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???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
---|---|---|
dc.contributor.advisor | 吳焜裕(Kuen-Yuh Wu) | |
dc.contributor.author | Hung-Wei Chao | en |
dc.contributor.author | 趙泓威 | zh_TW |
dc.date.accessioned | 2021-06-17T08:25:45Z | - |
dc.date.available | 2023-02-01 | |
dc.date.copyright | 2021-02-23 | |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-02-04 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74238 | - |
dc.description.abstract | 異環胺(Heterocyclic amines, HCAs)為高溫烹飪肉類衍生的化學物質,目前已純化鑑定出30多種HCAs。其中10種在動物實驗中導致多器官腫瘤,流行病學研究結果呈現HCAs暴露增加結直腸癌風險;IARC已將這10種HCAs分別歸類為2A及2B級致癌物質。民眾食用高溫烹飪肉品時,可能同時暴露多種HCAs,因此許多人關切日常飲食暴露HCAs的潛在健康威脅。為探討多種HCAs暴露對健康潛在的影響,需要執行HCAs累積性風險評估(Cumulative risk assessment),為完成這個評估需要有每個HCA致癌係數或是毒性相對效力因子(Relative potency factors, RPFs)。目前文獻上主要利用體外基因毒性試驗結果以估算致癌的RPFs以執行致癌風險評估,為減少推估致癌的RPFs不確定性,本研究的目的在於建立In-cell Western (ICW)方法分析組磷酸化的H2AX組蛋白(γH2AX)作為染色體基因傷害的生物標誌物,以定量分析18種HCAs的基因毒性。分別針對單一HCA使用多劑量在96孔盤高通量(high-throughput)處理人肝癌HepG2細胞,總共處理18種HCAs,處理24小時後,利用MTT試驗檢測細胞毒性,使用ICW免疫螢光法定量γH2AX檢測每種HCA的基因毒性,以ICW γH2AX讀值代表之。實驗結果顯示,在18種HCAs中,Trp-P-1是具最強基因毒性,接著依序為Trp-P-2、Glu-P-2、PhIP、Glu-P-1,相對地,MeIQ、DMIP、TMIP、4,8-DiMeIQx,Harman是產生微弱的基因毒性。利用基準劑量軟體(Benchmark dose software; BMDS)針對每一種HCA估算額外增加10% ICW讀值的95%信賴區間的下限 (Lower bound of 95% confidence interval of ICW; ICWL10)。同時也利用BMDS方法針對7種有多劑量致癌數據的HCAs,估算致癌斜率因子(CSF)。並利用統計回歸方法分析7 HCAs的CSFs與其相對的1/ICWL10的關係式,結果得到CSF與1/ ICWL10間有很好的線性關係(R2 = 0.914),再將剩下11 HCAs的1/ ICWL10帶入這個函數,便可估算這11種 HCAs的CSFs。結果發現將PhIP的RPF當作1,則Trp-P-1的RPF大於27,MeIQ、AαC、Trp-P-2等RPFs則與PhIP類似接近1.0,其餘HCAs的RPFs都小於0.5,代表基因毒性比PhIP低;研究顯示熱裂解型HCAs的基因毒性是比熱生成型HCAs更強。這些結果顯示高通量的γH2AX ICW試驗能用於篩選化學物質的基因毒性,這些基因毒性數據進一步可以用來估算RPFs與預測CSFs,本研究也是國際上首次估算出18種HCAs的CSFs,將可提供執行多種HCAs共暴露的累積性健康風險評估,以探討國人經飲食暴露的的多種HCAs對健康的影響。 | zh_TW |
dc.description.abstract | Heterocyclic amines (HCAs) are dietary carcinogens spontaneously formed in high temperature processed meats. There are about 30 HCAs have been identified. Ten of them were carcinogenic to rodents and causes tumors in multiple organs, including colorectal, pancreatic, and breast cancers. Numerous epidemiological studies revealed exposures to HCAs causing increases in the incidences of colorectal cancer (CRC). The International Agency for Research on Cancer (IARC) has classified 10 HCAs as 2A and 2B carcinogens, respectively. The adverse health effects due to co-exposures to HCAs have been of great concerns. To assess cumulative risk assessment of HCAs usually use relative potency factor (RPFs) approach. No RPFs of HCAs is currently available, instead of using cancer potencies, which were estimated by using tumor data of HCAs. γH2AX is a phosphorylated histone and were used as a biomarker of genotoxicity. In this study, a high-throughput and cell-base assay with a highly specific antibody combined with the immunofluorescence, In-Cell Western (ICW) assay, was established to analyze γH2AX and used to determine genotoxicity of 18 HCAs, and then used to derive genotoxicity RPFs and cancer slope factors (CSFs) for HCAs risk assessment. Human liver hepatocellular cells, HepG2 cells were used in the in vitro assay. The cells were treated with individual HCA at different doses for 24 hours. Cytotoxicity was determined by the MTT assay. Genotoxicity measured by using the γH2AX ICW assay in a 96‐well plate. The lower bound of 95% confidence interval of ICWL (ICWL10) was estimated by using the Benchmark dose software (BMDS), according to the dose-respnse relationship between γH2AX and dose of each HCA. The CSFs of HCAs were aslo estimated with the BMDS method. Regression analysis was used to fit the animal carcinogenicity data and 1/ ICWL10 with a linear function with R2= 0.914. The γH2AX ICW assay reveals that Trp-P-1 was the strongest genotoxicant, followed by Trp-P-2, Glu-P-2, PhIP, and Glu-P-1. MeIQ, DMIP, TMIP, 4,8-DiMeIQx, and Harman are very weak genotoxicants. By using the linear function, the CSFs and RPFs were derived for their corresponding HCAs. The RPF of Trp-P-1 was the highest among the 18 HCAs, 27 times greater than that of PhIP. The RPFs of MeIQ, AαC, and Trp-P-2 are appromiate to that of PhIP. The RPFs of other 13 HCAs were less than 0.5, suggesting that they are much weaker genotxicants than PhIP. Studies also shown that genotoxicity of pyrolytic HCAs are stronger than thermic HCAs. Our results demonstrated that the high-throughput γH2AX ICW assay can be used to determine genotoxicity of chemicals and used to derive RPFs and CSFs for the cumulative cancer risk assessment even though they lack animal carcinogenicity data. This study is also the first to study the genotoxicity of 18 HCAs. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T08:25:45Z (GMT). No. of bitstreams: 1 U0001-2401202122584500.pdf: 6518974 bytes, checksum: af51308bc8a5363278e3c005e9a8dfcd (MD5) Previous issue date: 2021 | en |
dc.description.tableofcontents | 誌謝 i 中文摘要 ii 英文摘要 iv 圖目錄 viii 表目錄 xii 第一章 前言 1 1.1. 異環胺介紹及分類 1 1.2. 異環胺代謝 3 1.3. 異環胺致突變性及致癌性 3 1.4. 異環胺流行病學研究 6 1.5. 異環胺累積性風險評估及等毒性當量因子 9 1.6. 基因傷害 11 1.7. 基因傷害指標 12 1.8 γH2AX In-Cell Western (ICW) 技術 12 第二章 研究目標 15 第三章 材料與方法 16 3.1 細胞培養及實驗相關試劑 16 3.2 異環胺藥品 16 3.3 動物致癌資料劑量反應關係評估 18 3.4 致癌斜率因子(Cancer slope factor, CSF ) 18 3.5 HepG2細胞培養及繼代 19 3.6 MTT存活率試驗 19 3.7 γH2AX In Cell Western試驗條件優化 21 3.8 γH2AX In Cell Western試驗 22 3.9 體外試驗之基因傷害指標劑量反應關係評估 23 3.10 相對效力因子計算 23 3.11 體外試驗指標預測致癌斜率因子 24 3.12 統計方法 24 第四章 結果 25 4.1 異環胺基準劑量10%下限值(BMDL10)及致癌斜率評估結果 25 4.2 以MTT試驗分析異環胺的致細胞毒性 31 4.3 γH2AX In Cell Western試驗條件優化 37 4.4 以γH2AX In Cell Western試驗定量分析異環胺的致基因毒性 38 4.5 異環胺劑量反應關係模型評估及相對效力因子 45 4.6 體外試驗指標預測致癌斜率因子 49 第五章 討論 51 第六章 結論 57 參考文獻 136 | |
dc.language.iso | zh-TW | |
dc.title | 利用高通量體外試驗評估異環胺的基因毒性及其應用 | zh_TW |
dc.title | Genotoxic potencies of Heterocyclic Amines: Assessing genotoxicity using high-throughput in vitro assay | en |
dc.type | Thesis | |
dc.date.schoolyear | 109-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 江素瑛(Su-Yin Chiang),陳炳輝(Bin-Huei Chen),鄭尊仁(Tsun-Jen Cheng),林靖愉(Ching-Yu Lin) | |
dc.subject.keyword | 異環胺,基因毒性,γH2AX,In-cell western試驗,致癌斜率因子,健康風險評估, | zh_TW |
dc.subject.keyword | Heterocyclic amines,Genotoxicity,γH2AX,In-cell western assay,Cancer slope factor,Risk assessment, | en |
dc.relation.page | 158 | |
dc.identifier.doi | 10.6342/NTU202100142 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2021-02-04 | |
dc.contributor.author-college | 公共衛生學院 | zh_TW |
dc.contributor.author-dept | 食品安全與健康研究所 | zh_TW |
Appears in Collections: | 食品安全與健康研究所 |
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