Snait Tamir

THE ROLE OF NITRIC OXIDE (NO) IN THE CARCINOGENIC PROCESS

The inflammatory process has long been known to be a risk factor for human cancers, particularly of the lung, bladder, colon, stomach, and female breast. Earlier hypothesis cited production of oxygen radicals, release of cytokines, and synthesis of prostaglandins and leukotrienes as biochemical modulators of the carcinogenic process. The discovery of NO. as a product of cells in the immune system has implicated this chemical in the mechanism of carcinogenesis, particularly when NO. is overproduced over a long period of time. After briefly reviewing the important chemical reactions of NO. under physiological conditions, we examine how the chemistry of its key reactants toward biologically important molecules relate to DNA damage and cytotoxicity. In these two processes, NO may play an important role in currently accepted models of multistage carcinogenesis.

No-induced oxidative stress and glutathione metabolism in rodent and human cells

Nitric oxide (NO.), a radical species produced by many types of cells, is known to play a critical role in both regulatory processes and cell defense, yet it may also participate in collateral reactions, leading to DNA damage and cell death in both NO-generating and neighboring cells. Glutathione has been shown to protect cells from the toxic effects of free radicals and reactive oxygen species. The goal of this study was to investigate whether differences in glutathione metabolism could account for the resistance or sensitivity to cell killing by NO.. The cytotoxic effect of NO. was examined in CHO-AA8 (Chinese Hamster Ovary) cells and TK6 (human lymphoblastoid) cells pretreated with L-buthionine SR-sulfoximine (BSO), a potent inhibitor of gamma-glutamylcysteine synthetase, and with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), an irreversible inhibitor of glutathione reductase. The consequences resulting from the depletion of glutathione levels and from the arrest of oxidoreduction allowed us to show the involvement of glutathione in protecting cells from NO. and to investigate the importance of changes in glutathione metabolism on NO-induced toxicity. In CHO-AA8 cells, we found that treatment with NO. resulted in the oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG) and to mixed glutathione disulfides (GSSR). The resulting depletion of GSH stimulated its de novo synthesis, enabling the cells to resist killing by NO.. A slight difference in GSH metabolism was observed in TK6 cells. NO. led to an increase in GSSG levels similar to that observed in CHO-AA8 cells, however, a decrease in GSH levels, no change in GSSR levels, and higher levels of toxicity were also found, suggesting that NO-treated TK6 cells are not as competent in GSH homeostasis as CHO cells. We conclude that GSH is involved in protecting cells from killing by NO. and that both de novo synthesis of GSH and GSSG reduction are important in maintaining an adequate level of protection for the cells.

The effect of oxidative stress on ERα and ERβ expression

Abstract The formation of intracellular reactive oxygen and nitrogen species (ROS and RNS) has been implicated in the pathogenesis of a variety of diseases. In excess, ROS and their byproducts may cause oxidative damage and be cytotoxic to cells. Recently, it has been established that these oxidants can also act as subcellular messengers in gene regulatory and signal transduction pathways. Estrogen, on the other hand, is known to offer protection from coronary artery diseases in post-menopausal women and to be involved in various ROS-related diseases, such as Alzheimer’s and Parkinson’s diseases, diabetes and aging. The existence of estrogen receptors in these tissues lead us to investigate whether ROS can regulate their expression. We demonstrated here, for the first time, that oxidative stress induced by hydrogen peroxide (H2O2), Fe2+, 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) and activated macrophages, affect the expression of estrogen receptors α and β (ERα and ERβ) differently, demonstrating cell-specific response which can be blocked by antioxidants. This data suggest that oxidative stress and the production of ROS/RNS function as physiological regulators of ERα and ERβ expression. This may provide a new insight into the ERβ-dependent protective action of estrogen and phytoestrogens in inflammation involving diseases, and may contribute to the development of novel therapeutic treatment strategies.

DNA damage and genotoxicity by nitric oxide.

Publisher Summary This chapter discusses the DNA Damage and genotoxicity by nitric oxide (NO). The murine macrophages disclosed a novel enzymatic pathway that involved the oxidation of guanido nitrogen of L-arginine to nitric oxide. NO. undergoes subsequent oxidations and is excreted as urinary nitrate. The cell-signaling capability of NO. is related to its ability to diffuse from one cell to a neighboring cell. Although a variety of mechanisms may ultimately prove to be involved in nitric oxide-related damage to DNA, three types of reactions are focused: (1) indirect interactions with electrophiles arising from metabolism of endogenously formed N-nitrosamines, (2) direct nitrosation of primary amines on nucleic acid bases, and (3) attack by active species arising from reactions of NO with endogenous oxygen radicals. The chemistry of cellular damage by NO, although potentially complex to the overall mechanisms and the actual alterations of the nucleic acid, arise from two fundamental processes such as reaction with oxygen to form nitrosating species that may lead to deamination of DNA and the formation of N-nitroso compounds, and the reaction with superoxide to form peroxynitrite that may lead to oxidation of DNA.

The Relation Between the Chemical Structure of Flavonoids and Their Estrogen-Like Activities

Estrogens are steroid hormones exhibiting a broad range of physiological activities, which are important in the homeostatic regulation of many cellular and biochemical events. Estradiol (E(2)) is an estrogen which is active in the development of the mammary glands and the uterus, in the maintenance of pregnancy and bone density, in protection from cardiovascular diseases, and in the relief of menopausal symptoms. However, it can also stimulate malignant growths, and hence contribute to the development of estrogen-dependent tumors, such as cancer of the breast and uterus. Phytoestrogens are plant-derived chemicals with estrogen-like activities, which could have a beneficial role in humans against estrogen deficiency. Several studies relate the lower incidence of estrogen deficiency-related disease among women in the Eastern word to a diet rich in phytoestrogens. This compound comprises a variety of structurally diverse chemicals, with flavonoids as their largest group. In the present review we summarize knowledge gained on the relationship between the structure of flavonoids to their ability to mimic estrogen activity, together with the methods adapted to perform these studies. The methods reviewed are qualitative and quantitative measurements of the estrogen-like activities of phytoestrogens, superposition analysis, docking analysis and quantitative structure-activity relationship (QSAR) models. The potential of various new flavonoid derivatives in hormone replacment therapy is discussed.

Estrogen-like activity of licorice root constituents: glabridin and glabrene, in vascular tissues in vitro and in vivo.

Post-menopausal women have higher incidence of heart diseases compared to pre-menopausal women, suggesting a protective role for estrogen. The recently Womens Health Initiative (WHI) randomized controlled trial concluded that the overall heart risk exceeded benefits from use of combined estrogen and progestin as hormone replacement therapy for an average of five years among healthy postmenopausal US women. Therefore, there is an urgent need for new agents with tissue-selective activity with no deleterious effects. In the present study, we tested the effects on vascular tissues in vitro and in vivo of two natural compounds derived from licorice root: glabridin, the major isoflavan, and glabrene, an isoflavene, both demonstrated estrogen-like activities. Similar to estradiol-17beta (E2), glabridin (gla) stimulated DNA synthesis in human endothelial cells (ECV-304; E304) and had a bi-phasic effect on proliferation of human vascular smooth muscle cells (VSMC). Raloxifene inhibited gla as well as E2 activities. In animal studies, both intact females or after ovariectomy, gla similar to E2 stimulated the specific activity of creatine kinase (CK) in aorta (Ao) and in left ventricle of the heart (Lv). Glabrene (glb), on the other hand, had only the stimulatory effect on DNA synthesis in vascular cells, with no inhibition by raloxifene, suggesting a different mechanism of action. To further elucidate the mechanism of action of glb, cells were pre-incubated with glb and then exposed to either E2 or to gla; the DNA stimulation at low doses was unchanged but there was abolishment of the inhibition of VSMC cell proliferation at high doses as well as inhibition of CK stimulation by both E2 and by gla. We conclude that glb behaved differently than E2 or gla, but similarly to raloxifene, being a partial agonist/antagonist of E2. Glabridin, on the other hand, demonstrated only estrogenic activity. Therefore, we suggest the use of glb with or without E2 as a new agent for modulation of vascular injury and atherogenesis for the prevention of cardiovascular diseases in post-menopausal women.

Inhibition of serotonin re-uptake by licorice constituents

Ovarian steroid hormones, estrogen and progestin, affect the function of the serotonin neural system by inhibiting serotonin re-uptake through allosteric interaction with the serotonin transporter (SERT) in a nongenomic mechanism. Blocking or reducing serotonin re-uptake at the synapse alleviates depression. The aim of this study was to test the effect of compounds of the isoflavan and isoflavene groups, subclasses of the flavonoids family, on serotonin re-uptake and to compare the results with the effect of other known phytoestrogens like genistein and daidzein to relate the activity of these compounds to their structure. The effect of these compounds on the re-uptake of radioactive serotonin was assayed in HEK-293 cells stably expressed the recombinant human serotonin transporter (hSERT). The results demonstrated that the isoflavans glabridin and 4′-O-methylglabridin (4′-OMeG) and the isoflavene glabrene inhibited serotonin re-uptake by 60, 53 and 47%, respectively, at 50 µM, whereas resorcinol, the isoflavan 2′-O-methylglabridin (2′-OMeG), and the isoflavones genistein and daidzein were inactive. The inhibition of serotonin re-uptake is dose dependant with glabridin and estradiol. These results emphasize the importance of the lipophilic part of the isoflavans, as well as the hydroxyl at position 2′ on ring B. In conclusion, this study showed that several isoflavans are unique phytoestrogens, which like estradiol, affects the serotonergic system and inhibits serotonin re-uptake and, thus, potentially may be beneficial for mild to moderate depression in pre- and postmenopausal women.

Estrogenic activity of glabridin and glabrene from licorice roots on human osteoblasts and prepubertal rat skeletal tissues

Data from both in vivo and in vitro experiments demonstrated that glabridin and glabrene are similar to estradiol-17beta in their stimulation of the specific activity of creatine kinase, although at higher concentrations, but differ in their extent of action and interaction with other drugs. In pre-menopausal human bone cells, the response to estradiol-17beta and glabridin (at higher concentration) was higher than in post-menopausal cells; whereas, glabrene (at higher concentration) was more effective in post-menopausal cells. At both ages, the response to estradiol-17beta and glabridin was enhanced by pretreatment with the less-calcemic Vitamin D analog CB 1093 (CB) and the demonstrably non-calcemic analog JK 1624 F(2)-2 (JKF). The response to glabrene was reduced by this pretreatment. Both glabridin and glabrene stimulated creatine kinase specific activity in diaphyseal bone and epiphyseal cartilage of prepubertal female rats. Daily feeding (3-14 days) of prepubertal female rats with glabridin, estradiol-17beta or their combination, also stimulated creatine kinase specific activity. Glabridine, similarly to estradiol-17beta, also stimulated creatine kinase specific activity in ovariectomized female rats. Raloxifene, in combination with glabridin or estradiol-17beta, demonstrated the phenomenon of mutual annihilation of stimulation of creatine kinase specific activity in both epiphysis and diaphysis. Glabrene activity was not inhibited by raloxifene. Therefore, glabridin shows greater similarity to estradiol-17beta and thus greater potential, with or without Vitamin D, to modulate bone disorders in post-menopausal women.

Human atherosclerotic plaque lipid extract promotes expression of proinflammatory factors in human monocytes and macrophage-like cells

OBJECTIVE The potential of the atherogenic human carotid plaque to stimulate the inflammatory process was examined in human monocytes and macrophages, in vitro. METHODS AND RESULTS Exposure of monocytes to human carotid plaque lipid extract (LE) elevated the transcription level of the proinflammatory cytokines, interleukin (IL)-1β and tumor necrosis factor (TNF)-α, by 2.9 and 100.2 fold, respectively (as determined by real time PCR), and induced TNF-α secretion (as measured by enzyme-linked immunosorbent assay). Furthermore, LE caused an increase of 1.3-3.1 fold in the mRNA expression of the proinflammatory factors, IL-1β, IL-6, TNF-α, cyclooxygenase-2 and intercellular adhesion molecule-1, in macrophage-like cells. In order to investigate the proinflammatory components in the extract, two fractions, obtained by silica gel separation of LE, were characterized. The cholesterol-oxysterol rich fraction was found to have the most significant proinflammatory effect. It caused an increase in TNF-α expression in monocytes, and upregulated IL-6, TNF-α, intercellular adhesion molecule-1 and cyclooxygenase 2 by 1.5-2.5 fold in macrophages. The triglyceride fraction had almost no effect on the cells. CONCLUSIONS The human carotid plaque lipid extract was demonstrated to promote inflammation, in vitro. These data support the atherogenic character of the plaque and imply that its lipid composition may have ramifications on the progress of atherosclerosis.

Glabridin, a phytoestrogen from licorice root, up-regulates manganese superoxide dismutase, catalase and paraoxonase 2 under glucose stress.

The risk of death from cardiovascular diseases (CVDs), which are exacerbated by oxidative stress, is higher in diabetic women. This phenomenon has been attributed to the loss of estradiol‐vascular protection. Such knowledge led us to examine the potential of glabridin, a phytoestrogen, to substitute estradiol up‐regulation of antioxidant enzymes under high glucose conditions. Chronic glucose stress was found to down‐regulate catalase (CAT) and paraoxonase 2 (PON2) mRNA expression by 20% and 17%, respectively, and to decrease PON2 activity by 83% in macrophages. Inflammatory conditions had an additive effect on PON2 expression in a time‐dependent manner. Treatment with glabridin, under high glucose stress, increased PON2 activity by 60% and up‐regulated its mRNA expression by 3.5 fold. Furthermore, glabridin up‐regulated the expression of manganese superoxide dismutase (Mn‐SOD) and CAT in monocytes. In conclusion, glabridin has the potential of strengthening the antioxidant defense mechanism and may serve as an antiatherogenic agent in diabetes. Copyright