open access

https://hdl.handle.net/2115/53897

トキシコゲノミクス的アプローチを用いた下水処理水の包括的影響評価及び下水応答性バイオマーカーの開発 [全文の要約]

英語

2013-09-25

Hokkaido University

https://www.lib.hokudai.ac.jp/dissertations/copy-guides/

The reclaimed wastewater has been served as an alternative water source in some countries but uncertain impacts of wastewater residues including vast categories of micropollutatns remains as a challenge to increase the public acceptance. Due to its comprehensive and rapid nature, transcriptome analysis such as DNA microarray and quantitative RT-PCR (qPCR) assay, was applied to human hepatocacinoma cells (HepG2) exposed to the effluents from membrane bioreactors (MBRs), and the activated sludge process (AS), to better understand the whole effluent impacts on humans, and develop genetic markers for such impacts. The effective reclamation processes were further suggested for the selected wastewater effeluents based on the toxicity resduction evaluation study using the genetic markers. The DNA microarray was first applied to MBR and AS effluents withouth any enrichment processes. In parallel, the conventional bioassays (i.e., cytotoxicity tests and bioluminescence inhibition test), which were wellestablished for the evaluation of the overall effluent toxicity were also performed for the same samples. The transcriptome analysis identified 2 to 926 differentially expressed genes after exposure to the effluents and the raw wastewater, which were categorized to 0 to 225 biological processes. Among the tested effluents, the MBR operated at a relatively long solid retention time (i.e., 40 days) and small membrane pore size (i.e., 0.03 μm) was suggested to have the least impacts on the HepG2 even at the level comparable to tap water. The observed gene expression responses were in good agreement with the results of cytotoxicity tests, and provided additional molecular mechanistic information on adverse effects occurred in the sub-lethal region. To select the effluent-responsive genetic markers, dose-response relationship between limited number of genes and effluent concentrations were studied by using MBR and AS effluents concentrated 10-15 times by reverse osmosis (RO), RO concentration successfully achieved sample enrichment with more than 80% recovery of organic content in the effeluents. The qPCR assay demonstrated that four out of nine candidate marker genes, which were selected from the DNA microarray data obtained for the concentrated effluents (i.e., 30 mg/L), had clear concentration-dependency, and therby suggested relevant as effluent marker genes. Based on the reduction in gene expression levels of marker genes, effectivity of the selected physicochemical treatment processes were investigated for both simulation of actual reclamation system and identification of responsible fraction. The qPCR assay of four marker genes (i.e., AKR1B10, CYP1A1, GCLM, and GPX2) before and after four typical treatments (i.e., aeration, solid phase extraction with C18, chelating, and ion exchange) together with the detail chemical analysis suggested that hydrophobic organic content, which may be forming complex with metals were responsible for a part of gene expression response observed in HepG2 exposed to the effluents. In addition, it was indicate the possibility that the responsible fraction behave together with humic substances or their building blocks falling in a size range from 300 to thousands of Da. Thus, activated carbon adsorption or reverse osmosis were suggested as key processes in reclamation system of the selected MBR and AS effluents. At the end, the arsenic-modulated gene expressions were investigated from inorganic arsenic exposure (5 nM to 40 micro M as arsenic trioxide for 48 hours). The concentration dependent modulation of gene expression (induction of cell cycle genes, suppression of DNA repair genes, induction of cell cycle arrest genes and apoptotic genes) following exposure to arsenic was further supported by acceleration of cell proliferation, ROS generation, and cytotoxicity. These results indicated the potential pro-carcinogenic actions of inorganic arsenic occur in environmentally relevant exposures. Comparison with gene expression profiles between effluents and model toxicicants revealed the uniqueness of the gene expression profiles of the effluents were suggested. In conclusion, the transcriptome analysis with human HepG2 cells is a powerful tool to rapidly and comprehensively evaluate impacts of whole wastewater effluents. The effluent-responsivbe genetic markers were also proposed to quantitatively evaluate the treatability of the wastewater effluents. Based on the response of genetic markers, the activated carbone adsorption was suggested as an effective process to remove responsible fraction, and the importance of final polishing with RO was also supported. Since there is still a gap between the gene expression response and the manifestation of toxicities, the gene expression response should be considered as “potential” of toxicity and thereby, further accumulation of DNA microarray data with wellestablished toxicity data is indispensible for development of this technology. In addition, due to the change of raw wastewater quality, seasonal variations of effluent impacts are to be investigated based on the gene expression response.

甲第11137号

博士(工学)

(主査) 教授 岡部 聡, 教授 高橋 正宏, 教授 船水 尚行

工学院（環境創生工学専攻）

博士論文

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