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Part I
以十四種胺基酸 （Ala, Gly, Ser, Tyr, Trp, Arg, Asp, Lys, Pro, Leu, Ile,
Glu, Phe,Thr) 與三種單醣 (Rib,Glc,Fru) 配合肌酸酐 (creatinine) 組成各種胺基酸/
單醣/肌酸酐模式系以探討形成 IQ 型致突變物之可能前驅物。 各模式系中以 Thr/單醣/
肌酸酐模式系產生致突變物之能力較強。 Thr/單醣/肌酸酐模式系中之 IQ 型致突變物經
HPLC 分析及質譜鑑定主要有 IQx、 MeIQx、 4,8-DiMeIQx、 IQ 與 MeIQ 等五種。此模
式系中 IQ 型致突變物之含量以 IQx 與 MeIQx 最高，約佔總含量之 33.6~59.2%。三種
醣所構成之 Thr/單醣/肌酸酐模式系之致突變性與 IQ 型致突變物之含量依序為 Rib>Glc
>Fru。為解釋此模式系中所生成的 MRPs 和 IQ 型致突變物形成之關係，進一步以 GC-MS
分析Thr/單醣/肌酸酐模式系中所生成之 MRPs，結果顯示妣阱類之生成量最高佔總 MRPs
量的 25~37%，其生成量與致突變性間有正相關性。因此推測 Thr/單醣/肌酸酐模式系形
成 IQ 型致突變物可能取決於形成妣阱類之能力。 為更確定那些妣阱類對 Thr/單醣/肌
酸酐模式系形成 IQ 型致突變物最有影響，因此在各模式系中添加四種不同的妣阱類，結
果發現四種妣阱類 ( 2-methylpyrazine , 2,3-dimethylpyrazine , 2,6-dimethyl
pyrazine, trimethylpyrazine) 均能促進模式系之致突變性10~49%，但其中以 2-methyl
pyrazine(29~49%) 與 2,3-dimethylpyrazine (24~31%) 具有較強的促進性。因此推測該
兩種妣阱類可能參與 IQx 及 MeIQx 的形成。
Part II
以八類十四種 MRPs (pyridines 、 pyrazines、thiazoles 、 thiophenes 、
furans 、 pyrroles、 imidazoles、 oxazoles) 與 acetylformaldehyde 配合肌酸酐
組成各種 MRPs/醛類/肌酸酐模式系以探討 IQ 型致突變物形成之可能前驅物。結果發現
八類 MRPs 中妣阱類、妣啶類與 塞 坐類三類所產生之致突變物以 IQ 型為主，且均佔
總致突變性之 83.9% 以上。另外醛類/肌酸酐與醛類/塞 坐類之二種模式於加熱反應後，
均會產生 妣阱類 。由此推測， 妣阱類在形成 IQ型致突變物上扮演比其它 MRPs 更為
重要之角色。因此進一步以四種妣阱類與醛類/肌酸酐組成模式系探討不同化學結構之妣
阱類對 IQ 型致突變物形成之關係。結果所得之萃取物經 HPLC 純化分析得知各妣阱類模
式系中的主要致突變物為 IQx、 MeIQx、7,8-DiMeIQx、 IQ 與 MeIQ 等五種，而前三者
已由質譜確定。再比較四種 妣阱類/醛類/肌酸酐模式系所形成的 IQ 型致突變物之含量
，發現 2-methylpyrazine 與 2,3-dimethylpyrazine 主要是形成IQx； 2,6-dimethyl-
pyrazine 主要是形成 MeIQx；而 trimethylpyrazine 主要是形成 7,8-DiMeIQx。因此
推測，具有甲基之妣阱類化合物可能參與 imidazoquinoxaline 致突變物之形成。
Part III
六種蔥屬植物含硫化合物 (diallyl sulfide, DAS; diallyl disulfide,DADS;
dipropyl sulfide,DPS; dipropyl disulfide, DPDS; allyl methyl sulfide,AMS;
allyl mercaptan,AM) 與兩種抗氧化劑 (flavone,BHA) 均能分別抑制豬肉汁與甘胺酸/
葡萄糖/肌酸酐模式系之致突變性與褐變程度。為研究其作用機制，進一步以氣相層析儀
分析 MRPs 之形成，而發現含硫化合物均能抑制豬肉汁中妣阱啶類、妣啶類、塞 坐類與
塞酚類等MRPs 之形成，其中總 MRPs 及塞酚類之降低量與抑制豬肉汁致突變性之間有顯
著相關性(r=0.64,P<0.05； r=0.87,P<0.01) 。 另外， BHA 和 flavone 兩種抗氧化
劑亦能抑制甘胺酸/葡萄糖/肌酸酐模式系之致突變性與 MRPs 之生成，而 flavone 之抑
制能力較 BHA 為強。 MRPs 中妣阱啶類受抑制量較其它 MRPs 為顯著。兩種抗氧化劑在
模式系加熱過程中所生成之五種妣阱啶類之含量變化與致突變性之抑制程度間具有顯著
的相關性，但 BHA 對 2,3-dimethylpyrazine 之抑制量與致突變性間則無相關性。另外
在添加各種妣阱啶類於豬肉汁模式系之研究中又發現 2,3-dimethylpyrazine 之促進致
突變性的能力最強。 因此推測 2,3-dimethylpyrazine 於上述之二模式系在形成
imidazoquinoxaline(IQx) 類致突變物上可能扮演重要的角色。
Part IV
本研究選取八種抗突變性較高之胺基酸/單醣 (Glc/Trp、Xyl/Arg、Xyl/Lys、
Xyl/Gly、Glc/Gly、Glc/Arg、Fru/Arg、Fru/Gly) 模式系進行其中抗突變物之探討。結
果發現八種胺基酸/單醣模式系之二氯甲烷萃取物對 IQ 之抗突變性強度依序為 Glc/Trp>
Xyl/Arg> Xyl/Lys > Fru/Arg > Xyl/Gly > Glc/Arg> Fru/Gly > Glc/Gly ，而具有最強
抗突變性之 Glc/Trp 則有 Glc/Gly 的 50 倍以上之抗突變性。為瞭解是何種 MRPs 參與
抗突變作用，以氣相層析儀配合質譜儀分析二氯甲烷萃取物中之 MRPs， 結果顯示主要含
有妣阱類與砆喃類兩類 MRPs，其總量與抗突變性有顯著相關性，尤其是妣阱類
(r=0.762,P<0.01) 。因此進一步比較八種妣阱類對 IQ 之抗突變性，結果發現
2-methylpyrazine 具有最強之抗突變性 (ID50,0.09μmol) 。妣阱類之抗突變性主要是
抑制 ECD 活性而阻斷 IQ 被活化成 N-OH-IQ 之代謝活化路徑。因此推測胺基酸/單醣模
式系反應液中主要抑制 IQ 致突變性的 MRPs 可能是妣阱類。
Part V
本研究將省產四川品之辣椒 (Capsicum annuum L.) 以八種不同極性溶劑 (水、
甲醇、乙醇、異丙醇、丙酮、二氯甲烷、乙醚及正己烷) 萃取精油樹脂，並以安氏試驗
法檢測其對 IQ 之抗突變性。八種辣椒精油樹脂對 IQ 具有不同程度之抗突變性，其中
以正己烷辣椒精油樹脂最強 (ID50,20μg/plate) ，其抗突變性為最低的水辣椒精油樹
脂的 100 倍以上的強度。為瞭解正己烷辣椒精油樹脂中主要的抗突變性成分，以 TLC
及 HPLC 分析精油樹脂中之類番椒素 (capsaicinoids) 與類胡蘿蔔素 (carotenoids)
之含量並進行抗突變性分析。發現類番椒素對 IQ 不具抗突變性而類胡蘿蔔素具有
顯著的抗突變性。經由HPLC與UV光譜確定其中主要具有抗突變性之類胡蘿蔔素分別為
β-carotene、 β-cryptoxanthin 、 cryptocapsin、 zeaxanthin、capsolutin、
cis-capsanthin、 ateraxnthin、 capsanthin、 violoxanthin、 capsorubin 及四種
未鑑定出之化合物，其中以cryptocapsin對 IQ 之抗突變性最強 (ID50,1.58 nmol)。
經統計分析發現八種不同溶劑萃取之辣椒精油樹脂中之類胡蘿蔔素含量與其抑制 IQ 之
致突變性間有顯著的相關性 (r=0.85,P<0.05) 。因此推測類胡蘿蔔素為辣椒精油樹脂
中主要的抗突變性成分。進一步分析類胡蘿蔔素對 IQ 的活化路徑與其代謝活化物
N-OH-IQ 之影響以瞭解其作用機制，結果顯示辣椒類胡蘿蔔素抑制 IQ 之致突變性的作
用機制主要是作用於 IQ 的活化物 N-OH-IQ 上，而不會影響 IQ 之代謝活化路徑。
Part VI
氧化型類胡蘿蔔素 cryptocapsin 已知在安氏試驗法上會抑制 IQ 之致突變性。
本研究以 32P-postlabeling 方法首先分析 cryptocapsin 是否會抑制 N-OH-IQ 在小
牛胸腺 DNA 所造成的 DNA 鍵結物，結果發現cryptocapsin會有效抑制 N-OH-IQ 的
DNA 鍵結物之形成。然後進一步在大白鼠 (Sprague Dawely rat) 初代肝細胞中探討
cryptocapsin 是否會同樣的抑制 IQ 所造成的 DNA 鍵結物。結果 cryptocapsin 亦
能抑制初代鼠肝細胞中 IQ-DNA 鍵結物的生成。為瞭解其作用機制，以紫外線光譜儀
分析 cryptocapsin 與 N-OH-IQ 是否發生作用，結果顯示在反應過程中可能因生成
一新複合物，而 UV 最大吸收峰 254nm 轉移至 268nm 。此一新複合物進一步以質譜
儀鑑定為一分子量 765 之化合物。因此推測 cryptocapsin 抑制 IQ-DNA 鍵結物之生
成可能是cryptocapsin 直接與 N-OH-IQ 鍵結生成一新的複合物。本研究提出一個抑
制 N-OH-IQ 致突變性新的作用機制。
Part I
Various amino acid/monosaccharides/creatinine model systems
were used to study which amino acids or monosaccharides play important
contributions on the production of mutagenicity and IQ. Among 14
amino acids, the extracts of Thr/monosaccharide/creatinine model system
shows the highest mutagenicity examined with S. typhimurium TA98 in
the presence of S9 mix. Five major mutagenic IQ compounds in the
extracts of Thr/Rib/creatinine model system were purified through 3
HPLC columns and the mutagenicity of 1-min fraction eluted from each
HPLC purified step was monitores with TA98 strains. Three mutagenic
fraction corresponding to the peaks of the standard mutagens IQx, MeIQx,
and 4,8-DiMeIQx were confirmed by the comparison of UV and mass spectra.
To clarify which MRPs involved the formation of IQ in three model systems,
MRPs in dichloromethane extracts of Thr/monosaccharide/creatinine model
systems were determined by GC-MS. The pyrazines in the extracts of three
model sy stems have the greatest amounts among 8 kinds of MRPs.
Moreover, the production amounts of pyrazines in three model systems
correlated well with their mutagenicity and IQ mutagen contents.
In addition, four pyrazines separately added into the three model
systems to study the effects of pyrazines on the mutagenicity.
The result showed that 2-methylpyrazine and 2,3-dimethylpyrazine were
important for the mutagenicity production than 2,6-dimethylpyrazine and
trimethylpyrazine. Therefore, we proposed that 2-methylpyrazine and
2,3-dimethylpyrazine might participated in imidazoquinoxaline-type mutagen
formation in Thr/monosaccharide/creatinine model systems.
Part II
In order to elucidate the role of MRPs on the IQ-type mutagen
formation , the mutagenicity of extracts of eight kinds of MRPs including
forteen MRPs/ acetylformaldehyde/creatinine model systems were examined
with Salmonella typhimurium TA 98 in the presence of S9 mix. After
nitrite treatment experiment, data showing three kinds of MRPs,
pyrazines,pyridines, and thiazoles in model systems contributed to
the IQ-type mutagen formation were important than other kinds of MRPs.
In addition, p yrazines were produced in acetylformaldehyde/creatinine
and acetylformaldehyde/thiazole model systems which were comfirmed by
mass spectrometry. We therefore suggested that pyrazines may play an
important role on the IQ-type mutagen formation. Furtherly, the IQ-type
mutagens were purified from different four kinds of pyrazines/
acetylformaldehyde/creatinine model systems by a seris of HPLC column.
Five major mutagen compounds were found to be IQx, MeIQx, 7,8-DiMeIQx, IQ,
and MeIQ. Among these formers were identified by mass spectrometry.
From the IQ-type mutagen yielded, we suggested that 2-methylpyrazine,
2,3-dimethylpyrazine 2,6-dimethylpyrazine, and trimethylpyrazine were
important for the IQx, MeIQx, and 7,8-DiMeIQx rpoduction, respectively.
Part III
Three heterocyclic aromatic amines, 2-amino-3-methylimidazo[4,5-f]
quinoline(IQ), 2-amino-3,4-dimethylimidazo[4,5-f] quinoline(MeIQ),
2-amino-3-methylimidazo[4,5-f] quinoxaline(MeIQx) have been found in
boiled pork juice. We have investigated the effect of naturally
occurring organosulfur compounds, which are present in garlic and onion,
on the mutagen formation in boiled pork juice. Six organosulfur
compounds were added separately to the pork juice before reflux boiling
and then the mutagenicity of each sample was examined with Salmonella
typhimurium TA98 in the presence of S9 mix. All of six organosulfur
compounds including diallyl disulfide(DADS), diallyl sulfide(DAS),
dipropyl disulfide (DPDS), dipropyl sulfide(DPS), allyl methyl sulfide(AMS)
and allyl mercaptan(AM) inhibited the mutagenicity of boiled pork juice.
The greastest inhibitory effect was observed with DADS and DPDS and this
was 111-fold higher than that of the lowest cysteine. To elucidate the
inhibitory effect of DADS on mutagen formation in boiled pork juice,
the major mutagenic fraction were monitored after HPLC separation by
their mutagenicity with S. typhimurium TA98. By comparison of the
retention time of authentic IQ compounds from boiled pork juice with
those following the addition of DADS , we showed that the mutagenicity
of three major fraction was significantly inhibited compared to those
same fraction in boiled pork juice alone. Moreover, the Maillard
reaction products(MRPs) in the boiled pork juice with and without
the addition of DADS were quantified and identified by capillary gas
chromatography and gas chromatography-mass spectrometry. The results
show that the reduction in the total amounts of MRPs(pyridines,pyrazines,
thiophenes, and thiazoles) in boiled pork juice after boiling 12hr was
correlated with their mutagenicity. Among the MRPs, tetrahydrothiophene-
3-one(THT) exhibited the strongest correlation . These data suggest
that the inhibition of IQ mutagen formation by DADS is mediated through the
reduction of MRPs production. Furthermore, 7 pyrazines were separately
added into th boiled pork juice to study the influence on the
mutagenicity of BPE. The greastest enhancement was observed with the
addion of 2,3-dimethylpyrazine in the boiled pork juice model systems.
On the other hands, we studied the inhibitory effects of addition of
BHA and flavone on the mutagenicity and IQ-mutagen formation of glucose/
glycine/creatinine model system. The reduction capability
of flavone on the mutagenicity and IQ-mutagen formation in the model
system was higher than that of BHA. To elucidate the mechanism ,
the correlation between the changed amounts of MRPs in the model system
during heating periods and their mutagenicity was also investigated .
Except 2,3-dimethylpyrazine in the model system with the addition of
BHA , significant correlation was observed between the amounts of
each of five pyrazines produced in both model system.(with the addition
of BHA and flavone ) and the mutagenicity. These suggest that
2,3-dimethylpyrazine might play an important role on the formation of
imidazoquinoxaline in boiled pork juice and glucose/glycine/creatinine
model system.
Part IV
The antimutagenicity of dichloromethane extracts from eight amino
acid/monosarrcharide model systems was determined using Salmonella
typhimurium TA 98 against 2 - amino -3 - methyl imidazo[4,5-f]quinoline
(IQ) in the presence of Aroclor 1254-induced rat hepatic S9. The
Maillard reaction products(MRPs) in the dichloromethane extracts were
then quantified and qualified by capillary gas chromatography and gas
chromatography-mass spectrometry，respectively‧ Pyrazines and furans
were found to be the major MRPs yielded in the extracts.
Furans showed the antimutagenicity in several investigator reports.
Moreover, the antimutagenicity of dichloromethane extracts correlated
positively with the total amounts of pyrazines (r=0.762, P<0.01).
Thus we concluded that pyrazines in dichloromethane extracts from eight
amino acid/monosarrcharide model systems play an important role in
the antimutagenicity of IQ. To elucidate the mechanism of
dichloromethane extracts, the inhibitory effect of pyrazines on
ethoxylcoumarin deethylase activity in aroclor 1254-induced hepatic
microsomes was examined. We also studied the effects of pyrazines on
IQ metabolism by Aroclor 1254-induced microsomes using high-performance
liquid chromatography. The antimutagenicity of pyrazines correlated
positively with the inhibition of cytochrome P-450 IA2-linked
ethoxylcoumarin deethylase in hepatic microsomes and the inhibition
of N-hydroxy-IQ formation from IQ metabolism by hepatic microsomes.
Moreover, we concluded that the modifying effect of pyrazines on the
mutagenicity of IQ is mediated through interaction with microsomal
activating enzymes to inhibit the major active metbolite in N-hydroxy-IQ
formation.
Part V
The inhibitory effects of chili (Capsicum annum L.) oleoresins
extracted with eight solvents on the mutagenicity of IQ were examined
with Salmonella typhimurium TA98 with the presence of S9 mix. The
highest antimutagenicity was observed on n-hexane oleoresin followed
by diether ether, dichloromethane, acetone, isopropanol, ethanol,
methanol and water olepresins in decreasing order. To determine the
major antimutagenic compound in chili oleoresin, carotenoids in
n-hexane oleoresin were purifie d and analyzed by TLC and HPLC.
The result showed that ten carotenoids were isolated and identified
such as β-carotene,β-cryptoxanthin, cryptocapsin, zeaxanthin,
capsolutin, cis-capsanthin , alteraxanthin, capsanthin, violaxanthin,
capsorubin . The remaining four unknown compounds presented in n-hexane
oleoresin need further investigation. Interestingly, the
antimutagenicity of egiht chili oleoresins correlated positively
with their total amounts of carotenoids(r=0.85,P<0.01). To elucidate
the mechanism of antimutagenicity of chili oleoresins, the
inhibitory effect of carotenoids on ECD activity in Aroclor
1254-induced hepatic microsomes was examined. We also studied the
effects of carotenoids on the direct mutagenicity of N-hydroxy-IQ
using TA98 strains. Thus we concluded that carotenoids in chili
oleoresins may play an important role in the antimutagenicity of IQ .
Moreover, the modifying effects of carotenoids on the mutagenicity
of IQ is mediated through direct interaction with N-hydroxy-IQ,
but not by alter the metabolic activation of IQ.
Part VI
Cryptocapsin is one of naturally occurring oxgenated carotenoids
in chili fruit. The effect of cryptocapsin on the formation of DNA
adducts of N-hydroxyamino-3-methylimidazo[4,5-f]quinoline(N-OH-IQ),
in Sprague Dawley rat liver primary cultured cells was examined by
32P-postlabeling assay. The N-OH-IQ-DNA adducts formed in rat primary
hepatocytes was inhibited in a dose-dependent manner by cryptocapsin.
The interaction between cryptocapsin and N-OH-IQ was monitored by UV
spectroscopic study to ascertain the mechanism . The absorption
maxinum shifts toward longer wavelength resulted from the interaction
between cryptocapsin and N-OH-IQ. Furthermore, the
N-OH-IQ-cryptocapsin adducts has been identified by mass spectrometry .
These results indicate that cryptocapsin inhibited the mutagenicity
and DNA adduct of N-OH-IQ was mediated through the formation of a
novel molecule. The mechanism of action against the mutagenicity
of N-OH-IQ is discussed.
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