50
Chem. Res. Toxicol. 1991, 4 , 50-57
Identification and Characterization of Deoxyguanosine Adducts of Methyl Vinyl Ketone and Ethyl Vinyl Ketone. Genotoxicity of the Ketones in the SOS Chromotest Erwin Eder,* Christian Hoffman, and Christoph Deininger Institute of Toxicology, University of Wiirzburg, Versbacherstrasse 9, 0-8700 Wurzburg, Germany Received June 29, 1990
The reaction of the a,@unsaturated ketones methyl vinyl ketone (MVK) and ethyl vinyl ketone (EVK) with nucleosides and 5’-mononucleotides was studied. The genotoxic activity of MVK and EVK in the SOS Chromotest was investigated. Three different types of adducts with deoxyguanosine were found and their structures elucidated: the cyclic 1,N2adducts, the linear N7 adducts with one still-unreacted carbonyl function, and the cyclic 1,N2,linear N7, bis adducts. The spectroscopic and other relevant characterization data for the deoxyguanosine adducts and the corresponding guanine adducts are presented here together with details of the chromatographic methods used for isolation. The adducts described could also be isolated in the reactions of MVK and EVK with 2’-deoxyguanosine 5’-monophosphate. No adducts could be isolated either with nucleosides other than deoxyguanosine or with nucleotides other than 2’-deoxyguanosine 5’-monophosphate, indicating that the guanine moiety is the most reactive DNA constituent for MVK and EVK. MVK and EVK were clearly genotoxic in the SOS Chromotest according to the criteria of Quillardet and Hofnung. The formation of these adducts was proposed as the mechanism for the genotoxicity of MVK and EVK: all data available support the assumption that MVK and EVK represent a mutagenic and carcinogenic risk for mankind.
Introduction a,@-Unsaturated carbonyl compounds are mutagens (1-4) and potential carcinogens (5,6),occurring as environmental pollutants (7, 8 ) , natural products (3, 9, IO), important technical chemicals (7,8,11,12),and metabolites of industrial chemicals and pesticides (13-15). Recently, we reported on the mutagenic activities of the a,@-unsaturatedketones methyl vinyl ketone (MVK) (2, 11, 16) and ethyl vinyl ketone (EVK) (17). Both compounds were unambiguously mutagenic in Salmonella typhimurium TAlOO without addition of S9 mix. They were, however, also rather toxic toward the bacteria so that higher doses of these substances could not be tested. MVK is a widely used industrial chemical and one of the strongest known irritants of mucous membranes in man and animals (18). EVK is widespread in our environment, in particular in food. It has been found in orange juice (19), in apple juice (20),in defatted soy bean flour (21),in fresh tomato aroma volatiles (22), in kiwi fruits (23),in blended endive (24), in black teas (25), in oxidized dairy products (26), in oxidized frozen white fish (27), in freshly cooked ground beef (28),in roasted chicken fat (29),in many other foodstuffs, and in burning cigarettes (30). As has been proposed for other a,@-unsaturatedcarbonyl compounds (31-35), the underlying primary mechanism of the genotoxicity of MVK and EVK is presumably the formation of DNA adducts. Indeed, Chung et al. (36) recently reported the formation of a cyclic N1,N2-deoxyguanosine adduct with MVK, and we could confirm their findings (16). We now present a detailed description and characterization of the deoxyguanosine adducts and guanine adducts formed in the reaction with these &-unsaturated ketones. * To whom correspondence should be addressed. 0893-228~/91/2704-0050$02.50/0
Furthermore, we have investigated the genotoxic activities of these ketones in the SOS Chromotest to extend the data base beyond the mutagenic activity demonstrated in S. typhimurium TA100, most probably also a result of adduct formation with the nucleosides in the Salmonella DNA.
Materials and Methods Chemicals. Methyl vinyl ketone (MVK) and ethyl vinyl ketone (EVK) were purchased from Aldrich Chemie, Steinheim, FRG. Both were distilled with a Fisher Spalt column (MVK 81 “C/760 mm, EVK 98 “C/760 mm) immediately before use. Their identities were checked by ‘H NMR spectroscopy and the purities shown to be 99.9% for MVK and 99.8% for EVK as determined by capillary gas chromatography. The nucleosides 2’-deoxyguanosine, 2’-deoxyadenosine, and 2’-deoxythymidine were bought from Serva Biochemica, Heidelberg, FRG, in the highest purity available, and 2’-deoxycytidine monohydrate was bought from Sigma Chemie GmbH, Deisenhofen, FRG. The nucleotides 2’deoxyadenosine 5’-monophosphate (free acid), 2’-deoxythymidine 5’-monophosphate (sodium salt), 2’-deoxyguanosine 5’-mOnOphosphate (sodium salt), and 2’-deoxycytidine 5’-monophosphate (free acid) were purchased from Sigma Chemie GmbH, Deisenhofen, FRG. All chemicals, solvents, and reagents for the preparation of the buffers, the eluents, etc., and for the SOS Chromotest were bought in the highest purity available from Merck, Darmstadt, FRG; Aldrich, Steinheim; Serva, Heidelberg; Sigma, Deisenhofen; Roth GmbH, Karlsruhe, FRG; or Boehringer Mannheim GmbH, Mannheim, FRG. Alkaline phosphatase from bovine intestinal mucosa was purchased from Sigma. Reaction of the Ketones with Nucleosides. (1) Reaction in Phosphate Buffer, pH 7. To a solution of 110 mg (0.412 mmol) of deoxyguanosine in 20 mL of 0.1 M sodium phosphate buffer, pH 7 , was added 140 mg (2.0 mmol) of MVK. The mixture was stirred either for 22 h a t 37 “C or for 4 days a t 90 “C under nitrogen. Another reaction of this type was carried out with 168 mg (2.0 mmol) of EVK and the mixture stirred for 22 h at 70 “C. Then the solutions were filtered through a 0.45-wm filter and the un0 1991 American Chemical Society
Chem. Res. Toxicol., Vol. 4, No. I , 1991 51
Deoxyguanosine Adducts of MVK and EVK reacted ketone was removed in a vacuum evaporator a t the indicated temperatures (37,70, or 90 "C). The residue was dissolved in 10 mL of water and filtered again. The workup procedures used to isolate the various adducts, i.e., MPLC, semipreparative HPLC, Sephadex LH-20 gel chromatography, and radial chromatography, are described later. In additional experiments, 0.4 mmol of the other deoxynucleotides were used instead of deoxyguanosine. (2) Reactions in Dimethyl Sulfoxide (DMSO). To 500 mg (1.87 mmol) of deoxyguanosine dissolved in 10 mL of DMSO was added either 1.68 g (24.0 mmol) of MVK or 1.69 g (20.1 mmol) of EVK. The mixtures were stirred in the dark a t room temperature. The progress of the reactions was followed by HPLC analysis of 10-pL aliquots of the reaction mixtures. After completion of the reactions, the surplus ketone and DMSO were removed in vacuo (0.01 mm) a t room temperature. The resulting pale yellow residue was redissolved in 40 mL of distilled water and filtered through a 0.45-pm filter, and the adducts were isolated by using chromatographic methods. Acid Hydrolysis of the Modified Adducts To Yield the Modified Nucleobases. Solutions of the nucleoside adducts (50-100 pmol) in 8 mL of 0.1 M HCl were heated at 65 "C for 45 min. After cooling to room temperature, the solution was neutralized by dropwise addition of 0.1 M potassium hydroxide solution. The modified nucleobases were isolated and purified on a Sephadex LH-20 column. Chromatographic Methods. Radial chromatography (centrifugally assisted preparative thin-layer chromatography) was performed with a Harrison Research Chromatotron Model 79241' on a 24-cm plate with a 2-mm layer of silica gel 60 PFz54 "containing gypsum". The eluent was a mixture of chloroform/methanol, 8:2, and the flow rate was 6-8 mL/min. Sephadex LH-20 chromatography was carried out in a 100 X 1.6 cm glass column, wet-filled with LH-20, 25-100 pm, from Pharmacia, using double-distilled water as eluent. A 20 mL/h flow was regulated with an Ismatec MV-CA-4 pump. The chromatogram was recorded a t 254 nm with an LKB 2138 Unicord UV detector and a Metrawatt Servogor 5b recorder. Thirty-minute fractions were collected with an LKB 2111 Multi Rac fraction collector. MPLC (medium-pressure liquid chromatography) was performed on a Labochrom "FPGC-prepacked" column, 18.5 mm X 48 cm, HD-Sil-18-30-60, 35-70 pm, reversed-phase silica gel C8, and a Labomatic MD 80-100 fitted with an Labomatic PC-100 pressure control unit at 254 nm with an Isco detector, Model 248, using the following stepwise gradient: methanol/water, 11% methanol for 22 min, then 30% methanol for 18 min, and finally 100% methanol for 10 min, a t a flow rate of 4 mL/min. For HPLC, the Waters system consisted of a U6K injection system or Rheodyne injection valve 1265, two Model 6000 A pumps, a Model 660 gradient solvent programmer, and a Model 450 variable-wavelength detector connected to a Hewlett/Packard 3385A integrator. A reversed-phase C18 column, 5 pm, 0.46 X 25 cm, from Knauer was used for the analysis, and a Bondapak reversed-phase, lO-pm, 0.78 X 30 cm column was used for semipreparative purposes. Analysis and Characterization. The substances were analyzed and characterized with the following instruments. (1) UV Spectroscopy. Kontron Uvikon Model 860 and Model 800 plotter a t pH 1 (0.1 N HCl); pH 7 (water); pH 13 (0.1 N NaOH). (2) FT-IR Spectroscopy. Nicolet 5DXC. (3) 'H NMR Spectroscopy. Bruker AC 250 (250.1 MHz) or Bruker WM 400 (400.1 MHz) as indicated, internal tetramethylsilane (6 = 0.00 ppm), trichloromethane (6 = 7.26 ppm), or dimethyl sulfoxide ( 6 = 2.54 ppm). (4) '3c NMR Spectroscopy. Bruker AC 250 (62.9 MHz) and a Bruker WM 400 (100.6 MHz); tetramethylsilane (6 = 0.00 ppm), trichloromethane (6 = 77.0 ppm), or dimethyl sulfoxide (6 = 39.7 PPd. (5) Mass Spectroscopy. (a) Electron Impact Mass Spectrometry (EI). Varian MAT CH7 (70 eV) fitted with a data system SS200. Fast atom bombardment (FAB)mass spectra were recorded on a 8200 Finnigan VAT (70-eV) spectrometer. Xenon and argon were used as ionization gases and glycerol and tri- or tetraethylene glycol as FAB matrix.
(b) Thermospray Mass Spectrometry (HPLC/MS). Finnigan Mat 450 quadrupole mass spectrometer with Finnigan thermospray LC-MS interface connected with a Waters Model 6500 A HPLC pump; water/methanol eluent; flow rate 1.2 mL/min. Ionization was initiated with a 17 mM ammonium acetate solution. The C, H, N elementary analysis was performed at the Institute of Inorganic Chemistry; UV spectroscopy and thermospray mass spectrometry were performed a t our Institute. The other spectra were recorded at the Institute of Organic Chemistry, University of Wiirzburg. Characterization Data of the Isolated Adducts. Spectroscopic data, melting points, and elementary analysis of the adducts are presented here. The description of the adducts and the chromatographic data are given under Results. Adduct Ia. Colorless powder; mp 162-164 "C dec. FT-IR (KBr): v = 3325 cm-' (OH), 2939 (CH), 1685 (C=O), 1560 (C=N, C=C), 1419,1338,1096,1055,930,779,722,642.UV (HZO): A, (log e) = 258 nm (4.23), 268 (sh); A- = 227 (3.56) (sh = shoulder). UV (0.1 N HCl): A, (log t) = 202 nm (4.40), 260 (4.21),278 (sh); ,,,,A,, = 232 (3.60). UV (0.1 N NaOH): A- (log t) = 215 nm (4.071, 258 (4.33); & = 228 (3.69). 'H NMR (400.1 MHz, [DZIDMSO): 6 = 1.48 (S, 3 H, II-H), 1.74 (tm, J7,7b = 13.5 HZ, J7,Bb = 13.5 HZ, n
OH
Ia 1 H, 7a-H), 1.99 (wide, d (dm), 1 H, 7b-H), 2.19 (m, 1 H, 2'a-H), 2.51 (m, 1 H, 2'P-H), 3.46-3.58 (m, 3 H, 8b-H, 5'-H), 3.82 (m, 1 H, 4'-H), 4.35 (m, 1H, 3'-H), 4.43 (dm, J&+, = 13.5 Hz, 1H, 8a-H), 4.95* (wide, s, 1H, 5'-OH), 5.30* (wide, s, 1 H, 3'-OH), 5.71 very = 6.0 Hz, 1 H, 1'-HI, 7.94 wide, s, 1 H, 6-OH), 6.12 (ps d, J1f,2t (d, J2,1' = 1.3 Hz, 1 H, 2-H), 8.31* (d, JNH= 5.3 Hz, N(5)-H). An asterisk (*) indicates the signal disappears after shaking with DzO. 13C NMR (100.6 MHz, [DcIDMSO): 6 = 28.54 (9, C - l l ) , 33.27 (t,C-7), 35.78 (t, C-8), 61.86 (t,C-5'), 70.87 (d, C-3'), 76.81 (s,C-6), 82.59 (d, C-1'), 87.67 (d, C-49, 115.98 (s, C-loa), 135.62 (d, C-2), 149.37 (s, C-3a), 150.49 (s, C-4a), 156.15 (s, C-10). The signal of C-2' is overlapped by the solvent signal. Thermospray MS: m / z (%) = 338 (9, MH'), 320 (20, MH' - HZO), 222 (10, MH' - dR), 204 (65, MH+ - dR - H,O), 166 (21), 152 (100, guanine H'), 134 (36). FAB-MS (70 eV, triethylene glycol): m / z (%) = 488.0 (7, MH+ triethylene glycol), 470.5 (31, MH+ - H,O + triethylene glycol), 354.3 (18, MH+ - H,O - dR + triethylene glycol), 338.3 (88, MH+), 320.2 (20, MH+ - HZO), 222.2 (85, MH' - dR), 204.2 (67, MH+ - H20- dR). Elementary analysis [Cl4Hl9N5O6(337.3) X 1H,O]: calcd, C 47.32, H 5.96, N 19.71; found, C 46.78, H 5.84, N 19.61.
+
Characterization of the Acid Hydrolysate of Adduct Ia. Colorless crystalline powder; mp 210 "C dec. FT-IR (KBr): v = 3240, 3172 (OH), 2985, 2945 (CH), 1696, 1695 (C=O), 1559 (C=N, C=C), 1473,1216,1132,924,778,719,638,611. UV (HZO): A, (log t ) = 251 nm (4.15), 266 (sh); Ami, = 228 (3.78). UV (0.1 N HC1): A, (log t) = 202 nm (4.45), 252 (4.20), 272 (sh); Amin = 229 (3.73). UV (0.1 N NaOH): A, (log e ) = 223 nm (4.46), 261 (4.02) 279 (4.01);A- = 243 (3.86),270 (3.99). 'H NMR (400.1 MHz, [DcIDMSO): 6 = 1.47 (s,3 H, 11-H), 1.74 (td, J7a,7b = 13.5
Hz, J,a,8b = 13.5, J7a,Bb = 5.0 Hz, 1 H, 7a-H), 2.00 (wide, d, 1 H, 7b-H), 3.51 (td, J 8 b , h = 13.5 Hz, JBb,7b = 4.0 HZ, 1 H, 8b-H), 4.40 (ddd, J&,,b = 3.0 Hz, 1 H, 8a-H), 5.66* (wide, s, 1 H, 6-OH), 7.70 (wide, s, 1 H, 2-H), 8.06* (wide, s, 1H, N(5)-H), 12.42* (very wide, s, 1 H, N(3)-H). An asterisk (*) indicates the signal disappears
Eder et al.
52 Chem. Res. Toxicol., Val. 4, No. 1, 1991 after shaking with D2O. 13CNMR (100.6 MHz, [DBIDMSO): 6 = 28.45 (4, C-ll), 33.21 (t, C-7), 35.51 (t, C-8), 76.57 (8, C-6), 136.20 (d, C-3), 149.81 (s, C-4a), 155.48 (8, C-10). The signal of C-lOa,Bb is not observed. ELMS (70 eV): m / z (%) = 204.2 (16, M+ - H 2 0 l),203.1 (100, M+-HzO), 202.1 (99),188.1 (15), 175.1 (ll),148.2 (12), 135.1 (13), 120.1 (14), 94.2 (20), 79.2 (9), 66.1 (8), 53.3 (17). Thermospray MS: m / z ( 5 ) = 222 (4, MH'), 204 (100, MH+ H,O), 190 (l), 152 (8, guanine H+). Elementary analysis [CBH11N50z (221.2) X 1.5 H20]: calcd, C 43.55, H 5.68, N 28.21; found, C 42.88, H 4.74, N 27.74. Characterization of Adduct Ib. Pale yellow powder; mp 141-143 "C dec. FT-IR (KBr): u = 3362 cm-' (OH), 2973,2938 (CH), 1686 (C=O), 1561 (C=N, C=C), 1462,1339,1096,1057, (log e ) = 258 nm (4.16), 268 943,780,723,644. UV (H20): A,, (sh); Amin = 229 (3.69). UV (0.1 N HC1): A,, (log t) = 204 nm (4.28), 259 (4.14), 277 (sh); Amin = 232 (3.69). UV (0.1 N NaOH): A, (log C) = 215 nm (4.11),258 (4.18); A- = 230 (3.72). 'H NMR (400.1 MHz, [DpJDMSO): b = 0.90 (t,J11,lz = 7.0 Hz, 3 H, 12-H),
+
a
7
I
9
/-.NI\/N.r
= 236 (1, MH+), 218 (100, MH+ - H20). Elementary analysis [C10H13N502 (235.2) X 0.5 HZO]: calcd, C 49.17, H 5.78, N 28.67; found, C 48.98, H 5.80, N 28.52. Characterization of Adduct IIa. Colorless crystalline powder. 'H NMR (250.1 MHz, [D6]DMSO): 6 = 2.12 (s,3 H, 13-H), 3.00 (t,Jll,lo = 6.9 Hz, 2 H, 11-H), 4.10 (t,2 H, 10-H), 6.57* (wide,
IIa
s, 2 H, N(2)H2),7.63 (s, 1 H, 8-H), 9.15* (wide, s, 1 H, N(l)-H). An asterisk (*) indicates the signal disappears after shaking with DzO. Thermospray MS: m / z (%) = 222 (100, MH'). Characterization of Adduct IIb. Colorless crystalline powder. '€1 NMR (250.1 MHz, [D6]DMSO): 6 = 1.06 (t, J14,13 = 7.0 Hz, 3 H, 14-H), 2.44 (4, 2 H, 13-H), 3.00 (t, Jll,lo = 7.0 Hz, in
OH Ib 1.63-1.83 (m, 3 H, 7a-H, 11-H), 1.92 (wide, d, J7b,7, = 13.0 Hz, 1 H, 7b-H), 2.19 (m, 1 H, 2'a-H), 3.44-3.58 (m, 3 H, 8b-H, 5'-H), 3.80 (m,1 H, 4'-H), 4.35 (wide, s, 1 H, 3'-H), 4.44 (dm, J&,8b = 13.0 Hz, 1 H, 8a-H), 4.95* (wide, s, 1 H, 5'-OH), 5.29* (wide, s, 1 H, 3'-OH), 5.66* (wide, s, 1H, 6-OH), 6.14 (m, 1 H, 1'-H), 7.95 (s, 1 H, 2-H), 8.19* (d, JNH,?= 4.0 Hz, 1H, N(5)-H). The signal of 2'0-H is overlapped by the solvent signal. An asterisk (*) indicates the signal disappears after shaking with DzO. 13CNMR (100.6MHz, [DBIDMSO): b = 8.01 (4, C-12), 29.52 (t, C-11), 33.23 (t, C-7), 35.28 (t,C-8), 61.59 (t,C-5'),70.60 (d, C-3'), 78.92 (s,C-6), 82.18 (d, C-l'), 87.39 (d, C-49, 115.75 (s, C-loa), 135.56 (d, C-2), 149.11 (s, C-3a), 150.57 (s, C-4a), 155.91 (s, C-lo). The signal of C-2' is overlapped by the solvent signal. Thermospray MS: m/z (%) = 334 (3, MH+ - HzO), 276 (13), 218 (100, MH+ - HzO - dR), 166 (4,152 (35, guanine H+), 134 (22). Elementary analysis [Cl5HZ1N5O5 (351.4) X HzO]: calcd, C 48.78, H 6.28, N 18.96; found, C 48.48, H 6.00, N 17.45.
Characterization of the Acid Hydrolysate of Adduct Ib. Colorless crystalline powder; mp 195-197 "C dec. FT-IR (KBr): u = 3316 (NH), 3230, 3101 (OH), 2977, 2939, 2882 (CH), 1707 (C=O), 1559 (C=N, C=C), 1210,1152,937,791,780,607. UV (log t) = 251 nm (4.09), 266 (sh); Amin = 228 (3.72). (H20): A,, UV (0.1 N HCl): A,, (log e ) = 202 nm (4.29), 252 (4.12); Ami,, = 229 (3.67). UV (0.1 N NaOH): A,, (log e ) = 213 nm (4.60), 221 (sh), 261 (3.94), 275 (3.92); Amin = 243 (3.00), 271 (3.92). 'H NMR (250.1 MHz, [DGIDMSO): 6 = 0.90 (t, J12,11 = 7.5 Hz, 3 H, 12-H), 8 9 01' 12 11
1
??+N\,
1.67 (td, J7,,7b = 13.5 HZ, J7,,8b = 13.5 HZ, J7a,& = 5.0 HZ, 1 H, 7a-H), 1.70 (dq, J l l a , l l b = 14.0 Hz, 1 H, lla-H), 1.79 (dq, 1 H, llb-H), 1.92 (wide, d, 1 H, 7b-H), 3.50 (td, J8b,& = 13.5 Hz, J8b,7b = 3.8 Hz, 1H, 8b-H), 4.43 (ddd, 1 H, J&,7b = 2.5 Hz, 1 H, 8a-H), 5.57* (wide, s, 1 H, 6-OH), 7.70 (wide, s, 1 H, 2-H), 7.98* (wide, s, 1 H, N(5)-H), 12.34* (very wide, s, 1 H, N(3)-H). An asterisk (*) indicates the signal disappears after shaking with DzO. 13C NMR (62.9 MHz, [DeIDMSO): 6 = 8.14 (4, C-12), 29.67 (t, C-14), 33.60 (t, C-7), 35.25 (t,C-8), 78.90 (9, C-6), 135.57 (d, C-21, 150.27 (s, C-4a), 156.05 (s,C-10). The signals of C-lOa,3a are not observed. ELMS (70 eV): m / z (%) = 218.2 (15, M+ - H 2 0 + l),217.1 (100, M+ - HZO), 202.1 (38), 188.0 (21), 161.2 (17), 135.1 (16), 134.1 (lo), 108.1 (8),80.1 (9), 67.1 (7), 53.0 (15). Thermospray MS: m / z (%)
0
IIb 2 H, 11-H), 4.13 (t,2 H, 10-H), 6.52* (wide, s, 2 H, N(2)H2),7.12 (s, 1H, &H), 9.12* (wide,s, 1H, N(1)-H). An asterisk (*) indicates the signal disappears after shaking with DzO. Thermospray MS: m / z (%) = 236 (100, MH'). Characterization of the Bis Adduct IIIa. Pale yellow powder. UV (H20): ,A, = 259 nm; Amin = 223. FAB-MS (70 eV, glycerol): m / z (%): 292.0 (18, 1 guanine + 2 MVK), 273.9 (27,l guanine + 2 MVK - H20),221.9 ( 2 1 , l guanine + 1 MVK), 203.9 (13, 1 guanine + 1 MVK - HzO). 'H NMR (250.1 MHz, [D,JDMSO): 6 = 1.46 (s, 3 H, l l - H ) , 1.72 (td, J7,,7b = 13.5 Hz,
IIIa = 13.5 Hz, J7a,8b = 5.0 Hz, 1 H, 7a-H), 1.99 (wide, d, 1 H, 7b-H), 2.11 (s,3 H, 15-H), 3.01 (t, J13,14 = 6.9 Hz, 2 H, 13-H), 3.48 (td, = 13.5 Hz, J8b,% = 4.3 HZ, 1 H, 8b-H), 4.10 (t, 2 H, 12-H), 4.40 (dm, 1H, 8a-H), 5.69* (wide, s, 1H, 6-OH), 7.64 (s, 1 H, 2-H), 8.31* (wide, s, 1 H, N(5)-H). An asterisk (*) indicates the signal disappears after shaking the D20. Reaction of the Ketones with 5'-Mononucleotides. Two hundred micromoles of MVK or EVK was added to a solution of 36.5 pmol of the respective 5'-mononucleotide in 2.0 mL of 10 mM sodium phosphate buffer, pH 7, and the mixture was stirred under nitrogen for 22 h at 90 "C in the case of MVK and 2 days a t 37 "C with EVK. After cooling to room temperature, the solution was fiitered through a 0.45-pm fiter and chromatographed on a Sephadex LH-20 column as described above with a flow rate of 14 mL/h. The unreacted nucleotides and modified nucleotides are eluted first, between 6 and 8 h, then the polymerized and oligomerized ketones, and lastly the unreacted ketones, nucleosides, and modified nucleosides formed during the reaction. The fractions obtained were analyzed by UV spectroscopy and by HPLC with a Bondapak 10-pm column (H20/methanol, 89:11, 1 mL/min) or a Bondapak 5-pm column (H20/methanol, 9010, 1 mL/min) as described under HPLC. All fractions containing modified nucleotides were lyophilized, and the colorless or yellowish powders obtained were subjected to further analysis. Enzymatic Hydrolyses of the Modified Nucleotides. Either 100 or 50 pL of alkaline phosphatase was added to 2 mg of the lyophilized nucleotide fractions dissolved in 2 mL of 10 mM sodium phosphate buffer. The mixture was incubated a t 37 "C for 22 h in an incubator-shaker. After cooling, the mixture was
J7,,8b
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 53
Deoxyguanosine Adducts of MVK and EVK
/1.4-1.8
11,719-21
d
HOCHZ
OH
Type A
d
HO CH2
OH
Type B
Figure 1. Regioisomen of the 1,N2-cyclicdeoxyguanosineadducts A and B formed by acrolein. In contrast to acrolein, MVK and EVK form only adduct A.
filtered through a 0.45-wm filter and analyzed by HPLC as described for the nucleosides. SOS Chromotest. The SOS Chromotest was performed with the Escherichia coli strain PQ37 according to the standard protocol of Quillardet and Hofnung (37). The strain was kindly provided by Professor Herzberg, Yavne, Israel. The sfiA gene linked &galactosidase activity (@-Gal) is determined as a measure of the induction of the SOS repair system. The alkaline phosphatase activity is used as a measure of protein synthesis and toxicity. Dimethyl sulfoxide was the solvent, and the SOS inducing potency (SOSIP)of nitroquinoline oxide (=43)was routinely determined as positive control. The SOSIP was calculated according to Quillardet and Hofnung from the linear part of the dose-response curve:
2.0T2.2
i!
4 90
/+HO 8 0 - 8 5
-34-37
3 4-3 7
5 6-6 0
[TY Pe A
I
LH
/6.0-6.3
9 16-79
J
~ T Y BP ~
f o u n d w i t h MVK a n d EVK
not f o u n d w i t h MVK a n d E V K
Figure 2. Differentiation between the type A and type B re-
gioisomers with ‘H NMR spectroscopy. 7n
H
I
OH
Bb
Figure 3. Illustration of the vicinal axial, axial coupling between the 7a and the 8b protons with a coupling constant of 13.5 Hz.
A and B have been identified with acrolein in other studies (16, 33) (Figure 1). Evidently for steric reasons, only the one regioisomer, type A, Ia and Ib, is formed with MVK and EVK. As can be seen in Figure 2, we could distinguish unambiguously between the two isomers by lH NMR spectroscopy: (a) and The hydroxy proton of the regioisomer type A appears at 6 = 5.6-6.0 ppm whereas that of type B appears a t a sig&Gal units R= nificantly lower field range, 6 = 6.4-6.7 ppm. (b) A strong alkaline phosphatase units low-field shift is observed for the equatorial 8-Hproton due to the influence of the hydroxy group and due to the In these equations Ro is the ratio of P-Gal units to alkaline phosphatase units at the concentration 0 (background)and Rc carbonyl function in the guanine moiety, so that the 8-H is the ratio at a given concentration (c). is the SOS inducing proton of type B is found a t 6 = 6.0-6.3 whereas the 8-H factor. The SOSIP is expressed as Zlnmol. The test result is proton of type A is found at 6 = 4.2-4.5. (c) The resonance considered to be significant if is at least 1.5 times the of the amine proton of the type A isomer is found at 6 = background, which is 1.0 for dimethyl sulfoxide. 8.0-8.5 due to the interaction of the vicinal hydroxy group whereas that of the amino proton of type B appears at a Results higher field, 6 = 7.6-7.9 ppm. The structure is also confirmed by the results of a nuclear Overhauser effect exResults of the Reactions of MVK and EVK with periment. In the difference spectrum the signal of the Deoxynucleosides. Three different types of deoxyN(5)-H proton is clearly increased when irradiating at the guanosine adducts could be found for the a,B-unsaturated frequency of the protons of the vicinal C-11 methyl group. ketones in this study: This effect demonstrates that the methyl group must be (1) l,N2-CyclicAdducts of MVK and EVK. Adduct in vicinal position to the N(10) atom. Ia [(6R /5)-3-(2’-Deoxy-j3-~-erythro -pentofuranosyl)-5,6,7,8-tetrahydro-6-hydroxy-6-methyl- The conformation of the tetrahydropyrimidine ring can pyrimido[ 1,2-a ]purin-l0(3H)-one] and Adduct Ib be derived from consideration of the coupling constants. In general, three different couplings can be differentiated [ (6R /S )-3-( 2’-deoxy-j3-~-erythro -pentofuranosyl)-6in the chair configuration: the axial, axial coupling (3J,), ethyl-5,6,7,8-tet rahydro-6-hydroxypyrimido[ 1,2-a 3the equatorial, equatorial (3Jee),and the axial, equatorial purin-l0(3H)-one]. The formulas of Ia and Ib are coupling (3Jae). In the case of adducts Ia and Ib (Figure presented together with the NMR data under Characterization data of the isolated adducts under Materials and 31,besides the geminal couplings only one vicinal axial, axial coupling was observed, namely, that between the 7a Methods. and 8b protons with a coupling constant of 13.5 Hz. In The adducts Ia and Ib could be isolated from all reacgeneral, vicinal axial, axial coupling constants of 12-14 Hz tions of deoxyguanosine with MVK and EVK irrespective were measured in the tetrahydropyrimidine ring of the solvent used, the reaction temperature, or the rethroughout our studies (unpublished results). No vicinal action time. They were the main products of the reactions coupling between the 7H and 6H protons can be seen carried out in buffer. Although only one set of signals was because the methyl (ethyl) group and the OH group are found in the lH NMR spectra, a rather wide signal in the located a t carbon atom 6. HPLC consisting of two unresolved peaks indicated the Acid hydrolysis of adducts Ia and Ib yields the respective presence of a pair of diastereomers for each single adduct 1,N2-cyclicguanine adducts. The chemical and spectroIa and Ib. Evidently, the diastereomers could not be scopic data for the guanine adducts are described under separated due to rapid epimerization. No indication of the Materials and Methods. occurrence of another possible regioisomer of 1,N2-cyclic adducts (A) as shown in the formulas could be found in (2) N7-Linear Adducts. The N7-linear adducts IIa the case of MVK or EVK, although the two regioisomers [7-(3’-oxobutyl)guanine]for MVK and IIb [7-(3’-oxo-
54
Chem. Res. Toxicol., Vol. 4, No. 1, 1991
pentyl)guanine] for EVK could be isolated after reactions of MVK or EVK with deoxyguanosine in buffer, irrespective of whether the reaction products were subjected to acid hydrolysis or not. Evidently, on N7-alkylation the deoxyribose residue is spontaneously cleaved (depurination). The presence of the resonance of the amine protons in the 'H NMR spectrum a t 6 = 6.57 and 9.15 ppm as well as that of the 8-H proton at 6 = 7.63 ppm (see Materials and Methods) clearly demonstrates the structure of the linear N7-substituted adduct. The signals of the methyl group of the side chain at N7 appear at 6 = 2.12 ppm, low field shifted by interaction of the vicinal carbonyl group. The methylene protons 10- and 11-H are observed as triplets (J = 6.8 Hz) at 6 3.00 and 4.10 ppm. In the thermospray MS the molecular peak MH+ is found. The 'H NMR spectrum, the thermospray MS (see Materials and Methods), and the comparison with the spectra of IIa also confirm the linear N7 structure of the adduct IIb. The 'H NMR spectrum of IIb is very similar to that of IIa. Only the 8C proton (6 = 7.12) of IIb shows an upfield shift when compared with the 8C proton of IIa (6 = 7.63). We did not further investigate this phenomenon and cannot provide a conclusive explanation. (3) 1,N2-Cyclic-7 Bis Adducts. 1-(3-Oxobutyl)5,6,7,8-tetrahydro-6-hydroxy-6-methylpyrimido[ 1,2a]purin-l0(3H)-one. IIIa for MVK was isolated from the reactions of MVK with deoxyguanosine in phosphate buffer a t higher temperatures (90 "C MVK). It was also present in traces in the reactions of MVK a t 30 "C. The 'H NMR spectrum (see above) shows the presence of a bis adduct between 2 molecules of MVK and guanine. The structure of the bis adduct IIIa can be derived unambiguously by comparing its 'H NMR spectrum with those of Ia and IIa. The signals of the amine protons N(l)-H and N2-H2of the parent compound guanine have disapeared, the resonance of the tetrahydropyrimidine ring is also observed, and the data for the ketone side chain are in agreement with those of IIa. Besides the adducts 1-111 clearly identified in this study, there were also trace amounts of other deoxyguanosine adducts formed; the amounts were, however, very small, so that the adducts could not be isolated and characterized. No adducts could be isolated from the reaction mixtures of MVK or EVK with deoxynucleosides other than deoxyguanosine. Results of Chromatographic Isolation of the MVK Adducts la, IIa, and IIIa and of the Hydrolysis Products. When dimethyl sulfoxide was used as solvent, exclusively adduct Ia in 46% yield (yields based on deoxyguanosine) was characterized after isolation by MPLC as described above (at a retention time of 15 min). With phosphate buffer as solvent at 90 "C the adducts Ia, IIa, and IIIa were isolated by semipreparative HPLC. Many impurities were, however, present in the fractions containing the adducts Ia, IIa, and IIIa. To obtain pure adducts, the lyophilized reaction products were subjected to Sephadex LH-20 gel chromatography in which oligomeric and polymeric impurities are separated and the adduct-containing fractions 13-30 were used for HPLC. The semipreparative HPLC (Bondapak column) fractions 15-17 were purified by radial chromatography. Fraction 1with an R, of 0.41 contained 27% IIIa and IIa in the ratio 3:1, and fraction 2 contained 11% Ia with an Rr of 0.23. Also with phosphate buffer at 37 "C the adducts Ia, IIa, and IIIa were found. The lyophilized residue of the reaction was subjected to acid hydrolysis. The lyophilized hydrolysate was subjected to Sephadex LH-20 gel chro-
Eder et al.
matography, and two fractions were obtained. Fractions 21-25 contained a mixture of 3% IIa and 12% cyclic, 1,N2-guanineadduct (hydrolyzed product of Ia). The two adducts from fractions 21-25 were separated by HPLC (Knauer column, 5 pm,H20/methanol, 89:11, isocratic, flow rate 1 mL/min). Under these conditions IIa had a retention time of 11.9 min, and the 1,N2-guanineadduct, of 15.8 min. Fractions 18-20 of the LH-20 Sephadex chromatography contained 3.5% IIIa. Results of Chromatographic Isolation of the EVK Adducts Ib, IIb, and IIIb and the Hydrolysis Products. With dimethyl sulfoxide as solvent at room temperature, exclusively adduct I (6.9%) was isolated by MPLC with a retention time of 12.8 min and a yield of 80% deoxyguanosine. Acid hydrolysis yielded the respective cyclic 1,N2-guanineadduct. The residue obtained from the reaction in phosphate buffer at 70 "C was purified by MPLC with a retention time of 28 min and then analyzed by HPLC. The product obtained from the MPLC purification (see above) was also subjected to acid hydrolysis. The hydrolysis product was filtered, lyophilized, and analyzed by Sephadex LH-20 gel chromatography. A mixture of the cyclic 1,N2-guanine adduct (hydrolysis product of Ib) (26.3%) and the N7 adduct IIb (8.7%) was eluted in fractions 23-26 and 25% IIIb in fractions 19-21. Results of the Reaction of MVK and EVK with 5'-DeoxynucleosideMonophosphate. Reaction of MVK with 2'-deoxyguanosine 5'-monophosphate under the conditions described under Materials and Methods yields a series of modified nucleotides with an absorption maximum at 258 nm. Most of these cannot be isolated or further identified because they are formed only in trace amounts. The main adduct is the 1,N2-cyclicadduct. A fraction with the retention time of the N7 adduct was also found. Enzymatic hydrolysis of the nucleotide fractions isolated from the Sephadex LH-20 chromatography yielded a mixture of deoxyguanosine and the cyclic 1,N2-deoxyguanosine adduct diastereomers Ia. Presumably due to the epimerization of the diastereomers of Ia and some remaining impurities, a rather wide peak with extensive tailing was observed in the HPLC chromatogram (retention time 22.4 min). The 'H NMR spectrum of this fraction confirms the presence of Ia. The reaction of EVK with 2'-deoxyguanosine 5I-monophosphate at 37 "C as described under Materials and Methods also yields 1,N2-cyclicadducts. A peak with the characteristic retention time of an N7 adduct could not be found in the HPLC analysis. After enzymatic hydrolysis, the HPLC shows a peak for deoxyguanosine a t a retention time of 4.3 min, a small amount of an adduct with a retention time of 11 min, which was not identified, and a wide signal at 19.9 min consisting of the unresolved peaks for the epimerizing diastereomers of the 1,N2-cyclic adduct Ib. Results of the SOS Chromotest. The results of the SOS Chromotest for MVK are shown in Figure 4. The results for EVK have been published recently, and in principle, the patterns were similar to those presented for MVK in Figure 4. Both compounds are significantly genotoxic, according to the criteria of Quillardet and Hofnung (37).
Discussion Our results clearly demonstrate that the a,@-unsaturated ketones MVK and EVK react with the same genetic targets as do other a,/?-unsaturated aldehydes, e.g., acrolein or crotonaldehyde (16,33). Evidently, the guanine moiety
Deoxyguanosine Adducts of MVK and EVK l9
13
-
11
-
9-
7-
,.
v
l v 25 50 75 10 Fmolr?;
Figure 4. Dose-response curves with MVK in the SOS Chromotest: ( X ) alkaline phosphatase activity; (A)@-galactosidase activity; (0)SOS-inducing factor I. is the most reactive target in the DNA. We could not isolate adducts from the reactions of MVK or EVK with nucleosides or nucleotides other than 2’-deoxyguanosine or 2’-deoxyguanosine 5’-monophosphate. Besides the main adducts, Ia and Ib, two other types of adducts, IIa, IIb, IIIa, and IIIb could be isolated and identified. The main adduct of MVK, Ia, was recently also found in binding studies by Chung et al. (36). The authors described, however, only the guanine adduct resulting from acid hydrolysis and not the deoxyguanosine adduct itself. Chung too found only one regioisomer. Our results with MVK confirm the findings of Chung et al. and demonstrate that the same types of adducts are formed in the reaction between EVK and deoxyguanosine. The other adducts identified in this study, adducts IIa, IIb, IIIa, and IIIb, have not been described before. Shapiro et al. (38)recently described bis adducts 1,N2,7,8of acrolein and guanine which are similar to our adducts IIIa and IIIb. To our knowledge this was the first time that the isolation and the ‘Hspectroscopic characterization of such bis adducts have been reported. We also found 7,&cyclic adducts and 1,N2,7,8bis adducts of a,@-unsaturatedaldehydes and guanine (unpublished results) and can confirm the results of Shapiro et al. In the case of the ketones MVK and EVK, 7,EJ-cyclic adducts or 1,N2,7,8bis adducts could, however, not be found. We found only the 1,N2,7bis adducts IIIa and IIIb. The influence of the structure of adducts Ia and Ib (see Results) on the genotoxicity of MVK and EVK is not yet clear. Nevertheless, a careful characterization of the adducts is a necessary basis for further studies. It is also not clear whether the adducts Ia and Ib found as main adducts in the in vitro study are also formed to such an extent in double-stranded DNA or in the in vivo situation and whether and to what extent the formation of the adducts Ia and Ib contributes to the induction of genotoxicity or the initiation of cancer. Nevertheless, we consider the formation of the cyclic adducts Ia and Ib to be a potentially significant DNA lesion at hot spots responsible for the induction of genotoxicity. The detection of such adducts in cellular systems can be regarded as an
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 55 indicator for the participation of such compounds in a genotoxic or carcinogenic process. The formation of N7 adducts can result in strand breaks (39), and indeed, spontaneous depurination to the N7 adducta IIa, IIb, IIIa, and IIIb was observed in our studies. Cross-linking is also conceivable because the linear N7 adduct still possesses a reactive carbonyl function which can react with other nucleophilic sites in the DNA. Such cross-linking products were not, however, identified in our studies. Another possibility is that the adduct IIIa can intercalate and induce frame shift. We did not, however, observe mutagenic activity of MVK and EVK in frameshift-sensitive strains such as his D3052 or TA98. In contrast to MVK and EVK, malondialdehyde (3) and 3-chloroacrolein (16), two other a,@-unsaturatedcarbonyl compounds, are clearly mutagenic in S. typhimurium his D3052. Our studies with 2’-deoxyguanosine 5’-monophosphate reveal qualitative and quantitative adduct formation which is similar to that with deoxyguanosine. At higher temperatures, trace amounts of other adducts with 2’-deoxyguanosine 5’-monophosphate were formed; they could, however, not be isolated. The results show that the isolation and detection of adducts of these ketones is not easy in vitro and is nearly impossible in cellular systems with the methods described here. Possible ways of increasing the detection sensitivity include the 32P-postlabeling technique and the antibody technique. The development of both kinds of techniques is made easier if well-characterized adducts are available as reference substances. The studies with nucleotides were performed not only to allow the quick identification of deoxyguanine adducts but also in order to develop a 32P-postlabelingtechnique using HPLC for the separation of the 32P-postlabelednucleotides. In general, it is rather difficult to detect genotoxic effects of a,@-unsaturated carbonyl compounds in the SOS Chromotest due to the high bacterial toxicity of these compounds. Similar difficulties have been observed when testing these compounds in the S. typhimurium reversion assay (2,11,12,17). MVK and EVK produced, however, clearly significantly positive results in this test, and the SOSIP value of MVK, 1.6 X lo2, and that of EVK, 2.7 X lo2, were clearly higher than that of crotonaldehyde, 7.3 X which did not yield clear positive results according to the criteria of Quillardet and Hofnung (37). The genotoxicities are not high when compared with nitroquinoline oxide, which was used as positive standard in the SOS Chromotest and for which we found a SOSIP of 43 (see Materials and Methods). They are, however, in the same range as those of alkylating compounds, e.g., methyl methanesulfonate (40). The genotoxic activities of MVK and EVK measured in the SOS Chromotest are consistent with the mutagenicities determined in the S. typhimurium reversion assay. As we have shown in recent publications (40,41) the two test systems are comparable to a certain extent. Although the molecular genetic mechanisms of the SOS system induction in bacteria are well understood, it is not known which DNA lesions induce this process (42). As discussed above, all the adducts described in this study must be considered as serious DNA lesions which potentially contribute to the induction of the SOS repair system. We have shown that both a,@-unsaturatedketones MVK and EVK interact with components of DNA and form adducts which can induce genotoxicity in bacterial tests. In their reactivity toward deoxyguanosine in their genotoxic activities, MVK and EVK are very similar to croto-
56 Chem. Res. Toxicol., Vol. 4, No. 1, 1991
naldehyde which induces liver tumors in the rat (5). The genotoxic activity of MVK and EVK in the SOS Chromotest is even higher than that of crotonaldehyde. All data available from these and other studies are consistent with the proposal that MVK and EVK possess mutagenic and carcinogenic potency and that MVK, due to its importance as an industrial chemical, and EVK, due to its widespread occurrence in foodstuffs, present a mutagenic and carcinogenic risk to mankind.
Acknowledgment. We are grateful to Dr. G. Lange for recording the mass spectra, Mrs. J. Colberg for performing the thermospray mass spectroscopy, and Mrs. E. Ruckdeschel for recording the NMR spectra. We thank Mrs. C. Grimm, Mrs. E. Weinfurtner, and Mrs. D. Muth for excellent technical assistance. We are indebted Dr. A. Dunlop for linguistic assistance. Registry No. Ia, 130548-32-2;Ib, 130410-23-0; IIa, 130410-24-1; IIb, 130410-25-2; IIIa, 130433-83-9;MVK, 78-94-4; EVK, 1629-58-9; deoxyguanosine, 961-07-9 2'-deoxyguanosine 5'-monophosphate, 902-04-5.
References (1) Eder, E., Henschler, D., and Neudecker, T. (1982) Mutagenic
properties of allylic and a,@-unsaturated carbonyl compounds: consideration of alkylating mechanisms. Xenobiotica 12,831-848. (2) Lutz, D., Eder, E., Neudecker, T., and Henschler, D. (1982) Structure mutagenicity relationship in a,@-unsaturatedcarbonyl compounds and their corresponding allylic alcohols. Mutat. Res. 93, 305-315. (3) Marnett, L., Hurd, H. K., Hollstein, M. C., Levin, D. E., Esterbauer, H., and Ames, B. N. (1985) Naturally occurring carbonyl compounds are mutagens in Salmonella tester strain TA104. Mutat. Res. 148, 25-34. (4) Henschler, D., and Eder, E. (1986) Structure-activity relationship of a,@-unsaturated carbonyl compounds (Singer, B., and Bartsch, H., Eds.) IARC Scientific Publication 70, pp 197-205, IARC Scientific, Lyon. (5) Chung, F. L., Tanaka, T., and Hecht, S. S. (1986) Induction of liver tumors in F34 rats by crotonaldehyde. Cancer Res. 46, 1285-1289. (6) Robinson, M., Bull., R. J., Olson, G. R., and Stober, J. (1989) Carcinogenic activity associated with halogenated acetones and acroleins in the mouse skin assay. Cancer Lett. 48, 197-203. (7) IARC (1985) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 36, pp 133-177, IARC, Lyon, France. (8) Draminski, W., Eder, E., and Henschler, D. (1983) New pathway of acrolein metabolism in rats. Arch. Toxicol. 52, 243-247. (9) Schoental, R., and Gibbard, S. (1972) Nasal and other tumors in rats eiven 3.4.5-trimethoxvcinnamaldehvde.a derivative of sinapaldihyde and of other a,j-unsaturated aldehyde wood lignine constituents. Br. J. Cancer 26, 504-505. 3) Coleman, W. E., Munch, J. W., Kaylor, W. H., Streicher, R. P., Ringhand, H. P., and Meier, J. R. (1984) Gas chromatography/ mass spectroscopy analysis of mutagenic extracts of aqueous chlorinated humic acid. A comparison of the by-products to drinking water contaminants. Environ. Sci. Technol. 18,674481. 1) Neudecker, T., Eder, E., Deininger, C., and Henschler, D. (1989) Crotonaldehyde is mutagenic in Salmonella typhimurium TA100. Enuiron. Mol. Mutagen. 14, 146-148. 2) Neudecker, T., Eder, E., Deininger, C., Hoffman, C., and Henschler, D. (1989) Mutagenicity of methylvinyl ketone in Salmonella tvDhimurium TA100-indication for eDoxidation as an activation mechanism. Mutat. Res. 227, 131-1'34. (13) Eder, E., Dornbusch, K., and Fischer, G. (1987) The role of biotransformation in the genotoxicity of allylic compounds. Arch. Toricol. 60, 182-186. (14) Eder, E., and Dornbusch, K. (1988) Metabolism of 2,3-dichloro-I-propene in the rat: consideration of bioactivation mechanisms. Drug. Metab. 16, 60-68. (15) Schuphan, J., and Cassida, J. E. (1979) S-Chloroallyl thiocarbamate herbicides: chemical and biological formation and rearrangement of diallate and triallate sulfoxides J . Agric. Food Chem. 27, 1060-1067.
Eder et al. (16) Eder, E., Hoffman, C., Bastian, H., Deininger, C., and Schekkenbach, S. (1990) Molecular mechanisms of DNA damage initiated by a,@-unsaturatedcarbonyl compounds as criteria for genotoxicity and mutagenicity. Enuiron. Health Perspect. 88, 99-106. (17) Deininger, C., Eder, E., Neudecker, T., and Hoffman, C. (1990) Mutagenicity and genotoxicity of ethylvinyl ketone in bacterial tests. J. Appl. Toxicol. 10 (3), 167-171. (18) Henschler, D., Ed. (1987) Gesundheitsschddliche Arbeitstoffe, VCH-Verlag, Weinheim. (19) Shaw, P. E., Ahmed, E. M., and Dennison, R. A. (1977) Orange juice flavor: contribution of certain volatile components as evaluated by sensory panels. Proc. Int. SOC.Citric 3, 804-807. (20) Bloeck, S., Kreis, A., and Stanek, 0. (1986) Comparative determination of aldehydes and ketones in apple juice after 2 years storage in inner protected aluminum and tin cans of 4-20 "C. Alimenta 25, 23-28. (21) Hsieh, 0. A. L., Huang, A. S., and Chang, S. S. (1981) Isolation and identification of objectionable volatile flavor components in defatted soy bean flour. J. Food Sci. 47, 16-18. (22) Buttery, R. G., Teraniski, R., and Ling, L. C. (1987) Fresh tomato aroma volatiles: a quantitative study. J. Agric. Food Chem. 35, 540-544. (23) Takeoka, G. R., Gunlert, M., Flath, R. A., Wurz, R. E., and Jennings, W. (1986) Volatile constituents of kiwi fruit (Actinida chinesis planch). J. Agric. Food Chem. 34, 576-578. (24) Goetz-Schmidt, E. M., and Schreier, P. (1986) Neutral volatiles from blended endive (Cichorium endivia, L). J. Apric. Food Chem. 34, 212-215. (25) Mick, W., and Schreier, P. (1984) Additional volatiles of black tea aroma. J. Apric. Food Chem. 32, 924-929. (26) Stark, W., Smith, J. F., and Foss, D. A. (1967) Pent-I-en-3-01 and pent-1-en-3-one in oxidized dairy products. J . Dairy Res. 34, 123-129. (27) Josephson, D. B., Lindsay, R. C., and Stuiber, D. A. (1984) Identification of volatile aroma compounds from oxidized frozen white fish (Cargonus dupea formis). Can. Inst. Food Sci. Technol. J. 17, 178-182. (28) MacLeod, G., and Ames, J. M. (1986) The effect of heat on beef aroma: comparison of chemical composition and sensory properties. Flavour Fragrance J. 1, 91-104. (29) Noleau, J., and Toulemonde, B. (1987) Volatile components of roasted chicken fat. Lebensm.- Wiss. Technol. 20, 37-41. (30) Baker, R. R., Dymond, H. F., and Shilabear, P. K. (1984) Determination of a,@-unsaturatedcompounds formed by burning cigarette. Anal. Proc. (London) 21, 135-137. (31) Chung, F. L., Young, R., and Hecht, S. S. (1984) Formation of cyclic 1,N2-propanodeoxyguanosineadducts in DNA upon reaction with acrolein or crotonaldehyde. Cancer Res. 44, 990-995. (32) Basu, A. K., O'Hara, S. M., Valladier, P., Stone, K., Mols, O., and Marnett, L. J. (1988) Identification of adducts formed by reaction of guanine nucleosides with malondialdehyde and structurally related aldehydes. Chem. Res. Toxicol. 1, 53-59. (33) Hoffman, C., Bastian, H., Wiedenmann, M., Deininger, C., and Eder, E. (1989) Detection of acrolein congener DNA adducts isolated from cellular systems. Arch. Toxicol. Suppl. 13, 219-223. (34) Foiles, P. G., Akerkar, S. A., and Chung, F. L. (1989) Application of an immunoassay for cyclic acrolein deoxyguanosine adducts to assess their formation in DNA of Salmonella typhimurium under conditions of mutation induction by acrolein. Carcinogenesis 10, 87-90. (35) Meerman, J. H. N., Smith, T. R., Pearson, P. G., Meier, G. P., and Nelson, S. D. (1989) Formation of cyclic 1,N2-pro anodeoxyguanosine and thymidine adducts in the reaction of t l e mutagen 2-bromoacrolein with calf thymus DNA. Cancer Res. 49, 6174-6179. (36) Young, R., Chung, F. L., and Hecht, S. S. (1983) Modification of deoxyguanosine by simple a,@-unsaturated carbonyl compounds. 74th Annual Metting of the American Association for Cancer Research, San Diego, CA, Abstract 269, p 68. (37) Quillardet, P., and Hofnung, M. (1985) The SOS Chromotest, a colorimetric bacterial assay for genotoxins: procedures. Mutat. Res. 147, 65-78. (38) Shapiro, R., Sodum, R. S., Everett, D. W., and Kundu, S. K. (1986)Reactions of nucleosides with glyoxal and acrolein (Singer, B., and Bartsch, H., Eds.) IARC Scientific Publication 70, pp 165-173, IARC Scientific, Lyon. (39) Moschel, R. C., Hudgins, W. R., and Dipple, A. (1979) Selectivity in nucleoside alkylation and alkylation in relation to chemical carcinogens. J . Org. Chem. 44, 3324-3328.
Deoxyguanosine Adducts of MVK and EVK (40) Eder, E.,Deininger, C., and Kutt, W. (1989)Genotoxicity of monofunctional methanesulphonates in the SOS chromotest as a function of alkylation mechanisms. A comparison with the mutagenicity in S. typhimurium TA100. Mutat. Res. 211, 51-65. (41) Eder, E.,Favre, A,, Deininger, C., Hahn, H., and Kutt, W. (1989) Induction of SOS repair by monofunctional methane-
Chem. Res. Toxicol., Vol. 4, No. I, 1991 57 sulphonates in various Escherichia coli strains. Structure-activity relationships in comparison with mutagenicity in Salmonella typhimurium. Mutagenesis 4, 179-186. (42) Boiteux, S., Huisman, O., and LaVal, J. (1984)3-Methyladenine residues in DNA induce the SOS function sfii in Escherichia coli. EMBO 3, 2569-2573.
