J. Am. Chem. Soc. 1999, 121, 9231-9232
9231
DNA Oxidation as a Source of Endogenous
Electrophiles: Formation of Ethenoadenine Adducts
in γ-Irradiated DNA
Wendelyn R. Jones and Peter C. Dedon*
DiVision of Bioengineering and EnVironmental Health
Massachusetts Institute of Technology, 56-787
Cambridge, Massachusetts 02139
ReceiVed May 7, 1999
DNA damage resulting from exposure to reactive oxygen
1
species plays a role in mutagenesis and aging. The genotoxicity
Figure 1. (A) Etheno adducts of DNA bases. (B) Proposed mechanism
for the formation of ꢀA from a 3′-phosphoglycoaldehyde residue and
chloroacetaldehyde.
of reactive oxygen species, which are formed during cellular
metabolism, inflammation, and exposure to ionizing radiation,1
may arise from direct damage to DNA or from reactions with
other biomolecules that lead to the formation of DNA-reactive
b,2
3
electrophiles. Lipid peroxidation products have been presumed
4
to be an example of the latter indirect pathway. For instance, an
epoxide metabolite of 4-hydroxynonenal has been proposed to
react with DNA to form the four exocyclic etheno adducts shown
4c
in Figure 1A. Among these adducts, which also arise by reaction
5
6
of DNA bases with chloroacetaldehyde, 1,N -ethenoadenine (ꢀA)
6
has been shown to be mutagenic, and it has been detected at
9
7
levels of 2-2600 adducts per 10 nt in mammalian cells. We
now report that ꢀA adducts also form during oxidation of DNA
by γ-radiation.
The premise for these experiments is that oxidation of
deoxyribose leads to the formation of electrophilic species capable
of forming adducts with nucleobases. As proof-of-concept, we
reported that base propenals arising from C4′-oxidation of
Figure 2. Analysis of ꢀA in a reaction of 2-phosphoglycoaldehyde with
dA using HPLC with UV (solid line) and fluorescence (dashed line)
detection.
1
deoxyribose react with dG to form the mutagenic M G adduct,
To test this hypothesis, we treated dA with synthetic 2-phos-
phoglycoaldehyde and subjected depurination products to reversed
phase HPLC.10 The reaction resulted in the quantitative formation
of ꢀA (Figure 2). A UV-absorbing, fluorescent species coeluted
with standard ꢀA and produced an ESI-MS molecular ion peak
which also arises in a reaction with the structurally analogous
malondialdehyde albeit less efficiently.8
On the basis of a similar structural analogy, we hypothesized
that 3′-phosphoglycoaldehyde residues derived from deoxyribose
3′-oxidation (Figure 1B) would react with nucleobases to form
+
11,12
(M+H) of 160 as expected.
etheno adducts. The phosphoglycoaldehyde residue is produced
9a
9b
in DNA treated with γ-radiation and certain rhodium complexes,
We next tested the hypothesis that an oxidizing agent capable
and it is a structural analogue of chloroacetaldehyde, which
readily reacts with guanine, cytidine and adenine to form etheno
adducts (Figure 1A,B).5
of causing formation of 3′-phosphoglycolaldehyde would also
produce ꢀA. As shown in Figure 3, HPLC analysis of γ-irradiated
13,14
DNA
revealed a UV-absorbing and fluorescent species that
coeluted at 17-18 min (panel B) with standard ꢀA (not shown)
*
Corresponding author: tel. 617-253-8017, fax 617-258-0225, E-mail
pcdedon@mit.edu.
1) (a) Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Proc. Natl. Acad.
and with ꢀA generated in chloroacetaldehyde-treated DNA (panel
A).5
,7,15b
ESI-MS analysis of the material eluting at 17-18 min
(
Sci. U.S.A. 1993, 90, 7915-7922. (b) Halliwell, B.; Gutteridge, J. M. C.;
Cross, C. E. J. Lab. Clin. Med. 1992, 119, 598-620.
(10) (a) 2-Phosphoglycoaldehyde was synthesized by treating R-glycerol-
phosphate (1 µmol) with 50 mM NaIO (1 h, 25 °C). Following Sep-Pak
(Waters) purification, product purity was assessed by paper chromatography.
(b) dA was treated with 5-fold molar excess of phosphoglycoaldehyde in 100
mM K HPO buffer, pH 7.4, for 30 min at ambient temperature. (c) The sample
was depurinated (0.1 N HCl, 70 °C, 1 h), and nucleobase products were
(
2) (a) Saran, M.; Bors, W. Radiat. EnViron. Biophys. 1990, 29, 249-262.
4
10d,e
(
b) Riley, P. A. Int. J. Radiat. Biol. 1994, 65, 27-33. (c) Clayson, D. B.;
Mehta, R.; Iverson, F. Mutat. Res. 1994, 317, 25-42.
3) (a) Chaudhary, A. K.; Nokubo, M.; Reddy, G. R.; Yeola, S. N.; Morrow,
(
2
4
J. D.; Blair, I. A.; Marnett, L. J. Science 1994, 265, 1580-1582. (b) Ames,
B. N.; Gold, L. S. Mutat. Res. 1991, 250, 3-16. (c) Dix, T. A.; Aikens, J.
Chem. Res. Toxicol. 1993, 6, 2-18.
1
1
analyzed by HPLC. (d) Johnson A. W.; Demple, B. J. Biol. Chem. 1988,
263, 18017-18022. (e) Levin, J. D.; Johnson, A. W.; Demple, B. J. Biol.
Chem. 1988, 263, 8066-8071.
(
4) (a) Marnett, L. J.; Burcham P. C. Chem. Res. Toxicol. 1993, 6, 771-
7
85. (b) Esterbauer, H.; Schaur, R. J.; Zollner. H. Free Rad. Biol. Med. 1991,
4
(11) Depurination products neutralized with NH OH were analyzed by
1
1, 81-128. (c) Chen, H. J.; Zhang, L.; Cox, J.; Cunninigham, J. A.; Chung,
HPLC using a C-18 reverse-phase column (250 mm × 4.6 mm) and tandem
UV diode array and fluorescence (310 nm excitation, 420 nm emission)
detectors. The products were eluted with 50 mM ammonium acetate (pH 7.0)
with an acetonitrile gradient (0-25%) over 30 min at 25 °C. Peaks were
F. L. Chem. Res. Toxicol. 1998, 11, 1472-1480.
(
5) Bartsch, J.; Barbin, A.; Marion, M.-J.; Nair, J.; Guichard, Y. Drug
Metab. ReV. 1994, 26, 349-371.
6) (a) Pandya, G. A.; Moriya, M. Biochemistry 1996, 35, 170-175. (b)
Barbin, A.; Laib, R.; Bartsch, H. Cancer Res. 1985, 45, 2440-2444.
7) (a) Nair, J.; Sone, H.; Nagao, M.; Barbin, A.; Bartsch, H. Cancer Res.
996, 56, 1267-1271. (b) Nair, J.; Vaca, C. E.; Velic, I.; Mutamen, M.; Valsta,
L. M.; Bartsch, H. Cancer Epidemiol., Biomarkers PreV. 1997, 6, 597-601.
e) Nair, J.; Gal, A.; Tamir, S.; Tannenbaum, S. R.; Wogan, G. N.; Bartsch,
H. Carcinogenesis 1998, 19, 2081-2084.
8) Dedon, P. C.; Plastaras, J. P.; Rouzer, C. A.; Marnett, L. J. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 11113-11116.
9) (a) Deeble, D. J.; VonSonntag, C. Int. J. Radiat. Biol. 1986, 49, 927-
36. (b) Sitlani, A.; Long, E. C.; Pyle, A. M.; Barton, J. K. J. Am. Chem.
Soc. 1992, 114, 2303-2312.
1
2a
(
identified by coelution with authentic markers and by ESI-MS.
(12) (a) Putative ꢀA in HPLC fractions was characterized by electrospray
(
ionization mass spectrometry (ESI-MS) using an HP 59987 ESI-MS system
1
2b
1
2
with 70 psi nebulizer gas (N ). The CID mass spectrum showed fragment
ions at 106, 119, and 133 in addition to the molecular ion peak of 160. (b)
Yen, T.-Y.; Holt, S.; Sangaiah, R.; Gold, A.; Swenberg, J. A. Chem. Res.
Toxicol. 1998, 11, 810-815.
(
(
(13) LaMarr, W. A.; Sandman, K. M.; Reeve, J. N.; Dedon, P. C. Chem.
Res. Toxicol. 1997, 10, 1118-1122.
(
(14) DNA (50 µL, 1 µg/µL) in 0.1 M K HPO buffer (pH 7.4) was subjected
2 4
60
9
to 0-1000 Gy of γ-radiation in a Co source (3 Gy/min). The irradiated
DNA was left at ambient temperature for 30 min before depurination.
1
0.1021/ja991517h CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/16/1999