J. Am. Chem. Soc. 2000, 122, 2373-2374
2373
The Trap Depth (in DNA) of
8-Oxo-7,8-dihydro-2′deoxyguanosine as Derived from
Electron-Transfer Equilibria in Aqueous Solution
Steen Steenken,* Slobodan V. Jovanovic,1
Massimo Bietti,2 and Klaus Bernhard
Max-Planck-Institut fu¨r Strahlenchemie
D-45413 Mu¨lheim, Germany
ReceiVed September 29, 1999
ReVised Manuscript ReceiVed January 9, 2000
8-Oxo-7,8-dihydro-2′deoxyguanosine (pKa1 ) 8.6, pKa2
)
11.7),3 frequently called 8-hydroxy-2′deoxyguanosine (≡ 8-O-
HdG), is probably the most important and best-documented
product of “oxidative stress” 4-7 in biological systems. Its
concentration in the cellular DNA is, in fact, a quantitative
measure of the degree of damage that an organism has
undergone.8-12 Unless 8-OHdG is built by nature into DNA on
purpose, it is the product of oxidative decomposition of 2′deoxy-
guanosine (dG). A large number of oxidants/oxidizing environ-
ments leading from dG to its 8-hydroxy derivative have been
identified, some of the more important ones being singlet
oxygen,13,14 the OH radical (produced by ionizing radiation or
transition metal catalyzed decomposition of hydroperoxides),15,16
and photo-oxidation17 (which is believed18-21 to proceed via the
guanosine radical cation).
8-OHdG is a mutagenic lesion involved in carcinogenesis and
aging,8,22 but as such it does not lead to DNA strand breakage.
However, it is much more easily oxidized than its natural “parent”,
dG,14,21,23-26 and its oxidation product is a candidate for (piperi-
dine-induced) strand breakage.27-29 In view of this pronounced
sensitivity, it is necessary to fully understand the radical chemistry
Figure 1. Oxidation of 8-OHdG by G(-H)• at pH 7. The pulse (200 ns
3 MeV electrons)-irradiated solution contained 2 mM guanosine, 0.6 mM
8-OHdG, 20 mM K2S2O8 and 0.5 M tert-butyl alcohol. Absorption spectra
(G(radical) ≡ 3.3) of G(-H)•, observed at 1.5 µs: O, and of 8-OHdG-
(-H)•, observed at 15 µs after the pulse: b. In insets d and e are shown
the determinations of the pKa-values of 8-OHdG(-H)• using, however,
•
N3 to oxidize 8-OHdG.
of 8-OHdG and particularly the one-electron redox properties of
this physiologically important molecule. With this aim, the pulse
radiolysis method with optical and conductance detection was
applied. 8-OHdG30 in 0.01 to 1 mM aqueous solutions was one-
electron-oxidized with the radiation-chemically produced inor-
•
ganic radicals Br2•-, N3 , Tl2+, or SO4•- (Table 1). From the pH-
dependent absorption spectra31 (Figure 1, insets d, e) are derived
the (de)protonation equilibria (Scheme 1)32 with pKa1 ) 6.6 and
pKa2 ) 12.3. These values are higher than those for dGuo (3.9
and 10.8)33 which reflects the increase in electron density due to
the oxygen at C8.
(1) Helix International, 381 Viewmount Dr., Nepean, ON, Canada K2E
7R9.
(2) Chemistry Department, Universita´ “Tor Vergata”, I-00133 Roma, Italy.
(3) Culp, S. J.; Cho, B. P.; Kadlubar, F. F.; Evans, F. E. Chem. Res. Toxicol.
1989, 2, 416.
(4) Sies, H. OxidatiVe Stress; Academic Press: Orlando, FL, 1985.
(5) Helbock, H. J.; Beckman, K. B.; Shigenaga, M. K.; Walter, P. B.;
Woodall, A. A.; Yeo, H. C.; Ames, B. N. Proc. Natl. Acad. Sci. U.S.A. 1998,
95, 288.
(6) Boiteux, S.; Radicella, J. P. Biochimie 1999, 81, 59.
(7) Lloyd, D. R.; Phillips, D. H. Mutat. Res. 1999, 424, 23.
(8) Kasai, H.; Nishimura, S. In OxidatiVe Stress; Sies, H., Ed.; Academic
Press: London, 1991; p 99.
The same conclusion can be drawn from the reduction potential
(0.74 V/NHE, see later) of 8-OHdG(-H)• as compared to the
1.29 V/NHE34 of G(-H)•.
8-OHdG can also be oxidized with various organic radicals
(Table 1), such as tyrosyl or tryptophyl or enolether radical
cations,35 peroxyls, and even with the deprotonated radical cation
of 2′deoxyguanosine-5′-monophosphate or that of guanosine,
G(-H)•. The protocol for this reaction is shown in Figure 1.
The initial spectrum (Figure 1, O) is that of G(-H)• (produced
via SO4•- from the excess guanosine present). Due to the presence
of 8-OHdG, G(-H)• disappears (inset b) to give rise to 8-OHdG-
(-H)• (see b and inset a). The rate constant for the oxidation of
8-OHdG by G(-H)• (obtained via inset c) is 4.6 × 108 M-1 s-1
at pH 7.
(9) Cundy, K. C.; Kohen, R.; Ames, B. N. In Oxygen Radicals in Biology
and Medicine; Simic, M. G., Taylor, K. A., Ward, J. F., von Sonntag, C.,
Eds.; Plenum Press: New York, London, 1988; Vol. 49, p 479.
(10) Bergtold, D. S.; Simic, M. G.; Alessio, H.; Cutler, R. G. In Oxygen
Radicals in Biology and Medicine; Simic, M. G., Taylor, K. A., Ward, J. F.,
von Sonntag, C., Eds.; Plenum Press: New York, London, 1988; Vol. 49, p
483.
(11) Kasai, H. Mutat. Res. 1997, 387, 147.
(12) The natural abundance of 8-OHdG is 1 per 104-5 dG units, see ref 8
and Shigenaga, M.; Park, J.-W.; Cundy, K. C.; Gimeno, C. J.; Ames, B. N.
Methods Enzymol. 1990, 186, 521.
(27) Kochevar, I. E.; Dunn, D. A. In Bioorganic Photochemistry; Morrison,
H., Ed.; Wiley and Sons: New York, 1990; Vol. 1, p 273.
(28) Koizume, S.; Inoue, H.; Kamiya, H.; Ohtsuka, E. Chem. Commun.
1996, 265.
(29) Muller, J. G.; Duarte, V.; Hickerson, R. P.; Burrows, C. J. Nucleic
Acids Res. 1998, 26, 2247.
(13) Devasagayam, T. P. A.; Steenken, S.; Obendorf, M. S. W.; Schulz,
W. A.; Sies, H. Biochemistry 1991, 30, 6283.
(14) Sheu, C.; Foote, C. S. J. Am. Chem. Soc. 1995, 117, 6439.
(15) Cadet, J.; Delatour, T.; Douki, T.; Gasparutto, D.; Pouget, J.-P.;
Ravanat, J.-L.; Sauvagio, S. Mutat. Res. 1999, 424, 9.
(16) Candeias, L. P.; Steenken, S. Chem. Eur. J. 2000, 6, 475.
(17) Candeias, L. P.; Steenken, S. J. Am. Chem. Soc. 1992, 114, 699.
(18) Kasai, H.; Yamaizumi, Z.; Berger, M.; Cadet, J. J. Am. Chem. Soc.
1992, 114, 9692.
(30) From Berry Co., Dexter, MI, purity “100%” (HPLC).
(31) The spectra of oxidized 8-OHdGuo at pH 3, 7.7, and 13.3 are available
in Supporting Information.
•-
(32) With the time-resolved AC-conductance method (using SO4 at pH
4.5 to one-electron-oxidize 8-OHdG and, for calibration, anisole radical cation
(O’Neill, P.; Steenken, S.; Schulte-Frohlinde, D. J. Phys. Chem. 1975, 79,
2773)) under the same conditions, it was established (the conductance signals
had the same shape and amplitude within (4%) that the species existing at
pH < 6 is the radical cation of 8-OHdG.
(19) Angelov, D.; Spassky, A.; Berger, M.; Cadet, J. J. Am. Chem. Soc.
1997, 119, 11373.
(20) Spasski, A.; Angelov, D. Biochemistry 1997, 36, 6571.
(21) Burrows, C. J.; Muller, J. G. Chem. ReV. 1998, 98, 1109.
(22) Shigenaga, M. K.; Hagen, T. M.; Ames, B. N. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 10771.
(23) Sheu, C. M.; Foote, C. S. J. Am. Chem. Soc. 1995, 117, 474.
(24) Adam, W.; Saha-Moeller, C. R.; Schoenberger, A. J. Am. Chem. Soc.
1997, 119, 719.
(25) Gasper, S. M.; Schuster, G. B. J. Am. Chem. Soc. 1997, 119, 12762.
(26) Prat, F.; Houk, K. N.; Foote, C. S. J. Am. Chem. Soc. 1998, 120, 845.
(33) Candeias, L. P.; Steenken, S. J. Am. Chem. Soc. 1989, 111, 1094.
(34) Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617.
(35) The reactions of enolether radical cations with the guanine moiety
are relevant because enolether radical cations are formed in DNA as a result
of C4′-radical initiated chain breaks (Dizdaroglu, M.; von Sonntag, C.; Schulte-
Frohlinde, D. J. Am. Chem. Soc. 1975, 97, 2277; Giese, B.; Beyrich-Graf,
X.; Erdmann, P.; Petretta, M.; Schwitter, U. Chem. Biol. 1995, 2, 367).
10.1021/ja993508e CCC: $19.00 © 2000 American Chemical Society
Published on Web 02/29/2000