Regulation of colony stimulating factor-1 (CSF-1) in endometrial cells: glucocorticoids and oxidative stress regulate the expression of CSF-1 and its receptor c-fms in endometrial cells.

OBJECTIVE: To evaluate the regulation and expression of CSF-1 and its receptor c-fms in endometrial cells. DESIGN: In vitro study. SETTING: Research and teaching institution. PATIENT(S): None. INTERVENTION(S): In vitro experimental study. MAIN OUTCOME MEASURE(S): The effect of glucocorticoid and oxidative stress on the expression of CSF-1 and c-fms in endometrial cells. RESULT(S): Cultured nonmalignant EM42 cells not only express CSF-1 and c-fms but are also capable of responding to exogenous CSF-1. We have also seen that glucocorticoids can regulate the expression of CSF-1/c-fms in endometrial cells. Furthermore, this study shows that oxidative stress plays a significant role in the induction of CSF-1 and its receptor c-fms. CONCLUSION(S): The results suggest that CSF-1 may promote the growth of nonmalignant endometrial cells in both an autocrine and paracrine manner and that endometrial cells under oxidative stress induce CSF-1 and c-fms.

Cholesterol depletion inhibits epidermal growth factor receptor transactivation by angiotensin II in vascular smooth muscle cells: role of cholesterol-rich microdomains and focal adhesions in angiotensin II signaling.

Angiotensin II (Ang II) induces transactivation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules in vascular smooth muscle cells (VSMCs). Cholesterol and sphingomyelin-enriched lipid rafts are plasma membrane microdomains that concentrate various signaling molecules. Caveolae are specialized lipid rafts that are organized by the cholesterol-binding protein, caveolin, and have been shown to be associated with EGF-Rs. Angiotensin II stimulation promotes a rapid movement of AT(1) receptors to caveolae; however, their functional role in angiotensin II signaling has not been elucidated. Here we show that cholesterol depletion by beta-cyclodextrin disrupts caveolae structure and concomitantly inhibits tyrosine phosphorylation of the EGF-R and subsequent activation of protein kinase B (PKB)/Akt induced by angiotensin II. Similar inhibitory effects were obtained with other cholesterol-binding agents, filipin and nystatin. In contrast, EGF-R autophosphorylation and activation of Akt/PKB in response to EGF are not affected by cholesterol depletion. The early Ang II-induced upstream signaling events responsible for transactivation of the EGF-R, such as the intracellular Ca(2+) increase and c-Src activation, also remain intact. The EGF-R initially binds caveolin, but these two proteins rapidly dissociate following angiotensin II stimulation during the time when EGF-R transactivation is observed. The activated EGF-R is localized in focal adhesions together with tyrosine-phosphorylated caveolin. These findings suggest that 1) a scaffolding role of caveolin is essential for EGF-R transactivation by angiotensin II and 2) cholesterol-rich microdomains as well as focal adhesions are important signal-organizing compartments required for the spatial and temporal organization of angiotensin II signaling in VSMCs.

Increased glycodelin levels in gynecological malignancies.

Glycodelin, an immunosuppressive protein with contraceptive properties, is synthesized by a variety of tissues and cell types. The ability of reproductive tissues to synthesize glycodelin is of major interest in pregnancy and disease conditions. We studied glycodelin levels in subjects with malignant gynecological tumors and in control subjects. Using a polyclonal glycodelin antibody against the synthetic glycodelin peptide sequence, an enzyme-linked immunosorbent assay (ELISA) was devised to measure plasma glycodelin levels. The assay detected as much as 5 ng/ml of glycodelin. There was a significant increase in plasma glycodelin levels in endometrial > ovarian > cervical cancer subjects when compared to those of controls. Strong expression of mRNA and protein were found in the ovarian and endometrial tumor tissues. Given glycodelin's immunosuppressive abilities, increased level of glycodelin may facilitate tumor growth in gynecological malignancies.

Irbesartan, an angiotensin type 1 receptor inhibitor, regulates markers of inflammation in patients with premature atherosclerosis.

OBJECTIVES: This study assessed the role of angiotensin II type 1 (AT1) receptor antagonists on inflammatory mechanisms involved in atherogenesis. Specific inflammatory markers included solubilized tumor necrosis factor-alpha receptor II (sTNF-alphaRII), vascular cell adhesion molecule-1 (VCAM-1) and superoxide. In addition, the AT1 receptor blocker irbesartan was evaluated for its ability to suppress these markers in individuals with atherosclerosis. BACKGROUND: Mechanisms involved in the complex process of atherogenesis include alterations in the inflammatory responses. The use of compounds that suppress these responses may reduce the degree of damage seen in atherosclerosis. METHODS: With a cross-sectional study design, 33 normotensive patients with stable coronary artery disease (CAD) were treated with irbesartan for a 24-week period. These patients were compared against a control population with no known coronary atherosclerosis. Marker levels were measured by enzyme-linked immunosorbent assay technique and lucigenin chemiluminescence assay and statistically evaluated by two-way repeated measures analysis of variance. RESULTS: All patients with coronary artery disease had increased levels of inflammatory molecules over those of control patients. Treatment with irbesartan in these patients significantly reduced levels of inflammatory molecules measured. Soluble VCAM-1 levels were reduced by 36%; soluble TNF-alpha levels were reduced by 54% and superoxide level decreased by 52%. Maximal suppression of inflammatory markers by irbesartan therapy in patients with CAD was seen at 12 weeks. CONCLUSIONS: The effect of irbesartan on each inflammatory marker is significant. Our results show that use of irbesartan may retard the inflammatory process seen in premature forms of atherosclerosis.

Increased myeloperoxidase and lipid peroxide-modified protein in gynecological malignancies.

Oxidative stress has been implicated in several diseases, including cancer. Oxidants induce oncogenes and their products associated with cell growth. Even though epidemiological studies implicate oxidants in promoting cancer, there is still a lack of in vivo evidence for the same. In this study, we measured the levels of myeloperoxidase (MPO), an enzyme associated with oxidation and autoantibodies to lipid peroxide-modified protein (LOOH-RSA), in the plasma of subjects with gynecological cancers. The gynecological cancer subjects (n = 201) had higher plasma MPO and LOOH-RSA levels compared with control subjects (n = 60). Immunohistochemical analysis of tissues revealed that immunostaining for MPO and LOOH-RSA was higher in cancer tissues compared with controls. The staining was specific to cell types and not ubiquitously present. Neutrophils, monocytes/macrophages, and natural killer cells have been proposed to play a role in cancer promotion and progression. This study proposes a role for oxidative stress and especially MPO in cancer.

RU486-induced growth inhibition of human endometrial cells.

OBJECTIVE: To determine the direct action of RU486 on endometrial cell proliferation and to differentiate whether the antioxidant or the antiprogesterone property of RU486 is predominately responsible for its effect on cell growth. DESIGN: In vitro study comparing the effects of RU486 (antiprogesterone and antioxidant), reduced RU486 (antioxidant), ZK112,993 (antiprogesterone), and lazaroid U74,500A (antioxidant) on endometrial cell growth. The human endometrial cell line EM42 was used in transient transfection assays to confirm the relative antiprogesterone potency of the various compounds. SETTING: Academic medical center PATIENT(S): Women presenting with pelvic pain or infertility and diagnosed with endometriosis at time of surgery or women desiring tubal ligation with a normal pelvis (controls). INTERVENTION(S): Endometrial cell cultures were treated with RU486, reduced RU486, lazaroid U74,500A, and ZK112,993. MAIN OUTCOME MEASURE(S): Tritiated thymidine incorporation was used to assess cell growth. Inhibition of progesterone induction of transiently transfected reporter plasmids was used to measure antiprogesterone activity of compounds studied. RESULT(S): RU486 reduced cell growth in a dose-dependent fashion of the endometrial cell lines EM42 and RL95-2 and of endometrial and endometriosis cells from primary culture. After being reduced, RU486 lost most of its antiprogesterone activity but retained its antiproliferative properties. ZK112,993 was similar in potency to RU486 as a progesterone antagonist but did not significantly modify endometrial cell growth. Lazaroid U74,500A was devoid of antiprogesterone activity but was shown to be a potent antiproliferative agent. CONCLUSION(S): RU486 has a direct inhibitory effect on human endometrial cell growth. This activity appears to be at least partly mediated through its antioxidant property.

Potential role of oxidized lipids and lipoproteins in antioxidant defense.

The atherogenic oxidative modification of low-density lipoprotein is suggested to occur in the aortic intima. There is reasonable evidence to suggest that antioxidants might be beneficial in preventing or retarding the progression of atherosclerosis. Exercise, estrogens, and substitution of polyunsaturated fat for saturated fat are beneficial in the prevention of atherosclerosis. Yet, paradoxically, they are capable of inducing an oxidative stress. To reconcile with this paradox, we postulate that under certain conditions an oxidative stress might be beneficial by inducing antioxidant enzymes in arterial cells. However, those with genetic deficiency in antioxidant enzymes or those who poorly respond to oxidative stress or those with overwhelming plasma oxidative stress might need additional antioxidant protection.

Autoantibodies to markers of oxidative stress are elevated in women with endometriosis.

OBJECTIVE: To measure autoantibodies that recognize oxidatively modified proteins in the sera of women with surgically proven endometriosis. DESIGN: Prospective study. SETTING: Tertiary care academic medical center. PATIENT(S): Women undergoing surgery for endometriosis or tubal ligation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Serum and peritoneal fluid autoantibody titers to malondialdehyde-modified low-density lipoprotein, oxidized low-density lipoprotein, and lipid peroxide-modified rabbit serum albumin determined by ELISA. Correlation of autoantibody titers with revised American Fertility Society staging classification, symptoms, and morphologic type of endometriosis. RESULT(S): Mean (+/-SEM) serum autoantibody titers (in optical density units) to the three antigens were as follows: [1] lipid peroxide-modified rabbit serum albumin, 0.49 +/- 0.12 units in the patients with endometriosis and 0.2 +/- 0.02 units in the controls; [2] oxidized low-density lipoprotein, 0.22 +/- 0.005 units in the patients with endometriosis and 0.18 +/- 0.006 units in the controls; and [3] malondialdehyde-modified low-density lipoprotein, 0.21 +/- 0.005 units in the patients with endometriosis and 0.16 +/- 0.003 units in the controls. There was no correlation between autoantibody titers and revised American Fertility Society stage, symptoms, or morphologic type of endometriosis. Peritoneal fluid did not contain autoantibodies to any of the three antigens. CONCLUSION(S): Autoantibodies to markers of oxidative stress were significantly increased in women with endometriosis. These findings strongly support our data demonstrating that women with endometriosis have enhanced oxidative stress.

Estradiol as an antioxidant: incompatible with its physiological concentrations and function.

Estradiol has been documented to inhibit the oxidation of low density lipoprotein (LDL). We show that physiological concentrations of estradiol do not inhibit the oxidation of LDL by copper. LDL samples isolated from a) premenopausal and postmenopausal women and from b) women at different time periods during their menstrual cycle, who differ vastly in plasma estradiol levels, were also oxidized at the same rates by copper. In contrast, LDL samples isolated from c) women who were hyperstimulated during in vitro fertilization (IVF), with estradiol concentrations above 2000 pg/ml, were resistant to oxidation by copper. However, these LDL samples were also oxidized at a higher rate by peroxidases. More importantly, subjects with high estradiol levels also showed an increase in myeloperoxidase (MPO) protein in the plasma. Based on these results, we conclude that at physiologic concentrations, it is unlikely that estradiol could act as an antioxidant. In fact, the ability of estradiol to induce MPO and become a prooxidant might instead suggest that MPO-mediated oxidative clearance of LDL from plasma by liver might favorably influence the outcome of atherosclerosis.

Oxidized low-density lipoprotein, a two-faced Janus in coronary artery disease?

The word antioxidant has become a household term, and every day we are bombarded with claims of antioxidant protection against a host of diseases. Atherosclerosis, cancer, gastric ulcers, memory loss, rheumatoid arthritis, endometriosis, pregnancy complications, hypertension, stroke, and a host of other diseases have been suggested to be induced by oxidative stress, and antioxidants have been suggested to be beneficial in the prevention and treatment of these disorders. While some of these may be exuberant claims, atherosclerosis is one disease in which the oxidation hypothesis has taken firm roots. The oxidation of low-density lipoprotein (LDL) has been suggested to be a key step in the initiation of the early atherosclerotic lesion. A number of proatherogenic effects have been described for both the protein and lipid components of oxidized low-density lipoprotein. In this commentary, a brief description of the involvement of oxidation and the potential for antioxidant treatment for cardiovascular disease will be provided. However, there are innumerable questions plaguing the hypothesis; this commentary, therefore, will also serve as a devil's advocate and propose that some form of oxidation might actually be beneficial.

Regulation of monocyte to macrophage differentiation by antiglucocorticoids and antioxidants.

OBJECTIVE: Our purpose was to examine RU 486 and related compounds on monocyte to macrophage differentiation through scavenger receptors and cellular adhesion. STUDY DESIGN: Human monocytes were isolated, cultured, and treated with dexamethasone, levonorgestrel, RU 486, and other structurally related compounds alone or in combination. Macrophage scavenger receptor activity, inhibited by glucocorticoids and associated in the current literature with macrophage cellular adhesion, was determined in this study by counting the number of adherent cells after treatment. In addition, fluorescent-labeled acetyl-low-density lipoprotein uptake was determined as a function of scavenger receptor biologic activity. RESULTS: Dexamethasone, levonorgestrel (antiglucocorticoid only) and RU 486 (antiglucocorticoid and antioxidant) all significantly decreased adherent macrophages (4%, 52%, and 74% of control). Levonorgestrel, however, demonstrated a marked uptake of fluorescent-labeled scavenger receptor ligand. RU 486 and dexamethasone were antagonistic when combined (P < .001); levonorgestrel was less antagonistic but, however, still significant (P < .05). Reduced RU 486 (antioxidant but loses antiglucocortioid activity) decreased cellular adhesion, yet scavenger receptor function was enhanced. Both probucol (extracellular mechanism of action) and probucol analog (intracellular action) markedly up-regulated scavenger function, but once again a separation of adhesion from scavenger activity was noted. Vitamin E (antioxidant) and onapristone (antioxidant and antiglucocorticoid) had virtually little to no effect on adhesion and scavenger receptor activity. Finally, pyrrolidine dithiocarbamate, a potent oxygen-free radical quencher, was toxic to all cells examined. CONCLUSIONS: RU 486 is a known antiglucocorticoid with novel antioxidant properties first demonstrated by our laboratories. Levonorgestrel has antiglucocorticoid but no antioxidant activity. RU 486 antagonized the inhibitory effect of dexamethasone on scavenger receptor development, whereas levonorgestrel was stimulatory. A separation of scavenger receptor-induced cellular adhesion and scavenger receptor internalized ligand was demonstrated by (1) reduced RU 486, which loses its antiglucocorticoid activity but retains its antioxidant activity, and (2) probucol analog, which is chemically altered to allow intracellular entry. Glucocorticoids decrease the development of scavenger receptors, whereas antioxidants regulate inflammatory cytokines by intracellular mechanisms. It is therapeutically important to up-regulate scavenger receptor activity by antiglucocorticoids in the peritoneal cavity of women with endometriosis. However, because these mechanisms also induce inflammatory cytokines, a balance of antioxidants and antiglucocorticoids such as those demonstrated in the above study may prove beneficial.

Generation and characterization of a polyclonal antipeptide antibody to human glycodelin.

OBJECTIVE: To develop and characterize an antiglycodelin antibody using a 15-amino acid synthetic peptide as antigen, derived from the sequence of human glycodelin. DESIGN: We have developed a chicken antiglycodelin-derived peptide antibody and have characterized the antibody with the use of endometrial and ovarian cell lines. The antibody was also tested for its ability to detect glycodelin by ELISA assay, immunocytochemistry, and by Western blot. SETTING: Various cell lines, cell culture medium, and amniotic fluid were used in the experiments. PATIENT(S): Amniotic fluid was collected from pregnant patients in their first trimester of pregnancy. INTERVENTION(S): No intervention. MAIN OUTCOME MEASURE(S): Detection of glycodelin. RESULT(S): The cell lines RL95-2 (human endometrial carcinoma cells), OVCAR-3 (human ovarian adenocarcinoma cells), HeLa (human cervical epitheloid carcinoma cells), and EM42-D (human endometrial epithelial cells) reacted with the antibody, indicating the presence of glycodelin. A specific 45-kd protein representing glycodelin was detected by Western blot in the amniotic fluid. CONCLUSION(S): Antipeptide antibodies can be successfully used to detect and quantify the presence of glycodelin in cells and fluids.

Endometriosis: a disease of oxidative stress?

Our central hypothesis proposes that oxidatively damaged red blood cells (RBCs), apoptotic endometrial cells or undigested endometrial tissue may signal the recruitment and activation of mononuclear phagocytes. Women with endometriosis are prone to respond to this stimulus with an inadequate macrophage scavenger receptor response although the secretory response is not impaired. Activated macrophages in the peritoneal cavity generate an oxidative stress, which consists of lipid peroxides, their degradation products, and products formed from their interaction with low-density lipoprotein (LDL) apoprotein and other proteins. The lipoproteins of the peritoneal fluid (interstitial fluid) have been shown to have lower vitamin E levels and to be more readily oxidized than plasma, so peritoneal fluid may actually contribute to the disease process actively rather than as a passive carrier of mediators of inflammation and growth. As a result of such a stress, a sterile, inflammatory reaction with secretion of growth factors, cytokines, and chemokines is generated, which is deleterious especially to successful reproduction. We propose that such a pro-oxidant environment (peritoneal fluid as well as activated macrophages) promotes growth of ectopic endometrium. The data presented in this review are just the beginning of exploring the role of oxidative stress in mediating the pathophysiology of endometriosis. Only by understanding the mechanisms involved in the pathogenesis of endometriosis can we develop the basis for new diagnostic and therapeutic approaches.

Aqueous extracts of cigarette smoke promote the oxidation of low density lipoprotein by peroxidases.

Oxidation of low density lipoprotein (LDL) by cigarette smoke has been considered a potential mechanism by which smoking may promote atherosclerosis. We report in this study that cigarette smoke extract (CSE) inhibited copper-induced oxidation of LDL suggesting the presence of antioxidants in CSE. It is currently believed that peroxidases may oxidize LDL in vivo and during such oxidations antioxidants become pro-oxidants. Accordingly, when LDL was oxidized by peroxidase in the presence of CSE there was an increase in the oxidation of LDL. This is the first study suggesting that smoking may promote atherosclerosis by enhancing peroxidase-catalyzed lipid peroxidation.

Generation of a polyclonal antibody against lipid peroxide-modified proteins.

A specific polyclonal antibody against the lipid peroxide (LOOH)-modified rabbit serum albumin (RSA) was generated in rabbits. The antibody selectively recognized the modified protein in a concentration-dependent manner and did not cross react with aldehyde-modified proteins or proteins directly oxidized with the free radical generator 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH). Oxidized low-density lipoprotein (Ox-LDL), but not native LDL, was also recognized by the antibody in a concentration-dependent manner. The antibody also cross reacted with several other proteins modified by LOOH suggesting that the antibody is directed towards a common epitope and not towards the protein sequence. Western blot analysis of normal human plasma showed that at least three different proteins are recognized by the antibody. RAW cells, preincubated with LOOH, were immunostained with the antibody and the antigenic epitopes were present intracellularly, while controls lacking in the primary antibodies failed to show immunoreactivity. Atherosclerotic arteries from cholesterol-fed monkeys and human atherosclerotic lesions were also immunostained by the antibody. The immunoreactivity was co-localized in areas rich in foam cell macrophages. These results suggest that LOOH-modified proteins present an unique antigenic epitope that may represent a primary product of interaction of LOOH with proteins.

Interaction of Interceed oxidized regenerated cellulose with macrophages: a potential mechanism by which Interceed may prevent adhesions.

OBJECTIVE: The objective of the study was to determine whether Interceed oxidized regenerated cellulose (Johnson & Johnson Medical, Arlington, Tex.), because of its polyanionic nature, may compete for the macrophage scavenger receptor. STUDY DESIGN: RAW macrophages were incubated with Interceed oxidized regenerated cellulose and known scavenger receptor ligands. The production of interleukin-1beta by mouse peritoneal macrophages was measured in the presence of Interceed cellulose. RESULTS: When macrophages were incubated with Interceed cellulose, increasing concentrations inhibited the uptake of fluorescent acetyl low-density lipoprotein. In the presence of Interceed cellulose there was a decrease in the production of interleukin-1beta by mouse macrophages. CONCLUSION: These results suggest that the interaction of Interceed oxidized regenerated cellulose with macrophages with scavenger receptors may result in a decreased secretion of matrix components, inflammatory mediators, and cellular growth factors. Thus Interceed cellulose may function as a biologic barrier in preventing adhesions.

The effect of RU 486 and related compounds on cultured macrophage differentiation and function.

OBJECTIVE: Our purpose was to examine RU 486 and related compounds on macrophage scavenger receptors and cellular adhesion. STUDY DESIGN: THP-1 cells were activated with phorbol myristate acetate and treated with dexamethasone, levonorgestrel, and RU 486 alone or in combination. Scavenger receptor activity was determined by counting adhered cells. In addition, fluorescently labeled acetyl low density lipoprotein uptake was determined. RESULTS: Both dexamethasone and RU 486 significantly decreased activated macrophages (81% and 26% of control). Levonorgestrel stimulated adherent cells in activated monocytes (130% of control). RU 486 and dexamethasone were antagonistic when combined (p < 0.001). In contrast, dexamethasone could not overcome the stimulatory effect of levonorgestrel (p < 0.001). Fluorescent studies yielded similar results. CONCLUSIONS: RU 486 is a known antiglucocorticoid with novel antioxidant properties. Levonorgestrel has antiglucocorticoid but no antioxidant activity. Glucocorticoids decrease scavenger receptors and antioxidants regulate inflammatory cytokines. RU 486 antagonized the inhibitory effect of dexamethasone on scavenger receptors, whereas levonorgestrel was stimulatory. It is therapeutically important to up-regulate scavenger receptor activity by antiglucocorticoids in the peritoneal cavity of women with endometriosis. However, because these mechanisms also induce inflammatory cytokines, a balance of antioxidants and antiglucocorticoids may prove beneficial.

Cellular cysteine generation does not contribute to the initiation of LDL oxidation.

It has been suggested that the generation of cysteine (Cys-SH) by cells may play a role in the initiation of oxidation of low density lipoprotein (LDL). Cysteine has long been considered as an antioxidant. We studied the effect of Cys-SH on the oxidation of LDL by copper. The presence of Cys-SH had a profound inhibitory effect on the formation of conjugated dienes when fresh LDL was used. However, when we used LDL samples that were subjected to pre-incubation with copper, a progressive decrease in the inhibition and an actual enhancement of oxidation by Cys-SH could be demonstrated. The oxidation of freshly prepared LDL by RAW macrophages as compared to older LDL was considerably less. The addition of Cys-SH inhibited the oxidation of LDL by cells. In contrast, the addition of cystine (Cys-S-S) enhanced the oxidation of older LDL preparations while such additions had no effect on the oxidation of freshly prepared LDL. When pre-incubated LDL was subjected to oxidation by cells an enhancement of oxidation by Cys-S-S could be noted. These results demonstrate that the role of Cys-SH generated as a result of cellular recycling of Cys-S-S in the oxidation of LDL may not relate to the initiation of oxidation reactions. However, Cys-SH may enhance the rate of oxidation of LDL that may contain peroxides.

Inhibition of macrophage-dependent low density lipoprotein oxidation by nitric-oxide donors.

We have previously shown that nitric oxide donors inhibit the oxidation of low density lipoprotein (LDL) initiated by copper ions or by azo-bis-amidinopropane (Hogg et al., 1993. FEBS Lett. 334: 170-174). In this study, the nitric oxide donors S-nitroso-N-acetylpenicillamine (SNAP), spermine NONOate, and sodium nitroprusside were tested for their ability to inhibit macrophage-dependent oxidation of LDL. SNAP and spermine NONOate inhibited macrophage-dependent oxidation of LDL in a time- and concentration-dependent manner. We propose that nitric oxide is acting as a chain-breaking antioxidant that can inhibit the progression of lipid peroxidation in cell dependent-oxidation of LDL. By this mechanism nitric oxide could be an endogenous defense against atherogenesis. In contrast, sodium nitroprusside enhanced cell-mediated oxidation of LDL by a mechanism dependent on superoxide production and transition metal ions. Sodium nitroprusside also enhanced LDL oxidation by cell culture medium alone by a similar mechanism. The use of sodium nitroprusside as a nitric oxide donor in cellular systems appears to be complicated by the release of iron leading to an enhanced oxidative stress. Thus the effects of sodium nitroprusside in such systems may be unrelated to nitric oxide release.