In vitro reactions of 2-cyanoethylene oxide with calf thymus DNA

Chem.-Biol. Interactions, 88 (1993) 115-135

115

Elsevier Scientific Publishers Ireland Ltd.

IN VITRO REACTIONS OF 2-CYANOETHYLENE OXIDE WITH CALF THYMUS DNA

JEROME J. SOLOMON, UDAI S. SINGH and ALVIN SEGAL Department of Environmental Medicine, New York University Medical Center, 550 First Avenue, New York, N Y 10016 (USA)

(Received January 5th, 1993) (Revision received February 10th, 1993) (Accepted February llth, 1993)

SUMMARY

Acrylonitrile oxide (CEO) is a direct-acting mutagen and the postulated proximate carcinogenic form of acrylonitrile (AN). We have studied the reactions of CEO with 2'-deoxyribonucleosides and in vitro with calf thymus DNA at pH 7.0- 7.5 and 37°C for 3 h. Reaction of CEO with dAdo gave 2 adducts, N 6(2-hydroxy-2-carboxyethyl)-dAdo (NS-HOCE-dAdo) (2% yield) and 1,TV6-ethenodAdo (&dAdo) (11%); reaction with dCyd resulted in the isolation of 3-HOCEdUrd (22%); reaction with dGuo gave 7-(2-oxoethyl)-Gua (7-OXE-Gua) (31%) and reaction with dThd yielded 3-OXE-dThd (3%). Structural elucidation of adducts was accomplished by ultraviolet spectroscopy, high-field proton NMR spectroscopy and mass spectrometry. Structural confirmation was provided by an accurate mass measurement technique where diagnostic ions in the electron impact mass spectra of trimethylsilyl derivatives were measured to within 0.0007 atomic mass units. The facile Dimroth rearrangement of 1-HOCE-dAdo to N 6HOCE-dAdo and hydrolytic deamination of a dCyd adduct to 3-HOCE-dUrd is postulated to be catalyzed by the hydroxyl group on the 3-carbon side chain of the adduct. Reaction of CEO with calf thymus DNA yielded (nmol/mg DNA) N 6HOCE-dAdo (2); &dAdo (11); 3-HOCE-dUrd (80); 7-OXE-Gua (110) and 3-OXEdThd (1). Thus CEO, like its metabolic precursor AN, directly alkylates DNA in vitro but at a much more rapid rate. Correspondence to: J.J. Solomon, Department of Environmental Medicine, New York University

Medical Center, 550 First Avenue, New York, NY 10016, USA. Abbreviations: AN, acrylonitrile; CEO, 2-cyanoethylene oxide; BSTFA, bis(trimethylsilyl)-trifluoro-

acetamide; CE, 2-carboxyethyl; CI, chemical ionization; mmu, millimass units; CNE, 2-cyanoethyl; g-Ado, LN6-etheno-2'-adenosine; g-dAdo, 1,N6-etheno-2'deoxyadenosine; EI, electron impact; HOCE, 2-hydroxy-2-carboxyethyl; HOCNE, 2-hydroxy-2-cyanoethyl; HP, 2-hydroxypropyl; HE, 2hydroxyethyl; HA, 2-hydroxyalkyl; HPLC, high-pressure liquid chromatography; OXE, 2-oxoethyl; PFK, perfluorokerosene; PO, propylene oxide; EO, ethylene oxide; TMS, trimethylsilyl. 0009-2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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Key words: 2-Cyanoethylene oxide -- DNA adducts -- Hydrolytic deamination Dimroth rearrangement

INTRODUCTION

Acrylonitrile (AN, CH2~CHCN), is extensively employed as a chemical intermediate in the chemical, pharmaceutical, plastics and rubber industries. The major uses of AN are in the production of acrylic and modacrylic fibers and in the manufacture of acrylonitrile-butadiene-styrene and styrene-acrylonitrile resins [1]. OSHA has estimated that approximately 278 000 workers are potentially exposed to AN in the workplace [1]. Production of AN in the USA in 1991 was 2.65 × 109 lb. [2]. Morphologically transformed foci were observed following treatment in culture of Syrian hamster embryo cells [3], mouse fibroblast C3H/10T1/2 cells and NIH/3T3 cells [4] with AN, and in the latter experiment, growth of morphologically transformed cells in soft agar was demonstrated [4]. AN was reported to be carcinogenic in male and female Sprague- Dawley rats following administration by inhalation and ingestion (drinking water) [5- 7]. The central nervous system was most noticeably affected, especially so after ingestion [7]. AN is also a suspected carcinogen in humans [8-11]. AN is mutagenic in Escherichia coli in the absence of exogenous metabolic activation [12], while metabolic activation was required for mutagenicity in Salmonella typhimurium test strains [13]. Neither bone marrow cytogenic changes in rats and mice [14] nor dominant lethal mutations in mice [15] were observed following administration of AN. It has been shown that AN is weakly mutagenic in human lymphoblasts [16]. The metabolism and genotoxic properties of AN have been reviewed [17]. The major route of AN metabolism involves its direct conjugation via Michael addition (cyanoethylation) to glutathione leading to the formation of N-acetyl-S-(2cyanoethyl)cysteine which has been identified as the major urinary metabolite of AN in rat, rabbit and rhesus monkey [17]. On the basis of the structure of other urinary metabolites of AN, it has been postulated that a fraction of AN is metabolically oxidized to the epoxide 2-cyanoethylene oxide (CEO, CH2(O)CHCN) prior to conjugation with glutathione [17]. Thus, metabolites such as glycolaldehyde, cyanoethanol, cyanoacetic acid, N-acetyl-S-(2-hydroxyethyl)cysteine and thiocyanate are thought to arise via that route [17]. Studies on the metabolism of AN by isolated rat hepatocytes revealed that the major metabolic pathway involved direct alkylation by AN of cysteinyl residues in glutathione and proteins [18]. Alkylation of hepatocellular DNA and RNA and extracellular DNA was not observed to a greater extent than one base in 3.5 × 105 [18]. CEO accumulated in the hepatocytic incubations but did not appear to contribute extensively to alkylation of glutathione or protein [18]. CEO formation and cyanide release have been shown to occur in liver microsomal systems [19,20]. Cyanide ion has been hypothesized to be liberated from an unstable cyanohydrin arising from CEO [21,22]. In comparing the non-enzymatic binding to DNA (in

117

an in vitro system) of labeled AN and CEO, it was found that binding of AN was barely detectable while a significant level of binding of CEO occurred rapidly [20]. CEO has been shown to be an active mutagen in human lymphoblasts [16] and recent studies have revealed the presence of CEO in the blood for at least 2 h after oral administration of AN to mice and for 4 h after oral administration of AN to rats [23]. This latter study indicates that CEO may be transported to target tissues once it has been formed in the liver by the metabolic activation of AN [23]. We have shown that AN can react in vitro extremely slowly with calf thymus DNA at pH 7.0 and 37°C for 40 d to form Michael addition adducts with all bases in DNA [24,25]. The adducts whose structures were identified were 1-(2carboxyethyl)-dAdo, (1-CE-dAdo), 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNEGua) and 3-CNE-dThd [24,25]. Subsequently, the major adduct found at the end of the 40-d reaction, 7-CNE-Gua, was identified and quantitated after 24 h reaction [25]. In this paper we report the structures of the adducts (Fig. 1) formed following reactions of CEO with dAdo, dCyd, dGuo and dThd and in vitro with calf thymus DNA at pH 7.0 - 7.5 and 37°C after 3 h. A preliminary report of this work has been published [26]. MATERIALS AND METHODS

Materials Compounds dAdo, dCyd, ~-dAdo, dThd, dGuo, 3-Me-dCyd, dUrd and calf thymus DNA were obtained from Sigma Chemical Co., St. Louis, MO. Compound 7-OXE-Gua was a gift from Dr. J.A. Miller of the McArdle Laboratory, University of Wisconsin (Madison, WI). AN, (99 + % inhibited with 35-45 ppm hydroquinone monomethyl ether) was obtained from Aldrich Chemical Co., Milwaukee, WI. Sodium hypochlorite solution (10.4% available chlorine) was purchased from Mallinkrodt Chemicals, St. Louis, MO. Bis-Tris buffer and Nuclease P1 from Penicillium citrium were obtained from Calbiochem-Behring Corp., La Jolla, CA. Nuclease P1 was stored at -20°C in 1 mM ZnC12 at a protein concentration of 1 mg/ml (phosphodiesterase activity: 270 units/ml). Alkaline phosphatase from E. coli (360 units/ml) was purchased from Pharmacia P-L Biochemicals, Milwaukee, WI. Acid phosphatase (Type 1) from wheat germ (0.36 units/mg, Sigma Chemical Co.) was stored at -20°C at a concentration of 242 mg/ml in water. Whatman 3 MM paper sheets for chromatography and phosphate buffers, pH 7.0 and pH 8.0 (potassium phosphate monobasic-sodium hydroxide, 0.05 M) were from Fisher Scientific Co., Springfield, NJ.

Paper chromatography Analytical and preparative paper chromatograms were run in a descending manner, and compounds were isolated from paper chromatograms as described previously [27]. UV or fluorescent bands were immersed in H20 (pH 6.3) at 23-25°C for 18 h to insure complete extraction. Paper was removed by filtration and the extract concentrated in vacuo (flash evaporator) at a water bath temperature of 40°C. Rf values of compounds analyzed by paper chromatography are summarized in Table I.

118

HOOC~,,,'%~ NH OH . , ~

_....N

OH

OH

OH

E-dAdo

N6-HOCE-dAdo

OH

O

7-OXE-Gua

O OH o

O

~OH

0 o- -N~_~OH

OH 3-HOCE-dUrd

OH 3-OXE-dThd

Fig. 1. Structure of adducts of 2-cyanoethyleneoxide (CEO) with 2'-deoxynucleosides.

High-pressure liquid chromatography (HPLC) Analytical and preparative HPLC were performed on a Waters Associates instrument with a #Bondapak C-18 column (7.8 x 300 mm) using a linear gradient in 30 min from 100% H20 to 100% methanol at a flow rate of 2 ml/min. Fractions were monitored simultaneously by both a Waters Model 440 UV detector and a Waters Model 420 fluorescence detector. Rt values of compounds analyzed by HPLC are summarized in Table I. Areas of peaks were analyzed by a Waters 840 Chromatography Data System.

119 TABLE I CHROMATOGRAPHIC CHARACTERISTICS DETERMINED BY HPLC CHROMATOGRAPHY (Rf) Compound

Gua dGuo 7-OXE-Gua Ade dAdo N6-HOCE-dAdo N6-HOCE-Ade 1-CE-dAdo N6-CE-dAdo 6-dAdo dCyd dUrd 3-HOCE-dUrd 3-CE-dCyd 3-Me-dCyd 3-Me-dUrd 3-OXE-dUrd dThd 3-OXE-dThd

Solvent systems a (Rf) values) A

B

C

0.49 0.65 0.63 0.63 0.70 0.65 0.58 0.63 0.72 0.77 0.72 0.76 0.70 0.73 0.80 0.93 0.81 0.78 0.79

0.39 0.48 0.43 0.58 0.62 0.35 0.32 0.31 0.45 0,64 0.63 0.68 0.39 0.45 0.74 0.84 0.73 0.70 0.79

0.18 0.22 0.30 0.40 0.35 0.08 (10 cm)b 0.09 (11 cm) 0.08 (9.5 cm) 0.12 (12.5 cm) 0.37 0.28 0.37 0.07 (7.2 cm) 0.16 {14.5 cm) 0.53 0.70 0.53 0.50 0.72

(Rt) AND PAPER HPLC, Rt (min)

12.8 15.4 15.0 16.7 18.1 11.4 10.2 13.2 12.4 19.1 12.5 13.1 6.4 12.1 14.4 16.7 16.5 15.7 18.4

aSolvent systems: A, ammonium acetate (1 M, pH 7.5)/100% ethanol (1:1.9, v/v); B, isopropanol/H20 (2.3:1); C, 1-butanol/100% ethanol/H20 (8:1:2.5). bDistances in parenthesis signify migration by adducts following continuous elution with solvent system C for 72 h.

Instrumentation UV spectra. UV spectra of aqueous solutions were recorded at pH 1 (HC1), pH 6.3 (H20) and pH 13 (NaOH) in a Beckman Model 25 spectrophotometer. Mass spectra. Electron impact (EI) and chemical ionization (CI) mass spectra were analyzed on a VG-70SE high resolution mass spectrometer interfaced to a VG 11/250 data system (VG, Manchester, UK). The source temperature was kept at 210°C and the ionizing voltage was 100 eV. Approximately 1 ~g of material was added to the solids probe in glass capillary tubes and gradually heated until spectra were obtained. Trimethylsilyl (TMS) derivatives were prepared by reacting approximately 25 ~g of dry adducts with bis(trimethylsilyl)trifluoroacetamide (BSTFA) at 100-120°C for 1 h. Isobutane (Matheson research grade, 99.995%) was used as the reagent gas for CI experiments at a source housing pressure of 3 x 10 -5 mbar. Spectra were recorded at 2 seconds/decade over the mass range m/z 900-45 (EI); 900-65 (CI). Data presented here have been background subtracted. Accurate mass measurement. The exact mass of the molecular ion, M +, and

120

the loss of methyl, M-CH3, in the EI mass spectra of the TMS derivatives of the reaction products of CEO with dAdo, dCyd, and dGuo were determined by an accurate mass measurement technique. Mass spectra were acquired at 5400-10 000 resolution while scanning the accelerating voltage linearly for 10 s over a narrow mass range which included three perfluorokerosene (PFK) reference peaks and the mass spectral peaks whose accurate mass was to be determined. Two of the PFK peaks which spanned the mass of interest were used to establish the mass scale and the third reference peak was used to verify the accuracy of mass measurement. In all experiments, the accuracy was found to be better than 0.0007 atomic mass units. Data were collected for at least 10 scans of the reference plus unknown in the multichannel analysis mode. NMR spectra. 1HNMR spectra were obtained on a Bruker AM 360 (360 MHz) spectrometer and all chemical shifts (6) are reported utilizing tetramethylsilane as an internal standard (0.0).

Methods Preparation of CEO. CEO was prepared from the reaction of sodium hypochlorite with AN as previously described [28] with the exception that 160 ml of sodium hypochlorite solution (instead of 122 ml) was stirred with AN (200 ml) due to the smaller amount of available chlorine (10.4% vs. 13%o). The reaction mixture containing CEO in AN was dried over anhydrous MgS04 and could be stored in this condition at -20°C for at least 2 weeks. However, the neat form of CEO which is a colorless liquid undergoes rapid polymerization and was therefore freshly distilled from the stored mixture before each reaction. The identity of the synthesized CEO was checked by mass spectrometry (CI, MH ÷ at m/z 70) and the purity checked by gas chromatography. Yields of CEO were in the range of 10-12% compared to 35% reported in the literature [28]. This method of synthesis produces CEO with a purity of greater than 99%, as established by gas chromatography and NMR [28].

Preparation and characterization of marker compounds reactions of CEO with dAdo, dCyd, dGuo, dThd and dUrd. CEO (50 retool, 3.5 g) and 2'deoxyribonucleoside (0.5 mmol) in 25 ml phosphate buffer, pH 7.0, were shaken in a glass stoppered Erlenmeyer flask at 37°C for 3 h. A major portion of CEO was insoluble in water and formed globules with a density close to that of water. The globules dissolved within the first 20 min of the 3 h incubation period. At the end of the incubation period the pH of the reaction mixtures had risen to between 7.3 and 7.5. An exception to the above reaction protocol was the reaction between CEO and dGuo. At the end of the reaction, the reaction mixture was extracted with 6 × 25 ml portions of chloroform to remove CEO. The mixture was then made pH i by addition of aqueous 2 N HC1, heated at 70°C for 20 min and then adjusted to pH 3 - 4 by addition of 5 N NaOH. Higher pH's result in the precipitation of Gua and possibly Gua adducts. The hydrolysis was performed to convert any 7-alkyl-dGuo present to 7-alkyl-Gua and 06-alkyl-dGuo to 06. alkyl-Gua. Under the hydrolysis conditions used Gua and Gua adducts are released and 06-alkyl-Gua is not hydrolyzed to Gua [29]. Adducts were isolated by preparative paper chromatography and further purified by preparative HPLC.

121

Aliquots of the reaction mixtures were also applied to HPLC for qualitative and quantitative analysis. The reaction mixtures could be stored at -20°C for at least 2 weeks without detectable changes. Quantitative analysis of the reactions between CEO and 2 'deoxyribonucleosides. With the exception of the reaction between CEO and dCyd, the quantitation of adducts formed by the reactions of CEO with 2'deoxyribonucleosides was performed in the following manner. Aliquots of the 3 h reaction mixtures (and the CEO-dGuo acid hydrolysis product) were applied to HPLC. For each reaction mixture, the areas of the adduct(s) and the 2'deoxyribonucleoside or base were determined on HPLC at 254 rim. Known mole % mixtures of the 2'-deoxyribonucleoside or base and marker adducts or authentic compounds were prepared and the peak areas on HPLC were used to generate calibration curves relating mole % to area. These curves were then used to relate HPLC areas of the reaction mixtures to yields. The quantitation of adducts formed by reactions between CEO and 2'-deoxyribonucleosides and in vitro between CEO and calf thymus DNA (see below), was determined using the extinction coefficients of the following compounds: N6-Me-dAdo, e 15 400, [30] for N6-HOCE-dAdo; 7-CE-Gua, e 7200 [31] for 7-OXE-Gua; 3-Me-Urd, 9500 [30] for 3-OXE-dThd, 3-OXEd-Urd and 3-HOCE-dUrd; E-dAdo, e 6000 [32] for ~-dAdo. The yield of the product of the reaction between CEO and dCyd was determined as follows. An aliquot (1 ml) of the freshly prepared CEO-dCyd reaction mixture was applied to a paper chromatogram (paper 1) and at the end of the 3 h reaction another i ml aliquot was applied to another paper chromatogram (paper 2). Both papers were eluted with solvent system B. Paper i contained only dCyd which was extracted with H20 and concentrated to a known volume. Paper 2 contained adduct and unreacted dCyd and the adduct was extracted with H20 and concentrated to a known volume. The yield of adduct was then determined following applications of aliquots of adduct and dCyd solutions to HPLC. Reaction of CEO with dAdo. The reaction of CEO with dAdo gave two major products when analyzed by paper chromatography with solvent systems A, B and C and HPLC. One product was a UV absorbing compound, designated CEOdAdo, and the other, a fluorescent compound (when viewed on paper chromatograms with UV light at 254 nm), was designated CEO-dAdo-F. Compound CEO-dAdo was isolated by preparative paper chromatography using solvent system C and further purified by HPLC. Due to the highly polar nature of the compound and its consequent slow mobility on paper chromatograms eluted with solvent system C, the best resolution from trace fluorescent components of the reaction mixture was obtained by eluting the chromatogram for 72 h at which time the CEO-dAdo band had migrated 10 cm. Compound CEO-dAdo-F was isolated by preparative paper chromatography using solvent system A and further purified by preparative HPLC. The UV spectra of CEO-dAdo was identical to reported spectra of N6-CE dAdo [33] and N6-Me-dAdo [30]. When CEO-dAdo was incubated at pH 1 and 70°C for 20 rain [29] it was completely converted to a new UV absorbing compound which was isolated by preparative paper chromatography using solvent

122

system C and was designated CEO-Ade. The UV spectra of CEO-Ade was identical to the reported spectra of N6-CE-Ade [33] and N6-Me-Ade [34]. Attempts to obtain EI and CI spectra of CEO-dAdo were unsuccessful due to the polarity of the adduct. Derivatization with BSTFA resulted in a volatile product which gave excellent spectra with a molecular ion, M ÷, in EI at m/z 627 and a protonated molecular ion MH ÷, in CI (isobutane, data not shown) at m/z 628 indicating a molecular weight of 627. The isotopic pattern of the molecular ion revealed a tetra-TMS derivative. The electron impact mass spectrum is given in Fig. 2A. The identity of the major ions are labelled. The structure assigned to this derivative was N6-HOCE-dAdo-(TMS)4. To verify this assignment, we determined the accurate mass of M ÷ and the methyl fragment, M-CH3. Table II gives the results of these experiments and indicated measured masses for these ions within 0.4 millimass units (mmu) of that expected. 1HNMR (360 MHz; D20) ~8.21 (s, 1H, HS), 8.19 (s, 1H, H2), 6.40 (t, 1H, J = 6.9 Hz, HI'), 4.58 (m, 1H, H3'), 4.24 (m, 1H, --CH(OH)CO2H), 4.11 (m, 1H, H4'), 3.79-3.68 (m, 4H, H5' 0 II

A

lOO

'-I

-

73 3

50

"

I

N I - HOCE - dA¢lo - ~ 1 4

I ~7o 1103 ,

0

OTMS I

CH=(OTMS)COTMS

155J

,.[[J,.AI.,..J .......... 100

B10O _

II

TMSO~

M-CH(OTMS)COTMS 408

I

200

300

400

M .. 627 6,,: M'CH3 / j

II

500

600

S~CH3b

o

73"

TMSOC__CH_CH 2~ N3

~

50 147

0 C

LII ,, L ~..

329

~t~.,

~

,,,., J . .

......

T M S O ~ ~9

M-CH3 Si(CH3~ 73

M-CH3 -TMSNHCN 28O

M 409

L~

~r

394

100

50

0 II M-COTMS 487 x 5

bH.CH 3

TMSO

CH ~.CH-- OTMIS I

7 -OXE - Ou~ - (1MS) 3 M409

m/z Fig. 2. Electron impact mass spectra of trimethylsilyl (TMS) derivatives of CEO-DNA adducts.

(A) N6-HOCE-dAdo-(TMS)4, (B) 3-HOCE-dUrd-(TMS)4, (C) 7-OXE-Gua-(TMS)3.

123 TABLE II ACCURATE MASS MEASUREMENT-TRIMETHYLSILYL (TMS) DERIVATIVES OF CEODNA ADDUCTS Compound

Elemental Composition

Ionic Species

Calculated Mass

Measured Mass

Ns-HOCE-dAdo-(TMS)4

C25H49NsO68i4

M MM MM MM M-

627.2760 612.2525 419.1809 404.1574 604.2488 589.2253 409.1786 394.1551

627.2764 612.2528 419.1814 404.1574 604.2490 589.2251 409.1779 394.1552

~-dAdo(TMS)2

ClsH29N503Si2

3-HOCE -dUrd-(TMS)4

C24H48N208Si4

7-OXE-Gua-(TMS)~

C16H31NsO2Si3

CH 3 CH a CH a CH a

and --CH2--), 2.78 (m, 1H, H2 '-B), 2.49 (m, 1H, H2 '-~). The structure assigned to CEO-dAdo was N~-HOCE-dAdo. Compound CEO-dAdo-F had identical Rf values on paper chromatograms with solvent systems A, B and C and an identical retention time on HPLC as an authentic sample of E-dAdo. The UV spectra of CEO-dAdo-F was identical to the spectra of commercial E-dAdo and to the reported spectra of E-Ado [32]. The EI mass spectra of CEO-dAdo-F was essentially identical to the spectra of E-dAdo reported by Green and Hathaway [35]. Trimethysilylation of CEO-dAdo-F and authentic E-dAdo gave similar spectra consistent with the spectra previously obtained [35]. Verification of the authenticity of the reference E-dAdo was established by measuring the accurate mass of the molecular ion of the TMS derivative at m/z 419.1814 (Table II) in excellent agreement with 419.1809 predicted for the M ÷ of E-dAdo-(TMS)2, (ClsH29NsOsSi2+). 1HNMR (360 MHz; D6-DMSO) ~9.30 (s, 1H, HS), 8.54 (s, 1H, H2), 8.08 and 7.56 (2s, 2H, etheno), 6.48 (t, 1H, J -- 6.7 Hz, HI'), 5.38 (d, 1H, J -- 4.1 Hz, 3'-OH), 4.99 (t, 1H, J = 5.5 Hz, 5'-OH), 4.44 (m, 1H, H3'), 3.90 (m, 1H, H4'), 3.59 (m, 2H, H5'), 2.74 (m, 1H, H2'-8), 2.37 (m, 1H, H2'-a). Compound CEO-dAdo-F was assigned the structure E-dAdo. The yields of N6-HOCE-dAdo and E-dAdo were 2 and 11%, respectively (Table III). Preparation of 1-CE-dAdo and N6-CE-dAdo. Compounds 1-CE-dAdo and N 6CE-dAdo were prepared from the reaction of ~-propiolactone with 2'deoxyadenosine-5 '-phosphate [33,36]. Dimroth rearrangement studies on 1-CE-dAdo. A solution of 1-CE-dAdo in phosphate buffer, pH 7.0, was incubated at 37°C for 3 h, then made pH 6.3 by addition of aqueous HC1 and incubated at 23-25°C for an additional 18 h. The solution was analyzed by HPLC for the presence of the Dimroth rearrangement product, N6-CE-dAdo at 3 h and then after the 18 h incubation. Reaction of CEO with dCyd. Paper chromatographic analysis of the reaction mixture with solvent systems A, B and C and analytical HPLC demonstrated a

124 TABLE III YIELDS OF ADDUCTS FOLLOWING REACTIONS OF CEO WITH dAdo, dCyd, dGuo, dThd AND dUrd (pH 7.0, 37°C, 3 h) 2 'Deoxynucleoside

Products

Yields (%)

dAdo

N6-HOCE-dAdo ~-dAdo 3-HOCE-dUrd 7-OXE-Gua 3-OXE-dThd 3-OXE-dUrd

2 11 22 31 3 6

dCyd dGuo dThd dUrd

new UV absorbing compound in addition to unreacted dCyd. The new compound was designated CEO-dCyd. Compound CEO-dCyd was isolated by preparative paper chromatography using solvent system B. The UV spectra of CEO-dCyd differed from that of a 3-alkyl-dCyd adduct, e.g., 3-CE-dCyd [37], 3-ethyl-Cyd [34] or 3-HE-dCyd [38] but was identical to the reported UV spectra of 3-methylUrd [30,39] and 3-hydroxyalkyl-dUrd (3-HA-dUrd) [38,40]. The compound was further purified by HPLC. Here again involatility prevented direct EI and CI mass spectral analysis. The EI spectra of the TMS derivative is given in Fig. 2B which clearly indicates a molecular weight of 604 which was confirmed by MH ÷ in CI (data not shown) at m/z 605. Accurate mass measurement (Table II) of M and M-CH3 ions established the TMS derivative as 3-HOCE-dUrd-(TMS)4. 1HNMR (360 MHz; D6-DMSO) 87.86 (d, 1H, J -- 8.1 Hz, H6), 6.19 (t, 1H, J = 6.1 Hz, HI'), 5.71 (d, 1H, J~8.1Hz, H5), 5.32 (br, 1H, 3'-OH), 5.10 (br, 1H, 5'OH), 4.24 (m, 1H, H3'), 4.17 (m, 1H, --CH(OH)CO2H), 3.92 and 3.83 (2 m, 2H, --CH2-CH), 3.79 (m, 1H, H4'), 3.57 (m, 2H, H5'), 2.11 (m, 2H, H2'-~fl). The structure assigned to compound CEO-dCyd was 3-HOCE-dUrd (yield, 22%; Table III). Reaction of CEO with dGuo. Paper chromatographic and HPLC analysis of the acid-hydrolyzed reaction mixture revealed the presence of a new UV absorbing compound, designated CEO-Gua, in addition to Gua. Compound CEO-Gua was isolated by preparative paper chromatography with solvent system B and further purified by preparative HPLC. The new adduct had UV spectra, Rf values on paper chromatography with solvent systems A, B and C and a retention time on HPLC which were identical with those of an authentic sample of 7-OXE-Gua. The UV spectra of CEO-Gua was identical to the reported spectra of 7-CE-Gua [31]. The EI spectrum of the TMS derivative (Fig. 2c) indicated a molecular weight of 409. Accurate mass measurement of M and M-CH3 ions (Table II) established the TMS derivative as 7-OXE-Gua-(TMS)3. The structure assigned to CEO-Gua was therefore 7-OXE-Gua (yield 31%, Table III). Reactions of CEO with dThd and dUrd. The reaction of CEO with dThd and with dUrd each yielded a single UV absorbing product by paper chromatographic analysis with solvent systems A, B and C and by analytical HPLC. The product

125

of the CEO-dThd reaction was designated CEO-dThd while the product of the CEO-dUrd reaction was designated CEO-dUrd. The UV spectra at pH 1, 6.3 and 13 of CEO-dThd exhibited a Xma~at 267 nm and a Xmi, at 237 nm while the spectra of CEO-dUrd exhibited a Xm~ at 262 nm and a Xmi, at 231 nm which indicated the absence of a dissociable proton at N3 in both products [41]. The UV spectra of CEO-dThd was essentially identical to 3-CE-dThd [42] and 3ethylthymidine [34] while the UV spectra of CEO-dUrd closely resembled that of 3-Me-Urd [34] and 3-HA-dUrd [38,40]. The CI spectra of these compounds are given in Fig. 3. The structure revealed in Fig. 3A for CEO-dUrd is 3-OXE-dUrd of molecular weight 270. A protonated molecular ion is seen at m/z 271 and typical glycosidic fragmentation to base plus two hydrogens, bH2, is observed at m/z 155. Sugar fragmentation to S at m/z 117 and S-H20 at m/z 99 are as expected. The structure determined for CEOdThd by the CI spectra given in Fig. 3B is 3-OXE-dThd of molecular weight 284. As discussed above MH is seen at 285 with bH2 at m/z 169 and sugar ions at m/z 117 and 99, respectively. 1HNMR (360 MHz; D6-DMSO) a9.54 (s, 1H, --CI4 O), 7.86 (s, 1H, H6), 6.19 (t, 1H, J = 6.7 Hz, HI'), 5.20 (br, 2H, 3',5'-0H), 4.69 (s, 2H, --CH2-CHO), 4.25 (m, 1H, H3'), 3.79 (m, 1H, H4'), 3.59 (m, 2H, H5'), 2.12 (m, 2H, H2'-aft), 1.84 (s, 3H, CI-I3). The structures assigned to CEO-dThd and CEO-dUrd were 3-OXE-dThd and 3-OXE-dUrd respectively. The yields of 3OXE-dThd and 3-OXE-dUrd were 3 and 6%, respectively (Table III).

100 -A

O

55 bH 2

O

HC --CH2--N

3

O

50-

b

HOCH2 99

153 270

s 117 127 113 |

,.!d.L

0 , I,J,, 100 5O

.

HO"

MH 271 xl0 , ,,-4b i .

.

,_.,...

.

,,,

,. ........

200

150

250

360

350

0

169

1001- B

3 - OXE -dUrd

0

HC--CH2--N 3

bH 2

OH 3

o s 117

O2 _ ~

HOCH

]

sb 167 t17 M 284

H

I 127++o 0

209

_ LJ ,I

50

16o

igo

200 m/z

3 - OXE - dThd

MH - H20 xl0 267 MH 285

250

I

3(~0

350

Fig. 3. Chemical ionization (i-C4Hlo) mass spectra of oxoethyl (OXE) adducts of CEO with dUrd (A) and dThd (B).

126

Preparation of 3-CE-dCyd. Compound 3-CE-dCyd was prepared from reaction of ~-propiolactone with dCyd [37]. Hydrolysis studies on 3-CE-dCyd. Solutions of 3-CE-dCyd were incubated in phosphate buffer, pH 7.0 and pH 8.0 at 37°C for 48 h. In vitro reaction of CEO with calf thymus DNA. A solution containing 75 mg of calf thymus DNA (1800 A260units) and 34 mmol (2.3 g) of CEO in 25 ml phosphate buffer, pH 7.0; was shaken in a glass stoppered Erlenmeyer flask at 37°C for 3 h. The reaction was monitored with a pH meter at 10 - 15 min intervals and the pH had risen to 7.5 after 3 h. Determination of depurinated 7-OXE~Gua in the solution of CEO-reacted DNA. An aliquot of the reaction mixture containing 9 mg of CEO-reacted DNA was applied to a paper chromatogram and eluted with solvent system C. DNA remained at the origin and that portion of the paper chromatogram between the DNA band and the solvent front (40 cm from the origin) was extracted with H20 and the extract concentrated in vacuo (flash evaporator) at 40°C to 2 ml. The 7-OXE-Gua peak on HPLC had an identical Rt a s marker 7-OXE-Gua and was quantitated using marker 7-OXE-Gua. Determination of 7-OXE-Gua remaining in CEO-reacted DNA. Immediately following the 3 h reaction of CEO with DNA, a portion of the reaction solution was dialyzed against phosphate buffer, pH 7.0, at 0 - 4 ° C for 18 h (4 ml of reaction solution dialyzed against 200 ml phosphate buffer, 3 changes of buffer) to remove unreacted CEO and spontaneously depurinated 7-OXE-Gua. The CEOreacted DNA was then heated at 70°C for 2 h [36] to completely release 7-OXEGua. An aliquot of the solution containing 9 mg of CEO-reacted DNA was applied to a paper chromatogram and eluted with solvent system C. Depurinated DNA remained at the origin and that portion of the paper chromatogram between the DNA band and the solvent front (42 cm from the origin) was extracted with H20 and the extract concentrated in vacuo at 40°C to 3 ml. The 7-OXEGua peak on HPLC was identified and quantitated using an authentic sample of 7-OXE-Gua. The UV spectra of the concentrated aqueous extract was identical to the spectra of the authentic sample of 7-OXE-Gua (obtained from J. Miller) and to the reported spectra of 7-CE-Gua [31].

Determination of other 2'-deoxyribonucleoside adducts by enzymatic hydrolysis ofCEO-reactedDNA. Immediately following the 3 h reaction between CEO and DNA, an aliquot of the solution containing CEO-reacted DNA was dialyzed against bis-Tris buffer, pH 6.5 (50 mM bis-Tris/1 mM MgC12) [27] at 0-4°C for 18 h. The CEO-reacted DNA in bis-Tris buffer was enzymatically hydrolyzed to 2'deoxyribonucleosides at 37°C for 18 h using Nuclease P1 (24 U; phosphodiesterase), bacterial alkaline phosphatase (2.4 U) and wheat germ acid phosphatase (0.3 U) per ml of DNA solution [29]. The concentration of CEOreacted DNA was 1.5 mg/ml. The reaction was stopped by heating the sample at 100°C for 5 min and then centrifuged to remove the denatured protein [29]. A portion of the hydrolysate representing 9 mg of CEO-reacted DNA was applied to a paper chromatogram and eluted with solvent system C. That portion of the chromatogram between the origin and the dGuo band was extracted with H20, concentrated in vacuo at 40°C to 3 ml and examined via HPLC. Com-

127

pounds N6-HOCE-dAdo and 3-HOCE-dUrd were identified and quantitated using the markers N6-HOCE-dAdo and 3-HOCE-dUrd, respectively. In an identical manner that portion of the chromatogram between dThd and the solvent front was extracted with water and 3-OXE-dThd was identified and quantitated using marker 3-OXE-dThd. Another portion of the hydrolysate representing 9 mg of CEO-reacted DNA was applied to a paper chromatogram and eluted with solvent system A. That portion of the chromatogram between the dAdo and dThd bands was extracted with H20 and concentrated in vacuo at 40°C to 3 ml. Compound ~-dAdo was then identified and quantitated on HPLC using a commercial sample of E-dAdo. RESULTS AND DISCUSSION

In this study we have characterized the reaction products of CEO with 2'deoxynucleosides and in vitro with calf thymus DNA at pH 7 - 7.5 and 37°C for 3h. Previous work in our laboratory had established the structures of DNA adducts formed by the metabolic precursor of CEO, namely AN [24,25], and two structural analogs of AN, namely acrylamide [43] and acrylic acid [44]. The fact that the adducts identified during our present studies resulted from attack by the most nucleophilic nitrogen atoms in the nucleosides is commensurate with CEO acting as an SN2 alkylating agent. In order to provide reference (marker) compounds to help identify adducts isolated from CEO-reacted DNA we reacted CEO with dAdo, dCyd, dGuo and dThd at pH 7.0-7.5 and 37°C for 3 h. The reaction time chosen was based on a reported half-life of 2 h for CEO in phosphate buffer, pH 7.4 at 37°C [20]. Reaction of CEO with dAdo yielded 2 products 8-dAdo (11% yield) and N 6HOCE-dAdo (2% yield) (Table III). Compound 8-dAdo was characterized on the basis of chromatographic properties and UV and mass spectral data which were identical to those of an authentic sample. The proton NMR spectrum revealed the presence of two singlets at 68.08 and 7.56 in addition to the signals arising from dAdo. These signals did not exhibit any splitting, possibly due to a small coupling constant (J < 1 Hz), and are in agreement with published data [45]. The structure of N6-HOCE-dAdo was assigned on the basis of chemical properties (acid hydrolysis), UV spectroscopy and mass spectra of its TMS derivative. The proton NMR spectrum displayed two multiplets at 64.24 and 3.74, corresponding to the methyne and methylene protons of the side chain, respectively, with the latter partially obscured by the 5'-sugar protons. The formation of N6-HOCE-dAdo may be understood with reference to the products obtained in our previous studies on the reactions of AN [25] and acrylamide [43] with dAdo using essentially identical reaction conditions of temperature and pH. In essence the formation of N6-HOCE-dAdo is a three step process involving initial alkylation at N1, rapid hydrolysis of the cyano group to a carboxy functionality, followed by a Dimroth rearrangement (Fig. 4). Reaction of AN with an endocyclic nitrogen on dAdo (N1) which was adjacent to an exocyclic nitrogen atom (N 6) resulted in a facile hydrolysis of the nitrile moiety to a carboxylic acid yielding 1-CE-dAdo instead of the expected Michael addition

128

NH2 ~lC~

~

N~,~ _N

CEO pH7, 37°C

I

dR

dR dR.2'-clloxyribole

1

÷2HzO - N~

÷NH2

I

H~

1 *

Dirnroth Rearrangement

HOOC~

NH

f

I

dR

dR

E-dAdO

Na.HOCE.dAdo

*brackets indicate postulatedintermediates

Fig. 4. Proposed pathways for formation of dAdo adducts with CEO. Brackets indicate intermediates which were not detected, dR, 2'-deoxyribose.

cyanoethyl adduct [24,25]. This hydrolysis was probably intramolecularly acidcatalyzed by the exocyclic nitrogen atom which possesses a full positive charge at physiological pH [46] once the endocyclic nitrogen atom is alkylated. Since the nitrile group hydrolyzes to a carboxylic acid via an amide intermediate, we hypothesized [43] that binding of acrylamide, like AN, to an endocyclic nitrogen on a base adjacent to an exocyclic nitrogen atom would afford similar results. This hypothesis was confirmed when following reaction of acrylamide with dAdo the adduct 1-CE-dAdo was identified [43]. Thus the rapid hydrolysis of the nitrile to the carboxylic acid, observed following alkylation at N1 of dAdo by CEO, is consistent with our previous findings with AN and acrylamide. When propylene oxide (PO) was reacted with dAdo at pH 7.0 and 37°C for 10 h, the only product

129 detected was N6~2-hydroxypropyl)-dAdo (N6-HP-dAdo) [40]. We hypothesized that the presence of the hydroxyl group on the hydroxypropyl sidechain catalyzed the Dimroth rearrangement of an initially formed 1-HP-dAdo to N6-Hp dAdo. Binding of CEO to a nucleophilic center such as N1 of dAdo would result in an epoxide ring opening with the formation of a hydroxyl group on the second carbon of the three-carbon sidechain, analogous to the reaction with PO. We should thus expect that Dimroth rearrangement of an N1 adduct of dAdo would occur. Indeed the product isolated from the reaction between CEO and dAdo was N6-HOCE-dAdo rather than 1-HOCE-dAdo. Under conditions similar to the reaction and extraction procedures used for the reaction of CEO with dAdo (incubation at pH 7.0 and 37°C for 3 h followed by incubation at pH 6.3 and 23-25°C for 18 h) Dimroth rearrangement of 1-CE-dAdo to N6-CE-dAdo could not be detected at a level of 1%. This lends support to our suggestion that the hydroxyl group assists in the Dimroth rearrangement of Nl-dAdo adducts since the only difference between CE and HOCE adducts at N1 of dAdo is the presence of an hydroxyl moiety on the side chain. Reaction of CEO with Ade to make a marker 3-HOCE-Ade adduct for DNA studies were unsuccessful. Trace UV absorbing bands were observed by paper chromatography which, when extracted and analyzed, did not yield a 3-alkyl-Ade UV spectra. The isolation of E-dAdo following-reaction of CEO with dAdo and the failure to detect 3~V4-ethenodeoxycytidine following reaction of CEO with dCyd is consistent with the observation of Guengerich et al., [20] who detected ~-Ado following reaction of CEO with Ado but failed to detect 3~4-ethenocytidine following reaction of CEO with Cyd. The postulated mechanism for the formation of ~dAdo and N6-HOCE-dAdo (Fig. 4) from CEO and dAdo involves the initial formation of the cyanohydrin, 1-HOCNE-dAdo (not detected). At this juncture the pathway diverges. N6-HOCE-dAdo is formed via the two-step mechanism postulated earlier (vide supra) while ~-dAdo results from the loss of HCN to form the aldehyde, 1-OXE-dAdo (not detected), followed by cyclization via loss of H20. The rapid loss of HCN from 1-HOCNE-dAdo is consistent with previous observations. The cyanohydrin, mandelonitrile, undergoes loss of HCN to form benzaldehyde [47]. The reaction is rapid at pH 6, 8 and 9 (105 min incubation at 30°C) with yields of benzaldehyde of about 55, 65 and 75%, respectively. Loss of HCN is considerably slower at pH 4 and 5, after 105 min at 30°C where between 10-15% of mandelonitrile is converted to benzaldehyde. The cyclization of the aldehyde intermediate, 1-OXE-dAdo, to E-dAdo is also supported by the observation that in vitro reaction of chloroacetaldehyde (a metabolite of vinyl chloride) with calf thymus DNA results in the formation of E-dAdo [35]. Reaction of CEO with dCyd yielded a single product assigned the structure 3HOCE-dUrd (22% yield, Table III) on the basis of UV, 1HNMR and mass spectra. The 1HNMR spectrum of this compound exhibited three multiplets at 84.17, 3.92 and 3.83 with the most downfield signal corresponding to the proton alpha to the carboxyl moiety and the two upfield signals, which exhibited geminal coupling, corresponding to the methylene protons adjacent to N3 of the pyrimidine ring. Reaction between CEO and dCyd, like that observed previously for the reaction between the epoxides PO [40], EO [38] or glycidol [48] and dCyd, yield-

130

ed a 3-hydroxyalkyl-dUrd (3-HA-dUrd) product (3-HOCE-dUrd). As was observed for the reaction between CEO and dAdo the Michael addition cyanoethyl adduct was not detected. Alkylation of dCyd at N3 results in the formation of an adduct which is protonated at physiological pH [38]. Similar to the scenario we suggested for the formation of N6-HOCE-dAdo, here the protonated amino group at N 4 is responsible for the conversion of the cyano moiety to a carboxylic acid via intramolecular acid-catalysis (Fig. 5). Once this transformation is complete we suggest that the rapid hydrolytic deamination of cytosine to uracil is catalyzed by the presence of the hydroxyl group on the 3-carbon side chain (Fig. 5), as has been observed with EO, PO and glycidol [38,40,48]. Compound 3-CEdCyd, which has an identical side chain as 3-HOCE-dCyd, (the postulated precursor of 3-HOCE-dUrd; Fig. 5) but lacks the side-chain hydroxyl group, remained unchanged following incubation at pH 7.0 or 8.0 and 37°C for 48 h, supporting the postulated catalytic effect of the side-chain hydroxyl group. Based on these observations we postulate that the side-chain hydroxyl group attacks C4 of the pyrimidine ring to form a transient five-membered ring. This is followed by the hydrolysis of the C4-N 4 bond resulting in the loss of ammonia and ring-opening of the five-membered moiety to afford the adducted dUrd compound. Reactions of CEO with dGuo, dThd and dUrd resulted in the alkylation of endocyclic nitrogen atoms not adjacent to an exocyclic amino group and the adducts isolated were 7-OXE-Gua (31% yield), 3-OXE-dThd (3% yield) and

NH2

CEO +

+ NH2

<

,~.~1 O N

pH7, 37°C

0~

I

dR

-N

I

~

dR

dR-2'-deoxydbose + 2H20 NH:I

o HOOC~ ~

HOOC~ A

T OH

-.3

I

o A l l )

o2i)

+ H=O - NH3

I

I

dR

dR 3.HOCE-dUrd

•

3-HOCE-dCyd "brackets indicate postulated intermediates

Fig. 5. Postulated mechanism for the formation of 3-HOCE-dUrd from the reaction of CEO with dCyd. Brackets indicate intermediates which were not detected, dR, 2 '-deoxyribose.

131

3-OXE-dUrd (6% yield), respectively (Table III). The reactions probably proceed through the formation of a cyanohydrin intermediate which loses HCN to form the aldehyde, OXE, adducts (vide supra) [47]. The reaction between CEO and dUrd was conducted to verify the role of the adjacent exocyclic nitrogen in the formation of HOCE adducts. With dUrd the lack of a positively charged adjacent exocyclic nitrogen did not permit formation of a 3-HOCE-dUrd adduct but instead resulted in the formation of an initial cyanohydrin, 3-HOCNE-dUrd, which as expected lost HCN to form the aldehyde, 3-OXE-dUrd. The adducts detected following in vitro reaction of CEO with calf thymus DNA were [nmol/mg DNA) N6-HOCE-dAdo (2), E-dAdo (11), 3-HOCE-dUrd (80), 7OXE-Gua (110) and 3-OXE-dThd (1). The order of reactivity with CEO was Gua > Cyt > Ade > Thy. Table IV compares the results of this study of CEO with calf thymus DNA and our previous results [24,25] with AN and calf thymus DNA. Both experiments were carried out at pH 7 and 37°C. CEO reacts much more quickly with DNA and the results are given after 3 h reaction time. AN reacts much more slowly and the data are given after a 40-d reaction time. The major adduct could be detected after 24 h reaction. Both AN and CEO react with N7 of guanine to form the major adduct, but CEO reacts to a greater extent with cytosine residues resulting in the detection of another major adduct, 3HOCE-dUrd. Compounds E-dAdo and 7-OXE-Gua were isolated by preparative HPLC and their assigned structures where further confirmed by UV and mass spectra. The major adduct formed following in vitro reaction of CEO with calf thymus DNA was 7-OXE-Gua (Table IV). Small amounts of 7-OXE-Gua were detected in the liver DNA of rats exposed to CEO [49]. Compound 7-OXE-Gua was reported to

TABLE IV YIELDS OF DNA ADDUCTS FOLLOWING IN VITRO REACTION OF CEO AND AN WITH CALF THYMUS DNA CEO + DNA a

Yield nmol/mg DNA

AN + DNA b

Yield nmol/mg DNA

N6-HOCE-Ado ~-dAdo 3-HOCE-dUrd c 7-OXE-Gua 3-OXE-dThd

2 11 80 110 1

N6-CE-Ade 1-CE-Ade 3-CE-Cyt 7-CNE-Gua 3-CNE-Thy

29 100 5 190d 64

aA solution containing 75 mg of calf thymus DNA (1800 A260 units) and 34 mmol (2.3 g) of CEO in 25 ml phosphate buffer, pH 7.0, was incubated at 37°C for 3 h. bA solution containing 150 mg of calf thymus DNA and 68 mmol of AN in 50 ml phosphate buffer, pH 7.0, was incubated at 37°C for 40 d [24,25]. cCompound 3-HOCE-dUrd was detected in the enzyme hydrolysate of CEO-reacted DNA. See Fig. 5. dThe amount of 7-CNE-Gua detected after 10, 5 and 1 d reactions was 38, 24 and 4 nmol/mg calf thymus DNA respectively [25].

132

be the major product of base alkylation in liver DNA following inhalation of labeled vinyl chloride, a structural analog of acrylonitrile (vinyl cyanide), by mice [50] and rats [51]. It was not identified in rat liver DNA following oral administration (drinking water) of vinyl chloride for 2 years [35]. Compound 7-OXE-Gua was not mutagenic in vitro [52]. However 7-OXE-Gua could still exert a mutagenic effect in vivo due to depurination [53]. 8-Ado was detected following the reaction of CEO with Ado [20]. The presence of 8-dAdo in liver DNA has been established following oral administration of vinyl chloride for 2 years [35]. However the adduct could not be identified in rat liver DNA following inhalation exposure to labeled vinyl chloride for 5 h [51]. The authors suggested that long term exposure to vinyl chloride may be necessary for the build-up of 8-dAdo to detectable levels. Our results indicate that CEO produces 8-dAdo in DNA in vitro after 3 h. Polynucleotides containing 8-dAdo have been utilized for in vitro replication studies and the results revealed no miscoding properties in that no incorporation of G, C or A was observed opposite the etheno lesion [54,55]. In single-stranded DNA 8-Ade was shown to be a very weak mutagen [56]. To the best of our knowledge the mutagenic effects of the other adducts formed by CEO in calf thymus DNA are not known. Studies on the in vivo binding of AN and CEO to proteins and nucleic acids following i.p. administration in rats have been reported [49]. Following administration of labeled CEO, binding to liver and brain protein was found, but no covalent binding to brain (target) DNA or liver DNA could be detected at a level of 0.3 alkylations per 106 bases [49]. The liver is the major site of the biotransformation of AN to CEO [49] but is not a target organ. CEO was shown to be formed in perfused rat liver as long as AN was available to the organ [49] and was present in blood for at least 2 - 4 h after oral administration to mice and rats [23]. AN is mutagenic only following metabolic activation in S. typhimurium [13] and its activated metabolite, CEO, is a direct-acting mutagen in the same system [57]. AN and CEO have also been tested for mutagenic activity in vitro in human lymphoblasts [16] with the suggestion made that CEO may be the ultimate mutagenic metabolite of AN. On the other hand, AN is a direct acting mutagen in E. coli [12]. In addition, attempts to find the epoxide adducts in DNA have met with limited success [49,58,59]. The major adducts formed following in vitro reactions of AN [24,25] and CEO with DNA have now been identified and their use as reference (marker) compounds will hopefully help to determine the identity of the DNA adducts formed following in vivo exposure to AN, i.e., AN and/or CEO adducts. This will help to assess the role of metabolism in the biological activity of AN. ACKNOWLEDGEMENTS

We are grateful for the assistance provided by Dr. J. Lin of the American Health Foundation with 1HNMR experiments. We also wish to acknowledge Dr. F. Mukai, Dr. F. Li and K. Decker-Samuelian for technical assistance. This work was supported by Research Grants ES05694 and ES03043 from the

133

National Institute of Environmental Health Sciences (NIEHS), Grant 9 0 - 1 2 from the Center for Indoor Air Research and by Center Grants ES00260 and CA13343 from the National Institutes of Health. Dr. Udai S. Singh was partially supported by a training grant ESO 7065 from NIEHS. REFERENCES 1 NIOSH (National Institute for Occupational Safety and Health), Current Intelligence Bulletin 18: Acrylonitrile, July 1, 1977, Available from NIOSH, Rockville, MD 21701, USA. 2 Chemical and Engineering News, Anonymous (April 13, 1992) 17. 3 R.A. Parent and B.C. Casto, Effect of acrylonitrile on primary Syrian golden hamster embryo cells in culture: transformation and DNA fragmentation, J. Natl. Cancer Inst., 62 (1979) 1025-1029. 4 S. Banerjee and A. Segal, In vitro transformation of C3H/10T1/2 and NIH/3T3 cells by acrylonitrile and acrylamide, Cancer Lett., 32 (1986) 293-304. 5 C. Maltoni, A. Cilborti and V. DiMaio, Carcinogenicity bioassays on rats of acrylonitrile administered by inhalation and ingestion, Med. Lavaro, 68 (1977) 401- 411. 6 J.F. Quast, B.A. Schwetz, M.F. Balmer, I.S. Gushow, C.N. Park and M.J. McKenna, A two-year toxicity and oncogenicity study with acrylonitrile following inhalation exposure of rats, Report prepared by the Toxicology Research Laboratory, Health and Environmental Sciences, Dow Chemical Company, Midland, MI for the Chemical Manufacturers Association, 2501 M-Street, N.W., Washington, DC-20037. December 9, 1980, pp. 1 - 279; may be obtained from the Chemical Manufacturers Association. 7 J.F. Quast, C.E. Wade, C.G. Hurniston, R.M. Carreon, E.A. Hermann, C.N. Park and B.A. Schwetz, A two-year toxicity and oncogenicity study with acrylonitrile incorporated in the drinking water of rats, Report prepared by the Toxicology Research Laboratory, Health and Environmental Sciences, Dow Chemical Company, Midland, MI for the Chemical Manufacturers Association, 2501 M. Street, N.W., Washington, DC 20037, January 22, 1980, pp. 1 - 315; may be obtained from the Chemical Manufacturers Association. 8 United States Department of Labor, OSHA Safety and Health Standards (29CFR 1910), OSHA 2206, Revised November 7, 1978, pp. 701-720. 9 M.T. O'Berg, Epidemiologic study of workers exposed to acrylonitrile, J. Occup. Med., 22 (1980) 245 - 252. 10 E. Delzell and R.R. Monson, Mortality among rubber workers: VI. Men with exposure to acrylonitrile, J. Occup. Med., 24 (1982) 767-769. 11 W. Koerselman and M. van der Graaf, Acrylonitrile: a suspected human carcinogen, Int. Arch. Occup. Environ. Health, 54 (1984) 317-324. 12 S. Venitt, C.T. Bushell and M. Osborne, Mutagenicity of acrylonitrile (Cyanoethylene) in Escherichia coli, Mutat. Res., 45 (1977) 283-288. 13 M. Duverger, C. Lambotte, C. DeMeester, M. Roberfroid, F. Poncelet and M. Mercier, Mutagenicity of acrylonitrile: A new experimental approach to investigate the activation mechanism, Arch. Int. Physiol. Biochim., 88 (1980) B29-B30 14 M.M. Rabello-Gay and A.E. Ahmed, Acrylonitrile: In vivo cytogenic studies in mice and rats, Mutat. Res., 79 (1980) 249-255. 15 A. Leonard, V. Gamey, F. Poncelet and M. Mercier, Mutagenicity of acrylonitrile in mice, Toxicol. Lett., 79 (1980) 249-255. 16 L. Recio and T.R. Skopek, Mutagenicity of acrylonitrile and its metabolite 2-cyanoethylene oxide in human lymphoblasts in vitro, Mutat. Res., 206 (1988) 297-305. 17 M. Lambotte-Vandepaer, M. Duverger-van Bogaert, Genotoxic properties of acrylonitrile, Mutat. Res., 134 (1984) 49-59. 18 L.E. Geiger, L.L. Hogy and F.P. Guengerich, Metabolism of acrylonitrile by isolated rat hepatocytes, Cancer Res., 43 (1983) 3080-3087. 19 M.E. Abreu and A.E. Ahmed, Metabolism of acrylonitrile to cyanide: in vitro studies, Drug Metab. Dispos., 8 (1980) 376-379.

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