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  • 學位論文

順丁烯二酸於大鼠中之藥物動力學,氧氮化壓力和代謝體學研究

Pharmacokinetics, Oxidative/Nitrosative stress and Metabolomics of Maleic Acid in Sprague-Dawley Rats

指導教授 : 吳焜裕

摘要


順丁烯二酸為一種人造有機化合物,主要用在各種工業用途及個人護理用品如牙膏、藥劑、洗髮精等。然而,食品製造商為求增加口感、彈性以及延長賞味期,在各種澱粉基底的美食中惡意參雜順丁烯二酸,造成了台灣嚴重的食品安全問題。此種未經許可將順丁烯二酸用作食物添加劑的作法,以及順丁烯二酸由包裝轉移至商品上的持續曝露風險,使得人體攝入順丁烯二酸的可能風險研究更有其必要。 雖然已知順丁烯二酸會造成動物不同程度的腎毒性傷害,但其機制尚屬未知。本論文先藉由液相層析串聯質譜儀(LC-MS/MS),開發一種新的測量生物流體中順丁烯二酸濃度的分析方法。接續以此分析方法,對攝入順丁烯二酸在SD公母大鼠血液及尿液中進行動力學及生體可用率研究。藥物動力結果證實順丁烯二酸被快速吸收、代謝並移除,以及低生體可用率。 本論文的下一部分則檢視順丁烯二酸的攝入和發生氧化攻擊與分子改變之間的關聯性。本研究開發出一個快速且敏感度高的離線液相層析串聯質譜儀 (off-line LC-MS/MS)分析方法,可同時分析多個生物標記。這些生物標記(biomarkers)可指示出氧化壓力,體內不同生物標記的量增加關係著各種不同疾病的風險增加。以此項分析方法進行後,顯示出單次口服順丁烯二酸曝露會引發DNA氧化傷害以及脂質過氧化作用。在尿液中,8-NO2Gua, 8-OHdG, and 8-isoPGF2α的濃度有著統計上顯著的增加,尤其是在12小時內攝入高劑量的公鼠體內。研究並顯示在攝入後,這些生物標記濃度的增加會維持數天;公鼠對於這些氧化作用的體內反應較母鼠強烈。 最後本論文藉由液相層析串聯質譜儀及核磁共振(LC-MS/MS and 1H NMR)進行亞慢性毒理分析;同時進行病理解剖及器官採樣後,對於收集的樣本進行組織分析。結果顯示順丁烯二酸造成了尿液中8-OHdG, 8-NO2Gua and 8-isoPGF2α濃度的上昇;acetoacetate, hippurate, alanine, and acetate的分析也在各自實驗組顯示出時間相關、劑量反應相關的不同結果。在個體體重以及腎臟/體重比等項目有著顯著變動;組織分析也顯示出腎臟和肝臟有著細胞結構上的改變。研究指示出攝入低劑量的順丁烯二酸提升了體內氧化負擔,引發膜內脂質崩壞,且干擾體內能量代謝。 這些分析方法有著高敏感性、高特異性及高穩定性,能在生物流體中偵測到接近十億分之一濃度等級(ppb)的順丁烯二酸及多種生物標記。結果顯示無論單次或重複性的順丁烯二酸曝露都會引發細胞毒性,且體內吸收移除皆快速,雖然並無臨床上病徵。本項實驗方法可用在其他食物污染或環境毒素的毒性確認及風險評估。

並列摘要


Maleic anhydride is an anthropogenic organic chemical reagent used in an array of industrial and personal care products. The purposeful adulteration of maleic anhydride by foods manufacturers into starch-based delicacies to improve texture, elasticity and prolong storage time instigated food safety concerns in Taiwan. This unapproved use of maleic anhydride as a food additive, combined with the continuous risk of exposure from packaging migration, underscore the need to investigate the potential risks upon ingestion. Despite prior knowledge indicating maleic anhydride can lead to varying degrees of nephrotoxicity in animals, its mechanisms of toxicity remain elusive. This dissertation began to develop a new analytical method using liquid chromatography tandem-mass spectrometry (LC-MS/MS) to determine maleic acid concentrations in biofluids. Subsequently, the kinetic fate and bioavailability of ingested maleic acid in serum and urine in both male and female Sprague-Dawley rats were studied using the aforementioned analytical protocol. Pharmacokinetic results confirmed that maleic acid is absorbed, metabolized and eliminated rapidly, along with low bioavailability. The next portion of this thesis examined the association between maleic acid consumption and the occurrence of oxidant attacks and molecular changes. A rapid and highly sensitive off-line LC-MS/MS method was developed to simultaneously quantify multiple biomarkers indicative of oxidative stress, with elevations associated with increases in risks of various diseases. Application of the abovementioned method demonstrated that oral single-dose exposure to maleic acid induced oxidative DNA damage and lipid peroxidation, as demonstrated by the statistically significant increases in urinary levels of 8-NO2Gua, 8-OHdG, and 8-isoPGF2α, particularly in high-dosed male rats within 12 h of consumption. Our findings also demonstrated that increases in concentration of these biomarkers persist for days after consumption; male rats appear to be more sensitive to oxidative burden compared to their counterparts. Finally, a subchronic toxicity study employing both LC-MS/MS and proton nuclear magnetic resonance spectroscopy (1H NMR) spectroscopy was conducted to elucidate the molecular events; organs were collected at necropsy and underwent histology analysis. Our results demonstrated that maleic acid increases urinary concentrations of 8-OHdG, 8-NO2Gua and 8-isoPGF2α; analysis of acetoacetate, hippurate, alanine, and acetate exhibited time- and dose-dependent variations in the treatment groups. Physio-morphological alterations were evident in body weights, relative kidney weights; histology analysis revealed cellular changes in the kidneys and liver. Our findings suggest that maleic acid consumption escalates oxidative burden, triggers membrane lipid destruction, and disrupts energy metabolism. The analytical methods demonstrated high sensitivity, specificity and stability capable of detecting maleic acid in biofluids and multiple urinary biomarkers at parts-per-billion concentrations. Our results demonstrated that with rapid absorption and elimination, as well as absence of clinical manifestations, both single and repeat exposures to maleic acid trigger cytotoxicity. The experimental approaches can be applied to clarify toxicity of other food contaminants or environmental toxicants for risk assessment. Additional studies targeting inhalation exposure and dermal absorption may further increase the merit of risk assessment.

參考文獻


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[8] USEPA, "Reassessment of the one exemption from the requirement of a tolerance for maleic anhydride(CAS#108-31-6) and maleic acid (CAS#110-16-7)," EPA, Ed., ed. Washington, D.C.: USEPA, 2006, p. 16.
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