Reaction of dehydropyrrolizidine alkaloids with valine and hemoglobin

Article abstract of DOI:10.1021/tx5002139

Pyrrolizidine alkaloid-containing plants are probably the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids exert toxicity through metabolism to dehydropyrrolizidine alkaloids that bind to cellular protein and

Full text of DOI:10.1021/tx5002139

Article
pubs.acs.org/crt
Reaction of Dehydropyrrolizidine Alkaloids with Valine and
Hemoglobin
Yuewei Zhao, Shuguang Wang, Qingsu Xia, Gonca̧
lo Gamboa da Costa, Daniel R. Doerge,†
†
,§
†,§
†
†
‡
,†
Lining Cai, and Peter P. Fu*
†National Center for Toxicological Research, Jefferson, Arkansas 72079, United States
‡Biotranex LLC, Monmouth Junction, New Jersey 08852, United States
*
S Supporting Information
ABSTRACT: Pyrrolizidine alkaloid-containing plants are
probably the most common poisonous plants affecting
livestock, wildlife, and humans. Pyrrolizidine alkaloids exert
toxicity through metabolism to dehydropyrrolizidine alkaloids
that bind to cellular protein and DNA, leading to
hepatotoxicity, genotoxicity, and tumorigenicity. To date, it
is not clear how dehydropyrrolizidine alkaloids bind to cellular
constituents, including amino acids and proteins, resulting in
toxicity. Metabolism of carcinogenic monocrotaline, riddel-
liine, and heliotrine produces dehydromonocrotaline, dehyroriddelliine, and dehydroheliotrine, respectively, as primary reactive
metabolites. In this study, we report that reaction of dehydromonocrotaline with valine generated four highly unstable 6,7-
dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived valine (DHP−valine) adducts. For structural elucidation,
DHP−valine adducts were derivatized with phenyl isothiocyanate (PITC) to DHP−valine−PITC products. After HPLC
1
1
1
separation, their structures were characterized by mass spectrometry, UV−visible spectrophotometry, H NMR, and H− H
COSY NMR spectral analysis. Two DHP−valine−PITC adducts, designated as DHP−valine−PITC-1 and DHP−valine−PITC-
3, had the amino group of valine linked to the C7 position of the necine base, and the other two DHP−valine−PITC products,
DHP−valine−PITC-2 and DHP−valine−PITC-4, linked to the C9 position of the necine base. DHP−valine−PITC-1 was
interconvertible with DHP−valine−PITC-3, and DHP−valine−PITC-2 was interconvertible with DHP−valine−PITC-4.
Reaction of dehydroriddelliine and dehydroheliotrine with valine provided similar results. However, reaction of valine and
dehydroretronecine (DHR) under similar experimental conditions did not produce DHP−valine adducts. Reaction of
dehydromonocrotaline with rat hemoglobin followed by derivatization with PITC also generated the same four DHP−valine−
PITC adducts. This represents the first full structural elucidation of protein conjugated pyrrolic adducts formed from reaction of
dehydropyrrolizidine alkaloids with an amino acid (valine). In addition, it was found that DHP−valine-2 and DHP−valine-4,
with the valine amino group linked at the C7 position of the necine base, can lose the valine moiety to form DHP.
INTRODUCTION
Metabolism of toxic pyrrolizidine alkaloids generates
dehydropyrrolizidine alkaloids as the primary pyrrolic metab-
olites that react with cellular protein and DNA in vivo to exert
toxicity, including hepatotoxicity, mutagenicity, and tumor-
■
Pyrrolizidine alkaloids and their N-oxide derivatives are
common phytochemical constituents of hundreds of plant
species of different botanical families widely distributed in many
geographical regions of the world.
pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides have
been identified in over 6000 plants, and about half of them
exhibit hepatotoxic activity.
about 3% of the world’s flowering plants contain toxic
pyrrolizidine alkaloids. Humans are exposed to toxic
pyrrolizidine alkaloids through staple foods, food contaminants,
herbal teas, herbal medicines, and dietary supplements.
As early as 1920, it was reported that people in South Africa
who ingested the pyrrolizidine alkaloid-containing plant Senecio
ilicifolius were poisoned. In 1954, retrorsine, a pyrrolizidine
alkaloid, was determined to induce liver tumors in rats. To
date, it is believed that pyrrolizidine alkaloid-containing plants
are possibly the most common poisonous plants affecting
4
,6,7,18−22
1
−11
igenicity (Figure 1).
Dehydropyrrolizidine alkaloids are highly unstable, with half-
To date, at least 660
23
lives about 0.3−5.1 s in aqueous medium, facilely hydrolyzed
to the necine base, (±)-6,7-dihydro-7-hydroxy-1-hydroxymeth-
yl-5H-pyrrolizine (DHP), and easily dimerized and polymer-
1
,7−9
It has been determined that
7
1
1
ized. They also react with cellular proteins, DNA, and
4
,7,24
glutathione (GSH) to form protein−DHP,
DNA−DHP
4
,25−28
4,7,29
4
,7,12−16
adducts,
and GSH−DHP adducts.
Similar to the
primary dehydropyrrolizidine alkaloid metabolites, all secon-
dary pyrrolic metabolites (protein−DHP, DNA−DHP, GSH−
DHP, and DHP) containing a necine (DHP) moiety are also
very unstable in aqueous medium, particularly under acidic
7
17
Received: May 28, 2014
Published: September 2, 2014
4
,7
livestock, wildlife, and humans.
©
2014 American Chemical Society
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biomarker of pyrrolizidine alkaloid-induced liver tumor
36
formation.
Although a role for DHP−protein adducts formed in vivo in
the liver has been proposed for pyrrolizidine alkaloid-induced
4
,7,37,38
hepatotoxicity,
the structures of these protein-DHP
adducts have not been characterized and it is not clear how
these protein−DHP adducts can affect hepatotoxicity. Our
success in determining the structures of DHP−DNA adducts in
vivo suggested that the structures of protein−DHP adducts
could also be characterized. Following our strategy on the
structural determination of DHP−dG and DHP−dA adducts,
we determined the products formed from the reaction of valine
with dehydroriddelliine, dehydromonocrotaline, and dehydro-
heliotrine, which are metabolites of monocrotaline, riddelliine,
and heliotrine, respectively (Figure 2). Both monocrotaline and
riddelliine are retronecine-type pyrrolizidine alkaloids, helio-
trine is a heliotridine-type tumorigenic pyrrolizidine alkaloid,
and all are among the most studied pyrrolizidine alkaloids
7
regarding cytotoxicity, genotoxicity, and tumorigenicity. Va-
line, an aliphatic branched-chain amino acid involved in the
promotion of cell normal growth, tissue repair, and providing
the body with energy, is a neutral and extremely hydrophobic
essential amino acid. In this study, we report the character-
ization of the main products derived from the in vitro reaction
of valine with dehydroriddelliine, dehydromonocrotaline, and
dehydroheliotrine and the formation of these adducts in the N-
terminal valine of hemoglobin of rats treated with dehydro-
monocrotaline.
Figure 1. Metabolic activation of pyrrolizidine alkaloids leading to
toxicity.
conditions. Consequently, mechanistic studies of pyrrolizidine
alkaloid-induced hepatotoxicity and tumorigenicity are difficult
to conduct at the molecular level. We recently determined that
riddelliine, a tumorigenic pyrrolizidiene alkaloid, induces liver
tumors through a DNA adduct-mediated genotoxic mecha-
EXPERIMENTAL PROCEDURES
■
Caution: These chemicals are dangerous. Monocrotaline, riddelliine,
heliotrine, dehydrmonocrotaline, dehydroriddelliine, dehydroheliotrine, and
DHR are carcinogenic in laboratory animals. They should be handled with
extreme care, using proper personal protective equipment and a well-
ventilated hood.
1
,30−35
nism.
The DNA adducts are a pair of epimers of 7-
2
hydroxy-9-(deoxyguanosin-N -yl) dehydrosupinidine (DHP−
dG-3 and DHP−dG-4) and a pair of epimers of 7-hydroxy-9-
6
Chemicals. Monocrotaline, o-chloranil, valine, rat hemoglobin,
acetonitrile, potassium carbonate, chloroform, and diethyl ether were
purchased from Sigma-Aldrich (St. Louis, MO). Dimethylformamide
(
deoxyadenosin-N -yl)dehydrosupinidine (DHP−dA-3 and
3
5
DHP−dA-4). Further studies demonstrated that DHP−dG-
, DHP−dG-4, DHP−dA-3, and DHP−dA-4 were also formed
in the liver of rats treated with seven hepatocarcinogenic
3
(DMF) and phenyl isothiocyanante (PITC) were obtained from
Fisher Scientific (Pittsburgh, PA). AX 1-X8 anion exchange resin was
purchased from Bio-Rad (Hercules, CA). Heliotrine was purchased
from Accurate Chemical & Scientific Corporation (Westbury, NY).
36
pyrrolizidine alkaloids and riddelliine N-oxide. These results
indicate that this set of DNA adducts is a common biological
Figure 2. Names and structures of pyrrolizidine alkaloids.
1
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Riddelliine was obtained from Dr. Po-Chuen Chan, National
Toxicology Program (NTP). Dehydromonocrotaline, dehydroriddel-
liine, dehydroheliotrine, and DHR were synthesized by dehydrogen-
ation of monocrotaline, riddelliine, heliotrine, and retronecine,
respectively, in chloroform with o-chloranil as previously de-
(1H, m, H6a), 4.03−4.06 (2H, m, H5a, H5b), 4.39 (1H, d, H5′, J = 3
Hz), 4.45−4.50 (2H, m, H9a, H9b), 6.08 (1H, m, H7), 6.26 (1H, d,
H2, J = 2.5 Hz), 6.74 (1H, d, H3, J = 2.5 Hz), 7.30−7.32 (2H, m, H8′,
H9′), 7.49−7.55 (3H, m, H12′, H13′, H14′).
1
DHP−Valine−PITC-4. H NMR (acetonitrile-d ) δ 0.97 (3H, d,
3
3
4,39,40
18
scribed.
H O (isotopic purity 97%) was purchased from
2
H11′/H10′, J = 7 Hz), 1.17 (3H, d, H10′/H11′, J = 7 Hz), 2.32−2.38
(1H, m, H6b, overlapped with solvent peak), 2.52−2.57 (1H, m, H7′),
2.73−2.81 (1H, m, H6a), 3.30 (1H, w, 7-OH), 3.91−3.95 (1H, m,
H5b), 4.08−4.13 (2H, m, H5a, H5′), 4.39 (1H, d, H9b, J = 15 Hz),
Cambridge Isotope Laboratories, Inc. (Andover, MA).
Blood Collection from Rats Treated with Pyrrolizidine
Alkaloids. Female F344 rats were treated by gavage with riddelliine
or monocrotaline at daily doses of 24 μmol/kg body weight in 0.5 mL
5
.11−5.12 (1H, m, H7), 5.72 (1H, d, H9a, J = 15 Hz), 6.22 (1H, d,
of 10% DMSO in water for 3 consecutive days and sacrificed by CO
2
H2, J = 2.5 Hz), 6.64 (1H, d, H3, J = 2.5 Hz), 7.27−7.30 (2H, m, H8′,
H9′), 7.48−7.54 (3H, m, H12′, H13′, H14′).
3
6
inhalation 24 h after the last dose, as reported previously. Blood
samples from each animal were collected by cardiac puncture into
heparinized tubes, and red blood cells were collected by centrifugation
and stored at −80 °C before use.
Stability of DHP−Valine Adducts. Each DHP−valine adduct was
dissolved in acetonitrile/distilled water (v/v 1:20) to afford a solution
with an absorbance of approximately 0.5 absorbance units at 268 nm.
The solution was incubated at room temperature, and aliquots were
taken at different time intervals for analysis by HPLC. For the
Reaction of Valine with Dehydropyrrolizidine Alkaloids. A
mixture of 2 mg of valine (2.8 equiv to dehydromonocrotaline), 2 mg
of K CO , 80 μL of distilled water, and 160 μL of DMF was sonicated
2
3
mechanistic study, pure DHP−valine-2 and DHP−valine-4 adducts in
until the solution turned clear. The valine solution was added dropwise
into a solution of 2 mg of dehydromonocrotaline in 240 μL of DMF.
The reaction was monitored at different time intervals by HPLC
monitored at 218 nm. After reaction for 48 h, the resulting reaction
mixture was filtered through a 0.4 μm RC membrane, and the reaction
products were separated by HPLC with a 250 × 10 mm Prodigy
column (Phenomenex, Torrance, CA) using the following chromato-
graphic conditions: 0−5 min, 2% acetonitrile in water; 5−45 min, 2−
18
acetonitrile/H O (v/v, 1:20) were incubated separately at room
2
temp for 24 h. The resulting DHP−valine-2 and DHP−valine-4
adducts were collected by HPLC for LC/MS analysis.
Interconversion between DHP−Valine−PITC-1 and DHP−
Valine−PITC-3 Adducts. Four DHP−valine−PITC adducts, des-
ignated as DHP−valine−PITC-1, DHP−valine−PITC-2, DHP−va-
line−PITC-3, and DHP−valine−PITC-4, were identified from the
reaction. To determine the possible interconversion among these
adducts, a 0.5 mL solution of DHP−valine−PITC-1 in acetonitrile/
8% acetonitrile in water; flow rate: 1 mL/min. The eluate was
monitored by UV absorbance at 218 nm.
The material eluted from 2−8% acetonitrile in water was further
purified by HPLC with a Phenomenex C18 Luna (2) column (250 ×
H O (v/v, 1:1) at room temp was monitored by HPLC at 0, 1, 2, and
2
3
days. The HPLC analytical conditions were as follows: ACE C18 AR
column (4.6 × 250 mm, 5 μ, from Mac-Mod Analytical, Inc., Chadds
Ford, PA), monitored at 268 nm, flow rate, 1 mL/min; gradient
program: 0−5 min, 10% acetonitrile in water; 5−50 min, 10−18%
acetonitrile in water. The percentage of DHP−valine−PITC-1 and the
resulting DHP−valine−PITC-3 was calculated on the basis of the ratio
of their HPLC peak areas. The sum of peak areas was stable (±3%)
over the incubation period. No additional new peaks were found
during the HPLC analysis, indicating that under these conditions both
the DHP−valine−PITC-1 and DHP−valine−PITC-3 adducts were
stable.
4
1
3
5
.6 mm). The chromatographic conditions were as follows: 0−10 min,
00% water; 10−13 min, 0−2% acetonitrile in water; 13−15 min, 2−
% acetonitrile in water; 15−18 min, 3−4% acetonitrile in water; 18−
0 min, 4−8% acetonitrile in water. The flow rate was 1 mL/min.
To obtain sufficient amount of valine−DHP adducts for further
experiments, reactions were repeated with a larger scale (up to 50 mg
of dehydromonocrotaline).
Reactions of valine with dehydroriddelliine, dehydroheliotrine, and
DHR were conducted similarly.
Reaction of DHP−Valine Adducts Mixture with Phenyl
Isothiocyanate (PITC). To the DHP−valine adducts mixture
described above, dissolved in 1 mL of acetonitrile, was added 2 μL
of phenyl isothiocyanate (PITC). The reaction was kept under shaking
in a water bath at 45 °C for 2 h. The resulting isothiohydantion
adducts (DHP−valine−PITC) were isolated by HPLC, using a
Phenomenex C18 Luna (2) column (250 × 4.6 mm) eluted at a flow
rate of 1 mL/min with a mobile phase of 43% acetonitrile in water and
monitored at 268 nm. The resulting isomeric DHP−valine−PITC
adducts were characterized by HPLC-ES-MS/MS and NMR analysis.
The full 1H NMR spectral assignments of DHP−valine−PITC
An experiment with pure DHP−valine−PITC-3 in acetonitrile/
H O (v/v, 1:1) was similarly conducted and monitored by HPLC daily
2
up to 6 days. The percentage of DHP−valine−PITC-1 and DHP−
valine−PITC-3 was similarly calculated.
Interconversion between DHP−Valine−PITC-2 and DHP−
Valine−PITC-4. The study of interconversion between DHP−valine−
PITC-2 and DHP−valine−PITC-4 was conducted under the
conditions described above for DHP−valine−PITC-1 and DHP−
valine−PITC-3, with the exception that the reactions were monitored
by HPLC at 0, 1, 2, and 3 h (or up to 7 h) respectively.
adducts follow.
Reaction of Dehydromonocrotaline and DHR with Hemo-
globin Followed by Reaction with Phenyl Isothiocyanate. A
solution of 20 mg of rat hemoglobin, 350 μL of deionized water, and
1
DHP−Valine−PITC-1. H NMR (acetonitrile-d ) δ 0.74 (3H, d,
H11′/H10′, J = 7 Hz), 1.11 (3H, d, H10′/H11′, J = 7 Hz), 1.38−1.45
3
1
00 μL of 0.5 M K CO was added dropwise into a 50 μL solution of 2
(
1H, m, H7′), 2.72−2.82 (2H, m, 9-OH, H6b), 3.02−3.10 (1H, m,
2 3
mg of dehydromonocrotaline in DMF and kept with shaking at 37 °C
for 3 h, followed by the addition of 2 equiv of PITC (in relation to the
dehydromonocrotaline). The reaction continued at 37 °C with shaking
for 16 h. After 3 mL of acetonitrile was added, the reaction solution
was centrifuged (15 000g, 5 min), the supernatant was collected, and
solvent was removed in a rotary evaporator under reduced pressure.
The resulting DHP−valine−PITC adducts were purified by HPLC
(following the conditions previously described for separation of
DHP−valine−PITC) and analyzed by LC/MS/MS.
H6a), 4.04−4.09 (1H, m, H5b), 4.12−4.18 (1H, m, H5a), 4.30 (1H, d,
H5′, J = 3 Hz), 4.43 (1H, two sets of d, H9b, J = 5.5 Hz), 4.55 (1H,
two sets of d, H9a, J = 4 Hz), 6.20 (H, d, H2, J = 2.5 Hz), 6.41−6.44
(
1H, m, H7), 6.67 (1H, d, H3, J = 2.5 Hz), 7.24−7.32 (2H, m, H8′,
H9′), 7.49−7.55(3H, m, H12′, H13′, H14′).
1
DHP−Valine−PITC-2. H NMR (acetonitrile-d ) δ 0.94 (3H, d,
3
H11′/H10′, J = 7 Hz), 1.20 (3H, d, H10′/H11′, J = 7 Hz), 2.29−2.37
(
3
H5a), 4.20 (1H, d, H5′, J = 3.5 Hz), 4.40 (1H, d, H9b, J = 15 Hz),
5
H2, J = 2.5 Hz), 6.62 (1H, d, H3, J = 2.5 Hz), 7.26−7.30 (2H, m, H8′,
H9′), 7.46−7.55 (3H, m, H12′, H13′, H14′).
1H, m, H6b), 2.62−2.65 (1H, m, H7′), 2.73−2.82 (1H, m, H6a),
.41 (1H, w, 7-OH), 3.88−3.93 (1H, m, H5b), 4.09−4.14 (1H, m,
Reaction of DHR with rat hemoglobin was conducted under similar
experimental conditions.
.19−5.20 (1H, m, H7), 5.62 (1H, d, H9a, J = 15 Hz), 6.28 (1H, d,
Analysis of DHP−Valine−PITC Adducts in Hemoglobin of
Rats Treated with Monocrotaline and Riddelliine. To identify
DHP−hemoglobin adducts formed in blood of rats treated with
monocrotaline or riddelliine, blood samples were treated with PITC to
convert DHP−hemoglobin adducts to phenylthiohydantoin (DHP−
1
DHP−Valine−PITC-3. H NMR (acetonitrile-d ) δ 0.91 (3H, d,
3
H11′, J = 7 Hz), 0.96 (3H, d, H10′, J = 7 Hz), 1.40−1.44 (1H, w,
H7′), 2.70−2.73 (1H, m, H6b), 2.94−2.96 (1H, m, 9-OH), 3.12−3.17
1
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valine−PITC) products through the Edman rearrangement reac-
4
1,42
tion.
In brief, red blood cells (100 μL) collected from rats treated with
monocrotaline or riddelliine were lysed by adding 120 μL of dH O
2
and reacted with 5 μL of PITC in 20 μL of 0.5 M K CO with shaking
2
3
at 37 °C for 16 h. The reaction was stopped by adding 1.5 mL of
acetonitrile. After centrifugation and concentration, the resulting
sample was analyzed by LC/MS/MS under similar conditions as those
described for the identification of DHP−valine−PITC adducts.
LC/MS/MS Analysis of DHP−Valine and DHP−Valine−PITC
Adducts. DHP−valine and DHP−valine−PITC adducts were
dissolved in water for LC/MS/MS analysis. The liquid chromatog-
raphy system consisted of a Shimadzu Prominence HPLC system,
including a CBM-20A system controller, two LC-20AD pumps, a SIL-
20AC HT autosampler, a SPD-20A UV/vis detector (Shimadzu
Scientific Instrument, Columbia, MD 21046), and an automated
switching valve (TPMV, Rheodyne, Cotati, CA). The switching valve
was used to divert the column effluent to either waste or to MS
instrument. The Shimadzu Prominence HPLC system was used for
sample injection and separation. Each sample (10−30 μL) was loaded
onto a reverse-phase column (ACE 3 C18, 2.1 mm × 100 mm, 3 μm,
Mac-Mod Analytical, Inc., Chadds Ford, PA) with a water/acetonitrile
gradient at 0.2 mL/min, and the sample components were eluted into
the mass spectrometer. The column chamber’s temperature was set to
4
5 °C. The mobile phases were water and acetonitrile. The initial
gradient consisted of 0% acetonitrile for 0.5 min followed by a linear
gradient up to 15% acetonitrile over 50 min; acetonitrile was increased
to 95% in 1.5 min. After holding 95% acetonitrile for 5 min, the
instrument was reset to the initial conditions in 1 min. The analytical
column was equilibrated with the starting mobile phase for 12 min.
The total run time for analysis was 70 min.
The HPLC eluate was coupled with an AB Sciex 4000 QTrap LC/
MS/MS system (AB Sciex, Foster City, CA) equipped with a Turbo V
ion source and a desolvation temperature of 500 °C. Nitrogen was
used as curtain, nebulizer, heater, and collision gases. The samples
were acquired in positive ion spray mode using MS full scan and
enhanced product ion scan (EPI) as well as using multiple reaction
monitoring methods (MRM). The ion transitions of the MRM
method for specific detection of DHP−valine and DHP−valine−PITC
adducts in the samples were m/z 253 → 136 and m/z 392 → 136,
respectively. The ion spray voltage was 4500 V; ion source gases 1 and
Figure 3. Reversed-phase HPLC profiles of (A) four DHP−valine
adducts formed from the reaction of dehydromonocrotaline and valine
and (B) four DHP−valine−PITC adducts formed from reaction of
DHP−valine mixture with phenyl isothiocycnate. HPLC conditions:
Phenomenex C18 Luna (2) column (250 × 4.6 mm); flow rate: 1 mL/
min; mobile phase (linear gradient): (A) 0−10 min, 100% water; 10−
2
5
were set to 60 and 50, respectively. The declustering potential was
0 V, and the collision energy was 25 eV for all transitions.
Instrumentation. For HPLC analysis, a Waters (Milford, MA)
1
3 min, 0−2% acetonitrile in water; 13−15 min, 2−3% acetonitrile in
HPLC system, consisting of a model 600 controller, a model 996
photodiode array detector, and a 600 pump, was used for separation
water; 15−18 min, 3−4% acetonitrile in water; 18−50 min, 4−8%
acetonitrile in water; (B) 43% acetonitrile in water at flow rate 1 mL/
min.
1
and purification of the DHP-derived DNA adducts. H nuclear
magnetic resonance (NMR) experiments were carried out at 301 K on
a Bruker Avance III spectrometer equipped with a Bruker BBFO Plus
Smart Probe (Bruker Instruments, Billerica, MA) operating at 500
MHz. Samples were dissolved in acetonitrile-d or acetonitrile-d with
3
3
membrane, and products were separated by HPLC with a
Prodigy column (250 × 10 mm). The prepurified material was
further purified by HPLC with a Phenomenex C18 Luna (2)
column (250 × 4.6 mm). An initial elution with water removed
the unreacted valine and necic acid formed from hydrolysis of
dehydromonocrotaline; further elution with acetonitrile in
water afforded a mixture of reaction products (Figure 3A).
LC/MS analysis of the reaction mixture showed that there
were four products, each having a mass spectrum with
a trace of D O. Chemical shifts are reported in parts per million
2
downfield from tetramethylsilane, and coupling constants are reported
in hertz. Two-dimensional NMR homonuclear decoupling (COSY)
experiments were conducted to assist in assigning proton resonances.
RESULTS
■
Reaction of Valine with Dehydropyrrolizidine Alka-
loids. The difficulty of the synthesis is that, while
dehydropyrrolizidine alkaloids are highly unstable in water-
containing solvent system, the reaction has to be conducted in
water-containing medium because valine is soluble only in
water. Therefore, to synthesize DHP−valine adducts, reaction
of dehydromonocrotaline and valine was repeated under several
different experimental conditions. The optimal conditions used
are described in the Experimental Procedures.
+
protonated molecule ions (M + H) at m/z 253. These results
suggested that the reaction mixture contained four isomeric
DHP−valine adducts (m/z 253, designated as DHP−valine-1,
DHP−valine-2, DHP−valine-3, and DHP−valine-4, respec-
tively). Because these adducts are unstable and decomposed
during HPLC purification, we were unable to obtain these
products in a sufficient quantity for structural identification by
1
After reaction of valine with dehydromonocrotaline for 48 h,
the resulting reaction mixture was filtered through a 0.4 μm RC
H NMR spectral analysis. Thus, the product mixture was
treated with phenyl isothiocyanate (PITC) to convert DHP−
1
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3
52 corresponding to the loss of water by the protonated
adduct (Figure S2 in the Supporting Information), which
suggested that they were four isomeric DHP−valine−PITC
adducts.
These adducts were further purified by HPLC to obtain
1
sufficient quantity for structural identification by H NMR
(
Figure 4). The four chromatographic peaks eluted at 16.9,
2
3.9, 26, and 28.1 min are designated as DHP−valine−PITC-1,
DHP−valine−PITC-2, DHP−valine−PITC-3, and DHP−va-
line−PITC-4 adducts, respectively.
1
The H NMR data of these four DHP−valine−PITC adducts
1
were compared with H NMR data of phenylthiohydantoin
adducts
42,43
and DHR, the necine base (present study,
assignments shown in Table 1). In accordance with the
reported data of a phenylthiohydantoin moiety, these adducts
had five phenyl protons at 7.2−7.6 ppm and six valine methyl
protons at 0.94−1.17 ppm. These adducts had the H5′ proton
as a doublet at 4.15−4.40 ppm. While DHP−valine−PITC-1
and DHP−valine−PITC-3 had the H7′ proton at 1.38−1.45
and 1.40−1.44 ppm, respectively, DHP−valine−PITC-2 and
DHP−valine−PITC-4 had the H7′ proton at 2.52−2.57 ppm.
Their NMR resonances of H2 and H3 were nearly identical to
those of the DHR moiety. Both DHP−valine−PITC-2 and
DHP−valine−PITC-4 had the pairs of geminal H5 and H6
protons almost unaffected by adduct formation. DHP−valine−
PITC-1 and DHP−valine−PITC-3 had their H7 proton
resonance at 6.41−6.44 and 6.08 ppm, respectively, whereas
DHP−valine−PITC-2 and DHP−valine−PITC-4 had their H7
proton resonance at 5.19−5.20 ppm and 5.11−5.12 ppm,
respectively (Table 1). The H7 of DHR resonates at 4.99 ppm.
Taken together, these data indicate that the DHP−valine−
PITC-1 and DHP−valine−PITC-3 adducts had their phenyl-
thiohydantoin group located at the C7 position of the DHP
moiety (the necine base) and that they were a pair of epimers.
1
Similar comparison of the CH (C9 position) group of the
Figure 4. H NMR spectra of products identified as (A) DHP−
2
DHP moiety of these adducts with that of DHP (DHR)
valine−PITC-1 and DHP−valine−PITC-3 adducts and (B) DHP−
valine−PITC-2 and DHP−valine−PITC-4 adducts.
indicated that DHP−valine−PITC-2 and DHP−valine−PITC-
4
adducts had their phenylthiohydantoin group located at the
C9 position of the DHP moiety and that they were a pair of
epimers. Thus, comparison of the chemical shifts of the necine
base of these four DHP−valine−PITC adducts with those of
DHR (Table 1) helped to determine the structures of these
phenylthiohydantoin adducts. The NMR assignments were
further confirmed by COSY NMR analysis (Figures S3−S4 in
the Supporting Information).
valine adducts to the phenylthiohydantoin (DHP−valine−
42
PITC) products through Edman rearrangement reaction.
Reaction of DHP−Valine Adducts with Phenyl
Isothiocyanate (PITC). After reaction of DHP−valine adducts
with PITC, the resulting phenylthiohydantoin adducts (DHP−
valine−PITC adducts) were separated by reversed-phase
HPLC (Figure 3B). Chromatographic peaks eluted at 16.9,
2
(
3.9, 26, and 28.1 min had similar UV absorption spectra
Figure S1 in the Supporting Information) and mass spectra
with preponderant sodiated ions at m/z 392 and ions at m/z
The NMR data indicated that DHP−valine−PITC-3 and
DHP−valine−PITC-1 are a pair of epimers and that DHP−
valine−PITC-2 and DHP−valine−PITC-4 are another pair of
Table 1. Proton NMR Spectroscopic Data (500 MHz) of the Necine Base in the Synthetically Prepared DHP−Valine−PITC
Adducts Measured in Acetonitrile-d
3
assignment
DHR
DHP−val−PITC-1
DHP−val−PITC-2
DHP−val−PITC-3
DHP−val−PITC-4
H6b
H6a
H5b
H5a
H9b
H9a
H2
2.15−2.20 (1H, m)
2.57−2.65 (1H, m)
3.75−3.79 (1H, m)
3.94−3.98 (1H, m)
4.27 (1H, d, J = 12)
4.33 (1H, d, J = 12)
6.02 (1H, d, J = 2.5)
4.99 (1H, dd, J = 6.5, 2.5)
6.54 (1H, d, J = 2.6)
2.75−2.82 (1H, m)
3.02−3.10 (1H, m)
4.04−4.09 (1H, m)
4.12−4.16 (1H, m)
4.43 (1H, dd, J = 5.5)
4.63 (1H, dd, J = 4)
6.20 (H, d, J = 2.5)
6.41−6.44 (1H, m)
6.67 (1H, d, J = 2.5)
2.29−2.41 (1H, m)
2.73−2.82 (1H, m)
3.88−3.93 (1H, m)
4.09−4.14 (1H, m)
4.40 (1H, d, J = 15)
5.62 (1H, d, J = 15)
6.28 (1H, d, J = 2.5)
5.19−5.20 (1H, w)
6.30 (1H, d, J = 2.5)
2.70−2.73 (1H, m)
3.12−3.17 (1H, m)
4.03−4.06 (2H, m)
Overlapped with solvent peak (HOD)
2.73−2.81 (1H, m)
3.91−3.95 (1H, m)
4.08−4.13 (1H, m)
4.45−4.50 (2H, m)
4.39 (1H, d, J = 15)
5.72 (1H, d, J = 15)
6.22 (1H, d, J = 2.5)
5.11−5.12 (1H, w)
6.26 (1H, d, J = 2.5)
6.08 (1H, m)
H7
H3
6.74 (1H, d, J = 2.5)
6.64 (1H, d, J = 2.5)
1
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Figure 5. Interconversion between DHP−valine−PITC-1 and DHP−valine−PITC-3 and between DHP−valine−PITC-2 and DHP−valine−PITC-
4
in acetontrile/water (v/v, 1:1): (A) starting from pure DHP−valine−PITC-1, (B) starting from pure DHP−valine−PITC-2, (C) starting from
pure DHP−valine−PITC-3, and (D) starting from pure DHP−valine−PITC-4. The relative percentages were calculated on the basis of their HPLC
peak area ratios.
Figure 6. HPLC chromatogram of derivatives formed from the reaction of each DHR−valine adduct with PITC: (A) from DHP−valine-1, (B) from
DHP−valine-2, (C) from DHP−valine-3, and (D) from DHP−valine-4. In the figure, P1, P2, P3, and P4 designate DHP−valine−PITC-1, DHP−
valine−PITC-2, DHP−valine−PITC-3, and DHP−valine−PITC-4, respectively. HPLC conditions: Phenomenex C18 Luna (2) column (250 × 4.6
mm); flow rate: 1 mL/min; mobile phase: 43% acetonitrile in water.
1.46 × 10 M cm−1 (at 270 nm) in acetonitrile. Accordingly,
the reaction yields of DHP−valine−PITC-1, DHP−valine−
PITC-2, DHP−valine−PITC-3, and DHP−valine−PITC-4
were estimated to be 1.2, 1.4, 1.8, and 1.6%, respectively.
These results indicate that reaction of valine and dehydromo-
nocrotaline was nonregioselective, with the reaction at the C7
and C9 positions of the necine base of dehydromonocrotamine
proceeding at a near equal ratio.
4
−1
epimers. As expected, the experimentally determined UV molar
extinction coefficients of DHP−valine−PITC-1 and DHP−
valine−PITC-3 were identical, and the UV molar extinction
coefficients of DHP−valine−PITC-2 and DHP−valine−PITC-
1
4
were also identical. On the basis of H NMR spectroscopic
analysis in the presence of a known quantity of tert-butanol as
an internal standard, the UV molar extinction coefficients of
DHP−valine−PITC-1 and DHP−valine−PITC-3 were deter-
4
−1
−1
mined to be 1.66 × 10 M cm (at 268 nm) in acetonitrile,
and those of DHP−valine−PITC-2 and DHP−valine−PITC-4,
Interconversion between DHP−Valine−PITC-1 and
DHP−Valine−PITC-3 Adducts. We determined that at
1
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Figure 7. Structures of four DHP−valine adducts and the relationship with the four DHP−valine−PITC adducts.
room temperature DHP−valine−PITC-1 and DHP−valine−
PITC-3 dissolved in 10% acetonitrile in water (v/v, 1:1) were
interconvertible. HPLC analysis indicated that DHP−valine−
PITC-1 was converted into DHP−valine−PITC-3 gradually
and reached an approximately equal ratio in 3 days (Figure 5A).
Similar results occurred when the experiment was started with
pure DHP−valine−PITC-3, but the rate of interconversion was
slower, with an equal ratio of both epimers being reached at 6
days of incubation (Figure 5C). These results indicate that
conversion of DHP−valine−PITC-1 is faster than that of
DHP−valine−PITC-3.
Interconversion between DHP−Valine−PITC-2 and
DHP−Valine−PITC-4 Adducts. A study of interconversion
between DHP−valine−PITC-2 and DHP−valine−PITC-4 was
similarly conducted. It was found that the rate of
interconversion between DHP−valine−PITC-2 and DHP−
valine−PITC-4 was much faster than that between DHP−
valine−PITC-1 and DHP−valine−PITC-3. Interconversion of
DHP−valine−PITC-2 to DHP−valine−PITC-4 to an equal
ratio required only 3 h (Figure 5B). Interconversion of DHP−
valine−PITC-4 to a near equal ratio of DHP−valine−PITC-2
took 7 h (Figure 5D), a little longer time than that of DHP−
valine−PITC-2 but much shorter than those between DHP−
valine−PITC-1 and DHP−valine−PITC-3 (Figure 5A,C).
Stability of DHP−Valine−PITC Adducts. It was deter-
mined that DHP−valine−PITC adducts dissolved in acetoni-
trile with and without a trace of water were stable for up to a
week. However, in fully aqueous medium or in a solvent
consisting of 20% acetonitrile in water during HPLC
Figure 8. Kinetic analysis of decomposition of DHP−valine adducts in
acetontrile/water (v/v, 1:20). Decomposition of (A) DHP−valine-2
adduct to DHP and (B) DHP−valine-4 adduct to DHP.
1
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Figure 9. HPLC chromatogram of four DHP−valine adducts formed from the reaction valine with (A) dehydroriddelliine, (B)
dehydromonocrotaline, (C) dehyroheliotrine, and (D) dehyroretronecine (DHR). In the figure, P1, P2, P3, and P4 designate DHP−valine-1,
DHP−valine-2, DHP−valine-3, and DHP−valine-4, respectively. HPLC conditions: column, Phenomenex C18 Luna (250 × 4.6 nm); flow rate: 1
mL/min; mobile phase (linear gradient): 0−10 min, 100% water; 10−13 min, 0−2% acetonitrile in water; 13−15 min, 2−3% acetonitrile in water;
15−18 min, 3−4% acetonitrile in water; 18−50 min, 4−8% acetonitrile in water.
separation, the DHP−valine−PITC adducts gave rise to many
decomposition products.
4, and 5 h. As shown in Figure 8A, the amount of DHP−valine-
2 adduct decreased gradually, and DHP formed increasingly in
a time-dependent manner. The sum of the integrated peak
areas of the two peaks, DHP−valine-2 and DHP, in each
analysis is almost equal. No other peak was found in the HPLC
analysis, which suggested that the DHP−valine-2 adduct
decomposed to DHP and valine. The same phenomenon was
found for DHP−valine-4 adduct under similar experimental
conditions. As shown in Figure 8B, the amount of DHP−
valine-4 adduct decreased gradually and DHP formed
increasingly in a time-dependent manner.
Structural Identification of DHP−Valine Adducts. To
determinate the structures of DHP−valine adducts (DHP−
valine-1, DHP−valine-2, DHP−valine-3, and DHP−valine-4)
formed from reaction of dehydromonocrotaline and valine, we
isolated the materials contained in each of the chromatographic
peaks shown in Figure 3A to react with PITC, and we then
compared their HPLC retention times and UV spectra with
those of the four DHP−valine−PITC adducts, whose structures
1
had been elucidated by mass spectrometry and H NMR. Upon
derivatization with PITC, each of the four peaks labeled as
DHP−valine-1 (eluted at 24.1 min), DHP−valine-2 (26.7 min),
DHP−valine-3 (29.2 min), and DHP−valine-4 (33.5 min) in
Figure 3A formed one major and one minor DHP−valine−
PITC adducts (Figure 6). As described above, interconversion
occurred between DHP−valine−PITC-1 and DHP−valine−
PITC-3 as well as between DHP−valine−PITC-2 and DHP−
valine−PITC-4. Therefore, it is reasonable to assume that the
major DHP−valine−PITC was generated from its parent
DHP−valine adduct and that the minor DHP−valine−PITC
was formed by interconversion from the major one. As a result,
DHP−valine−PITC-4 was derived from DHP−valine-1
Reaction of Dehydromonocrotaline, Dehydroriddel-
liine, Dehydroheliotrine, and DHR with Valine. As already
described, DHP−valine adducts are unstable, undergoing
interconversion between epimers and, as other dehydropyrro-
lizidine alkaloids, are quickly hydrolyzed into DHP (the
racemic form of DHR) in aqueous medium. To compare the
relative yield, reactions of dehydromonocrotaline, dehydror-
iddelliine, dehydroheliotrine, and DHR with valine were
conducted for 30 min. It was found that reaction of
dehydromonocrotaline, dehydroriddelliine, and dehydrohelio-
trine all produced DHP−valine adducts, but with different
yields and patterns. In contrast, reaction of DHR and valine did
not generate DHP−valine at a detected level (Figure 9).
Reaction of Dehydromonocrotaline and DHR with
Hemoglobin Followed by Reaction with Phenyl Iso-
thiocyanate. Similar to the reaction of valine with
dehydromonocrotaline, a reaction of hemoglobin (Hb) with
dehydromonocrotaline in K CO and DMF was conducted,
(
Figure 6A), DHP−valine−PITC-1 was derived from DHP−
valine-2 (Figure 6B), DHP−valine−PITC-2 was derived from
DHP−valine-3 (Figure 6C), and DHP−valine−PITC-3 was
derived from DHP−valine-4 (Figure 6D).
Because the structures of DHP−valine−PITC adducts have
been characterized, the structures of four DHP−valine adducts
are subsequently deduced as shown in Figure 7.
2
3
followed by reaction with phenyl isothiocynante. The reaction
products were analyzed by HPLC, and the eluate was analyzed
by ultraviolet detection and mass spectrometry. There were
four chromatographic peaks, each of which had retention time,
Stability of DHP−Valine Adducts. A sample of DHP−
valine-2 adduct was incubated in acetonitrile/H O (v/v, 1:20)
2
at room temperature and was monitored by HPLC at 0, 1, 2, 3,
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Figure 10. HPLC chromatogram and mass spectrometric profiles of DHP−valine−PITC adducts formed from reaction of dehydromonocrotaline
with hemoglobin followed by reaction with PITC.
UV−visible absorption spectra, and mass spectra evidencing
ions at m/z 352 that corresponded to the loss of water by the
protonated molecules and (M + Na) at 392 m/z (Figure 10)
identical to those of the four DHP−valine−PITC adducts
formed from the reaction of dehydromonocrotaline with valine.
In contrast, on the basis of similar spectral analyses, the reaction
of DHR with Hb did not generate DHP−valine−PITC adducts.
Analysis of DHP−Hemoglobin Adducts Formed in
Vivo. The hemoglobin samples of rats treated with riddelliine
and monocrotaline by gavage were similarly treated with phenyl
isothiocyanate. LC/MS/MS analysis of the reaction mixtures
indicated that no DHP−valine−PITC adducts were detected in
the samples using LC/MS full scan and MS/MS methods as
well as using MRM.
+
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Chemical Research in Toxicology
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DISCUSSION
may severely underestimate the total reactive metabolite
produced or that the reactive metabolite does not get into
circulation. Consistently, as reported in the present study, no
DHP−valine−PITC adducts were detected from the hemoglo-
bin of rats administered riddelliine or monocrotaline by gavage.
■
Probably because of their highly unstable nature, limited
information on the structures of DHP−protein adducts and
DHP−amino acid adducts have been reported. 7-Cysteine-
DHP, prepared from reaction of DHR with cysteine by
35
4
4
In a previous study, we determined that calf thymus DNA,
dG, and dA bind to dehydroriddelliine preferentially at the C9
position of the necine base. Furthermore, in an in vivo study, we
determined that female rats gavaged with riddelliine, mono-
crotaline, retrorsine, heliotrine, lasiocarpine, clivorine, senkir-
kine, and riddelliine N-oxide afforded the same pattern of DNA
adducts, forming two sets of epimeric dG and dA adducts with
the nucleic bases linked to the C9 position of the necine base.
We previously hypothesized that the regioselectivity of these
Robertson et al., is the only DHP−amino acid adduct with
the structure fully elucidated by MS and NMR analysis. Estep et
4
5
al. isolated N-acetylcysteine−DHP in the urine of rats
administered monocrotaline and senecionine. Its identity was
based on an Ehrlich-reagent-positive result, which indicates the
presence of a pyrrolic moiety, and its molecular weight was
determined by MS. In this present study, the reaction of
dehydromonocrotaline, dehyroriddelliine, and dehydrohelio-
trine with valine and the reaction of dehydromonocrotaline
with hemoglobin were studied. The structures of the resulting
DHP−valine-1, DHP−valine-2, DHP−valine-3, and DHP−
valine-4 were elucidated through the full structural character-
ization of the four corresponding DHP−valine−PITC deriva-
tives. This represents the first full structural elucidation of an
amino acid−DHP adduct that was prepared from the reaction
between an amino acid and dehydropyrrolizidine alkaloids, the
primary reactive metabolites of pyrrolizidine alkaloids.
36
reaction is mainly due to steric hindrance at the C7 position.
In contrast, it was determined that dehydopyrrolizidine
alkaloids and DHR bind with nucleophiles preferentially at
1,7,34
the C7 position of the necine base.
The binding of DHR
with cysteine and glutathione (GSH) also preferentially
occurred at the 7-position to form 7-cysteine−DHP and 7-
GSH−DHP; neither 9-cysteine−DHP nor 9-GSH−DHP were
4
4,48
formed.
On the other hand, our findings in the present
In aqueous medium, DHP−valine adducts proved to be
highly unstable, facilely decomposed, and interconverted
between DHP−valine epimers. Moreover, both DHP−valine-
study clearly indicate that reactions of valine with dehydromo-
nocrotaline and dehydroriddelliine are not regioselective,
although reaction at the C7 position to form DHP−valine-2
and DHP−valine-4 proceeded at higher yields than that at the
C9 position, which yields DHP−valine-1 and DHP−valine-3
adducts. Overall, in order to better understand the mechanism
of pyrrolizidine alkaloid-induced hepatotoxicity and liver tumor
formation, determination of the reaction of dehydropyrrolizi-
dine alkaloids with cellular macromolecules warrants further
investigation.
2
and DHP−valine-4 can facilely lose the valine moiety at the
C7 position of the necine base to form DHP in a time-
dependent manner (Figures 8). These findings are highly
significant because they suggest that DHP−protein adducts
may also dissociate into protein and DHP in vivo and in vitro.
This observation may shed light on the role of DHP−protein
adducts on hepatotoxicity and carcinogenesis induced by
pyrrolizidine alkaloids.
In the field of chemical carcinogenesis, hemoglobin adducts
can provide noninvasive biomarkers of reactive species involved
in liver tumorigenicity, if there is a correlation between the liver
tumor potency and the level of hemoglobin adducts in the
blood. The most commonly employed method for quantitation
of metabolite-bound protein adducts in vivo is the use of Edman
ASSOCIATED CONTENT
■
*
S
Supporting Information
1
1
Ultraviolet, LC/MS, and H− H COSY NMR spectra of
DHP−valine−PITC products. This material is available free of
charge via the Internet at http://pubs.acs.org.
46
rearrangement reaction. Edman reaction involves the use
phenyl isothiocyanate and derivatives, such as pentafluor-
ophenyl isothiocyanate, to react with the valine terminal amino
group of the hemoglobin adducts, resulting in the replacement
of the hemoglobin moiety by a phenylthiohydantoin group.
AUTHOR INFORMATION
■
*
Corresponding Author
Tel.: +1-870-543-7207. Fax: +1-870-543-7136. E-mail: peter.
fu@fda.hhs.gov.
47
Quantitation can then be accomplished by LC/MS/MS.
There are limitations as to whether a specific hemoglobin
adduct can be validated as a biomarker. At minimum, these
include the yield and the stability of the hemoglobin adduct
that is formed by linking the valine terminal amino group of the
hemoglobin to the metabolite, the yield of the Edman
rearrangement reaction, the stability of the final products, the
ease of the separation of final products by HPLC, the detection
efficiency of the final products by LC/MS/MS, and the
availability of the deuterated final products used as internal
standards for detection and quantification by LC/MS/MS.
In the present study, we demonstrate that DHP−valine
adducts are highly unstable in aqueous media, which suggests
that DHP−protein adducts that are formed through linking the
valine terminal amino group to the DHP moiety should be
highly unstable as well. Thus, our data indicate that
quantification of DHP−hemoglobin adducts in vivo via the
Edman rearrangement reaction pathway may not be feasible or
Author Contributions
Y.Z. and S.W. contributed equally to this work.
§
Funding
This research was supported in part by appointments (Y.Z. and
S.W.) to the Postgraduate Research Program at the NCTR
administered by the Oak Ridge Institute for Science and
Education through an interagency agreement between the U.S.
Department of Energy and the U.S. Food and Drug
Administration (FDA).
Notes
This article is not an official U.S. Food and Drug
Administration (FDA) guidance or policy statement. No
official support or endorsement by the U.S. FDA is intended
or should be inferred.
The authors declare no competing financial interest.
1
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Chemical Research in Toxicology
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(
15) Huxtable, R. J. (1980) Herbal teas and toxins: novel aspects of
ACKNOWLEDGMENTS
■
pyrrolizidine poisoning in the United States. Perspect. Biol. Med. 24, 1−
4.
16) Stegelmeier, B. L., Edgar, J. A., Colegate, S. M., Gardner, D. R.,
We thank Dr. Frederick A. Beland for critical review of this
manuscript and Dr. Po-Chuen Chan of NTP for supplying
riddelliine.
1
(
Schoch, T. K., Coulombe, R. A., and Molyneux, R. J. (1999)
Pyrrolizidine alkaloid plants, metabolism and toxicity. J. Nat. Toxins 8,
95−116.
ABBREVIATIONS
■
(17) Schoental, R., head, M. A., and Peacock, P. R. (1954) Senecio
DHR, dehydroretronecine or (−)-R-6,7-dihydro-7-hydroxy-1-
hydroxymethyl-5H-pyrrolizine; DHP, (±)-6,7-dihydro-7-hy-
droxy-1-hydroxymethyl-5H-pyrrolizine; PITC, phenyl isothio-
chanate; DHP−valine-1 and DHP−valine-3, an epimeric pair of
alkaloids: primary liver tumours in rats as a result of treatment with (1)
a mixture of alkaloids from S. jacobaea lin.; (2) retrorsine; (3) isatidine.
Br. J. Cancer 8, 458−465.
(18) IARC (1976) Monocrotaline. IARC Monogr. Eval. Carcinog. Risk
Chem. Man 10, 291−302.
7
-hydroxy-9-valine-dehydrosupinidine; DHP−valine-2 and
DHP−valine-4, an epimeric pair of 7-valine-dehydrosupinidine;
DHP−valine−PITC-2 and DHP−valine−PITC-4, an epimeric
pairs of 7-hydroxy-9-valine-dehydrosupinidine; DHP−valine−
PITC-1 and DHP−valine−PITC-3, an epimeric pairs of 7-
valine-dehydrosupinidine; Hb, hemoglobin; LC/MS/MS, high-
performance liquid chromatography electrospray ionization
tandem mass spectrometry; NCTR, National Center for
Toxicological Research
(19) Chan, P. C., Haseman, J. K., Prejean, J. D., and Nyska, A. (2003)
Toxicity and carcinogenicity of riddelliine in rats and mice. Toxicol.
Lett. 144, 295−311.
(
20) Galloway, S. M., Armstrong, M. J., Reuben, C., Colman, S.,
Brown, B., Cannon, C., Bloom, A. D., Nakamura, F., Ahmed, M., Duk,
S., et al. (1987) Chromosome aberrations and sister chromatid
exchanges in Chinese hamster ovary cells: evaluations of 108
chemicals. Environ. Mol. Mutagen. 10, 1−175.
(
21) Li, N., Xia, Q., Ruan, J., Fu, P. P., and Lin, G. (2011)
Hepatotoxicity and tumorigenicity induced by metabolic activation of
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