臺灣博碩士論文加值系統

I.鼠體內亞硝基反應對AAF致肝癌作用之影響以及活化致癌基因的角色.
乙醯胺基二苯駢伍圜 ( 2-acetylaminofluorene 簡稱 2-AAF ) 加上sodium nitr-ite ,在酸性狀態下,經 nitrosation 會產生亞硝基乙醯胺基二苯駢伍圜 ( N-notroso-2-acetylaminofluorene 簡稱 N-NO-AAF ),過去已經證實其基因毒性和致突變性比 2-AAF更強.在本篇研究,為了證明 NaNO2可能促進 AAF致肝癌之作用,利用大白鼠同時餵 AAF 與NaNO2,來觀察 NaNO2對 AAF導致肝腫瘤生成的影響.因此本篇的論文研究,將Wistar 雄性大白鼠分成五組: 第一組做對照組 (control),給予普通飼料. 第二組餵予含 0.3% NaNO2之飼料, 第三組餵予含 0.02% AAF之飼料,第四組餵予含 0.02% AAF 加上 0.2% NaNO2之飼料, 第五組餵予含 0.02%AAF 加上 0.3% NaNO2之飼料,為期 12週. 實驗結果發現第 3,4,5 組的大白鼠皆發生早期肝癌, 包括大小不同病灶的肝腫大和新生的結節. 大白鼠餵食 AAF 和 NaNO2 會誘發肝細胞癌之生成, 但是不同劑量的 NaNO2對 AAF 致肝癌生成有不同的影響, 較高劑量之NaNO2 有顯著的促進 AAF 致肝癌之作用,但 NaNO2 本身並不會有致肝癌作用.
當大白鼠餵食 0.02% AAF 三個月後,在鼠肝內的 c-Fos, c-Jun, c-Myc 蛋白量會上升.然而由 AAF 誘發的c-Fos, c-Jun, c-Myc 的表現,會因為加入NaNO2 而有意義的增高(p<0.001).這樣的結果證實了 NaNO2 加強 AAF 誘發肝癌生成的初期變化是經由促進誘發c-Fos, c-Jun, c-Myc表現量的增加.

II.原兒茶酸對鼠體內LPS所誘發內生性 NO 產生和 NO-AAF 形成的抑制作用.
亞硝基化合物由於直接攻擊 DNA 形成 DNA adducts,所以會造成癌症,而且在in vivo也會產生,表示內生性的 NO 可能扮演著重要的角色. NO ,不管是內生性或外生性,都可以將二級amine亞硝基化,如此會造成癌症. 內生性的 NO 在微生物感染時會induce macrop-hage 內的iNOS 活化,然後release NO,所以NO量增加.在肝臟內也有iNOS存在,當受到感染時Kupffer cell會產生cytokine來活化iNOS. 因此可以假設減少感染時期的內生性 NO產生可以避免像nitroso compound所引起的化學致癌作用.Hibiscus PCA 本身帶有catecholstructure,是oxygen free radical 捕捉劑,即抗氧化劑.假設 PCA藉由free radical捕捉的能力,抑制LPS所誘發的內生性NO,就可能預防亞硝基化進行.
當大白鼠事先處理PCA,可以證明會特異性的降低內生性 NO的產生 (表一).當大白鼠用 LPS 處理三天後,NO的量增加約 8倍.當大白鼠用 LPS 和 AAF處理三天後, NO 的量是745.18 +/- 17.45,如果先以各種不同濃度的 PCA(50mg/kg. 100mg/kg. 250mg/kg.)處理的大白鼠,再處理 LPS 和 AAF, 第七組的 NO 量是 455.33 +/- 50.48; 第八組的 NO量是403.13 +/-30.40;第九組的量是 320.00 +/- 37.37 ,顯然 PCA 會有意義的降低 NO的量(p<0.005).
除此之外, PCA還會稍微抑制由 AAF 和 LPS 所引起的肝炎 (表二), LPS與 AAF 都會引起肝臟指數ALT升高 (89.2 +/- 28.5; 99.15 +/- 19.3比59.93 +/-16.3) (p<0.01).如果大白鼠同時以 LPS 與 AAF 處理,其肝功能指數明顯上升 (212.50 +/- 14.8). 如果先以 PCA (50mg/kg.;100mg/kg.; 250mg/kg.)處理後,再處理 LPS ,AAF,則肝功能指數可以降至138.98 +/- 27.1 ; 116.44 +/- 16.9 ; 128.86 +/- 24.6 , 顯然 PCA會有意義降低指數.(p<0.005)
以 LPS 活化 inducible NO synthase (iNOS)使內生性 NO 增加, 在生理情況之下,內生性的 NO 有其攻擊的靶分子 (像 Heme proteins, Fe-Sproteins, SH groups, Metalloproteins, Oxygen, DNA, Amines......)所以 NO 要讓它在生理狀況下與外來的 AAF結合並不容易.本篇實驗以光電比色法 (spectrophotometer) 與高效率液相層析法去分析血清內的 N-NO-AAF ,不論那一組的老鼠血清都無法偵測到 N-NO-AAF.以 LPS 與 AAF 處理過的大白鼠可以看到各種不同程度的壞死現象 : 第三組 (LPS)可見到局部性壞死; 第四組 (AAF)可以見到凝固性壞死; 第五組 (LPS + AAF)顯現出嚴重的液化性壞死; 第七組(PCA 50mg/kg.+ LPS +AAF),第八組(PCA 100mg/kg.+ LPS + AAF),第九組 (PCA 250mg/kg.+ LPS+ AAF)都可以見到液化性的壞死.

I.Potential effect of sodium nitrite on the expression of
nuclear proto-oncogenes during 2-acetylaminofluorene-induced
hepatocarcinogenesis in rats 2-acetylaminofluorene(AAF) reacts
in acidic conditions with nitrous fume yiel-ding N-nitrso-AAF
(N-NO-AAF),as previously described,that exerts more toxic and
mutagenic effects than its parental compound. In this study, the
effect of sodiumnitrite (NaNO2) on the tumorgenicity of AAF in
rats fed with AAF and NaNO2 was observed. Wistar rats were
divided into five groups: group I served as control;group II
treated with NaNO2(0.3%); group III was given 0.02% AAF alone;
group IVand group V received both AAF and NaNO2 (0.2% and 0.3%
respectively) in diet for12 weeks. At the end of experimental,
all rats in group III,IV and V developed early stage phenomena
of hepatocellular carcinoma.including hepatomegaly with
variable-size foci and neoplastic nodules. Severe damages were
observed in the rats treated with AAF and NaNO2. Feeding with
AAF(0.02%) for 3 months elevated the le-vels of c-Jun,c-Fos and
c-Myc proteins in the rats livers. The AAF-induced c-Jun,c-Fos
and c-Myc expressions were significantly magnified (p<0.001) by
NaNO2. These data confirmed that the strengthening of AAF-
induced hepatocarcinogenesis by NaNO2 should be associated with
its enhancing effect on the AAF-induced incre-ases in the
expressions of c-Jun,c-Fos and c-Myc. II.Inhibition by Hibiscus
protocatechuic acid of Lipopolysaccharide-induced endo-genous
Nitric oxide production and NO-AAF formation in rats
Nitrosocompound ,which can cause cancer by directly attacking
DNA to form DNAadducts,can be produced in vivo,indicating that
endogenous nitric oxide (NO) plays an important role. NO,either
exogenous or endogenous,can nitrosate secondary amines,thus
causing cancer. Endogenous NO would response to infection via
inducible NOsynthase which could be activated by various
microorganisms. Inducible NO synthasealso exists in hepatocyte
and is activated by cytokines from Kupffer cells in infe-ction.
Therefore,it was hypothesized that reduced endogenous NO in
infection may contribute to prevention in chemical
carcinogenesis caused by nitroso compounds.Hibiscus
protocatechuic acid (PCA),an antioxidant,can scavenge oxygen
free radicals with the catechol structure. It was hypothesized
if PCA could inhibit the elevationof endogenous NO induced by
Lipopolysaccharide (LPS),via scavenging free radicals,it would
prevent nitrosation reaction progression. Significantly
reduced endogenous NO production was demonstrated in rats
treated with PCA. In addition,rats pretreated with PCA inhibited
slightly hepatitis caused by AAF, Thus,PCA can be used as an
efficient chemopreventor in carcinogenesis caus-ed by
nitrosation of endogenous NO via free radical scavenging action.
Endogenous NO production,induced by Lipopolysaccharide,in
physiological condi-tions,reacts with target molecules (Heme
proteins,Fe-S proteins,SH groups,Other non-heme Fe and
metalloproteins,Amines,DNA,Oxygen,Superoxide anion, Hydrogen
peroxide),so that NO reacted with AAF may be difficult to
detect. Detect N-NO-AAF by Spectrophotometer method and by
HPLC method showed not detect-able. Rats treated with LPS and
AAF showed variable necrosis: in group III(LPS only) showed
focal necrosis;in group V (LPS and AAF) showed severe
liquefactive necrosis;in group VII (PCA 50mg/Kg.andLPS and AAF),
in group VIII (PCA 100mg/Kg.and LPS andAAF), in group IX (PCA
250mg/Kg. and LPS and AAF) showed liquefactive necrosis in these
groups.