全 文 ：Metabolism of clivorine in female rat liver microsomes
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:The metabolism of clivorine , an othonecine-
type hepatotoxic pyrrolizidine alkaloid , was investigated in
female rat liver microsomes.The major in vitro metabo-
lites of clivorine found in the incubation mixture are two
non-pyrrolic metabolites(M1 and M2)and the formation of
hepatotoxic pyrrolic metabolites from clivorine is only the
minor in vitro metabolic pathway of clivorine in female
rats.It suggests that the major in vitro metabolic pathway
of clivorine in female rats is different from that in male
rats.Selective CYP450 inhibitors pilocarpine (Pil ,
CYP2A1), diethyldithiocarbamate (DDC , CYP2E1),
sulfaphenazole (Sul , CYP2C)and ketoconazole (Ket ,
CYP3A)had no significant effects on the formation of M1
and M2. Selective flavin-containing monooxygenase(FMO)inhibitor methimazole could inhibit the formation
of M2 , but had no inhibitory effect on the formation of M1
and the formation of M1 was NADPH independent.The
above results suggests that the hydrolase and FMO in
microsomes are involved in the formation of M1 and M2 re-
spectively .On the other hand , Selective CYP450 in-
hibitors Pil , DDC , Sul had no significant effects on the
formation of pyrrolic metabolites from clivorine ,but selec-
tive CYP3A inhibitor Ket could significantly inhibited the
formation of pyrrolic metabolites of clivorine whereas
clivorine was not metabolized by recombinant rat
CYP2C12 and CYP2E1 , but could be metabolized by
recombinant rat CYP3A1 and CYP3A2 to produce its cor-
responding hepatotoxic pyrrolic metabolites.The above re-
sults indicate that rat CYP3A1 and CYP3A2 are the pri-
mary CYP450 involved in the formation of hepatotoxic
　
　　Received date:2001-03-06　Accepted date:2001-07-04
　　Foundation item:The project supported by National Natural
Science Foundation of China(39970862);and National 973 Project
(G1998051119).
　　Biographies:LIU Xiao-Quan(1960-), male , native of Wu-
jiang, Jiangsu Province , Doctor of Pharmacy , associate professor ,
main research field is pharmacokinetics;WANG Guang-Ji(1953-),
male, native of Yangzhou , Jiangsu Province , Doctor of Pharmacy ,
professor , main research field is pharmacokinetics.
　　＊Corresponding author.Tel:(025)3271347 , Fax:(025)
3302827 , E-mail:Liuxq@Jlonline.com
pyrrolic metabolites.The difference in clivorine-induced
toxicity can be partly attributed to this metabolic differ-
ence in sex.
Key words:clivorine;microsomes , liver;metabolism
CLC number:R963
Document code:A
Article ID:1000-3002(2001)06-0413-05
Clivorine is an othonecine-type pyrrolizidine
alkaloid(PA)found in various Ligularia species
including some traditional Chinese medicinal
herbs.The majority of naturally occurring PA is
found to be hepatotoxic to both man and animals
and the consumption of PA-containing plants ,
herbal teas and herbal medicines may cause liver
damage
[ 1-5] .The hepatotoxic PA can be classi-
fied structurally into two types:retronecine-type
and othonecine-type.The mechanism of hepato-
toxicity induced by retronecine-type PA has been
extensively investigated.It is established that
the oxidative biotransformation of this type of PA
to the corresponding unstable reactive pyrrolic
esters which rapidly form covalent adducts with
cellular macromolecules in the liver and therefore
produce hepatotoxicity[ 6] .Previous studies have
found that the metabolic pattern of clivorine in
male rat liver microsomes is to form corresponding
hepatotoxic pyrrolic metabolites , namely dehy-
droretronecine (DHR), 7-glutathionyl-dehy-
droretronecine (7-GSH-DHR), 7 , 9-diglutathionyl-
dehydroretronecine(7 , 9-diGSH-DHR)and clivoric
acid , which have been unequivocally character-
ized[ 7] .The objective of the current study is to
investigate the metabolic difference of clivorine in
female rat as possible explanation for the observed
sexual difference in clivorine-induced toxicity.
·413·中国药理学与毒理学杂志　　2001 年 12月;15(6):413-417
1　MATERIALS AND METHODS
1.1　Chemicals
The reduced form of glutathione(GSH),
retrorsine , glucose-6-phosphate(G6P), glucose-
6-phosphate dehydrogenase (G6PDH), sul-
faphenazole(Sul), ketoconazole (Ket), pilo-
carpine(Pil), diethyldithiocarbamate(DDC), me-
thimazole were purchased from Sigma Chemical
Co.Clivorine , DHR , 7-GSH-DHR , 7 , 9-diGSH-
DHR and clivoric acid were generous gifts from
Dr.Lin Ge in Department of Pharmacology of the
Chinese University of Hong Kong.All other
chemicals were commercially available and of the
highest grade.
1.2　Preparation of rat liver microsomes
Adult female Sprague-Dawley rats (body
weight 220-250 g)were utilized in the present
study.Microsomes were prepared as described
previously
[ 8]
and suspended in 0.1 mol·L-1
potassium phosphate buffer (pH 7.4).After
determination of protein concentration[ 9] , the
suspended microsomes were frozen , and kept at
-70℃ for use.Recombinant rat CYP2C12 ,
CYP2E1 , CYP3A1 and CYP3A2 were obtained
from Gentest (Woburn , MA , USA)and stored at
-70℃ for use.
1.3　In vitro metabolism of clivorine
A typical incubation mixture consisted of 100
mmol·L-1 potassium phosphate buffer(pH 7.4),
2.0 mmol·L-1GSH , an NADPH generating sys-
tem(5 mmol·L-1MgCl2 , 10 mmol·L-1G6P , 1
mmol·L-1 NADP , 1 unit·mL-1 G6PDH), 250
mmol·L-1 clivorine and 1 g·L-1microsomal pro-
tein , in a final 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 retrorsine
(25 mg·L-1)was added as an internal standard.
Incubation mixture used for recombinant CYP450
was of the same composition as described above.
1.4　Chemical inhibition
Selective inhibitors
[ 10-12]
of rat CYP iso-
forms were added to the incubation mixtures and
incubated as previously described.Incubations
with form-selective inhibitors for each of the major
rat liver P450s at a concentration of 250 mmol·
L
-1
clivorine were performed with rat micro-
somes.Inhibitors and inhibitor concentrations for
each of the following specific forms have been
shown previously to offer selectivity for that partic-
ular form:CYP2A1 (Pil 10 μmol · L-1),
CYP2C9(Sul 30 μmol·L-1), CYP2E1(DDC 30
μmol·L-1), CYP3A(Ket 2μmol·L-1).Methi-
mazole (1 mmol·L-1)was used to inhibit micro-
somal flavin-containing monooxygenase[ 13] .
1.5 　HPLC analysis of the metabolites of
clivorine
The metabolites of clivorine in the incuba-
tions were determined by HPLC
[ 14] .The separa-
tion of the metabolites of clivorine in the incuba-
tions was performed on a PRP-1 reverse-phase
column(5 μm , 150 mm×4.1 mm , Hammilton
Co.Reno , NV)coupled with a PRP-1 guard col-
umn(5 μm , 50 mm ×4.1 mm , Hamilton Co ,
Reno , USA).The mobile phase consisted 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 35-40min ,
linear change from 75%A to 70%A.Flow rate
was kept constant at 0.8 mL·min-1 throughout
the whole course of analysis.Peak responses were
measured at 230 nm by UV detector.The resul-
tant incubates were centrifuged at 105 000×g at
2℃ for 30 min and aliquots(200 μL)of the su-
pernatant were directly injected on to the column
for quantitative analysis.
2　RESULTS
2.1　Metabolism of clivorine in female rat liv-
er microsomes
After incubation of clivorine with female rat
liver microsomes in the presence of GSH , five
metabolites were formed with the corresponding
decrease in concentration of clivorine.Direct
HPLC/MS analysis using an electrospray ioniza-
tion interface under positive and negative ionmode
resolved five drug-related compounds in the incu-
·414· ChineseJournal of Pharmacology and Toxicology　2001　Dec;15(6)
bations.The five metabolites found in the incuba-
tions are:DHR , 7-GSH-DHR , clivoric acid and
two novel metabolites with the highest mass unit at
m/ z 363(M1)and m/ z 347(M2), respectively.
In the case of in vitro metabolism of clivorine in
female rat liver microsomes , the metabolic rate of
clivorine was similar to that in male rat liver mi-
crosomes.Apparently , the metabolites M1 and M2
accounted for the conversion of clivorine to some
extent.M1 and M2 were the major metabolites of
clivorine in the female liver microsomes based on
a comparison of the apparent chromatographic in-
tensity.The results suggest that the in vitro
metabolic pathway of clivorine in female rats is
different from that in male rats.The formation of
M1 from the microsomal incubation was NADPH-
generating system-independent while the formation
of M2 was NADPH-generating system-dependent.
The M1 levels were increased in the microsomal
incubation that did not contain the NADPH-gener-
ating system.The formation of M2was significant-
ly inhibited by selective flavin-containing
monooxygenase(FMO)inhibitor(Tab 1).
2.2　Inhibition analysis
To determine the specific CYP isozyme(s)
involved in the biotransformation of clivorine in fe-
male rats , incubations were conducted using
chemical inhibitors that are specific for various
CYP isozymes.The results showed that Pil
(CYP2A1), Sul (CYP2C9), DDC (CYP2E1)
have little or no effects on the metabolism of
clivorine and the formation of pyrrolic metabolites
in female rat liver microsomes as shown in Tab 2 ,
whereas Ket caused a significant reduction of for-
mation for pyrrolic metabolites.All four pyrrolic
metabolites were not detectable in the incubation
with the corresponding increase in clivorine level.
Tab 1.　Effects of methimazole and gene rating system on the metabolism of clivorine in female rat liver
microsomes
Group
AS/AIS
M1 M2
Metabolite/μmol·L-1
DHR 7-GSH-DHR Clivoric acid
Clivorine
/mmol·L-1
Buffer - - - - - 　　 239±15
With NADPH 0.090±0.031 0.847±0.058 9.2±1.1 1.6±0.6 8.8±0.3 138±16
Without NADPH 0.350±0.059 - - - - 149±20
Methimazole 0.229±0.012 0.466±0.026 14.2±1.1 1.6±0.4 11.1±0.5 121±4
AS:peak area of sample , AIS:peak area of internal standard;(-):not detectable.DHR:dehydroretronecine;7-GSH-DHR:7-glu-
tathionyl-dehydroretronecine. x±s , n=3.
Tab 2.　Effects of selective CYP450 inhibitors on the metabolism of clivorine in the liver microsomes of female
rat
Drug
/μmol·L-1
AS/AIS
M1 M2
Metabolite/μmol·L-1
DHR 7-GSH-DHR Clivoric acid
Clivorine
/ mmol·L-1
Control 0.125±0.019 1.00±0.15 10.6±1.5 - 9.0±0.1 　 178±21
Pilocarpine 10 0.152±0.016 1.16±0.14 10.0±0.8 - 10.8±1.6 180±10
Sulfaphenazole 30 0.167±0.007 0.87±0.08 9.9±0.5 - 9.2±1.4 164±4
Diethyldithiocarbamate 30 0.163±0.007 0.98±0.15 10.4±1.0 - 9.8±0.6 173±5
Ketoconazole 2 0.146±0.011 0.98±0.07 - - - 181±9
x±s , n=3.
·415·中国药理学与毒理学杂志　　2001 年 12月;15(6)
2.3 　Metabolism of clivorine by specific
recombinant P450s
The metabolism of clivorine by recombinant
rat CYPs (CYP2C12 , CYP2E1 , CYP3A1 and
CYP3A2)was investigated .Rat CYP2C12 and
CYP2E1 showed extremely low activity for the
metabolism of clivorine and no metabolites could
be measured in the incubations of CYP2C12 and
CYP2E1 , whereas clivorine was metabolized by
CYP3A1 and CYP3A2 to its corresponding
pyrrolic metabolites as shown in Tab 3.
Tab 3.　Metabolism of clivorine by recombinant rat
CYP isoforms
Sample
Metabolite/ nmol·min-1·g-1
DHR 7-GSH-DHR Clivoric acid
Clivorine
/mmol·L-1
CYP2C12 　　- 　　- 　　- 　231±17
CYP2E1 　　- 　　- 　　- 246±8
CYP3A1 　99±5 　107±2 　364±2 135±10
CYP3A2 109±13 134±6 312±21 147±3
x±s , n=3.
3　DISCUSSION
Previous studies have found that the major
in vitro metabolic pathway of clivorine in male rat
liver microsomes was to form hepatotoxic pyrrolic
metabolites and female rats were less susceptible
to PA toxicity compared with male rats
[ 15] .The
present investigation is to study if there is signifi-
cant metabolic difference of clivorine in male and
female rats and the role of CYP450 in the forma-
tion of hepatotoxic pyrrolic metabolites.The re-
sults of our experiment showed that the formation
of pyrrolic metabolites of clivorine was markedly
inhibited by selective CYP3A inhibitor Ket , fur-
thermore recombinant CYP3A1 and CYP3A2 cat-
alyzed the metabolism of clivorine and the forma-
tion of pyrrolic metabolites.All of the above re-
sults strongly suggest rat CYP3A is involved in the
formation of hepatotoxic pyrrolic metabolites.It is
interesting to note that the metabolism of clivorine
in female rat liver microsomes was different from
that in male′s.The formation of pyrrolic metabo-
lites was much lower in female rat liver micro-
somes than in male ones and the metabolic rate of
clivorine was the similar for both male and female
rats.At the same time two novel metabolites were
found in female rat liver microsomes.The above
observation suggests that the formation of pyrrolic
metabolites is only a minor metabolic pathway in
female rats.This is partly due to the lack of
CYP3A in the female rat liver microsomes
[ 16] .
The major metabolites of clivorine in female rat
liver microsomes were two novel non-pyrrolic
metabolites M1 and M2.The selective CYP in-
hibitors have no significant effects on the formation
of the two novel metabolites either.Recombinant
CYP2C12 , CYP2E1 , CYP3A1 and CYP3A2 had
little discernible effect with regard to the two novel
metabolites.While selective FMO inhibitor methi-
mazole inhibited the formation of M2 from
clivorine , but did not inhibited the formation of
M1 .Both M1 and M2 are generated in microsomal
incubation from clivorine , but the formation of M1
is NADPH-generating system-independent while
the formation of M2 is NADPH-generating system-
dependent.The above results suggest that hydro-
lase and FMO are involved in the formation of M1
and M2 respectively.
In conclusion , rat CYP3A plays the key role
in the formation of hepatotoxic pyrrolic metabo-
lites.But the formation of pyrrolic metabolites
mediated by CYP3A is only a minor metabolic
pathway in female rat due to the lack of CYP3A in
their liver microsomes.The major metabolic path-
way of clivorine in female rat liver microsomes is
to generate two non-pyrrolic metabolites mediated
by both hydrolase and FMO.The difference in
clivorine-induced toxicity can be partly attributed
to this metabolic difference in sex.
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山冈橐吾碱在雌性大鼠肝微粒体内的代谢
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
药理学系 ,沙田 ,香港)
摘要:研究了山冈橐吾碱(clivorine)在雌性大鼠肝微
粒体内的代谢.山冈橐吾碱在雌性大鼠肝微粒体内
的主要代谢物为两个非吡咯代谢物 M1 和 M2.与雄
性大鼠不同 ,生成肝毒性的吡咯代谢物为其次要的
代谢途径.文献报道山冈橐吾碱在雄性大鼠肝微粒
体内的主要代谢方式是形成相应的吡咯代谢物.这
提示山冈橐吾碱在雌雄大鼠肝微粒体内的主要代谢
方式不同.CYP450 特异性抑制剂黄胺苯吡唑
(CYP2C), 毛果芸香碱(CYP2A1), 二乙基二硫代氨
基甲酸钠(CYP2E1)和酮康唑(CYP3A)对 M1 和 M2
的形成无明显的影响.黄素单氧化酶的特异性抑制
剂甲巯咪唑可以显著地抑制M2的形成 ,但对M1 的
形成无明显的抑制作用 ,且 M1 在肝微粒体中的形
成为NADPH 非依赖性 ,上述结果提示参与M1 和M2
代谢的酶分别为肝微粒体中的水解酶和黄素单氧化
酶.另一方面 ,毛果芸香碱 , 黄胺苯吡唑和二乙基
二硫代氨基甲酸钠对山冈橐吾碱的吡咯代谢物的形
成无明显的影响 ,而CYP3A的特异性抑制剂酮康唑
可以显著地抑制吡咯代谢物的生成 ,且山冈橐吾碱
在重组的大鼠肝 CYP2C12 , CYP2E1温孵液中无代
谢 ,而在重组的大鼠肝 CYP3A1和CYP3A2的温孵液
中山冈橐吾碱被代谢成相应的吡咯代谢物.这提示
CYP3A作为主要的 CYP450酶参与了山冈橐吾碱的
肝毒性吡咯代谢物的形成.山冈橐吾碱在雌雄大鼠
体内的毒性差异可能与其在雌雄大鼠体内的代谢存
在差异有关.
关键词:山冈橐吾碱;微粒体 , 肝;代谢
　
　　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)
·417·中国药理学与毒理学杂志　　2001 年 12月;15(6)