Development of a system for the detection of the inflammatory response induced by airborne fine particulate matter in rat tracheal epithelial cells.

Exposure to airborne particulate matter (PM) is related to the increased risk of several diseases, including chronic and allergic rhinitis. We have previously shown that atmospheric endotoxin level was positively associated with the number of emergency department visits for asthma even after adjusting for meteorological factors, suggestive of the significant association between atmospheric endotoxin level and asthma exacerbation. Whether atmospheric endotoxin level is related to inflammatory response induction is, however, unclear. Here, we established stable cell lines to determine the promoter activity of the genes encoding pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin 6 (IL6), and IL33 by transfection of each reporter plasmid into rat tracheal epithelial EGV-4 T cells. These cells could measure the inflammatory response induced by endotoxin treatment more easily, rapidly, and sensitively than the conventional system using immunodetection assays. Furthermore, we revealed a relationship between atmospheric endotoxin level and inflammatory response induction. Thus, the system established herein may serve as a promising tool to monitor inflammatory response induced upon PM exposure.

Development of a system for the detection of the inflammatory response induced by airborne fine particulate matter in rat tracheal epithelial cells.

Exposure to airborne particulate matter (PM) is related to the increased risk of several diseases, including chronic and allergic rhinitis. We have previously shown that atmospheric endotoxin level was positively associated with the number of emergency department visits for asthma even after adjusting for meteorological factors, suggestive of the significant association between atmospheric endotoxin level and asthma exacerbation. Whether atmospheric endotoxin level is related to inflammatory response induction is, however, unclear. Here, we established stable cell lines to determine the promoter activity of the genes encoding pro-inflammatory cytokines such as tumor necrosis factor alpha, interleukin 6 (IL6), and IL33 by transfection of each reporter plasmid into rat tracheal epithelial EGV-4 T cells. These cells could measure the inflammatory response induced by endotoxin treatment more easily, rapidly, and sensitively than the conventional system using immunodetection assays. Furthermore, we revealed a relationship between atmospheric endotoxin level and inflammatory response induction. Thus, the system established herein may serve as a promising tool to monitor inflammatory response induced upon PM exposure.

Genotoxicity and estrogenic activity of 3,3'-dinitrobisphenol A in goldfish.

3,3'-Dinitrobisphenol A (dinitro-BPA) is formed in a mixture of bisphenol A (BPA) and nitrite under acidic conditions. It shows genotoxicity in male ICR mice on a micronucleus test, but its estrogenic activity has not been examined in vivo. We examined its estrogenic activity using goldfish (Carassius auratus) by measuring plasma levels of vitellogenin (VTG) by the ELISA method. Expression of VTG didn't increase in the plasma of goldfish intraperitoneal injected with dinitro-BPA at a dose of 10 mg/kg of body weight. We also examined the genotoxicity of dinitro-BPA by single-cell gel electrophoresis (comet assay) and a micronucleus test using goldfish. The DNA tail moment of blood cells increased after intraperitoneal injection of dinitro-BPA. Dinitro-BPA at the same dose significantly increased micronucleus frequency in gills of goldfish. On the other hand, BPA did not significantly increase the frequency of micronucleated cells. In conclusion, we found that dinitro-BPA did not show estrogenic activity, but had genotoxic potency stronger than that of BPA.

Application of a new bioassay technique using goldfish for assessment of water toxicity.

There are a variety of chemicals in aquatic environment, so it is important to assess the toxicity. The biomarkers such as induction of DNA damage, micronuclei, vitellogenin, and hepatic P450 in fish are known to be effective for monitoring genotoxic and/or estrogenic chemicals. However, there is little study to use these biomarkers in same fish. Goldfish (Carassius auratus) is widely used and is suitable in size to collect blood or organs. In this study, validity of multiple-biomarkers in goldfish was checked using standard chemicals and applied in the river water. Ho River, which flows through the textile dyeing factory in Shizuoka Prefecture, Japan, was reported to show genotoxicity toward Salmonella typhimurium TA98 and YG1024. When the goldfish were exposed to Ho River, DNA damage, estrogenic activity, and CYP1A induction were observed. Through the study, it was assumed that not only mutagens/carcinogens but also endocrine disrupting chemicals and poly aromatic hydrocarbons were present in Ho River. Therefore, chemical identification should be required. We could evaluate both genotoxicity and estrogenic activity simultaneously, so goldfish might be a good experimental model for estimation of chemical contamination levels in aquatic environment.

Evaluation of mutagenic activities of leachates in landfill sites by micronucleus test and comet assay using goldfish.

To develop a simple system for monitoring the presence of mutagens/carcinogens in the leachates from landfill sites, we used a micronucleus test and a single cell gel electrophoresis (comet) assay originally developed for mice and rats on goldfish (Carassius auratus). The goldfish were exposed for 9 days to the leachate with chemical and biological treatment (treated leachate) or without treatment (raw leachate). The goldfish exposed to several samples died because of the high concentrations of NaCl or ammonium ion (NH4+). In the comet assay using peripheral erythrocytes, the raw leachates showed higher mutagenic activity than the treated leachates. In the micronucleus test, it was difficult to detect the micronuclei in peripheral erythrocytes. On the other hand, the frequency of micronuclei was high in gill cells of goldfish exposed to the raw leachates compared to the treated leachates. A combination of the two bioassays was shown to be useful to evaluate the mutagenic activity of the leachates. We also propose a new scoring method for determination of water quality by using acute toxicity and mutagenic activity.

Changes in the mutagenic and estrogenic activities of 17beta-estradiol after treatment with nitrite.

We determined the changes in the mutagenic and estrogenic activities of 17beta-estradiol after a nitrite treatment. Nitrite-treated 17beta-estradiol showed mutagenic activities toward Salmonella typhimurium strains TA 100 and TA 98. We confirmed that nitrite-treated 17beta-estradiol generated radicals from the results of an analysis of electron spin resonance. By applying an instrumental analysis, we identified 2-nitro-17beta-estradiol to have been formed in the reaction mixture. 2-Nitro-17beta-estradiol did not exhibit mutagenic activities toward Salmonella typhimurium strains, suggesting that other mutagens might have been formed in the reaction mixture. The clastogenic properties of nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol were analyzed by a micronucleus test with male ICR mice. Nitrite-treated 17beta-estradiol and 2-nitro-17beta-estradiol induced a significantly higher frequency of micronucleated reticulocytes in mice. The estrogenic activity of 2-nitro-17beta-estradiol was found to be lower than that of 17beta-estradiol. These data suggest that a daily oral intake of 17beta-estradiol and nitrite might induce the formation of mutagenic compounds in our body.