (1) i...

tert-Butyl hydroperoxide (TBHP) mobilizes arachidonic acid (AA) from membrane phospholipids in rat hepatocytes under cytotoxic conditions, thus leading to an increase in intracellular AA, which precedes cell death. In the present work, the involvement of lipid peroxidation, thiol status, and reactive oxygen species (ROS) in the intracellular AA accumulation induced by 0.5 mM TBHP was studied in rat hepatocytes. Cells treated with TBHP maintained viability and energy status at 10 min. However, TBHP depleted GSH, as well as inducing lipid peroxidation and ROS formation, detected by dichlorofluorescein (DCF) fluorescence. TBHP also significantly increased (32.5%) the intracellular [14C]-AA from [14C]-AA-labelled hepatocytes. The phospholipase A2 (PLA2) inhibitor, mepacrine, completely inhibited the [14C]-AA response. The addition of antioxidants to the cell suspensions affected the TBHP-induced lipid response differently. The [14C]-AA accumulation correlated directly with ROS and negatively with endogenous GSH. No correlation between [14C]-AA and lipid peroxidation was found. Promethazine prevented lipid peroxidation and did not affect the [14C]-AA increase. We conclude that TBHP stimulates the release of [14C]-AA from membrane phospholipids through a PLA2-mediated mechanism. Endogenous GSH and ROS play a major role in this effect, while lipid peroxidation-related events are unlikely to be involved. Results suggest that specific ROS generated in iron-dependent reactions, different from lipid peroxyl radicals, are involved in PLA2 activation, this process being important in TBHP-induced hepatocyte injury.

Estrogens are effective antioxidants in diverse biological systems. Despite their antioxidant activities, it is not known yet whether estrogens prevent or alleviate liver toxicity induced by oxidative stress. In the present work, we studied this possibility by examining in vitro the protective potential of different estrogen compounds (17beta-estradiol, 2-hydroxyestradiol, and diethylstilbestrol) against tert-butyl hydroperoxide-induced hepatocyte damage. Various parameters such as cell viability, lipid peroxidation, adenine nucleotide content, and thiol status were measured as an index of cytotoxicity. The protective effects of estrogens were compared to those of the iron chelator deferoxamine. The molecules tested prevented oxidant-induced cell death differently, showing variable degrees of protection. Deferoxamine was the most potent agent, followed by diethylstilbestrol and 2-hydroxyestradiol, 17beta-estradiol being the least efficient. The inhibitory effects on lipid and thiol oxidations paralleled the effects on cell viability. The molecules also reduced the oxidant-induced ATP depletion, except for 17beta-estradiol which had no effect on the decreased ATP levels. Our results suggest that the mechanisms of the preventive actions of estrogens may be related not only to their antioxidant activity against free radicals, but also and to a lesser extent to the maintenance of the normal redox status of the cell, which partially recovers the intracellular GSH levels.

Objective To investigate whether the Ala16Val polymorphism in the SOD2 gene, encoding for mitochondrial manganese superoxide dismutase (SOD), is associated with [1] infertility and [2] the pregnancy rate (PR) in IVF cycles. Design Prospective case-control study. Setting Public university and public university hospital. Patient(s) A total of 362 newborns (nonselected population) and 148 infertile women undergoing an IVF cycle, from which 44 became pregnant and 104 did not. Intervention(s) Blood samples extracted from the patients and newborn umbilical cord. Main Outcome Measure(s) Genotype and allele distribution of the Ala16Val polymorphism in the SOD2 gene using the tetra-primer amplification refractory mutation system–polymerase chain reaction (PCR). Result(s) The polymorphism distribution of the subfertile women was similar to that of a nonselected population. The SOD2 Ala allele frequency was 49% both in controls and IVF patients. In IVF population the Ala/Ala SOD2 genotype was 25%, with a 28% Val/Val homozygous. In contrast, the Ala/Ala genotype was associated with higher PRs in IVF (47% in Ala/Ala vs. 23% in no Ala/Ala). A multivariate logistic regression analysis revealed that the Ala/Ala genotype was an independent predictor of pregnancy (odds ratio [OR] = 3.29), followed by the number of transferred embryos (OR = 2.37) and age (OR = 0.84). Conclusion(s) The Ala/Ala SOD2 genotype is a significant independent predictor of the occurrence of pregnancy in IVF. Data also support a role for antioxidant defense, particularly in the mitochondria, in conception in IVF.

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 1 h 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 2 h, but did not affect cell viability, ATP or malondialdehyde (MDA) levels at any time. DOX did not modify the intracellular levels of [3H]-ceramide during the first 90 min of exposure, but increased it significantly at 2 h. [3H]-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 [3H]-oleate into intracellular DAG from 60 min. In contrast, DOX reduced the incorporation of [3H]-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.

Objective To evaluate the serum oxidizability and antioxidant status in women undergoing an in vitro fertilization (IVF) cycle and to assess the possible relationship of the oxidizability indexes with the pregnancy rate. Design Prospective, longitudinal study. Setting Public university and public university hospital. Patient(s) Systematically recruited cohort of 125 women undergoing either IVF or intracytoplasmic sperm injection (ICSI). Intervention(s) Serum samples were collected before the beginning of the use of gonadotropins (basal) and the day of human chorionic gonadotropin (hCG) administration (final) during an IVF cycle. Main Outcome Measure(s) The Cu 2+ -induced serum oxidation in terms of the oxidation rate in the lag ( V lag ) and propagation ( V max ) phases and the time at which the oxidation rate is maximal ( t max ), and measurements of serum total antioxidant activity (TAA), tocopherol, hydrophilic antioxidants, malondialdehyde, and nitric oxide. Result(s) Albumin, urate, bilirubin, α-tocopherol and γ-tocopherol, TAA, and t max statistically significantly decreased after the IVF cycle. Conception cycles were associated with a serum more prone to oxidation compared with nonconception cycles. In multivariate logistic regression analysis, the difference (final-basal) of the oxidation index V lag (OR 1.394) and the body mass index (OR 0.785) were independent predictors of pregnancy. Conclusion(s) Treatment with IVF induces the production of reactive oxygen species (ROS), which is reflected in a serum less protected against oxidation. The results also suggest a role for ROS in the occurrence of conception in IVF.

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 α-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.

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 17β-estradiol (E 2 ), during the menstrual cycle were associated with changes in the low-density lipoprotein (LDL) particle size, fatty acyl composition, α-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, α-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), α-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.

Ferryl heme proteins may play a major role in vivo under certain pathological conditions. Catecholestrogens, the estradiol-derived metabolites, can act either as antioxidants or pro-oxidants in iron-dependent systems. The aim of the present work was (1) to determine the effects of ferrylmyoglobin on hepatocyte cytotoxicity, and (2) to assess the pro/antioxidant potential of a series of estrogens (phenolic, catecholic and stilbene-derived) against ferrylmyoglobin induced lipid peroxidation in rat hepatocytes. Cells were exposed to metmyoglobin plus hydrogen peroxide to form ferrylmyoglobin in the presence of the transition metal chelator diethylentriaminepentaacetic acid. Results showed that ferrylmyoglobin induced an initial oxidative stress, mainly reflected in an early lipid peroxidation and further decrease in GSH and ATP. However, cells gradually adapted to this situation, by recovering the endogenous ATP and GSH levels at longer incubation times. Phenolic and stilbene-derived estrogens inhibited ferrylmyoglobin-induced lipid peroxidation to different degrees: diethylstilbestrol>estradiol>resveratrol. Catecholestrogens at concentrations higher than 1 μM also inhibited lipid peroxidation with similar efficacy. The ability of estrogens to reduce ferrylmyoglobin to metmyoglobin may account for their antioxidant activity. In contrast, physiological concentrations (100 pM–100 nM) of the catecholestrogens exerted pro-oxidant activities, 4-hydroxyestradiol being more potent than 2-hydroxyestradiol. The implications of these interactions should be considered in situations where local myoglobin or hemoglobin microbleeding takes place.

Female infertility involves an emotional impact for the woman, often leading to a state of anxiety and low self-esteem. The assisted reproduction techniques (ART) are used to overcome the problem of infertility. In a first step of the in vitro fertilization therapy women are subjected to an ovarian stimulation protocol to obtain mature oocytes, which will result in competent oocytes necessary for fertilization to occur. Ovarian stimulation, however, subjects the women to a high physical and psychological stress, thus being essential to improve ART and to find biomarkers of dysfunction and fertility. GSH is an important antioxidant, and is also used in detoxification reactions, catalysed by glutathione S-transferases (GST). In the present work, we have investigated the involvement of GST in follicular maturation. Patients with fertility problems and oocyte donors were recruited for the study. From each woman follicles at two stages of maturation were extracted at the preovulatory stage. Follicular fluid was separated from the oocyte by centrifugation and used as the enzyme source. GST activity was determined based on its conjugation with 3,4-dichloronitrobenzene and the assay was adapted to a 96-well microplate reader. The absorbance was represented against the incubation time and the curves were adjusted to linearity (R(2)>0.990). Results showed that in both donors and patients GST activity was significantly lower in mature oocytes compared to small ones. These results suggest that GST may play a role in the follicle maturation by detoxifying xenobiotics, thus contributing to the normal development of the oocyte. Supported by FIS/FEDER (PI11/02559), Gobierno Vasco (Dep. Educacion, Universiades e Investigacion, IT687-13), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01).

The aim of this study was to explore the possible modifications induced by 17beta-estradiol (E(2)) in vivo on low-density lipoprotein (LDL) lipid composition, particle size, and oxidizability. For this purpose, women were recruited from an in vitro fertilization program, ranging their plasma E(2) levels from less than 12 pg/ml to more than 2000 pg/ml at the end of the treatment. The LDL lipid constituents were analyzed by thin layer chromatography and image analysis, and the LDL diameter was calculated from the lipid data. The results showed that high plasma E(2) levels were associated with smaller LDL particles, with lower amounts of free and esterified cholesterol and an increased relative content of alpha-tocopherol. The hormonal treatment produced a remodelation of the LDL acyl composition, rendering a lipoprotein enriched in saturated fatty acids, with a poorer polyunsaturated fatty acid content. These alterations in the physicochemical properties of LDL paralleled changes in the susceptibility of LDL to in vitro oxidation induced by both Cu(2+) and the peroxyl radical generator, 2,2'-azobis (2-amidinopropane), these changes being mainly reflected in a reduced maximum oxidation rate. The in vivo changes in the physicochemical properties of LDL induced by E(2) could explain some of the antiatherogenic actions of estrogens.

Natural estrogens have much greater radical-scavenging antioxidant activity than has previously been demonstrated, with activities up to 2.5 times those of vitamin C and vitamin E. The biological significance of this finding remains to be elucidated. In this work the antioxidant activity of a range of estrogens (phenolic, catecholic and stilbene-derived) has been studied. The activity of these substances as hydrogen-donating scavengers of free radicals in an aqueous solution has been determined by monitoring their relative abilities to quench the chromogenic radical cation 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS +). The results show that the order of reactivity in scavenging this radical in the aqueous phase is dependent on the precise estrogenic structure, with phenolic estrogens being more potent antioxidants than catecholestrogens or diethylstilbestrol. The ability of the same estrogens to scavenge lipid phase radicals has also been assessed, determined by the ex vivo enhancement of the resistance of low-density lipoprotein (LDL) to oxidation; the order of efficacy is different from that in the aqueous phase, with the phenolic estrogens estriol, estrone and 17β-estradiol being less potent than 2-hydroxyestradiol, 4-hydroxyestradiol, or diethylstilbestrol. In this lipid-based system, phenolic estrogens were found to be unable to regenerate α-tocopherol from LDL subjected to oxidative stress, while at the same time 2- and 4-hydroxyestradiol significantly delayed α-tocopherol loss. These results indicate that the various estrogens are good scavengers of free radicals generated in both the aqueous and the lipophilic phases. The antioxidant activity of an estrogen depends not only on the hydrophilic or lipophilic nature of the scavenged radical, but also on the phenol and catechol structures of the estrogen compound.

Oxidative stress is associated with alterations in arachidonic acid (AA) metabolism. The present work was performed to assess the effect of the oxidant tert-butyl hydroperoxide on the release of AA from rat hepatocytes, and the possible preventive actions of estrogens on this effect. The exposure of [14C]-prelabeled cells to tertbutyl hydroperoxide produced the mobilization of [14C]-AA from hepatocyte lipids, both an intracellular [14C]-AA accumulation and an increased release of [14C]-products into the medium being observed. The formation of lysophospholipids was also enhanced significantly in the presence of the oxidant, thus suggesting the involvement of phospholipase A2 (E.C. 3.1.1.4) in the hepatocyte response to tert-butyl hydroperoxide. Estradiol and 2-hydroxyestradiol (25-100 microM) added in vitro to cell suspensions prevented significantly the oxidant- mediated stimulation of AA release, this effect probably being caused by the estrogen inhibitory actions against cellular lipid peroxidation.

The in vitro addition of 17 beta-estradiol (0-100 microM) to isolated rat hepatocytes efficiently prevented cellular lipid oxidation induced by the Fe(III)/ADP complex. 17 beta-estradiol was found to be less effective than its metabolic derivative 2-hydroxyestradiol. The presence of specific inhibitors of cytochrome P450 activity significantly diminished the antioxidant capacity of estradiol. These observations support the hypothesis that estradiol, in the micromolar range, inhibits iron-induced lipid peroxidation in liver cells by diverting reducing equivalents from the peroxidative process to its own metabolism.

The antioxidant effects of natural estrogens (estrone, E1; 17 beta-estradiol), synthetic estrogens (17 alpha-ethynylestradiol, EE2; mestranol, MES; diethylstilbestrol, DES) and catecholestrogens (2-hydroxyestradiol; 4-hydroxyestradiol, 4-OHE2) on lipid peroxidation induced by different means in rat liver microsomes were investigated. The extent of lipid peroxidation was determined by measuring thiobarbituric acid reactive substances. Prooxidants included Fe3+/ADP/reduced NADPH, Fe2+/ascorbate, tert-butyl hydroperoxide (t-BOOH) and 2,2'-azobis(2-amidinopropane) (AAPH). Estrogens and catecholestrogens decreased lipid peroxidation in all four systems tested. In the iron/ascorbate model it was shown that (i) 4-OHE2 and DES had analogous patterns of inhibition, irrespective of the presence of NADPH or the functional integrity of the microsomes, and (ii) the antioxidant activities of E1, EE2 and MES were dependent on the assay conditions with the activity being markedly higher when estrogen metabolism was favored. When peroxidation was initiated by the peroxyl radical generator AAPH, the inhibitory effects observed were least pronounced. Our data also showed that, in each of the systems, all inhibitors displayed the same order of inhibitory potency with DES and catecholestrogens being the most potent antioxidants under all experimental conditions used. The present results confirm earlier findings and point toward a link between estrogen metabolism and estrogen antioxidant activity. The data also indicate that estrogens and catecholestrogens interact with the peroxidative process at different levels with their interactions with iron or the metal-derived species being the most important modes of inhibition.