Sodium Nitroprusside Toxicity Research Articles

Blockade of TRPM7 Alleviates Chondrocyte Apoptosis and Articular Cartilage Damage in the Adjuvant Arthritis Rat Model Through Regulation of the Indian Hedgehog Signaling Pathway.

Articular cartilage damage with subsequent impairment of joint function is a common feature of articular diseases, in particular, rheumatoid arthritis and osteoarthritis. While articular cartilage injury mediated by chondrocyte apoptosis is a known major pathological feature of arthritis, the specific mechanisms remain unclear at present. Transient receptor potential melastatin-like seven channel (TRPM7) is reported to play an important regulatory role in apoptosis. This study focused on the effects of TRPM7 on arthritic chondrocyte injury and its underlying mechanisms of action. Sodium nitroprusside (SNP)-induced rat primary chondrocyte apoptosis and rat adjuvant arthritis (AA) were used as in vitro and in vivo models, respectively. Blockage of TRPM7 with 2-APB or specific siRNA resulted in increased chondrocyte viability and reduced toxicity of SNP. Moreover, treatment with 2-APB enhanced the Bcl-2/Bax ratio and reduced cleaved PARP and IL-6, MMP-13 and ADAMTS-5 expression in SNP-treated chondrocytes. Activation of Indian Hedgehog with purmorphamine reversed the protective effects of 2-APB on SNP-induced chondrocyte apoptosis. Blockage of TRPM7 with 2-APB relieved the clinical signs of AA in the rat model and reduced the arthritis score and paw swelling. Similar to findings in SNP-treated chondrocytes, 2-APB treatment increased the Bcl-2/Bax ratio and suppressed cleaved PARP, IL-6, MMP-13, ADAMTS-5, TRPM7, and Indian hedgehog expression in articular cartilage of AA rats. Our collective findings suggest that blockade of TRPM7 could effectively reduce chondrocyte apoptosis and articular cartilage damage in rats with adjuvant arthritis through regulation of the Indian Hedgehog signaling pathway.

Ameliorative effect of Parinari curatellifolia seed extracts on sodium nitroprusside–induced cardiovascular toxicity in rats

The pharmacological potential of Parinari curatellifolia has remained largely unexplored by the scientific community and little information is available to support the folkloric and ethnobotanical uses of the plant. This present study was carried out to investigate the ameliorative effect of P. curatellifolia seed extracts on sodium nitroprusside (SNP) toxicity on the heart and artery in rats. P. curatellifolia seed crude aqueous-methanolic extract (CMPC) as well as n-hexane (HFPC), dichloromethane (DFPC), ethyl acetate (EFPC), and n-butanolic (BFPC) fractions was prepared and screened for phytoconstituents. In the in vitro test, crude extract and fractions were assessed for antiperoxidative potential in SNP-induced lipid peroxidation (LPO) in rat heart and artery. For in vivo assessment, CMPC (400, 500, and 600 mg/kg bwt) and the fractions (5 and 6 mg/kg bwt) were co-administered with 5 mg/kg SNP to Wistar rats for 7 days. Extent of LPO in the heart and artery and serum activities of aspartate transaminase (AST) and alanine transaminase (ALT) as well as concentrations of bilirubin, creatinine, urea, and uric acid were evaluated. The result of phytochemical screening revealed the presence of alkaloids, tannins, saponins, flavonoids, terpenoids, phlobatannins, and cardiac glycosides. SNP toxicity significantly (p < 0.05) increased LPO, bilirubin, creatinine, urea, and uric acid levels as well as the activities of AST and ALT. However, the extracts reversed the effect of SNP toxicity. The study revealed the protective effect of P. curatellifolia against SNP-induced cardiovascular toxicity in rats. The study plant could serve as a good therapeutic agent to combat cardiovascular-related complications.

THE INFLUENCE OF SODIUM NITROPRUSSIDE, POTASSIUM FERROCYANIDE AND 2,4–DINITROPHENOL ON THE METABOLISM AND URIC ACID CONTENT IN DROSOPHILA MELANOGASTER

The toxicity of potassium ferrocyanide (PFC) and sodium nitroprusside (SNP) and protective effects of 2.4–dinitrophenol (DNP) under PFC or SNP treatment were tested on the Drosophila melanogaster model system. Fly larvae were bred on food supplemented with PFC or SNP at concentrations of1.0 mM and mixtures with DNP in concentrations of 0.50 and1.25 mM, either alone or in combination with1.0 mM PFC or SNP. Larval treatment with PFC or SNP resulted in higher uric acid and lower triglycerides content in adult flies, while DNP in mixtures with PFC or SNP relieved these effects. Furthermore, SNP treatment resulted in lower ALT and AST activities either alone or in mixtures with DNP. The potential mechanisms of protective DNP effects against PFC or SNP toxicity are discussed.

Sodium Nitroprusside Toxicity in a Young Infant Following Cardiac Surgery

Adverse effects associated with sodium nitroprusside (SNP) administration are rarely observed in children. Monitoring of metabolic changes appears to be the most sensitive and accurate indicator of early toxicity. We report a case of acute toxicity in a 3-month-old boy treated with high-dose SNP infusion for systemic hypertension after elective coarctectomy, who developed seizures and severe lactic acidosis. We suggest blood lactate levels and base excess levels should be carefully monitored during SNP treatment in children, in order to detect early signs of toxicity, particularly when using high infusion rates. Case Study Silvagni et al.; IJMPCR, 2(5): 122-125, 2015; Article no.IJMPCR.2015.024 123

NecroX‐5 suppresses sodium nitroprusside‐induced cardiac cell death through inhibition of JNK and caspase‐3 activation

Although sodium nitroprusside (SNP) is an effective hypotensive drug and is often used in pediatric intensive care units and to treat acute heart failure, clinical application of SNP is limited by its cardiotoxicity. NecroX-5 (NX-5) was recently developed and has the capacity to inhibit necrotic cell death. No current literature addresses whether NX-5 suppresses SNP-induced cell death or its mechanism of action. We have investigated the protective role of NX-5 against SNP-induced cell death in cardiomyocyte-like H9c2 cells. SNP treatment induced severe cell death, possibly through phosphorylation of stress-activated protein kinase/c-Jun NH₂-terminal kinase (JNK) and activation of the apoptotic signaling pathway, including downregulation of Bcl-2 and cleavage of caspase-3. However, NX-5 suppresses SNP-induced cell death through inhibition of JNK activation and suppression of both downregulation of Bcl-2 protein expression and caspase-3 cleavage. These findings will provide insights and facilitate development of antidotes to SNP toxicity in cardiac cells.

The mitochondrial uncoupler 2,4-dinitrophenol attenuates sodium nitroprusside-induced toxicity in Drosophila melanogaster: Potential involvement of free radicals

The toxicity of sodium nitroprusside (SNP) (an inducer of oxidative/nitrosative stress) and the attenuation of SNP effects by 2,4-dinitrophenol (DNP) (that induces mild uncoupling of respiration) were evaluated in the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with 1.0mM SNP, 0.5 or 1.25mM DNP, or with mixtures 1.0mM SNP plus 0.5 or 1.25mM DNP. Food supplementation with SNP decreased larval viability and pupation height whereas supplementation with DNP substantially reversed these changes. Biochemical analyses of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies emerged from control larvae and larvae fed on food supplemented with SNP, DNP, or SNP/DNP mixtures. Larval exposure to SNP lowered activities of aconitase, while the presence of DNP reduced the negative impact of SNP by raising aconitase activity back to near control levels. Larval treatment with SNP also elevated the contents of carbonyl protein, uric acid and low molecular mass thiols and produced higher activities of superoxide dismutase, glutathione S-transferase, glucose-6-phosphate dehydrogenase and thioredoxin reductase in adult flies. However, the presence of DNP in the food mixtures prevented SNP-induced changes in thioredoxin reductase and glucose-6-phosphate dehydrogenase activities, as well as uric acid and low-molecular-mass thiol content. The potential mechanisms by which DNP exerts protective effects against SNP toxicity are discussed.

The protective effects of guaraná extract (Paullinia cupana) on fibroblast NIH-3T3 cells exposed to sodium nitroprusside

The antioxidant effects of the hydro-alcoholic guaraná extract (Paullinia cupana var. sorbilis Mart.) on nitric oxide (NO) and other compounds generated from the degradation of sodium nitroprusside (SNP) in an embryonic fibroblast culture (NIH-3T3 cells) were evaluated. The guaraná bioactive compounds were initially determined by high-performance liquid chromatography: caffeine=12.240mg/g, theobromine=6.733mg/g and total catechins=4.336mg/g. Cells were exposed to 10μM SNP during a 6h period because the cells exhibited >90% mortality at this concentration. Guaraná was added to the cultures in five concentrations (0.5, 1, 5, 10 and 20mg/mL). The guaraná antioxidant effect was evaluated by viability assays, biochemical oxidation [lipid peroxidation, catalase and superoxide dismutase (SOD) activity] and genotoxicity (DNA Comet assay) analysis. Additionally, oxidative stress was evaluated by a 2,7-dihydrodichlorofluorescein diacetate fluorescence assay. Guaraná reverted the SNP toxicity mainly at lower concentrations (<5mg), which decreased cell mortality, lipid peroxidation, DNA damage and cell oxidative stress as well as increased the SOD levels. These results demonstrate that guaraná has an antioxidant effect on NO metabolism in situations with higher cellular NO levels.

Central nervous system toxicity of sodium nitroprusside in treatment of patients with aortic dissection

This study examined the sodium nitroprusside (SNP) toxicity to central nervous system (CNS) in treatment of patients with aortic dissection (AD). The medical records of 191 AD patients who were admitted to Tongji Hospital, China, from Jan. 1998 to Feb. 2009 were retrospectively analyzed. There were 140 cases of hypertension (73.3%) and 13 cases of Marfan syndrome (6.8%) among the 191 AD patients. A total of 157 patients were given SNP treatment. The toxic reactions of CNS occurred in 18 subjects (11.5%). Most of the adverse reactions occurred on the fifth day following SNP injection. SNP infusion rate was significantly higher in patients who developed CNS toxicity. It was suggested that systemic hypertension is the most common predisposing factor for AD. The combination of SNP with a β-receptor blocker is a medical therapy commonly used in patients with AD. Cyanide and thiocyanate toxicity from SNP treatment is always the consequence of prolonged drug infusion or relatively high dose administration.

SODIUM NITROPRUSSIDE TOXICITY INDrosophila melanogaster: DELAYED PUPATION, REDUCED ADULT EMERGENCE, AND INDUCED OXIDATIVE/NITROSATIVE STRESS IN ECLOSED FLIES

The toxicity of sodium nitroprusside (SNP) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with SNP at concentrations of 0.01-1.5 mM. Food supplementation with SNP caused a developmental delay in flies and reduced adult eclosion. Biochemical analyses such as levels of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies emerged from control and SNP-fed larvae. Larval exposure to SNP resulted in lower activities of aconitase and catalase in adult flies relative to the control cohort. However, larval treatment with SNP led to higher carbonyl protein content and higher activities of superoxide dismutase, glucose-6-phosphate dehydrogenase, thioredoxin reductase, and glutathione-S-transferase in flies. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by SNP. The study also suggests that the toxicity of SNP may arise not just from its direct effects, but also from its decomposition products such as nitric oxide and iron ions.

ERK activation increases nitroprusside induced apoptosis in human melanoma cells irrespective of p53 status: Role of superoxide dismutases

Constitutive ERK activation, superoxide dismutases (SOD) and p53 mutations are implicated in modulating tumor apoptotic response. We now investigated whether human melanoma survival in response to sodium nitroprusside (SNP) is modulated by: a) stable introduction of a DN- mutant p53 ; b) pharmacologically inhibiting ERK activation with UO126 ; c) addition of exogenous SOD. Nitroprusside releases nitric oxide (NO) when intact , or acts in a NO-independent manner via iron and residual cyanide after light exposure (lex-SNP) . When tested at 300 µM in 72 hour treatments by cytometric live-dead assays , intact SNP caused a 50 % lethality versus a 30 % lethality induced by lex-SNP. No protection from SNP toxicity was seen when inhibiting the PI3-kinase pathway with LY294002 or c-Jun NH(2) kinase signaling with SP600125. However , pretreatment with UO126 protected from SNP –mediated cell death including counteracting apoptosis-associated Bax expression and PARP cleavage, plus reversing loss of Cu,Zn-SOD. Moreover , addition of exogenous SOD also protected cells from SNP toxicity. In spite of the greater earlier effects of intact SNP, cells treated with single doses of either intact or lex-SNP, revealed about a 90 % mortality in longer 120 hour treatments, and these were also counteracted by UO126 or exogenous SOD. This report is the first to show that: constitutive ERK activation characteristic of cancer cells, increases a nitroprusside-induced apoptosis modulated by SOD.

Evaluation of Sodium Nitroprusside Toxicity in Pediatric Cardiac Surgical Patients

Sodium nitroprusside (SNP) is often used in postoperative pediatric cardiac surgical patients. Cyanide toxicity may occur with the use of SNP. There is a paucity of literature describing dosing parameters or physical signs and symptoms of toxicity with SNP. To determine the incidence of cyanide toxicity in postoperative pediatric cardiac surgical patients treated with SNP and identify dosing parameters and physical signs and symptoms that may predict elevated cyanide concentrations. Medical records of patients who received SNP in the pediatric cardiac intensive care unit from January 2002 through December 2002 were identified and evaluated for cyanide and thiocyanate levels, dosing, and signs and symptoms of toxicity. Patients were included if they had received SNP after cardiac surgery, were 18 years of age or less, and had at least one cyanide or thiocyanate level determined while receiving therapy. Patients were excluded if they had received sodium thiosulfate. The Mann-Whitney U test was used to determine significant differences in mean dose, duration of infusion, renal function, serum lactate, and acid-base status between groups with elevated or nonelevated levels. Logistic regression and receiver operator curve were used to determine variables associated with elevated levels. Relationships between signs and symptoms of toxicity and elevated levels were evaluated with Fisher's exact test. Cyanide concentrations were in the toxic range in 7 of 63 (11%) patients. Patients with elevated concentrations had significantly higher mean dose, cumulative dose, and acid-base excess values. Elevated cyanide levels were independently predicted by mean dose, cumulative dose, and acid-base excess values, and a dose of 1.8 microg/kg/min predicted an elevated cyanide concentration with 89% sensitivity and 88% specificity. Adverse events were not reliable predictors of elevated cyanide levels. Mean dose of SNP is the best predictor of elevated cyanide levels. Adverse events commonly associated with cyanide toxicity may not be reliable indicators of elevated cyanide concentrations.

Protective effects of lipopolysaccharide preconditioning against nitric oxide neurotoxicity

We have characterized lipopolysaccharide (LPS) preconditioning-induced neuroprotective mechanisms against nitric oxide (NO) toxicity. Pretreatment of rat cortical cultures with LPS attenuated neurotoxicity of NO donors, including sodium nitroprusside (SNP) and diethylamine NONOate (NONOate). A transiently increased expression of endothelial nitric oxide synthase (eNOS) accompanied by an increase in NO production was observed during LPS preconditioning. Application of NOS inhibitors including L-N(5)-(1-iminoethyl)-ornithine (L-NIO) and L-nitroarginine methylester (L-NAME) abolished LPS-dependent protection against SNP toxicity. The LPS effect was also blocked by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). Consistently, application of 8-bromo-cyclic GMP (8-Br-cGMP), a slowly degradable cGMP analogue capable of PKG activation, was neuroprotective. LPS preconditioning resulted in a heightened neuronal expression of Bcl-2 protein that was abolished by L-NAME and KT5823, the respective inhibitors of NOS and PKG. Together, our results reveal the signaling cascade of "LPS --> eNOS --> NO --> cGMP/PKG --> Bcl-2" that might have contributed to the LPS protective effects in cortical neurons.

Nitric oxide influences the meiotic maturation of porcine oocytes cultured in hypoxanthine‐supplemented medium

Nitric oxide (NO) plays an important role in many pathological processes. The present investigation was undertaken to ascertain the effects of NO at high level on porcine oocyte meiotic development. Cumulus-enclosed oocytes (CEOs) and denuded oocytes (DOs) were obtained from the follicles (2-6 mm diameter). Exogenous NO was provided with sodium nitroprusside (SNP). Control (0 mM), 0.1 mM, 1 mM or 10 mM SNP was introduced to the medium. Meanwhile, 0.1 mM, 1 mM and 10 mM preincubated SNP was also added to the medium respectively. The cumulus expansion, cumulus cells DNA fragmentation, oocyte meiotic maturation and degeneration were determined 44 h after incubation. SNP inhibited cumulus expansion and cumulus cells DNA fragmentation in a dose-dependent manner. Significantly fewer CEOs treated by any level of SNP resumed meiosis than control (p < 0.05) and more CEOs exposed to 0.1 mM or 1 mM SNP were arrested at GV stage. Moreover, SNP increased the percentage of CEOs at metaphase I (MI) stage but decreased the percentage of CEOs at metaphase II (MII) stage. 0.1 mM or 1 mM SNP showed no influence on the proportion of germinal vesicle breakdown of DOs and only 10 mM SNP reduced the proportion. SNP accelerated degeneration of both CEOs and DOs, especially at higher concentrations. DOs were more sensitive to SNP toxicity. Preincubated SNP showed no effect on cumulus expansion, cumulus cells DNA fragmentation, oocyte meiotic maturation or degeneration at lower concentration, but higher concentrations had effect, especially on the viability of porcine oocytes. Taken together, high level of NO inhibits porcine oocyte meiotic maturation, especially the transition from MI to MII, by mediating the functions of cumulus cells.

Chimeric Vitreoscilla hemoglobin (VHb) carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: new insight into the functional role of VHb.

Dimeric hemoglobin (VHb) from the bacterium Vitreoscilla sp. strain C1 displays 30 to 53% sequence identity with the heme-binding domain of flavohemoglobins (flavoHbs) and exhibits the presence of potential sites for the interaction with its FAD/NADH reductase partner. The intersubunit contact region of VHb indicates a small interface between two monomers of the homodimer, suggesting that the VHb dimers may dissociate easily. Gel filtration chromatography of VHb exhibited a 25 to 30% monomeric population of VHb, at a low protein concentration (0.05 mg/ml), whereas dimeric VHb remained dominant at a high protein concentration (10 mg/ml). The structural characteristics of VHb suggest that the flavoreductase can also associate and interact with VHb in a manner analogous to flavoHbs and could yield a flavo-VHb complex. To unravel the functional relevance of the VHb-reductase association, the reductase domain of flavoHb from Ralstonia eutropha (formerly Alcaligenes eutrophus) was genetically engineered to generate a VHb-reductase chimera (VHb-R). The physiological implications of VHb and VHb-R were studied in an hmp mutant of Escherichia coli, incapable of producing any flavoHb. Cellular respiration the of the hmp mutant was instantaneously inhibited in the presence of 10 microM nitric oxide (NO) but remained insensitive to NO inhibition when these cells produced VHb-R. In addition, E. coli overproducing VHb-R exhibited NO consumption activity that was two to three times slower in cells overexpressing only VHb and totally undetectable in the control cells. A purified preparation of VHb-R exhibited a three- to fourfold-higher NADH-dependent NO uptake activity than that of VHb alone. Overproduction of VHb-R in the hmp mutant of E. coli conferred relief from the toxicity of sodium nitroprusside, whereas VHb alone provided only partial benefit under similar condition, suggesting that the association of VHb with reductase improves its capability to relieve the deleterious effect of nitrosative stress. Based on these results, it has been proposed that the unique structural features of VHb may allow it to acquire two functional states in vivo, namely, a single-domain homodimer that may participate in facilitated oxygen transfer or a two-domain heterodimer in association with its partner reductase that may be involved in modulating the cellular response under different environmental conditions. Due to this inherent structural flexibility, it may perform multiple functions in the cellular metabolism of its host. Separation of the oxidoreductase domain from VHb may thus provide a physiological advantage to its host.

Sodium Nitroprusside-Induced Mitochondrial Apoptotic Events in Insulin-Secreting RINm5F Cells Are Associated with MAP Kinases Activation

Exposure of insulin-secreting RINm5F cells to the chemical nitric oxide donor sodium nitroprusside (SNP) resulted in apoptotic cell death, as detected by cytochrome c release from mitochondria and caspase 3 activation. SNP exposure also leads to phosphorylation and activation of enzymes involved in cellular response to stress such as signal-regulated kinase 2 (ERK2) and c-Jun NH2-terminal kinase 46 (JNK46). Both cytochrome c release and caspase 3 activation were abrogated in cells exposed to MEK and p38 inhibitors. Treatment of cells with the NO donors SNP, DETA–NO, GEA 5024, and SNAP resulted in phosphorylation of the antiapoptotic protein Bcl-2, which was resistant to blockade of MEK, p38, and JNK pathways and sensitive to phosphoinositide 3-kinase (PI3K) inhibition. In addition, transient transfection of cells with the wild-type PI3K γ gene mimics the increased rate of Bcl-2 phosphorylation detected in NO-treated cells. The generation of phosphoinositides seems to participate in the process since Bcl-2 phosphorylation was not observed in cells overexpressing lipid-kinase-deficient PI3Kγ. The potential of SNP toxicity directly from NO was supported by our finding that the NO scavenger carboxy-PTIO prevented cell death. We found no evidence to support the contention that oxygen radicals generated during cellular SNP metabolism mediate cell toxicity in RINm5F cells, since neither addition of catalase/superoxide dismutase nor transfection with superoxide dismutase prevented SNP-induced cell death. Thus, we propose that exposure to apoptotic concentrations of NO triggers ERK- and p38-dependent cytochrome c release, caspase 3 activation, and PI3K-dependent Bcl-2 phosphorylation.

Taurine is a weak scavenger of peroxynitrite and does not attenuate sodium nitroprusside toxicity to cells in culture.

Many studies have suggested an antioxidant role for taurine, but few studies have directly measured its free radical scavenging activity. The aim of the present study was to directly determine the action of taurine and taurine analogs to inhibit peroxynitrite-mediated oxidation of dihydrorhodamine 123 (DHR) to rhodamine. Taurine was also tested to determine if it could attenuate the toxicity of sodium nitroprusside (SNP) to neuronal cultures. Taurine at concentrations above 30 mM had a modest ability to inhibit peroxynitrite formation derived from SIN-1. Hypotaurine could inhibit peroxynitrite formation from both SIN-1 (decrease 75%) and SNP (decrease 50%) at 10 mM. Other taurine analogs (homotaurine, beta-alanine & isethionic acid) slightly potentiated DHR oxidation by SIN-1. Short-term (1-hour) treatment of PC12 cultures with either SNP (1-2mM) or taurine (20-40 mM) appeared to induce cellular proliferation. In contrast, 24-hour treatment with SNP (1 mM) induced cell death. Combination treatments with taurine and SNP appeared to interact in an additive fashion for both cell proliferation and neurotoxic actions. It appears unlikely that taurine is a major endogenous scavenger of peroxynitrite.

S-nitroso- N-acetylpenicillamine and nitroprusside induce apoptosis in a neuronal cell line by the production of different reactive molecules

CHP212 neuroblastoma cells were exposed to two different nitric oxide (NO) donors, S-nitroso- N-acetylpenicillamine and sodium nitroprusside. Apoptosis and necrosis were determined with flow cytometric analysis of annexin V binding and propodium iodide uptake. Both S-nitroso- N-acetylpenicillamine and sodium nitroprusside induced apoptosis, but with a different time dependency. Oxyhemoglobin (NO scavenger) attenuated the toxicity of S-nitroso- N-acetylpenicillamine, but had no effect on the toxicity of sodium nitroprusside. By contrast, deferoxamine (iron chelator) attenuated the toxicity of sodium nitroprusside, but had no effect on the toxicity of S-nitroso- N-acetylpenicillamine. Urate (ONOO − scavenger) did not influence the toxicity of either S-nitroso- N-acetylpenicillamine or sodium nitroprusside, but protected from SIN-1 (3-morpholinosydnonimine, ONOO − donor). It was shown that both dithiothreitol and ascorbic acid affected the toxicity of S-nitroso- N-acetylpenicillamine and sodium nitroprusside in opposite ways. In the presence of dithiothreitol, superoxide dismutase and catalase decreased the toxicity of sodium nitroprusside. In the presence of cells, but not in their absence, S-nitroso- N-acetylpenicillamine decomposed with a half-life of about 4 h as assessed by the production of nitrite and absorbance reduction at 335 nm. Sodium nitroprusside decomposed very slowly in the presence of cells as assessed by the production of ferrocyanide. It can be concluded that (1) slow and sustained release of NO from S-nitroso- N-acetylpenicillamine at the cell surface causes apoptosis in CHP212 cells, probably without the involvement of ONOO −, (2) sodium nitroprusside causes apoptosis by the production of H 2O 2 and/or iron, rather than NO, and probably has to be taken up by the cell for decomposition.

Differential induction of cell death in human glioma cell lines by sodium nitroprusside.

High grade gliomas represent very aggressive and lethal forms of human cancer, which often exhibit recurrence after surgical intervention and resistance to conventional chemotherapeutic and radiologic treatment. The clinically approved antihypertensive agent sodium nitroprusside (SNP) has been shown to induce cytotoxicity toward a number of carcinoma cell lines in vitro. Three human glioma cell lines were examined for susceptibility to the cytotoxic effects of SNP. The role of the protein kinase C (PKC)alpha gene in mediating resistance to SNP-induced killing in U343 cells was investigated using antisense oligonucleotide inhibition. Stable transfection and overexpression of the PKCalpha gene in the SNP-susceptible cell line U251 was performed to further implicate PKCalpha as a mediating factor in SNP cytotoxicity. In addition, the presence of bcl-2 protein in these cells was examined for possible correlation(s) with resistance to SNP. Exposure of U251 cells and LN-Z308 cells to 0.5 mM SNP resulted in significant cytotoxicity over a 72-hour period. U343 cells were resistant to SNP killing. U343 cells were shown to exhibit higher basal levels of PKCalpha and bcl-2 than either U251 or LN-Z308 cells. bcl-2 expression and resistance to SNP toxicity both were decreased by the introduction of PKCalpha antisense oligonucleotides into U343 cells. Conversely, enhanced PKC activity in PKCalpha-transfected U251 clones was associated with increased bcl-2 expression and greater resistance to SNP-induced toxicity relative to control transfected cells. SNP can induce cytotoxicity in glioma cells. The susceptibility of these glioma cells to nitroprusside-induced killing appears to be correlated inversely with bcl-2 and PKC activity. bcl-2 levels in these cells can be altered through modulation of PKC signaling, specifically, by induction or inhibition of PKCalpha. These in vitro results provide an interesting basis for further study into the potential use of SNP for treatment of human gliomas in patients receiving combination therapy with conventional chemotherapeutic agents that exhibit PKC inhibitory activity.

Mesencephalic neuron death induced by congeners of nitrogen monoxide is prevented by the lazaroid U-83836E

We explored the effects of congeners of nitrogen monoxide (NO) on cultured mesencephalic neurons. Sodium nitroprusside (SNP) was used as a donor of NO, the congeners of which have been found to exert either neurotoxic or neuroprotective effects depending on the surrounding redox milieu. In contrast to a previous report that suggests that the nitrosonium ion (NO+) is neuroprotective to cultured cortical neurons, we found that the nitrosonium ion reduces the survival of cultured dopamine neurons to 32% of control. There was a trend for further impairment of dopamine neuron survival, to only 7% of untreated control, when the cultures were treated with SNP plus ascorbate, i.e. when the nitric oxide radical (NO.) had presumably been formed. We also evaluated the effects of an inhibitor of lipid peroxidation, the lazaroid U-83836E, against SNP toxicity. U-83836E exerted marked neuroprotective effects in both insult models. More than twice as many dopamine neurons (75% of control) survived when the lazaroid was added to SNP-treated cultures and the survival was increased eight-fold (to 55% of control) when U-83836E was added to cultures treated with SNP plus ascorbate. We conclude that the congeners of NO released by SNP are toxic to mesencephalic neurons in vitro and that the lazaroid U-83836E significantly increases the survival of dopamine neurons in situations where congeners of NO are generated.

Interaction of ascorbic acid with the neurotoxic effects of nmda and sodium nitroprusside

We have previously shown that ascorbic acid (AA) protects cortical neurons in culture from the toxic effects of NMDA. In the present study, we examined the interactions of AA with toxicity produced by nitric oxide (NO) that is generated from the breakdown of sodium nitroprusside (SNP) and with NMDA toxicity measured 24 h later. AA enhanced SNP toxicity, but it reduced toxicity of NMDA. We propose that these data support a model wherein AA produces neuroprotection by an action at the NMDA receptor and indirectly with respect to NO. This effect occurs probably by antagonizing Ca 2+ influx starting the cascade of biochemical events that lead to the production of NO.