Shizuo Hasegawa

Effects of β- and γ-carboline derivatives on DNA topoisomerase activities

Abstract β-Carbolines, harman (1-methyl-9 H -pyrido[3,4- b ]indole) and norharman (9 H -pyrido[3,4- b ]indole) and γ-carbolines, 3-amino-1,4-dimethyl-5 H -pyrido[4,3- b ]indole (Trp-P-1) and 3-amino-4-methyl-5 H -pyrido[4,3- b ]indole (Trp-P-2), are present in cooked foods and cigarette smoke. We studied the effects of these heterocyclic amines on the activity of DNA topoisomerases. Trp-P-1 and Trp-P-2 inhibited topoisomerase I (topo I) activity with ED 50 values of 1.48 and 1.55 μg/ml, respectively, in a relaxation assay. Harman and norharman inhibited topo I activity but with much higher ED 50 values, 23.8 and 34.4 μg/ml, respectively. Trp-P-1 and Trp-P-2 also inhibited topoisomerase II (topo II) activity at about 50 μg/ml, in a decatenation assay. Harman and norharman showed a much lower inhibitory effect on topo II activity. None of these compounds stabilized the cleavable complex mediated by topo II. Trp-P-1 and Trp-P-2 intercalated into DNA at concentrations inhibitory to topoisomerases. We considered that the intercalation with DNA and the inhibition of DNA topoisomerases by heterocyclic amines might be partly related to their inhibition of DNA excision repair and their enhancing effect on UV- or chemically induced mutagenic activity.

ET-1 released histamine from guinea pig pulmonary but not peritoneal mast cells

Endothelin(ET)-1 triggered histamine release of mast cells from pulmonary tissue but not from the peritoneal cavity of guinea pigs. The observed difference in response to ET-1 was attributable to a quantitative difference in ET-1 binding sites between both cells. The concentrations of ET-1 required for half maximal release of histamine and half maximal binding of [125I]ET-1 were approximately 0.05 and 0.08 nM, respectively. The release of histamine by ET-1 was a Ca(2+)-dependent but not a cytotoxic process. These observations, taken together, suggest that ET-1 induces histamine release from mast cells in a receptor-dependent fashion.

Elimination of Neutrophils by Apoptosis During the Resolution of Acute Pulmonary Inflammation in Rats

Abstract. We evaluated the apoptosis of neutrophils during the resolution of acute pulmonary inflammation induced by exposure to ozone. The inflammatory response was assessed in rat lungs 0, 1, 3, and 7 days after 4-h exposure to air or 2 ppm ozone. Analysis of bronchoalveolar lavage fluid demonstrated significant increases in albumin concentrations on days 0 and 1 and in the number of lavageable neutrophils on days 0, 1, and 3, indicating the presence of acute pulmonary inflammation. These parameters returned to control values on day 7, which suggests that the acute pulmonary inflammation induced by ozone was reversible. On days 1 and 3, but not on day 0, the neutrophils showed morphologic evidence of apoptosis. Based on morphologic analysis, the proportion of apoptotic neutrophils was 23.3 ± 2.2% on day 1 and 55.7 ± 3.2% on day 3. Terminal deoxynucleotidyl transferase-mediated dUTP end labeling (TUNEL), in contrast, revealed that the proportion of apoptotic cells was 59.7 ± 9.1% on day 1 and 68.0 ± 4.3% on day 3. On day 3, light microscopy and electron microscopy demonstrated engulfment of the neutrophils by macrophages. These findings indicate that the apoptosis of neutrophils followed by their engulfment by macrophages contributes to the clearance of neutrophils from the sites of inflammation. Moreover, TUNEL detected apoptotic neutrophils with greater sensitivity compared with morphologic assessment.

Hypersensitivity Pneumonitis Induced by Toluene Diisocyanate: Sequelae of Continuous Exposure

A 41-year-old automobile paint sprayer showed the clinical features of hypersensitivity pneumonitis 1 week after he had begun to work with paint materials containing toluene diisocyanate. His symptoms began 6 to 8 hours after exposure to the agent and spontaneously disappeared by the next morning. He had diffuse, fine reticulonodular shadows on a chest roentgenogram and a restrictive impairment of pulmonary function. Immunoglobulin G antibody to toluene diisocyanate-human serum albumin was present in bronchoalveolar lavage fluid and sera: IgA antibody was present only in bronchoalveolar lavage fluid. Also, the patient had sensitized bronchoalveolar lymphocytes to toluene diisocyanate-human serum albumin. The histologic findings suggested hypersensitivity pneumonitis. The results of bronchoalveolar lavage, which was repeated on four separate occasions, showed lymphocytosis and a predominance of suppressor-cytotoxic T cells. The findings from serial determinations of humoral antibodies showed no changes consistent with the results of clinical and laboratory studies. In contrast, blastogenic responses of bronchoalveolar lymphocytes to toluene diisocyanate markedly decreased, and the patient showed clinical improvement despite continued exposure to the agent.

Cysteine proteinases in bronchoalveolar epithelial cells and lavage fluid of rat lung.

We examined the presence of cathepsins B, H, and L in bronchoalveolar epithelial cells, including alveolar macrophages, and in bronchoalveolar lavage fluid (BALF), using immunocytochemistry and immunoblotting. By light and electron microscopy, immunoreactivity for cathepsins B, H, and L was detected in lysosomes of ciliated and non-ciliated epithelial cells of bronchi and bronchioles, and in macrophages. Immunodeposits for cathepsin H only were demonstrated in lamellar bodies of Type II alveolar epithelial cells, suggesting the cosecretion of surfactants and cathepsin H from the cells into the alveolar space. By immunoblotting, cathepsins B and H were found to be present in BALF. To further investigate the origin of these enzymes in BALF, alveolar macrophages obtained from BALF were cultured for 6 hr in a serum-free medium. Immunoblotting revealed that protein bands corresponding to the pro-form and mature form of cathepsin B and the mature form of cathepsin H were present in the culture medium. From these results, the presence of cathepsins B and H in BALF can be explained by the fact that cathepsin B is secreted from alveolar macrophages and cathepsin H is secreted mainly with surfactants from Type II cells and also from macrophages.

Clinical usefulness of CYFRA assay in diagnosing lung cancer: measurement of serum cytokeratin fragment.

We evaluated the diagnostic usefulness of measurement of the soluble cytokeratin 19 fragment, a new tumor marker, in 391 patients with lung cancer and in 424 patients with benign lung diseases. Serum concentrations of cytokeratin 19 fragment were measured by a sandwich ELISA (CYFRA). The cut‐off value was defined as 3.5 ng/ml, which is associated with a specificity of 85% for benign lung diseases. CYFRA had a high sensitivity (57.5%) in all subjects with lung carcinoma, and had a higher sensitivity for squamous cell carcinoma (73.1%, n = 141) than squamous cell carcinoma‐related antigen (61.0%). CYFRA was associated with a relatively high sensitivity (42.1%) in early‐stage squamous cell carcinoma (stage I, based on the classification of the Japan Lung Cancer Society), but the CYFRA titer was higher in advanced squamous cell carcinoma than in early‐stage squamous cell carcinoma. Our findings suggest that CYFRA is potentially useful for diagnosis and monitoring of lung carcinoma, especially for squamous cell carcinoma.

Clinical Evaluation of CYFRA 21-1 in Malignant Pleural Fluids

The diagnostic value of the novel tumor marker CYFRA 21-1 in malignant pleural fluid was assessed in comparison to carcinoembryonic antigen (CEA). CYFRA 21-1 and CEA were measured in pleural fluid obtained from patients with 108 malignant and benign diseases. The levels of pleural fluid CYFRA 21-1 in malignant diseases (median: 84.5 ng/ml) were statistically higher than those in benign diseases (median: 13.9 ng/ml; p < 0.01). The CYFRA 21-1 test was able to discriminate significantly between squamous cell lung cancer and pneumonia (p < 0.01), while pleural fluid CEA levels could not. Receiver operating characteristic (ROC) curve analysis showed that the CYFRA 21-1 test has an advantage over CEA because of its higher specificity. These results indicate that measurement of CYFRA 21-1 in pleural fluid is a new tool, in addition to cytologic examination, to discriminate between malignant and benign diseases.

Cloning of rat eotaxin: ozone inhalation increases mRNA and protein expression in lungs of brown Norway rats.

The C-C chemokine eotaxin is thought to be important in the selective recruitment of eosinophils to the site of inflammation in guinea pigs, mice, and humans. We isolated the rat eotaxin gene to determine whether a similar molecule might play a role in the pulmonary infiltration of eosinophils during acute inflammation in the rat. The cDNA for rat eotaxin encoded a 97-amino acid protein containing a 74-amino acid mature eotaxin protein with 97.3% identity to mouse eotaxin. The recombinant protein encoded by this gene displayed specific chemotactic activity for eosinophils when analyzed with a microchemotactic chamber. The expression of eotaxin mRNA increased approximately 1.6-fold immediately after exposure to ozone and was 4-fold higher after 20 h. The number of lavageable eosinophils at the same time points were 3- and 15-fold greater, respectively, than control eosinophils. Immunocytochemistry revealed that alveolar macrophages and bronchial epithelial cells were positive for eotaxin. These results suggest that eotaxin may be involved in the recruitment of eosinophils into the air spaces during certain inflammatory conditions in rats.The C-C chemokine eotaxin is thought to be important in the selective recruitment of eosinophils to the site of inflammation in guinea pigs, mice, and humans. We isolated the rat eotaxin gene to determine whether a similar molecule might play a role in the pulmonary infiltration of eosinophils during acute inflammation in the rat. The cDNA for rat eotaxin encoded a 97-amino acid protein containing a 74-amino acid mature eotaxin protein with 97.3% identity to mouse eotaxin. The recombinant protein encoded by this gene displayed specific chemotactic activity for eosinophils when analyzed with a microchemotactic chamber. The expression of eotaxin mRNA increased ∼1.6-fold immediately after exposure to ozone and was 4-fold higher after 20 h. The number of lavageable eosinophils at the same time points were 3- and 15-fold greater, respectively, than control eosinophils. Immunocytochemistry revealed that alveolar macrophages and bronchial epithelial cells were positive for eotaxin. These results suggest that eotaxin may be involved in the recruitment of eosinophils into the air spaces during certain inflammatory conditions in rats.

The significance of complement activation in the pathogenesis of hypersensitivity pneumonitis: sequential changes of complement components and chemotactic activities in bronchoalveolar lavage fluids.

Hypersensitivity pneumonitis (HP) is believed to be induced by immunological mechanisms, the details of which remain to be clarified. While a role for cellular immunity is accepted in the pathogenesis of HP, several clinical observations also suggest a role for immune-complex-mediated lung injury. We have previously demonstrated the presence of chemotactic factors for polymorphonuclear cells (PMNs) in bronchoalveolar lavage (BAL) fluids of acutely ill patients with the summer type of HP found in Japan. The present study correlated chemotactic factors for PMNs with the level of C5a des Arg in BAL fluids obtained from patients with summer type HP. Furthermore, this study demonstrated that PMNs were increased in BAL fluids obtained after 2 days of avoidance of exposure to the presumptive causative agent. The percentage of PMNs in the BAL increased in proportion to the activity of the chemotactic factors. Finally, leukotriene B4 was not detected in concentrated BAL or supernatant fluids of cultured macrophages. These results suggest that complement activation in the respiratory tract may occur as the early event in the pathogenesis of HP.

Role of endogenous nitric oxide in allergen‐induced airway responses in guinea‐pigs

1 Endogenous nitric oxide (NO) can be detected in exhaled air and accumulates in inflamed airways. However its physiological role has not been fully elucidated. In this study, we investigated a role for endogenous NO in allergen‐induced airway responses. Sensitised guinea‐pigs were treated with NG‐nitro‐l‐arginine methyl ester l‐NAME (2.0 mm) or aminoguanidine (AG) (2.0 mm) 30 min before the allergen challenge, and 3 and 4 h after the challenge. Alternatively, l‐arginine (2.4 mm) treatment was performed 30 min before, and 2 and 3 h after the challenge. In all groups, ovalbumin (OVA) challenge (2 mg ml−1 for 2 min) was performed, and airway responses, NO production, infiltration of inflammatory cells, plasma exudation and histological details were examined. 2 Allergen‐challenged animals showed an immediate airway response (IAR) and a late airway response (LAR), which synchronised with an increase in exhaled NO. Treatment with l‐NAME and AG did not affect IAR while they significantly blocked LAR (72% and 80% inhibition compared to vehicle) and production of NO (35% and 40% inhibition). On the other hand, treatment with l‐arginine did not affect IAR but potentiated LAR (74% augmentation). 3 In bronchoalveolar lavage (BAL) fluid, allergen‐induced increases in eosinophils were reduced by 48% for l‐NAME treatment compared to vehicle, and increased by 56% for l‐arginine treatment. 4 Treatment with l‐NAME significantly decreased airway microvascular permeability to both Monastral blue (MB) and Evans blue (EB) dye (50.6% and 44% inhibition). 5 We conclude that allergen‐induced LAR is closely associated with NO production, and that NO plays a critical role in inflammatory cell infiltration and plasma exudation in the allergic condition.