Styrene oxide-induced 2′-deoxycytidine adducts: implications for the mutagenicity of styrene oxide

Chemico-Biological Interactions 126 (2000) 201–213

Styrene oxide-induced 2%-deoxycytidine adducts:implications for the mutagenicity of styrene

oxide

Mikko Koskinen *, Davide Calebiro, Kari HemminkiCenter for Nutrition and Toxicology, Department of Biosciences at No6um, Karolinska Institute,

S-141 57 Huddinge, Sweden

Received 20 January 2000; received in revised form 14 March 2000; accepted 17 March 2000

Abstract

The reaction between 2%-deoxycytidine and styrene 7,8-oxide (SO) resulted in alkylation atthe 3-position and at the O2-position through the a- and b-carbons of the epoxide but at theN4-position only through the a-carbon. The 3-alkylated adducts were found to deaminate tothe corresponding 2%-deoxyuridine adducts (37°C, pH 7.4) with half-lives of 6 min and 2.4 hfor the a- and b-isomers, respectively. The N4-alkylated products were stable at neutral pH.The O2-alkylated products were unstable being prone to depyrimidation and to isomerisationbetween a- and b-isomers. In SO-treated double-stranded DNA, enzymatic hydrolysisallowed the identification of the b3-deoxyuridine and aN4-deoxycytidine adducts (1.9 and0.5% of total alkylation, respectively), in addition to the previously identified DNA-adducts.The 3-substituted uracil may have implications for the mutagenicity of SO. © 2000 ElsevierScience Ireland Ltd. All rights reserved.

Keywords: Cytosine; Hydrolytic deamination; DNA adducts; Styrene oxide

www.elsevier.com/locate/chembiont

Abbre6iations: BMO, butadiene monoxide; cyt, cytosine; dAdo, 2%deoxyadenosine; dCyd, 2%-deoxycy-tidine; dGuo, 2%deoxyguanosine; dUrd, 2%deoxyuridine; ESI-MS, electrospray ionization mass spectrome-try; SO, styrene 7,8-oxide.

* Corresponding author. Tel.: +46-8-608-9245; fax: +46-8-608-1501.E-mail address: [email protected] (M. Koskinen)

0009-2797/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0009 -2797 (00 )00165 -4

M. Koskinen et al. / Chemico-Biological Interactions 126 (2000) 201–213202

1. Introduction

Styrene 7,8-oxide (SO) is a major metabolite of styrene, a widely used chemicalmonomer. SO has been classified as a probable human carcinogen [1]. It has beenshown to form DNA adducts in vitro and in vivo which may be the cause of themutagenic and carcinogenic properties of the chemical [2,3]. SO can bind tonucleophilic positions in DNA either through the a- or b-carbon of the epoxidemoiety. Since the a-carbon is chiral the reaction with a racemic SO results in adiastereomeric pair of alkylation products. When double-stranded DNA wastreated with SO, the main alkylation sites were found to be 7-guanine and3-adenine comprising 93 and 4% of total alkylation, respectively. A total of 1.5% ofthe alkylation was shown to take place at the N2-position of guanine and ca. 1.2%at the 1- and N6-positions of the adenine residues [4]. A dominating type ofmutation found in the hypoxanthine-guanine phosphoribosyl transferase (hprt)gene in SO-treated cultured human lymphocytes was the AT�GC transition [5].This transition is thought to be related to the N6-adenine adducts [6] or to thedeaminated 1-adenine adducts [7]. The GC�TA and AT�TA transversions werealso more frequent in SO-treated hprt mutant clones than in the controls [5]. Thesemutations were suggested to be related to apurinic sites originating from depuri-nated 7-guanine and 3-adenine adducts of SO, respectively [5].

Hydroxyalkyl adducts at the 3-position of 2%-deoxycytidine (dCyd), formed bye.g. ethylene oxide, can undergo rapid spontaneous deamination to the correspond-ing 2%-deoxyuracil (dUrd) adducts, which may be of great biological importance[8–13]. 3-(2-hydroxypropyl)-uracil, formed from the corresponding propylene oxideinduced cytosine adduct, has been postulated to be a mutagenic lesion [14]. Theadduct occupies a central Watson-Crick base pairing position thus disruptingnormal hydrogen bonding. If not repaired, this may lead to GC�AT transitionsand to a lesser extent to GC�TA and GC�CG transversions [15]. Recently it hasbeen shown using protein extracts from mammalian cells that an enzymatic repairmechanism exists for the removal of 3-(2-hydroxypropyl)-cytosine but not for thecorresponding uracil adduct. However they are not substrates for uracil glycosylase[16]. Furthermore, a mutagenicity study applying a site-specific incorporation of a3-hydroxyethyluridine adduct in a 55-nucleotide template suggested that the adductmay be a critical mutagenic lesion induced by ethylene oxide, leading to GC�ATand GC�TA mutations [17].

Since the GC�TA transitions were identified also in SO-treated cell lines it maybe that the 3-dUrd adducts are involved in the mutagenicity induced by SO. Thereports describing dCyd alkylation by SO have been somewhat contradictory. Inthe report by Savela et al. [18] dCyd was shown to be alkylated at the 3-, N4- andO2-positions of the base. However, only limited characterisation of the adducts wasgiven. Recently, Barlow and Dipple [19] identified only the diastereomeric pairs ofthe b-isomer of the 3-alkylated dUrd and the a-isomer of the N4-alkylated dCyd.So far dCyd adducts have not been reported in DNA. In order to characterise dCydadducts we determine here the deamination rates for 3-alkylated dCyd adducts.These adducts are also studied in DNA. They may play an important role inSO-induced mutagenicity.

M. Koskinen et al. / Chemico-Biological Interactions 126 (2000) 201–213 203

2. Materials and methods

2.1. Chemicals

Chemicals were used as purchased from the manufacturers. dCyd, cytosine(Cyt), dUrd, salmon testis DNA (sodium salt), alkaline phosphatase and snakevenom phosphodiesterase were purchased from Sigma Chemical Co (St Louis,MO). Nuclease P1 was from Boehringer Mannheim Chemica (Mannheim, Ger-many) and DNase I from Fluka Chemie AG (Buchs, Switzerland). Racemicstyrene oxide (\97% pure) was from Aldrich Chemie (Steinheim, Germany),methanol was gradient grade from Merck (Darmstadt, Germany). All otherchemicals were either from Sigma or Merck.

2.2. Reactions between SO and dCyd, dUrd or Cyt. Identification of the adducts

A total of 12 mg of dCyd and 8 mg of Cyt were treated in 4 ml of 50 mMTris–HCl (pH 7.4) and 30% methanol with 100 mM SO, and 12 mg of dUrdwas treated in 4 ml of 20 mM NH4HCO3/NH3 buffer (pH 10.5) and 30%methanol with 100 mM SO. The mixtures were incubated overnight at 37°C. Theexcess SO was extracted twice with ethyl acetate (1 vol.), and the mixtures wereevaporated to dryness in freeze dryer. The dried mixtures were redissolved inwater and the reaction products were separated by high-performance liquid chro-matography (HPLC, Beckman system Gold with 168 diode array UV-detectormodule), equipped with a C-18 column (Phenomenex, Kromasil, 250×4.5 mm, 5mm), using a binary gradient consisting of 50 mM ammonium formate (pH 4.6)and methanol. Initial elution was with 10% methanol in the buffer for 10 min,followed by an increase of the proportion of methanol first to 30% in 60 minand then to 46% in 20 min and further to 100% in 20 min. Methanol (100%)was maintained for 5 min and decreased to 10% in 10 min. The peaks weredetected by UV-absorption at 262 nm.

The collected reaction products were evaporated to dryness in a freeze-dryerand characterised by electrospray ionization mass spectroscopy (ESI-MS, Finni-gan LCQ LC/MSn system) in the negative ion mode, and by UV-spectroscopy inwater, 0.1 M NaOH and 0.1 M HCl. The isomerism of the hydroxyphenylethylmoiety was differentiated by fragmentation obtained in the ESI-MS/MS experi-ments on the deoxynucleoside adducts or on the depyrimidated adducts. Thea-isomer was identified as a MS/MS product in which the CH2O-fragment wascleaved from the ion corresponding to the alkylated base. The characteristicfragment of the b-isomer was the species devoid of C6H5CHO [20]. Depyri-midation of the dCyd and dUrd adducts was performed in 0.2 M HCl byincubating the product overnight at 95°C, except those reacted at O2-position ofdCyd (see results). The depyrimidation products were purified by HPLC asabove.


2.3. Kinetics of deamination of 3-SO-dCyd

The deamination half-life (t12) of the a-isomer of the 3-alkylated dCyd was

determined at room temperature (�22°C) by UV-spectroscopy and at 37°C byHPLC. By the UV-method the adduct fraction was incubated in 100 ml of 50 mMTris–HCl (pH 7.4) in the cuvette inside the spectrophotometer (Beckman DU-640)and the absorbance at 284 nm was followed for 30 min. The velocity constant (k)was calculated from the slope of a semilogaritmic graph plotting absorbance versustime, and t1

2was derived from k. By the HPLC method, four adduct fractions were

incubated for 0, 10, 20 and 30 min in 50 mM Tris–HCl (pH 7.4) at 37°C and wereanalysed immediately after incubation by HPLC, as above. The t1

2was calculated as

above from the areas of the peaks corresponding to either the disappearing3-SO-dCyd adduct or the forming 3-SO-dUrd. The deamination rate of theb-isomer of the 3-alkylated dCyd was determined by HPLC, as above, except thefractions were incubated 0, 2, 3, 4, and 6.5 h.

2.4. DNA alkylation and hydrolysis

4 mg of salmon testis DNA (3.3 mg/ml) was incubated in 10 mM Tris buffer (pH7.4), 0.15 M NaCl with 14 ml SO at 37°C for 63 h. After incubation, the excess SOwas extracted twice with ethyl acetate, and the 7-guanine and 3-adenine adductswere liberated from the DNA by keeping the solution in boiling water bath for 30min. The DNA was precipitated at −20°C with 4 M sodium acetate (0.1 vol.) andethanol (1.4 vol.). After centrifugation for 20 min (14 000 r.p.m.) the supernatantcontaining the 7-guanine and 3-adenine adducts was evaporated to dryness andanalysed by HPLC, as described [4]. The pellet was washed with 70% ethanol andwas dissolved in 1.5 ml of 50 mM Tris pH 7.2, 1 mM MgCl2. The enzymatichydrolysis was performed as described by Selzer and Elfarra [13] using nuclease P1(24 U/ml), alkaline phosphatase (3 U/ml), DNase I (10 U/ml) and snake venomphosphodiesterase (0.3 U/ml). After incubation for 26 or 48 h at 37°C (both onesgiving similar yields of adducts), the reaction was stopped by keeping the mixturein boiling water bath for 5 min. The mixture was cooled in an ice bath, centrifugedand filtered to another tube. After evaporation to dryness in a freeze-dryer thehydrolysate was analysed by HPLC as for the dCyd alkylation products above. Theadduct concentration in DNA hydrolysate was determined by collecting the adductsfractions from the HPLC separation. After evaporation to dryness in a freeze-dryer,the fractions were dissolved in water and measured by UV-spectrophotometer usingpublished extinction coefficients for corresponding methylation products [21].

3. Results

3.1. dCyd and dUrd adducts of SO

SO was reacted with dCyd and dUrd in order to confirm the adduct assignment.3-, N4- and O2-substituted products were identified on the treatment of dCyd with


racemic SO (Table 1). Fig. 1A shows a chromatogram of the reaction products.Initially, N3 was the highest alkylated site at dCyd. The two diastereomers of theb-isomer (b3-SO-dCyd) were resolved by the chromatographic system used, whileonly one fraction of the a-isomer (a3-SO-dCyd) was detected, the twodiastereomers obviously co-eluting in the same fraction. The isomerism was verifiedby ESI-MS/MS on the adducted base after depyrimidation (Table 2). Thus, in the

Table 1The yields for the SO-dCyd and SO-Cyt adducts

Cyt (%)aAdduct dCyd (%)a

0.14b,c 0.42b,c30.14N4 0.08

0.03c,d 0.12cO2

a The proportion of the adducts formed as compared to the amount of dCyd or Cyt in the reaction.b Contains also the deaminated fractions. The true proportion of 3-SO-dCyd is higher, because some

of the deaminated product is not included due to the interfering products.c The proportions contain both a- and b-isomers.d True proportion is higher because the depyrimidated O2-products are not included.

Table 2UV-spectroscopic and ESI-MS/MS data on the identified adductsa

ESI-MS/MS fragmentAdduct UV-maxima in [M-H]−

ions (m/z)bwater/0.1 M NaOH/ (m/z)0.1 M HCl

230.3, 212.0, 200.1346.3a3-SO-dCyd 281/269/281281/269/281 346.4b3-SO-dCyd 230.6, 212.1, 124.1,

212.1230.3, 212.1, 200.0aN4-SO-dCyd 273/274/285 346.1231.0, 214.0, 200.7aO2-SO-dCyd 268/271/261 347.3

268/271/261 347.3bO2-SO-dCyd 230.9347.1263/264/263 231.1, 213.0, 200.7a3-SO-dUrd

263/264/263 231.1, 213.1, 125.1,n.d.b3-SO-dUrd111.1

277/299/277 230.4b3-SO-Cyt 212.0, 124.1, 110.1268/285/282 n.d.aN4-SO-Cyt n.d.

n.d.n.d.aO2-SO-Cyt 266/269/259266/269/259 n.d. n.d.bO2-SO-Cyt262/289/262a3-SO-Ura n.d. n.d.

n.d.262/289/262b3-SO-Ura n.d.

a n.d.=not determined.b MS/MS fragments are obtained either by fragmentation of [M-H]− or from MS/MS experiments of

the depyrimidated product.


Fig. 1. HPLC separations of the products formed in the reaction between SO and (A) dCyd; (B) dUrd;and (C) Cyt. Peaks in (A): 1, dCyd; 2, phenylethylene glycol; 3, b3-SO-dCyd; 4, a3-SO-dCyd; 5, theshoulder in the front is b3-SO-dCyd, the main peak unknown; 6 and 7, aN4-SO-dCyd; 8, bO2-SO-dCyd;9, aO2-SO-dCyd; 10 and 11, 3-SO-dUrd. Peaks in (B): 1, dUrd; 2 and 3, a3-SO-dUrd; 4 and 5,b3-SO-dUrd. Peaks in (C): 1, Cyt; 2, phenylethylene glycol; 3 b3-SO-Cyt; 4, aN4-SO-Cyt; 5, a3-SO-Ura;6, b3-SO-Ura; 7 and 8, O2-SO-Cyt.


case of the a-isomer, the ion corresponding to adducted base m/z 230 was cleavedto a product ion m/z 200, that is the adducted base lacking CH2O. For the b-isomerthe characteristic MS/MS product ion originating from m/z 230 was 124 indicatingcleavage of C6H5CHO. Also incubation of the 3-SO-dCyd adducts at neutral pHsupported identification, because the treatment converted the adducts to newproducts that were chromatographically, and by the UV-spectra obtained by diodearray detector, identical to 3-substituted dUrds. dUrd, that was used as a referencecompound to identify the deamination products, was reacted at the 3-positionthrough b- (b3-SO-dUrd) and a-carbon (a3-SO-dUrd) (see Fig. 1B). Alkylation waslow at neutral pH but at pH 10.5 the nucleoside became very reactive. Reactionthrough the b-carbon was favoured. When the rates of the spontaneous deamina-tion at pH 7.4 of the 3-SO-dCyd adducts were studied, a3-SO-dCyd was found tobe highly unstable. The t1

2of the adduct was 6 min at 37°C and 10 min at room

temperature (ca. 22°C). The t12

of the deamination of the b-isomer was markedlylonger, being 2.4 h at 37°C.

The N4-position of dCyd opened the epoxide only at the a-carbon (aN4-SO-dCyd), and two diastereomeric forms were resolved. The aN4-adduct was stablewhen treated at pH 7.4 and 100°C for 30 min. In the case of O2-substituted dCydstwo fractions were isolated (aO2-SO-dCyd and bO2-SO-dCyd). The two isomerseluted each in single fraction, obviously containing both diastereomers of theisomer (peaks 8 and 9 in Fig. 1A). MS-analysis on the O2-alkylated products gavem/z with one mass unit more than in the 3- and N4-alkylated products. TheESI-MS/MS analysis of the peak 9 gave a product ion characteristic of thea-isomer. For the b-isomer no characteristic MS/MS product was obtained. It can,however, be assumed that the peak 8 corresponds to the b-isomer because of thedifferences in the stability of the adducts. Thus, when the b-isomer was incubatedat pH 7.4 and 37°C for 30 min the original deoxynucleoside product was completelydisappeared. Instead, in the HPLC analysis two later eluting products wereobserved whose UV-spectra obtained by diode array detector were identical tothose of the O2-alkylated cytosines, which suggests that the adduct is prone todepyrimidation. For the a-isomer, a small fraction of the aO2-SO-dCyd wasdetected by HPLC after 2 h of incubation at similar conditions. In addition, afraction eluting at the same retention time as bO2-SO-dCyd was observed indicat-ing isomerisation of the a- to the b-isomer. When the a-isomer was incubated in 0.1M NaOH for 2 h and 37°C, no trace of the original adduct was observed, indicatinga base catalysed isomerisation.

3.2. Cyt adducts of SO

We reacted SO also with cyt to verify the assignment of the depyrimidationproducts of the nucleoside adducts (Fig. 1C, Table 1). The 3-alkylated productthrough the b-carbon (b3-SO-Cyt) was a major product (Table 1). The correspond-ing uracil adduct was also observed (b3-SO-Ura). The a-isomer of 3-cytosine(a3-SO-Cyt) was not detected obviously because of the rapid deamination to theuracil adduct (a3-SO-Ura). N4-alkylated Cyt (aN4-SO-Cyt) as well as the productscorresponding to O2-alkylation (O2-SO-Cyt) were also identified.


Fig. 2. HPLC separation of the enzymatically hydrolysed SO-treated salmon testis DNA. Peaks: 1 and2, aN4-SO-dCyd; 3 and 4, aN2-SO-dGuo; 5 and 6, b3-SO-dUrd; 7 and 8, aN6-SO-dAdo.

Table 3The yields of SO-induced DNA adducts in salmon testis DNA determined by neutral depurination andenzymatic hydrolysisa

Proportion of total alkylation (%)Adduct Alkylation levelb

38.1a7-SO-guanineNeutral depurination 514349.1b7-SO-guanine 66262.8379a3-SO-adenine1.4b3-SO-adenine 185

4.4aN6-SO-dAdo 589Enzymatic hydrolysis1.9aN2-SO-dGuo 2541.9258b3-SO-dUrd0.5aN4-SO-dCyd 70

a Alkylation level as mean of three determinations.b The alkylation levels are expressed as adducted nucleotides/106 normal nucleotides.

3.3. DNA adducts of SO

The enzymatic hydrolysis and subsequent HPLC analysis of the SO-treateddouble-stranded DNA allowed the detection of the previously unidentified dCyd-re-lated DNA adducts, i.e. b3-SO-dUrd and aN4-SO-dCyd (Fig. 2; Table 3). Theproportion of the b3-SO-dUrd adducts was 1.9% of the total alkylation and that ofaN4-SO-dCyd 0.5%. These adducts in DNA were identified by their co-elution withthe standards and by the UV-spectral properties at neutral, alkaline and acidicconditions. Depyrimidation of the two diastereomic forms of b3-SO-dUrd andaN4-SO-dCyd resulted in single peaks in the HPLC analysis that co-eluted with the


corresponding Ura and Cyt adducts, respectively. The a-isomers of N2-substituted2%-deoxyguanosine (aN2-SO-dGuo) and N6-substituted 2%-deoxyadenosine (aN6-SO-dAdo) adducts were identified by their UV spectral properties at different pHs andby coelution of the depurination products with previously described standards[4,22]. O6-substituted dGuo was detected as minor shoulders after the late elutingaN6-SO-dAdo peak and the following unknown peak. Other products related toSO-alkylation were also detected at ca. 53 min and around the later elutingaN2-SO-dGuo, some of the latter obviously being 1-SO-deoxyinosine adducts afterdeamination of the 1-SO-dAdo adducts [6].

4. Discussion

We have studied the properties of the main SO-induced dCyd adducts, i.e. thosereacted at the 3-, O2- and N4-positions of the base. In addition, this study is thefirst to report SO- adducts at dCyd in double-stranded DNA, i.e. b3-SO-dUrd andaN4-SO-dCyd. The t1

2for deamination of a3-SO-dCyd and b3-SO-dCyd were found

to be 6 min and 2.4 h, respectively, at physiological pH and temperature. This ismarkedly faster than the deamination of 3-hydroxyethyl-dCyd and 3-(2-hydrox-ypropyl)-dCyd, which have t1

2of 10 and 6 h, respectively, at similar conditions [11].

The hydroxyethyl and 2-hydroxylpropyl dUrd-adducts have been suggested to bemutagenic precursors, because they are stable in DNA and no repair activity hasbeen found [14,17]. Furthermore, 3-(2-hydroxypropyl)-dCyd has been shown to beefficiently repaired while the deaminated adduct is persistent and not repaired [16].This could also be the case for 3-SO-dCyd and should be considered when themutagenicity of SO is of concern.

The properties of butadiene monoxide (BMO) induced 3-dCyd adducts [12] arevery similar to those we report now on the 3-SO-dCyd adducts. For BMO induced3-(2-hydroxy-3-buten-1-yl)-dCyd adducts, i.e. those reacted through the terminalepoxide carbon, the deamination t1

2for the two different diastereomers were 2.3 and

2.5 h. By contrast, the deamination of BMO adducts which were bound through2-carbon was so fast that the dCyd adduct was not detected. Thus, for both SO-and BMO-adducts the faster deaminating isomer is the one with a primaryhydroxyl group. Therefore it is tempting to speculate that the nucleophilicity is thefactor that determines the rate of deamination, because the primary hydroxyl isexpected to have a higher nucleophilicity as compared to the sterically hinderedsecondary hydroxyl. The hydroxyethyl adduct has also a primary hydroxyl group,but the rate of deamination is rather slow as compared to the SO and BMOadducts. In these the p-electron system similarly positioned with respect of thehydroxyl group probably causes an increase in the deamination rates. Based on theobservation that 3-methyl and 3-ethyl alkylated dCyds deaminate much slower thanthe 3-hydroxyalkyl analogues, Solomon et al. [8] presented a mechanism of deami-nation involving intramolecular catalysis of the adjacent imine bond by thehydroxyl on the hydroxyalkyl moiety. This can also be adapted to the 3-SO-dCydadducts (pathway A in Fig. 3). However, another pathway can also be presented


involving intramolecular nucleophilic attack (pathway B in Fig. 3). The mechanismB have been proposed for the 1-adenine adducts of SO, and it involves a formationof an oxazolinium ring followed by an attack of water to the C-4 atom of dCyd[23,24]. Similarity of the deamination mechanisms between 1-dAdo and 1-dCydadducts is supported by the observation that for the 1-SO-dAdo adduct thea-isomer was reported to deaminate ca. 35-times faster than the b-isomer [25].Similarly, in the case of the 3-SO-dCyd the deamination of the a-isomer is 24-timesfaster than that of the b-isomer.

Recently, Barlow and Dipple [19] detected for the reaction between dCyd and SOonly the deaminated b3-SO-dUrd but no corresponding a-isomer or any 3-substi-tuted dCyd. In the present study, both isomers of 3-dCyd adducts were isolateddespite their fast deamination in an aqueous solution. These adducts turned out tobe very sensitive to the reaction conditions, and further studies are needed to findout the factor that allowed the detection of a3-SO-dCyd. The exact estimation ofthe relative proportions of the a- and b-isomers, initially reacted at the 3-positionof dCyd, is difficult due to the interfering products eluting at similar retention timesas the 3-SO-dUrd products. Most likely the b-isomer is dominating becausering-nitrogens with relatively high nucleophilicity generally prefer SN2 attack at theless hindered b-carbon [22,25]. Also, the 3-alkylation of dUrd and Cyt proceeded toa higher extent through the b-carbon of SO, being, in the case of dUrd, twice thatof the a-carbon. Unlike BMO, ethylene oxide and propylene oxide, SO reacted alsoat the exocyclic N4-group. The reaction proceeded exclusively through the a-car-bon, being similar to the reaction at exocyclic amino-groups in guanine and adenineresidues [22,25].

Another interesting mechanistic aspect observed in the present study was theinterconversion between the a- and b-isomers of O2-SO-dCyd. Similar interconver-sion has been observed for SO induced O6-guanosine adducts [26]. The base-catalysed mechanism presented for the O6-guanosine adducts can also be suggestedfor O2-SO-dCyd (Fig. 4). It involves an abstraction of a proton from the hydroxyl

Fig. 3. Possible deamination mechanisms for a3-SO-dCyd.


Fig. 4. Interconversion between the two isomeric forms of O2-SO-dCyd adducts.

group of the hydroxyphenylethyl moiety followed by an intramolecular addition toC-2 to form a Meisenheimer complex, which could rearomatize by expulsion of thea-alkoxide function [26]. The isomerization takes place both in alkylated cytosine aswell as the deoxynucleoside. The glycosidic bond of the O2-SO-adduct turned outto be labile similarly to O2-ethyl-dCyd [27], the b isomer of the SO-adduct beingmore labile. In less than 30 min the adduct was depyrimidated completely, beingmarkedly faster than the depurination of the 7-substituted deoxyguanosinemonophosphates (t1

27.4 h and 4.7 h for a- and b-isomer, respectively) [28]. Thus, if

the O2-cytosine adducts are formed in DNA they may also introduce possiblymutagenic abasic sites. This would probably contribute to the CG�TA transitions.

In double-stranded DNA the b3-SO-dUrd adduct was found to constitute aconsiderable proportion of the total alkylation. The method is not informativeregarding 3-SO-dCyd adducts because the DNA hydrolysis procedure involved aheating step to remove the 7-guanine and 3-adenine adducts during which all3-SO-dCyd adducts were deaminated. Unlike alkylepoxides, SO was found to formN4-substituted dCyds at low concentrations in DNA. aN4-SO-dCyd may be apotential mutagenic precursor because it occupies a Watson-Crick hydrogen bond-ing position and is chemically stable. The alkylation levels reported in this study aresomewhat different from our recently published report where the purine adductswere released by acid hydrolysis [4]. In the present study, the proportions of7-guanine adducts, aN6-SO-dAdo and aN2-SO-dGuo were 87.2, 4.3 and 1.9%,respectively. In the previous study they were 93, 0.7 and 1.5%, respectively [4]. Inthe present study DNA was incubated with SO for 63 h while in the previous studythe incubation lasted 23 h. Therefore, the most probable explanation to thedifference is that the 7-alkyl guanine depurinates and thus reaches an apparentsaturation level faster than the positions involved in the base-pairing. Further


studies are in progress to investigate the kinetics of formation the different adducts,and to identify all the SO-alkylation products in the enzyme hydrolysate.

In conclusion, we have detected two new types of SO-induced pyrimidine adductsin DNA. Even though these b3-SO-dUrd and aN4-SO-dCyd adducts are formed toa lesser extent than, e.g. 7-guanine adducts they might have an important contribu-tion to the mutagenicity because of their stability. Furthermore, they may also beuseful in the biomonitoring of styrene.

Acknowledgements

The project was supported by the Swedish Council for Work Life Research.

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Styrene oxide-induced 2′-deoxycytidine adducts: implications for the mutagenicity of styrene oxide