Chem. Res. Toxicol. 1993,6, 486-494
486
Identification and Characterization of Deoxyguanosine Adducts of Mutagenic @-Alkyl-SubstitutedAcrolein Congeners Erwin Eder' and Christian Hoffman Institute of Toxicology, University of Wiirzburg, Versbacher Strasse 9, 0-8700 Wiirzburg, FRG Received January 18,1993
The reaction of the mutagenic 6-alkyl-substituted acroleins (E)-a-pentenal, (E)-2-hexenal, (E&)-2,4-hexadienal, and 3,3-dimethylacrolein with nucleosides and 5'-mononucleotides was studied. We found two different types of adducts with deoxyguanosine and 2'-deoxyguanosine 5'-monophosphate. No adducts could be isolated with either nucleosides other than deoxyguanosine or nucleotides other than 2'-deoxyguanosine 5'-monophosphate. With pentenal, hexenal, and 3,3-dimethylacrolein, we identified and characterized 1,N2-cyclic adducts and 7,8-cyclic adducts. The 1,N2-adductsof pentenal and hexenal were mixtures of diastereomers, one pair in which the substituents in the newly formed ring were trans (6S,8S and 6R,8R) and another in which they were cis. The cis isomers were formed to a much lesser extent. In all cases, the regioisomer is formed in which the OH group is vicinal to the N-1 atom of the guanine moiety. In the case of the 7,8 adducts, the ribose cleaved spontaneously during the reaction, and a mixture of isomers in which the substituents were cis and trans in the newly formed tetrahydropyrrole ring was observed. Since these compounds form adducts in a similar way to crotonaldehyde and are also mutagenic like crotonaldehyde, it was proposed to regard them as carcinogenic, like crotonaldehyde, unless experimental examination demonstrates nonmutagenicity.
Introduction cu,B-Unsaturated carbonyl compounds are a class of substances to which mankind, animals, and plants are ubiquitously exposed. As potential toxic and genotoxic compounds, these substances are of increasing significance for human health. The research on the biological interactions of these compoundsis a vast and rapidly expanding field involving not only mutagenicityand cancer initiation but also tumor promotion and many other biological processes such as aging (1). Acrolein and the lower alkylsubstituted congeners (e.g., crotonaldehyde or methyl vinyl ketone) are of high importance in industry. These compounds are mutagenic (2,3)and form DNA adducts (4-8), and crotonaldehyde was shown to be carcinogenic (9). The higher members of this class are, however, no less interesting since they are found as natural products in plants, foodstuffs, soil, and water (I, 10, 11)and they possess manifold biological functions in nature (e.g., as repellents, attractants, fragrance substances, or natural fungicides and insecticides) (10). Some of them are also usedin technical processes (e.g., hexenal for the production of perfumes or hexadienal for the synthesis of sorbic acid). In particular, the 8-alkyl-substitutedand the @-hydroxysubstituted acroleins are produced endogenously in mammals during some physiological and pathophysiological processes (e.g., lipid peroxidation, arachidonic acid oxidation), or during reactions of reactive oxygen species (12, 13),especially after inflammatory processes and ischemia (12). Thus these compoundsrepresent a possible constant source of DNA damage and are therefore considered to play an important role in human carcinogenicity (13). However, the severity of this damage and whether or to *To whom correspondence and requests for reprints should be addreeeed.
what extent these compounds are involved in the induction of tumors in general, and in the induction of endogenous cancer in particular, have not yet been determined experimentally. We have recently published structure-mutagenicity relationships for 8-alkyl-substituted acroleins and have proposed that the mutagenicity of these compounds depends on the formation of DNA adducts (3). Our results with Salmonella typhimurium strain TA 100 are in line with those obtained with strain TA 104 by Marnett et al. (14).
We now present a detailed description and characterization of the deoxyguanosine and guanine adducts formed by 8-alkyl-substituteden& with deoxyguanosine and with 5'-deoxyguanosine monophosphate.
Materials and Methods Chemicals. Caution: The enuls are toxic, irritating to eyes and mucosa, and mutagenic. (EI-a-Pentenal and (E)-2-hexenal were purchased from Aldrich (Steinheim, FRG) (gold label), (E,E)-2,4-hexadienalwas from EGA-Chemie (Steinheim, FRG), and 3-methyl-2-butenal (3,3-dimethylacrolein)was from Merck (Darmstadt, FRG). They were distilled under nitrogen, with a 'Fischer Spaltrohr" column MMS 255 from Fischer (Mechenheim, FRG) immediately before use; their identity was checked by f H NMR spectroscopy and their purity by capillary gas chromatography: pentenal(l20 "C/760 Torr, >99.5% 1; hexenal (47 OC/17 Torr, >99%); 3,3-dimethylacrolein (72 OC/15 Torr, >99.3%); and 2,4-hexadienal (72 OC/15 Torr, 95% containing 5 % (E,Z)-isomer). 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
0893-228~/93/2706-0486$04.oo/o 0 1993 American Chemical Society
Chem. Res. Toxicol., Vol. 6, No. 4, 1993 487
dG Adducts of 8-Alkyl-Substituted Enals salt), 2'-deoxyguanosine 5'-monophosphate (sodium salt), and 2'-deoxycytidine 5'-monophosphate (free acid) were purchased from Sigma Chemie GmbH. Alkaline phosphatase from bovine intestinal mucosa was purchased from Sigma Chemie. All chemicals, solvents, and reagents for the preparation of the buffers, the eluents, etc., were obtained in the highest purity available from Merck, Darmstadt; Aldrich (Steinheim); Serva (Heidelberg); or Boehringer (Mannheim, FRG). Reactions of the Test Substances with Nucleosides and Nucleotides. To a solution of 0.412 mmol of nucleoside (365 pmol of nucleotide) in 20 mL (2 mL) of 0.1 M sodium phosphate buffer (pH 7) was added 2.0 mmol (0.2 mmol) of the respective end. The mixture was stirred at 90 "C for 5 days with pentenal, for 6 days with hexenal and 3,3-dimethylacrolein,and for 4 days with 2,4-hexadienal. The reaction solutionswere colored yellow, orange, or yellowish brown. The details of the reaction have been reported elsewhere. (7,8,15). Acid Hydrolysis of t h e Modified Adducts To Yield t h e Modified Nucleobases. Solutions of the nucleoside adducts (50-100 pmol) in 8 mL of 0.1 M HC1 were heated at 65 OC for 45 min. After cooling to room temperature, the solution was neutralized by dropwise addition of 0.1 M potassium hydroxide solution. (For isolation see "Chromatographic Isolation of the Adducts", part e). Enzymatic Hydrolysis of the Nucleotides To Yield t h e Modified Nucleosides. Either 100 or 50 pL of alkaline phosphatase was added to 2 mg of the lyophilized nucleotide in 2 mL of 10 mM sodium phosphat buffer. The mixtures were incubated at 37 OC for 22 h in an incubator-shaker. After cooling, the mixtures were filtered through a 0.45-pm filter and the resulting nucleosidesseparated and analyzed as described below. The nucleotide fractions were obtained from Sephadex LH-20 chromatography (0.23 mL/min). Nucleotides and modified nucleotides elute first and than polymeric and oligomeric products. Chromatographic Methods. Sephadex LH-20 chromatography (LH-20)' was carried out in a 100 X 1.61 cm glass column, wet-filled with Sephadex LH-20, 20-100 pm, from Pharmacia (Freiburg, FRG), using double-distilled water as eluent. The flow rates indicated below were regulated with an Ismatec MVCA-4 pump (Wertheim,FRG). The chromatogram was recorded at 254 nm with an LKB 2138 Uvicord UV detector and a Metrawatt Servogar 5b recorder. Thirty-minute fractions were collected with an LKB 2111 Multi Rac fraction collector. Medium-presure liquid chromatography (MPLC) was performed on a Labochrom "FPGC-prepacked" column, 18.5 mm X 28 cm, HD-Sil-18-30-60,35-70 pm, reversed-phase silica gel C8, and a Labomatic MD 80-100 pump fitted with a Labomatic PC100 pressure control unit at 254 nm with an Isco detector, Model 248, using a stepwise gradient as indicated below. For HPLC, the Waters system consisted of a U6K injection system or a Rheodyne injection valve 1265, two Model 6000 A pumps, a Model 660 gradient solvent programmer, and a Model 450 variable-wavelengthdetector connectedto a Hewlett/Packard 3385 A integrator. A reversed-phase C18 column, 5 pm (and another column with 10 pm), 0.46 X 25 cm, from Knauer (Homburg, FRG), was used for the analysis and a Bondapak reversed-phase, 10-pm, 0.78 X 30 cm column was used for semipreparative purposes. The solvents, gradients, etc., are described below. Chromatographic Isolation of the Adducts. The chromatographic purification and isolation of the adducts were complex. In general, the reaction mixtures obtained with deoxyguanosine were filtered through a 0.45-pm filter and then (in portions) subjected to LH-20. (a) Adducts of Pentenal. The LH-20 fractions 8-12 contained the adducts. The adducts were isolated with MPLC. The l Abbreviations: LH-20, SephadexLH-20gel chromatography;MPLC, medium-pressureliquid chromatography;EI, electron impact; FAB, faat atom bombardment.
a) Sephedex LH 20
b) HPLC
UV Detection (254 nm) 30 min I Fradlon
Sephadex LH 20 Fr. 27
Of
dGuo lla diestereomera
lla (trans) 118
/
(as)
L
Figure 1. Chromatographic properties of the hexenal adducts. (a) Sephadex LH-20 gel column chromatography of the reaction mixture with deoxyguanosine: 1,N2-cyclicadducts are in fractions 23-27 and the 7,8-cyclic adducts in fractions 31-37. (b) HPLC of Sephadex LH-20 fraction 27. The pair of cis diastereomers had retention times of 10.1 and 11.5 min, and the trans diastereomers, 17.3 and 20.0 min. Conditions: Knauer column reversed-phase C 18,5pm, 0.46-cm i.d., length 25 cm; methanol/ water, 9010, flow rate 1 mL/min, isocratic; UV detection, 254 nm. fraction with t~ = 21 min contains 2.4% adducts Ib (7,&cyclic adducts, cis:trans = 41), a colorless crystalline powder which decomposesat 230 OC, and the fraction with t~ = 22 min contains 19% adducts Ia, a pair of diastereomers of Ia trans l,Na-cyclic adducts (18.5%) and a pair of diastereomers of la cis (0.5%) (crystalline colorless powder with a melting point of 144-147 "C). The Ia (trans) diastereomers have retention times of t R = 20.7 and 25.1 min, and the Ia (cis) diastereomers, t~ = 12.5 and 13.7 min, in the HPLC using a Bondapak 10-pm column (HzO/ methanol, 90:10, flow rate 1 mL/min). Ib trans and cis had retention times of tR = 9.2 and 11.4 min. (b) Adductsof Hexenal. Adducts IIa (trans/&) (1,N2-cyclic adducts) were in the LH-20 fractions 23-27 and adducts IIb (trawlcis) in fractions 31-37 (see also Figure 1). In this case, a flow rate of 0.283mL/min was used instead of the standard rate of 0.5 mL/min in order to achieve an optimal separation. The adducts IIa were purified by MPLC and had a retention time of t~ = 23 min under the following conditions: stepwise gradient, HzO/methanol, 16% methanol for 16 min, 40% methanol for 13 min, and then 100% methanol for 8 min and a flow rate of 6 mL/min. We obtained 12% colorless powder, melting point 132-134 "C. For HPLC see Figure lb. Adducts IIb were purified by an additional LH-20 chromatography run, and we obtained 1.2% colorless crystalline powder (&/trans 4:l) which decomposed at 270 OC. (c) Adducts of 3,3-Dimethylacrolein. Adduct IIIa (l,Nacyclic adduct) was in the LH-20 fractions 25-26 and adduct IIIb (7,8-cyclic adduct) in the fractions 33-37 (flow rate 0.25 mL/ min). Adduct IIIa was purified by MPLC and was isolated in a yield of 1.2 % colorless crystalline powder, melting point 152154 OC,andhadaretentiontimeoft~=12minunderthefollowing conditions: stepwise gradient, HzO/methanol, 9010 for 10 min, 4060 for 15 min; flow rate 8 mL/min. Retention time in HPLC was t R = 16.1 min under the following conditions: Bondapak 10 pm, methanol/water, 9010, flow rate 1 mL/min. The diastereomers could not be separated. Adduct IIIb was isolated in a yield of 1 mg as colorless crystalline powder. The 1H NMR spectrum showed that there were some impurities. Additional purification by LH-20 resulted in trace amounts sufficient only for UV spectroscopy and not for an NMR spectrum. (d) Adducts of (E,J3')-2,4-Hexadienal. Adducts of hexadienal were detected in fractions 17-34 of LH-20 (0.283mL/min). HPLC analysis (Knauer 10 pm, HgO/methanol, 80.20, flow rate 1mL/ min) showed a pair of adducts with retention times of t~ = 17.9
Eder and Hoffman
488 Chem. Res. Toxicol., Vol. 6, No. 4, 1993
Table I. FT-IR-UV Spectroscopy, Mass Spectrometry, and Elemental Analysis of 1,NWyclic Deoxyguanosine Adducts with Pentenal (Ia), Hexenal (IIa),3.3-Dimethylacrolein (IIIa). and Hexadienal (IVa) ~
FT-IR (KBr) Y, cm-1
UV A,, nm (lo e) (0.1 N HCl, 0.1 N fiaOH)
elemental analysis MS (5%) thermospray: mlz 352 (8,MH?, 3323 (OH), 2967,2931, HzO: 258 (4.16), 277;' C16HZlN606 (351.4)*2.5HzO 236 (100, BHz+),218 (18, BHz+ A- 229 (3.60) calcd, 45.45, H 6.61, N 17.67; 2881 (CH), 1690 (C=O), - HzO), 166 (5), 152 (67, GuaH+), found, C 45.64; H, 5.95, N 17.33 Ia, cis 1570,1539 (C=N, C=C), HCI: 202 (4.231,261 (4.12), 134 (4); FAB (70 eV, glycerol): 283;O A- 234 (3.55) 1334,1095,1056,782,642 m/z352.3 (52, MH+), 236.2 (100, NaOH 216 (4.11),260 (4.11), BHz+),207.2 (68),192.1 (36) 2.77;O A- 233 (3.68) HzO: 259 (4.18), 2.77;' Athermospray: mlz 401 (3), 366 (34, IIa, trans 3402 (OH), 2960,2930, CieHzsNsOs (365.4).0.6Hzo: MH+), 250 (100, BHz+),232 227 (3.46) calcd, C 51.33, H 6.46, N 18.71; 2874 (CH), 1690 (C=O), (BH2+ HzO), 166 (12), 152 (51, found, C 50.98, H 6.42, N 18.37 1570,1538 (C=N, C=C), HCI: 262 (4.10), 283;' AGuaH+),134 (40); FAB.170 ev): 234 (3.43) 1333,1096,1055,941, m/z 366.2 (54, MH+), 351.3 (W, NaOH: 216 (3.97), 260 (4.12), 781,644 315.3 (6), 250.2 (100, BHz+) 277;' A- 231 (3.54) HzO: 260 IIa, cis thermospray: m/z 352 (100, MH+), HzO 258 (4.20), 279;' AIIIa 3402 (OH), 2967 (CH), C ~ & Z I N ~(351.4).1.0H~o: O~ 258 (30),236 (64, BHz+), 192 (lo), 227 (3.50) calcd, C 48.78, H 6.28, N 18.96; 1690 (C=O), 1566,1539, 166 (78), 152 (47, GuaH+);FAB found, C 48.27, H 6.23, N 19.06 (C=N, C=C), 1339,1104, HCI: 262 (4.17); A- 234 (3.55) (70 eV, glycerol): mlz 352.3 (65, NaOH: 215 (4.10),260 (4.19), 944,782,645,473,447 277;' A- 230 (3.62) MH+), 299.3 (6), 236.3 (100,BHz+), 207.2 (49), 192.1 (19) HzO 258; A- 231 IVa, trans HzO: 260, A- 229 IVa, cis adduct
Ia, trans
-
Shoulder. and 19.1min. Many other smaller HPLC signals were, however, also present. The LH-20 fractions 25-27 were further purified by semipreparative HPLC under the following conditions: Bondapak 10pm, HzO/methanol, 9010, isocratic for 45 min, then gradient 6 (Waters programmer) up to 100% methanol in 10 min, flow rate 5 mL/min. Two pairs of adducts IVa were detected with retention times of t R = 24.0 and 28.3 min [uv (H2O): A, = 258 nm and A- = 231 nm] and t R = 30.6 and 36.9 min [uv (HzO): A, = 260 nm and A- = 229 nm] in the ratio 1:1:7:7. The trace amounts were not sufficient to record an NMR spectrum. According to the UV spectra and to the chromatographic properties these compounds are pairs of diastereomers of 1,NZ-cyclic adducts, cis and trans. (e) lJP-Guanine Adducts Iaf, IIaf, and IIIaf (Acid Hydrolysates of Ia,IIa,and IIIa). The 1,N2-cyclicdeoxyguanosine adducts were hydrolyzed as described above, and the resulting guanine adducts were then isolated by LH-20 chromatography. Ia' was obtained as crystalline powder, melting point 180-183 "C, in a yield of 82% from LH-20 fractions 33-40 (0.267 mL/ min) and had a retention time of t R = 11.9 min in HPLC (Knauer 10 pm, H2O/methanol, 9010, flow rate 1 mL/min). IIa' was obtained from LH-20 fractions 37-46 (flow rate 0.283 mL/min) as a colorless crystalline powder, melting point 175177 "C, in a yield of 83%. The retention time in HPLC was t R = 24.2 min (Knauer 10 pm, HzO/methanol, 9010, flow rate 1 mL/min). IIIa' was obtained from LH-20 fractions 24-27 as a colorless crystalline powder, melting point 208-210 "C. The retention time in HPLC was t~ = 12.1min (Bondapak 5pm, HzO/methanol, 9010, flow rate 1 mL/min). Analysis and Characterization. The substances were analyzed and characterized with the following instruments: UV Spectroscopy: Kontron Uvikon Model 860 and Model 800 plotter, at pH 1 (0.1 N HC1); pH 7 (water); pH 13 (0.1 N NaOH). FT-IR Spectroscopy: Nicolet 5 DXC. 1H NMR Spectroscopy: Bruker AC 250 (250.1 MHz) and Bruker WM 400 (400.1 MHz); tetramethylsilane. 13CNMR Spectroscopy: Bruker AC 250 (62.9 MHz) and Bruker WM 400 (100.6 MHz); tetramethylsilane. Mass Spectroscopy: (a) Electron impact mass spectrometry (EI): Varian MAT CH7 (70eV) fitted with a data system 200. (b) Fast atom bombardment (FAB)mass spectra were recorded on a 8200 Finnigan VAT C 70-eV spectrometer. Xenon and argon were used as ionization gases and glycerol and triethylene or tetraethylene glycol as FAB matrix. (c) 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 witha 17mM ammonium acetate solution. The C, H, N elemental analysis was performed at the Institute of Inorganic Chemistry (University of Wbzburg). Characterization of t h e Adducts. The spectroscopic data of FT-IR, UV, mas8 spectrometry, and the elemental analysis of ddducta Ia, IIa, IIIa, and IVa are shown in Table I. For adduct IVa, only UV data are given. The 1H NMR data of adducts Ia, IIa, and IIIa are presented in Table 11, and the 18C NMR data, in Table 111. For better comparsion the lH NMR data of the guanine adducts Ia', IIa', and IIIa' are included in Table I1 and the 1W NMR data in Table 111. The FT-IR, UV, mass spectrometry, and elemental analysis of Ia', IIa', and IIIa' are shown in Table IV. The spectroscopic data of FT-IR, W, and mass spectrometry of the 7,8-cyclic adducts Ib and IIb are presented in Table V and the lH NMR data in Table VI. For adduct IIIb, only UV data are given in Table V.
Results After reaction of the j3-alkyl-substituted acrolein (E)2-pentenal, (E)-2-hexenal, and 3,3-dimethylacrolein with deoxyguanosine and its 5'-monophosphate, we isolated two different types of adducts (shown in Scheme I as an example for pentenal). Acid hydrolyses of the respective 1,W-deoxyguanosineadducts resulted in the cleavage of deoxyribose, and the respective 1,W-guanineadduch were obtained. The guanine moiety is, most probably, the most reactive DNA target for these enals since we were not able to isolate any adducts with other nucleosides or nucleotides.
1 3 - C y c l i c Adducts. With deoxyguanosine, we isolated as main products pairs of diastereomers of trans Ia, IIa, IIIa, and IVa and lower amounts of the cis diastereomers (see chromatographic isolation). According to the IUPAC nomenclature we propose the following names: Adducts of pentenal: Ia, (6S,8S)- and (6R,8R)-3-(2'deoxy-~-~erythro-pent~furanosyl)-6-ethyl-5,6,7,&tetrahydro-8-hydroxypyrimido[l,2-u]purin-l0(3H)-one;Ia', (6S,8S)- and (6R,8R)-6-ethyl-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purin- 10-(3H)-one. Adducts of hexenal: IIa, (6S,8S)- and (6R,8R)-3-(2'deoxy-~-~-erythro-pentofuranosyl)-5,6,7,8-tetrahydro-8-
Chem. Res. Toxicol., Vol. 6, No. 4, 1993 489
dG Adducts of 8-Alkyl-Substituted Enals
Table 11. 1H NMR Data of l,N*-Cyclic Deoxyguanosine Adducts and Guanine Adducts of Pentenale Ia, and Ia', of Hexenale IIa and IIa', and of 3,3-Dimethylacroleine IIIa and IIIa' (400.1 MHz, [DeIMelSO) proton
7a-H coupling
7b-H coupling
adduct IIIa adduct IIa' 1.92, dt, 1H 1.30-1.46, m, 4H 1.30-1.46, m, 4H 7a-H, lla-H, 12a,b-H 7a-H, lla-H, 12a,b-H J7.,7b 3 14.0 HZ J7,6 = 14.0 HZ J,.J 3.5 HZ J,.? = 3.5 HZ 2.15, dd, 1H 2.04, br d, 1H 2.06, br d, 1H 2.05, br d, 1H 2.07, br d, 1H J7b.7. 13 Hz Jn,,. = 12.8 HZ Jna 2.0 HZ Jn,,. = 13.0 HZ 2.18, m, 1H 2.24, m, 1A 2.20, m, 1H d 2.50,O m, 1H 2.51,O m, 1H 3.51-3.60, m," 2H 3.47-3.56, m, 2H 3.48-3.60, m, 2H 3.56-3.63, m, 1H 3.59, m, 1H 3.48-3.60m, m, 1H 3.53, m, 1H 3.80, m, 1H 3.80, m, 1H 3.81, m, 1H 4.34, m,1H 4.34, br s, 1H 4.36, br 8, 1H 4.95,b br 8 , 1H 4.93: t, 1H 4.95, t, 1H J O H =, ~5.0 HZ JOHT 4.5 HZ 5.25: d, 1H 5.28: br 8 , 1H 5.28: d, 1H JoH= , ~3.3 ~ HZ JOHJ 3.0 HZ 6.12, ps t, 1H 6.13, dd, 1H 6.12, dd, 1H J1.3 6.0 HZ J19,y = 7.8,6.0 HZ J1,z = 8.0,6.0 HZ 6.23, br s (m), 1H 6.21, bra, 1H 6.22, br 8 , 1H 6.20, m, 1H 6.23, br 8 , 1H 6-63: d, 1H 6.46," br s, 1H 6.58, d, 1H 6.58: br 8, 1H 6.64: br s, 1H JOHB 4.5 HZ J o H ,=~4.3 HZ 7.90,s,lH 7.90: s 7.60,' br 8, 1H 7.66, br 8 , 1H 7.91, 8, 1H 7.80, br 8 , 1H 7.60: br 8, 1H 7.56,b br s, 1H 7.90,O s 7.84: br 8, 1H 12.20: very br 8 , 1H 12-35: very br 8, 1H
adduct Ia 1.41, m, 1H J,&n 13.0 HZ J,,e 13.0 HZ
2'~-H 2'8-H 5'-H 6-H 4'-H 3'-H 5'-OH coupling 3'-OH coupling 1'-H coupling 8H 8-OH coupling 2-H N(5)-H N(3)-H side-chain protons lla-H 1.43-1.52, m, 1H coupling llb-H 12-H coupling 13-H coupling
1.76. m. 1H 0.92; t, 3H 5 1 2 ~ 1= 7.3 HZ
1.45, m, 1H 13.5 HZ 13 HZ 1.69, m, 1H 0.90, t, 3H Jl1,12 7.3 HZ
adduct IIIa' (250 MHz)
adduct IIa
adduct Ia' 1.39, br t, 1H J,&n 13.5 HZ J7,,6 13.5 HZ
1.77, dd, 1H 14.0 Hz J74e = 4.5 HZ J7.,7b
2.08, br d, 1H
6.17, m, 1H 6.38: very br 8,lH 7.63,s,lH 7.59: br 8, 1H C
see 7a
see 7a
1.26,8,3H
1.24,8,3H
1.69, m, 1H see 7a
1.64-1.72, m, 1H see 7a
1.42,s,3H
1.41, e., 3H
0.91, t, 3H = 7.1 HZ
0.90, t, 3H Jl3,12 7.0 HZ
J11.,lp, J11.,6 =
Jls,12
*
The signal is partly overlapped either by the solvent signal or by other signals. The signal disappears after shaking with DzO.e The N(3)-H signal of IIIa' is not observed. The 2'a-H of IIIa is completely overlapped by the solvent signal.
la
II a
hydroxy-6-propylpyrimido[1,2-alpurin-9(3H)-one; IIa', (6S,8S)- and (6R/8R)-5,6,7,&tetrahydro-8-hydroxy-6-propylpyrimido[1,2-a]purin- 10(3H)-one. Adducts of 3,3-dimethylacrolein: IIIa, (BRIS)-3-(2'deoxy-j3-~-erythro-pentofuranosyl) -5,6,7,8-tetrahydro-Bhydroxy-6,6-dimethylpyrimido1,2-a1purin- 10(3H)-one; IIIa', 5,6,7,&tetrahydro-8-hydroxy-6,6-dimethylpyrimido[l,2-alpurin-10(3H)-one. In these adducts, the newly formed 1,N2-ring(9,5-ring in the new nomenclature) is in a chair-like configuration in which the OH group is in the axial position and the alkyl group is in the equatorial position. In general, the equatorial position is favored; however, polar groups in the a-position to a hetero ring atom frequently take the axial position (anomericeffect) (16). The described trans adducts are the conformers with the lowest energy level (17).Figure 2 shows the lH NMR spectrum of adduct Ia' (trans) as an example to demonstrate the conformation via the couplingconstants. Axial, axial couplingconstants which were 12-14 Hz throughout our experiment were only observed between the 6-H proton and the 7-Ha proton ( J q= 13.5 Hz) of the newly formed tetrahydropyrimidine ring. Geminal coupling constants which were also 13.5 Hz were found between 7-Ha and 7-Hb and the exocyclic
111 a
l l - H protons. The axial, equatorial couplings Ja, and equatorial, equatorial coupling constants (J-1 were about 2.3 Hz. Sinceno axial, axial coupling was observed between the 8-H proton and the 7-H protons, the OH group must be in the axial position of 8, and since axial, axial coupling was observed between the 6-H and the 7-Ha proton, the alkyl group must take the equatorial position. From the IH NMR data (Figure 2 and Table 11)one can also derive that there is in no case a change in conformation and that no ring inversion of the tetrahydropyrimidine ring takes place at room temperature. Otherwise the coupling constants should possess an average value between JBB and J,, of about 7-9 Hz. The cis diastereomers of Ia, IIa, and IVa are formed to a much lesser extent because the alkyl group is in this case in the energetically less favored axial position. In principle, two different regioisomers, one in which the OH group is vicinal to the N-1 amino group of the guanine moiety (N-9in the new nomenclature) and one in which the OH group is adjacent to the N2atom (N-5 in the new nomenclature), can be formed. We have recently demonstrated how the two regioisomers can be distinguished by 'H NMR spectroscopy and have shown that unsubstituted acrolein forms both regioisomers, whereas crotonaldehyde forms exclusively the first type
Eder and Hoffman
490 Chem. Res. Toxicol., Vol. 6, No. 4, 1993
Table 111. ‘42NMR of 1,NWyclic Deoxyguanosine Adducts and Guanine Adducts (100.6 MHz, [DtlMeBO) proton aide chain C-13 c-12
c-11 C-llb cyclic carbons c-7 C-6 C-8 C-lob
c-2 C-3b C-4b c-10
Ia
Ia’
9.00, q 26.81, t
9.13, q 27.14, t
38.81, t 45.50, d 69.72, d 115.38, s 135.24, d 149.80,s 150.80, s 155.54, a
32.30, t 45.62, d 69.84, d
IIa
a
135.45, d
a 150.79, s 156.2, a
IIa’
13.92, q 17.59, t 32.44, tb
14.04 q 17.68, t 32.90, tb
36.23, tb 44.10, d 69.27, d 115.39, s 135.16, d 149.78, s 150.76, s 155.52, s
36.58, tb 44.16, d 69.47, d a 137.12, d a 150.55, a 155.60, a
IIIa
39.73, q 31.81, q C
49.29, a 72.02, d 115.46, a 136.46, d 150.80, a 150.87, a 156.96,s
d-Rib 61.61, t 70.63, d 82.14, d 87.39, d c
c-5’ (2-3’
c-1’ (2-4’ (2-2’ a
62.33, t 71.46, d 83.29, d 87.96, d
61.63, t 70.66, d 82.19, d 87.40, d
c
These signals were not observed. The assignment is not clear. The signals are overlapped by the solvent signal.
lla 1l b
CH3CI 12
1
la
II a
111 a
Table IV. FT-IR, UV Spectra, Mass Spectrometry, and Elemental Analysis of l,N*-Cyclic Guanine Adducts (Acid Hydrolysates of the Deoxyguanosine Adducts) of Pentenal (Ia’), Hexenal (IIa’), and 3,3-Dimethylacrolein(IIIa‘) adduct Ia’
IIa’
IIIa’
FT-IR(KBr). .~~~Y., cm-1 ~
~
~~
I.
UV A nm (lo e) (0.1 N Hmdi.)0.1 N fiaOH)
MS (.% ),
elemental analysis
Cl&IlsNsOZ (235.2)*1HzO HzO: 252 (4.10). 282 (3.86): thermosmav: mlz 236 (100. MH+), 218 (i4, MH+ - H~o),152 (12, calcd, C 47.43, H 5.97, N 27.65; -A230 (3.76) 272 (3.833 GuaH+);E1 (70 eV): m/z236.1 found, C 47.43, H 5.94, N 28.14 HCI:202 (4.25), 256 (4.09); (15, M+ + 11, 235.1 (38, M+),206.1 A- 230 (3.64) (24), 192.1 (42), 188.1 (71), 178.1 (43), NaOH 222 (4.38), 280 (3.96); 151.1 (100, Gua+),135.1 (42), 110.1 A- 247 (3.69) (30), 109.1 (23), 84.3 (69), 83.2 (32), 66.1 (62),55.2 (72),43.2 (59) CllHl&4sOz (249.2)*1.25H20: HzO 252 (4.08), 283 (3.84); thermospray: m/x 250 (100, MH+), 3382 (NH), 3248,3180 (OH), 232 (7, MH+ - HzO), 190 (l), 152 calcd, C 48.61, H 6.49, N 25.77; A- 230 (3.73), 273 (3.81) 2961,2933,2874 (CH), found, C 48.11, H 5.59, N 25.64 (18, GuaH+);E1 (70 eV) m/z 250.1 HCI:202 (4.26), 256 (4.09); 1689 (M), 1569 (C-N, (2, M+- l), 249.1 (5, M’), 207.1 (3), A b 230 (3.60) C=C), 1332,1179,784, 206.1 (9), 188.0 (9), 178.1 (8),151.1 NaOH 222 (4.38), 280 (3.96); 776,638 (29, Gus+), 135.1 (9), 83.2 (34), A- 248 (3.69) 629 (53), 55.3 (41), 41.1 (100) E1 (70 eV): m / z 236.2 (12, M+ + l), HzO: 253 (4.09)s 283 (3.95); 3537,3415 (NH), 3260,3170 235.2 (56, M+),207.2 (12, M+ + l), A d 231 (3.91), 273 (3.93) (OH), 3001,2975,2935 (CH), 235.2 (56, M+),207.2 (9), 202.1 (27), HCI:207 (4.16), 256 (4.09); 1684,1647 (C=O), 1571 192.1 (81), 175.1 (lo), 151.2 (100, A- 231 (3.83) (C-N, e-C), 1473,1194, Gua+),135.1 (29), 110.7 (26),84.2 1175,954,784,768,720,631 NaOH 223 (4.51),281 (4.09); (46),55.1 (43) A b 247 (3.80) 3528.3395 (NHI. 3230 (OH). . . 2967,2935,2876 (CH), 1685 (C=O),1575 (C=N, C=C), 1172,786,772,641
of regioisomers (8). Interestingly,the ketones methyl vinyl ketone and ethyl vinyl ketone only form that regioisomer in which the OH group is vicinal to the N2atom (7).Like crotonaldehyde the other 8-alkyl-substitutedacroleins also form the first regioisomer, probably due to steric reasons. In the case of hexadienal we were not able to obtain sufficient amounts for recording an NMR spectrum; however, the HPLC chromatogram and the UV spectrum indicate that the same regioisomer is formed as with the other enals studied here. 7,8-CyclicAdducts. In addition to l,N2-cyclic adducts, also 7,&cyclic adducts of pentenal Ib and hexenal IIb were isolated. In the case of 3,3-dimethylacrolein the amounts of IIIb were only sufficient for recording UV spectra. The
HPLC chromatogram and the UV spectrum, however, indicate that this enal forms 7,8-cyclic adducts as well. In the case of hexadienal we found many smaller signals in the HPLC chromatogram which could not be clearly attributed to adducts and which could not be isolated in substantial amounts. So we are not entirely sure whether or not such adducts were also formed by hexadienal. We propose the following nomenclature: Adducts of pentenak Ib, cisltrans-2-amino-7-ethyl-5,6dihydro-5-hydroxy-1H-pyrrolo[l,2-dl purin-9(3H)-one. Adducts of hexenal: IIb, cisltrans-2-amino-5,6-dihydro5-hydroxy-7-propyl- lH-pyrrolo[1,2-d1purin-9(3H)-one. The conformation of the 7,8-tetrahydropyrrole ring in adducts Ib and IIb is probably an envelope type in which
Chem. Res. Toxicol., Vol. 6,No. 4, 1993 491
dG Adducts of 8-Alkyl-Substituted Enals
Table V. FT-IR-Spectroscopy, UV Spectroscopy, and Mass Spectrometry of 7,8-Cyclic Adducts of Pentenal Ib, cis,truns, Hexenal ID, cis,trans, and 3.3-Dimethylacrolein IIIb UV, nm (HzO) MS (%) adduct FT-IR (KBr) v, cm-1 thermospray: mlz 236 (100, MH+);ELMS (70 eV): ,A 217,249,285 Ib 3340,3210 (OH, NH), 2968, A- 237,264 m/z 236.1 (9, M+ + l),2352 (74, M+),217.1 (31, 2880 (CH), 1685 (C=O), 1541, M+ - HzO), 206.9 (41), 201.9 (30), 180.0 (87), 1465,1337,1090,776,578 163.0 (261,151.0 (71, Gus+), 138.0 (16), 135.1 (17), 69.1 (la), 55.2 (36), 53.0 (30), 43.0 (100) thermospray: mlz 250 (100, MH+);ELMS (70 eV): A, 220,250,284 IIb 3317,3167 (OH, NH), 2961,2934, A- 236,264 m/z 250.3 (16, M+ + l),249.3 (100, MH+),232.2 (13, 2875,2757 (CH), 1680 (C=O), M+- H2O + l),220.3 (23), 206.1 (531,180.1 (83), 1541,1465,1378,1076,777,577 163.2 (24), 151.2 (38), 138.1 (14), 135.3 (E),110.2 (ll), 69.1 (lo), 55.1 (17), 43.1 (54) A, 214,250 IIIb A- 283
CH3CHz I HOCH,
%CH2
:k>
CH3 a \O
2
1
Ia (cis)
40
\:j
CH3CH, I H
I H
OH
Ia (uans)
Ia' (85%)
HOCH2
\:j
the C-6 is not in the plane of the ring. In the cis configuration both substituents are in the energetically favored (pseudo-) equatorial position. In the case of the
~ z c H 3
CH2CH3
trans isomers the OH group presumably takes the equatorial position; however, the pseudoaxial position (anomeric effect) cannot be excluded.
492 Chem. Res. Toxicol., Vol. 6, No. 4, 1993
Eder and Hoffman
,.
11
c
IBH21+ 50.0
. .
I
1
1
1
0
I
I
I
,
I
I
,
,
I
I
0
8
7
6
5
4
3
2
1
0
[2B+HJt 499 2
366 2
1520
PPM
Figure 2.
lH NMR spectrum of the pentenal adduct Ia' (trans).
Illustration of the coupling between the 7a,b-H and the 6-H as well as lla,b-H and 6-H.
100.0
1.5
E
I
OH I ,
250 3
Figure 4. Thermospray E1 mass spectra of the diastereomers of the hexenal trans l,N*-cyclicadduct IIa.
- la
I
0.75
0 200
h 230
260
290
320
350 nm
adducts. Another possibility is the utilization of HPLCthermospray mass spectrometry. Figure 4 shows the thermospray mass spectra of the two diastereomers of the hexenal IIa (trans) adduct. Figure 4 also shows that a dimeric mass fragment [2B + HI+of mlz = 499 is formed. The mechanisms of the formation of this mass fragment are unclear. It may result from dimerization of M and the ion [M + HI+ to [2M + HI+ and loss of the two deoxyribose moieties. Dimerizations of mass fragments (e.g., those of [BHz+]) do not occur because of Coulombic repulsion. We frequently observed such dimeric mass fragments when applying the thermospray mass spectrometry technique. Both techniques can be used for a first but reliable identification of adducts in rather low amounts.
Figure 3. UV spectra of the pentenal adducts Ia, Ia', and Ib.
The structure of the 7,8 adducts can be derived from the lH NMR spectrum as we have shown recently for the 7,8-cyclic adducts of crotonaldehyde (8). Since the 'H NMR spectra of Ib and IIb are very similar to that of the crotonaldehyde adduct, therefore details of the structural enlightenment need not be repeated here for adducts Ib and IIb. As we have shown for the respective crotonaldehyde adduct, the proposed structure is also expected from the reaction mechanism in that the first step must be a Michael addition of the guanine N-7 atom to the double bond of the respective enal. Assignment of the Adducts via Other Spectroscopic Data. The two different types of adducts exhibit some characteristic spectroscopic properties and can be easily distinguished by their chromatographic data (see above) and their spectra (Tables I-VI). We have recently demonstrated that FT-IR techniques can be utilized to determine the substitution patterns of deoxyguanosine with crotonaldehyde (8).A combination of HPLC analysis with spectroscopic methods provides the possibility of fast assignment of the adducts. Figure 3 shows as an example the different UV spectra of the pentenal adducts Ia, Ia', and Ib. Thus HPLC analysis in combination with a diode array detector allows a quick and reliable assignment of
Discussion Relatively little was known about adduct formation of higher substituted cr,&unsaturated carbonyl compounds, Chung et al. (18) recently described deoxyguanosine adducts of cyclohexen-l-one, and Winter et al. (19) reported on the formation of cyclic deoxyguanosine adducts with truns-4-hydroxy-2-hexenaland trans-4hydroxy-2-nonenal. Here we have demonstrated that the mutagenic, higher 8-alkyl-substituted enals form similar adducts as does crotonaldehyde,the lowest member of 8-alkyl-substituted acroleins. We found the same type of regioisomersof 1,N2cyclic adducts with identical configuration in the newly formed tetrahydropyrimidine ring with pentenal, hexenal, and 3,3-dimethylacrolein as with crotonaldehyde. In general, pairs of diastereomers were found for the respective trans and cis 1,N2-cyclicadducts. According to the HPLC and UVspectra this was also the casefor hexadienal. Furthermore, the same type of 7,8-cyclic adducts as with crotonaldehyde was found with pentenal and hexenal. (For 3,3-dimethylacrolein see Results.) However, we did not isolate 1,N2,7,8-bis-cyclicguanine adducts as with crotonaldehyde (8). It may well be that such bis-cyclic adducts are formed with the other 8-alkyl-substitutedenals as well,
dG Adducts of 8-Alkyl-Substituted Enals
but to a much lesser extent so that we were not able to isolate them. In general, a clear tendency was found that reactivity toward deoxyguanosine and its 5’-monophosphate decreases with increasing degree of substitution. The decrease in reactivity can be explained by steric hindrance of the Michael addition a t the @-carbonatom, the first step of adduct formation, due to alkylsubstitution at this position. Problems in isolating sufficient amounts of adducts with hexadienal did not, however, arise from reduced reactivity. With this compound we even estimated a higher reactivity than with hexenal. Many adductcontaining fractions could be seen in the LH-20 chromatography and in the HPLC analysis of the reaction mixtures obtained with hexadienal; however, the amounts were so low that we could not isolate adducts in substantial amounts. Evidently, the additional double bond in conjugation to the other double bond and the C=O group is responsible for increased reactivity and a higher number of different adducts. In general, we found a good relationship between the estimated reactivities of the @-alkylsubstituted enals and their mutagenic activities determined recently ( 3 ) . Since these adducts are considered as premutagenic DNA lesions (15, 18,201, we propose that adduct formation is the primary mechanism underlying mutagenicity. Because the higher members of @-alkylsubstituted enals form similar adducts as does crotonaldehyde and are also mutagenic like crotonaldehyde, these higher substituted compounds should also be considered to be carcinogeniclike crotonaldehyde unless experimental examination demonstrates noncarcinogenicity. The relationship between adduct formation and mutagenic and carcinogenic activities of a,@-unsaturated carbonyl compounds in mammals and humans is sometimes denied (21) since adduct formation is expected to be low and it is believed that these reactive compounds are inactivated fast in vivo by reactions with nucleophiles such as glutathione or proteins. Indeed, such inactivation via scavenging with glutathione are observed and can lead to a local glutathione depletion in cases in which high local tissue concentrations of enals are formed (22). On the other hand, Witz ( 1 ) has shown that aldehyde-thiol adducts can still possess biological activity, and it has been suggested that the orginal aldehyde can be regenerated near the target by dissociation of the adduct (23). Protein interactions are also described; however, they can also indirectly lead to genotoxicity, e.g., by interactions with tubulin, inhibition of DNA synthesis, or inhibition of DNA repair enzymes (22,241. In general, the stability of a,@unsaturated carbonyl compounds is higher than that of many other reactive alkylating intermediates proposed as ultimate mutagens and ultimate carcinogens (e.g., cations formed metabolicallyfrom nitrosamines). Reactive oxygen species (e.g., OH radicals) are usually also more reactive and more unstable than enals. a,@-Unsaturatedcarbonyl compounds, which are often considered as second toxic messengers of reactive oxygen species,could therefore have a better chance to reach DNA and form adducts than other more reactive and less stable species (25). Brambilla et al. (21) recently emphasized that enals can remain associated with the lipid core of the endoplasmatic reticulum where they are formed endogenously and can diffuse within the lipid membrane to the cell nucleus, without encountering cytosolic detoxification. Considering these arguments it is well conceivable that the @-alkylsubstituted enals are a constant source of DNA damage
Chem. Res. Toxicol., Vol. 6, No. 4, 1993 493 due to their ubiquitous occurrence in food and environment and due to their endogenous formation, in particular, after inflammation and ischemia (26). The highly sensitive detection of such adducts in mammalian and in human tissue would provide a more reliable estimation of the extent of adduct formation and of the role of a,@-unsaturatedcarbonyl compounds. The synthesis and the characterization of the adducts and the enlightenment of their structures as well as the presentation of their spectroscopic data and chromatographic properties are prerequisites for the development of such sensitive detection techniques. Synthesis, description, and characterization of these adducts can also provide an insight into the mechanisms of adduct formation in vivo as well as a better understanding of the biological significance of these adducts; they are also prerequisites for further studies for final clarification of these questions.
Acknowledgment. We are grateful to Mrs. E. Weinfurtner for excellent technical assistance and are indebted to Mrs. Mertel for linguistic assistance. This work was supported by Deutsche Forschungsgemeinschaft SFB 172 and by BG-Chemie.
References (1) Witz, G. (1989) Biologicalinteractions of a,&unsaturated aldehydes. Free Radical Biol. Med. 7, 333-349. (2) 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. (3) Eder, E., Deininger, C., Neudecker, T., and Deininger, D. (1992) Mutagenicity of 8-alkylsubstituted acrolein congeners in the Salmonella typhimurium strain TA 100 and genotoxicity testing in the SOS-chromotest. Enuiron. Mol. Mutagen. 19, 338-345. (4) Lutz,D.,Eder,E.,Neudecker,T.,and Henschler,D. (1982) Structuremutagenicity relationship in a,&unsaturated carbonylic compounds and their corresponding allylic alcohols. Mutat. Res. 93,305-315. (5) Chung, F. L., Young, R., and Hecht., S. S. (1984) Formation of cyclic l,N2-propanodeoxyguanosineadducts in DNA upon reaction with acrolein or crotonaldehyde. Cancer Res. 44, 990-995. (6) Shapiro, R., Sodum, R. S., Everett, D. W., and Kundu, S. K. (1986) Reaction of nucleosides with glyoxal and acrolein. InZARCScientific Publications No. 20 (Singer, B., and Bartach, H., Eds.) pp 165-173, IARC, Lyon. (7) Eder, E., Hoffman, C., and Deininger, C. (1991) Identification and characterization of deoxyguanosine adducts of methyl vinyl ketone and ethyl vinyl ketone. Genotoxicity of the ketones in the 50s chromotest. Chem. Res. Toxicol. 4, 50-57. (8) Eder, E., and Hoffman, C. (1992) Identification and characterization of deoxyguanosine-crotonaldehyde adducts. Formation of 7,&cyclic adductsand 1,Nz,7,8bis-cyclicadducts. Chem. Res. Toxicol. 5,802808. (9) Chung, F. L., Tanaka, T., and Hecht, S. S. (1986) Induction of liver tumors in F 344 rats bycrotonaldehyde. Cancer Res. 46,1285-1289. (10) Eder, E., Scheckenbach, S., Deininger, C., and Hoffman, C. (1993) The possible role of a,B-unsaturated carbonyl compounds in mutagenesis and carcinogenesis. Toxicol. Lett. 67, 87-103. (11) Schauenstein, E., Esterbauer, H., and Zollner, H. (1977) Aldehydes withspecific biological functions. In Aldehydes in biologicalsystem. Their natural occurrence and biological actiuities, pp 172-200, Methuen Inc., New York. (12) Esterbauer, H., Cheesman, K. H., Dianzani, M. K., Poli, G., and Slater, T.F. (1982) Separation and characterization of the aldehydic products of lipid peroxidation stimulated by ADP-FeZ+ in rat liver microsomes. Biochem. J . 208, 129-140. (13) Pederson, T. C., and A u t , S. (1973) The role of superoxide and singulet oxygen in lipid peroxidation by xanthine oxidase. Biochem. Biophys. Res. Commun. 52, 1071. (14) Marnett, L., Hurd, H. K., Hollstein, M. C., Levin, D. E., Esterbauer, H., and Ames, B. N. (1985) Naturally occurring carbonylcompounds are mutagens in Salmonella tester strain TA 104. Mutat. Res. 148, 25-34. (15) Eder,E.,Hoffman,C.,Bastian,H.,Deininger,C.,andScheckenbach, S. (1990) Molecular mechanisms of DNA damage initiated by a,&
unsaturated carbonyl compounds as criteria for genotoxicity and mutagenicity. Enuiron. Health Perspect. 88, 99-106.
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(16) Kirby, A. J. (1983) The anomeric effect and relatedstereoelectronic effects at oxygen, Springer Verlag, New York. (17) Vierhapper, F. W. (1981) Preference for a conformation with axial t-butyl group: S&t-cis dfcahydroquinoline. Tetrahedron Lett. 22, 5161-5164. (18) Chung, F. L., Kalpana, R. R., and Hecht, S. S. (1988) A study of reactions of ru,&unsaturated carbonyl compounds with deoxyguanosine. J. Org. Chem. 53, 14-17. (19) Winter, C. K., Segall, H., and Haddon, W. F. (1986) Formation of cyclic adducta of deoxyguanosine with aldehydes trans-4hydroxy2-hexenal and trans-4-hydroxy-2-nonenal in vitro. Cancer Res. 46, 5682-5686. (20) Marinelli, E. R., Johnson, F., Iden, C. R., and Yu, P.-L. (1990) Synthesis of 1,N*-(1,3-propano)-2deoxyguanosine: A model for exocyclic acrolein-DNA adducta. Chem. Res. Toxicol. 3,4948. (21) Brambilla, G., Sciaba, L., Faggin, P., Maura, A. A., Marinari, U. M., Ferro, M., and Esterbauer, H. (1986) Cytotoxicity, DNA fragmentation and sister chromatid exchange in Chinese hamster ovary cells
Eder and Hoffman exposed to the lipid peroxide product Chydroxynonenal and homologous aldehydes. Mutat. Res. 171, 169-176. (22) Krokan, H., Grafstr6m, R. C., Sundquist, K., Esterbauer, H., and Harris, C. C. (1985) Cytotoxicity, thiol depletion and inhibition of 06-methylguanine-DNA-methyl transferase by various aldehydes in cultured human bronchial fibroblasta. Carcinogenesis 6,17551759. (23) Esterbauer,H.,Ertl,A.,andScholz,N. (1976)Thereactionofcysteine with cY,@unsaturated aldehydes. Tetrahedron 32, 285-289. (24) Gabriel, L., Bonelli, G., and Dianzani, M. U. (1977) Inhibition of colchicine binding to rat liver tubulin by aldehydes and by linoleic acid hydroperoxide. Chem.-Biol. Interact. 19,101-109. (25) Esterbauer, H., Schaur R. J., and Zollner H. (1990) Chemistry and biochemistry of Chydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biol. Med. 11,81-128. (26) Esterbauer, H., Eckl, P., and Ortner, A. (1989) Possible mutagens derived from lipids and lipid precursors. Mutat. Re8.238,223-233.
