Molecular Requirements for the Mutagenicity of Malondialdehyde and Related Acroleins1

[CANCER RESEARCH 44, 2848-2854, July 1984]

Ashis K. Basu and Lawrence J. Marnett2

Department of Chemistry, Wayne State University, Detroit, Michigan 48202

ABSTRACT genieactivityare not understood.MDAexistsas its /9-hydroxy- acroleintautomer in polar solvents,and as an enolateion at Malondialdehyde, a product of lipid peroxidation and prosta- physiologicalpH(Chart1). glandin biosynthesis, is mutagenic in Salmonella. To determine the molecular requirements for its mutagenicity, we tested a serÃes of ^-substituted acroleins in Salmonella typhimurlum hisD3052. Mutagenicity is dependent on the steric bulk of the pKa - 4.46 substituent (revertants/Mmol) at the ÃŸposition: 0-methoxyacrotein, 220; 0-ethoxyacrolein, 110; and /3-isobutoxyacrolein, 40. A good leaving group at the ÃŸposition substantially increases the mutagenic activity (revertants/ixmol): /3-(p-nitrophenoxy)acrolein, The enolateis much less reactiveto nucleophilesthan is ÃŸ- 620; 0-benzoyloxyacrolein, 320; /3-chloroacrolein, 890; and di-7- hydroxyacrolein.Consequently,verylittleis knownof the struc oxopropenyl ether, 870. These data suggest that nucleophilic ture of MDA-nucleicacidadductsformed under physiological attack on the 0-carbon followed by elimination of the ÃŸsubstit conditions,andvirtuallynothingis knownof the relationshipof uent is important for mutagenicity. Substitution of a methyl group adductformationto biologicalactivity.Wehavereportedthat ÃŸ- at the a-carbon abolishes mutagenicity of these compounds. methoxyacroleinand/3-ethoxyacroleinare,respectively,40and This effect can be explained by the lack of chemical reactivity of the Â«-methylanalogues toward oxygen or nitrogen nucleophiles. 25 timesmoremutagenicthanMDAinS. typhimuriumhisD3052 (18).Thissuggeststhatrelativelysmallchangesinthe molecular Propynal, which can add nucleophiles to generate a substituted structureof MDAmay havesignificanteffectson its biological acrolein, exhibits the highest mutagenicity (1370 revenants/ activity.Wehave,therefore,synthesizedaseriesof/3-substituted /Â¿mol)inthis series. The importance of the aldehyde functionality acroleinsand havedeterminedtheir mutagenicityin hisD3052. is suggested by the nonmutagenicity of propiolonitrile, ethyl propicÃate, 4-benzoyloxy-3-buten-2-one, and 4-methoxy-3-bu- The resultsof the experimentsprovideimportantclues to the ten-2-one. Aldehyde addition subsequent to the formation of the chemicalbasisfor MDAmutagenicity. Michael adduci is, therefore, important for mutagenesis. An investigation of the toxicity of the present series indicates that MATERIALS AND METHODS toxicity and mutagenicity are independent events based on dif ferent chemical reactions. Acetylacetone, acrolein, ethyl chloroformate, ethyl propicÃate, isobutyl chloroformate, 4-methoxy-3-buten-2-one, methyl chloroformate, 4-nitro- phenol, propargyl alcohol, and tetramethoxypropane were purchased INTRODUCTION from Aldrich. Chemical Co. Propionaldehyde and triethylorthoformate ' MDA3 is widely formed in mammalian organisms during the were supplied by MCB. Benzoyl chloride was obtained from Mallinckrodt Chemical Works. Phenyl chloroformate was from Tridom/Fluka. Sepha- metabolism of prostaglandin endoperoxides and as a result of dex LH-20 and Dowex 50 W-X-4 were purchased from Sigma Chemical lipid peroxidation (3, 6,12, 29). It is present in a number of food Co. and J. T. Baker Chemical Co., respectively. products and its concentration is increased by irradiation (4, 22, NMR spectra were recorded on a VarÃanT-60. Mass spectra were 26). It has been found to be mutagenic to a murine L5178Y run at Michigan State University, Lansing, Ml. UV spectroscopy was lymphoma cell line cultured in vitro (32) and in several strains of performed on a Cary 210 and an Aminco DW2a spectrophotometer. 3-Ethoxy-2-propenal (/3-ethoxyacrolein) (18), 3-ethoxy-2-methyl-2-pro- Salmonella typhimurium (20, 28). Because of its nearly universal penal (/3-ethoxy-a-methylacrolein) (18), 3-methoxy-2-propenal(/3-meth- occurrence, others have speculated that MDA may be a mediator oxyacrotein) (14), 3-methoxy-2-methyl-2-propenal(|8-methoxy-a-meth- of spontaneous and chemically induced carcinogenesis. Al ylacrolein) (15), and 2'-methylpropoxy-2-propenal(/3-isobutoxyacrolein) though MDA is weakly mutagenic, it is not carcinogenic following (15) were prepared by the published literature procedures, and were topical administration to mouse skin (11). An evaluation of its purified by preparative gas chromatography on a 6' column of 10% OV- carcmogenicity following p.o. administration to mice and rats is 17 on Chromosorb W-HP at 110-120Â°. All the isolated materials exhib in progress. ited single peaks when reinjected under the same conditions, and the The molecular mechanisms by which MDA exerts its muta- respective proton NMR spectra did not show any detectable organic 1This work was supported by Research Grant CA 22206 from the National impurity. 3-(4-Nitrophenoxy)-2-propenal[/3-(p-nitrophenoxy)acrolein] (2), Cancer Institute. gave acceptable combustion analysis, and exhibited a single peak in 2 Recipient of an American Cancer Society Faculty Research Award (FRA 243). reverse phase high-pressure liquid chromatography (45% methyl alcohol To whom requests for reprints should be*addressed. in water with a flow rate of 2 ml/min). Propiolamide (31), propiolonitrile 3 The abbreviations used are: MDA, makxidiaktehyde; NaMDA, sodium maton- (30), propynal (24), sodium acetylacetaldehydate (13), NaMDA (2), so dialdehyde: NK/IR, nuclear magnetic resonance; d, doublet; dd, doublet of doublet; s, singlet; DMSO, dimethyl sulfoxide; NBP, 4-

2848 CANCER RESEARCH VOL. 44

Hshed literature procedures. All these compounds were routinely checked Mutagenicity by proton NMR and exhibited purities in excess of 95%. S. typhimurium strains were kindly provided by Professor Bruce Ames, /3-Benzoyloxyacrolein University of California, Berkeley, CA. Mutagenicity assays were carried out by the standard plate incorporation method, as well as by a liquid The synthetic procedure of Protopopova and Skoldinov (23) was preincubation procedure (1). In the plate incorporation assay, 2 ml of molten top agar at 45Â°(containing 50 MMhistidine and 50 /UMbiotin) and modified as follows: 0.99 g (10.5 mmol) anhydrous NaMDA (dried under 0.2 mm pressure for 24 hr) was suspended in 10 ml of anhydrous 0.1 ml of an overnight nutrient broth culture of the strain were added to tetrahydrofuran; 1.4 g (10 mmol) benzoyl chloride were slowly added 0.1 ml solution of the compound in DMSO. The contents were mixed by with vigorous stirring at ambient temperature. The mixture was stirred vortexing at low speed, and the resulting mixture was poured onto a for 1 hr, the solid was filtered off, the filtrate was evaporated, and the minimal agar plate. Liquid preincubation was done by mixing 10 /Â¿Iofa residue was redissolved in 10 ml ether. The ether solution was washed solution of the compound in DMSO to 390 rf 0.1 M phosphate buffer (pH twice with aqueous NaHCO3 and once with water, and was dried (K2CO3). 7.4) and 100 ÃŸ\of the bacterial culture, followed by shaking at 37Â°for Ether evaporation left a white crystalline solid that was recrystallized 20 min. Addition of top agar, vortexing, and plating were done immedi from ether.hexane. NMR (CDCI3), 5 6.35 dd, J = 8.13 Hz (1 H); 7.58 ately after the preincubation. Macroscopic colonies were counted after multiplet (3 H); 8.17 multiplet (2 H); 8.45 d, J = 13 Hz (1 H); 9.66 d, J = incubation at 37Â°for 48 hr. Triplicate plates were poured for each dose 8 Hz (1 H). of the compound. The effect of metabolic activation was assessed, using S-9 prepared from Aroclor 1254-treated Long-Evans rats. The sponta 0-Benzoyloxy-or-methylacrolein and 4-Benzoyloxy-3-buten-2-one neous reversion rate, determined in parallel incubation, was subtracted from each experimental value. A similar method to the above was used with sodium methylmalondi- Toxicity aldehydate as the starting material for /3-benzoyloxy-o-methylacrolein and NaMDA for 4-benzoyloxy-3-buten-2-one. One-tenth ml of an overnight culture of hisD3052 was added to 0.1 /S-Benzoyloxy-a-methylacrolein, NMR (CDCI3); 5 1.93 s (3 H); 7.54 ml solution of the compound in DMSO mixed with 2 ml sterile 0.1 M multiplet (3 H); 8.13 multiplet (3 H); 9.57 s (1 H). phosphate buffer (pH 7.0). The mixtures were incubated with shaking at 4-Benzoyloxy-3-buten-2-one, NMR (CDCI3); Â«2.37 s (3 H); 6.19 d, J 37Â°for 30 min. The number of surviving bacteria were determined on = 13 HZ (1 H); 7.55 multiplet (3 H); 8.16 multiplet (2 H); 8.45 d, J = 13 rich media plates after a 104 dilution of the incubation mixtures in 0.9% Hz (1 H). sterile sodium chloride solution.

Di-7-oxopropenyl Ether Rate Measurements

A modification of the procedure of Kalinina et al. (14) was used. To a The hydrolysis of Â«,/3-unsaturated carbonyl compounds was followed stirred suspension of 1.2 equivalents of NaMDA (1.13 g) in 10 ml dry by MDA, CH3MDA, or acetylacetaldehyde release at 267, 275, or 280 ether was gradually added 1 equivalent (1.57 g) phenyl chloroformate at nm, respectively, following the addition of NaOH. Absorbances were ambient temperature. After stirring at this temperature for 1 hr, salts and corrected for overlaps of starting materials at these wavelengths. All excess NaMDA were filtered. Evaporation of the solvent yielded about 1 experiments were carried out with a large excess of NaOH and followed g of a pale yellow oil. The phenyl-0-formylvinyl carbonate thus prepared pseudo-first-order kinetics. Second-order rate constants were obtained was kept at 30Â°until completely solidified (usually 4 to 5 hr). During this by dividing each observed first-order rate constant by the concentration period it undergoes a spontaneous disproportionation reaction with of NaOH, according to the equation liberation of COj and formation of diphenyl carbonate and di-7-oxopro- In2 penyl ether. The solid was repeatedly washed with ether until all the ether-soluble diphenyl carbonate was extracted. The ether-insoluble fl/2-[B]o white crystalline di-7-oxopropenyl ether was recrystallized from where [B]0 was the concentration of NaOH used. DMSO:chloroform. NMR (DMSO-d6), & 5.97 dd, J = 8,13 Hz (2 H); 8.09 d, J = 13 Hz (2 H); 9.49 d, J = 8 Hz (2 H). Mass spectrum (70 eV) m/e Alkylating Activity (intensity) 126 (19%), 97 (74%), 71 (100%), 69 (59%), 43 (71%). Alkylating activities were determined by the NBP test (9,10). The test /S-Chloroacrolein was performed under 2 different conditions. Alkylating Activity at Room Temperature. One ml of a 30 ITIMsolution of the compound in ethylene glycol and 1 ml of 200 RIM NBP in the same Propynal (5.4 g; 100 mmol) in 20 ml dry ether was slowly added to solvent were mixed, and 1 ml ethylene glycol was added. The mixture 120 mmol HCI in dry ether with vigorous stirring at ambient temperature, was stirred for 1 hr at room temperature. Absorbance at 487 nm (497 and the mixture was stirred for 10 min. Excess sodium bicarbonate was nm for 4-benzoyloxy-3-buten-one) was determined after appropriate added, and stirring was continued for 20 min. The mixture was filtered dilution. and the ether was evaporated under vacuum; 3.8 g of a clear oil were Alkylating Activity at 100Â°.This test was only performed with com obtained that was found to be a mixture of c/s- and frans-/3-chloroacrolein pounds that did not produce any color in the test at room temperature. with predominant (>80%) formation of the frans isomer. NMR (CDCI3) (trans); Ã±6.47 dd, J = 7, 13 Hz (1 H); 7.37 d, J = 13 Hz (1 H); 9.55 d, J One ml of a 30 mw solution of the compound in ethylene glycol and 1 ml of 200 mw NBP in the same solvent were refluxed at 100Â°for 10 min. = 7 Hz (1 H). Mass spectrum (70 eV) m/e (intensity) 93 (11%), 92 (6%), After cooling in ice water, 1 ml of triethylamine:ethylene glycol (1:1) was 91 (39%), 90 (14%), 64 (36%), 63 (30%), 62 (100%). added. Absorbance of the resulting solution at 487 nm was determined.

Purity of Compounds RESULTS The purity of all the compounds was routinely assayed by NMR and/ or gas chromatography before making solutions in DMSO for the muta- Highly purified MDA was found to be a weak mutagen that genicity tests. NMR analysis indicated that all the compounds tested induces -5 revertants/i/mol in S. typhimurium hisD3052, the were greater than 95% pure. strain with the highest reported sensitivity to MDA mutagenesis

JULY 1984 2849

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. A. K. Basu and L. J. Marnett (2). All .^'-substituted acroleins and related compounds were, therefore, also tested in this strain. Nu- Chart 2 presents a comparison of the dose-response curves in hisD3052 for 3 /3-alkoxyacroleins and NaMDA by the plate incorporation method. The specific mutagenicity seems to be dependent on the steric bulk of the alkoxy group at the ÃŸposition of the acrolein moiety. /3-Methoxyacrolein is more mutagenic than /3-ethoxyacrolein, which is, in turn, more mutagenic than ÃŸ- isobutoxyacrolein; MDA has the lowest mutagenicity in this Nu group. Mechanistically, this pattern can be explained by Chart 3. Michael addition of a nucleic acid base at the ÃŸposition, followed by elimination of the leaving group, would form a mono adduci that could be mutagenic, or could undergo further reac tion to generate a mutagenic lesion. This mechanism predicts Charts. Proposed mechanism of reaction of .(-substituted acroleins with nu- deophiles. that the mutagenicity should be dependent on the strength of the leaving group, and consistent with this scheme, we found Table 1 that an extremely poor leaving group like tryptophan methyl Mutagenicity ot ÃŸ-substitutedacroleins;effect of leaving group ester on the /8-carbon completely abolishes mutagenicity. Such Muta a mechanism also implies that the substitution of a better leaving Compound Structure genicity group on the 0-carbon should enhance the mutagenicity of this structure. In an attempt to establish a relationship between the leaving group ability of the ÃŸsubstituent and the mutagenicity of H >=0 the respective compound, we synthesized and tested several HXhisD3052 compounds in hisD3052 with substantially better leaving groups at the ÃŸposition, specifically, /3-(p-nitrophenoxy)acrolein, /3-ben- (revenants/ zoyloxyacrolein, ÃŸ-chloroacrolein, and di-7-oxopropenyl ether. Mutagenicity is, indeed, dependent on the leaving group ability /3-Methoxyacrolein HSCO- 220 of the ÃŸsubstituent, as shown in Table 1. All of the compounds with better leaving groups are more mutagenic than are the /3-(p-Nitrophenoxy)acrolein 620 alkoxyacroleins and MDA. However, the correlation is not a

400 - /3-Benzoytoxyacrotein 320

Di-y-oxopropenyl ether 870

/3-Chloroacrolein CI â€” 890 Enaminalof tryptophan and MDA Trp â€”NH â€” 0 a Estimated from the linear portion of the dose-response curve. 6 Extremely unstable to hydrolysis.

perfect one. /3-Benzoyloxyacrolein, possessing an excellent leav ing group, is less mutagenic than #-{p-nitrophenoxy)acrolein. It should also be pointed that /3-benzoyloxyacrolein is extremely unstable to hydrolysis, even at neutral pH. This compound, therefore, may not survive long enough under the conditions of the experiment to exert its complete mutagenic effect. In addi tion, all of these compounds, with the exception of ^-alkoxyacro leins, are extremely toxic. As a result, the mutagenicities in this table are estimated from the linear portions of the dose-response curves (Chart 4) and may be prone to slight errors. It should also be noted that the mutagenicity in this series of compounds is not strongly dependent on the strength of the leaving group. For example, improving the leaving group ability by 7 orders of magnitude by substituting p-nitrophenoxy for methoxy only tri ples the mutagenicity. In contrast to the relative insensitivity of the mutagenicity to /Â¿mol/plate the strength of the leaving group, the presence of a methyl group Chart 2. Mutagenicity of alkoxyacroleins and MDA in hisD3052 by plate incor poration method. The spontaneous reversion rate (-18), determined in parallel on either the carbonyl carbon or the carbon a to the aldehyde incubation was subtracted from each experimental value. functionality completely abolishes mutagenicity (Table 2). This is

2850 CANCER RESEARCH VOL. 44

true whether the compounds are assayed by the standard plate Table2 incorporation method or by the liquid preincubation procedure. Mutagenicity ot ÃŸ-substitutedacroleins;effect of methyl substitution The methyl effect is observed, regardless of the strength of the Mutagenicity of his- unsubstituted acrolein as a mutagen. Substitution of a methyl Compound D3052(revertants/Mmol) group on to MDA, /3-methoxyacrolein, or ÃŸ-benzoyloxyacrolein, which differ in mutagenicity by 40- and 60-fold, eliminates mutagenicity. To investigate the basis for the dramatic effect of methyl substitution on mutagenicity, we determined second-order rate constants of hydrolysis for some /3-substituted acroleins and related compounds (Table 3). Marked reduction in reactivity is observed following substitution of a methyl group at the a position. The rate of hydrolysis of 0-benzoyloxy-a-methylacrolein is 790 times slower than the unsubstituted analogue. This ac counts for the lack of reactivity of the Â«-substituted compound toward a nucleophile, and is consistent with the lack of muta genicity of the compound. The rates of hydrolysis of /S-methoxyacrolein and ,o-(p-nitrophenoxy)acrolein are also in good agree 220 ment with their relative mutagenicity. However, the lack of mu tagenicity of 4-benzoyloxy-3-buten-2-one does not correlate with ,OCH, its reactivity. This ketone is almost as reactive as its aldehyde analogue, 0-benzoyloxyacrolein. This suggests that a reaction of the aldehyde functionality subsequent to the formation of the Michael adduct is important for mutagenesis. Theoretically, it should be possible to form the same adduct by nucleic acid addition to propynal that is formed from ÃŸ- substituted acroleins by Michael addition at the /3-carbon, fol lowed by elimination of the leaving group (Chart 5). We have, therefore, determined the mutagenicity of propynal and several 320 analogues (Table 4). In fact, propynal is the most mutagenic compound tested in the series, inducing 1370 revertants/Mmol. This is not unexpected, because propynal, with no ÃŸsubstituent, should be more reactive toward a nucleophile than any of the ÃŸ- substituted acroleins. Analogues of propynal with different func-

CHj H 100 - Tabte3 Second-order rate constant of hydrolysis of some a,0-unsaturatedcarbonyI compounds Method described in "Materials and Methods." ÃŸ-Chloroacroletn Compound k (liter mor1 sec'1) 75 /3-Benzoytoxyacrotein 79 4-Benzoyloxy-3-buten-2-one 32 /3-Benzoytoxy-a-methylacrotein 0.1 0-(p-Nitrophenoxy)acrolein 3 D 0-Methoxyacrolein Q. 0.9 Di-Y/OKOpropenylether 50 ^~Â° ÃŸ-(p-Nitrophenoxy) acrolein tional groups substituted for the aldehyde functionality are, how >Â» -o ever, not mutagenic. The low mutagenicity of propargyl alcohol Methoxyacrotein is probably due to its partial oxidation to propynal by bacterial -â€”o enzymes. Oxidation has been invoked to explain the weak 25 - BenzoykMyacrotein mutagenicity of allyl alcohol relative to acrolein in TA100 (8). The aldehyde group of propynal, like the mutagenic ^-substituted acroleins, is, therefore, absolutely essential for mutagenicity. Interestingly, acrolein, the monosaturated analogue of propynal and the parent molecule for all the substituted acroleins de 50 100 150 200 scribed here is not mutagenic in hisD3052. nmol/plate We have also tested the present series of compounds in TA98 and TA100, the more sensitive and widely used strains of S. Chart 4. Mutagenicity of /3-substitutedacroleins in hisD3052 by plate incorpo ration method. The spontaneous reversion rate, determined in parallel incubation, typhimurium. None of the compounds was mutagenic in these was subtracted from each experimental value. strains.

2851

XH * Di-Y-oK>propenylÂ«thÂ« o ÃŸ-chloroocrolein â€¢Propiolonitrile 4 p-(p-NilrophenoÂ«y)ocfolein Nu o ÃŸ-Benzoyloxyacrolem o Propynal

Nu-

H Chart 5. .(-Substituted acroleins as propynal equivalents.

Table4 Mutagenicityofpropynalanalogues Mutagen icity of his- D3052 (revenants/ Compound Structure ^mol) 100200 400 800 1600 H-CSC-C nmol compound/ml Propynal 1370 Chan, complete 6. Toxicity bactericidal of some .(-substituted effect on theacrolems, higher dosage propynal (for methodsand propiolonitnle. see "Ma H 0 terials and Methods'). I //

Acrolem H \ OH c=c H-CSC-CH, PropargyI alcohol 15 _/ \ \ (RI.RZ = HorCHj) H-CzC â€”CmN 0 Propiolonitrile Chart?. NBP-ComptexX. 0 H-CSC-C propiolonitrile and 4-benzoyloxy-3-buten-2-one have strong al \ kylating potency and exhibit high bacterial toxicity. Interestingly, OCtHs Ethyl propicÃate however, both these compounds lack an aldehyde functionality. On the other hand, /3-methoxyacrolein,which is quite mutagenic, H-CSC â€”C has low alkylating activity. The relative inertness of /3-benzoyloxy-

Propiolamide a-methylacrolein toward nucleophiles is further substantiated by the NBP reaction. In general, reactivities of ÃŸ-substitutedacro leins to water and NBP are similar. H-CsCâ€”C \ Propiolicacid OH DISCUSSION

While performing the mutagenicity assays, we noted that The present study demonstrates that MDA and acroleins different compounds exhibit widely divergent toxicity to bacteria. substituted at the ÃŸpositionwith good leaving groups are direct- We have therefore systematically investigated the toxicity of the acting mutagens in S. typhimurium hisD3052, a strain with intact present series of compounds to S. typhimurium hisD3052. The excision repair but without plasmid pKM101 that codes for the .i-alkoxyacroleins are not toxic, whereas the .--aryloxyacrolems. error-prone repair enzyme of the SOS system. In the presence /3-chloroacrolein,di--y-oxopropenylether, and propynal are highly of a metabolic activation system (' S-9").the specific mutagenicity toxic (Chart 6). The nonmutagenic compound propiolonitrile also of each compound does not increase, and in some cases de exhibits very potent bacterial toxicity. It is, therefore, evident that creases (data not shown). mutagenicity and toxicity do not correlate. Covalent interaction of chemicals with DNA is one of the The alkylating activity of some representative compounds of primary events in mutagenesis. The precise nature and mecha the present series was determined using NBP (Chart 7) as a nism of mutation induced by MDA and the /3-substitutedacroleins nucleophile. /3-Substituted acroleins react with NBP to yield a is yet to be determined. However, our structure-activity studies salt X with an intense red color absorbing at 487 nm (when R2 indicate that a key step in the mutagenic response is Michael = H) or 497 nm (R2 = CH3).The more reactive compounds of addition of a nucleic acid base to the 0-carbon of the acrolein the series yield the color at room temperature, whereas others moiety, followed by elimination of the leaving group. This gen require more vigorous conditions such as reflux, followed by erates an acrolein substituted at the .; position, with the nucleic addition of alkali (Table 5). Alkylating activity appears to correlate acid base as shown in Chart 3. This is suggested by the decrease well with toxicity. All of the compounds with potent alkylating in mutagenicity-attendant increased steric hindrance in the alk- activity are very toxic. Two nonmutagenic compounds such as oxy-acrolein series, and by the increase in mutagenicity observed

2852 CANCER RESEARCH VOL. 44

Tables Alkylating activity of ^-substituted acroleins, their analogues,and some related "V acetylenic compounds activityCompoundPropynal Alkylating :0 Â»â€¢ Nu- R + room tem perature"285 H H Chart 8. Altemate scheme to explain mutagenicity of 0-alkoxyacroleins.

Propiolonitrile 55 tÃ-Benzoyloxyacrolein 82 4-Benzoyloxy-3-buten-2-one 57e ide, and propiolic acid are all negative in mutagenicity tests. fi-Benzoyloxy-a-methylacrolein 0 Especially significant is the fact that propiolonitrile and 4-benzo- /S-Chloroacrolein 124 Di-f-oxopropenyl ether 116 yloxy-3-buten-2-one are chemically very reactive, exhibit potent ii-Methoxyacrotein 0 alkylating activity, and are highly toxic. The rate of hydrolysis of MOAAt 0AMOO000.290.780 4-benzoyloxy-3-buten-2-one is almost as rapid as that of ÃŸ- * One ml of 30 mw compound and 1 ml of 200 mw NBP were stirred with 1 ml benzoyloxyacrolein. This strongly suggests that a second step of ethylene glycol for 1 hr at room temperature. Absorbance at 487 nm of the resulting solution was determined after appropriate dilution. in mutagenesis, but not toxicity, may be a nucleophilic addition 6 One ml of 30 mw compound and 1 ml of 200 mm NBP were refluxed at 100Â° to the aldehyde carbon. Indeed, enals have been suggested to for 10 min. Then 1 ml Methylamine was added and absorbance at 487 nm was be toxic to cells by virtue of their ability to react with sulfhydryl determined. c Absorbance at 497 nm (Aâ„¢,forthis complex). groups of proteins (25), whereas their mutagenicity is probably due to covalent interaction with nucleic acid bases. by substituting better leaving groups at the ÃŸposition. MDA mutagenicity has often been attributed to its ability to The decrease in mutagenicity of the alkoxyacroleins with in cross-link DNA (20); the mutagenic /3-substituted acroleins and creasing steric hindrance could be interpreted in terms of an propynal may cross-link in a similar fashion. Cross-linking agents alternate mechanism for mutagenesis (Chart 8). Nucleophilic cannot be detected as mutagens by Salmonella strains that do displacement could occur on the alkoxyl carbon, resulting in not have excision repair (17). The nonmutagenicity of MDA and alkylation. The enolate of MDA is a good leaving group (pKa of ^-substituted acroleins in TA98 and TA100, both of which lack /3-hydroxyacrolein 4.5), and hence this mechanism is theoretically uvrB, is an indication that these compounds may cross-link DNA. possible. However, one can discount this mechanism, based on However, a recent study that we have completed indicates that the observation that methyl substitution at the Â«position abol the ability of substituted acroleins to form interstrand cross-links ishes the mutagenicity of ^-alkoxyacroleins (Table 2). Substitu with DNA does not correlate to their mutagenicity (33).4 A second tion of a methyl group on the Â«-carbon should have minimal possibility is that both aldehyde equivalents of the acroleins react effect on nucleophilic attack at the alkoxyl carbon. Furthermore, with nucleophiles provided by a single base. Moschel and Leon attack on either of the 0-carbons of di-7-oxopropenyl ether ard (19) reported that Â«-substituted malondialdehydes form a should result in the same adduct that is being formed by Chart tricyclic adduci when incubated with guanine at pH 4.5. Small 3. The mutagenic activity of this compound supports the transfer amounts of a similar adduct were also isolated after prolonged of an enal moiety by a Michael addition, rather than alkylation. treatment of MDA with guanosine at acidic pH (27). Chung and Substitution of a methyl group at the Â«position abolishes Hecht (5) isolated a related adduct from the reaction of croton- mutagenicity. This effect can be explained by the lack of reactiv aldehyde with deoxyguanosine at neutral pH. It may be, there ity of the Â«-methyl analogues toward oxygen (~OH) or nitro fore, that MDA and /3-substituted acroleins are mutagenic as a gen (NBP) nucleophiles. The rates of hydrolysis, as well as the result of their ability to form similar adducts with the nucleic acid measurement of alkylating activity, indicate a tremendous reduc bases. It is interesting to note, though, that the parent molecule tion in reactivity following substitution of a methyl group at the of this series, acrolein, is not mutagenic in hisD3052, but is Â«position. Such a strong electronic effect of a methyl group is mutagenic in TA100 (8). This suggests a fundamental difference not unprecedented. A similar effect has been reported for hydro- in the mechanism of action of acrolein that is detected by the cyanation of acrylonitrile and acrylates compared to their Â«- use of different strains of Salmonella. methyl analogues. Acrylonitrile (CH2=CHâ€”C=N) and acrylates Although the mutagenic 0-substituted acroleins and propynal O can theoretically form the same adduct on reaction with nucleic II acid bases, different adducts may be formed. It is well known (CHÃ¼=CHâ€”Câ€”OR)areconverted to their 1,4-adducts in 73 to that electronic differences can significantly alter the site of alkyl- 93% yields, whereas methacrylonitrile [CH2=C(CH3)â€”C^N] ation on the same nucleic acid base (e.g., guanine) (7). The 0 present series of mutagens may also induce different adducts. II These sÃ©lectivitÃ©s,inturn, will probably trigger differential re and methyl methacrylate [CH2=C(CH3)â€”Câ€”OCH3] under simi sponses in repair enzymes or polymerases. We are attempting lar or more vigorous conditions afford only 2 to 5% of their 1,4- to identify the nucleic acid adducts formed from 0-substituted adducts (21). acroleins, and correlate the levels of individual adducts with the The importance of the aldehyde functionality in mutagenesis mutagenicity reported in the present paper. The range of muta is indicated by the lack of mutagenicity of acetylacetaldehyde, genic responses exhibited by these compounds makes them a 4-methoxy-3-buten-2-one, and 4-benzoyloxy-3-buten-2-one, powerful set of probes with which to investigate molecular where only the aldehyde hydrogen of MDA, 0-methoxyacrolein mechanisms of mutagenesis. and /3-benzoyloxyacrolein, respectively, were replaced by methyl groups. Similar results are observed when the aldehyde group of propynal is altered; propiolonitrile, ethyl propiolate, propiolam- 4This study does not preclude the possibility that intrastrand cross-links form.

JULY 1984 2853

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2854 CANCER RESEARCH VOL. 44

Ashis K. Basu and Lawrence J. Marnett

Cancer Res 1984;44:2848-2854.

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