IGF-I and IGF-II are potent mitogens, postulated to exert autocrine/paracrine effects on growth regulation in human lung cancer. Their proliferative effects are modulated by IGF-binding proteins (IGFBPs), which are found in conditioned medium (CM) of lung cancer cell lines. The biological role of the IGFBPs, which are ontogenetically and hormonally regulated, is not fully understood. Both inhibitory and stimulatory effects on cell growth have been demonstrated. Exogenous IGFBP-3 has been consistently shown to block IGF action, inhibiting cell growth in vitro. In order to evaluate the action of endogenously produced IGFBP-3 on cell growth in lung cancer, we stably transfected the non-small cell lung cancer cell line NCI-H23 with human IGFBP-3 cDNA (resulting in NCI-H23 pOPI3/BP-3) or with the vector pOPI3CAT as control (resulting in NCI-H23 pOPI3CAT). RT-PCR confirmed expression of IGFBP-3-specific mRNA in NCI-H23 pOPI3/BP-3, but not in NCI-H23 or NCI-H23 pOPI3CAT. Western ligand blot and Western immunoblot analysis of CMs yielded strong signals of the characteristic 40-44 kDa human IGFBP-3 protein in NCI-H23 pOPI3/BP-3. An IGFBP-3 ELISA demonstrated a 20-fold increase in IGFBP-3 protein expression in NCI-H23 pOPI3/BP-3 as compared with NCI-H23. The growth of NCI-H23 pOPI3/BP-3 in serum-containing medium was significantly slower (1·7-fold) than that of NCI-H23 or the vector-transfected control NCI-H23 pOPI3CAT. While the proliferation rate of parental and vectortransfected cells could be stimulated by IGF-I, IGF-II, IGF-I analog Long R 3 IGF-I or insulin, that of NCI-H23 pOPI3/BP-3 could not. Xenotransplantation in nude mice resulted in marked tumor growth after the injection of NCI-H23 or NCI-H23 pOPI3CAT, but absent or minimal growth for the IGFBP-3-transfected cell line.These data suggest that IGFBP-3 is a potent inhibitor of cell growth in human lung cancer cell lines and may impair tumorigenicity in vivo.

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Carbon black nanoparticle (CBNP) applications in high doses have been shown to be harmful to the lung. It is postulated that even small, environmentally relevant concentrations induce changes on lung homeostasis. The present study determined the impact of low-dose single and multiple CBNP (Printex 90) applications on mouse alveolar cell metabolism, especially inflammatory and oxidative stress parameters. Nanoparticles were administered to mice by a single or 8 oropharyngeal aspirations at wk 1, 2, 3, 5, 7, 9, 11, and 12 using 7 μg Printex 90, 7 μg DQ12 quartz (positive control), with water vehicle and saline as negative controls. After 2 d or 3 mo lung function was analyzed. Further lung histology, bronchoalveolar lavage fluid (BALF) parameters, and mRNA expression of cytokines and antioxidants enzymes in type II pneumocytes were measured on d 3 or after 3 mo. Single low-dose Printex 90 application induced no marked alterations in lung functions or BALF phospholipid levels but significant decrease in superoxide dismutase 2 and numerically elevated glutathione peroxidase 3 mRNA expression levels in type II pneumocytes. Multiple CBNP applications produced reduced lung function, collagen accumulation, elevated phospholipid levels in BALF, and a massive infiltration of macrophages. Type II pneumocyte mRNA expression of antioxidative enzymes remained unchanged throughout the subchronic experiment, but showed a significant decrease in interleukin (IL)-6Rα mRNA expression. This study demonstrates that an environmentally relevant CBNP concentration induced an acute inflammatory response, an effect that is exacerbated throughout the subchronic duration.

Inhalation of nitrogen dioxide (NO2) is known to alter the composition of the bronchoalveolar lavage (BAL) and to impair the surfactant metabolism of type II pneumocytes. However, information is sparse as to whether application of the widely used antioxidant N-acetylcysteine (NAC) is capable of preventing or reducing these alterations. The aim of the study was to investigate if in vivo administration of NAC to NO2-inhaling rats protected BAL parameters and physiology of type II pneumocytes from impairment. For this purpose, rats were exposed to 720 p.p.m. h-1 NO2, that was applied continuously, intermittently or repeatedly. During inhalation one group of rats received saline and the other group received NAC antioxidant (200 mg kg-1, intraperitoneally) once a day. The BAL protein and phospholipid content increased most in the continuously and repeatedly NO2-exposed rats when compared to the controls, while the intermittent exposure did not change these parameters. Application of NAC led to a marked decrease of the protein elevation for the continuously and intermittently exposed groups, but exhibited no influence on the BAL phospholipid. Surprisingly, all NO2 exposure modes elevated the glutathione content (reduced and oxidized) in the BAL. Application of NAC clearly decreased the content of both forms of glutathione in the continuously and the repeatedly NO2-exposed groups. Phospholipid synthesis, measured by choline uptake into type II cells, was increased most after continuous NO2 inhalation. The NAC reduced this increase moderately. Whereas choline uptake by type II cells was obviously stimulated by NO2, the stimulated secretion of phosphatidylcholine from these cells was decreased by this oxidant. Only continuous exposure reduced this activity markedly. The NAC clearly restored the impaired secretion activity in the cells from the continuously NO2-exposed animals. Since the efficacy of NAC in the prevention of NO2-induced impairments in the surfactant system is striking mainly in the continuously exposed group, we suggest that administration of NAC to NO2-induced lung injury partially restores altered BAL components and the impaired physiology of type II pneumocytes.

Inhalation of nitrogen and reactive oxygen species (ROS) is known to induce lung inflammation, which is prevented by enzymatic and nonenzymatic antioxidant systems. These agents form nitrated allergens that were shown to enhance allergenicity. The aim of this study was to examine the influence of nitrated proteins on inflammation and antioxidant status of the lung. Ovalbumin (OVA) in nitrated form (nOVA) was intraperitoneally (ip) injected in mice for sensitization and in nitrated or unmodified form for challenge to induce allergic bronchial inflammation. To study the allergen potential of unrelated protein and verify cross-reactivity, nitrated and unmodified keyhole limpet hemocyanin (nKLH, KLH) was used for challenge. Challenge with OVA or nOVA reduced lung function and increased eosinophilia and protein content in bronchoalveolar lavage fluid (BALF). Challenge with nitrated or native OVA or KLH elevated glutathione (GSH) ratio in type II pneumocytes. Reduced mRNA expression of glutathione peroxidase (GPX) 3, glutathione reductase (GR), superoxide dismutase (SOD) 2, and catalase (CAT) was most prominent after challenge with nitrated OVA and nitrated KLH, respectively. Challenge with nOVA enhanced SOD1 mRNA reduction. Immunostaining of GPX 3 and SOD2 increased after challenge with OVA or nOVA, while reactivity of GR and reactivity of SOD2 were reduced after challenge with KLH or nKLH. SOD1 immunostaining was diminished after challenge with nonnitrated OVA or KLH. CAT immunoreaction was similar in all groups. Nitrated proteins without allergenic potential triggered mRNA reduction of antioxidants in type II cells after sensitization with a nitrated allergen but did not induce bronchial inflammation.