High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of pyrrolizidine alkaloid-derived DNA adducts in vitro and in viwo

Chem. Res. Toxicol. 2010, 23, 637–652

637

High-Performance Liquid Chromatography Electrospray Ionization

Tandem Mass Spectrometry for the Detection and Quantitation of

Pyrrolizidine Alkaloid-Derived DNA Adducts in Vitro and in ViWo

Peter P. Fu,*^,†Ming W. Chou,^†Mona Churchwell,^†Yuping Wang,^†Yuewei Zhao,^†

Qingsu Xia,^†Gonc¸alo Gamboa da Costa,^†M. Matilde Marques,^‡Frederick A. Beland,^†and

Daniel R. Doerge^†

National Center for Toxicological Research, Jefferson, Arkansas 72079, and Centro de Qu´ımica Estrutural,

Complexo I, Instituto Superior Te´cnico, UniVersidade Te´cnica de Lisboa, P-1049-001 Lisboa, Portugal

ReceiVed NoVember 2, 2009

Pyrrolizidine alkaloid-containing plants are widespread in the world and are probably the most common

poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids require metabolism to

exert their genotoxicity and tumorigenicity. We have determined that the metabolism of a series of

tumorigenic pyrrolizidine alkaloids in Vitro or in ViVo generates a common set of (()-6,7-dihydro-7-

hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts that are responsible for tumor

induction. The identiﬁcation and quantitation of the DHP-derived DNA adducts formed in ViVo and in

Vitro were accomplished previously by ³²P-postlabeling/HPLC methodology. In this article, we report

the development of a sensitive and speciﬁc liquid chromatography-electrospray ionization-tandem mass

spectrometry (HPLC-ES-MS/MS) method to detect DHP-derived DNA adducts formed in Vitro and in

ViVo. The method is used to quantify the levels of DHP-2′-deoxyguanosine (dG) and DHP-2′-

deoxyadenosine (dA) adducts by multiple reaction monitoring (MRM) analysis in the presence of known

quantities of isotopically labeled DHP-dG and DHP-dA internal standards. This HPLC-ES-MS/MS method

is accurate and precise. When applied to liver samples from rats treated with the pyrrolizidine alkaloids

riddelliine and monocrotaline, the method provided signiﬁcant new information regarding the mechanism

of DNA adduct formation.

Central Africa, the West Indies, China, Jamaica, Canada,

Europe, New Zealand, Australia, and the U.S., rendering it

highly possible that pyrrolizidine alkaloid-containing plants are

the most common poisonous plants affecting livestock, wildlife,

and humans (4, 5, 8, 9, 13-15).

Introduction

Pyrrolizidine alkaloids are heterocyclic compounds, most of

which are derived from esters of basic alcohols, known as the

necine bases. The hydrolyzed products of pyrrolizidine alkaloids

are a necine base and a necic acid. The structures and numbering

system of the several representative necine bases, platynecine,

retronecine, heliotridine, and otonecine that form the basis of a

variety of commonly studied pyrrolizidine alkaloids are shown

in Figure 1.

Pyrrolizidine alkaloids are phytochemicals that are constitu-

tively produced by plants as secondary metabolites for exerting

a defense mechanism against insect herbivores (1-3). As a

consequence, pyrrolizidine alkaloids are common constituents

of hundreds of plant species of different unrelated botanical

families distributed in many geographical regions in the world

(1, 4-11). Pyrrolizidine alkaloids are found in more than 12

higher plant families of the Angiosperms, among which three

families, Compositae (Asteraceae), Boraginaceae, and Legu-

minosae (Fabaceae), contain the most toxic pyrrolizidine

alkaloids. It has been reported that about 3% of the world’s

ﬂowering plants contain toxic pyrrolizidine alkaloids (8, 11, 12).

More than 660 pyrrolizidine alkaloids and pyrrolizidine alkaloid

N-oxides have been identiﬁed in over 6,000 plants, and about

half of them exhibit hepatotoxic activity (4, 5, 13). Toxic

pyrrolizidine alkaloid-containing plants grow in South Africa,

Pyrrolizidine alkaloids are among the ﬁrst naturally occurring

carcinogens identiﬁed in plants. In 1954, a pure pyrrolizidine

alkaloid, retrorsine, was found to induce liver tumors in

experimental animals (16). Subsequently, lasiocarpine and

riddelliine were found to induce liver tumors in National

Toxicology Program (NTP¹) chronic tumorigenicity bioassays

(17, 18). Because of the concern about human exposure to

genotoxic and tumorigenic pyrrolizidine alkaloids, in 1989, the

International Programme on Chemical Safety (IPCS) determined

that pyrrolizidine alkaloids are a threat to human health and

safety (10). To date, all of the known tumorigenic pyrrolizidine

alkaloids are based upon a retronecine, heliotridine, or otonecine

structure (5) (Figure 2).

¹Abbreviations: DHR, dehydroretronecine or (-)-R-6,7-dihydro-7-

hydroxy-1-hydroxymethyl-5H-pyrrolizine; DHP, (()-6,7-dihydro-7-hydroxy-

1-hydroxymethyl-5H-pyrrolizine; PNK, cloned T4 polynucleotide kinase;

MN, micrococcal nuclease; SPD, spleen phosphodiesterase; dG, 2′-

deoxyguanosine; dA, 2′-deoxyadenosine; DHP-dG-1 and DHP-dG-2,

epimeric pairs of 7-(deoxyguanosin-N²-yl)dehydrosupinidine; DHP-dG-3,

9-(deoxyguanosin-N²-yl)dehydrosupinidine; DHP-dG-4, 5-(deoxyguanosin-

N²-yl)dehydrosupinidine; DHP-dA-1 and DHP-dA-2, epimeric pairs of

7-(deoxyadenosin-N⁶-yl)dehydrosupinidine; DHP-dA-3, x- (deoxyadenosin-

N⁶-yl)-dehydrosupinidine; DHP-dA-4, y-(deoxyadenosin-N⁶-yl)dehydrosu-

pinidine; HPLC-ES-MS/MS, high-performance liquid chromatography-

electrospray ionization-tandem mass spectrometry; NCTR, National Center

for Toxicological Research; NTP, National Toxicology Program; MRM,

multiple reaction monitoring; LOD, limit of detection.

* Corresponding author. Tel: +1 870-543-7207. Fax: +1 870-543-7136.

E-mail: peter.fu@fda.hhs.gov.

^†National Center for Toxicological Research.

^‡Universidade Te´cnica de Lisboa.

10.1021/tx900402x  2010 American Chemical Society

Published on Web 01/15/2010

638 Chem. Res. Toxicol., Vol. 23, No. 3, 2010

Fu et al.

Figure 1. (A) Representative necine bases of pyrrolizidine alkaloids and (B) structures of (()-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine

(DHP); dehydroretronecine [(-)-(R)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHR)]; and dehydroheliotridine [(+)-(S)-6,7-dihydro-

7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHH)].

Figure 2. Nomenclature and structures of representative tumorigenic retronecine-, heliotritridine-, and otonecine-type pyrrolizidine alkaloids.

Pyrrolizidine alkaloids themselves are not toxic and require

metabolic activation to exert their acute and chronic toxicity.

The mechanism of tumor formation induced by pyrrolizidine

alkaloids was not known until 2001, when it was determined

that riddelliine induces liver tumors (i.e., hemangiosarcoma in

male and female rats and male mice, and a lower incidence of

hepatocellular adenoma and carcinoma in male and female rats)

through a genotoxic mechanism mediated by (()-6,7-dihydro-

7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA

adduct formation (19).

with the preferential induction of liver hemangiosarcomas by

riddelliine. Furthermore, metabolism of riddelliine by human

liver microsomes resulted in a metabolic pattern and DNA

adduct proﬁle that were very similar to those formed in rat liver,

indicating that the results of in ViVo and in Vitro mechanistic

studies with experimental rodents are highly relevant to humans

(22). Since riddelliine induces liver tumors in rodents and the

DHP-derived DNA adducts are responsible for liver tumor

induction, these results suggest that riddelliine can be genotoxic

to humans via DHP-derived DNA adduct formation. Partly

because of these mechanistic ﬁndings, the NTP has classiﬁed

riddelliine as “reasonably anticipated to be a human carcinogen”

(23).

The levels of the DHP-derived DNA adducts correlated

closely with the tumorigenic potencies in rats fed different doses

of riddelliine (19-21). We subsequently determined that the

DHP-derived DNA adduct levels formed in ViVo in hepatic

endothelial cells, the cells of origin for the hemangiosarcomas,

were signiﬁcantly greater than those in parenchymal cells (20).

These results indicated that the levels of riddelliine-induced

DNA adducts in speciﬁc populations of liver cells correlated

Subsequent mechanistic studies on the tumorigenic pyrrolizi-

dine alkaloids, retrorsine, monocrotaline, retronecine, clivorine,

lasiocarpine, and heliotrine, conducted under similar experi-

mental conditions, also generated a common set of DHP-derived

DNA adducts (24-29). While riddelliine, retrorsine, monocro-

DNA Adducts from Pyrrolizidine Alkaloids

Chem. Res. Toxicol., Vol. 23, No. 3, 2010 639

HPLC grade. (-)-(R)-6,7-Dihydro-7-hydroxy-1-hydroxymethyl-5H-

pyrrolizine (DHR, dehydroretronecine) (Figure 1) was prepared by

barium hydroxide-catalyzed hydrolysis of monocrotaline followed

by dehydrogenation of the resulting retronecine with o-bromanil

(19). The 7R stereochemistry of DHR was established previously

by circular dichroism (CD) spectroscopic analysis (26).

Instrumentation. A Waters HPLC system (Milford, MA),

consisting of a Model 600 controller, a Model 996 photodiode array

detector, and a 600 pump, was used for the separation and

puriﬁcation of the DHP-derived DNA adducts.

taline, and retronecine are retronecine-type pyrrolizidine alka-

loids, clivorine is an otonecine-type, and heliotrine and lasio-

carpine are heliotridine-type pyrrolizidine alkaloids (Figure 2).

The formation of the same DHP-derived DNA adducts from

the metabolism of all of these tumorigenic pyrrolizidine alkaloids

indicates that these DHP-derived DNA adducts are potential

biomarkers of pyrrolizidine alkaloid exposure and tumorigenicity

(5, 25, 30).

Pyrrolizidine alkaloids have long been found as contaminants

in many human food sources and may potentially cause

worldwide human health problems (1, 5, 6, 13, 31-39). A

particular concern is that tumorigenic pyrrolizidine alkaloids

have been found in Chinese herbal plants and herbal dietary

supplements sold in the U.S. (40), including comfrey, coltsfoot,

and borage. We recently reported that the DHP-derived DNA

adducts are formed in the livers of female F344 rats gavaged

orally with three dietary supplements (comfrey root extract,

comfrey compound oil, and coltsfoot root extract) or an extract

of a Chinese herbal plant, Flos farfara (Kuan Tong Hua) (40).

This indicates that pyrrolizidine alkaloid-containing herbal plants

and herbal dietary supplements may pose a human health risk.

DNA adducts are considered to be a biomarker for assessing

the risk of chemically induced carcinogenesis. We previously

developed a ³²P-postlabeling/HPLC method that enabled us to

establish that the DHP-derived DNA adducts formed in ViVo

are potential biomarkers of pyrrolizidine alkaloid exposure and

tumorigenicity (41). With this method, we identiﬁed and

quantiﬁed the DHP-derived DNA adducts in rodents treated with

pure pyrrolizidine alkaloids (e.g., riddelliine, retrorsine, and

monocrotaline), Chinese herbal plants, and herbal dietary

supplements purchased in the U.S. and Canada. These DNA

adducts were also formed from the rat and mouse liver

microsomal metabolism of several different pyrrolizidine alka-

loids (22, 24-29, 40). We also determined that the same DHP-

derived DNA adducts were present in leukocytes of rats fed

riddelliine, suggesting that DHP-derived DNA adducts in blood

can serve as potential biomarkers of pyrrolizidine alkaloid

exposure (30).

¹H-Nuclear magnetic resonance (NMR) experiments were carried

out at 301 K on a Bruker AM 500 spectrometer (Bruker Instruments,

Billerica, MA) operating at 500 MHz. Samples were dissolved in

DMSO-d₆or DMSO-d₆with a trace of D₂O. NOE difference and

homonuclear decoupling experiments were conducted to assist in

assigning proton resonances.

Treatment of Rats with Pyrrolizidine Alkaloids. Procedures

involving the care and handling of rats were reviewed and approved

by the National Center for Toxicological Research (NCTR)

Laboratory Animal Care and Use Committee. Female F344 rats

were obtained from the NCTR breeding colony as weanlings and

maintained on a 12-h light-dark cycle. At 8-10 weeks of age,

the rats were orally gavaged for 3 consecutive days with riddelliine,

at daily doses of 0, 0.1, 1, 2, and 5 mg/kg body weight in 0.5 mL

of 75% DMSO, or with monocrotaline, at daily doses of 1 and 5

mg/kg in the same solvent and volume. Twenty-four hours after

the last dose, the rats were sacriﬁced by exposure to CO₂followed

by exsanguination. Livers were removed, rinsed with cold saline,

and stored at -70 °C before DNA was isolated for DNA adduct

analysis. DNA samples were extracted using RecoverEase DNA

Isolation Kits (Stratagene, Cedar Creek, TX) according to the

manufacturer’s instructions. The concentration of the DNA was

determined spectrophotometrically.

National Toxicology Program (NTP) Female F344 Rat

Liver Samples. Riddelliine-modiﬁed rat liver DNA was obtained

from female F344 rats treated by gavage with riddelliine in a study

conducted by the NTP (42). The rats had been treated under the

same experimental conditions used for the two year chronic

tumorigenicity study, with the exception that there were six animals

per dose group and that the animals were sacriﬁced after six months

of treatment. Brieﬂy, female F344/N rats (7-8 weeks old) were

gavaged with 0, 0.01, 0.033, 0.1, 0.33, or 1.0 mg of riddelliine/kg

body weight in 0.1 M phosphate buffer ﬁve times a week and

continued until sacriﬁce at six months after the ﬁrst dosing day.

After sacriﬁce, liver tissue was collected, stored at -78 °C, and

shipped by the NTP to NCTR for DNA adduct analysis. Rat liver

DNA was isolated according to the method of Beland et al. (43).

Synthesis of DHP-dG and DHP-dA Adducts. DHP-dG adducts

were prepared by an adaptation of the method of Robertson (44).

Brieﬂy, to a solution of dG (53 mg, 0.2 mmol) in 10 mL of 25

mM potassium phosphate (pH 7.4) was added DHR (15 mg, 0.1

mmol) under argon over a period of 5 min. The reaction mixture

was heated at 37 °C for 15 h. After cooling, the reaction mixture

was ﬁltered through a 0.22-µm Millipore ﬁlter to remove insoluble

materials. The solvent was removed under vacuum, and the resulting

residue was separated by HPLC using a Prodigy 5 µm ODS column

(Phenomenex, Torrance, CA, 4.6 mm × 250 mm), by eluting with

a methanol and water linear gradient (0-20 min, 20-30%

methanol; 30-50 min 30-50% methanol). The ﬂow rate was 1.0

mL/min, and UV absorbance was monitored at 254 nm. DHP-dA

adducts were prepared similarly, with the exception that for the

HPLC separation, the UV wavelength was set at 266 nm.

The yield of DHP-dA adduct formation is very low. In order to

obtain sufﬁcient amounts of pure DHP-dA adducts for structural

There are several limitations associated with ³²P-postlabeling/

HPLC methods for DNA adduct analysis. These include (i) the

risk from exposure to high levels of radioactivity, (ii) DNA

adduct instability under the experimental conditions, (iii)

frequent problems with enzymatic reactions, and (iv) the lack

of structural information. In view of these limitations, it is

desirable to develop alternate methods for the detection and

quantitation of DHP-derived DNA adducts in ViVo and in Vitro,

in particular from human samples. In this article, we report the

successful development of an HPLC-ES-MS/MS methodology

for the identiﬁcation and quantitation of DHP-derived DNA

adducts in ViVo and in Vitro.

Experimental Procedures

Caution: Riddelliine, monocrotaline, and DHP are carcinogenic

in laboratory animals. They should be handled with extreme care,

using proper personal protectiVe equipment and a well-Ventilated

hood.

Chemicals. Monocrotaline, o-bromanil, calf thymus DNA (so-

dium salt, type I), 2′-deoxyguanosine (dG), 2′-deoxyadenosine (dA),

glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and nic-

otinamide adenine dinucleotide phosphate (NADP⁺) were purchased

from the Sigma Chemical Co. (St. Louis, MO). Riddelliine was

obtained from the National Toxicology Program (NTP). [¹⁵N₅]dG

1

determination by H NMR spectroscopic analyses and for deter-

mination of their UV molar extinction coefﬁcients, 8.0 g of

monocrotaline was used to prepare retronecine (2.8 g, 75% yield)

(19). Dehydrogenation of the retronecine (2.8 g) with o-bromoanil

produced DHR (2.14 g, 77% yield) (19), which when reacted with

dA (14 g) generated a mixture of four DHP-dA adducts. Upon

HPLC puriﬁcation, pure DHP-dA-1 and DHP-dA-2 were obtained.

13

and [¹⁵N₅, C₁₀]dA were obtained from Spectra Stable Isotopes

(Columbia, MD). Alkaline phosphatase was acquired from Roche

Diagnostics Corporation (Indianapolis, IN). All solvents used were

640 Chem. Res. Toxicol., Vol. 23, No. 3, 2010

Fu et al.

On the basis of ¹H NMR spectroscopic analysis in the presence of

a known quantity of tert-butanol as internal standard, 3.8 mg of

DHP-dA-1 (0.07% yield) and 3.6 mg of DHP-dA-2 (0.067% yield)

were obtained.

DHP-H6a), 3.82 (1H, m, dG-H4′), 3.84-3.90 (1H, m, DHP-H7b),

3.92-3.97 (1H, m, DHP-H7a), 4.26 (1H, t, J ) 14, DHP-H9b),

4.35 (1H, two sets of d, J ) 14.2, DHP-H9a), 4.35 (1H, m, dG-

H3′), 4.74 (1H, dd, J ) 6.4, 7.6, DHP-H5), 6.11 (1H, d, J ) 2.7,

DHP-H2), 6.17 (1H, m, dG-H1′), 6.68 (1H, two sets of d, J ) 2.7,

DHP-H3), 7.89 (1H, s, dG-H8).

¹H NMR spectroscopic data for each of the isolated adducts

follow.

DHP-dA-1. ¹H NMR (DMSO-d₆) δ 2.23-2.31 (1H, m, dA-H2′′),

2.35-2.47 (1H, m, DHP-H6b), 2.68-2.78 (1H, m, dA-H2′), 2.85-2.93

(1H, m, DHP-H6a), 3.49-3.56 (1H, two sets of d, J ) 4.5, dA-H5′′),

3.60-3.66 (1H, two sets of d, J ) 4.5, dA-H5′), 3.85-3.93 (2H, m,

DHP-H5b, dA-H4′), 4.01-4.25 (3H, m, DHP-H5a, H9a, H9b),

4.38-4.45 (1H, m, dA-H3′), 4.48 (1H, bs, OH), 5.10-5.45 (2H, bs,

overlapped, OH), 5.78 (1H, bs, DHP-H7), 6.04-6.10 (1H, two sets

of d, J ) 2.5, DHP-H2), 6.35-6.38 (1H, dd, dA-H1′), 6.60-6.67 (1H,

two sets of d, J ) 2.5, DHP-H3), 8.07 (1H, bs, dA-N6H), 8.27 (1H,

bs, dA-H8), 8.35 (1H, s, dA-H2).

1

DHP-dG-1. H NMR (DMSO-d₆) δ 2.21-2.25 (1H, m, dG-

H2′′), 2.34-2.38 (1H, m, DHP-H6b), 2.62-2.68 (1H, m, dG-H2′),

2.86-2.90 (1H, m, DHP-H6a), 3.49-3.54 (1H, m, dG-H5′′),

3.57-3.60 (1H, m, dG-H5′), 3.80-3.83 (1H, m, dG-H4′), 3.89-3.94

(1H, m, DHP-H5b), 3.99-4.05 (1H, m, DHP-H5a), 4.28 (1H, bd,

DHP-H9b), 4.32 (1H, bd, DHP-H9a), 4.38-4.40 (1H, m, dG-H3′),

4.51 (1H, bs, OH/NH), 4.87 (1H, bt, OH/NH), 5.25-5.28 (2H, m,

DHR-H7, OH/NH), 6.09 (1H, d, J ) 2.6, DHP-H2), 6.20 (1H, dd,

J ∼ 7, dG-H1′), 6.66 (1H, d, J ) 2.6, DHP-H3), 6.73 (1H, bs,

OH/NH), 7.93 (1H, s, dG-H8), 10.31 (1H, s, dG-NH1).

DHP-dA-1. ¹H NMR (DMSO-d₆/D₂O) δ 2.25-2.31 (1H, m, dA-

H2′′), 2.33-2.47 (1H, m, DHP-H6b), 2.67-2.73 (1H, m, dA-H2′),

2.87-2.96 (1H, m, DHP-H6a), 3.48-3.56 (1H, two sets of d, J )

4.5, dA-H5′′), 3.59-3.62 (1H, two sets of d, J ) 4.5, dA-H5′),

3.84-3.92 (2H, m, DHP-H5b, dA-H4′), 4.04-4.23 (3H, m, DHP-

H5a, H9a, H9b), 4.37-4.43 (1H, m, dA-H3′), 5.72 (1H, bs, DHP-

H7), 6.04-6.06 (1H, two sets of d, J ) 2.5, DHP-H2), 6.34 (1H,

dd, dA-H1′), 6.59-6.65 (1H, two sets of d, J ) 2.5, DHP-H3)

8.25 (1H, s, dA-H8), 8.32 (1H, s, dA-H2).

DHP-dG-1. ¹H NMR (DMSO-d₆/D₂O) δ 2.21-2.26 (1H, m, dG-

H2′′), 2.31-2.37 (1H, m, DHP-H6b), 2.61-2.67 (1H, m, dG-H2′),

2.83-2.90 (1H, m, DHP-H6a), 3.80-3.83 (1H, m, dG-H4′),

3.87-3.91 (1H, m, DHP-H5b), 3.99-4.03 (1H, m, DHP-H5a), 4.25

(1H, d, J ) 12, DHP-H9b), 4.29 (1H, d, J ) 12, DHP-H9a),

4.37-4.39 (1H, m, dG-H3′), 5.25-5.27 (1H, m, DHP-H7), 6.08

(1H, d, J ) 2.5, DHP-H2), 6.19 (1H, dd, J ∼ 7, dG-H1′), 6.65

(1H, d, J ) 2.5, DHP-H3), 7.91 (1H, s, dG-H8).

1

DHP-dG-2. H NMR (DMSO-d₆) δ 2.21-2.26 (1H, m, dG-

DHP-dA-2. ¹H NMR (DMSO-d₆) δ 2.23-2.32 (1H, m, dA-H2′′),

2.34-2.45 (1H, m, DHP-H6b), 2.68-2.78 (1H, m, dA-H2′), 2.84-2.94

(1H, m, DHP-H6a), 3.47-3.56 (1H, m, dA-H5′′), 3.57-3.66 (1H, m,

dA-H5′), 3.81-3.94 (2H, m, DHP-H5b, dA-H4′), 4.04-4.25 (3H, m,

DHP-H5a, H9a, H9b), 4.41 (1H, bs, dA-H3′), 4.47 (1H, bs, OH), 5.25

(1H, bs, OH), 5.33 (1H, bs, OH), 5.78 (1H, bs, DHP-H7), 6.06 (1H,

d, J ) 2.0, DHP-H2), 6.35-6.38 (1H, dd, dA-H1′), 6.60-6.67 (1H,

two sets of d, J ) 2.0, DHP-H3), 8.06 (1H, bs, dA-N6H), 8.27

(1H, bs, dA-H8), 8.35 (1H, s, dA-H2).

H2′′), 2.34-2.39 (1H, m, DHP-H6b), 2.62-2.68 (1H, m, dG-H2′),

2.86-2.90 (1H, m, DHP-H6a), 3.49-3.54 (1H, m, dG-H5′′),

3.57-3.60 (1H, m, dG-H5′), 3.81-3.83 (1H, m, dG-H4′), 3.89-3.94

(1H, m, DHP-H5b), 4.00-4.05 (1H, m, DHP-H5a), 4.29-4.34 (1H,

m, DHP-H9b), 4.32-4.34 (1H, m, DHP-H9a), 4.38-4.40 (1H, m,

dG-H3′), 4.51 (1H, t, OH/NH), 4.87 (1H, t, OH/NH), 5.25-5.28

(2H, m, DHP-H7, OH/NH), 6.09 (1H, d, J ) 2.5, DHP-H2), 6.20

(1H, dd, J ∼ 7.1, dG-H1′), 6.66 (1H, d, J ) 2.5, DHP-H3), 6.73

(1H, bs, OH/NH), 7.92 (1H, s, dG-H8), 10.28 (1H, s, dG-NH1).

DHP-dG-2. ¹H NMR (DMSO-d₆/D₂O) δ 2.21-2.23 (1H, m, dG-

H2′′), 2.34-2.36 (1H, m, DHP-H6b), 2.63-2.67 (1H, m, dG-H2′),

2.83-2.90 (1H, m, DHP-H6a), 3.80-3.83 (1H, m, dG-H4′),

3.87-3.92 (1H, m, DHP-H5b), 4.00-4.03 (1H, m, DHP-H5a), 4.26

(1H, d, J ) 12, DHP-H9b), 4.30 (1H, d, J ) 12, DHP-H9a),

4.36-4.38 (1H, m, dG-H3′), 5.25-5.27 (1H, m, DHP-H7), 6.08

(1H, d, J ) 2.5, DHP-H2), 6.19 (1H, dd, J ∼ 7, dG-H1′), 6.65

(1H, d, J ) 2.5, DHP-H3), 7.91 (1H, s, dG-H8).

DHP-dA-2. ¹H NMR (DMSO-d₆/D₂O) δ 2.24-2.32 (1H, m, dA-

H2′′), 2.33-2.44 (1H, m, DHP-H6b), 2.62-2.74 (1H, m, dA-H2′),

2.85-2.96 (1H, m, DHP-H6a), 3.47-3.56 (1H, two sets of d, J )

4.0, dA-H5′′), 3.65 (1H, m, dA-H5′, overlapped with D₂O),

3.84-3.92 (2H, m, DHP-H5b, dA-H4′), 4.02-4.23 (3H, m, DHP-

H5a, H9a, H9b), 4.37-4.43 (1H, m, dA-H3′), 5.74 (1H, bs, DHP-

H7), 6.03-6.09 (1H, two sets of d, J ) 2.0, DHP-H2), 6.32-6.35

(1H, dd, dA-H1′), 6.60-6.65 (1H, two sets of d, J ) 2.0, DHP-

H3), 8.25 (1H, s, dA-H8), 8.32 (1H, s, dA-H2).

DHP-dG-3. ¹H NMR (DMSO-d₆) δ 2.19-2.26 (2H, m, dG-H2′′,

DHP- H6b), 2.54-2.68 (2H, m, dG-H2′, DHP-H6a), 3.48-3.53

(1H, m, dG-H5′′), 3.57-3.59 (1H, m, dG-H5′), 3.81-3.85 (2H,

m, dG-H4′, DHP-H5b), 3.99-4.03 (1H, m, DHP-H5a), 4.23-4.28

(1H, m, DHP-H9b), 4.31-4.37 (1H, m, DHP-H9a), 4.35-4.38 (1H,

m, dG-H3′), 4.81 (1H, m, OH/NH), 5.06-5.08 (1H, m, DHP-H7),

5.14-5.15 (1H, m, OH/NH), 5.26-5.27 (1H, m, OH/NH), 6.09

(1H, d, J ) 2.6, DHP-H2), 6.18 (1H, m, dG-H1′), 6.34 (1H, m,

OH/NH), 6.61 (1H, d, J ) 2.5, DHP-H3), 7.91 (1H, s, dG-H8),

10.40 (1H, s, dG-NH1).

Synthesis of Isotopically Labeled DHP-dG and DHP-dA

13

Adducts. DHP-[¹⁵N₅]dG and DHP-[¹⁵N₅, C₁₀]dA adducts were

prepared as described above by reacting DHR (3.0 mg, 0.02 mmol)

with [¹⁵N₅]dG (5 mg, 0.02 mmol) and [¹⁵N₅,¹³C₁₀]dA (5 mg, 0.02

mmol), respectively.

Chemical Reaction of DHR with Calf Thymus DNA. To

prepare DNA standards modiﬁed with DHR, 5 mg of calf thymus

DNA in 2 mL of 5 mM Tris-HCl buffer, pH 7.1, containing 0.1

mM EDTA (Tris-EDTA) was reacted with 0, 0.1, 0.5, 1, 5, and 50

µg of DHR in 100 µL of methanol at 37 °C for 3 h. After the

incubation, the reaction mixture was extracted twice with 5 mL of

a chloroform/isoamyl alcohol mixture (24/1, v/v). The DNA in the

aqueous phase was precipitated by adding 250 µL of 3 M sodium

acetate followed by an equal volume of cold 2-propanol and washed

with 70% ethanol. After the DNA was dissolved in Tris-EDTA,

the DNA concentration was determined spectrophotometrically. The

DNA solution was stored at -78 °C prior to hydrolysis and LC-

ES-MS/MS analysis.

The deoxynucleoside adducts were quantiﬁed by HPLC-ES-MS/

MS after standard enzymatic hydrolysis of the DNA to deoxy-

nucleosides (43). The hydrolysis mixtures from 100 µg aliquots of

the DHR-modiﬁed DNA were analyzed directly, without any adduct

enrichment procedure.

HPLC-ES-MS/MS Analysis of DHP-Derived DNA Adducts

Formed in Vitro and in ViWo. Quantiﬁcation of DHP-DNA

Standards. Puriﬁed samples of the DHP-dG-1 and DHP-dG2

adduct standards were quantiﬁed spectrophotometrically on the basis

of the UV molar extinction coefﬁcients 1.70 × 10⁴and 1.72 × 10⁴

DHP-dG-3. ¹H NMR (DMSO-d₆/D₂O) δ 2.18-2.25 (2H, m, dG-

H2′′, DHP- H6b), 2.55-2.66 (2H, m, dG-H2′, DHP-H6a), 3.78-3.83

(2H, m, dG-H4′, DHP-H5b), 3.98-4.03 (1H, m, DHP-H5a), 4.24

(1H, two sets of d, J ) 14, DHP-H9b), 4.32 (1H, two sets of d,

DHP-H9a), 4.33-4.38 (1H, m, dG-H3′), 5.03-5.08 (1H, m, DHP-

H7), 6.07 (1H, d, J ) 2.7, DHP-H2), 6.19 (1H, m, dG-H1′), 6.60

(1H, d, J ) 2.5, DHP-H3), 7.89 (1H, s, dG-H8).

1

DHP-dG-4. H NMR (DMSO-d₆) δ 2.18-2.24 (1H, m, dG-

H2′′), 2.34-2.37 (1H, m, DHP-H6b), 2.56-2.65 (2H, m, dG-H2′,

DHP-H6a), 3.49-3.53 (1H, m, dG-H5′′), 3.54-3.57 (1H, m, dG-

H5′), 3.82 (1H, m, dG-H4′), 3.86-3.92 (1H, m, DHP-H7b),

3.95-4.00 (1H, m, DHP-H7a), 4.25-4.32 (1H, m, DHP-H9b),

4.33-4.36 (1H, m, DHP-H9a), 4.33-4.37 (1H, m, dG-H3′), 4.76

(1H, dd, DHP-H5), 4.85 (1H, bs, OH/NH), 5.25 (1H, d, OH/NH),

6.12 (1H, d, DHP-H2), 6.18 (1H, m, dG-H1′), 6.43 (1H, bs, OH/

NH), 6.70 (1H, two sets of d, DHP-H3), 7.90 (1H, s, dG-H8), 8.56

(1H, s, OH/NH), 10.38 (1H, bs, dG-NH1).

DHP-dG-4. ¹H NMR (DMSO-d₆/D₂O) δ 2.19-2.24 (1H, m, dG-

H2′′), 2.34-2.39 (1H, m, DHP-H6b), 2.56-2.65 (2H, m, dG-H2′,

DNA Adducts from Pyrrolizidine Alkaloids

Chem. Res. Toxicol., Vol. 23, No. 3, 2010 641

Scheme 1. Synthetic Preparation of DHP-dG Adducts^a

^aThe absolute conﬁguration assignments at the C7 positions of the necine base of DHP-dG-1 and DHP-dG-2 are arbitrary.

M^-1cm^-1at 256 nm (pH 7.0) reported by Robertson (44). DHP-

dA-1 and DHP-dA-2 adduct standards were also quantiﬁed spec-

trophotometrically using the following experimentally determined

UV molar extinction coefﬁcients: 2.47 ( 0.26 × 10⁴and 2.48 (

0.14 x10⁴M^-1cm^-1, respectively, at 271 nm in water and 2.21 (

0.23 × 10⁴and 2.18 ( 0.12 × 10⁴M^-1cm^-1, respectively, in

methanol at 270 nm. The concentrations of the isotopically labeled

adduct standards for DHP-dG-1, DHP-dG-2, DHP-dA-1, and DHP-

dA-2 were quantiﬁed by LC-UV (256 nm) by comparison to the

unlabeled standards. Isotopically labeled standards were not avail-

able for DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4;

isotopically labeled DHP-dG-2 was used as an internal standard

for DHP-dG-3 and DHP-dG-4, and isotopically labeled DHP-dA-2

was used as an internal standard for DHP-dA-3 and DHP-dA-4.

Response factors (i.e., unlabeled standard response/internal standard

response) were determined by adding known amounts of labeled

and unlabeled standards to DNA hydrolysates.

DNA Hydrolysis. DNA samples (typically ∼100 µg) obtained

from a reaction of DHR with calf thymus DNA, rats treated with

riddelliine or monocrotaline, and rats from the NTP study were

enzymatically hydrolyzed to nucleosides with micrococcal nuclease,

spleen phosphodiesterase, and nuclease P1 as previously described

(43). After adding approximately 50 fmol of each internal standard,

the samples were injected on the HPLC for ES-MS/MS analyses.

Liquid Chromatography. The liquid handling system consisted

of an Alliance 2795 separations module (Waters, Milford, MA), a

Dionex GP40 quaternary gradient pump (Dionex, Sunnyvale, CA),

and an automated switching valve (TPMV, Rheodyne, Cotati, CA).

The switching valve allowed for online sample cleanup. The valve

was used to divert the trap column efﬂuent to either waste or to

the analytical column. The Alliance 2795 system was used for

sample injection, sample cleanup, and regeneration of the trap

column. The Dionex pump was used to back-ﬂush the trap column

to the analytical column during analysis and to keep a constant

ﬂow of mobile phase going into the mass spectrometer during the

sample loading and preparation periods.

column was equilibrated with the starting mobile phase. The total

run time for sample preparation and analysis was 34 min.

Mass Spectrometry. A Quattro Ultima quadrupole mass spec-

trometer (Waters, Milford, MA.), equipped with an ES interface,

was used with a source block of 100 °C and a desolvation

temperature of 400 °C. Nitrogen was the desolvation (750 L/h),

cone gas (100 L/h), and nebulizing gas. Argon was the collision

gas, at a collision cell pressure of 2.2 × 10^-3mBar. Positive ions

were acquired in the multiple reaction monitoring (MRM) mode

(dwell time of 0.2 s and interchannel delay of 0.03 s). The DHP-

dG adducts were monitored at the [(M + H)⁺] (m/z 403) to m/z

269 transition and the dG internal standards at the (M + H)⁺(m/z

408) to m/z 274 transition. The DHP-dA adducts were monitored

at the [(M + H)⁺] (m/z 387) to m/z 253 transition and the dA

internal standards at the (M + H)⁺(m/z 402) to m/z 263 transition.

The cone voltage was 25 V, and the collision energy was 14 eV

for all of the transitions. Samples were quantiﬁed by comparing

the areas of the unlabeled chromatogram peaks to those of the

corresponding labeled internal standard chromatogram peaks.

Results

Synthesis of DHP-dG Adducts. DHP-dG adducts were

synthesized by the reaction of DHR with dG (Scheme 1). The

resulting reaction products were puriﬁed by HPLC, ﬁrst with a

Whatman ODS-3 column, to remove excess dG (data not

shown), and then with a Prodigy 5 µm ODS column (Figure

3A). The materials contained in the chromatographic peaks

eluted at 18.2 (DHP-dG-1) and 19.5 (DHP-dG-2) min showing

baseline separation and exhibited UV-visible absorption spectra

(Figure 4A) that were identical to those reported by Robertson

(44). The whole scan mass spectra of these compounds revealed

that both had protonated molecules (M + H)⁺at m/z 403 (data

not shown). These products were also characterized by ES/MS

positive product ion spectra, which showed transition ions at

m/z 269 (DHP-guanine-OH), m/z 152 (guanine), and m/z 136

(DHP) (Figure 5).

Each sample was loaded onto a reverse phase trap column [Luna

C18(2), 2 mm × 30 mm, 3 µm, Phenomenex, Torrance, CA] with

95% water and 5% acetonitrile at a ﬂow rate of 0.2 mL/min for

3.5 min. After switching the divert valve, the concentrated sample

zone was back-ﬂushed from the trap column onto the analytical

column [Luna C18(2), 2 mm × 150 mm, 3 µm, Phenomenex] with

a water/acetonitrile gradient at 0.2 mL/min, and the sample

components were eluted into the mass spectrometer. The gradient

consisted of 10% acetonitrile for 2 min followed by a linear gradient

up to 20% acetonitrile over 20 min, and then reset to the initial

conditions. After 3 min, the valve was switched back to the load

position, and the trap column was cleaned of waste with 70% of

acetonitrile for 10 min at a ﬂow rate of 0.5 mL/min. Then the trap

The CD spectra of DHP-dG-1 and DHP-dG-2 were similar

to those reported by Robertson (44). The CD Cotton effects of

DHP-dG-1 were approximate mirror images of those of DHP-

dG-2 (Figure 6A), indicating that these adducts were a pair of

epimers differing in the conﬁguration of the stereocenter of the

1

DHR chromophore. The H NMR spectra of DHP-dG-1 and

DHP-dG-2 showed characteristic downﬁeld shifts (ca. 0.27 ppm)

of H7 compared to H7 in DHR itself (Table 1), as reported by

Robertson (44). By contrast, the location of the resonances for

H2, H3, H5a, H5b, H9a, and H9b showed smaller downﬁeld

642 Chem. Res. Toxicol., Vol. 23, No. 3, 2010

Fu et al.

Figure 3. Reversed-phased HPLC proﬁles of (A) DHP-dG adducts and (B) DHP-dA adducts formed from reactions of DHR with 2′-deoxyguanosine

(dG) and 2′-deoxyadenosine (dA), respectively. For HPLC conditions, see Experimental Procedures.

ppm) compared to the corresponding protons in DHR, which

indicated proximity to the substitution site. These data were

consistent with adduct formation through substitution at H7 of

the DHP moiety. Each of the deoxyribose protons was clearly

evident in both DHP-dG-1 and DHP-dG2. In addition, reso-

nances could be assigned to H8 and N1H of dG, which excluded

substitution at C8, N1, or O⁶of the dG moiety. On the basis of

the UV-visible absorption, mass, CD, and NMR spectra, the

structures of DHP-dG-1 and DHP-dG-2 were characterized as

a pair of epimeric DHP-dG adducts in which reaction had

occurred between C7 of DHP and N²of dG (i.e., (()-7-

(deoxyguanosin-N²-yl)dehydrosupinidine) (Scheme 1).

The materials eluting at 25.5 (DHP-dG-3) and 25.8 (DHP-

dG-4) min (Figure 3A) had UV-visible absorption spectra

(Figure 4A) identical to those of DHP-dG-1 and DHP-dG-2.

Whole scan mass spectra revealed that the adducts had proto-

nated molecules (M + H)⁺at m/z 403 (data not shown). Similar

to the mass spectrometric pattern of DHP-dG-1 and DHP-dG-

2, product ion mode mass spectrometric analysis indicated that

both DHP-dG-3 and DHP-dG-4 had transition ions at m/z 269

(DHP-guanine-OH), m/z 152 (guanine), and m/z 136 (DHP)

(Figure 5).

1

In contrast to DHP-dG-1 and DHP-dG-2, the H NMR

spectrum of DHP-dG-3 had a resonance for H7 of the DHP

moiety that was nearly identical to H7 of DHR (Table 1 and

Figure 7); likewise, H2, H3, and both pairs of geminal H5

and H6 protons were almost unaffected by adduct formation.

In addition, both H9a and H9b appeared as a set of mutually

coupled doublets (Table 1), which suggested signiﬁcant

prochirality. As with DHP-dG-1 and DHP-dG-2, resonances

could be assigned to each of the deoxyribose protons, H8,

and N1H of dG. These NMR data indicated that reaction had

occurred at C9 of DHR through the exocyclic nitrogen of

dG, an interpretation supported by a fragment at m/z 164 in

the product ion mass spectrum, which is consistent with a

protonated guanine bearing a methylene group at the N²

position. Thus, on the basis of the UV-visible, mass, and

Figure 4. UV-visible absorption spectra of (A) 1, DHP-dG-1; 2, DHP-

dG-2; 3, DHP-dG-3; and 4, DHP-dG-4; and (B) 1, DHP-dA-1; 2, DHP-

dA-2; 3, DHP-dA-3; and 4, DHP-dA-4. Spectra were obtained in 25%

methanol in water.

shifts from the same resonances in DHR. Moreover, both

geminal H6 protons were substantially deshielded (ca. 0.18-0.26

DNA Adducts from Pyrrolizidine Alkaloids

Chem. Res. Toxicol., Vol. 23, No. 3, 2010 643

Figure 5. Electrospray positive product ion mass spectra of the four DHP-dG adduct standards (A ) DHP-dG-1, B ) DHP-dG-2, C ) DHP-dG-3,

and D ) DHP-dG-4). The product ions of the (M + H)⁺m/z 403 were acquired with a cone voltage of 25 V and a collision energy of 30 eV.

The ¹H NMR spectrum of DHP-dG-4 had a single C-5 proton,

which was shifted downﬁeld by approximately 0.8-1 ppm

relative to H5 in DHR and the other DHP-dG adducts (Table 1

and Figure 7). In contrast to the other adducts, there were two

H7 resonances in DHP-dG-4, and these were shifted upﬁeld by

approximately 1-1.3 ppm compared to that of the H7 resonance

in DHR and the other DHP-dG adducts. As with the other DHP-

dG adducts, resonances were clearly evident for each of the

deoxyribose protons, H8, and N1H of dG. Thus, on the basis

of its UV-visible, mass, and ¹H NMR spectroscopic data, DHP-

dG-4 was characterized as 5-(deoxyguanosin-N²-yl)dehydrosu-

pinidine.

The UV molar extinction coefﬁcients of DHP-dG-1 and DHP-

dG-2 were previously reported by Robertson (44). On the basis

of the similarity of the UV spectra (Figure 4A) and assuming

that the molar extinction coefﬁcients of DHP-dG-3 and DHP-

dG-4 are identical to those of DHP-dG-1 and DHP-dG-2, the

yields of these four adducts in the crude reaction mixture of

DHR with dG were estimated from the corresponding HPLC

peak areas as follows: DHP-dG-1, 0.89%; DHP-dG-2, 0.69%;

DHP-dG-3, 0.15%; and DHP-dG-4, 0.06%. Because of the

unstable nature of pyrrolic compounds, these adducts, particu-

larly DHP-dG-3 and DHP-dG-4, decomposed during HPLC

puriﬁcation.

Synthesis of DHP-dA Adducts. DHP-dA adducts were

synthesized by the reaction of DHR with dA (Scheme 2) and

puriﬁed by HPLC, ﬁrst with a Whatman ODS-3 column (not

shown) and then with a Prodigy 5 µm ODS column (Figure

3B). Whole scan mass spectrometric analysis revealed that the

materials contained in the chromatographic peaks eluted at 28.5

(DHP-dA-1), 29.5 (DHP-dA-2), 33.4 (DHP-dA-3), and 33.9

(DHP-dA-4) min had protonated molecules (M + H)⁺at m/z

Figure 6. CD spectra of (A) DHP-dG-1 and DHP-dG-2 in methanol,

387 (data not shown); electrospray positive product ion mass

spectrometric analysis indicated transition ions at m/z 253 (DHP-

adenine-OH) and m/z 136 (DHP) (Figure 8), which suggested

that they were DHP-dA adducts.

and (B) DHP-dA-1 and DHP-dA-2 in water.

¹H NMR spectroscopic data, DHP-dG-3 was characterized

as 9-(deoxyguanosin-N²-yl)dehydrosupinidine (Figure 7).

644 Chem. Res. Toxicol., Vol. 23, No. 3, 2010

Fu et al.

DHP-dA-1 and DHP-dA-2 had identical UV-visible absorp-

1

tion spectra (Figure 4B) and highly similar H NMR spectra

(Figure 9). As shown in Figure 6B, the CD Cotton effects of

DHP-dA-1 were an approximate mirror image of those of DHP-

dA-2, indicating that these adducts were a pair of epimers

differing in the conﬁguration of one stereocenter in the DHR

1

chromophore. Similar to DHP-dG-1 and DHP-dG-2, the H

NMR spectra of DHP-dA-1 and DHP-dA-2 showed character-

istic downﬁeld shifts (ca. 0.73 ppm) of H7 compared to that of

H7 in DHR (Figure 9 and Table 1). Resonances could be

assigned to H2, H3, H5a, H5b, H6a, H6b, H9a, and H9b. With

the exception of H9a and H9b, which were shielded by

approximately 0.2 ppm, these resonances were very similar to

those observed for DHP-dG-1 and DHP-dG-2. These data are

consistent with substitution through C7 of the DHR moiety, with

positioning of the methylene C9 protons in the shielding zone

of the adenine ring. Each of the deoxyribose protons was clearly

evident for the dA moiety. In addition, resonances could be

assigned to H2 and H8, and the N⁶H resonance appeared as a

single proton. On the basis of the UV-visible absorption, mass,

CD, and NMR spectra, the structures of DHP-dA-1 and DHP-

dA-2 were characterized as a pair of epimeric DHP-dA adducts

in which the reaction had occurred between C7 of DHP and N⁶

of dA [i.e., (()7-(deoxyadenosin-N⁶-yl)dehydrosupinidine]

(Scheme 2).

1

There was an insufﬁcient amount of material for H NMR

spectroscopic characterization of DHP-dA-3 and DHP-dA-4. On

the basis of their mass and UV-visible spectroscopic charac-

teristics, DHP-dA-3 and DHP-dA-4 were tentatively assigned

as dA-dehydrosupinidine adducts. With the assumption that

DHP-dA-3 and DHP-dA-4 have molar extinction coefﬁcients

similar to those of DHP-dA-1 and DHP-dA-2, upon HPLC

analysis, the crude yields of these adducts were estimated as

follows: DHP-dA-1, 0.1%; DHP-dA-2, 0.1%; DHP-dA-3,

0.01%; and DHP-dA-4, 0.01%.

Synthesis of Isotopically Labeled DHP-[¹⁵N₅]dG and

DHP-[¹⁵N₅, ¹³C₁₀]-dA. DHP-[¹⁵N₅]dG and DHP-[¹⁵N₅, ¹³C₁₀]dA

adducts were similarly prepared by reacting DHR with [¹⁵N₅]dG

13

and [¹⁵N₅, C₁₀]dA, respectively. DHP-[¹⁵N₅]dG-1, DHP-

[¹⁵N₅]dG-2, DHP-[¹⁵N₅]dG-3, and DHP-[¹⁵N₅]dG-4 showed a

(M + H)⁺at m/z 408 and a transition ion at m/z 274 (DHR-

13

[¹⁵N₅]guanine-OH) (data not shown). DHP-[¹⁵N₅, C₁₀]dA-1,

DHP-[¹⁵N₅, ¹³C₁₀]dA-2, DHP-[¹⁵N₅, ¹³C₁₀]dA-3, and DHP-[¹⁵N₅,

13

C ]dA-4 had a (M + H)⁺at m/z 402 and a transition ion at

10

m/z 263 (data not shown).

Standard Characterization and Calibration Curves. HPLC-

ES-MS/MS calibration curves were generated for synthetically

prepared DHP-dG-1, DHP-dG-2, DHP-dA-1, and DHP-dA-2

versus their respective [¹⁵N₅] and [¹⁵N₅, ¹³C₁₀] internal standards.

Response factors were generated from these curves. The curves

for the unlabeled compounds went from 10 to 500 fmol on-

13

column versus 50 fmol of the [¹⁵N₅] and [¹⁵N₅, C₁₀] internal

standards. The curves were linear with r²> 0.999.

[¹⁵N₅] adduct standards did not exist for DHP-dG-3, DHP-

dG-4, DHP-dA-3, and DHP-dA-4. To generate response factors

for these adducts, 100 µg aliquots of hydrolyzed DNA were

fortiﬁed with the following mixtures: 50 fmol DHP-dG-3 and

55 fmol DHP-[¹⁵N₅]dG-2; 25 fmol DHP-dG-4 and 55 fmol

DHP-[¹⁵N₅]dG-2; 50 fmol DHP-dA-3 and 82 fmol DHP-[¹⁵N₅,

13

C ]dA-2; and 134 fmol DHP-dA-4 and 82 fmol DHP-[¹⁵N₅,

10

13

C ]dA-2. The ﬁnal response factors obtained were as follows:

DHP-dG-1 vs DHP-[¹⁵N₅]dG-1 ) 1.18; DHP-dG-2 vs DHP-

[¹⁵N₅]dG-2 ) 1.20; DHP-dG-3 vs DHP-[¹⁵N₅]dG-2 ) 2.10;

DHP-dG-4 vs DHP-[¹⁵N₅]dG-2 ) 1.50; DHP-dA-1 vs DHP-

DNA Adducts from Pyrrolizidine Alkaloids

Chem. Res. Toxicol., Vol. 23, No. 3, 2010 645

1

Figure 7. H NMR spectra of DHP-dG-3 and DHP-dG-4 measured in DMSO-d₆.

Scheme 2. Synthetic Preparation of DHP-dA Adducts^a

^aThe absolute conﬁguration assignments at the C7 positions of the necine base of DHP-dA-1 and DHP-dA-2 are arbitrary.

[¹⁵N₅, ¹³C₁₀]dA-1 ) 0.93; DHP-dA-2 vs DHP-[¹⁵N₅, ¹³C₁₀]dA-

The levels of DHP-dG and DHP-dA adducts formed from

13

2 ) 0.91; DHP-dA-3 vs DHP-[¹⁵N₅, C₁₀]dA-2 ) 6.50; and

each of the reactions are tabulated in Table 2. These results

indicate that (i) DHP-dG and DHP-dA adducts were formed in

a dose-responsive manner; (ii) DHP-dA-3 and DHP-dA-4 were

the most abundant, followed by all of the DHP-dG adducts,

which were formed to a similar extent, and DHP-dA-1 and DHP-

dA-2 were formed to the least extent; and (iii) DHP-dA and

DHP-dG adducts could be detected even at the lowest treatment

group (0.1 µg of DHR reacted with 5 mg of calf thymus DNA).

13

DHP-dA-4 vs DHP-[¹⁵N₅, C₁₀]dA-2 ) 4.2.

HPLC-ES-MS/MS Analysis of DHP-dG and DHP-dA

Adducts Formed from the Reaction of Calf Thymus DNA

and DHR. DHP-DNA adducts were prepared by reactions of 5

mg of calf thymus DNA in 2 mL of 5 mM Tris-HCl buffer, pH

7.1, containing 0.1 mM EDTA with 0, 0.1, 0.5, 1, 5, and 50 µg

of DHR in 100 µL of methanol at 37 °C for 3 h. After enzymatic

hydrolysis of the resulting reaction mixtures, the quantity of

DHP-dG and DHP-dA adducts in each reaction was determined

by HPLC-ES-MS/MS through multiple reaction monitoring

(MRM) combined with the use of DHP-[¹⁵N₅]dG and DHP-

HPLC-ES-MS/MS Analysis of DHR-dG and DHR-dA

Adducts Formed in ViWo. Female rats were dosed daily with

riddelliine (0.1, 1.0, 2.0, and 5.0 mg/kg body weight) or

monocrotaline (1.0 and 5.0 mg/kg body weight) for three

consecutive days. One day after the last treatment, DNA was

isolated from the livers for the analysis of DHP-dG and DHP-

dA adducts by HPLC-ES-MS/MS. Representative MRM

chromatograms of liver DNA from a rat dosed with 5 mg of

monocrotaline/kg body weight/day and a rat dosed with 5

mg of riddelline/kg body weight/day are shown in Figure

13

[¹⁵N₅, C₁₀]dA adducts as internal standards. Representative

multiple reaction monitoring (MRM) measurements for DHP-

dG and DHP-dA adducts formed by reacting 5 mg of calf

thymus DNA with 5 µg of DHR are shown in Figure 10, which

clearly indicates the presence of all four DHP-dG and DHP-

dA adducts.

646 Chem. Res. Toxicol., Vol. 23, No. 3, 2010

Fu et al.

Figure 8. Electrospray positive product ion mass spectra of the four DHP-dA adduct standards (A ) DHP-dA-1, B ) DHP-dA-2, C ) DHP-dA-3,

and D ) DHP-dA-4). The product ions of the MH⁺m/z 387 were acquired with a cone voltage of 25 V and a collision energy of 30 eV.

1

Figure 9. H NMR spectra of DHP-dA-1 and DHP-dA-2 measured in DMSO-d₆.

11. The data, which are summarized in Table 3, indicate that

for both riddelliine and monocrotaline (i) DHP-dG-3 and

DHP-dG-4 were formed in a dose-responsive manner and to

a greater extent than DHP-dA-3 and DHP-dA-4; and that (ii)

with the exception of DHP-dA-2, which was found in liver

DNA from rats treated with 5 mg of riddelliine/kg body

weight/day, DHP-dG-1, DHP-dG-2, DHP-dA-1, and DHP-

dA-2 were not detected in any treatment groups.

HPLC-ES-MS/MS Analysis of DHP-dG and DHP-dA

Adducts Formed in Rat Liver Samples from an NTP

Study. In a previous study, we used the ³²P-postlabeling

methodology to determine the levels of DHP-derived DNA

DNA Adducts from Pyrrolizidine Alkaloids

Chem. Res. Toxicol., Vol. 23, No. 3, 2010 647

Figure 10. Representative MRM chromatograms (relative signal intensity vs time) for DHP-dG and DHP-dA adducts formed during the in Vitro

reaction of 5 mg of calf thymus DNA with 5 µg of DHR. Internal standards (IS) were added in amounts of 50, 55, 50, and 80 fmol for DHP-dG-1,

DHP-dG-2, DHP-dA-1, and DHP-dA-2, respectively.

Table 2. Levels of DHP-dG and DHP-dA Adducts Formed from Reactions of 5 mg of Calf Thymus DNA in 2 mL of 5

mMTris-HCl Buffer, pH 7.1, Containing 0.1 mM EDTA and 0, 0.1, 0.5, 1, 5, or 50 µg of DHR in 100 µL of Methanol for 3 h

sample

levels of DHP-dG and DHP-dA/10⁸nucleotides

DHR DNA^a

DHP-dG-1

DHP-dG-2

DHP-dG-3

DHP-dG-4

DHP-dA-1

DHP-dA-2

DHP-dA-3

DHP-dA-4

50 µg

5 µg

213.5

24.6

15.3

<LOD

1.5

164.6

15.5

10.4

<LOD

1.5

143.0

23.0

19.4

5.4

1.3

<LOD

1.2

180.6

30.1

23.1

5.8

<LOD

1.5

20.1

2.3

1.9

<LOD

0.3

21.9

3.5

3.0

0.3

<LOD

0.1

266.0

42.7

27.3

4.4

1.2

<LOD

0.1

366.0

60.7

37.8

6.3

1.6

<LOD

0.1

1 µg

0.5 µg

0.1 µg

0 µg

LOD^b

0.1

^aThe reaction consisted of the amount of DHR indicated in the presence of 5 mg of DNA. ^bLOD ) limit of detection based upon the analysis of

100 µg of DNA by HPLC-ES-MS/MS.

adducts in liver samples from an NTP study in which female

F344 rats were treated with riddelliine for six months (41). In

the current study, we assessed the same samples by HPLC-ES-

MS/MS. Representative chromatograms are shown in Figure

12, and the data are summarized in Table 4. As with the DNA

samples from the rats treated for three days with either riddelliine

or monocrotaline, DHP-dG-3- and DHP-dG-4 were formed in

a dose-responsive manner and to a greater extent than DHP-

dA-3 and DHP-dA-4. Likewise, DHP-dG-1, DHP-dG-2, DHP-

dA-1, and DHP-dA-2 were not detected in any treatment groups.

To develop the method, the eight DHP-dG and DHP-dA adducts,

namely, DHP-dG-1, DHP-dG-2, DHP-dG-3, DHP-dG-4, DHP-

dA-1, DHP-dA-2, DHP-dA-3, and DHP-dA-4, as well as their

13

[¹⁵N₅]dG and [¹⁵N₅, C₁₀]dA adducts, were prepared syntheti-

cally. Upon determination of the optimal HPLC and mass

spectrometric conditions, we are able to detect these eight DHP-

derived dG and dA adducts in Vitro and/or in ViVo and quantify

the formation of these adducts in the multiple reaction monitor-

ing (MRM) mode. The DNA adducts assessed included those

formed from the reaction of DHR with calf thymus DNA in

Vitro, in liver DNA from female rats treated with different doses

of riddelliine or monocrotaline for three consecutive days, and

in liver DNA from female rats treated with riddelliine for six

months.

Discussion

Pyrrolizidine alkaloids are a class of genotoxic and tumori-

genic naturally occurring phytochemicals (3-6, 8, 26). In this

article, we report the development of a sensitive HPLC-ES-

MS/MS method for the detection of DHP-derived DNA adducts

and the utilization of this method for the detection and

quantiﬁcation of these adducts from in Vitro and in ViVo samples.

In this developed HPLC-ES-MS/MS method, the sensitivity

for different DHP-DNA adducts varies in the range of 0.1-1.5

adducts in 10⁸nucleotides for the analysis of 100 µg samples.

This level of sensitivity is potentially adequate for measuring

648 Chem. Res. Toxicol., Vol. 23, No. 3, 2010

Fu et al.

Figure 11. Representative MRM chromatograms (relative signal intensity vs time) for rat liver DNA samples. The right panels show a 100 µg

aliquot of DNA from a rat dosed daily with 5 mg of riddelline/kg body weight for three days, and the left panels show a 100 µg sample of DNA

from a rat dosed daily with 5 mg of monocrotaline/kg body weight for three days. Internal standards (IS) were added in amounts of 50, 55, 50, and

80 fmol for DHP-dG-1, DHP-dG-2, DHP-dA-1, and DHP-dA-2, respectively.

Table 3. Levels of DHP-dG and DHP-dA Adducts Formed in the Livers of Female Rats Dosed with Riddelliine or

Monocrotaline for Three Consecutive Days

levels of DHP-dG and DHP-dA/10⁸nucleotides^a

treatment

(mg/kg body weight/day)

DHP-dG-1

DHP-dG-2

DHP-dG-3

DHP-dG-4

DHP-dA-1

DHP-dA-2

DHP-dA-3

DHP-dA-4

riddelliine

5

2

1

0.1

<LOD

7.5

5.0

2.0

<LOD

10.2

4.9

2.6

<LOD

0.5

1.0

0.5

0.3

0.1

1.4

0.6

0.3

0.1

<LOD

monocrotaline

5

1

<LOD

5.2

<LOD

6.9

<LOD

0.6

0.1

0.9

0.1

control

<LOD

^aAliquots of DNA (100 µg) were assayed by HPLC-ES-MS/MS. The LOD for each of the adducts is given in Table 2.

adduct levels in exposed humans, provided that sufﬁcient DNA

samples can be collected (e.g., lymphocyte DNA is present in

whole blood at 6 µg/mL), that the ingested doses are high

enough (e.g., detectable in rat liver from 3 doses of 0.1 mg/kg

bw riddelliine), and that samples are collected soon enough after

ingestion so that adduct loss through DNA repair does not occur.

and DHP-dA-4 (Table 2). In contrast, rats dosed with either

riddelliine or monocrotaline formed DHP-dG-3 and DHP-dG-4

as the major adducts, accompanied by small amounts of DHP-

dA-3 and DHP-dA-4 (Tables 3 and 4).

On the basis of the results of our previous mechanistic studies

conducted by ³²P-postlabeling/HPLC, we had proposed a general

mechanism for DHP-derived DNA adduct formation from the

metabolism of all three types of tumorigenic pyrrolizidine

alkaloids (3, 5, 22, 24-26). Since the HPLC-ES-MS/MS method

provides further insight regarding adduct formation, we have

revised the proposed general metabolic activation mechanism,

which is presented in Scheme 3. In this Scheme, the retronecine-,

heliotridine-, and otonecine-type pyrrolizidine alkaloids are

metabolized to reactive pyrrolic esters (3, 5, 22, 24-26).

Hydrolysis and the subsequent loss of a hydroxyl group of the

There were marked differences in regioselectivity depending

upon the system being investigated. Reactions of DHR with

dG produced a pair of epimeric DHP-dG-1 and DHP-dG-2 as

the predominant adducts, with DHP-dG-3 and DHP-dG-4 being

formed in a much lower yield (Figure 3A). Likewise, DHR

reacted with dA in a regioselective manner to produce pre-

dominantly DHP-dA-1 and DHP-dA-2 (Figure 3B). When DHR

was reacted with calf thymus DNA, all four DHP-dG adducts

were formed to a similar but to a lower extent than DHP-dA-3

DNA Adducts from Pyrrolizidine Alkaloids

Chem. Res. Toxicol., Vol. 23, No. 3, 2010 649

Figure 12. Representative MRM chromatograms (relative signal intensity vs time) for liver DNA samples from an NTP study in which female

F344 rats were administered riddelliine. The left panel is a 100 µg aliquot of liver DNA from a control rat, and the right panel shows a 100 µg

sample of DNA from a rat dosed at 1 mg of riddelliine/kg body weight ﬁve days/week for six months. Internal standards (IS) were added in

amounts of 50, 55, 50, and 80 fmol for DHP-dG-1, DHP-dG-2, DHP-dA-1, and DHP-dA-2, respectively. The differences in retention times between

the left and right panels are due to the use of two different HPLC columns.

Table 4. Levels of DHP-dG and DHP-dA Adducts Formed in the Livers of NTP Female Rats Dosed with Riddelliine for Six

Months

levels of DHP-dG and DHP-dA/10⁸nucleotides^a

riddelliine (mg/kg b.w./day)

DHP-dG-1

DHP-dG-2

DHP-dG-3

DHP-dG-4

DHP-dA-1

DHP-dA-2

DHP-dA-3

DHP-dA-4

1

<LOD

5.9 ( 2.4

2.3 ( 1.2

<LOD

8.0 ( 2.4

2.7 ( 1.2

<LOD

0.5 ( 0.1

0.3 ( 0.1

0.1 ( 0.0

<LOD

0.8 ( 0.2

0.3 ( 0.1

0.1 ( 0.1

<LOD

0.33

0.1

0.033

0.01

0

<LOD

^aAliquots of DNA (100 µg) were assayed by HPLC-ES-MS/MS. The LOD for each of the adducts is given in Table 2. The data are presented as the

mean ( s.d., n ) 5.

pyrrolic ester lead to the formation of carbonium ions I and II.

Reaction of carbonium ion I with DNA produces C7-substituted

DHP-DNA adducts (i.e., DHP-dG-1, DHP-dG-2, DHP-dA-1,

and DHP-dA-2). Alternatively, carbonium ion I can undergo a

1,3-hydride shift to form carbonium ion III, which is stabilized

through resonance with iminium ion IV. The reaction of

carbonium ion III with DNA forms C5-substituted DHP-DNA

adducts (i.e., DHP-dG-4), whereas the reaction of carbonium

ion II with DNA leads to C9-substituted DHP-DNA adducts

(i.e., DHP-dG-3).

The pyrrolic ester (shown in Scheme 3) can also undergo

two consecutive hydrolysis steps to form DHP, which upon loss

of a hydroxyl group forms carbonium ions V and VI. Reaction

of these carbonium ions with DNA directly or after conversion

into carbonium ion VII through a 1,3-hydride shift generates

both DHP-C7 DNA adducts and DHP-C5 DNA adducts. The

DHP-C7 DNA adducts formed from carbonium ion I should

be kinetically controlled adducts, while the DHP-C5 DNA

adducts, mediated by the 1,3-hydride shift of carbonium ion I,

should be thermodynamically controlled adducts.

A comparison of the ratio of adducts obtained from in Vitro

reactions with DHR indicates that steric hindrance plays a

critical role in the regioselectivity of the reaction. When

reactions are conducted between DHR and the nucleosides dG

and dA, adducts arising from the reaction at C7 of DHP are

formed to a greater extent than adducts arising from substitution

at C5 or C9 of DHP. When DHR is reacted with DNA, the

ratio between C7-substituted adducts remains the same, as is

observed in the reactions with deoxynucleosides (DHP-dG-1/

2:DHP-dA-1/2; ∼10:1), while the proportion of adducts from

reaction at C5 and/or C9 (i.e., DHP-dG-3/4 and DHP-dA-3/4)

increases substantially (Table 2). This suggests that with the

deoxynucleosides, reactions occur primarily with carbonium ion

V, while with DNA, because of steric constraints, the equilib-

rium is shifted in favor of carbonium ions VI and VII (Scheme

3). The situation in ViVo appears to be more complex. The only

650 Chem. Res. Toxicol., Vol. 23, No. 3, 2010

Fu et al.

Scheme 3. Proposed General Mechanism Leading to DHP-Derived DNA Adduct Formation from the Metabolism of the Three

Types of Carcinogenic Pyrrolizidine Alkaloids^a

^aRiddelliine, lasiocarpine, and clivorine are used as examples for the retronecine-type, heliotridine-type, and otonecine-type pyrrolizidine alkaloids,

respectively.

adducts detected in animals treated with riddelliine or mono-

crotaline are those obtained from the reaction at C5 and/or C9

of DHP (i.e., DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-

dA-4; Tables 3 and 4). The lack of adducts arising from

substitution at C7 of DHP indicates that adduct formation in

ViVo does not occur with free DHR but rather must result from

pathways involving carbonium ions I (which rearranges to

carbonium ion III) and II, which leads to DHP-dG-3, DHP-

dG-4, DHP-dA-3, and DHP-dA-4. The other possibility to

explain the lack of DHP-dG-1, DHP-dG-2, DHP-dA-1, and

DHP-dA-2 adduct formation in ViVo is that these adducts are

indeed formed but are readily repaired.

It is worthy to note that pyrrolizidine alkaloids are capable

of binding at the C7 and the C9 positions of the necine base to

two sites in DNA or protein to form DNA or protein cross-

linking. The antimitotic, toxic, and carcinogenic activities of

pyrrolizidine alkaloids are thought to be caused, at least in part,

by these cross-links (45-50). At present, it is not known whether

or not DNA-interstrand cross-linking is involved, at least in part,

in the observed DNA adduct formation proﬁle in ViVo and in

Vitro. It warrants further investigation.

On the basis of our comprehensive studies on the mech-

anisms of pyrrolizidine alkaloid carcinogenesis, we previously

proposed that DHP-derived DNA adducts are potential

DNA Adducts from Pyrrolizidine Alkaloids

Chem. Res. Toxicol., Vol. 23, No. 3, 2010 651

(16) Schoental, R., Head, M. A., and Peacock, P. R. (1954) Senecio

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Doerge, D. R., and Fu, P. P. (2003) Identiﬁcation of DNA adducts

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Acknowledgment. We thank Dr. Po-Chuen Chan of NTP

for supplying riddelliine and the NTP rat livers. This research

was supported in part by appointments (Y.W. and Y.Z.) 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. 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.

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TX900402X

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High-performance liquid chromatography electrospray ionization tandem mass spectrometry for the detection and quantitation of pyrrolizidine alkaloid-derived DNA adducts in vitro and in viwo

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