飼料污染三聚氰胺與三聚氰酸引起急性腎毒性作用機制與基因毒理之研究

Abstract

本實驗已完成2004年疑似污染飼料經長期餵食後，對實驗動物(大鼠)誘發腎毒性作用之再現性，並證實與三聚氰胺與三聚氰酸污染有關。本實驗目的為進一步探討三聚氰胺和三聚氰酸腎毒性毒理試驗及其可能作用機制研究，擬分為3年完成以下實驗：第1年：三聚氰胺與三聚氰酸混合對腎急性毒性動物模式之研究，探討混合使用之口服半致死毒性劑量，試圖找出致死性時間及毒性閥值，並分析血液學、肝腎血清生化學、尿液學、腎臟抗氧化酵素分析、腎臟細胞激素分析、組織病理學檢查、組織免疫化學染色包括增殖細胞核酸抗原(Proliferating cellnuclear antigen, PCNA)、造骨蛋白(Osteoponin, OPN) Nucleolin-related-protein(NRP)、Annexin-2 (ANX2)、OX-1 (CD45, leukocyte common antigen)以及ED-1(CD68,monocyte/macrophage marker)等測定腎小管上皮細胞增殖及結晶形成、腎臟細胞電子顯微鏡等超顯結構改變之觀察，以期建立混合使用對腎毒性傷害之病理形態學診斷， 找出主要毒性作用細胞(target cells) 及偵測生物指標(biomarkers)，進行後續研究方向正確性；第2年：三聚氰胺與三聚氰酸(1:1)混合長期食用對大鼠28天及90天動物餵食安全性之研究，分析項目包括體重、飲水及飼料消耗量之變化、各種血液及血清生化值、尿液學、肉眼及組織病理等動物毒理評估，探討兩者聯合混合連續食用後之無毒害作用劑量(no observed adverseeffect level, NOAEL)，可作為日後訂定三聚氰胺與三聚氰酸聯合毒性每日可攝取量(acceptable daily intake, ADI; Tolerance daily intake, TDI)之安全性閥值(thethreshold dose)之參考依據；第3年：三聚氰胺與三聚氰酸混合對腎細胞及大鼠腎毒性作用及致基因毒理機制之研究，本實驗將分別探討兩者混合於不同酸鹼值之結晶析出量，對不同部位腎小管上皮細胞之細胞毒性差異性，分析造成腎臟細胞死亡途徑、找出致死性時間及毒性閥值。進一步以大鼠腎毒性動物模式，測試調節酸鹼值之藥物進行抑制三聚氰胺與三聚氰酸混合使用對，作為臨床中毒預防及治療參考。最後評估三聚氰胺與三聚氰酸(1:1)致基因毒理試驗，以期建立混合中毒對腎毒性傷害之致癌潛在毒性風險性之評估。

producing in experimental rats the nephrotoxicities similar to the outbreak in 2004.Although low acute toxicity of melamine or cyanuric acid; however, the pathogenicmechanisms and toxic thresholds of synergistic nephrotoxicity of melamine andcyanuric acid combination are unclear. The objective of this study will investigatethe pathogenic mechanisms and toxic thresholds of synergistic nephrotoxicity ofmelamine and cyanuric acid combination in the next three years. Firstly, the acuteanimal toxicity of melamine and cyanuric acid combination in vivo (rats) model willbe conducted for the threshold dose and time course, including hematology,biochemistry, urinary, antioxidative, cytokines, pathology, immunohistochemistricalbiomarkers for proliferating cell nuclear antigen (PCNA) and osteoponin (OPN),Nucleolin-related-protein (NRP)、Annexin-2 (ANX2), OX-1 (CD45, leukocytecommon antigen), ED-1 (CD68,monocyte/macrophage marker), and electronicmicroscopy to find the target cells of kidney. Secondary, we will determine the 28and 90-day feeding toxicity in rats base on the level of acute LD50, variousparameters including body weight, water and food consumptions, hematology,biochemistry, urinary, and pathology will be examined and to set the no observedadverse effect level (NOAEL) in rats for safety reference of acceptable daily intake(ADI) or tolerance daily intake (TDI) of melamine and cyanuric acid combination infoods. Finally, we will evaluate the MC crystals formation and cytotoxicity of renalcells in different species and the potential genetic toxicity. Four different renal cellswill be coincubated with melamine and cyanuric acid combination at ratio of 1:1 tocompare the acute cytotoxicity for renal cells. Additionally, the inhibitory effect ofurinary alkalization in the melamine and cyanuric acid (MC) toxicity will beevaluated and provided the validity of therapy for the acute renal toxicity of MCintoxication. Furthermore, three tests of genetic alterations (in vitro and in vivo) ofmelamine and cyanuric acid combination at ratio of 1:1 will be performed for thepotential cancer risk assessment in human.