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b-Carotene, Lycopene and Oxidation: In Vitro Antioxidant and Pro-Oxidant Effects and the Inhibition of Gap
|關鍵字:||beta-carotene;beta-胡蘿蔔素;lycopene;antioxidant;pro-oxidant;gap junction intercellular communicaiton;茄紅素;抗氧化性;促氧化性;細胞間隙聯繫||出版社:||食品科學系||摘要:||
利用分別預培養b-胡蘿蔔素及茄紅素的Hs68細胞（人類纖維母細胞）再分別以三種氧化劑（ferric nitrilotriacetate (Fe/NTA); 2,2’-azobis(2,4-dimethyl -valeronitrile) (AMVN); 2,2’-azobis(s-amidinopropane)dihydrochloride(AAPH)）處理，結果發現茄紅素及b-胡蘿蔔素均具抗氧化性及促氧化性，依不同的氧化條件而不同，以親脂性氧化劑AMVN處理，細胞中茄紅素及b-胡蘿蔔素消耗最快並表現出促氧化性。為進一步瞭解此二種類胡蘿蔔素之促氧化性是否與其氧化產物有關，我們以預先加熱氧化的茄紅素及b-胡蘿蔔素（OLP及OBC）分別與Hs68 細胞及小牛胸線DNA培養，結果顯示20 mM OBC 及OLP均會造成細胞死亡，但同濃度下b-胡蘿蔔素及茄紅素則不影響細胞生長，單細胞電泳（慧星影像分析）及8-OH-dG結果證明OBC及OLP會誘發DNA傷害。
由於8-OH-dG的產生，一般認為是活性氧（reactive oxygen species; ROS）例如羥自由基，氧化修飾DNA鹼基的結果，但以DCFH-DA與ROS反應生成螢光物質的方法偵測，並未發現OBC 或OLP促使細胞中ROS含量增加，此矛盾有可能是DCFH-DA易受細胞內過氧化酵素及光線干擾之故。為證實產生活性氧是OBC及OLP誘發8-OH-dG及DNA傷害的機制之一，我們利用小牛胸線DNA為模式，將DNA與OBC及各種金屬螯合劑或活性氧捕捉劑混合共同培養，結果發現金屬螯合劑（Desferal）及某些活性氧捕捉劑可以強烈抑制8-OH-dG的產生，顯示b-胡蘿蔔素自氧化的確會產生活性氧，而此反應可能藉環境中金屬離子催化b-胡蘿蔔素過氧化物斷裂產生。
除了抗氧化性，b-胡蘿蔔素還有許多的生理活性，其中之一即可能藉由增加Cx43蛋白質表現而增強細胞間的聯繫（gap junction intercellular communication; GJIC），並可能藉此機制抑制腫瘤細胞生長，然而近來有一個動物實驗顯示，給予實驗動物服用高劑量b-胡蘿蔔素可能抑制細胞的GJIC，此一結果是否由於受試大鼠體內有較多OBC形成之故，並不清楚。因此利用人類肺癌細胞株（A549細胞）與b-胡蘿蔔素及OBC分別培養，我們發現5 mMOBC明顯抑制細胞GJIC，反之，同濃度下的b-胡蘿蔔素卻顯著增加細胞GJIC。OBC抑制A549細胞GJIC會伴隨著Cx43蛋白高磷酸化及細胞內位置錯置現象發生，此一結果支持我們的假說，即OBC誘發的細胞GJIC下降與Cx43高磷酸化與位置紊亂有關。
Epidemiological studies demonstrate an inverse relationship between the diets rich in fruits and vegetables and the risk of cancer and cardiovascular disease especially in smokers. These effects have been attributed to the antioxidant properties of carotenoids, especially b-carotene (BC). Surprisingly, two famous clinical trials of supplemental BC suggested that BC may be harmful to smokers, and many researches attempted to clarify the apparently contradictory results of intervention and epidemiologic studies. It has been suggested that the carcinogenic response to high dose BC supplementation is related to the instability of the BC molecule in the free radical-rich environment in the lungs of cigarette smokers. At the same time, lycopene (LP), another carotenoid, has become a new focal point for being a natural antioxidant. However, the structures of these two carotenoids are similar. Whether LP also has pro-oxidant activity under certain conditions, as has BC, is unclear.
Using BC or LP-enriched human foreskin fibroblast Hs68 incubated with three oxidants (ferric nitrilotriacetate (Fe/NTA); 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN); 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH)), we found that BC and LP can be either antioxidant or pro-oxidant depending on the oxidant used. Employing Hs 68 cells and calf thymus DNA, we further examined the hypothesis that oxidized b-carotene (OBC) and oxidized lycopene (OLP) are pro-oxidants while b-carotene and lycopene themselves are antioxidants. The results demonstrated that OBC and OLP, rather than BC and LP, induced DNA damage measured as formation of 8-OH-dG and as strand breaks by the comet assay. The Hydroxylation of C-8 of guanine is known to result from the attack of DNA by ROS. However, by measuring intracellular ROS using 2',7'- dichlorofluorescein diacetate (DCFH-DA) assay, we found that neither OBC or OLP increased the levels of ROS. The lack of ROS induction by OBC and OLP may be due to the interruption of DCFH fluorescence by cellular endogenous peroxidase, a problem inherited from the DCFH-DA assay (Appendix I.1). To investigate whether ROS are involved the damage to DNA induced by OBC, we used a cell-free system, that is, calf thymus DNA, by adding iron chelators and radical scanvengers during incubation with OBC. The results showed that the level of 8-OH-dG was strongly inhibited by desferal and several radical scanvengers. Take together, these finding suggest that ROS produced by BC autoxidation, possibly by metal ion-catalyzed breakdown of pre-formed BC peroxides, damage DNA to form 8-OH-dG.
In addition to its antioxidant effect, b-carotene has various biologic effects, such as up-regulation of gap junction intercellular communication (GJIC) and the possible control of tumor cell growth. In contrast, a recent animal study indicated that a very high dose of BC (50 mg/Kg body weight per day) decreased GJIC of rat liver. We were interested to know whether OBC contributes to the effect of high doses BC to disturb GJIC in the tissue and, thereby, to increase the carcinogenic potential. Using lung cancer cells (A549) incubated with OBC or BC, we found that OBC added at 5 mM inhibited GJIC, whereas BC added at 5 mM strongly increased GJIC. Loss of GJIC in A549 induced by OBC accompanied the hyper-phosphorylation and aberrant localization of Cx43. The results support our hypothesis that the decrease of GJIC in A549 induced by OBC is associated with aberrant phosphorylation level and dislocalization of Cx43.
In summary, this dissertation research demonstrates that b-carotene and lycopene can be a pro-oxidant in vitro, and part of the pro-oxidant properties can be attributed to the oxidation of BC or LP. OBC induces the damage to intercellular DNA and calf thymus DNA by the generation of ROS during autoxidation of BC, possibly catalyzed by metal ions. In addition to its pro-oxidative effect, OBC strongly inhibits GJIC. However, the inhibition of GJIC by OBC appears to be independent of the pro-oxidant effect of OBC. Theses results support the hypothesis that the carcinogenic response to high-dose BC supplementation reported in clinical trials is related to the oxidation of BC in the lung of smokers.
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