Search

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. This work was supported by research grants from the University of the Basque Country UPV/EHU (General Support to Research Groups, refs. GIU16/62 and GIU20/021), and Basque Government (pre-doctoral grant to I.P.-R.).

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.

Objective To investigate the association between serum golimumab (GLM) trough levels, clinical disease activity and treatment response during the first year of therapy in patients with axial spondyloarthritis (axSpA), as well as determining an optimal concentration range of GLM in axSpA. Methods This was an observational prospective study including 49 patients with axSpA monitored during 52 weeks (W52). Serum GLM trough levels were measured by capture ELISA and antidrug antibodies by bridging ELISA at baseline, W24 and W52. Disease activity was assessed by the Ankylosing Spondylitis Disease Activity Score (ASDAS) and clinical improvement by ΔASDAS. The association between serum GLM trough levels and disease activity was assessed using univariable and multivariable regression. In case of drop-out or missing data before W52, the last observation carried forward (LOCF) was performed. ASDAS values and GLM levels at W24 were available for 42 patients and 38 patients at W52. Results In the univariable analyses, serum GLM trough levels were inversely associated with ASDAS at W24 (n=42, r =-0.445; p < 0.01), at W52 (n=38, r=-0.330; p < 0.05) and W52LOCF (n=49, r=-0.309; p < 0.05). In the multivariable analysis, this significant association remained. Serum trough GLM levels above the 0.7-1.4mg/L range did not contribute to additional clinical improvement. Conclusion In patients with axSpA, serum GLM trough levels are associated with disease activity during the first year of treatment. A concentration range of 0.7-1.4mg/L appears to be useful to achieve clinical response to GLM.

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.

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.

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.

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.

Introduction Several methods are used to measure infliximab (IFX), adalimumab (ADL), antibodies to IFX (ATI) and antibodies to ADL (ATA). These agents elicit an unwanted immune response leading to loss of response. Measurement of drug levels (DL) and anti-drug antibodies (ADA) are often required for routine patient monitoring. The aim of this study was to compare different commercially available ELISA tests for the measurement of DL and ADA for IFX and ADL. Method A total of 109 samples (69 IFX and 40 ADL) from 71 patients with IBD were included in the study. DL and ADA trough levels were measured with Promonitor kits (Progenika, Spain) and LISA-TRACKER (LT) Duo IFX and ADL kits (Theradiag, France). In addition, drug-spiked samples were analysed to ascertain trueness. Pearson and Bland-Altman analysis was used to study the association and agreement between DL methods. Cohen´s kappa was used to study agreement of ADA assays. Results Correlation coefficients between each Promonitor test and the corresponding LT assay were 0.99 and 0.83 (p Conclusion Performances of both assays were comparable for all measurements. However, the systematic differences in the magnitude of DL and limited measurement ranges observed for LT tests should be taken with caution since this could impact patient management. Disclosure of interest D. Nagore Employee of: Progenika, A. Ruiz del Agua Employee of: Progenika, J. Pascual Employee of: Progenika, F. Llinares-Tello: None Declared, B. Herreros: None Declared, A. Martinez Employee of: Progenika, S. Martin Grant/ Research Support from: Progenika, R. Navarro: None Declared, L. del Rio Employee of: Progenika.

Background Golimumab (GLM) is a fully human monoclonal antibody to TNFa and it is an inhibitor that binds to both transmembrane and soluble bioactive human TNFa and blocks their action. This TNF-inhibitor is used in the treatment of the rheumatoid arthritis (RA), ankylosing spondilitis (AS) and psoriatic arthritis (PsA). Recently it has been reported that there exists a significant relationship between GLM levels circulating in serum and the patients9 response. Therapeutic drug monitoring (TDM) with serum level measurement can be a useful tool to optimization of the anti-TNF treatment, but the lack of standardization can hinder the comparison. Objectives To determine the correlation and agreement between two different Enzyme-Linked Immunosorbent Assay (ELISA) assays for measuring golimumab. Methods A total of 121 sera from a cohort of 44 patients under golimumab treatment with different rheumatic diseases (RA, AS and PsA) were analyzed. The golimumab trough levels were analyzed at the beginning of the treatment and every 6 months to 2 years of treatment. The serum levels were analyzed in blind manner by the Immunology Unit from the La Paz University Hospital (Madrid, Spain) using the commercial Kit Promonitor®-GLM (Progenika, Biopharma SA, Derio, Vizcaya) and by Sanquin Diagnostic Services (Amsterdam, The Netherlands) using an own developed ELISA. The statistical analysis to quantify the correlation and the agreement between GLM levels were made with SPSS version 20 (IBM, New York, NY, USA) and with GraphPad Prism version 6.0 for Windows (GraphPad Software, San Diego, CA, USA). Results The correlation between GLM levels of the different two assays was r Pearson of 0.963 (p Conclusions There is an excellent correlation and agreement between GLM levels obtained with the commercial Kit Promonitor®-GLM and the levels obtained in the Sanquin laboratory, both of them with ELISA technology. These results may help us to standardize the GLM levels and the comparisons between different data reported with either of these two assays. Disclosure of Interest None declared

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.

Doxorubicin (DOX) is an antibiotic used in the treatment of various cancers. However, its clinical use is limited due to the toxic effects it causes. In several biological systems reactive oxygen species (ROS) derived from DOX metabolism have been associated with its toxicity because they can alter several signal transduction pathways. One of the major signal transduction pathways activated in response to oxidant stress is that of the nuclear transcription factor ?B (NF-?B), which is crucial for cell survival, cell proliferation, and immune responses. Previous studies from our laboratory have described that DOX triggersthe activation of the MAPK (ERK, p38, and JNK) pathways, NF-?B mobilization and caspase activation in rat hepatocytes. These events were indirectly ascribed to ROS generation by pharmacological approaches. Although NF-?B mobilization is usually accompanied by up-regulation of the genes with regulatory ?B elements, our gene expression analysis rendered a quite different scenario. The aim of this work was to verify the implication of ROS in the DOX-mediated signaling and to explore the transcriptional regulatory behaviour of NF-?B mobilized by DOX. Results showed that DOX treatment induces ROS generation, particularly H2O2. DOX-induced caspase-3 activation was ROS dependent, whereas the NF-?B mobilization was a process independent of H2O2. Furthermore, DOX-mobilized NF-?B complexes were dimmers formed by p65 with all the members of the Rel family, excepting Rel B. The analysis of the NF-?B transcriptional activity performed by reporter gene assays suggests that these DOX-mobilized NF-?B complexes are not transcriptionally competent.

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.