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Excretion of urinary N7 guanine and N3 adenine DNA adducts in mice after inhalation of styrene
Toxicology Letters 184 (2009) 33–37
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Excretion of urinary N7 guanine and N3 adenine DNA adducts in mice after inhalation of styrene Petr Mikeˇs a , Marek Koˇrínek a , Igor Linhart b,∗ , Jan Krouˇzelka b , Emil Frantík c , L’udmila Vodiˇcková c , Lenka Neufussová c a b c
RE&D VUFB, Podˇebradská 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, Sˇ robárova 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 (N3A), 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 (N7G) 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 + N3A) excreted amounted to nearly 0.8 × 10−5 % of the absorbed dose while urinary N7 guanine adducts (N7␣G + N7G) 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
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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 (N7G) 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 (N7G) were described in Novák et al. (2004), those for 3-(2hydroxy-1-phenylethyl)adenine (N3␣A) and 3-(2-hydroxy-2-phenylethyl)adenine (N3A) are described in Krouˇzelka 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 N3A 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 N7G 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 + N7G or N3␣A + N3A) 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 N3A 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 N3A. 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
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Fig. 2. Excretion profile of urinary adenine adducts N3␣A + N3A (mean ± S.D.; n = 2).
Fig. 1. Mass chromatograms of blank urine and exposed sample, transition m/z 256 → 136. Retention time of N3A 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:N3A 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 N7G 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 N7G
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 N7G). 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 N7G. The limit of quantification was 0.08 ng/mL and 0.05 ng/mL for N7␣G and N7G, 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 N7G. 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 N3A and N3␣A. Urinary excretion, mean ± S.D. (pmol kg−1 day−1 )
Exposure (mg m−3 )
Day
N3␣A
N3A
N3␣A + N3A
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 N7G is 5.95 min.
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Fig. 4. Excretion profile of urinary guanine adducts N7␣G + N7G (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:N7G 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 N7G, 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
N7G
N7␣G + N7G
† 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 N3A were 10 h and 20 h, respectively, while both N7␣G and N7G 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
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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. References Arms, A.D., Travis, C.C., 1988. Reference physiological parameters in pharmacokinetic modeling. US Environmental Protection Agency, Washington, DC, Report No. EPA/600/6-88/004. Bhattacharya, S., Barbacci, D.C., Shen, M., Liu, J., Casale, G.P., 2003. Extraction and purification of depurinated benzo[a]pyrene-adducted DNA bases from human
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urine by immunoaffinity chromatography coupled with HPLC and analysis by LC/quadrupole ion-trap MS. Chem. Res. Toxicol. 16, 479–486. Bond, J.A., 1989. Review of the toxicology of styrene. CRC Crit. Rev. Toxicol. 19, 227–249. Casale, G.P., Singhal, M., Bhatacharya, S., Ramanathan, R., Roberts, K.P., Barbacci, D.C., Zhao, J., Jankowiak, R., Gross, M.L., Cavalieri, E.L., Small, G.J., Rennard, S.I., Mumford, J.L., Shen, M., 2001. Detection and quantification of depurinated benzo[a]pyrene-adducted DNA bases in the urine of cigarette smokers and women exposed to household coal smoke. Chem. Res. Toxicol. 14, 192– 201. Filser, J.G., Schwegler, U., Csanády, G., Creim, H., Kreuzer, P.E., Kessler, W., 1993. Species-specific pharmacokinetics of styrene in rat and mouse. Arch. Toxicol. 67, 517–530. Fujii, T., Saito, T., Nakasaka, T., 1980. Synthesis, ring opening and glycosidic bond cleavage of 3-methyl-2 -deoxyadenosine. Chem. Commun., 758–759. Hemminki, K., Koskinen, M., Rajaniemi, H., Zhao, C., 2000. DNA adducts, mutations and cancer 2000. Regul. Toxicol. Pharm. 32, 264–275. IARC, 2002. Monograph on the Evalution of Carcinogenic Risks to Humans: Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene, vol. 82. International Agency for Research on Cancer, Lyon, p. 437. Koskinen, M., Plna, K., 2000. Specific DNA adducts induced by some monosubstituted epoxides in vitro and in vivo. Chem. Biol. Interact. 129, 209– 229. Koskinen, M., Vodicka, P., Hemminki, K., 2000. Adenine N3 is a main alkylation site of styrene oxide in double stranded DNA. Chem. Biol. Interact. 124, 13–27. Koskinen, M., Vodickova, L., Vodicka, P., Warner, S.C., Hemminki, K., 2001. Kinetics of formation of specific styrene oxide adducts in double stranded DNA. Chem. Biol. Interact. 138, 111–124. Krouˇzelka, J., Linhart, I., Tobrman, T., 2008. Synthesis of 3-(2-hydroxy-1phenylethyl)- and 3-(2-hydroxy-2-phenylethyl)adenine. DNA adducts derived from styrene. J. Heterocycl. Chem. 45, 789–795. Margison, G.P., Capps, M.J., O’Connor, P.J., Craig, A.W., 1973. Loss of 7-methylguanine from rat liver DNA after methylation in vivo with methyl methanesulphonate or dimethylnitrosoamine. Chem. Biol. Interact. 6, 119–124. Mueller, N., Risenbrand, G., 1985. The influence of N7 substituents on the stability of N7 alkylated guanosines. Chem. Biol. Interact. 53, 173–181. Novák, J., Linhart, I., Dvoˇráková, H., 2004. Syntheses of 7-(2-hydroxy-1-phenylethyl)and 7-(2-hydroxy-2-phenylethyl)guanine, DNA adducts derived from styrene 7, 8-oxide. Eur. J. Org. Chem., 2738–2746. Pauwels, W., Vodicka, P., Severi, M., Plna, K., Veulemans, H., Hemminki, K., 1996. Adduct formation on DNA and haemoglobin in mice intraperitoneally administered with styrene. Carcinogenesis 17, 2673–2680. Prevost, V., Shuker, D.E.G., 1996. Cigarette smoking and urinary 3-alkyladenine excretion in man. Chem. Res. Toxicol. 9, 439–444. Shuker, D.E.G., Bailey, E., Gorf, S.M., Lamb, J., Farmer, P.B., 1984. Determination of N7-[2 H3 ] methyl guanine in rat urine by gas chromatography–mass spectrometry following administration of trideuteromethylating agents or precursors. Anal. Biochem. 140, 270–275. Shuker, D.E.G., Farmer, P., 1992. Relevance of urinary DNA adducts as markers of carcinogen exposure. Chem. Res. Toxicol. 5, 450–460. Shuker, D.E.G., Bartsch, H., 1994. Detection of human exposure to carcinogens by measurement of alkyl-DNA adducts using immunoaffinity clean-up in combination with gas chromatography–mass spectrometry and other methods of quantitation. Mutat. Res. 313, 263–268. Timbrell, J.A., 1998. Biomarkers in toxicology. Toxicology 129, 1–12. ˇ etina, R., Smerák, ˇ Vodicka, P., Koskinen, M., Vodickova, L., Stˇ P., Barta, I., Hemminki, K., 2001. DNA adducts, trand breaks and micronuclei in mice exposed to styrene by inhalation. Chem. Biol. Interact. 137, 213–227. Vodicka, P., Koskinen, M., Arand, M., Oesch, F., Hemminki, K., 2002a. Spectrum of styrene-induced DNA adducts: the relationship to other biomarkers and prospects in human biomonitoring. Rev. Mutat. Res. 511, 239–254. Vodicka, P., Stetina, R., Koskinen, M., Soucek, P., Vodickova, L., Hlavac, P., Kuricova, M., Necasova, R., Hemminki, K., 2002b. New aspects in the biomonitoring of occupational exposure to styrene. Int. Arch. Occup. Environ. Health 75 (Suppl), S75–S85. Vodicka, P., Linhart, I., Novak, J., Koskinen, M., Vodickova, L., Hemminki, K., 2006. 7Alkylguanine adduct levels in urine, lung and liver of mice exposude to styrene by inhalation. Toxicol. Appl. Pharmacol. 210, 1–8. Yen, T.-Y., Holt, S., Sangaiah, R., Gold, A., Swenberg, J.A., 1998. Quantitation of 1, N6 ethenoadenine in rat urine by immunoaffinity extraction combined with liquid chromatography/electrospray ionization mass spectrometry. Chem. Res. Toxicol. 11, 810–815.
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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 Mikeˇs a , Marek Koˇrínek a , Igor Linhart b,∗ , Jan Krouˇzelka b , Emil Frantík c , L’udmila Vodiˇcková c , Lenka Neufussová c a b c
RE&D VUFB, Podˇebradská 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, Sˇ robárova 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 (N3A), 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 (N7G) 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 + N3A) excreted amounted to nearly 0.8 × 10−5 % of the absorbed dose while urinary N7 guanine adducts (N7␣G + N7G) 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
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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 (N7G) 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 (N7G) were described in Novák et al. (2004), those for 3-(2hydroxy-1-phenylethyl)adenine (N3␣A) and 3-(2-hydroxy-2-phenylethyl)adenine (N3A) are described in Krouˇzelka 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 N3A 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 N7G 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 + N7G or N3␣A + N3A) 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 N3A 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 N3A. 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. Mikeˇs et al. / Toxicology Letters 184 (2009) 33–37
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Fig. 2. Excretion profile of urinary adenine adducts N3␣A + N3A (mean ± S.D.; n = 2).
Fig. 1. Mass chromatograms of blank urine and exposed sample, transition m/z 256 → 136. Retention time of N3A 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:N3A 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 N7G 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 N7G
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 N7G). 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 N7G. The limit of quantification was 0.08 ng/mL and 0.05 ng/mL for N7␣G and N7G, 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 N7G. 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 N3A and N3␣A. Urinary excretion, mean ± S.D. (pmol kg−1 day−1 )
Exposure (mg m−3 )
Day
N3␣A
N3A
N3␣A + N3A
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 N7G is 5.95 min.
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Fig. 4. Excretion profile of urinary guanine adducts N7␣G + N7G (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:N7G 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 N7G, 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
N7G
N7␣G + N7G
† 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 N3A were 10 h and 20 h, respectively, while both N7␣G and N7G 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. Mikeˇs 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. References Arms, A.D., Travis, C.C., 1988. Reference physiological parameters in pharmacokinetic modeling. US Environmental Protection Agency, Washington, DC, Report No. EPA/600/6-88/004. Bhattacharya, S., Barbacci, D.C., Shen, M., Liu, J., Casale, G.P., 2003. Extraction and purification of depurinated benzo[a]pyrene-adducted DNA bases from human
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