Liat Lomnitski

The effect of natural antioxidants, NAO and apocynin, on oxidative stress in the rat heart following LPS challenge

Oxidative damage plays a key role in septic shock induced by lipopolysaccharide (LPS) which is known to enhance the formation of reactive oxygen species (ROS). In this study, biochemical parameters indicative of oxidative stress were tested in the rat heart following LPS challenge, with and without pretreatment with the antioxidants NAO (natural antioxidant) and apocynin. NAO is a natural antioxidant isolated and purified from spinach and its main components are flavonoids and coumaric acid derivatives. Treatment with LPS alone significantly (P<0.05) increased the malondialdehyde (MDA) level in heart, both in cytosolic and mitochondrial fractions by 1.5- and 2.4-fold, respectively, and in plasma (2.66 fold). In the heart homogenate, the level of hydroperoxides also increased significantly (P<0.05). In addition, LPS treatment significantly (P<0.05) increased NADPH oxidase activity in the heart microsomal fraction by approximately 10-fold compared to control. Pretreatment for 7 days with either apocynin or NAO prior to the LPS challenge significantly (P<0.05) improved rat survival, decreased MDA levels in both fractions and decreased microsomal NADPH-oxidase activity, compared to LPS alone. Catalase (CAT) activity slightly increased at 24 h post-LPS injection in LPS group and returned to the control level in the apocynin treated group. No meaningful changes were indicated for glutathione peroxidase activity among all the treatment groups. The activities of cytosolic and mitochondrial superoxide dismutase (SOD) enzymes significantly (P<0.05) increased approximately 20% in the LPS-treated group, compared to control. Apocynin significantly (P<0.05) decreased SOD level in the mitochondrial fraction with no effect on the cytosolic fraction; whereas, NAO had no important effect on SOD level in both fractions. The beneficial pretreatment effects of the antioxidants against oxidative stress in the rat heart presented in this study may suggest a potential chemopreventive effect of this compound in sepsis prevention.

Composition, Efficacy, and Safety of Spinach Extracts

Spinach leaves, containing several active components, including flavonoids, exhibit antioxidative, antiproliferative, and antiinflammatory properties in biological systems. Spinach extracts have been demonstrated to exert numerous beneficial effects, such as chemo- and central nervous system protection and anticancer and antiaging functions. In this review article, we present a compilation of data generated in our laboratories and those of other investigators describing the chemical composition of spinach, its beneficial effects, relative safety information, and its recommended inclusion in the human diet. A powerful, water-soluble, natural antioxidant mixture (NAO), which specifically inhibits the lipoxygenase enzyme, was isolated from spinach leaves. The antioxidative activity of NAO has been compared to that of other known antioxidants and found to be superior in vitro and in vivo to that of green tea,N-acetylcysteine (NAC), butylated hydroxytoluene (BHT), and vitamin E. NAO has been tested for safety and is well tolerated in several species, such as mouse, rat, and rabbit. NAO has been found to be nonmutagenic and has shown promising anticarcinogenic effects in a few experimental models, such as skin and prostate cancer; it has not shown any target-organ toxicity or side effects. The current review provides epidemiological and preclinical data supporting the efficacy of extracts of spinach and the safety of its consumption.

Slowing tumorigenic progression in TRAMP mice and prostatic carcinoma cell lines using natural anti-oxidant from spinach, NAO--a comparative study of three anti-oxidants.

The TRAMP model and human prostatic cancer (PCA) cell lines DU145 and PC3 are useful for chemopreventive studies. We compared the efficacy of 3 anti-oxidants [a water-soluble natural anti-oxidant, NAO (200 mg/kg), found in spinach leaves; epigallocatechin-3 gallate, EGCG (200 mg/kg), a major green tea polyphenol; and N-acetylcysteine, NAC (125 mg/kg)] plus vehicle in slowing spontaneous tumorigenic progression in TRAMP and wild-type male mice. Sacrifices occurred on weeks 5, 9, and 13. Prostatic histopathology and oxidative-stress blood markers were evaluated. Hyperplasias were ranked by a combination of severity grade and distribution (focal, multifocal, and diffuse). The effectivity of each tested compound in reducing the severity/focalness of hyperplasia varied from lobe to lobe. NAO exerted a significant effect on the dorsal and lateral lobes; NAC, on the anterior and ventral lobes, and EGCG, on the ventral lobe. When the most severe hyperplasia in all 4 lobes of TRAMPs was evaluated, only NAO reduced hyperplasia at weeks 9 and 13. Plasma peroxide levels in TRAMPs were reduced following oral administration of NAO or NAC for 13 weeks; EGCG only slightly reduced these levels. In NAO-treated DU145 and PC3 PCA cells, inhibition of cellular proliferation occurred in a dose-dependent manner, increasing numbers of G1 cells and reducing ROS levels. The anti-oxidative and antiproliferative properties of NAO may explain its efficacy in slowing the spontaneous prostatic carcinogenic process in the TRAMP and its effects in the cell lines.

The interaction between β-carotene and lipoxygenase in plant and animal systems

Abstract The effect of β-carotene (BC) on the activity of lipoxygenase (LOX) from plant and animal sources has been examined. Soybean lipoxygenase L-2 activity towards linoleate was inhibited by BC by a maximum of 70% at pH 6.5, whereas L-1 activity was little affected at pH 9.0. Lineweaver-Burk plots indicated that BC inhibited LOX activity by mixed competitive/non-competitive mechanisms. Other hydrophobic compounds also inhibited LOX activity; oleic acid and retinol were competitive inhibitors whereas tocopherol acetate and 5,8,11,14-eicosatetraynoic acid (ETYA) were non-competitive inhibitors. Binding studies with L-2 LOX bound to Sepharose indicated BC-binding and inhibition with the immobilized LOX. Activity of LOX from animal sources was also inhibited by BC both towards linoleate and arachidonate.

Effects of water-soluble antioxidant from spinach, NAO, on doxorubicin-induced heart injury

Doxorubicin (DOX) produces clinically restorative responsesinnumeroushumancancers,butitscardiotoxicity has limited its usefulness. Because reactive oxygen species may affect DOX-induced antitumor activity and cardiotoxicity, weevaluated the prophylactic effect ofspinachnatural antioxidant (NAO) on DOX-induced cardiotoxicity and oxidative stress in female Balb/c mice using histological, electron microscopical and biochemical parameters. Mice were treated with NAO for 7 days prior to and/or for 6 days after DOX administration. Pretreatment with NAO (cumulative dose: 130 mg/kg) did not hinder the effectiveness of DOX. Light and electron microscopy of DOX-treated heart revealed myocardial degeneration. When administered combined before and after DOX, NAO conferred the most significantcardiacprotection.TheeffectsofNAOonthelipid peroxidation product, malondialdehyde, and on H2O2 / hydroperoxides were examined on day 6 following DOX administration; levels of both were elevated in DOX-treated mice, compared to control. Pretreatment with NAO prevented these changes. Pretreatment with NAO before DOX administration decreased catalase and increased super oxide dismutase activities compared to the DOX group. Our results suggest usage of NAO in combination with DOX as a prophylactic strategy to protect heart muscle from DOX inducedcellulardamage.

Effects of Antioxidants Apocynin and the Natural Water-Soluble Antioxidant from Spinach on Cellular Damage Induced by Lipopolysaccaride in the Rat

Oxidative damage plays a key role in septic shock induced by the endotoxin lipopolysaccaride (LPS) by enhancing the formation of reactive oxygen species such as superoxide anion radicals, peroxides, and their secondary product, malondialdehyde, especially in the liver. In this study, histopathologic changes in several organs were compared among groups of male Wistar rats that had been injected with LPS following prophylactic pretreatment with either of 2 antioxidants, a group that had been injected with LPS without pretreatment with antioxidants, an untreated control group, and groups that had been injected with either of the 2 antioxidants only. The antioxidants used were a water-soluble natural antioxidant from spinach (NAO) and the NADPH oxidase inhibitor apocynin. Hematoxylin-and-eosin-stained slides were prepared, and lesions were semiquantitatively scored. Exposure to LPS alone was associated with multifocal hepatocellular necrosis and acute inflammation, thymic and splenic lymphoid necrosis, ocular retinal hemorrhage and acute endophthalmitis, adrenal medullary vacuolation and necrosis and acute inflammation, and decreased adrenal cortical cytoplasmic vacuolation (consistent with depletion of steroidal hormone contents). Results indicated that pretreatment with both antioxidants for 8 days reduced, in some organs, the necrotic and inflammatory changes associated with the LPS challenge. These findings suggest a potential therapeutic application for these antioxidants in clinical sepsis.

The prophylactic effects of natural water-soluble antioxidant from spinach and apocynin in a rabbit model of lipopolysaccharide-induced endotoxemia

Radical-scavenging antioxidants, as part of the cellular defense system, function to inhibit the formation and propagation of free radicals and active oxygen species formation. In previous studies we demonstrated that endotoxin lipopolysaccharide (LPS) promotes oxidative stress and associated pathological changes in a rat model and that use of selected antioxidants was effective in reducing LPS-related lipid peroxidation product formation in the liver, as well as LPS-related pathological changes in different organs. In this study, several toxicological parameters (ie, clinical signs, blood chemistry, and histopathological changes) were compared among groups of male New Zealand rabbits injected with LPS following prophylactic pretreatment with either of 2 antioxidants, a group injected with LPS without pretreatment with antioxidants, groups injected with either of the 2 antioxidants only, and an untreated control group. The antioxidants used were a water-soluble natural antioxidant (NAO) from spinach and the NADPH oxidase inhibitor, apocynin. Exposure to LPS alone was associated clinically with depression, tachypnea, outer ear vasodilation, and iris congestion; biochemically with a significant increase in blood total bilirubin, transaminase activity, and glucose, total cholesterol, and triglyceride levels; macroscopically with multiple whitish areas in the liver; and histologically with hepatocellular focal necrosis and acute inflammation, thymic and splenic lymphoid necrosis and depletion, acute uveitis and hemorrhages in the ciliary processes, and decreased adrenal cortical cytoplasmic vacuolation considered consistent with depletion of steroidal hormone contents. The NAO had more effective prophylactic capacities than the apocynin. The protective effects were obvious in all investigated parameters. The results indicate the possible therapeutic efficacy of NAO in the treatment of clinical endotoxemia associated with gram-negative bacterial sepsis that is known to be associated with oxidative stress.

Topical and oral administration of the natural water-soluble antioxidant from spinach reduces the multiplicity of papillomas in the Tg.AC mouse model

The Tg.AC mouse carrying the v-Ha-ras structural gene is a useful model for the study of chemical carcinogens, especially those acting via non-genotoxic mechanisms. This study evaluated the efficacy of the non-toxic, water-soluble antioxidant from spinach, natural antioxidant (NAO), in reducing skin papilloma induction in female hemizygous Tg.AC mice treated dermally five times over 2.5 weeks with 2.5 microg 12-O-tetradecanoylphorbol-13-acetate (TPA). The TPA-only group was considered as a control; the other two groups received, additionally, NAO topically (2 mg) or orally (100 mg/kg), 5 days/week for 5 weeks. Papilloma counts made macroscopically during the clinical observations showed a significant decrease in multiplicity (P<0.01) in the NAO topically treated group. According to histological criteria, papilloma multiplicity were lower in both topical-NAO and oral-NAO groups, but significantly so only in the oral-NAO mice (P<0.01). The beneficial effect of NAO in the Tg.AC mouse is reported.

The effect of dietary vitamin E and ß-carotene on oxidation processes in the rat testis

The effects of dietary vitamin E and beta-carotene were studied on enzymes involved in arachidonic acid metabolism and other related enzymes in the rat testis. Groups of rats were fed various soybean oil-based semi purified diets. Group 1 was fed a vitamin E-supplemented diet (+E - beta); Group 2 was fed a beta-carotene-supplemented diet (-E + beta); Group 3, the control group (-E - beta) was fed a vitamin E-deficient diet; and Group 4, the standard diet group (S), was fed vitamin E plus beta-carotene-standard diet. Soybean oxidized oil was added to the three diet groups - (+E - beta), (- E + beta) and (- E - beta), whereas the diet of S group contained non-oxidized oil. After 8 weeks rats were killed, blood and testis samples were collected for biochemical determinations. Vitamin E deficiency caused significant increase in testis thiobarbituric acid value and activities of testis NADPH oxidase, testis 15-lipoxygenase and in plasma pyruvate kinase. In contrast, significant decreases were observed in activity of testis prostaglandin synthetase, compared with antioxidant-supplemented diet groups. We also found a significant increase in 15-lipoxygenase activity in (- E + beta) diet group, compared with (- E - beta) diet group. Fatty acid analysis of testis parenchyma indicated decrease in palmitate (16:0) and arachidonate (20:4(n - 6)), and increase in oleate (18:1(n-6)) linoleate (18:2(n - 6)) and linolenate (18:3(n - 3)), when compared (-E - beta) diet group with vitamin E-supplemented diet groups. The results suggest that dietary vitamin E has a role in both enzymatic and non-enzymatic peroxidation of polyunsaturated fatty acids in the testis.

Glutathione S-transferase pi expression in forestomach carcinogenesis process induced by gavage-administered 2,4-hexadienal in the F344 rat.

Abstract. 2,4-Hexadienal (2,4-Hx), an unsaturated aldehyde formed by in vivo and in vitro peroxidation of unsaturated lipid induced, in National Toxicology Program (NTP) gavage studies of F344 rats, forestomach hyperplasia in 13-week and 2-year exposures and squamous papilloma and carcinoma in 2-year studies. Hyperplasia was characterized by thickening of all layers of epithelium with particularly prominent proliferation of the basal cells. The present investigation describes the nature and potential significance of glutathione-S-transferase-Pi (GST-Pi) immunoexpression of normal forestomach epithelium, compared to that of 2,4-Hx-related basal cell hyperplasia and squamous cell papilloma and carcinoma. Paraffin-embedded forestomachs from these NTP studies were used to investigate possible correlations between the carcinogenic process and expression of GST-Pi, a physiological metabolic barrier and an inducible phase II detoxifying enzyme suggested to decrease the responsiveness of reactive oxygen species (ROS) and organic electrophilic compounds. The amount of immunopositive staining was graded on a scale of 0 (no staining) to 4 (marked staining). The simple basal epithelium of control rats showed strong immunopositivity. In cases of basal cell hyperplasia from the 13-week and 2-year studies, these cells usually expressed strong immunopositivity for GST-Pi (grade 3 to 4). In the 2-year treated animals only, occasional focal reduction (grade 0 to 2) in immunoreactivity for GST-Pi was noted. In papillomas and squamous cell carcinomas, a wide range of GST-Pi expression was observed, perhaps indicating irregularities in its induction or change in the phenotype of these cells compared to normal or hyperplastic ones. Reduced expression of GST-Pi by the foci of basal cell hyperplasia and in tumor cells may suggest changes in cellular protection from oxidative or electrophilic DNA damage; these changes may result in genetic alterations and be the precursor to clonal expansion.