奈米銀粒子誘導CL5肺腺癌細胞株細胞激素表現及環境毒物之生物效應

Induction of cytokines by silver nanoparticles in CL5 human lung adenocarcinoma cells and biological effects of environmental toxicants

薛翔予(Hsiang-Yu Hsueh)

指導教授 ： 翁祖輝

國立臺灣大學/醫學院/毒理學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.10159

摘要

本篇論文主要目的為探討：一、奈米銀粒子對肺腺癌細胞株 CL5 細胞激素表現之影響；二、農藥愛殺松對雄性大鼠肝臟細胞色素 P450 表現之影響；三、機車廢氣對雄性大鼠及其子代肝臟細胞色素 P450 表現之影響。近年來，奈米銀粒子已經被應用於生物科技以及生命科學領域，奈米物質的粒徑為1到100 nm，這種粒徑的銀粒子可能具有特殊的物理、化學以及毒物動力學性質。本篇主要探討不同粒徑大小的奈米級的銀粒子Ag (7 nm)、Ag (50 nm) 對肺腺癌細胞 CL5 mRNA表現的影響，從 reverse transcriptase-polymerase chain reaction (RT-PCR) 分析結果顯示，Ag (7 nm) 會增加 fibroblast growth factor (FGF)-9 以及 interleukin (IL)-1β 的 mRNA 表現； Ag (50 nm) 增加 IL-1β 的 mRNA 表現，而 FGF-9 則沒有影響；AgNO3 會增加 FGF-9 mRNA 的表現，而 IL-1β 則沒有影響。在酵素部份 Ag (7 nm)、AgNO3 會使 ethoxyrcoumarin O-deethylase (ECOD) 活性、peroxide production 上升，glutathione S-transferase (GST) 活性與 total glutathione content 下降，lipid peroxidation (LPO) 沒有影響；Ag (50 nm) 使 ECOD 活性上升，而 GST、total glutathione content、LPO 則沒有影響；AgNO3 會使 ECOD 活性、LPO 上升，GST、total glutathione (GSH) content 下降。低粒徑的奈米粒子會對 CL5 細胞造成較強的氧化性傷害，高粒徑則較弱，對 FGF-9 與 IL-1β 的 mRNA 誘導表現也較強，氧化性傷害與 peroxide 的生成有關。
愛殺松是屬於有機磷類神經毒性殺蟲，用於防治蚜蟲、蝨、介殼蟲、葉蟬、蛆等病蟲害，它可以使用於許多種類的食物、纖維、裝飾類的作物，以及溫室作物。本研究的主要目的試探討愛殺松在雄性大鼠肝臟細胞色素 P450 的表現，分為低劑量與高劑量兩個部份，雄性大鼠以腹腔注射愛殺松，低劑量處理為 1、10 mg/kg；高劑量處理為 20 mg/kg，1 天 1 次進行 4 天，於肝臟微粒體，低劑量組 10 mg/kg 愛殺松使 pentoxyresorufin O-dealkylase (PROD) 活性增加，cytochrome P450、NADPH -cytochrome P450 reductase、methoxyresorufin O-demethylase 與 aniline hydroxylation 則是下降；高劑量組 20 mg/kg 愛殺松使 PROD 活性上升。在西方墨點法和 RT-PCR 分析中 CYP2B 的蛋白量和 mRNA 都有增加，且具有劑量效應關係。研究結果發現愛殺松是雄性大鼠肝臟內 CYP2B 的誘導物。
機車在台灣是最主要的交通工具之一，其排放的廢氣造成嚴重的空氣汙染，因此廢氣的為害對於人體的影響是我們所必須重視的。本實驗中藉由觀察大鼠肝臟內 P450 mRNA 的改變來了解機車廢氣對生物體及其後代的影響。實驗結果顯示雄性大鼠肝臟內的 CYP1A1、CYP2B、CYP2E1 mRNA 表現有上升，CYP2C12 則是下降，這顯示機車廢氣會對肝臟內的 P450 mRNA 產生影響。暴露機車廢氣的雄性大鼠與未暴露的雌性大鼠交配之後產下了第二代，第二代雄性大鼠肝臟內的 CYP1A2、CYP3A mRNA 表現於出生後 56、98 天時有下降，140 天則沒有影響；第二代雌性大鼠肝臟內的 CYP1A2 mRNA 於出生後 56 天時有下降，98、140 天時則沒有影響，CYP3A1 mRNA 於56、98 天時有下降，140 天沒有影響；CYP2B、CYP2C11 mRNA 只有雄性子代於 98 天時有下降。研究結果發現機車廢氣對雄性大鼠及其子代肝臟 P450 mRNA 表現有影響，因此應進一步針對其機制做探討。

關鍵字

奈米銀粒子 ； 愛殺松 ； 機車廢氣 ； 纖維母細胞生長因子 9 ； 白細胞介素 1β ； 細胞色素 P450

並列摘要

The major purposes of these studies were to determine the effects of silver nanoparticles on cytokine expression in humane lung adenocarcinoma cells CL5. the ability of ethion to induce cytochrome P450 expression in male rat liver, and the effect of paternal exposure to motorcycle exhaust on cytochrome P450 expression in rat liver.
Nanoparticles are applied to the domains of life science and biotechnology. The diameter of nanoparticle is 1 to 100 nm, so it may have special physics and toxicokinetic properties. The major objective of this study was to investigate the effects of different scale diameter nanoparticles on mRNA expression in human lung adenocarcinoma cells CL5. The result of reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that Ag (7 nm) increased fibroblast growth factor (FGF)-9 and interleukin (IL)-1β mRNA expression. Ag (50 nm) increased IL-1β mRNA expression but no effect on FGF-9. AgNO3 increased FGF-9 mRNA expression but had no effects on IL-1β. Ag (7 nm) and AgNO3 increased ethoxyrcoumarin O-deethylase (ECOD) activity and peroxide production, decreased glutathione S-transferase (GST) activity and total glutathione content, had no effects on lipid peroxidation. Ag (50 nm) increased ECOD activity but had no effects on GST activity, total glutathione content, and lipidperoxidation. The present finding shows that the smaller diameter nanoparticles cause more oxidative damage and FGF-9 and IL-1β mRNA expression than the larger diameter one did. Peroxide production may cause oxidative damage.
Ethion is an organophosphate pesticide used to kill aphids, mites, leafhoppers, and maggots . It is used on a wide variety of food, fiber, ornamental crops, and greenhouse crops. The major objective of the present study was to investigate the ability of ethion to modulate CYP-dependent monooxygenase in rat liver. Male rats were treated with 1, 10, and 20 mg/kg ethion intraperitoneally once daily for 4 days. In liver microsome, 10 mg/kg ethion treatment increased pentoxyresorufin O-dealkylase (PROD) activity but decreased cytochrome P450 content, NADPH -cytochrome P450 reductase, methoxyresorufin O-demethylase activity and aniline hydroxylase activity. Treatment with 20 mg/kg ethion increased PROD activity. The result of immunoblot and RT-PCR analyses showed that ethion dose dependently induced CYP2B protein and mRNA in the liver. The present finding shows that ethion is an inducer of CYP2B in male rat liver .
Motorcycle is a popular means of transportation in Taiwan. The exhaust causes air pollution seriously and human expose to the exhaust is an important health issue. Thus, we have studied the effect of motorcycle exhaust on cytochrome P450 mRNA in male rats and its offspring. Motorcycle exhaust increased CYP1A1, CYP2B and CYP2E1 mRNA expression but decreased CYP2C11 mRNA expression in the liver. Motorcycle exhaust-exposed male rats were mated with untreated female rats and pregnant female rats were allowed to deliver. In the F1 generation, CYP1A2 and CYP3A mRNA expression was decreased in male rats liver on postnatal day (PND) 56 and 98 but no effect was observed on 140. CYP1A2 and mRNA expression was decreased in female offspring liver on PND 56 but no effects were observed on PND 98 and 140. CYP3A1 mRNA expression was decreased in female offspring liver on PND 56 and 98 but not on PND 140. CYP2B and CYP2C11 mRNA was only decreased in male rat liver on PND 98. The present finding shows that motorcycle exhaust changes P450 mRNA expression in male rats and offspring liver, so we should find out the mechanism in the future.

並列關鍵字

silver nanoparticles ； ethion ； motorcycle exhaust ； FGF-9 ； IL-1β ； CYP

參考文獻

勞工安全衛生技術。行政院勞工安全研究所編制

Afsharian P., Terelius Y., Hassan Z., Nilsson C., Lundgren S., Hassan M. (2007). "The effect of repeated administration of cyclophosphamide on cytochrome P450 2B in rats." Clinical Cancer Research 13: 4218-4224.

Agardh C.D., Israelsson B., Thuesen-Olesen B., Agardh E. (2002). "Application of quantitative competitive polymerase chain reaction for measurements of mRNA from antioxidative enzymes in the diabetic rat retina and kidney." Metabolism 51: 1279-1284.

Agency for toxic substances and disease registry (1990). "Toxicological profile for silver." U.S. Department of Health and Human Services, Public Health Service.

Baggiani C., Anfossi L., Giovannoli C. (2007). "Solid phase extraction of food contaminants using molecular imprinted polymers." Analytica Chimica Acta 591: 29-39.