Blood plasma and urinary biomarkers of oxidative stress in cats with urethral obstruction.

BACKGROUND: This study aimed to investigate variations of the oxidative status in cats affected by urethral obstruction (UO) under Feline Idiopathic Cystitis (FIC) and Bacterial Cystitis (BC), in comparison with a group of healthy subjects. In both groups, the levels of several markers (either direct or indirect) indicative of the oxidative attack and of the antioxidant response were analyzed on plasma and urine samples. In particular, the plasma samples were evaluated for nitric oxide (NO), hydroperoxides derived by reactive oxygen activity (d-ROMs test), superoxide anion (O2-), glutathione peroxidase activity (GPx), superoxide dismutase activity (SOD), and ferric reducing antioxidant power (FRAP test); while on urine the levels of NO, d-ROMs, FRAP, SOD, malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) were measured. Urine of UO patients was also subjected to urine-culture test. RESULTS: The analytical data on plasma showed that UO, independently of the FIC or BC etiology, induced the insurgence of oxidative stress conditions at the systemic level. In the urine of the UO patients, except for SOD that increased, the markers of redox status were markedly decreased due probably their compromised filtration, thus suggesting involvement of renal function (assessed also by the high levels of plasma creatinine and proteinuria) with no oxidative damage of the lower urinary tract. Moreover, the adoption of a novel oxidative stress index' (OSI) allowed to establish, by means of a numerical value, the different degrees of oxidative stress conditions for single UO patients, both in terms of oxidative attack and antioxidant response. CONCLUSIONS: Feline urethral obstruction, induced by Idiopathic Cystitis and Bacterial Cystitis, causes oxidative stress conditions at the systemic level that do not interest the lower urinary tract. Despite to the high variability of the profiles of oxidative stress indexes both in healthy and UO patients, the determination of OSI made possible the evaluation of their single degrees of oxidative stress. Possibly the results of this investigation can be compared with those of correspondent pathologies both in humans and in other animal species.

Mesenchymal Stromal Cells Derived from Canine Adipose Tissue: Evaluation of the Effect of Different Shipping Vehicles Used for Clinical Administration.

Mesenchymal Stromal Cells (MSCs)-based therapies are rapidly gaining interest in veterinary medicine. Cellular therapy represents a new challenge for practitioners and requires precise coordination between the cell processing laboratory and the veterinary clinic. Cryopreservation is the best method to provide fast, in-time, and long-distance delivery of cells for therapeutic applications. However, potentially toxic cryoprotectants and xenobiotic products make the direct administration of cells impracticable for patients. Alternatively, the cells may be resuspended in a ready-to-use vehicle and shipped to the veterinary clinic. In this study, two nutrient-poor vehicles (physiologic saline and ringer lactate solutions) and two nutrient-rich vehicles (the releasate derived from autologous Platelet Poor Plasma and Platelet Rich Plasma) were tested on adipose tissue-derived canine MSCs (AD-MSCs). AD-MSCs stored for 2, 4, or 24 h in the different media were compared regarding mortality, metabolic activity, and replicative capacity. Furthermore, antioxidant activity and the pattern of expression of genes related to AD-MSCs function were performed following 24 h of storage. The results showed that all the different vehicles preserve cell vitality and replication following short-term storage. In long-term storage, the vehicle and cell density affect cell vitality, proliferation, and gene expression (CCL-2, CXCR-4, and TSG-6). Nutrient-rich vehicles seem better suited to preserve cell functionalities in this contest.

Nanoplastics induced oxidative stress and VEGF production in aortic endothelial cells.

Plastic is an important environmental issue and a more critical aspect concerns plastic fragments, mainly in term of nanoplastics (NPs). We demonstrated that NPs interfere with reproductive and adipose stromal cells. Since several research underlined an increased cardiovascular risk due to NPs, present study was undertaken to investigate their effect on aortic endothelial cells (AOC). We explored the specificity of their interaction with endothelial cells, quantifying their load in treated cells. Then, NPs effect was assessed on cell growth, generation of free radicals and antioxidant defence. Our data demonstrate that NPs colocalize with AOC. We found a significant (p < 0.01) increase both in metabolic activity and Vascular Endothelial Growth Factor (VEGF) production (p < 0.01). Redox status appeared to be disrupted (p < 0.05) by NPs. Taken together, the normal function of cultured AOC appeared negatively affected by AOC. Since NPs have been detected in blood, our present data appear of particular interest.

Perfluorooctanoic acid (PFOA) affects steroidogenesis and antioxidant defence in granulosa cells from swine ovary.

PFOA is mainly employed in products with water and oil repellent properties. Due to its persistence, bioaccumulation and critical effects on health, its use has been restricted in several countries. This research was intended to explore PFOA action on the main functions of swine ovarian granulosa cells, a valuable model for translational medicine. Moreover, since we previously demonstrated a disruptive effect on free radical generation we sought to explore PFOA effects on the main antioxidant enzymes. PFOA inhibited cell proliferation (p < 0.001), assessed by BrdU uptake. Steroidogenesis was disrupted: PFOA also stimulated 17ß-estradiol production (p < 0.05), increased progesterone production (p < 0.05) at the lowest dose while it displayed an inhibitory effect at higher concentrations (p < 0.05). SOD (p < 0.001), catalase (p < 0.05) and peroxidase (p < 0.01) activities were stimulated. Therefore, our study supports a disruptive effect of PFOA in cultured swine granulosa cells.

Effects of the Myokine Irisin on Stromal Cells from Swine Adipose Tissue.

Irisin is a hormone able to reproduce some of the positive effects of physical activity and diet. Recently, we demonstrated the presence of Irisin at the ovarian level as a potential physiological regulator of follicular function. Adipose tissue is crucial for reproductive function through its metabolic activity and the production of adipokines. At present, the exact nature of adipocyte precursors is still under debate, but an important role has been assigned to the population of adipose tissue mesenchymal stromal cells (ASCs) of perivascular origin. It should be noted that, when appropriately stimulated, ASCs can differentiate into preadipocytes and, subsequently, adipocytes. Therefore, this present study was undertaken to explore the potential effect of Irisin on ASCs, known for their high differentiative potential. Since Irisin expression in ASCs was confirmed by PCR, we tested its potential effects on the main functional activities of these cells, including proliferation (BrdU uptake); metabolic activity (ATP production); redox status, evaluated as the generation of free molecules such as superoxide anion and nitric oxide; and scavenger activities, assessed as both enzymatic (superoxide dismutase) and non-enzymatic antioxidant power. Moreover, we tested the effect of Irisin on ASCs adipogenic differentiation. BrdU uptake was significantly (p < 0.001) inhibited by Irisin, while ATP production was significantly (p < 0.05) increased. Both superoxide anion and nitric oxide generation were significantly increased (p < 0.001) by Irisin, while scavenger activity was significantly reduced (p < 0.05). Irisin was found to significantly (p < 0.05) inhibit ASCs adipogenic differentiation. Taken together, the present results suggest a potential local role of Irisin in the regulation of adipose tissue function.

Evaluation of Oxidative Stress in Blood of Domestic Chickens and Eurasian Magpies (Pica pica).

A physiological equilibrium exists between pro- and antioxidant factors. When the oxidant factors exceed the capacity of their removal or inactivation, oxidative stress (OS) occurs. The OS levels were assayed in plasma obtained from 2 bird species. Blood samples were collected from 20 healthy domestic chicken hens, 10 living in an intensive farming environment and 10 free-range, and from 18 healthy Eurasian magpies (Pica pica; 7 females and 11 males, with an estimated age of >1 year of age). For OS biomarker assessment, the determinable reactive oxygen metabolites (d-ROMs) were measured, and the plasmatic antioxidant test (PAT) was performed; the OS index (OSI) was then calculated (d-ROMs/PAT × 1000) as a parameter of overall oxidative stress. Moreover, lipid peroxidation was assessed by measuring plasmatic malondialdehyde (MDA) levels. A hematological evaluation was also performed on each bird with a hemocytometer, on which a blood sample was placed to obtain both a total and differential white blood cell (WBC) count. In hens, OSI and MDA levels were significantly higher (P = .04, and P = .004) in subjects from intensive farming (14.7 ± 7.1 and 27.2 ± 10.4 nmol/mL) than in those bred in rural conditions (5.6 ± 10.3 and 8.2 ± 13.3 nmol/mL). In magpies, a positive correlation between the total WBC count and OS was found, and both d-ROMs and OSI were significantly higher (P = .03) in subjects with a total WBC count greater than the median value (20.4 × 103 cells/µL) with respect to those with a total WBC count less than the median value. The results generated from this study indicate that higher OS levels occurred in hens bred in an intensive indoor farm environment compared with outdoor free-range conditions. Possibly the higher OS levels could be related to the higher stocking density and dust levels found in the indoor facility. Additionally, the correlation between OS biomarker levels in magpies and total WBC count suggests that OS level is influenced by immune response, in agreement with previous studies. Collectively, present data seem to be promising for the application of OS measurement in avian medicine for health and animal welfare monitoring.

Evaluation of Triclosan Effects on Cultured Swine Luteal Cells.

Triclosan is a chlorinated phenolic, used in many personal and home care products for its powerful antimicrobial effect. Several studies have shown triclosan toxicity and the American Food and Drug Administration (FDA) in 2016 has limited its use. It has been recently included in endocrine-disrupting chemicals (EDCs), a list of chemicals known for their ability to interfere with hormonal signaling with particular critical effects on reproduction both in animals and humans. In order to deepen the knowledge in this specific field, the present study was undertaken to explore the effect of different concentrations of triclosan (1, 10, and 50 µM) on cultured luteal cells, isolated from swine ovaries, evaluating effects on growth Bromodeoxyuridine (BrDU) incorporation and Adenosine TriPhosphate (ATP) production, steroidogenesis (progesterone secretion) and redox status (superoxide and nitric oxide production, enzymatic and non-enzymatic scavenging activity). A biphasic effect was exerted by triclosan on P4 production. In fact, the highest concentration inhibited, while the others stimulated P4 production (p < 0.05). Triclosan significantly inhibited cell proliferation, metabolic activity, and enzymatic scavenger activity (p < 0.05). On the contrary, nitric oxide production was significantly increased by triclosan (p < 0.01), while superoxide anion generation and non-enzymatic scavenging activity were unaffected.

Melatonin modulates swine luteal and adipose stromal cell functions.

Based on our previous study in follicles, the first aim of this work was to evaluate the effect of melatonin in the swine corpus luteum (CL). Luteal cells were exposed to 10 and 20pg mL-1 melatonin. We evaluated the effect on proliferation (bromo-deoxy-uridine uptake), steroidogenesis (progesterone) and redox status by means of Griess test (nitric oxide production), WST-1 test (superoxide anion generation) and FRAP test (non-enzymatic antioxidant power). The results showed a significant increase in antioxidant power, as well as a reduction in the other parameters analysed. These data and the expression of MT2 observed in luteal cells allow us to hypothesise a physiological role of melatonin in the regulation of CL functionality. The reproductive function is dependent on energy reserves stored in adipose tissue. Therefore, we sought to verify the effect of melatonin on adipose stromal cells (ASCs). MT2 receptor expression was detected in ASCs and the presence of gene markers (PPARγ and leptin) before and after adipogenic differentiation was verified. The differentiation was significantly inhibited by melatonin, as well as cell viability. In conclusion, present results suggest that melatonin exerts a potential inhibitory action on luteal function and adipogenesis, possibly mediated by MT2.

Xenobiotic-Free Medium Guarantees Expansion of Adipose Tissue-Derived Canine Mesenchymal Stem Cells Both in 3D Fibrin-Based Matrices and in 2D Plastic Surface Cultures.

Mesenchymal stem cells (MSCs) have been recently introduced in veterinary medicine as a potential therapeutic tool for several pathologies. The large-scale in vitro expansion needed to ensure the preparation of a suitable number of MSCs for clinical application usually requires the use of xenogeneic supplements like the fetal bovine serum (FBS). The substitution of FBS with species-specific supplements would improve the safety of implanted cells, reducing the risk of undesired immune responses following cell therapy. We have evaluated the effectiveness of canine adipose tissue-derived stromal vascular fraction (SVF) and MSCs (ADMSCs) expansion in the presence of canine blood-derived supplements. Cells were cultured on traditional plastic surface and inside a 3D environment derived from the jellification of different blood-derived products, i.e., platelet-poor plasma (PPP), platelet-rich plasma (PRP), or platelet lysate (PL). PPP, PRP, and PL can contribute to canine ADMSCs in vitro expansion. Both allogeneic and autologous PPP and PL can replace FBS for ADMSCs culture on a plastic surface, exhibiting either a similar (PPP) or a more effective (PL) stimulus to cell replication. Furthermore, the 3D environment based on homospecific blood-derived products polymerization provides a strong stimulus to ADMSCs replication, producing a higher number of cells in comparison to the plastic surface environment. Allogeneic or autologous blood products behave similarly. The work suggests that canine ADMSCs can be expanded in the absence of xenogeneic supplements, thus increasing the safety of cellular preparations. Furthermore, the 3D fibrin-based matrices could represent a simple, readily available environments for effective in vitro expansion of ADMSCs using allogeneic or autologous blood-products.

The effect of pathogen-associated molecular patterns on the swine granulosa cells.

Recent studies have demonstrated the surprising ability of reproductive endocrine cells to express receptors of innate immunity useful to sense danger in order to avoid disruption of tissue homeostasis. Present research demonstrates the presence of pattern recognition receptors, i.e. toll like receptors (TLR) TLR2, TLR4 and TLR 5 and NOD like receptors (NLR) NOD1 and NOD2 in swine granulosa cells from ovarian follicles> 5 mm. Therefore, our second goal was to expose granulosa cells to different concentrations (1000, 100 and 10 ng/ml) of lipopolysaccharide (LPS) and N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)- propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]- lysine (Pam3CSK4), two substances associated with pathogen molecular patterns. Their potential effects on the main functional parameters were monitored: proliferation (through the incorporation of Bromo-deoxy-Uridine), cell viability (by testing the metabolization of MTT salt), steroidogenic activity (by immunoenzymatic examination) and redox status (evaluating the production of superoxide anion by means of the WST test, production of nitric oxide through the use of the Griess test, and the non-enzymatic reducing power, by FRAP test). The data collected show a significant inhibition (p < 0.01) of cell proliferation after treatment with both LPS and Pam3CSK4, while cell viability has not been modified. As for steroidogenesis, treatment with both LPS and Pam3CSK4 significantly inhibited (p < 0.05) the production of 17ß-estradiol and progesterone. LPS and Pam3CSK4 stimulated (p < 0.05) the production of superoxide anion and nitric oxide, while inhibited (p < 0.05) the antioxidant power. In conclusion, the study shows that the functionality of granulosa cells is compromised by the exposure to molecular profiles associated with pathogens; the knowledge gathered could lay the theoretical basis for the definition of therapeutic treatments related to diseases that can affect normal reproductive processes.

The Contribution of Adipose Tissue-Derived Mesenchymal Stem Cells and Platelet-Rich Plasma to the Treatment of Chronic Equine Laminitis: A Proof of Concept.

Laminitis, a highly debilitating disease of the foot in ungulates, is characterized by pathological changes of the complex lamellar structures that maintain the appendicular skeleton within the hoof. Laminitis is a multifactorial disease that involves perturbation of the vascular, hematological, and inflammatory homeostasis of the foot. Interestingly, the pathogenesis of the disease resembles what is observed in metabolic syndromes and sepsis-induced organ failure in humans and animals. We hypothesized that local administration of mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) might contribute to establishing an anti-inflammatory and pro-angiogenic environment, and could stimulate the injured tissue in order to restore its functional integrity. According to this assumption, an experimental protocol based on the local intravenous administration of adipose tissue-derived MSCs (aMSCs) in combination with PRP was developed for the treatment of horses affected by chronic laminitis. Nine horses with severely compromised venograms (showing grade III and IV laminitis) that had been unsuccessfully treated with conventional therapies were enrolled. aMSCs and PRP (15 × 106 cells resuspended in 15 mL of PRP) were injected into the lateral or medial digital vein three times, at one-month intervals. The first administration was performed with allogeneic aMSCs, while for the following administrations, autologous aMSCs were used. There was no adverse short-term reaction to the intravenous injection of aMSCs. In the long term, venograms outlined, in all subjects, a progressive amelioration of the vascularization of the foot. An improvement in the structure and function of the hoof was also observed. No adverse events were reported during the follow-up, and the horses returned to a comfortable quality of life. Although the number of animals enrolled in the study is limited, both clinical observations and venography demonstrated an enhancement in the condition of all horses, suggesting that the regenerative therapies in chronic laminitis could be useful, and are worthy of further investigation.

Melatonin potentially acts directly on swine ovary by modulating granulosa cell function and angiogenesis.

Melatonin exerts well-known reproductive effects, mainly acting on hypothalamic gonadotrophin-releasing hormone release. More recent data suggest that melatonin acts directly at the ovarian level, even if, at present, these aspects have been only partly investigated. Swine follicular fluid contains melatonin and its concentration is significantly reduced during follicular growth. Therefore, the present study was undertaken to examine the effects of melatonin, used at physiological concentrations, on cultured swine granulosa cells collected from small (<3mm) and large (>5mm) follicles on the main parameters of granulosa cell function such as proliferation and steroidogenesis, namely oestradiol 17ß and progesterone (P4) production. Moreover, the effects of melatonin on superoxide anion and nitric oxide (NO) generation by swine granulosa cells were also investigated. Finally, since angiogenesis is crucial for follicle growth, the effects of melatonin on new vessel growth were studied. Collected data indicate that melatonin interferes with cultured granulosa cell proliferation and steroidogenesis, specifically in terms of P4 production and NO output. In addition, the events of physiological follicular angiogenesis were stimulated by melatonin as evidenced by angiogenesis bioassay. Therefore, we suggest that physiological melatonin concentrations could potentially be involved in local modulation of swine ovarian follicle function.

Bisphenol A interferes with swine vascular endothelial cell functions.

Several studies have demonstrated that the endocrine disruptor bisphenol A (BPA) negatively affects animal and human health. An angiogenic process has been suggested among the events disrupted by this molecule, but the underlying mechanisms have not yet been clarified. The effect of BPA on angiogenesis was investigated by means of a bioassay previously validated in our laboratory. Using immortalized swine aortic endothelial cell line (AOC), the development of new blood vessels through a three-dimensional in vitro angiogenesis assay was evaluated. Subsequently, since vascular endothelial growth factor (VEGF) and nitric oxide (NO) are key players in the regulation of the angiogenic process, the effect of BPA on the production of these molecules by AOC was examined. BPA (10 µmol/L) stimulated AOC growth (p < 0.05) and VEGF production (p < 0.05), but did not modify NO levels. Our data suggest that the endocrine-disrupting effects of BPA could also be associated with the promotion of vascular growth, thus interfering with a physiologically finely tuned process resulting from a delicate balance of numerous molecular processes. The stimulatory effects of BPA on VEGF production may have negative implications, potentially switching the balance toward uncontrolled neovascularization. Moreover, since angiogenesis is involved in several pathologies, including cancer growth and progression, potential health risks of BPA exposure should be carefully monitored.

Swine Granulosa Cells Show Typical Endothelial Cell Characteristics.

Granulosa cells, which belong to the somatic compartment of the ovarian follicle, are actively involved as endocrine cells in follicle growth. Recently, it has been proposed that these cells are not terminally differentiated and possess multipotency. Therefore, we cultured swine granulosa cells in specific endothelial cell culture medium (EBM-2), and phenotypic and functional characteristics of endothelial cells were assessed. The collected data suggest that these endocrine cells can also behave as endothelial cells, therefore potentially contributing to follicular angiogenesis, a crucial process in follicle growth and selection.

Effects of a ferulate-derived dihydrobenzofuran neolignan on angiogenesis, steroidogenesis, and redox status in a swine cell model.

In the ongoing search for new therapeutic compounds, lignans and neolignans, which are widely distributed in plants, deserve special attention because of their interactions with several biological targets. Searching for potential antiangiogenic agents related to natural lignans/neolignans, we were attracted by a previously studied synthetic dihydrobenzofuran neolignan. We synthesized the compound by means of an eco-friendly, enzyme-mediated biomimetic coupling of the methyl ester of ferulic acid, and the present study was aimed to deeply investigate its effect in angiogenesis bioassays validated in our laboratory. In addition, a previously well-defined granulosa cell model was employed to evaluate the effect of dihydrobenzofuran neolignan on cell viability, steroidogenesis, and redox status. Present data support the antiangiogenic effect of this neolignan. Moreover, we demonstrate that, at least at the highest concentrations tested, dihydrobenzofuran neolignan affects granulosa cell viability and steroidogenesis. In addition, the compound inhibits generation of free radicals and stimulates scavenger enzyme activities. The present data, which are a further deepening of the evaluation of the biological activities of the dihydrobenzofuran lignan in well-defined cell models, are of interest and worthy of special attention.

Atrazine disrupts steroidogenesis, VEGF and NO production in swine granulosa cells.

Atrazine is one of the most widely employed herbicides. Due to its environmental persistence, it can be detected in ground and water thus becoming the subject of a serious concern because of its potential endocrine disrupting activity. In particular, several in vitro and in vivo studies point out adverse effects on reproduction. However, these data were mainly collected in the male, while studies on females are lacking. Present work was therefore set up on swine ovarian granulosa cells to investigate the effect of atrazine on steroidogenesis and proliferation. Moreover, since vessel growth is fundamental for reproductive function, we evaluated the herbicide's effect on two of the main angiogenesis signaling molecules, VEGF and NO. Our data show that atrazine markedly interferes with steroidogenesis while it does not modify cell proliferation; in addition, the herbicide has also been found to affect the production of the examined angiogenesis molecules. Collectively, these results indicate for the first time a potential negative effect of atrazine on ovarian functions in the swine species.

Antiangiogenic properties of an unusual benzo[k,l]xanthene lignan derived from CAPE (caffeic acid phenethyl ester).

Angiogenesis is normally a highly regulated process that occurs during development, reproduction, and wound repair. However, angiogenesis can also become a fundamental pathogenic process in cancer and several other diseases. To date, the synthesis of angiogenesis inhibitors has been researched in several ways also starting from bioactive plant compounds. In the present study, we tested both in an angiogenesis bioassay and in ovarian cell culture, the potential antiangiogenic effect of a natural-derived benzo[k,l]xanthene lignan (5). This unusual compound was synthesized through the biomimetic dimerization of CAPE (caffeic acid phenethyl ester), a bioactive component of honeybee propolis. The lignan showed a significant, dose-related inhibitory effect on new vessel growth in the angiogenesis bioassay and it inhibited Vascular Endothelial Growth Factor secretion in ovarian cell culture. Therefore, we indicate the natural-derived benzo[k,l]xanthene lignan 5 as a potential new angiogenesis inhibitor.

Hypoxia stimulates the production of the angiogenesis inhibitor 2-methoxyestradiol by swine granulosa cells.

We previously demonstrated the presence of 2-methoxyestradiol (2-ME) in swine follicular fluid. Present study was aimed first of all to investigate if swine granulosa cell produce 2-ME; in addition, we tried to assess a potential effect of hypoxia in modulating 2-ME output. Finally, we explored the effect of 2-ME in an angiogenesis bioassay set up in our lab. Our data show that cultured granulosa cells are able to produce 2-ME; interestingly, the secretion of the hormone appeared to be stimulated by hypoxia. Angiogenesis bioassay points out that 2-ME displays an inhibitory effect on neovascularisation. Therefore our data suggest that 2-ME could be a local effector in determining the fine tuning responsible for follicle angiogenesis. These data deserve special attention since the ovary is a valuable experimental model in angiogenesis research.

Biological effects on granulosa cells of hydroxylated and methylated resveratrol analogues.

Several resveratrol analogues have been designed to improve bioactivity: among these polymethoxystilbenes appear to be particularly promising. The present study was set up to investigate the biological functions of polymethoxystilbenes 2 and 3, recently found in our lab as antiangiogenic agents, on a well-defined swine granulosa cell model. Proliferative activity and effects on steroidogenesis were evaluated, as well as the effect on granulosa cell vascular endothelial growth factor (VEGF) production, since these cells in basic conditions synthesize the main proangiogenic peptide. Moreover, we considered the effect of these two resveratrol analogues on granulosa cell redox status. Analogue 3 inhibited granulosa cell growth, while it stimulated steroidogenesis. A similar effect was displayed by 2 on estradiol 17beta production and cell proliferation at the highest concentration tested. On the other hand, at the same dosage 2 decreased progesterone levels. Both analogues inhibited VEGF output. Granulosa cell redox status was unaffected by resveratrol analogue 2 while the highest concentration of 3 stimulated free radicals generation and scavenging enzyme activities. The overall results indicate that analogue 3 is the more powerful compound, thus suggesting that a slight modification in the structure markedly increases effectiveness. These data could be useful to develop more active resveratrol analogues for therapeutic use.

An SPME-GC-MS method using an octadecyl silica fibre for the determination of the potential angiogenesis modulators 17beta-estradiol and 2-methoxyestradiol in culture media.

A simple and easily automable method based on solid-phase microextraction followed by gas chromatographic-mass spectrometric analysis was developed for the determination of two potential angiogenesis modulators 17beta-estradiol (17-BE) and 2-methoxyestradiol (2-MEOE) in culture media. Trifluoroacetic anhydride was used as the derivatising agent. A homemade octadecyl silica coating, characterised by a coating thickness of 72 +/- 10 microm and a good thermal stability until 250 degrees C, was prepared. Experimental design was used to optimise the extraction conditions in terms of derivatisation time, derivatisation temperature and time of extraction. As for method validation, lower limits of quantification of 0.17 and 0.015 microg/l for 17beta-estradiol and 2-methoxyestradiol, respectively, were obtained. Finally, the capabilities of the developed fibres were evaluated for the analysis of the investigated analytes developed by granulosa cells in culture media maintained under normoxic, hypoxic and anoxic conditions, in order to better elucidate their possible role in the angiogenic process. An increase of the production of both 17-BE and 2-MEOE in hypoxic and anoxic conditions seems to be related to the effect of oxygen deprivation.