Development of a 32p-postlabeling/HPLC method for detection of dehydroretronecine-derived DNA adducts in vivo and in vitro

Article abstract of DOI:10.1021/tx000149o

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a ³²P-postlabeling/HPLC method for detection of (i) two DHR-3′-dGMP and four DHR-3′-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR-3′-dGMP, DHR-3′-dAMP, and DHR-3′,5′dG-bisphosphate standards and characterization of their structures by mass and ¹H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and ³²P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR-3′,5′-dG-bisphosphate adducts both in vitro and in vivo.

Full text of DOI:10.1021/tx000149o

Chem. Res. Toxicol. 2001, 14, 91-100
91
Develop m en t of a 32P -P ostla belin g/HP LC Meth od for
Detection of Deh yd r or etr on ecin e-Der ived DNA Ad d u cts
in Vivo a n d in Vitr o
Ya-Chen Yang,† J ian Yan,† Mona Churchwell,† Richard Beger,† Po-Cheun Chan,‡
Daniel R. Doerge,† Peter P. Fu,† and Ming W. Chou*,†
National Center for Toxicological Research, J efferson, Arkansas 72079, and National Institute of
Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received J uly 11, 2000
Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large
number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates
dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the
development of a 32P-postlabeling/HPLC method for detection of (i) two DHR-3′-dGMP and
four DHR-3′-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in
vivo. The approach involves (1) synthesis of DHR-3′-dGMP, DHR-3′-dAMP, and DHR-3′,5′-
1
dG-bisphosphate standards and characterization of their structures by mass and H NMR
spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct
enrichment, and 32P-postlabeling, and (3) development of optimal HPLC conditions. Using this
methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric
DHR-3′,5′-dG-bisphosphate adducts both in vitro and in vivo.
activities of the parent pyrrolizidine alkaloids (1, 2, 6,
11-14, 23-27). Many of the more tumorigenic pyrolizi-
dine alkaloids are macrocyclic diesters of the necine base,
in particular of the retronecine base. Dehydropyrroliz-
idines are highly electrophilic and also highly unstable.
Thus, like most dehydropyrrolizidines, retronecine-
derived dehydropyrrolizidines bind to DNA to form DNA
adducts which undergo hydrolysis to release the corre-
sponding necic acids and produce the dehydroret-
roneciene (DHR)1-derived DNA adducts. Alternatively,
retronecine-derived dehydropyrrolizidines are first hy-
drolyzed to DHR which subsequently binds to DNA,
forming the DHR-derived DNA adducts. Consequently,
the resulting DHR-modified DNA adducts could be
responsible for tumor initiation of a number of tumori-
genic pyrrolizidine alkaloids. DHR has been found as a
common metabolite of a large number of pyrrolizidine
alkaloids in vitro and in vivo, including retrorsine (13,
28), monocrotaline (13, 28-31), senecionine (13, 32, 33),
and indicine (13). DHR itself is a tumorigen that induces
rhabdomyosarcomas in rats (29, 34) and skin tumors in
mice (30). DHR has also been demonstrated to be capable
of covalently binding to DNA, nucleosides, and nucle-
In tr od u ction
Pyrrolizidine alkaloids are a class of heterocyclic
compounds that are common constituents of hundreds
of plant species around the world (1-3). Many pyrrolizi-
dine alkaloids are highly toxic, causing tremendous
livestock loss due to liver and pulmonary lesions (4-9).
A number of pyrrolizidine alkaloids, including monocro-
taline, retrorsine, isatidine, lasiocarpine, clivorine, and
riddelliine, have been found to induce liver tumors in rats
(2, 10-17). Nevertheless, the mechanisms leading to
carcinogenesis have not been established. Human food-
stuffs, such as herbs, milk, and honey, may also be
contaminated by pyrrolizidine alkaloids, which can cause
human health problems (2, 5). The herbal tea “gordolobo
yerba”, popular in the American southwest, and “bush
tea”, used to treat children for colds in J amaica, may
contain pyrrolizidine alkaloids (11, 18, 19). In addition,
pyrrolizidine alkaloids have been found in dietary supple-
ments, such as comfrey and coltsfoot, available from
commercial sources (20-22). Comfrey, a popular herbal
tea in the world and used for healing broken bones,
ulcers, bruises, and the digestive tract (21), was found
by Betz et al. to contain pyrrolizidine alkaloids in ranges
from 0.1 to 400 ppm (21).
1 Abbreviations: MN, micrococcal nuclease; SPD, spleen phosphodi-
esterase; DHR, dehydroretronecine [7-hydroxy-1-(hydroxymethyl)-6,7-
dihydro-5H-pyrrolizine]; 3′-dGMP, 2′-deoxyguanosine 3′-monophos-
phate; 5′-dGMP, 2′-deoxyguanosine 5′-monophosphate; DHR-3′-dGMP
adducts (I and II), 3′-monophosphate of 7-(deoxyguanosin-N2-yl)-
dehydrosupinidine; DHR-3′,5′-dG-bisphosphate adducts (I and II),
3′,5′-bisphosphate of 7-(deoxyguanosin-N2-yl)dehydrosupinidine; 3′-
dAMP, 2′-deoxyadenosine 3′-monophosphate; DHR-3′-dAMP adduct,
3′-monophosphate of 7-(deoxyadenosin-N6-yl)dehydrosupinidine; DHR-
3′,5′-dA-bisphosphate adduct, 3′,5′-bisphosphate of 7-(deoxyadenosin-
N6-yl)dehydrosupinidine; PNK, cloned T4 polynucleotide kinase; DTT,
dithiothreitol; PB microsomes, liver microsomes of female F344 rats
pretreated with phenobarbital; NTP, National Toxicology Program;
NCTR, National Center for Toxicological Research.
Pyrrolizidine alkaloids require metabolic activation to
exert their toxicities (5, 6). The pyrrole metabolites
(dehydropyrrolizidines), resulting from hydroxylation of
pyrrolizidine alkaloids followed by dehydration, have
been found to be capable of binding to DNA, and these
compounds are responsible for most of the genotoxic
* To whom correspondence should be addressed. Telephone: (870)
543-7661. Fax: (870) 543-7719. E-mail: mchou@nctr.fda.gov.
† National Center for Toxicological Research.
‡ National Institute of Environmental Health Sciences.
10.1021/tx000149o This article not subject to U.S. Copyright. Published 2001 by the American Chemical Society
Published on Web 12/12/2000

92 Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Yang et al.
Syn th esis of th e 3′-Mon op h osp h a te of 7-(Deoxygu a -
n osin -N2-yl)d eh yd r osu p in id in e Ad d u cts (DHR-3′-d GMP ).
A solution of 3′-dGMP (20 mg, 60 µmol) in 4 mL of 20 mM
K2CO3 (pH 8.0) was purged with argon for 5 min. DHR (18 mg,
120 µmol) was added, and the resulting solution was stirred
anaerobically at 60 °C for 6 h. The reaction mixture was then
filtered through a 0.22 µm Millipore filter. The filtrate was
concentrated to half of the volume under reduced pressure, and
the adducts were purified by HPLC on a Whatman Partisil
ODS-3 (4.6 mm × 250 mm) column eluted with 20 mM K2CO3
(pH 8.0) isocratically at a flow rate of 1 mL/min, and monitored
at 254 and 220 nm. The DHR-3′-dGMP adducts were further
purified on an analytical Prodigy 5 µm ODS column (Phenom-
enex, 4.6 mm × 250 mm) eluted isocratically with 10% methanol
in 20 mM NH4OAc (pH 7.0) at a flow rate of 1 mL/min. The
synthesis was repeated on a larger scale so that a greater
quantity of DHR-3′-dGMP adducts was obtained for structural
determination and for use as external standards for 32P-
postlabeling and HPLC.
otides (25-27, 35-39). In this paper, we report the
development of a 32P-postlabeling/HPLC methodology for
detecting and quantifying DHR-derived DNA adducts,
including DHR-3′-dGMP and DHR-3′-dAMP adducts,
and use of this method in quantifying DHR-derived DNA
adducts obtained in vitro and in vivo.
Ma ter ia ls a n d Meth od s
Ma ter ia ls. Riddelliine was obtained from the National
Toxicology Program (NTP). Phenobarbital (sodium salt), calf
thymus DNA (sodium salt, type I), 2′-deoxyguanosine 3′-
monophosphate (sodium salt) (3′-dGMP), 2′-deoxyguanosine 5′-
monophosphate (free acid) (5′-dGMP), adenosine 5′-triphosphate
(disodium salt) (ATP), glucose 6-phosphate, glucose-6-phosphate
dehydrogenase, nicotinamide adenine dinucleotide phosphate
(NADP+), nuclease P1, micrococcal nuclease (MN), spleen phos-
phodiesterase (SPD), bicine, spermidine, and dithiothreitol were
purchased from Sigma Chemical Co. (St. Louis, MO). Monocro-
taline, o-bromanil, and barium hydroxide octahydrate were
purchased from Aldrich Chemical Co. (Milwaukee, WI). Cloned
T4 polynucleotide kinase (PNK) was obtained from U.S. Bio-
chemical Corp. (Cleveland, OH). Adenosine [γ-32P]-5′-triphos-
phate ([γ-32P]ATP) (specific activity of >7000 Ci/mmol) was
purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). All
other reagents were obtained through commercial sources and
were the highest quality available. All solvents were HPLC
grade.
Retronecine was prepared via barium hydroxide-catalyzed
hydrolysis of monocrotaline using the procedure of Hoskins and
Crout (40). A solution of monocrotaline (1 g, 3.1 mmol) and
barium hydroxide octahydrate (2 g, 6.3 mmol) in water (10 mL)
was heated at reflux for 2 h. The solution was cooled, treated
with carbon dioxide (dry ice), filtered, acidified with 1 N
hydrochloric acid to adjust the pH to 3-4, and then extracted
repeatedly with ethyl ether. The aqueous phase was collected
and concentrated under reduced pressure. The residue was then
passed through a column of AG 1X8 ion-exchange resin (20 g,
OH- form, 200-400 mesh, Bio-Rad Laboratories, Hercules, CA)
and eluted with H2O until the eluate was neutral. The combined
eluates were evaporated and extracted three times with hot
acetone. After filtration to remove the precipitate, the filtrate
was collected and the solvent removed under reduced pressure.
The resulting residue was crystallized from acetone to give 392
mg of retronecine in an 81% yield.
According to Mattocks et al. (41, 42), DHR can be prepared
by either dehydrogenation of retronecine or dehydration of
retronecine N-oxide. Dehydrogenation of retronecine was chosen
for the DHR preparation. Briefly, to a solution of retronecine
(100 mg, 650 µmol) in chloroform (30 mL) in an ice bath was
added o-bromanil (300 mg, 708 µmol) in CHCl3 (6 mL) drop-
wise with stirring, over the course of 2 min. The hydroquinone
byproduct was removed by extraction with anion exchange AG
1X8 resin (200-400 mesh, OH- form). The organic phase was
separated and concentrated under reduced pressure, providing
practically pure DHR, which was recrystallized from ether and
light petroleum giving pure DHR as white prisms in a 40%
yield.
An im a ls. Female F344 rats were obtained from the NCTR
breeding colony as weanlings. Liver microsomes of female F344
rats treated with phenobarbital (PB microsomes) were prepared
according to published procedures (43). The rats were injected
intraperitoneally with phenobarbital (75 mg/kg of body weight/
day, in 0.5 mL of H2O) for three consecutive days. Twenty-four
hours following the final injection, rats were sacrificed by CO2
inhalation. The livers were perfused with cold 1.15% KCl via
the portal vein and immediately stored at -78 °C. The liver
microsomes were prepared from thawed tissue by differential
centrifugation methods (44) and stored at -78 °C prior to use.
Protein concentrations were determined using a protein assay
based on the Bradford method using a Bio-Rad protein detection
kit (Bio-Rad Laboratories).
Syn th esis of DHR-2′-Deoxygu a n osin e 3′,5′-Bisp h os-
p h a te Ad d u cts (DHR-d G 3′,5′-Bisp h osp h a te). A solution of
10 nmol of DHR-3′-dGMP in 50 µL of water and a 50 µL
reaction mixture containing 0.4 µmol of ATP, 150 units of PNK,
40 mM bicine-NaOH (pH 9.5), 20 mM MgCl2, 2 mM spermidine,
and 20 mM dithiothreitol (DTT) was incubated at 37 °C for 40
min. The resulting products were separated by HPLC using a
Prodigy 5 µm ODS column (Phenomenex, 4.6 mm × 250 mm)
and eluted isocratically with 10% methanol in 20 mM NH4OAc
at a flow rate of 1 mL/min, and monitored at 254 nm with a
Waters 996 photodiode array detector.
Syn th esis of DHR-2′-Deoxygu a n osin e 5′-Mon op h os-
p h a te Ad d u cts (DHR-5′-d GMP ). Following the procedure of
Wickramanayake et al. (37), the DHR-5′-dGMP adduct was
synthesized by reaction of DHR (18 mg, 120 µmol) with 5′-dGMP
(64 mg, 180 µmol) in aqueous K2CO3 at pH 7.4 and 60 °C for 6
h. The precipitate was removed by filtration, and the products
in the filtrate were separated by semipreparative HPLC. The
crude DHR-5′-dGMP adducts were isolated using a Prodigy 5
µm ODS column (Phenomenex, 10 mm × 250 mm) equilibrated
with 20 mM NH4OAc (pH 7.0). After the sample had been
applied, the column was eluted with a linear gradient from 20
mM NH4OAc to 50% methanol in 20 mM NH4OAc for 40 min
with a flow rate of 2 mL/min. The adducts were further purified
with an analytical Prodigy 5 µm ODS column (Phenomenex, 4.6
mm × 250 mm) eluted isocratically with 20% methanol in 8 mM
NH4OAc (pH 7.0) at a flow rate of 1 mL/min. The collected
fractions were lyophilized and stored at -70 °C until they were
used.
Syn t h esis of t h e 3′-Mon op h osp h a t e of 7-(Deoxya d -
en osin -N6-yl)deh ydr osu pin idin e Addu cts (DHR-3′-dAMP ).
Like the DHR-3′-dGMP adduct, the DHR-3′-dAMP adduct was
prepared by reaction of 3′-dAMP (43 mg, 130 µmol) in 8 mL of
20 mM K2CO3 (pH 8.0) with DHR (40 mg, 267 µmol) at 60 °C
for 40 h. The resulting products were separated by HPLC using
a Prodigy 5 µm ODS column (Phenomenex, 4.6 mm × 250 mm)
eluted at a flow rate of 1.0 mL/min with a linear gradient from
20 mM NH4OAc to 50% methanol in 20 mM NH4OAc in 40 min.
The collected adducts were further purified by reversed-phase
HPLC using the same conditions for the purification of the
DHR-3′-dGMP adduct.
Ch em ica l R ea ct ion of DHR w it h Ca lf Th ym u s DNA.
Purified calf thymus DNA (2.5 mg, 7.5 µmol) in 2.5 mL of 20
mM K2CO3 (pH 7.5) was reacted with 64 nmol of DHR at 37 °C
for 40 min. After incubation, the reaction mixture was extracted
twice with 2.5 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 had been redissolved in 20 mM K2CO3 (pH 7.5), the
DNA concentration and purity were analyzed spectrophoto-
metrically. The DNA was stored at -78 °C prior to 32P-
postlabeling/HPLC analysis.

Detection of Dehydroretronecine-Derived DNA Adducts
Chem. Res. Toxicol., Vol. 14, No. 1, 2001 93
Develop m en t of 32P -P ostla belin g/HP LC Meth od ology
for An a lysis of DHR-3′-d GMP a n d DHR-3′-d AMP Ad -
d u cts. (1) En zym a tic Digestion of DHR-Der ived DNA.
Initially, conventional 32P-postlabeling enzymatic digestion
procedures were employed (45-59). Briefly, 10 µg of DNA (in
10 µL of distilled water) from the reaction of DHR and calf
thymus DNA was enzymatically hydrolyzed to the corresponding
2′-deoxyribonucleoside 3′-monophosphates at 37 oC for 4 h by
1.25 units of MN and 62 milliunits of SPD contained in a 20 µL
solution of 20 mM sodium succinate and 10 mM calcium chloride
(pH 6). Two enrichment methods, nuclease P1 treatment and
n-butanol extraction, were employed. For the nuclease P1
method, the MN/SPD-digested DNA solutions were incubated
at 37 °C for 40 min with nuclease P1 (8 µg, in 4 µL of buffer
containing 0.24 M sodium acetate and 2 mM ZnCl2 at pH 5) to
remove the normal 3′-monophosphate of 2′-deoxyribonucleosides.
The resulting incubation mixture was then evaporated to
dryness under reduced pressure and redissolved in 10 µL of
distilled water for 32P-postlabeling.
Sch em e 1. Syn th etic P r ep a r a tion of
DHR-3′-d GMP Ad d u cts
Cronex films, and the radioactivity on the TLC spots was
quantitated by Cerenkov counting.
(6) An a lysis of [32P ]DHR-5′-d GMP Ad d u cts by HP LC.
The labeled mixture containing [32P]DHR-3′,5′-dG-bisphos-
phate adducts obtained above was adjusted to pH 5.0 with 4
µL of 0.4 M acetic acid and then 3′-dephosphorylated with 17.5
µg of nuclease P1 (5 µg/µL in 0.42 M sodium acetate and 2 mM
ZnCl2) at 37 °C for 5 h. The dephosphorylated mixture was then
subjected to HPLC analysis under the conditions described in
part 3.
For the enrichment by n-butanol extraction, the incubation
mixture (30 µL) was extracted with water-saturated n-butanol
(2 × 100 µL) in the presence of phase-transfer agent tetrabu-
tylammonium chloride. A buffer solution (4 µL) containing 50
mM bicine (pH 9.0) and 0.1 M DTT (2/1, v/v) was added, and
the n-butanol was removed under reduced pressure. The
samples were redissolved in 25 µL of water for 32P-postlabeling.
In str u m en ta tion . A Waters HPLC system consisting of a
model 600 controller, a model 996 photodiode array detector,
and a pump was used for the separation and purification of
DHR-derived DNA adducts. Electrospray (ES) mass spectrom-
etry was performed using a Platfoum II single-quadrupole
instrument (Micromass, Inc., Altrincham, U.K.). ES tandem
mass spectrometry was performed using a Quattro liquid
chromatograph (Micromass). Separate MS functions were used
to acquire full scan data at a low and high cone voltages in a
single chromatographic run (e.g., 20 and 40 V, respectively, for
m/z 100-600). ES tandem mass spectrometry was performed
using the negative and/or positive ion mode with a source
temperature of 80 °C for infusion with a syringe pump. Product
ion scans were obtained from CID of selected ions using a cone
voltage between 37 and 40 V and collision energies between 24
and 31 eV. The collision gas was Ar at pressures between 2 and
4 × 10-3 mbar. LC/MS samples (5 µL injection volume) were
introduced into the ES probe following separation with a Prodigy
5 µm ODS column (Phenomenex, 4.6 mm × 250 mm) and eluting
with the conditions previously described, split to approximately
0.2 mL/min entering the probe. The 1H nuclear magnetic
resonance (NMR) experiments were carried out on a Bruker AM
500 MHz spectrometer (Bruker Instruments, Billerica, MA) at
301 K. Samples were dissolved in 0.6 mL of deuterated water
(D2O). The D2O peak was assigned a resonance of 4.7 ppm.
Typical NMR spectral acquisition parameters were as follows:
data size, 32K; flip angle, 90°; sweep width, 6000 Hz; and
relaxation delay, 1 s. NOE difference and homonuclear coupling
NMR experiments were conducted for assisting in proton
resonance assignment. Circular dichroism (CD) spectra of
DHR-3′-dGMP adducts were determined with a quartz cell with
a path length of 1 cm at ambient temperature on a J asco 500A
spectropolarimeter. CD spectra are expressed by ellipticity (in
millidegrees) for 20 mM NH4OAc solutions that read 1.0
(2) Effect of MN a n d SP D on DHR-Der ived DNA Ad d u ct
Detection . To determine the optimal conditions for DNA
digestion and adduct enrichment, enzymatic digestion of DNA
from reaction of DHR and calf thymus DNA was also conducted
with different quantities of digestion enzymes, including using
1
1/4, 1/8, 1/16, and /32 of the MN and SPD quantity employed above.
To determine whether MN and SPD enzymes would affect the
yield of DHR-derived DNA adducts, incubation of the syntheti-
cally prepared DHR-3′-dGMP and DHR-3′-dAMP adducts
(30-60 fmol) with MN and SPD was compared.
(3) An a lysis of [32P ]DH R-3′,5′-d G-Bisp h osp h a t e a n d
[
32P ]DHR-3′,5′-d A-Bisp h osp h a te Ad d u cts by HP LC. Each
of the MN/SPD-digested DNA samples was dissolved in 10 µL
of distilled water and 32P-phosphorylated by incubating with
10 µL of PNK mix containing 100 µCi of [γ-32P]ATP (specific
activity of >7000 Ci/mmol), 12 units of PNK, and 2 µL of 10×
PNK buffer [200 mM bicine-NaOH (pH 9.6), 100 mM DTT, 100
mM MgCl2, and 10 mM spermidine] at 37 °C for 40 min. The
labeled mixture was injected onto a Prodigy 5 µm ODS column
(Phenomenex, 4.6 mm × 250 mm) and eluted isocratically with
20 mM NaH2PO4 (pH 4.5) for 10 min, followed by a linear
gradient of 20 mM NaH2PO4 (pH 4.5) to 15% methanol in 20
mM NaH2PO4 for 60 min. The HPLC flow rate was 1.0 mL/min,
and the scintillation fluid flow rate was 3.0 mL/min. To avoid
interference by the high level of radioactivity of the free 32P and
the unreacted [γ-32P]ATP, the on-line FLO-ONE radioactivity
detector (Radiomatic Instruments, Tampa, FL) was equipped
with a diverter, and the eluent from the first 40 min was
diverted away from the radioactivity detector.
(4) 32P -P ostla belin g/HP LC An a lysis of th e DHR-3′-
d GMP Ad d u ct Usin g Sm a ller Am ou n t s of DNA. Besides
using 10 µg of DNA, 32P-postlabeling/HPLC analysis of the
DHR-3′-dGMP adduct was also conducted with 5, 3, 1, and
0.5 µg of DNA under conditions similar to those described
above.
absorbance unit in
a UV/visible spectrophotometer at the
wavelength of maximum absorption in a quartz cell with a path
length of 1 cm.
Resu lts
(5) 32P -P ostla belin g/TLC An a lysis of th e DHR -3′-d GMP
Ad d u ct. An aliquot containing 10 µg of DNA from the reaction
of DHR and calf thymus DNA was digested by MN and SPD,
enriched by nuclease P1, and 32P-postlabeled as described above.
As a control experiment, 8.3 fmol of the synthetic DHR-3′-
dGMP adduct was also 32P-postlabeled in parallel under the
same conditions. To analyze the [32P]DHR-3′,5′-dG-bisphos-
phate adducts, the labeled mixture was applied onto 10 cm ×
Syn th esis of Retr on ecin e a n d Deh yd r or etr on e-
cin e (DHR). Retronecine was prepared from barium
hydroxide-catalyzed hydrolysis of monocrotaline by the
procedure of Hoskins and Crout (40). DHR (Scheme 1)
was synthesized by dehydrogenation of retronecine with
o-bromanil in CHCl3 (41). Its structure was confirmed
1
by H NMR spectral analysis: 1H NMR (DMSO-d6): δ
6.53 (d, J 2,3 ) 2.5 Hz, 1, H3), 6.01 (d, J 2,3 ) 2.5 Hz, 1,
H2), 4.99 (m, 1, H7), 4.90 (d, 1, OH7), 4.36 (m, 1, H9), 4.32
(m, 1, H9), 3.97 (m, 1, H5), 3.77 (m, 1, H5), 2.61 (m, 1,
10 cm PEI-cellulose plates (Macherey-Nagel) and
a two-
dimensional development was carried out as previously de-
scribed (45, 46). Autoradiography was performed on Dupont

94 Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Yang et al.
F igu r e 1. Reversed-phase HPLC purification of the syntheti-
cally prepared DHR-3′-dGMP adducts I and II, employing a
Prodigy 5 µm ODS column (4.6 mm × 250 mm) eluted isocrati-
cally with 10% methanol in 20 mM NH4OAc (pH 7.0) at a flow
rate of 1 mL/min.
F igu r e 3. Negative ion electrospray mass spectra of the
synthetically prepared (A) DHR-3′-dGMP adduct I and (B)
DHR-3′-dGMP adduct II.
F igu r e 2. (A) Ultraviolet/visible absorption and (B) CD spectra
of the synthetically prepared epimeric DHR-3′-dGMP adduct
I and DHR-3′-dGMP adduct II obtained from the HPLC
purification described in the legend of Figure 1. These adducts
eluted at 21.9 and 25.5 min, respectively.
F igu r e 4. Proton NMR spectrum (500 MHz) of the syntheti-
cally prepared DNA adduct standards identified as the mixture
of DHR-3′-dGMP adducts I and adduct II.
H6), 2.17 (m, 1, H6). The NMR chemical shift assign-
ment of DHR was determined by nuclear Overhauser
enhancement (NOE) difference experiments, homonucle-
ar coupling experiments, peak splitting, and peak inte-
gration.
graphic peaks at 21.9 and 25.5 min shown in Figure 1
were racemic DHR-3′-dGMP adducts.
The CD spectra of both adducts were determined
(Figure 2B). The CD Cotton effects of the first adduct
were a mirror image of those of the second adduct,
indicating that these two adducts were a pair of epimers.
These CD spectra also had Cotton effects similar to those
of 7-(deoxyguanosin-N2-yl)dehydrosupinidine previously
Syn th esis of DHR-3′-d GMP Ad d u cts. DHR-3′-
dGMP adducts were synthesized by the reaction of DHR
with 3′-dGMP (Scheme 1). The resulting reaction prod-
ucts were purified by HPLC, first onto a Whatman ODS-3
column to remove most of the excess 3′-dGMP (data not
shown) and further with a Prodigy 5 µm ODS column
(Phenomenex) (Figure 1). The materials contained in the
chromatographic peaks that eluted at 21.9 and 25.5 min
exhibited a baseline separation and exhibited identical
UV/visible absorption spectra (Figure 2A) which were
identical to that of the DHR-5′-dGMP adduct reported
by Wickramanayake et al. (37). These products were also
characterized from the ES-MS negative product ion
spectra, which were identical (Figure 3). The deproto-
nated molecule, (M - H)-, was at m/z 481 and product
ions corresponding to the deprotonated base (m/z 285),
deoxyribose - monophosphate (m/z 195), deoxyribose -
monophosphate - H2O (m/z 177), and ions derived from
phosphate ion (m/z 97 and 79). The positive ion product
spectrum (not shown) contained the protonated molecule,
(M + H)+, at m/z 483, loss of H2O (m/z 465), the
protonated DHR - guanine (m/z 269), dGMP (m/z 348),
guanine (m/z 152), and DHR - H2O (m/z 136). These data
suggested that the materials contained in the chromato-
1
reported by Wickramanayake et al. (37). The H NMR
spectrum of the adduct mixture was determined by NMR
decoupling and NOE techniques as well as by comparison
of the NMR data of the DHR and 3′-dGMP measured
under identical conditions (Figure 4 and Table 1).
Consequently, on the basis of mass, CD, and NMR
spectral analysis, the structures of these two products
were characterized as a pair of epimeric DHR-3′-dGMP
adducts.
On the basis of the analysis by proton NMR using an
internal standard (1,4-dioxane), the quantity of these
adducts obtained from the organic reaction followed by
repeated purification was 0.91 mg. Using the UV/visible
absorbance measurement in water, the molar extinction
coefficient of DHR-3′-dGMP adducts at 254 nm was
determined to be 4.7 × 104 M-1 cm-1
.
Reactions of DHR and 3′-dGMP were carried out three
times, with the molar ratio of DHR to 3′-dGMP being 1/1,
2/1, and 3/1. The reaction with a ratio of 2/1 provided
the highest yield (2.5%).

Detection of Dehydroretronecine-Derived DNA Adducts
Chem. Res. Toxicol., Vol. 14, No. 1, 2001 95
Ta ble 1. 1H NMR Sp ectr a l Da ta (500 MHz) of DHR,
3′-d GMP , th r DHR-3′-d GMP Ad d u ct, 5′-d GMP , a n d th e
DHR-5′-d GMP Ad d u ct
chemical shift (ppm)
DHR 3′-dGMP DHR-3′-dGMP 5′-dGMP DHR-5′-dGMP
H2
H3
H5
H5
H6
H6
H7
H9
H9
OH7 4.90
H8
H1′
H2′
H2′
6.01
6.53
3.97
3.77
2.61
2.17
4.99
4.36
4.32
6.29
6.76
4.13
3.99
2.96
2.51
5.56
4.41
4.36
6.31
6.73
4.17
4.15
3.02
2.59
5.57
4.71
4.24
F igu r e 7. HPLC separation of DHR-3′,5′-dG-bisphosphate
adducts formed from the reaction of the DHR-3′-dGMP adduct
with cold ATP catalyzed by PNK following the HPLC conditions
described in the legend of Figure 1.
7.97
6.27
2.62
2.74
4.82
4.21
3.76
3.77
8.01
6.41
2.88
2.59
4.75
4.25
3.83
3.83
8.77
6.35
2.72
2.60
4.63
4.25
4.04
4.09
8.14
6.46
2.87
2.59
4.77
4.24
4.49
4.49
1
The H NMR spectrum was assigned by NOE differ-
H3′
H4′
H5′
H5′
ence experiments, homonuclear coupling experiments,
peak splitting, and peak integration. H1′ (6.35 ppm) to
H5′′ (4.04 ppm) and H8 (8.77 ppm) were assigned by NOE
techniques. The chemical shift and coupling constant
assignments are shown in Table 1.
coupling constant (Hz)
DHR 3′-dGMP DHR-3′-dGMP 5′-dGMP DHR-5′-dGMP
7.0 7.0
There was a very strong similarity between the chemi-
cal shifts of 3′-dGMP and the DHR-3′-dGMP adduct, and
between 5′-dGMP and the DHR-5′-dGMP adduct. The
biggest difference between the 3′-dGMP and 5′-dGMP
chemical shifts was seen along the backbone H3′, H5′,
and H5′′ NMR chemical shifts. There was a very strong
similarity between the DHR NMR chemical shifts of the
DHR-3′-dGMP adduct and the DHR-5′-dGMP adduct.
These adducts had UV/visible absorption spectra iden-
tical to those of the DHR-5′-dG adduct reported by
Wickramanayake et al. (37). Thus, on the basis of UV/
visible absorption and mass and NMR spectral analysis,
the compounds contained in chromatographic peaks that
eluted at 25.1 and 27.7 min in Figure 5 were identified
as DHR-5′-dGMP adducts. The reaction yield was 0.9%.
Syn th esis of th e DHR-3′,5′-d G-Bisp h osp h a te Ad -
d u ct. The DHR-3′,5′-dG-bisphosphate adduct was syn-
thesized by 5′-phosphorylation of the synthetically pre-
pared epimeric DHR-3′-dGMP adducts with cold (non-
radioactive) ATP catalyzed by PNK. The resulting reac-
tion mixture was purified by reversed-phase HPLC
(Figure 7). The chromatographic peaks that eluted at 19.2
and 23.8 min contained the two recovered substrates,
epimeric DHR-3′-dGMP adduct I and adduct II, respec-
tively. The materials contained in chromatographic peaks
that eluted at 6.2 and 7.3 min, respectively, had UV/
visible absorption spectra similar to that of the DHR-
3′-dGMP adduct (Figure 2A). These materials were
characterized by analysis of their identical negative
product ion ES mass spectra (Figure 8). The deprotonated
molecule, (M - H)-, was at m/z 561 and product ions
corresponding to loss of H2O (m/z 543), loss of one
H3PO4 (m/z 463), loss of the DHR moiety (m/z 426), loss
of two H3PO4 (m/z 365), the ribose 3′,5′-bisphosphate ion
(m/z 275), loss of H2O from the ribose 3′,5′-bisphosphate
ion (m/z 257), ribose - the monophosphate ion (m/z 195),
J 1′,2′
J 2,3 2.5
J 9,9
2.5
12.1
2.5
Syn th esis of DHR-5′-d GMP Ad d u cts. Like the
synthesis and HPLC purification of the DHR-3′-dGMP
adduct, the DHR-5′-dGMP adduct was prepared by
reaction of DHR with 5′-dGMP followed by two HPLC
separations. The HPLC profile of the second HPLC
purification provided a baseline separation of the DHR-
5′-dGMP adducts (Figure 5). Negative ion ES mass
spectral analysis showed that both these adducts had
product ion spectra that were identical to those of the
DHR-3′-dGMP adducts (Figure 6). The positive ion
product spectrum (not shown) contained the protonated
molecule, (M + H)+, at m/z 483, loss of H2O (m/z 465),
the protonated DHR - guanine (m/z 269), dGMP (m/z
348), guanine (m/z 152), and DHR - H2O (m/z 136).
F igu r e 5. HPLC purification of the DHR-5′-dGMP adduct
with a Prodigy 5 µm ODS column (4.6 mm × 250 mm) eluted
isocratically with 8 mM NH4OAc (pH 7.0) at a flow rate of 1
mL/min.
-
-
-
H3P2O7 (m/z 177), HP2O6 (m/z 159), H2PO4 (m/z 97),
and PO3- (m/z 79). On the basis of UV/visible absorption
and mass spectral analysis, the structures of these two
reaction products were identified as DHR-3′,5′-dG-
bisphosphate adduct I and DHR-3′,5′-dG-bisphosphate
adduct II, respectively.
Syn th esis of DHR-3′-d AMP Ad d u cts. Like the
DHR-3′-dGMP adducts, the DHR-3′-dAMP adducts
F igu r e 6. Negative ion electrospray mass spectrum of the
DHR-5′-dGMP adduct.

96 Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Yang et al.
F igu r e 8. Negative ion electrospray proton ion mass spectrum
of the DHR-3′,5′-dG-bisphosphate adduct.
F igu r e 9. HPLC separation of DHR-3′-dAMP adducts with a
Prodigy 5 µm ODS column (Phenomenex, 4.6 mm × 250 mm)
eluted isocratically with 10% methanol in 20 mM NH4OAc (pH
7.0) at a flow rate of 1 mL/min.
F igu r e 10. Negative ion electrospray mass spectra of four
isomeric DHR-3′-dAMP adducts prepared from the reaction of
DHR and 3′-dAMP followed by HPLC purification as described
in the legend of Figure 1.
were prepared by reaction of DHR with 3′-dAMP followed
HPLC separations. The HPLC profile of HPLC purifica-
tion is shown in Figure 9. The chromatographic peak that
eluted at 18.2 min contained the recovered substrate,
DHR. The materials contained in the chromatographic
peaks that eluted at 13.2, 15, 20.5, and 23.5 min were
analyzed using LC/ES-MS. As shown in Figure 10A-D,
these four products had identical mass spectra with the
(45-48). No DHR-3′,5′-dG-bisphosphate adducts were
detected by HPLC (Figure 11A). With a concern that the
digestion enzymes may interact with DHR-3′-dGMP
and/or [32P]DHR-3′,5′-dG-bisphosphate adducts, optimal
quantities of MN and SPD and an optimal digestion time
were then pursued. Digestion of DNA was repeated by
using (i) 312 milliunits of MN and 16 milliunits of SPD,
(ii) 156 milliunits of MN and 8 milliunits of SDP, (iii) 78
milliunits of MN and 4 milliunits of SDP, and (iv) 39
milliunits of MN and 2 milliunits of SPD (panels B-E of
Figure 11, respectively). As shown in panels C and D of
Figure 11, there are eight chromatographic peaks that
eluted at 47.6, 48.3, 51.4, 53.9, 55.3, 60.1, 61.0, and 62.6
min are designated as P1-P8, respectively. These chro-
matographic peaks were not detected from 32P-postlabel-
ing/HPLC analysis of the untreated calf thymus DNA
(Figure 11F) or from incubation of riddelliine with calf
thymus DNA in the absence of rat liver microsomes (data
not shown). Thus, the eight chromatographic peaks, P1-
P8, are all DHR-derived DNA adducts. As compared with
the HPLC profile from 32P-postlabeling/HPLC analysis
of the synthetically prepared DHR-3′-dGMP adducts
(Figure 12A), the DNA adducts designated as P4 and P6
are [32P]DHR-3′,5′-dG-bisphosphate adducts derived
from DHR-3′-dGMP adduct I and adduct II, respectively
(each indicated with an arrow in Figure 11A-E). Thus,
the results shown in Figure 11 clearly indicate that the
use of 78 milliunits of MN and 4 milliunits of SPD
provided the highest yield of DHR-3′,5′-dG-bisphosphate
adducts (Figure 11D).
-
(M - H)- ion (m/z 465), M - deoxyribose - PO4 (m/z
269), deoxyribose - PO4- (m/z 195), and H2PO4- (m/z 97).
Thus, on the basis of mass spectral analysis, they were
all DHR-3′-dAMP adducts.
Develop m en t of a 32P -P ostla belin g/HP LC Meth od
for An a lysis of DHR-3′-d GMP Ad d u cts. 32P-postla-
beling methodology was selected for detecting and quan-
tifying DHR-modified DNA adducts in vitro and in vivo.
The synthesized epimeric DHR-3′-dGMP adducts with
a specific quantity ranging from 1 to 60 fmol were first
employed to develop optimal conditions for the entire
process, including DNA enzyme digestion, adduct enrich-
ment, 32P-postlabeling, and HPLC separation. To confirm
the 32P-postlabeling products, [32P]DHR-3′,5′-dG-bispho-
sphate, the cold synthetically prepared DHR-3′,5′-dG-
bisphosphate adducts I and II were cochromatographed
by HPLC with the 32P-postlabeling reaction product
mixture.
(1) Effect of MN/SP D on 32P -P ostlabelin g of DHR-
3′-d GMP a n d DHR-3′-d AMP Ad d u cts. Initially, 10 µg
of DNA from the reaction of DHR and calf thymus DNA
was digested with 1.25 units of MN and 62 milliunits of
SPD, quantities commonly reported for enzyme digestion

Detection of Dehydroretronecine-Derived DNA Adducts
Chem. Res. Toxicol., Vol. 14, No. 1, 2001 97
F igu r e 12. Effect of micrococcal nuclease (MN) and spleen
phosphodiesterase (SPD) concentration on 32P-postlabeling/
HPLC analysis of a mixture of 8.3 fmol of DHR-3′-dGMP and
20 fmol of DHR-3′-dAMP adducts. The DNA samples were
digested with (A) 1250 milliunits of MN and 62 milliunits of
SPD, (B) 312 milliunits of MN and 16 milliunits of SPD, (C)
156 milliunits of MN and 8 milliunits of SPD, (D) 78 milliunits
of MN and 4 milliunits of SPD, and (E) 39 milliunits of MN
and 2 milliunits of SPD.
F igu r e 11. Effect of micrococcal nuclease (MN) and spleen
phosphodiesterase (SPD) concentration on 32P-postlabeling/
HPLC analysis of DHR-modified DNA adducts contained in
DNA from the reaction of DHR and calf thymus DNA. Ten
micrograms of DNA was digested by MN and SPD and postla-
beled with 32P by enzymatic phosphorylation in the presence of
[γ-32P]ATP and polynucleotide kinase. Various enzyme concen-
trations were used for DNA digestion. The 32P-postlabeled
adduct formation from the enzymatic digestion with (A) 1250
milliunits of MN and 62 milliunits of SPD, (B) 312 milliunits of
MN and 16 milliunits of SPD, (C) 156 milliunits of MN and 8
milliunits of SPD, (D) 78 milliunits of MN and 4 milliunits of
SPD, and (E) 39 milliunits of MN and 2 milliunits of SPD and
(F) 32P-postlabeling/HPLC analysis of untreated calf thymus
DNA (10 µg) using 78 milliunits of MN and 4 milliunits of SPD.
monophosphate deoxyribonucleoside adducts formed from
enzymatic digestion of DNA were fortified with the
phase-transfer agent tetrabutylammonium chloride and
then extracted with n-butanol four times. The adducts
collected from the n-butanol fraction were 32P-postlabeled
followed by HPLC analysis (data not shown). On the basis
of three trials, the yields of [32P]DHR-3′,5′-deoxyribo-
nucleoside bisphosphate adducts were erratic and much
lower than those determined by nuclease P1 enrichment.
Therefore, this approach was found to be less satisfactory
than nuclease P1 enrichment.
(3) HP LC Sep a r a tion of th e [32P ]DHR-3′,5′-De-
oxyr ibon u cleosid e Bisp h osp h a te Ad d u cts. After the
(i) synthetically prepared DHR-3′-dGMP adducts, (ii)
combined synthetically prepared DHR-3′-dGMP and
DHR-3′-dAMP adducts, and (iii) DNA from the reaction
of DHR and calf thymus DNA were 32P-postlabeled under
the optimal conditions described above, the resulting
[R-32P]DHR-3′,5′-deoxyribonucleoside bisphosphate ad-
ducts were separated by HPLC. After a number of trials
using different HPLC columns, solvent systems, and
elution profiles (data not shown), an optimal separation
condition for separation of the DHR-3′,5′-bisphosphate
adducts was developed. The developed HPLC profile for
separation of the two epimeric DHR-3′,5′-dG-bisphos-
phate adducts is shown in Figure 12A. The optimal HPLC
conditions for separation of the mixture of DHR-3′,5′-
dG-bisphosphate and DHR-3′,5′-dA-bisphosphate ad-
ducts are like those shown in Figure 12D. Similarly, the
The conditions for analysis of DHR-3′-dAMP adducts
by 32P-postlabeling were then pursued using different
amounts of MN and SPD and different incubation times.
As shown in panels A-E of Figure 12, like DHR-3′-
dGMP adducts, the optimal conditions for analysis of
DHR-3′-dAMP adducts were also the use of 78 milliunits
of MN and 4 milliunits of SPD. These were also the
optimal conditions for 32P-postlabeling a mixture of
DHR-3′-dGMP and DHR-3′-dAMP adducts (Figure
12A-E).
(2) En r ich m en t of DHR-3′-d GMP Ad d u cts by
Nu clea se P 1 a n d n -Bu ta n ol Extr a ction . DHR-derived
DNA was obtained from the reaction of DHR with calf
thymus DNA; 10 µg was enzymatically digested under
optimal conditions (same conditions described in Figure
11D), and the resulting DHR-3′-monophosphate deoxy-
ribonucleoside adducts were enriched with various
amounts of nuclease P1 and with an incubation time
ranging from 20 to 40 min. It was found that use of 8 µg
of nuclease P1 and incubation for 20 min at 37 °C
provided the best enrichment.
Enrichment by n-butanol was similarly studied. Fol-
lowing the conventional procedure (46), the DHR-3′-

98 Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Yang et al.
optimal HPLC conditions for separation of the DNA
adducts from the reaction of DHR and calf thymus DNA
are the same as shown in Figure 11D.
derived DNA adducts and utilization of this method for
detection and quantification of these adducts from in vitro
and in vivo samples.
32P-postlabeling methodology is a highly sensitive
method for detecting and quantifying carcinogen-modi-
fied DNA adducts (45-59). Because 32P-postlabeling is
conducted on a small scale, the products, [32P]-3′,5′-
bisphosphate of carcinogen-modified 2′-deoxyribonucleo-
sides, are always formed in an extremely small quantity,
and thus, they are usually not characterized. In our
study, however, the postlabeling products, DHR-3′,5′-
dG-bisphosphate adducts, were characterized by mass
spectrometry and confirmed by co-hromatography with
synthetic DHR-3′,5′-dG-bisphosphate standards.
Although determination of recovery between different
trials performed at different times can be normalized on
the basis of a concentration-response relationship (curve),
possible variation in enzyme activity and experimental
conditions can result in quantitative deviations between
trials. Thus, without 32P-postlabeling of a cold authentic
standard of known quantity in parallel with the tested
samples, quantitative comparison of 32P-postlabeling
products obtained from different trials is not reliable. For
our study, in each experiment, external standards, a pair
of epimeric DHR-3′-dGMP synthetic adducts at a known
level that closely matched the range of the modification
level of the biological DNA samples, were analyzed in
parallel with the biological samples.
The necine base of DHR and other dehydropyrroliz-
idines contain a pyrrole moiety that facilitates polymer-
ization under acidic conditions (6). Similarly, the DHR-
derived DNA adducts, including DHR-3′-dGMP, DHR-
3′-dAMP, DHR-5′-dGMP, and DHR-3′,5′-dG-bisphos-
phate, are all unstable. We have found that 25% of the
DHR-3′-dGMP adducts decomposed on storage at room
temperature for 48 h, and that more than 50% of these
adducts decomposed on storage in buffer at pH <7.0 and
37 °C for 48 h. This instability seriously handicapped the
development of 32P-postlabeling conditions. Because of
adduct decomposition in the presence of MN and SPD,
it was necessary to use quantities of MN and SPD for
digestion of DNA that were much smaller than those
conventionally employed for 32P-postlabeling of other
types of carcinogenic chemicals. The optimal conditions
that were developed included the use of shorter incuba-
tion times in both DNA enzyme digestion (reduced from
the commonly used 4 h to 20 min) and nuclease P1
enrichment (reduced from 40 to 20 min).
(4) E va lu a t ion of In t r a - a n d In t er exp er im en t a l
Rep r od u cibility of 32P -P ostla belin g/HP LC Resu lts.
On the basis of our experience in employing or developing
a
32P-postlabeling/HPLC methodology for detection of
carcinogen-modified DNA adducts (49-60), we first at-
tempted to establish reliable conditions for validating
interexperimental reproducibility. However, the DNA
enzyme digestion products (e.g., DHR-3′-dGMP) and/or
32P-postlabeling products (e.g., DHR-3′,5′-dG-bisphos-
phate) are highly unstable to the DNA digestion enzymes,
incubation media, and experimental conditions. As shown
in Figure 11, the yield of 32P-postlabeling is dependent
on the amount of MN and SPD. It was also found that
the storage of DNA, the use of [γ-32P]ATP from different
batches or the same batch but on a different day, and
re-preparation of the buffer media can all result in poor
interexperimental reproducibility (data not shown). Con-
sequently, we decided to develop a 32P-postlabeling/HPLC
methodology that allowed us to detect and quantify DNA
adducts from a large number of samples (up to 30
samples) on the same day.
Thus, intraexperimental reproducibility was deter-
mined using the synthetic DHR-3′-dGMP adduct stan-
dards and DNA from the reaction of DHR with calf
thymus DNA for 32P-postlabeling/HPLC analysis. DNA
samples in triplicate were enzymatically digested, adduct
enriched (by nuclease P1), and 32P-postlabeled at the
same time (on the same day). The levels of modification
of the DNA from the reaction of DHR with calf thymus
DNA were 615.6, 577.4, and 578.0 adducts/108 nucle-
otides (SD ) 590.4 ( 21.6, 4% of relative standard
deviation). Among these adducts, the levels of the two
enantioneric DHR-3′,5′-dG-bisphosphate adducts I (P4)
and II (P6) were 33.7 ( 2.1 and 23.9 ( 1.2 adducts/108
nucleotides, respectively.
(5) An a lysis of [32P ]DH R-5′-d GMP Ad d u ct s b y
HP LC. Attempts were made to enzymatically 3′-dephos-
phorylate [32P]DHR-3′,5′-dG-bisphosphate adducts to the
corresponding [32P]DHR-5′-dGMP adducts followed by
HPLC analysis. On the basis of comparison of the HPLC
retention time of the synthetic DHR-5′-dGMP standard,
no DHR-5′-dGMP adduct was detected (data not shown).
(6) 32P -P ost la b elin g/TLC An a lysis of DHR -3′-
d GMP Ad d u cts. After 32P-postlabeling of the DHR-3′-
dGMP adducts as described above, the resulting [32P]-
DHR-3′,5′-dG-bisphosphate adducts were separated using
TLC on PEI-cellulose plates (45, 46). Comparison of the
autoradiography results with those from controls indi-
cated no adducts were detected (data not shown).
Presumably because of the high lability of the products
under the conditions used for enzymatic digestion, sat-
isfactory absolute interexperimental reproducbility could
not be obtained. However, optimal conditions were
developed that allowed us to detect and quantify DHR-
modified DNA adducts with a good intraexperimental
reproducibility. With this approach, a total of 30 DNA
samples could be analyzed at the same time. As such,
the quantity of the 32P-postlabeling products obtained
from different trials could be reliably compared.
In addition to the two DHR-3′-dGMP adducts, another
six chromatographic peaks are detected in calf thymus
DNA from the reaction with DHR. These six chromato-
graphic peaks were not observed in control samples, but
were also formed in liver DNA of rats treated with
riddelliine. Due to the lack of synthetic standards and
insufficient quantities for structural determination, their
identities were not determined. As shown in Figure 11,
Discu ssion
Pyrrolizidine alkaloids are a class of genotoxic natu-
rally occurring phytochemicals, a number of which have
been found to induce tumors in experimental animals (2,
3, 6, 10, 61-75). Pyrrolizidine alkaloids are biologically
inert and require metabolism to exert their tumorige-
nicity. However, the mechanisms that lead to tumorige-
nicity are not clear. No pyrrolizidine alkaloid-derived
DNA adducts have been identified in vivo or in vitro, and
no established methodologies are available for their
detection. In this paper, we report the development of a
32P-postlabeling/HPLC method for detection of DHR-

Detection of Dehydroretronecine-Derived DNA Adducts
Chem. Res. Toxicol., Vol. 14, No. 1, 2001 99
(11) White, I. N. H., and Mattocks, A. R. (1972) Reaction of dihydro-
pyrrolizidines with deoxyribonucleic acids in vitro. Biochem. J .
128, 291-297.
(12) Eastman, D. F., Dimenna, G. P., and Segall, H. J . (1982) Covalent
binding of two pyrrolizidine alkaloids, senecionine and seneci-
phylline, to hepatic macromolecules and their distribution, excre-
tion, and transfer into milk of lactating mice. Drug Metab. Dispos.
10, 236-240.
(13) Mattocks, A. R., and Bird, I. (1983) Alkylation by dehydroret-
ronecine, a cytotoxic metabolite of some pyrrolizidine alkaloids:
an in vitro test. Toxicol. Lett. 16, 1-8.
(14) Petry, T. W., Bowden, G. T., Huxtable, R. J ., and Sipes, I. G. (1984)
Characterization of hepatic DNA damage induced in rats by the
pyrrolizidine alkaloid monocrotaline. Cancer Res. 44, 1505-1509.
(15) Petry, T. W., Bowden, G. T., Huxtable, R. J ., and Sipes, I. G. (1986)
Genotoxicity of the pyrrolizidine alkaloid jacobine in rats. Toxicol.
Lett. 32, 275-281.
the total quantity of these six adducts was much higher
than those of the two DHR-3′-dGMP adducts, with the
later accounting for less than 10% of the total DNA
adducts.
DHR has been found as a metabolite commonly formed
from metabolism of a number of pyrrolizidine alkaloids
in vitro and in vivo, including retrorsine (13, 28), mono-
crotaline (13, 28-31), senecionine (13, 32, 33), and
indicine (13). As described in the following paper, these
eight DHR-derived DNA adducts have been observed in
livers of rats fed riddelliine. Thus, these DNA adducts
may also be formed from rats, and probably other
mammals, fed other retronecine-derived tumorigenic
pyrrolizidine alkaloids, such as monocrotaline, retrorsine,
senecionine, and indicine. As such, these eight DHR-
derived adducts may be able to serve as biomarkers of
retronecine-derived pyrrolizidine alkaloid-associated car-
cinogenesis. Consequently, our development of sensitive
and reliable 32P-postlabeling/HPLC methodology for de-
tecting and quantifying DHR-derived DNA adducts will
facilitate the risk assessment of human exposure to
pyrrolizidine alkaloids.
(16) Griffin, D. S., and Segall, H. J . (1986) Genotoxicity and cytotoxicity
of selected pyrrolizidine alkaloids, a possible alkenal metabolite
of the alkaloids, and related alkenals. Toxicol. Appl. Pharmacol.
86, 227-234.
(17) MacGregor, J . T., Wehr, C. M., Henika, P. R., and Shelby, M. D.
(1990) The in vivo erythrocyte micronuclleus test: Measurement
at steady-state increases assay efficiency and permits integration
with toxicity studies. Fundam. Appl. Toxicol. 14, 513-522.
(18) Hincks, J . R., Kim, H.-Y., Segall, H. J ., Molyneux, R. J ., Stermitz,
F. R., and Coulombe, R. A., J r. (1991) DNA cross-linking in
mammalian cells by pyrrolizidine alkaloids: structure-activity
relationships. Toxicol. Appl. Pharmacol. 111, 90-98.
(19) Muller, L., Kasper, P., and Kaufmann, G. (1992) The clastogenic
potential in vitro of pyrrolizidine alkaloids employing hepatocyte
metabolism. Mutat. Res. 282, 169-176.
Ack n ow led gm en t. We thank Linda S. Von Tungeln
for technical assistance and Frederick A. Beland for
critical review of the manuscript. This research was
supported in part by Interagency Agreement 224-93-0001
between the Food and Drug Administration/National
Center for Toxicological Research (FDA/NCTR) and the
National Institute for Environmental Health Sciences/
National Toxicology Program (NIEHS/NTP). Through
this agreement, this research was supported by appoint-
ments (Y.-C.Y. and J .Y.) to the Postgraduate Research
Program at the NCTR administered by the Oak Ridge
Institute for Science and Education through an inter-
agency agreement between the U.S. Department of
Energy and the FDA.
(20) Roeder, E. (1995) Medicinal plants in Europe containing pyr-
rolizidine alkaloids. Pharmazie 50, 83-98.
(21) Betz, J . M., Eppley, R. M., Taylor, W. C., and Andrzejewski, D.
(1994) Determination of pyrrolizidine alkaloids in commercial
comfrey products (Symphytum sp.). J . Pharm. Sci. 83, 649-653.
(22) Hirono, I., Mori, H., and Culvenor, C. C. J . (1976) Carcinogenic
activity of coltsfoot, Tussilago Farfara L. Gann 67, 125-129.
(23) Hincks, J . R., and Coulombe, R. A. (1989) Rapid detection of DNA-
interstrand and DNA-protein cross-links in mammalian cells by
gravity-flow alkaline elution. Environ. Mol. Mutagen. 13, 211-
217.
(24) Hincks, J . R., Kim, H.-Y., Segall, H. J ., Molyneux, R. J ., Stermitz,
F. R., and Coulombe, R. A., J r. (1991) DNA cross-linking in
mammalian cells by pyrrolizidine alkaloids: structure-activity
relationships. Toxicol. Appl. Pharmacol. 111, 90-98.
(25) Kim, H. Y., Stermitz, F. R., and Coulombe, R. A., J r. (1995)
Pyrrolizidine alkaloid-induced DNA-protein cross-links. Carcino-
genesis 16, 2691-2697.
Refer en ces
(1) Bull, L. B., Culvenor, C. C. J ., and Dick, A. J . (1968) The
Pyrrolizidine Alkaloids. Their Chemistry, Pathogenicity and other
Biological Properties, North-Holland, Amsterdam.
(2) International Agency for Research on Cancer (1976) Pyrrolizidine
alkaloids. In IARC Monograph on the Evaluation of Carcinogenic
Risk of Chemicals to Man: Some Naturally Occurring Substance,
Vol. 10, pp 265-342, International Agency for Research on
Cancer, Lyon, France.
(26) Coulombe, R. A., J r., Drew, G. L., and Stermitz, F. R. (1999)
Pyrrolizidine alkaloid crosslink DNA with actin. Toxicol. Appl.
Pharmacol. 154, 198-202.
(27) Kim, H. Y., Stermitz, F. R., Li, K.-K. J ., and Coulombe, R. A., J r.
(1999) Comparative DNA cross-linking by activated pyrrolizidine
alkaloids. Food Chem. Toxicol. 37, 619-625.
(28) Hsu, I. C., Allen, J . R., and Chesney, C. F. (1973) Identification
and toxicological effects of dehydroretronecine, a metabolite of
monocrotaline. Proc. Soc. Exp. Biol. Med. 144, 834-838.
(29) Shumaker, R. C., Robertson, K. A., Hsu, I. C., and Allen, J . R.
(1976) Neoplastic transformation in tissues of rats exposed to
monocrotaline or dehydroretronecine. J . Natl. Cancer Inst. 56,
787-789.
(3) Schoental, R. (1976) Carcinogens in plants and microorganisms.
In Chemical Carcinogens (Searle, C. E., Ed.) ACS Monograph 173,
American Chemical Society, Washington, DC.
(4) World Health Organization (1988) Pyrrolizidine Alkaloids. In
International Programme on Chemical Safety, Environmental
Health Criteria, Vol. 80, p 337, World Health Organization,
Geneva.
(5) Mattocks, A. R. (1968) Toxicology of pyrrolizidine alkaloids.
Nature 217, 723-728.
(6) Mattocks, A. R. (1986) Chemistry and Toxicology of Pyrrolizidine
(30) J ohnson, W. D., Robertson, K. A., Pounds, J . G., and Allen, J . R.
(1978) Dehydroretronecine-induced skin tumors in mice. J . Natl.
Cancer Inst. 61, 85-89.
Alkaloids, Academic Press, New York.
(31) Couet, C. E., Hopley, J ., and Hanley, A. B. (1996) Metabolic
activation of pyrrolizidine alkaloids by human, rat and avocado
microsomes. Toxicon 34, 1058-1061.
(32) Williams, D. E., Reed, R. L., Kedzierski, B., Dannan, G. A.,
Guengerich, F. P., and Buhler, D. (1989) Bioactivation and
detoxication of the pyrrolizidine alkaloid senecionine by cyto-
chrome P-450 enzymes in rat liver. Drug Metab. Dispos. 17, 387-
392.
(33) White, I. N. H., Mattocks, A. R., and Butler, W. H. (1973) The
conversion of the pyrrolizidine alkaloid retrorsine to pyrrolic
derivatives in vivo and in vitro and its acute toxicity to various
animal species. Chem.-Biol. Interact. 6, 207-218.
(34) Allen, J . R., Hsu, I. C., and Carstens, L. A. (1975) Dehydroret-
ronecine-induced rhabdomyosarcomas in rats. Cancer Res. 35,
997-1002.
(7) Noble, J . W., Crossley, J ., Hill, B. D., Pierce, R. J ., McKenzie, R.
A., Debritz, M., and Morley, A. A. (1994) Pyrrolizidine alkaloidosis
of cattle associated with senecio lautus. Aust. Vet. J . 71, 196-
200.
(8) McLean, E. K. (1970) The toxic actions of pyrrolizidine (Senecio)
alkaloids. Pharmacol. Rev. 22, 429-483.
(9) Bah, M., Bye, R., and Pereda-Miranda, R. (1994) Hepatotoxic
pyrrolizidine alkaloids in the Mexican medicinal plant Packera
candidissima (Asteraceae senecioneae) J . Ethnopharmacol. 43,
19-30.
(10) Schoental, R., Head, M. A., and Peacock, P. R. (1954) Senecio
alkaloids: primary liver tumors in rats as a result of treatment
with (I) a mixture of alkaloids from S. jacobaea linn., (ii)
retrorsine, (iii) isatidine. Br. J . Cancer 8, 458-465.

100 Chem. Res. Toxicol., Vol. 14, No. 1, 2001
Yang et al.
(35) Hsu, I. C., Robertson, K. A., Shumaker, R. C., and Allen, J . R.
(1975) Binding of tritiated dehydroretronecine to marcomolecules.
Res. Commun. Chem. Pathol. Pharmacol. 11, 99-106.
(36) Reed, R. L., Ahern, K. G., Pearson, G. D., and Buhler, D. R. (1988)
Crosslinking of DNA by dehydroretronecine, a metabolite of
pyrrolizidine alkaloids. Carcinogenesis 9, 1355-1361.
(37) Wickramanayake, P. P., Arbogast, B. L., Buhler, D. R., Deinzer,
M. L., and Burlingame, A. L. (1985) Alkylation of nucleosides and
nucleotides by dehydroretronecine: Characterization of covalent
adducts by liquid secondary ion mass spectrometry. J . Am. Chem.
Soc. 107, 2485-2488.
of DNA adducts of nitro-polycyclic aromatic hydrocarbons in
mouse and rat liver microsomes and cytosols. Polycyclic Aromat.
Compd. 10, 187-194.
(56) Fu, P. P., Von Tungeln, L. S., Zhan, D.-J ., and Bucci, T. (1996)
Potent tumorigenicity of 7-chlorobenz[a]anthracene and 7-bro-
mobenz[a]anthracene in the neonatal B6C3F1 male mouse. Cancer
Lett. 101, 37-42.
(57) Zhan, D.-J ., Chiu, L.-H., Von Tungeln, L. S., Cheng, E., Herreno-
Saenz, D., Evans, F. E., Heflich, R. H., and Fu, P. P. (1997)
Characterization of DNA adducts in Chinese hamster ovary cells
created with mutagenic doses of 1- and 3-nitrosobenzo[a]pyrene
and the trans-7,8-diol-anti-9,10-epoxides of 1- and 3-nitrobenzo-
[a]pyrene. Mutat. Res. 379, 43-52.
(58) Fu, P. P., Von Tungeln, L. S., Chiu, L.-H., Zhan, D.-J ., Deck, J .,
and Wang, J .-C. (1998) Structure, tumorigenicity, microsomal
metabolism, and DNA binding of 7-nitrodibenz[a,h]anthracene.
Chem. Res. Toxicol. 11, 937-945.
(59) Yi, P., Zhan, D.-J ., Samokyszyn, V. M., Doerge, D. R., Evans, F.
E., and Fu, P. P. (1997) Synthesis and 32P-postlabeling/HPLC
separation of diastereomeric 1,N2-(1,3-propano)-2′-deoxyguanosine
3′-phosphate adducts formed from 4-hydroxy-2-nonenal. Chem.
Res. Toxicol. 10, 1259-1265.
(60) Yi, P., Sun, X., Doerge, D. R., and Fu, P. P. (1998) An improved
32P-postlabeling/high-performance liquid chromatography method
for the analysis of malondialdehyde derived 1,N2-propanodeoxy-
guanosine DNA adduct in animal and human tissues. Chem. Res.
Toxicol. 11, 1032-1041.
(61) Newbern, P. N., and Rogers, A. E. (1973) Nutrition, monocrotaline
and aflatoxin B1 in liver carcinogenesis. Plant Foods Man, 23-
31.
(62) Svoboda, D. J ., and Reddy, J . K. (1972) Malignant tumors in rats
given lasicarpine. Cancer Res. 32, 908-913.
(63) Schoental, R., and Head, M. (1957) progression of liver lesions
produced in rats by temporary treatment with pyrrolizidine
(senecio) alkaloids, and the effects of betaine and high casein diet.
Br. J . Cancer 11, 535-544.
(38) Robertson, K. A. (1982) Alkylation of N2 in deoxyguanosine by
dehydroretronecine, a carcinogenic metabolite of the pyrrolizidine
alkaloid monocrotaline. Cancer Res. 42, 8-14.
(39) Tomer, K. B., and Gross, M. L. (1986) Fast atom bombardment
and tandem mass spectrometry of covalently modified nucleosides
and nucleotides: adducts of pyrrolizidine alkaloid metabolites.
Anal. Chem. 58, 2527-2534.
(40) Hoskins, W. M., and Crout, D. H. G. (1977) Pyrrolizidine alkaloid
analogues. Preparation of semisynthetic esters of retronecine. J .
Chem. Soc., Perkin Trans 1, 538-543.
(41) Mattocks, A. R., J ukes, R., and Brown, J . (1989) Simple proce-
dures for preparing putative toxic metabolites of pyrrolizidine
alkaloids. Toxicon 27, 561-569.
(42) Christie, M. H., Kropman, M., Leisegang, E. C., and Warren, F.
L. (1949) The Senecio Alkaloids. Part III. The structure of
retrorsine and isatidine, and the isomerism of retronecic acid and
isatinecic acid. J . Chem. Soc., 1700-1702.
(43) Chou, M. W., Wang, B., Von Tungeln, L. S., Beland, F. A., and
Fu, P. P. (1987) Induction of rat hepatic cytochromes P-450 by
environmental nitropolycyclic aromatic hydrocarbons. Biochem.
Pharmacol. 36, 2449-2454.
(44) Miranda, C. R., Reed, R. L., Gguengerich, F. P., and Buhler, D.
R. (1991) Role of cytochrome P450IIIA4 in the metabolism of the
pyrrolizidine alkaloid senecionine in human liver. Carcinogenesis
12, 515-519.
(45) Reddy, M. V., and Randerath, K. (1986) Nuclease P1-mediated
enhancement of sensitivity of 32P-postlabelling test for structur-
ally diverse DNA adducts. Carcinogenesis 7, 1543-1551.
(46) Gupta, R. C. (1993) 32P-Postlabelling analysis of bulky aromatic
adducts. In Postlabelling Methods for Detection of DNA Adducts
(Phillips, D. P., Castegnaro, M., and Bartsch, H., Eds.) pp 11-
23, International Agency for Research on Cancer, Lyon, France.
(47) Nath, R. G., and Chung, F.-L. (1994) Detection of exocyclic 1,N2-
propanodeoxyguanosine adducts as common DNA lesions in
rodents and humans. Proc. Natl. Acad. Sci. U.S.A. 91, 7491-7495.
(48) Chung, F.-L., Chen, H.-Y. C., and Nath, R. G. (1996) Lipid
peroxidation as a potential endogenous source for the formation
of exocyclic DNA adducts. Carcinogenesis 17, 2105-2111.
(49) Zhan, D.-J ., Herreno-Saenz, D., Chiu, L.-H., Von Tungeln, L. S.,
Wu, Y.-S., Lewtas, J ., and Fu, P. P. (1995) Separation of
32P-labeled 3′,5′-bisphosphate nucleotides of polycyclic aromatic
hydrocarbon anti-diol-epoxides and derivatives. J . Chromatogr.,
A 710, 149-157.
(50) Herreno-Saenz, D., Evans, F. E., Beland, F. A., and Fu, P. P.
(1995) Identification of two N2-deoxyguanosinyl DNA adducts
upon nitroreduction of the environmental mutagen 1-nitropyrene.
Chem. Res. Toxicol. 8, 269-277.
(51) Herreno-Saenz, D., Evans, F. E., Lai, C. C., Fu, P. P., and Delclos,
K. B. (1993) Characterization of the products of the in vitro
reaction of metabolites of 3-aminochrysene with DNA. Chem.-
Biol. Interact. 86, 1-15.
(64) Hirono, I., Mori, H., Yamada, K., Hirata, Y., and Haga, M. (1977)
Carcinogenic activity of petasitenine, a new pyrrolizidine alkaloid
isolated from Petasites japonicus Maxim. J . Natl. Cancer Inst. 58,
1155-1157.
(65) Hirono, I., Ueno, I., Aiso, S., Yamaji, T., and Haga, M. (1983)
Carcinogenic activity of Farfugium japonicum and Senecio can-
nabifolius. Cancer Lett. 20, 191-198.
(66) Harris, P. N., and Chen, K. K. (1970) Development of hepatic
tumors in rats following ingestion of Senecio longilobus. Cancer
Res. 30, 2881-2886.
(67) Kuhara, K., Takanashi, H., Hirono, I., Furuya, T., and Asada, Y.
(1980) Carcinogenic activity of clivorine, a pyrrolizidine alkaloid
isolated from Ligularia dentata. Cancer Lett. 10, 117-122.
(68) Schoental, R., Fowler, M. E., and Coady, A. (1970) Islet cell tumors
of the pancreas found in rats given pyrrolizidiine alkaliods from
Amsinckia intermedia Fisch and Mey and from Heliotropium
supinum L. Cancer Res. 30, 2127-2131.
(69) Schoental, R., and Cavanagh, J . B. (1972) Brain and spinal cord
tumors in rats treated with pyrrolizidine alkaloids. J . Natl. Cancer
Inst. 49, 665-671.
(70) Schoental, R. (1975) Pancreatic islet-cell and other tumors in rats
given heliotrine, a monoester pyrrolizidine alkaloid, and nicoti-
namide. Cancer Res. 35, 2020-2024.
(71) Rao, M. S., and Reddy, J . K. (1978) Malignant neoplasms in rats
fed lasiocarpine. Br. J . Cancer 37, 289-293.
(52) Herreno-Saenz, D., Von Tungeln, L. S., Heflich, R. H., Lewtas,
J ., and Fu, P. P. (1994) Formation of N-(deoxyguanosin-8-yl)-2-
aminofluoranthene as a major 2-nitrofluoranthene induced DNA
adduct in Salmonella typhimurium and neonatal B6C3F1 mice.
Polycyclic Aromat. Compd. 6, 79-85.
(53) Herreno-Saenz, D., Evans, F. E., and Fu, P. P. (1994) Nitrore-
duction of 1- and 3-nitro-7,8,9,10-tetrahydrobenzopyrene resulting
in the N2-deoxyguanosinyl adducts through long-range migration.
Chem. Res. Toxicol. 7, 806-814.
(54) Ni, Y.-C., Kadlubar, F. F., and Fu, P. P. (1995) Formation of
malondialdehyde-modified 2′-deoxyguanosinyl adduct from me-
tabolism of chloral hydrate by mouse liver microsomes. Biochem.
Biophys. Res. Commun. 216, 1110-1117.
(55) Fu, P. P., Zhan, D.-J ., Von Tungeln, L. S., Yi, P., Qui, F.-Y.,
Herreno-Saenz, D., and Lewtas, J . (1996) Comparative formation
(72) Mattocks, A. R., and Cabral, J . R. (1982) Carcinogenicity of some
pyrrolic pyrrolizidine alkaloid metabolites and analogues. Cancer
Lett. 17 (1), 61-66.
(73) Chan, P. C., Mahler, J ., Bucher, J . R., Travlos, G. S., and Reid,
J . B. (1994) Toxicity and carcinogenicity of riddelline following
13 weeks of treatment to rats and mice. Toxicon 32, 891-908.
(74) Chan, P. C. (1993) NTP Technical Report on Toxicity Studies of
Riddelline (CAS No. 23246-96-0) Administered by Gavage to
F344/N Rats and B6C3F1 Mice. NIH Publication 94-3350, U.S.
Government Printing Office, Washington, DC.
(75) Huxtable, R. J . (1980) Herbal teas and toxins: novel aspects of
pyrrolizidine poisoning in the United States. Perspect. Biol. Med.
24, 1-14.
TX000149O