Pyrrolizidine Alkaloid Secondary Pyrrolic Metabolites Construct Multiple Activation Pathways Leading to DNA Adduct Formation and Potential Liver Tumor Initiation

Article abstract of DOI:10.1021/acs.chemrestox.8b00096

Pyrrolizidine alkaloids (PAs) and their N-oxide derivatives are hepatotoxic, genotoxic, and carcinogenic phytochemicals. PAs induce liver tumors through a general genotoxic mechanism mediated by a set of four (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts. To date, the primary pyrrolic metabolites dehydro-PAs, their hydrolyzed metabolite DHP, and two secondary pyrrolic metabolites 7-glutathione-DHP (7-GS-DHP) and 7-cysteine-DHP are the known metabolites that can generate these DHP-DNA adducts in vivo and/or in PA-treated cells. Secondary pyrrolic metabolites are formed from the reaction of dehydro-PAs with glutathione, amino acids, and proteins. In this investigation, we determined whether or not more secondary pyrrolic metabolites can bind to calf thymus DNA and to cellular DNA in HepG2 cells resulting in the formation of DHP-DNA adducts using a series of secondary pyrrolic metabolites (including 7-methoxy-DHP, 9-ethoxy-DHP, 9-valine-DHP, 7-GS-DHP, 7-cysteine-DHP, and 7,9-diglutathione-DHP) and synthetic pyrroles for study. We found that (i) many secondary pyrrolic metabolites are DNA reactive and can form DHP-DNA adducts and (ii) multiple activation pathways are involved in producing DHP-DNA adducts associated with PA-induced liver tumor initiation. These results suggest that secondary pyrrolic metabolites play a vital role in the initiation of PA-induced liver tumors.

Full text of DOI:10.1021/acs.chemrestox.8b00096

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Article
Pyrrolizidine Alkaloid Secondary Pyrrolic Metabolites
Construct Multiple Activation Pathways Leading to DNA
Adduct Formation and Potential Liver Tumor Initiation
Qingsu Xia, Xiaobo He, Liang Ma, Shoujun Chen, and Peter P. Fu
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00096 • Publication Date (Web): 01 Jun 2018
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Pyrrolizidine Alkaloid Secondary Pyrrolic Metabolites Construct Multiple Activation
Pathways Leading to DNA Adduct Formation and Potential Liver Tumor Initiation
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Qingsu Xia, Xiaobo He, Liang Ma, Shoujun Chen, and Peter P. Fu*
National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR
72079, United States
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* Corresponding Author: National Center for Toxicological Research, U.S.
Food and Drug Administration, Jefferson, AR 72079, United States
Tel: +1ꢀ870ꢀ543ꢀ7207. Fax: +1ꢀ870ꢀ543ꢀ7136.
Eꢀmail: peter.fu@fda.hhs.gov.
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Table of Content
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R
R
O
O
HO
OH
P450
O
O
H2O
O
O
O
O
N
DHP
N
N
PA
Dehydro-PA
Cellular Nucleophile
HO
Nu:
Nu:
OH
Nu:
N
Nu:
N
N
Secondary pyrrolic metabolites
DHP-dG-3 + DHP-dG-4
DHP-dA-3 + DHP-dA-4
Liver tumors
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ABSTRACT
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Pyrrolizidine alkaloids (PAs) and their Nꢀoxide derivatives are hepatotoxic, genotoxic, and
carcinogenic phytochemicals. PAs induce liver tumors through a general genotoxic mechanism
mediated by a set of four (±)ꢀ6,7ꢀdihydroꢀ7ꢀhydroxyꢀ1ꢀhydroxymethylꢀ5Hꢀpyrrolizine (DHP)ꢀ
derived DNA adducts. To date, the primary pyrrolic metabolites, dehydroꢀPAs, their hydrolyzed
metabolite, DHP, and two secondary pyrrolic metabolites, 7ꢀglutathioneꢀDHP (7ꢀGSꢀDHP) and
7ꢀcysteineꢀDHP, are the known metabolites that can generate these DHPꢀDNA adducts in vivo
and/or in PAꢀtreated cells. Secondary pyrrolic metabolites are formed from the reaction of
dehydroꢀPAs with glutathione, amino acids, and proteins. In this investigation, we determined
whether or not more secondary pyrrolic metabolites can bind to calf thymus DNA and to cellular
DNA in HepG2 cells resulting in the formation of DHPꢀDNA adducts by using a series of
secondary pyrrolic metabolites (including 7ꢀmethoxyꢀDHP, 9ꢀethoxyꢀDHP, 9ꢀvalineꢀDHP, 7ꢀ
GSꢀDHP, 7ꢀcysteineꢀDHP, and 7,9ꢀdiꢀglutathioneꢀDHP) and synthetic pyrroles for study. We
found that: (i) many secondary pyrrolic metabolites are DNA reactive, can form DHPꢀDNA
adducts; and (ii) multiple activation pathways are involved in producing DHPꢀDNA adducts
associated with PAꢀinduced liver tumor initiation. These results suggest that secondary pyrrolic
metabolites play a vital role in the initiation of PAꢀinduced liver tumors.
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Keywords: Pyrrolizidine alkaloid; secondary pyrrolic metabolites; DHP; DHPꢀDNA adducts
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INTRODUCTION
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Pyrrolizidine alkaloids (PAs) and their Nꢀoxides are phytochemicals present in thousands of
plant species worldwide; with approximately half of these compounds are hepatototoxic,
genotoxic, and tumorigenic.1ꢀ12 Many herbal plants, herbal dietary supplements, and staple foods
contain carcinogenic PAs and PA Nꢀoxides.2, 5, 7, 8, 10, 11, 13ꢀ17 It has been reported that a number
of PAs and PA pyrrolic metabolites are able to induce tumors, particularly liver tumors, in
rodents. Accordingly, the International Programme on Chemical Safety (IPCS) has concluded
that PAs are a threat to human health and safety.7 Monocrotaline, riddelliine, and lasiocarpine
are classified as possible human carcinogens by the International Agency for Research on Cancer
(IARC).18, 19 The U.S. National Toxicology Program (NTP) listed riddelliine as "reasonably
anticipated to be a human carcinogen” in the NTP 12th Report of Carcinogens in 2014.20
Upon metabolism, PAs and PA Nꢀoxides generate primary pyrrolic reactive metabolites,
dehydropyrrolizidine alkaloids (dehydroꢀPAs), which in the aqueous medium rapidly hydrolyze
to (+/ꢀ)ꢀ6,7ꢀdihydroꢀ7ꢀhydroxyꢀ1ꢀhydroxymethylꢀ5Hꢀpyrrolizine (DHP).2, 6, 8, 21 DehydroꢀPAs
and DHP are able to react with DNA, proteins, and amino acids resulting in forming DHPꢀDNA
and DHPꢀprotein adducts associated with cytotoxicity, genotoxicity, and carcinogenicity.2, 3, 6, 8
We recently determined that the metabolic activation of 12 tumorigenic PAs and PA Nꢀ
oxides is by the identical biochemical mechanism, mediated by the formation of four DHPꢀDNA
adducts (designated as DHPꢀdGꢀ3, DHPꢀdGꢀ4, DHPꢀdAꢀ3, and DHPꢀdAꢀ4) associated with liver
tumor initiation (Figure 1).3, 4, 22, 23 This set of DHPꢀDNA adducts has been formed in common,
including (i) in the livers of rats dosed seven hepatocarcinogenic PAs (retrorsine, riddelliine,
monocrotaline, lasiocarpine, heliotrine, clivorine, and senkirkine) and riddelliine Nꢀoxide;23 (ii)
from the rat liver microsomal incubation of the Nꢀoxides of riddelliine, retrorsine, monocrotaline,
lasiocarpine, and heliotrine with calf thymus DNA;22, 23 (iii) in the livers of male mice
administered retrorsine;24 and (iv) in the livers of cows accidently fed with hay containing PAs.25
Consequently, we have proposed that these DHPꢀDNA adducts are biomarkers of PA and PA Nꢀ
oxide exposure and PAꢀinduced liver tumor initiation.3, 22, 23, 26ꢀ28
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Figure 1. Structures of DHPꢀdG and DHPꢀdA adducts.
DehydroꢀPAs are reactive bifunctional alkylating agents, capable of binding to DNA,
proteins, amino acids, and glutathione at the nucleophilic sites carrying –OH, ꢀNH, and the –SH
functional groups. The resulting secondary pyrrolic metabolites, 7ꢀsubstitutedꢀDHP, 9ꢀ
substitutedꢀDHP, and 7,9ꢀdisubstitutedꢀDHP themselves are reactive bifunctional alkylating
agents as well, each possessing the oxygenꢀ, nitrogenꢀ, and/or sulfurꢀlinkage at the C7 and/or C9
position.8
7ꢀGlutathioneꢀDHP (7ꢀGSꢀDHP) and 7ꢀcysteineꢀDHP are the known secondary pyrrolic
metabolites that are formed from the reaction of dehydroꢀPAs with glutathione and cysteine,
respectively.27, 28 Very recently we found that 7ꢀGSꢀDHP and 7ꢀcysteineꢀDHP also produced
this set of four DHPꢀDNA adducts by rat and human liver microsomes in the presence of calf
thymus DNA and by incubation with human HepG2 cells.3, 26ꢀ28 Based on these findings, we
hypothesized that there could be more DNA reactive secondary pyrrolic metabolites.3
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To pursue this goal, in the present study, eleven secondary pyrrolic metabolites and pyrroles,
each of which possessing an Oꢀ (Oꢀethyl ether), an Nꢀ (Nꢀethyl ether), and/or an Sꢀ (thioether)
linkage at C7 and/or C9 positions, were synthesized and used to determine their ability to
produce DHPꢀDNA adducts from the reaction with calf thymus DNA and from incubation in
HepG2 cells. The structures of the selected secondary pyrrolic metabolites and pyrroles are
shown in Figure 2. These pyrrolic substrates include: (i) three 7ꢀmonoꢀsubstitutedꢀDHPs: 1ꢀ
hydroxymethylꢀ7ꢀmethoxyꢀ6,7ꢀdihydroꢀ5ꢀHꢀpyrrolizine (7ꢀMeOꢀDHP), 7ꢀGSꢀDHP, and 7ꢀ
cysteineꢀDHP; (ii) two 9ꢀmonoꢀsubstitutedꢀDHPs: 9ꢀvalineꢀDHP and 9ꢀethoxyꢀDHP (9ꢀEtOꢀ
DHP); and (iii) six 7,9ꢀdiꢀsubstitutedꢀDHPs: 7,9ꢀdiꢀvalineꢀDHP, 7,9ꢀdiꢀMeOꢀDHP, 7,9ꢀdiꢀEtOꢀ
DHP, 7,9ꢀdiꢀGSꢀDHP, 7,9ꢀdiꢀcysteineꢀDHP, and 7,9ꢀdiꢀNꢀacetylꢀcysteineꢀDHP (7,9ꢀdiꢀNACꢀ
DHP). Among these substrates, 7ꢀMeOꢀDHP is a known metabolite formed from the
metabolism of senecionine by mouse liver microsomes.29 Both 9ꢀvalineꢀDHP and 7,9ꢀdiꢀvalineꢀ
DHP are metabolites formed from the human liver microsomal metabolism of monocrotaline in
the presence of valine (results from this study). 7,9ꢀDiꢀGSꢀDHP was formed from the
metabolism of riddelliine by human liver microsomes in the presence of glutathione.27 As
described above, 7ꢀGSꢀDHP and 7ꢀcysteineꢀDHP have previously been studied.
Dehydromonocrotaline and DHR (the 7ꢀR enantiomer of the racemic DHP metabolite), which
have been studied previously under similar experimental conditions, were included for
comparison.4, 23
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Figure 2. Structures of monoꢀ and diꢀsubstituted secondary pyrrolic metabolites and pyrroles
synthesized for this study.
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EXPERIMENTAL PROCEDURES
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Note: Dehydromonocrotaline and DHR are carcinogens in laboratory animals and must be
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handled with safety precautions.
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Chemicals. Monocrotaline, glutathione (GSH), cysteine, Nꢀacetylcysteine, calf thymus DNA,
micrococcal nuclease (MN), nuclease P1, spleen phosphodiesterase (SPD), NADPH, 2'ꢀ
deoxyguanosine (dG), and 2'ꢀdeoxyadenosine (dA) were obtained from SigmaꢀAldrich (St.
Louis, MO). Dimethylformamide (DMF), acetonitrile, potassium carbonate, chloroform, and
diethyl ether were purchased from Fisher Scientific (Pittsburg, PA, USA). Absolute ethanol was
purchased from AAPER Alcohol and Chemical Co (Shelbyville, KY). All solvents were LC/MS
or HPLC grade. All DHPꢀDNA adducts (DHPꢀdGꢀ1, DHPꢀdGꢀ2, DHPꢀdGꢀ3, DHPꢀdGꢀ4, DHPꢀ
dAꢀ1, DHPꢀdAꢀ2, DHPꢀdAꢀ3, and DHPꢀdAꢀ4) and their isotopeꢀlabelled adducts (DHPꢀ
[15N5]dGꢀ1, DHPꢀ[15N5]dGꢀ2, DHPꢀ[15N5]dGꢀ3, DHPꢀ[15N5]dGꢀ4, DHPꢀ[15N5 C10]dAꢀ1, DHPꢀ
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[15N5 C10]dAꢀ2, DHPꢀ[15N5 C10]dAꢀ3, and DHPꢀ[15N5 C10]dAꢀ4) were synthesized as
described previously, and the purity of each was checked by HPLC before use.4, 23, 30
The human hepatocarcinoma HepG2 cell line was purchased from American Type Culture
Collection (ATCC, Manassas, VA). Dulbecco's Modified Eagle's Medium (DMEM), fetal
bovine serum (FBS), phosphate buffered saline (PBS, pH 7.4), trypsinꢀEDTA, penicillin, and
streptomycin were purchased from Life Technologies (Grand Island, NY). Blood & Cell Culture
DNA kits were purchased from QIAGEN (Valencia, CA). Mixed 50 Donorꢀpooled Human liver
microsomes were purchased from Bioreclamation IVT (Baltimore, MD).
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Dehydromonocrotaline and DHR were prepared from the reaction of monocrotaline and
retronecine with oꢀchloroanil (or oꢀbromanil) in chloroform as described previously.31, 32 7ꢀGSꢀ
DHP was synthesized and purified as previously described.28, 33 7,9ꢀDiꢀglutathioneꢀDHP (7,9ꢀ
DiꢀGSꢀDHP) was similarly synthesized, with glutathione in a 5ꢀfold excess,28, 33 and was
separated by HPLC (Phenomenex Luna C18 (2) column, 250 x 10 mm) using 20% methanol in
water containing 0.1% formic acid with flow rate at 3 ml/min.33
7ꢀCysteineꢀDHP was prepared by reaction of cysteine in PBS (pH = 7.4) and
dehydromonocrotaline in 2 mL DMF for 1 h followed by HPLC separation.22 7,9ꢀDiꢀcysteineꢀ
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DHP was synthesized under similar experimental conditions, using cysteine in 5ꢀfold excess.22, 27
7,9ꢀDiꢀNꢀacetylcysteine (7,9ꢀDiꢀNACꢀDHP) was similarly prepared, with Nꢀacetylcysteine
(NAC) in a 7ꢀfold excess.28, 33
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Culvenor et al.34 reported the synthesis of 7ꢀMeOꢀDHP by reacting dehydroheliotridine
(DHH), the enantiomer of DHR, with methanol under refluxing. In our study, reaction of DHR
in methanol under refluxing overnight produced a mixture of 7ꢀMeOꢀDHP, 9ꢀMeOꢀDHP, and
7,9ꢀdiꢀMeOꢀDHP in 61.8, 7.8, and 6.8% yields, respectively. From the reaction mixture, 7ꢀ
MeOꢀDHP was isolated by HPLC, with a C18 Prodigy column eluted with the following
program: 0 – 5 min: 15% MeOH in H2O; 5 – 45 min: 15 – 50% MeOH in H2O at 1 mL/min.
Reaction of DHR in methanol containing one drop of 2 N HCl with shaking for 2 min produced
7,9ꢀdiꢀMeOꢀDHP in near quantitative yield.
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9ꢀEtOꢀDHP was prepared by the reaction of absolute ethanol with dehydroheliotrine, which
was synthesized by the reaction of heliotrine with oꢀchloroanil in chloroform, under
experimental conditions similar to that for the synthesis of dehydromonocrotaline from
monocrotaline.32 The 1H NMR spectrum of 9ꢀEtOꢀDHP is shown in Figure S1 of Supporting
Information. 7,9ꢀDiꢀEtOꢀDHP was synthesized by reacting dehydromonocrotaline with ethanol
at ambient temperature for 24 h following the method of 35 with modification.36
9ꢀValineꢀDHP was synthesized following the method we previously described37 with
modification. A mixture of 140 mg of valine (9.8 equiv of dehydromonocrotaline), 40 mg of
K2CO3, 0.6 mL of distilled water, and 2.4 mL of DMF was added dropwise into a solution of 40
mg of dehydromonocrotaline in 3 mL of DMF. After reaction for 168 h, the mixture was
separated by HPLC with a 250 × 10 mm Prodigy column (Phenomenex, Torrance, CA). The
reaction products contained both 7ꢀvalineꢀDHP (previously assigned as DHPꢀvalineꢀ2 and DHPꢀ
valineꢀ4) and 9ꢀvalineꢀDHP (previously assigned as DHPꢀvalineꢀ1 and DHPꢀvalineꢀ3).37, 38 9ꢀ
ValineꢀDHP (DHPꢀvalineꢀ3) was collected for the present study. DiꢀvalineꢀDHP was similarly
prepared with a 20ꢀfold excess of valine. The resulting product was separated by HPLC and
characterized by mass and 1H NMR spectral analyses. The full 1H NMR spectral assignments of
7,9ꢀdiꢀvalineꢀDHP are: 1H NMR (acetonitrileꢀd3): δ 0.73 (3H, d, H7’/H8’/H15’/H16’, J = 7 Hz),
0.85 (3H, d, H7’/H8’/H15’/H16’, J = 7 Hz), 0.89 (3H, d, H7’/H8’/H15’/H16’, J = 7 Hz), 1.05
(3H, d, H7’/H8’/ H15’/H16’, J = 7 Hz), 1.17−1.20 (1H, m, H4’/H12’), 1.38−1.41 (1H, m,
H12’/H4’), 2.38−2.45 (1H, m, H6b), 2.61−2.69 (1H, m, H6a), 3.59 (1H, d, H2’/H10’, J = 3.3
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Hz), 3.71 (1H, d, H10’/H2’, J = 3.3 Hz), 3.78−3.84 (1H, m, H5b), 4.02−3.95 (1H, m, H5a), 4.16
(1H, H9b, dd, J = 14 Hz), 4.25 (1H, H9a, dd, J = 14 Hz), 5.70−5.76 (1H, m, H7), 6.10 (1H, d,
H2, J = 2.5 Hz), 6.51 (1H, d, H3, J = 2.5 Hz).
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Human Liver Microsomal Metabolism of Riddelliine in the Presence of Valine. The
metabolism was conducted in a 1 mL incubation volume, consisting of 100 mM sodium
phosphate buffer (pH 7.6), 1 mM NADPH, 5 mM MgCl2, 500 µM riddelliine, 2 mM valine, and
1 mg of human liver microsomal protein at 37 °C for 1 h. After incubation, the incubation
mixture was centrifuged at 105,000 g for 30 min at 4 °C and microsomal proteins removed, the
supernatant was analyzed by LCꢀMS/MS.
Reaction of PA Pyrrolic Metabolites and Pyrroles with Calf Thymus DNA. A solution
containing 0.5 mM 7ꢀMeOꢀDHP and 1 mg calf thymus DNA in 1 mL acetonitrile and 0.01%
formic acid water solution (pH 8 adjusted with ammonium hydroxide) at 1:1 volume was stirred
at 37 ◦C for 72 h. The reaction mixture was then enzymatically hydrolyzed as previously
described.39 The resulting samples were spiked with internal standards DHPꢀ[15N5]dGs and
DHPꢀ[15N5 C10]dAs for LCꢀESI/MS/MS analysis.
13
The reactions of vehicle control and other pyrroles were similarly conducted in parallel.
DHP-DNA Adducts Formed in Cultured HepG2 Cells Exposed to PA Pyrrolic Metabolites.
Human HepG2 cells were cultured in DMEM supplemented with 10% (v/v) FBS and antibiotics
(100 U/mL penicillin and 100 µg/mL streptomycin) at 37 °C in a humidified atmosphere
containing 5% CO2. One day before each experiment, cells were seeded at a density of 3 × 106
cell/mL in 150 cm2 culture dishes. The cells were then exposed to vehicle control (0.1% DMSO
in DMEM) and pyrrolic metabolites and analogs at 50 µM for 24 h.
After exposure, the cells were collected and DNA was extracted using a Blood & Cell
Culture DNA Isolation kit following manufacturer's instructions. The concentration of the DNA
was determined spectrophotometrically. DNA samples from treated or vehicle cells (200 ꢁg)
were then enzymatically hydrolyzed to nucleosides as described previously.39
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Quantitation of DHP-dG and DHP-dA Adducts by LC/MS/MS Analysis. The LC/MS/MS
identification and quantitation of the DHPꢀdG and DHPꢀdA adducts formed in each sample were
conducted following the procedures previously published.28
7
8
9
Positive ions were acquired in the selected reaction monitoring (SRM) mode (dwell time of
100 ms). DHPꢀdG adducts were monitored at the [M+H] + m/z 403 to m/z 269 [MꢀH2O+HꢀdR]
transition;DHPꢀ[15N5]dG internal standards at the [M+H] + m/z 408 to m/z 274 [MꢀH2O+Hꢀ
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dR];DHPꢀdA adducts at the [M+H] + m/z 387 to m/z 253 [MꢀH2O+HꢀdR]; DHPꢀ[15N5 C10]dA
internal standards at the [M+H] + m/z 402 to m/z 263 [MꢀH2O+HꢀdR] transition. Samples were
quantified by comparing the areas of the unlabeled chromatogram peaks to those of the
corresponding labeled internal standard chromatogram peaks. The Thermo Xcalibur 2.0 SR2
software was used for the data acquisition and reprocessing.
Standard characterization and calibration curves were obtained by plotting the amounts of
standard compounds against peak area. In a 10 µL injection volume, each sample contained 40
fmol DHPꢀ[15N5]dG and 10 fmol DHPꢀ[15N5,13C10]dA.
Statistical Analysis. Assay data are presented as mean ± SD, the results represent two separate
experiments conducted in triplicate. A twoꢀsample tꢀtest, assuming equal variances, in Excel
was used to determine the significance between different treatments or doses. The difference
was considered statistically significant when the pꢀvalue was less than 0.05.
RESULTS
Synthesis of Secondary Pyrrolic Metabolites and Pyrrole Analogs. Secondary pyrrolic
metabolites and pyrrole analogs (Figure 2) used in this study were synthesized in our laboratory.
Dehydromonocrotaline and DHR were the starting materials used for the synthesis of these
secondary pyrrolic products. Because both dehydromonocrotaline and DHR are highly unstable,
they had to be prepared in a highly pure form. These chemicals readily decomposed during
reaction, resulting in erratic yields and difficulty in purification of the products by HPLC. The
resulting pyrroles are reactive bifunctional alkylating agents, and are also highly unstable and
easily decomposed. For example, 7,9ꢀdiꢀEtOꢀDHP in acetonitrile decomposed into 7ꢀEtOꢀDHP
and 9ꢀEtOꢀDHP even when stored at ꢀ80 ºC. Consequently, all these substrates had to be
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synthesized a highly pure form; and after storage, had to be purified again before use. For
illustration, the dehydromonocrotaline that we prepared is highly pure, as demonstrated by its 1H
NMR shown in Figure S2 of Supporting Information.
7
8
9
Human Liver Microsomal Metabolism of Riddelliine in the Presence of Valine. After the
metabolism, 9ꢀvalineꢀDHP and 7,9ꢀdiꢀvalineꢀDHP were identified based on comparing their
HPLC retention times, UVꢀvisible absorption spectra, and mass spectra with those of the
synthetic standards. The formation of these two metabolites was further confirmed by
comparison of their LC/MS/MS selected reaction monitoring (SRM) mode profiles (Figure S3 of
Supporting Information) with those of the synthetic standards.
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Standard Characterization and Calibration Curves. HPLCꢀESꢀMS/MS calibration curves
were obtained from DHPꢀdGs and DHPꢀdAs versus their respective isotopeꢀlabelled (Figures S4
and S5 of Supporting Information).
Reactions of Pyrrolic Metabolites and Pyrroles with Calf Thymus DNA. DNA samples
isolated from each reaction were enzymatically hydrolyzed to nucleosides from which the DHPꢀ
dG and DHPꢀdA adducts were quantified.
The quantitation of these adducts formed from each reaction are summarized in Table 1.
LC/MS/MS SRM chromatograms of DHPꢀdG and DHPꢀdA adducts generated from the reaction
of calf thymus DNA with vehicle control (solvent) and with 7ꢀMeOꢀDHP are shown in Figure
3A and Figure 3B, respectively. Figure 3Aꢀ1 and Figure 3Bꢀ1 show the isotopeꢀlabeled internal
standards DHPꢀ[15N5]dGꢀ1, DHPꢀ[15N5]dGꢀ2, DHPꢀ[15N5]dGꢀ3, and DHPꢀ[15N5]dGꢀ4 adducts,
each involving the transition of the [M+H]+ precursor ion m/z 408 to the selected product ion m/z
274. Figure 3Aꢀ3 and Figure 3Bꢀ3 are DHPꢀ[15N5, 13C10]dAꢀ1, DHPꢀ[15N5,13C10]dAꢀ2, DHPꢀ
[15N5,13C10]dAꢀ3, and DHPꢀ[15N5,13C10]dAꢀ4 adducts involving the transition of m/z 402 to m/z
263. Figure 3Aꢀ2 and Figure 3Aꢀ4 present the DHPꢀdG and DHPꢀdA adducts formed from the
control, which indicate that these DHPꢀdG and DHPꢀdA adducts were not detected (Table 1).
The results shown in Figure 3Bꢀ2 and Figure 3Bꢀ4 indicate that the reaction of 7ꢀMeOꢀDHP with
calf thymus DNA generated all the four DHPꢀdG and four DHPꢀdA adducts and that the total
level of DHPꢀdG adducts are higher than those of DHPꢀdA adducts (Table 1).
SRM chromatograms from reactions of 7ꢀvalineꢀDHP and 7,9ꢀdiꢀvalineꢀDHP with calf
thymus DNA for 72 h are shown in Figure 4. All the four DHPꢀdG and four DHPꢀdA adducts
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were generated from both 7ꢀvalineꢀDHP and 7,9ꢀdiꢀvalineꢀDHP. Also, the level of DHPꢀdG
adducts formation was higher than that of DHPꢀdA adducts (Table 1).
As summarized in Table 1, the eleven secondary pyrrolic metabolites and pyrrolic analogs,
together with dehydromonocrotaline and DHR, all generated the four DHPꢀdG adducts and the
four DHPꢀdA adducts. With the exceptions of 7,9ꢀdiꢀGSꢀDHP, 7,9ꢀdiꢀcysteineꢀDHP, and 7,9ꢀdiꢀ
NACꢀDHP, all the compounds produced high levels of DHPꢀDNA adducts. The levels of DHPꢀ
dGꢀ3/4 adducts were significantly higher than those of DHPꢀdAꢀ3/4 adducts. 7,9ꢀDiꢀGSꢀDHP,
7,9ꢀdiꢀcysteineꢀDHP, and 7,9ꢀdiꢀNACꢀDHP produced much lower levels of DHPꢀDNA adducts.
No DHPꢀdG or DHPꢀdA adducts were detected from the vehicle control group.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
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Table 1. LCꢀMS/MS Measurements of DHPꢀdG and DHPꢀdA Adducts Formed from Reaction of
Monoꢀ and DiꢀSubstituted Pyrrolic Metabolites and Analogs with Calf Thymus DNA for 72
Hours.
Chemicals
Levels of DHPꢀdG and DHPꢀdA / 108 nucleosidesc
DHPꢀ
dGꢀ1
DHPꢀ
dGꢀ2
<LOD
DHPꢀ
dGꢀ3/4
<LOD
DHPꢀ
dAꢀ1
<LOD
DHPꢀ
dAꢀ2
<LOD
DHPꢀ
dAꢀ3/4
<LOD
Total DHPꢀdG
and DHPꢀdA
<LOD
Vehicle Control
7,9ꢀDiꢀEtOꢀDHPa
9ꢀEtOꢀDHPa
<LODd
16010
± 1495
16740
± 1382
15029
± 703
13220
± 1722
13768
± 1419
13046
± 251
16193
± 1718
20022
± 1759
15715
± 510
2830
± 126
4282
± 328
2736
± 108
1955
± 146
2925
± 349
1949
± 32
3499
± 271
4610
± 395
3110
±91
53707
62346
51586
7,9ꢀDiꢀMeOꢀDHPa
7ꢀMeOꢀDHPa
7,9ꢀDiꢀValineꢀDHPa
9ꢀValineꢀDHPa
7ꢀGSꢀDHP
6036
± 434
9847
± 1201
10977
± 988
16948
± 929
19326
± 1034
5325
± 398
5051
± 542
5762
± 630
13982
± 723
14769
± 714
9409
± 570
11525
± 1372
26040
± 1679
26220
± 1146
33443
± 1238
12.0 ±
0.7
17.1 ±
1.5
22.0 ±
2.0
25529
± 1819
1335
± 198
1583
± 226
1947
± 170
6064
± 417
7059
± 392
1050
± 166
1510
± 145
1823
± 168
3950
± 422
4604
± 487
2717
± 300
5516
± 370
5804
± 500
8006
± 382
9313
± 554
25872
35032
52354
75169
88514
41.5
7ꢀCysteine
7,9ꢀDiꢀGSꢀDHPb
7,9ꢀDiꢀcysteineꢀDHPb
7,9ꢀDiꢀNACꢀDHPb
DHRa
8.2 ± 0.8 2.7 ± 0.3
2.2 ± 0.4 2.0 ± 0.2 14.3 ± 1.3
3.5 ± 0.3 3.0 ± 0.2 10.7 ± 1.0
8.0 ± 0.6 3.5 ± 0.2 11.5 ± 0.8
11.3 ±
0.8
16.2 ±
1.5
13859
± 1476
6.7 ± 0.7
52.4
17.7 ±
1.6
11433
± 960
78.8
5478
± 490
3771
± 366
7039
± 446
67109
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Dehydromonocrotalinea
12363
10285
21043
4034
3063
6915
57703
± 1065
± 1110
± 1538
± 331
± 219
± 523
a Aliquots of DNA (0.13 ꢁg) were assayed by HPLCꢀESꢀMS/MS. b Aliquots of DNA (39.6 ꢁg) were assayed by
HPLCꢀESꢀMS/MS. cThe data are presented as the mean ± SD, n=3. dLOD = limit of detection based upon the
analysis of 45 ꢁg DNA by HPLCꢀESꢀMS/MS. Under experimental conditions, the LOD of DHPꢀdGꢀ1/2 is 0.71,
DHPꢀdGꢀ3/4 is 0.71, DHPꢀdAꢀ1/2 is 0.14, and DHPꢀdAꢀ3/4 is 0.14 adducts per 108 nucleosides, separately.
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Time (min)
Figure 3. LC/MS/MS SRM chromatograms of DHPꢀdG and DHPꢀdA adducts formed from (A)
vehicle control; and (B) reaction of 50 µM 7ꢀMeOꢀDHP with calf thymus DNA for 72 h. IS:
DHPꢀ[15N5]dG and DHPꢀ[15N5,13C10]dA labeled internal standards.
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Figure 4. LC/MS/MS SRM chromatograms of DHPꢀdG and DHPꢀdA adducts formed from
reaction of (A) 50 µM 9ꢀvalineꢀDHP and (B) 7,9ꢀdiꢀvalineꢀDHP with calf thymus DNA for 72 h.
LC-MS/MS Analysis of DHP-dG and DHP-dA Adducts in HepG2 Cells Exposed to Mono-
and Di-Substituted Pyrrolic Metabolites. The levels of DHPꢀdG and DHPꢀdA adducts formed
in HepG2 cells treated with individual pyrrolic metabolites and pyrrolic analogs are summarized
in Table 2. As examples, LC/MS/MS SRM chromatograms of DHPꢀdG and DHPꢀdA adducts
formed from HepG2 cells exposed to 50 µM of 7ꢀvalineꢀDHP (Figure 5ꢀA) and 7,9ꢀdiꢀvalineꢀ
DHP (Figure 5ꢀB) for 24 h are shown in Figure 5.
Compared with the results from the reaction with calf thymus DNA (Table 1), only DHPꢀdGꢀ
3/4 and DHPꢀdAꢀ3/4 adducts were formed; DHPꢀdGꢀ1, DHPꢀdGꢀ2, DHPꢀdAꢀ1, and DHPꢀdAꢀ2
were not detected (Table 2). This DHPꢀDNA adduct pattern corresponds to that observed in vivo
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3, 4, 23. The levels of DHPꢀdAꢀ3/4 adducts were lower than those of DHPꢀdGꢀ3/4 adducts. No
DHPꢀdG or DHPꢀdA adducts were detected from the control incubations. No DHPꢀDNA
adducts were detected in the HepG2 cells treated with 7,9ꢀdiꢀGSꢀDHP, 7,9ꢀdiꢀcysteineꢀDHP, or
7,9ꢀdiꢀNACꢀDHP.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
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58
59
60
Table 2. LCꢀMS/MS Analysis of DHPꢀdG and DHPꢀdA Adducts for HepG2 Cells Exposed to
Monoꢀ and DiꢀSubstituted Pyrrolic Metabolites and Analogs at 50 µM for 24 hours.
Levels of DHP-dG and DHP-dA / 108 nucleosidesa
Total DHPꢀ
Chemicals
DHPꢀdGꢀ
3/4
DHPꢀdAꢀ
3/4
DHPꢀdGꢀ1 DHPꢀdGꢀ2
DHPꢀdAꢀ1 DHPꢀdAꢀ2
dG and
DHPꢀdA
<LOD
1.7
14.6
2.9
4.8
4.9
9.8
11.7
13.4
<LOD
<LOD
<LOD
7.8
Control incubation
7,9ꢀdiꢀEtOꢀDHP
9ꢀEtOꢀDHP
7,9ꢀdiꢀMeOꢀDHP
7ꢀMeOꢀDHP
7,9ꢀdiꢀValineꢀDHP
7ꢀValineꢀDHP
7ꢀGSꢀDHP
7ꢀcysteineꢀDHP
7,9ꢀdiꢀGSꢀDHP
7,9ꢀdiꢀcysteineꢀDHP
7,9ꢀdiꢀNACꢀDHP
DHR
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
1.3 ±0.3
12.3 ±1.1
2.3 ± 0.4
4.0 ± 0.3
4.1 ± 0.5
8.4 ± 1.0
10.0 ± 1.0
11.4 ± 1.4
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
0.4 ± 0.1
2.3 ± 0.3
0.6 ± 0.1
0.8 ± 0.1
0.8 ± 0.1
1.4 ± 0.2
1.7 ± 0.1
2.0 ± 0.2
<LOD
<LOD
<LOD
6.6 ± 0.7
11.3 ± 1.2
<LOD
<LOD
1.2 ± 0.1
1.9 ± 0.2
Dehydromonocrotaline
13.2
aAliquots of DNA (45 ꢁg) were assayed by HPLCꢀESꢀMS/MS. The data are presented as the mean ± SD, n=3. bThe
LOD values are identical to those described in the note of Table 1.
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Figure 5. LC/MS/MS SRM chromatograms of DHPꢀdG and DHPꢀdA adducts formed from
HepG2 cells exposed to (A) 50 µM 9ꢀvalineꢀDHP and (B) 7,9ꢀdiꢀvalineꢀDHP for 24 h.
DISCUSSION
We proposed that DHPꢀDNA adducts are biomarkers of PA exposure and PAꢀinduced liver
tumor initiation. To validate this hypothesis and determine if additional metabolites can generate
these DHPꢀDNA adducts, the ability of 13 secondary pyrrolic metabolites and pyrrolic analogs to
bind calf thymus DNA and cellular DNA in HepG2 cells was studied. We found that all these
secondary pyrroles can bind to calf thymus DNA and produce the same set of DHPꢀDNA
adducts (Table 1).
When incubated with HepG2 cells, with the exception that no DHPꢀDNA adducts were
detected from the 7,9ꢀdiꢀGSꢀDHP, 7,9ꢀdiꢀcysteineꢀDHP, and 7,9ꢀdiꢀNACꢀDHP groups, the
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remaining 10 pyrroles generated these DHPꢀDNA adducts (DHPꢀdGꢀ3, DHPꢀdGꢀ4, DHPꢀdAꢀ3,
and DHPꢀdAꢀ4 adducts) (Table 2). The DNA adduct profile obtained from these secondary
pyrrolic metabolites is similar to that produced in the livers of rats, mice, and cattle in vivo.3, 4, 23,
24 The uniqueness of this profile is that: (i) no DHPꢀdGꢀ1, DHPꢀdGꢀ2, DHPꢀdAꢀ1, and DHPꢀdAꢀ
2 were detected; and (ii) the levels of DHPꢀdGꢀ3/4 adduct formation were significantly higher
than those of DHPꢀdAꢀ3/4 adducts (Table 2).
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In this study all secondary pyrroles possess an Oꢀether linkage, an Nꢀsecondary amine
linkage, or a thioetherꢀlinkage located at the C7 and/or C9 position of the necine base (Figure 2).
Removal of the Oꢀ, Sꢀ, or Nꢀsubstituent produces carbonium ions located at the C7 or C9
position that can be stabilized by resonance with the πꢀelectrons at the two conjugated double
bonds and the nitrogen atom of the necine base.8 These carbonium ions bind to other
nucleophiles producing new alkylatedꢀDHP products. When the attacked nucleophile is DNA,
the resulting alkylated DHP products are DHPꢀDNA adducts. Based on the results obtained in
this present study, many secondary pyrrolic metabolites, which are produced from the reactions
of dehydroꢀPAs with glutathione, amino acids, and proteins, are able to produce DHPꢀDNA
adducts through the reaction with DNA. 2, 8, 22, 27, 28, 40, 41 Consequently, there are multiple
activation pathways that result in the formation of DHPꢀDNA adducts associated with PAꢀ
induced liver tumor initiation. Based on these findings, we propose a general multiple metabolic
activation pathway (Figure 6) to mediate the formation of these DHPꢀDNA adducts. Dehydroꢀ
PAs are highly unstable8, 42 while the secondary pyrrolic metabolites are relatively more stable.8
In consideration of this fact, secondary pyrrolic metabolites should play a vital role in the PAꢀ
induced liver tumor initiation.
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Figure 6. Proposed PA secondary pyrrolic metabolites construct multiple activation pathways
leading to DNA adduct formation and potential liver tumor initiation.
Among the three atoms, oxygen has the highest electronegativity (3.44), possessing the most
electron attracting ability. As such, oxygen attracts electrons easier than the nitrogen atom,
which has an electronegativity number 3.04, and much easier than the sulfur atom which has a
lower electronegativity number (2.58). Consequently, the –Oꢀether linkage at the C7 or C9ꢀ
position of a pyrrole is most unstable, followed by the Nꢀetherꢀlinkage, and –Sꢀlinkage is most
stable. Based on our experience in handling these compounds, we have found that 7ꢀGSꢀDHP
and 7ꢀcysteineꢀDP are more stable than 9ꢀvalineꢀDHP, 7ꢀMeOꢀDHP, and 9ꢀEtOꢀDHP. These
observations are in agreement with the report by Mattocks8 that stated “those the nucleophile
bound to the DHP moiety through a sulfuhydryl linkage are more stable than those linked by an
oxygen atom or a nitrogen atom”.
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It has been well established that the reaction of pyrrolic metabolites with nucleophilic
cellular substituents is an SN1 type mechanism,4, 8 mediated by the formation of an oxygenium
ion, an nitronium ion, or a sulfonium ion. In order to produce DHPꢀDNA adducts, 7,9ꢀdiꢀGSꢀ
DHP, 7,9ꢀdiꢀcysteineꢀDHP, and 7,9ꢀdiꢀNACꢀDHP need to remove two sulfonium ions, and the
removal of the first sulfonium ion should be the rateꢀdetermining step. Consequently, 7,9ꢀdiꢀGSꢀ
DHP, 7,9ꢀdiꢀcysteineꢀDHP, and 7,9ꢀdiꢀNACꢀDHP exhibit low or nonꢀbinding activity in the
production of DHPꢀDNA adducts (Tables 1 and 2), while 7,9ꢀdiꢀMeOꢀDHP, 7,9ꢀdiꢀEtOꢀDHP,
and 7,9ꢀdiꢀvalineꢀDHP can bind to DNA.
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The results obtained in this present study indicate that the set of DHPꢀDNA adducts (DHPꢀ
dGꢀ3, DHPꢀdGꢀ4, DHPꢀdAꢀ3, and DHPꢀdAꢀ4) could be generated through the binding of a
variety of metabolism products, including: (i) primary pyrrolic metabolites; (ii) secondary
pyrrolic metabolites; and (iii) DHPꢀprotein adducts. If this is the case, these DHPꢀDNA adducts
provide useful common biomarkers of PA exposure and PAꢀinduced liver tumor initiation. This
warrants further investigation.
Concerning the study of the metabolic activation of chemical carcinogens, our findings
represent the first case that a series of reactive secondary pyrrolic metabolites are generated of
which some are expected to form DNA adducts in inducing mutations leading to liver
tumorigenesis.
ASSOCIATED CONTENT
Supporting Information
Figures S1−S2 show the NMR spectra of the synthesized 9ꢀEtOꢀDHP, monocrotaline, and
dehydromonocrotaline. Figures S3 are the LC/MS SRM chromatograms of monoꢀvalineꢀDHP
and 7,9ꢀdiꢀvalineꢀDHP, and Figures S4−S5 show the standard characterization and calibration
curves of DHPꢀDNA adducts. This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Funding
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This research 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.
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Qingsu Xia and Xiaobo He equally contribute the work and share the first authorship.
ACKNOWLEDGMENTS
We thank Drs. Frederick A. Beland and Danial R. Doerge for critical review of this manuscript.
This article is not an official U.S. 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.
ABBREVIATIONS
DMF, dimethylformamide; DHR, dehydroretronecine or (ꢀ)ꢀRꢀ6,7ꢀdihydroꢀ7ꢀhydroxyꢀ1ꢀ
hydroxymethylꢀ5Hꢀpyrrolizine; DHP, (+/ꢀ)ꢀ6,7ꢀdihydroꢀ7ꢀhydroxyꢀ1ꢀhydroxymethylꢀ5Hꢀ
pyrrolizine; 7ꢀGSꢀDHP, 7ꢀglutathioneꢀDHP; 7,9ꢀdiꢀGSꢀDHP, 7,9ꢀdiglutathioneꢀDHP; NAC, Nꢀ
acetylcysteine; PNK, cloned T4 polynucleotide kinase; MN, micrococcal nuclease; SPD, spleen
phosphodiesterase; dG, 2’ꢀdeoxyguanosine; dA, 2’ꢀdeoxyadenosine; DHPꢀdGꢀ1 and DHPꢀdGꢀ2,
a pair of epimers of 7ꢀ(deoxyguanosinꢀN2ꢀyl)dehydrosupinidine adducts; DHPꢀdGꢀ3 and DHPꢀ
dGꢀ4, a pair of epimers of 7ꢀhydroxyꢀ9ꢀ(deoxyguanosinꢀN2ꢀyl)dehydrosupinidine adducts; DHPꢀ
dAꢀ1 and DHPꢀdAꢀ2, a pair of epimers of 7ꢀ(deoxyadenosinꢀN6ꢀyl)dehydrosupinidine adducts;
DHPꢀdAꢀ3 and DHPꢀdAꢀ4, a pair of epimers of 7ꢀhydroxyꢀ9ꢀ(deoxyadenosinꢀN6ꢀ
yl)dehydrosupinidine adducts; LCꢀESꢀMS/MS, highꢀperformance liquid chromatography
electrospray ionization tandem mass spectrometry; SRM, selected reaction monitoring; LOD,
limit of detection; NCTR, National Center for Toxicological Research.
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