Toxicology Letters 184 (2009) 33–37
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Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet
Excretion of urinary N7 guanine and N3 adenine DNA adducts in mice after
inhalation of styrene
Petr Mikeš a , Marek Kořínek a , Igor Linhart b,∗ , Jan Krouželka b , Emil Frantík c ,
L’udmila Vodičková c , Lenka Neufussová c
a
b
c
RE&D VUFB, Poděbradská 186, CZ-180 66 Prague, Czech Republic
Institute of Chemical Technology, Prague, Department of Organic Chemistry, Technická 5, CZ-166 28 Prague, Czech Republic
National Institute of Public Health, Šrobárova 48, CZ-100 42 Prague, Czech Republic
a r t i c l e
i n f o
Article history:
Received 9 September 2008
Received in revised form 15 October 2008
Accepted 16 October 2008
Available online 25 October 2008
Keywords:
3-Alkyladenines
7-Alkylguanines
Urinary DNA adducts
Styrene
Biomarkers of effective dose
a b s t r a c t
New urinary adenine adducts, 3-(2-hydroxy-1-phenylethyl)adenine (N3␣A), 3-(2-hydroxy-2phenylethyl)adenine (N3␤A), were found in the urine of mice exposed to styrene vapour. These styrene
7,8-oxide derived adenine adducts as well as previously identified guanine adducts, 7-(2-hydroxy1-phenylethyl)guanine (N7␣G) and 7-(2-hydroxy-2-phenylethyl)guanine (N7␤G) were quantified
by HPLC–ESI–MS2 and the excretion profile during and after a repeated exposure to 600 mg/m3 or
1200 mg/m3 of styrene for 10 consecutive days (6 h/day) was determined. The excretion was dose
dependent. Total N3 adenine adducts (N3␣A + N3␤A) excreted amounted to nearly 0.8 × 10−5 % of the
absorbed dose while urinary N7 guanine adducts (N7␣G + N7␤G) amounted to nearly 1.4 × 10−5 % of
the dose. No accumulation of the adducts was observed. Due to rapid depurination from the DNA, the
excretion of both N3 adenine and N7 guanine adducts ceased shortly after finishing the exposure. Both
N3 adenine and N7 guanine adducts may be used as non-invasive biomarkers of effective dose reflecting
only a short time exposure to styrene.
© 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Electrophilic compounds are bound to nucleophilic sites in the
DNA yielding DNA adducts. Some of these adducts are cleaved off
the DNA molecule and excreted in urine indicating a damage to
DNA. Positions N7 of guanine and N3 of adenine represent major
sites of the attack by DNA alkylating agents (Shuker and Farmer,
1992; Koskinen and Plna, 2000). An alkylation at these sites results
in a positively charged imidazole ring of the purine making the
N-glycosidic bond more labile and prone to hydrolysis, so that
both N7 guanine and N3 adenine adducts depurinate easily yielding 7-alkylguanines and 3-alkyladenines, respectively, which are
then excreted in urine (Mueller and Risenbrand, 1985; Margison
et al., 1973; Fujii et al., 1980). Therefore, both N7 guanine and N3
adenine adducts are useful non-invasive biomarkers of both exposure and response to mutagenic and carcinogenic agents (Shuker
and Farmer, 1992; Timbrell, 1998) reflecting spontaneous depurination and base excision repair of specific DNA adducts. Hitherto
the most versatile method for the detection and quantification of
∗ Corresponding author. Fax: +420 220 443 288.
E-mail address: [email protected] (I. Linhart).
0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2008.10.010
DNA adducts is that of 32 P-postlabeling (Hemminki et al., 2000).
However, this method cannot be used for the analysis of urinary
nucleobase adducts. Recent development in the analytical techniques based on mass spectrometry made possible to detect and
quantitate nucleobase DNA adducts in the urine at concentrations
which are relevant for toxicological studies and potentially also for
biological monitoring of human exposure to some environmental
mutagens and carcinogens. Various mass spectrometric methods
have been developed for analysis of DNA adducts in urine formed
as a result of exposure to mutagens or carcinogens (Shuker et al.,
1984; Shuker and Bartsch, 1994; Prevost and Shuker, 1996; Casale
et al., 2001; Yen et al., 1998; Bhattacharya et al., 2003).
For styrene, an important industrial monomer, which has
been classified by IARC as a possible human carcinogen (2B)
(IARC, 2002), N7 guanine and N3 adenine adducts comprise 93%
and 4%, respectively, of the total alkylation in double stranded
DNA in vitro (Koskinen et al., 2000). These adducts are derived
from styrene 7,8-oxide, an electrophilic metabolic intermediate
of styrene (Bond, 1989), which undergoes a ring opening reaction at both ␣- and ␤-carbon by nucleophilic attack of nitrogen
atoms in the DNA nucleobases leading to two types of adducts, 2hydroxy-1-phenylethyl- and 2-hydroxy-2-phenylethyl-derivatives.
A number of other DNA adducts derived from styrene 7,8-oxide has
34
P. Mikeš et al. / Toxicology Letters 184 (2009) 33–37
been reported in experimental animals and humans (Pauwels et
al., 1996; Vodicka et al., 2001, 2002a,b). However, only the N7 guanine and N3 adenine adducts mentioned above depurinate readily
to release corresponding modified nucleobases (Koskinen et al.,
2001). Therefore, these adducts are likely to be excreted in urine
at much higher concentrations than any other styrene derived
adducts.
In a previous study styrene N7 guanine adducts of both types, i.e.,
7-(2-hydroxy-1-phenylethyl)guanine (N7␣G) and 7-(2-hydroxy-2phenylethyl)guanine (N7␤G) were found in the urine of mice after
repeated inhalation exposure to high doses of styrene (Vodicka
et al., 2006). In this study we describe urinary excretion of both
N7 guanine and N3 adenine adducts in mice exposed to styrene
in a subacute inhalation experiment. To follow the excretion profile of urinary adducts sensitive and structurally specific LC/MS/MS
methods for both N7 guanine and N3 adenine adducts have been
developed.
2. Materials and methods
2.1. Animal treatment
Adult male NMRI mice (mean weight 29 g) were exposed to styrene in a
dynamic exposure chamber with controlled level of concentration of 600 mg/m3
and 1200 mg/m3 in the inhaled air for 10 consecutive days, 6 h/day. Animals were
divided into five groups, six animals per group. Each group was placed into a glass
metabolic cage with free access to food and water. To enhance diuresis, sucrose
(8 mg/mL) was added to the drinking water. Animals were exposed by inhalation,
two groups at each concentration level, one additional group remained unexposed.
Urine was collected each day of exposure and then for another day after last exposure. During sample collection the urine was filtered through a gauze filter to remove
pieces of faeces and crumbs of food pellets. The walls of the metabolic cages were
rinsed with distilled water and resulting solution was added to the main portion
of collected urine. Fractions collected on days 3–4, 5–7 and 8–10 were combined
within each group. All samples were stored at −20 ◦ C until analysed. Animal experiments were approved by the Central Committee for Animal Protection of the Czech
Republic.
2.2. Authentic standards
Authentic samples of the analytes were prepared in our laboratory. Synthetic
procedures for 7-(2-hydroxy-1-phenylethyl)guanine (N7␣G) and 7-(2-hydroxy-2phenylethyl)guanine (N7␤G) were described in Novák et al. (2004), those for 3-(2hydroxy-1-phenylethyl)adenine (N3␣A) and 3-(2-hydroxy-2-phenylethyl)adenine
(N3␤A) are described in Krouželka et al. (2008).
2.3. Sample preparation, procedure for adenine adducts
Aliquots (900 ␮L) of urine diluted with 900 ␮L of water were mixed with 200 ␮L
of 66% trichloroacetic acid solution and vortexed for 10 min. The calibration samples were prepared in the same way from 900 ␮L of blank urine spiked with 18 ␮L
of standard solution containing both N3␣A and N3␤A to provide final concentrations ranging from 0.01 ng/mL to 2 ng/mL. The samples were centrifuged (8000 rpm,
10 min) and 1.5 mL of the supernatants were mixed with 300 ␮L of 3 M sodium
hydroxide solution and 200 ␮L of 5% sodium hydrogen carbonate solution to adjust
pH to 10.5. The 2 mL samples were then poured onto OasisTM HLB 60 mg SPE columns
(Waters), which were previously conditioned with 2 mL of methanol followed by
2 mL of 1% sodium hydrogen carbonate solution (pH 10.5). The sample vials were
washed twice with 1 mL of 1% sodium hydrogen carbonate solution and the washes
were applied onto the cartridges. The cartridges were then washed with 2 mL of
20% methanol in 1% sodium hydrogen carbonate solution to eliminate acidic components of the urine and the retained liquid was removed by suction. Adenine adducts
were eluted with 2 mL of acetonitrile:methanol (70:30) solution during 5 min, the
eluates were evaporated to dryness under a stream of nitrogen at 70 ◦ C and the
residues were re-dissolved in 150 ␮L of 30% aqueous methanol. After mixing shortly
the solutions were transferred into a microtiter plate, the plate was sealed and the
samples analysed by HPLC/MS.
2.4. Sample preparation, procedure for guanine adducts
Aliquots (1 mL) of urine were mixed with 0.5 mL of 2% formic acid solution and
the acidic solutions formed were extracted by solid phase extraction. The calibration
samples were prepared in the same way from 1 mL of blank urine and 400 ␮L of 2%
formic acid solution spiked with 100 ␮L of standard solution containing both N7␣G
and N7␤G to provide final concentrations ranging from 0.05 ng/mL to 5 ng/mL. Sep-
PakTM Vac 3 mL, tC18, 200 mg columns (Waters) were conditioned with 2 mL of
methanol and 2 mL of 2% formic acid solution (pH 2.0). The 1.5 mL acidic urine or
standard samples were passed through the conditioned cartridges. The sample vials
were washed twice with 1 mL of 2% formic acid solution and the washes were applied
onto the cartridges which were then washed with additional 2 mL of 10% methanol
in 2% formic acid solution and the retained liquid was removed by suction. Guanine
adducts were eluted by 2 mL of 22% methanol in 2% formic acid solution during
5 min, the eluates were evaporated to dryness under a stream of nitrogen at 70 ◦ C
and the solid residues were re-dissolved in 500 ␮L of 25% methanol. After a short
mixing the solutions were transferred into a microtiter plate, the plate was sealed
and the samples analysed by HPLC/MS.
2.5. HPLC/MS/MS analyses
The LC/MS system consisted of a Micromass triple quadrupole Quattro Premier
XE (Waters Micromass, USA) interfaced with Agilent 1200 Series binary gradient
pump (Agilent, USA) and CTC Analytics Autosampler (Switzerland). A silica based
column SunFireTM C18, 4.6 × 100 mm, 5 ␮ (Waters, USA) was used for all analyses.
The mobile phase A was 10 mM ammonium acetate (pH 6.7) and the mobile phase
B was methanol. The flow rate was 0.8 mL/min. For separation of adenine adducts
the initial concentration 32% of methanol was kept constant for first 0.33 min then
changed linearly to 38% in additional 8 min and subsequently to 95% in another
1 min. After a short isocratic elution with 95% methanol the initial mobile phase
composition was re-adjusted. Total analyses time was 13.3 min.
For guanine adducts isocratic conditions were used with 37% methanol in the
ammonium acetate buffer. After 7 min when both adducts were eluted the column
was washed by increasing methanol concentration up to 97%. Total analysis time
was 13.3 min.
The analytes were detected by single reaction monitoring (SRM) after eslectrospray ionisation (ESI). The transitions for guanine and adenine adducts in positive
mode were m/z 272 → 152 and 256 → 136, respectively, which corresponds to the
main fragmentation reaction, the loss of PhCH2 CH2 O from the (M + H)+ ions. The
continuous flow injections of standard solutions of analytes were performed to tune
and establish MS/MS operating conditions. Since parent ions of both adenine and
guanine adducts fragmented spontaneously in the ion source, the capillary voltage
was set to only 1.1 kV. The cone voltage was set to 23 V, the source temperature to
110 ◦ C and the temperature of desolvation gas to 400 ◦ C. Desolvation gas flow was
set to 1000 L/h. Collisions of parent ions were performed using argon as a collision
gas at a flow rate of 0.3 mL/min and collision energy of 23 V.
2.6. Calculations
The total uptake of inhaled styrene was calculated by multiplying the inhaled air
concentration by the duration of exposure, the retention of styrene in lungs (0.55)
and the minute ventilation volume of mice related to kg of body weight (25 mL/min
in a mouse of 25 g i.e. 1 L/min kg body weight; Arms and Travis, 1988). The excretion
rates were calculated by multiplying the concentration of the adduct in urine by
the urine sample volume divided by the sum of body weights of animals in the
metabolic cage and the collection time in days. Standard deviations for the sum of
adducts (N7␣G + N7␤G or N3␣A + N3␤A) were calculated as the total of standard
deviations of the summands.
3. Results
3.1. N3 adenine adducts
The adenine adducts were pre-separated by solid phase extraction (SPE) of deproteinized urine on macroporous copolymer of
divinylbenzene with vinylpyrrolidone (Waters HLB columns) under
basic conditions, at pH nearly 10.5. At these conditions urinary
carboxylic acids are in their ionized form and can be therefore
washed out from the column while retaining weakly basic analytes. The extraction recovery for N3␣A and N3␤A was more than
97%. Samples were concentrated to a sixth of their original volume
affording the limit of quantification of 0.01 ng/mL and the limit of
detection of nearly 0.003 ng/mL for both N3␣A and N3␤A. Calibration curves were linear (R2 > 0.99) within the concentration range
of 0.01–2 ng/mL. A closely eluting peak was found in blank urine,
which we were able to separate from N3␣A by a very flat gradient,
so that it did not interfere with the analysis within the concentration range found in the exposed samples (Fig. 1). The excretion
was dose dependent although during first two days the differences
between the high and low exposure group were not statistically
significant due to large variations between the two groups at each
P. Mikeš et al. / Toxicology Letters 184 (2009) 33–37
35
Fig. 2. Excretion profile of urinary adenine adducts N3␣A + N3␤A (mean ± S.D.;
n = 2).
Fig. 1. Mass chromatograms of blank urine and exposed sample, transition m/z
256 → 136. Retention time of N3␤A is 7.91 min and that of N3␣A is 8.80 min.
exposure level (Table 1 and Fig. 2). After the end of exposure adenine adducts in urine decreased rapidly so that on the first day post
exposure no adducts were detected. Total styrene related N3 adenine adducts accounted for about 0.8 × 10−5 % of the absorbed dose
(Table 3). Isomer ratio N3␣A:N3␤A based on the total cumulative
excretion was 27:73 and 29:71 for low and high exposure group,
respectively.
3.2. N7 guanine adducts
An improved HPLC/MS/MS method for the determination of
urinary N7␣G and N7␤G was developed. Most of the LC/MS/MS
measurements of biological samples suffer from significant matrix
effects especially when m/z values of analytes are below 300 m.
Strong eluent system, which was necessary for the elution of N7
guanines from HLB columns, did not enable to reduce strong interferences from the matrix occurring at low concentration levels.
Moreover, guanine adducts exhibited much lower response than
adenine adducts and thus the sample clean up procedure used for
the adenine adducts was not sufficient for removal of co-eluting
compounds which interfered with the analysis of N7␣G and N7␤G
even when a highly specific SRM detection was used. Therefore,
we developed another SPE clean-up procedure on reversed phase
extraction columns (tC18) with a very narrow washing/eluting window. Cartridges were washed with 10% methanol leaving more
than 97% of the analytes retained and than eluted quantitatively
with 22% methanol in 2% aqueous formic acid (20% of methanol in
2% aqueous formic acid was sufficient to give quantitative elution
of both N7␣G and N7␤G). Carrying out SPE at acidic conditions
enabled us to elute guanine adducts while retaining most of the
urinary carboxylic acid fraction. Detection by SRM using the main
fragmentation reaction (m/z 272 → 152) provided a structurally
specific and sensitive way to determine both N7␣G and N7␤G. The
limit of quantification was 0.08 ng/mL and 0.05 ng/mL for N7␣G
and N7␤G, respectively, the corresponding limits of detection were
about 0.03 ng/mL and 0.02 ng/mL. Calibration curves were linear
with R2 > 0.99 for both N7␣G and N7␤G. No co-eluting peaks were
found in blank urine (see Fig. 3). Adduct excretion was dose dependent. At the high exposure level a maximum of excretion was
observed on second day of exposure whereas at the low exposure
level a slightly lower excretion was found on the first day followed
by an increase on second day and stabilization during following
days (Fig. 4 and Table 2). After finishing the exposure the urinary
Table 1
Excretion of urinary N3 adenine adducts N3␤A and N3␣A.
Urinary excretion, mean ± S.D. (pmol kg−1 day−1 )
Exposure
(mg m−3 )
Day
N3␣A
N3␤A
N3␣A + N3␤A
600
1
2
3–4
5–7
8–10
11
29 ± 9
35 ± 9
13 ± 2
19 ± 1
21 ± 1
n.d.
104 ± 44
100 ± 43
29 ± 17
49 ± 7
52 ± 16
n.d.
133 ± 53
135 ± 52
42 ± 19
68 ± 8
73 ± 17
n.d.
1200
1
2
3–4
5–7
8–10
11
69 ± 63
84 ± 29
51 ± 4
52 ± 1
46 ± 1
n.d.
196 ± 171
220 ± 193
101 ± 19
125 ± 1
99 ± 2
n.d.
265 ± 234
304 ± 222
152 ± 23
177 ± 2
145 ± 3
n.d.
Fig. 3. Mass chromatograms of blank urine and exposed sample, transition m/z
272 → 152. Retention time of N7␣G is 5.51 min and that of N7␤G is 5.95 min.
36
P. Mikeš et al. / Toxicology Letters 184 (2009) 33–37
Fig. 4. Excretion profile of urinary guanine adducts N7␣G + N7␤G (mean ± S.D.;
n = 2).
adduct level decreased rapidly so that on the first day post exposure
(day 11) the adduct excretion decreased nearly by half of its average value. No accumulation of urinary adducts during the exposure
was observed. Total styrene related N7 guanine adducts accounted
for about 1.4 × 10−5 % of the absorbed dose (Table 3). The ratio of
N7␣G:N7␤G isomers accounted for 51:49 for low exposure and
47:53 for high exposure group when total cumulative values were
used.
4. Discussion
The values of excretion of urinary guanine adducts found in this
study were very similar to those found in our previous study, i.e.,
Table 2
Excretion of urinary N7 guanine adducts N7␤G, N7␣G.
Urinary excretion, mean ± S.D. (pmol kg−1 day−1 )
Exposure
(mg m−3 )
Day
600
1
2
3–4
5–7
8–10
11
40
84
94
83
86
27
±
±
±
±
±
±
19†
4
33
1
12
2†
73
107
88
72
72
37
±
±
±
±
±
±
4
26
3
6
2
5
113
191
182
155
158
64
±
±
±
±
±
±
22
30
36
7
14
8
1200
1
2
3–4
5–7
8–10
11
111
194
159
124
148
76
±
±
±
±
±
±
52
57
55
4
47
3
200
287
187
132
135
74
±
±
±
±
±
±
83
73
29
8
28
3
311
481
346
256
284
149
±
±
±
±
±
±
135
130
84
11
75
6
N7␣G
N7␤G
N7␣G + N7␤G
†
Concentration of N7␣G in these samples was above the detection limit but below
the limit of quantification so that the obtained values are less accurate.
1.0–1.7 × 10−5 % of the absorbed dose compared to 0.8–3.1 × 10−5 %.
Method used for the determination of these adducts was a modification of the one used previously (Vodicka et al., 2006). Sample
work-up was simplified by replacement of the two step extraction
procedure by a single step SPE process.
Urinary adenine adducts derived from styrene have not been
found as yet. Their level of excretion was unexpectedly high as compared to that of guanine adducts. When styrene 7,8-oxide (SO) was
reacted with double stranded DNA under physiological conditions,
N3 adenine adducts were the second most abundant ones after N7
guanines, yet they amounted only to 4% of the total adducts formed.
In the same experiment, N7 guanine adducts amounted to 93% of
the total alkylation (Koskinen et al., 2000). In other in vitro studies, somewhat higher proportion of N3 adenine adducts (nearly 9%
of the total DNA alkylation) was reported (Koskinen et al., 2001;
Vodicka et al., 2002a,b). In contrast to these in vitro results, urinary adenine adducts ranged from 0.8 to 1.2 × 10−5 % of absorbed
dose, i.e. more than 50% of those of N7 guanines. Higher proportion
of urinary N3 adenines than expected from in vitro studies cannot be explained by differences in depurination or base excision
repair. Both N3 adenine and N7 guanine adducts are eliminated
rapidly from DNA. Reported half lives of N3␣A and N3␤A were 10 h
and 20 h, respectively, while both N7␣G and N7␤G were eliminated
with a half life of 51 h (Koskinen et al., 2000). Despite longer persistence of N7 guanine adducts in in vitro experiments, no cumulative
effect was observed in vivo. On the first day post exposure excretion
of adenine adducts decreased below the limit of detection while
excretion of guanine adducts decreased nearly to a half of the average value. Similarly, during previous 21 long inhalation exposure
no tendency to increase urinary N7 guanine adduct excretion was
observed (Vodicka et al., 2006).
The reason for higher proportion of urinary adenine adducts
than expected from in vitro studies may reside in a higher proportion of N3 adenines formed in the native DNA than in those used
in the in vitro experiments as isolated double stranded DNA may
contain a unwinded fraction. Moreover, guanine adducts may be
oxidatively metabolised after they are cleaved off the DNA molecule
and this may lead to dealkylation. However, the ratio of ␣:␤ isomers for both urinary adenine and guanine adducts found is not
significantly different from that reported when SO was reacted with
double stranded DNA, i.e. 34:66 for N3 adenine adducts and 44:56
for guanines (Koskinen et al., 2000).
Urinary excretion of both N7 guanine and N3 adenine adducts
showed a higher variability among groups of the animals during
first two days of exposure than on subsequent days. The differences in the adduct excretion found at various time intervals
during the exposure period were not statistically significant. They
are obscured by variability between the two groups of animals.
Table 3
Styrene uptake and N7 guanine adduct excretion in urine (cumulative values).
Exposure (mg m−3 )
Day
Styrene uptake
(mmol kg
−1
)
Guanine adducts excreted
(pmol kg
−1
)
Adenine adducts excreted
(% of dose)
−5
(pmol kg−1 )
(% of dose)
600
1
2
4
7
10
11
1.14
2.28
4.56
7.98
11.4
11.4
113
304
668
1123
1607
1671
1.0 × 10
1.3 × 10−5
1.5 × 10−5
1.4 × 10−5
1.4 × 10−5
1.5 × 10−5
133
268
352
556
775
775
1.2 × 10−5
1.2 × 10−5
0.8 × 10−5
0.7 × 10−5
0.7 × 10−5
0.7 × 10−5
1200
1
2
4
7
10
11
2.28
4.56
9.12
16.0
22.8
22.8
311
792
1484
2252
3104
3253
1.4 × 10−5
1.7 × 10−5
1.6 × 10−5
1.4 × 10−5
1.4 × 10−5
1.4 × 10−5
265
569
873
1404
1839
1839
1.2 × 10−5
1.2 × 10−5
1.0 × 10−5
0.9 × 10−5
0.8 × 10−5
0.8 × 10−5
P. Mikeš et al. / Toxicology Letters 184 (2009) 33–37
However, a uniform tendency to increase the excretion of adducts
on the second day followed by its decrease and stabilization on subsequent days of exposure is apparent for both types of adducts at
both exposure levels (Figs. 2 and 4). These differences in excretion and its variability can be explained by changes in styrene
uptake due to locomotor activity of the animals rather than in
styrene metabolism. In fact, on the first day a marked depression
of motoric activity was observed in all exposed animals as compared to the unexposed ones. This effect subsided after adaptation.
At both exposure levels styrene uptake should be the limiting factor in styrene metabolism (Filser et al., 1993) so that changes in the
uptake should have a marked influence on the formation of metabolites and adducts. Physical activity and lung ventilation became
settled after an adaptation period so that interindividual variations diminished leading to a steady excretion rate. Nevertheless,
an induction of SO detoxifying enzymes by styrene exposure, which
would explain a decrease in the formation of adducts after certain
induction period, cannot be excluded.
Human exposure to styrene in highly exposed professional
groups such as lamination workers may reach up to 200 mg/m3
during work shift. These levels are much lower than those used
in this animal study. Duration of repeated exposure period, which
is commonly much higher in humans, will probably not have any
significant effect on urinary N7 guanine and N3 adenine adduct
levels because these adducts are rapidly cleaved off the DNA and
excreted in urine. Therefore, the methods developed in this study
may be directly applicable only for very high exposure groups of
occupationally exposed individuals. However, a further improvement in sensitivity is possible by modifying the pre-concentration
process or, more markedly, by application of specific antibody based
immunoaffinity methods.
In conclusion, both N7 guanine and N3 adenine DNA adducts
derived from styrene are rapidly depurinated and eliminated in
urine. Proportion of urinary N3 adenines was much higher than
those expected from in vitro studies. Both N7 guanines and N3
adenines are promising biomarkers of effective dose of styrene
reflecting, however, only a short-term exposure.
Conflict of interest statement
None.
Acknowledgements
This work was supported by grant No. 203/06/0888 from the
Grant Agency of the Czech Republic. The authors would like to thank
Mrs. Nad’a Kellerová for her skilful technical assistance with animal
exposure and handling.
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