1-Formyl-7-hydroxy-6,7-dihydro-5 H-pyrrolizine (1-CHO-DHP): A Potential Proximate Carcinogenic Metabolite of Pyrrolizidine Alkaloids

Article abstract of DOI:10.1021/acs.chemrestox.9b00038

Pyrrolizidine alkaloids (PAs) are phytochemicals present in more than 6000 plant species worldwide; about half of the PAs are hepatotoxic, genotoxic, and carcinogenic. Because of their wide exposure and carcinogenicity, the International Programme on Chemical Safety (IPCS) concluded that PAs are a threat to human health and safety. We recently determined that PA-induced liver tumor initiation is mediated by a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts and proposed that these DHP-DNA adducts are biomarkers of PA exposure and liver tumor initiation. To validate the generality of this metabolic activation pathway and DHP-DNA adducts as biomarkers, it is significant to identify reactive metabolites associated with this metabolic activation pathway. Segall et al. (Segall et al. (1984) Drug Metab. Dispos. 12, 68-71) previously reported that 1-formyl-7-hydroxy-6,7-dihydro-5H-pyrrolizine (1-CHO-DHP) is generated from the metabolism of senecionine by mouse liver microsomes. In the present study, we examined the metabolism of seven hepatocarcinogenic PAs (senecionine, intermedine, retrorsine, riddelliine, DHR, heliotrine, and senkirkine) and one noncarcinogenic PA (platyphylline) by human, rat, and mouse liver microsomes. 1-CHO-DHP was identified as a common metabolite from the metabolism of these hepatotoxic PAs, but not from platyphylline. Incubation of 1-CHO-DHP with HepG2 and A549 cells produced the same set of DHP-DNA adducts, which were identified by both LC/MS MRM mode and selected ion monitoring analyses through comparison to synthetic standards. In the incubation medium of 1-CHO-DHP treated HepG2 cells, both DHP and 7-cysteine-DHP were formed, which were capable of binding to cellular DNA to produce DHP-DNA adducts. These results suggest that 1-CHO-DHP is a proximate DNA metabolite of genotoxic and carcinogenic PAs.

Full text of DOI:10.1021/acs.chemrestox.9b00038

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Article
1-Formyl-7-hydroxy-6,7-dihydro-5H-pyrrolizine (1-CHO-DHP) - A
Potential Proximate Carcinogenic Metabolite of Pyrrolizidine Alkaloids
Xiaobo He, Qingsu Xia, Gonçalo Gamboa da Costa, Ge Lin, and Peter P. Fu
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00038 • Publication Date (Web): 23 May 2019
Downloaded from http://pubs.acs.org on May 30, 2019
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1
-Formyl-7-hydroxy-6,7-dihydro-5H-pyrrolizine (1-CHO-DHP) - A Potential
Proximate Carcinogenic Metabolite of Pyrrolizidine Alkaloids
†
†
†
§
†,
Xiaobo He, Qingsu Xia, Gonçalo Gamboa da Costa, Ge Lin, and Peter P. Fu *
†National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson,
AR 72079, United States
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§School of Biomedical Sciences, Faculty of Medicine, the Chinese University of Hong Kong,
Hong Kong
Running Title: Pyrrolizidine alkaloids, 1-CHO-DHP, potential tumorigenic metabolites
*
Corresponding Author: National Center for Toxicological Research, U.S.
Food and Drug Administration, Jefferson, AR 72079, United States
Tel: +1-870-543-7207. Fax: +1-870-543-7136.
E-mail: peter.fu@fda.hhs.gov.
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Graphic Abstract
H
Senecionine, Riddelliine,
Retrorsine, Intermedine,
Heliotrine, Senkirkine,
Dehydroretronecine
HO
C
O
P450
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-CHO-DHP
Platyphylline
HO
HO
H2C dG
HO
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H2C dG
N
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Liver Tumor
Initiation
?
H2C dA
H2C dA
N
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DHP-dA-4
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ABSTRACT:
Pyrrolizidine alkaloids (PAs) are phytochemicals present in more than 6000 plant species
worldwide; about half of the PAs are hepatotoxic, genotoxic, and carcinogenic. Because of their
wide exposure and carcinogenicity, the International Programme on Chemical Safety (IPCS)
concluded that PAs are a threat to human health and safety. We recently determined that PA-
induced liver tumor initiation is mediated by a set of four (±)-6,7-dihydro-7-hydroxy-1-
hydroxymethyl-5H-pyrrolizine (DHP)-DNA adducts, and proposed that these DHP-DNA
adducts are biomarkers of PA exposure and liver tumor initiation. To validate the generality of
this metabolic activation pathway and DHP-DNA adducts as biomarkers, it is significant to
identify reactive metabolites associated with this metabolic activation pathway. Segall et al.
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(Drug Metab Dispos 12, 68-71, 1984) previously reported that 1-formyl-7-hydroxy-6,7-dihydro-
5
H-pyrrolizine (1-CHO-DHP) is generated from the metabolism of senecionine by mouse liver
microsomes. In the present study, we examined the metabolism of seven hepatocarcinogenic
PAs (senecionine, intermedine, retrorsine, riddelliine, DHR, heliotrine, and senkirkine) and one
non-carcinogenic PA (platyphylline) by human, rat, and mouse liver microsomes. 1-CHO-DHP
was identified as a common metabolite from the metabolism of these hepatotoxic PAs, but not
from platyphylline. Incubation of 1-CHO-DHP with HepG2 and A549 cells produced the same
set of DHP-DNA adducts, which were identified by both LC/MS MRM mode and selected ion
monitoring analyses through comparison to synthetic standards. In the incubation medium of 1-
CHO-DHP treated HepG2 cells, both DHP and 7-cysteine-DHP were formed, which were
capable of binding to cellular DNA to produce DHP-DNA adducts. These results suggest that 1-
CHO-DHP is a proximate DNA metabolite of genotoxic and carcinogenic PAs.
Key words: pyrrolizidine alkaloids; secondary pyrrolic metabolite; DNA adducts; biological
biomarker; LC/MS/MS
INTRODUCTION
In the plant kingdom, plants produce phytochemical secondary metabolites as a constitutive
plant defense mechanism against the selective pressure of herbivory.1-4 Most phytochemicals
exhibit therapeutic and/or toxic activities. Pyrrolizidine alkaloids (PAs) and PA N-oxides are
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widespread and toxic phytochemicals, rendering PA-containing plants as being the most
common poisonous plant species affecting livestock, wildlife, and humans.1, 3-5 To date, many
PAs have been found to exhibit cytotoxicity and genotoxicity, and to induce liver tumors in
experimental animals.1-4 Monocrotaline, riddelliine, and lasiocarpine are classified as Group 2B,
possible human carcinogens by the IARC.6, 7 Through the U.S. National Toxicology Program
(NTP) carcinogenicity bioassays, both lasiocarpine and riddelliine were determined to be rodent
carcinogens.8, 9 Riddelliine is also listed a "reasonably anticipated to be human carcinogen” in
the 13rd Report on Carcinogens.10
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PAs require metabolic activation to exert toxicity. Metabolism of toxic PAs generates
primary pyrrolic metabolites (dehydro-PAs) and the hydrolyzed metabolite DHP, which are able
to bind to proteins and DNA to form pyrrole-protein adducts,11-14 DNA-DNA cross-links, DNA-
protein cross-links,15-17 endogenous DNA adducts,18, 19 and exogenous DNA adducts2, 18, 20-22
leading to hepatocytoxicity and liver tumor initiation.
Recently, we established a common genotoxic mechanism mediated by a set of DHP-DNA
adducts, designated as DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4, leading to PA-
induced liver tumor initiation (Figure 1).2, 18, 20, 21 This common mechanism was involved in the
metabolism of seven hepatocarcinogenic PAs and riddelliine N-oxide in rats in vivo,2, 18, 20, 21 in
2
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the liver of male mice administered retrorsine, in the livers of cows accidently fed with PA-
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contaminated hay, and in a series of liver microsomal metabolism in vitro. Based on these
findings, we proposed that these DHP-DNA adducts are potential biomarkers of PA and PA N-
oxide exposure and liver tumor initiation.2, 21, 23-27 From the liver microsomal metabolism, four
additional DHP-DNA adducts, designated as DHP-dG-1, DHP-dG-2, DHP-dA-1, and DHP-dA-2
adducts, were also generated (Figure 1); however, to date these adducts have not been detected in
vivo.
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R
O
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O
O
HO
HO
H2C dG
HO
HO
H2C dG
N
PA
N
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CYP450
H2C dA
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H C dA
R
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R1
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CH -R
calf
CH2OH
CH2OH
CH2OH
in
thymus vitro
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dA
dG
HO
HO
H2C dG
HO
HO
H2C dG
N
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DNA
N
N
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N
N
+
dA
CH OH
2
H2C dA
2
H C dA
N
N
N
DHP-dA-2
DHP-dA-4
Figure 1. Metabolic activation of PAs mediated by the formation of DHP-dG-3, DHP-dG-4,
DHP-dA-3, and DHP-dA-4 adducts in vivo, and the additional formation of DHP-dG-1, DHP-
dG-2, DHP-dA-1, and DHP-dA-2 adducts in vitro.
We recently determined that besides dehydro-PAs and DHP, a series of secondary pyrrolic
metabolites can also bind to calf thymus DNA or cellular DNA in HepG2 and A549 cells to form
DHP-DNA adducts. 25, 27-29 These results suggest that secondary pyrrolic metabolites are
potential DNA reactive metabolites, and that there exist multiple activation pathways leading to
the formation of DHP-DNA adducts (Figure 1). Consequently, it is important to determine if
additional secondary pyrrolic metabolites are activated through this metabolic activation
pathway.
30
In 1984, Segall et al. reported that 1-formyl-7-hydroxy-6,7-dihydro-5H-pyrrolizine
(hydroxydanadal; 1-CHO-DHP) was formed from the metabolism of senecionine by mouse liver
1
4
microsomes. 1-CHO-DHP was also produced from the metabolism of [ C]monocrotaline in
perfused rat liver.31, 32 In the present study, we determined that 1-CHO-DHP is a general
metabolite, formed from the metabolism of a series of PAs, senecionine, intermedine, retrorsine,
riddelliine, DHR, heliotrine, senkirkine, and platyphylline, by human, rat, and mouse liver
microsomes. The structures of these PAs are shown in Figure 2. Incubation of 1-CHO-DHP in
HepG2 and A549 cells produced DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts.
These results suggest that 1-CHO-DHP is a DNA-reactive pyrrolic metabolite.
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HO CH2OH
O
HO CH OH
2
H
H
HO Me
HO CH(Me)2
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H3C
H C
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H
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H
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HO
CH OH
H3C
H
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CH3
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CH2
O
N
O
CH2
HO
CH2
H
N
H
N
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DHR
CH3
Platyphylline
Heliotrine
Senkirkine
Figure 2. Structures of senecionine, intermedine, retrorsine, riddelliine, DHR, heliotrine,
senkirkine, and platyphylline.
EXPERIMENTAL PROCEDURES
Caution: Riddelliine, retrorsine, monocrotaline, senecionine, intermedine, senkirkine,
heliotrine, dehydromonocrotaline, and dehydroretronecine (DHR) are carcinogenic in
laboratory animals, and thus should be handled with extreme care.
Chemicals. Monocrotaline, retrorsine, calf thymus DNA (sodium salt, type I), micrococcal
nuclease (MN), spleen phosphodiesterase (SPD), nuclease P1, β-nicotinamide adenine
dinucleotide 2’-phosphate, reduced (NADPH), 2'-deoxyguanosine (dG), and 2'-deoxyadenosine
(
dA) were purchased from Sigma-Aldrich (St. Louis, MO).
Retronecine was synthesized by the barium hydroxide-catalyzed hydrolysis of monocrotaline
as described previously.33 Dehydroretronecine (DHR) was prepared by dehydrogenation of
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retronecine with o-chloranil.33 DHP and dehydromonocrotaline were prepared as described
previously.34,35 7-GS-DHP and 7-cysteine-DHP were prepared as previously described.25,27
Riddelliine and platyphylline were obtained from America Custom Chemicals Corp. (San
Diego, CA, USA). Heliotrine was purchased from Accurate Chemical & Scientific Corp.
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(Westbury, NY). Intermedine was purchased from Cerilliant Corp. (Round Rock, TX).
Senkirkine was purchased from ChromDex (Irvine, CA, USA). Senecionine was purchased from
Extrasynthese (Genay, France).
The human hepatocarcinoma cell line HepG2 and human bronchoalveolar carcinoma cell line
A549 were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human,
rat, and mouse liver microsomes were purchased from BioreclamationIVT (Baltimore, MD).
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DHP-dG, DHP-dA, DHP-[ N ]dG, and DHP-[ N , C ]dA were synthesized as previously
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described.20, 36 All solvents used were HPLC grade or LC/MS grade.
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Synthesis of 1-CHO-DHP. Following the method of Culvenor et al. (1970), a reaction mixture
containing retronecine (100 mg) and manganese dioxide (2 g) in 10 mL chloroform at 4 °C was
stirred in a water-ice bath for 3.5 h. After the reaction, the manganese dioxide was filtered, and
the chloroform was removed under reduced pressure. The resulting residue was dissolved in
acetonitrile and separated by HPLC using a Luna 5 μm C18 (2) column (10 x 250 mm,
Phenomenex, Torrance, CA), eluting with 9% acetonitrile in water. The material contained in
the HPLC peak that eluted at 20.5 min had a UV-visible absorption spectrum with the highest
+
absorption at 260 nm (data not shown), a mass spectrum having the [M+H] ions at m/z 152
1
(
Figure S1), and the H NMR spectrum indicating containing an aldehyde functional group and a
necine base (Figure S2).
1
1
The H NMR assignments are: 1-CHO-DHP. H NMR (acetonitrile-d ): δ 2.30−2.37 (1H, m,
3
H6b), 2.76−2.84 (1H, m, H6a), 3.75 (1H, d, 7-OH, J = 3.4 Hz), 3.93 (1H, ddd, H5b, J = 8.9, 6.6,
4
.1 Hz), 4.13 (1H, ddd, H5a, J = 8.9, 6.4, 4.5 Hz), 5.32 (1H, dt, H7, J = 6.0, 3.2 Hz), 6.55 (1H, d,
H2, J = 2.3 Hz), 6.66 (1H, d, H3, J = 2.3 Hz), 9.74 (1H, s, CHO)
1
Thus, this product was unequivocally identified as 1-CHO-DHP. Also, the H NMR
spectrum (Figure S2) indicated that the synthetically prepared 1-CHO-DHP is highly pure.
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Metabolism of PAs by Human, Rat and Mouse Liver Microsomes. The metabolism of seven
hepatocarcinogenic PAs (senecionine, intermedine, retrorsine, riddelliine, DHR, heliotrine, and
senkirkine) and one non-carcinogenic PA (platyphylline) by pooled human liver microsomes,
male F344 rat, and male ICR/CD-1 mouse liver microsomes was conducted as previously
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described. HPLC conditions: flow rate: 1 mL/min; gradient program: 0-10 min: 2 mM formic
acid buffer, pH 8.0 adjusted with ammonia solution; 10-30 min: from 2 mM buffer to 20%
acetonitrile in the buffer; 30-40 min: from acetonitrile 20% to 80% in the buffer.
DHP-DNA Adducts Formation in Cultured HepG2 and A549 Cells Exposed to 1-CHO-
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DHP. Human HepG2 or A549 cells were cultured and then exposed to vehicle control (0.1%
DMSO in DMEM), 1-CHO-DHP, dehydromonocrotaline, DHR, or platyphylline at 50 µM for
2
4 h. After exposure, the cells were collected. DNA was extracted using Blood & Cell Culture
DNA Isolation kits (QIAGEN; Valencia, CA). The DNA was adjusted to 2 mg/mL and
enzymatically hydrolyzed to nucleosides for LC/MS/MS analysis as described previously.29
Quantitation of DHP-dG and DHP-dA Adducts by LC/MS/MS MRM Analysis.
The LC/MS/MS MRM analysis was under experimental conditions as previously described.23
Calibration Curves. Calibration curves for quantitation were generated by plotting the amounts
of standard compounds against the peak-area ratios of DHP-dG/dA standards to the
corresponding labeled internal standards.28,35
The calibration curves were linear over the range of 4-500 fmol on-column for DHP-dG-1,
DHP-dG-2, DHP-dA-1, and DHP-dA-2; 2-500 fmol for DHP-dG-3/4, and 0.5-500 fmol for
1
5
15
13
DHP-dA-4. Forty fmol of the DHP-[ N ]dG-3/4 and 10 fmol of the DHP-[ N , C ]-dA 3/4
5
5
10
were used as internal standards. The best curve fit for the calibration curves were achieved with
2
a weighting factor of 1/X, with the coefficients of determination (r ) for all analyses > 0.99.
Further Identification of DHP-dG and DHP-dA Adducts by LC/MS/MS Analysis. The
enzymatically hydrolyzed DNA sample was concentrated 9-fold under reduced pressure, and
subjected to LC/MS analysis in Selected Ion Monitoring (SIM) mode and Product Ion Scanning
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(
PIS) mode. Chromatographic profiles were obtained by LC-SIM-MS analysis of m/z 403
(DHP-dG) and m/z 387 (DHP-dA), then the retention time and fragmentation spectra of
chromatographic peaks for test samples were compared with those of the corresponding synthetic
standards by product ion scan analysis.
1
1
1
1
1
1
1
1
1
1
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RESULTS
Metabolism of PAs by Human, Rat and Mouse Liver Microsomes. Metabolism of 7
individual carcinogenic PAs and one non-carcinogenic platynecine-type PA by pooled human
liver microsomes, male F344 rat, and male ICR/CD-1 mouse liver microsomes were conducted
at 37 °C for 30 min. These PAs include: five retronecine-type PAs (riddelliine, senecionine,
retrorsine, intermedine, and DHR), one heliotridine-type PA (heliotrine), one otonecine-type PA
(senkirkine), and one platynecine-type PA (platyphylline).
From each metabolism, the resulting incubation mixture (60 µL) was analyzed by reversed-
phase HPLC coupled with a diode array detector and/or LC/MS. The resulting metabolites,
including 1-CHO-DHP, PA N-oxide, and DHP, as well as the unreacted substrate (PA), were
identified by comparison of their HPLC retention times, UV-visible absorption spectra, and mass
spectra with those of the synthetically prepared or purchased authentic standards. The HPLC
chromatographic profiles from the metabolism of riddelliine by male mouse liver microsomes
are shown in Figure 3 Panel A and B (monitored at 220 and 260 nm respectively). The riddelliine
metabolite eluting at 26.6min (Figure 3A) had its retention time, UV-visible absorption spectrum
(Figure 3B, inserted spectrum), and mass spectrum identical with those of the synthetically
prepared 1-CHO-DHP, and thus this metabolite was identified as 1-CHO-DHP.
The chromatographic peaks eluted at 24.0, 31.5, and 36.0 min were similarly determined to
be DHP, riddelliine N-oxide, and the recovered riddelliine substrate, respectively.
The HPLC chromatographic profiles from the metabolism of senecionine, intermedine, and
retrorsine detected at 260 nm are each shown in Figure 3 Panel C, D, and E, respectively, from
which the 1-CHO-DHP metabolite was similarly identified. 1-CHO-DHP was also formed from
the metabolism of DHR, senkirkine, and heliotrine by human, male F344 rat, or male ICR/CD-1
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mouse liver microsomes. In contrast, 1-CHO-DHP was not detected from the metabolism of
platyphylline by rat, mouse, or human liver microsomes (data not shown).
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Figure 3. HPLC profiles of the 1-CHO-DHP metabolite produced from the mouse liver
microsomal metabolism of (A) riddelliine measured at 220 nm; (B) riddelliine measured at 260
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nm and UV-visible spectrum of 1-CHO-DHP (inset); (C) senecionine measured at 260 nm; (D)
intermedine measured at 260 nm; and (E) retrorsine measured at 260 nm.
Identification of DHP-DNA Adducts Formed in HepG2 Cells Exposed to 1-CHO-DHP.
1
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1
1
1
1
1
1
1
1
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1
. LC/MS/MS in MRM Mode Analysis. The DHP-dG and DHP-dA adducts formed in the HepG2
cells exposed to 1-CHO-DHP were identified and quantified by the LC/MS/MS MRM mode and
1
5
15
13
using the DHP-[ N ]dG and DHP-[ N , C ]dA as labeled internal standards. The HPLC
5
5
10
elution conditions used a linear gradient of 15 - 65% methanol in water (containing 2 mM
ammonium acetate, pH 5) over 35 min. The resulting LC/MS/MS MRM chromatograms, shown
in Figure 4, demonstrated that DHP-dG-3/4 and DHP dA-3/4 were formed.
Figure 4A and Figure 4C show the mass chromatogram traces of the isotope-labeled internal
standards. The mass chromatograms on Figure 4B and Figure 4D provide evidence for the
formation of DHP-dG-3/4 and DHP-dA-3/4, but not DHP-dG-1/2 nor DHP-dA-1/2 adducts in
HepG2 cells exposed to 1-CHO-DHP.
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Figure 4. LC/MS/MS MRM chromatograms. (A) the isotope-labeled internal standards DHP-dGs
with the transition of the [M+H] precursor ion m/z 408 to the selected product ion m/z 274. (C)
the labeled internal standards DHP-dAs with the transition of m/z 402 to m/z 263. (B) and (D)
the DHP-dG-3/4 and DHP-dA-3/4 adducts formed in HepG2 cells exposed to 1-CHO-DHP with
+
elution condition: 5 – 40 min, 15-65% CH OH.
3
2
. Product Ion Scan Analysis of DHP-dG and DHP-dA Adducts. The identity of the DHP-dG-3/4
adducts formed in HepG2 cells exposed to 1-CHO-DHP was further confirmed by comparison of
their fragmentation pattern (Figure 5A) with the fragmentation pattern of the authentic synthetic
standard of these adducts (Figure 5B), obtained by daughter scan analysis of theprotonated
molecular ion at m/z 403. The fragmentation patterns were essentially identical, with prominent
ions at m/z 385 ascribable to the loss of water from the pyrrolizidine moiety, followed by the loss
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of the deoxyribose moiety to afford ions at m/z 269. Likewise, the nature of the DHP-dA-3/4
adducts formed in HepG2 cells exposed to 1–CHO-DHP was also confirmed by comparison of
the fragmentation of their protonated molecular ions (m/z 387), affording prominent ions at m/z
3
69 resulting from the loss of water from the pyrrolizidine moiety, followed by loss of the
1
1
1
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0
deoxyribose moiety to afford ions at m/z 253 (Figure 6A, 6B).
269.3
100
90
80
70
60
A
50
40
30
20
10
403.0
384.8
0
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z
2
68.7
100
O
N
[
MH]+
B
90
80
70
60
50
40
30
20
10
m/z 269N
m/z 403
NH
N
O
HO
NH
385.4
N
m/z 385
HO
OH
403.2
0
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z
Figure 5. (A) Product ion mass spectrum (10 eV) of m/z 403 ions stemming from DHP-dG
adducts eluting at 28.5 min formed in HepG2 cells exposed to 1-CHO-DHP; B) product ion mass
spectrum (15 eV) of synthetic DHP-dG-3/4 standards and proposed main fragmentation
mechanisms (inset).
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53.0
1
00
9
0
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A
8
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6
387.3
368.9
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100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z
[MH]+
387
253.4
100
m/z 369
N
B
9
0
80
70
60
NH
N
HO
N
N
368.7
5
4
3
2
1
0
0
0
0
0
387.3
HO
N
O
m/z 253
OH
0
1
00 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z
Figure 6. (A) Product ion mass spectrum of m/z 387 ions stemming from DHP-dA adducts
eluting at 35.0 min formed in HepG2 cells exposed to 1-CHO-DHP; B) product ion mass
spectrum of synthetic DHP-dA-3/4 standards and proposed main fragmentation mechanisms
(
inset).
Identification of DHP-DNA adducts formed in A549 cells exposed to 1-CHO-DHP. Similar
to the adducts formed from HepG2 cells (Figure 4), the identification and quantitation of DHP-
dG-3/4 and DHP-dA-3/4 adducts formed in A549 cell exposed to 1-CHO-DHP were determined
(
Figure 7).
Levels of DHP-dG and DHP-dA adducts formed in HepG2 and A549 cells treated with
individual dehydromonocrotaline, 1-CHO-DHP, DHR, and platyphylline are summarized in
Table 1.
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9
0
Figure 7. LC/MS/MS MRM chromatograms. (A) the isotope-labeled internal standards DHP-
+
dGs with the transition of the [M+H] precursor ion m/z 408 to the selected product ion m/z 274.
(C) the labeled internal standards DHP-dAs with the transition of m/z 402 to m/z 263. (B) and
(D) the DHP-dG-3/4 and DHP-dA-3/4 adducts formed in A549 cells exposed to 1-CHO-DHP
with elution condition: 5 – 40 min, 15-65% CH OH.
3
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Table 1. Levels of DHP-dG and DHP-dA Adducts Formed in HepG2 and A549 Cells Treated
with 1-CHO-DHP at 50 µM for 24 Hours.
8
a
Levels of DHP-dG and DHP-dA / 10 nucleotides in HepG2 cells
Total
DHP-dG &
DHP-dA
chemicals
DHP-dG-1 &
DHP-dG-2
DHP-dG-3 &
DHP-dG-4
DHP-dA-1 &
DHP-dA-2
DHP-dA-3 &
DHP-dA-4
Vehicle Control
Dehydromonocrotaline
1-CHO-DHP
<LODb
<LOD
<LOD
<LOD
<LOD
<LOD
11.3 ± 1.2
18.0 ± 1.6
6.6 ± 1.0
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
13.2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1.9 ± 0.2
2.3 ± 0.3
1.2 ± 0.1
20.3
DHR
7.8
Platyphylline
<LOD
<LOD
<LOD
8
a
Levels of DHP-dG and DHP-dA / 10 nucleotides in A549 cells
chemicals
DHP-dG-1 &
DHP-dG-2
DHP-dG-3 &
DHP-dG-4
DHP-dA-1 &
DHP-dA-2
DHP-dA-3 & DHP-dG &
DHP-dA-4
DHP-dA
Vehicle Control
<LODb
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
9.54
Dehydromonocrotaline
8.08 ± 0.85
7.70 ± 1.19
3.95 ± 0.42
<LOD
1.46 ± 0.16
1.44 ± 0.15
0.73 ± 0.20
<LOD
1
-CHO-DHP
DHR
9.14
4.68
Platyphylline
<LOD
a
Aliquots of DNA (45 μg) were assayed by HPLC-ES-MS/MS. The data are presented as the mean ± SD of three
b
separate experiments, each performed in triplicate. LOD = limit of detection based upon the analysis of 45 μg DNA
by HPLC-ES-MS/MS. Under experimental conditions, the LOD of DHP-dG-1/2: 0.71, DHP-dG-3/4: 0.71, DHP-
8
dA-1/2: 0.14, and DHP-dA-3/4: 0.14 adducts per 10 nucleotides, separately.
LC/MS/MS detection of DHP and 7-cysteine-DHP metabolites formed in the incubation
medium of HepG2 and A549 cells exposed to 1-CHO-DHP. In addition to the quantitation of
DHP-DNA adducts in HepG2 and A549 cells exposed to 1-CHO-DHP, the metabolites DHP and
7
-cysteine-DHP in the intracellular medium were identified by comparison of their LC/MS/MS
MRM mode profiles with those of the synthetic standards (Figure 8). The LC/MS MRM
chromatograms of the synthetic standards DHP, 7-cysteine-DHP, and 7-GS-DHP are shown in
Figure 8A. The LC/MS MRM chromatograms of the intracellular medium of the HepG2 cells
exposed to 50 µM 1-CHO-DHP indicate that both DHP and 7-cysteine-DHP, but not 7-GS-DHP,
were produced (Figure 8B). The identical results were also obtained from the medium of A549
cells exposed to -CHO-DHP (Figure 8C).
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9
0
Figure 8. LC/MS MRM chromatograms of DHP, 7-GS-DHP, and 7-cysteine-DHP from (A)
organic synthesis; (B) intracellular detection on HepG2 cells exposed to 50 µM 1-CHO-DHP;
and (C) intracellular detection on A549 cells exposed to 100 µM 1-CHO-DHP. The MRM
+
scheme was employed involving transitions of the [M+H] precursor ions to selected product
ions, with the following values: m/z 154 → 106 for DHP; m/z 443 → 118 for 7-GS-DHP; and
m/z 257 → 151 for 7-cysteine-DHP.
DISCUSSION
In this study, we demonstrated that 1-CHO-DHP is a common metabolite formed from the
human, rat, and mouse liver microsomal metabolism of a series of PAs, and that incubation of 1-
CHO-DHP in HepG2 and A549 cells produced the same set of DHP-DNA adducts (DHP-dG-3/4
and DHP-dA-3/4), biomarkers of PA-induced liver tumor initiation. The reactive dehydro-PA
metabolites are either 7,9-diesters or a 7- and 9-monoesters with the ester group linked to the
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necine base.1,4 On the other hand, the secondary pyrrolic metabolites so far identified, stemming
from mammalian metabolism of dehydro-PAs with amino acids or glutathione, carry an S-ether
linkage, an O-ether linkage, or an N-ether linkage at the C7 or C9 position of the necine base.2,4,
2
8
We have now demonstrated that 1-CHO-DHP, a novel pyrrolic metabolite carrying a different
0
1
2
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7
8
9
0
1
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8
9
0
reactive functional group at the C1 position of the necine moiety, is also capable of reacting with
DNA to afford stable covalent DNA adducts.
The mechanism of the formation of DHP-DNA adducts in HepG2 and A549 cells treated
with 1-CHO-DHP remains to be elucidated. In order to form DHP-dG-3/4 or DHP-dA-3/4
adducts, the aldehyde carbon needs to react with the exocyclic nitrogen of 2’-deoxyguanosine, or
2’-deoxyadenosine, presumably through an electrophilic reaction. In order to exclude the
possibility that under our experimental conditions DHP-DNA adducts might have been formed
from other reactive pyrrolic metabolites, such as DHR, present in an impure 1-CHO-DHP
substrate, we have ensured the purity of the 1-CHO-DHP by an exhaustive analysis conducted by
1
HPLC with UV detection, mass spectrometry, and H NMR (Figure S2).
In the incubation medium of HepG2 cells treated with 1-CHO-DHP, we identified DHP and
7
-cysteine-DHP, metabolites that have previously been demonstrated to be capable of reacting
with DNA to afford covalent DNA adducts.25,28,29 In the present study, these two metabolites
were also identified in the incubation medium of A549 cells treated with 1-CHO-DHP. Thus,
one of the possible activation pathways that 1-CHO-DHP may lead to the formation of covalent
DHP-DNA adducts is the reductive metabolism of 1-CHO-DHP into DHP and 7-cysteine-DHP,
which are capable of reacting with DNA. Since DHP-DNA adducts are known to be involved in
PA-induced liver tumor initiation, we hypothesize that 1-CHO-DHP may be a proximate
metabolite involved in PA hepatocarcinogenesis, as proposed in Figure 9.
At the present, it is not known whether or not there are other mechanisms by which 1-CHO-
DHP may bind to DNA leading to the formation of the DHP-dG-3/4 or DHP-dA-3/4 adducts. For
instance, an alternative mechanism is that these DHP-DNA adducts are formed by reacting the –
CHO group of 1-CHO-DHP with the -NH groups of deoxyguanosine and deoxyadenosine to
2
form –C=N– linkages which are then reduced to form the DHP-DNA adducts. This warrants
further investigation.
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Carcinogenic PAs
P450
R
HO
CH OH
2
O
N
O
DHP
O
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O
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Dehydro-PA
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1-CHO-DHP
?
?
HO
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NH2
HO
CH OH
2
Cysteine
S
CH OH
2
N
DHP
N
DNA
7-Cysteine-DHP
DNA
HO
HO
H2C dG
HO
HO
H2C dG
N
N
Liver Tumor
Initiation
H2C dA
H2C dA
N
N
DHP-dA-4
Figure 9. Proposed metabolic activation pathway mediated by the 1-CHO-DHP as a proximate
carcinogenic metabolite.
We previously reported that there are a series of secondary pyrrolic metabolites that can bind
to DNA leading to the formation of DHP-dG-3/4 and DHP-dA-3/4 adducts.28
These DNA reactive secondary pyrrolic metabolites include: DHP, 7-methoxy-DHP, 7-GS-DHP,
7-cysteine-DHP, and N-acetylcysteine-DHP.24, 25, 27-29 The data reported herein support the
hypothesis that there may exist multiple concurrent activation pathways that can lead to the
formation of DHP-DNA adducts in vitro (Figure 1). With the inclusion of 1-CHO-DHP, there
are in total six DNA reactive secondary pyrrolic metabolites that may be involved in the
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metabolic activation of PA, leading to cytotoxicity, genotoxicity, and carcinogenicity. In
contrast with all other previously identified PA metabolites, 1-CHO-DHP possesses the
aldehyde group in the C1 position of the necine base28 Thus, for the study of structure-activity
relationship of PAs, it is significant to investigate the metabolic activation pathway mediated by
this metabolite.
0
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It is noteworthy that1-CHO-DHP is used by the males of a number of species of butterflies
and moths as a courtship pheromone. The butterflies and moths also convert a small amount of
the PAs ingested to 1-CHO-DHP and several other volatile DHP metabolites (e.g. danaidal,
danaidone). Apparently, the butterflies and moths are unaware of the likely health risks posed by
using 1-CHO-DHP and the other volatile DHP metabolites as pheromones.38-40
ASSOCIATED CONTENT
Supporting Information
1
positive mass spectra in full scan mode and H NMR spectrum of 1-CHO-DHP prepared from
organic synthesis. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Funding
Funding was provided by an internal FDA fund and was supported in part by appointment (X.H.)
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 FDA.
ACKNOWLEDGMENTS
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.
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ABBREVIATIONS
DHP, (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine; DHP-dG-1 and DHP-dG-2, a
pair of epimers of 7-(deoxyguanosin-N2-yl)dehydrosupinidine adducts; DHP-dG-3 and DHP-
dG-4, a pair of epimers of 7-hydroxy-9-(deoxyguanosin-N2-yl)dehydrosupinidine adducts; DHP-
dA-1 and DHP-dA-2, a pair of epimers of 7-(deoxyadenosin-N6-yl)dehydrosupinidine adducts;
DHP-dA-3 and DHP-dA-4, a pair of epimers of 7-hydroxy-9-(deoxyadenosin-N6-
1
1
1
1
1
1
1
1
1
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5
5
5
5
5
5
6
0
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2
3
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5
6
7
8
9
0
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2
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6
7
8
9
0
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2
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9
0
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9
0
yl)dehydrosupinidine adducts; 1-CHO-DHP, 1-formyl-7-hydroxy-6,7-dihydro-5H-pyrrolizine;
DHR (dehydroretronecine), (+)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine; SIM,
selected ion monitoring; MRM, selected reaction monitoring; LOD, limit of detection.
REFERENCES
(1)
Edgar, J. A., Molyneux, R. J., and Colegate, S. M. (2015) Pyrrolizidine alkaloids:
potential role in the etiology of cancers, pulmonary hypertension, congenital anomalies,
and liver disease. Chem. Res. Toxicol. 28, 4-20.
(
2)
3)
Fu, P. P. (2017) Pyrrolizidine Alkaloids: Metabolic activation pathways leading to liver
tumor initiation. Chem. Res. Toxicol. 30, 81-93.
(
Fu, P. P., and Xia, Q. (2014) Assessment of safety and quality assurance of herbal dietary
supplements, In Nutraceutical and Functional Food Regulations in the United States and
Around the World (Bagchi, D., Ed.) pp 151-168, Elsevier/Academic Press, New York.
Mattocks, A. R. (1986) Chemistry and Toxicology of Pyrrolizidine Alkaloids., Academic
Press, London, NY.
(
4)
5)
(
Smith, L. W., and Culvenor, C. C. J. (1981) Plant sources of hepatotoxic pyrrolizidine
alkaloids. J. Nat. Prod. 44, 129-152.
(6)
IARC. (1976) Pyrrolizidine alkaloids, In IARC Monograph on the evaluation of
carcinogenic risk of chemicals to man - Some naturally occurring substance,
International Agency for Research in Cancer, Lyon, France.
(
7)
8)
IARC. (2002) Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and
Styrene, In IARC Monographs on the evaluation of carcinogenic risks of chemicals to
humans pp 153-168, International Agency for Research in Cancer, Lyon, France.
NTP. (1978) Bioassay of lasiocarpine for possible carcinogenicity. Natl Cancer Inst
Carcinog Tech Rep Ser 39, 1-66.
(
(9)
NTP. (2003) Toxicology and carcinogenesis studies of riddelliine (CAS No. 23246-96-0)
in F344/N rats and B6C3F1 mice (gavage studies). Natl Toxicol Program Tech Rep Ser,
1
-280.
(10) NTP. (2014) Riddelliine in Report on Carcinogens 13th Edition National Toxicololgy
Program, Research Triangle Park, NC.
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Chemical Research in Toxicology
Page 22 of 23
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
11) Lin, G., Wang, J. Y., Li, N., Li, M., Gao, H., Ji, Y., Zhang, F., Wang, H., Zhou, Y., Ye,
Y., Xu, H. X., and Zheng, J. (2011) Hepatic sinusoidal obstruction syndrome associated
with consumption of Gynura segetum. J. Hepatol. 54, 666-673.
(12) Ma, J., Xia, Q., Fu, P. P., and Lin, G. (2018) Pyrrole-protein adducts - A biomarker of
pyrrolizidine alkaloid-induced hepatotoxicity. J Food Drug Anal. 26, 965-972.
(13) Ruan, J., Gao, H., Li, N., Xue, J., Chen, J., Ke, C., Ye, Y., Fu, P. P.-C., Zheng, J., Wang,
J., and Lin, G. (2015) Blood pyrrole-protein adducts–A biomarker of pyrrolizidine
alkaloid-induced liver injury in humans. J. Environ. Sci. Health, Part C 33, 404-421.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
14) Xia, Q., Zhao, Y., Lin, G., Beland, F. A., Cai, L., and Fu, P. P. (2016) Pyrrolizidine
alkaloid-protein adducts-potential non-invasive biomarkers of pyrrolizidine alkaloid-
induced liver toxicity and exposure. Chem. Res. Toxicol. 29, 1282-1292.
(15) Coulombe, R. A., Jr., Drew, G. L., and Stermitz, F. R. (1999) Pyrrolizidine alkaloids
crosslink DNA with actin. Toxicol. Appl. Pharmacol. 154, 198-202.
(
16) Kim, H.-Y., Stermitz, F. R., and Coulombe, R. A. J. (1995) Pyrrolizidine alkaloid-
induced DNA-protein cross-links. Carcinogenesis 16, 2691-2697.
17) Kim, H.-Y., Stermitz, F. R., Li, J. K.-K., and Coulombe, R. A. J. (1999) Comparative
DNA cross-linking by activated pyrrolizidine alkaloids. Food Chem. Toxicol. 37, 619-
625.
(
(18) Fu, P. P., Xia, Q., Lin, G., and Chou, M. W. (2004) Pyrrolizidine alkaloids--genotoxicity,
metabolism enzymes, metabolic activation, and mechanisms. Drug Metab. Rev. 36, 1-55.
(
19) Miranda, C. L., Reed, R. L., Cheeke, P. R., and Buhler, D. R. (1981) Protective effects of
butylated hydroxyanisole against the acute toxicity of monocrotaline in mice. Toxicol.
Appl. Pharmacol. 59, 424-430.
(
20) Fu, P. P., Chou, M. W., Churchwell, M., Wang, Y., Zhao, Y., Xia, Q., Gamboa da Costa,
G., Marques, M. M., Beland, F. A., and Doerge, D. R. (2010) High-performance liquid
chromatography electrospray ionization tandem mass spectrometry for the detection and
quantitation of pyrrolizidine alkaloid-derived DNA adducts in vitro and in vivo. Chem.
Res. Toxicol. 23, 637-652.
(21) Xia, Q., Zhao, Y., Von Tungeln, L. S., Doerge, D. R., Lin, G., Cai, L., and Fu, P. P.
(2013) Pyrrolizidine alkaloid-derived DNA adducts as a common biological biomarker of
pyrrolizidine alkaloid-induced tumorigenicity. Chem. Res. Toxicol. 26, 1384-1396.
(22) Zhu, L., Xue, J., Xia, Q., Fu, P. P., and Lin, G. (2017) The long persistence of
pyrrolizidine alkaloid-derived DNA adducts in vivo: kinetic study following single and
multiple exposures in male ICR mice. Arch. Toxicol. 91, 949-965.
(
23) Fu, P. P., Xia, Q., He, X., Barel, S., Edery, N., Beland, F. A., and Shimshoni, J. A. (2017)
Detection of pyrrolizidine alkaloid DNA adducts in livers of cattle poisoned with
heliotropium europaeum. Chem. Res. Toxicol. 30, 851-858.
(24) He, X., Ma, L., Xia, Q., and Fu, P. P. (2016a) 7-N-Acetylcysteine-pyrrole conjugate—A
potent DNA reactive metabolite of pyrrolizidine alkaloids. J Food and Drug Analysis 24,
6
82-694.
(
25) He, X., Xia, Q., Ma, L., and Fu, P. P. (2016b) 7-Cysteinyl-pyrrole conjugate-a new
potential DNA reactive metabolite of pyrrolizidine alkaloids. J Environ. Sci.Health, Part
C 34, 57-76.
(
26) He, X., Xia, Q., Woodling, K., Lin, G., and Fu, P. P. (2017) Pyrrolizidine alkaloid-
derived DNA adducts are common toxicological biomarkers of pyrrolizidine alkaloid N-
oxides. J Food Drug Anal. 25, 984-991.
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Chemical Research in Toxicology
1
2
3
4
5
6
7
8
9
(
(
(
(
27) Xia, Q., Ma, L., He, X., Cai, L., and Fu, P. P. (2015) 7-Glutathione pyrrole adduct: a
potential DNA reactive metabolite of pyrrolizidine alkaloids. Chem. Res. Toxicol. 28,
6
15-620.
28) Xia, Q., He, X., Ma, L., Chen, S., and Fu, P. P. (2018) Pyrrolizidine alkaloid secondary
pyrrolic metabolites construct multiple activation pathways leading to DNA adduct
formation and potential liver tumor initiation. Chem. Res. Toxicol. 31, 619-628.
29) He, X., Xia, Q., and Fu, P. P. (2017) 7-Glutathione-pyrrole and 7-cysteine-pyrrole are
potential carcinogenic metabolites of pyrrolizidine alkaloids. J Environ. Sci. Health, Part
C 35, 69-83.
30) Segall, H. J., Dallas, J. L., and Haddon, W. F. (1984) Two dihydropyrrolizine alkaloid
metabolites isolated from mouse hepatic microsomes in vitro. Drug Metab. Dispos. 12,
68-71.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(31) Lame, M. W., Jones, A. D., Morin, D., and Segall, H. J. (1991) Metabolism of
[14C]monocrotaline by isolated perfused rat liver. Drug Metab. Dispos. 19, 516-524.
(32) Lame, M. W., Jones, A. D., Morin, D., Segall, H. J., and Wilson, D. W. (1995) Biliary
excretion of pyrrolic metabolites of [14C]monocrotaline in the rat. Drug Metab. Dispos.
2
3, 422-429.
(33) Yang, Y., Yan, J., Churchwell, M., Beger, R., Chan, P., Doerge, D. R., Fu, P. P., and
Chou, M. W. (2001) Development of a (32)P-postlabeling/HPLC method for detection of
dehydroretronecine-derived DNA adducts in vivo and in vitro. Chem. Res. Toxicol. 14,
9
1-100.
(
34) Yang, Y.-C., Yan, J., Doerge, D. R., Chan, P.-C., Fu, P. P., and Chou, M. W. (2001)
Metabolic activation of the tumorigenic pyrrolizidine alkaloid, riddelliine, leading to
DNA adduct formation in vivo. Chem. Res. Toxicol. 14, 101-109.
(35) Zhao, Y., Xia, Q., Yin, J. J., Lin, G. and Fu, P. P. (2011) Photoirradiation of
dehydropyrrolizidine alkaloids-Formation of reactive oxygen species and induction of
lipid peroxidation. Toxicol. Lett. 205, 302-309.
(
36) Zhao, Y., Xia, Q., Gamboa da Costa, G., Yu, H., Cai, L., and Fu, P. P. (2012) Full
structure assignments of pyrrolizidine alkaloid DNA adducts and mechanism of tumor
initiation. Chem. Res. Toxicol. 25, 1985-1996.
(
37) Culvenor, C. C. J., Edgar, J. A., Smith, L. W., and Tweeddale, H. J. (1970)
Dihydropyrrolizines III. Preparation and reactions of derivatives related to pyrrolizidine
alkaloids. Aust. J. Chem. 23, 1853-1867.
(
38) Edgar, J. A., Culvenor, C. C., and Smith, L. W. (1971) Dihydropyrrolizine derivatives in
the ‘hair-pencil’ secretions of danaid butterflies. Experientia 27, 761-762.
39) Culvenor, C., and Edgar, J. (1972) Dihydropyrrolizine secretions associated with
coremata ofUtetheisa moths (family Arctiidae). Experientia 28, 627-628.
(
(40) Edgar, J. A., and Culvenor, C. C. (1974) Pyrrolizidine ester alkaloid in danaid butterflies.
Nature 248, 614-616.
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