Omega-3 fatty acids differentially alter the expression of detoxification enzymes and nitric oxide bioavailability in endothelial cells during IL-6 exposure

Background Atherosclerotic plaques can elaborate reactive oxygen species (ROS) that reduce nitric oxide (NO) bioavailability. Cellular detoxification enzymes including various peroxiredoxin (PRDX) and superoxide dismutase (SOD) isoforms can inactivate ROS. The omega-3 fatty acid (n3-FA) eicosapentaenoic acid (EPA) reduced cardiovascular (CV) events in high-risk patients (REDUCE-IT), a benefit not observed with mixed n3-FAs containing docosahexaenoic acid (DHA). Purpose The purpose of this study was to compare the effects of EPA and DHA on NO bioavailability and expression of detoxification enzymes in the vascular endothelium in vitro. Methods Human umbilical vein endothelial cells (HUVECs) were pretreated with EPA or DHA at equimolar levels (10 μM) for 2 h, then challenged with IL-6 at 12 ng/ml for 24 h. Proteomic analysis was performed using LC/MS to measure relative protein expression. Only significant (p<0.05) changes between treatment groups >1-fold were analyzed. Cells were stimulated with calcium ionophore to measure NO and peroxynitrite (ONOO-) release using a porphyrinic nanosensor. Results EPA, but not DHA, augmented PRDX-2 and SOD1 expression in HUVECs relative to IL-6 alone (1.2-fold and 1.6-fold, respectively, p=0.03). EPA also significantly lowered other isoforms unlike DHA. Either EPA or DHA increased thioredoxin expression by 1.5-fold (p=0.001) and 1.3-fold (p=0.02), respectively and decreased SOD2 expression by 1.5-fold (p=8.75E-11) and 1.6-fold (p=6.03E-9), respectively. IL-6 alone only increased expression of 6 detoxification enzymes by at least 1.2-fold, relative to vehicle. Unlike DHA, EPA also increased the NO to ONOO- release ratio by 36% (p<0.05) relative to IL-6 alone, without changes in NO synthase (eNOS) expression. Conclusions n3-FAs differentially influenced NO bioavailability and expression of ROS detoxification proteins, including peroxiredoxin and SOD isoforms. The net benefits of EPA on eNOS function and ROS detoxification may contribute to reduced atherothrombotic risk compared to DHA. FUNDunding Acknowledgement Type of funding sources: Private company. Main funding source(s): Amarin Pharma Inc., Elucida Research

THE ACTIVITY AND ISOFORMS OF SUPEROXIDE DISMUTASE IN TISUES OF REPRODUCTIVE ORGANS OF RATS

The aсtivity of superoxide dismutase, the content of its isoforms in testis, epididimis, and in epididimal spermatozoa of pubescent rat males were studied. The experiments were performed on adult male rats (age 5 - 6 months). After decapitation, the testes and the epididimis were taken, from which the spermatozoa were washed with 0.9 % sodium chloride solution. The total protein and activity of superoxide dismutase (SOD) were determined in the supernatant and the sperm suspension. SOD isoforms were detected after electrophoresis in 10 % polyacrylamide gel by specific staining of gel plates with nitrosine tetrazolium. The highest activity was found to be in the tissue of the testes (18.2 ± 2.59 IU/mg of protein), less on 34.6% in the epididymis and the lowest on 46.7% in epididymal sperm. Electrophoresis in 10 % polyacrylamide gel and specific staining in the tissues of testis, epididymis, and epididymal sperm of rats revealed five major SOD isoforms, which, depending on the speed of movement in PAAG, were labeled, from maximum to least mobile, such as S1, S2, S3, S4 and S5. The specifity of testicular tissue is the presence of two minor isoforms (S1a- and S1b-isoforms), which are characterized by high electrophoretic mobility and lower intensity of manifestation, and the color disappears over time, unlike other proteins of the enzyme. It was found that the SOD isoenzyme spectrum is characterized by tissue specificity and depended on the physiological and functional features of the tissue of the reproductive organs of males and spermatozoa. In the testis tissue, S2 isoform was in the largest amount– 36.7 ± 1.91%, less and almost the same content of S1-, S3- and S4-isoforms (18.0 ± 3.27, 19.3 ± 3.98 and 15.7 ± 2.15%) and the least – S5-, S1a- and S1b-isoform (6.2 ± 1.20%; 2.2 ± 0.03 and 1.9 ± 0.01%). In the epididimis tissue, the content of the S3-isoform was higher on 17.9% and S2- and S1- respectively on 7.9% and 4.9%, respectively, compared to the testis. SOD isoenzymes of epididymal spermatozoa are characterized by a high content of S2 isoforms, which is more on 11.2% than in the testis, and the content of S1 isoforms, by contrast, is lower on 10.0%. The established differences in the content and activity of individual isoforms of the enzyme indicate the existence of tissue specificity of SOD proteins, due to the functional characteristics of the studied tissues. Optimal protection of sperm from O2• - in the process of their differentiation and maturation is ensured by the activity of different SOD isoforms, confirming the established differences in the isoenzyme spectrum.

Activity of Antioxidant Enzymes in the Tumor and Adjacent Noncancerous Tissues of Non-Small-Cell Lung Cancer

Lung tissue is directly exposed to high oxygen pressure, as well as increased endogenous and exogenous oxidative stress. Reactive oxygen species (ROS) generated in these conditions play an important role in the initiation and promotion of neoplastic growth. In response to oxidative stress, the antioxidant activity increases and minimizes ROS-induced injury in experimental systems. The aim of the present study was to evaluate the activity of antioxidant enzymes, such as superoxide dismutase (SOD; isoforms: Cu/ZnSOD and MnSOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST), along with the concentration of malondialdehyde (MDA) in tumor and adjacent noncancerous tissues of two histological types of NSCLC, i.e., adenocarcinoma and squamous cell carcinoma, collected from 53 individuals with surgically resectable NSCLC. MDA concentration was similar in tumors compared with adjacent noncancerous tissues. Tumor cells had low MnSOD activity, usually low Cu/ZnSOD activity, and almost always low catalase activity compared with those of the corresponding tumor-free lung tissues. Activities of GSH-related enzymes were significantly higher in tumor tissues, irrespective of the histological type of cancer. This pattern of antioxidant enzymes activity could possibly be the way by which tumor cells protect themselves against increased oxidative stress.

Abstract 376: Prolonged Excess of Superoxide in Mouse Afferent Arterioles Causes Remodeling and Enhances Myogenic and Angiotensin II Contractions

Background: Superoxide (O 2 .- ) is associated with cardiovascular disease (CVD) and inactivating SNIPs of the EC-SOD gene increased CVD, but the mechanisms are unclear. Superoxide dismutases (SODs) have three isoforms: cytoplasmic Cu/ZnSOD (SOD1), mitochondrial MnSOD (SOD2) and extracellular Cu/ZnSOD (SOD3). Complete SOD2-/- was lethal. We tested the hypothesis that these isoforms of SOD modulate angiotensin II (Ang II), myogenic responses (MRs) and remodeling of afferent arterioles (Affs). Methods and Results: MRs were assessed from the slope of the regression of active wall tension on perfusion pressure (PP) and Ang II contractions from incubation with 10 -6 M Ang II. O 2 .- was assessed from PEG-SOD inhibitable ethidium:dihydroethidium fluorescence and remodeling from media:lumen area ratio (M:L). Results: 1. Compared to +/+, increasing PP from 40 to 80 mmHg or applying 10 -6 M Ang II in Affs increased O 2 .- more in SOD1-/- (PP, 8 ± 1% vs 5 ± 1% , P<0.05; Ang II, 29 ± 7 vs 10 ± 2%, P<0.05), in SOD2+/- (PP, 15 ± 2% vs 7 ± 1%, P<0.01; Ang II, 32 ± 5 % vs 7 ± 1%, P<0.01), and in SOD3-/- ( PP, 20 ± 4% vs 7 ± 1%, P<0.05; Ang II, 32 ± 5% vs 12 ± 3%, P<0.05). However, O 2 .- did not increase in SOD1+/- and SOD3+/-. 2. Compared to +/+, MRs (dynes.cm -1 .mmHg -1 ) were significantly increased in SOD1-/- (3.49 ± 0.30 vs 2.30 ± 0.27, P<0.05), in SOD2+/- (3.85 ± 0.48 vs 1.80 ± 0.34, P<0.05), and in SOD3-/- (3.51 ± 0.20 vs 2.43 ± 0.15, P<0.01), with no increase in SOD1+/- and SOD3+/-. 3. Compared to +/+, Ang II contractions were stronger in SOD1-/- (-68 ± 2% vs -33 ± 3%, P<0.0001), in SOD2+/- (-66 ± 6% vs -42 ± 3%, P<0.01), and in SOD3-/- (-75 ± 3% vs -34 ± 3%, P<0.0001) 4. Compared to +/+, M:L ratio of Affs were greater in SOD1-/- (4.0 ± 0.2 vs 1.8 ± 0.2, P<0.0001), in SOD2+/- (3.7 ± 0.4 vs 1.8 ± 0.4, P<0.001), and in SOD3-/- (4.5 ± 0.2 vs 1.9 ± 0.1, P<0.0001), but were unchanged in SOD1+/- and SOD3+/-. M:L ratio was strongly correlated to MR (r 2 =0.32, P<0.005) and Ang II contractions (r 2 = 0.56, P<0.005) across genotypes. 5. There were no compensatory changes in expression of other SOD isoforms. Conclusions: Lifetime increases in cytosolic, mitochondrial or extracellular O 2 .- enhanced afferent arteriolar myogenic and Ang II contractions that were closely related to vascular remodeling.

Profiling of superoxide dismutase isoenzymes in compartments of the developing bovine antral follicles

The antral follicle constitutes a complex and regulated ovarian microenvironment that influences oocyte quality. Oxidative stress is a cellular state that may play a role during folliculogenesis and oogenesis, although direct supporting evidence is currently lacking. We thus evaluated the expression of the three isoforms (SOD1, SOD2, and SOD3) of the enzymatic antioxidant superoxide dismutase in all the cellular (granulosa cells, cumulus cells, and oocytes) and extracellular (follicular fluid) compartments of the follicle. Comparisons were made in bovine ovaries across progressive stages of antral follicular development. Follicular fluid possessed increased amounts of SOD1, SOD2, and SOD3 in small antral follicles when compared with large antral follicles; concomitantly, total SOD activity was highest in follicular fluids from smaller diameter follicles. SOD1, SOD2, and SOD3 proteins were expressed in granulosa cells without any fluctuations in follicle sizes. All three SOD isoforms were present, but were distributed differently in oocytes from small, medium, or large antral follicles. Cumulus cells expressed high levels of SOD3, some SOD2, but no detectable SOD1. Our studies provide a temporal and spatial expression profile of the three SOD isoforms in the different compartments of the developing bovine antral follicles. These results lay the ground for future investigations into the potential regulation and roles of antioxidants during folliculogenesis and oogenesis.

Lipid composition of pea (Pisum sativum L.) and maize (Zea mays L.) root plasma membrane and membrane-bound peroxidase and superoxide dismutase

Plasma membrane was isolated from roots of pea and maize plants and used to analyze POD and SOD isoforms, as well as lipid composition. Among lipids, phospholipids were the main lipid class, with phosphatidylcho?line being the most abundant individual component in both pea and maize plasma membranes. Significant differences between the two plant species were found in the contents of cerebrosides, free sterols, and steryl glycosides. Most maize POD isoforms were with neutral and anionic pI values, but the opposite was observed in pea. While both anionic and cationic SOD isoforms were isolated from maize, only two anionic SOD isoforms were detected in pea.