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J. Biol. Chem., Vol. 275, Issue 51, 40601-40604, December 22, 2000
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,,, and
From the Laboratoire "Lésions des Acides
Nucléiques" Service de Chimie Inorganique et Biologique, UMR
CNRS 5046, Département de Recherche Fondamentale sur la
Matière Condensée, CEA Grenoble, 17 Avenue des Martyrs,
F-38054 Grenoble Cedex 9, France and the Departamento de
Bioquímica, Instituto de Química, Universidade de
São Paulo, CP 26077, CEP 05513-970, São
Paulo, SP, Brazil
Received for publication, July 26, 2000, and in revised form, September 18, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The aim of the present work was to evaluate the
potential for 1O2 to induce oxidation of
cellular DNA. For this purpose cells were incubated in the presence of
a water-soluble endoperoxide whose thermal decomposition leads to the
formation of singlet oxygen. Thereafter, DNA was extracted and the
level of several modified DNA bases was determined by HPLC analysis
coupled to a tandem mass spectrometric detection. A significant
increase in the level of 8-oxo-7,8-dihydro-2'-deoxyguanosine was
observed upon incubation of the cells with the chemical generator of
1O2, whereas the level of the other DNA bases
measured remained unchanged. To demonstrate that singlet oxygen is
directly involved in the formation of
8-oxo-7,8-dihydro-2'-deoxyguanosine, the corresponding 18O-labeled endoperoxide was used. Incubation of the cells
with such a generator of 18O-labeled singlet oxygen results
in the formation of 18O-labeled
8-oxo-7,8-dihydro-2'-deoxyguanosine in the nuclear DNA. This result
clearly demonstrates that singlet oxygen, when released within cells,
is able to directly oxidize cellular DNA.
Modification of cellular DNA upon exposure to reactive oxygen and
nitrogen species is the likely initial event involved in the induction
of the mutagenic and lethal effects of various oxidative stress agents
(1-3). As an example, the deleterious effects of UVA radiation are, at
least partly, explained in terms of photooxidation of cellular DNA (4,
5). The mechanism of UVA-mediated photooxidative damage to DNA is not
completely established. Evidence has been accumulated over the years
for the significant implication of singlet oxygen, as the result of UVA
activation (4, 6) of endogenous photosensitizers (porphyrins, flavins,
... ) not yet characterized. However, a type I mechanism
involving the initial formation of a DNA radical cation, that could be
predominantly located at guanine sites due the lowest ionization
potential of the latter base and/or to the possibility of charge
transfer in DNA (7), could not be excluded. To our knowledge, no clear evidence has been provided to demonstrate that singlet oxygen is able
to oxidize cellular DNA. It should be added, however, that
1O2 is known to be mutagenic and genotoxic (2,
3, 8). In addition, singlet oxygen has been identified as the reactive oxygen species involved in numerous biological processes. Among others
we may cite neutrophils phagocytosis (9) and enzymatic processes (10).
Reactions of singlet oxygen with nucleosides and isolated DNA are well
documented. Interestingly, it was shown that
1O2 oxidizes, among the nucleosides, almost
exclusively the guanine base. Singlet oxygen reacts with free
dGuo1 and short
oligonucleotides to give rise to the overwhelming formation of the
4R and 4S diastereomers of
4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine, together with a small
amount of 8-oxodGuo (11-15). In contrast, 8-oxodGuo was found to be
the major oxidation product formed upon exposure of isolated DNA to
1O2 (6, 11, 16). However, 8-oxodGuo cannot be
considered as a specific biological marker of
1O2, since this DNA lesion could be formed
under various conditions of oxidative stress, including those generated
by one-electron process (17), hydroxyl radical (18), and Fenton-type
reactions (19). On one hand, this explains why 8-oxodGuo could be used as an ubiquitous biomarker of DNA oxidation (20-22). On the other hand, the formation of 8-oxodGuo in cellular DNA could not be attributed to the initial formation of 1O2.
During the recent past, water-soluble generators of
1O2, that consist of aromatic hydrocarbon
endoperoxides, became available (23-25). Thermal decomposition of the
latter compounds is used to efficiently produce singlet oxygen (26).
Interestingly, the water-soluble non-ionic endoperoxide
DHPNO2 (23) was recently found to be incorporated into
cells upon incubation (27). Using such a chemical generator of singlet
oxygen, Klotz et al. (27) were able to show the activation
of specific genes upon intracellular release of singlet oxygen.
The purpose of the present work was to use DHPNO2 to
determine whether intracellular singlet oxygen is able to oxidize
nuclear DNA. Emphasis was placed on the determination of the mechanism of DNA oxidation. For this purpose, the 18O-labeled
endoperoxide DHPN18O2 was prepared to assess if
the formation of oxidative damage results from the direct reaction of
1O2 within cellular DNA. It was clearly shown
that intracellular formation of singlet oxygen is able to efficiently
produce 8-oxodGuo.
Cell Culture and Preparation--
The THP1 monocyte cell line
used for this study was obtained as described previously (28). Before
treatment with the endoperoxide, the cells were recovered by
centrifugation (250 × g, 4 min) and washed twice with PBS buffer.
Preparation of the Endoperoxide--
The water-soluble non-ionic
endoperoxide DHPNO2 was prepared by methylene blue-mediated
photosensitization of DHPN as reported previously (29). The
corresponding labeled DHPN18O2 endoperoxide,
whose synthesis is described in detail elsewhere (30), was obtained
using the same protocol, in the presence of 18O-labeled
molecular oxygen.
Incubation of the Cells with Either DHPNO2 or
DHPN18O2--
Cells (30 × 106 per sample) were collected by centrifugation prior to
being suspended in either 200 or 400 µl of PBS buffer, to which
different amounts of either 210 mM DHPNO2 or
130 mM DHPN18O2 were added.
For control experiment, complete deactivation of DHPNO2 was
obtained by heating the endoperoxide for 40 min at 70 °C. The cell
suspension was incubated at either 4 or 37 °C for increasing periods
of time. After incubation, the cells were recovered by centrifugation
and washed with 5 ml of PBS buffer to remove the excess of endoperoxide
prior to DNA extraction.
DNA Extraction--
Extraction of cellular DNA was performed
according to a previously reported procedure (14, 28) with one major
modification. Typically, 10 mM sodium azide, a well known
singlet oxygen quencher, was added to the buffered solutions (14, 28)
used to isolate DNA (buffer A, 320 mM sucrose, 5 mM MgCl2, 10 mM Tris-HCl, 0.1 mM deferoxamine, 1% Triton X-100, 10 mM
NaN3, pH 7.5; and buffer B, 10 mM Tris-HCl, 5 mM EDTA-Na2 0.15 mM
deferoxamine, 10 mM NaN3, pH 8.0).
Sodium azide addition was made to prevent oxidation due to the presence
of residual endoperoxide during cell lysis and subsequent DNA isolation
(see below).
8-OxodGuo Measurement--
The level of 8-oxodGuo in cellular
DNA was assessed using the recently available HPLC-MS/MS assay (31,
32). This required a quantitative enzymatic digestion of DNA into
nucleosides following a reported optimized procedure (31, 33). Accurate
quantification of the level of 8-oxodGuo was obtained by using an
isotopically labeled M + 5 internal standard (31, 32). Typically, 1.5 pmol of [M + 5] 8-oxodGuo was added to the DNA sample prior to its enzymatic digestion. The resulting hydrolysate was directly injected onto the HPLC column coupled to the mass spectrometric detector. The
output of the column was also connected to a UV detector set up at 260 nm for quantification of the DNA amount through the measurement of dGuo
(31, 32). Both 284 to 168 and 289 to 173 transitions were used
for the detection of 8-oxodGuo and the corresponding [M + 5]-labeled
internal standard, respectively (31, 32). In addition, the 286 to 170 transition was recorded to monitor the formation of [M + 2] 18O-labeled 8-oxodGuo. The measurement of the other DNA
lesions, including thymidine glycols,
5-(hydroxymethyl)-2'-deoxyuridine, 5-formyl-2'-deoxyuridine, and
8-oxo-7,8-dihydro-2'-deoxyadenosine, was performed as described
previously (32). Results, expressed as the number of lesion
formed per 106 DNA bases, represent the average and S.D.
of, at least, three independent determinations.
Incubation of cell suspension for 1 h at 37 °C with
DHPNO2 results in the formation of 8-oxodGuo in cellular
DNA (Fig. 1) as determined using the
HPLC-MS/MS assay. The background level of 8-oxodGuo in cellular DNA was
estimated to be close to 0.2 modification per 106 DNA bases
(Fig. 1, Control). Incubation of the cells with increasing amounts of DHPNO2 (conditions A, B,
C, and D) results in a significant increase (up
to 10 times) in the level of 8-oxodGuo. No significant increase in the
level of 8-oxodGuo was observed when cells were incubated with 50 µl
of deactivated DHPNO2 (Fig. 1) nor when the incubation was
performed at 4 °C (Fig. 2).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (7K):
[in a new window]
Fig. 1.
Measurement of 8-oxodGuo in DNA of cells
treated in the presence of varying amounts of DHPNO2.
Cells (30 × 106 per sample) suspended in 400 µl of
PBS buffer were treated for 1 h at 37 °C with either 5 µl
(A), 10 µl (B), 20 µl (C), or 50 µl (D) of DHPNO2. Control cells were not
treated or treated with 50 µl of heat-deactivated DHPNO2
(D heated).
View larger version (8K):
[in a new window]
Fig. 2.
Measurement of 8-oxodGuo in DNA of cells
treated for increasing periods of time in the presence of
DHPNO2. Cells (30 × 106 per sample,
four samples per condition) suspended in 200 µl of PBS buffer were
treated by 50 µl of DHPNO2 at either 4 °C for 60 min
or at 37 °C for 30 and 60 min.
In the absence of the endoperoxide the HPLC-MS/MS assay is sensitive
enough (Fig. 3) to measure endogenous
8-oxodGuo and to quantify its amount by isotope dilution using
15N5-labeled [M + 5] 8-oxodGuo as the
internal standard (Fig. 3, Control). When cells are
incubated with DHPNO2 an increase in the level of 8-oxodGuo
is observed (Fig. 3.). Furthermore, as expected, no [M + 2]
18O-labeled 8-oxodGuo could be detected in either
DHPNO2-treated cells or in the control experiment. When
cells were incubated with the labeled endoperoxide
DHPN18O2 a slight increase of 8-oxodGuo is
observed, and [M + 2] 18O-labeled 8-oxodGuo is detected
(Fig. 3). The results obtained after treatment of the cells with two
different concentrations of the labeled endoperoxide are summarized in
Fig. 4.
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An attempt was also made to evaluate if the formation of 8-oxodGuo is
mainly due to the intracellular production of singlet oxygen. For this
purpose, the cells were first incubated with DHPN18O2 at 4 °C to allow the penetration of
the endoperoxide into cells. Thereafter, the cells were collected by
centrifugation to remove the excess of DHPN18O2
and resuspended in PBS buffer before incubation at 37 °C. Under these conditions (Fig. 5, column
C) the formation of [M + 2] 8-oxodGuo could be detected.
However, without removing the excess of endoperoxide (Fig. 5,
column D) an about 3-fold higher amount of the lesion is
measured.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Incubation of the cell suspension for 1 h at 37 °C in the presence of DHPNO2 results in a significant formation of 8-oxodGuo. No increase is observed if the endoperoxide is deactivated (by heating) confirming the involvement of singlet oxygen in the formation of the lesion. The background level of 8-oxodGuo was determined to be close to 0.2 lesion per 106 DNA bases. This relatively low value is in agreement with recent data (14, 34) and confirms that former measurements have led to an overestimation of the level of 8-oxodGuo in cellular DNA due to an artifactual oxidation during the work-up (35). No detectable increase in the formation of other DNA lesions, including thymidine glycols, 5-(hydroxymethyl)-2'-deoxyuridine, 5-formyl-2'-deoxyuridine, and 8-oxo-7,8-dihydro-2'-deoxyadenosine (data not shown), was observed upon incubation of the cells in the presence of DHPNO2 as determined by HPLC-MS/MS measurement (32). The results clearly show that 1O2 is able to selectively generate 8-oxodGuo in cellular DNA, in agreement with the known reactivity of 1O2 toward the guanine moiety of isolated DNA.
An experiment was designed to demonstrate that 8-oxodGuo is not produced after cell lysis. For this purpose, cells were incubated in the presence of DHPNO2 at 4 °C. Under these conditions, since the endoperoxide is stable at 4 °C, the production of singlet oxygen during the incubation is very low, and the amount of residual DHPNO2 left is maximum during cell lysis. Under these conditions, no increase in 8-oxodGuo was observed (Fig. 3). This demonstrates that 8-oxodGuo is not formed during cell lysis and DNA isolation. This could be explained, at least partly, by the fact that the first step of the DNA extraction protocol consists in the purification of the nuclei (14), which is accompanied by the elimination of a large amount of the endoperoxide. In addition, sodium azide, a well known singlet oxygen quencher, was added to the different isolation buffers to scavenge singlet oxygen and thus to prevent DNA oxidation due to the presence of the residual generator of 1O2 (DHPNO2). The results (Figs. 1 and 2) show that the release of 1O2 from the thermal decomposition of DHPNO2 is able to induce oxidation to the guanine base of cellular DNA.
However, such an oxidation could result from either the direct reaction
of 1O2 within cellular DNA (Scheme
1) or the oxidative stress induced by the
intracellular production of singlet oxygen. Therefore, a chemical
generator of labeled singlet oxygen DHPN18O2
(30) was used to distinguish between the two possible mechanisms. For
this purpose, the cell suspension was incubated with either DHPNO2 or DHPN18O2, and the amount
of 8-oxodGuo was assessed by using the accurate and highly sensitive
HPLC-MS/MS assay. The latter assay allows the simultaneous
quantification of endogenous 8-oxodGuo, together with [M + 2]-labeled
8-oxodGuo. The latter compound is formed by the reaction of dGuo with
18O-labeled 1O2 generated by the
thermal decomposition of DHPN18O2. When cells
are incubated with DHPNO2 an increase in the level of
8-oxodGuo is observed (Fig. 3). Furthermore, as expected, no [M + 2]
18O-labeled 8-oxodGuo could be detected in either
DHPNO2-treated cells or in the control experiment (Fig. 3).
A non-significant variation in the level of unlabeled 8-oxodGuo and an
important formation of 18O-labeled 8-oxodGuo is measured
when cells were incubated with the labeled endoperoxide. The results,
summarized in Fig. 4, indicate that, as already observed with the
unlabeled endoperoxide, the formation of 8-oxodGuo is almost linear
with the amount of endoperoxide used. Interestingly, taking into
account that the isotopic enrichment of the endoperoxide is about 85%
(30), the result indicates that the formation of 8-oxodGuo observed
when cells are treated with the labeled endoperoxide could be almost
entirely attributed to the direct reaction of singlet oxygen within
nuclear DNA, following the reaction described in Scheme 1.
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An attempt was also made to evaluate if the formation of 8-oxodGuo is mainly due to the intracellular production of singlet oxygen. For this purpose, the excess of endoperoxide was removed by centrifugation after incubation of the cells with the labeled endoperoxide. However, without removing the excess of endoperoxide (Fig. 5) an about 3-fold higher amount of the lesion is measured. This would indicate that 8-oxodGuo could be formed by intracellular as well as extracellular generated singlet oxygen. However, the difference in the formation of 8-oxodGuo could be probably explained by the fact that an equilibrium exists between the intra- and extracellular localization of the endoperoxide. Removing extracellular DHPN18O2 probably results in a decrease of its intracellular concentration that may explain the lower amount of 8-oxodGuo measured. In addition, penetration of the endoperoxide into cells within a 15- min period at 4 °C is probably not complete. Therefore, it may be concluded that the formation of 8-oxodGuo is due almost exclusively to the intracellular formation of 1O2. This is in agreement with the fact that the endoperoxide used has been shown to have predominantly an intracellular localization due to its non-ionic chemical character (27).
A rapid estimation could be made to determine the yield of formation of
the lesion. From data reported in Fig. 4, about two 18O-labeled 8-oxodGuo per 106 DNA bases could
be formed when 30 × 106 cells were treated in 200 µl of PBS buffer with 50 µl of 130 mM
DHPN18O2. Two 8-oxodGuo per 106
bases represent 2 × 6000 lesions per cell (cell having 6 × 109 DNA bases) corresponding to 2 × 6000 × 30 × 106 = 3.6 × 1011
8-oxodGuo formed. Since the concentration of
DHPN18O2 was 130 mM and the volume
used was 50 µl, it may be inferred that 50 × 10
6 × 130 × 103 × 6.02 × 1023 = 3.9 × 1018 molecules of
DHPN18O2 were available. Taking into account
that about half of the oxygen release by the thermal decomposition of
the endoperoxide is in its singlet state (23, 36), this indicates that,
under the conditions used, about 2 × 1018 molecules
of 1O2 were able to give rise to 3.6 × 1011 molecules of 8-oxodGuo. Therefore, 5.4 × 106 molecules of 1O2 are required
for the formation of one 8-oxodGuo. Similar results could be obtained
if the estimation is performed using the results presented in the
different figures. However, the yield of formation is higher when the
final concentration of the endoperoxide is increased. For example, the
amount of 8-oxodGuo in the experiment reported in Fig. 2 is two times
higher than that obtained in the experiment reported in Fig. 1. In the
first experiment 50 µl of DHPNO2 were used to treat
30 × 106 cells in 400 µl of PBS buffer, whereas, in
the second one, the same experiment was carried out but in using only
200 µl of PBS buffer.
The low yield of formation of the lesion highlights the low efficiency for singlet oxygen to oxidize cellular DNA. This might be due, at least partly, to the powerful protection afforded by the cellular defenses to prevent the formation of mutagenic DNA damage. In this respect the scavenging or deactivating properties of carotenoids and polyamines toward singlet oxygen have already been demonstrated (37).
The present results have demonstrated that singlet oxygen is able to
induce oxidation of cellular DNA. Such an oxidation reaction results in
the selective formation of 8-oxodGuo. The use of a pure chemical
generator of 18O-labeled singlet oxygen allows us to
demonstrate that the formation of the lesion is due to the direct
reaction of 1O2 within cellular DNA. These
experiments, however, do not define the origin of the 8-oxodGuo present
in cellular DNA before treatment with DHPNO2, which is
likely to arise from oxygen metabolism.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Laboratoire "Lésions des Acides Nucléiques," DRFMC/SCIB, CEA/Grenoble, F-38054 Grenoble Cedex 9, France. Tel.: 33-4-76-88-49-87; Fax: 33-4-76-88-50-90; E-mail: jcadet@cea.fr.
Published, JBC Papers in Press, September 27, 2000, DOI 10.1074/jbc.M006681200
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ABBREVIATIONS |
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The abbreviations used are: dGuo, 2'-deoxyguanosine; 8-oxodGuo, 8-oxo-7,8-dihydro-2'-deoxyguanosine; DHPN, N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide; DHPNO2, 1,4-endoperoxide of DHPN; DHPN18O2, [18O2]1,4-endoperoxide of DHPNO2; HPLC-MS/MS, high performance liquid chromatography coupled to tandem mass spectrometry.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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F. Coste, M. Ober, T. Carell, S. Boiteux, C. Zelwer, and B. Castaing Structural Basis for the Recognition of the FapydG Lesion (2,6-Diamino-4-hydroxy-5-formamidopyrimidine) by Formamidopyrimidine-DNA Glycosylase J. Biol. Chem., October 15, 2004; 279(42): 44074 - 44083. [Abstract] [Full Text] [PDF]
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M. L. Estevam, O. R. Nascimento, M. S. Baptista, P. Di Mascio, F. M. Prado, A. Faljoni-Alario, M. d. R. Zucchi, and I. L. Nantes Changes in the Spin State and Reactivity of Cytochrome c Induced by Photochemically Generated Singlet Oxygen and Free Radicals J. Biol. Chem., September 17, 2004; 279(38): 39214 - 39222. [Abstract] [Full Text] [PDF]
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 J.-L. Ravanat, T. Douki, P. Duez, E. Gremaud, K. Herbert, T. Hofer, L. Lasserre, C. Saint-Pierre, A. Favier, and J. Cadet Cellular background level of 8-oxo-7,8-dihydro-2'-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up Carcinogenesis, November 1, 2002; 23(11): 1911 - 1918. [Abstract] [Full Text] [PDF]
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Y. Zhang, W.-Y. Ma, A. Kaji, A. M. Bode, and Z. Dong Requirement of ATM in UVA-induced Signaling and Apoptosis J. Biol. Chem., January 25, 2002; 277(5): 3124 - 3131. [Abstract] [Full Text] [PDF]
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