Time-dependent effect of inhaled cigarette smoke exposure in the bleomycin-induced lung injury rat model.

Cigarette smoke (CS) substances are known to induce diverse ailments such as cancer, decreased immunity, and lung diseases. Although some studies have been actively conducted to evaluate cigarette toxicity, the current animal exposure methods, that is, exposure of 28- or 90-days, require considerable research cost and lead to obscure results of the CS effects. In a previous study, we compared the effects of CS in a rat model of bleomycin (BLM) and lipopolysaccharide (LPS) induced lung disease. We determined that compared to the LPS-induced rat model, the BLM-induced rat model was more sensitive to alterations in secreting cytokines and total cell number. In the current study, we further confirmed the time-point of effective inhalation exposure by CS in the BLM-induced lung injury rat model. Using an automatic video instillator, rats were administered a single dose of 2.5 mg/kg BLM (day 1), and subsequently exposed to CS via inhalation (nose-only) 4 h/day, for 1, 2, 3, and 4 weeks. The bronchoalveolar lavage fluid (BALF) was obtained from the right lung lobes, total cell numbers were counted, and chemokine and cytokine expressions were evaluated using Enzyme-Linked Immunosorbent Assay. For the 1-week exposure, we observed a greater increase of neutrophils in the BLM + CS 300 µg/L group than in the BLM or CS 300 µg/L groups. Exposure of CS in the BLM-induced lung injury rat model enhanced the secretions of chemokines and cytokines, such as CCL2/MCP-1, CXCL2/MIP-2 and TNF-α, at 1 week. Immunohistochemistry and Hematoxylin and Eosin staining of lungs at 1-2 weeks after exposure clearly confirmed this tendency in the increased levels of CCL2/MCP-1 and TNF-α. Taken together, these results indicate that the rat model of BLM-induced lung injury is more sensitive to CS exposure than other rat models, and may be an appropriate model to evaluate the effect of CS exposure at 1-2 weeks.

Effect of the phenylpyrrole fungicide fludioxonil on cell proliferation and cardiac differentiation in mouse embryonic stem cells.

Fludioxnil is extensively used as a fungicide in agricultural application, but its possible impact on embryonic development is not yet well understood. In this study, the potential effect of fludioxonil on cardiac differentiation was evaluated in mouse embryonic stem cells (mESCs). The water-soluble tetrazolium (WST) and colony formation assays were conducted to confirm the effect of fludioxonil on proliferation of mESCs. The effect of fludioxonil on the ability of mESCs to form mouse embryoid bodies (mEBs) was determined by the hanging drop assay, whereas the ability of cardiomyocyte differentiation in the early stage was evaluated by determining the beating ratio (ratio of the number of contracting cells to the number of attached EBs) of cardiomyocytes. The viability of mESCs was significantly decreased (less than 50 %) at 10-5 M fludioxonil. Results of the colony formation assay revealed suppressed colony formation at 10-5 M fludioxonil (about 50 % at 5 days). Furthermore, the expressions of cell-cycle related proteins, i.e., cyclin D1, cyclin E, p21 and p27, were altered and trending towards inhibiting cell growth. Exposure to fludioxonil also resulted in reduced size of the mEB and induced increasing expression levels of the pluripotency markers Oct4, Sox2 and Nanog. Development of the beating ratio in the process of differentiation to cardiomyocytes derived from mESCs was completely inhibited after exposure to 10-5 M fludioxonil during the early stage of differentiation (day 5), whereas the beating ratio gradually increased after 5-day treatment. Simultaneously, expressions of the cardiomyocyte-related proteins, Gata4, Hand1 and cTnI, were inhibited after exposure to 10-5 M fludioxonil. Taken together, these results imply that fludioxonil may impact on the developmental process of mESCs, particularly the cardiac lineage.

Inhalation exposure by cigarette smoke: Effects on the progression of bleomycin- and lipopolysaccharide-induced lung injuries in rat models.

The toxic substances of cigarette smoke (CS) induce inflammatory responses in the lung by recruiting inflammatory cells. In this study, we investigated the effects of CS on the progression of lung disease in bleomycin (BLM) and lipopolysaccharide (LPS)-induced lung injury rat models. Briefly, rats were exposed to CS via inhalation (nose-only) for 28 consecutive days, for 4 h per day. Using an automatic video instillator, rats were administered a single dose of 2.5 mg/kg BLM (day 1) or 0.5 mg/kg LPS (day 26), prepared in 50 µL phosphate-buffered saline (PBS) solution. Examination of the bronchoalveolar lavage fluid (BALF) revealed that the number of neutrophils increased in a concentration-dependent manner of CS. Exposure to CS also enhanced the expression of cytokines, i.e., CCL2 (MCP-1), CCL3 (MIP-1α), CXCL2 (CINC3), CXCL10 (IP-10), TNF-α, IFN-γ, IL-2, IL-4 in the BALF of the vehicle (VC) and BLM groups in a concentration-dependent manner. In particular, the expressions of CCL2, CXCL10 and TNF-α were remarkably upregulated in the BLM + CS 300 treatment as compared to VC, while there were no differences in these cytokine levels in the serum following CS exposure. Exposure to CS resulted in compacted alveolar spaces and macrophage aggregation in the lung tissues following BLM and LPS treatments. Compared to VC, pulmonary fibrosis and chronic inflammation of bronchioloalveoli were observed in the BLM + CS treatment and inflammatory cell infiltration of bronchioloalveoli was observed in the LPS + CS treatment in a concentration-dependent manner by CS. The expression levels of CCL2 and IFN-γ in the lung tissues were increased similar to the levels obtained in BALF, in a concentration-dependent manner by CS. Taken together, these results indicate that repeated exposure to CS may exacerbate the lung injury initially caused by BLM and LPS.

Fenhexamid induces cancer growth and survival via estrogen receptor-dependent and PI3K-dependent pathways in breast cancer models.

Fenhexamid (Fen), a fungicide used to treat gray mold of fruits and vegetables, is reported to function as an endocrine disrupting chemical via the estrogen receptors (ER), despite low-toxicity of the pesticide. In this study, we elucidated that the disrupting effects of Fen are exerted via the ER and phosphatidylinositol 3-kinase (PI3K) pathways in breast cancer models. The WST assay, live cell monitoring, cell cycle analysis, colony formation assay, apoptotic analysis by JC-1 dyeing, and Western blot analysis were applied in ER positive MCF-7 and ER negative MDA-MB-231 breast cancer cells, after exposure to 17ß-estradiol (E2), Fen, ICI 182,780 (ICI; an ER antagonist) and/or Pictilisib (Pic; a PI3K inhibitor). Exposure to E2 and Fen induced the cell growth and survival ability of MCF-7 cells by increasing the S-phase cells and regulating the cell cycle-related proteins (Cyclin D1 and E1, p21 and p27). In addition, E2 and Fen treatment resulted in elevated levels of the survival-related proteins (Survivin and PCNA), and inhibited apoptosis by increasing the mitochondrial membrane potential and regulating the apoptosis-related proteins (BAX, BCL-2, and Caspase-9). These changes were reversed to the same level as the control group when exposed to their respective inhibitors, thereby indicating that the changes are exerted via the ER and PI3K pathways. In particular, co-treatment with these inhibitors induced greater inhibition than single treatment. Conversely, no alterations were observed in the ER-negative MDA-MB-231 breast cancer cells. Taken together, these results indicate that Fen promotes the growth of breast cancer cells via the ER and/or PI3K pathways, similar to the E2 mechanism. Although a relatively safe pesticide, Fen possibly exerts its influence as an endocrine disrupting chemical in ER-positive breast cancer cells via the ER and PI3K pathways.

Inhibitory effects of cigarette smoke extracts on neural differentiation of mouse embryonic stem cells.

Maternal smoking during the perinatal period is linked to adverse neonatal outcomes such as low birth weight and birth defects. Numerous studies have shown that cigarette smoke or nicotine exposure has a widespread effect on fetal nerve development. However, there exists a lack of understanding of what specific changes occur at the cellular level on persistent exposure to cigarette smoke during the differentiation of embryonic stem cells (ESCs) into neural cells. We previously investigated the effects of cigarette smoke extract (CSE) and its major component, nicotine, on the neural differentiation of mouse embryonic stem cells (mESCs). Differentiation of mESCs into neural progenitor cells (NPCs) or neural crest cells (NCCs) was induced with chemically defined media, and the cells were continuously exposed to CSE or nicotine during neural differentiation and development. Disturbed balance of the pluripotency state was observed in the NPCs, with consequent inhibition of neurite outgrowth and glial fibrillary acidic protein (Gfap) expression. These inhibitions correlated with the altered expression of proteins involved in the Notch-1 signaling pathways. The migration ability of NCCs was significantly decreased by CSE or nicotine exposure, which was associated with reduced protein expression of migration-related proteins. Taken together, we concluded that CSE and nicotine inhibit differentiation of mESCs into NPCs or NCCs, and may disrupt functional development of neural cells. These results imply that cigarette smoking during the perinatal period potentially inhibits neural differentiation and development of ESCs cells, leading to neonatal abnormal brain development and behavioral abnormalities.