Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/50805
標題: Oxidative damage of human fibroblasts by ferric nitrilotriacetate: the relationship between lipid peroxidation and protein oxidation
Ferric nitrilotriacetate對人類纖維母細胞之氧化傷害:脂質過氧化和 蛋白質氧化傷害之關係
作者: 李佳裕
Lee, ChiaYu
關鍵字: lipid peroxidation;脂質過氧化;human fibroblast;protein oxidative damage;cytotoxicity;人類纖維母細胞;蛋白質氧化傷害;細胞毒性
出版社: 食品科學系
摘要: 
Ferric nitrilotriacetate (Fe(III)-NTA)為清潔劑中的一種物質,已證
實會引起癌症。Human foreskin fibroblast (HFF)為一株正常之人類細
胞。脂質過氧化的過程中,會產生許多產物,包括小分子醛類之4-
Hydroxyl-2-nonenal (HNE)和Malondialdehyde (MDA)。雖然HNE和MDA在
in vitro的試驗中發現能造成蛋白質的損傷,但在體內的情形則不明朗。
在一些病理或動物試驗中發現脂質過氧化和蛋白質carbonyl groups的形
成沒有密切的相關性。因此脂質過氧化和細胞內蛋白質的氧化傷害之間的
關係有待進一步的研究。本研究以HFF和Fe(III)-NTA探討:1) Fe(
III)-NTA對HFF細胞之毒性?2) 細胞膜或細胞質的蛋白質之氧化傷
害是否與脂質過氧化有關?本研究針對上述問題提出下述假說:1)
Fe(III)-NTA會對HFF細胞造成脂質過氧化、蛋白質氧化傷害之廣泛性的傷
害。2) 脂質過氧化之產物與細胞膜或細胞質的蛋白質之氧化傷害有
相關。本研究以trypan blue、lactate dehydrogenase(LDH)活性和顯微
照相評估細胞毒性,以Thiobarbituric acid-reactive substances
(TBARS)、Cyclohexanedione-reactive substances (CHDRS)測定質脂過
氧化,以蛋白和carbonyl groups、細胞內glutathione (GSH)含量、細胞
膜酵素(5''-nucleotidase)、細胞質酵素(Glutamine synthetase)、微粒
體酵素(NADPH cytochrome P-450 reductase)和抗氧化酵素(Catalase,
superoxide dismutase (SOD), GSSG reductase, GSH peroxidase, GSH
S-transferase)的活性評估蛋白質的氧化損傷。研究結果顯示:Fe(III)-
NTA會對HFF細胞造成細胞毒性,而使細胞的存活率降低,細胞外觀的變形
,脂質過氧化,細胞內GSH含量的降低,抑制細胞內特定的酵素活性,但
不影響蛋白質carbonyl groups的增加。其傷害可能是Fe(III)-NTA對細胞
所造成的氧化壓力。GSH含量的降低反應出細胞確實遭受到了氧化壓力。
GSH含量的降低,可能與Fe(III)-NTA被還原的機制有關。Glutamine
synthetase和NADPH cytochrome P-450 reductase和抗氧化酵素之活性,
如catalase、SOD和GSH peroxidase活性的沒有顯著變化,反應出細胞尚
能承受Fe(III)-NTA初期所產生的氧化壓力。GSSG reductase和GSH S-
transferase的活性隨脂質過氧化的上升而降低,然而控制組(HFF細胞不
經Fe(III)-NTA處理)之酵素活亦降低,推測酵素活性的抑制主要不是來自
脂質過氧化。HFF細胞的Glutamine synthetase和NADPH-cytochrome
P-450 reductase之活性不受Fe(III)-NTA的影響。由此得Fe(III)-NTA雖
造成脂質過氧化,但對於細胞質內蛋白質和抗氧化酵素沒有顯著的影響。
換句話說:脂質過氧化的產物不會造成顯著的細胞質內蛋白質氧化傷害和
抗氧化酵素活性降低。Butylated hydroxytoluene和diphenylamine可抑
制Fe(III)-NTA所引起的脂質過氧化,但細胞膜上的5''-nucleotidase活性
未受到保護,因此推測5''-nucleotidase是受Fe(III)-NTA之作用而改變活
性。研究中曾嚐試以EDTA和desferal螯合Fe(III)-NTA中Fe(III),雖降低
了膜脂質過氧化,但仍然無法防止5''-nucleotidase活性的減少,可能是
這些螯合劑與5''-nucleotidase所需的金屬鋅螯合,所以未有效的保護酵
素。Fe(III)-NTA所造成的5''-nucleotidase活性降低可能原因有二:一、
受到Fe(III)-NTA所催化產生的H攻擊;二、NTA與5''-nucleotidase活性
作用所要的Zn螯合。
Fe(III)-NTA is a component of detergent, and had been identified
as a carcinogen. HFF is a normal human cell line. HNE and MDA
formed from lipid peroxidation can damage protein in vitro, but
whether this will occur in vivo is still unclear. Some animal
and pathological studies indicate that there is no correlation
between lipid peroxidation and protein carbonyl levels,
whilestudies show that protein carbonyl occur concurrently with
lipid peroxidation. Hence, the relationship between lipid
peroxidation and protein oxidation remains to be resolved.This
study used HFF cells and Fe(III)-NTA to investigate: 1) The
cytotoxicity of Fe(III)-NTA on HFF cells.2) Whether the damage
to membrane-bound or cytosolic proteins by Fe(III)-NTA is
associated with lipid peroxidation.Two hypotheses were proposed
in this study:1) Fe(III)-NTA induce both lipid peroxidation
and protein damage to HFF cells.2) There is a correlation
in the oxidative damage between lipid peroxidation and membrane
and cytosolic protein.This study used trypan blue, LDH activity
and microphotography to evaluate cytotoxicity. Biochemical
assays include lipid peroxidation, protein carbonyl groups,
cellular GSH content, activities of a plasma membrane enzyme
5''-nucleotidase, microsome membrane enzyme NADPH cytochrome
P-450 reductase, and antioxidant enzymes (catalase, SOD, GSSG
reductase, GSH peroxidase, GSH S-transferase). A protein
oxidative damage marker, glutamine synthetase, was also
determind.The results show that: Fe(III)-NTA resulted in
cytotoxicity, as evidenced by morphologic changes and decreased
cell viability. In addtion, Fe(III)-NTA increased lipid
peroxidation, decreased cellular GSH content, and inhibited some
enzymes activities in the cell. Fe(III)-NTA did not
significantly increase protein carbonyl levels in the cell. The
damage induced by Fe(III)-NTA may be relate to oxidative stress.
The decreased cellular GSH content reflected the increasing
oxidative stress. The decreased GSH content may be due to the
reduction of Fe(III)-NTA by cysteine and cystine-glucine.
Glutamine synthetase, NADPH chtochrome P-450 reductase, and
antioxidant enzymes activities, including catalase and SOD, did
not significantly change. The unchanged enzyme activities
suffest that the cytosolic defense system in not overwhelmed by
the oxidative stress induced by Fe(III)-NTA. The activities of
GSSG reductase and GSH S-transferase were decreased with or
without Fe(III)-NTA, indicating that oxidative stress is not the
cause of such decrease. Thus, Fe(III)-NTA induced lipid
peroxidation did not significantly damage cytosolic proteins
ordecrease the antioxidant enzyme activities.Butylated
hydroxytoluene and diphenylamine inhibited lipid peroxidation
induced by Fe(III)-NTA, but the activity of 5''-nucleotidase, a
plasma membrane bound enzyme, was not resotred. The result
indicates that lipid peroxidation did not affect
5''-nucleotidase. The activity of 5''-nucleotidase was affected by
Fe(III)-NTA. EDTA and desferal chelated the Fe(III) of Fe(III)-
NTA, decreased lipid peroxidation but did not prevent the loss
of 5''-nucleotidase activity. Two possible mechanisms for the
loss of 5''-nucleotidase activity induced by Fe(III)-NTA were
suggested. One was that H formed by Fe(III)-NTA attack
5''-nucleotidase. The other mechanism was that NTA chelated the
metal need for 5''-nucleotidase activity.
URI: http://hdl.handle.net/11455/50805
Appears in Collections:食品暨應用生物科技學系

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