奈米鈦分子之腎臟毒理學研究及奈米化槲皮素之急性腎損傷療效與毒性評估

Abstract

第一部分:奈米鈦之腎臟毒理學研究 科技文明愈是進步，毒理的傷害就越貼進人類生活。奈米(nanometer,nm)的定義，是規範物質的直徑大小，或是任何一面截面積在100nm以下。過去研究發現毒理的表現則與直徑大小相關，直徑越小，接觸的表面積相對增加，則毒理作用就愈明顯。奈米鈦(Nano-Titanium Dioxide, Nano-TiO2)是一種低溶解度的白色粉末，由國際癌症研究中心 (The International Agency for Research on Cancer; IARC) 將其歸類在2B組(具有致癌的風險性)。常見的曝露方式是由呼吸道吸入，皮膚接觸或是經由口腔誤食入，再藉由顆粒性白血球吞噬或因其大小直徑足以自由通透細胞而進入血流循環，藉由血液循環分佈至全身沉積在各個器官產生不同程度的傷害。奈米鈦是金屬元素，不溶解，經由血液循環至組織、器官後，具有累積的現象，進而衍生發炎或是細胞訊息傳遞的機制。以呼吸道曝露模式，過去主要使用氣管滴注及固定氣體噴霧等方式模擬。當奈米鈦經由呼吸道曝露後，肺臟為其第一標的器官，會引起肺臟吞噬細胞的移行及浸潤，促使杯狀細胞分泌黏液，造成細胞發炎及肺臟纖維化，進而促成為結節、斑痕化，最後形成腫瘤及癌症。腎臟為身體的排泄器官，凡身體各器官代謝後的產物，都需藉由腎臟形成尿液排出體外，因此推斷腎臟同為奈米鈦之標的器官，但截至目前奈米鈦是否對腎臟造成傷害及其相關機制，尚未明瞭其啟動的機轉。本研究是利用鼷鼠曝露於三種不同的濃度的奈米鈦(0.1毫克、0.25毫克、0.5毫克)，曝露方式以滴注方式，每週一次，連續四週(1)。四週後犧牲，收集鼷鼠之血液及肝、腎、脾、肺等器官。由實驗結果顯示；曝露奈米鈦沉積而產生活性氧化物質(ROS, Reactive oxygen species)傷害近端腎小管，使得小管內層刷狀毛脫落，細胞凋亡，進而造成腎臟功能的損壞。我們以掃描式電子顯微鏡發現於近端腎小管細胞的細胞質內有鈦元素沉澱現象，血液中尿素氮(blood uria nitrogen,BUN)濃度增加。而活性氧化物質的阿爾發型誘發性缺氧因子(hypoxia inducible factor-1α, HIF-1α) 表現，促使下游的血紅素氧化酶(Heme Oxygenase -1,HO-1) 及貝他型轉化誘導因子(transforming growth factor-β, TGFβ)表現，造成纖維蛋白堆積。若給予活性氧物質清除者乙醯基半胱胺酸(N-acetylcysteine, NAC)及誘導型一氧化氮合成酶抑制劑(iNOS inhibitor):氨基胍碳酸氫鹽(aminoguanidine)，則可有效減輕奈米氧化鈦所造成之腎臟損傷。根據本研究發現，奈米鈦會經血液循環於腎臟累積，誘發活性氧物質產生並進一步造成氧化傷害。未來也許可以應用此訊息傳遞路徑減少氧化鈦所造成之腎臟傷害。 第二部份:奈米化槲皮素之急性腎損傷療效與毒性評估 槲皮素(Quercetin)是一種自然界中存在的可食用黃酮醇(flavonol)，是類黃酮家族(flavonoid family)的成員。槲皮素是多環酚類 (polyphenol)的架構，其主要的結構是中間具有苯環的相連接。於食物中通常是與單醣、寡醣體(Mono-, or Oligo -saccharide)相結合而形成醣苷鍵(Glycosidic bond)，增加水溶性。一般可經由食物攝取而獲得的有蘋果、洋蔥、西洋芹及可可亞等食物中均含有。身體中或是腸道中細菌所產生的β-glycosidase可幫助水解成不同的甲基化或是硫化基的型式。槲皮素可經由肝臟的代謝、腸道的吸收、及腎臟的排泄而增進人類的使用。槲皮素具有抗發炎、抗氧化、甚至也有研究報導具有抗癌的功效，所以每日所攝取的量會因食物的種類及數量不同而異。由於是歸類為食物補充品，其建議攝取量為；每日每人200至1200毫克(200-1200 mg/day)。身體中所產生的過氧化氫(H2O2)或是自由基(free radical)，可經由槲皮素做結合、清除。由於槲皮素不易溶於水，需以有機類溶劑增加其溶解度，往往會造成攝取的不便及毒性的傷害。本實驗嘗試以新合成的化合物奈米化槲皮素-線性聚乙烯亞胺合成物〔(nano-scale quercetin- PEI )〕來改善增加槲皮素的水溶解度。對於新合成奈米化的槲皮素，雖增加了水溶性的功能，但對於線性聚乙烯亞胺的接合進入生物體，是否會引起毒性的反應或併發症則是未知，因此必須研究其生物的相容性與價值。因黃酮醇(槲皮素)俱有光化學效應(phytochemical reaction)，其代謝的中間產物與細胞結合是否會造成傷害?也需進一步的研究證實。實驗設計是以單一劑量的急毒性及連續口服28天的亞急毒性的測試。單一劑量急毒性是以5公克/每公斤的濃度換算，以餵食管的方式注入動物口腔。而在亞急毒性則是分為三種不同濃度，分別為；低(150毫克/每公斤)、中(500毫克/每公斤)及高( 1000毫克/每公斤)劑量，連續餵食28天。期間觀察動物的行為狀態、活動力、外觀皮膚、毛髮是否脫落、眼睛出血、飲食、排泄物是否異常，及死亡率等。急毒性組於14 天後犧牲，觀察內臟器官是否重量改變，充血、發炎、壞死等現象。亞急毒性組攜牲後，收集血液、及心、肝、脾、肺、腎，睪丸、子宮、卵巢等器官。然而，在抗氧化傷害測試，以腎臟做缺血/再灌流模式(ischemia / reperfusion)。將鼷鼠單側的腎臟血管用血管夾夾住，缺血時間設定為25分鐘，再將夾子鬆開，讓血流再灌流通過。於實驗期間將另一側的腎臟作摘除的動作。經由實驗的觀察；對於急毒性結果，動物沒有發生死亡，皮膚毛髮外觀無改變，及行為活動力降低等現象。而在28天亞急毒性的結果；動物也沒有死亡，皮膚毛髮沒有脫落，眼睛無充血及行為活動力也無異常。犧牲後體重沒有差異，內臟器官絕對及相對重量比較上也沒有統計學上的改變。血液學常規檢查及臨床生化學檢查也沒有統計上的差異。組織切片H E染色，於高劑量組別各器官形態上均沒有明顯的損傷與變化。所以對於新合成奈米化槲皮素應用在動物體的毒性測試顯示；不具有生物毒性的反應。另外，對於臨床的急性腎臟缺血性/再灌流模式，其結果在尿素氮(blood uria nitrogen, BUN)及肌酸酐(creatinine)的數據與對照組比較，均有顯著的下降。且Cystatine C與其估計之腎絲球廓清率(Estimated Glomerular filtration ratio, eGFR)在使用奈米化槲皮素後也有改善，具有統計上的意義。蘇木精-伊紅染色(hematoxylin and eosin stain, H E stain)組織染色與血清檢測雖有變化，但不具有劑量上的相對效應。為提升吸收效率及增加對水的溶解率而新合成的化合物，經由急毒性及亞急毒性的測試，對於生物體的使用是不具有生物性的毒性反應。

Part I: To Evaluate Renal Function in Toxicological study of Nano -Titanium Dioxide (Nano-TiO2) The more advanced science and civilization, the more toxicity closed in ours daily life. Nanometer definition is; below the 100nm in any section of the particle surfaces. The toxicity value and the particle diameter are opposite, the more advanced toxicity is related to the particle size smaller in diameter, which increased the contact surfaces. Nano-Titanium Dioxide (Nano-TiO2) is a low solubility white powder. The International Agency for Research on Cancer (IRAC), therefore, has classified TiO2 as group 2B carcinogen (possibly carcinogenic to human). The most exposure way of nano-TiO2 is by respiratory tract, skin contact or oral feeding. It can pass the cell membrane, induce target organ injury, and enter into blood circulation that induced the inflammatory signal by accumulation. The intra-tracheal instillation and gas spray were the conventional way for studying of nano-TiO2 model. Lung injury is the target commonly inflammation result. It induced alveoli macrophage migration, phargocytosis, goblet cell secretion and developed nodules, scaring, fibrosis into carcinogenesis of the particles exposure. Kidney are metabolic and excretion organ, also a target of nano-TiO2, but induced pathologic injury were still controversy. We used different concentration of nano-TiO2 (0.1mg , 0.25mg and 0.5 mg) by intra- tracheal instillation weekly course . After 4 weeks later, we collect whole blood for blood routine and biochemistry test. Liver, spleen, lung, and kidney organs for histopathology staining. As result shown that TEM (Transmission Electronic Microscopy) figure shown nano-TiO2 had deposited in renal proximal tubular cell cytoplasm. In hematoxylin and eosin (H E staining), tubular lumen brush border loss and cast formation. In biochemistry test, blood urea nitrogen (BUN) increased. In Immunohistochemistry stain (IHC stain), cell signaling HIF-1α (hypoxia induced factor-1α), HO-1 (heme oxygenase-1), and TGF-β (transforming growth factor-β) were activated, and developed collagen-I accumulation. We tried to use the ROS (reactive oxygen species) scavenger NAC (N-acetylcysteine) and iNOS inhibitor (aminoguanidine, AQ) for decreased ROS-induced renal injury, and it had decreased in IHC staining. Conclusion: In the present study, the nano-TiO2 particle will trans-through lung alveoli into the circulation system by respiratory tract exposure. It will induced the inflammation and oxidative stress by nanoparticle deposit in target organs. Part II: Toxic assessment and therapeutic effects of nano-scale quercetin PEI in acute kidney injury Quercetin is a naturally food supplement of flavonol, which is a member of the flavonoid family of compounds. The basic structure of quercetin is consisting of two benzene rings linked a heterocyclic pyran or pyrone ring. Quercetin is bound to mono- or oligosaccharides via a glycosidic bond on the oxygen-containing ring for increasing aqueous solubility. In our daily uptake, the sources of quercetin are usually from apple、cooked onion、cocoa and raw celery. Both mammalian-encoded enzyme or intestinal microflora produced β-glycosidase hydrolysis quercetin into the metabolites such as methylated or sulfated forms. In the present studied showed that quercetin has anti-oxidant, anti-inflammatory, even the anti-carcinogenic effects. The daily recommended dosages is 200-1200 mg/day. Although, it can scavanged the superoxide and free redical, it also induced the toxicity by organic solvent for solubility, which could caused inconvenience and harmful injury. We try to synthesis a new compound of nano-scale quercetin- PEI (polyethyleneimine) to increase water solubility. It's also need to evaluate the toxicity and side-effect. The experimental model are acute and sub-acute toxicity models. The concentration for acute toxicity test is 5g/kg by oral gavage once time, and for sub-acute group are 150mg/kg/day, 500mg/kg/day and 1000mg/kg/day for 28 days consecutively. In period time, we observed the skin, hair, eyeball bleeding, food feeding, activity, and motality. After 14 and 28 days, animal were sacrificed and blood, visceral organs, such as heart, liver, spleen, lung, kidney, testis, uterus and ovary were collected. Biochemical and histopathology test were used to evaluate the toxicity of quercetin-PEI. Moreover, we also want to investigate the effects of quercetin-PEI in kidney, so we choose the renal ischemia/reperfusion (I/R) model to evaluate the therapeutic possibility of quercetin-PEI. In H E stain, Quercetin-PEI could reversed renal I/R induced tubule dilation, cast formation and cell death. The values of BUN, creatinine, Cystatin C and eGFR which represent kidney function were also reversed by queretin-PEI. Similar results were shown in the lipid peroxidation and the expression of COX-2. Taken together, our results showed that quercetin-PEI could reversed acute renal injury and its no-observed-adverse-effect level (NOEAL) is 1000 mg/kg/day.