Laura Dugo

A role for superoxide in gentamicin-mediated nephropathy in rats

Gentamicin is an antibiotic effective against Gram-negative infection, whose clinical use is limited by its nephrotoxicity. Oxygen free radicals are considered to be important mediators of gentamicin-mediated nephrotoxicity, but the exact nature of the radical in question is not known with certainty. We have investigated the potential role of superoxide in gentamicin-induced renal toxicity by using M40403, a low molecular weight synthetic manganese containing superoxide dismutase mimetic, which selectively removes superoxide. Administration of gentamicin at 100 mg/kg, s.c. for 5 days to rats induced a marked renal failure, characterised by a significant decrease in creatinine clearance and increased plasma creatinine levels, fractional excretion of sodium, lithium, urine gamma glutamyl transferase (gamma GT) and daily urine volume. A significant increase in kidney myeloperoxidase activity and lipid peroxidation was also observed in gentamicin-treated rats. M40403 (10 mg/kg, i.p. for 5 days) attenuated all these parameters of damage. Immunohistochemical localisation demonstrated nitrotyrosine formation and poly(ADP-ribose) synthetase (PARS) activation in the proximal tubule of gentamicin-treated rats. Renal histology examination confirmed tubular necrosis. M40403 significantly prevented gentamicin-induced nitrotyrosine formation, poly(ADP-ribose) synthetase activation and tubular necrosis. These results confirm our hypothesis that superoxide anions play an important role in gentamicin-mediated nephropathy and support the possible clinical use of low molecular weight synthetic superoxide dismutase mimetics in those conditions that are associated with over production of superoxide.

Increased levels of malondialdehyde and nitrite/nitrate in the blood of asphyxiated newborns : reduction by melatonin

Free radicals have been implicated in the pathogenesis of neonatal asphyxia and its complications. This study measured a product of lipid peroxidation, malondialdehyde, and the nitrite/nitrate levels in the serum of 20 asphyxiated newborns before and after treatment with the antioxidant melatonin given within the first 6 hr of life. Ten asphyxiated newborns received a total of 80 mg of melatonin (8 doses of 10 mg each separated by 2‐hr intervals) orally. One blood sample was collected before melatonin administration and two additional blood samples (at 12 and 24 hr) were collected after giving melatonin. A third group of healthy newborn children served as controls. Serum malondialdehyde and nitrite+nitrate concentrations in newborns with asphyxia before treatment were significantly higher than those in infants without asphyxia. In the asphyxiated newborns given melatonin, there were significant reductions in malondialdehyde and nitrite/nitrate levels at both 12 and 24 hr. Three of the 10 asphyxiated children not given melatonin died within 72 hr after birth; none of the 10 asphyxiated newborns given melatonin died. The results indicate that the melatonin may be beneficial in the treatment of newborn infants with asphyxia. The protective actions of melatonin in this study may relate to the antioxidant properties of the indole as well as to the ability of melatonin to increase the efficiency of mitochondrial electron transport.

Pyrrolidine dithiocarbamate attenuates the development of acute and chronic inflammation

The nuclear factor‐κB (NF‐κB) is a transcription factor which plays a pivotal role in the induction of genes involved in physiological processes as well as in the response to injury and inflammation. Dithiocarbamates are antioxidants which are potent inhibitors of NF‐κB. We postulated that pyrrolidine dithiocarbamate (PDTC) would attenuate inflammation. In the present study we investigate the effects of PDTC in animal models of acute and chronic inflammation (carrageenan‐induced pleurisy and collagen‐induced arthritis). We report here for the first time that PDTC (given at 100, 30 or 10 mg kg−1 i.p. in the pleurisy model or at 10 mg kg−1 i.p. every 48 h in the arthritis model) exerts potent anti‐inflammatory effects (e.g. significant reduction of (A) pleural exudate formation, (B) polymorphonuclear cell infiltration, (C) lipid peroxidation, (D) inducible nitric oxide synthase (iNOS) activity and nitric oxide production (E) plasma and pleural exudates levels of interleukin‐1β and tumour necrosis factor‐α, (F) histological injury and (G) delayed development of clinical indicators). Furthermore, PDTC reduced immunohistochemical evidence of (A) formation of nitrotyrosine, (B) activation of poly (ADP‐ribose) polymerase (PARP), (C) expression of iNOS and (D) expression of cyclo‐oxygenase‐2 (COX‐2) in the lungs of carrageenan‐treated mice and in the joints from collagen‐treated mice. Additionally, Western blotting and immunohistochemical analysis of lung tissue revealed that PDTC prevented degradation of IKB‐α and translocation of NF‐κB from the cytoplasm into the nucleus. Taken together, our results clearly demonstrate that prevention of the activation of NF‐κB by PDTC reduces the development of acute and chronic inflammation. Therefore, inhibition of NF‐κB may represent a novel approach for the therapy of inflammation.

Protective effects of cyanidin-3-O-glucoside from blackberry extract against peroxynitrite-induced endothelial dysfunction and vascular failure

Anthocyanins are a group of naturally occurring phenolic compounds as colorants in several plants, flowers and fruits. These pigments have a great importance as quality indicators, as chemotaxonomic markers and antioxidants. The content of blackberry (Rubus species) juice was investigated by HPLC/ESI/MS using narrow bore HPLC columns. Using this method we demonstrated that cyanidin-3-O-glucoside represents about 80% of the total anthocyanin contents in blackberry extract. Here we investigated antioxidant activity of the blackberry juice and cyanidin-3-O-glucoside on the endothelial dysfunction in cells and in vascular rings exposed to peroxynitrite. In human umbilical vein endothelial cells (HUVEC) in vitro, peroxynitrite caused a significant suppression of mitochondrial respiration (38 +/- 2.1% of control cells), as measured by the mitochondrial-dependent conversion of the dye MTT to formazan. Peroxynitrite caused DNA strand breakage (63 +/- 1.9% single strand vs 3 +/- 0.9% single strand in control cells), as measured by the alkaline unwinding assay, and caused an activation of PARS, as measured by the incorporation of radiolabeled NAD(+) to nuclear proteins. Blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM; 0.028 microM; 0.0085 microM) reduced the peroxynitrite-induced suppression of mitochondrial respiration, DNA damage and PARS activation in HUVECs. Vascular rings exposed to peroxynitrite exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine as well as a vascular contractility dysfunction in response to norepinephrine. The development of this peroxynitrite-induced vascular dysfunction was ameliorated by the blackberry juice (different dilutions that contained 80 ppm;40 ppm;14.5 ppm of cyanidin-3-O-glucoside) and cyanidin-3-O-glucoside (as chloride) (0.085 microM;0.028 microM;0.0085 microM). In conclusion our findings clearly demonstrate that blackberry juice containing cyanidin-3-O-glucoside is a scavenger of peroxynitrite and that exert a protective effect against endothelial dysfunction and vascular failure induced by peroxynitrite.

Gsk-3β inhibitors attenuate the organ injury/dysfunction caused by endotoxemia in the rat

Objective:Serine-threonine protein kinase glycogen synthase kinase (GSK)-3 is involved in regulation of many cell functions, but its role in regulation of inflammatory response is unknown. Here we investigate the effects of GSK-3β inhibition on organ injury/dysfunction caused by lipopolysaccharide or coadministration of lipopolysaccharide and peptidoglycan in the rat. Design:Prospective, randomized study. Setting:University-based research laboratory. Subjects:Ninety-nine anesthetized male Wistar rats. Interventions:Study 1: Rats received either intravenous Escherichia coli lipopolysaccharide (6 mg/kg) or vehicle (1 mL/kg; saline). Study 2: Rats received either intravenous E. coli lipopolysaccharide (1 mg/kg) and Staphylococcus aureus peptidoglycan (0.3 mg/kg) or vehicle. The potent and selective GSK-3β inhibitors TDZD-8 (1 mg/kg intravenously), SB216763 (0.6 mg/kg intravenously), and SB415286 (1 mg/kg intravenously) or vehicle (10% dimethyl sulfoxide) was administered 30 mins before lipopolysaccharide or lipopolysaccharide and peptidoglycan. Measurements and Main Results:Endotoxemia resulted in increases in the serum levels of creatinine (indicator of renal dysfunction), aspartate aminotransferase, alanine aminotransferase (markers for hepatocellular injury), lipase (indicator of pancreatic injury), and creatine kinase (indicator of neuromuscular injury). Coadministration of lipopolysaccharide and peptidoglycan resulted in hepatocellular injury and renal dysfunction. All GSK-3β inhibitors attenuated the organ injury/dysfunction caused by lipopolysaccharide or lipopolysaccharide and peptidoglycan. GSK-3β inhibition reduced the Ser536 phosphorylation of nuclear factor-&kgr;B subunit p65 and the messenger RNA expression of nuclear factor-&kgr;B-dependent proinflammatory mediators but had no effect on the nuclear factor-&kgr;B/DNA binding activity in the lung. GSK-3β inhibition reduced the increase in nuclear factor-&kgr;B p65 activity caused by interleukin-1 in human embryonic kidney cells in vitro. Conclusions:The potent and selective GSK-3β inhibitors TDZD-8, SB216763, and SB415286 reduced the organ injury/dysfunction caused by lipopolysaccharide or lipopolysaccharide and peptidoglycan in the rat. We propose that GSK-3β inhibition may be useful in the therapy of the organ injury/dysfunction associated with sepsis, shock, and other diseases associated with local or systemic inflammation.

Protective effects of a new stable, highly active SOD mimetic, M40401 in splanchnic artery occlusion and reperfusion

Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of M40401, a new S,S‐dimethyl substituted biscyclohexylpyridine Mn‐based superoxide dismutase mimetic (SODm, kcat=1.2×10+9 M−1 s−1 at pH=7.4), in rats subjected to SAO shock. Treatment of rats with M40401 (applied at 0.25, 2.5 or 25 μg kg−1, 15 min prior to reperfusion), attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO‐shock. M40401 also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in ileum from SAO‐shocked rats. The degree of staining for nitrotyrosine was markedly reduced in tissue sections obtained from SAO‐shocked rats which had received M40401. Reperfused ileum tissue sections from SAO‐shocked rats showed positive staining for P‐selectin and for anti‐intercellular adhesion molecule (ICAM‐1) in the vascular endothelial cells. M40401 treatment markedly reduced the intensity and degree of P‐selectin and ICAM‐1 in tissue sections from SAO‐shocked rats. M40401 treatment significantly improved survival. Additionally, the very high catalytic activity of this new mimetic (comparable to the native human Cu/Zn SOD enzyme and exceeding the activity of the human Mn SOD enzyme) translates into a very low dose (∼μg kg−1) required to afford protection in this SAO model of ischemia reperfusion injury. Taken together, our results clearly demonstrate that M40401 treatment exerts a protective effect, and part of this effect may be due to inhibition of the expression of adhesion molecules and peroxynitrite‐related pathways with subsequent reduction of neutrophil‐mediated cellular injury.

LC-MS for the identification of oxygen heterocyclic compounds in citrus essential oils

The oxygen heterocyclic compounds (coumarins, psoralens and polymethoxylated flavones) present in the nonvolatile residue of the essential oils of Mandarin, Sweet Orange, Bitter Orange, Bergamot and Grapefruit were analysed with an HPLC/API/MS system equipped with an APcI probe in positive mode. The use of hyphenated techniques, such as LC/MS provides a great information about the content and nature of constituents of natural complex matrices, such as essential oils. In this work, MS spectra were recorded at different voltages, to obtain structural information in addition to molecular weight information. The different response of the compounds identified has been also evaluated. The method allowed the confirmation of the identification of the main components of the fraction, previously reported for the different oils. MS characteristics of coumarins, psoralens and polymethoxylated flavones with different substitution patterns were determined on the basis of the response obtained with the APcI interface. Interface parameters were optimised to obtain a contemporaneous response for all the three classes of components.

Protective effects of n-acetylcysteine on lung injury and red blood cell modification induced by carrageenan in the rat

Oxidative stress has been suggested as a potential mechanism in the pathogenesis of lung inflammation. The pharmacological profile of n‐acetylcysteine (NAC), a free radical scavenger, was evaluated in an experimental model of lung injury (carrageenan‐induced pleurisy). Injection of carrageenan into the pleural cavity of rats elicited an acute inflammatory response characterized by fluid accumulation in the pleural cavitythat contained manyneutrophils (PMNs), an infiltration of PMNs in lung tissues and subsequent lipid peroxidation, and increased production of nitrite/nitrate, tumor necrosis factor α, and interleukin 1β. All parameters of inflammation were attenuated by NAC treatment. Furthermore, carrageenan induced an up‐regulation of the adhesion molecules ICAM‐1 and P‐selectin, as well as nitrotyrosine and poly (ADP‐ribose) synthetase (PARS), as determined by immuno‐histochemical analysis of lung tissues. The degree of staining for the ICAM‐1, P‐selectin, nitrotyrosine, and PARS was reduced by NAC. In vivo NAC treatment significantly reduced peroxynitrite formation as measured by the oxidation of the fluorescent dihydrorho‐damine‐123, prevented the appearance of DNA damage, an decrease in mitochondrial respiration, and partially restored the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan‐induced pleurisy. A significant alteration in the morphology of red blood cells was observed 24 h after carrageenan administration. NAC treatment has the ability to significantly diminish the red blood cell alteration. Our results clearly demonstrate that NAC treatment exerts a protective effect and clearly indicate that NAC offers a novel therapeutic approach for the management of lung injury where radicals have been postulated to play a role.—Cuzzocrea, S., Mazzon, E., Dugo, L., Serraino, I., Ciccolo, A., Centorrino, T., De Sarro, A., Caputi, A. P. Protective effects of n‐acetylcysteine on lung injury and red blood cell modification induced by carrageenan in the rat. FASEB J. 15, 1187–1200 (2001)

Rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2, ligands of the peroxisome proliferator-activated receptor-γ (PPAR-γ), reduce ischaemia/reperfusion injury of the gut

The peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is a member of the nuclear receptor superfamily of ligand‐dependent transcription factors related to retinoid, steroid and thyroid hormone receptors. The thiazolidinedione rosiglitazone and the endogenous cyclopentenone prostaglandin (PG)D2 metabolite, 15‐deoxy‐Δ12,14‐PGJ2 (15d‐PGJ2), are two PPAR‐γ ligands, which modulate the transcription of target genes. The aim of this study was to investigate the effect of rosiglitazone and 15d‐PGJ2 on the tissue injury caused by ischaemia/reperfusion (I/R) of the gut. I/R injury of the intestine was caused by clamping both the superior mesenteric artery and the coeliac trunk for 45 min, followed by release of the clamp allowing reperfusion for 2 or 4 h. This procedure results in splanchnic artery occlusion (SAO) shock. Rats subjected to SAO developed a significant fall in mean arterial blood pressure, and only 10% of the animals survived for the entire 4 h reperfusion period. Surviving animals were killed for histological examination and biochemical studies. Rats subjected to SAO displayed a significant increase in tissue myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels, significant increases in plasma tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β levels and marked injury to the distal ileum. Increased immunoreactivity to nitrotyrosine was observed in the ileum of rats subjected to SAO. Staining of sections of the ileum obtained from SAO rats with anti‐intercellular adhesion molecule (ICAM‐1) antibody resulted in diffuse staining. Administration at 30 min prior to the onset of gut ischaemia of the two PPAR‐γ agonists (rosiglitazone (0.3 mg kg−1 i.v.) and 15d‐PGJ2 (0.3 mg kg−1 i.v.)) significantly reduced the (i) fall in mean arterial blood pressure, (ii) mortality rate, (iii) infiltration of the reperfused intestine with polymorphonuclear neutrophils (MPO activity), (iv) lipid peroxidation (MDA levels), (v) production of proinflammatory cytokines (TNF‐α and IL‐1β) and (vi) histological evidence of gut injury. Administration of rosiglitazone and 15d‐PGJ2 also markedly reduced the nitrotyrosine formation and the upregulation of ICAM‐1 during reperfusion. In order to elucidate whether the protective effects of rosiglitazone and 15d‐PGJ2 are related to the activation of the PPAR‐γ receptor, we also investigated the effect of a PPAR‐γ antagonist, bisphenol A diglycidyl ether (BADGE), on the protective effects of rosiglitazone and 15d‐PGJ2. BADGE (1 mg kg−1 administered i.v. 30 min prior to the treatment of rosiglitazone or 15d‐PGJ2) significantly antagonised the effect of the two PPAR‐γ agonists and thus abolished the protective effect against gut I/R. These results demonstrate that the two PPAR‐γ agonists, rosiglitazone and 15d‐PGJ2, significantly reduce I/R injury of the intestine.

Glycogen synthase kinase 3beta as a target for the therapy of shock and inflammation.

After the discovery that glycogen synthase kinase (GSK) 3&bgr; plays a fundamental role in the regulation of the activity of nuclear factor &kgr;B, a number of studies have investigated the effects of this protein kinase in the regulation of the inflammatory process. The GSK-3&bgr; inhibition, using genetically modified cells and chemically different pharmacological inhibitors, affects the regulation of various inflammatory mediators in vitro and in vivo. Insulin, an endogenous inhibitor of GSK-3 in the pathway leading to the regulation of glycogen synthase activity, has recently been clinically used in the therapy for septic shock. The beneficial anti-inflammatory effects of insulin in preclinical and clinical studies could possibly be due, at least in part, to the inhibition of GSK-3 and not directly correlated to the regulation of blood glucose. We describe the latest studies describing the effects of GSK-3 inhibition as potential target of the therapy for diseases associated with inflammation, ischemia/reperfusion, and shock.