Optimization of the workup procedure for the analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine with electrochemical detection

Article abstract of DOI:10.1021/tx015573j

The artifactual generation of the biomarker for oxidative stress, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), during the workup procedure for its analysis is a difficult problem to solve, and the responsible factors are unclear. Here, peroxide removal and other antioxidant procedures during workup were compared using a limited amount of rat liver (50 mg) as starting material, with subsequent hydrolysis of 50 μg of DNA. A cold (0 °C) high salt GTC (4 M guanidine thiocyanate) nonphenol DNA extraction method was developed where DNA is quickly isolated. GSH (reduced glutathione) generated artifactual formation of 8-oxodG during the workup procedure, whereas H₂O₂ removal using catalase, Fe³⁺ removal and passivation using desferal, peroxide removal using glutathione peroxidase, ebselen and a peroxidase mimic lowered the 8-oxodG levels, all identifying peroxides as the responsible oxidants. Desferal was more protective when excluding Mg²⁺ and Ca²⁺ from buffers but was found to disturb the electrochemical detector when repeatedly injected five to six times, even at 100 μM. Addition of the OH-scavenger ethanol in all steps at 2% v/v had no protective effect. Zn²⁺ was found necessary for efficient DNA hydrolysis using nuclease P₁, which was poor below 37 °C. Use of water substitutes was tested but inhibited DNA hydrolysis completely. H₂¹⁸O could, however, work for mass spectrometry methods. Long-term (38 days) storage of 0.5% v/v Triton X-100 generated more 8-oxodG than Tween 20 when incubated with free dG. The cold GTC DNA extraction method was used for analysis of freshly isolated human lymphocytes/monocytes from 60 healthy men using catalase and TEMPO as antioxidants, giving a background level of 0.074 ± 0.027 8-oxodG/10⁵ dG (or 16 8-oxodG/10⁸ nucleotides or 1943 8-oxodG/nuclei) which is probably the lowest value obtained yet. No increase with age was seen. Oxidation of dG to 8-oxodG during workup was found to fit a mathematically defined curve, and a calculated background level of 0.047 8-oxodG/10⁵ dG was obtained. To obtain more reliable results it is recommended that control samples are included during the workup procedure, having an equal amount of cells (or DNA) as the exposed samples.

Full text of DOI:10.1021/tx015573j

4
26
Chem. Res. Toxicol. 2002, 15, 426-432
Op tim iza tion of th e Wor k u p P r oced u r e for th e An a lysis
of 8-Oxo-7,8-d ih yd r o-2′-d eoxygu a n osin e w ith
Electr och em ica l Detection
Tim Hofer* and Lennart M o¨ ller
Unit for Analytical Toxicology, Department of Biosciences, Karolinska Institute, 141 57 Huddinge,
Stockholm, Sweden
Received October 16, 2001
The artifactual generation of the biomarker for oxidative stress, 8-oxo-7,8-dihydro-2′-
deoxyguanosine (8-oxodG), during the workup procedure for its analysis is a difficult problem
to solve, and the responsible factors are unclear. Here, peroxide removal and other antioxidant
procedures during workup were compared using a limited amount of rat liver (50 mg) as starting
material, with subsequent hydrolysis of 50 µg of DNA. A cold (0 °C) high salt GTC (4 M
guanidine thiocyanate) nonphenol DNA extraction method was developed where DNA is quickly
isolated. GSH (reduced glutathione) generated artifactual formation of 8-oxodG during the
3
+
2 2
workup procedure, whereas H O removal using catalase, Fe removal and passivation using
desferal, peroxide removal using glutathione peroxidase, ebselen and a peroxidase mimic
lowered the 8-oxodG levels, all identifying peroxides as the responsible oxidants. Desferal was
2
+
2+
more protective when excluding Mg and Ca from buffers but was found to disturb the
electrochemical detector when repeatedly injected five to six times, even at 100 µM. Addition
•
2+
of the OH scavenger ethanol in all steps at 2% v/v had no protective effect. Zn was found
necessary for efficient DNA hydrolysis using nuclease P
water substitutes was tested but inhibited DNA hydrolysis completely. H
1
, which was poor below 37 °C. Use of
1
8
2
O could, however,
work for mass spectrometry methods. Long-term (38 days) storage of 0.5% v/v Triton X-100
generated more 8-oxodG than Tween 20 when incubated with free dG. The cold GTC DNA
extraction method was used for analysis of freshly isolated human lymphocytes/monocytes
from 60 healthy men using catalase and TEMPO as antioxidants, giving a background level of
5
8
0
.074 ( 0.027 8-oxodG/10 dG (or 16 8-oxodG/10 nucleotides or 1943 8-oxodG/nuclei) which is
probably the lowest value obtained yet. No increase with age was seen. Oxidation of dG to
-oxodG during workup was found to fit a mathematically defined curve, and a calculated
8
5
background level of 0.047 8-oxodG/10 dG was obtained. To obtain more reliable results it is
recommended that control samples are included during the workup procedure, having an equal
amount of cells (or DNA) as the exposed samples.
In tr od u ction
Exposure of living cells to various oxidizing agents such
by the temperature, purity of solutions, incubation times,
etc. (5). Small amounts of cells or DNA have been shown
to give higher levels of 8-oxodG while keeping volumes
and concentrations of enzymes constant (6-8), suggesting
that for methodological comparisons the amount of cells
and/or DNA used should be indicated.
1
as peroxides, singlet oxygen ( O ), UV- and γ-irradiation
2
leads to an elevation of 8-oxo-7,8-dihydro-2′-deoxygua-
1
nosine (8-oxodG) in DNA resulting from oxidation of 2′-
deoxyguanosine (dG). However, analyzing the true back-
ground 8-oxodG level in DNA and measuring small
differences in 8-oxodG levels is difficult since dG can be
oxidized during the workup procedure with artifactual
formation of 8-oxodG (1, 2). Coordinated comparisons of
methodologies for analysis of 8-oxodG among mainly
European laboratories are ongoing within ESCODD (3,
).
The workup procedure takes several hours and in-
volves heat-dependent steps, such as the enzymatic
hydrolysis of RNA, proteins, and DNA. The generation
of artifactual 8-oxodG during workup is also influenced
The aim of this study was to develop a cold (0 °C)
workup procedure, which to our knowledge has not yet
been developed, and compare various antioxidant proce-
dures for 8-oxodG analysis with electrochemical detection
from a limited constant amount of cells (50 mg of rat
liver) and DNA (50 µg) using the same set of calibration
standards. It was considered important to evaluate the
effect of peroxide removal, cold DNA extraction, and
4
•
addition of a hydroxyl radical (OH ) scavenger molecule
during the workup procedure for analysis of 8-oxodG. In
addition, using the optimized analytical method, the
background 8-oxodG levels in lymphocytes/monocytes
from healthy men would be measured.
*
To whom correspondence should be addressed.
Abbreviations: 8-oxodG, 8-oxo-7,8-dihydro-2′-deoxyguanosine; dG,
1
2
′-deoxyguanosine; dA, 2′-deoxyadenosine; rA, adenosine; EC, electro-
Exp er im en ta l P r oced u r es
chemical detection; HPLC, high-performance liquid chromotography;
GTC, guanidine thiocyanate; PLG, phase lock gel; TEMPO, 2,2,6,6-
tetramethylpiperidine-N-oxyl; UV, ultraviolet; GSH, glutathione (re-
duced form).
Ma ter ia ls. The chemicals were stated low in transition
metals by the suppliers. Sodium acetate, methanol, zinc chloride,
magnesium chloride, calcium chloride, sodium chloride, glycine,
1
0.1021/tx015573j CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/12/2002

Analysis of 8-Oxo-7,8-dihydro-2′-deoxyguanosine
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 427
glacial acetic acid, ascorbic acid, isoamyl alcohol, chloroform,
dimethyl sulfoxide (DMSO), and 2-propanol were from Merck
Sch em e 1. Cold GTC Meth od
(
Darmstadt, Germany). 8-OxodG, dG, dA, rA, calf thymus DNA,
deferoxamine mesylate (desferal), 2-phenyl-1,2-benzisoselenazol-
[2H]-one (ebselen), glutathione (reduced form), Triton X-100
X-100), RNase A (R-6513), protease (P-6911), catalase (As-
3
(
pergillus niger, C-3515), and glutathione peroxidase (GPx,
G-6137) were from Sigma (St. Louis, MO). Proteinase K and
nuclease P were from Roche Diagnostics (Mannheim, Ger-
1
many). Glycerol and ammonium formate were from BDH (Poole,
England). 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and
ethylene glycol were from Aldrich (Milwaukee, WI). Sucrose was
purchased from J . T. Baker (Deventer, Holland), redistilled
phenol from Eastman Kodak (New Haven, CT), Tris from
Amresco (Solon, OH), guanidine thiocyanate (GTC) from Fluka
and incubated at 37 °C for 30 min (shaken after 15 min). A total
of 14 µL of proteinase K was added (20 mg/mL proteinase K, 2
mM calcium chloride, 20 mM Tris, pH 7.5) and incubated at 37
(Buchs, Switzerland), Tween 20 (20605) from United States
°
C for 45 min (shaken after 15 and 30 min). The solution was
Biochemical (Cleveland, OH), Chelex 100 resin (100-200 mesh,
sodium form) from Bio-Rad Laboratories (Hercules, CA), and
Phase Lock Gel (PLG) tubes (prespun at 13000g for 2 min) from
Eppendorf-Netheler-Hinz (Hamburg, Germany). 3-[4-(N,N-Di-
methylamino)benzenetellurenyl]propanesulfonic acid sodium
salt (“compound x”) was kindly supplied by Lars Engman,
Uppsala University, Sweden. Water was of Milli-Q grade. The
software package MacCurveFit from Kevin Raner Software
transferred to a prespun 2.0 mL PLG (light) tube, and 560 µL
of sevag was added. The tubes were handshaken using a
“multishaker” for 1 min, and proteins/fat were removed at
1
3000g for 5 min. Another 560 µL of sevag was added, and the
tubes were shaken and spun again (13000g for 5 min). After
transferring the upper DNA-containing phase to a new 2.0 mL
tube, 75 µL of sodium chloride (5 M) was added together with
6
35 µL of 2-propanol. After shaking, the DNA was precipitated
(Victoria, Australia) was used for mathematical curve fits.
at -20 °C for 15 min and spun down at 20800g for 10 min.
DNA Extr a ction /Cold Meth od s. 4 M GTC. The crude nuclei
pellets were completely dissolved in 850 µL of cold (0 °C) 4 M
GTC (20 mM Tris, pH 7.5) using a pipet, and the solution was
transferred to a 2.0 mL PLG (heavy) tube (Scheme 1). The tubes
were filled with sevag (≈850 µL), shaken by hand for 1 min and
spun at 13000g to remove proteins/fat. The upper phase was
transferred to a new 2.0 mL tube, and 850 µL of 2-propanol was
added to precipitate DNA at -20 °C for 15 min. DNA was
collected by spinning at 20800g for 10 min.
Density Gradient Nuclei Isolation Followed by 4 M GTC.
OptiPrep (contains 60% w/v iodixanol) from Nycomed (Nycomed
Pharma, Oslo, Norway) was used according to the manufactur-
er’s instructions (application sheet S8), but at 0 °C. Frozen rat
liver was cut into approximately 50 mg pieces and gently
homogenized separately using a 7 mL Dounce glass-glass
homogenizer (pestle A + B) in 4.5 mL of iso-osmotic buffer (0.25
M sucrose, 25 mM potassium chloride, 5 mM magnesium
chloride, 20 mM tricine, pH 7.8). After filtration through a 40
µm Falcon cell strainer (Becton Dickinson, NJ ), crude nuclei
were spun down at 1000g for 10 min. The crude nuclei were
dissolved in 500 µL iso-osmotic buffer and mixed with 500 µL
P r ep a r a tion s. Aqueous solutions were chelex treated by
stirring for 1 h and then filtered through a CN 0.45 µm filter
Nalgene, Rochester, NY) and stored in the dark in plastic
(
bottles at +8 °C. Catalase and alkaline phosphatase were stored
at +8 °C, and the other enzymes were frozen in small aliquots.
Sevag consisted of isoamyl alcohol and chloroform (1:24). All
steps and centrifugations were made at 0 °C if otherwise not
stated. Ebselen was dissolved in 50% v/v ethanol (insoluble in
pure water) and the other antioxidants in water prior to use.
Solutions containing potential peroxide removal agents (cata-
lase, GPx, ebselen, LE 1 and GSH) were preincubated at room
temperature for at least 1 h in the dark immediately before
chilling for 30 min on ice before use. Antioxidants were added
to all solutions except 2-propanol and 70% v/v ethanol. To 4 M
GTC (cold method) was added only TEMPO.
Test of Lon g-Ter m Stor a ge of Deter gen ts. Buffer con-
taining 5 mM magnesium chloride and 20 mM Tris was set to
pH 7.5 with hydrochloric acid and split into three parts. Triton
X-100 and Tween 20 were added to 0.5% v/v, and the solutions
were stored in the dark in plastic bottles at +8 °C for 38 days
and were occasionally aerated. Incubations with 100 µM dG at
3
7 °C for 1.5 h tested for 8-oxodG formation.
5
0% w/v iodixanol and transferred to a 2.0 mL tube. The solution
Hom ogen iza tion . Control male Sprague-Dawley rats (∼275
(25% w/v iodixanol) was underlayered with 665 µL of 30% and
g, corresponding to 49 days of age) were sacrificed, the livers
3
2
35 µL of 35% w/v iodixanol, respectively. Spinning (10000g for
0 min, swing-out rotor) caused the purified nuclei to collect at
(
9-12 g) removed and rinsed with ice-cold PBS, and then cut
into 1 g pieces which were immediately frozen under dry ice
and stored at -80 °C. Twelve samples (two groups of six) were
simultaneously extracted followed by DNA hydrolysis during
each round of workup. Two pieces of liver (320-350 mg) were
cut frozen on aluminum foil, put on ice, and separately homog-
enized in 10 mL of ice-cold homogenization buffer (20 mM Tris,
pH 7.5, 5 mM magnesium chloride) using a 10 mL Potter-
Elvehjem homogenizer with a PTFE pestle. A total of 50.0 mg
was transferred to 6.0 mL tubes. Buffer was added to 4.5 mL,
and the crude nuclei were pelleted at 1000g for 10 min at 0 °C.
After withdrawing all the supernatant (contains membranes,
proteins, mitochondria, and most of the RNA), the crude nuclei
were dissolved in 1 mL of Tween 20 buffer (0.5% v/v Tween 20,
the 30%/35% iodixanol interface; they were then carefully
withdrawn using a pipet. Preparation of the 50%/35%/30%
iodixanol solutions was done with 150 mM potassium chloride,
3
0 mM magnesium chloride, and 120 mM tricine, pH 7.8. The
purified nuclei were further processed using 4 M GTC as above.
TRI REAGENT. TRI REAGENT from Sigma which contains
GTC/acid phenol was used according to the manufacturer’s
instructions but at 0 °C. A total of 320 mg of rat liver (cut frozen)
was immediately homogenized for approximately 10 min in 3.2
mL of TRI REAGENT with a 10 mL Potter-Elvehjem homog-
enizer (PTFE pestle), and 550 µL (50.0 mg of liver) was
transferred to 2.0 mL tubes. A total of 110 µL of chloroform was
added, and the tubes were shaken and put on ice for 10 min.
Spinning at 12000g for 15 min separated the liquid into three
phases. After withdrawal of the top aqueous RNA-containing
phase, 165 µL of ethanol was added to precipitate DNA
2
0 mM Tris, pH 7.5, 5 mM magnesium chloride) with a pipet,
and buffer was added to 4.5 mL. After 5 min on ice, the crude
nuclei/chromatin were pelleted at 1000g (must not be too slow)
for 10 min at 0 °C. After withdrawal of the supernatant this
procedure was repeated once with fresh 4.5 mL of Tween 20
buffer.
(intermediate phase). The tubes were shaken and left on ice for
5
min, and DNA was spun down at 2000g for 5 min. All the
supernatant (containing phenol and chloroform) was carefully
removed, and the DNA was washed for 30 min in 0.1 M sodium
citrate, 10% v/v ethanol. The DNA was spun down (2000g for 5
min) and the supernatant withdrawn.
DNA Extr a ction /Wa r m Meth od . The crude nuclei pellets
were dissolved in 540 µL of RNase A-buffer (100 µg/mL RNase
A, 2 mM calcium chloride, 20 mM Tris, pH 7.5) using a pipet,

4
28 Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Hofer and M o¨ ller
DNA Wa sh a n d Hyd r olysis. The DNA pellets were loosened
from the tube wall and washed in 1.8 mL of 70% v/v ethanol by
vortexing and spun down for 3 min at 20800g. After withdrawal
of all the solution using a pipet, the DNA was dissolved by
pipetting in 60 µL water (2 µL was taken to 118 µL of 10 mM
Tris buffer, pH 7.4, for spectrophotometric concentration deter-
mination at 260 and 280 nm). A total of 50.0 µg of DNA was
hydrolyzed in 100 µL of solution (final concentrations: 10 µg of
nuclease P
acetate, 0.1 mM zinc chloride, pH 5.3) for 60 min at 50 °C in a
.5 mL of prespun PLG (light) tube. A total of 100 µL of sevag
1
, 1 unit of alkaline phosphatase, 25 mM sodium
0
was added, and the tubes were briefly shaken. Proteins were
removed at 13000g for 5 min, and the upper phase was
transferred to a new 0.5 mL tube which was stored at -80 °C.
To test DNA hydrolysis at different temperatures, 50.0 µg of
rat liver DNA (purified by the warm RNase A/proteinase K
method) was hydrolyzed with 25 µg of nuclease P1 and 1 unit
of alkaline phosphatase concurrently in 100 µL at the indicated
temperatures.
F igu r e 1. DNA hydrolysis curves. A total of 50 µg of rat liver
DNA was hydrolyzed concurrently with 25 µg of nuclease P
1
and 1 unit of alkaline phosphatase in 100 µL of 25 mM sodium
acetate, 0.1 mM zinc chloride, pH 5.3, at the indicated temper-
atures. Proteins were removed with sevag using PLG tubes.
Hydrolysis overnight (21 h) at +4.0 °C and 0.0 °C gave 10.8 (
Lym p h ocyte/Mon ocyte An a lysis. Blood from 60 healthy
men (average age: 52 years) was collected in two 8 mL sodium
heparin Vacutainer CPT tubes (Becton Dickinson, Franklin
Lakes, NJ ) which were spun at 1500g for 15 min at room
temperature. The lymphocytes/monocytes were withdrawn,
pooled into a 50 mL tube which was filled with ice-cold PBS,
and spun down at 300g for 15 min at 0 °C. The cells were then
washed twice by dissolving them in 50 mL of PBS with a pipet
and spun down. After storage at -80 °C, approximately 20
samples were processed during each round of workup and
analyzed the following day. Crude nuclei were prepared by
pipetting in 1.8 mL of 0.5% v/v fresh Triton X-100 (20 mM Tris,
pH 7.5, 5 mM magnesium chloride), leaving them for 5 min on
ice and spinning the crude nuclei down at 1000g for 10 min.
The supernatant was discarded and this procedure was re-
peated. DNA was extracted using 4 M GTC as described. After
the PBS step, all the solutions contained 1 mM TEMPO and 50
units/mL catalase as antioxidants.
0
.1 and 7.3 ( 0.0 nmol, respectively. Duplicate analysis.
50-100 µg of DNA) were 11 and 16 min, respectively. Calibra-
tion curves for dG and 8-oxodG were made by injection three
times of each standard (100 µL, different concentrations).
Preparation of dG and 8-oxodG standards was done by three
separate weighings each before separate dilution. Correction
was made for both the purity and water content using the
following equation: true quantity ) weighed amount × purity
× (1 - water content). After use, the EC-detector was switched
off and the buffer recirculated at 0.05 mL/min. Overnight HPLC-
column washing (0.08 mL/min) using pure methanol (having
the EC-detector disconnected), followed by 20 min HPLC-buffer
reequilibration using fresh buffer at 0.5 mL/min before recon-
necting the EC-detector, resulted in a lowering of the baseline
current from 5-15 nA to 0.7-3.0 nA and fewer baseline
fluctuations.
Ch eck for RNA Con ta m in a tion Usin g HP LC/UV. On a
separate HPLC system, nucleosides from 5 µg of DNA were
separated on a Genesis reversed-phase column (250 × 4.6 mm
i.d., 4 µm) from J ones Chromatography (Hengoed, U.K.) using
a linear gradient at 0.7 mL/min (Solvent Module 126, Beckman
System Gold, Beckman Instruments, Fullerton, CA) changing
from 98% v/v 50 mM ammonium formate, pH 4.6: 2% v/v
methanol to 60% v/v 50 mM ammonium formate, pH 4.6: 40%
v/v methanol over 30 min. rA and dA were measured at 254
nm (Diode Array Detector 168, Beckman System Gold) giving
sharp separated peaks with retention times of 22.3 and 23.3
min, respectively.
Lym p h ocyt e/Mon ocyt e An a lysis (exist in g p r ot ea se/
TEMP O m eth od ). After PBS washing and crude nuclei prepa-
ration by pipetting in 0.5% v/v Triton X-100 buffer, the warm
RNase A/protease method using phenol/sevag was used as
previously described (2), however, with fewer cells and less
extensive DNA solubilization. A total of 20 mL of blood was
collected from 15 healthy humans in two 10 mL sodium heparin
VENOJ ECT tubes (Terumo Europe, Leuven, Belgium). The
blood was diluted in PBS, layered onto Ficoll-Paque (Amersham
Pharmacia Biotech, Uppsala, Sweden), and spun at 400g for 30
min according to the manufacturer’s instructions. After collect-
ing the lymphocyte/monocyte layer, the cells were washed in
PBS twice, split into three aliquots (each from 6.6 mL of blood)
and processed as described (2). TEMPO (100 µM) was added to
all solutions after the PBS wash steps.
Test of Wa ter Su bstitu tes. DNA pellet solubilization of 50
µg of calf thymus DNA in 100 µL of pure methanol, ethanol,
ethyleneglycol, glycerol, and DMSO, all set to pH 4.5-6.0 with
glacial acetic acid, was attempted by extensive pipetting. Also,
HP LC/EC/UV An a lysis. The HPLC system consisted of a
zirconium mobile-phase filter (Elsico Labs, Moscow, Russia), an
isocratic Scantec 650 pump (Scantec, Partille, Sweden) set at
1
DNA hydrolysis using nuclease P and alkaline phosphatase was
attempted after dissolving the DNA in aqueous hydrolysis buffer
and adding each water substitute to 50% v/v, respectively. After
attempted hydrolysis (50 °C, 1.5 h) the solutions were ultrafil-
trated (UFC3IPH00, Millipore, Bedford, MA) at 12000g for 15
min to remove enzymes.
0
.8 mL/min with an extra PEEK pulse damper (Scientific
Instruments, Inc., State College, PA), an injector (7725i, Rheo-
dyne, Cotati, CA) with a 200 µL PEEK loop, a 1 mm (C-18) Opti-
Guard column (Optimize, Portland, OR), and two Delta-Pak
(
150 × 3.9 mm id, 5 µm) reversed-phase columns (Waters,
Milford, MA). 8-OxodG was detected with an electrochemical
detector (Coulochem II, ESA, Chelmsford, MA) with a graphite
filter protected 5011 analytical cell (ESA, screen electrode: +200
mV, analytical electrode: +350 mV), and dG was measured
with a 486 UV detector (Waters) set at 290 nm. Plastic and
PEEK tubing was used throughout. The HPLC buffer consisted
of 10% v/v methanol, water of Milli-Q grade (Millipore), 20 mM
sodium acetate set to pH 5.3 with acetic acid and was filtered
through a CN 0.2 µm filter from (Nalgene). A total of 100 µL
Resu lts
DNA Hyd r olysis. The tested temperature dependence
for efficient DNA hydrolysis (Figure 1) using nuclease
P
1
and alkaline phosphatase concurrently shows that
DNA hydrolysis was very poor below 37 °C, consequently
0 °C for 60 min was selected for further experiments.
5
2
+
Removal of Zn from the hydrolysis buffer resulted in a
4% drop in hydrolysis efficiency, so it was retained.
Lowering the nuclease P amount from 25 to 10 µg had
no major effect on DNA (50 µg) hydrolysis, and 10 µg was
7
(
tubes were handthawed immediately prior to injection) was
injected, and the retention times of dG and 8-oxodG (detection
limit approximately 5 fmol for pure standard and 20 fmol for
1

Analysis of 8-Oxo-7,8-dihydro-2′-deoxyguanosine
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 429
F igu r e 2. Test for 8-oxodG formation by incubation of 100 µM
dG in long-term (38 days, +8 °C, dark, occasionally aerated)
stored Triton X-100 and Tween 20 solutions at 0.5% v/v in 5
mM magnesium chloride and 20 mM Tris, pH 7.5, at 37 °C for
1
.5 h. Triplicate incubation.
F igu r e 3. Analysis of Sprague-Dawley rat liver with different
workup conditions. Each bar represents five to six separately
processed samples and analyses from 50 mg of rat liver, each
with hydrolysis of 50 µg of DNA. Data are shown as mean (
SD.
Ta ble 1. RNA Con ta m in a tion a n d DNA Yield for th e
Tested Meth od s
method
rA/dA (%)
µg of DNA/mg of livera
RNase A/proteinase
GTC (0 °C workup)
Optiprep + GTC (0 °C) 10.5 ( 1.5
TRI REAGENT (0 °C)
0.56 ( 0.07
17.7 ( 0.90
1.71 ( 0.01
1.57 ( 0.11
1.07 ( 0.12
1.63 ( 0.12
µM desferal during DNA hydrolysis resulted in immedi-
ate EC baseline disturbances. For 100 µM, severe dis-
turbance was noticed after 5-6 injections with difficulties
in quantifying the 8-oxodG peak. Preincubation of solu-
tions with catalase at 50 units/mL lowered the 8-oxodG
levels (0.26 ( 0.031 8-oxodG/105 dG) compared to no
preincubation (0.33 ( 0.027) by direct comparison using
the warm method. Catalase at 50 units/mL gave lower
53.5 ( 1.3
a
RNA has been subtracted. Triplicate DNA extraction from 50
mg of rat liver.
also sufficient for >100 µg of DNA (Figure 5, panel B2).
DNA pellet solubilization by extensive pipetting in pure
methanol, ethanol, ethyleneglycol, glycerol, and DMSO
was difficult, and DNA hydrolysis in all these water
substitutes at 50% v/v resulted in no detectable dG peaks
by HPLC/UV.
Deter gen t Tests. After the 38 days of storage, Triton
X-100 was more oxidizing than Tween 20 when incubated
with free dG (Figure 2). Catalase did not inhibit the
5
levels than 10 units/mL (0.32 ( 0.023 8-oxodG/10 dG)
5
or 200 units/mL (0.34 ( 0.045 8-oxodG/10 dG). Inclusion
•
of the OH scavenger ethanol at 2% v/v (0.34 M) in all
the solutions had no significant effect on the 8-oxodG
levels.
An a lysis of Hu m a n Lym p h ocytes/Mon ocytes. Fig-
ure 5, panel A1, shows the 8-oxodG levels in lymphocytes/
monocytes from 15 healthy humans using the existing
protease/TEMPO method, with an average ratio of 1.03
8
-oxodG formation caused by the detergents. During
workup, use of either 0.5% v/v Triton X-100 or 0.5% v/v
Tween 20 gave equally pure DNA.
5
( 0.47 8-oxodG/10 dG (triplicate analysis each from 6.6
Ra t Liver DNA Extr a ction Meth od s. The yield of
DNA using Optiprep density gradient nuclei isolation
followed by 4 M GTC was considerably lower than with
the warm and the cold GTC methods (Table 1) and was
not further used. Of the cold DNA-extraction methods
tested, 4 M GTC worked best and also worked for rat
brain and rat spinal cord (data not shown). RNA at these
low concentrations was not found to disturb the detection
of 8-oxodG or dG by HPLC/EC/UV (8-oxodG has a specific
retention time and a specific oxidation potential). The
DNA pellet from the warm RNase A/proteinase K workup
mL blood). Figure 5, panel A2, shows the data in Figure
5, panel A1, plotted against the amount of dG (nmole)
detected. In all cases, high 8-oxodG/10 dG ratios were
5
correlated with a low amount of dG (nmole). The con-
centration-dependence data fitted the curve y ) a + b/x.
If it is assumed that a relatively constant amount of
8-oxodG forms during the workup procedure then the
5
following can be assigned: y ) (8-oxodG/10 dG)measured,
5
a ) (8-oxodG/10 dG)background, b ) 8-oxodGworkup, and x )
dGmeasured. This gives the average amount of 8-oxodG
generated by the workup to 20 fmol () b) for the existing
protease/TEMPO method.
2
procedure dissolved within a minute by pipetting in H O.
For the cold GTC method, more extensive pipetting (5
min) was required, but the DNA was equally well
hydrolyzed as detected by HPLC/UV. The PLG tubes
made the upper phase withdrawal easy and very fast and
increased the amount of liquid that could be withdrawn.
Effect of An tioxid a n t P r oced u r es. The cold GTC
The average background level in lymphocytes/mono-
cytes from 60 healthy men was 0.074 ( 0.027 8-oxodG/
5
8
10 dG (or 16 8-oxodG/10 nucleotides or 1943 8-oxodG/
cell nucleus, calculating with 22% dG in DNA and 12 ×
9
10 nucleotides/diploid genome) using the cold GTC
method starting from 16 mL of blood with hydrolysis
of all the extracted DNA, and TEMPO/catalase as anti-
oxidants (the average 8-oxodG value for each individual
was used from 2 to 4 separate workup procedures and
analyses; total number of analyses: 214). No increase
in 8-oxodG levels with age was observed (Figure 5,
5
method gave lower 8-oxodG levels (0.27 8-oxodG/10 dG)
than the warm RNase A/proteinase K method (0.36
5
8
-oxodG/10 dG) not using antioxidants (Figure 3). Inclu-
sion of the tested antioxidants during the workup pro-
cedure had no significant effect on DNA yield, purity, or
DNA hydrolysis efficiency. Use of 1 mM desferal during
the initial DNA extraction steps significantly reduced the
5
panel B1). Plotting the 8-oxodG/10 dG values from all
the points of analysis (n ) 214) against the dG amount
measured by HPLC/UV (Figure 5, panel B2) and
using the curve fit method (y ) a + b/x) gave 0.047
artifactual 8-oxodG formation when Ca2 and Mg were
+
2+
omitted (Figure 3). However, the use of 1 mM and 200

4
30 Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Hofer and M o¨ ller
8
(
-oxodG/105 dG () a) where the curve flattened out
or 10 8-oxodG/108 nucleotides or 1238 8-oxodG/cell
nucleus) and 8-oxodGworkup ) 8.4 fmol () b). This method
gave 84.5 ( 32.8 µg of DNA (RNA subtracted), with an
rA/dA ratio of 3.0 ( 0.82% from 16 mL of blood using
Vacutainer CPT tubes from 10 individuals.
F igu r e 4. Structure of 3-[4-(N,N-dimethylamino)benzene-
tellurenyl]propanesulfonic acid sodium salt called “compound
x” in the text to avoid acronyms.
Discu ssion
Ta ble 2. Mea su r ed Con tr ol Levels in F r esh ly Isola ted
Hu m a n Lym p h ocytes/Mon ocytes
DNA hydrolysis was very poor below 37 °C using
5
nuclease P
1
(Figure 1). An extensive hydrolysis of DNA
method
8-oxodG/10 dG
(SD
ref
at low temperatures could be tested for using other
enzymes, but is likely to require a long time and may
not therefore lower the 8-oxodG levels.
Long-term storage of the commonly used Triton X-100
was more oxidizing than Tween 20 when incubated with
free dG for 1.5 h at 37 °C, although relatively weakly
HPLC/EC
HPLC/EC
HPLC/EC
HPLC/EC
HPLC/EC
HPLC/EC
1
(0.35
(0.10a
(0.18
(0.8
17
18
19
20
21
0.52a
2.77
6.8
0.17
0.074
(0.1
(0.027
this study
oxygen removal during DNA extraction:
8
-oxodG generating (Figure 2). Catalase did not inhibit
HPLC/EC
HPLC/EC
HPLC/EC
1.157
0.188
0.237
(0.0414
(0.126
(0.121
22
23
24
the detergent generated 8-oxodG formation (Figure 2).
Polyether detergents are peroxidized both in the pure
state and in aqueous solution and contain hydroperoxides
a
Calculated from Table 1 in this reference.
(9). Triton X-100 has been found to be considerably more
oxidizing that Tween 20 (10).
The switching from protease to proteinase K during
the warm method and chelex treatment of solutions
solutions before use. The increase in 8-oxodG when using
GSH indicate the presence of peroxides being reduced in
the buffers, but should not be seen as a proof of OH , as
•
5
resulted in low background 8-oxodG/10 dG levels (Figure
two-electron reductions of peroxides may also occur (11).
The alkyl aryl telluride “compound x” (Figure 4) has been
shown to have thiol peroxidase activity (12). The use of
GPx during routine workup is not realistic due to its very
high cost even at 1 unit/mL. Figure 5, panels A1 and A2,
shows that the inclusion of 100 µM TEMPO in the
solutions did not eliminate the artifactual 8-oxodG
formation. Also, the free radical TEMPO increased the
8-oxodG levels when incubated with proteinase K (Figure
3), but not when using the high salt method. TEMPO
has previously been found to be protective against
8-oxodG formation (2, 11). The measured background of
3
), even when using a limited amount of tissue and DNA.
With no antioxidant added, the cold GTC method reduced
the 8-oxodG levels compared to the warm RNase A/pro-
teinase K method. The cold GTC method has the poten-
tial to work below 0 °C with the addition of glycerol. The
use of TRI REAGENT to extract DNA was the fastest
method (no isolation of crude nuclei pellets) but resulted
in considerable RNA contamination (Table 1) and el-
evated 8-oxodG levels (Figure 3). With this method it is
difficult to remove all the phenol from the DNA, and it
may be more suitable for RNA extraction. Although the
initial crude nuclei preparation steps removed most of
the RNA from the RNA-rich liver sample (Table 1), the
cold GTC method (Scheme 1) could, although not neces-
sary for HPLC/EC/UV measurement of 8-oxodG, be
further optimized to remove more protein/RNA (facili-
tates DNA solubilization) by quick filtration of the DNA
containing GTC solution using Micropure-EZ filters
5
∼0.25 8-oxodG/10 dG from 50 mg of rat liver with
hydrolysis of 50 µg of DNA (Figure 3) is slightly lower
than a recently reported background level of 0.34 ( 0.06
8-oxodG/105 dG from 100 mg of rat liver (13), using
desferal as antioxidant and the commonly used chao-
tropic method (14, 15).
Switching to a solvent other than H O during the
2
(
(
Millipore), or using spin columns separating on size
such as Sigma S-3420 columns), selective DNA binding,
workup procedure could, based on knowledge on how
8-oxodG forms (11), be successful. However, as 50% v/v
methanol, ethanol, DMSO, etc., were already found to
inhibit DNA hydrolysis and as DNA was difficult to
dissolve in these solvents, water substitutes are unlikely
to work. Although expensive, isotopically labeled water
use of spin columns separating on size (such as Sigma
S-3420 columns) or selective DNA binding, acid phenol
and selective RNA precipitation using lithium chloride
or ammonium acetate after GTC removal. For GC/MS
where DNA is hydrolyzed into free base and sugar
constituents, a nearly complete removal of RNA is
required as the same bases are present in RNA.
1
8
17
H2 O or H2 O (both nonradioactive) could be an inter-
esting choice for groups analyzing 8-oxodG (or 8-oxoG)
by mass spectrometry methods such as LC/MS and GC/
The lowering in 8-oxodG levels using desferal suggests
that free catalytic iron is responsible for a major part of
the oxidation of 2′-deoxyguanosine during the workup
procedure. However, the necessity of Zn2 for effective
DNA hydrolysis and the EC baseline noise disturbances
MS. After removal of dissolved O
2
, any artifactual oxida-
•+
tion of dG (by formation of dG with subsequent hydrox-
1
8
18
•
ylation in H
2
O, or OH attack (11) would be expected
+
18
16
to give 8-( O)oxodG (instead of 8-( O)oxodG), which,
having the mass M + 2, can be separated from 8-oxodG
using mass spectrometry. The initial cytoplasmic H
could be removed by dilution or freeze-drying. Using D
[similar to those observed by H. J . Helbock, et al. (7)]
2
O
O
suggests that desferal should be excluded from the DNA
hydrolysis step for good EC detection. The use of catalase,
TEMPO, or combinations thereof, can be a good alterna-
tive to desferal during DNA hydrolysis to avoid this
problem. The lowering in 8-oxodG levels using preincu-
bation of solutions with catalase compared to no prein-
2
2
instead of H O would not solve the problem as the protons
in -OH, -SH, and, -NH groups are interchangeable
with the solvent (16) [8-oxodG (keto form) is inter-
convertible with its tautomer 8-OH-dG (enol form)].
For the lymphocyte/monocyte samples, TEMPO/cata-
lase was chosen as antioxidant due to possible EC-
2 2
cubation, gave evidence for the existence of H O in the

Analysis of 8-Oxo-7,8-dihydro-2′-deoxyguanosine
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 431
F igu r e 5. Freshly isolated human lymphocyte/monocyte background 8-oxodG levels by two different methods. (A1) DNA extraction
5
from 6.6 mL of blood (triplicate) using the exising protease/TEMPO method (1.03 ( 0.47 8-oxodG/10 dG, n ) 15 individuals). (A2)
The data in panel A1 plotted against the measured amount of dG. The data could be fitted to the equation y ) a + b/x. (B1) DNA
extraction from 16 mL of male blood with hydrolysis of all the extracted DNA using the cold GTC method with TEMPO and catalase
5
as antioxidats (average 0.074 ( 0.027 8-oxodG/10 dG). No increase with age was seen. Each point (n ) 6.0) is the average of two to
four analyses each from 16 mL of blood. (B2) Plot of the 8-oxodG values from all the analysis points (n ) 214, each from 16 mL of
5
blood) against the dG amount measured by HPLC/UV. Use of the curve fit (y ) a + b/x) gave 0.047 8-oxodG/10 dG () a) where the
curve flattened out.
detector disturbances using desferal (1, 7). The average
background level of 0.074 (0.027 8-oxodG/105 dG in
human lymphocytes/monocytes is lower than previously
reported levels (see Table 2). No indication of further
oxidation of 8-oxodG into other species during the workup
procedure was found. The absence of increased 8-oxodG/
dG levels with age in lymphocytes/monocytes (Figure 5,
panel B1) may be due to a large fraction of young cells.
In conclusion, the results give evidence that peroxides,
with H specifically proven, are involved in the workup
formation of 8-oxodG, and that the background 8-oxodG
level in lymphocytes/monocytes from healthy men is
lower than previously reported.
2
O
2
Ack n ow led gm en t. The authors thank Prof. Lars
Engman (Uppsala University) for the kindly supplied
“compound x”.
The concentration dependence in Figure 5, panels A2
and B2, means, mathematically, that at higher dG
concentrations the 8-oxodG formed during workup will
Refer en ces
5
be “diluted out” and the level of 8-oxodG/10 dG measured
will approach the true background level. The difference
in dG content is likely due to the large variance in
lymphocyte/monocyte concentration among individuals.
Comparison of curves for control and exposed samples
(1) Helbock, H. J ., Beckman, K. B., Shigenaga, M. K., Walter, P. B.,
Woodall, A. A., Yeo, H. C., and Ames, B. N. (1998) DNA oxidation
matters: the HPLC-electrochemical detection assay of 8-oxo-
deoxyguanosine and 8-oxo-guanine. Proc. Natl. Acad. Sci. U.S.A.
9
5, 288-293.
could in many cases be better than comparison of
(2) Hofer, T., and M o¨ ller, L. (1998) Reduction of oxidation during the
preparation of DNA and analysis of 8-hydroxy-2′-deoxyguanosine.
Chem. Res. Toxicol. 11, 882-887.
5
8
-oxodG/10 dG ratios. In a perfect method, no 8-oxodG
will be formed during the workup, and the curve y ) a +
b/x should become a straight line: y ) a when b ) 0,
starting from a common source of material. Simultaneous
workup and analysis of exposed and control tissue in
equal amounts is recommended to reduce the influence
of artifactual 8-oxodG formation (storage of cells/tissue
at -80 °C has not been found to give 8-oxodG formation).
(
3) Lunec, J . (1998) ESCODD: European Standards Committee on
Oxidative DNA Damage. Free Radical Res. 29, 601-608.
4) ESCODD (2000) Comparison of different methods of measuring
8-oxoguanine as a marker of oxidative DNA damage. Free Radical
Res. 32, 333-341.
(
(
5) M o¨ ller, L., Hofer, T., and Zeisig, M. (1998) Methodological
considerations and factors affecting 8-hydroxy-2′-deoxyguanosine
analysis. Free Radical Res. 29, 511-524.
The recently developed "Fpg enzyme-HPLC/EC" assay
25) for 8-oxoG analysis gives cleaner chromatograms but
requires the complete removal of RNA, a 100% dissolu-
tion of DNA to access all the 8-oxodG sites and may suffer
from variability in enzyme activity.
(6) Beckman, K. B., and Ames, B. N. (1996) Detection and quantifica-
tion of oxidative adducts of mitochondrial DNA. Methods Enzymol.
(
2
64, 442-453.
(
7) Helbock, H. J ., Beckman, K. B., and Ames, B. N. (1999) 8-Hy-
droxydeoxyguanosine and 8-hydroxyguanine as biomarkers of
oxidative DNA damage. Methods Enzymol. 300, 156-166.

4
32 Chem. Res. Toxicol., Vol. 15, No. 3, 2002
Hofer and M o¨ ller
(
8) Zeisig, M., Hofer, T., Cadet, J ., and M o¨ ller, L. (1999) 32P-
oxidative damage during isolation. Free Radical Biol. Med. 20,
93-98.
(18) Collins, A. R., Gedik, C. M., Olmedilla, B., Southon, S., and
Bellizzi, M. (1998) Oxidative DNA damage measured in human
lymphocytes: large differences between sexes and between
countries, and correlation with heart disease mortality rates.
FASEB J . 12, 1397-1400.
(19) Mecocci, P., Polidori, M. C., Ingegni, T., Cherubini, A., Chionne,
F., Cecchetti, R., and Senin, U. (1998) Oxidative damage to DNA
in lymphocytes from AD patients. Neurology 51, 1014-1017.
(20) Bashir, S., Harris, G., Denman, M. A., Blake, D. R., and Winyard,
P. G. (1993) Oxidative DNA damage and cellular sensitivity to
oxidative stress in human autoimmune diseases. Ann. Rheum.
Dis. 52, 659-666.
(21) Collins, A. R., Dusinska, M., Gedik, C. M., and Stetina, R. (1996)
Oxidative damage to DNA: do we have a reliable biomarker.
Environ. Health Perspect. 104, 465-469.
(22) Takeuchi, T., Nakajima, M., Ohta, Y., Mure, K., Takeshita, T.,
and Morimoto, K. (1994) Evaluation of 8-hydroxydeoxyguanosine,
a typical oxidative DNA damage, in human leukocytes. Carcino-
genesis 15, 1519-1523.
(23) Nakajima, M., Takeuchi, T., and Morimoto, M. (1996) Determi-
nation of 8-hydroxydeoxyguanosine in human cells under oxygen-
free conditions. Carcinogenesis 17, 787-791.
(24) Nakajima, M., Takeuchi, T., Takeshita, T., and Morimoto, K.
(1996) 8-Hydroxydeoxyguanosine in human leukocyte DNA and
daily health practice factors: effects of individual alcohol sensitiv-
ity. Environ. Health Perspect. 104, 1336-1338.
(25) Beckman, K. B., Saljoughi, S., Mashiyama, S. T., and Ames, B.
N. (2000) A simpler, more robust method for the analysis of
8-oxoguanine in DNA. Free Radical Biol. Med. 29, 357-367.
3
2
postlabeling high-performance liquid chromatography ( P-HPLC)
adapted for analysis of 8-hydroxy-2′-deoxyguanosine. Carcino-
genesis 20, 1241-1245.
(
9) Lever, M. (1977) Peroxides in detergents as interfering factors
in biochemical analysis. Anal. Biochem. 83, 274-284.
10) Ashani, Y., and Catravas, G. N. (1980) Highly reactive impurities
in Triton X-100 and Brij 35: partial characterization and removal.
Anal. Biochem. 109, 55-62.
(
(
11) Hofer, T. (2001) Oxidation of 2′-deoxyguanosine by H
2
O
2
-ascor-
bate: evidence against free OH and thermodynamic support for
two-electron reduction of H . J . Chem. Soc., Perkin Trans. 2,
10-213.
•
2
O
2
2
(
12) Kanda, T., Engman, L., Cotgreave, I. A., and Powis, G. (1999)
Novel water-soluble diorganyl tellurides with thiol peroxidase and
antioxidant activity. J . Org. Chem. 64, 8161-8169.
13) Ramon, O., Wong, H.-K., J oyeux, M., Riondel, J ., Halimi, S.,
Ravanat, J .-L., Favier, A., Cadet, J ., and Faure, P. (2001)
(
2
′-Deoxyguanosine oxidation is associated with decrease in the
DNA-binding activity of the transcription factor SP1 in liver and
kidney from diabetic and insulin-resistant rats. Free Radical Biol.
Med. 30, 107-118.
(
14) Cadet, J ., Douki, T., Pouget, J .-P., and Ravanat, J .-L. (2000)
Singlet oxygen DNA damage products: formation and measure-
ment. Methods Enzymol. 319, 143-153.
15) Wang, L., Hirayasu, K., Ishizawa, M., and Kobayashi, Y. (1994)
Purification of genomic DNA from human whole blood by isopro-
panol-fractionation with concentrated NaI and SDS. Nucleic Acids
Res. 22, 1774-1775.
16) Friebolin, H., Ed. (1991) Basic one- and two-dimensional NMR
spectroscopy, VCH Verlagsgesellschaft mbH, Weinheim, Germany.
17) Finnegan, M. T. V., Herbert, K. E., Evans, M. D., Griffiths, H.
R., and Lunec, J . (1996) Evidence for sensitation of DNA to
(
(
(
TX015573J