Search

Enhancement of lung antioxidant capacity has been proposed in the therapy of acute lung injuries involving local accumulation of reactive oxygen species (ROS). We have studied in the female Sprague-Dawley rat the effect of intratracheal administration of catalase (CAT) on the acute lung response induced by different ROS generating systems. The lung response was assessed at several time intervals (60-360 min) by monitoring in bronchoalveolar fluid (BALF) the activity of lactate dehydrogenase and the levels of total protein, albumin, and glucose. While CAT (50,000 IU/rat) significantly reduced the biochemical changes induced by hydrogen peroxide produced by a glucose/glucose oxidase system, it markedly exacerbated the lesions induced by phorbol myristate acetate (PMA). Several observations indicate that a particular chemical species formed during the catalase inactivation process is responsible for this effect. Parallel to the development of the lung damage, we noted a rapid reduction of CAT activity (80%) in the BALF of animals treated with PMA and CAT. In vitro an inhibition of CAT activity was observed in the presence of a superoxide anion generating system, and this inhibition was prevented by superoxide dismutase (SOD). A dose of 10,000 IU superoxide dismutase did not prevent the development of the lung lesions induced by PMA plus CAT. Administered alone or in association with PMA, CAT inactivated by heat or 3-aminotriazole also caused severe lung damage. In conclusion, the present study indicates that exogenous catalase may not always protect against the inflammatory reaction resulting from an oxidative stress. In the presence of superoxide anions, catalase may aggravate the lesions, and this possibility should be kept in mind when considering an antioxidant therapy. [ABSTRACT FROM AUTHOR]

For repeated dose oral gavage studies in rodents, OECD guidelines do not include the nasal turbinates in the list of recommended tissues for histopathologic examination. During the past few years, unexpected high mortality was noted with two different compounds in rodents for which the cause of death was mainly based on histological evaluation of the nose. For compound A (a solution in PEG 400), unexpected high mortality was encountered early in the carcinogenicity studies. The dose-related increase in mortality correlated with an increase in inflammatory and necrotic or ulcerative changes within the respiratory tract of the preterminal deaths, often involving the nose and sinuses. For several animals rhinitis/sinusitis (mainly involving the lower half of the nose and most prominently affecting the respiratory epithelium) was the most important finding. The respiratory tract findings were consistent with irritation. The irritant nature of the formulation was confirmed in a BCOP-test. It was concluded that a part of the irritant formulation entered into the nose/airways via aspiration during gavage-dosing. This was considered to be related to the high viscosity of the vehicle (PEG400). The problem was further prevented by adapting the gavage procedure and by reducing the volume and/or concentration of the formulation. Compound B (also a solution in PEG 400) resulted in high mortality at the high dose in the 3m mouse study, from the 1st week onwards. At necropsy, the preterminally dead/sacrificed animals showed gastrointestinal dilatation with test formulation being present in the stomach. Acute, necrotizing rhinitis/sinusitis was the cause of death for the majority of these mice. Only a minority of these animals showed lesions in other parts of the respiratory tract. The test article and PEG 400 (to a lesser extent) were demonstrated to delay gastric emptying in a separate pharmacology study. This effect, together with the high viscosity of the vehicle and the irritant nature of the compound formulation resulted in entry of the formulation in the respiratory tract (probably due to regurgitation/aspiration) with subsequent inflammatory/necrotizing changes. For the majority of mice, the cause of death would not have been evident without evaluation of the nose. In conclusion, evaluation of the nose can be valuable to determine the cause of death in rodents. [Copyright &y& Elsevier]

We investigated the effect of intratracheally instilled coal fly ash(FA) and copper smelter dust (CU) on the lung integrity and on the ex vivo release of tumor necrosis factor alpha (TNF-alpha) by alveolarphagocytes. Groups of female NMRI mice received a single intratracheal administration of different particles normalized for the arsenic content (20 mu g/kg body weight, i.e., 600 ng arsenic/mouse) and the particle load (100 mg/kg body weight, i.e., 3 mg/mouse). Mice receivedtungsten carbide (WC) alone (100 mg/kg), FA alone (100 mg/kg, i.e., 20 mu g arsenic/kg), CU mixed with WC (CU, 13.6 mg/kg, i.e., 20 mu g arsenic/kg, WC, 86.4 mg/kg) and Ca3(AsO4)2 mixed with WC (20 mu g arsenic/kg; WC, 100 mg/kg). Animals were sacrificed at 1, 6, or 30 d posttreatment and analyzed by bronchoalveolar lavage for total protein (TP) content, inflammatory cell number and type, and TNF-alpha production. Additional mice were studied toevaluate particle retention by measuring total arsenic retention in the lung at appropriate times. Instillation of WC induced a mild and transient (d 1) inflammatory reaction characterized by an increase ofTP and an influx of polymorphonuclear leukocytes in the alveolar compartment. Compared to WC, Ca3(AsO4)2produced a significant increase of TP content in BALF. CU particles caused a severe but transient inflammatory reaction, while a persisting alveolitis (30 d) was observed after treatment with FA. Compared to control saline, a marked inhibition of TNF-alpha release was observed in response to LPS in all groups at d 1. Cytokine production was upregulated in WC- and Ca3(AsO4)2- treated animals after 6 and 30 d respectively. However, a 90% inhibition of TNF-alpha production was still observed at d 30 after administration of CU and FA. Although arsenic was cleared from the lung tissue 6 d after Ca3(AsO4)2 administration,a sign [ABSTRACT FROM AUTHOR]

The European legislation on plant protection products (Regulation (EC) No. 1107/2009) and biocides (Directive 98/8/EC), as well as the regulation concerning chemicals (Regulation (EC) No. 1907/2006 'REACH') only support the marketing and use of chemical products on the basis that they do not induce endocrine disruption in humans or non-target species. However, there is currently no agreed guidance on how to identify and evaluate endocrine activity and disruption. Consequently, an ECETOC task force was formed to provide scientific criteria that may be used within the context of these three legislative documents. Specific scientific criteria for the determination of endocrine disrupting properties that integrate information from both regulatory (eco)toxicity studies and mechanistic/screening studies are proposed. These criteria combine the nature of the adverse effects detected in studies which give concern for endocrine toxicity with an understanding of the mode of action of toxicity so that adverse effects can be explained scientifically. The criteria developed are presented in the form of flow charts for assessing relevant effects for both humans and wildlife species. In addition, since not all chemicals with endocrine disrupting properties are of equal hazard, assessment of potency is also proposed to discriminate chemicals of high concern from those of lower concern. The guidance presented in this paper includes refinements made to an initial proposal following discussion of the criteria at a workshop of invited regulatory, academic and industry scientists., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background & Aims: The search for targeted anti-hepatitis C virus (HCV) drugs is driven by the adverse effect profile and limited efficacy of the current standard of care (pegylated interferon-alpha/ribavirin). In a first-in-human trial, we tested the safety, tolerability, and pharmacokinetics of the macrocyclic HCV NS3/4A protease inhibitor TMC435 in healthy volunteers, followed by HCV genotype 1-infected patients to assess antiviral activity., Methods: The TMC435350-C101 study was a phase I, randomized, double-blind, placebo-controlled trial in 49 healthy volunteers, followed by an open-label, nonplacebo-controlled panel in 6 genotype 1 hepatitis C patients. Healthy volunteers received oral, single, ascending doses (up to 600 mg) or 5-day multiple ascending doses (200 mg twice daily or 100, 200, or 400 mg once daily). Patients received 200 mg once daily for 5 days. Pharmacokinetics and safety were evaluated for all panels, and plasma HCV-RNA levels were determined in patients., Results: There were no serious adverse events, no grade 3 reactions, and no treatment-related discontinuations; pharmacokinetics supported a once daily dosing regimen. Plasma HCV-RNA levels dropped rapidly in all patients, with a median maximal reduction of 3.9-log(10) IU/mL and a median of 6 days to maximal reduction. The initial steep reduction of HCV-RNA (median 3.5-log(10) IU/mL at day 3) was followed by a more gradual decline that was maintained over the dosing period. No viral breakthroughs (>1-log(10) IU/mL HCV-RNA increase from nadir) were observed during treatment nor in the 3 days posttreatment; HCV-RNA returned to pretreatment levels by week 4., Conclusions: Once daily TMC435 given orally was generally safe and well tolerated and demonstrated potent antiviral activity., (Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Very few biomarkers are available for the non-invasive detection of effects of urban air pollution on the respiratory tract. The objective was to evaluate whether Clara cell protein (CC16) and surfactant-associated protein-A (SP-A), two pulmonary secretory proteins, were useful in the detection of effects of urban air pollutants on the pulmonary epithelium. These proteins were determined in the serum of 53 policemen working in Brussels, Belgium, and a control group of 59 foresters working in the countryside. Except for ozone (O(3)), annual concentrations of the main air pollutants (PM(10), NO(2), CO, SO(2) and benzene) were significantly higher in Brussels than in the country. The proportion of smokers was lower in urban policemen compared with foresters, but they smoked on average a similar number of cigarettes per day as confirmed by their urinary excretion of cotinine. Muconic acid, a marker of benzene exposure, was significantly higher in urban policemen than in foresters, in both smokers and non-smokers. Multiple regression analysis showed that the type of work, smoking habits and time spent outdoors and in a car were significant determinants of benzene uptake. Tobacco smoking impaired lung function to a similar extent in urban policemen and foresters. The serum levels of SP-A were significantly increased in smokers but were not different between policemen and foresters. Serum CC16 was significantly reduced by tobacco smoking and slightly decreased in policemen compared with foresters. Interestingly, the reduction of serum CC16 was more pronounced in the subgroup of traffic compared with survey policemen, the latter being also less exposed to benzene. The results suggest that serum pneumoproteins and especially serum CC16 could be useful in the detection of chronic effects of urban air pollutants on the respiratory epithelium of populations particularly at risk.

Clara-cell protein (CC16), the predominant protein secreted by bronchiolar Clara cells, increasingly appears to protect the respiratory tract against oxidative stress and inflammation. The aim of this study was to test in inbred strains of mice whether the lung susceptibility to O3 correlates with the transepithelial leakage of CC16, with the mRNA and protein levels of CC16, and possibly with specific isoforms of the protein in the respiratory tract. Five strains of mouse with increasing sensitivity to O3 (C3H, AKR, SJL, CBA, and C57Bl) were exposed to 1.8 ppm O3 for 3 h and examined at 0 and 6 h postexposure. The most sensitive (C57Bl) and resistant (C3H) mice were also continuously exposed to 0.11 ppm O3 for up to 3 days. Lung injury was evaluated by measuring in bronchoalveolar lavage fluid (BALF) the levels of total protein, albumin, lactate dehydrogenase (LDH), and inflammatory cells. The patterns of proteins in BALF were also analyzed by two-dimensional electrophoresis (2-DE). Exposure to 1.8 or 0.11 ppm O3 caused a transient elevation of CC16 in serum that was maximal immediately after exposure and closely correlated with the extent of lung injury evaluated by BALF markers. The epithelial damage assessed on the basis of serum CC16 or BALF markers showed an inverse relation with the preexposure levels of CC16 in BALF. Since preexposure levels of CC16 mRNA were similar between the strains and since lung epithelium damage was also negatively correlated with preexposure levels of albumin in BALF, these findings identify basal lung epithelium permeability as a determinant of susceptibility to O3. The 2-DE mapping of proteins in BALF of these two strains revealed the existence of two distinct isoforms of CC16 with pI values of 4.9 and 5.2. The most acidic form was significantly less concentrated in the C57Bl strain, the most sensitive to O3, a difference that might be related to the higher permeability of the lung epithelium or to some post-transcriptional variations. In conclusion, these results suggest that the permeability of the lung epithelial barrier may be an important determinant of the lung susceptibility to O3, controlling the intrapulmonary levels of CC16 and possibly of other antioxidant/inflammatory proteins.

Previous studies have shown that the pulmonary response to ozone (O(3)) varies greatly among strains of mice, but the factor(s) and the mechanism(s) that are responsible for this differential susceptibility have not yet been clearly identified. The present study explores the molecular bases for this differential O(3) susceptibility by studying the expression of proteins associated to the epithelial lining fluid (ELF) from two strains of mice, C57BL/6J and the C3H/HeJ, respectively described as O(3)-sensitive and O(3)-resistant. The ELF proteins of these two strains were displayed by two-dimensional gel electrophoresis (2-DE) of bronchoalveolar lavage fluids (BALFs) and the protein patterns obtained with BALF samples of both strains were compared. Two major differences were observed between the BALF 2-DE protein maps obtained from C57BL/6J and C3H/HeJ strains. First, two isoforms of the antioxidant protein 2 (AOP2) were detected in a strain-dependent manner: C3J/HeJ possesses only AOP2a (isoelectric point 5.7) and C57BL/6J exhibits only AOP2b (isoelectric point 6.0). Second, the levels of anti-inflammatory and immunosuppressive Clara cell protein-16 (CC16) were 1.3 times higher in the BALF from resistant C3H/HeJ than from sensitive C57BL/6 mice. Moreover, two 6 kDa isoforms of CC16 with isoelectric points of 4.9 (CC16a) and 5.2 (CC16b) are detected in both strains. Interestingly, the C57BL/6J strain had a twice decreased level of the acidic isoform of CC16 compared to C3H/HeJ. Our results suggest that AOP2 and CC16 might participate in the protection of the pulmonary tract to O(3)-induced lung injury. The possible differential contribution of specific protein isoforms in the differential susceptibility to oxidative stress is discussed.

Triallate is a selective herbicidal chemical used for control of wild oats in wheat. It has an extensive genotoxicity database that includes a variety of in vitro and in vivo studies. The chemical has produced mixed results in in vitro assay systems. It was genotoxic in bacterial mutation Ames assays, predominantly in Salmonella typhimurium strains TA100 and TA1535 in the presence of S9. Weaker responses have been observed in TA100 and TA1535 in the absence of S9. Mixed results have been observed in strain TA98, whereas no genotoxicity has been observed in strains TA1537 and TA1538. The presence and absence of S9 and its source seem to play a role in the bacterial response to the chemical. There have also been conflicting results in other test systems using other bacterial genera, yeast, and mammalian cells. Chromosome effects assays (sister-chromatid exchange and cytogenetics assays) have produced mixed results with S9 but no genotoxicity without S9. Triallate has not produced any genotoxicity in in vitro DNA damage or unscheduled DNA synthesis assays using EUE cells, human lymphocytes, and rat and mouse hepatocytes. In a series of in vivo genotoxicity assays (cytogenetics, micronucleus, dominant lethal, and unscheduled DNA synthesis), there has been no indication of any adverse genotoxic effect. Metabolism data indicate that the probable explanation for the differences observed between the in vitro studies with S9 and without S9 and between the in vitro and the in vivo studies is the production of a mutagenic intermediate in vitro at high doses of triallate is expected to be at most only transiently present in in vivo studies. The weight of evidence strongly suggests that triallate is not likely to exert mutagenic activity in vivo due to toxicokinetics and metabolic processes leading to detoxification.

The Belgian PCB incident occurred at the end of January 1999 when a mixture of polychlorinated biphenyls (PCBs) contaminated with dioxins was accidentally added to a stock of recycled fat used in the production of animal feeds. Although signs of poultry poisoning were noticed by February, 1999, the source and the extent of the contamination were discovered only in May 1999, when it appeared that more than 2500 farms could have been supplied with contaminated feeds. This resulted in a major food crisis, which rapidly extended to the whole country and could be resolved only by the implementation of a large PCB/dioxin food monitoring program. Screening for PCB contamination was based on the determination of the seven PCB markers. When PCB concentrations exceeded the tolerance levels of 0.1 (milk), 0.2 (poultry, bovine, and pig meat), or 1 (animal feed) microg/g fat, dioxins (17 PCDD/Fs congeners) were also determined. At the end of December 1999, the database contained the results of more than 55,000 PCB and 500 dioxin analyses. The study of PCB levels and profiles in contaminated feeds delivered to poultry or pig farms confirmed that the Belgian PCB incident was due to a single source of PCB oil introduced into the food chain at the end of January 1999. This PCB oil had a congeners pattern closely matched to a mixture of Aroclor 1260/1254 in the proportion 75/25. The total amount of PCBs added to recycled fats was estimated at 50 kg (sum of the seven markers) or approximately 150 kg total PCBs, which corresponds to about 100 liters of PCB oil. This PCB mixture contained about 1g TEQ dioxins (more than 90- contributed by PCDFs) and about 2g TEQ dioxin-like PCBs. The proportions of PCB 52 and 101 congeners were fairly constant in animal feeds, excluding the possibility of secondary contamination due to fat recycling from contaminated animals. The highest concentrations of PCBs and dioxins were found in poultry and especially in the reproduction animals (hens and chicks), which showed the classical manifestations of chick edema disease. The pigs were also affected but to a lesser extent and no sign of intoxication was observed. The study of PCB/dioxin patterns and of the PCB:dioxin ratios revealed major differences in the metabolism of these compounds by farm animals. Whereas the PCBs:dioxins ratio was fairly constant in all poultry products with a mean value similar to that found in contaminated feeds (50,000), in pigs this ratio was both much higher and more variable (values up to 10,000,000), reflecting a faster elimination of dioxins than PCBs in these animals. These metabolic differences also emerged from the PCB and dioxin patterns which were altered much more in pigs than in poultry. Although the most contaminated food products (chicken meat) had PCB and dioxin levels more than 100 times above maximal recommended values, it is unlikely that this incident could have caused adverse effects in the general population of Belgium. A doubling of the PCB and dioxin burden of the young adult population would require the consumption of, respectively, 10 and 20 highly contaminated meals. In view of the very limited proportion of the poultry chain effectively contaminated during the incident (around 2%), such an extreme scenario was quite improbable for the general population except perhaps for farmers consuming their own products. But even in that case, it would have meant going back to the levels in the 1980s or attaining the body burden of subjects regularly eating contaminated seafood.

The role of the genetic polymorphism of NAD(P)H:quinone oxidoreductase (NQO1) and glutathione-S-transferase micro-1 (GSTM1) in the responsiveness to O(3)-induced acute effects was investigated in 24 healthy nonsmokers performing 2-h bike rides at ambient O(3) varying from 32 to 103 ppb. Before and after rides, each subject performed spirometric tests and provided a blood sample for the measurement of the Clara cell protein CC16. NQO1 and GSTM1 polymorphisms were characterized by polymerase chain reaction- based methods. The 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct was also measured in DNA of peripheral leukocytes. Rides at O(3) > 80 ppb resulted in significant decrements of pulmonary function tests and increased levels of serum CC16, consistent with mild impairment in respiratory function and increased lung epithelial permeability, respectively. Whereas NQO1wt and GSTM1null subjects showed both functional changes and increased serum CC16 after acute O(3) exposure, people with other haplotypes showed a rise in serum CC16 but no changes in lung function tests. In NQO1wt and GSTM1null subjects, partial correlation analysis showed that functional decrements and increased serum CC16 are closely associated with each other and with O(3) levels, whereas no such relationships were found among subjects bearing other haplotypes. An increased reaction rate between O(3) and hydroquinones would be consistent with the greater increase in 8-OHdG after O(3) exposure in this "susceptible" group.

Clara cell secretory protein (CC16, CC10, or CCSP), the major secretory protein of the Clara cell, presents several biologic properties, suggesting that it may play a protective role against intrapulmonary inflammatory processes. The aim of the present study was to investigate the changes of CC16 concentrations in the lung, bronchoalveolar lavage fluid (BALF), and serum of rats with acute lung injury induced by lipopolysaccharide (LPS). These changes were compared with Clara cell density, CC16 mRNA level in the lung and classic indices of inflammation in BALF. Injected at doses of 10, 100, or 200 microgram/100 g body weight, LPS induced an acute lung inflammation as estimated by an increased influx of cells and albumin in the BALF. This inflammatory response was associated with a marked reduction of CC16 concentrations in BALF and lung homogenate as well as of the CC16 mRNA levels in the lung. At the highest dose of LPS, the CC16-positive cell density in the bronchiolar epithelium was also decreased. In serum, by contrast, the concentration of CC16 was elevated as a consequence of increased airway permeability. Pretreating rats intraperitoneally with dexamethasone (2 mg/kg) significantly lowered the leukocyte influx and attenuated the albumin increase in BALF. Dexamethasone, however, failed to prevent the increased airway permeability to CC16, suggesting that during inflammation different mechanisms regulate the leakage of proteins across the alveolocapillary barrier depending on the direction of passage and/or the size of the protein. Our results show a marked decrease of the secretion and synthesis of CC16 during LPS-induced acute lung inflammation.

Since tumor growth factor beta (TGF-beta) and its receptor are ubiquitously expressed and because latent TGF-beta cannot bind to the cell surface receptor, the ability of a cell to activate latent TGF-beta upon secretion represents an important regulatory mechanism of TGF-beta action. In vivo, the protease plasmin is considered to be one of the main enzymes operative in the proteolytic cleavage of the latency-associated peptide moiety from TGF-beta, which converts it into the biologically active form. The TGF-beta response was characterized in alveolar macrophages during pulmonary inflammation (d 3) and fibrosis (d 120) induced by a single intratracheal instillation of silica particles (5 mg/mouse). To appreciate the role of urokinase-type plasminogen activator (uPA) in the activation of TGF-beta, the production of total, active and latent TGF-beta by explanted alveolar macrophages was compared in uPA-deficient (uPA-/-) mice and their normal counterparts (uPA+/+). At d 3 and 120 after silica treatment, a significant increase in cell-associated PA activity was found in uPA+/+ mice compared to that of saline controls. As expected, this response was almost totally absent in uPA-/- mice. Alveolar macrophages from uPA+/+ controls were found to release TGF-beta mainly expressed in a biologically active form. In response to silica treatment, inflammatory cells were found to upregulate, especially at the fibrotic stage, their secretion of total and bioactive TGF-beta. No significant difference was found between uPA-/- and uPA+/+ silica-treated animals for the expression of total, active, or latent TGF-beta. Although it has previously been reported that macrophage surface activation of TGF-beta is dependent on both plasmin generation and uPA cell surface receptor, no evidence was found to support this hypothesis in the present study.

The lung plasminogen activator (PA) response was examined in four different models of particle-induced pulmonary lesions in NMRI mice (single intratracheal administration, 0.75 to 5 mg/mouse). Sequential changes in cellular (total and differential counts) and biochemical markers of alveolitis (lactate dehydrogenase [LDH], total proteins) were monitored in bronchoalveolar fluid (BALF) and the fibrotic lung response was assessed histologically. An intense but spontaneously resolving alveolitis was produced by manganese dioxide (MnO2) and a fibrosing alveolitis was elicited by crystalline silica (DQ12). Minimal and noninflammatory responses were obtained after instillation of titanium dioxide (TiO2) and tungsten carbide (WC), respectively. The comparison between the resolving and the fibrosing alveolitis model was especially taken into consideration in an attempt to identify fibrinolytic changes associated with the development of fibrosis. At the alveolitis stage, similarly increased BALF PA activities were measured in both the resolving and the fibrosing alveolitis models whereas only slight and no PA modifications were noted after administration of TiO2 and WC, respectively. Persistently (up to 120 d) increased BALF PA activity was selectively associated with the progression to fibrosis (DQ12), suggesting that PA is involved in the fibrotic process. ELISA measurements demonstrated that the changes in BALF PA activity were exclusively related to changes in urokinase (uPA), not tissue-type PA. A rapid and persisting (up to Day 30) upregulation of cell-associated PA activity occurred after DQ12, MnO2, and TiO2 treatment only. Cellular PA activity was however significantly higher in fibrogenic inflammatory cells recovered from DQ12 than from MnO2-treated mice suggesting that the intensity of cellular PA upregulation may represent an early indicator of the progression to fibrosis. The implication of urokinase in the pathogenesis of silica-induced fibrosis was demonstrated by the use of a uPA knockout mice. The acceleration of the fibrotic process in uPA-deficient compared with the wild type animals demonstrated the contribution of uPA to limit the fibrotic process.