Butadiene diolepoxide- and diepoxybutane-derived DNA adducts at N7-guanine: a high occurrence of diolepoxide-derived adducts in mouse lung after 1,3-butadiene exposure
Pertti Koivisto,
Ilkka Kilpeläinen1,
Ilpo Rasanen2,
Ilse-Dore Adler3,
Francesca Pacchierotti4 and
Kimmo Peltonen5
Department of Industrial Hygiene and Toxicology, Chemistry Laboratory, Finnish Institute of Occupational Health, Topeliuksenkatu 41 aA, FIN-00250 Helsinki,
1 Institute of Biotechnology, University of Helsinki, FIN-00560 Helsinki,
2 Department of Forensic Medicine, PO Box 40, University of Helsinki, FIN-00014 Helsinki, Finland,
3 GSF-Institut für Säugetiergenetik, D-85764 Neuherberg, Germany and
4 ENEA, CR CasacciaVia Anguillarese 301, I-00060 Roma, Italy
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Abstract
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Butadiene (BD) is a high production volume chemical and is known to be tumorigenic in rodents. BD is metabolized to butadiene monoepoxide (BMO), diepoxybutane (DEB) and butadiene diolepoxide (BDE). These epoxides are genotoxic and alkylate DNA both in vitro and in vivo, mainly at the N7 position of guanine. In this study, a 32P-post-labeling/thin-layer chromatography (TLC)/high-pressure liquid chromatography (HPLC) assay for BDE and DEB adducts at the N7 of guanine was developed and was used in determining the enantiomeric composition of the adducts and the organ dose of BD exposure in lung. Exposure of 2'-deoxyguanosine (dGuo), 2'-deoxyguanosine-5'-phosphate (5'-dGMP) and 2'-deoxyguanosine-3'-phosphate (3'-dGMP) to racemic BDE followed by neutral thermal hydrolysis gave two products (products 1 and 2) that were identified by MS and UV and NMR spectroscopy as a diastereomeric pair of N7-(2,3,4-trihydroxybutan-1-yl)-guanines. Exposure of dGuo nucleotides to RR/SS DEB (also referred to as dl DEB) followed by thermal depurination resulted in a single product coeluting with the BDE product 1. If the reaction mixture of BDE and 5'-dGMP was analyzed by HPLC before hydrolysis of the glycosidic bond, four major nucleotide alkylation products (A, B, C and D) with identical UV sepectra were detected. The products were isolated and hydrolyzed, after which A and C coeluted with product 1 and B and D coeluted with the product 2. The major adduct of DEB-exposed 5'-dGMP was N7-(2-hydroxy-3,4-epoxy-1-yl)-dGMP (product E). A 32P-post-labeling assay was used to detect BDE- and DEB-derived N7-dGMP adducts in DNA. Levels of adducts increased with a dose of BDE and DEB and exhibited a half life of 30 ± 3 (r = 0.98) and 31 ± 4 h (r = 0.95), respectively. Incubation of DEB-modified DNA at 37°C at neutral pH for up to 142 h did not lead to an increase of N7-(2,3,4-trihydroxybutan-1-yl)-dGMP in the DNA. These observations led to the conclusion that the N7-(2,3,4-trihydroxybutan-1-yl)-dGMP adducts in DNA can be used as a marker of BDE exposure and that N7-(2-hydroxy-3,4-epoxy-1-yl)-dGMP adducts are related to DEB exposure. Dose-related levels of BDE- and DEB-derived adducts were detected in lungs of mice inhaling butadiene. Most of the N7-dGMP adducts (73%; product D) were derived from the 2R-diol-3S-epoxide of 1,3-butadiene. The data presented in this paper indicate that in vivo, 98% of N7-dGMP alkylation after BD exposure is derived from BDE, and ~2% of the adducts were derived from DEB and BMO.
Abbreviations: 3'-dGMP, 2'-deoxyguanosine-3'-phosphate; 5'-dGMP, 2'-deoxyguanosine-5'-phosphate; AF, ammonium formate; BD, 1,3-butadiene; BDE, butadiene diolepoxide; BMO, butadiene monoepoxide; Ct-DNA, calf thymus DNA; CYP, cytochrome P450; DEB, diepoxybutane; dGuo, 2'-deoxyguanosine; DMSO, dimethyl sulfoxide; HPLC, high-performance liquid chromatography; MN, micrococcal nuclease; MS, mass spectrometry; ODS, octadecylsilane; Rf, retention factor; SAX, strong anion exchange; SPD, spleen phosphodiesterase; TLC, thin layer chromatography.
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Introduction
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1,3-Butadiene (BD) is a high production volume chemical mainly used in the chemical industry. BD is also a ubiquitous environmental pollutant detected in urban air, tobacco smoke and car exhausts (1).
BD is mainly oxidized by CYP 2E1 and 2A6 to electrophilic butadiene monoepoxide (BMO) enantiomers, which can be further oxidized to diastereomeric diepoxides (diepoxybutane; DEB) (25). Butadiene diolepoxide (BDE) can be formed from butene diol, the hydrolysis product of BMO or a partial hydrolysis of DEB (3,6) (Figure 1
). Even though the qualitative composition of the metabolites are the same in mice and rats, the quantity of different metabolites varies between the species (7,8). BMO and DEB formation has also been observed in human liver microsome preparations if they were exposed to BD (4,5).

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Fig. 1. The metabolism of 1,3-butadiene to stereochemically active epoxy metabolites: BMO, DEB and BDE.
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BD has a low acute toxicity but the epoxy metabolites are reported to be genotoxic (912). Long term animal exposures have shown that BD is a potent rodent carcinogen, mice being more sensitive than rats (13). Recently, Nieusma et al. (14) demonstrated that the stereochemistry of BMO and DEB affected the hepatotoxic response in vivo.
Recent retrospective follow-up studies of men employed in the styrene-butadiene rubber industries in North America and Canada indicated an increased risk of leukemia which was related to exposure to BD (15,16). BD has been evaluated by the IARC, and it was classified as a group 2A carcinogen (probably carcinogenic to humans) (1).
BMO-derived purine adducts have been characterized and the adducts have been detected in experimental animals exposed to BD (1721). Recently, it has been demonstrated in vitro that an initial alkylation product of DEB is the N7-(2-hydroxy-3,4-epoxy-1-yl)-guanine (22). In this particular adduct, the remaining epoxy moiety can hydrolyze to form a diol resulting in adducts that have the same chemical structures as the adducts derived from BDE. Alternatively, the epoxy moiety can react with other bases in DNA to form intra- and/or interstrand cross-links (2325) (Figure 2
). A monoadduct derived from DEB at adenine residues has been observed in both in vitro and in vivo experiments (2628). To our knowledge, no studies of DNA adduct formation specific to BDE or DEB have been published so far.

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Fig. 2. The structure of N7-dGuo adducts derived from BDE and DEB. The trihydroxy derivative (1), the epoxy derivative (2) and the possible N7 guanine cross-link products (3) are shown.
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Exposure of mice and rats to 14C-labeled BD caused increased radioactivity at about the same level in the liver DNA of both mice and rats. However, in the nucleoproteins, the level was higher in mice (29). The chromatographic analysis of mouse liver DNA after in vivo [14C]BD exposure suggested that both BMO and DEB adducts were formed (30).
In this paper, we describe the preparation of markers of BDE- and DEB-alkylated guanine N7 DNA adducts for 32P-post-labeling purposes. An analytical method that is based on 32P-postlabeling and a subsequent HPLC analysis with a radioactivity detector was developed. The method was applied to samples from animals exposed to BD by inhalation. By using the markers and the method developed, we were able to demonstrate for the first time, that the adducts under study were formed in mouse lung in a dose-dependent manner and that the adduct levels derived from BDE were unexpectedly high.
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Materials and methods
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Chemicals
Micrococcal nuclease (MN), nuclease P1 and RNase A were from Sigma (St Louis, MO). HPLC grade MeOH was purchased from LAB-SCAN (Dublin, UK). Spleen phosphodiesterase (SPD), T4 polynucleotide kinase and alkaline phosphatase (AP) from calf intestine were from Boehringer Mannheim (Indianapolis, IN). Proteinase K and calf thymus DNA (Ct-DNA) were from Merck (Germany). RR/SS (dl) DEB was obtained from Aldrich (Germany), crude [
-32P]ATP, with 7000 Ci/mmol specific activity, was from ICN (Belgium) and scintillation liquid Ultima flo ammonium formate (AF) was from Packard (Holland). DNA was isolated and purified using Qiagen DNA extraction cartridges (Qiagen, Chatsworth, CA). Racemic 3,4-epoxybutane-1,2-diol was synthesized as described previously (31).
All HPLC analyses were carried out with a Waters 717 autosampler, a Waters 600 controller and a Waters 486 Tunable absorbance detector. A UV detector was connected to a Radiomatic Flo-One ß 150 TR flow scintillation analyzer. The cuvette in the Radiomatic detector was a plastic LQ TR cell (500 µl) and the measured energy range was set to 01710 KeV. Scintillation fluid was pumped at a flow rate of 1.1 ml/min. Reverse-phase chromatography of the BDE- and DEB-derived 2'-deoxyguanosine-3'-phosphate or 2'-deoxyguanosine-5'-phosphate (3'- or 5'-dGMP, respectively) nucleotide adducts was performed using a linear MeOH gradient in 50 mM AF (500 mM if radioactivity detector was applied) buffer at pH 4.5. The gradient consisted of 5 min of 0% MeOH followed by a linear increment of MeOH to 15% during 30 min. From 35 to 40 min, MeOH was further increased to 50%, and linearly decreased from 50 to 0% during 5 min. Between the injections, the column was equilibrated for 20 min. A column used was an Inertsil ODS-3 (4x150 mm) at a flow rate of 0.45 ml/min.
NMR spectra were measured at 500 MHz using a Varian Unity 500 spectrometer. Samples were dissolved in d6-dimethyl sulfoxide (DMSO) and D2O was added to exchange labile protons. The spectra were recorded at 27°C and referenced to the DMSO.
Mass spectra were recorded with a Perkin Elmer (Norwalk, CT) API 365 bench-top Triple-Quad Mass Spectrometer with the TurboIonSpray ionization probe. Experiments were performed in a positive ion mode with the orifice voltage set at 30 V. The resolution was set on both quadrupoles at 0.1 a.m.u. (measured at 1/2 height) in all experiments. The mass spectrometer was programmed to transmit the protonated molecular ions [M+H]+ (m/z 255.9) through the first quadrupole, followed by collision-induced fragmentation in the Q2 collision cell operating with nitrogen as the collision gas and a collision energy of 2050 V. Full scan product ion spectra were recorded in Q3. Mass spectra were collected in a continuous flow mode by connecting a Harward infusion pump directly to the TurboIonSpray probe. A 1 µg/ml solution of guanine adduct in methanol:water (1:1 v/v) was infused at 8 µl/min.
Preparation of adducts
Reactions with deoxyguanosine.
Deoxyguanosine was exposed to BDE overnight in aqueous solution at neutral pH. After exposure, the mixture was heated for 30 min at 100°C. An aliquot was injected to HPLC and peaks with retention times of 18 (peak 1) and 19.5 min (peak 2) were collected. Approximately 1 mg of each product was isolated from the reaction mixture and the UV spectra of the collected fractions were recorded. The two products (peak 1 and peak 2) separated by HPLC from the neutral hydrolysis were desalted by using reversed phase HPLC and 2% MeOH in 0.1% formic acid as an eluent. Using an Inertsil ODS-2 column (4.6x250), peak 1 eluted at 3 min and peak 2 eluted at 5 min, at a flow rate of 0.6 ml/min. After desalting, the fractions were lyophilized.
Reactions with deoxyguanylic acids.
3'- or 5'-dGMP nucleotides were exposed to BDE or DEB for 6 h and adducted N7-BDE and -DEB products were enriched by using strong ion exchange (SAX) solid phase extraction and applied as described previously (32). An aliquot of the solutions was taken and subjected to neutral thermal hydrolysis for comparison of HPLC retention times with those of spectroscopically identified guanine adducts. For quantitative purposes, a calibration graph was generated using commercially available 7-methylguanine. Simply, BDE- and DEB-derived adducts at the N7 position of 3'-dGMP were depurinated, the peak area integrated and products quantitated using the calibration graph of 7-methylguanine. Analysis of 7-methyl- and 7-hydroxyethyl-5'-dGMP adducts in DNA was performed as reported (33,34).
Reactions with DNA.
Ct-DNA was exposed to various concentrations of BDE or DEB (100, 200, 400, 1000 nmol/ml/mg DNA) for 6 h in neutral solutions (37°C and pH 7.4 in 50 mM TrisHCl). DNA was then precipitated with ethanol/NaCl, dissolved in H2O and stored in a freezer (70°C). The stability of DEB- and BDE-alkylated DNA was studied by incubating DEB- and BDE-modified, but purified, DNA in TrisHCl buffer (50 mM, pH 7.4) at 37°C. An aliquot was taken after 0, 3, 6, 12, 24, 33, 48, 57, 72 and 142 h incubation and the samples were stored in a freezer (20°C) until analysis using 32P-post-labeling/thin-layer chromatography (TLC)/high-pressure liquid chromatography (HPLC) assay.
DNA digestion and adduct enrichment
DNA (1 mg/ml) was digested to nucleotides with MN (1 U/10 µg DNA in 2 mM TrisHCl/0.2 mM CaCl2 at pH 8.8 for 2 h at 37°C) followed by SPD (10 mU/10 µg DNA in 5 mM ammonium acetate at pH 4 for 2 h at 37°C) (33). After digestion, the samples were stored in a freezer (20°C). An aliquot of sample (10100 µl) was taken, the volume adjusted to 300 µl with AF buffer (2 mM, pH 8) and subsequently subjected to ion exchange solid phase extraction (SAX, 1 cc; Bond Elut, Varian). N7 adducts were eluted with loading buffer (500 µl of 2 mM AF, pH 8) and normal nucleotides with AF buffer with higher molarity and different pH than loading buffer (600 µl of 200 mM, pH 4).
Postlabeling
An aliquot of an enriched sample was evaporated to dryness using a centrifugal evaporator. N7-3'-dGMP adducts were labeled using 5 U phosphatase-free T4 kinase in 50 mM TrisHCl, 10 mM MgCl2, 10 mM mercaptoethanol, pH 9, followed by a 15 min nuclease P1 incubation (1 U in 1 µl of 0.4 M sodium acetate and 20 mM ZnCl2, pH 4.5). Samples were applied to a TLC plate and eluted in one direction using AF buffer (0.2 M, pH 8). After exposure, TLC plates were dried and a film was exposed for 1 h using a film cassette equipped with intensifying screens. The area that contained the adducts (Rf = 0.3) was extracted two times with AF buffer (1 M, pH 6.5, 90 µl) and the extracts were pooled and subsequently injected into a high-pressure liquid chromatograph equipped with UV and radioactivity detectors (18).
The amount of DNA successfully digested was quantitated by analyzing normal nucleotides liberated enzymatically. Normal nucleotides were analyzed by using a Shimadzu LC-6A pump with a SPD 6AV detector at 254 nm, and samples were injected with a Gilson 234 autoinjector. A guard column used was a biocompatible Upchurch reverse-phase ODS (2x10 mm) and an analytical column was Inersil ODS-2 (2.1x150 mm). An isocratic eluent was acetonitrile (1%) in a phosphate buffer (50 mM, pH 4) (18).
Cell treatments
MCF-7 cells were grown in medium containing 500 ml RPM 1640 medium with 25 mM HEPESL-glutamate, 55 ml inactivated fetal bovine serum, 5 ml penicillinstreptomycin (10 000 IU/ml10 000 µg/ml) and 5 ml of 200 mM L-glutamine, all from GibcoBRL. About 108 cells were treated with R- and S-butene diols overnight (10 mg/ml). Treated cells were then harvested and washed with phosphate-buffered saline (Ca and Mg free; Orion Diagnistica, Finland). DNA was isolated from cell pellets by using Qiagen DNA extraction columns.
Animal exposures
Animals were exposed as described previously (35). Briefly, the animals were exposed for 5 days and 6 h/day to 50, 200, 500 and 1300 p.p.m. of BD. The animals were killed after exposure and organs were taken and stored in a freezer (70 °C). Lungs were powdered in a cold mortar using liquid nitrogen as a coolant, and DNA was extracted by using a Qiagen DNA extraction column. DNA was extracted by following the manufacturer's instructions, except that 3 h proteinase K treatment at 37°C was applied. An approximate DNA concentration was measured spectrophotometrically.
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Results and Discussion
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Synthesis, characterization and post-labeling of BDE- and DEB-derived N7-guanine adducts
Overnight reaction of 2'-deoxyguanosine (dGuo) with BDE resulted in the formation of several products. However, after thermal neutral hydrolysis, only two products with retention times of 18 (labeled 1) and 19.5 min (labeled 2) were obtained (Figure 3
). These guanine products labeled 1 and 2 exhibited pH-dependent absorptions characteristic of 7-alkyl guanines (36,37). Furthermore, by MS using a low collision energy (20V), material from peaks 1 and 2 gave a molecular ion at 256 m/z, consistent with a 7-substituted guanine structure (22). The intensity of the molecular ion decreased strongly as a function of increasing energy, and was not detectable at 40V (data not shown). The fragmentation of the products in peaks 1 and 2 were identical, each showing ions at 152, 135 and 110 m/z. Identical mass spectrum indicates a similar chemical structure of the adducts, because regioisomeric adducts are reported to have differences in their mass spectrum (17,21,38). The NMR data indicated seven protons in each of the products 1 and 2. In both cases, six of the protons were assigned to the alkyl chain of butane and one to the proton on the C-8 of guanine. The chemical shifts of the C-8 protons were identical in both products, suggesting that the products isolated were not C-1 and C-2 regioisomers, since in the case of BMO and styrene oxide, these have different chemical shifts for the C-8 protons (39,40). Chemical shifts and the assignment of the protons are presented in Table I
. All the spectroscopic data are consistent with previously published data and indicate that the products 1 and 2 are 7-(2,3,4-trihydroxybutan-1-yl)guanines, and are diastereomeric to each other (22).

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Fig. 3. A typical HPLC chromatogram of the reaction mixture of BDE and dGuo after neutral thermal hydrolysis. The two products, which had the retention times of 18 (peak 1) and 19.5 min (peak 2) were collected and identified as N7-(2,3,4-trihydroxybutan-1-yl)-guanine using spectroscopic techniques. Guanine is indicated, but deoxyguanosine would have eluted at 31 min and is not displayed.
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It can be seen from Figure 4
that the racemic diol epoxide can give rise to two pairs of enantiomers and that peak 1 in Figure 3
presumably represents one pair and peak 2 represents the other. By exposure of dGuo to racemic RR/SS DEB, it was possible to determine which peak in Figure 3
represented which enantiomer pair. This reaction also resulted in the formation of several products but after thermal hydrolysis, the major product coeluted with product 1. Again, as seen in Figure 4
the RR/SS DEB can only give rise to the RR and SS guanine adducts. Thus, peak 1 must represent the pair of RR and SS enantiomers, whereas peak 2 must contain the SR and RS enantiomers.
HPLC analysis of the reaction mixture of racemic BDE and 5'-dGMP after 6 h of exposure revealed four major nucleotide products (Figure 5
, peaks AD). After isolation and neutral thermal hydrolysis, the products from peak A and peak C coeluted with the product 1 (i.e. the original nucleotide adducts A and C must have had RR and SS configurations at the two chiral trihydroxybutane carbons). The hydrolysis products from peaks B and D both coeluted with product 2 and, thus, nucleotide adducts B and D had SR and RS configurations.

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Fig. 5. Reverse-phase HPLC chromatogram of four N7-(2,3,4-trihydroxybutan-1-yl)-5'-dGMP adducts detected with a UV detector. Letters A, B, C and D correspond to structures shown in Figure 4 .
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Exposure of 5'-dGMP to RR/SS DEB produced one major peak with a retention time of 30 min (product E). This was isolated and subjected to thermal hydrolysis. As for the reaction of this DEB with dGuo, the hydrolysis product showed an identical retention time and UV spectrum to the product 1 derived from BDE (i.e. peak 1 in Figure 3
). Since the thermal hydrolysis converts the epoxy group to vicinal diol, the original product E was probably 7-(2-hydroxy-3,4-epoxy-1-yl)-5'-dGMP a product previously spectroscopically identified by Tretjakova et al. (22).
32P-labeled BDE-5'-dGMP products were prepared by reacting 3'-dGMP with BDE. The resultant products could then be labeled on the 5'-position with T4 kinase and dephosphorylated at the 3' hydroxyl group with nuclease P1. This procedure gave four products that could be identified by comparison with the non-radioactive 5'-dGMP BDE markers (Figure 5
, peaks AD) that were known to be derived from the RR or SS diol epoxides (peaks A and C) and from the RS or SR diol epoxides (peaks B and D). If RR/SS DEB-alkylated 3'-dGMP products were post-labeled, as above, a major peak was detected at a retention time of 30 min (E) and two minor peaks at retention times of 15 and 21 min. Products at the retention times of 15 and 21 min had the same retention time as the BDE-derived adducts A and C, and they are suggested to originate from hydrolysis of the epoxy group during post-labeling or a partial hydrolysis of DEB to BDE during the storage of DEB.
DNA adducts in vitro
Six hours exposure of Ct-DNA with BDE and DEB clearly showed adduct formation, but the amount of modification of DNA by BDE was lower than by DEB (Table II
). The HPLC profile of the post-labeled N7 nucleotide adducts resulting from BDE and DEB exposures is shown in Figure 6A and B
, where it can be seen that the product profiles resulting from these two chemical exposures are quite different. The half life of N7 adducts derived from BDE measured as trihydroxy adducts in DNA was found to be 30 ± 4 h (r = 0.98) and that for the DEB-derived N7-(2-hydroxy-3,4-epoxy-1-yl)-dGMP adduct was similar at 31 ± 3 h (r = 0.95). The incubation of DEB-modified DNA at neutral conditions for up to 142 h did not increase the amount of N7-(2,3,4-trihydroxybutan-1-yl)-dGMP in DNA, presumably because the rate of hydrolysis was lower than the rate of depurination. In addition to depurination, the number of DEB-derived monoadducts can also decrease because of DNADNA cross-link formation (23,24,41). Our data indicate that the amount of cross-links is probably low, because the monoadduct formation [measured as N7-(2-hydroxy-3,4-epoxy-1-yl)-5'-dGMP] is high (Table II
). Summing up the data of in vitro DNA experiments, we suggest that N7-(2,3,4-trihydroxybutyl)-dGMP adducts are related to BDE exposure only and cannot be used to monitor DEB exposure.

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Fig. 6. 32P-postlabeling/TLC/HPLC analysis of Ct-DNA exposed to BDE (A) and DEB (B). The products AD are derived from (R,R), (S,R), (S,S) and (R,S) BDEs. Product E is derived from RR/SS DEB. X1 demonstrates the endogenous N7-hydroxyethyl-guanine adduct.
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To identify the enantiomeric origin of each nucleotide product, MCF-7 cells were treated with R- and S-butene diols. The treatment of cells with the R enantiomer could result in two post-labeled adducts in DNA that were derived either from SS or SR BDE (Figure 1
). Similarly, treatment of cells with the S enantiomer of butene diol could only result in DNA adducts that originated from the RR or RS diastereomers of BDE (Figure 1
). Since the S enantiomer of butene diol gave only adducts that ran with the nucleotide adducts A and D, and A was already known to be derived from either the SS or RR diol epoxide, it follows that A arises from the RR diol epoxide and D from the RS diol epoxide. Similarly, since the R enantiomer of butene diol gave only post-labeled adducts B and C, and it was known that C arises from the SS or RR diol epoxide, then C must be derived from the SS diol epoxide and B from the SR diol epoxide, all as illustrated in Figure 6
. In summary, the elution order of the adducts was 2R,3R (A), 2R,3S (B), 2S,3S (C) and 2S,3R (D) (Figure 6
).
DNA adducts in vivo
Inhalation exposure of BD by mice resulted in DNA adducts similar to the adducts derived from BDE in vitro, except the amount of different isomers varied (Figure 7
). Differences in isomeric composition may be due to the differences in the production ratio of BDE isomers, differences of their detoxification or differences in a DNA repair process to fix the lesions (35,42). N7-hydroxyethyl- and 5'-dGMP-methyl adducts were qualitatively analyzed and they eluted at 19 (X1) and 24 min (X2), respectively (Figure 6A
) (34). A weak HPLC signal, which has the same retention time as the N7-(2-hydroxy-3,4-epoxy-1-yl)-5'-dGMP, was detected and is shown in an insert in Figure 7
, but we also noticed that one of the adducts of BMO eluted very close to that product. A doseresponse curve of the BDE-derived N7-dGMP adducts is shown in Figure 8
. At the exposure level of 500 p.p.m., the peaks A (2R,3R) and B (2R,3S) account for ~23% of the adducts (formed from 2R,3R and 2S,3R diastereomers of BDE). The product C (2S,3S) accounts for ~2% of the adducts. The product D (2S,3R) is formed from 2R,3S diastereomer of BDE and represents 73% of the adducts derived from BDE. Thus, in combination with our previous report of BMO adducts in the same lung samples it can be concluded that BDE adducts constituted 98% of guanine N7 adducts of BMO. This is in accordance with our paper that reported that BDE formed most of the adducts in hemoglobin in vivo (43).

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Fig. 7. 32P-postlabeling/TLC/HPLC analysis of DNA samples isolated from lung samples of sham exposed (A) and 500 p.p.m. exposed (B) animals (AE correspond to the structures shown in Figure 4 ). X1 coelutes with N7-hydroxyethyl-5'-dGMP and X2 coelutes with N7-methyl-5'-dGMP. The insert demonstrates adducts derived from DEB and BMO.
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Fig. 8. The doseresponse of BDE-derived DNA adducts formed in mice after inhalation exposure to 1,3-butadiene.
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Acknowledgments
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We thank Ms Nina Tamminen for technical assistance with cell culture, and Dr Anthony Dipple for donating MCF-7 cells and for his kind help. Financial support for the study was obtained from the EV5V-0543 of DG XII of the European Communities, coordinated by Dr Francesca Pacchierotti.
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Notes
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5 To whom correspondence should be addressedEmail: kimmo.peltonen{at}occuphealth.fi 
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Received August 11, 1998;
revised March 22, 1999;
accepted March 24, 1999.