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Mass Spectrometric Studies of DNA Adducts from a Reaction with Terpenoids.

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Angewandte
Chemie
DNA Adducts
Mass Spectrometric Studies of DNA Adducts
from a Reaction with Terpenoids**
Wolfgang Schrader,* Sven Dring, and Werner Joppek
The phenomenon of blue haze above forests was described in
the 1960s by Went,[1] who correlated the formation of organic
aerosols with biogenic plant emissions. Biogenic emissions,
especially of mono- and sesquiterpenes, react with atmospheric oxidants to form a large number of products,[2] which
are thought to undergo a gas-to-particle conversion to form
the organic aerosol. These particles in the atmosphere behave
differently than the gaseous compounds, because they absorb
or scatter the solar radiation, serve as cloud condensation
nuclei, and are involved in multiphase atmospheric chemistry.[3]
In addition to their atmospheric impact, terpenes and
terpenoids are considered to be involved in plant–insect
interactions.[4] Indoor emissions of terpenes from furniture[5]
have been investigated as well as their impact on the nasal and
respiratory system, and the risks of respiratory cancer for
exposed woodworkers.[6] The implication of terpenes and
terpenoids in the generation of chronic pulmonary disease
and acute bronchitis is well established.[7] Their impact on the
human organism is otherwise still unclear. Even pharmocokinetic data are not specific, and systematic studies on the
metabolism have not been reported.[7]
One marker used to observe the impact of xenobiotic
compounds on organisms is the formation of DNA adducts,
which in theory can lead to tumor development.[8] Studies on
DNA damage caused by oxidative stress or other natural
compounds are of great interest to scientists.[9] Randerath
et al.[10] were studying DNA modifications caused by polycyclic hydrocarbons using a 32P-postlabeling assay when they
found a significant number of DNA modifications in control
samples from untreated animals. The authors suggested that
these adducts arose from indigenous compounds and therefore called them “i-spots”. The pattern of these i-spots is
dependent on tissue, species, gender, and diet,[11] and the
number of i-spots increases with age.[12] Recently, i-spots have
been associated with cancer development.[13] Unfortunately,
the causes are still not fully understood. Some results indicate
that compounds responsible for oxidative stress could cause
some of the i-spots,[9] although this does not sufficiently
explain the complex pattern of the indigenous DNA modifications. Therefore other chemical compounds must be
responsible for these lesions. One source that could have a
substantial influence on base-level DNA damage is unsatu[*] Dr. W. Schrader, S. Dring, W. Joppek
Max-Planck-Institut f!r Kohlenforschung
Kaiser-Wilhelm-Platz 1, 45470 M!lheim/Ruhr (Germany)
Fax: (+ 49) 208-306-2982
E-mail: wschrader@mpi-muelheim.mpg.de
[**] The authors thank Prof. Dr. A. F!rstner and Dr. J. Geiger for helpful
discussions.
Angew. Chem. Int. Ed. 2004, 43, 6657 –6660
rated biogenic hydrocarbons, because they are ubiquitous.
The only biogenic hydrocarbon that has been investigated in
detail with regard to DNA-adduct formation is ethylene.[14]
Ethylene metabolizes to ethylene oxide, which is considered a
strong carcinogen. Until now, there have been no studies on
the impact of unsaturated hydrocarbons with regard to DNA
adducts. Here, we report on a new class of xenobiotics and
their effect in terms of base-level DNA damage.
Identification of xenobiotics contributing to base-level
DNA damage is difficult, because the level is very low (about
one adduct per 108 nucleotides),[15] which presents a challenge
for analytical methods. The most sensitive technique is the
32
P-postlabeling assay, which allows detection of one adduct
per about 108–1010 bases.[16] Since the adducts are detected by
cochromatography, a synthetic standard is required and
unknown adducts cannot be identified.
We have been studying the reactivity of terpenoids
tentatively identified as products of the gas-phase ozonolysis
of monoterpenes with regard to their reactivity with DNA
and DNA constituents in vitro by applying enzymatic
digestion followed by mass-spectrometric analysis to characterize the reaction products. Mass spectrometry is not as
sensitive as 32P-postlabeling, but the DNA adducts can be
detected in small quantities and information about the
structure of the adduct molecule can be extracted.[17] For
this purpose we use both an ion trap and a Fourier transform
ion cyclotron resonance mass spectrometer (FTICR-MS) to
study the reaction of a-pinene oxide with calf thymus DNA.
While the ion trap MS can be used for fragmentation
experiments induced through collision activation (CAD),
FTICR-MS provides high-resolution mass data for the
reaction products, which can be used to calculate the
empirical formulas of the products in complex reaction
mixtures.
Enzymatic DNA degradation is a standard tool for the
study of these macromolecules. For a better understanding of
the reaction, the DNA was digested using two different
approaches: with the enzyme nuclease P1 and, alternatively,
with a combination of the enzymes benzonuclease with an
alkaline phosphatase. While nuclease P1 digests DNA to form
mononucleotides, the benzonuclease produces a number of
oligonucleotides of different chain lengths. The alkaline
phosphatase removes the 5’-phosphates from the oligonucleotides, leading to oligonucleotides composed of n nucleosides and n 1 phosphate groups.[17] Because MS provides only
mass information about a nucleotide, the sequence specificity
cannot be determined. Together, the two approaches provide
an overview of possible DNA adducts resulting from the
reaction with terpenoids.
Here, we report the first results obtained from the
reaction of DNA with a-pinene oxide, an early product of
a-pinene oxidation. Epoxides are reactive towards DNA,[17]
and their generation, by enzymatic oxidation through the
cytochrome P 450, is often the first metabolic step in the
removal of insoluble xenobiotics from the organism. A
number of strong carcinogens are also activated in this way.[18]
In addition to the DNA experiments we studied reactions
of the four mononucleotides with a-pinene oxide in order to
compare the results and facilitate the interpretation. The
DOI: 10.1002/anie.200461022
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
6657
Communications
reaction of a-pinene oxide with DNA leads to a number of
different adducts. Although their abundance is rather low,
some specific DNA adducts could be detected and characterized. The DNA was detected as mononucleotides after
nuclease P1 digestion; besides the not alkylated mononucleotides two adducts were detected. A signal at m/z = 498 was
recorded for the deoxyguanylmonophosphate (dGMP)
adduct and one at m/z = 482 for the deoxyadenylmonophosphate (dAMP) adduct. The other two bases did not form
adducts under these conditions. MS/MS experiments with
these adducts indicate that a-pinene oxide is coupled directly
to the nucleobase. Corresponding results from FTICR-MS
experiments confirm the findings and make it possible to
obtain the empirical formulas of the respective adducts. When
the reaction was conducted at 60 8C rather than at 37 8C, the
dCMP and dTMP adducts with signals at m/z = 473 and 458,
respectively, were observed. In comparison, pure mononucleotides showed adduct formation at 37 8C for all four
nucleotides.
Spectra of the products of DNA degradation with
benzonase and alkaline phosphatase show signals of oligonucleotides with different chain lengths. The most intensive
signals were those of the dinucleotides, some of which also
formed adducts. A total of seven different adducts with apinene oxide were characterized (Figure 1), and they are
listed with the mononucleotide adducts in Table 1.
MS/MS and MS3 spectra obtained from collision activation revealed that the adduct formation occurs primarily at
the purine bases within the dinucleotides. No fragment ion
could be detected that would indicate a reaction of a-pinene
oxide with a pyrimidine base, while the fragments at m/z =
286 and m/z = 302 are characteristic for the adenine and
guanine adducts, respectively. All the adducts were also
characterized by FTICR-MS; the spectra confirmed the
results from the ion trap measurements and the empirical
formulas of the adducts were also determined (see Table 1).
The results indicate that a-pinene oxide reacts with DNA at
more or less random positions. The reaction seems to occur
primarily with purine bases, while pyrimidine bases are less
frequently involved.
In addition to the exocyclic sites in DNA, such as the N2
and O6 positions of guanine, the N7 position of guanine seems
to be especially reactive. The FTICR spectrum in Figure 2
Figure 2. FTICR mass spectrum of enzymatically digested DNA showing the mononucleotides and the N7 guanine/pinene oxide adduct.
Figure 1. FTICR mass spectrum of enzymatically digested DNA. DNA
adducts of a-pinene oxide shown as their dinucleotides are shown;
seven of the possible ten dinucleotides are alkylated. The empirical formulas can be calculated from accurate mass determinations.
Table 1: DNA adducts resulting from the reaction with a-pinene oxide.
DNA adduct
m/z
Formula[a]
Accuracy [ppm]
dAMP
dGMP
dApdC
dApdT
dGpdC
dApdA
dGpdT
dApdG
dGpdG
482.18072
498.17611
691.26342
706.26088
707.25712
715.27209
722.25681
731.26880
747.26350
C20H29N5O7P
C20H29N5O8P
C29H40N8O10P
C30H41N7O11P
C29H40N8O11P
C30H40N10O9P
C30H41N7O12P
C30H40N10O10P
C30H40N10O11P
0.6
0.38
3.4
0.23
1.6
0.26
1.6
2.2
1.9
[a] Calculated formulas are based on the negatively charged ions
[M H] .
6658
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
shows the four unreacted mononucleotides as well as the
guanine adduct at m/z = 302.16225 (0.6 ppm). The accurate
mass measurement led to a formula of C15H20N5O2, which
corresponds to the a-pinene oxide/guanine adduct.
The enzymatic digestion used here should result in
mononucleotides or oligonucleotides depending on the
enzymes. The a-pinene oxide/guanine adduct in Figure 2
presumably results from reaction at the N7 position of
guanosine, because alkylation at this site weakens the Nglycosidic bond, subsequently leading to bond cleavage and
removal of the alkylated base from the DNA strand. This
effect has been reported previously in studies of styrene oxide
alkylation.[19] Reaction at any other position of guanosine
would not weaken the N-glycosidic bond and but would lead
to either the corresponding mono- or oligonucleotide adduct
depending on the enzyme. As a result of the hydrolytic
cleavage of DNA, the very viscous reaction solution becomes
fluid. A possible reaction mechanism is suggested in
Scheme 1. No cleavage products could yet be identified for
this hydrolysis of the remaining DNA. In addition to the
guanine adduct, an adenine adduct could be found at m/z =
286.16733 (0.46 ppm), but the signal has lower intensity.
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 6657 –6660
Angewandte
Chemie
were dissolved in triply distilled water, combined with a-pinene oxide
(75 mL), and also incubated at 37 8C for 24 h. All reaction mixtures
were analyzed without additional purification.
Mass spectrometric detection: A Bruker Esquire 3000 ion trap
mass spectrometer was used. The mass range was scanned from m/z =
50 to 2000, and two scans were added for each spectrum. MS/MS and
MS3 experiments were carried out inside the ion trap after the desired
ion had been isolated; helium was used as the collision gas. Ions were
isolated using a width of 0.5 Da. All samples were dissolved in
acetonitrile in a ratio of 1:2 (v/v) and were introduced into the mass
spectrometer using the direct-infusion mode with a flow rate of
2 mL s 1. In general, scans were recorded for 2 min and afterwards
averaged for a better signal-to-noise ratio. High-resolution mass
measurements were obtained from a Bruker APEX III FTICR-MS
employing a 7 T magnet. The instrument was equipped with an
Agilent electrospray ion source. After ionization ions were stored
inside of a hexapole for sampling times between 0.3 and 0.8 s and
afterwards transferred to the cyclotron cell. The mass range was
scanned from m/z = 80 to 2000 with 512k data points. For detection of
DNA adducts up to 100 scans were accumulated for a better signal-tonoise ratio.
Received: June 21, 2004
Revised: September 1, 2004
.
Keywords: DNA · i-spots · mass spectrometry · terpenoids
Scheme 1. The proposed reaction of a-pinene oxide at the N7 position
of guanine, which leads to cleavage of the N-glycosidic bond and subsequent formation of the adduct.
The results reported here clearly indicate a reaction
between a-pinene oxide and DNA. Although the concentrations used here are higher than in natural circumstances,
the aim was to observe DNA adducts and provide sufficient
structural information about a new class of xenobiotics that
could have an effect on base-level DNA damage.
Experimental Section
Reaction: DNA (4–5 mg) was dissolved in 1 mL triply distilled water,
combined with a-pinene oxide (25–100 mL), and then incubated at
37 8C for 24–72 h. The reaction mixture was enzymatically digested
using a) a combination of benzonase (25 U mg 1 DNA; Merck,
Darmstadt, Germany) and alkaline phosphatase (1.75 U mg 1 DNA;
Roche Diagnostics, Mannheim, Germany) or b) nuclease P1
(6 U mg 1 DNA; Roche Diagnostics, Mannheim, Germany) and
incubated for 24 h at 37 8C. The mononucleotides (c = 1 mg mL 1)
Angew. Chem. Int. Ed. 2004, 43, 6657 –6660
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