Chem. Res. Toxicol. 1994, 7, 62-67
62
Formation of the Acrolein-Derived 1,N2-PropanodeoxyguanosineAdducts in DNA upon Reaction with 3- (N-Carbethoxy-N-nitrosamino)propionaldehyde Fung-Lung Chung,' Jacek Krzeminski, Mingyao Wang, Hauh-Jyun C. Chen, and Bogdan Prokopczyk American Health Foundation, 1 Dana Road, Valhalla, New York 10595 Received August 23, 1 9 9 3
3-(Methy1nitrosamino)propionaldehyde(MNPA) is a carcinogenic nitrosamine formed by nitrosation of arecoline, a major alkaloid in areca nut which is a constituent of betel quid. While DNA adducts of its analogue, 3-(methylnitrosamino)propionitrile, have been characterized, little is known about the structures of DNA adducts by MNPA. In this paper, we report that the acrolein-derived 1,N2-propanoguanineadducts are formed upon incubating deoxyguanosine or DNA with 3-(N-carbethoxy-N-nitrosamino)propionaldehyde, a stable carbamate precursor of the metabolically activated MNPA. The identities of these adducts were confirmed by HPLC co-migration, by their NMR and UV spectra, and by chemical properties as compared with those of the synthetic standards. Analogous results were obtained from the reaction of the carbamate with calf thymus DNA. Upon acid or enzyme hydrolysis of the carbamate-modified DNA, acrolein-guanine adducts were detected, and the levels were quantitated. Again, the identities of the adducts were verified by co-chromatography with the standards, by UV spectroscopy, or by the ring-opening with NaOH/N&H4. Consistent with its ability to modify DNA, the carbamate was found to be mutagenic in Salmonella tester strains. These results show that acrolein is a likely metabolite from the activation of MNPA and that the formation of 1,N 2-propanoguanineadducts may contribute to the mutagenicity of the carbamate of MNPA.
Introduction 3-(Methy1nitrosamino)propionaldehyde(MNPAY is a nitrosamine formed by nitrosation of arecoline, a major alkaloid in areca nut (1). Areca nut is a major ingredient used in betel quid. This nitrosamine may be formed during betel quid chewing because saliva is known to contain nitrite and thiocyanate. A recent bioassay showed that MNPA was highly toxic and induced lung tumors in F344 rats when administered subcutaneously (2). Because of its toxicity and carcinogenicity, MNPA is thought to be involved in oral cavity cancers commonly associated with the habit of betel quid chewing. For most nitrosamines, a-hydroxylation is an important activation pathway which is mediated mainly by cytochrome P-450 enzymes (3). This pathway yields alkyl diazohydroxide ions which alkylate DNA bases. The alkylated bases such as 06-methylguanine and 04methylthymine have been shown to cause miscoding during DNA replication and are, therefore, recognized as critical lesions in nitrosamine carcinogenesis (4, 5). Previous studies have shown that treatment of rats with the potent arecoline-derived nitrosamine 3-(methylnitrosamin0)propionitrile (MNPN) resulted in the formationof methyl and cyanoethyl guanines in the target tissue DNA of rats (6). Unlike MNPN, however, little is known about the structures of DNA adduct by MNPA. Both MNPA and MNPN share the common structural features as dialkylnitrosamines possessing a methyl and a propyl moiety. e Abstract published in Adoance ACS Abstracts, December
15,1993. MNPA, 3-(methylnitrosamino)propionaldehyde; MNPN, 3-(methylnitrosamino)propionitrile; DMSO-de, dimethyl sulfoxide-de. 1 Abbreviations:
The difference between MNPA and MNPN lies in the terminal aldehyde group in MNPA vs the nitrile group in MNPN. The structure of MNPA suggests that upon metabolic activation via a-hydroxylation it should yield a methylating and a formylethylating agent. Although MNPA is cytotoxic and causes significant DNA damage, so far there is no evidence for the in vivo alkylation of DNA bases by MNPA2 (7). Acrolein is a likely metabolite of MNPA, since the formylethyl diazohydroxide ion can undergo further decomposition through the loss of nitrogen and H2O. As the simplest enal, acrolein is highly toxic, mutagenic, and potentially tumorigenic (8,9). It reacts readily with DNA bases by forming various cyclic propano adducts (10-13). Site-specific mutagenesis studies showed that the model 1,N 2-propanodeoxyguanosineadduct causes either frameshift or base substitution mutation (14,15). In order to elucidate the structures of DNA adducts by MNPA, in this study we characterized the products from the reaction of 3-(N-carbethoxy-N-nitrosamino)propionaldehyde (11, a stable carbamate precursor of a-hydroxyMNPA, with deoxyguanosine and DNA (Scheme l), and we also examined its mutagenicity in Salmonella typhimurium tester strains.
Materials and Methods Chemicals. Calf thymus DNA, porcine liver esterase, and deoxyguanosine were purchased from Sigma (St. Louis, MO). The 1JV2-propanodeoxyguanosine adducts of acrolein were prepared by the reaction of acrolein with deoxyguanosine according to a published procedure (10). The ring-opened 2
B. Prokopczk et al., unpublished data.
0893-228~/94/2101-0062$04.50/0 0 1994 American Chemical Society
Chem. Res. Toxicol., Vol. 7,No. 1, 1994 63
Acrolein-dGuo Adducts from a Precursor of MNPA
Scheme 1. Formation of the Acrolein-Derived 1,N2-PropanodeoxyguanosineAdducts from Reaction of Carbamate (1) with Deoxyguanosine or DNA 0
II
CH3NCH2CH2CH
I N=O
1
MNPA a - h y F l a l i o n at CH3
0
0
11
II
esterase
CH3CH20CNCH2CH2CH v
I
N=O
[
II
NCH2CH2CH
II
]
1
CHz=CHCH acrolein
-
derivative of the 1,N 2-propanoguanineadduct 3 (Scheme 1)was prepared by a previously described procedure (10). 3-(NCarbethoxy-N-nitrosamino)propionaldehydewas synthesizedby a method similar to that described for MNPN (16). The synthesis is described as follows: 3-aminopropanol(l5g, 0.2 mol) in benzene (90 mL) was stirred with a 10% NaHC03 solution (40 mL). To this mixture was added ethyl chloroformate (21.6 g, 0.2 mol) dropwise over a period of 1h. After stirring at room temperature for 4 h, phases were separated, and the water layer was extracted withCH2Clz (2 X 100mL). All organicphases were combined, dried over MgSOr, and concentrated. The resulting oil was distilled (118 "C/0.3 mmHg) to afford 3-(Ncarbethoxyamino)propanol (yield: 15.2 g, 52 % ). 'H-NMR (DMSO-& 6 1.14 (t, 3H, CH3), 1.55 (m, 2H, CHz), 3.0 (4, 2H, N-CHz), 3.4 (q,2H, CHzOH), 3.97 (q,2H, CHzCHs), 4.48 (t, lH, OH), 7.0 ( 8 , lH, NH). To a mixture of 3-(N-carbethoxyamino)propanol (18.8 g, 0.13 mol) in 40 mL of ether and NaNOz (40 g, 0.58 mol) in 65 mL of water was added HN03(35%,75 g) dropwise from a long-stem addition funnel with stirring over a period of 40 min at 0 "C. The reaction mixture was kept below 5 "C for 2 h and was then diluted with ether. The aqueous layer was extracted with ether (2 x 100 mL). The ether extracts were combined and washed with H20, dried over K2CO3, and concentrated to give an oil (19.1 g, 85%) which was used in the subsequent step without further purification. The pure 3-(Ncarbethoxy-N-nitrosamino)propanolcan be obtained after chromatography on a silica column (ch1oroform:ether151). 'H-NMR (DMSO-&) 6 1.35 (t, 3H, CHs), 1.48 (m, 2H, CH2), 3.33 (t, 2H, N-CHz), 3.77 (t,2H, CHzOH), 4.47 (q, 2H, CH2CHs). 'H-NMR spectra reveals a trace amount of another stereoisomer (E or Z). These chemical shift values are for the major isomer. A solution of 3-(N-carbethoxy-N-nitrosamino)propanol(l2g, 0.068 mol) in methylene chloride (80 mL) was added dropwise with stirring, over a period of 2 h, to a suspensionof pyridinium chlorochromate (44 g, 0.2 mol) and powdered molecular sieves 3 8, (12 g) in methylene chloride (300 mL) at 0 "C. The mixture was stirred at 0-2 "C for 5 h and decanted into 1 L of ether. The residue was extracted once with methylene chloride (150 mL). The extract was combinedwith the ether solutionand filtered through a silicagel bed with 8 cm thickness. The filtrate was concentrated in U ~ C U Oto afford a dark oil which was applied on a silica gel column and eluted with ch1oroform:ether (15:l) to afford 4.1 g (35%) of yellow oil as crude product. The column chromatog-
OH 0
dR
raphy was repeated twice to provide pure compound 1. 'H-NMR (DMSO-&) 6 1.35 (t, 3H, CH3), 2.55 (9, 2H, CH&HO), 3.94 (t, 2H, N-CH2),4.48 (q,2H, CH2CHd,9.56 (8, lH, CHO). Chemical ionization MS (mle): 175 (M + 1); 144 (M - NO); 103 (M C3H602). Instrumentation. NMR spectra were run on a Bruker Model AM360WB spectrometer. Mass spectra were obtained on a Hewlett-Packard Model 5988A mass spectrometer. UV spectra were obtained on a Waters 994 programable photodiode array detector. Chromatography. The following HPLC systems were used for analysis: System 1,a SupelcosilLC-18-DB 25 cm X 4.6 mm, 5-pm column (Supelco,Bellefonte, PA) with H20/CHsCN (95/5) eluted isocratically at a flow rate of 1 mL/min. System 2, a Supelcosil SPL C-18-DB 25 cm X 10 mm, 5-pm column eluted isocratically with H20/CH&N (95/5) at a flow rate of 4.0 mL/ min. System 3, two Whatman 25 cm X 4.6 mm Partisil-10 SCX strong cation-exchangecolumns (Whatman,Clifton, NJ) in series eluted isocratically with 50 mM ammonium phosphate, pH 2.0, at a flow rate of 1.0 mL/min with detection by a Perkin-Elmer 650-10s fluorescence spectrophotometer (excitation at 290 nm and emission at 380 nm). System 4, two Waters 30 cm X 3.9 mm C18 pBondapak reverse-phase columns (Waters, Milford, MA) inserieselutedwithsolvent Afor 6min, thenagradientof0-100% solvent B in 60 min, at a flow rate of 1.0 mL/min. Solvent A, 0.1 M phosphate buffer, pH 5.7; solvent B, 20% methanol in HzO. The detector was a Waters 994 programable photodiode array detector. System 5, a 25-cm, 5-pm, B&J 005 octadecyl column (Baxter, Edison, NJ) eluted with a gradient of 5-20% methanol in H2O in 30 min, at a flow rate of 1mL/min using the photodiode array detector described above. Reaction with Deoxyguanosine. To a 10-mL solution of 1 (92 mg, 0.53 mmol) in 0.1 M sodium phosphate (pH 8.0) was added deoxyguanosine (70 mg, 0.26 mmol) and esterase (50 pL, 144 units). The reaction mixture was incubated at 37 "C for 161 h. An aliquot of the reaction mixture was analyzed by HPLC system 1. For the preparative scale, the reaction mixture was filtered through a Gelman Nylon Acrodisc 13filter, and products were collected from HPLC system 2. Reaction with DNA. Carbamate 1 (170 mg, 1.0 mmol) was incubated with calf thymus DNA (23 mg) and esterase (51 pL,
64
Chem. Res. Toxicol., Vol. 7, No. 1, 1994
144 units) in 3 mL of 0.1 M phosphate buffer pH 7.0 at 37 "C for 15 h. An equal volume of HzO was added to the reaction mixture, and DNA was isolated by extracting the mixture with 10 mL of ch1oroform:isoamylalcohol (24:l) followed by precipitating with 0.1 vol of 5 M NaCl and an equal volume of cold ethanol and then was washed twice each with ethanol, ethanol: ether (l:l),and ether. (i) Acid Hydrolysis. DNA (2 mg) was hydrolyzed in 1 mL of 0.1 N HC1 at 85 OC for 45 min to release the acrolein-guanine adducts. An aliquot of acid hydrolysate (10-20 pL) was analyzed with HPLC system 3. (ii) Enzyme Hydrolysis. DNA (2.6 mg) was dissolved in 5 mL of 10 mM Tris-HC1 and 5 mM MgC12 at pH 7.0, incubated with DNase (1000 units) a t 37 "C for 10 min, and then incubated with phosphodiesterase (0.06 unit) and alkaline phosphatase (400 units) for 60 min. After incubation, the mixture was cooled to 0 "C in an ice bath and fiitered through a Centri-freefiiter (Amicon Division, W. R. Grace and Co., Danver, MA). The filtrate was concentrated and analyzed with HPLC system 4. Fractions corresponding to the adducts were collected, concentrated, and analyzed again using HPLC system 3. Confirmation of Identities of Adducts. The identity of the guanine adduct was verified by conversionto the ring-opened derivative (10). Briefly, portions of the collected acrolein-guanine adduct 3 were treated with 0.5 N NaOH and NaBHl(22 mg/mL) at 100 "C for 30 min. This treatment converts the propano ring in adduct 3 to a hydroxypropyl group. The resulting mixture was analyzed on HPLC system 3. The identities of adducts 1 and 2 in DNA acid hydrolysate were confirmed by co-migration in HPLC system 3. The identities of adducts in the enzyme hydrolysate were confirmed by co-migration in HPLC systems 3 and 5 with standards and by their UV spectra as compared to standards. Mutagenicity Assay. The mutagenicity of carbamate 1 was examined in S. typhimurium TA 104, TA 100, TA 1535,and TA 1538 using pre-incubation modification of the standard assay conditions (17). Briefly, 1 was dissolved in DMSO to provide a concentration range from 0.1 to 0.4 rmol/plate. There was no significant toxicity of the test compound a t these concentrations. The assays were carried out with a 20-min pre-incubation in the absence of rat liver S-9 fraction.
Chung et al.
(7d.d ,
200 250
deoxyguanosine
5
0
I
10 15 Retention Time (min)
20
Figure 1. HPLC chromatogram and UV spectra of producta obtained from analysis of the reaction mixture of carbamate 1 and deoxyguanosine in the presence of esterase using HPLC system 1.
adducts 1 and 2
Results Previously, we have shown that three 1,N2-cyclic deoxyguanosine adducts were formed in the reaction with acrolein, designated as adducts 1-3 (10). Adduct 3 (Scheme 1)is derived from an initial nucleophilic addition to acrolein by the N-2 of guanine via Michael addition, followed by ring closure between the aldehyde group and the N-1 of guanine. The resulting adduct possesses the hydroxy group adjacent to the N-1 position of guanine. Adducts 1 and 2 (Scheme 1)are formed from ring closure of the opposite direction and they are interconvertible and exist in equilibrium with an equal amount of each isomer. Upon incubating 1 with deoxyguanosine at 37 "C and pH 8 in the presence of esterase, three major peaks were formed which eluted at 12.3,15.2, and 16.7 min. These peaks co-chromatographed with the synthetic standards of the 1, W-propanodeoxyguanosine adducts 1-3 of acrolein. A typical HPLC obtained from analysis of the mixture is shown in Figure 1. The identities of these adducts were verifed by their proton NMR and UV spectra and by comparing with those of the adduct standards. The proton NMR spectra of adducts 1-3, given in Figure 2, are in agreement with those reported previously (10). The 1,N 2-propanoguanineadducts were also detected in calf thymus DNA incubated with 1 under similar conditions. The HPLC obtained from analysis of the acid
1
1
1
1
1
1
1
1
1
1
1
1
I
I
I
adducts 3
I
C.4
'1 Ce-H
M A L l
8.0
l
l
7.0
l
l
6.0
l
l
l
5.0 4.0 PPM
l
l
3.0
l
l
2.0
l
~
1.0
Figure 2. 360-mHzproton NMRspectra of adducts 1-3 isolated from the reaction mixture of carbamate 1 and deoxyguanosine in the presence of esterase using HPLC system 2. hydrolysate of the modified DNA is shown in Figure 3. The earlier peak, eluting at 12.3 min, co-migrates with the corresponding guanine adducts of adducts 1 and 2. The later peak, eluting at 14.5 min, co-migrates with adduct 3. Further confirmation of the identity of the later peak
l
Chem. Res. Toxicol., Vol. 7,No. 1, 1994 65
Acrolein-dGuo Adducts from a Precursor of MNPA
I 1
Adduct 3
Adducts 1 and 2
A 0
4
8
12 16 20 24 Retention Time (min)
28
32
Figure 3. HPLC chromatogram obtained from analysis of acid hydrolysate of calf thymus DNA modified with carbamate 1. The peak at 12.3min is assigned as the guanine adducts of adducts 1 and 2, and the peak at 14.5 min is the guanine adduct of adduct 3. HPLC system 3 was used.
A
B
Standard
Standard
I
al v)
a
6 8 10 12 14 16 Retetion Time (min)
6 8 10 12 14 16 Retetion Time (min)
Figure 4. Co-migrationwith standard before (A) and after (B) ring-opening of guanine adduct 3 with NaOH/N&Hd.
as adduct 3 came from chemical conversion with sodium borohydride in base (Scheme 1). This treatment converts the propano ring of adduct 3 to a 3-hydroxypropyl group (10).The product from this treatment showed aretention time identical to that of the ring-opened derivative obtained from the guanine adduct 3 standard (Figure 4). The combined yield of adducts 1 and 2 in the carbamate-
200 220 240 260 280 300 320 340 360 380 400 Wavelength (nm) Figure 5. Comparison of the UV spectra of adduct 3 isolated from the enzyme hydrolysate with that of standard.
treated DNA was approximately 100 mmol/mol guanine, and the yield of adduct 3 was 223 mmol/mol guanine. Analogous results were obtained from enzyme hydrolysis of the modified DNA. However, the analysis of the enzyme hydrolysate was complicated by co-elution of adduct 3 with deoxyadenosine in HPLC system 4. Thus, the fraction corresponding to adduct 3 was collected and further analyzed with HPLC system 3. The final chromatography showed the formation of adduct 3, as verified by comparing its retention time with that of the standard. A comparison of UV spectra of this adduct with that of the standard is shown in Figure 5. The ability of 1 to yield adducts with DNA suggests that carbamate 1 is a mutagen. Using standard assay conditions, we tested the mutagenicity of 1 in Salmonella tester strains TA 104, TA 100, TA 1535, and TA 1538. With the exception of TA 1538, a strain sensitive to frameshift mutation, carbamate 1 induced a significant number of revertants in TA 104, TA 100, and TA 1535 without S-9. However, the parent nitrosamine MNPA was inactive with rat liver S-9fraction in these strains. The mutagenicities of 1 in T A 104, T A 100, and T A 1535 are shown in Figure 6.
Discussion Upon a-hydroxylation, certain nitrosamines such as N-nitrosopyrrolidine, N-nitrosopiperidine, N-nitrosomorpholine, and diallyl nitrosamine yield, beside alkylating intermediates, the reactive a,@-unsaturatedaldehydes or ends (18-21). For example, N-nitrosopyrrolidinereleases
66 Chem. Res. Toxicol., Vol. 7,No. 1, 1994
Chung et al. "00[:
TA 100
1200
pmoles / Plate Figure 6. Mutagenicity of carbamate 1 in S. typhimurium TA 104, TA 100, and TA 1535.
both a formylpropylating agent and crotonaldehyde upon a-hydroxylation (18). The formylpropylating agent yields 7-substituted guanines as major products, while crotonaldehyde gives l,N 2-propanoguanines (22). Although dialkyl nitrosamines possessing a formylethyl or hydroxybutyl moiety also permit the formation of enals as metabolites, this pathway has not been previously investigated. The fact that enals are themselves mutagenic, carcinogenic, and capable of damaging DNA by forming propano exocyclic adducts with guanine and other bases in DNA suggests that this pathway may contribute to the carcinogenicity of these nitrosamines. The structure of MNPA suggests that it could act as a methylating and formylethylating agent upon metabolic activation. However, MNPA has not been shown to methylate or formylethylate DNA, or if it does, the levels of alkylated bases must be too low to be detected.2 The reason for the lack of apparent alkylation by MNPA is unclear. It is conceivable, however, that the aldehyde group of MNPA is metabolized to the more polar hydroxy or carboxylicgroup by alcohol or aldehyde dehydrogenase. This transformation would facilitate the excretion of the nitrosamine by conjugation reactions. It is also possible that the formylethylating agent rapidly loses a proton to yield acrolein. A study of the solvolysis products from the carbamate may provide some insights. Results of this study showed that acrolein is formed when a-hydroxylation occurs at the methyl group of MNPA. The acroleinreleased reacts readily with guanines of DNA forming the 1,N2-cyclic adducts, and this DNA lesion may be involved in the mutagenicity of 1. Since acrolein is highly toxic, this pathway may also be partially responsible for the observed toxicity of MNPA. In the present study, we have focused on guanine adducts, although adducts of other bases such as adenine and cytosine are also likely to be formed by acrolein (11-13). The reaction with DNA showed several products besides l,N 2-propanoguanine adducts. However, the identities of these products were not determined in this study. As expected, carbamate 1 is mutagenic without S-9 activation in all the strains tested except TA 1538. Acrolein is not mutagenic in Salmonella his D 3052, a strain responsive to frameshift mutation, while it causes base pair mutation in T A 104 and TA 100 (8,23,24). This is consistent with our results since TA 1538is the only strain tested that detects frameshift mutation. MNPA is not active in these tester strains in the presence of the S-9 fraction. Several factors could explain the lack of mutagenicity of MNPA. It is possible that MNPA is only poorly activated by rat liver S-9. Furthermore, it is known that the pH of the assay medium could affect the half-life
of the a-hydroxylated nitrosamines (25). A greater stability, thus, the mutagenicity, could be observed in the medium of a lower pH. Since our assays were carried out at pH 7.0, a-hydroxyMNPA may be too short-lived to reach the nuclear DNA of bacteria. Our findings are in agreement with the general observation that nitrosamines are relatively weaker mutagens in bacterial mutation assays. The results of this study provide a basis for studying in vivo formation of the 1fl 2-propanodeoxyguanosine adducts in tissue DNA of rodents treated with MNPA. The use of a synthetic stable precursor of the activated carcinogen is a practical means to study the activation pathways leading to adduction. It allows us to obtain a sufficient quantity of adducts for structural characterization. With a better understanding of the chemical mechanism of the formation of these adducts, a highly sensitiveand specific 32P-postlabelingmethod for detection of the exocyclic DNA adducts in uiuo is likely to be developed. We would like to eventually apply this method to studying DNA damage in oral tissues of betel quid chewers as a means to assess the potential risk of cancer development. Acknowledgment. This work is supported by Grants CA-51830 and CA-29580 from the National Cancer Institute.
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Acrolein-dGuo Adducts from a Precursor of MNPA (9) Cohen, S.M., Garland,E. M., St. John, M., Okamura,T., andsmith, R. A. (1992) Acrolein initiates rat bladder carcinogenesis. Cancer Res. 52, 3577-3581. (10) Chung, F.-L., Young, R., and Hecht, S. S. (1984)Formation of cyclic 1,N 2-propanodeoxyguanosineadducts in DNA upon reaction with acrolein or crotonaldehyde. Cancer Res. 44, 990-995. (11) Smith, R. A., Williamson,D. S.,andCohen, S. M. (1989) Identification of 3 8 4-propanodeoxycytidine 5-monophosphate formed by the reaction of acrolein with deoxycytidine 5’-monophosphate. Chem. Res. Toricol. 2, 267-271. (12) Smith, R. A,, Williamson, D. S., Cerny, R. L., and Cohen, S. M. (1990) Detection of 1JV8-propanodeoxyadenosine in acroleinmodified polydeoxyadenylic acid and DNA by s2P-postlabeling. Cancer Res. 50,3005-3012. (13) Sodum, R. S., and Shapiro, R. (1988) Reaction of acrolein with cytosine and adenine derivatives. Bioorg. Chem. 16, 272-282. (14) Moriya, M., Marinelli, E., Shibutani, S., and Joseph, J. (1989) Sitespecific mutagenesis using the model exocyclicDNA adduct, 1JV2propanodeoxyguanosine. Roc. Am. Assoc. Cancer Res. 30, A555. 115) Benamira, M., Singh, U., and Marnett, L. J. (1992) Site-specific frameshift mutagenesis by a propanodeoxyguanoaine adduct positioned in the (CpG), hot-spot of Salmonella typhimurium his D3052 carried on an M13 vector. J. Biol. Chem. 267, 22392-22400. (16) Prokopczyk, B., Bertinato, P., and Hoffmann, D. (1988) Synthesis and kinetics of decomposition of 7-(2-cyanoethyl)guanineand 0 6(2-cyanoethyl)guanine, markers for reaction of acrylonitrile and 3-(methylnitrosamino)propionitrilewith DNA. Carcinogenesis 9, 2125-2128.
Chem. Res. Toxicol., Vol. 7, No. 1, 1994 67 (17) Maron, D., and Ames, B. (1983)Revised method for the Salmonella mutagenicity test. Mutat. Res. 113, 173-215. (18) Wang, M. Y., Chung, F.-L., and Hecht, S. S.(1988) Identification of crotonaldehyde as a hepatic microsomal metabolite formed by a-hydroxylation of the carcinogenN-nitroeopyrrolidine. Chem. Res. Toxicol. 1, 28-31. (19) Hecht, S. S., Young-Sciame, R., and Chung, F.-L. (1992) Reaction of a-acetoxy-N-nitrosopiperidinewith deoxyguanosine: oxygen dependent formation of 4-oxo-2-pentenal and a 1,N 2-ethenodeoxyguanosine adduct. Chem. Res. Toxicol. 5,706-712. (20) Chung, F.-L., and Hecht, S. S. (1985) Formation of the cyclic 1,N *glyoxaldeoxyguanosine adduct upon reaction of N-nitroso-2-hydroxymorpholine with deoxyguanosine. Carcinogenesis 6, 16711673. (21) Farelly,J.G.,Steward,M.L.,andLijinsky, W. (1984)TheMetabolbm of nitrosodi-n-propylamine,nitrosodiallylamine and nitroeodiethylamine. Carcinogenesis 5,1015-1019. (22) Wang, M.-Y., Chung, F.-L., and Hecht, S. S. (1989) Formation of acyclic and cyclic guanine adducts in DNA reacted with a-acetoxyN-nitrosopyrrolidine. Chem. Res. Toxicol. 2, 423-428. (23) Basu, A. K., and Marnett, L. J. (1984) Molecular requirements for the mutagenicity of malondialdehyde and related acroleins. Cancer Res. 44, 2848-2854. (24) Lutz,D.,Eder,E.,Neudecker,T.,andHenechler,D. (1982)Structuremutagenicity relationship in a,fl-unsaturated carbonyl compounds and their corresponding allylic alcohols. Mutat. Res. 93,305-315. (25) Guttenplan, J. B. (1980)Enhanced mutagenic activity of N-nitroso compounds in weakly acidic media. Carcinogenesis 1, 439-444.
