Toxicology

Letters
ELSEVIER Toxicology Letters 88 (1996) 85-90

Sensitive analysis of oxidative DNA damage in mammalian cells:

use of the bacterial Fpg protein in combination with
alkaline unwinding

Andrea Hartwig *, Heike Dally, Regina Schlepegrell

University of Bremen. Depurtment of Biology und Chemistry, Postfuch 330440, D-28334 Bremen, Germany

Abstract
The measurement of oxidative DNA base modifications by different methods has received special attention in
recent years. Here we describe a procedure to quantify DNA lesions recognized by the bacterial formamido-
pyrimidine-DNA glycosylases (Fpg protein). These include 7,8-dihydro-8-oxoguanine (8-hydroxyguanine) as well
as some other forms ‘ofimidazole ring-opened purines, which are converted into abasic sites and subsequently into
DNA single-strand breaks by the associated endonuclease activity. The frequency of DNA strand breaks is
determined by the alkaline unwinding technique. The procedure provides a fast and sensitive tool to assess the
extent of spontaneous as well as induced oxidative DNA damage in mammalian cells.

Keywords: Oxidative DNA damage; 8-Hydroxyguanine; Fpg protein; Alkaline unwinding; Ferric nitrilotriacetate;
Mammalian cells

1. Introduction review see [1,2]). Among these, 7,8-dihydro-8-

oxoguanine (&hydroxyguanine) is one of the
Oxidative stress plays an important role in major DNA base modifications and has attracted
various diseases including cancer and in aging. special attention, since it is mutagenic by causing
Cellular damage is mediated by reactive oxygen G to T transversions [3] and has been suggested
species derived from endogenous and exogenous to play an important role in carcinogenesis [4].
sources, which attack all types of macromolecules Therefore, much effort has been made to measure
including the DNA as perhaps the most critical the extent of 8-hydroxyguanine during the past
target. While neither O,- nor H,O, reacts direc- years and, especially, its determination by high
tly with DNA, in vivo transition metal ions such performance liquid chromatography (HPLC)
as Fe’+ or Cu’+ are believed to catalyse their with electrochemical detection has been widely
conversion into the highly reactive OH . -radical, applied. Furthermore, a broad spectrum of DNA
which in turn provokes a broad spectrum of base modifications including 8-hydroxyguanine
DNA lesions. These include DNA single- and can be quantified by the technique of gas
double-strand breaks, abasic sites, DNA-protein chromatography/mass spectrometry (GC/MS)
cross-links and DNA base modifications (for (for review see [S]).
An alternative procedure to analyse DNA
*Corresponding author. Tel.: +49 421 218 3259; Fax: f49 damaged by reactive oxygen species is the appli-
421 218 7433. cation of purified repair enzymes in combination

037%4274/96/$15.00 $1 1996 Elsevier Ireland Ltd. All rights reserved

PII SO378-4274(96)037:2-S
86 A. Hartwig et al. I Toxicology Letters 88 (19%) 85-90

with assays to detect DNA single strand breaks

(e.g. [a). Here we describe a test system where 1.2 -
the frequency of DNA lesions recognized by the
bacterial formamidopyrimidine-DNA glycosy-
lase (Fpg protein) is quantified by the alkaline
.[ ‘.O
unwinding technique. This enzyme isolated from
E. coli specifically removes 8-hydroxyguanine,
1 0.8 -
the imidazole ring-opened purines 2,6diamino-
4-hydroxy-S-formamidopyrimidine (FapyGua) 5

and 4,6-diamino-5formamidopyrimidine (Fapy- i 0.6 -

Ade), as well as small amounts of 8-hy-
droxyadenine, and the resulting abasic sites are P

converted into DNA single-strand breaks by the 0.4

associated endonuclease activity [7]. Among
the DNA base modifications, 8-hydroxyguanine
exerts the highest affinity and - due to its 0.2

mutagenic potential-is also believed to be the

biologically most relevant substrate for this re- 1
0
pair enzyme [8]. Therefore, the frequency of A B C D
Fpg-sensitive sites provides a valuable tool to Fig. 1. Induction of DNA strand breaks (0) and Fpg-
assess the extent of oxidative DNA damage. sensitive sites Q by methylene blue plusvisible light in PM2
DNA. A, Control; B, DNA modified with methylene blue; C,
DNA modified with methylene blue plus 20 s visible light; D,
DNA modified with methylene blue plus 40 s visible light.
2. Materials and methods Mean values from two independent determinations.

2.1. Cell culture and chemicals

Chinese hamster V79 cells or HeLa cells were sodium acetate, left for 30 min at room tempera-
grown as monolayers in minimal essential me- ture, centrifuged, and the supernatant was dis-
dium (MEM), amodified, or minimal essential carded. Up to this point, all steps were conducted
medium (MEM), respectively, containing 10% in the dark. The DNA was redissolved in buffer
fetal bovine serum, 100 units penicillin/ml and (pH 7.5) containing 50 mM sodium phosphate,
100 pg streptomycin/ml. The cultures were incu- 10 mM EDTA and 100 mM NaCl and the Fpg
bated at 37°C with 5% COZ in air and 100% protein was added where indicated to a final
humidification. concentration of 1 clg/ml for 30 min at 37°C. The
Methylene blue was always prepared freshly reaction was stopped by the addition of 3 ~1 10%
before use and kept in the dark. The preparation sodium dodecylsulphate and the frequency of
of Fe-NTA has been described previously [9]. DNA strand breaks was analysed on a neutral
agarose gel as described previously [lo].
2.2. Substrate speciJicity of the Fpg protein on
PM2 DNA 2.3. Detection of DNA strand breaks and
Fpg-sensitive sites in intact cells
To determine the substrate specificity of the
Fpg protein, PM2 DNA (0.16 pg in 50 mM DNA strand breaks and Fpg-sensitive sites
sodium phosphate buffer, pH 7.4) was treated were determined by a modification of a pro-
with methylene blue (10 pg/ml) and irradiated on cedure described previously [lo]. V79 cells
ice with visible light (60 W, at a distance of 25 (2 x 10’) were seeded and allowed to attach for
cm) for the times indicated in Fig. 1. Subsequent- at least 5 h before treatment with the test chemi-
ly, the DNA was precipitated with ethanol- cals. At the end of treatment, the culture medium
 A. Hurtwig et al. I Toxicology Letters 88 (19%) M-90 87

was removed and a lysis buffer was added con- (O-50 Gy). Subsequently, the lysis and further
taining 0.006 M Na,HPO,, 0.001 M KH,PO,, procedure were carried out as described above,
0.137 M NaCI, 0.003 M KC1 and 0.1% Triton X but omitting the Fpg protein. According to Flihe
100. After 5 min on ice, the solution was sucked and Dikomey [ll], the decrease in double-
off and the cells were treated with a high salt stranded DNA after irradiation with increasing
solution containing 2 M NaCI, 0.01 M EDTA doses of X-rays is described by the formula
and 0.002 M Tris (pH 8.0) for 2 min on ice,
whereafter the cells were left on ice for additional -In (F/F& = c x D,
8 min. The nucleoids were then incubated with
the Fpg protein (1 pg/ml) in enzyme buffer (0.05 where F is the fraction of double-stranded DNA
M sodium phosphate, pH 7.5,O.Ol M EDTA, 100 of irradiated cells, F0 is the fraction of double-
mM NaCI) for 30 min at 37°C. For the detection stranded DNA of unirradiated control cells, c is
of DNA strand breaks, the Fpg protein was the slope of the calibration curve and D is the
omitted. At the end of incubation, an alkaline dose applied in Gy. Based on a value of lo3 DNA
solution was added yielding a final concentration strand breaks per Gy and cell [12-141, the
of 0.07 N NaOH, 0.013 M EDTA, and 0.37 M number of enzyme sensitive sites and/or DNA
NaCI, pH 12.3, and the DNA was allowed to strand breaks per cell induced by the DNA
unwind for 30 min in the dark. The further steps damaging agents was calculated from
of unwinding, neutralization and separation of
N = -ln(F/F,) x 1000
single- and double-stranded DNA were per-
C
formed as describe’d previously [9]. Briefly, the
solution was neutralized with HCl, sonicated and
where Fa is the fraction of double-stranded DNA
SDS was added to a final concentration of 0.05%. in control cells and F the fraction of double-
Separation of single- and double-stranded DNA stranded DNA after the respective treatment.
was performed on 1 ml hydroxyapatite columns
(Calbiochem, high resolution) at 60°C where
single- and double-stranded DNA were eluted 3. Results and discussion
with each 3 ml of 0.15 M and 0.35 M potassium
3. I. Detection of Fpg-sensitive sites in isolated
phosphate buffer, respectively.
DNA
The DNA content of both fractions was deter-
mined by adding Hoechst 33258 dye to a final Prior to the use of intact cultured cells, the
concentration of 7.5 x lo-’ M to 1 ml of each substrate specificity of the Fpg protein has been
sample and measuring the fluorescence with a tested in isolated DNA. We applied supercoiled
spectrophotofluorometer (Aminco-Bowman) at PM2 DNA, which is converted into the closed
an excitation wav’elength of 360 nm and an circular or linear form by the introduction of one
emission wavelength of 450 nm. The fraction of or more DNA strand breaks, respectively. These
double-stranded DNA was calculated as de- three forms were separated on a neutral agarose
scribed in [9]. gel and the frequency of DNA strand breaks was
quantified densitometrically. To assess the sub-
2.4. Quantzjication of Fpg-sensitive sites strate specificity of the Fpg protein, the DNA
was modified with methylene blue plus visible
In order to quantitate the frequency of Fpg- light, which has been shown to yield 8hy-
sensitive sites, the fraction of double-stranded droxyguanine [15] as well as small amounts of
DNA was correlated with the amount of DNA FapyGua [7], presumably due to the generation
strand breaks by calibration with X-rays using a of singlet oxygen. The result is shown in Fig. 1.
200 keV X-ray source at a dose-rate of 4 Gy/min In the case of unmodified DNA, the frequency of
with an additional 0.5 mm copper filter (Sie- DNA strand breaks is only slightly enhanced on
mens). Cells covered with growth medium were the addition of the Fpg protein, indicating a
irradiated on ice with different doses of X-rays small number of Fpg-sensitive sites present in the
88 A. Hurtwig rr ~1.I Toxicology Letters 88 (19%) 85-90

PM2 DNA. The treatment of the DNA with htect cdl8

methylene blue in the absence of visible light

adds no appreciable amount of both types of
Treatment with cbemiul or physicalagents
DNA lesions. However, if the modified DNA is
irradiated with visible light, the frequency of
Fpg-sensitive modifications increases in a dose-
dependent manner, demonstrating the selective
recognition of the induced DNA lesions by the
Fpg protein. The low frequency of DNA strand
breaks as compared to the high number of Fpg-
sensitive sites confirms results obtained by
Schneider et al. [16] and Epe et al. [17]; it
resembles the damage distribution of both types
of lesions induced by singlet oxygen as opposed
to OH. -radicals [17].

3.2. Detection of Fpg-sensitive sites in intact

mammalian cells

To quantify Fpg-sensitive sites in intact mam-

malian cells, we adapted a procedure originally l-l---
established in our laboratory for the detection of UU--[III
UV-induced cyclobutane pyrimidine dimers [lo]. Separation
of single- sod doobiestraaded DNA
The principle of the applied method is shown in (hydroxyqutite cbroma~pby)
Fig. 2. The cells are grown as monolayers and
treated with the respective chemical or physical Fiuorimetic determination of sin&- and doublcstrtied DNA
agent. Afterwards, the cells are gently lysed with
Triton X 100, and subsequently the histones are Coledationof lesion frequency (calibration nitb X-rays)
removed by high salt treatment. This treatment Fig. 2. Flow diagram illustrating the steps involved in the
generates nucleoids, which are still attached to detection of Fpg-sensitive sites in mammalian cells.
the cell culture dishes and where the DNA is
accessible to enzymatic attack due to the de-
pletion of most nuclear proteins. The subsequent In order to quantitate the lesion frequency, the
incubation with the Fpg protein specifically in- whole procedure of cell lysis and alkaline un-
troduces DNA strand breaks at the sites of 8-hy- winding has been calibrated with X-rays as de-
droxyguanine and some other forms of ring- scribed in Materials and methods. As shown in
opened purines, as described above, by the Fig. 3, the extent of double-stranded DNA de-
glycosylase and associated endonuclease activity. creases exponentially with the X-ray dose, and no
These DNA strand breaks are detected and difference is seen between Chinese hamster V79
quantified by the alkaline unwinding method: the and human HeLa cells. Based on a frequency of
DNA is allowed to unwind at pH 12.3 for 30 min lo3 breaks per Gy [12-141, the slope of the
at room temperature in the dark; this is stopped calibration curve is 0.06 for both cell lines on the
by neutralization and sonication. Single- and unwinding conditions applied. According to the
double-stranded DNA are separated on small formulas presented in Materials and methods, it
hydroxyapatite columns, and the respective is now possible to calculate the frequency of
amounts are quantified fluorimetrically by the Fpg-sensitive sites from the amount of double-
addition of the fluorescence dye Hoechst 33258. stranded DNA.
 A. Hartwig et al. I Toxicology Letters 88 (19%) 85-90 89

By applying this procedure, we observed an

background level of about 1000 Fpg-sensitive
sites in V79 Chinese hamster cells and we inves-
tigated whether different complexes of ferric iron
can enhance the extent of oxidative DNA dam-
age in intact cells [18]. As shown in Fig. 4, a
pronounced dose-dependent increase in Fpg-
sensitive sites is observed after 48 h incubation of
V79 Chinese hamster cells with ferric nitrilot-
riacetate (Fe-NTA).

4. Conclusions

0 5 10 15 20 25 The procedure presented in this paper provides

0c-G~)
a sensitive method to detect spontaneous as well
as induced oxidative DNA damage in intact
Fig. 3. Reduction of double-stranded DNA after irradiation
with X-rays, determined by alkaline unwinding. V79 cells (0) mammalian cells. It combines the substrate spe-
or HeLa cells (0) were irradiated with X-rays, doses as cificity of the bacterial Fpg protein with the high
indicated, and the fractions of single- and double-stranded sensitivity of the alkaline unwinding technique to
DNA were determined as described in Materials and detect DNA strand breaks. The lysis, the removal
methods. Mean values from triplicate determinations +I SD.
of the histones and the enzyme incubation take
place directly on the cell culture dishes without
trypsinization of the cells and isolation of the
DNA. This is favourable in several respects: the
number of DNA strand breaks induced by the
handling of the cells is low [lo]. The procedure
is fast and allows the analysis of many samples in
parallel; this might also help to prevent the
unintended induction of oxidative DNA damage
during DNA isolation procedures. Furthermore,
comparatively few cells are needed (about
1 x lo6 cells per sample) and no radioactivity is
required, making the method suitable also for the
detection of oxidative DNA damage in human
lymphocytes.

OJ- A-250
Fe-NTA [phl’j
500
Acknowledgements

The Fpg protein was a kind gift of Dr. Serge

Boiteux, Institut Gustave Roussy, Villejuif,
France. We would like to thank Dr. J. Dahm-
Daphi, University of Hamburg, for his support in
Fig. 4. Frequencies of DNA strand breaks (0) and Fpg-
sensitive sites (W) in V79 cells after incubation with 250 PM
calibrating the alkaline unwinding method with
or 500 PM Fe-NTA for 48 h. Bars indicate mean values from X-rays. This work was supported by the Univer-
triplicate determinations; error bars indicate S.D. (from [18]). sity of Bremen.
90 A. Hartwig et al. I Toxicology Letters 88 (19%) 85-90

References lationship between lipid peroxidation, DNA strand

breaks and sister-chromatid exchanges. Carcinogenesis
[1] Halliwell, B. and Gutteridge, J.M.C. (1990) Role of free 14, 107-112.
radicals and catalytic metal ions in human disease: an Cl01Kasten, U., Beyersmann, D., Dahm-Daphi, J. and Har-
overview. In: L. Packer and A.E. Glazer (Eds.), twig, A. (1995) Sensitive nonradioactive detection of
Methods in Enzymology, Vol. 186. Academic Press, UV-induced cyclobutane pyrimidine dimers in intact
New York, pp. l-85. mammalian cells. Mutat. Res. 336, 143-152.
iI21Teebor, G.W., Boorstein, R.J. and Cadet, J. (1988) The Cl11Fiihe, C. and Dikomey, E. (1994) Induction and repair
repairability of oxidative free radical mediated damage of DNA base damage studied in X-irradiated CHO cells
to DNA: a review. Int. J. Radiat. Biol. 54, 131-150. using the M. luteus extract. Int. J. Radiat. Biol. 66,
697-704.
c31Wood, M.L., Dizdaroglu, M., Gajewski, E. and Essig-
man, J.M. (1990) Mechanistic studies of ionizing radi- Cl21Ahnstrom, G. and Edvardsson, K.A. (1974) Radiation-
ation and oxidative mutagenesis: genetic egects of a induced single-strand breaks in DNA determined by
single I-hydroxyguanine (7-hydro-8-oxoguanine) resi- rate of alkaline strand separation and hydroxyapatite
due inserted at a unique site in a viral genome. Bio- chromatography: an alternative to velocity sedimenta-
chemistry 29,7024-7032. tion. Int. J. Radiat. Biol. 26,493-497.
[13] Goodhead, D.T. (1989) The initial physical damage
c41Floyd, R.A. (1990) The role of 8-hydroxyguanine in
carcinogenesis. Carcinogenesis 11, 1447- 1450. produced by ionizing radiations. Int. J. Radiat. Biol. 56,
623-634.
c51Halliwell, B. and Dizdaroglu, M. (1992) The measure-
ment of oxidative damage to DNA by HPLC and Cl41Roots, R., Holley, W., Chatterjee, A., Irizarry, M. and
GC/MS techniques. Free Rad. Res. Commun. 16, 75- Kraft, G. (1990) The formation of strand breaks in
87. DNA after high-LET irradiation: a comparison of data
from in vitro and cellular systems. Int. J. Radiat. Biol.
WI Epe, B. and Hegler, J. (1994) Oxidative DNA damage:
endonuclease fingerprinting. In: L. Packer (Ed.), 58, 55-69.
Methods in Enzymology, Vol. 234. Academic Press, Cl51Floyd, R.A., West, MS., Eneff, K.L. and Schneider, J.E.
New York, pp. 122-131. (1989) Methylene blue plus light mediates 8-hy-
droxyguanine formation in DNA. Arch. Biochem. Bi-
c71Boiteux, S., Gajewski, E., Lava], J. and Dizdaroglu, M.
(1992) Substrate specificity of the Escherichia co/i Fpg ophys. 273, 106-l 11.
protein (formamidopyrimidine-DNA glycosylase): ex- Cl61Schneider, J.E., Price, S., Maidt, M.L., Gutteridge,
cision of purine lesions in DNA produced by ionizing J.M.C. and Floyd, R.A. (1990) Methylene blue plus light
radiation or photosensitization. Biochemistry 31, 106- mediates 8-hydroxy-2’-deoxyguanosine formation in
110. DNA preferentially over strand breakage. Nucl. Acids
Res. 18, 631-635.
PI Tchou, J., Kasai, H., Shibutani, S., Chung, M.-H., Lava],
J., Grollman, A.P. and Nishimura, S. (1991) 8- Cl71Epe, B., Pflaum, M. and Boiteux S. (1993) DNA damage
Oxoguanine (I-hydroxyguanine) DNA glycosylase and induced by photosensitizers in cellular and cell-free
its substrate specificity. Proc. Natl. Acad. Sci. USA 88, systems. Mutat. Res. 299, 135-145.
4690-4694. Cl81Hartwig, A. and Schlepegrell, R. (1995) Induction of
oxidative DNA damage by ferric iron in mammalian
c91Hartwig, A., Klyszcz-Nasko, H., Schlepegrell, R. and
Beyersmann, D. (1993) Cellular damage by ferric ni- cells. Carcinogenesis, 16, 30093013.
trilotriacetate and ferric citrate in V79 cells: interre-