[CANCER RESEARCH 44, 990-995, March 1984]

Formation of Cyclic 1,/V2-Propanodeoxyguanosine Adducts in DMA upon

Reaction with Acrolein or Crotonaldehyde1 2

Fung-Lung Chung,3 Ruth Young, and Stephen S. Hecht

Naylor Dana Institute tor Disease Prevention, American Health Foundation, Valhalla, New York 70595

ABSTRACT

Acrolein reacted with deoxyguanosine at pH 7 and 37°to give

three major products, Adducts 1 to 3, which were separated byhigh-performance liquid chromatography. They were identified

by their ultraviolet, mass, and nuclear magnetic resonance spectra, by the spectra of the corresponding guanine derivatives, andby chemical transformations. Adducts 1 and 2 were two rapidlyequilibrating diastereomers of 3-(2-deoxy-/8-D-e/yf/7ro-pentofu-ranosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-a]purine-10(3H)one, and Adduci 3 was 3-(2-deoxy-/3-D-e/yf/7ro-pentofu-ranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-

10(3H)one. Adducts 1 and 2 were formed by Michael addition ofN-1 of deoxyguanosine to C-3 of acrolein, followed by ringclosure between A/2 of deoxyguanosine and C-1 of acrolein.

Adduct 3 was formed by ring closure in the opposite direction.Adduci 3 was analogous to the major crotonaldehyde-deoxy-

guanosine adducts which were previously characterized. Adduct3 (0.2 mmol/mol DNA-P) or the corresponding crotonaldehydeadduct (0.03 mmol/mol DNA-P) was formed when either acrolein

or crotonaldehyde was allowed to react with DNA at pH 7 and37°.These results demonstrate that cyclic 1,A/2-propanodeoxy-

guanosine adducts are formed by reaction of acrolein and crotonaldehyde with DNA.

INTRODUCTION

Acrolein (CH2=CHCHO) is the simplest member of the classof a,/3-unsaturated carbonyl compounds. These compounds are

ubiquitous in the human environment, occurring in a wide rangeof natural and commercial products. Recent studies have shownthat simple a,/3-unsaturated carbonyl compounds such as acro

lein, crotonaldehyde, and methylvinyl ketone are mutagenic toward Salmonella typhimurium in the absence of an activatingsystem (5, 18, 20). It is apparent from these and other studiesthat they can interact directly with DNA (13). However, thechemical structures of the DNA adducts have not been established.

In an earlier study we showed that crotonaldehyde (CH3CH=CHCHO) can react with deoxyguanosine to yield cyclic 1,/V2-

propanodeoxyguanosine adducts (3). These results and the mu-

tagenicity studies cited above encouraged us to investigate thepotential generality of the reaction of «.ß-unsaturatedcarbonyl

compounds with deoxyribonucleosides and DNA. Acrolein is notonly chemically a logical starting point for this investigation, butis also important in terms of potential human exposure. Its annual

1This study was supported by National Cancer Institute Grant CA 23901. Thisis Paper 61 of the series, "A Study of Chemical Carcinogenesis."

2 Presented in part at the 74th Annual Meeting of the American Association for

Cancer Research, San Diego, CA, May 1983 (27).3To whom requests for reprints should be addressed.

Received September 8, 1983; accepted November 29,1983.

production in the United States is estimated at 60 million pounds,excluding production as an unisolated intermediate for production of acrylic acid. It occurs in relatively high concentrations incigarette smoke, and has been detected in automobile exhaustand in occupational settings (11). It is a primary metabolite of thewidely used chemotherapeutic drug, cyclophosphamide (12).

Evaluation of the potential carcinogenicity of acrolein andcrotonaldehyde is complicated by their irritant properties andtoxicity (4, 11). An inhalation bioassay of acrolein in hamstersdid not show an increase in tumor incidence (6). However,acrolein released in vivo as a metabolite of cyclophosphamideappears to be at least partially responsible for the teratogenicityand bladder toxicity of this drug (12). It is not known whetheracrolein may also be involved in the bladder carcinogenicity ofcyclophosphamide. The purpose of the present study was todetermine the chemical structures of the major adducts formedupon reaction of acrolein with deoxyguanosine under physiological conditions, and to establish whether or not acrolein andcrotonaldehyde react with DNA to give deoxyguanosine adducts.These studies are necessary to gain a better understanding ofthe potential adverse physiological effects of acrolein and othera,/3-unsaturated carbonyl compounds.

MATERIALS AND METHODS

HPLC4 Analysis

HPLC was performed with a Waters Associates Model ALC/GPC-204high-speed liquid Chromatograph (Waters Associates, Milford, MA)

equipped with a Model 6000A solvent delivery system, a Model 660solvent programmer, a Model U6K septumless injector, a Model 440 UV/visible detector operated at 254 nm, or a Perkin-Elmer Model 650-1 OSfluorescence detector (Perkin-Elmer Corp., Oak Brook, IL). The following

solvent elution systems were used:System 1. A 50-cm x 9.4-mm (inside diameter) Partisil ODS-3 Mag

num 9 column (Whatman, Inc., Clifton, NJ) programmed from 0 to 20%methanol in H2O in 50 min, using Curve 5 or 6, and a flow rate of 5 ml/min.

System 2. Two 25-cm x 4.6-mm Partisil PXS 10/25 ODS columns

(Whatman) programmed from 20 to 50% methanol in H2O in 60 min,using Curve 8 and a flow rate of 1.5 ml/min.

System 3. Two 25-cm x 4.6-mm Partisil PXS 10/25 ODS columns

eluted isocratically with 10% methanol in 0.085 M ammonium phosphate,pH 3.0, at a flow rate of 1.5 ml/min.

System 4. A 25-cm x 4.6-mm Partisil-10 SCX strong cation exchange

column (Whatman) eluted isocratically with 2% methanol in 0.085 Mammonium phosphate, pH 2.0, at a flow rate of 1 ml/min.

The following wavelengths in nm (excitation, emission) were used forfluorescence detection: acrolein nucleoside Adducts 1 and 2 of Table 1,290, 380; acrolein nucleoside Adduct 3, 300, 387; crotonaldehyde nucleoside adducts, 280, 392; guanine derivative from acrolein nucleoside

4The abbreviations used are: HPLC, high-performance liquid chromatography;

NMR, nuclear magnetic resonance; MS, mass spectrum.

990 CANCER RESEARCH VOL. 44

on March 11, 2020. © 1984 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

Adducts 1 and 2, 287, 360; guanine derivative from acrolein nucleosideAdduct 3, 295, 363; guanine derivative from crotonaldehyde nucleosideadducts, 297, 370.

Reaction of Acrolein and Deoxyguanosine

Small-scale reactions were carried out by adding acrolein (112 mg, 2

mmol; Aldrich Chemical Co., Milwaukee, Wl) to 20 ml of phosphatebuffer, pH 7, containing deoxyguanosine (53 mg, 0.2 mmol). The solutionwas incubated at 37°with shaking, and then analyzed by HPLC, using

System 1.For larger-scale preparation of acrolein-deoxyguanosine adducts,

deoxyguanosine (2 g, 7.6 mmol) was dissolved in 200 ml of phosphatebuffer, pH 7, by heating at 50°.The solution was then cooled to 37°,

and acrolein (8.4 g, 0.15 mol) was added in one batch. The reactionmixture was incubated for 2 hr at 37°with shaking. Another 4.2 g ofacrolein were added, and the reaction was continued at 37°for 1 hr

more. The reaction mixture was extracted 3 times with 50 ml of CHCI3.The CHCI3 extracts were discarded, and the aqueous phase was concentrated to 30 ml by rotary evaporation at room temperature. Methanolwas added until the solution became cloudy. After the solution had stoodat room temperature, an oil separated, which was discarded. Silica gelwas added to the aqueous solution, and the suspension was concentrated to dryness by rotary evaporation. The residue was applied to thetop of a dry-packed silica gel column. The column was eluted withCHCbimethanol (4:1), and fractions were examined by thin-layer chro-matography on silica with development by CHC^methanohNI-UOH

(95:15:2); Ft,,Adducts 1 and 2 of Table 1,0.24; Adduct 3, 0.29. Fractionscontaining Adducts 1 and 2 or Adduct 3, respectively, were combinedand the solvents were removed. The residue containing Adducts 1 and2 was dissolved in a small amount of methanol:H2O (1:1) and acetonewas added until the solution became turbid. After standing at 5°over

night, pure Adducts 1 and 2 precipitated and were collected. Thecombined yields of Adducts 1 and 2 were 60 mg (2.5%). Adduct 3 wasfurther purified by HPLC, using System 1, yielding 45 mg (1.8%).

Hydrolysis of Acrolein-Deoxyguanosine Adducts

Adducts were incubated at 90°for 45 min in 1 to 2 ml of 0.1 N HCI.

Analysis by thin-layer chromatography on silica, CHC^methanokNI-UOH

(95:5:2), showed disappearance of the nucleoside adducts. R, of theresulting guanine derivatives: from Adducts 1 and 2, 0.27; from Adduct3, 0.27. The guanine adducts were further analyzed by HPLC, usingSystem 1 with Programmer Curve 5. Retention volumes: guanine derivative from Adducts 1 and 2, 110 ml; guanine derivative from Adduct 3,117ml.

Reaction of Adducts with NaOH and NaBH,

The appropriate nucleoside or base was dissolved in 1 to 2 ml of 0.5N NaOH, and an excess of NaBH.«was added. The resulting mixture washeated under reflux for 30 min, cooled, and neutralized with 1 N HCI. Rfvalues using CHCI3:methanol:NH4OH (95:5:2): A/2-(3-hydroxypropyl)gua-

nine, 0.20; 5,6,7,8-tetrahydropyrimido[1,2-a]purine-10(3H)one, 0.35.HPLC retention volumes, using System 1 with Programmer Curve 5: N2-

(3-hydroxypropyl)guanine, 27.5 ml; 5,6,7,8-tetrahydropyrimido[1,2-a]purine-10(3H)one, 35.9 ml.

Reaction of Acrolein or Crotonaldehyde with DMA

Calf thymus DNA (Sigma Chemical Co., St. Louis, MO), 20 mg, wasdissolved in 2 ml of phosphate buffer, pH 7, at 37°.Acrolein (116 mg,

2.1 mmol) or crotonaldehyde (43 mg, 0.61 mmol) was added and themixture was incubated with shaking for 3 hr at 37°(acrolein), or for 16

hr (crotonaldehyde). Control incubations were carried out without thealdehydes. The DNA was precipitated by addition of 0.1 volume of 3 Msodium acetate and 2 volumes of cold ethanol. The isolated DNA wasreprecipitated twice more using this procedure, and was then dissolved

Formation of Cyclic 1,N2-Propanodeoxyguanosine Adducts

in a solution of 5 ITIMTris-HCI (pH 7.1) and 5 mw MgCI2 to make a final

DNA concentration of 0.5 to 1 mg/ml. The resulting solution was incubated sequentially with enzymes (Sigma Chemical Co.) as follows: DNaseI (300 Mg/mg DNA; 3 hr), alkaline phosphatase from Escherichia coli (2units/mg DNA; 18 hr), spleen phosphodiesterase (0.2 unit/mg DNA; 1hr), crystalline snake venom phosphodiesterase (0.005 unit/mg DNA; 1hr), and alkaline phosphatase from E. coli (0.4 unit/mg DNA; 1 hr). Thefinal incubation mixture was concentrated to dryness by rotary evaporation at room temperature, and the residue was thoroughly extractedwith three 10-ml portions of methanol:ethanol (1:1). The extracts were

combined, concentrated to 0.3 to 0.5 ml, and the volume was broughtto 1.0 ml with H2O. Samples were analyzed by HPLC, using System 1and UV detection. The band with the correct retention volume for theappropriate adducts was collected, concentrated to dryness, redissolvedin H20:methanol (4:1) and analyzed, using System 2, with UV detection.The appropriate band was collected, concentrated to dryness, redissolved in H20:methanol (4:1) and analyzed, using System 3, with fluorescence detection. The appropriate band was collected, concentrated,and analyzed, using System 4, with quantitation by fluorescence detection. In this sequential analysis procedure, correct retention volumes hadto be established by injection of standards on each column prior tosample injections. To guard against contamination, the injector waswashed after each standard injection. A blank sample was then run tocheck for contamination. Using this procedure, no evidence for samplecontamination with standards was found.

RESULTS

Chart 1 illustrates a chromatogram obtained by HPLC analysisof the reaction of acrolein and deoxyguanosine; 3 major adductpeaks were observed. When either Adduct 1 or Adduct 2 wascollected and immediately reanalyzed under the same HPLCconditions, a mixture of Adducts 1 and 2 was obtained, indicatingthat these 2 products rapidly equilibrated. Adduct 3 remained asa single peak when reanalyzed.

The UV spectrum of Adduct 3 is illustrated in Chart 2A. This

UJV)

iMUer

o>

0 75 150

VOLUME(ml)

Chart 1. HPLC chromatogram obtained upon analysis of the reaction mixturefrom acrolein and deoxyguanosine (dg). System 1 was used, with Curve 5.

MARCH 1984 991

on March 11, 2020. © 1984 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

F-L Chung et al.

spectrum resembled those of 1,A/2-dimethylguanosine and the1,A/2-propanodeoxyguanosine adducts formed upon reaction of

crotonaldehyde with deoxyguanosine (3, 26). The chemical de-

sorption ionization MS of Adduci 3 showed an M + 1 ion atm/e 324, and a base peak of m/e 208, corresponding to loss ofdeoxyribose. These data are consistent with the addition of onemolecule of acrolein to deoxyguanosine. The 90-MHz NMR

spectrum of Adduct 3 is summarized in Table 1. This spectrumwas analogous to that of the 1,A/2-propanodeoxyguanosine ad-

duct from crotonaldehyde, in which C-6 of the structure of

Adduct 3 shown in Table 1 is substituted with a methyl group.The important differences were the lack of a methyl resonance,and an upfield shift of approximately 0.3 ppm for the C-6 méthylène protons, compared to the C-6 methine proton in the

crotonaldehyde adduct.To obtain more evidence on the structure of Adduct 3, it was

hydrolyzed to the corresponding guanine derivative. Its UV spectrum was essentially identical to that of the 1,A/2-propanoguanine

adduct from crotonaldehyde. The MS of the guanine derivativehad a molecular ion of m/e 207 and a base peak at m/e 151(guanine). The 300-MHz NMR spectrum of the modified guanine

derivative is illustrated in Chart 3A. These data are entirelyconsistent with the structure shown in Chart 3A.

Treatment of the guanine derivative of Adduct 3 with NaBH4and NaOH converted it in high yield to A/2-(3-hydroxypro-

pyl)guanine. The UV spectrum of this compound was characteristic of an A/2-substituted guanine, and its MS showed a molec

ular ion at m/e 209. Its NMR spectrum, summarized in Table 2,was entirely consistent with the structure illustrated. This productresulted from base-catalyzed ring opening, followed by reduction

of the intermediate aldehyde. These data establish the structureof Adduct 3 as 3-(2-deoxy-|8-D-eryf/7ro-pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10(3W)one, as illustrated in Table 1. Since C-8 is a chiral center, Peak 3 is presumably a mixture of 2 diastereomers. It showed no circular dichro-

ism spectrum, in agreement with this assumption.The UV spectra of Adducts 1 and 2 were identical and were

similar to that of Adduct 3, as illustrated in Chart 2B. The 90-

TabtelNMRspectral data (90 MHz) for acrolein-deoxyguanosineadducts

220 260WAVELENGTH In

300

•)

220 260 300WAVELENGTH<nm)

Chart 2. UV spectra, measured in H2O, of Adduct 3 of Chart 1(A), and Adduct 2 of Chart 1(B).The spectra of Adducts 1 and 2 were identical. , pH 7;

,pH 1; ,pH 13.

M o

tYrv.kMA.^~.NiHO Hj "4"»H

H0£•0vj,•^y/^'g*OHAdducts

1 and 2i*

«i5H„o^OJ,.«A

/jt—tfOHAdduct

3Chemical

shifts(ppm)Position25678OH1'2'3'4'5'3'-OH,5'-OHAdducts

1 + 2(combined)7.90,s"8.35,d65.0,

m1.9,m4.35,

m(1H)C5.89,d"6.12,

tu = 8Hz)OOm"Z.2,m4.33,

m3.80,m3.5,m5.25,

d; 4.90, m"Adduc137.86,87.85,

s"3.40,

m1.9,m6.3,bs6.3,bs66.09,

t U = 8 Hz)2.2,rri4.31,m3.79,m3.5,m4.8-5.4,

broad6

"s, singlet; d, doublet; t. triplet; m, multiplet; bs, broad singlet."Disappeared upon D2Otreatment.C1Hpartially obscured by H20.dPartiallyobscured by dimethyl sulfoxide.

14 12 10 8 6PPM

14

Chart 3. NMR spectra (300 MHz) measured in dimethyl sulfoxide-d«,(DMSO),of acid hydrolysis product of Adduct 3 of Chart 1(X>);and acid hydrolysis productof Adducts 1 and 2 of Chart 1(B).

992 CANCER RESEARCH VOL. 44

on March 11, 2020. © 1984 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

Formation of Cyclic 1,N2-Propanodeoxyguanosine Adducts

MHz NMR spectrum of combined Adducts 1 and 2 is summarizedin Table 1. The spectral data are consistent with the structureillustrated in Table 1. Treatment of Adducts 1 and 2 with acidgave a single guanine derivative. Its UV spectrum is illustrated inChart 44. Its MS did not give the expected molecular ion at m/e207, but instead gave a peak at m/e 189 (M+—H2O; relative

intensity, 80) and a base peak at m/e 188. The NMR spectrumof the guanine derivative is shown in Chart 3fî.The chemicalshifts in the NMR spectrum in Chart 36 are consistent with thoseof the nucleoside Adducts 1 and 2, summarized in Table 1, butthe spectrum in Chart 38 is easier to interpret, because there isno interference from the deoxyribose protons. The resonancesat 5.9,8.1, and 12.5 ppm disappeared upon treatment with D20,which is in agreement with the assigned structure. The ABpattern observed for the protons attached to C-7 is similar tothat seen in the spectrum illustrated in Chart 3A, and appears tobe characteristic of 5,6,7,8-tetrahydropyrimido[1,2-a]purine-10(3H)ones substituted at C-6 or C-8 with a hydroxy group.

Treatment of nucleoside Adducts 1 and 2 with NaBH4 andNaOH gave a single compound with a UV spectrum similar tothat of 1,A/2-dimethylguanosine. Hydrolysis of this product with

Table2NMR spectral data (90 MHz) for Nz-(3-hydroxypropyl)guanine

0

Position1

23OHN'H

8N«H"t,

triplet; qui, quintet; m,Chemical

shifts(ppm)3.40,t" (J = 7.2 Hz)

1.66, qui (J = 7.2 Hz)3.45, m4.55, t" (J = 5 Hz)6.3,m"10.4,08"7.55,

s12.4,bs6multiplet;

bs, broad singlet; s, singlet.

A.

220 260 300WAVELENGTH(nm)

220 260 300

WAVELENGTH(nm)

acid gave a guanine derivative having the UV spectrum illustratedin Chart 4B. The same guanine derivative was obtained by acidhydrolysis of Adducts 1 and 2, followed by reaction with NaBH4and NaOH. The UV spectrum of this compound was similar tothat shown in Chart 4A, but was different from that of 1-

methylguanine (7). The MS of the product showed a molecularion at m/e 191 (relative intensity, 100) and an MM peak (relativeintensity, 58) at m/e 190. Its NMR spectrum ¡ssummarized inTable 3. These data can be explained only by the structureillustrated in Table 3. Thus, treatment of nucleoside Adducts 1and 2, or of the corresponding guanine derivative, with NaOHand NaBH4, resulted in elimination of H20, followed by reduction.The ring structure was maintained, in contrast to the resultsobtained upon treatment of the base derived from nucleosideAdduci 3 with NaOH and NaBH4. Taken together, these chemicaland spectral data establish the structures of nucleoside Adducts1 and 2 as a pair of rapidly interconverting diastereomers of 3-(2-deoxy-/3-D-eryfftro-pentofuranosyl)-5,6,7,8-tetrahydro-6-hy-droxypyrimido[1,2-a]purine-10(3H)one(see structure in Table 1).

DNA which had been allowed to react with acrolein or croton-aldehyde at pH 7 and 37° was enzymatically hydrolyzed to

deoxyribonucleosides. The resulting mixtures were purified bysequential HPLC in 3 different systems, as described in "Materials and Methods." The retention volumes of nucleoside Adducts

1 to 3 of Chart 1, and of the crotonaldehyde-deoxyguanosine

adducts in these systems, are summarized in Table 4. Thefractions containing the appropriate adducts were finally analyzed by HPLC, using a strong cation exchange column andfluorescence detection. The resulting chromatogram from theacrolein-DNA reaction is illustrated in Chart 5A. Chart 5B illus

trates the chromatogram obtained by identical treatment of acontrol DNA sample, which was isolated from an incubationcarried out in the absence of acrolein. The peak in Chart 5Amarked with an asterisk had the same retention time, establishedby coinjection, as nucleoside Adduct 3 from reaction of acroleinwith deoxyguanosine. The minor peak eluting just prior to theAdduct 3 peak corresponded in retention time to nucleosideAdducts 1 and 2. The peak marked with the asterisk in Chart 5Awas collected and treated with acid to convert it to the corresponding base. The resulting material (Chart 5C) was chromat-

ographically indistinguishable from the standard prepared by acidhydrolysis of nucleoside Adduct 3 from the reaction of acrolein

TablesNMR spectral data (90 MHz) for 5,6,7,8-tetrahydropyrimido[1,2-a]purine-

10(3H)one

» o,•ÇQQ'Position2N3H

N5H

6(8)78(6)Chemical

shifts(ppm)7.62,s"

12.3, bs7.48, s"3.30, tc (J =1.90, qui0 (J3.90, r (J =5

Hz)= 5 Hz)5 Hz)

Chart 4. UV spectra, measured in H2O,of acid hydrolysis product of Adducts 1and 2 of Chart 1(/4);and product obtained upon treatment of the acid hydrolysisproduct with NaBH4and NaOH (B). , pH 7; , pH 1; , pH 13.

as, singlet; bs, broad singlet; t, triplet; qui, quintet.

Disappearedupon addition of D20.c Collapsed to singlet upon irradiation at 1.9 ppm." Collapsedto triplet upon irradiationat 3.9 ppm.

MARCH 1984 993

on March 11, 2020. © 1984 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

F-L Chung et al.

Table 4

HPLC retention volumes of adducts

H HOY-" °-J

OH

Retention volumes (ml) of adducts from

CHî=CHCHO

HPLCsystem81

234R,

= OH; R2 =HDiastereomer

16185

28.533

6.1Diastereomer

2o200

31.541.3

6.1R,

- H; RÃŽ-OHC205

35.342.0

7.0ori3on=ononu

(«i==on3; HI =OH)Diastereomer

1d273

63.539.8

9.2Diastereomer

2d290

69.544.3

9.28 See "Materials and Methods" for conditions6 Chart 1, Peaks 1 and 2.c Chart 1, Peak 3.d For structure determination, see Ref. 3.

•J*

2 4 6 8 10 12 2 4 6 8 10 12 2 4 6 8 10 12

VOLUME(ml)

Chart 5. HPLC chromatogram, using System 4, of the purified fraction obtainedupon analysis of DMA that had been treated with acrolein. See "Materials andMethods" for details. A, modified nucleosides from acrolein-treated DMA. •,peak

that corresponded in retention volume to Adduci 3 (see structure in Table 1). Thepeak eluting immediately after this peak corresponded to the hydrolysis product ofAdduci 3, which was partially formed under the acidic conditions of HPLC System4. B, analysis of control DNA, treated identically as the sample in A, except thatacrolein was omitted from the initial reaction mixture. C, chromatogram obtainedby treating the peak with the asterisk in A with acid. The retention volume wasidentical to that of the hydrolysis product of Adduci 3.

and deoxyguanosine. The ratio of the peak heights detectedsimultaneously by fluorescence and UV was 4:1 for the standardguanine adduci, and 4:1 for that isolated from the DNA reaction.These results demonstrate that one of the adducts formed uponreaction of acrolein with DNA is the nucleoside adduct corresponding to Peak 3 of Chart 1. The minimum level of modificationwas 0.2 mmol/mol DNA phosphate. Entirely analogous resultswere obtained upon analysis of the crotonaldehyde-DNA reac

tion. The diastereomeric adducts, Isomer 1 and Isomer 2 ofTable 4, were detected as one peak in the final HPLC analysis,corresponding to a minimum level of modification of 0.03 mmol/mol DNA phosphate.

DISCUSSION

The results of this study demonstrate that under physiologicalconditions, acrolein and crotonaldehyde can modify DNA by

forming cyclic 1,A/2-propanodeoxyguanosine adducts. We are

not aware of any previous studies in which the DNA adductsformed from acrolein or crotonaldehyde have been chemicallycharacterized. Since the 1 and A/2 positions of guanine are

involved in base pairing, it seems possible that the adductsdescribed in this study may be involved in the mutagenicity ofacrolein and crotonaldehyde. However, adducts with other DNAbases or possibly cross-linked adducts might also form upon

reaction of DNA with these compounds. These possibilities werenot investigated in the present study.

The concentrations of acrolein and crotonaldehyde used in thereactions with DNA were 102 to 10" times higher than those

used in the mutagenicity assays (18). These high concentrationswere used because the extents of DNA modification by acroleinand crotonaldehyde were relatively low, and the limits of sensitivity of the fluorescence detection method would have beenexceeded if significantly lower concentrations were used. Nowthat the structures and chemical properties of the deoxyguanosine adducts have been established, it should be possible todesign more sensitive methods to allow the detection of theseadducts under the conditions of mutagenicity assays or of invivo exposure. Postlabeling techniques with NaB3H4or 32Pwould

seem appropriate.The reaction of acrolein with deoxyguanosine was more com

plicated than the reaction of crotonaldehyde with deoxyguanosine. Crotonaldehyde gave diastereomeric adducts resulting exclusively from Michael type addition of the A/2-amino group ofdeoxyguanosine to C-3 of crotonaldehyde, followed by ringclosure between the N-1 of deoxyguanosine and C-1 of croton

aldehyde. This mode of addition was also observed for acrolein,giving rise to Peak 3 of Chart 1 However, major acrolein adductswere also formed by Michael addition in the opposite direction,with initial bond formation between N-1 of deoxyguanosine andC-3 of acrolein, followed by ring closure between the A/2-amino

group and C-1 of acrolein. Examination of models suggests that

the selectivity in the crotonaldehyde reaction is due to stericcrowding caused by the methyl group.

The chemical properties of the acrolein Adducts 1 and 2, inwhich C - 6 of the 5,6,7,8 - tetrahydropyrimido [ 1,2 - a ] purine-

994 CANCER RESEARCH VOL. 44

on March 11, 2020. © 1984 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

•Formationof Cyclic 1,Nz-Propanodeoxyguanosine Adducts

10(3H)one system bears a hydroxyl group, seem quite differentfrom those of Adduci 3, or of the crotonaldehyde adducts inwhich the hydroxyl group is attached to C-8. For example,

interconversion of the diastereomeric crotonaldehyde adductswas never observed (3), but Adducts 1 and 2 interconvertedrapidly. The interconversion seems to result from loss and read-dition of H2O, but further studies are necessary to clarify themechanism (1). The relative reactivity of the 6-hydroxy-substi-

tuted acrolein adducts may be responsible for our observationthat their levels in DMA were much lower than those of the 8-hydroxy-substituted adducts.

The number of compounds that can form cyclic adducts withdeoxyribonucleosides or DNA is increasing rapidly. In our studies, we have documented this type of interaction for a-acetoxy-/V-nitrosopyrrolidine, 4-(carbethoxynitrosamino)-butanal, croton

aldehyde, and acrolein (3). Other compounds which form cyclicadducts directly, or upon metabolism, include glyoxal, 1,3-bis(2-chloroethyl)-1 -nitrosourea, ethyl carbamate, chloroacetaldehyde,vinyl chloride, /J-propiolactone, glycidaldehyde, trióse reductone,misonidazole, and substituted malondialdehydes (2, 8-10, 14-17, 19, 21-25). This class of nucleoside adducts may have

general importance in mutagenesis and carcinogenesis.

ACKNOWLEDGMENTS

Chemical desorption ¡onizationMSs were obtained at the Rockefeller UniversityMass Spectrometric Biotechnology Resource. NMR spectra (300 MHz) wereobtained using the 7T spectrometer at the Rockefeller University, purchased inpart with funds from the National Science Foundation (PCM-7912083), and fromthe Camille and Henry Dreyfus Foundation.

REFERENCES

1. Anderson, G. L., Rizkalla, B. H., and Broom, A. D. Synthesis of some tricyclicnucleosides related to the "Y" base of tRNA. J. Org. Chem., 39: 937-939,

1974.2. Chen, R., Mieyal, J. J., and Goldthwait, D. A. The reaction of /3-propiolactone

with derivatives of adenine and with DNA. Carcinogenesis (Lond.), 2: 73-80,1981.

3. Chung, F-L, and Hecht, S. S. Formation of cyclic 1,W2-adducts upon reactionof deoxyguanosine with a-acetoxy-N-nitrosopyrrolidine, 4-(carbethoxynitrosa-mino)butanal, and crotonaldehyde. Cancer Res., 43:1230-1235, 1983.

4. Committee on Aldehydes, Board on Toxicology and Environmental HealthHazard, Assembly of Ufe Sciences, National Research Council, Formaldehydeand other Aldehydes, pp. 221 -254. Washington, DC: National Academy Press,1981.

5. Eder, E., Henschler, D., and Neudecker, T. Mutagenic properties of allylic anda,i8-unsaturated compounds: consideration of alkylating mechanisms. Xenobiotica, 72: 831-848, 1982.

6. Feron, V. J., and Kruysse, A. Effects of exposure to acrolein vapor in hamsters

simultaneously treated with benzo[a]pyrene or diethylnitrosamine. J. Toxico).Environ. Health, 3: 379-394, 1977.

7. Friedman, O. M., Mahapta, G. N., Dash, B., and Stevenson, R. The action ofdiazomethane on deoxyribonucleosides. Biochim. Biophys. Acta, 703: 286-297, 1965.

8. Goldschmidt, B. M., Biazej, T. P., and van Duuren, B. L. The reaction ofguanosine and deoxyguanosine with glycidaldehyde. Tetrahedron Lett., 73:1583-1586,1978.

9. Gombar, C. T., Tong, W. P., and Ludlum, D. B. Reactions of bis-chloroethylnitrosourea and chloroethyl cydohexy! nitrosourea with deoxyribonucleic acid.Biochem. Pharmacol., 29: 2639-2643,1980.

10. Hemminki, K. Nucleic acid adducts of chemical carcinogens and mutagens.Arch. Toxicol., 52: 249-285,1983.

11. International Agency for Research on Cancer. IARC Monographs on theEvaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 19, pp.479-494. Lyon, France: International Agency for Research on Cancer, 1979.

12. International Agency for Research on Cancer. IARC Monographs on theEvaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 26, pp.165-202. Lyon, France: International Agency for Research on Cancer, 1981.

13. Izard, C., and Libermann, C. Acrolein. Mutât.Res., 47:115-138,1978.14. Laib, R. J., and Bolt, H. M. Alkylation of RNA by vinyl chloride metabolites in

vitro and in vivo; formation of 1-A/'-ethenoadenosine. Toxicology, fl: 185-195,

1977.15. Laib, R. J., Gwinner, L. M., and Bolt, H. M. DNA alkylation by vinyl chloride

metabolites: etheno derivatives or 7-alkylation of guanine? Chem.-Biol. Interact., 37: 219-231,1981.

16. Lee, J-H., Shinohara, K., Murakami, H., and Omura, H. Intermediates in thebrowning reaction of trióse reductone with guanine, guanosine, or guanylicacid. Agrie. Biol. Chem., 43: 279-286, 1979.

17. Ludlum, D. B., Kramer, B. S., Wang, J., and Fenselau, C. Reaction of 1,3-bis(2-chloroethyl)-1 -nitrosourea with synthetic polynucleotides. Biochemistry,74:5480-5485,1975.

18. Lutz, D., Eder, E., Neudecker, T., and Henschler, D. Structure-mutagenicityrelationship in o,#-unsaturated carbonylic compounds and their correspondingallylic alcohols. Mutât.Res., 93: 305-315, 1982.

19. Moschel, R. C., and Leonard, N. J. Fluorescent modification of guanine.Reaction with substituted malondialdehydes. J. Org. Chem., 41: 294-300,1976.

20. Neudecker, T., Lutz, D., Eder, E., and Henschler, D. Crotonaldehyde ismutagenic in a modified Salmonella typhimurium mutagenicity testing system.Mutât.Res., 97: 27-31,1981.

21. Ribovich, M. L., Miller, J. A., Miller, E. C., and Timmins, L. G. Labeled 1,We-ethenocytidine in hepatic RNA of mice given (ethyl-1,2-3H or ethyl-1-14C)ethylcarbamate (urethan). Carcinogenesis (Lond.), 3: 539-546,1982.

22. Sattsangi, P. D., Leonard, N. J., and Frihart, C. R. 1JV2-Ethenoguanine andN2,3-ethenoguanine. Synthesis and comparison of the electronic spectral

properties of these linear and angular triheterocycles related to the Y bases.J. Org. Chem., 42: 3292-3296, 1977.

23. Seto, H., Okuda, T., Takesue, T., and Ikemura, T. Reaction of malondialdehydewith nucleic acid. I. Formation of fluorescent pyrimido[1,2a]purin-10(3H)-onenucleosides. Bull Chem. Soc. Jpn., 56: 1799-1802,1983.

24. Shapiro, R., and Hachmann, J. The reaction of guanine derivatives with 1,2-dicarbonyl compounds. Biochemistry, 5: 2799-2807,1966.

25. Varghese, A. J., and Whitmore, G. F. Modification of guanine derivatives byreduced 2-nitroimidazoles. Cancer Res., 43: 78-82,1983.

26. Yamazaki, A., Kumashiro, I., and Takeniski, T. Synthesis of some A/'-methyl-2-substituted inosines and their 5'-phosphates. Chem. Pharm. Bull. (Tokyo),

76, 1561-1566,1968.27. Young, R., Chung, F-L., and Hecht, S. S. Modification of deoxyguanosine by

simple a,0-unsaturated carbonyl compounds. Proc. Am. Assoc. Cancer Res.,

24:68, 1983.

MARCH 1984 995

on March 11, 2020. © 1984 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1984;44:990-995. Cancer Res   Fung-Lung Chung, Ruth Young and Stephen S. Hecht  DNA upon Reaction with Acrolein or Crotonaldehyde

-Propanodeoxyguanosine Adducts in2NFormation of Cyclic 1,

  Updated version

  http://cancerres.aacrjournals.org/content/44/3/990

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/44/3/990To request permission to re-use all or part of this article, use this link

on March 11, 2020. © 1984 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from