Human liver microsomal metabolism and DNA adduct formation of the tumorigenic pyrrolizidine alkaloid, riddelliine

Article abstract of DOI:10.1021/tx025605i

Riddelliine, a widespread naturally occurring genotoxic pyrrolizidine alkaloid, induced liver tumors in rats and mice in an NTP 2-year carcinogenicity bioassay. We have determined that riddelliine induces liver tumors in rats through a genotoxic mechanism involving the formation of (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), which reacts with DNA to form a set of eight DNA adducts. To determine the relevance to humans of the results obtained in experimental animals, the metabolism of riddelliine was conducted using human liver microsomes. As with rat liver microsomes, DHP and riddelliine N-oxide were major metabolites in incubations conducted with human liver microsomes. The levels of DHP and riddelliine N-oxide were 0.20-0.62 and 0.03-0.15 nmol/min/mg protein, respectively, which are comparable to those obtained from rat liver microsomal metabolism. When metabolism was conducted in the presence of calf thymus DNA, the same set of eight DHP-derived DNA adducts was formed. Both the metabolism pattern and DNA adduct profile were very similar to those obtained from rat liver microsomes. When metabolism was conducted in the presence of the P450 3A4 enzyme inhibitor triacetyleandomycin, the formation of DHP and riddelliine N-oxide was reduced 84 and 92%, respectively. For DHP formation, the Km values were determined to be 0.37 ± 0.05 and 0.66 ± 0.08 mM from female rats and female humans; the V_max values from female rat and human liver microsomal metabolism were 0.48 ± 0.03 and 1.70 ± 0.09 nmol/min/mg protein, respectively. These results strongly indicate the mechanistic data on liver tumor induction obtained for riddelliine in laboratory rodents is highly relevant to humans.

Full text of DOI:10.1021/tx025605i

6
6
Chem. Res. Toxicol. 2003, 16, 66-73
Hu m a n Liver Micr osom a l Meta bolism a n d DNA Ad d u ct
F or m a tion of th e Tu m or igen ic P yr r olizid in e Alk a loid ,
Rid d elliin e
†
†
†
‡
Qingsu Xia, Ming W. Chou, Fred. F. Kadlubar, Po-Cheun Chan, and
,
†
Peter P. Fu*
National Center for Toxicological Research, J efferson, Arkansas 72079, and National Institute of
environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received August 25, 2002
Riddelliine, a widespread naturally occurring genotoxic pyrrolizidine alkaloid, induced liver
tumors in rats and mice in an NTP 2-year carcinogenicity bioassay. We have determined that
riddelliine induces liver tumors in rats through a genotoxic mechanism involving the formation
of (()-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), which reacts with DNA
to form a set of eight DNA adducts. To determine the relevance to humans of the results
obtained in experimental animals, the metabolism of riddelliine was conducted using human
liver microsomes. As with rat liver microsomes, DHP and riddelliine N-oxide were major
metabolites in incubations conducted with human liver microsomes. The levels of DHP and
riddelliine N-oxide were 0.20-0.62 and 0.03-0.15 nmol/min/mg protein, respectively, which
are comparable to those obtained from rat liver microsomal metabolism. When metabolism
was conducted in the presence of calf thymus DNA, the same set of eight DHP-derived DNA
adducts was formed. Both the metabolism pattern and DNA adduct profile were very similar
to those obtained from rat liver microsomes. When metabolism was conducted in the presence
of the P450 3A4 enzyme inhibitor triacetyleandomycin, the formation of DHP and riddelliine
N-oxide was reduced 84 and 92%, respectively. For DHP formation, the K
determined to be 0.37 ( 0.05 and 0.66 ( 0.08 mM from female rats and female humans; the
max values from female rat and human liver microsomal metabolism were 0.48 ( 0.03 and
m
values were
V
1
.70 ( 0.09 nmol/min/mg protein, respectively. These results strongly indicate the mechanistic
data on liver tumor induction obtained for riddelliine in laboratory rodents is highly relevant
to humans.
In tr od u ction
N-oxides; and (iii) dehydrogenation of the necine base,
through initial hydroxylation at the C-3 or C-8 position
to form 3- or 8-hydroxynecine derivatives followed by
dehydration, to form the corresponding dehydropyrrolizi-
dine (pyrrolic) derivatives. Metabolism of pyrrolizidine
alkaloids to dehydropyrrolizidines is mainly catalyzed by
cytochromes P450, specifically both the P450 3A and
P450 2B6 enzymes (26-36). Metabolism of pyrrolizidine
alkaloids to the corresponding N-oxides is catalyzed by
both cytochrome P-450 and flavin-containing monooxy-
genase (28, 33, 37, 38).
Pyrrolizidine alkaloids are naturally occurring hetero-
cyclic compounds that are found worldwide in more than
2 plant families, among which three families, Boragi-
naceae, Compositae (Asteraceae), and Legumionsae (Fa-
baceae), contain most toxic pyrrolizidine alkaloids (1-
). More than 660 pyrrolizidine alkaloids have been
identified in over 6000 plants of these three families, and
about half of them exhibit toxic activities (7). It has been
recognized since the 18th century that many pyrrolizidine
alkaloids are highly toxic, causing numerous poisonings
in livestocks and humans (1, 2, 4, 5). Pyrrolizidine
alkaloids exhibit a variety of genotoxicities, including
DNA binding, DNA cross-linking, DNA-protein cross-
linking, mutagenicity, and carcinogenicity (1, 2, 6, 7-25).
1
7
Dehydropyrrolizidine metabolites have been shown to
be the principle metabolites responsible for genotoxicities
of the parent pyrrolizidine alkaloids (2). Because of high
instability and easy hydrolysis into (()-6,7-dihydro-7-
1
hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP), dehy-
Pyrrolizidine alkaloids require metabolic activation in
order to exert their toxicities (2). In general, there are
three principal metabolic pathways: (i) hydrolysis of the
ester functional group to form the necine bases; (ii)
oxidation of the necine bases to the corresponding necine
dropyrrolizidines cannot be detected in vivo or in vitro.
Thus, it is probable that the high genotoxicities of
1
TAO, triacetyloleandomycin (troleandomycin, oleandomycin tri-
acetate); ATP, adenosine 5′-triphosphate; MN, micrococcal nuclease;
SPD, spleen phosphodiesterase; DHP, (R/S)6,7-dihydro-7-hydroxy-1-
hydroxymethyl-5H-pyrrolizine; DHR, dehydroretronecine (or R-6,7-
dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine; or R-DHP); DHR-
3′-dGMP adducts (I and II), 3′-monophosphate of 7-(deoxyguanosin-
*
To whom correspondence should be addressed. National Center
2
for Toxicological Research, J efferson, AR 72079. Telephone: (870) 543-
N -yl)dehydrosupinidine; DHR-3′,5′-dG-bisphosphate adducts (I and
2
7
207. Fax: (870) 543-7136. E-mail: pfu@nctr.fda.gov.
II), 3′,5′-bisphosphate of 7-(deoxyguanosin-N -yl)dehydrosupinidine;
†
National Center for Toxicological Research.
PNK, cloned T4 polynucleotide kinase; NTP, National Toxicology
Program; NCTR, National Center for Toxicological Research.
‡
National Institute of environmental Health Sciences.
1
0.1021/tx025605i This article not subject to U.S. Copyright. Published 2003 by the American Chemical Society
Published on Web 12/10/2002

Metabolism and DNA Adduct Formation of Riddelliine
Chem. Res. Toxicol., Vol. 16, No. 1, 2003 67
dehydropyrrolizidines in vivo are exerted through a
combination of dehydropyrrolizidines and DHP (10).
from trauma victims or patients with metastatic disease. Female
and male F344 rats were obtained from the National Center
for Toxicological Center (NCTR) breeding colony as weanlings.
Female and male rat liver microsomes were prepared by
combining liver tissues of five female rats and six male F344
rats, respectively.
P r ep a r a tion of Hu m a n a n d Ra t Liver Micr osom es.
Human liver microsomes were prepared and stored as previ-
ously described (41). Protein concentrations were determined
using a Bio-Rad protein detection kit (Bio-Rad Laboratories,
Hercules, CA) based on the Bradford method (42). For compari-
son, liver microsomes of female and male F344 rats were
similarly prepared.
Develop m en t of Op tim a l Con d ition s for Meta bolism of
Rid d elliin e by Ra t a n d Hu m a n Liver Micr osom es. Incuba-
tion conditions for rat and human liver microsomal metabolism
were optimized to ensure that product formation (DHP or
riddelliine N-oxide) was linear with respect to time of incubation
and microsomal protein concentration. Microsomal protein
concentrations (0.1-2.0 mg/mL of incubation volume), substrate
(riddelliine) concentrations (0.05-2.0 mM), and incubation times
(5-60 min) were employed for determining optimal incubation
conditions.
Riddelliine, a representative genotoxic pyrrolizidine
alkaloid, produced in plants growing in range lands of
the western United States (2, 23, 24). Riddelliine exhibits
both acute toxicity and a large spectrum of genotoxic
activities, including tumorigenicity. Human foodstuffs,
such as grains, herbs, milk, honey, herbal teas, and
herbal medicine, may be contaminated by pyrrolizidine
alkaloids including riddelliine (2, 6). Due to its genotox-
icity and potential for human exposure, riddelliine was
nominated by the U.S. Food and Drug Administration
to the National Toxicology Program (NTP) for genotox-
icity and carcinogenicity testing (23, 24). In the NTP
2
-year carcinogenicity bioassay, riddelliine induced liver
tumors in laboratory male and female rats and male mice
24). As part of the NTP study, we investigated the
(
mechanism by which riddelliine induces liver tumors in
laboratory rats and mice (39) and found that metabolism
of riddelliine by liver microsomes of F344 female rats
generated DHP as a reactive metabolite (6, 39). A set of
eight DHP-derived DNA adducts was detected and
quantified in liver of rats fed riddelliine (39). Two of these
adducts were identified as DHP-derived 7-deoxygua-
Met a bolism of R id d elliin e by R a t a n d Hu m a n Liver
Micr osom es. The metabolism of riddelliine by rat and human
liver microsomes of both sexes was conducted in a 1.0 mL
2
incubation volume containing 100 mM sodium phosphate buffer
nosin-N -yl epimers (DHP-3′-dGMP), and the other six
+
(
pH 7.6), 5 mM magnesium chloride, 1 mM NADP , 8 mM
were DHP-derived dinucleotide adducts (39, 40). It was
also shown that the levels of DNA adduct formation
correlated with liver tumor potency (24, 39). Our results
suggest that riddelliine induces liver tumors in laboratory
male and female rats and male mice through a genotoxic
mechanism and the eight DHP-derived DNA adducts are
likely to contribute to the liver tumor development (24).
glucose 6-phosphate, 2 units glucose 6-phosphate dehydroge-
nase, 0.1 mM riddelliine, and 0.5 mg of microsomal protein at
3
7 °C for 30 min. After incubation, the incubation mixture was
centrifuged at 105000g for 30 min at 4 °C to remove microsomal
protein. The supernatant was collected and the metabolite
mixture was separated by reversed-phase HPLC employing a
Prodigy 5 µm ODS column (10 × 250 mm, Phenomenex,
Torrance, CA) eluted isocratically with 20 mM ammonium
acetate buffer (pH 7) at 5 mL/min for 10 min followed with a
linear gradient from 20 mM ammonium acetate buffer (pH 7.0)
to 50% methanol in the buffer for 30 min.
For obtaining kinetic parameters of riddelliine metabolism
by rat and human liver microsomes, incubations were similarly
conducted as described above, with the exception that 1.0 mg
of microsomal protein/mL and different substrate concentrations
To determine whether the mechanistic studies in
experimental animals are relevant to humans, in this
paper we report the results of metabolism and DNA
adduct formation of riddelliine by male and female
human liver microsomes and compare the results with
those from rat liver microsomal metabolism.
(
from 0.1 to 1.6 mM) were used. After incubations, each of the
Ma ter ia ls a n d Meth od s
incubation mixtures was centrifuged and the resulting metabo-
lite mixture was separated by reversed-phase HPLC as de-
scribed above. Values of Vmax and K
incubation results were determined by GraphPad Prism, Graph-
Pad Software, Inc. (San Diego, CA).
Meta bolism of Rid d elliin e by Hu m a n Liver Micr osom es
in th e P r esen ce of P 450 3A4 En zym e In h ibitor . The
metabolism of riddelliine by human liver microsomes of both
sexes in the presence of the enzyme inhibitor, TAO, was
similarly conducted as described above. First, 0.5 mL of 100 mM
sodium phosphate buffer (pH 7.6), containing 5 mM magnesium
Ma ter ia ls. Riddelliine was obtained from the NTP (23, 24).
DHR and riddelliine N-oxide were prepared following published
procedures (39). Troleandomycin (triacetyloleandomycin, TAO),
phenobarbital (sodium salt), calf thymus DNA (sodium salt, type
I), 2′-deoxyguanosine-3′-monophosphate (sodium salt) (3′-
dGMP), glucose-6-phosphate, glucose-6-phosphate dehydroge-
m
of each microsomal
+
nase, nicotinamide adenine dinucleotide phosphate (NADP ),
nuclease P1, micrococcal nuclease (MN), spleen phospho-
diesterase (SPD), bicine, spermidine, and dithiothreitol were
purchased from the Sigma Chemical Co. (St. Louis, MO). Cloned
T4 polynucleotide kinase (PNK) was obtained from U.S. Bio-
+
chloride, 1 mM NADP , 8 mM glucose 6-phosphate, 1 unit
3
2
chemical Corp. (Cleveland, OH). [γ P]Adenosine 5′-triphosphate
glucose 6-phosphate dehydrogenase, and 0.5 mg microsomal
protein was incubated with 20 nmol of TAO (in DMSO) at 37
°C for 30 min, control incubations contained DMSO. After
incubation, 0.5 mL of 100 mM sodium phosphate buffer (pH 7.6),
3
2
(
[γ- P]ATP) (sp. act. >7000 Ci/mmol) was purchased from ICN
Biomedicals, Inc. (Costa Mesa, CA). All other reagents were
obtained through commercial sources and were the highest
+
quality available. All solvents used were of HPLC grade.
containing 5 mM magnesium chloride, 1 mM NADP , 8 mM
2
3
′-Monophosphate of 7-(deoxyguanosin-N -yl)dehydrosupinidine
glucose 6-phosphate, 1 units glucose 6-phosphate dehydroge-
nase, and 0.5 µmol of riddelliine was added into above inhibition
reaction mixture, and incubated at 37 °C for another 30 min.
Meta bolism of Rid d elliin e by Hu m a n Liver Micr osom es
in th e P r esen ce of Ca lf Th ym u s DNA. The metabolism of
riddelliine by human liver microsomes of both sexes was
conducted in a 1.0 mL incubation volume containing 100 mM
sodium phosphate buffer (pH 7.6), 5 mM magnesium chloride,
adducts (DHR-3′-dGMP) were prepared in our laboratory (39).
DNA from reaction of DHR with calf thymus DNA was prepared
as previously described (39).
Human livers were obtained from organ donor samples. Four
male (no. 4886, 80 years old Caucasian; no. 9504, 62 years old
Caucasian; no. 9603, 57 yeas old Caucasian; and no. 9310, 50
years old white) and four female (no. 5807, 69 years old white;
no. C055G, 52 years old Caucasian; no. 9603, 50 years old white,
and no. 9502, 30 years old African American) liver samples were
used for study. All these human liver samples were obtained
+
1 mM NADP , 8 mM glucose 6-phosphate, 2 units of glucose
6-phosphate dehydrogenase, 0.1 mM riddelliine, 1.0 mg of
purified calf thymus DNA, and 0.5 mg of microsomal protein at

6
8
Chem. Res. Toxicol., Vol. 16, No. 1, 2003
Xia et al.
3
7 °C for 30 min. After incubation, the reaction was terminated
by cooling with ice-water, then sequentially extracted with 1.0
mL of phenol, 1.0 mL of phenol/chloroform/isoamyl alcohol (v/
v/v, 25/24/1), and 1.0 mL of chloroform/isoamyl alcohol (v/v, 24/
1
0
). The DNA in the aqueous phase was precipitated by adding
.1 mL of 5 M sodium chloride followed by equal volume of cold
ethanol and washed three times with 70% ethanol. After
redissolving in 300 µL of distilled water, the DNA concentration
and purity were analyzed spectrophotometrically, and DNA was
3
2
stored at -78 °C prior to P-postlabeling/HPLC analysis.
3
2
P -P ostla belin g/HP LC An a lysis of DHP -Der ived DNA
Ad d u cts. The DHP-derived DNA adducts formed from metabo-
lism of riddelliine by human or rat liver microsomes in the
presence of calf thymus DNA were analyzed employing the 32P-
postlabeling/HPLC methodology previously developed in our
laboratories (29, 43). Ten micrograms of the DNA dissolved in
1
0 µL of distilled water was enzymatically hydrolyzed at 37 °C
for 20 min with 78 milliunits (mU) of MN and 4 mU of SPD
contained in a 20 µL solution of 20 mM sodium succinate and
1
0 mM of calcium chloride (pH 6). The MN/SPD digested DNA
solutions were incubated with nuclease P1 (8 µg, in 4 µL of
buffer containing 0.24 M sodium acetate and 2 mM ZnCl at
2
pH 5) at 37 °C for 20 min to remove normal 3′-monophosphate
of 2′-deoxyribonucleosides. The resulting incubation mixture was
then evaporated under reduced pressure, redissolved in 10 µL
of distilled water and incubated with 10 µL of PNK mixture
3
2
F igu r e 1. Reversed-phase HPLC analysis of riddelliine me-
tabolites formed from metabolism of riddelliine by (A) female
rat liver microsomes, (B) male rat liver microsomes, (C) female
human liver microsomes, and (D) male human liver microsomes.
HPLC analysis was conducted on a Prodigy 5 µm ODS column
(10 × 250 mm, Phenomenex) eluted isocratically with 20 mM
ammonium acetate buffer (pH 7) at 5 mL/min for 10 min
followed with a linear gradient from 20 mM ammonium acetate
buffer (pH 7.0) to 50% methanol in the buffer for 30 min.
containing 100 µCi of [γ- P]ATP (sp. act., 7000 Ci/mmol), 12
units of PNK, and 2 µL of 10 × PNK buffer (200 mM bicine-
2
NaOH, pH 9.6, 100 mM dithiothreitol, 100 mM MgCl , and 10
mM spermidine) at 37 °C for 40 min. The 32P labeled mixture
was injected onto a Prodigy 5 µ ODS column (Phenomenex, 4.6
×
250 mm) and eluted isocratically with 20 mM sodium
phosphate buffer (pH 4.5) for 10 min, followed by a 60-min linear
gradient of 20 mM sodium phosphate buffer (pH 4.5) to 15%
methanol in 20 mM sodium phosphate buffer. The HPLC flow
rate was 1.0 mL/min and the scintillation fluid flow rate was
substrate, riddelliine. On the basis of comparison of their
HPLC retention times and UV-vis absorption spectra
with those of synthetic standards and the metabolites of
previous study (39), the materials contained in the
chromatographic peaks eluting at 25.4 and 30.1 min were
identified as DHP and riddelliine N-oxide, respectively.
The HPLC profiles obtained from metabolism of riddel-
liine by male rat liver microsomes (Figure 1B), female
human liver microsomes (F3) (Figure 1C), and male
human liver microsomes (M2) (Figure 1D) were similarly
analyzed. As shown in Figure 1, metabolism of riddelliine
by male and female rat and human liver microsomes
produced similar metabolism pattern. The chromato-
graphic peaks eluted prior to 22 min were also found from
incubation in the absence of riddelliine and, therefore,
are presumably from microsomal proteins.
3
.0 mL/min. To avoid interference by the high radioactivity of
3
2
32
the free P and the unreacted [γ- P]ATP, the on-line FLO-ONE
radioactivity detector (Radiomatic Instruments, Tampa, FL) was
equipped with a diverter, and the eluent from the first 40 min
was diverted from the radioactivity detector.
In str u m en ta tion . A Waters HPLC system consisting of a
Model 600 controller, a Model 996 photodiode array detector,
and pump was used for the separation and purification of DHP-
derived DNA adducts. For radiochromatography analysis of the
3
2
P-postlabeled reaction mixtures, an HPLC system with on-
line detection of both radioactivity and UV absorbance was
conducted consisting of a solvent gradient programer (Waters
Model 680), two HPLC pumps (Waters 510), a UV detector
(Waters Model 440), a radio-chromatography detector (Hewlett-
Packard FLO-ONE /Beta A-500) equipped with a diverter and
an autosampler (Waters 717).
In this study, metabolism of riddelliine by four male
samples M1-M4) and four female (samples F1-F4)
Resu lts
(
Ra t a n d Hu m a n Liver Micr osom a l Meta bolism of
Rid d elliin e. To compare riddelliine metabolism profiles
between human and rat liver microsomal metabolism,
optical incubation conditions were determined by incuba-
tions with different concentrations of microsomal protein
human liver microsomes was conducted, and all the
resulting metabolism patterns were similar. The rates
of metabolite formation from all 10 rat and human liver
microsomal incubations were determined. While the rat
data are based on triplicate results, due to limit amount
of human tissues available, human data are based on a
single experiment (Table 1). The levels of DHP formation
from female and male human liver microsomal metabo-
lism were within a range of 0.20-0.62 nmol/min/mg
protein. These levels are similar to or higher than those
from male and female rat liver microsomal metabolism.
The levels of riddelliine N-oxide formed from human liver
microsomal incubations were in the range of 0.03-0.15
nmol/min/mg protein, which are similar to those from
female rat liver microsomal metabolism, but significantly
lower than those from male rat liver microsomal me-
(
(
0.1-2.0 mg/mL of incubation volume) and substrate
riddelliine) (0.05-2.0 mM), and with different incubation
times (5-60 min). Under the established conditions,
product formation (DHP and riddelliine N-oxide) was
linear with respect to time of incubation and microsomal
protein concentration.
After incubation, the metabolism products were sepa-
rated by reversed-phase HPLC. Figure 1A shows the
reversed-phase HPLC profile of riddelliine metabolized
by female rat liver microsomes. The chromatographic
peak eluting at 35.7-39.2 min contained the recovered

Metabolism and DNA Adduct Formation of Riddelliine
Chem. Res. Toxicol., Vol. 16, No. 1, 2003 69
Ta ble 1. Ra tes of Meta bolic F or m a tion of DHP a n d Red d elliin e N-Oxid e fr om Meta bolism of Rid d elliin e by Liver
Micr osom es of Ra t a n d Hu m a n
rate of formation (nmol/min/mg protein)
male rata
MR
female rata
FR
male humanb
female humanb
metabolite
DHP
M1
M2
M3
M4
F1
F2
F3
F4
0.21 ( 0.01
0.48 ( 0.02
0.14 ( 0.02
0.06 ( 0.01
0.31
0.06
0.62
0.15
0.49
0.11
0.32
0.05
0.29
0.06
0.20
0.03
0.27
0.05
0.50
0.12
riddelliine N-oxide
a
Data based on triplicate results and expressed as means ( SD. b Data based on a single experiment due to the limited amount of
human tissues available.
Ta ble 2. In h ibition Effect of Tr ia cetylolea n d om ycin (TAO) of P 450 3A4 on Rid d elliin e Meta bolism by Hu m a n Liver
Micr osom es
DHP
riddelliine N-oxide
liver
microsomes
P450 3A4
inhibitor
nmol/min/mg
protein
percentage of
inhibition (%)
nmol/min/mg
percentage of
inhibition (%)
protein
male (M2)
1.50 ( 0.03
0.23 ( 0.02
1.00 ( 0.03
0.16 ( 0.01
0.36 ( 0.02
0.03 ( 0.004
0.25 ( 0.01
0.02 ( 0.002
TAO
TAO
85
84
92
92
female (F4)
Ta ble 3. En zym e Kin etic P a r a m eter s of Rid d elliin e Oxid a tive Meta bolism in Ra t a n d Hu m a n Liver Micr osom esa
kinetic parameters
Vmax (nmol/min/mg protein)
Km (mM)
riddelliine
riddelliine
liver microsomes
DHP
N-oxide
DHP
N-oxide
b
female rat (FR)
0.48 ( 0.03
1.12 ( 0.04
1.70 ( 0.09
0.95 ( 0.02
0.30 ( 0.01
2.17 ( 0.08
0.43 ( 0.03
0.26 ( 0.01
0.37 ( 0.05
0.28 ( 0.03
0.66 ( 0.08
0.24 ( 0.02
0.44 ( 0.04
0.25 ( 0.03
0.71 ( 0.12
0.44 ( 0.06
c
male rat (MR)
human, female
human, male (M4)
d
a
Kinetic parameters, represented as means ( SD (n ) 3), were determined by employing GraphPad Prism software. b Liver microsomes
c
were prepared by combining liver tissues of six female rats. Liver microsomes were prepared by combining liver tissues of five male
rats. Combining equal amount of liver microsomal protein from the four female human liver microsomes samples (F1, F2, F3, and F4).
d
tabolism. Male rat liver microsomal metabolism is the
only example where the rate of riddelliine N-oxide
formation is higher than that of DHP (Table 1).
The rates of formation of DHP vs riddelliine N-oxide
from human liver microsomal metabolism were com-
pared. A linear relationship exists between the rates of
DHP formation and the rates of riddelliine N-oxide
formation from the four female human liver microsomal
2
metabolism, with a correlation coefficient of r > 0.99. A
similar relationship was obtained from male human liver
2
microsomal metabolism (r > 0.99). A linear relationship
2
(
r ) 0.99) was also obtained on the rates of formation
from all the eight male and female human liver microso-
mal metabolism.
Meta bolism of Rid d elliin e by Hu m a n Liver Mi-
cr osom es in th e P r esen ce of P 450 3A4 In h ibitor
Tr ia cetylea n d om ycin (TAO). To determine whether
P450 3A4 is the principal metabolizing enzyme that
catalyzes metabolism of riddelliine to DHP and riddel-
liine N-oxide, metabolism of riddelliine by male (M2) and
female (F4) human liver microsomes was conducted in
the presence TAO, a specific P450 3A4 inhibitor. As
shown in Table 2 and Figure 2, the DHP metabolite
formed from the male and the female human microsomal
metabolism was 85 and 84% reduced, respectively, and
formation of riddelliine N-oxide was 92% inhibited in both
male and female human microsomal metablism.
Kin etic P a r a m eter s of Rid d elliin e Meta bolism by
Ra t a n d Hu m a n Liver Micr osom es. Kinetic param-
F igu r e 2. Inhibition of human liver microsomal metabolism
of riddelliine by the P450 3A4 inhibitor TAO. Male human liver
microsomes M2 and female human liver microsomes F4 were
used for this study. The HPLC analysis of the resulting DHP
and riddelliine N-oxide metabolites was conducted under condi-
tions as those in the legend of Figure 1.
rats, (iii) combining equal amount of liver microsomes
from the four female human liver samples (F1, F2, F3,
and F4), and (iv) one male human (M4) liver microsomes.
The Vmax and K
m
values from male and female rat and
human liver microsomal metabolism are summarized in
Table 3. As an illustration, the substrate concentration-
dependent initial velocity of DHP and riddelliine N-oxide
formation from female rat liver microsomal metabolism
is shown in Figure 3A. The Lineweaver-Burke double-
reciprocal plots for DHP and riddelliine N-oxide are
shown in Figure 3, panels B and C, respectively. These
data indicate that DHP was formed at a higher Vmax rate
m
eters (Vmax and K ) of metabolism of riddelliine were
determined. The determinations include those from
metabolism with liver microsomes prepared by (i) pooling
livers of six female rats, (ii) pooling livers of five male

7
0
Chem. Res. Toxicol., Vol. 16, No. 1, 2003
Xia et al.
F igu r e 4. 32P-Postlabeling/HPLC analysis of DHP-derived
DNA adducts formed from metabolism of riddelliine by liver
microsomes of male rat liver microsomes, female rat liver
microsomes, four individual male human liver microsomes, and
four individual female human liver microsomes in the presence
of calf thymus DNA. HPLC analysis was conducted on a Prodigy
5
µm ODS column (Phenomenex, 4.6 × 250 mm) and eluted
isocratically with 20 mM sodium phosphate buffer (pH 4.5) for
0 min, followed by a 60-min linear gradient of 20 mM sodium
1
phosphate buffer (pH 4.5) to 15% methanol in 20 mM sodium
phosphate buffer. The HPLC flow rate was 1.0 mL/min, and the
scintillation fluid flow rate was 3.0 mL/min. To avoid interfer-
3
2
ence by the high radioactivity of the free P and the unreacted
3
2
[
γ- P]ATP, the on-line FLO-ONE radioactivity detector was
equipped with a diverter, and the eluent from the first 40 min
was diverted from the radioactivity detector.
H u m a n Liver Micr osom a l-Med ia t ed DH P -De-
r ived DNA Ad d u ct F or m a tion . To determine the
relevance of our mechanistic study on rodents to humans,
DHP-derived DNA adduct formation mediated by human
liver microsomal metabolism of riddelliine was studied,
and the results were compared with those from female
F344 rat liver microsomal metabolism in the presence
of calf thymus DNA. Incubation conditions are similar
to those described above for metabolism of riddelliine by
rat and human liver microsomes. The levels of DHP-
derived DNA adducts formed by female and male rat and
human liver microsomal metabolism were analyzed by
F igu r e 3. Female rat liver microsomal metabolism of riddel-
liine, (A) the substrate concentration-dependent initial velocity
of DHP and riddelliine N-oxide formation, and the Lineweaver-
Burke double-reciprocal plots for (B) DHP and (C) riddelliine
N-oxide.
3
2
P-postlabeling/HPLC.
As previously determined (39, 40), reaction of DHR and
than riddelliine N-oxide. The results of female rat and
human liver microsomal metabolism are compared. The
calf thymus DNA resulted in a set of eight DHP-derived
DNA adducts (Figure 4A). These eight DHP-derived DNA
adducts contained in the chromatographic peaks eluted
at 47.6, 48.3, 51.4, 53.9, 55.3, 60.1, 61.0, and 62.6 min
are designated as P1, P2, P3, P4, P5, P6, P7, and P8,
respectively. The DNA adducts designated as P4 and P6
are DHP-3′-dGMP adducts, and the other six DHP-
derived adducts (P1, P2, P3, P5, P7, and P8) were
characterized as DHP-derived dinucleotides (40). Me-
tabolism of riddelliine by female and male rat and human
K
m
values of the DHP formation are 0.37 ( 0.05 and 0.66
(
0.08 mM from female rats and female humans,
respectively (Table 3). The Vmax values of the female rat
and human liver microsomal metabolism are 0.48 ( 0.03
and 1.70 ( 0.09 nmol/min/mg protein, respectively. It is
necessary to note that because the metabolism is via
multienzyme complex (microsomes rather than purified
enzyme), the kinetics parameters addressed are apparent
rather than absolute.

Metabolism and DNA Adduct Formation of Riddelliine
Chem. Res. Toxicol., Vol. 16, No. 1, 2003 71
F igu r e 5. Formation of total DHP-derived DNA adducts from
male F344 rat liver microsomes (MR), female F344 rat liver
microsomes (FR), male human liver microsomes (M1, M2, M3,
and M4), and female human liver microsomes (F1, F2, F3, and
F4) in the presence of calf thymus DNA. Data represent the
mean ( SD (n ) 3).
liver microsomes in the presence of calf thymus DNA also
produced the same set of eight DHP-derived DNA ad-
ducts. The HPLC profiles resulting from liver microsomal
metabolism of female rats, male rats, female human (F2),
and male human (M4) are shown in Figure 4, panels B
and E. The levels of the total DHP-derived DNA adducts
formed from all the 10 male and female rat and human
liver microsomes are shown in Figure 5. These results
clearly indicate that the same set of eight DHP-derived
DNA adducts was formed in all the cases. Furthermore,
the quantities of DNA adducts formed from human
microsomal metabolism are either similar to or 2-3-fold
higher than those from rat liver microsomal metabolism.
F igu r e 6. Proposed metabolic activation of riddelliine leading
to DHP-derived DNA adduct formation in rodent and human
liver microsomal metabolism.
enzyme first provides 8-hydroxyriddelliine and/or 3-hy-
droxyriddelliine as the primary metabolites, which upon
enzymatic dehydration produced dehydroriddelliine (Fig-
ure 6). As previously discussed, there are two possible
pathways that lead to DHP-derived DNA adducts. The
first pathway is the covalent binding of dehydroriddel-
liine to cellular DNA followed by hydrolysis, either
enzymatically or chemically, to the eight DHP-derived
DNA adducts (Figure 6). The second pathway is that
dehydroriddelliine is hydrolyzed, catalyzed by esterases
and/or other hepatic enzymes, to form DHP, which
subsequently binds to DNA. To date, it is not known
which pathway is predominant in DHP-derived DNA
adduct formation. It warrants further investigations.
Our finding that the formation of DHP and riddelliine
N-oxide from riddelliine are principally catalyzed by the
P450 3A4 enzyme is consistent with that published by
Miranda et al. (28) that P450 3A4 is the major metaboliz-
ing enzyme catalyzing the metabolism of senecionine, a
tumorigenic pyrrolizidine alkaloid, to DHP and senecion-
ine N-oxide in male and female human liver microsomes.
However, the metabolizing enzymes in catalyzing these
human metabolism are different from those in laboratory
animals. For example, it has been shown that P450 2B
was involved in the metabolism of a number of pyrrolizi-
dine alkaloids to the DHP metabolite in guinea pig,
hamsters, and sheep (33, 35, 37). It has also been
determined that flavin-containing monooxygenase is
involved in the conversion of pyrrolizidine alkaloids to
the corresponding N-oxides (2, 38).
Although a number of pyrrolizidine alkaloids have been
found to induce tumors in laboratory animals (3, 4, 6,
39), the mechanisms of these compounds leading to
tumorigenicity are not clear. Our previous study on
riddelliine provides the first established genotoxic mech-
anism by which a pyrrolizidine alkaloid (riddelliine)
induces liver tumors mediated by the formation of eight
DHP-derived DNA adducts. We anticipate that this
activation pathway may be general and the eight DHP-
derived DNA adducts may be formed in vivo and in vitro
when exposure occurs to other genotoxic pyrrolizidine
alkaloids. Since this present study has shown that the
same genotoxic mechanism of metabolic activation of
riddelliine exists in humans, further investigations are
Discu ssion
In this study, we report that riddelliine is metabolized
by male and female human liver microsomes to form
DHP which leads to the formation of a set of eight DHP-
derived DNA adducts. The metabolism pattern and DNA
adduct profiles from human liver microsomes are very
similar to those formed in rat liver in vitro and in vivo
m
(39). The kinetic parameters, Vmax and K , from human
liver microsomal metabolism are also comparable to those
from rat liver microsomal metabolism. Taken together,
these results strongly indicate that our previous in vivo
and in vitro mechanistic studies with laboratory rodents
(39) are highly relevant to humans.
Since riddelliine induces liver tumors in male and
female rats and male mice (24) and the DHP-derived
DNA adducts are responsible for liver tumor induction,
the results in the present study suggest that riddelliine
can be highly genotoxic to humans and the genotoxic
mechanism is mediated by the eight DHP-derived DNA
adducts. The proposed metabolic activation of riddelliine
leading to genotoxicity mediated by DNA adduct forma-
tion is shown in Figure 6.
The metabolism study in the presence of TAO, a
human P450 3A4 inhibitor, strongly suggests that the
formations of DHP and riddelliine N-oxide from metabo-
lism of riddelliine are both principally catalyzed by the
P450 3A4 enzyme or by two or more closely related
enzymes which under a combined condition produce
similar results. It has been reported that hydroxylation
of pyrrolizidine alkaloids at the necine base, particularly
at the C-8 and C-3 positions to form 8- and 3-hydroxyne-
cine derivatives, followed by dehydration to form the
corresponding dehydropyrrolizidine (pyrrolic) derivatives,
is a general metabolism pathway (2). Thus, we propose
that metabolism of riddelliine catalyzed by P450 3A4

7
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Chem. Res. Toxicol., Vol. 16, No. 1, 2003
Xia et al.
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Ack n ow led gm en t. We thank Dr. Paul Howard for
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research was supported in part by an Interagency Agree-
ment 224-93-0001 between the Food and Drug Admin-
istration/National Center for Toxicological Research (FDA/
NCTR) and the National Institute for Environmental
Health Sciences/National Toxicology Program (NIEHS/
NTP). Through this agreement, this research was sup-
ported by appointment (Q.X.) to the Postgraduate Re-
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Ridge Institute for Science and Education through an
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