Significant Differences In Reactive Oxygen Species Levels Research Articles

Imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species in stallion sperm.

Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been debated by researchers over the last decade. The objective of this study was to evaluate the relationship between various sperm morphologies and ROS production in fresh and cooled stallion semen by employing the novel method of imaging flow cytometry for stallion semen assessment. For evaluation of fresh semen, single ejaculates (n=5) were collected from four resident stallions at the University of California, Davis. For the evaluation of 24-h cool-stored semen, single ejaculates were collected from stallions at Texas A&M University (n=5) and shipped to the University of California, Davis overnight for evaluation. Ejaculate volume, sperm concentration and motility parameters were recorded. Samples were co-stained for viability and ROS detection with SytoxGreen™ and dihydroethidium (DHE), respectively, and evaluated with the Amnis® ImageStream® system (Luminex Corporation). Antimycin, an electron transport chain inhibitor that triggers ROS production (1μM), was used as a positive control for DHE, while dead cells (2× snap frozen in liquid nitrogen) served as a positive control for SytoxGreen™. Unstained samples were also evaluated as controls. Imaging flow cytometric analysis was performed with the ideas® software (Luminex Corporation). Evaluated morphologies included abnormal head (AH), abnormal midpiece (AM), abnormal tail (AT), proximal cytoplasmic droplet (PD), or distal cytoplasmic droplet (DD), and morphologically normal (MN) cells. For fresh semen, an additional abnormality, coiled tail and midpiece (CTM) was assessed; 24-h cool-stored semen did not contain enough viable CTM cells for analysis. Only cells with obvious, single abnormalities were selected for the first portion of analysis to minimize subjectivity. Mixed effects modelling was used to evaluate the relationship between each morphologic classification and the corresponding DHE fluorescence intensity. Compared to the MN population, ROS production was significantly higher in viable cells with AH, PD and AM (p < .0001) in both fresh and cooled semen. CTM cells had significantly higher levels of ROS production compared to MN cells in fresh semen (p < .0001). There was no significant difference in ROS levels between MN cells and AT and DD cells in either fresh or cooled semen (p > .05). These results suggest that ROS generation is indicative of abnormal cell morphology and function and confirm that imaging flow cytometry is a valuable tool for the assessment of stallion semen.

Evaluation of Oxidative Stress as a Contributor of Anesthetic Agents' Effects on Embryo Quality and ICSI Outcome in Iraqi Patients

Infertility is described as a couple's failure to conceive for at least a year, using unprotected sex. Even though all anesthetic chemicals have been revealed in the follicular fluids, general anesthesia is still used in many IVF centers for patients who want to get pregnant. This study included 60 infertile women randomly divided into two groups under general anesthesia. 30 patients were given a Ketamine dose of 0.5 mg/Kg and the other 30 patients were received Remifentanil dose of 0.5 µg/Kg for induction of anesthesia. We collected serum for Reactive Oxygen Species (ROS) levels assessment for all patients before and after starting general anesthesia. Routine ICSI procedures was performed on all participants, including clinical evaluation (history, examination, and investigation), controlled ovarian and ovulation stimulation, oocyte retrieval under general anesthesia, follicular fluid collection for postoperative anesthesia medication concentration (Remifentanil and Ketamine), oocyte stripping, oocyte maturity assessment, intra-oocyte sperm injection into mature cells (MII), fertilization and division evaluation and embryo categorization, embryo selection and transfer, luteal phase support, and beta-hCG determination. ROS concentrations were compared between the Remifentanil and Ketamine groups. There was no significant difference in embryo features between Remifentanil and Ketamine, indicating that neither one is superior to the other in this regard. When Remifentanil or Ketamine was taken, there was no significant difference in ROS levels in serum or follicular fluid.

Evaluation of Oxidative Stress as a Contributor of Anesthetic Agents' Effects on Embryo Quality and ICSI Outcome in Iraqi Patients

Infertility is described as a couple's failure to conceive for at least a year, using unprotected sex. Even though all anesthetic chemicals have been revealed in the follicular fluids, general anesthesia is still used in many IVF centers for patients who want to get pregnant. This study included 60 infertile women randomly divided into two groups under general anesthesia. 30 patients were given a Ketamine dose of 0.5 mg/Kg and the other 30 patients were received Remifentanil dose of 0.5 µg/Kg for induction of anesthesia. We collected serum for Reactive Oxygen Species (ROS) levels assessment for all patients before and after starting general anesthesia. Routine ICSI procedures was performed on all participants, including clinical evaluation (history, examination, and investigation), controlled ovarian and ovulation stimulation, oocyte retrieval under general anesthesia, follicular fluid collection for postoperative anesthesia medication concentration (Remifentanil and Ketamine), oocyte stripping, oocyte maturity assessment, intra-oocyte sperm injection into mature cells (MII), fertilization and division evaluation and embryo categorization, embryo selection and transfer, luteal phase support, and beta-hCG determination. ROS concentrations were compared between the Remifentanil and Ketamine groups. There was no significant difference in embryo features between Remifentanil and Ketamine, indicating that neither one is superior to the other in this regard. When Remifentanil or Ketamine was taken, there was no significant difference in ROS levels in serum or follicular fluid.

Reactive Oxygen Species (ROS)-Responsive Prodrugs, Probes, and Theranostic Prodrugs: Applications in the ROS-Related Diseases.

Elevated levels of reactive oxygen species (ROS) have commonly been implicated in a variety of diseases, including cancer, inflammation, and neurodegenerative diseases. In light of significant differences in ROS levels between the nonpathogenic and pathological tissues, an increasing number of ROS-responsive prodrugs, probes, and theranostic prodrugs have been developed for the targeted treatment and precise diagnosis of ROS-related diseases. This review will summarize and provide insight into recent advances in ROS-responsive prodrugs, fluorescent probes, and theranostic prodrugs, with applications to different ROS-related diseases and various subcellular organelle-targetable and disease-targetable features. The ROS-responsive moieties, the self-immolative linkers, and the typical activation mechanism for the ROS-responsive release are also summarized and discussed.

Oxidative stress, glutathione, and gene expression as key indicators in SH-SY5Y cells exposed to zearalenone metabolites and beauvericin

The co-presence of mycotoxins from fungi of the genus Fusarium is a common fact in raw food and food products, as trace levels of them or their metabolites can be detected, unless safety practices during manufacturing are carried out. Zearalenone (ZEA), its metabolites α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL) and, beauvericin (BEA) are co/present in cereals, fruits or their products which is a mixture that consumer are exposed and never evaluated in neuronal cells. In this study the role of oxidative stress and intracellular defense systems was assessed by evaluating reactive oxygen species (ROS) generation and glutathione (GSH) ratio activity in a human neuroblastoma cell line, SH-SY5Y cells, treated individually and combined with α-ZEL, β-ZEL and BEA. It was further examined the expression of genes involved in cell apoptosis (CASP3, BAX, BCL2) and receptors of (endogenous or exogenous) estrogens (ERβ and GPER1), by RT-PCR in those same conditions. These results demonstrated elevated ROS levels in combinations where α-ZEL was involved (2.8- to 8-fold compared to control); however, no significant difference in ROS levels were detected when single mycotoxin was tested. Also, the results revealed a significant increase in GSH/GSSG ratio at all concentrations after 24 h. Expression levels of CASP3 and BAX were up regulated by α-ZEL while CASP3 and BCL2 were down regulated by β-ZEL, revealing how ZEA´s metabolites can induce the expression of cell apoptosis genes. However, BEA down-regulated the expression of BCL2. Moreover, β-ZEL + BEA was the only combination treatment which was able to down regulate the levels of cell apoptosis gene expression. Relying to our findings, α-ZEL, β-ZEL and BEA, induce injury in SH-SY5Y cells elevating oxidative stress levels, disturbing the antioxidant activity role of glutathione system and finally, causing disorder in the expressions and activities of the related apoptotic cell death genes.

Study on anti-inflammatory and antioxidant mechanism of compound huoxue ling in preventing deep vein thrombosis after fracture

Objective To explore the mechanism of anti-inflammatory and anti-oxidation effects of prescription Huoxueling (HXL) in preventing deep vein thrombosis after fracture. Methods Sixty Wistar rats aged one month were randomly divided into blank group (purified water 10 ml/kg), control group (purified water 10 ml/kg), positive drug group (aspirin 116.9 mg/kg), and Chinese medicine HXL. Low-, medium-, and high-strength (containing crude drug amounts of 0.625, 1.230 and 2.460 g/ml aqueous solutions) of 10 rats in each group were used to prepare rat deep venous thrombosis using a traumatic model; 7 days before the injection of the mould, once a day, on the 7th day after the injection, the blood of the rats in each group was used to measure the blood rheology and related coagulation indexes, and the interleukin in the tissue was detected by enzyme-linked immunosorbent assay (ELISA). interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) levels were changed; biochemical methods were used to detect the level of reactive oxygen species (ROS) in the venous venous tissue. Superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px), malondialdehyde (MDA) content. Results The activated partial thromboplastin time (APTT), plasma prothrombin time (PT), thrombin time (TT), and D-dimer were (19.10±0.54) s in the positive drug group [(8.61±0.56), (17.76±0.58) s, (52.82±2.66) μg/L) and HXL high-dose groups [(21.53±0.34), (9.16±0.30), (18.75±0.41) s, (46.89±1.64) μg/L] Significance (P=0.003, 0.001, 0.002, 0.001), HXL high-dose group had better improvement of coagulation indexes APTT, PT, TT, and D-dimer than positive drug group; positive drug group IL-1, TNF-α, IL-6 levels were 356.27±24.41, 250.89±24.77, 152.71±11.36 and HXL high-dose groups (297.32±17.86, 240.23±23.15, 145.83±10.94), respectively. High-and low-dose HXL groups were positive for IL. There was a significant difference between the-1 and the two groups (P=0.000); the ROS, SOD, GSH-Px and MDA values of the positive drug group were (34 297.38±302.44), (110.40±11.32), (221.05±19.67) U/L, respectively, (11.02±1.15) mmol/L and HXL high-dose groups [(31 704.24±272.12), (114.92±11.72), (228.65±15.18) U/L, (8.97±0.97) mmol/L], HXL high-dose group and positive drug There was a statistically significant difference in ROS levels between the two groups (P=0.000). The high-dose HXL group showed better improvement in oxidative stress response than the positive drug group. Conclusion Compound Huoxue Lingfang may have anti-inflammatory and anti-oxidant effects. To prevent deep vein thrombosis after fracture. Key words: Huoxueling; Deep vein thrombosis; Inflammation; Oxidative stress; Prevention

Diagnosis of prostate cancer by analyzing oxidative stress in human seminal plasma: developing unsophisticated tools for noninvasive prostate cancer diagnosis.

Prostate-specific antigen blood testing has improved early detection of prostate cancer (PCa); however, PCa mortality has not decreased accordingly and a prostate biopsy is still required for a definitive diagnosis. Proteomic biomarker screening in easily available body fluids such as seminal plasma is now increasingly being proposed as a solution to improve PCa detection and prognosis. PCa cells typically produce high levels of reactive oxygen species (ROS). In this study, we therefore investigated ROS levels in semen samples from patients with a negative or a positive prostate biopsy to predict PCa diagnosis. Multiple clinicopathological parameters (digital rectal examination, prostate-specific antigen scoring, prostate biopsy, and ROS levels) of patients examined for PCa were measured. No significant differences in ROS levels were detected in relation to PCa diagnosis. Although seminal plasma is a well-suited medium for prostate-related biomarkers, no significant differences in ROS levels were observed between the patient groups. Comparison with ROS levels encountered in semen of larger patient groups is the next logical step.

Oxidative and Inflammatory Responses of Monocytes or Macrophages to Ambient Particulate Matter

Oxidative stress and inflammation are two major responses to ambient particulate matter (PM) and are thought to be responsible for the majority of its adverse health impacts. The hypothesis of this study is that monocytes and macrophages will have different responses to PM and that the oxidative and inflammatory responses will be linked. A human monocytic cell line (THP‐1) was used as a model for monocytes. These cells were also differentiated to macrophages via exposure to phorbol ester (PMA) for 48 hours. These cells were then exposed to three different size fractions of particulate matter: PM10 (coarse), PM2.5 (fine), and PM0.1 (ultrafine) for 24 hours. Oxidative stress was measured using the DCFH‐DA assay. Reactive oxygen species (ROS) increased compared to the control, but the increase was not dependent on PM size. There was no significant difference in ROS levels in monocytes compared to macrophages. Inflammatory cytokines TNF‐α and IL‐1β were measured using ELISA. In both monocytes and macrophages, fine and coarse particles elicited a significant increase. No significance occurred after exposure to ultrafine particles and there was no difference in the response to monocytes compared to macrophages. This study suggests that monocytes and macrophages respond similarly to particulate matter. Also, PM‐induced oxidative and inflammatory responses may not be directly linked. The mechanism by which PM causes these observed responses in immune competent cells is the subject of future studies in the laboratory.Support or Funding InformationBritish Petroleum/ Southern California Air Quality Management District (BP/SCAQMD Award #11527) and Western University of Health Sciences

Efficacy of erdosteine 900 versus 600 mg/day in reducing oxidative stress in patients with COPD exacerbations: Results of a double blind, placebo-controlled trial

BackgroundAcute exacerbations of chronic obstructive pulmonary disease (AECOPD) are associated with increased airway and systemic inflammation. There is evidence that erdosteine accelerates recovery from AECOPD by reducing airway inflammation. AimTo investigate the dose-dependent antioxidant/anti-inflammatory activity of erdosteine in COPD patients. MethodsIn this single-centre, double blind, double dummy study, patients with mild-to-moderate COPD (GOLD stage II–III), were randomised to receive either placebo or two dosages of oral erdosteine (300 mg tid or 300 mg bid + 1 capsule of indistinguishable placebo) for 28 days in addition to their standard treatment. Primary variables were plasma reactive oxygen species (ROS) and 8-isoprostane levels, while secondary variable was lung function (FEV1; FEV1/FVC; FEV1 short-term reversibility), all assessed in baseline; every two weeks during the study, and one week after the end of the study. ResultsBaseline demographic characteristics, plasma ROS and 8-isoprostane levels and lung function were not significantly different in the 24 eligible patients (14 males, aged 38–75 years). At 2 weeks, there was a dose-dependent decrease in ROS in the erdosteine groups. By week 4 there were significant differences in ROS levels compared to baseline between patients receiving 900 mg/day (p < 0.003) and those receiving 600 mg/day (p < 0.04). This effect continued in the follow-up week (p < 0.021). Erdosteine also lowered 8-isoprostane plasma levels after 4 weeks (p < 0.01), and this effect lasted over the post-treatment week. Moreover, % FEV1 reversibility after salbutamol 400 mcg obtained after a 4 -week treatment of erdosteine 900 mg/day was significantly higher than that obtained after 600 mg/day (p < 0.01). Erdosteine was well tolerated and no treatment-related adverse event was reported. ConclusionsResults confirm the antioxidant dose- and time-dependent activity of erdosteine, and support the utility of including erdosteine it in the therapeutic strategy for the prevention and treatment of oxidative stress-induced inflammation, which frequently leads to AECOPD occurrence.

Reactive oxygen species and sperm DNA damage in infertile men presenting with low level leukocytospermia.

BackgroundLeukocytes contribute directly and indirectly to reactive oxygen species (ROS) production. Although leukocytospermia is defined as the presence of ≥1 × 106 white blood cells/mL (WBC/mL) in a semen sample, the presence of less than 1×106 WBC/mL (low-level leukocytospermia) can still produce a detectable amount of ROS, impairing sperm function and lowering the chances of pregnancy. Our objective was to assess the effect of low-level leukocytospermia on semen quality, ROS levels, and DNA damage in infertile men.MethodsSemen samples were examined from 472 patients and divided into 3 groups: no seminal leukocytes; group 2, men with low-level leukoctyospermia (0.1-1.0 × 106 WBC/mL); and group 3, frank leukocytospermia, (>1.0 × 106. WBC/mL). Semen analysis, leukoctyospermia, reactive oxygen species and DNA fragmentation was tested.ResultsConventional semen parameters between the 3 groups were similar. Group 2 patients had significantly higher levels of ROS and sperm DNA fragmentation (1839.65 ± 2173.57RLU/s; DNA damage: 26.47 ± 19.64%) compared with group 1 (ROS: 1101.09 ± 5557.54 RLU/s; DNA damage: 19.89 ± 17.31%) (ROS: p = 0.002; DNA damage: p = 0.047). There was no significant difference in ROS levels between groups 2 and 3.ConclusionsPatients presenting with low-level leukocytospermia have seminal oxidative stress. Although these patients are not categorized as leukocytospermic by current World Health Organization (WHO) guidelines, these men may benefit by treatment with antibiotics, testing for bacterial cultures, or antioxidant supplements to reduce ROS-induced sperm DNA fragmentation and improve their chances of fertility. The WHO guidelines for leukocytospermia may need to be revised accordingly.

Inhibition of micro RNA-9 expression promotes UV-induced ROS damage in nasopharyngeal carcinoma cells

To investigate the effects of down-regulated miR-9 expression on ultraviolet rays (UV)-induced reactive oxygen species (ROS) damage in nasopharyngeal carcinoma (NPC) cells. The NPC cells were transfected with inhibitors of miR-9 by lipofectamine to decrease the expression of miR-9, and the cells transfected with inhibitor control as the control. ROS levels following UV exposure were examined with DCF-DA method and the concentration of glutathione was analyzed via the benzoic acid method; DNA damage and apoptosis also were evaluated. There was significant difference in ROS levels between miR-9 expression-inhibited cells and control cells (26 895 ± 218 vs 15 765 ± 927, t = 39.754, P < 0.001), and also there were significant differences in DNA damage rates (28.0% ± 10.0% vs 23.6% ± 9.2%) and in apoptosis rates (8.0% ± 0.9% vs 4.5% ± 0.8%) following UV exposure between two groups of cells. The miR-9 expression-inhibited cells showed lower level (1.87 ± 0.15) µmol/L of glutathione compared with the control cells (9.85 ± 0.15) µmol/L (t = -48.832, P < 0.001). Inhibition of miR-9 expression promoted UV-induced ROS damage in nasopharyngeal carcinoma cells.

1,25-Dihydroxyvitamin D3 and the aging-related Forkhead Box O and Sestrin proteins in osteoblasts

Forkhead Box O (FoxO) transcription factors and Sestrins (SESN) are highly conserved and related stress-responsive proteins that protect the organism against age-related pathologies. For FoxOs, growing evidence shows a crucial role in osteoblast function. Here we investigated the role of different FoxO and SESN isoforms in 1,25(OH)2D3-treated MC3T3-E1 osteoblasts. 1,25(OH)2D3 rapidly and strongly induced the expression of SESN1 and FoxO3a but down-regulated the expression of SESN3 and FoxO1. SESN2 and FoxO4 levels were hardly affected by 1,25(OH)2D3. Chromatin Immunoprecipitation (ChIP)-sequencing revealed significant VDR/RXR binding to a DR3-type VDRE in SESN1 but not in the genomic region where FoxO3a is located. Mutation of the SESN1 VDRE abolished responsiveness to 1,25(OH)2D3 in luciferase-based transfection assays. siRNA-mediated knock-down of SESN1, SESN3, FoxO1 or FoxO3a did not prevent 1,25(OH)2D3 from reducing the expression of cell cycle markers like Cyclin D1 and Cdc6 and from exerting its characteristic antiproliferative effect on MC3T3-E1 osteoblasts. Accordingly, the 1,25(OH)2D3-induced reduction in the number of S-phase cells was also maintained. The antiproliferative effect was still present in primary osteoblast in which all three FoxO isoforms were deleted (TKOpOB). Interestingly, both MC3T3-E1 osteoblasts in which FoxO1 was knocked-down and TKOpOBs accumulated significantly more reactive oxygen species (ROS) after treatment with 1,25(OH)2D3 than control cells. siRNA-mediated knock-down of individual SESN isoforms did not result in significant differences in ROS levels. In conclusion, 1,25(OH)2D3 directly and indirectly alters the expression levels of different FoxO and SESN isoforms in osteoblasts, presumably not to exert its antiproliferative action but to control ROS levels.This article is part of a Special Issue entitled ‘Vitamin D Workshop’.

Heparin or EDTA; anticoagulant of choice in free radical estimation?

Physiological levels of reactive oxygen species (ROS) are needed for various normal processes like cellular immunity, sperm function, capacitation, acrosome reaction, proper embryonic development and implantation, cell signalling, DNA repair activation and gene transcription. Hence anything that creates imbalance in the ROS homeostasis would affect these essential physiological processes. Heparin is a sulfated anionic glycosaminoglycan widely used as an anticoagulant in conditions such as acute coronary syndrome, atrial fibrillation, deep-vein thrombosis and pulmonary embolism, cardiopulmonary bypass for heart surgery and hemofiltration. Ethylenediaminetetraacetic acid (EDTA) is a widely used anticoagulant which is also used in medical conditions like heavy metal poisoning for chelation therapy. To measure the ROS levels in the whole blood we employed a simple chemiluminescence assay using luminol as a chemiluminescent probe. To avoid clotting of blood while estimating free radical levels we used heparin and EDTA as anticoagulants. We observed a significant difference in ROS levels between the two readings using heparin and EDTA. We wish to state that though heparin and EDTA are used as anticoagulants they cause a marked reduction (false low) in the readings due to their antioxidant properties. EDTA caused a much more drastic and significant decline in ROS levels as compared to heparin. Thus in clinical setting they should be used with caution; after assessment of ROS levels so that the redox sensitive physiological reactions may not be impaired and it may not decrease rate of transcription of several genes.

Naturally high plasma glucose levels in mourning doves ( Zenaida macroura) do not lead to high levels of reactive oxygen species in the vasculature

Plasma glucose (P Glu) concentrations in birds are 1.5–2 times higher than those of mammals of similar body mass. In mammals, sustained elevations of P Glu lead to oxidative stress and free radical-mediated scavenging of endogenous vasodilators (e.g., nitric oxide), contributing to elevated blood pressure. Despite the relatively high P Glu levels in birds, they appear resistant to the development of oxidative stress in tissues such as the heart, brain and kidneys. To our knowledge no information exists on oxidative stress susceptibility in the resistance vasculature of birds. Therefore, we compared endogenous antioxidant mechanisms in the resistance vasculature of mourning doves (MODO; Zenaida macroura) and rats ( Rattus norvegicus). Reactive oxygen species (ROS) were assessed with the fluorescent indicator 7′-dichlorodihydrofluorescein diacetate, acetyl ester in mesenteric arteries from rats and wild-caught MODO. Despite having significantly higher P Glu than rats, there were no significant differences in ROS levels between mesenteric arteries from rats or doves. Although superoxide dismutase and catalase activities were lower in the plasma, total antioxidant capacity, uric acid, vitamin E (α-tocopherol), and carotenoids (lutein and zeaxanthin) were significantly higher in MODO than in rats. Thus, compared to rats, MODO have multiple circulating antioxidants that may prevent the development of oxidative stress in the vasculature.

Abstract 3363: Identification and characterization of stem-like cells in human esophageal epithelial and adenocarcinoma cells

Abstract The aim of this study was to identify and characterize stem-like cells in human normal esophageal epithelial and adenocarcinoma cell lines. The immortalized esophageal epithelial cell line, Het-1A, and the primary esophageal adenocarcinoma (EADC) cell line, OE33, were first studied by flow cytometry and immunofluorescence using a panel of antibodies to putative stem cell biomarkers which included CD34, CD44, CD44/CD24, CD117/integrin α6, Musashi I, EpiCam, and Oct 4. Fluorescence activated cell sorting (FACS) was then used to isolate 15% of cells with the highest CD44 staining (CD44high) or reactive oxygen species (ROS) (ROShigh) and 15% of cells with the lowest (including negative) CD44 staining (CD44low) or ROS (ROSlow). Each cell population was further characterized using sphere-forming assays, soft agar clonogenic assays, flow cytometry for ROS, and synchrotron mid-infrared (IR) spectromicroscopy. CD44 positivity was found in 90% of Het-1A and 75% of OE33 cells overall. For both Het-1A and OE33 cell lines, spheroid formation (mean±SD/5000 cells) was significantly higher in cells expressing high levels of CD44 (Het-1A:138.33±2.08 vs. 97.67±1.53, p&amp;lt;0.01; OE33: 150.00±31.19 vs. 100.67±7.02, p=0.056) and low levels of ROS (Het-1A: 149.33±20.43 vs. 92.00±8.89, p=0.011; OE33: 199.67±31.63 vs. 119.33±14.47, p=0.016). Colony formation (mean±SD/1000 cells) was also found to be significantly higher in CD44high (Het-1A: 15.33±4.51 vs. 8.00±1.00, p=0.051; OE33: 92.67±11.02 vs. 18.00±1.73, p&amp;lt;0.01) and ROSlow (Het-1A: 45.00±1.00 vs. 31.67±3.79, p&amp;lt;0.01; OE33: 33.00±5.29 vs. 14.67±5.03, p=0.012) cell populations. Whereas OE33 CD44high cells were found to express consistently lower levels of ROS compared with OE33 CD44low cells (p&amp;lt;0.01), no differences in ROS expression were found in Het-1A cells sorted by CD44 expression. Spectromicroscopic analysis revealed only small non-significant differences between cell populations sorted by CD44 and ROS expression. We conclude that the higher rates of spheroid formation and clonogenicity of Het-1A and OE33 cells expressing high levels of CD44 (CD44high) and low levels of ROS (ROSlow) reflect an enrichment of stem-like cell populations in esophageal cell lines. However, the significant differences in ROS levels between CD44high OE33 cells and CD44high Het-1A cells suggests unique and biologically distinct pathways in stem-like cells derived from normal and malignant esophageal epithelia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3363.

Protection of Cells in Physiological Oxygen Tensions against DNA Damage-induced Apoptosis

Oxygen availability has important effects on cell physiology. Although hyperoxic and hypoxic stresses have been well characterized, little is known about cellular functions in the oxygen levels commonly found in vivo. Here, we show that p53-dependent apoptosis in response to different DNA-damaging agents was reduced when normal and cancer cells were cultured at physiological oxygen tensions instead of the usual atmospheric levels. Different from what has been described in hypoxia, this was neither determined by decreases in p53 induction or its transactivation activity, nor by differences in the intracellular accumulation of reactive oxygen species. At these physiological oxygen levels, we found a constitutive activation of the ERK1/2 MAPK in all the models studied. Inhibition of this signaling pathway reversed the protective effect in some but not all cell lines. We conclude that a stress-independent constitutive activation of prosurvival pathways, including but probably not limited to MAPK, can protect cells in physiological oxygen tensions against genotoxic stress. Our results underscore the need of considering the impact of oxygen levels present in the tissue microenvironment when studying cell sensitivity to treatments such as chemotherapy and radiotherapy.

Analysis of Calcium Ion homeostasis and mitochondrial function in cerebellar granule cells of adult Cav 2.1 Calcium Ion channel mutant mice

CaV 2.1 voltage-gated calcium channels (VGCC) are highly expressed by cerebellar neurons, and their dysfunction is linked to human disorders including familial hemiplegic migraine, episodic ataxia type 2 and spinocerebellar ataxia type 6. Altered calcium homeostasis, due to dysfunctional Ca(V 2.1 VGCC can severely affect mitochondrial function, eventually leading to neuronal cell death. We study leaner and tottering mice, which carry autosomal recessive mutations in the gene coding for the alpha 1A pore-forming subunit of CaV 2.1 VGCC. Both leaner and tottering mice exhibit cerebellar ataxia and epilepsy. Excessive leaner cerebellar granule cell (CGC) death starts soon after postnatal day 10, but it is not known whether the degree of CGC cell death observed in adult leaner mice is significantly different from wild type mice. We used Fluoro-Jade and TUNEL staining to quantify apoptotic cell death in leaner and wild type CGC. We investigated calcium homeostasis, mitochondrial function and generation of reactive oxygen species (ROS) in isolated CGC, using indicator dyes Fura-2AM, TMRM and CMH2DCFDA, respectively. We observed a small but significant increase in number of apoptotic adult leaner CGC. Calcium homeostasis and mitochondrial function also were altered in leaner CGC. However, no significant differences in ROS levels were observed. It is possible that CGC death in leaner mice may be related to mitochondrial dysfunction but may not be directly related to decreased basal intracellular calcium.

Effect of antioxidant supplementation on leucocyte expression of reactive oxygen species in athletes

Objective. Previous studies have shown that leucocytes, in particular granulocytes, have an enormous capacity for reactive oxygen species (ROS) production, and that this can be influenced by the physical activity of the individual. Theoretically, endurance‐trained athletes could profit by increasing their intake of antioxidants, thus neutralizing increased ROS production. The aim of the present investigation was to study the effect of dietary antioxidant supplementation on leucocyte ROS expression and total plasma antioxidant status (TAS) in endurance‐trained athletes over the course of 4 weeks. Methods. Eighteen athletes were recruited for the study. A randomized, double‐blinded, placebo‐controlled crossover study of 4 weeks of antioxidant supplementation (BiO‐Antioxidant 2.1 (400 mg vitamin C and 180 mg vitamin E d−1) and BiO‐Quinon Q10 (200 mg d−1)) was performed. Flow cytometry was applied to examine the leucocyte expression of ROS using the ROS‐sensitive probes dihydroethidium and dihydrorhodamine 123. The Randox Total Antioxidant Status kit was used to measure the plasma TAS of the athletes. Results. After 4 weeks of antioxidant supplementation, we observed no significant differences in ROS levels in granulocytes and monocytes either basally or after in vitro stimulation with phorbol myristate acetate. Plasma TAS did not change significantly during the intervention period. Conclusions. We observed no influence of 4 weeks of dietary antioxidant supplementation on oxidative stress status. Based on these findings, there is no rationale advising athletes to ingest antioxidant supplements in addition to their regular diet if that includes daily recommended doses of vitamins.

Impact of clinical varicocele and testis size on seminal reactive oxygen species levels in a fertile population: a prospective controlled study

To investigate: 1) the impact of clinical varicocele on reactive oxygen species (ROS) levels in neat and washed semen in a proven fertile population; and 2) the correlation between ROS levels, testicular volume, and varicocele grade in the same population of fertile men. Prospective controlled clinical study. Andrology laboratory at tertiary-care hospital. One hundred fourteen healthy fertile men (81 normal fertile and 33 fertile with clinical varicocele) and 30 infertile patients (control subjects). Standard semen analysis and measurement of sperm ROS production. Seminal parameters, seminal ROS levels, seminal leukocyte levels, clinical varicocele, and testis size. Thirty-three of the 114 (29%) fertile men had clinical varicocele (grade 1, n = 14; grade 2, n = 11; and grade 3, n = 8), and the remaining 81 (71%) had a normal physical examination. Levels of ROS and semen quality did not differ significantly between the fertile men with or without varicocele. No significant differences in ROS levels in neat and washed semen were observed compared with fertile men with grades 2 and 3 varicocele and with fertile men with varicocele grade 1. The ROS levels in neat and washed semen were not significantly correlated with varicocele grade in fertile men. No significant correlations between ROS levels and testis volume were observed between the fertile groups. The presence of clinical varicocele in fertile men is not associated with higher seminal ROS levels or abnormal semen parameters. Levels of ROS are not correlated with varicocele grade or testis volume in the same population of fertile men.