SYNTHESIS OF 1,N6-ETHENO-20-DEOXYADENOSINE AND 1,N2-ETHENO-20-DEOXYGUANOSINE
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alkylnitronaphthalenes (100 folds, Tang et al., 2005). Nitro-
MATERIALS AND METHODS
Chemicals
phenols, such as 2,4-dinitrophenol, are also significant
atmospheric pollutants. The molecule 2,4-dinitrophenol has
been detected at ꢀg Lꢀ1 levels in rain (Asman et al., 2005)
and is thought to be a contributing factor of the forest
decline. Surprisingly, its genotoxic capacity has not been
fully addressed yet. The occurrence of this compound is
thought to result from the nitration of mononitrophenols in
the aqueous phase of the atmosphere (Vione et al., 2005b).
Nitroreduction is regarded as a relevant pathway in the
metabolic activation of nitro-PAHs to mutagenic species
(Carroll et al., 2002). Nitroreduction of nitro-PAHs, cata-
lyzed by two-electron transfer reaction (e.g., diaphorase),
yields N-hydroxy arylamines. These molecules react,
directly or upon esterification of the N-hydroxy function,
with DNA to form covalent adducts (Ritter et al., 2002).
The reduction rate increases with the number and the posi-
tion of the nitro groups (Fu et al., 1998). In contrast to
two-electron reaction, single electron reactions of flavoen-
zyme electron transferase (e.g. NADPH: cytochrome P-450
reductase or ferredoxin: NADPþ reductase) can initiate a
redox cycling of nitrocompounds and challenge the for-
mation of N-hydroxylamine. This type of reactions pro-
motes also the formation of reactive oxygen species (ROS)
and the ROS induce oxidative DNA damage (Murata et al.,
2004).
2,4-Dinitrophenol (purity >99%), ꢁ-nicotinamide adenine
dinucleotide reduced disodium salt hydrate (NADH, purity
>98%), nitroreductase (E. coli, 2.46 units/mg protein), lino-
leic acid (purity >99%), 1,N6-etheno-20-deoxyadenosine, 4-
hydroxy-2-nonenal (4-HNE) dimethylacetal were pur-
chased from Sigma-Aldrich (St. Quentin Fallavier, France).
20-Deoxyguanosine (purity >99%), 20-deoxyadenosine (pu-
rity >99%), sodium phosphate dibasic (purity >99%), and
potassium phosphate monobasic (purity >99%) from Acros
Organics (Noisy le Grand, France). All reactants were used
without further purification. Methanol was LiChrosolv gra-
dient grade from Merck (Fontenay Sous Bois, France). Sol-
utions and HPLC eluents were prepared with Milli-Q water.
FeSO4 (puriss.p.a.) and formic acid (>98%) were from
Fluka (St. Quentin Fallavier).
Synthesis of 4-Oxo-2-nonenal
4-Oxo-2-nonenal (4-ONE) dimethylacetal was prepared by
MnO2 oxidation of 4-hydroxy-2-nonenal (4-HNE) dimethy-
lacetal. The latter was synthesized according to the method
of Esterbauer and Weger (1967). 4-ONE was obtained by
acid treatment of 4-ONE dimethylacetal.
Unsubstituted etheno-DNA adducts ("-adducts), such
as 1,N6-etheno-20-deoxyadenosine ("dA), 1,N2-etheno-20-
deoxyguanosine ("dG), and 3,N4-etheno-20deoxycytosine
("dC) are highly mutagenic, because they are chemically
stable and are slowly removed in case of impairment or
imbalance of cellular DNA repair pathways. The persist-
ence of such DNA adducts might lead to the increase in
mutations and genomic instability (Bartsh and Nair, 2005;
Bolt, 2005). These chemicals were originally considered to
originate from exposure to environmental carcinogens,
such as the occupational carcinogen vinyl chloride. They
are now recognized to be produced endogenously by lipid
peroxidation. Lately, "-adducts have been examined as use-
ful markers to assess oxidative stress and lipid peroxidation
during the early stages of carcinogenesis, particularly dur-
ing the inflammatory process. However, the direct link
between the exposure to environmental NACs and the for-
mation of "-adducts through a lipid oxidation process has
not been clearly evidenced until now. Thus, the major aims
of this work were the following:
Synthesis of 1,N2-etheno-20-deoxyguanosine ("dG)
The compound "dG was synthesized according to the pro-
cedure of Sattsangi et al. (1977) by using a phosphate
buffer (0.1 M; pH 6.4). The molecule "dG was isolated by
collecting fractions during HPLC for further characteriza-
tion by nuclear magnetic resonance (NMR).
Analytical Determinations
The identifications of dA, dG, "dA, "dG, and 4-ONE were
carried out by liquid chromatography multistage mass spec-
trometry (LC/MS2). Positive electrospray ((þ) ESP) was
performed on an Esquire 6000 ion trap system (Bruker,
Bremen, Germany), with a potential of 4 kV applied to the
electrospray needle. Nitrogen was used as drying and nebu-
lizing gas. The capillary temperature was held at 250 8C.
Full scanning analysis was performed in the range of 100–
500 m/z. The relative collision energy was set at 30% of the
maximum (1 V). The [MþH]þ ion at m/z 276, m/z 292, m/z
252, and m/z 268 was selected as the precursor ion for "dA,
"dG, dA, and dG, respectively. All compounds showed the
same fragmentation pattern. Daughter ion spectra were
only detected as one intense base peak at m/z 160, m/z 176,
m/z 136, and m/z 152 for "dA, "dG, dA and dG, respec-
tively. All fragments corresponded to the loss of the neutral
20-deoxyribose moiety (116 uma) formed by the rupture of
the N-glycoside bond. The MS2 mass spectrum of 4-oxo-2-
i. To assess the ubiquity of the in vitro 1,N6-etheno-20-
deoxyadenoside and 1,N2-ethano-20-deoxyguanosine for-
mation upon the presence of nitroreductase and NACs.
ii. To study the kinetics of elimination of 20-deoxyadeno-
sine (20-dA) and 20-deoxyguanosine (20-dG) and the
kinetics of production of "dA and "dG.
iii. To discuss the possibility to use "-adducts as NACs ex-
posure biomarkers.
Environmental Toxicology DOI 10.1002/tox