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研究生:王維譽
論文名稱:WUVA及NNK誘發之DNA傷害的促進作用以及類黃酮的抑制作用
論文名稱(外文):Enhancement by b-carotene and suppression by flavonoids on DNA damage induced by UVA or NNK
指導教授:胡淼琳胡淼琳引用關係葉姝蘭
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:b-胡蘿蔔素類黃酮DNA傷害近紫外線NNK
外文關鍵詞:UVANNKb-caroteneflavonoidDNA damage
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摘要
b-胡蘿蔔素是否有助於減少日照或抽煙所造成的傷害,各種試驗的結果並不一致;許多研究也顯示其他抗氧化成分同時存在時,可能會影響b-胡蘿蔔素的反應。由於類黃酮是深色蔬菜水果中另一大量存在的抗氧化成分,其與b-胡蘿蔔素之相關性因此受到注意,但目前仍少有研究結果可支持此種論點。因此本研究分別以細胞模式探討照射UVA或暴露於香菸中致癌成分4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)時,β-胡蘿蔔素及naringin、quercetin、rutin等三種類黃酮對DNA傷害的影響及其交互作用。
β-胡蘿蔔素及類黃酮對UVA傷害之影響
此部分研究將小鼠胚胎細胞(C3H 10T1/2)與β-胡蘿蔔素及三種類黃酮分別或共同預培養1小時,再照射3.8及7.6 kJ/m2 UVA,探討β-胡蘿蔔素及類黃酮分別及共同作用對UVA誘發DNA傷害的影響,並探討其可能機制。結果顯示,β-胡蘿蔔素明顯增加UVA誘發之DNA傷害達35%,而naringin、quercetin和rutin三種類黃酮則顯著降低UVA的傷害,效果依序為naringin>rutin>quercetin。而併用類黃酮及β-胡蘿蔔素與細胞預培養,亦可抑制β-胡蘿蔔素的促氧化作用,三種類黃酮的效果順序與前面一致。分析類黃酮對UVA的吸收光譜,發現在波長範圍320-380 nm間的吸收量大小依序為quercetin>rutin>naringin。但分析單重態氧的捕捉情況,則又依序為naringin>rutin>quercetin。另外以HPLC分析UVA照射下,β-胡蘿蔔素在細胞內的消耗量,結果以naringin減少β-胡蘿蔔素氧化效果較好,而quercetin與rutin效果較差。以上結果顯示這些類黃酮確能抑制UVA誘發的DNA傷害及β-胡蘿蔔素促氧化性,而其效果應不只來自對UVA的遮蔽作用,可能與類黃酮捕捉UVA誘發的單重態氧或其他活性氧有關,因而減少β-胡蘿蔔素被UVA氧化,進而抑制β-胡蘿蔔素對細胞DNA促氧化現象。
β-胡蘿蔔素及類黃酮對NNK傷害之影響
此部分研究以NNK誘發人類肺癌細胞A549之DNA傷害為模式,探討β-胡蘿蔔素及類黃酮分別及共同作用對DNA傷害的影響,並探討其可能機制。做法為:將A549細胞與β-胡蘿蔔素及類黃酮分別或共同預培養1小時之後,以PBS洗掉,加入700mM NNK於HBSS緩衝液繼續培養4小時,分析細胞DNA彗星影像,評估DNA傷害程度;此外我們也以活性氧的捕捉劑、DCFH-DA分析法及CYP450酵素的抑制劑來分析活性氧及CYP450酵素在NNK誘發傷害過程中扮演的角色。結果顯示NNK會誘發細胞DNA傷害,並具劑量關係,而β-胡蘿蔔素不會造成DNA傷害,但會增加NNK對DNA的傷害達1.8倍,其中機制與β-胡蘿蔔被NNK氧化及增加CYP450酵素活性有關。反之,Naringin、quercetin、rutin則可以顯著降低NNK誘發的DNA傷害,其效果依序為quercetin>naringin>rutin。DCFH-DA分析可以看到三種類黃酮化合物均可明顯抑制NNK誘發活性氧的產生,其抑制效果與保護DNA傷害一致。我們更進一步觀察到,這類黃酮亦能抑制β-胡蘿蔔素暴露在NNK下的促傷害作用,其抑制效果仍與前面一致。類黃酮的這種保護作用可能與其抗氧化性及對CYP450酵素活性的抑制有關。
Abstract
Inconsistent results have been reported regarding the effect of β-carotene on UV-induced skin damage and on the lung cancer incidence in smoker. It has been suggested that the existence of other antioxidants may affect the behavior of β-carotene under various oxidative stress. Flavonoids are another group of natural antioxidants ubiquitously found in fruits and vegetables. However, the interactions between β-carotene and flavonoids are unclear. Therefore, this thesis work was divided in to two parts. Using cell culture model, one was to investigate the individual and combined effects of β-carotene and flavonoids, naringin, quercetin and rutin, on DNA damage induced by UVA exposure; the other was to investigate the effects of these compounds on DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) NNK, a smoke-related carcinogen.
Effects of b-carotene and flavonoids on UVA induced DNA damage
The C3H 10T1/2 cells, a cell line of mouse embryo fibroblasts, were first incubated with b-carotene and/or flavonoids for 1 hr followed by irradiation with UVA (3.8 and 7.6 KJ/m2). The aims of this part were to investigate the individual and combined effects of β-carotene and flavonoids on DNA damage induced by UVA exposure and to investigate the possible mechanisms. The results showed that b-carotene significantly enhanced the DNA damage after irradiation with UVA by about 35%. The flavonoids employed in this study significantly decreased the raise of DNA damage induced by UVA combined with or without b-carotene. The effects of these flavonoids are in an order naringin, quercetin and rutin. In contrary, the absorption of UVA (320-380 nm) was in an order quercetin, rutin and naringin. However, the activity of scavenging singlet oxygen and the prevention of loss of b-carotene induced by UVA were in the same order, i.e., naringin, rutin and quercetin. These results suggest that flavonoids may through scavenge singlet oxygen or other reactive oxygen species to inhibit DNA damage and to inhibit the prooxidation of β-carotene induced by UVA irradiation.
Effects of b-carotene and flavonoids on NNK induced DNA damage
The aim of this part was to investigate the individual and combined effects of b-carotene and certain common flavonoids, naringin, quercetin and rutin, on DNA damage in A549 cells induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-related carcinogen in humans. In addition, we investigated the possible mechanisms. A549 cells were first pre-incubated with either b-carotene, flavonoid, or both of them for 1h, followed by incubation with NNK for 4 h. The DNA damage was determined by comet assay (single-cell DNA gel electrophoresis). In addition, using some reactive oxygen species (ROS) scavengers, DCFH-DA assay and one cytochrome P450 (CYP) inhibitor, we investigated the role of ROS and CYP in NNK-induced DNA damage. NNK induced DNA damage in A549 cells in a dose dependent manner. b-carotene significantly increased DNA damage induced by NNK up to 1.8-fold. The mechanisms likely included the increase in oxidation of β-carotene and the increased activity of CYP by β-carotene. In contrast, naringin, quercetin, rutin individually enriched in cells significantly inhibited NNK-induced DNA damage. The inhibitive effects of flavonoids were in an order quercetin, naringin and rutin. The flavonoids markedly inhibited the formation of ROS (determined by DCFH assay) induced by NNK in cells in an order as mentioned above. Furthermore, the flavonoids suppressed the enhancing effect of β-carotene on NNK-induced DNA damage. The effects of flavonoids may be attributed to the antioxidant activity and possibly, the suppression of CYP.
目錄
縮寫表…………………………………………………...……………..….……….1
摘要………………………………………………………………………………...2
1. 前言…………………………………………………………………………...7
1.1 緒論 …………………………………………………………………………..7
1.2 β-carotene簡介………………………………………………………………8
1.3 類黃酮 ……………………………………………………………………….10
1.4 Ultraviolet A (UVA)……………………………………………………….....13
1.5 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)………….……….14
1.6 研究目標……………………………………………………………………...15
1.7 實驗架構.………………………………………………………………….….16
2. 材料與方法………………………………………………...……………….17
2.1 材料…………………………………………………………………...……….17
2.2 方法………………………………………………………...………………….18
2.2.1 細胞解凍與保存…………………………………………...…………..18
2.2.2 細胞培養………………………………….………………...………….18
2.2.2.1 C3H細胞培養………...……………………….………………18
2.2.2.2 A549細胞培養………………………………………………..19
2.2.3 細胞的處理……………………………………….……………………19
2.2.3.1 β-胡蘿蔔素與類黃酮併入細胞…………….…………………19
2.4.3.2 UVA照射………………………………….………….……….19
2.4.3.3 NNK處理………………………………………….….……….20
2.2.4 DNA傷害測定-Comet assay(single cell gel electrophoresis assay)……………………………………………………...………...20
2.2.5 b-胡蘿蔔素含量分析……….……...………………………………...21
2.2.6 電子順磁共振光譜 (electron paramagnetic resonance spectrometer, EPR)測定單重態氧捕捉能力 (1O2)………………...………...….….21
2.2.7 流式細胞分析法測定細胞中ROS含量…...………………………....21
2.2.8 統計分析……………………………………………….…..…………..22
3. β-胡蘿蔔素及類黃酮對UVA傷害之影響………………….…..…..23
3.1 結果……………………………………………………...………………….....23
3.1.1 β-胡蘿蔔素促進UVA所誘發之DNA傷害…………...…………....23
3.1.2 類黃酮抑制UVA所誘發之DNA傷害與β-胡蘿蔔素的促氧化性....23
3.1.3 細胞中β-胡蘿蔔素濃度……………………………………………….24
3.1.4. 類黃酮的吸收光譜及對單重態氧的吸收…………………….……...25
3.2 討論…………………………………………………………………….……...26
3.2.1 β-胡蘿蔔素促進UVA所誘發之DNA傷害………………………...26
3.2.2 類黃酮抑制UVA誘發之DNA傷害………………………….………27
圖表………………………………………………………………………………...30
4. β-胡蘿蔔素及類黃酮對NNK傷害之影響……..…….……………..37
4.1結果…………………………………………………………..….……………...37
4.1.1 NNK誘發之DNA傷害……………………………..……………….37
4.1.2 β-胡蘿蔔素對DNA傷害的促進及類黃酮的抑制….……….………37
4.1.3 細胞中β-胡蘿蔔素含量……………………………….………………38
4.1.4 1-aminobenzotriazole (ABT)抑制β-胡蘿蔔素促傷害性……………39
4.1.5 類黃酮抑制ROS產生…………………………………………………39
4.2 討論……………………………………………………………...……………40
4.2.1 NNK誘發DNA傷害…………………………………..……………40
4.2.2 β-胡蘿蔔素促進NNK所誘發的傷害…………………..……...…...41
4.2.3 類黃酮抑制NNK的DNA傷害及β-胡蘿蔔對NNK之促傷害性....43
4.2.4 類黃酮抑制b-胡蘿蔔素的促傷害…………………………………….44
圖表……………………………………………………………………....………...46
5. 結論…………………………………………………………………………..54
6. 參考文獻………...………………………………………………………….55
7. 附錄…………………………………………………………………………..66
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