Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury.

Activated alveolar macrophages and epithelial type II cells release both nitric oxide and superoxide which react at near diffusion-limited rate (6.7 x 10(9) M-1s-1) to form peroxynitrite, a potent oxidant capable of damaging the alveolar epithelium and pulmonary surfactant. Peroxynitrite, but not nitric oxide or superoxide, readily nitrates phenolic rings including tyrosine. We quantified the presence of nitrotyrosine in the lungs of patients with the adult respiratory distress syndrome (ARDS) and in the lungs of rats exposed to hyperoxia (100% O2 for 60 h) using quantitative immunofluorescence. Fresh frozen or paraffin-embedded lung sections were incubated with a polyclonal antibody to nitrotyrosine, followed by goat anti-rabbit IgG coupled to rhodamine. Sections from patients with ARDS (n = 5), or from rats exposed to hyperoxia (n = 4), exhibited a twofold increase of specific binding over controls. This binding was blocked by the addition of an excess amount of nitrotyrosine and was absent when the nitrotyrosine antibody was replaced with nonimmune IgG. In additional experiments we demonstrated nitrotyrosine formation in rat lung sections incubated in vitro with peroxynitrite, but not nitric oxide or reactive oxygen species. These data suggest that toxic levels of peroxynitrite may be formed in the lungs of patients with acute lung injury.     

Role of nitric oxide and peroxynitrite in the cytokine-induced sustained myocardial dysfunction in dogs in vivo.

Studies in vitro suggested that inflammatory cytokines could cause myocardial dysfunction. However, the detailed mechanism for the cytokine-induced myocardial dysfunction in vivo remains to be examined. We thus examined this point in our new canine model in vivo, in which microspheres with and without IL-1beta were injected into the left main coronary artery. Left ventricular ejection fraction (LVEF) was evaluated by echocardiography for 1 wk. Immediately after the microsphere injection, LVEF decreased to approximately 30% in both groups. While LVEF rapidly normalized in 2 d in the control group, it was markedly impaired in the IL-1beta group even at day 7. Pretreatment with dexamethasone or with aminoguanidine, an inhibitor of inducible nitric oxide synthase, prevented the IL-1beta-induced myocardial dysfunction. Nitrotyrosine concentration, an in vivo marker of the peroxynitrite production by nitric oxide and superoxide anion, was significantly higher in the myocardium of the IL-1beta group than in that of the control group or the group cotreated with dexamethasone or aminoguanidine. There was an inverse linear relationship between myocardial nitrotyrosine concentrations and LVEF. These results indicate that IL-1beta induces sustained myocardial dysfunction in vivo and that nitric oxide produced by inducible nitric oxide synthase and the resultant formation of peroxynitrite are substantially involved in the pathogenesis of the cytokine-induced sustained myocardial dysfunction in vivo.     

Nitric oxide production and perivascular nitration in brain after carbon monoxide poisoning in the rat.

Nitric oxide is a short-lived free radical and physiological mediator which has the potential to cause cytotoxicity. Studies were conducted to investigate whether nitric oxide, and the potent oxidant peroxynitrite, were generated in brain during experimental carbon monoxide (CO) poisoning in the rat. Nitric oxide production was documented by electron paramagnetic resonance spectroscopy, and found to be increased by ninefold immediately after CO poisoning. Evidence that peroxynitrite was generated was sought by looking for nitrotyrosine in the brains of CO-poisoned rats. Nitrotyrosine was found deposited in vascular walls, and also diffusely throughout the parenchyma in inummocytochemical studies. The affinity and specificity of an anti-nitrotyrosine antibody was investigated and a solid phase immunoradiochemical assay was developed to quantity nitrotyrosine in brain homogenates. A 10-fold increase in nitrotyrosine was found in the brains of CO-poisoned rats. Platelets were involved with production of nitrotyrosine in the early phase of exposure to CO. However, nitrotyrosine formation and leukocyte sequestration were not decreased in thrombocytopenic rats poisoned with CO according to the standard model. When rats were pre-treated with the nitric oxide synthase inhibitor, L-nitroarginine methyl ester, formation of both nitric oxide and nitrotyrosine in response to CO poisoning were abolished, as well as leukocyte sequestration in the microvasculature, endothelial xanthine dehydrogenase conversion to xanthine oxidase, and brain lipid peroxidation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events which lead to brain oxidative stress in CO poisoning.     

Peroxynitrite is an important mediator in thermal injury-induced lung damage

OBJECTIVE: Intestinal ischemia and reperfusion injury was known to cause postinjury multiple organ failure by neutrophil and unclear nonneutrophil factors. Peroxynitrite formed by the rapid reaction between superoxide and nitric oxide, is a toxic substance that contributes to tissue injury in a number of biological systems. In this study, the role of nitric oxide and neutrophils on lung damage after burn was investigated. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Thermal injury models in the rat. INTERVENTIONS: In experiment 1, specific pathogen-free Sprague-Dawley rats underwent 35% total body surface area burn. At 4, 8, 16, and 24 hrs after burn, intestinal mucosa and lung tissue were harvested for myeloperoxidase (MPO) assay, blood was collected for measurement of peroxynitrite-mediated oxidation of dihydrorhodamine 123, and pulmonary microvascular dysfunction was quantified by measuring the extravasation of Evans blue dye. In experiment 2, polymorphonuclear granulocyte antibody (0.12 mL/100 g administered intraperitoneally 16 hrs before burn), S-methylisothiourea (7.5 mg/kg, intraperitoneally, immediately after burn), a specific inducible nitric oxide synthase inhibitor, and sterile saline (15 mL/kg, intraperitoneally, immediately after burn) were given to different groups of thermally injured animals individually. The plasma dihydrorhodamine 123 oxidation level, intestinal and lung MPO activity, lung permeability, and lung histology were evaluated at 8 hrs after burn. The cellular localization of nitrotyrosine, a marker for peroxynitrite reactivity, was also examined by immunostaining. In experiment 3, 3-morpholinosydnonimine (10 mM, intraperitoneally), a peroxynitrite donor, was given to nonburned rats to examine the peroxynitrite effect on lung inducible nitric oxide synthase expression. MEASUREMENTS AND MAIN RESULTS: The level of MPO activity in intestine and lung, blood dihydrorhodamine 123 oxidation, and lung permeability were increased up to 2-fold, 2.5-fold, 2-fold, and 2-fold of normal, respectively, at 8 hrs after burn. S-methylisothiourea injection significantly decreased (p <.05) 31% of the lung MPO activity, 41% of the blood peroxynitrite level, 54% of the lung permeability, and the lung peroxynitrite production in burned rats. Polymorphonuclear granulocyte antibody pretreatment significantly decreased 60% of the intestinal MPO, 92% of the blood peroxynitrite level, and 56% the lung MPO activity in burned rats, but the lung permeability was only slightly decreased by polymorphonuclear granulocyte antibody pretreatment. Furthermore, 3-morpholinosydnonimine increased the lung inducible nitric oxide synthase messenger RNA levels. CONCLUSIONS: Thermal injury induces blood dihydrorhodamine 123 oxidation, intestinal and lung neutrophil deposition, lung nitrotyrosine production, and lung damage. Both specific inhibition of inducible nitric oxide synthase and polymorphonuclear granulocyte antibody pretreatment decrease blood dihydrorhodamine 123 oxidation and intestinal and lung neutrophil deposition, but only inducible nitric oxide synthase inhibition with S-methylisothiourea reduces lung peroxynitrite production and thermal injury-induced lung damage. Nitric oxide and the ensuing peroxynitrite production in lung play a more important role than neutrophil in contributing to thermal injury-induced lung damage.     

Oxidative inactivation of nitric oxide and endothelial dysfunction in stroke-prone spontaneous hypertensive rats

This study tested the hypothesis that increased nitric oxide (NO) inactivation and concurrent peroxynitrite formation is responsible for endothelial dysfunction in the spontaneously hypertensive stroke-prone rat (SHRSP). In SHRSP, the aortic vasorelaxation to acetylcholine (ACh) was decreased (p < 0.05), but NO production was unchanged. Nitrotyrosine staining, a footprint of peroxynitrite (ONOO(-)) formation, was detected. Exposure of SHRSP to a high-salt, high-fat diet (SFD) further exacerbated hypertension and accelerated end-organ disease. A severe endothelial dysfunction [maximal ACh relaxation: 49.8 +/- 2.1 versus 94.5 +/- 1.8% in Wistar-Kyoto rats (WKY), p < 0.01], increased basal NO production (482 +/- 17 versus 356 +/- 21 nM, p < 0.01), decreased ACh-stimulated NO production (57 +/- 6 versus 112 +/- 6 nM, p < 0.01), extensive inducible NO synthase and nitrotyrosine staining, elevated nitrotyrosine content (21-fold increase over WKY), and a high percentage of cells with DNA damage were observed in the aortic tissues from these animals. Treatment of SHRSP on SFD with carvedilol restored ACh-induced vasorelaxation and NO production, inhibited nitrotyrosine formation, reduced vascular cell DNA damage, and reduced end-organ injury. These data demonstrate that endothelial dysfunction was caused by increased NO inactivation alone (SHRSP) or in combination with decreased NO production from endothelial NO synthase (SHRSP on SFD). Antioxidant treatment with carvedilol exerted significant vascular protective effects, attenuated end-organ damage, and decreased mortality under these conditions.     

Inducible nitric oxide synthase and nitric oxide production inLeishmania infantum-infected human macrophages stimulated with interferon-γ and bacterial lipopolysaccharide

Nitric oxide produced by an inducible nitric oxide synthase constitutes one of the main microbicidal mechanisms of murine macrophages and its importance is now being recognized for human macrophages. In this study we evaluated inducible nitric oxide synthase expression, nitric oxide release, and parasitocidal ability of Leishmania infantum-infected monocyte-derived human macrophages. The inducible nitric oxide synthase was detected by immunofluorescence and western blotting and nitric oxide production was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Experiments were performed on macrophages with or without previous stimulation with recombinant human interferon-gamma and bacterial lipopolysaccharide. Inducible nitric oxide synthase expression and nitric oxide release were higher in Leishmania-infected stimulated macrophages than in uninfected cells or infected cells without previous stimulation. Nitric oxide production and parasitocidal activity against Leishmania infantum were reduced in macrophages treated with the nitric oxide synthase inhibitor L-N(G) monomethylarginine. These results suggest a microbicidal role for nitric oxide in human leishmaniasis, with the possible practical application of immunological or pharmacological regulation of nitric oxide synthesis in the treatment of this infection.     

Arginine reverses ethanol-induced inflammatory and fibrotic changes in liver despite continued ethanol administration.

We investigated the potential of arginine to reverse pathological changes in alcohol-induced liver injury. Four groups (six rats/group) of male Wistar rats were fed a fish oil-ethanol diet for 6 (group 2) or 8 (group 1) weeks. Rats in group 3 were fed fish oil-ethanol for 6 weeks, after which they were administered arginine with fish oil-ethanol for an additional 2 weeks. Rats in group 4 were fed fish oil-dextrose for 8 weeks. Liver samples were analyzed for histopathology, lipid peroxidation, cytochrome P4502E1 activity, nuclear factor-kappaB, and levels of messenger RNA for tumor necrosis factor-alpha, cyclooxygenase-2, and inducible nitric oxide synthase. Concentrations of endotoxin were measured in plasma. The most severe inflammation and fibrosis was detected in groups 1 and 2, as were the highest levels of endotoxin, lipid peroxidation, cytochrome P450 2E1 activity, activation of nuclear factor-kappaB, and mRNA levels for tumor necrosis factor-alpha, cyclooxygenase-2, and inducible nitric oxide synthase. Plasma nitric oxide was also increased as was nitrotyrosine in liver. After arginine was administered, there was marked improvement in the pathological changes accompanied by decreased levels of endotoxin, lipid peroxidation, activation of nuclear factor-kappaB, tumor necrosis factor-alpha, cyclooxygenase-2, inducible nitric oxide, and nitrotyrosine staining. The therapeutic effects of arginine are probably secondary to increased levels of nitric oxide but other effects of arginine cannot be excluded.     

Skin nitric oxide and its metabolites are increased in nonburned skin after thermal injuries

Local and systemic inflammation can lead to progression of burn wounds, converting second- to third-degree wounds or extending the burn to adjacent areas. Previous studies have suggested that the skin is an important site of production of nitric oxide (NO), synthesized by inducible nitric oxide synthase (iNOS) activation after injury. NO increases in burned wounds, but its formation in noninjured skin has not been investigated. We hypothesized that after severe burns, NO and cytotoxic peroxynitrite would increase in noninjured skin. We also tested the hypothesis that BBS-2, a specific inhibitor of iNOS, would impair NO formation after burn. Thirteen female sheep were randomized into burn injury and smoke inhalation (n = 5, group 1), burn and smoke treated with BBS-2 (n = 3, group 2), and sham (saline treatment, no injury) (n = 5, group 3). All the animals, including the sham-injury group, were mechanically ventilated for 48 h. Samples of nonburned skin and plasma were collected from each animal, and levels of NO and its metabolites were evaluated using a NO chemiluminescent detector. Nitrotyrosine and iNOS expression were determined in the skin by Immunoperoxidase staining, and scoring of masked slides (epidermis, hair follicles, vessels, glands, and stroma) was performed. Skin NO and metabolites significantly increased in the burn and smoke injury group, and this was inhibited by BBS-2. Nitrotyrosine expression also increased significantly in the skin of burned animals. BBS-2 prevented the increase of NOx but not the increase of nitrotyrosine expression in skin. Plasma levels of NO increased in burned animals when compared with sham, but this increase was not significant. The increase of NO and its metabolites after burn in noninjured skin is followed by a significant increase in peroxynitrite, a potent cytotoxic mediator.     

Activation of alveolar macrophages in acid-injured lung in rats: different effects of pentoxifylline on tumor necrosis factor-alpha and nitric oxide production

OBJECTIVE: To determine whether acid instillation augments tumor necrosis factor-alpha and nitric oxide production by alveolar macrophages in rats, and to study the effects of treatment with pentoxifylline before acid instillation on the production of these inflammatory mediators. DESIGN: Controlled laboratory investigation on tumor necrosis factor-alpha and nitric oxide production by alveolar macrophages of rats that had acid-induced lung injury. SETTING: University research laboratory. SUBJECT: Alveolar macrophages of rats. INTERVENTIONS: Alveolar macrophages were recovered by bronchoalveolar lavage at 4, 10, 16, 24, and 72 hrs after unilateral hydrochloric acid (pH, 1.0; volume, 0.1 mL) instillation into the lungs of rats. Alveolar macrophages then were cultured with or without lipopolysaccharide. One group of rats was pretreated with pentoxifylline before acid instillation. MEASUREMENTS AND MAIN RESULTS: Alveolar macrophages from both acid-instilled and contralateral lungs, which had recovered 24 hrs after acid instillation, produced significantly greater tumor necrosis factor-alpha and nitric oxide. Subsequent exposure to lipopolysaccharide, as a surrogate for bacterial infection, further promoted tumor necrosis factor-alpha and nitric oxide release. Alveolar macrophages from rats pretreated with pentoxifylline before acid instillation produced significantly less tumor necrosis factor-alpha and did not overproduce tumor necrosis factor-alpha when exposed to lipopolysaccharide. In contrast, pretreatment with pentoxifylline had no effect on nitric oxide production by alveolar macrophages. CONCLUSIONS: Acid instillation stimulates alveolar macrophages to produce tumor necrosis factor-alpha and nitric oxide. Pentoxifylline preserved innate production of tumor necrosis factor-alpha to lipopolysaccharide and did not inhibit the production of bactericidal nitric oxide. This may partly explain why pentoxifylline reduces acid aspiration-induced lung injury while maintaining the host's ability to combat bacterial infection after acid aspiration.     

Okanin, a chalcone found in the genus Bidens, and 3-penten-2-one inhibit inducible nitric oxide synthase expression via heme oxygenase-1 induction in RAW264.7 macrophages activated with lipopolysaccharide

Excess production of nitric oxide by activated macrophages via inducible nitric oxide synthase leads to the development of various inflammatory diseases. Heme oxygenase-1 expression via activation of nuclear factor-erythroid 2-related factor 2 inhibits nitric oxide production and inducible nitric oxide synthase expression in activated macrophages. Okanin is one of the most abundant chalcones found in the genus Bidens (Asteraceae) that is used as various folk medications in Korea and China for treating inflammation. Here, we found that okanin (possessing the α-β unsaturated carbonyl group) induced heme oxygenase-1 expression via nuclear factor-erythroid 2-related factor 2 activation in RAW264.7 macrophages. 3-Penten-2-one, of which structure, as in okanin, possesses the α-β unsaturated carbonyl group, also induced nuclear factor-erythroid 2-related factor 2-dependent heme oxygenase-1 expression, while both 2-pentanone (lacking a double bond) and 2-pentene (lacking a carbonyl group) were virtually inactive. In lipopolysaccharide-activated RAW264.7 macrophages, both okanin and 3-penten-2-one inhibited nitric oxide production and inducible nitric oxide synthase expression via heme oxygenase-1 expression. Collectively, our findings suggest that by virtue of its α-β unsaturated carbonyl functional group, okanin can inhibit nitric oxide production and inducible nitric oxide synthase expression via nuclear factor-erythroid 2-related factor 2-dependent heme oxygenase-1 expression in lipopolysaccharide-activated macrophages.     

Role of nitric oxide in experimental obliterative bronchiolitis (chronic rejection) in the rat.

The role of nitric oxide in obliterative bronchiolitis development, i.e., chronic rejection, was investigated in the heterotopic rat tracheal allograft model. An increase in the intragraft inducible nitric oxide synthase (iNOS) mRNA and mononuclear inflammatory cell iNOS immunoreactivity was demonstrated during progressive loss of respiratory epithelium and airway occlusion in nontreated allografts compared to syngeneic grafts. In nontreated allografts, however, intragraft nitric oxide production was decreased, most likely because of loss of iNOS epithelial expression. Treatment with aminoguanidine, a preferential inhibitor of inducible nitric oxide synthase, was associated with enhanced proliferation of alpha-smooth muscle actin immunoreactive cells and the intensity of obliterative bronchiolitis early after transplantation. Aminoguanidine treatment did not affect iNOS mRNA synthesis or intragraft nitric oxide production, but decreased iNOS immunoreactivity in smooth muscle cells. Treatment with L-arginine, a precursor of nitric oxide, significantly reduced obliterative changes. L-arginine supplementation enhanced intragraft iNOS mRNA synthesis and iNOS immunoreactivity in capillary endothelial and smooth muscle cells as well as intragraft nitric oxide production. Immunohistochemical analysis of allografts showed that neither iNOS inhibition nor supplementation of the nitric oxide pathway affected the number of graft-infiltrating CD4+ and CD8+ T cells, ED1+ and ED3+ macrophages, immune activation with expression of IL-2R or MHC class II, or production of macrophage or Th1 cytokines. In contrast, L-arginine treatment was associated with increased staining for Th2 cytokines IL-4 and IL-10. In conclusion, this study demonstrates that nitric oxide has a protective role in obliterative bronchiolitis development in this model, and suggests that nitric oxide either directly or indirectly inhibits smooth muscle cell proliferation and modulates immune response towards Th2 cytokines.     

Effects of chronic nitric oxide activation or inhibition on early hepatic fibrosis in rats with bile duct ligation

Hepatic fibrosis or increased liver collagen contents drive functional abnormalities that, when extensive, may be life threatening. The purpose of this study was to assess the effects of the chronic stimulation or inhibition of nitric oxide synthesis in rats with hepatic fibrosis induced by permanent common bile duct ligation (3 weeks) and the role of expression of the different nitric oxide synthase isoforms. Bile duct ligation led to an important accumulation of collagen in the hepatic parenchyma, as shown both histologically and by the hydroxyproline contents of livers. Bilirubin and serum enzyme activities (measured as markers of cholestasis) increased several-fold after bile duct ligation. The area of fibrotic tissue, liver hydroxyproline content and serum markers of cholestasis were clearly related in obstructed rats. The absence of modifications in haemodynamic parameters excludes circulatory changes from being responsible for the development of liver alterations. In animals treated with NG-nitro-L-arginine methyl ester (L-NAME) the area of fibrosis was similar to that of untreated animals, the signs of cholestasis and cellular injury being more evident. In rats treated with L-arginine the area of fibrosis was almost three times larger than that found in bile duct ligated rats and in L-NAME-treated bile duct ligated rats, although the observed biochemical changes were similar to those seen in rats treated with L-NAME. Our results with inducible nitric oxide synthase, obtained by Western blots and immunohistochemistry, indicate a greater expression of the inducible enzyme in bile duct ligated and L-arginine-treated animals and a lower expression in the L-NAME and control groups. Constitutive nitric oxide synthase expression, obtained by Western blots, was very similar in all groups, except for the L-arginine-treated rats in which it was lower. These results suggest that nitric oxide production may be a key factor in the development of fibrosis in bile duct ligated rats. They also support the hypothesis of a dual role for nitric oxide; one beneficial, mediated by its circulatory effects, and the second negative, through its local toxic effects.     

Oral spermine administration inhibits nitric oxide-mediated intestinal damage and levels of systemic inflammatory mediators in a mouse endotoxin model

Enhanced intestinal nitric oxide production observed during sepsis is thought to play a central role in lipopolysaccharide-induced intestinal damage. In contrast intestinal polyamines, both from endogenous and exogenous origin, are essential for the maintenance of mucosal integrity. Polyamines have been shown to inhibit lipopolysaccharide-induced nitric oxide release in vitro and have been claimed to exert additional antiinflammatory actions. In this study, the effect of the polyamine spermine on the release of the proinflammatory mediators nitric oxide and tumor necrosis factor-alpha by a murine macrophage cell line was investigated. Furthermore, we investigated whether oral spermine administration inhibits lipopolysaccharide-induced intestinal inducible nitric oxide synthase and nitrotyrosine expression and modulates the release of inflammatory mediators. Our results show that although spermine inhibited lipopolysaccharide-induced nitric oxide release in a murine macrophage cell line, no effect on tumor necrosis factor-alpha release was observed. In addition, oral spermine administration inhibited intestinal inducible nitric oxide synthase and nitrotyrosine expression suggesting a protective effect of spermine on lipopolysaccharide-induced intestinal damage. In parallel a decrease in serum levels of the proinflammatory mediators nitrate, nitrite, and interferon-gamma and an increase in the antiinflammatory cytokine interleukin-10 was observed, although tumor necrosis factor-alpha levels were unaffected. These results indicate that spermine inhibits lipopolysaccharide-induced nitric oxide release in vitro as well as in vivo. Further, intraluminally derived polyamines modulate the systemic immune response. It is concluded that oral spermine administration might have therapeutic perspectives for several disorders characterized by systemic inflammation and intestinal damage.     

Continuous inhalation of nitric oxide protects against development of pulmonary hypertension in chronically hypoxic rats.

Exposure to hypoxia and subsequent development of pulmonary hypertension is associated with an impairment of the nitric oxide (NO) mediated response to endothelium-dependent vasodilators. Inhaled NO may reach resistive pulmonary vessels through an abluminal route. The aim of this study was to investigate if continuous inhalation of NO would attenuate the development of pulmonary hypertension in rats exposed to chronic hypoxia. In conscious rats previously exposed to 10% O2 for 3 wk, short-term inhalation of NO caused a dose-dependent decrease in pulmonary artery pressure (PAP) from 44 +/- 1 to 32 +/- 1 mmHg at 40 ppm with no changes in systemic arterial pressure, cardiac output, or heart rate. In normoxic rats, acute NO inhalation did not cause changes in PAP. In rats simultaneously exposed to 10% O2 and 10 ppm NO during 2 wk, right ventricular hypertrophy was less severe (P < 0.01), and the degree of muscularization of pulmonary vessels at both alveolar duct and alveolar wall levels was lower (P < 0.01) than in rats exposed to hypoxia alone. Tolerance to the pulmonary vasodilator effect of NO did not develop after prolonged inhalation. Brief discontinuation of NO after 2 wk of hypoxia plus NO caused a rapid increase in PAP. These data demonstrate that prolonged inhalation of low concentrations of NO induces sustained pulmonary vasodilation and reduces pulmonary vascular remodeling in response to chronic hypoxia.     

Right man,right time,right place? - On the time course of the mediator orchestra in septic shock

Appropriate timing of treatment assumes particular importance in critical care. Lange and colleagues recently reported on the time course of the different nitric oxide synthase (NOS) isoforms, nitrosative stress, and poly(ADP-ribosylation) during Pseudomonas aeruginosa pneumonia-induced ovine septic shock. Initially, endothelial NOS expression was increased together with markers of peroxynitrite formation, DNA damage, and nuclear factor-kappa-B activation. Later on, measurable NOS activity and nitric oxide production resulted mainly from inducible NOS activation. These results emphasize the need for long-term, large-animal studies investigated over days so that future therapeutic interventions can be better tailored and matched to the exact time course of the activation of the mediator orchestra.     

Intensified regression of colon cancer liver metastases in mice treated with irinotecan and the immunomodulator JBT 3002

The authors recently reported that tumoricidal activation of macrophages by a new synthetic bacterial lipopeptide, JBT 3002, can augment chemotherapy-mediated tumor-cell killing. The aim of this study was to identify the mechanism responsible for the destruction of metastatic cells. Three daily oral doses of JBT 3002 before once-weekly intraperitoneal injections of 100 mg/kg irinotecan for 3 weeks significantly increased the eradication of established CT-26 murine colon cancer liver metastases compared with treatment with irinotecan alone. Immunohistochemical analyses revealed that the hepatic metastases in mice given combination therapy contained infiltrating CD8+ lymphocytes and a dense infiltrate of macrophages expressing both inducible nitric oxide synthase (iNOS) and interleukin-15. In vitro treatment of peritoneal macrophages with JBT 3002 plus interferon-gamma induced the expression of iNOS and the production of nitric oxide. In the presence of a low (subtoxic) dose of irinotecan, these activated macrophages produced significant lysis of CT-26 cells. The high level of cytotoxicity was inhibited by the specific inducible nitric oxide synthase inhibitor, NG-methyl-L-arginine. In contrast, irinotecan-mediated lysis of normal intestinal epithelial IEC-6 cells was not increased by activated macrophages. Scanning electron microscopy revealed that activated macrophages bound to CT-26 tumor cells but not to normal IEC-6 cells, confirming that nitric oxide-mediated cytotoxicity is specific for tumor cells. Collectively, the results suggest that augmentation of the therapeutic efficacy of irinotecan against colon cancer liver metastases by immunomodulation with JBT 3002 may be associated with elevated inducible nitric oxide synthase and endogenous interleukin-15 in tumor-infiltrating macrophages.     

Cardiac peroxynitrite formation and left ventricular dysfunction following doxorubicin treatment in mice

Selective cardiotoxicity of doxorubicin remains a significant and dose-limiting clinical problem. The mechanisms involved have not been fully defined but may involve the production of reactive oxygen species and/or alteration of cardiac energetics. Here, we tested the hypotheses that doxorubicin causes left ventricular dysfunction in mice and is associated with dysregulation of nitric oxide in cardiac tissue, leading to the accumulation of 3-nitrotyrosine (a biomarker of peroxynitrite formation). Animals were dosed with doxorubicin (20 mg/kg i.p.), and left ventricular performance was assessed in vivo using M-mode and Doppler echocardiography. Five days after doxorubicin administration, left ventricular fractional shortening, cardiac output, and stroke volume parameters were significantly reduced relative to control values (30.0 +/- 3.6 versus 46.1 +/- 1. 6%, 8.9 +/- 0.9 versus 11.5 +/- 0.6 ml/min, and 21.2 +/- 0.1 versus 29.5 +/- 0.1 microl for doxorubicin versus control, P <.05). Statistically significant (P <.05) increases in the immunoprevalence of myocardial inducible nitric oxide synthase (33 +/- 18 versus 9 +/- 2%, via quantitative image analysis) and 3-nitrotyrosine formation (56 +/- 24 versus 0.3 +/- 0.4%) were also observed after doxorubicin. Correlation analyses revealed a highly significant inverse relationship between left ventricular fractional shortening and cardiac 3-nitrotyrosine immunoprevalence (P <.01). No such relationship was observed for inducible nitric oxide synthase. Western blot analyses of cardiac myofibrillar fractions revealed extensive nitration of an abundant 40-kDa protein, shown to be the myofibrillar isoform of creatine kinase. These data demonstrate that alteration of cardiac nitric oxide control and attendant peroxynitrite formation may be an important contributor to doxorubicin-induced cardiac dysfunction. Furthermore, nitration of key myofibrillar proteins and alteration of myocyte energetics are implicated.