三聚氰胺及三聚氰酸合併加強腎小管細胞傷害及大鼠腎毒性作用之研究

Abstract

在2004及2007年國內外爆發犬貓食用受污染之寵物飼料造成腎衰竭事件，其病因證實為寵物飼料遭三聚氰胺(melamine；M)及三聚氰酸(cyanuric acid；C)之聯合汚染毒性所造成。雖然已知單一三聚氰胺(LD50：3,161 mg/kg)或三聚氰酸(LD50：7,700 mg/kg)對動物之急毒性低，但當兩者同時存在其對腎毒性卻明顯增強，唯其誘發腎毒性作用機制及毒性等級，仍有待進一步探討。本實驗分成活體外(in vivtro)及活體內(in vivo)探討三聚氰胺和三聚氰酸合併加強腎毒性作用，在In vitro部份：比較不同來源腎小管上皮細胞，如近端腎小管(NRK52E及LLC-PK1)、遠端腎小管(MDCK及PK-15)與三聚氰胺及三聚氰酸在偏弱酸(pH 6.5及pH 6.0)或弱鹼(pH 7.5及pH 7.0)培養液中作用，結晶形成數量性質及對腎小管細胞毒性之差異。結果顯示，三聚氰胺及三聚氰酸隨pH值愈低結晶析出愈多，同時對細胞毒性也愈強，且以近端腎小管較敏感，其細胞死亡路徑以壞死為主。在In vivo部分：則以50、100、200、300及400 mg/kg三聚氰胺與三聚氰酸(M+C, 1:1)混合劑量，進行大鼠腎臟急毒性指標試驗。此外，為尋找臨床中毒緩解方法，嘗試單次投予400 mg/kg三聚氰胺與三聚氰酸(M+C, 1:1)後，同時投予低(500 mg/kg)或高(2000 mg/kg)劑量檸檬酸鈉(sodium citrate)連續3天，評估其緩解腎毒性作用。結果顯示，三聚氰胺與三聚氰酸合併毒性之口服半致死劑量(LD50)雄、雌鼠分別為58.6 mg/kg及83.9 mg/kg，毒性等級分類為中等毒性，腎臟病變可見大量褐色放射狀結晶析出，並嚴重破壞腎小管細胞。而投予高劑量檸檬酸鈉則可有效緩解M+C組尿液pH值之酸化作用，降低血清BUN及Creatinine、尿液結晶、造骨蛋白(osteopontin)、增殖細胞核抗原(PCNA)及結晶數量，但腎臟中抗氧化酵素(GSH、SOD、MDA)及炎症前期細胞激素(IL-1β, IL-6, TNF-α)無顯著性差異(p>0.05)。綜合以上結果，三聚氰胺與三聚氰酸兩者化合物混合可析出大量結晶體，促使對大鼠腎急性毒性具有加強作用，投予檸檬酸鈉則可部份減緩腎毒性傷害，推測可能因檸檬酸鈉可降低三聚氰胺及三聚氰酸造成尿液酸化作用，導致腎結晶不易於腎小管中析出有關。

Two outbreaks of pet food associated renal failure in companion animals occurred in Asian, included Taiwan and North American countries in early 2004 and 2007. The combined toxicity of melamine (M) and cyanuric acid (C) are considered as responses in the pet food associated nephrotoxicosis. Although low acute toxicity of melamine (LD50: 3,161 mg/kg) or cyanuric acid(LD50: 7,700 mg/kg); however, the mechanisms of synergistic nephrotoxicity are unclear. This experiment was divided into In vivtro and In vivo to assay the melamine and cyanuric acid combination of synergistic nephrotoxicity. In vitro assay, the potential cytotoxicity of MC (1:1) combination in four different renal tubular cells in either weak acid (pH 6.0 and 6.5) or alkaline (pH 7.0 and 7.5) medium. Results revealed that melamine and cyanuric acid of combination easily precipitated and reformated crystals in a pH dependent, and significant enhanced cellular toxicity to the proximal tubular cells (NRK52E and LLC-PK1). Besides, cytotoxicity was mainly via necrosis pathway. In vivo assay, The acute renal toxicity (rat) model were given by oral gavage at dosage (50, 100, 200, 300 and 400 mg of each compound/kg) M+C mixture, Furthermore, the inhibitory effect of urine alkalinization by treating single dose of 400 mg/kg MC and accompanied with low (500 mg/kg) or high (2000 mg/kg) dose of sodium citrate for 3 days in rats. Results revealed that the acute oral LD50 of M+C is 58.6 mg/kg in male and 83.9 mg/kg in female, and as a moderate toxicity, and showed numerous of brownish radiate crystals formation and caused tubular necrosis. In addition, urine alkalinization with high dose of sodium citrate possed not only significantly decrease of urine acidity but also reduced serum BUN and creatinine parameters. Furthermore, histoscores of toxic nephropathy, immunohistochemical indices of proliferating cell nuclear antigen (PCNA), crystal related osteopontin (OPN) were also significantly decreased, but no effect on antioxidant enzymes (SOD, GSH, MDA), proinflammation cytokines (IL-1, IL-6, TNF-α) in injured kidneys. In conclusion, results demonstrate that synergistic nephrotoxicity of MC combination is found in rats; however, it can be partially attenuated by treatment of sodium citrate by deceasing crystallization in kidneys.