[CANCER RESEARCH 44, 2848-2854, July 1984] Molecular Requirements for the Mutagenicity of Malondialdehyde and Related Acroleins1 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 seríes of ^-substituted acroleins in Salmonella typhimurlum hisD3052. Mutagenicity is dependent on the steric bulk of the pKa - 4.46 substituent (revertants/Mmol) at the ßposition: 0-methoxyacro- tein, 220; 0-ethoxyacrolein, 110; and /3-isobutoxyacrolein, 40. A good leaving group at the ßposition substantially increases the mutagenic activity (revertants/ixmol): /3-(p-nitrophenoxy)acrolein, The enolateis much less reactiveto nucleophilesthan is ß- 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 ßsubstit conditions,andvirtuallynothingis knownof the relationshipof uent is important for mutagenicity. Substitution of a methyl group adductformationto biologicalactivity.Wehavereportedthat ß- 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 «-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 /¿mol)inthis series. The importance of the aldehyde functionality acroleinsand havedeterminedtheir mutagenicityin hisD3052. is suggested by the nonmutagenicity of propiolonitrile, ethyl propicíate, 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 propicíate, 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 VaríanT-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 110-120°. 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-<p-nitrobenzyl)-pyridine. dium methylmalondialdehydate (16), and the enaminal from the addition Received December 16,1983; accepted April 3,1984. of tryptophan methyl ester to MDA (22) were also prepared by estab- 2848 CANCER RESEARCH VOL. 44 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1984 American Association for Cancer Research. Mutagenlcity of MDA and Related Acroleins 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 45°(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 /¿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 ß\of the bacterial culture, followed by shaking at 37°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 37°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); «2.37 s (3 H); 6.19 d, J 37°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 «,/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,