Intensive Care Med (2003) 29:2016–2025 DOI 10.1007/s00134-003-1887-8

Salvatore Cuzzocrea Antonietta Rossi Barbara Pisano Rosanna Di Paola Tiziana Genovese Nimesh S. A. Patel Elisabetta Cuzzocrea Angela Ianaro Lidia Sautebin Francesco Fulia Prabal K. Chatterjee Achille P. Caputi Christoph Thiemermann Received: 24 December 2002 Accepted: 4 June 2003 Published online: 17 July 2003 © Springer-Verlag 2003 This work was supported by Ministero Publica Istruzione, Fondi 40%. P.K.C. is funded by The National Kidney Research Fund (Grant R41/2/2000), and C.T. is a Senior Fellow of the British Heart Foundation (FS96/018). An editorial regarding this article can be found in the same issue (http://dx.doi.org/10.1007/s00134-0031932-7)

S. Cuzzocrea (✉) · R. Di Paola T. Genovese · A. P. Caputi Institute of Pharmacology, School of Medicine, Torre Biologicala, Policlinico Universitario, University of Messina, Via C. Valeria Gazzi, 98100 Messina, Italy e-mail: [email protected] Tel.: +39-090-2213644 Fax: +39-090-694951 A. Rossi · B. Pisano · A. Ianaro L. Sautebin Department of Experimental Pharmacology, Università Federico II, Via D. Montesano 49, 80131 Naples, Italy N. S. A. Patel · P. K. Chatterjee C. Thiemermann Department of Experimental Medicine and Nephrology, William Harvey Research Institute, Queen Mary, University of London, Charterhouse Square, London, EC1 M 6BQ UK

E X P E R I M E N TA L

Pyrrolidine dithiocarbamate attenuates the development of organ failure induced by zymosan in mice

E. Cuzzocrea Anaesthetics and Intensive Care Department, University Hospital of Messina, Italy F. Fulia Neonatal Intensive Care Unit, Barone Romeo Hospital, Patti, Italy

Abstract Objective: Nuclear factor (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 anti-oxidants which are potent inhibitors of NFκB. We postulated that pyrrolidine dithiocarbamate (PDTC) would attenuate multiple-organ failure (MOF). Design and setting: Rats in a university research laboratory Interventions and measurements: We investigated the effects of PDTC (10 mg/kg) on the MOF caused by zymosan (500 mg/kg, administered i.p. as a suspension in saline) in mice. MOF in mice was assessed 18 h after administration of zymosan and/or PDTC and monitored for 7 days (for loss of body weight and mortality). Results: Treatment of mice with PDTC (10 mg/kg i.p., 1 and 6 h after zymosan) attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan. PDTC also at-

tenuated the lung, liver and pancreatic injury and renal dysfunction caused by zymosan as well as the increase in myeloperoxidase activity and malondialdehyde levels caused by zymosan in the lung, liver and intestine. Immunohistochemical analysis for inducible nitric oxide synthase, nitrotyrosine and poly(ADPribose) revealed positive staining in lung, liver and intestine tissues obtained from zymosan-treated mice. The degree of staining for nitrotyrosine and poly(ADP-ribose) were markedly reduced in tissue sections obtained from zymosan-treated mice which received PDTC. Furthermore, treatment of mice with PDTC significantly reduced the expression of nitric oxide synthase in lung, liver and intestine. Conclusions: This study provides the first evidence that PDTC attenuates the degree of zymosan-induced MOF in mice. Keywords Zymosan-induced multiple organ failure · Pyrrolidine dithiocarbamate · Nuclear factor-κB · Inducible nitric oxide synthase · Nitric oxide · Inflammation

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Introduction The expression of inducible genes leading to the formation of these proteins relies on transcription factors, which are either controlled by (other) inducible genes and hence require de novo protein synthesis or, alternatively, by so-called “primary transcription factors”. Among the latter nuclear factor (NF) κB has received a considerable amount of attention because of its unique mechanism of activation, its active role in cytoplasmic/nuclear signalling, and its rapid response to pathogenic stimulation [1]. NF-κB plays a central role in the regulation of many genes responsible for the generation of mediators or proteins in inflammation including the genes for tumor necrosis factor (TNF) α, interleukin (IL) 1, IL-6, IL-8, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, inducible nitric oxide synthase (iNOS) and cyclo-oxygenase 2, to name but a few [1]. The discovery in 1997 that inhibition of the activation of NF-κB may be useful in conditions associated with local or systemic inflammation stimulated the search for agents which prevent the activation of NF-κB [2]. The dithiocarbamates are a class of anti-oxidants reported to be potent inhibitors of NF-κB in vitro [3]. More recently it has been shown that the most effective NF-κB inhibitor is the pyrrolidine derivative of dithiocarbamate (pyrrolidine dithiocarbamate, PDTC) as a result of its ability to traverse the cell membrane and its prolonged stability in solution at physiological pH [4]. The potential for modulating both cell activation and the effects of oxidants with the dithiocarbamates suggests that these agents offer therapeutic benefit in acute and chronic inflammatory conditions in which activation of NK-κB plays a major role. This study investigates the effects of PDTC in an animal model of zymosan-induced multiple-organ failure (MOF) in the mice. To gain a better insight into the mechanism(s) of action of PDTC we also investigated its effects on the expression of iNOS (the expression of which is associated with NF-κB activation), the nitration of cellular proteins by peroxynitrite and the activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP).

Material and methods Animals Male CD mice (20–22 g; Charles River, Milan, Italy) were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192) and with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986)

Zymosan-induced generalized inflammation Animals were randomly divided into four groups (ten in each group). The first group were treated intraperitoneally (i.p.) with vehicle (saline) for PDTC and served as a sham group. The second group was treated with zymosan (500 mg/kg, suspended in saline solution, i.p.). In the third and fourth groups mice received PDTC (10 mg/kg, i.p.) 1 h and 6 h after zymosan or saline administration. At 18 h after administration of zymosan, animals were assessed for MOF as described below. In another set of experiments animals (ten in each group) were randomly divided into four groups (as described treated above) and monitored for loss of body weight and mortality for 12 days after administration of zymosan or saline. The dose of 10 mg/kg was based on those used in previous studies [5]. Assessment of acute peritonitis Eighteen hours after zymosan or saline injection all animals (n=10 for each group) were killed under ether anaesthesia to evaluate the development of acute inflammation in the peritoneum. Through an incision in the linea alba 10 ml phosphate buffer saline (PBS, composition in mM: NaCl 137, KCl 2.7, NaH2PO4 1.4, Na2HPO4 4.3, pH 7.4) was injected into the abdominal cavity. Washing buffer was removed with a plastic pipette and was transferred into a 10-ml centrifuge tube. The amount of exudate was calculated by subtracting the volume injected (10 ml) from the total volume recovered. Peritoneal exudate was centrifuged at 7000 g for 10 min at room temperature. Cells were suspended in PBS and counted using an optical microscope in a Burker’s chamber after vital staining with trypan blue. Measurement of nitrite+nitrate concentrations Nitrite/nitrate (NOx) production, an indicator of NO synthesis, was measured in plasma samples collected 18 h after zymosan or saline administration. Plasma was incubated with nitrate reductase (0.1 U/ml) and NADPH (1 mM) and flavin adenine dinucleotide (50 µM) at 37°C for 15 min followed by another incubation with lactate dehydrogenase (100 U/ml) and sodium pyruvate (10 mM) for 5 min. The nitrite concentration in the samples was measured by the Griess reaction by adding 100 µl Griess reagent [0.1% (w/v) naphthylethylene diamide dihydrochloride in H2O and 1% (w/v) sulphanilamide in 5% (v/v) concentrated H2PO4; vol. 1:1] to 100 µl sample. The optical density at 550 nm (OD550) was measured using an enzyme-linked immunosorbent assay microplate reader (SLT-Lab Instruments Salzburg, Austria). Nitrate concentrations were calculated by comparison with OD550 of standard solutions of sodium nitrate prepared in saline solution. Immunohistochemical localization of iNOS, nitrotyrosine and PAR Tyrosine nitration and PAR activation were detected as previously described [5] in lung and intestine sections using immunohistochemistry. At 18 h after zymosan or saline injection tissues were fixed in 10% (w/v) PBS-buffered formalin, and 8-µm sections were prepared from paraffin embedded tissues. After deparaffinization endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (v/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin (DBA, Milan, Italy). The sections were then incubated overnight with

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1:1000 dilution of primary anti-nitrotyrosine antibody (DBA), anti-poly(ADP-ribose) (PAR) antibody (DBA), primary anti-iNOS (DBA) or with control solutions. Controls included buffer alone or non-specific purified rabbit IgG. Specific labelling was detected with a biotin-conjugated specific secondary anti-igG and avidinbiotin peroxidase complex (DBA). Measurement of myeloperoxidase activity and malondialdehyde levels Myeloperoxidase (MPO) activity, which was used as an indicator of polymorphonuclear neutrophil (PMN) infiltration into in lung, liver and intestinal tissues, was measured as previously described [6]. Levels of malondialdehyde (MDA) in lung and intestine tissues were determined as an index of lipid peroxidation as described by Okhawa et al. [7]. Preparation of cytosolic fractions and western blot analysis for IκB-α Extracts of peritoneal macrophages collected 18 h after the zymosan administration from mice treated with or without PDTC were prepared as described [8]. Briefly, harvested cells (2×107) were washed twice with ice-cold PBS and centrifuged at 180 g for 10 min at 4°C. The cell pellet was resuspended in 100 µl ice-cold hypotonic lysis buffer (10 mM hydroxyethylpiperazine ethanesulfonic acid, 1.5 mM MgC12, 10 mM KCI, 0.5 mM phenylmethylsulfonyl fluoride, 1.5 µg/ml soybean trypsin inhibitor, pepstatin A 7 µg/ml, leupeptin 5 µg/ml, 0.1 mM benzamidine, 0.5 mM dithiothreitol) and incubated in ice for 15 min. The cells were lysed by rapid passage through a syringe needle five or six times, and the cytoplasmic fraction was then obtained by centrifugation for 1 min at 13 000 g for 1 min. Protein concentration was determined with the Bio-Rad protein assay kit. Immunoblotting analysis of IκB-α proteins was performed on cytosolic fraction. Cytosolic fraction proteins were mixed with gel loading buffer (50 mM Tris, 10% sodium dodecyl sulfate, 10% glycerol/10% 2-mercaptoethanol, 2 mg bromophenol per millilitre) at a ratio of 1:1, boiled for 3 min and centrifuged at 10000 g for 10 min. Protein concentration was determined and equivalent amounts (75 µg) of each sample were electrophoresed in a 12% discontinuous polyacrylamide minigel. The proteins were transferred onto nitrocellulose membranes according to the manufacturer’s instructions (Bio-Rad). The membranes were saturated by incubation at 4°C overnight with 10% non-fat dry milk in PBS and then incubated with anti-IκB-α (1:1000) for 1 h at room temperature. The membranes were washed three times with 1% Triton X-100 in PBS and then incubated with anti-rabbit immunoglobulins coupled to peroxidase (1:1000). The immune complexes were visualized by the enhanced chemiluminescence method (Amersham). Subsequently the relative expression of the proteins was quantified by densitometric scanning of the X-ray films with GS-700 Imaging Densitometer (Bio-Rad) and a computer program (Molecular Analyst, IBM).

Quantification of organ function and injury Blood samples were taken at 18 h after zymosan or saline injection. The blood sample was centrifuged (1610 g for 3 min at room temperature) to separate plasma. All plasma samples were analysed within 24 h by a veterinary clinical laboratory using standard laboratory techniques. The following marker enzymes were measured in the plasma as biochemical indicators of multiple organ injury/dysfunction: (a) Liver injury was assessed by measuring the rise in plasma levels of alanine aminotransferase (ALT, a specific marker for hepatic parenchymal injury) and aspartate aminotransferase (AST, a non-specific marker for hepatic injury) [9]. (b) Renal dysfunction was assessed by measuring the rises in plasma levels of creatinine (an indicator of reduced glomerular filtration rate and hence renal failure) [10]. (c) In addition, serum levels of lipase and amylase were determined as an indicator of pancreatic injury. Light microscopy Lung and small intestine sample were taken 18 h after zymosan or saline injection. The tissue slices were fixed in Dietric solution [14.25% (v/v) ethanol, 1.85% (w/v) formaldehyde, 1% (v/v) acetic acid] for 1 week at room temperature, dehydrated by graded ethanol and embedded in Paraplast (Sherwood Medical, Mahwah, N.J., USA). Sections (thickness 7 µm) were deparaffinized with xylene, stained with haematoxylin and eosin and observed in Dialux 22 Leitz microscope. Materials Unless stated otherwise, all reagents and compounds used were obtained from Sigma (Milan, Italy). Data analysis All values in the figures and text are expressed as mean ±standard error of the mean ofn observations. For the in vitro studies data represent the number of wells studied (6–9 wells from two or three independent experiments). For the in vivo studies n is the number of animals studied. In the experiments involving histology or immunohistochemistry the figures shown are representative of at least three experiments performed on different experimental days. The results were analysed by one-way analysis of variance followed by Bonferroni’s post-hoc test for multiple comparisons. A p value less than 0.05 was considered significant. Statistical analysis for survival data was calculated by Fisher’s exact probability test. For such analyses p<0.05 was considered significant. The MannWhitney test was used to examine differences between the body weight and organ weights of control and experimental groups. When this test was used, p<0.05 was considered significant.

Results Cytokine production The levels of TNF-α and IL-1β were evaluated in the plasma at 18 h after zymosan or saline administration. The assay was conducted by using a colorimetric commercial kit (Calbiochem-Novabiochem, La Jolla, Calif., USA).

Effect of zymosan on animal mortality and body weight Administration of zymosan caused a severe illness in the mice characterized by a systemic toxicity and significant loss of body weight (Table 1). The PDTC treatment prevented the development of systemic toxicity and the loss in body weight (Table 1). At the end of observation period (12 days) 80% of zymosan-treated mice were dead.

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Table 1 Effect of PDTC on median toxicity score and weight change during non-septic shock induced by zymosan (ZYM) Time (days) 0

3

Median toxicity score ZYM + vehicle ZYM+ PDTC (10 mg/kg)

0±0 0±0**

Weight change (%) Sham + vehicle ZYM + vehicle ZYM+ PDTC (10 mg/kg)

−1±2.1 0±1 −1±2.1

6

2.3±0.5 0.8±0.3** 0±2 −13±1.2* −2±2**

9

6.2±0.12* 3±0.12** −1±2.1 −9±1.2* −3±2.1**

12

9±0.1* 3±0.1** 2±2.4 −18±1.7* −5±2.4**

10±0.11* 3.5±0.11** 3±1.9 −18±1.9* −5±1.9**

* p<0.01 vs. sham, ** p<0.01 vs. ZYM

At the end of observation period (12 days) 30% of zymosan-treated mice which were treated with PDTC were dead. Sham animals injected only with saline appeared healthy and active through the entire observation period (data not shown). Effect of zymosan on acute peritonitis At 18 h after administration of zymosan increased formation of turbid exudate was detected (0.728±0.13 ml, Table 2). Trypan blue staining revealed a total PMN infiltration of 4.74±0.94×106/mouse in comparison to sham mice (0.13±0.005×106/mouse, Table 2). Sham animals demonstrated no abnormalities in the peritoneal cavity or fluid (Table 2). The degree of peritoneal exudation and PMN migration was significantly reduced in mice treated with PDTC (Table 2). PDTC treatment did not cause significant changes in sham mice (Table 2). Effect of zymosan administration on NO formation and nitrosative stress The biochemical and inflammatory changes observed in the peritoneal cavity of zymosan-treated mice were associated with a significant elevation in plasma NOx levels. Nitrite/nitrate levels were significantly elevated in zymosan-treated mice in comparison to sham mice (Table 2). PDTC treatment did not cause significant changes in NOx levels in sham mice (Table 2). Immunohistochemical analysis of lung and intestine sections obtained from zymosan-treated mice revealed positive staining for iNOS (Figs. 1A, 2A). In contrast, no staining for iNOS was found in the lung and intestine of zymosan-treated mice which had been treated with PDTC (10 mg/kg; Figs. 1B, 2B). Staining was absent in tissues obtained from sham mice administered PDTC only (data not shown). At 18 h following i.p. administration of zymosan, sections of the lung and intestine were also analysed for the

evidence of nitrotyrosine formation. Immunohistochemical analysis using a specific anti-nitrotyrosine antibody revealed positive staining in lung and intestine from zymosan-treated mice (Figs. 1C, 2C). A marked reduction in nitrotyrosine staining was found in the lung, liver and intestine of the zymosan-treated mice that had been treated with PDTC (10 mg/kg i.p.; Figs. 1D, 2D). Immunohistochemical analysis of lung, liver and intestine sections obtained from mice treated with zymosan also revealed a positive staining for PAR (Figs. 1E, 2E), indicating PARP activation. In contrast, no positive staining for PAR was found in the lung and intestine of zymosantreated mice which had been treated with PDTC (Figs. 1F, 2F). There was no staining for either nitrotyrosine or PAR in lung and intestine obtained from shamoperated mice (data not shown). Effect of zymosan on myeloperoxidase activity and malondialdehyde levels At 18 h after zymosan administration MPO activity, an indicator of PMN infiltration, was measured in lung and intestine tissue. In the same tissues the MDA levels were measured as an indicator of the degree of lipid peroxidation. As shown in Table 2, MPO activity and MDA levels were significantly increased in all organs (p<0.01) 18 h after administration of zymosan. MPO activity and MDA levels were significantly (p<0.01) reduced by PDTC treatment (Table 2). Effect of PDTC on cytokine production The levels of TNF-α and IL-1β were significantly elevated in the plasma from zymosan-treated mice. In contrast, the levels of these cytokines were significantly lower in the zymosan-treated mice that had been treated with PDTC (Table 2). No significant cytokine increase was observed in the plasma from sham mice (Table 2).

1.01±0.05* 11±0.51* 0.39±0.07** 4±0.62** 438.0±20* 285±30**

The appearance of IκB-α in the cytosolic fractions was investigated by immunoblotting analysis. A basal level of IκB-α was detectable in the cytosolic fraction of unstimulated cells whereas at 18 h after zymosan administration IκB-α disappeared. PDTC in vivo treatment prevented IκB-α degradation; in fact the IκB-α band remained unchanged at 18 h after zymosan administration (Fig. 3). Effects of PDTC on the multiple organ dysfunction syndrome caused by zymosan Effects on the liver injury In sham mice the administration of saline or PDTC did not result in any significant alterations in the plasma levels of AST (Table 3), ALT (Table 3), bilirubin (Table 3) or alkaline phosphatase (Table 3). Compared with sham mice, zymosan administration resulted in significant rises in the plasma levels of AST, ALT, bilirubin and alkaline phosphatase (Table 3), demonstrating the development of hepatocellular injury. Pre-treatment of zymosantreated mice with PDTC abolished the liver injury caused by zymosan (Table 3).

123.0±19* 50.0±11**

5724.0±1023* 1023.0±387**

Effect of PDTC on IκB-α degradation

Effects on the renal dysfunction In sham mice the administration of saline or PDTC did not result in any significant alterations in the plasma levels of creatinine (Table 3). Compared with sham mice, zymosan administration resulted in significant rises in the plasma levels of creatinine, demonstrating the development of renal dysfunction (Table 3). Pre-treatment of zymosan-treated mice with PDTC abolished the renal dysfunction caused by zymosan (Table 3). Effects on pancreatic injury * p<0.01 vs. sham, ** p<0.01 vs. ZYM

48.5±2.4* 24±1** 4.74±0.94* 1.88±0.121** 0.728±0.13* 0.08±0.01**

567.0±48* 320.0±32**

0.1±0.2 0.9±0.2 0.1±0.06 0.11±0.06 228.0±30 225.0±20 797.0±190 785.0±180 27.0±3 28.0±1.8 28.6±0.25 26.4±0.41 0.13±0.005 0.11±0.004

Sham + vehicle Sham + PDTC (10 mg/kg) ZYM + Vehicle ZYM+ PDTC (10 mg/kg)

0.04±0.02 0.03±0.01

324.0±16 328.0±18

Lung MPO activity (U/g wet tissue) Ileum MDA levels (µM/mg wet tissue) Lung MDA levels (µM/mg wet tissue) Nitrite/nitrate (nmol/wet) Leukocyte infiltration (106 cells/rat) Volume exudate (ml)

Table 2 Effect of PDTC on zymosan (ZYM) induced inflammation. Data are means ±SEM from ten rats in each group

IL-1β (pg/ml) TNF-α (pg/ml) Ileum MPO activity (U/g wet tissue)

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In sham mice the administration of saline or PDTC did not result in any significant alterations in the plasma levels of lipase and amylase (Table 3). Compared with sham mice, zymosan administration resulted in significant rises in the plasma levels of lipase and amylase, demonstrating the development of pancreatic injury (Table 3). Pre-treatment of zymosan-treated mice with PDTC abolished the pancreatic injury caused by zymosan (Table 3).

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Fig. 1 Immunohistochemical localization of iNOS, nitrotyrosine and PAR in intestine tissues. At 18 h following zymosan administration intestine section obtained from vehicletreated mice showed intense positive staining for iNOS (A), nitrotyrosine (arrows,C) and PAR (E). In the intestine of the PDTC-treated mice no positive staining for iNOS (B), nitrotyrosine (D) and PAR (F) was observed. Original magnification, ×128.5. Figure is representative of at least three experiments performed on different experimental days

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Fig. 2 Immunohistochemical localization of iNOS, nitrotyrosine and PAR in lung tissues. At 18 h following zymosan administration lung section obtained from vehicle-treated mice showed intense positive staining for iNOS (A), nitrotyrosine (arrows,C) and PAR (E). In the lungs of the PDTC-treated mice no positive staining for iNOS (B), nitrotyrosine (D) and PAR (F) was observed. Original magnification, ×128.5. Figure is representative of at least three experiments performed on different experimental days

Effects of PDTC on the injury (histological evaluation) of the lung, liver and intestine of zymosan-treated mice At 18 h after zymosan administration, the tissue injury in lung and small intestine was evaluated histologically. On histological examination the lung and intestine (see representative sections; Fig. 4) revealed pathological chang-

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Table 3 Effect of PDTC on zymosan (ZYM)-induced organ dysfunction. Data are means ±SEM from ten rats for each group AST (UI/L) Sham + Vehicle 66.0±22.0 Sham + PDTC (10 mg/kg) 65.0±9.5 ZYM + Vehicle 215.0±16.00* ZYM+ PDTC (10 mg/kg) 118.0±13.0**

ALT (UI/L)

Bilirubin (mg/dl)

Alkaline Phosphatase (UI/L)

16.0±2.7 19.0±2.1 58.0±7.00* 32.0±1.8**

0.33±0.002 88.0±2.7 0.17±0.058 86.0±2.1 0.7±0.033* 221.0±7.00* 0.36±0.003** 121.0±4.0**

Creatinine (mg/dl)

Amylase (UI/L)

Lipase (UI/L)

0.16±0.0012 0.15±0.0003 0.65±0.021* 0.31±0.00016

1653.0±150.0 1573.0±42.0 2528.0±65.00* 1621.0±36.0**

26.0±2.7 28.0±1.0 57.0±7.00* 35.0±2.0**

* p<0.01 vs. sham, ** p<0.01 vs. ZYM

Fig. 3 Effect of PDTC on IκB-α degradation. Representative western blot of IκB-α (A) and densitometric analysis (B) show the effect of PDTC on IκB-α protein expression evaluated in peritoneal macrophages collected at 18 h after zymosan administration. The data illustrated are from a single experiment and are representative of a total of three separate experiments. The results in B are expressed as mean ±SEM; five mice for each group.*p<0.01 vs. vehicle,°p<0.01 vs. zymosan

es. Examination of lung biopsy specimens revealed extravasation of red cells and PMN and macrophage accumulation (Fig. 4A). Sections from the distal ileum revealed significant oedema in the space bounded by the villus and epithelial separation from the basement membrane (Fig. 4C). Treatment with PDTC significantly reduced the organ injury, indicating a potent anti-inflammatory effect (Fig. 4B, D).

Discussion Animal models of multiple-organ dysfunction syndrome allow a systematic investigation of the processes and mechanisms of this disease and are likely to contribute to

a greater understanding of the relevant pathophysiology and ultimately will help to improve treatment. We have recently reported that zymosan administration to mice causes within 18 h both signs of peritonitis and of MOF [11]. The onset of the inflammatory response caused by zymosan in the peritoneal cavity was associated with systemic hypotension, high peritoneal and plasma levels of NO, maximal cellular infiltration, exudate formation and cyclo-oxygenase activity [11]. This study provides the first evidence that pre-treatment of mice with PDTC attenuates (a) the development of zymosan-induced peritonitis, (b) the infiltration of the lung and intestine with PMNs (histology and MPO activity), (c) the degree of lipid peroxidation in the lung, liver and intestine (MDA levels), (d) the renal dysfunction (biochemical analysis) and (e) the liver, lung, pancreatic and intestinal injury (biochemical and histological analysis) caused by injection of zymosan administration. All of these findings support the view that PDTC attenuates the degree of MOF induced by zymosan in the mice. What is the mechanism, however, by which PDTC reduces the organ injury associated with zymosan-induced non-septic shock? PDTC and other dithiocarbamates inhibit the activation of NF-κB and possess anti-oxidative properties [3].Recent evidence suggests that the activation of NFκB is also under the control of oxidant/anti-oxidant balance [12]. The exact mechanisms by which PDTC suppresses NF-κB activation in inflammation are not known. However, the results of this study demonstrate that PDTC inhibits the degradation of IκB-α, thus inhibiting NF-κB activation. Binding of NF-κB to the respective binding sequence on genomic DNA encoding for iNOS, cyclo-oxygenase 2 IL-1, IL-10 and TNF-α results in a rapid and effective transcription of these genes [13]. There is evidence that the pro-inflammatory cytokines TNF-α and IL-1 help to propagate the extension of MOF [14]. Nemeth and colleagues [15] have previously reported that PDTC inhibits TNF-α, macrophage inflammatory protein 1α and IL-12 production in an experimental model of endotoxin shock. Therefore the inhibition of the production of TNF-α and IL-1β by PDTC described in the present study is most likely due to the inhibitory effect the activation of NF-κB.

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Fig. 4 Morphological changes in lung and intestine. Representative lung sections from zymosan-treated mice demonstrate inflammatory cells infiltration (A). Lung sections from a zymosan-treated mice treated with PDTC (B) demonstrate reduced inflammatory cells infiltration. Representative ileum sections from zymosantreated mice demonstrate oedema in the space bounded by the villus and epithelial separation from the basement membrane (C). Ileum sections from zymosan-treated mice treated with PDTC (D) demonstrate reduced ileum injury. Original magnification, ×125. Figure is representative of at least three experiments performed on different experimental days

Activation of the transcription factor NF-κB plays an important role in the expression of iNOS [16].Enhanced formation of NO by iNOS may contribute to the inflammatory process [17]. This study demonstrates that PDTC attenuates the expression of iNOS in the lung and intestine from zymosan-treated mice. Thus the reduction in the expression of iNOS and/or the anti-oxidant property of PDTC may contribute to the attenuation by this agent of nitrotyrosine formation in the lung and intestine from zymosan-treated mice. There is recent evidence that the activation of PARP induced by reactive oxygen species and peroxynitrite

also plays an important role in zymosan-induced nonseptic shock (MOF) [18]. We demonstrate here that PDTC attenuates the increase in PARP activity in the lung, liver and intestine from zymosan-treated mice. In conclusion, this study demonstrates for the first time that PDTC attenuates (a) the renal dysfunction, (b) the hepatocellular dysfunction, (c) the lung injury, (d) the intestinal injury and (e) the pancreatic dysfunction caused by zymosan administration in mice. We also report that PDTC attenuates the expression of iNOS protein in the lung and intestine of mice subjected to zymosan-induced non-septic shock. Finally, PDTC also prevents the degree of reactive oxygen species formation and PARP activation in the lung and intestine of mice subjected to zymosan-induced non-septic shock. Acknowledgements We thank Giovanni Pergolizzi and Carmelo La Spada for their excellent technical assistance during this study, Mrs. Caterina Cutrona for secretarial assistance and Miss Valentina Malvagni for editorial assistance with the manuscript.

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