In vitro Cytotoxicity of the Nitric Oxide Donor,S-Nitroso-N-acetyl-peraciUamine,towards Cells from Human Oral Tissue

Abstract: The cytotoxicity of the nitric oxide donor, S-nitroso-N-acetyl-penicillamine (SNAP), towards cultured human cells from oral tissue was evaluated. The toxicity of SNAP to Smulow-Glickman gingival epithelial cells was correlated with the liberation of nitric oxide, as N-acetyl-D, L-penicilIamine, the SNAP metabolites, N-acetyl-D, L-penicillamine disulfide and nitrite, and preincubated (denilrosylated) SNAP did not affect viability. Comparing equimolar concentrations of various nitric oxide donors, cytotoxicity appeared to be inversely related to the relative stability (i.e., half-life) of the test compound; the sequence of cytotoxicity for a 4 hr exposure was S-nitrosoglutathione>>spermine NONOate> SNAP>DPTA NONOate>>DETA NONOate. Intracellular reduced glutathione (GSH) was lowered in S-G cells exposed to SNAP. Pretreatment of the cells with the GSH depleter, 1, 3-bis-(chloroethyl)-1-nitrosourea (BCNU), enhanced the toxicity of SNAP. Similar findings of enhanced sensitivity to SNAP were noted with gingival fibroblasts and periodontal ligament cells pretreated with BCNU. The toxicity of SNAP towards the gingival epithelial cells was decreased by cotreatment with the antioxidants, N-acetyl-L-cysteine, L-ascorbic acid, and (+)-catechin. Cells exposed to SNAP exhibited nuclear aberrations, including multilobed nuclei and multinucleation. SNAP-induced cell death was apparently by apoptosis, as noted by fluorescence microscopy and DNA agarose gel electrophoresis.     

In vitro cytotoxicity of glyco-S-nitrosothiols. a novel class of nitric oxide donors.

The cytotoxicities of the nitric oxide (NO) donors, S-nitroso-N-acetylpencillamine (SNAP) and three glyco-SNAPs, glucose-1-SNAP, glucose-2-SNAP, and fructose-1-SNAP, towards the human gingival epithelioid S-G cell line and three human carcinoma cell lines derived from tissues of the oral cavity were compared using the neutral red (NR) assay. In general, the glucose-SNAPs were more cytotoxic than SNAP, which, in turn, was more cytotoxic than fructose-1-SNAP. Further studies focused on the response of S-G cells to glucose-2-SNAP. The cytotoxicity of glucose-2-SNAP was attributed to NO, as glucose-2-SNAP (t1/2=20 h at 28 degrees C) aged for 4 days was nontoxic, toxicity was eliminated in the presence of hydroxocobalamin, a specific NO scavenger, and toxicity was not noted with glucose-2-AP (the parent compound used to construct glucose-2-SNAP). Exposure of cells to glucose-2-SNAP resulted in a lessening of the intracellular level of glutathione and cells pretreated with the glutathione-depleter, 1,3-bis-(chloroethyl)-1-nitrosourea, were more sensitive to a subsequent challenge with glucose-2-SNAP. Cytotoxicity of glucose-2-SNAP was lessened upon coexposure with the antioxidants, myricetin, N-acetyl-L-cysteine, and L-ascorbic acid. S-G cells exposed to glucose-2-SNAP exhibited bi- and multinucleation. Death of S-G cells exposed to glucose-2-SNAP apparently occurred by apoptosis, as demonstrated with fluorescence microscopy by the appearance of brightly stained, hypercondensed chromatin in spherical cells and of membrane blebbing and by the DNA-ladder of oligonucleosome-length fragments noted with gel electrophoresis. In comparison with other classes of NO donors the sequence of toxicity towards S-G cells was S-nitrosoglutathione>glucose-SNAPs>SNAP, sodium nitroprusside>spermine NONOate>DPTA NONOate>DETA NONOate>fructose-1-SNAP>SIN-1.     

In vitro toxicity of sodium nitroprusside to human endothelial ECV304 cells

The cytotoxicity of sodium nitroprusside (SNP) to the human endothelial cell line, ECV304, was studied. The cytotoxicity of SNP was primarily related to the liberation of nitric oxide (NO). S-nitroso-N-acetyl-d-penicillamine (SNAP), an NO donor, was highly toxic. Other degradation products of SNP either exerted much less toxicity (i.e. cyanide and nitrite) or were non-toxic (i.e. ferricyanide and ferrocyanide). SNP induced multinucleation, inhibited cell proliferation, lowered the endogenous level of reduced glutathione (GSH), and induced apoptotic cell death. The plasma membrane was not the prime site of toxic action, as leakage of lactic acid dehydrogenase (LDH) occurred only at a relatively high concentration of SNP. Cells treated with non-toxic levels of the glutathione-depleting agents, 1-chloro-2,4-dinitrobenzene (CDNB), dl-buthionine-[S,R]-sulfoximine (BSO), and 1,3-bis-(chloroethyl)-1-nitrosourea (BCNU), were hypersensitive to subsequent exposure to SNP. The GSH status of the cells was, therefore, a key factor in determining the cytotoxicity of SNP.     

The role of reduced glutathione and glutathione reductase in the cytotoxicity of chromium (VI) in osteoblasts.

It is accepted that to exert cytotoxicity and carcinogenicity chromium VI has to be reduced inside cells. The role of reduced glutathione (GSH) and glutathione reductase in the intracellular reduction of Cr VI was investigated using an immortalized rat osteoblast cell line, FFC. Alkaline phosphatase activity was the index of cytotoxicity measured. To investigate the role of GSH in Cr VI toxicity, GSH levels in the cells were elevated by pretreatment with L-cysteine, and depleted using buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. Intracellular GSH levels were not depleted during the metabolism of Cr VI. Depletion of GSH by BSO caused the cells to be more resistant to the toxicity of Cr VI, indicating that GSH is involved in reduction of the Cr VI. Inhibition of glutathione reductase by carmustine (BCNU) partially protected against the cytotoxicity of Cr VI irrespective of the intracellular GSH. The cytotoxic response was similar if cells were pretreated with BCNU plus L-cysteine, or with BCNU plus BSO, although the GSH levels were markedly different. The results indicate that glutathione reductase plays an important role in the intracellular reduction of Cr VI in osteoblasts.     

Role of copper ions and cytochrome P450 in the vasodilator actions of the nitroxyl anion generator, Angeli's salt, on rat aorta

Since copper ions catalyse the oxidation of nitroxyl anion to nitric oxide, we investigated whether this might explain the vasodilator actions of the nitroxyl generator, Angeli's salt, in rat aorta. Parallel studies were conducted with S-nitroso-N-acetyl-D,L-penicillamine (SNAP), since Cu ions catalyse the liberation of nitric oxide from this compound. Copper sulphate enhanced relaxation to Angeli's salt and SNAP but this resulted from reduced destruction of nitric oxide by superoxide rather than from enhanced generation of nitric oxide, since it was mimicked by superoxide dismutase and by the superoxide dismutase mimetic, MnCl2. Results with the selective Cu2+ chelators, neocuproine and bathocuproine disulfonate, and the Cu2+ chelators, EDTA, cuprizone and diethyldithiocarbamate, confirmed an important role for endogenous copper in mediating relaxation to SNAP but suggested only a minor role for Angeli's salt. Relaxation to Angeli's salt was, however, powerfully blocked by proadifen, suggesting an important role for cytochrome P450.     

In vitro response of human gingival epithelial S-G cells to resveratrol

WST-1 (mitochondrial dehydrogenase activities). Arrest of cell growth, due to inhibition of DNA synthesis, may explain the leveling of toxicity between day 2 and 3 for a 3-day continuous exposure to resveratrol. Irreversible damage to cell proliferation was noted in S-G cells exposed to 75-150 microM resveratrol for 2 days and then subsequently maintained for another 3 days in resveratrol-free medium. The cytotoxicity of resveratrol was neither potentiated nor ameliorated in the presence of an hepatic S9 microsomal fraction. The cytotoxicity of hydrogen peroxide to S-G cells was lessened by N-acetyl-L-cysteine and quercetin, but not by resveratrol. For nitric oxide, only N-acetyl-L-cysteine reduced toxicity. The ability of resveratrol to function as an antioxidant was, therefore, not noted under these test conditions.     

Cytotoxicity of Sanguinarine Chloride to Cultured Human Cells from Oral Tissue

Abstract: The in vitro cytotoxicity of sanguinarine chloride, a dental product used in the treatment of gingivitis and plaque, was compared using cell lines and primary cells from oral human tissues. For the established cell lines, sanguinarine chloride exhibited similar potencies to S-G gingival epithelial cells and to KB carcinoma cells, whereas HGF-1 gingival fibroblasts were more tolerant. However, a gingival primary cell culture was more sensitive to sanguinarine chloride than were the established cell lines. Detailed studies were performed with the S-G cells. The 24-hr midpoint (NR₅₀) cytotoxicity value towards the S-G cells was 7.6 uM, based on the neutral red cytotoxicity assay; vacuolization and multinucleation were noted. When exposed to sanguinarine chloride for 3 days, a lag in growth kinetics was first observed at 1.7 μM. Damage to the integrity of the plasma membrane was evident, as leakage of lactic acid dehydrogenase occurred during a 3 hr exposure to sanguinarine chloride at 0.1275 mM and greater. The cytotoxicity of sanguinarine chloride to the S-G cells was lessened in the presence of an S9 hepatic microsomal fraction from Aroclor-induced rats or by including fetal bovine serum (15%) in the exposure medium. Progressively increasing the pH from 6.0 to 7.8 enhanced the potency of sanguinarine chloride, presumably due to the enhanced uptake of the lipophilic alkanolamine form, as compared to that of the cationic iminium form.     

Nitric oxide donors prevent while the nitric oxide synthase inhibitor L-NAME increases arachidonic acid plus CYP2E1-dependent toxicity

Polyunsaturated fatty acids such as arachidonic acid (AA) play an important role in alcohol-induced liver injury. AA promotes toxicity in rat hepatocytes with high levels of cytochrome P4502E1 and in HepG2 E47 cells which express CYP2E1. Nitric oxide (NO) participates in the regulation of various cell activities as well as in cytotoxic events. NO may act as a protectant against cytotoxic stress or may enhance cytotoxicity when produced at elevated concentrations. The goal of the current study was to evaluate the effect of endogenously or exogenously produced NO on AA toxicity in liver cells with high expression of CYP2E1 and assess possible mechanisms for its actions. Pyrazole-induced rat hepatocytes or HepG2 cells expressing CYP2E1 were treated with AA in the presence or absence of an inhibitor of nitric oxide synthase L-N(G)-Nitroarginine Methylester (L-NAME) or the NO donors S-nitroso-N-acetylpenicillamine (SNAP), and (Z)-1-[-(2-aminoethyl)-N-(2-aminoethyl)]diazen-1-ium-1,2-diolate (DETA-NONO). AA decreased cell viability from 100% to 48+/-6% after treatment for 48 h. In the presence of L-NAME, viability was further lowered to 23+/-5%, while, SNAP or DETA-NONO increased viability to 66+/-8 or 71+/-6%. The L-NAME potentiated toxicity was primarily necrotic in nature. L-NAME did not affect CYP2E1 activity or CYP2E1 content. SNAP significantly lowered CYP2E1 activity but not protein. AA treatment increased lipid peroxidation and lowered GSH levels. L-NAME potentiated while SNAP prevented these changes. Thus, L-NAME increased, while NO donors decreased AA-induced oxidative stress. Antioxidants prevented the L-NAME potentiation of AA toxicity. Damage to mitochondria by AA was shown by a decline in the mitochondrial membrane potential (MMP). L-NAME potentiated this decline in MMP in association with its increase in AA-induced oxidative stress and toxicity. NO donors decreased this decline in MMP in association with their decrease in AA-induced oxidative stress and toxicity. These results indicate that NO can be hepatoprotective against CYP2E1-dependent toxicity, preventing AA-induced oxidative stress.     

Mechanisms of N-acetyl-p-benzoquinone imine cytotoxicity

N-Acetyl-p-benzoquinone imine (NAPQI), a reactive metabolite of acetaminophen, rapidly reacts at physiological pH with glutathione (GSH) forming an acetaminophen-glutathione conjugate and stoichiometric amounts of acetaminophen and glutathione disulfide (GSSG). The same reaction products are formed in isolated hepatocytes incubated with NAPQI. In hepatocytes which have been treated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) in order to inhibit glutathione reductase, the initial rise in GSSG concentration in the presence of NAPQI is maintained, whereas GSSG is rapidly reduced back to GSH in untreated hepatocytes. Oxidation by NAPQI of GSH to GSSG and the reduction of GSSG back to GSH by the NADPH-dependent glutathione reductase appear to be responsible for the rapid oxidation of NADPH that occurs in hepatocytes incubated with NAPQI in that the effect is blocked by pretreatment of cells with BCNU. When added to hepatocytes, NAPQI not only reacts with GSH but also causes a loss in protein thiol groups. The loss in protein thiols occurs more rapidly in cells pretreated with BCNU or diethylmaleate. Whereas both of these treatments enhance cytotoxicity caused by NAPQI, BCNU pretreatment has no effect on the covalent binding of [14C-ring]NAPQI to cellular proteins. Furthermore, dithiothreitol added to isolated hepatocytes after maximal covalent binding of [14C-ring]NAPQI but preceding cell death protects cells from cytotoxicity and regenerates protein thiols. Thus, the toxicity of NAPQI to isolated hepatocytes may result primarily from its oxidative effects on cellular proteins.     

Role of cyclic GMP on inhibition by nitric oxide donors of human eosinophil chemotaxis in vitro

1. This study was designed to investigate the effects of the nitric oxide (NO) donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) on N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP, 1 x 10(-7) M)-induced human eosinophil chemotaxis, cyclic guanosine-3',5'-monophosphate (cGMP) levels, protein nitration and cytotoxicity. 2. Human eosinophils were exposed to SNP, SIN-1 and SNAP (0.001-1.0 mM) for either short (10 min) or prolonged (90 min) time periods. Exposition of eosinophils with these NO donors significantly inhibited the eosinophil chemotaxis irrespective of whether cells were exposed to these agents for 10 or 90 min. No marked differences were detected among them regarding the profile of chemotaxis inhibition. 3. Exposition of eosinophils to SNP, SIN-1 and SNAP (0.001-1.0 mM) markedly elevated the cGMP levels above basal levels, but the 90-min exposition resulted in significantly higher levels compared with the 10-min protocols (5.3+/-0.6 and 2.6+/-0.2 nM 1.5 x 10(6) cells(-1), respectively). The cGMP levels achieved with SNAP were greater than SNP and SIN-1. 4. The NO donors did not induce cell toxicity in any experimental condition used. Additionally, eosinophils exposed to SNP, SIN-1 and SNAP (1.0 mM each) either for 10 or 90 min did not show any tyrosine nitration in conditions where a strong nitration of bovine serum albumin was observed. 5. Our findings show that inhibitory effects of fMLP-induced human eosinophil chemotaxis by NO donors at short or prolonged exposition time were accompanied by significant elevations of cGMP levels. However, additional elevations of cGMP levels do not change the functional profile (chemotaxis inhibition) of stimulated eosinophils.     

一氧化氮和白细胞介素—10对小鼠肺泡巨噬细胞产生肿瘤坏死因？…

目的 观察一氧化氮和ＩＬ－１０对肺泡巨噬细胞炎症反应的调节作用,方法：小鼠肺泡汇噬细胞（ＡＭ）受脂多糖（ＬＰＳ）１０ｍｇ．Ｌ＾－１刺激同时,加入一氧化氮合酶抑制剂Ｓ－硫酸甲基异硫脲（ＳＭＴ）或一氧化氮供体Ｓ－亚硝基乙酰青霉胺（ＳＮＡＰ）,ＥＬＩＳＡ法测定上清液中ＴＮＦα,ＩＬ－１β,ＩＬ－６和ＩＬ－１０浓度,结果：ＡＭ受ＬＰＳ刺激后,ＴＮＦα,ＩＬ－１β和ＩＬ－６释放峰值分别在６,１２和２４小时,     

GSH-dependent iNOS and HO-1 mediated apoptosis of human Jurkat cells induced by nickel(II)

The molecular mechanisms by which nickel compounds cause immune cytotoxicity are far from understood. Our preliminary data suggested that nickel(II) induced apoptosis in Jurkat cells by mitochondrial pathway, specifically via mitochondrial membrane potential dissipation and antiapoptotic gene bcl-2 down-regulation. The main goal of this study was to further investigate the toxicity of nickel, especially the induction of reactive oxygen species (ROS) on immune cells, which finally induced apoptosis. Nickel was found to induce glutathione (GSH) depletion in a dose- and time-dependent manner. When Jurkat cells were preincubated with antioxidant N-acetylcysteine (NAC), apoptosis was inhibited distinctly, which suggested that ROS played an initial role in nickel immune toxicity. Heme oxygenase-1 (HO-1) and Nitric oxide (NO) which may play an important role in regulatory and protective processes in cells were assayed upon nickel treatment. A significant increase in HO-1 mRNA levels was detected in nickel treated cells. We confirmed that reduction of Nitrate levels in Jurkat cells was due to down-regulation of inducible nitric oxide synthase (iNOS), not endothelial nitric oxide synthase (eNOS). Expression changes of HO-1 and iNOS were markedly blocked when Jurkat cells were preincubated with NAC, suggesting that ROS resulted in HO-1 and iNOS dysfunction in Jurkat cells. We supposed that the immune toxicity of nickel(II) was mainly due to GSH depletion and finally led to apoptosis, probably via changing the expression levels of HO-1 and iNOS in human T lymphocytes.     

Nitric Oxide Donors Enhance Rectal Absorption of Macromolecules in Rabbits

Purpose. The objective of this investigation is to evaluate the potential of nitric oxide (NO) donors as a new class of absorption enhancers which may act on intestinal epithelial cells through epithelial actions of the chemical mediator, NO. Methods. Suppositories containing NO donors and insulin were administered into the rabbit rectum. After administration of the suppository, blood samples were collected from the auricular vein. The plasma insulin and glucose concentrations were determined. Results. The NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP, 4 mg) induced a significant increase in the rate of insulin absorption from the rectum. Administration of a suppository containing SNAP without insulin affected neither the plasma insulin nor the plasma glucose concentration. Other NO donors, NOR1 and NOR4, also induced increases in the insulin absorption. The absorption enhancement effect of SNAP was inhibited by coadministration of the NO scavenger carboxy-PTIO. SNAP also enhanced FITC-dextran (MW 4,000) absorption. Little cytotoxicity of SNAP (3.0 mg/ml) as assessed in terms of the rate of lactate dehydrogenase (LDH) release from Caco-2 cells was detected for 2 h of incubation. Conclusions. These findings suggest that NO enhanced macromolecular absorption from the rectum without mucosal cell damage, and that NO donors can act as potent absorption enhancers.     

Aroclor 1254-induced cytotoxicity in catecholaminergic CATH.a cells related to the inhibition of NO production

The neuronal nitric oxide synthase (nNOS) specific inhibitor, 7-nitroindazole (7-NI), and the nitric oxide (NO) donor (S-nitroso-N-acetylpenicillarnine, SNAP) were used to study the role of NO in polychlorinated biphenyl (PCB: Aroclor 1254)-induced cytotoxicity in the immortalized dopaminergic cell line (CATH.a cells), derived from the central nervous system of mice. Treatment of the dopaminergic cells with various concentrations of Aroclor 1254 (0.5-10 microg/ml), a commercial PCB mixture, showed significant cytotoxicity as evaluated by lactate dehydrogenase (LDH) release and assessment of cell viability, depending on the concentration used. We also observed that Aroclor 1254 treatment reduced the level of nNOS expression. Furthermore, the cytotoxicity of Aroclor 1254 was augmented by 10 microM of 7-NI, which alone did not produce cytotoxicity, while it was protected by treatment with SNAP. Depending on the concentrations of Aroclor 1254 used, intracellular dopamine and dihydroxyphenylacetic acid (DOPAC) concentrations were significantly decreased. Therefore, these results suggest that PCBs have the potential for dopaminergic neurotoxicity, which may be related with the PCBs-mediated alteration of NO production originating from nNOS at least in part.     

Cytotoxicity and glutathione depletion by 1-methyl-2-nitrosoimidazole in human colon cancer cells

The biological effects of 1-methyl-2-nitrosoimidazole (INO), the 2 electron reduction product of biologically active 1-methyl-2-nitroimidazole, were examined in HT-29 human colon cancer cells by clonogenic assay and glutathione (GSH) determination. INO was very toxic towards HT-29 cells and was equally toxic under aerobic and hypoxic conditions. Cytotoxicity was highly dependent on cell concentration, decreasing as cell concentration increased. INO also resulted in an initial dose-dependent depletion of intracellular GSH which plateaued when the GSH content of INO-treated cells reached approximately 8% of control levels. As was the case for cytotoxicity, the magnitude of GSH depletion by any given INO dose was highly dependent on cell concentration, being greatest at low cell densities. Both toxicity and GSH depletion were more pronounced when cells were exposed in culture medium without the reducing agent, ascorbate. HT-29 cells preincubated with the GSH synthesis inhibitor, buthionine sulfoximine (BSO), to reduce GSH levels to approximately 10% of control levels were more sensitive to subsequent INO exposure. These data suggest that the nitroso- reduction product of 2-nitroimidazoles may be responsible for cytotoxicity and glutathione depletion associated with hypoxic exposure to 2-nitroimidazoles.     

Establishment of a human induced pluripotent stem cell derived alveolar organoid for toxicity assessment

Alveolar epithelial cells (AECs) are vulnerable to injury, which can result in epithelial hyperplasia, apoptosis, and chronic inflammation. In this study, we developed human induced pluripotent stem cell (hiPS) cell-derived AECs (iAECs) and the iAECs based organoids (AOs) for testing AEC toxicity after chemical exposure. HiPS cells were cultured for 14 days with differentiation medium corresponding to each step, and the iAECs-based AOs were maintained for another 14 days. SFTPC and AQP5 were expressed in the AOs, and mRNA levels of SOX9, NKX2.1, GATA6, HOPX, and ID2 were increased. The AOs were exposed for 24 h to nine chemical substances, and IC₅₀ values of the nine chemicals were determined using MTT assay. When the correlations between iAECs 2D culture and AOs 3D culture were calculated using Pearson's correlation coefficient r value, the nine chemicals that caused a significant decrease of cell viability in 3D culture were found to be highly correlated in 2D culture. The cytotoxicity and nitric oxide release in AO cultured with macrophages were then investigated. When AOs with macrophages were exposed to sodium chromate for 24 h, the IC₅₀ value and nitric oxide production were higher than when the AOs were exposed alone. Taken together, the AO-based 3D culture system provides a useful platform for understanding biological characteristics of AECs and modeling chemical exposures.     

Inhibition of bleomycin-induced cell death in rat alveolar macrophages and human lung epithelial cells by ambroxol

The mitochondrial permeability transition is recognized to be involved in toxic and oxidative forms of cell injury. In the present study, we investigated the effect of ambroxol against the cytotoxicity of bleomycin (BLM) by looking at the effect on the mitochondrial membrane permeability in alveolar macrophages and lung epithelial cells. Alveolar macrophages or lung epithelial cells exposed to BLM revealed the loss of cell viability and increase in caspase-3 activity. Ambroxol (10-100 microM) reduced the 75 mU/mL BLM-induced cell death and activation of caspase-3 in macrophages or epithelial cells. It reduced the condensation and fragmentation of nuclei caused by BLM in macrophages. Ambroxol alone did not significantly cause cell death. Treatment of alveolar macrophages with BLM resulted in the decrease in transmembrane potential in mitochondria, cytosolic accumulation of cytochrome c, increase in formation of reactive oxygen species (ROS) and depletion of GSH. Ambroxol (10-100 microM) inhibited the increase in mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to BLM in macrophages. Ambroxol exerted a scavenging effect on hydroxyl radicals and nitric oxide and reduced the iron-mediated formation of malondialdehyde and carbonyls in liver mitochondria. It prevented cell death due to SIN-1 in lung epithelial cells. The results demonstrate that ambroxol attenuates the BLM-induced viability loss in alveolar macrophages or lung epithelial cells. This effect may be due to inhibition of mitochondrial damage and due to the scavenging action on free radicals.     

Comparative effects of several nitric oxide donors on intracellular cyclic GMP levels in bovine chromaffin cells: correlation with nitric oxide production.

1. Sodium nitroprusside, S-nitroso-N-acetyl-D,L-penicillamine, Spermine NONOate and DEA NONOate raised cyclic GMP levels in bovine chromaffin cells in a time and concentration dependent manner with different potencies, the most potent being DEA/NO with an EC50 value of 0.38 +/- 0.02 microM. 2. Measurements of NO released from these donors revealed that DEA/NO decomposed with a half-life (t1/2) of 3.9 +/- 0.2 min. The t1/2 for SPER/NO was 37 +/- 3 min. SNAP decomposed more slowly (t1/2 = 37 +/- 4 h) and after 60 min the amount of NO produced corresponded to less than 2% of the total SNAP present. The rate of NO production from SNAP was increased by the presence of glutathione. 3. For DEA/NO and SPER/NO there was a clear correlation between nitric oxide production and cyclic GMP increases. Their threshold concentrations were 0.05 microM and maximal effective concentration between 2.5 and 5 microM. 4. For SNAP, threshold activation was seen at 1 microM, whereas full activation required a higher concentration (500-750 microM). The dose-response for SNAP increases in cyclic GMP was shifted nearly two orders of magnitude lower in the presence of glutathione. At higher concentrations an inhibition of cyclic GMP accumulation was found. This effect was not observed with either the nitric oxide-deficient SNAP analogue or other NO donors. 5. Although NO-donors are likely to be valuable for studying NO functions, their effective concentrations and the amount of NO released by them are very different and should be assessed in each system to ensure that physiological concentrations of NO are used.     

Role of oxidative stress and glutathione in busulfan toxicity in cultured murine hepatocytes

This study examines busulfan metabolism. Busulfan given in vivo or in vitro decreased hepatocyte glutathione (GSH) by 60 and 50%, respectively. In vitro, busulfan toxicity was prevented by glutathione S-transferase inhibitors or by antioxidants and led to increased production of oxidized GSH and thiobarbituric acid reactive substances. 'Rescue' from toxicity by GSH precursors was prevented by N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU). Depletion of GSH exacerbated toxicity. In GSH-depleted hepatocytes, busulfan decreased GSH by 95% and BCNU did not prevent rescue by GSH precursors. Conclusions: (1) In hepatocytes with normal GSH: busulfan toxicity requires GSH conjugation, does not cause profound GSH depletion and is mediated by oxidative stress. We postulate that a GSH conjugate promotes oxidative stress. (2) In GSH-depleted hepatocytes: busulfan profoundly depletes GSH; toxicity is mediated by oxidative stress and is prevented by restoring GSH levels; cell death may be due to unopposed endogenous oxidative stress.