機車排放污染物之毒理研究─機車廢氣對乙醯膽鹼酯活性之影響(3/3)

本研究的主旨在探討機車排放懸浮微粒萃 取物以及多環芳香烴對乙醯膽鹼酯活性 之影響。我們發現機車排放懸浮微粒萃取 物會抑制由電鰻所分離的乙醯膽鹼酯之 活性，其抑制百分之五十的濃度為20.72± 1.50 ppm。反之，柴油巴士排放懸浮微粒萃 取物抑制乙醯膽鹼酯活性百分之五十之 濃度遠大於300 ppm。我們亦發現苯環數目 在三環或三環以上的多環芳香烴， 如 anthracene， benzo(a)pyrene, chrysene 等， 對乙醯膽鹼酯具有明顯的抑制作用，其 抑制百分之五十的濃度約為2 ~ 6 ppm，而 低苯環數目的多環芳香烴或含氧的多環芳 香烴，則抑制乙醯膽鹼酯活性的情況較 不明顯。此外，機車排放懸浮微粒萃取物 及多環芳香烴亦會對丁醯膽鹼酯活性具 有抑制作用，其抑制百分之五十的濃度分 別為22.90±3.20 ppm 及4 ~ 9 ppm。The effect of the extract of motorcycle exhaust particles (MEPE) and polycyclic aromatic hydrocarbons (PAHs) on acetylcholinesterase (AChE) activity was examined. We have found that the MEPE dose-dependently inhibited the activity of AChE purified from electrical eel with IC50 of 20.72±1.50ppm. On the other hand, the IC50 of diesel exhaust particulate extract was greater than 300ppm. We have also found that PAHs with 3 or greater ring number, such as anthracene, benzo(a)pyrene, chrysene, inhibited the AChE activity in a competitive manner with IC50 values between 2~6ppm. The PAHs with lower molecular weight, such as acenaphthylene and naphthalene, and oxygenated PAHs, such as xanthone and anthraquinone, were ineffective. Both MEPE and PAHs inhibited the butylcholinesterase activity at similar concentrations

多環芳香烴對內皮細胞之影響

多環芳香烴類的化合物是廣泛存在於環境中的污染物，包括工業製程所排放的氣 體、交通運輸排放廢氣、化石燃料的不完全燃燒、拜香和樟腦的使用等，都是環境中多 環芳香烴的污染來源，這些污染物可以經由皮膚接觸、呼吸道或腸胃道吸收進入人體， 由循環系統擴散至各組織，經由細胞色素的代謝活化，引起遺傳物質的突變，或藉由氧 化自由基的生成對細胞產生毒性。血管內皮細胞是位於血管內壁的單層細胞，不僅為血 管壁的屏障，更可以經由EDRF/NO 的釋放，維持血流的順暢，在之前的研究中發現， 短時間暴露低環數多環芳香烴會活化內皮型一氧化氮合成(endothelial nitric oxide synthase)，合成並釋放NO，引發血管平滑肌的放鬆作用，然而內皮細胞長時間暴露在多 環芳香烴下的影響，卻不十分清楚，因此，本實驗採用人類臍靜脈內皮細胞(human umbilical vein endothelial cell)來進行更深入的探討。 我們發現高濃度(>50mM)的naphthalene、fluoranthene、fluorene 等多環芳香烴會抑制 細胞生長週期的進展，使細胞週期停頓在G0/G1 時期，而在低濃度(0.1-50mM)時對細胞 生長週期的影響較輕微，也不會造成細胞死亡。此外，多環芳香烴會促進細胞內eNOS mRNA 的表現，進而增加eNOS 的蛋白轉譯量，並增加eNOS 的活性和NO 的生合成量， 然而，這些多環芳香烴卻不會促進cyclooxygenase-1 的表現，顯示由多環芳香烴所調控 的基因表現具有專一性。在進一步探討多環芳香烴的作用機轉中， 我們發現 phosphatidylinositol 3-kinase 抑制劑wortmannin 和MAP kinase kinase 抑制劑PD98059，均無 法改變多環芳香烴對eNOS mRNA 的正向調節作用。反之當我們利用了鈣離子螯合劑 EGTA 和BAPTA-AM 分別螯合細胞內和外的鈣離子，結果發現在鈣離子被螯合的狀態 下，明顯的抑制由多環芳香烴所誘發的eNOS mRNA 表現，顯示鈣離子參與多環芳香烴 對eNOS mRNA 的調節。相較於低環數的多環芳香烴，高環數benzo(a)pyrene 卻無法促進 eNOS 的表現，因此aryl hydrocarbon receptor 可能不參與以上多環芳香烴對eNOS 基因表 現的影響。 綜合以上的結果，在我們的實驗中發現，長時間暴露多環芳香烴，會增加eNOS 的 蛋白表現，而其作用點主要是在eNOS mRNA 層次，而細胞內的鈣離子則扮演關鍵性的 角色。Polycyclic aromatic hydrocarbons (PAHs) are found in the exhausts of industrial manufacture, and transportation due to the incomplete combustion of fuel, or the usage of camphor balls and joss sticks which are common pollutants and wild spread in our environment. These pollutants can be absorbed into body via respiratory tract, gastrointestinal tract or skin contact, then distributed to the whole body through blood circulation. In biological system, PAHs have been found to induce mutagenic or carcinogenic effect after cytochrome P-450 bioactivation. In addition, PAHs also produced reactive oxygen species during metabolism and resulted in cytotoxic effects. Endothelial cells localized inside the blood vessels, and function as a vascular barrier between lumen and vascular smooth muscle. One of the important pathological and physiological function of endothelial cell is synthesis and releasing of endothelium-derived relaxating factor, nitric oxide, which can modulate vasotension and maintain the homeostasis of vascular system. Preliminary studies revealed that endothelial nitric oxide synthases were activated immediately after low-molecular weight PAHs exposure, then converted L-arginine to nitric oxide resulted to vasodilation. However, the long term effect of PAHs on endothelial cell is still unclear. In present research, the human umbilical vein endothelial cell was used. We found that several low-molecular weight PAHs such as naphthalene, fluoranthene, fluorene inhibited cell growth cycle progression at higher concentrations(>50mM), resulting in cell cycle arrest at G0/G1 phase. However, the effects on cell cycle were absent and no cytotoxicity was observed in lower concentrations(0.1-50mM). In addition, PAHs will enhance eNOS mRNA expression in HUVEC, increase eNOS protein content and activity, and NO production. In contrast, the constitutively expressed cyclooxygenase-1 wasn’t affected, suggesting that effect of PAHs on eNOS was specific. We also found that the phosphatidylinositol 3-kinase inhibitor, wortmannin and MAP kinase kinase inhibitor, PD98059 can not reverse the upregulation of eNOS mRNA levels by PAHs. However, when the calcium chelators, EGTA and BAPTA-AM were used to chelated extracellular and intracellular calcium, respectively the effect of PAHs on eNOS mRNA expression was abolished, suggesting that calcium might be involved. In contrast to low-molecular weight PAHs, benzo(a)pyrene, a high-molecular weight PAHs didn’t enhance eNOS expression, implying that the aryl hydrocarbon receptor might not be involved in the previous findings. In summary, we found that long term exposure of PAHs on HUVEC will upregulate eNOS mRNA and protein level in a calcium-dependent manner

Reactive Oxygen Specises Involved in Motorcycle-Exhaust Particles Induced Genotoxicity in Vivo and in Vitro

The genotoxic potency of mototcycle exhaust particles (MEP) were investigated by use of bacterial reversion assay, chromosome aberration and micronucleus test in this study. In bacterial reversion assay (Ames test), MEP dose dependently increase TA98, TA100 and TA102 revertants in the presence of metabolic activating enzymes. In chromosome aberration test, MEP dose dependently increased abnormal structure chromosomes in CHO-K1 cells both with and without S-9. Pretreatment with antioxidants (vitamin E, vitamin C, catalase and NAC) shown varying degrees of inhibitory effects in MEP induced mutagenic effect and chromosomes structure abnormality. In vivo micronucleus test, MEP dose-dependently induced micronucli formation in peripheral red blood cells 24h and 48h after treatment. Intraperitoneal injection of vitamin E and vitamin C can significantly inhibited MEP induced clastogen in mice. The fluorescence intensity of DCFH-DA loaded CHO-K1 cells were induced upon addition of MEP. Our data suggested MEP can induce genotoxicity through ROS dependent pathway, which could be augmented by metabolic activation. Vitamin E, vitamin C, catalase and NAC can inhibited MEP induced genotoxicity indicated ROS might be involved in this effect

機車排放懸浮微粒對細胞黏附分子表現之影響及其機制之 探討

依據行政院衛生署所公佈92 年台灣地區主要死亡原因資料顯示，國內台灣地區主要死 因中惡性腫瘤排行第1，其中又以肺癌居首，其他肺部疾病如肺炎排名第7，支氣管炎、 肺氣腫及氣喘則排名11，這顯示了肺部傷害對於國人健康影響極大。在台灣機車數量為 17,849,070 輛是最常使用的交通工具，其密度遠大於歐美及日本等國，而依據行政院環 境保護署公告監測的空氣污染指標中指出懸浮微粒PM10和臭氧是為最主要的污染物，而 其來源除了工廠排放為最大宗之外，第二污染源就是汽機車引擎燃燒後所排放的廢氣， 在文獻報告中指出柴油引擎懸浮微粒（diesel exhaust particle；DEP）會引發肺部發炎， 造成各種細胞激素(cytokine)和細胞黏著分子（cell adhesion molecule；CAM）中的 ICAM-1、VCAM-1 和E-selectin增加，這些細胞黏著分子普遍在發炎的上皮細胞和內皮 細胞膜上大量表現，造成免疫細胞如嗜中性白血球、巨噬細胞的聚集和活化，是引發發 炎反應中一重要的反應，但目前仍沒有機車引擎懸浮微粒（motorcycle exhaust particle； MEP）對於細胞黏著分子表現影響的研究，因此本研究利用氣管滴入（intratracheal instillation；IT）的方式將MEP滴入BALB/c鼷鼠的氣管中使其自然吸入肺中後，觀察發 現MEP會造成BALB/c鼷鼠肺部的細胞黏著分子ICAM-1 表現量上升。肺泡中主要的細胞 組成除了內皮細胞外還有兩型上皮細胞和一些巨噬細胞，因此在體外實驗中發現人類臍 帶靜脈內皮細胞（human umbilical vascular endothelial cells；HUVEC）經MEP處理後會 造成ICAM-1、VCAM-1 和E-selectin表現量的上升並且造成人類單核球細胞THP-1 的黏 著，進一步的研究中還發現HUVEC經MEP的處理之後其NF-κB會活化移動到細胞核 內，而IκB則會有降解的現象發生。MEP所誘導CAMs的表現及NF-κB的活化會被抗氧 化劑所拮抗顯示氧化性壓力可能扮演著重要的腳色

空氣污染物對血管作用之探討

The in vitro effect of motorcycle exhaust particles on blood vessel was studied in thoracic aorta isolated from rat. The extract of motorcycle exhaust particles (MEPE) relaxed the phenylephrine pre-contracted aorta with an EC50 value of 0.05 ± 0.004 mg ml-1. This relaxing effect of MEPE persisted in endothelium-denuded aorta suggesting that the relaxation induced by MEPE is endothelium-independent. The phenylephrine-induced vasocontraction and inositol 1,4,5-trisphosphate formation were inhibited dose-dependently in aorta pretreated with MEPE. However, the high- K+-induced vasocontraction and the Ca2+ sensitivity of the contractile proteins were not significantly affected by MEPE. In addition to the inhibitory effect on agonistinduced contraction, both the vasorelaxing effect of acetylcholine and sodium nitroprusside were impaired by MEPE. The inhibitory effect of MEPE on acetylcholine and sodium nitroprusside, but not phenylephrine, responses were reversed by co-treatment with superoxide dismutase, suggesting that the impairment of acetylcholine response by MEPE might be due to the production of superoxide anion by MEPE. These results showed that the extract of motorcycle exhaust particle, added in vitro caused relaxation of aortic rings precontracted with phenylephrine but not KCl and impaired the relaxation induced by acetylcholine in a superoxide-dependent manner

機車排放懸浮微粒對呼吸系統之影響(2/2)

許多調查及研究顯示因燃燒石化燃料所造成的環境污染會透過許多因子影響免疫系統的功能包 括加強過敏反應及呼吸道的傷害。在本計劃中，我們探討了機車排放懸浮微粒，台灣都市主要污 染之一，對實驗動物過敏反應之影響。鼷鼠BALB/c以氣管植入的方式處理機車排放懸浮微粒， 每隔週一次共三次。我們發現機車排放懸浮微粒的處理會造成動物之肺部發炎現象，肺泡浸潤液 裏有嗜酸性球、嗜中性球、淋巴細胞及巨噬細胞大量的累積。肺臟病理切片的結果亦顯示發炎細 胞的侵入。機車排放懸浮微粒的處理亦加強了IL-4, IFN-g 等細胞激素以及血清IgE的產生，同時 亦造成了呼吸道的過度反應。在將機車排放懸浮微粒以不同的有機溶液粹取後，重複以上之實 驗，我們則發現苯粹取液會造成類似的反應。至於是何種物質造成發炎及過敏的反應則有待進一 步的分析。Large survey and experiments have reported that environmental pollutants from fossil fuel combustion would cause immune system deleterious by enhancement of allergic reaction and damage to respiratory tract. In this study, we investigated the effect of motorcycle exhaust particles (MEP), a major pollutant in Taiwan urban area, on airway allergic reaction in laboratory animals. BALB/c mice were intratracheal instillation with MEP once every two weeks for three times. We found MEP would induce airway inflammation characterized by infiltration of eosinophils, neutrophils, lymphocytes, and macrophages in bronchoalveolar lavage fluid (BALF) and caused inflammation of lung. In addition, MEP treatment enhanced BALF IL-4, IFN-g cytokine and serum IgE production and induced airway hyperresponsiveness. The benzene-extracted fraction induced airway inflammation characterized by infiltration of eosinophils and neutrophils, increase of BALF IL-4 production and serum IgE, and induction of airway hyperresponsiveness as seen with MEP

血根鹼毒性及藥性之探討(2/2)

血根鹼被認為與造成人類流行病水腫有很大之關係。流行病水腫主要 是因為食用了污染的芥子油中毒而導致之結果，其臨床症狀包括了水腫﹑ 心衰竭﹑呼吸困難﹑巨肝等。血根鹼也已知會對脊椎動物產生毒性導致呼 吸麻痺而死亡，同時亦有報導指出血根鹼具細胞及肝毒性。除了毒性之外， 有許多報導證實血根鹼具多種藥理活性，且被用在許多產品，如漱口水﹑ 牙膏以及咳嗽和感冒成藥中。雖然有關血根鹼的毒性已有許多報導，但其 致毒機制仍不十分清楚。因此，我們將於本計畫中針對血根鹼之致毒機制 作一深入的研究。 在去年度的實驗中，我們以分離之橫膈膜、胸主動脈以及心肌來探討 血根鹼之毒性與藥性。我們證實了血根鹼確實會透過不同的機制造成橫膈 膜之巒縮而導至肌肉的麻痺，抑制血管之收縮作用，並觀察到血根鹼會誘 發心臟肌肉之巒縮。本年度，我們證實了將進一步探討血根鹼造成心臟肌 肉之巒縮主要是透過鈣離子內流的作用所引起的。同時，我們亦證實了血 根鹼確實會對肝細胞造成傷害，主要是透過對粒線體的傷害而來。Sanguinarine, a benzophenanthridine alkaloid, has been implicated in outbreaks of human poisoning known as epidemic dropsy which is characterized by edema of the legs, congestive heart failure, hepatomegaly, ataxia and glaucoma. Sanguinarine is toxic to vertebrates when administered orally or intraperitoneally with death resulting mainly from respiratory paralysis. Sanguinarine is shown to be cytotoxic and a possible liver toxin. Besides its toxicity, sanguinarine has been used in many over-the counter products including toothpaste, mouthwash, cough and cold remedies, and homeopathic preparations. Although much more with respect to the toxic effects of sanguinarine has been carried out, the mechanism of action of this compound is still unclear. The goal for current project is to 3 elucidate the toxic effect of sanguinarine on respiratory and cardiovascular system and liver and the possible underline mechanism of action. In previous year, by using the isolated diaphragm, thoracic aorta and cardiac muscle strips, we have found that sanguinarine could directly induce contracture of diaphragm and leading to the paralysis of the muscle, inhibit the agonist-induced vasocontraction of the aorta, and induce contracture of the cardiac muscle through different mechanisms. In this year, we have proof that the contracture induced by sanguinarine in the cardiac muscle is mainly due to the induction of calcium influx. We have also found that sanguinarine is cytotoxic to the hepatocyte possibly due to the inhibition of mitochondria