| 摘要: | 攝食紅肉與加工肉品在流行病學研究中，被發現會增加大腸直腸癌、乳癌與前列腺癌的風險。國際癌症研究組織在2015年將攝食紅肉和加工肉品分別歸類為2A級和1級人類致癌物。雜環胺類和亞硝胺類化合物是紅肉加熱或肉類加工(如醃製和煙燻)過程中所產生的致癌物質，其在體內會分別被CYP1A1、CYP1A2、CYP1B1或CYP2E1等酵素代謝成活性代謝物，進而造成基因毒性與致癌性。在台灣肉鬆與美式加工肉品中含量最高的雜環胺類分別是harman與2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)，在香腸中含有高量的亞硝胺類化合物N-nitrosodiethylamine (NDEA)。本論文探討這些化合物間是否有交互作用，進而增加或降低混合物的毒性。我們以體外肝細胞為模型，觀察單獨暴露harman或NDEA、及其與PhIP共暴露所造成的毒性效應，來探討共暴露時化合物間的交互作用。觀察的毒性指標包括細胞毒性、基因毒性、氧化傷害、和細胞凋亡，並分析CYP代謝酵素的基因與蛋白質表現，以探討毒性交互作用的可能分子作用機制。使用人類肝癌細胞株(HepG2)和大鼠正常肝細胞株(Clone 9)，單獨暴露或共暴露化合物24小時之後，以MTT試驗法檢測細胞毒性、以彗星試驗法檢測基因毒性、以細胞流式儀和螢光染色法檢測氧化傷害、以TUNEL染色法檢測細胞凋亡情形；另外，以定量即時聚合?鏈鎖反應(qPCR)和西方轉漬法檢測CYP代謝酵素的基因與蛋白質表現。MTT試驗結果顯示，無論在HepG2癌細胞與Clone 9正常細胞，單獨暴露harman、NDEA或PhIP都會造成具劑量依賴性的細胞毒性，且兩株細胞的毒性差異不大，所以之後的實驗只使用HepG2細胞。在HepG2細胞，harman、NDEA與PhIP誘導20%細胞生長抑制的濃度(IC20)，分別為100 ?M、100 ?M與40 mM；共暴露時，以細胞存活率計算藥物相互作用係數(coefficient of drug interaction, CDI)，發現harman或NDEA分別與PhIP (100 或200 ?M)共暴露時，具有協同作用(CDI < 1)。彗星試驗結果顯示，單獨暴露NDEA(20 或 40 mM)或100 ?M PhIP會誘導DNA損傷(P < 0.05)，但單獨暴露100 ?M harman不會；而共暴露時，PhIP會顯著增強harman與NDEA的致DNA損傷效應(P < 0.05)，結果顯示具有協同作用。此外，流式細胞儀結果顯示，單獨暴露harman會誘導活性氧族群的產生，單獨暴露PhIP則不會；共暴露時，螢光染色法結果顯示，PhIP會稍微增強harman的致氧化傷害效應。TUNEL染色法結果顯示，PhIP與harman皆會誘導細胞凋亡；共暴露時，PhIP會稍微增強harman的致細胞凋亡效應。qPCR試驗結果顯示，單獨暴露harman或PhIP都會誘導CYP1A1與CYP1A2基因的表現，共同暴露harman和PhIP時，會更顯著增強CYP1A1與CYP1A2基因的表現；另外，CYP1A1的蛋白質表現在單獨暴露harman的細胞有增加的趨勢。本論文在HepG2細胞的實驗結果顯示，與單獨暴露harman組相比較，共暴露PhIP時會增強harman的致細胞毒性、致基因毒性、致氧化傷害、與致細胞凋亡情形。民眾攝食肉類加工品極可能同時共暴露到harman和PhIP時，我們的結果表明兩化合物間可能存在致基因毒性的協同作用，可能歸因於harman與PhIP可誘導自身的代謝活化酵素CYP1A1和CYP1A2的表現。因此，建議進行加工肉品雜環胺類健康風險評估時，將化合物間可能的交互作用列入考量。
Epidemiological evidence reveals that consumption of red and processed meat significantly increases the risk of colorectal, breast and prostate cancers. Since 2015, the International Agent for Research on Cancer (IARC) has classified red meats as class IIA human carcinogens and processed meats as class I human carcinogens, due to the presence of heterocyclic amines (HCAs) and N-nitrosamines; chemical compounds that are generated by cooking procedures. These chemical compounds are metabolized by cytochrome P450 (CYP) enzymes CYP1A1, 1A2, 1B1, and CYP2E1 to active metabolites, which are associated with their genotoxicity and carcinogenicity. The highest content of HCAs in Taiwanese pork floss and western processed meat are harman and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), respectively. The nitrosamine compound N-nitrosodiethylamine (NDEA) is found in high amounts in sausages. This thesis explores whether these compounds interact with each other and thereby increase or decrease the toxicity of the mixture. We exposed the human liver cancer cell line HepG2 and cultured rat liver clone-9 cells to PhIP, NDEA, or harman, alone or in combinations of PhIP with either NDEA or harman to determine cytotoxicity, genotoxicity, oxidative stress, and apoptosis, over 24-h exposure periods. We also analyzed the mRNA and protein expression of CYP450. Cell viability was measured by the MTT assay; genotoxicity was tested with the Comet assay. Flow cytometry and fluorescence staining analyzed the effects of oxidative stress and was used to detect reactive oxygen species (ROS). The TUNEL assay determined levels of apoptosis. Quantitative polymerase chain reaction (qPCR) assay and Western blot assays measured levels of CYP gene and protein expression. MTT assay results showed that the exposure of HepG2 and clone-9 cells to harman, NDEA or PhIP alone causes dose-dependent cytotoxicity. We found that exposing HepG2 cells to 100 µM harman, 100 µM PhIP and 40 mM NDEA inhibited cell growth by 20% (IC20). Calculations of the coefficient of drug interaction (CDI) based on the relative survival data revealed synergistic effects after co-exposure of PhIP with harman or NDEA (CDI <1). Comet assay data shows DNA damage after exposure to NDEA or PhIP alone, but harman alone did not cause DNA damage. Combined exposure with 100 μM PhIP significantly enhanced the DNA damage induced by harman or NDEA (P < 0.05). Furthermore, flow cytometry results showed that exposure to harman but not PhIP alone induced the formation of ROS. The fluorescence staining results showed that PhIP slightly enhanced the oxidative damage induced by harman. TUNEL staining results showed that PhIP or Harman alone induced apoptosis; while co-exposure, PhIP slightly enhanced harman-induced apoptosis. The qPCR results showed that harman or PhIP alone induced the levels of CYP1A1 and CYP1A2 gene expression. Combined exposure to harman and PhIP markedly increased the expression of CYP1A1 and CYP1A2 genes. Besides, the protein expression of CYP1A1 slightly increased in cells exposed to harman alone. In this paper, results from HepG2 cells showed that as compared to exposure to harman alone, co-exposure with PhIP enhanced the cytotoxicity, genotoxicity, and oxidative damage, apoptosis induced by harman. People may be exposed to both harman and PhIP when ingest processed meat products. Our results indicate a possible synergistic effect between the two compounds, possibly due in part to the induction of CYP1A1 and CYP1A2 by harman and PhIP. Therefore, it is recommended that the possible interactions between compounds may be taken into account in conducting health risk assessment of HCAs in processed meat. |
