Regulation and control of nitric oxide (NO) in macrophages: Protecting the "professional killer cell" from its own cytotoxic arsenal via MRP1 and GSTP1.

We recently demonstrated that a novel storage and transport mechanism for nitric oxide (NO) mediated by glutathione-S-transferase P1 (GSTP1) and multidrug resistance protein 1 (MRP1/ABCC1), protects M1-macrophage (M1-MØ) models from large quantities of endogenous NO. This system stores and transports NO as dinitrosyl-dithiol-iron complexes (DNICs) composed of iron, NO and glutathione (GSH). Hence, this gas with contrasting anti- and pro-tumor effects, which has been assumed to be freely diffusible, is a tightly-regulated species in M1-MØs. These control systems prevent NO cytotoxicity and may be responsible for delivering cytotoxic NO as DNICs via MRP1 from M1-MØs, to tumor cell targets.

Nitric oxide potentiates hydrogen peroxide-induced killing of Escherichia coli.

Previously, we reported that nitric oxide (NO) provides significant protection to mammalian cells from the cytotoxic effects of hydrogen peroxide (H2O2). Murine neutrophils and activated macrophages, however, produce NO, H2O2, and other reactive oxygen species to kill microorganisms, which suggests a paradox. In this study, we treated bacteria (Escherichia coli) with NO and H2O2 for 30 min and found that exposure to NO resulted in minimal toxicity, but greatly potentiated (up to 1,000-fold) H2O2-mediated killing, as evaluated by a clonogenic assay. The combination of NO/H2O2 induced DNA double strand breaks in the bacterial genome, as shown by field-inverted gel electrophoresis, and this increased DNA damage may correlate with cell killing. NO was also shown to alter cellular respiration and decrease the concentration of the antioxidant glutathione to a residual level of 15-20% in bacterial cells. The iron chelator desferrioxamine did not stop the action of NO on respiration and glutathione decrease, yet it prevented the NO/H2O2 synergistic cytotoxicity, implicating metal ions as critical participants in the NO/H2O2 cytocidal mechanism. Our results suggest a possible mechanism of modulation of H2O2-mediated toxicity, and we propose a new key role in the antimicrobial macrophagic response for NO.

DNA deaminating ability and genotoxicity of nitric oxide and its progenitors.

Nitric oxide (NO), a multifaceted bioregulatory agent and an environmental pollutant, can also cause genomic alterations. In vitro, NO deaminated deoxynucleosides, deoxynucleotides, and intact DNA at physiological pH. That similar DNA damage can also occur in vivo was tested by treating Salmonella typhimurium strain TA1535 with three NO-releasing compounds, including nitroglycerin. All proved mutagenic. Observed DNA sequence changes were greater than 99% C----T transitions in the hisG46 (CCC) target codon, consistent with a cytosine-deamination mechanism. Because exposure to endogenously and exogenously produced NO is extensive, this mechanism may contribute to the incidence of deamination-related genetic disease and cancer.

Modulation of angiogenesis by dithiolethione-modified NSAIDs and valproic acid.

BACKGROUND AND PURPOSE: Angiogenesis involves multiple signaling pathways that must be considered when developing agents to modulate pathological angiogenesis. Because both cyclooxygenase inhibitors and dithioles have demonstrated anti-angiogenic properties, we investigated the activities of a new class of anti-inflammatory drugs containing dithiolethione moieties (S-NSAIDs) and S-valproate. EXPERIMENTAL APPROACH: Anti-angiogenic activities of S-NSAIDS, S-valproate, and the respective parent compounds were assessed using umbilical vein endothelial cells, muscle and tumor tissue explant angiogenesis assays, and developmental angiogenesis in Fli:EGFP transgenic zebrafish embryos. KEY RESULTS: Dithiolethione derivatives of diclofenac, valproate, and sulindac inhibited endothelial cell proliferation and induced Ser(78) phosphorylation of hsp27, a known molecular target of anti-angiogenic signaling. The parent drugs lacked this activity, but dithiolethiones were active at comparable concentrations. Although dithiolethiones can potentially release hydrogen sulphide, NaSH did not reproduce some activities of the S-NSAIDs, indicating that the dithioles regulate angiogenesis through mechanisms other than release of H(2)S. In contrast to the parent drugs, S-NSAIDs, S-valproate, NaSH, and dithiolethiones were potent inhibitors of angiogenic responses in muscle and HT29 tumor explants assessed by 3-dimensional collagen matrix assays. Dithiolethiones and valproic acid were also potent inhibitors of developmental angiogenesis in zebrafish embryos, but the S-NSAIDs, remarkably, lacked this activity. CONCLUSIONS AND IMPLICATION: S-NSAIDs and S-valproate have potent anti-angiogenic activities mediated by their dithiole moieties. The novel properties of S-NSAIDs and S-valproate to inhibit pathological versus developmental angiogenesis suggest that these agents may have a role in cancer treatment.

Hypoxic mammalian cell radiosensitization by nitric oxide.

The bioregulatory molecule, nitric oxide (NO), was evaluated as a hypoxic cell radiosensitizer. Authentic NO gas was nearly as effective as oxygen in radiosensitizing hypoxic Chinese hamster V79 lung cells as evaluated using clonogenic assays. When NO was delivered to hypoxic Chinese hamster V79 cells using the NO-releasing agent (C2H5)2N[N(O)-NO]- Na+, radiosensitization was also observed with a sensitizer enhancement ratio of 2.4 (1 mM (C2H5)2N[N(O)NO]-Na+). Aerobic radiosensitivity was not affected at this concentration. The hypoxic cell radiosensitization properties of (C2H5)2N[N(O)NO]-Na+, coupled with the vasodilatory effects of NO on tumor vasculature, suggest that such agents open a new avenue of research in radiation oncology.

In vivo radiation protection by nitric oxide modulation.

Drugs that affect blood flow have been shown to be whole body radiation protectors. Using NG-nitro-L-arginine, a specific inhibitor of nitric oxide synthase, and the NO-releasing agent (C2H5)2N[N(O)NO-]Na+ (DEA/NO), we have studied the ability of NO to modulate whole body radiation toxicity in C3H mice. NG-Nitro-L-arginine given to mice between 15 and 60 min prior to radiation afforded significant protection from whole body irradiation, e.g., the estimated whole body irradiation dose required to kill 50% of mice by 30 days after radiation (LD50/30) in mice treated with NG-nitro-L-arginine 60 min before irradiation was 1051 cGy compared with a whole body radiation LD50/30 of 822 cGy in control mice (P < 0.00001). Treatment of mice with DEA/NO prior to whole body irradiation also significantly reduced toxicity; the estimated whole body radiation LD50/30 was 1063 and 945 cGy in mice treated with DEA/NO 10 or 30 min before irradiation, respectively (P < 0.00001 for radiation LD50/30 of either DEA/NO-treated group compared with control). Measurement of [14C]etanidazole binding to bone marrow demonstrated that DEA/NO and NG-nitro-L-arginine exacerbated bone marrow hypoxia. Perturbations of NO levels have profound effects on in vivo radiosensitivity of normal tissues. We hypothesize that alterations in regional blood flow may underlie the changes in radiosensitivity that we have observed.

Regulation of transforming growth factor beta1 by nitric oxide.

Many tumor cells or their secreted products suppress the function of tumor-infiltrating macrophages. Tumor cells often produce abundant transforming growth factor beta1 (TGF-beta1), which in addition to other immunosuppressive actions suppresses the inducible isoform of NO synthase. TGF-beta1 is secreted in a latent form, which consists of TGF-beta1 noncovalently associated with latency-associated peptide (LAP) and which can be activated efficiently by exposure to reactive oxygen species. Coculture of the human lung adenocarcinoma cell line A549 and ANA-1 macrophages activated with IFN-gamma plus lipopolysaccharide resulted in increased synthesis and activation of latent TGF-beta1 protein by both A549 and ANA-1 cells, whereas unstimulated cultures of either cell type alone expressed only latent TGF-beta1. We investigated whether exposure of tumor cells to NO influences the production, activation, or activity of TGF-beta1.A549 human lung adenocarcinoma cells exposed to the chemical NO donor diethylamine-NONOate showed increased immunoreactivity of cell-associated latent and active TGF-beta1 in a time- and dose-dependent fashion at 24-48 h after treatment. Exposure of latent TGF-beta1 to solution sources of NO neither led to recombinant latent TGF-beta1 activation nor modified recombinant TGF-beta1 activity. A novel mechanism was observed, however: treatment of recombinant LAP with NO resulted in its nitrosylation and interfered with its ability to neutralize active TGF-beta1. These results provide the first evidence that nitrosative stress influences the regulation of TGF-beta1 and raise the possibility that NO production may augment TGF-beta1 activity by modifying a naturally occurring neutralizing peptide.

Direct measurement of the accumulation and mitochondrial conversion of nitric oxide within Chinese hamster ovary cells using an intracellular electron paramagnetic resonance technique.

We have developed an electron paramagnetic resonance (EPR) method for the nondestructive detection and quantification of intracellular NO in real time. Based upon this technique, we have obtained evidence for the metabolism of this bioregulatory molecule by mitochondria. Line-broadening of the EPR signal of a coal derivative, fusinite, was calibrated as a function of NO concentration in aqueous solution. The methodology was validated using two compounds which release NO in a controlled and predictable manner with first-order rate constants k1 = 5.0 x 0.10(-3) s-1 and k'1 = 3.4 x 10(-4) s-1 (35 degrees C). Fusinite was internalized in Chinese hamster ovary cells (CHO) by phagocytosis, after which the cells were allowed to consume the available O2, producing an hypoxic environment. The NO released from one of the NO donors, added to the culture fluid at an initial concentration of 50 microM, was directly measured in the intracellular environment as line-broadening of the fusinite EPR signal. The linewidth diminished with time, indicating that NO was being converted to a non-paramagnetic species by the cells with an apparent zero-order rate constant of 5 x 10(8) NO molecules cell-1 min-1 (20 degrees C). Addition of cyanide to the culture medium (5 mM final concentration) inhibited this disappearance of NO. NO also was converted in the presence of isolated mitochondria in the absence of oxygen. These observations suggest that under hypoxic conditions, there exists in CHO cells a metabolic pathway for the conversion of NO to diamagnetic species, which involves interactions with mitochondria.

Characterization of nitric oxide-stimulated ADP-ribosylation of various proteins from the mouse macrophage cell line ANA-1 using sodium nitroprusside and the novel nitric oxide-donating compound diethylamine dinitric oxide.

We examined the ability of nitric oxide (NO) to stimulate the ADP-ribosylation of proteins from the mouse macrophage cell line ANA-1. To demonstrate a specific effect of NO, we used a novel compound named diethylamine dinitric oxide (DEA/NO; 1,1-diethyl-2-hydroxy-2-nitrosohydrazine, sodium salt; [Et2NN(O)NO]Na), which releases NO in aqueous solution at neutral pH. DEA/NO stimulated the ADP-ribosylation of at least three cytosolic proteins (M(r) = 28,000, 33,000 and 39,000) from ANA-1 macrophages. The effect of DEA/NO on the ADP-ribosylation of the predominant target p39 was dose dependent (EC50 = 80 microM). Moreover, the effect of DEA/NO was attributed specifically to released NO rather than diethylamine or nitrite. Sodium nitroprusside (SNP) also stimulated the ADP-ribosylation of cytosolic proteins from ANA-1 mouse macrophages. However, SNP exhibited different time- and dose-dependent effects on the modification of p39. NO synthesized via the activity of interferon-gamma plus lipopolysaccharide-induced NO synthase also enhanced the ADP-ribosylation of p39, confirming that the effects of DEA/NO and SNP could be attributed to NO or reactive nitrogen oxide species. Neither pertussis toxin nor cholera toxin stimulated the ADP-ribosylation of p39; however, cholera toxin stimulated the ADP-ribosylation of proteins with approximate molecular weight of 28,000 and 33,000. These data suggest that the induced expression of NO synthase in tumoricidal macrophages may be associated with autocrine and paracrine effects of NO that include the ADP-ribosylation of various proteins. Moreover, these results indicate that DEA/NO and related compounds may be useful as pharmacologic tools for investigating the effects of NO and reactive nitrogen oxide species on macrophages.

Ionizing radiation potentiates the induction of nitric oxide synthase by IFN-gamma and/or LPS in murine macrophage cell lines: role of TNF-alpha.

Macrophages are activated to become cytotoxic by a highly coordinated set of cytokine signals. Ionizing radiation can mimic cytokine signals and lead to enhanced states of activation. We tested the ability of gamma-radiation, alone and with interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS), to induce nitric oxide (NO) production in J774.1 and RAW264.7 murine macrophages. NO was induced weakly, moderately, or strongly by IFN-gamma alone, LPS alone, or IFN-gamma + LPS, respectively. Radiation alone (0.5-50 Gy) did not induce NO, but enhanced NO production in a dose-dependent manner (0.5-5 Gy) when cells were exposed to IFN-gamma or LPS 24 h post-irradiation. Immunoblots showed parallel induction of nitric oxide synthase (NOS2). Application of anti-tumor necrosis factor alpha (TNF-alpha) antibody before irradiation blocked induction of NO by IFN-gamma. We conclude (1) that irradiated cells produce more NO in response to either IFN-gamma or LPS and (2) that the increase is mediated by induction of TNF-alpha.

Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide.

There has been confusion as to what role(s) nitric oxide (NO) has in different physiological and pathophysiological mechanisms. Some studies imply that NO has cytotoxic properties and is the genesis of numerous diseases and degenerative states, whereas other reports suggest that NO prevents injurious conditions from developing and promotes events which return tissue to homeostasis. The primary determinant(s) of how NO affects biological systems centers on its chemistry. The chemistry of NO in biological systems is extensive and complex. To simplify this discussion, we have formulated the "chemical biology of NO" to describe the pertinent chemical reactions under specific biological conditions. The chemical biology of NO is divided into two major categories, direct and indirect. Direct effects are defined as those reactions fast enough to occur between NO and specific biological molecules. Indirect effects do not involve NO, but rather are mediated by reactive nitrogen oxide species (RNOS) formed from the reaction of NO either with oxygen or superoxide. RNOS formed from NO can mediate either nitrosative or oxidative stress. This report discusses various aspects of the chemical biology of NO relating to biological molecules such as guanylate cyclase, cytochrome P450, nitric oxide synthase, catalase, and DNA and explores the potential roles of NO in different biological events. Also, the implications of different chemical reactions of NO with cellular processes such as mitochondrial respiration, metal homeostasis, and lipid metabolism are discussed. Finally, a discussion of the chemical biology of NO in different cytotoxic mechanisms is presented.

Autoxidation kinetics of aqueous nitric oxide.

Reports on the kinetics of the autoxidation of aqueous nitric oxide are discussed. It is concluded that the correct rate law is -d[NO]/dt = 4kaq[NO]2[O2] with kaq = 2 x 10(6) M-2.s-1 at 25 degrees c and that a recent report of a rate law zero order in NO is incorrect.

Electron-paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron cannot discriminate between nitric oxide and nitroxyl: implications for the detection of reaction products for nitric oxide synthase.

Purified neuronal nitric oxide synthase (NOS) does not produce nitric oxide (NO) unless high concentrations of superoxide dismutase (SOD) are added, suggesting that nitroxyl (NO(-)) or a related molecule is the principal reaction product of NOS, which is SOD-dependently converted to NO. This hypothesis was questioned by experiments using electron paramagnetic resonance spectroscopy and iron N-methyl-D-glucamine dithiocarbamate (Fe-MGD) as a trap for NO. Although NOS and the NO donor S-nitroso-N-acetyl-penicillamine produced an electron paramagnetic resonance signal, the NO(-) donor, Angeli's salt (AS) did not. AS is a labile compound that rapidly hydrolyzes to nitrite, and important positive control experiments showing that AS was intact were lacking. On reinvestigating this crucial experiment, we find identical MGD(2)-Fe-NO complexes both from S-nitroso-N-acetyl-penicillamine and AS but not from nitrite. Moreover, the yield of MGD(2)-Fe-NO complex from AS was stoichiometric even in the absence of SOD. Thus, MGD(2)-Fe directly detects NO(-), and any conclusions drawn from MGD(2)-Fe-NO complexes with respect to the nature of the primary NOS product (NO, NO(-), or a related N-oxide) are invalid. Thus, NOS may form NO(-) or related N-oxides instead of NO.

Comparison of control of Listeria by nitric oxide redox chemistry from murine macrophages and NO donors: insights into listeriocidal activity of oxidative and nitrosative stress.

The physiological function of nitric oxide (NO) in the defense against pathogens is multifaceted. The exact chemistry by which NO combats intracellular pathogens such as Listeria monocytogenes is yet unresolved. We examined the effects of NO exposure, either delivered by NO donors or generated in situ within ANA-1 murine macrophages, on L. monocytogenes growth. Production of NO by the two NONOate compounds PAPA/NO (NH2(C3H6)(N[N(O)NO]C3H7) and DEA/NO (Na(C2H5)2N[N(O)NO]) resulted in L. monocytogenes cytostasis with minimal cytotoxicity. Reactive oxygen species generated from xanthine oxidase/hypoxanthine were neither bactericidal nor cytostatic and did not alter the action of NO. L. monocytogenes growth was also suppressed upon internalization into ANA-1 murine macrophages primed with interferon-gamma (INF-gamma) + tumor necrosis factor-alpha (TNF-alpha or INF-gamma + lipid polysaccharide (LPS). Growth suppression correlated with nitrite formation and nitrosation of 2,3-diaminonaphthalene elicited by stimulated murine macrophages. This nitrosative chemistry was not dependent upon nor mediated by interaction with reactive oxygen species (ROS), but resulted solely from NO and intermediates related to nitrosative stress. The role of nitrosation in controlling L. monocytogenes was further examined by monitoring the effects of exposure to NO on an important virulence factor, Listeriolysin O, which was inhibited under nitrosative conditions. These results suggest that nitrosative stress mediated by macrophages is an important component of the immunological arsenal in controlling L. monocytogenes infections.

Mechanisms of the antioxidant effects of nitric oxide.

The Janus face of nitric oxide (NO) has prompted a debate as to whether NO plays a deleterious or protective role in tissue injury. There are a number of reactive nitrogen oxide species, such as N2O3 and ONOO-, that can alter critical cellular components under high local concentrations of NO. However, NO can also abate the oxidation chemistry mediated by reactive oxygen species such as H2O2 and O2- that occurs at physiological levels of NO. In addition to the antioxidant chemistry, NO protects against cell death mediated by H2O2, alkylhydroperoxides, and xanthine oxidase. The attenuation of metal/peroxide oxidative chemistry, as well as lipid peroxidation, appears to be the major chemical mechanisms by which NO may limit oxidative injury to mammalian cells. In addition to these chemical and biochemical properties, NO can modulate cellular and physiological processes to limit oxidative injury, limiting processes such as leukocyte adhesion. This review will address these aspects of the chemical biology of this multifaceted free radical and explore the beneficial effect of NO against oxidative stress.

Redox generation of nitric oxide to radiosensitize hypoxic cells.

PURPOSE: Previous studies have shown that nitric oxide (NO) delivered from NO donor agents sensitizes hypoxic cells to ionizing radiation. In the present study, nitroxyl (NO-), a potential precursor to endogenous NO production, was evaluated for hypoxic cell radiosensitization, either alone or in combination with electron acceptor agents. METHODS AND MATERIALS: Radiation survival curves of Chinese hamster V79 lung fibroblasts under aerobic and hypoxic conditions were assessed by clonogenic assay. Hypoxia induction was achieved by metabolism-mediated oxygen depletion in dense cell suspensions. Cells were treated with NO- produced from the nitroxyl donor Angeli's salt (AS, Na2N2O3, sodium trioxodinitrate), in the absence or presence of electron acceptor agents, ferricyanide, or tempol. NO concentrations resulting from the combination of AS and ferricyanide or tempol were measured under hypoxic conditions using an NO-sensitive electrode. RESULTS: Treatment of V79 cells under hypoxic conditions with AS alone did not result in radiosensitization; however, the combination of AS with ferricyanide or tempol resulted in significant hypoxic radiosensitization with SERs of 2.5 and 2.1, respectively. Neither AS alone nor AS in combination with ferricyanide or tempol influenced aerobic radiosensitivity. The presence of NO generated under hypoxic conditions from the combination of AS with ferricyanide or tempol was confirmed using an NO-sensitive electrode. CONCLUSION: Combining NO- generated from AS with electron acceptors results in NO generation and substantial hypoxic cell radiosensitization. NO- derived from donor agents or endogenously produced in tumors, combined with electron acceptors, may provide an important strategy for radiosensitizing hypoxic cells and warrants in vivo evaluation.

Complexes of .NO with nucleophiles as agents for the controlled biological release of nitric oxide. Vasorelaxant effects.

Selected nucleophile/nitric oxide adducts [compounds which contain the anionic moiety, XN(O-)N = O] were studied for their ability to release nitric oxide spontaneously in aqueous solution and for possible vasoactivity. The diversity of structures chosen included those in which the nucleophile residue, X, was that of a secondary amine [Et2N, as in [Et2NN(N = O)O]Na, 1], a primary amine [iPrHN, as in [iPrHNN(N = O)O]Na, 2], a polyamine, spermine [as in the zwitterion H2N(CH2)3NH2+(CH2)4N[N(N = O)O-](CH2)3NH2, 3], oxide [as in Na[ON(N = O)O]Na, 4], and sulfite [as in NH4[O3SN(N = O)O]NH4, 5]. The rate constants (k) for decomposition in pH 7.4 phosphate buffer at 37 degrees C, as measured by following loss of chromophore at 230-260 nm, were as follows: 1, 5.4 x 10(-3) s-1; 2, 5.1 x 10(-3) s-1; 3, 0.30 x 10(-3) s-1; 4, 5.0 x 10(-3) s-1; and 5, 1.7 x 10(-3) s-1. The corresponding extents of nitric oxide release (ENO) were 1.5, 0.73, 1.9, 0.54, and 0.001 mol/mol of starting material consumed, respectively, as determined from the integrated chemiluminescence response. Vasodilatory activities expressed as the concentrations required to induce 50% relaxation in norepinephrine-constricted aortic rings bathed in pH 7.4 buffer at 37 degrees C (EC50) were as follows: 1, 0.19 microM; 2, 0.45 microM; 3, 6.2 microM; 4, 0.59 microM; and 5, 62 microM. Vasorelaxant potency (expressed as 1/EC50) was strongly correlated with the quantity of .NO calculated from the physicochemical data to be released in the interval required to achieve maximum relaxation at the EC50 doses (r = 0.995). This suggests that such nucleophile/.NO adducts might generally be useful as vehicles for the nonenzymatic generation of nitric oxide, in predictable amounts and at predictable rates, for biological purposes. The particular significance for possible drug design is underscored in the very favorable potency comparison between several of these agents and the established nitrovasodilators sodium nitroprusside and glyceryl trinitrate (EC50 values of 2.0 and greater than 10 microM, respectively) in parallel aortic ring tests.

Oxidizing intermediates generated in the Fenton reagent: kinetic arguments against the intermediacy of the hydroxyl radical.

It has long been recognized that the aqueous mixture of hydrogen peroxide and ferrous ion, known as the Fenton reagent, generates powerful oxidants. Furthermore, the chemical intermediates and reaction pathways of the type generated by this reagent have been implicated in oxidative damage in biological systems. Although the subject continues to be debated, the hydroxyl radical (.OH) is generally invoked as the predominant oxidizing intermediate formed by the Fenton reagent. However, recent results from this laboratory have demonstrated that the principal pathway for the Fenton-mediated oxidation of N-nitrosodimethylamine does not involve .OH, but instead must involve the intermediacy of another strongly oxidizing species. This conclusion was based on stopped-flow spectrophotometric observation of a transient, A, believed to be an iron(II) nitrosyl adduct, which was formed at a rate five-fold faster than that predicted for formation of .OH. Subsequent experiments have shown that methanol and other organic compounds can quench the formation of A. This quenching procedure provides a unique spectrophotometric probe with which to examine the relative reactivities of putative Fenton-type oxidizing intermediates toward organic substrates. Presented here are the results of several such quenching studies, plus an overview of our mechanistic investigations of the Fenton reaction.

Review article: chronic inflammation and reactive oxygen and nitrogen metabolism--implications in DNA damage and mutagenesis.

It is well known that chronic inflammation of the digestive tract is associated with an increased risk of malignant transformation. Because phagocytic leukocytes and cytokine-activated parenchymal cells produce large amounts of reactive metabolites of oxygen and nitrogen, there has been substantial interest in ascertaining whether these reactive intermediates may mediate mutagenesis and malignant transformation in vivo. However, very little information is available regarding the basic chemistry of how these oxygen and nitrogen-derived species may interact to yield potentially carcinogenic agents. This review will discuss our present understanding of the chemical and biochemical interactions between superoxide and nitric oxide and provide a model by which these reactive species may damage DNA and mediate mutagenesis.

Role of antioxidants in the nitric oxide-elicited inhibition of dopamine uptake in cultured mesencephalic neurons. Insights into potential mechanisms of nitric oxide-mediated neurotoxicity.

Under aerobic conditions the addition of (C2N5)2N(N[O]NO)-.Na+(DEA/NO), S-nitroso-N-acetyl penicillamine and nitric oxide (NO)-saturated buffer, but not S-nitroso-L-glutathione, to dopamine solutions resulted in dopamine o-semiquinone formation that was dependent on the formation of a NO/oxygen intermediate. High pressure liquid chromatography (HPLC) electrochemical analysis of dopamine demonstrated that the DEA/NO-induced oxidation of dopamine was abrogated in the presence of the antioxidants, ascorbate and glutathione. NO spontaneously released from DEA/NO decreased [3H]dopamine accumulation in primary cultures of mesencephalic neurons in a dose-dependent fashion. In contrast, [3H] gamma-aminobutyric acid uptake by mesencephalic neurons tested under the same conditions was unchanged. When DEA/NO was added to incubation buffer that contained [3H]dopamine and the antioxidant, ascorbate or glutathione, [3H]dopamine uptake was also inhibited. These data excluded that oxidation of extracellular [3H]dopamine by the intermediates of the NO/O2 reaction could have caused this decrease. Instead, NO may have acted directly on a not yet identified target operative in the regulation of dopamine storage and release. Analysis of the rate constants for the NO reaction with ascorbate, glutathione and dopamine revealed that dopamine quinone formation was delayed by the presence of antioxidants. Since the formation of NO as well as neurotransmitter release are activated during ischemia reperfusion injury, it is possible that prolonged NO exposure could deplete antioxidants and facilitate the oxidation of dopamine and thereby cause neurotoxicity.