Radical Oxygen Species Levels Research Articles

P-399 Beyond chromosome screening: The impact of progesterone and in vitro oxidative stress on failed euploid blastocyst transfers

Abstract Study question To assess the effect of serum progesterone levels on the production of radical oxygen species (ROS) by endometrial immune cells in patients with non-genetic miscarriages. Summary answer Elevated serum pre-transfer progesterone levels favour the development of an endometrial microenvironment that is not conducive to a healthy pregnancy, by increasing ROS production. What is known already Endometrial immune cells, especially uterine Natural Killer (uNK) cells, play an essential role in the proper development of pregnancy, promoting angiogenesis and adequate placentation. The maturation of these cells is indirectly determined by progesterone levels, which, during the secretory phase, enhance the synthesis of key cytokines for uNKs’ functionality. Previous studies have already indicated that increases in progesterone levels during the follicular phase can alter the endometrial microenvironment, affecting its receptivity. However, the molecular effect that imbalances of this hormone may have on the endometrium on the day of embryo transfer and directly in relation to uNK is still unknown. Study design, size, duration We conducted a prospective exploratory study, between September 2022 and January 2024, including 15 patients with previous miscarriages after euploid single embryo transfer (SET), ruling out the genetic cause for such gestational losses. We obtained endometrial biopsies of all patients, after 5 days of progesterone, in a cycle with hormone replacement therapy. The same day of the biopsy, serum progesterone levels were measured in all the patients included. Participants/materials, setting, methods In vitro cultures enriched in uNK cells (CD56+CD16-) were established from the patients’ endometrial biopsies and cell enrichment was confirmed by flow cytometry. These cells were co-cultured with the JEG-3 cell line (placental cells), simulating the microenvironment of early pregnancy. Cellular ROS production was measured by fluorescence detection 40 hours post-culture using a commercial kit. A linear regression model was used to determine statistically significant correlations (p < 0.05) between ROS and progesterone levels. Main results and the role of chance The mean age of the participating patients was 38.9 years and the BMI was 23.2 kg/m2. The mean progesterone level the day of the biopsy was 15.6 ng/ml. The cell cultures obtained from patients were successfully enriched in the desired cell population, with an average percentage of 89.6% uNK cells (CD56+CD16-) of total leukocytes (CD45+) obtained by flow cytometry. After culturing the same number of cells from each patient with the same number of spheroids of the JEG-3 cell line, an average ROS production of 31341.2 arbitrary units (a.u.) was obtained. Taking into account the co-cultures stablished from the 15 patients, a positive correlation between cellular ROS production and progesterone level was observed (r = 0.6728, p = 0.006). Limitations, reasons for caution Although the results shown may provide clues about the molecular dynamics of the endometrium during early pregnancy, they should be taken with caution, as it is not yet known whether this progesterone elevation is the cause or rather the consequence of an unfavourable endometrial microenvironment with high oxidative stress. Wider implications of the findings This study provides new information on the endometrial microenvironment of patients suffering non-genetic miscarriages and, although much remains to be studied, this association between hormone levels and oxidative stress may point the way to future studies investigating a possible explanation for these undesirable reproductive outcomes. Trial registration number Not Applicable

Antioxidant and Anti-Inflammatory Properties of Recombinant Bifidobacterium bifidum BGN4 Expressing Antioxidant Enzymes.

Bifidobacterium bifidum BGN4-SK (BGN4-SK), a recombinant strain which was constructed from B. bifidum BGN4 (BGN4) to produce superoxide dismutase (SOD) and catalase, was analyzed to determine its antioxidant and anti-inflammatory properties in vitro. Culture conditions were determined to maximize the SOD and catalase activities of BGN4-SK. The viability, intracellular radical oxygen species (ROS) levels, intracellular antioxidant enzyme activities, and pro-inflammatory cytokine levels were determined to evaluate the antioxidant and anti-inflammatory activities of BGN4-SK in human intestinal epithelial cells (HT-29) and murine macrophage cells (RAW 264.7). Antioxidant enzymes (SOD and catalase) were produced at the highest levels when BGN4-SK was cultured for 24 h in a medium containing 500 μM MnSO4 and 30 μM hematin, with glucose as the carbon source. The viability and intracellular antioxidant enzyme activities of H2O2-stimulated HT-29 treated with BGN4-SK were significantly higher (p < 0.05) than those of cells treated with BGN4. The intracellular ROS levels of H2O2-stimulated HT-29 cells treated with BGN4-SK were significantly lower (p < 0.05) than those of cells treated with BGN4. BGN4-SK more significantly suppressed the production of interleukin (IL)-6 (p < 0.05), tumor necrosis factor-α (p < 0.01), and IL-8 (p < 0.05) in lipopolysaccharide (LPS)-stimulated HT-29 and LPS-stimulated RAW 264.7 cells compared to BGN4. These results suggest that BGN4-SK may have enhanced antioxidant activities against oxidative stress in H2O2-stimulated HT-29 cells and enhanced anti-inflammatory activities in LPS-stimulated HT-29 and RAW 264.7 cells.

Oxidative Effects during Irreversible Electroporation of Melanoma Cells-In Vitro Study.

Irreversible electroporation (IRE) is today used as an alternative to surgery for the excision of cancer lesions. This study aimed to investigate the oxidative and cytotoxic effects the cells undergo during irreversible electroporation using IRE protocols. To do so, we used IRE-inducing pulsed electric fields (PEFs) (eight pulses of 0.1 ms duration and 2–4 kV/cm intensity) and compared their effects to those of PEFs of intensities below the electroporation threshold (eight pulses, 0.1 ms, 0.2–0.4 kV/cm) and the PEFs involving elongated pulses (eight pulses, 10 ms, 0.2–0.4 kV/cm). Next, to follow the morphology of the melanoma cell membranes after treatment with the PEFs, we analyzed the permeability and integrity of their membranes and analyzed the radical oxygen species (ROS) bursts and the membrane lipids’ oxidation. Our data showed that IRE-induced high cytotoxic effect is associated both with irreversible cell membrane disruption and ROS-associated oxidation, which is occurrent also in the low electric field range. It was shown that the viability of melanoma cells characterized by similar ROS content and lipid membrane oxidation after PEF treatment depends on the integrity of the membrane system. Namely, when the effects of the PEF on the membrane are reversible, aside from the high level of ROS and membrane oxidation, the cell does not undergo cell death.

Mutual regulation of ROS accumulation and cell autophagy in wheat roots under hypoxia stress

Here, we explored the mutual regulation of radical oxygen species (ROS) and autophagy in wheat (Triticum aestivum L.) roots under hypoxia stress. We also analyzed differences between the responses of the stele and the cortex in the two wheat cultivars Huamai 8 (waterlogging-tolerant) and Huamai 9 (waterlogging-sensitive) to hypoxia stress. In situ detection and ultracytochemical localization analysis in wheat roots showed that hypoxia stress caused greater increases in ROS levels and the expression levels of alternative oxidase (AOX) and antioxidant enzymes in the stele than in the cortex. The analysis of exogenous ROS addition and the inhibition of its production revealed the pivotal roles played by ROS in autophagy. Moreover, transmission electron microscopy and qRT-PCR analysis indicated that the stele had a higher level of autophagy than the cortex and that the two wheat cultivars primarily differed in the type and number of autophagosomes. Additional research revealed that autophagy could remove excess ROS, as pre-treatment with the autophagy inhibitor 3-methyladenine increased ROS levels in roots and the addition of the autophagy inducer rapamycin reduced root ROS levels. In conclusion, hypoxia stress induced ROS accumulation in wheat roots where ROS acted as an autophagy signal. Furthermore, higher levels of autophagy and antioxidant enzyme expression in the stele facilitated the elimination of oxidative damage caused by excessive ROS and thereby increased cell survival; in the cortex, a large number of cells died and formed aerenchyma.

GATA6 enhances the stemness of human colon cancer cells by creating a metabolic symbiosis through upregulating LRH-1 expression.

Cancer stem cells play critical roles in tumor initiation, progression, and relapse. Since we previously found that GATA6 promotes the stemness in HCT‐116 and HT‐29 human colorectal cancer (CRC) cells, we aimed to identify the downstream mediator(s) of the stemness‐stimulating effect of GATA6 herein. LRH‐1 was found as a direct target of GATA6 and its upregulation promoted the stemness in both HCT‐116 and HT‐29 cells. Subsequently, hypoxia‐inducible factor‐1α (HIF‐1α) was identified as a direct target of LRH‐1 and its expression level and activity were significantly elevated in the LRH‐1‐overexpressing clones established from the aforementioned two CRC lines. Accordingly, the expression levels of several HIF‐1α targets were also markedly increased, resulting in a stronger glycolysis associated with dramatic elevations of the lactate levels in these cells. Strikingly, higher mitochondrial activities were also found in these clones which might be attributed to the increase of PGC‐1α stimulated by the lactate uptaken through the upregulated MCT‐1. Finally, significant increases in the self‐renewal ability, intracellular radical oxygen species levels and mitochondrial mass were detected in the CD133+/CD44+ subpopulations isolated from CRC cells regardless of their LRH‐1 expression levels. Together, our results suggest a novel metabolic symbiosis between different colorectal cancer stem cell subpopulations critical for maintaining their mutual stemness.

The HACE1-NRF2 Axis a Novel Target in Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is a genetically heterogenous disease characterized by clonal expansion of immature myeloid progenitors cells in the bone marrow (BM). Despite this genetic heterogeneity, AML patients share Leukemia associated oncogenes such as NF-E2-related factor 2 (Nrf2) (Rushworth SA et al.). NRF2 is a transcription factor that activates genes with antioxidant response elements (ARE)-containing promoters and protects cancer cells from apoptosis. Inhibition of NRF2 or antioxidant defense increases the level of Radical Oxygen Species (ROS), leading to tumor supression (Chio IIC et al.). Recently, the E3 Ubiquitin-Protein Ligase HACE1, a tumor suppressor in solid tumors, was demonstrated to promote the expression of NRF2 in Huntigton disease (Rotblat B et al.). Thus, we hypothesized a role for HACE1 as an oncogenic factor acting through NRF2 activation in myeloid malignancies and provide first data supporting the HACE1-NRF2 axis to be a novel target in acute myeloid leukemias.Material and methodsThe mRNA expression data from AML patients (296 samples) vs normal Hematopoietic Stem Cells (HSC) (6 samples) were exported from the bloodSpot database. HACE1 mRNA and protein expression was measured by q-RT-PCR and western blot in 12 commercially available Myeloid Malignancies cell lines. The HACE1 inducible knock down (KD) was carried out by Sleeping Beauty Transposon system in U937 and NOMO-1 cell lines. The cell viability was analyzed by Cell Titer Glo Luminescent assay. Apoptosis was measured by Annexin V (AV)/Propidium Iodide (PI) assay.Results and discussionHACE1 mRNA is downregulated in AML patients compared to HSC (***p<0.001, Bloodspot database). However mRNA and HACE1 protein expression do not correlate in AML cell lines, suggesting post translational modifications. High HACE1 protein expression was observed in most AML cell lines. HACE1 KD reduced drastically the cell viability of U937 cells through caspase activation and NRF2 degradation. However, no effect on cell viability was observed in NOMO-1 cells. Recently, non-programmed cell death necroptosis induction has been described by TNFR1 activation in HACE1 knock out Mouse Embrionic Fibroblast cells (Tortola L et al.). In line with this study, we observed that TNF induces strong cell death in HACE1 KD NOMO-1 cells within 48 hours.In addition HACE1 KD promotes autophagy through p62 degradation (late autophagy marker) in U937 cells. Autophagy has recently been described to contribute to the differentiation and death of AML cells, and to the promotion of immunostimulatory signals activating immune responses against cancer cells (Chen L et al.; Pietrocola F et al.). Thus HACE1 might be a potential target to induce autophagy, providing a novel therapeutical target in the treatment of myeloid malignancies.Finally, HACE1 KD in our hands promoted sensitization of U937 and NOMO-1 cells to cytatarabine, the backbone therapy in AML patients. This treatment promotes HACE1 protein expression at 24 and 48 hours in NOMO-1 cells, which may explain the better response rates of HACE1 KD cells to cytarabine.Taken together, we provide first evidence of HACE1 being a novel oncogene in AML and that the HACE1-NRF2 axis is a promising target in the treatment of Acute Myeloid Leukemias. DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The activation of group II metabotropic glutamate receptors protects neonatal rat brains from oxidative stress injury after hypoxia-ischemia.

Birth asphyxia resulting in brain hypoxia-ischemia (H-I) can cause neonatal death or lead to persistent brain damage. Recent investigations have shown that group II metabotropic glutamate receptor (mGluR2/3) activation can provide neuroprotection against H-I but the mechanism of this effect is not clear. The aim of this study was to investigate whether mGluR2/3 agonists applied a short time after H-I reduce brain damage in an experimental model of birth asphyxia, and whether a decrease in oxidative stress plays a role in neuroprotection. Neonatal H-I in 7-day-old rats was used as an experimental model of birth asphyxia. Rats were injected intra peritoneally with mGluR2 (LY 379268) or mGluR3 (NAAG) agonists 1 h or 6 h after H-I (5 mg/kg). The weight deficit of the ischemic brain hemisphere, radical oxygen species (ROS) content levels, antioxidant enzymes activity and the concentrations of reduced glutathione (GSH) were measured. Both agonists reduced weight loss in the ischemic hemisphere and mitigated neuronal degeneration in the CA1 hippocampal region and cerebral cortex. Both agonists reduced the elevated levels of ROS in the ipsilateral hemisphere observed after H-I and prevented an increase in antioxidant enzymes activity in the injured hemisphere restoring them to control levels. A decrease in GSH level was also restored after agonists application. The results show that the activation of mGluR2 and mGluR3 a short time after H-I triggers neuroprotective mechanisms that act through the inhibition of oxidative stress and ROS production. The prevention of ROS production by the inhibition of glutamate release and decrease in its extracellular concentration is likely the main mechanism involved in the observed neuroprotection.

Elevated levels of the small GTPase Cdc42 induces senescence in male rat mesenchymal stem cells.

Mesenchymal stem cells (MSCs) represent a promising cell source for cellular therapy and tissue engineering and are currently being tested in a number of clinical trials for various diseases. However, like other somatic cells, MSCs age, and this senescence is accompanied by a progressive decline in stem cell function. Several lines of evidence suggest a role for the Rho family GTPase Cdc42 activity in cellular senescence processes. In the present study, we have examined aging-associated Cdc42 activity in rat adipose-derived mesenchymal stem cells (ADMSCs) and the consequences of pharmacological inhibition of Cdc42 in ADMSCs from aged rats. We demonstrate that ADMSCs show a decreased rate of cell growth and a decreased ability to differentiate into chrodrogenic, osteogenic and adipogenic cell lineages as a function of rat age. This is accompanied with an increased staining for SA-β-Gal activity and increased levels of Cdc42 bound to GTP. Treatment of ADMSCs from 24-month old rats with three Cdc42 inhibitors significantly increased proliferation rates, decreased SA-β-Gal staining, and reduced Cdc42-GTP. The Cdc42 inhibitor CASIN increased adipogenic and osteogenic differentiation potential in ADMSCs from 24-month old rats, and decreased the levels of radical oxygen species (ROS), p16INK4a levels, F-actin, and the activity of the ERK1/2 and JNK signaling pathways that were all elevated in these cells. These data suggest that ADMSCs show increased rates of senescence as rats age that appear to be due to elevated Cdc42 activity. Thus, Cdc42 plays important roles in MSC senescence and differentiation potential, and pharmacological reduction of Cdc42 activity can, at least partially, rejuvenate aged MSCs.

C‐peptide and Insulin Work in Concert to Mitigate Hyperglycemia‐induced Oxidative Stress and Facilitate Cellular Recovery

The role of C‐peptide in the prevention and resolution of diabetes‐induced vascular injuries is not fully understood. C‐peptide is a 31 amino acid peptide that is co‐produced and co‐secreted with insulin by pancreatic beta islet cells. In insulin‐dependent diabetics C‐peptide levels correlate with the delayed onset of comorbidities that arise as a consequence of vascular compromise. These comorbidities include increased incidence of cardiovascular disease, retinopathies, renal failure, and compromised wound healing. In 2013, G‐protein‐coupled‐receptor 146 was identified by our group as the receptor for C‐peptide. GPR146 is highly expressed on endothelial cells, fibroblasts, red blood cells, and lymphocytes. We hypothesized that C‐peptide and insulin act in concert to regulate and reduce hyperglycemia‐induced oxidative stress and normalize cellular function in hyperglycemia. In this study, primary human dermal microvascular endothelial cells (HDMEC), primary normal human dermal fibroblasts (NHDF), and human microvascular endothelial cells (HMEC), were used to investigate cellular response to incubation in normal glucose(NG) or high glucose (25 mM)(HG) alone or with the addition of C‐peptide (1.5 nM), insulin (0.5 nM), or both C‐peptide and insulin. All cell lines were pre‐screened for the presence of the insulin receptor and GPR 146 by qPCR and the responses to C‐peptide and insulin by relative expression of cFOS mRNA. In cells incubated with 25 mM glucose for 3 days, the ratio of phosphorylated ERK1–2/ERK 1–2 was not significantly increased by C‐peptide or insulin alone but was significantly increased by incubation with both. Cells incubated in HG for 3 days demonstrated phosphorylated AKT/AKT levels which were not altered by C‐peptide alone, but were increased by insulin and markedly enhanced by the presence of both. In wound healing assays cells in HG demonstrated impaired migration relative to those in NG and the greatest improvement in migration was in the presence of paired C‐peptide and insulin. Endothelial cell nitric oxide (NO) production was significantly elevated in HG, and while there was no significant inhibition by C‐peptide or insulin alone, incubation with both resulted in complete restoration to normal levels. NO generated by ECs in response to HG is produced in conditions of mitochondrial oxidative stress and production of radical oxygen species (ROS), in this environment NO complexes with ROS to form peroxynitrites. Peroxynitrites are potent mediators of cellular damage, forming complexes with proteins, enzymes, and lipids. ROS and superoxide (SO) levels were increased in cells exposed to high glucose conditions, and incubation with both C‐peptide and insulin resulted in the reduction of ROS and SO levels. This study demonstrated the cytoprotective potential of C‐peptide when paired with insulin. These results support the hypothesis that co‐administration of C‐peptide with insulin to insulin‐dependent diabetics may reduce the onset of comorbidities.Support or Funding InformationHJ121456This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

ANALYSIS OF DL--TOCOPHEROL AS ANTIOXIDANT ON MALONDIALDEHYDE LEVEL IN PEDIATRIC PATIENTS WITH -THALASSEMIA MAJOR

Thalassemia is a hereditary form of anemia that affects the synthesis of hemoglobin. The management of therapy in patients with b-thalassemia major which patients should receive continuous blood transfusions and increased iron absorption from the digestive tract causes excess iron in the body. This will lead to an increase of free iron level that triggers Radical Oxygen Species (ROS). Increased level of ROS can initiate lipid peroxidation which used as an indicator of oxidative stress in cells and tissues and produce reactive carbonyl, mainly malondialdehyde (MDA). Thus, MDA measurement is widely used as an indicator of lipid peroxidation. On the other hand, the risk of oxidative damage can be reduced by antioxidant, one of them is Vitamin E that is a fat-soluble vitamin with high potential antioxidant. The objective of this study was to analyze the effect of the dl-a-tocopherol (Vitamin E) administration on decrease of MDA serum level on pediatric patients with b-thalassemia major. This was a longitudinal observational study design for one group without comparison was conducted to examine the use of vitamin E to decreased MDA serum level on children patients with b-thalassemia major. The inclusion criteria were patients who rely on blood transfusions, patients who received only one type of iron chelating agents during the study period, the clinical condition is stable, agrees, and has completed the informed consent. In the course of the study of 21 patients there were variations in patient compliance in taking vitamin E tablet dosage 200 IU once-daily for one month: only 11 out of 21 patients consumed 30 tablets of vitamin E 200 IU (total dose of 6000 IU) in the 1-month study, and only data from those 11 samples will be analysed further. MDA serum level was measured pre- and post-administration of vitamin E and patient’s characteristics of subjects was obtained for additional information. Pre-administration of vitamin E, serum level of MDA was 1239.4 ± 502.55 ng/mL with a range of 216.95 to 2297.3 ng/mL, whereas in the group post administration of vitamin E, MDA serum level was 786.49 ± 704.88 ng/mL with a range of 6.5380 to 1958.6 ng/mL. In conclusion, there was no significant difference in MDA serum level in the group pre- and post- administration of vitamin E (p = 0.15).

Abstract 2138: Tumor treatment fields downregulate the BRCA1/FA pathway genes leading to reduced DNA repair capacity, the inhibition of mitophagy and enhanced cell death

Abstract The application of new physical cancer treatment modality utilizing alternating electric fields termed tumor treatment fields (TTFields) has revolutionized the treatment of recurrent and newly diagnosed glioblastoma. This non-invasive exposure to low-intensity, intermediate frequency, alternating electric fields to the region of the tumor has resulted in a significant increase in overall survival when compared to standard therapy with very minimal side effects. Clinical trials are recruiting or ongoing at additional tumor sites including lung, pancreatic, and ovarian cancer. The primary mechanism of TTField cell killing is thought to be the disruption of mitosis; however, other potential mechanisms are under investigation. Using a panel of five NSCLC cell lines we found that TTFields treatment alone inhibits cell proliferation, and decreases survival, though the degree of inhibition varies between cell lines. To understand the molecular mechanisms underlying the biological effects of TTField exposure we studied temporal gene expression changes in the NSCLC cell lines after TTField treatment. We observed that most differentially expressed genes are part of cell cycle and proliferation pathways which is in agreement with earlier findings. Interestingly we found that the expression of BRCA1 DNA damage repair pathway genes were significantly downregulated (P &amp;lt; 0.05) upon TTField treatment. We confirmed the downregulation of BRCA1/FA pathway proteins by western blot. When examining the nuclear role of the BRCA1/FA pathway genes we found that TTField treatment slowed the repair of ionizing radiation-induced DNA damage compared to radiation alone which is evident by an increased number of DNA double strand break repair foci at any given time. Moreover, we found that TTField treatment increased the incidence of chromatid aberrations. We also examined the newly identified BRCA1/FA pathway genes cytosolic role in mitophagy where we observed alterations in mitophagy related gene (PINK1, OSCP1, ATP5 and DAPIT) expression and confirmed the same at the protein level by western blot. We hypothesized that TTFields disrupt the clearance of damaged mitochondria due to the downregulation of BRCA1/FA pathway players, causing an imbalance in oxygen metabolism leading to the production of high levels of radical oxygen species (ROS) and as a result, cell death. Using CellROX dye we found that TTField treatment did result in increased ROS production suggesting a new mechanism of action for TTField exposure. Novel chemotherapy agents, particularly PARP inhibitors, in combination with DNA damaging agents like radiation and TTFields may be advantageous through the conditional vulnerability of down-regulated BRCA1. Citation Format: Narasimha Kumar Karanam, Lianghao Ding, Brock Sishc, Debabrata Saha, Michael D. Story. Tumor treatment fields downregulate the BRCA1/FA pathway genes leading to reduced DNA repair capacity, the inhibition of mitophagy and enhanced cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2138. doi:10.1158/1538-7445.AM2017-2138

Fermented Brown Sugar Residue Prolongs the &amp;lt;i&amp;gt;Caenorhabditis elegans&amp;lt;/i&amp;gt; Lifespan &amp;lt;i&amp;gt;via&amp;lt;/i&amp;gt; DAF-16

Purification of biomass ethanol from the products of brown sugar yeast-fermentation produces a large amount of residue. This fermentation residue contains abundant brown sugar-derived nutrients and is mainly used as compost or livestock feed. However, the in vivo physiological effects of oral residue ingestion are not known. The purpose of this study was to elucidate the physiological action and molecular mechanism of fermented brown sugar residue in nematode stress tolerance, aging, and lifespan using Caenorhabditis elegans. Fermented brown sugar residue was divided into two layers, supernatant and precipitate, and each was given to nematodes. Analysis of motility and survival rate under thermal stress revealed reduced mobility and increased survival rate following treatment with fermented brown sugar residue. The survival rate of nematodes under 1% H2O2 was markedly increased by the residue and mitochondrial membrane depolarization was induced and mitochondrial radical oxygen species levels increased. Furthermore, aging dependent reduction of motility was suppressed, and the average life span of nematodes was extended by treatment with fermented brown sugar residue. Moreover, the effects of fermented brown sugar residue on stress tolerance, lifespan elongation, and decreased aging dependent momentum reduction were lost in the daf-16 mutant. Taken together, our results show that the various physiological actions of fermented brown sugar residue, including stress tolerance and lifespan extension, occur via DAF-16.

Hypoglycaemic, hypolipidaemic and antioxidant effects of blackberry beverage consumption in streptozotocin-induced diabetic rats

Abnormal glucose metabolism, hyperlipidaemia profiles and high levels of radical oxygen species (ROS) are classic features of diabetes. This study evaluates the effect of the consumption of two different blackberry beverages at 25 and 12.5% given orally for 40 days to rats with streptozotocin induced diabetes. The lower dose of blackberry (12.5%) non-statistically decreased glycaemia (−10.4%), triacylglycerols (−4.6%) and cholesterol (−21.0%). These differences were not statistically significant. The higher dose of blackberry (25%) significantly decreased glucose (−48.6%), triacylglycerols (−43.5%) and cholesterol (−28.6%). The higher dose of blackberry (25%) improved plasma antioxidant capacity, reduced the levels of lipid peroxidation in plasma (−19%) and in kidney (−23%). Blackberry intake did not improve catalase, suggesting that attenuation of oxidative stress via scavenging activities rather than improving the activities of antioxidant enzymes. These results provide promising data for this blackberry as a dietary adjuvant to the pharmacological management of diabetes.

Vitamin E Analogue Improves Rabbit Sperm Quality during the Process of Cryopreservation through Its Antioxidative Action.

The process of cryopreservation results in high concentration of reactive oxygen species which is detrimental to spermatozoa. The aim of this study was to investigate whether addition of vitamin E analogue to freezing extender can facilitate the cryosurvival of spermatozoa in rabbits, and how vitamin E protects spermatozoa against damages during the process of preservation. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of Trolox (a vitamin E analogue). The level of radical oxygen species (ROS) in spermatozoa that was exposed to Trolox was significantly lower than that of the control during each step of the process of preservation. The percentage of frozen-thawed spermatozoa with lipid peroxidation in the Trolox treatments was significantly lower than that of the control. The motility, intact acrosome, membrane integrity and mitochondrial potentials of the frozen-thawed spermatozoa in the treatment of 200 μM Trolox were significantly higher than those of the control. These observations suggest that addition of vitamin E to a freezing extender leads to higher integrity of acrosome, plasma membrane and mitochondrial membrane potential as well as higher motility. Vitamin E protects spermatozoa through its capacity to quench ROS accumulation and lipid peroxidation during the process of preservation. Addition of Trolox is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry.

Tumor Necrosis Factor-Alpha Up-Regulates ICAM-1 Expression and Release in Intestinal Myofibroblasts by Redox-Dependent and -Independent Mechanisms.

Intercellular adhesion molecule-1 (ICAM-1) is distributed and expressed on cell surface and is present in circulation as soluble form (sICAM-1). Tumor necrosis factor-alpha (TNFα) and radical oxygen species (ROS) up-regulate the expression of ICAM-1. This study demonstrates for the first time in 18 Co cells, a myofibroblast cell line derived from human colonic mucosa, an up-regulation of ICAM-1 expression and sICAM-1 release induced by oxidative stress and TNFα stimulation. The intracellular redox state was modulated by L-buthionine-S,R-sulfoximine (BSO) or N-acetylcysteine (NAC), inhibitor and precursor respectively of GSH synthesis. ROS production increases in cells treated with BSO or TNFα, and this has been related to an up-regulation of ICAM-1 expression and sICAM-1 release. The involvement of metalloproteinases in ICAM-1 release has been demonstrated. Moreover, also expression and activation of A disintegrin and metalloproteinase 17, a membrane-bound enzyme known as TNFα-converting enzyme (TACE), have been related to ROS levels. This suggests the possible involvement of TACE in the cleavage of ICAM-1 on cell surface in condition of oxidative stress. NAC down-regulates the expression and release of ICAM-1 as well as the expression and activation of TACE. However, in TNFα stimulated cells NAC treatment reduces only in part ICAM-1 expression and sICAM-1 release. Given this TNFα may also act on these events by a redox-independent mechanism.

The human ATF1 rs11169571 polymorphism increases essential hypertension risk through modifying miRNA binding

Activating transcription factor 1 (ATF1) may be involved in essential hypertension (EH) by induction of NADPH oxidase 1 (NOX1) and radical oxygen species (ROSs) production. Abnormal expression of ATF1 was found in EH in previous microarray analysis. Here we tested whether a single nucleotide polymorphism (SNP) located in the 3′-untranslated region (3′UTR) of ATF1 was associated with EH susceptibility by affecting microRNA (miRNA) binding. In silico analysis indicated that rs11169571 (T>C) was a candidate SNP to modulate miRNA: ATF1 mRNA complex, with the greatest changed energy for hsa-miR-1283, and the luciferase reporter analysis showed that miR-1283 inhibited the activity of the reporter vector carrying –T allele, but not the –C allele. In addition, inhibition of miR-1283 in HA-VSMCs enhanced the expression of ATF1 mRNA as well as the ROS levels. Further case-control study showed that rs11169571 was significantly associated with increased risk of EH. Finally, we observed an increased ATF1 protein level in peripheral blood of EH patients with CC carriers compared to TT carriers of rs11169571, with an intermediate ATF1 level in TC carriers. These results suggested that rs11169571 of ATF1 gene may be associated with EH, and the SNP-modified posttranscriptional gene regulation by miRNAs could be a potentially pathogenetic mechanism of EH.

Identification and characterization of antioxidant peptides from chickpea protein hydrolysates

Oxidative stress due to the excess of radical oxygen species (ROS) contribute to the development of different diseases. The use of antioxidants may prevent the development of these diseases by counteracting ROS levels. There is an increasing interest in natural antioxidants as they are safer for consumers than synthetic antioxidants. In this work, reducing power, free radical scavenging and cellular antioxidant activities of chickpea peptides fractions have been investigated. Peptide sequences included in fractions with antioxidant activity were identified. Main sequences, ALEPDHR, TETWNPNHPEL, FVPH and SAEHGSLH, corresponded to legumin, the main seed protein. Most peptides contained histidine, which has shown antioxidant activity. Two peptides also included tryptophan and phenylalanine, in which the phenolic group could also serve as hydrogen donor. These results show that legumin is a source of antioxidant peptides of high interest for food and pharmaceutical industries to develop new nutraceuticals and functional foods.

Influence of drought stress on photosynthetic, radical oxygen, respiration, assimilate partitioning, activities of enzymes, phytohormones and essential oils in crop plants

Water-deficit stresses occur in about 70% of arable land around the world and have been shown to have an effect on every aspect of plant growth. Drought is one of the most common environmental stresses that may limit agriculture production worldwide. Many crops have high water requirement and in most countries supplemental irrigation is necessary for successful crop production. Photosynthetic response to drought is a highly complex in plants. Water deficit inhibits photosynthesis by causing stomatal closure and metabolic damage. Stomata of the leaves that are slightly deficient in water opened more slowly in light and close more quickly in the dark. The activities of many enzymes are affected by drought conditions. For example, nitrate reductase activity has been shown to be highly sensitive to water stress, and a significant decrease in nitrate reductase activity was observed in many plant species under drought conditions. Drought stress also increases the levels of radical oxygen species (ROS) in plant cells, resulting in lipid peroxidation and protein damage.

Early stage of obesity potentiates nitric oxide reduction during the development of renal failure

Obesity is a serious health problem associated with the pathogenesis of various metabolic diseases. Nitric Oxide (NO) plays an important role in kidney function and altered NO levels have been associated with the pathogenesis of obesity. Therefore, we aimed to study whether an early stage of obesity contributes with progression of renal failure through further NO impairment. Male C57BL/6 mice were fed with a high-fat diet (HFD) or a normal diet (ND) during 2 weeks. All mice underwent either sham surgery (sham) or 5/6 nephrectomy (Np). One group of HFD Np mice was treated with antioxidants plus L-arginine. Kidney damage parameters were assessed and eNOS metabolism was evaluated. Mice on a HFD increased body weight, eNOS protein and mRNA expression, and radical oxygen species (ROS). Urine nitrites excretion, urine volume, and plasma BH4 were decreased. In HFD mice, 5/6 Np further increased BH2 and urine protein concentration, ROS levels, and eNOS mRNA expression. The decrease in BH4 plasma levels and urine nitrites excretion was accentuated. NO synthesis stimulation with the antioxidants + L-arginine treatment prevented all these changes. The early changes in NO metabolism are associated with an early stage of obesity. This effect on NO potentiates kidney damage development.

Phycocyanin Induces Apoptosis and Enhances the Effect of Topotecan on Prostate Cell Line LNCaP

C-phycocyanin (C-PC) from Spirulina has been previously shown to have anticancer properties. Here, we report on anticancer activity of C-PC that was isolated from the novel cyanobacterium Limnothrix sp. 37-2-1. C-PC from this organism exhibited anticancer properties in our in vitro systems; however, the required doses were well above the range of anticancer drugs normally used. Therefore, we conducted several experiments to test whether lower-than-usual doses of the anticancer drug topotecan (TPT) can offer the same level of cytotoxic effects as normal doses when combined with C-PC. For this purpose, cytotoxicities of C-PC and TPT were tested using the LNCaP (prostate cancer) cells. We found that when only 10% of a typical dose of TPT was combined with C-PC, the cancer cells were killed at a higher rate than when TPT was used alone at full dose. Similarly, we were also able to detect an increased level of radical oxygen species (ROS) generation as well as an increase in activities of caspase-9 and caspase-3 when these two compounds were used in combination. Taken together, our findings suggest that combining C-PC from Limnothrix sp. with the lower dose of TPT can induce apoptosis through generation of ROS and activation of caspases. In that respect, we suggest that C-PC can potentially improve the efficacy of the currently available anticancer drug, and therefore diminish its harsh side effects in the patient.