Oxidative/Nitrative Mechanism of Molsidomine Mitotoxicity Assayed by the Cytochrome c Reaction with SIN-1 in Models of Biological Membranes.

Molsidomine is currently used as a vasodilator drug for the treatment of myocardial ischemic syndrome and congestive heart failure, although still presenting some mitochondrial-targeted side effects in many human cells. As a model of molsidomine mitotoxicity, the reaction of cytochrome c with phosphatidylserine (PS)- and cardiolipin (CL)-containing liposomes was investigated in oxidative/nitrosative conditions imposed by SIN-1 decomposition, which renders peroxynitrite (ONOO-) as a main reactive product. In these conditions, the production of thiobarbituric acid-reactive substance (TBARs) and LOOH was affected by the lipid composition and the oxidative/nitrative conditions used. The oxidative/nitrative conditions were the exposure of lipids to SIN-1 decomposition, native cytochrome c after previous exposure to SIN-1, concomitantly to SIN-1 and native cytochrome c, native cytochrome c, and cytochrome c modified by SIN-1 that presents a less-rhombic heme iron (L-R cytc). TBARs and LOOH production by lipids and cytochrome c exposed concomitantly to SIN-1 differed from that obtained using L-R cytc and featured similar effects of SIN-1 alone. This result suggests that lipids rather than cytochrome c are the main targets for oxidation and nitration during SIN-1 decomposition. PS- and CL-containing liposomes challenged by SIN-1 were analyzed by Fourier transform infrared spectroscopy that revealed oxidation, trans-isomerization, and nitration. These products are consistent with reaction routes involving lipids and NOx formed via peroxynitrite or direct reaction of NO• with molecular oxygen that attacks LOOH and leads to the formation of substances that are not reactive with thiobarbituric acid.

Force generated by myosin cross-bridges is reduced in myofibrils exposed to ROS/RNS.

Skeletal muscle weakness is associated with oxidative stress and oxidative posttranslational modifications on contractile proteins. There is indirect evidence that reactive oxygen/nitrogen species (ROS/RNS) affect skeletal muscle myofibrillar function, although the details of the acute effects of ROS/RNS on myosin-actin interactions are not known. In this study, we examined the effects of peroxynitrite (ONOO-) on the contractile properties of individual skeletal muscle myofibrils by monitoring myofibril-induced displacements of an atomic force cantilever upon activation and relaxation. The isometric force decreased by ~50% in myofibrils treated with the ONOO- donor (SIN-1) or directly with ONOO-, which was independent of the cross-bridge abundancy condition (i.e., rigor or relaxing condition) during SIN-1 or ONOO- treatment. The force decrease was attributed to an increase in the cross-bridge detachment rate (gapp) in combination with a conservation of the force redevelopment rate (kTr) and hence, an increase in the population of cross-bridges transitioning from force-generating to non-force-generating cross-bridges during steady-state. Taken together, the results of this study provide important information on how ROS/RNS affect myofibrillar force production which may be of importance for conditions where increased oxidative stress is part of the pathophysiology.

Triosephosphate isomerase tyrosine nitration induced by heme-NaNO2 -H2 O2 or peroxynitrite: Effects of different natural phenolic compounds.

Peroxynitrite and heme peroxidases (or heme)-H2 O2 -NaNO2 system are the two common ways to cause protein tyrosine nitration in vitro, but the effects of antioxidants on reducing these two pathways-induced protein nitration and oxidation are controversial. Both nitrating systems can dose-dependently induce triosephosphate isomerase (TIM) nitration, however, heme-H2 O2 -NaNO2 was less destructive to protein secondary structures and led to more nitrated tyrosine residue than 3-morpholinosydnonimine hydrochloride (SIN-1, a peroxynitrite donor). Both of desferrioxamine and catechin could inhibit TIM nitration induced by heme-H2 O2 -NaNO2 and SIN-1 and protein oxidation induced by SIN-1, but promoted heme-H2 O2 -NaNO2 -induced protein oxidation. Moreover, the antagonism of natural phenolic compounds on SIN-1-induced tyrosine nitration was consistent with their radical scavenging ability, but no similar consensus was found in heme-H2 O2 -NaNO2 -induced nitration. Our results indicated that peroxynitrite and heme-H2 O2 -NaNO2 -induced protein nitration was different, and the later one could be a better model for anti-nitration compounds screening.

Development of Novel Warfarin-Silica Composite for Controlled Drug Release.

PURPOSE: The work is devoted to synthesis and study of warfarin composites with unmodified, methyl and phenyl modified silica in order to develop controlled release formulation of the anticoagulant. METHODS: The composites were prepared by two routes, adsorption and sol-gel, and characterized with FTIR spectroscopy, dynamic light scattering and DSC methods. The drug release behavior from the composites in media with pH 1.6, 6.8 and 7.4 was analyzed in vitro. The release kinetics of the warfarin - silica composites prepared by the two routes was compared among each other and with analogous silica composites with water soluble drug molsidomine. RESULTS: The comparative analysis showed that in general the kinetic regularities and mechanisms of release for both drugs are similar and determined by nonuniform distribution of the drugs over the silica matrixes and stability of the matrixes in the studied media for the adsorbed composites and uniformly distributed drug and more brittle structure for the sol-gel composites. CONCLUSIONS: The sol-gel composite of warfarin - phenyl modified silica is perspective for further development of novel warfarin formulation with controlled release because it releases warfarin according to zero-order kinetic law with approximately equal rate in the media imitating different segments of gastrointestinal tract.

Development of High-Throughput Method for Measurement of Vascular Nitric Oxide Generation in Microplate Reader.

BACKGROUND: Despite the importance of nitric oxide (NO) in vascular physiology and pathology, a high-throughput method for the quantification of its vascular generation is lacking. OBJECTIVE: By using the fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), we have optimized a simple method for the determination of the generation of endothelial nitric oxide in a microplate format. METHODS: A nitric oxide donor was used (3-morpholinosydnonimine hydrochloride, SIN-1). Different factors affecting the method were studied, such as the effects of dye concentration, different buffers, time of reaction, gain, and number of flashes. RESULTS: Beer's law was linear over a nanomolar range (1-10 nM) of SIN-1 with wavelengths of maximum excitation and emission at 495 and 525 nm; the limit of detection reached 0.897 nM. Under the optimized conditions, the generation of rat aortic endothelial NO was measured by incubating DAF-FM with serial concentrations (10-1000 µM) of acetylcholine (ACh) for 3 min. To confirm specificity, Nω-Nitro-l-arginine methyl ester (l-NAME)-the standard inhibitor of endothelial NO synthase-was found to inhibit the ACh-stimulated generation of NO. In addition, vessels pre-exposed for 1 h to 400 µM of the endothelial damaging agent methyl glyoxal showed inhibited NO generation when compared to the control stimulated by ACh. CONCLUSIONS: The capability of the method to measure micro-volume samples makes it convenient for the simultaneous handling of a very large number of samples. Additionally, it allows samples to be run simultaneously with their replicates to ensure identical experimental conditions, thus minimizing the effect of biological variability.

Oxidation and Tyrosine Nitration Induce Structural Changes and Inhibits Plasmodium falciparum Falcipain-2 Activity In Vitro.

Falcipain-2 (FP2) is an important hemoglobinase from the malaria parasite Plasmodium falciparum and a suitable target for the development of an antimalarial chemotherapy. Many reports have indicated that radical nitrogen species (RNS) including nitric oxide (NO) are inhibitors of P. falciparum growth and promoters of recovery from malaria symptoms. In this scenario, FP2 emerges as a potential target of RNS, since its inhibition partially hinders the parasite growth. We report that in vitro FP2 did not undergo S-nitrosylation when exposed to the NO-donor GSNO. However, it was modified by a combined mechanism of methionine oxidation and tyrosine nitration in response to SIN-1, and NaNO2- H2O2 treatment. The treatments with the nitrating agents caused a pronounced decrease in protease activity most likely induced by a disruption on the secondary and tertiary structure of FP2. Our data also demonstrate that at least four tyrosine residues were nitrated and found on the surface of the enzyme, partially or completely exposed to the solvent. Although performed in vitro, these results suggest that falcipain-2 may be a target of RNS activity and its inhibition could explain the hindering of the parasite growth when exposed to these radicals. The understanding of the molecular mechanisms involving free radicals and its inhibition activity towards FP2 may be effective in the development of antimalarial therapies.

Magnetic silica hybrids modified with guanidine containing co-polymers for drug delivery applications.

Guanidine containing co-polymers grafted onto silica nanoparticles to form core-shell structure were prepared by sol-gel method in the presence of γ-Fe2O3 nanoparticles. The morphological features for uncoated and coated silica particles have been characterized with scanning electron microscopy. The results show that the polymer coated silicas exhibit spherical morphology with rough polymeric surface covered by γ-Fe2O3 nanoparticles. The grafting amount of guanidine containing co-polymers evaluated by thermogravimetric analysis was in the range from 17 to 30%. Then, the drug loading properties and cumulative release of silica hybrids modified with guanidine containing co-polymers were evaluated using molsidomine as a model drug. It was shown that after polymer grafting the loading content of molsidomine could reach up to 3.42±0.21 and 2.34±0.14mg/g respectively. The maximum drug release of molsidomine is achieved at pH1.6 (approximately 71-75% release at 37°C), whereas at pH7.4 drug release is lower (50.4-59.6% release at 37°C). These results have an important implication that our magneto-controlled silica hybrids modified with guanidine containing co-polymers are promising as drug carriers with controlled behaviour under influence of magnetic field.

Design, green synthesis and pharmacological evaluation of novel 5,6-diaryl-1,2,4-triazines bearing 3-morpholinoethylamine moiety as potential antithrombotic agents (.).

The aim of this research work was to investigate a series of novel 5,6-diaryl-1,2,4-triazines (3a-3q) containing 3-morpholinoethylamine side chain, and to address their antiplatelet activity by in vitro, ex vivo and in vivo methods. All compounds were synthesized by environment benign route and their structures were unambiguously confirmed by spectral data. Compounds (3l) and (3m) were confirmed by their single crystal X-ray structures. Out of all the synthesized compounds, 10 were found to be more potent in vitro than aspirin; six of them were found to be prominent in ex vivo assays and one compound (3d) was found to have the most promising antithrombotic profile in vivo. Moreover, compound (3d) demonstrated less ulcerogenicity in rats as compared to aspirin. The selectivity of the most promising compound (3d) for COX-1 and COX-2 enzymes was determined with the help of molecular docking studies and the results were correlated with the biological activity.

Myeloperoxidase scavenges peroxynitrite: A novel anti-inflammatory action of the heme enzyme.

Peroxynitrite, a potent pro-inflammatory and cytotoxic species, interacts with a variety of heme containing proteins. We addressed the question whether (i) the interaction of myeloperoxidase (MPO, an enzyme generating hypochlorous acid from hydrogen peroxide and chloride ions) with peroxynitrite affects the clearance of peroxynitrite, and (ii) if peroxynitrite could modulate the chlorinating activity of MPO. Our results show that this interaction promotes the decomposition of the highly reactive pro-inflammatory oxidant, whereby MPO Compound II (but not Compound I) is formed. The efficiency of MPO to remove peroxynitrite was enhanced by L-tyrosine, nitrite and (-)-epicatechin, substances known to reduce Compound II with high reaction rate. Next, peroxynitrite (added as reagent) diminished the chlorinating activity of MPO in the presence of hydrogen peroxide. Alternatively, SIN-1, a peroxynitrite donor, reduced hypochlorous acid formation by MPO, as measured by aminophenyl fluorescein oxidation (time kinetics) and taurine chloramine formation (end point measurement). At inflammatory loci, scavenging of peroxynitrite by MPO may overcome the uncontrolled peroxynitrite decomposition and formation of reactive species, which lead to cell/tissue damage.

Peroxynitrite-mediated nitrosative stress decreases motility and mitochondrial membrane potential in human spermatozoa.

Nitrosative stress is produced by high levels of reactive nitrogen species (RNS). The RNS include peroxynitrite, a highly reactive free radical produced from a diffusion-controlled reaction between nitric oxide and superoxide anion. Peroxynitrite causes nitration and oxidation of lipids, proteins and DNA, and is thus considered an important pathogenic mechanism in various diseases. Although high levels of peroxynitrite are associated with astenozoospermia, few reports exist regarding the in vitro effect of high levels of this RNS on human sperm. The aim of this study was to evaluate the in vitro effect of nitrosative stress caused by peroxynitrite on the viability, motility and mitochondrial membrane potential of human spermatozoa. To do this, human spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a molecule that generates peroxynitrite. Incubations were done at 37°C for up to 4 h with SIN-1 concentrations between 0.2 and 1.0 mmol/l. Generation of peroxynitrite was confirmed using dihydrorhodamine 123 (DHR) by spectrophotometry and flow cytometry. Sperm viability was assessed by propidium iodide staining; sperm motility was analyzed by CASA, and the state of mitochondrial membrane potential (ΔΨm) by JC-1 staining. Viability and ΔΨm were measured by flow cytometry. The results showed an increase in DHR oxidation, demonstrating the generation of peroxynitrite through SIN-1. Peroxynitrite decreased progressive and total motility, as well as some sperm kinetic parameters. Mitochondrial membrane potential also decreased. These alterations occurred with no decrease in sperm viability. In conclusion, peroxynitrite-induced nitrosative stress impairs vital functions in the male gamete, possibly contributing to male infertility.

Impact of SIN-1-derived peroxynitrite flux on endothelial cell redox homeostasis and bioenergetics: protective role of diphenyl diselenide via induction of peroxiredoxins.

Increased production of reactive nitrogen (RNS) and oxygen (ROS) species and its detrimental effect to mitochondria are associated with endothelial dysfunction. This study was designed to determine the effect of a peroxynitrite flux, promoted by 1,3-morpholinosydnonimine (SIN-1), in mitochondrial function and some redox homeostasis parameters in bovine aortic endothelial cells (BAEC). Moreover, the effect of diphenyl diselenide (PhSe)2, a simple organic selenium compound, in preventing peroxynitrite-mediated cytotoxicity was also investigated. Our results showed that overnight exposure to SIN-1 (250 µM) caused a profound impairment of oxygen consumption, energy generation and reserve capacity in mitochondria of BAEC. Mitochondrial dysfunction resulted in an additional intracellular production of peroxynitrite, amplifying the phenomenon and leading to changes in redox homeostasis. Moreover, we observed an extensive decline in mitochondrial membrane potential (ΔΨm) induced by peroxynitrite and this event was associated with apoptotic-type cell death. Alternatively, the pretreatment of BAEC with (PhSe)2, hindered peroxynitrite-mediated cell damage by preserving mitochondrial and endothelial function and consequently preventing apoptosis. The protective effect of (PhSe)2 was related to its ability to improve the intracellular redox state by increasing the expression of different isoforms of peroxiredoxins (Prx-1-3), efficient enzymes in peroxynitrite detoxification.

Comparative study of adsorption capacity of mesoporous silica materials for molsidomine: effects of functionalizing and solution pH.

Adsorption capacities of mesoporous silica materials having various surface functional groups (hydroxyl, phenyl, mercaptopropyl, aminopropyl) at pH values of 4.8, 7.4, and 8.0 were studied. It was found that the maximum amount of adsorbed molsidomine is affected by method of preparation of the silica materials, chemistry of their surfaces and solution pH from where adsorption is carried out. The effects were explained by different states of the adsorbents and molsidomine in solution at the studied pH. The most efficient adsorption of molsidomine is observed onto phenyl modified silica prepared by grafting at pH4.8. Aminopropyl modified silica adsorbs the lowest amount of molsidomine and the adsorption was observed only at pH7.4. Interactions responsible for the adsorption were elucidated by spectroscopic studies.

Bicarbonate plays a critical role in the generation of cytotoxicity during SIN-1 decomposition in culture medium.

3-Morpholinosydnonimine (SIN-1) is used as a donor of peroxynitrite (ONOO(-)) in various studies. We demonstrated, however, that, the cell-culture medium remains cytotoxic to PC12 cells even after almost complete SIN-1 decomposition, suggesting that reaction product(s) in the medium, rather than ONOO(-), exert cytotoxic effects. Here, we clarified that significant cytotoxicity persists after SIN-1 decomposes in bicarbonate, a component of the culture medium, but not in NaOH. Cytotoxic SIN-1-decomposed bicarbonate, which lacks both oxidizing and nitrosating activities, degrades to innocuous state over time. The extent of SIN-1 cytotoxicity, irrespective of its fresh or decomposed state, appears to depend on the total number of initial SIN-1 molecules per cell, rather than its concentration, and involves oxidative/nitrosative stress-related cell damage. These results suggest that, despite its low abundance, the bicarbonate-dependent cytotoxic substance that accumulates in the medium during SIN-1 breakdown is the cytotoxic entity of SIN-1.

Evaluation of novel antioxidant triterpenoid saponins from the halophyte Salicornia herbacea.

As a part of an ongoing search for novel antioxidants from the salt marsh plants, bioactivity-isolation and structure determination of constituents from Salicornia herbacea were performed. One new triterpenoid saponin (4), along with three known saponins (1-3), has been isolated from n-BuOH fraction of S. herbacea. On the basis of the spectroscopic methods, the structure of the new saponin 4 was elucidated as 3ß-hydroxy-23-oxo-30-noroleana-12, 20(29)-diene-28-oic acid 3-O-ß-D-glucuronopyranosyl-28-O-ß-d-glucopyranoside. Scavenging effects of saponins 1-4 were examined on 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical and peroxynitrite. Particularly, saponin 3 exerted significant antioxidant activity on both authentic peroxynitrite and peroxynitrite generated from morpholinosydnonimine (SIN-1).

Comparative kinetics of thiol oxidation in two distinct free-radical generating systems: SIN-1 versus AAPH.

To study oxidative stress in biological systems, chemical compounds capable of producing free radicals have been widely used. Here, we compared two free-radical generators, 3-morpholinosydnonimine (SIN-1) and 2,2'-azo-bis(2-amidinopropane) hydrochloride (AAPH), by measuring the thiol oxidation kinetics of various thiols. We found that SIN-1 is > 30 times potent in causing thiol oxidation than AAPH. Kinetic simulations revealed that in the SIN-1 system (0.1 mM), superoxide, nitrogen dioxide and carbonate radicals are the major reactive species which, in combination, induce ∼50% of thiol molecules to undergo one-electron oxidation, thereby forming the thiyl radical which propagates further thiol oxidation by direct coupling with thiolates. Similarly, the alkyl peroxyl radical derived from AAPH (3 mM) initiates comparable extent of one-electron oxidation and formation of the thiyl radical. In conclusion, our study provides experimental and theoretical evidence that SIN-1 is mainly an one-electron oxidizing agent that can be functionally mimicked by AAPH.

(13Z)- and (9Z)-lycopene isomers are major intermediates in the oxidative degradation of lycopene by cigarette smoke and Sin-1.

The breakdown of lycopene in the presence of reactive oxygen and reactive nitrogen species has been studied in order to identify key in vitro intermediates. These compounds may in turn be produced as metabolites in the body and may have significant physiological properties, such as increased antioxidant capacity. We have studied the in vitro degradation of lycopene in solvent, in plasma and in low density lipoprotein, when challenged with freshly generated gaseous cigarette smoke or free radicals generated in situ by S-morpholinosydonimine at 37°C. The emphasis has been to establish the major intermediates and to compare the data with previous studies using different reactants. We have found that (13Z)-lycopene is the major intermediate in both cigarette smoke and S-morpholinosydonimine reactions (representing ≥60% of the converted (all-E)-lycopene at ∼50% depletion). Additionally, (9Z)-lycopene and various (all-E) and (Z)-lycopene epoxides were predominant. Notably, (5Z)-lycopene appeared to be the most stable form of lycopene under the stated conditions. Previous theoretical studies of isomer thermodynamics and rotational energy barriers for carbon double bonds fully support the pattern of isomer production and stability. In contrast to ß-carotene studies, nitro-derivatives of lycopene could not be detected. In conclusion, (Z)-lycopene production and (5Z)-lycopene stability may help explain elevated (Z)-lycopene in plasma over (Z)-lycopene content in lycopene-containing foods in the diet.

Nitrosation-modulating effect of ascorbate in a model dynamic system of coexisting nitric oxide and superoxide.

The coexistence of nitric oxide and superoxide leads to complex oxidative and nitrosative chemistry, which has been implicated in many pathophysiological conditions. The present study investigated the role of ascorbate in affecting the kinetics of nitrosative chemistry in a model dynamic snystem of coexisting nitric oxide and superoxide. SIN-1 (3-morpholinosydnonimine) was used to elicit various degrees of nitroxidative stress in a reaction buffer and DAN (2,3-diaminonaphthalene) was used as a probe for N-nitrosation reaction. The nitrosation kinetics in the absence and presence of ascorbate was followed by measuring the formation of the fluorescent product over time. Computational modelling was used to provide quantitative or semi-quantitative insights into the studied system. The results show that ascorbate effectively quenches N-nitrosation reaction, which could be partially attributed to the free radical scavenging and repairing effect of ascorbate. Computational modelling reveals an interesting temporal distribution of superoxide, nitric oxide and peroxynitrite. The model predicts that peroxynitrite is the most predominant species in the SIN-1 system. Furthermore, ascorbate might alter the system dynamics by removing superoxide and, thereby, increasing the availability of nitric oxide.

Examination of imprinting process with molsidomine as a template.

Eight different functional monomers were used with ethylene glycol dimethacrylate as a cross-linker and molsidomine as a template to obtain molecularly imprinted polymers (MIPs). Non-covalent interactions between molsidomine and each functional monomer in DMSO prior to thermal bulk polymerization were utilized. On the basis of calculated imprinting factors, MIP prepared with N,N'-diallyltartaramide was chosen for further investigations. Examination of interactions in the prepolymerization complex between molsidomine and N,N'-diallyltartaramide was performed using the Job method. The absorbance of isomolar solutions reaching a maximum for the molar ratio of template to monomer equal to 1:4. Scatchard analysis was used for estimation of the dissociation constants and the maximum amounts of binding sites. The polymer based on N,N'-diallyltartaramide has two classes of heterogeneous binding sites characterized by two values of K(d) and two B(max): K(d)(1) = 1.17 mM(-1) and B(max)(1) = 0.8 mumol/mg for the higher affinity binding sites, and K(d)(2) = 200 microM(-1) and B(max)(2) = 2.05 mumol/mg for the lower affinity binding sites. Furthermore, effects of pH and organic solvent on binding properties of MIP and NIP were investigated, together with release of molsidomine from both MIP and NIP.

Chemical model systems for cellular nitros(yl)ation reactions.

S-nitros(yl)ation belongs to the redox-based posttranslational modifications of proteins but the underlying chemistry is controversial. In contrast to current concepts involving the autoxidation of nitric oxide ((.)NO, nitrogen monoxide), we and others have proposed the formation of peroxynitrite (oxoperoxonitrate (1(-))as an essential intermediate. This requires low cellular fluxes of (.)NO and superoxide (UO2(-)), for which model systems have been introduced. We here propose two new systems for nitros(yl)ation that avoid the shortcomings of previous models. Based on the thermal decomposition of 3-morpholinosydnonimine,equal fluxes of (.)NO and UO2(-) were generated and modulated by the addition of (.)NO donors or Cu,Zn superoxide dismutase. As reactants for S-nitros(yl)ation, NADP+-dependent isocitrate dehydrogenase and glutathione were employed, for which optimal S-nitros(yl)ation was observed at nanomolar fluxes of (.)NO and UO2(-) at a ratio of about 3:1. The previously used reactants phenol and diaminonaphthalene (C- and Nnitrosation)demonstrated potential participation of multiple pathways for nitros(yl)ation. According to our data, neither peroxynitrite nor autoxidation of UNO was as efficient as the 3 (.)NO/1 UO2(-) system in mediating S-nitros(yl)ation. In theory this could lead to an elusive nitrosonium (nitrosyl cation)-like species in the first step and to N2O3 in the subsequent reaction. Which of these two species or whether both together will participate in biological S-nitros(yl)ation remains to be elucidated. Finally, we developed several hypothetical scenarios to which the described (.)NO/UO2-flux model could apply, providing conditions that allow either direct electrophilic substitution at a thiolate or S-nitros(yl)ation via transnitrosation from S-nitrosoglutathione.

Modification of surfactant protein D by reactive oxygen-nitrogen intermediates is accompanied by loss of aggregating activity, in vitro and in vivo.

Surfactant protein D (SP-D) is an important effector of innate immunity. We have previously shown that SP-D accumulates at sites of acute bacterial infection and neutrophil infiltration, a setting associated with the release of reactive species such as peroxynitrite. Incubation of native SP-D or trimeric SP-D lectin domains (NCRDs) with peroxynitrite resulted in nitration and nondisulfide cross-linking. Modifications were blocked by peroxynitrite scavengers or pH inactivation of peroxynitrite, and mass spectroscopy confirmed nitration of conserved tyrosine residues within the C-terminal neck and lectin domains. Mutant NCRDs lacking one or more of the tyrosines allowed us to demonstrate preferential nitration of Tyr314 and the formation of Tyr228-dependent cross-links. Although there was no effect of peroxynitrite or tyrosine mutations on lectin activity, incubation of SP-D dodecamers or murine lavage with peroxynitrite decreased the SP-D-dependent aggregation of lipopolysaccharide-coated beads, supporting our hypothesis that defective aggregation results from abnormal cross-linking. We also observed nitration, cross-linking of SP-D, and a significant decrease in SP-D-dependent aggregating activity in the lavage of mice acutely exposed to nitrogen dioxide. Thus, modification of SP-D by reactive oxygen-nitrogen species could contribute to alterations in the structure and function of SP-D at sites of inflammation in vivo.