Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/50806
標題: Oxidative damage to membrane lipids and DNA in HepG2 cells, rat liver tissue slice and nuclei: a comparison of methods and the relationship of lipid peroxidation and DNA damage
大鼠肝組織切片、細胞核和HepG2細胞之脂質及DNA氧化傷害:脂質過氧化 和DNA傷害測定法之比較及兩者之關係
作者: 王永發
Wang, Yung-Fa
關鍵字: DNA damage;脂質過氧化;lipid peroxidation;8-OH-dG
出版社: 食品科學系
摘要: 
ABSTRACTIs there a cause-and-effect relationship between
oxidative damage to membrane lipids and DNA?In order to answer
this question, this study employed rat liver tissue slice,
nuclei and HepG2 induced by oxidants including Fe(Ⅲ)-NTA
(ferric nitrilotriacetate), BrCCl3 (bromotrichloromethane), Brb
(bromobenzene) and 2-NP (2-nitropropane). At the same time,
tissue slice model was employed to assess the suitability of
oxidative damage to membrane lipids and DNA, and assess the
effect of decrease GSH (reduced glutathione) of rat liver tissue
slices on lipid peroxidation and DNA damage.Lipid peroxidation
was assessed by measurement of thiobarbituric acid reactive
substances (TBARS) with spectrophotometer, spectrofluorometer
and HPLC. DNA damage was assessed by measurement of base
modification, strand breakage and fragmentation. TBARS were
measured in the medium, and DNA damage was measured in
precipitated tissues or cells after incubation. Results showed
that the correlation of these three methods of measurement lipid
peroxidation was good, because of its simplicity and sensitivity
of TBARS by measured spectrophotometrically was used to
determine lipid peroxidation in the following experiments.Rat
liver tissue slices are suitable for the analysis of 8-OH-dG
(8-hydroxy-2*-deoxyguanosine) but not DNA strand breakage.
Injection of rats with DEM (diethyl maleate) decreased the
hepatic contents of GSH to 13% of control rats and significantly
increased both lipid peroxidation and 8-OH-dG induced by all
oxidants. Rat liver tissue slices, nuclei and HepG2 cells were
suitable for the analysis of lipid peroxidation and 8-OH-dG, and
HepG2 cells were also suitable for the analysis of DNA strand
breakage. Thus, HepG2 cells were used to study the relationship
of lipid peroxidation and DNA damage and to compare the
different types of DNA damage.BrCCl3, Brb, 2-NP and Fe(Ⅲ)-NTA
are environmental contaminants that are strongly hepatotoxic.
Fe(Ⅲ)-NTA strongly changed the morphology of HepG2 (cells
deformation) and formed vacuoles, indicating that Fe(Ⅲ)-NTA is
cytotoxic. BrCCl3, Brb and 2-NP caused lipid peroxidation and
single-stranded DNA breakage, whereas only BrCCl3 induced low
level of 8-OH-dG (near detection limit). Among these three
oxidants, BrCCl3 was the strongest and 2-NP was the weakest in
oxidative damage. Exposure of HepG2 cells to Fe(Ⅲ)-NTA
resulted in dose- and time-dependentlipid peroxidation and DNA
damage. 1 mM Fe(Ⅲ)-NTA caused 32% (p<0.001) DNA strand
breakage but did not significantly increase lipid peroxidation
or 8-OH-dG level. Addition of ascorbate to Fe(Ⅲ)-NTA system
resulted in significantly increased lipid peroxidation and 8-OH-
dG level. The results demonstrate that measurement of DNA
strand breakage is more sensitive than that of 8-OH-dG, but the
former has large variability (low precision). Therefore, to
study DNA damage two kinds of DNA damage should be analyzed.In
order to study the relationship of lipid peroxidation and DNA
damage, BHT (butylated hydroxytoluene) was used to inhibit the
formation of lipid peroxidation. Results showed thatBHT
inhibited (90%, P<0.001) lipid peroxidation and DNA strand
breakage induced by BrCCl3, Brb and 2-NP. BHT inhibited lipid
peroxidation (>70%, P<0.001), DNA strand breakage (>10%, P<0.05)
and 8-OH-dG (>34%, P<0.05) induced by Fe(Ⅲ)-NTA or Fe(Ⅲ)-NTA/
ascorbate. These results suggest a cause-and-effect
relationship between oxidative damage to membrane lipids and DNA
induced by all oxidants.To confirm this point, the stronger
oxidants, BrCCl3 and Fe(Ⅲ)-NTA/ascorbate, were used for the
time course experiment. Results showed that BHT completely
inhibited lipid peroxidation and partially inhibited DNA strand
breakage (25%, at 120 min, P<0.01) induced by BrCCl3.
BHTinhibited (60-70%, P<0.001) lipid peroxidation induced by Fe(
Ⅲ)-NTA/ascorbate in each time interval but only significantly
inhibited DNA strand breakage (20%, P<0.05) and 8-OH-dG (42%,
P<0.05) at 120 min. Induction of 8-OH-dG by Fe(Ⅲ)-NTA/
ascorbate was delayed by BHT until 60 min after incubation,
suggesting that formation of 8-OH-dG occurs only when membrane
lipid peroxidation is extensive. The result indicates that
lipid peroxidation is partially related to DNA strand breakage
induced by BrCCl3 and Fe(Ⅲ)-NTA/ascorbate, and that the
formation of 8-OH-dG induced by Fe-NTA/ascorbate may be a result
of lipid peroxidation.This study concludes that rat liver tissue
slices are suitable for the analysis of 8-OH-dG but not DNA
strand breakage. Depletion of GSH of rat liver tissue slices
significantly increases both lipid peroxidation and 8-OH-dG
induced by all oxidants. Measurement of DNA strand breakage is
more sensitive than that of 8-OH-dG, but the former has large
variability (low precision). Therefore, to study DNA damage,
more than one assay of DNA damage should be used. Lipid
peroxidation is partially related to DNA strand breakage induced
by BrCCl3 and Fe(Ⅲ)-NTA/ascorbate, and that the formation of
8-OH-dG induced by Fe-NTA/ascorbate may be a result of lipid
peroxidation. Thus, the relationship between lipid peroxidation
and DNA damage depends on the oxidative system and/or the type
of DNA damage that is measured.keywordsReactive oxygen species
(ROS), thiobarbituric acid reactive substances (TBARS),
8-hydroxydeoxyguanosine (8-OH-dG), electrochemical detection
(ECD).

摘要脂質過氧化與DNA傷害有因果關係嗎?為了回答這個問題,本研究選
擇大鼠肝組織切片、大鼠肝細胞核和HepG2細胞三種模式來比較脂質過氧
化與DNA傷害的關係,並評估組織切片在脂質過氧化與DNA傷害分析之適用
性及降低組織切片中GSH(reduced glutathione)含量之影響。脂質過氧
化以分光光度計、螢光光度計及HPLC三種方法分析。DNA傷害則以strand
breakage, base modification以及DNA fragmentation方法分析。實驗結
果顯示:三種分析脂質過氧化之方法所得到的結果都有很好的相關性,故
本研究後續試驗採用最簡單、便利的分光光度計法測定脂質過氧化。另外
,組織切片適用於DNA鹼基傷害的分析,但由於DNA不易從組織切片溶出,
故不適用於DNA strand breakage的測定。此外,大鼠腹腔注射DEM(
diethyl maleate)後可降低肝中GSH含量,以此大鼠肝組織切片與氧化劑
培養後可造成更嚴重的脂質和DNA傷害。本研究所用之三種模式中組織切
片及細胞核皆可測得脂質過氧化及DNA鹼基修飾,此外,由於HepG2細胞更
可用於DNA strand breakage的分析,故本研究乃選用細胞來探討脂質過
氧化與DNA傷害的關係及不同形式的DNA傷害之比較。本研究所選用的氧化
劑(bromotrichloromethane (BrCCl3), bromobenzene (Brb),
2-nitropropane (2-NP)和ferric nitrilotriacetate (Fe(Ⅲ)-NTA))皆
為環境之污染物,且皆有肝毒性。這些氧化劑都會造成細胞外觀的改變,
尤其是以Fe(Ⅲ)-NTA處理的組別,其細胞內充滿了大大小小的空泡,細胞
的輪郭也變的模糊不清,顯示其有強的細胞毒性。在生物分子的傷害上,
BrCCl3, Brb和2-NP三者之中,皆可造成脂質過氧化與single-strand DNA
breakage,但只有BrCCl3可造成微量(接近儀器之測定極限)的8-OH-dG
(8-hydroxy-2*-deoxyguanosine),其中以BrCCl3誘導的氧化壓力為最
強,Brb次之,2-NP最弱。Fe(Ⅲ)-NTA誘導之脂質過氧化與DNA傷害有濃度
和時間效應,1 mM Fe(Ⅲ)-NTA並未使脂質過氧化和8-OH-dG含量明顯增加
,但卻造成32% (由89%降到60%, P<0.001)的DNA strand breakage(包含
單股及雙股)。加入各種濃度的ascorbate強烈促進Fe(Ⅲ)-NTA造成的脂
質過氧化和8-OH-dG含量。結果顯示:strandbreakage比8-OH-dG靈敏,但
其變異性大,故分析DNA傷害時應該同時分析一種以上的DNA傷害形式較為
恰當其次,本研究利用BHT(butylated hydroxytoluene)抑制脂質過氧
化,以瞭解脂質過氧化和DNA傷害的關係。結果顯示:細胞經BrCCl3, Brb
和2-NP處理時,有加BHT之各組,其脂質過氧化抑制率皆可達90%
(P<0.001);ds DNA含量各上升53% (P>0.05), 25% (P<0.05)和22%
(P>0.05)。細胞經Fe(Ⅲ)-NTA及Fe(Ⅲ)-NTA/ascorbate處理時,有加BHT
之各組,其脂質過氧化抑制率大於70% (P<0.001);ds DNA則各上升17%
及10% (P<0.05)。在8-OH-dG方面,各種Fe(Ⅲ)-NTA/ascorbate之比例造
成之8-OH-dG皆可以顯著的被BHT所抑制(P<0.05)。上述結果顯示脂質過氧
化與DNA傷害有因果關係。為了更進一步確定這關係,本研究乃選擇可造
成最大的氧化傷害的BrCCl3及Fe(Ⅲ)-NTA/ascorbate來進行時間效應的實
驗。結果顯示:BHT完全抑制BrCCl3所誘發之脂質過氧化,並使ds DNA之
含量上昇(在120分鐘時上昇25%, P<0.01);Fe(Ⅲ)-NTA/ascorbate在各
培養時間所誘導的脂質過氧化約有60~70% (P<0.001)可被BHT抑制;ds
DNA的回升約20% (P<0.05)上下;在培養120分鐘時,8-OH-dG也有42%
(P<0.05)被抑制,且培養時間要到60分鐘後才會有8-OH-dG的產生,顯示
:脂質過氧化的產物要達到一定的程度才可能引起DNA的傷害。以上結果
說明:BrCCl3及Fe(Ⅲ)-NTA造成的脂質過氧化與DNA strand breakage只
有部份相關,Fe(Ⅲ)-NTA造成的脂質過氧化與8-OH-dG有因果關係。本研
究綜合之結果顯示:大鼠肝適用於DNA鹼基傷害的分析,但不適用於DNA
strand breakage的測定。此外,降低大鼠肝組織切片中GSH含量,可造成
更嚴重的脂質和DNA傷害。測定DNA strand breakage比測定8-OH-dG靈敏
,但其變異性大,故分析DNA傷害時應該同時分析一種以上的DNA傷害形式
較為恰當。BrCCl3及Fe(Ⅲ)-NTA/ascorbate造成的脂質過氧化與DNA
strand breakage只有部份相關,Fe(Ⅲ)-NTA/ascorbate 造成的脂質過氧
化與8-OH-dG有因果關係。因此,脂質過氧化和DNA傷害的關係依所使用的
氧化系統和DNA傷害的形式而定。
URI: http://hdl.handle.net/11455/50806
Appears in Collections:食品暨應用生物科技學系

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