全 文 ：Involvement of human CYP3A4 in the formation of hepatotoxic
metabolites of clivorine
LIU Xiao-Quan1＊ , LIN Ge2 , WANG Guang-Ji1 , QIAN Zhi-Yu3
(1.Center of Drug Metabolism and Pharmacokinetics;3.Department of Pharmacology , China Pharmaceutical
University , Nanjing　210009 , China;2.Department of Pharmacology , The Chinese University of
Hong Kong , Shatin , HongKong)
Abstract:AIM　This study was conducted to identify
the human CYP isoforms responsible for the biotransfor-
mation of clivorine in human liver microsomes and the
mechanism of metabolism-induced hepatotoxicity of
clivorine.METHODS 　Human liver microsomes were
used to investigate the metabolism of clivorine in vitro.
Selective CYP-450 inhibitors and cDNA expressed human
CYPs were used to study their effects on the formation of
hepatotoxic metabolites and the metabolism of clivorine
and the principal CYP-450 isoform involved in the forma-
tion of hepatotoxic metabolite.RESULTS　Four metabo-
lites , namely dehydroretronecine(DHR), 7-glutathionyl-
dehydroretronecine(7-GSH-DHR), 7 , 9-diglutathionyl-
dehydroretronecine(7 , 9-diGSH-DHR)and clivoric acid
were found in the microsomal incubations.Chemical inhi-
bition studies indicated that the metabolism of clivorine
and the formation of pyrrolic metabolites as well as the
bound pyrroles were strongly inhibited by CYP3A inhibitor
ketoconazole(Ket).Whereas α-naphthoflavone(Nap),
sulfaphenazole(Sulp), quinidine(Qui), diethyldithio-
carbamate(DDC) have no significant effects on the
metabolism of clivorine and the formation of pyrrolic
metabolites in human liver microsomes.The results of
metabolism of clivorine by cDNA expressed human CYPs
showed that only CYP3A4was found to be capable of cat-
alyzing the metabolism of clivorine , while CYP1A2 ,
CYP2C9 , CYP2D6 and CYP2E1 did not play significant
roles in the metabolism of clivorine and the formation
of pyrrolic metabolites.CONCLUSION　The results
　
　　Received date:2001-05-08　Accepted date:2001-09-15
　　Foundation item:The project supported by the National Natu-
ral Science Foundation of China (39970862);and National 973
Project(G1998051119)
　　Biographies:LIU Xiao-Quan(1960-), male , native of Wu-
jiang , Jiangsu Province , PhD , associate professor , main research
field is pharmacokinetics;WANG Guang-Ji(1953-), male , native
of Yangzhou , Jiangsu Province , PhD , professor , main research field
is pharmacokinetics.
　　＊Corresponding author.　Tel:(025)3271182 , Fax:(025)
3302827 , E-mail:Liuxq@Jlonline.com
demonstrated that the pyrrolic metabolites were the major
in vitro metabolites of clivorine and CYP3A4 was the ma-
jor CYP isoform involved in clivorine metabolism and the
formation of hepatotoxic pyrrolic metabolites in human
liver microsomes.CYP3A4 plays a key role in the
clivorine induced hepatotoxicity.
Key words:clivorine;liver;microsome;biotransforma-
tion
CLC number:R963
Document code:A
Article ID:1000-3002(2002)01-0015-06
Clivorine is one of the otonecine-type
pyrrolizidine alkaloids(PAs)found in awide vari-
ety of traditional Chinese medicinal herb and often
causes acute and chronic liver damages in man
through direct consumption of PA-containing
herbal teas and herbal medicines[ 1-3] .Many of
naturally occurring PAs are hepatotoxic
[ 4] .The
metabolism-induced hepatotoxicity by retronecine-
type PA has been extensively investigated and it is
bioactivated by hepatic cytochrome P450 to reac-
tive pyrrolic esters , which injure liver by a rapid
covalent binding with hepatic cellular macro-
molecules
[ 5 , 6] .Previous studies[ 7] have found that
like retronecine-type PA , a similar metabolic
pathway of clivorine was observed in rat microso-
mal incubation and four metabolites , namely dehy-
droretronecine (DHR), 7-glutathionyl-dehydro-
retronecine (7-GSH-DHR), 7 , 9-diglutathionyl-
dehydroretronecine(7 , 9-diGSH-DHR)and clivoric
acid , have been found and identified.The reac-
tive pyrrolic metabolites also play a key role in the
hepatotoxicity induced by otonecine-type PA.The
aim of the work presented here is to evaluate the
·15·中国药理学与毒理学杂志　　2002 年 2月;16(1):15-20
metabolic profiles of clivorine in human liver mi-
crosomes and the role of human hepatic CYP iso-
forms in the formation of reactive pyrrolic metabo-
lites of clivorine.
1　MATERIALS AND METHODS
1.1　Chemicals
The reduced form of glutathione (GSH),
retrorsine , glucose-6-phosphate (G6P), glucose-
6-phosphate dehydrogenase (G6PDH), quinidine
(Qui), α-naphthoflavone (Nap), ketoconazole
(Ket), sulfaphenazole (Sul), diethyldithio-
carbamate (DDC), troleandomycin (TAO)were
purchased from Sigma Chemical Co.Clivorine and
DHR , 7-GSH-DHR , 7 , 9-diGSH-DHR and
clivoric acid were generous gifts from Dr.Lin in
the Department of Pharmacology , the Chinese
University of Hong Kong.
1.2　Human liver microsomes
Human liver microsomes and microsomes
from human B-lymphoblastoid cell lines expressing
human CYP1A2 , CYP2C9 , CYP2D6 , CYP2E1
and CYP3A4 were obtained from Gentest
(Woburn , MA , USA)and stored at -70℃until
use.
1.3　In vitro metabolism of clivorine
A typical incubation mixture consisted of 0.1
mol·L-1 potassium phosphate buffer(pH 7.4),
2.0 mmol·L-1 GSH , NADPH generating system
(5 mmol·L-1MgCl2 10 mmol·L-1G6P , 1mmol·
L-1NADP , 1 kU·L-1 G6PDH), 250 mmol·L-1
clivorine and 1 g·L-1 microsomal protein , in a fi-
nal volume of 1 mL.The reaction was initiated by
addition of the NADPH generating system.After
incubation at 37℃ for 60 min , the reaction was
terminated in ice bath and retronecine (25 mg·
L-1)was added as an internal standard.Incuba-
tion mixture used for cDNA-expressed CYP450
was of the same composition as described above.
1.4　Chemical inhibition
Selective inhibitors of human CYP iso-
forms
[ 8 ,9]
were added to the incubation mixtures
and incubated as previously decribed.Incubations
with CYP form-selective inhibitors for each of the
major human liver P450s at a concentration of 250
mmol·L-1 clivorine were performed with micro-
somes from human liver samples.Inhibitors and in-
hibitor concentrations for each of the specific P450
forms are as following:1A2(Nap , 10μmol·L-1),
2C9(Sul , 100μmol·L-1), 2D6(Qui , 10μmol·
L-1), 2E1(DDC , 100μmol·L-1), 3A4(TAO ,
200μmol·L-1).Ket was added to microsomal in-
cubations at several concentrations ranging from
0.25 to 5.0μmol·L-1.
1.5 　HPLC analysis of the metabolites of
clivorine
The metabolites of clivorine in human liver
microsomes were determined by HPLC[ 10] .The
separation of the metabolites of clivorine in human
liver microsomes was performed on a PRP-1 re-
verse-phase column(5 μm , 150 mm×4.1 mm ,
Hammilton Co.Reno , NV)coupled with a PRP-1
guard column (5 μm , 50 mm×4.1 mm , Ham-
milton Co.Reno , NV).The mobile phase con-
sisted of 2%(V/ V)formic acid(solvent A)and
acetonitrile (solvent B).The gradient elution was
as follows:at 0-5 min 100%A;at 5-35 min ,
linear change from 100%A to 75%A;at 35-40
min , linear change from 75%A to 70%A.Flow
rate was kept constant at 0.8 mL·min-1 for the
complete analysis.Peak responses were measured
at 230 nm by UV detector.The resultant incubates
were centrifuged at 105 000×g at 2℃ for 30 min
and aliquots(200μL)of the supernatant were di-
rectly injected on to the column for quantitative
analysis.The resultant pellets of the incubations
were used for the determination of tissue-bound
pyrroles.
1.6　Determination of bound pyrroles
It is believed that the metabolites of PAs , not
the alkaloids themselves , are responsible for the
toxic effects[ 11] .The key step in metabolic activa-
tion of PAs is the formation of unstable pyrrolic es-
ters which may directly react with nucleophilic tis-
sue constituents such as proteins or nucleic acid to
form covalent bound adducts , called bound pyr-
role , and lead to specific cytotoxic effects.There-
fore in this experiment bound pyrroles were mea-
sured as an index of PAs induced hepatotoxicity.
·16· Chinese Journal of Pharmacology and Toxicology　2002　Feb;16(1)
The tissue-bound pyrrole formed after incubation
was measured by a modified method reported in
literature
[ 12] .
1.7　Statistical analysis
Data were expressed as x ±s and analyzed
using Student t test.
2　RESULTS
2.1　Metabolism of clivorine in human liver
microsomes
Metabolite profiles obtained from human liver
microsomes indicated that clivorine was oxidized to
four primary metabolites with the corresponding
level of clivorine decreased.Using chromatography
with authentic standard and HPLC mass spectro-
metry , all four metabolites were identified as being
the previously described primary metabolites of
clivorine in male rat liver microsomal incubation:
DHR , 7-GSH-DHR , 7 , 9-diGSH-DHR and
clivoric acid respectively.In addition , bound pyr-
role was also found in microsomal incubations and
the total bound pyrrole in microsomal incubations
was 1.026 μmol·L-1 .The results suggests that
the metabolic pattern of clivorine in human liver
microsomes is similar to that in male rat liver mi-
crosomes.
2.2　Inhibition analysis
To determine the specific CYP isozyme(s)
involved in the biotransformation of clivorine , the
CYP-isoform selective probes were screened for in-
hibitory effects on the metabolism of clivorine in
human liver microsomes and incubations were con-
ducted using chemical inhibitors that are specific
for various CYP isozymes.The effects of CYP-iso-
form selective inhibitors on the metabolism of
clivorine and the formation of pyrrolic metabolites
are shown in Tab 1.The results showed that Nap
(CYP1A2), Sul (CYP2C9), Qui (CYP2D6),
DDC (CYP2E1)had little or no significant in-
hibitory effects on both the metabolism of clivorine
and the formation of corresponding metabolites in
human liver microsomes , whereas TAO
(CYP3A4)significantly inhibited the metabolism
of clivorine in human liver microsomes.Ket
caused a significant reduction of formation rate for
DHR to 45%and for clivoric acid to 20%of con-
trol values and the levels of 7-GSH-DHR , 7 , 9-
diGSH-DHR was decreased to below the detect
limit.Whereas intact clivorine was increased from
56.8% to 87.4%.Both the formation of the
metabolites and the bound pyrrole were significant-
ly inhibited by Ket(Fig 1 and Fig 2), even at low
concentrations(0.5μmol·L-1).The formation of
both pyrrolic metabolites and bound pyrrole were
completely inhibited by high concentration (5
μmol·L-1)of Ket and both of them were not de-
tectable in the microsomal incubations , while the
Tab 1.　Effect of selective CYP inhibitors on the metabolism of clivorine in human liver microsomes
Group
CYP activity/nmol·min-1·g-1
DHR 7-GSH-DHR 7 , 9-diGSH-DHR Clivoric acid
Clivorine content
/mmol·L-1
Control 　　263±32 　　 86±12 　　　42±0.7 　　674±37 　　　　 142±13
Nap 271±8 78±6 45±2 607±61 146±9
Sul 257±32 83±17 39±2 662±35 150±9
Qui 265±27 90±10 45±3 632±39 150±2
DDC 259±22 90±14 44±2 633±93 145±5
TAO 119±2＊＊ 　　　 - 　　　　- 135±2＊＊ 223±8＊
Clivorine:250 mmol·L-1;Nap:α-naphthoflavone(10μmol·L-1);Sul:sulfaphenazole(100μmol·L-1);Qui:quinidine(10μmol·L-1);
DDC:diethyldithiocarbamate(100 μmol·L-1);TAO:troleandomycin (200 μmol·L-1);DHR:dehydroretronecine;7-GSH-DHR:7-glu-
tathionyl-dehydroretronecine;7 , 9-diGSH-DHR:7 , 9-diglutathionyl-dehydroretronecine.(-):not detectable. x±s , n=3.＊P <0.05 ,
＊＊P<0.01 , compared with control.
·17·中国药理学与毒理学杂志　　2002 年 2月;16(1)
Fig 1.　Effects of ketoconazole on the metabolism of
clivorine and the formation of its pyrrolic metabo-
lites.DHR(★):dehydroretronecine;7-GSH-DHR(○):7-glu-
tathiony l-dehydroretronecine;7 , 9-diGSH-DHR(■):7 , 9-diglu-
tathiony l-dehydroretronecine;clivoric acid(△); clivorine(◆).
x±s , n=3.
Fig 2.　Effects of ketoconazole on the formation of
bound pyrrole of clivorine in human liver micro-
somes.
intact clivorine was increased to 91%.These data
indicated that CYP3A4 was responsible for about
80% of the metabolism of clivorine into pyrrolic
metabolites.
2.3 　Metabolism of clivorine by cDNA ex-
pressed specific P450s
The metabolism of clivorine by cDNA ex-
pressed human CYPs(CYP1A2 , 2C9 , 2D6 , 2E1
and 3A4)was investigated .Among the five dif-
ferent recombinant human CYPs only CYP3A4
showed detectable catalytic activity for the
metabolism of clivorine.Incubations of clivorine
with cDNA-expressed CYP3A4 resulted in the for-
mation of all four pyrrolic metabolites and bound
pyrrole.But the other human CYPs(1A2 , 2C9 ,
2D6 and 2E1)showed extremely low activity for
the metabolism of clivorine and no metabolites
could be measured in the incubations.The results
are presented in Tab 2.
3　DISCUSSION
Previous investigation of the metabolism of
retronecine-type PAs has clearly indicated that the
major way of metabolism was to form hepatotoxic
pyrrolic metabolites
[ 13] .PAs naturally occur in
variety of plants and traditional herbs and often
cause hepatotoxicity inman through direct or indi-
rect consumption of PA-containing plants and
herbs. However , the metabolic profiles of
clivorine , an otonecine-type PA , in human liver
microsomes are poorly understood.Therefore such
information is essential to the prevention of ad-
verse events caused by PAs.The results of pre-
sent studies showed that the major way of
Tab 2.　Metabolism of clivorine by cDNA expressed human CYP isoforms
Group
CYP activity/nmol·min-1·g-1
DHR 7-GSH-DHR 7 , 9-diGSH-DHR Clivoric acid
Clivorine content
/mmol·L-1
Bound pyrrole content
/μmol·L-1
CYP1A2 - - - - 　　　　250±20 -
CYP2C9 - - - - 252±20 -
CYP2D6 - - - - 248±16 -
CYP2E1 - - - - 242±10 -
CYP3A4 160±18 53±5 32±3 327±21 203±4 0.288
Clivorine:250 mmol·L-1.(-):not detectable. x±s , n=3.
·18· Chinese Journal of Pharmacology and Toxicology　2002　Feb;16(1)
metabolism of clivorine was also to form hepato-
toxic pyrrolic metabolites.This suggests that the
major metabolic pathway of clivorine in human liv-
er microsomes is similar to that in male rat liver
microsomes.
　　The results of chemical inhibition showed
that both Ket and TAO , selective human CYP3A
inhibitor , significantly inhibited the formation of
hepatotoxic pyrrolic metabolites and bound pyrrole
and lowered the rate of metabolism for clivorine.
Therefore the formation of pyrrolic metabolites was
the major metabolic pathway in human liver mi-
crosomes.The inhibitory effect of Ket showed
concentration-related and the formations of
pyrrolic metabolites and bound pyrroles generated
from clivorine were abolished by high level of
Ket.Whereas other CYP inhibitors , i.e.Nap
(1A2), Sulp (2C9), Qin (2D6) and DDC
(2E1)had little or no significant inhibitory effect
on the metabolism and the formation of pyrrolic
metabolites. Furthermore cDNA expressed
CYP3A4 catalyzed the metabolism of clivorine and
while incubations with other cDNA expressed
CYP1A2 , CYP2C9 , CYP2D6 and CYP2E1 did
not produce detectable levels of metabolites.
These data strongly suggest that human CYP3A4 is
the primary isoform involved in the metabolism of
clivorine and the formation of reactive metabolites
and this is the major in vitro metabolic pathway of
clivorine in human liver microsomes.CYP3A4 is
known to be the major human CYP isoforms in-
volved in the oxidative metabolism of many clini-
cally used drugs as well as xenobiotics.In addi-
tion , CYP3A4 is inducible and inhibitable by a
lots of xenobiotics[ 14 , 15] .Pharmacokinetic pro-
cesses are regarded as the most important factors
that affect the levels of drugs in the body.The
findings obtained in this study suggests that there
is a possibility that the clivorine-induced hepato-
toxicity could be decreased by CYP3A inhibitors.
In conclusion , the in vitro metabolic path-
way of clivorine in human liver microsomes is to
generate hepatotoxic pyrrolic metabolites and bind
with hepatic tissues that leads to hepatotoxicity
and this is the major in vitro metabolic pathway of
clivorine in human liver microsomes.Human
CYP3A is the primary CYP isoforms involved in
the metabolism of clivorine and formation of hepa-
totoxic pyrrolic metabolites and plays a key role in
the clivorine induced hepatotoxicity.
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·19·中国药理学与毒理学杂志　　2002 年 2月;16(1)
[ 14] 　Periti P , Mazzei T , Mini E , Noveli A.Pharmacokinetic
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人肝 CYP3A参与了山冈橐吾碱的代谢及其肝毒性代谢物的形成
柳晓泉1 , 林　鸽2 , 王广基1 , 钱之玉3
(中国药科大学 1.药物代谢研究中心 , 3.药理学教研室 , 江苏 南京　210009;
2.香港中文大学药理系 , 沙田 香港)
摘要:目的　在体外研究山冈橐吾碱在人肝微粒体
内的代谢及参与其代谢的主要的 CYP450 酶 , 探讨
其代谢致毒机理 。方法　采用人肝微粒体研究山冈
橐吾碱的主要代谢方式和代谢物。在体外运用
CYP450酶的选择性抑制剂和 cDNA 表达的人肝
CYP450酶 ,探讨其对山冈橐吾碱的代谢及肝毒性的
吡咯代谢物形成的影响及参与山冈橐吾碱代谢的主
要的 CYP450酶 。结果　山冈橐吾碱在人肝微粒体
内的主要代谢物为肝毒性的吡咯代谢物:去氢倒千
里光裂碱 , 7-谷胱甘肽基-去氢倒千里光裂碱 , 7 , 9-
二谷胱甘肽基去氢倒千里光裂碱和山冈囊吾酸 。
CYP450特异性抑制剂α-萘黄酮(抑制 CYP1A2)、黄
胺苯吡唑(抑制 CYP2C)、奎尼丁(抑制CYP2D6)和二
乙基二硫代氨基甲酸钠(抑制 CYP2E1)对山冈橐吾
碱的代谢无明显的影响。但 CYP3A的特异性抑制
剂酮康唑和三乙酰竹桃霉素可以显著地抑制山冈橐
吾碱的代谢及其吡咯代谢物和结合型吡咯物的形
成 。此外 , 在 cDNA 表达的人肝 CYP3A4的温孵液
中 ,山冈橐吾碱被代谢成相应的吡咯代谢物 ,而山冈
橐吾碱在 cDNA 表达的人肝 CYP1A2 、CYP2C9 、
CYP2D6和CYP2E1温孵液中无代谢。结论　山冈
橐吾碱在人肝微粒体内的主要代谢方式是形成肝毒
性吡咯代谢物 , CYP3A 作为主要的 CYP450酶参与
了山冈橐吾碱的代谢及其肝毒性吡咯代谢物的形
成 。CYP3A在山冈橐吾碱所致的肝毒性中发挥了
重要的作用 。
关键词:山冈橐吾碱;肝脏;微粒体;生物转化
　
　　基金项目:国家自然科学基金资助项目(39970862);国
家 973 计划资助项目(G1998051119)
(本文编辑　石　涛)
告读者与作者
1.本刊从 2002年起改用铜版纸印刷 ,但售价暂不变。
2.本刊 2002年的投稿须知有重要变化。如:综述的要求 、摘要的结构 、中文标点符号的使用 、优秀
论文的稿酬及参考文献著录格式等。详情请见 2002年第 1期的投稿须知。
《中国药理学与毒理学杂志》编辑部
·20· Chinese Journal of Pharmacology and Toxicology　2002　Feb;16(1)