[CANCER RESEARCH 40, 276-282, February 19801 0008-5472/80/0040-0000$02.00 Comparison of the Mutagenicities of Malondialdehyde and the Side Products Formed during Its Chemical Synthesis1 Lawrence J. Marnett2 and Melissa A. Tuttle Department of Chemistry, Wayne State University. Detroit, Michigan 48202 ABSTRACT (9, 15). It is believed that the formation of these polymers can be avoided by using very dilute solutions of TEP or TMP for the Malondialdehyde, a product of polyunsaturated fatty acid hydrolyses (15). Since MDA is a relatively weak mutagen, high metabolism and degradation, has been reported to be muta concentrations must be used to detect a mutagenic response genic and carcinogenic. The malondialdehyde used for testing (16, 21). This necessitates the use of concentrated solutions was generated by the acidic hydrolysis of tetraalkoxypropanes. of TEP or TMP for the preparation of the MDA solutions. We have studied the production of compounds mutagenic to MDA is a moderatelyweak acid (pK@,,4.46)which exists as Sa!mone!!a typhimurium strain his D 3052 following the hy its conjugate base at physiological pH (Ref. 17; Equation A). drolysis of tetraalkoxypropanes. The major mutagenic com pound produced from tetraethoxypropane is 18-ethoxy-acrolein 0 0 (90 to 100 revertants4tmol) and not malondialdehyde (3 to 5 II @ revertants/jsmol). Hydrolysis of tetramethoxypropane pro H )@ H pKa duces two compounds, $-methoxy-acrolein (125 to 160 re _________ H@ ,,C@ vertants4tmol) and 3,3-dimethoxypropionaldehyde (105 to II 135 revertants/@mol), which are more mutagenic than is ma ,C@ @ londialdehyde. Using standard conditions for the hydrolysis of H H tetraethoxypropane, the yield of malondialdehyde is 25%, and the yield of ,6-ethoxyacrolein is 13%. Considering the differ The conjugate base of MDA is much less reactive than is MDA ences in specific mutagenicity, the compound which accounts toward nucleophiles and does not participate in self-conden for the bulk of the mutagenicity of a crude hydrolysate of sation reactions (4, 6). This suggests that the side products tetraethoxypropane is not malondialdehyde. The presence of formed during the acidic hydrolysis of TEP or TMP to MDA are these incomplete hydrolysis products may lead to a substantial unlikely to be formed under physiological conditions where overestimation of the actual mutagenicity and carcinogenicity MDAexistsexclusivelyas its conjugatebase.If the side prod of malondialdehyde. ucts are responsible for the reported initiating activity, then MDAitselfmaynotbea mutagenandcarcinogenandmayplay INTRODUCTION no role in spontaneous carcinogenesis. We have recently synthesized [‘4C]TEPforuse as a source MDA3 is formed in animal tissue as an end product of lipid of malondialdehyde-1 ,2,3-14C3in tracer studies (3). This has peroxidation and as a side product of prostaglandin and throm enabled us to quantitate the compounds produced during the boxane biosynthesis (2, 7, 10, 13, 22). Shamberger et a!. (20) acidic hydrolysis of TEP and to compare their specific muta have reported that MDA exhibits initiating activity in female genicities. Our studies show that (a) the sodium salt of the Swiss mice using the 2-stage assay. Mukai and Goldstein (16) conjugate base of MDA (NaMDA) is very weakly mutagenic, (b) and Shamberger et a!. (21) have reported that MDA is muta the polymeric decomposition products formed from MDA under genic in several strains of Salmonella typhimurium. The wide acidic conditions are not mutagenic, and (C) the major muta spread occurrence of MDA in the animal kingdom coupled with genic compound produced from TEP using literature hydrolysis its apparent initiating activity suggests that MDA may be an conditions is not MDA but, an intermediate hydrolysis product, important causative agent of spontaneous carcinogenesis. BEA. MDA is unstable and cannot be stored in the pure state (11). Therefore, solutions of it are usually prepared immediately prior MATERIALS AND METHODS to testing by the acidic hydrolysis of TEP or TMP (18). Previous investigators have noted that the hydrolysis of these com TMP was purchased from Aldrich Chemical Co. and distilled pounds is accompanied by the production of colored by-prod prior to use. TEP was synthesized according to literature ucts which are believed to be condensation polymers of MDA methods (12) or purchased from ICN Pharmaceuticals (Plain view, N. Y.). [14C]TEPwas synthesized as described and dem onstrated to be in excess of 95% radiochemically pure by I This investigation was supported by Grant CA-22206 awarded by the Na tional Cancer Institute, Department of Health, Education, and Welfare. radiogas chromatography (3). The synthesized material (25 2 To whom correspondence should be addressed. @Ci/mmol)was diluted with unlabeled TEP to produce the 3 The abbreviations used are: MDA, malondialdehyde; TEP, 1 ,1 ,3,3-tetra ethoxypropane; TMP, 1.1,3,3-tetramethoxypropane; [‘4CJTEP,tetraethoxypro specific activity desired for a given experiment. Dowex SOW pane-i ,2,3-'4C3;NaMDA, sodium malonaldehydate; BEA, f@-ethoxy-acroleln;GC, X4 was purchasedfromJ. T. BakerChemicalCo. and washed gas chromatography; NMR, nuclear magnetic resonance; t, triplet; q, quartet; exhaustively with water to remove colored impurities. Sepha dd, doublet of doublets; d, doublet; DMP, 3,3-dimethoxypropionaldehyde; BMA, $-methoxy-acrolein, 5, singlet. dex LH-2O was purchased from Sigma Chemical Co. Water Received July 18, 1979; accepted October 23, 1979. was distilled twice in glass. 276 CANCERRESEARCHVOL. 40 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1980 American Association for Cancer Research. MDA Mutagenicity NaMDA. TEP (1.1 g; 5 mmol) and 5 g Dowex 50 (5 mmol Aqueous solutions of compounds to be tested were sterilized Hi were suspended in 10 ml H20 and agitated in a shaking by passage through 0.2-zm Acrodiscs (Gelman Instrument water bath at 25°for 30 mm. The slurry was titrated to pH 7.0 Co.). Dimethyl sulfoxide, acetone, and ethanol solutions were with 5 and 1 N NaOH. The Dowex 50 was filtered off, and the sterile. filtrate was extracted 3 times with equal volumes of water Spectral Analysis of BEA and NaMDA in Hydrolyzed DMP saturated ethyl acetate. The aqueous phase was lyophilized, Solutions. Solutions of DMP incubated in neutral buffers exhibit and the residue was dissolved in 1.5 ml H20. This solution was absorbances at 245 and 267 nm due to the presence of BMA applied to a column of Sephadex LH-20 (1.8 x 40 cm) and and NaMDA, respectively. Assuming that these are the only 2 eluted with H20. Fractions (2.5 ml) were collected, and those absorbing species in solution and using the molar absorptivities containing the major 267-nm-absorbing material were pooled of BMA at 245 nm (18.4/mM/cm) (19) and NaMDA at 267 nm and lyophilized. NaMDA elutes from the LH-20 column at a (34.2/mM/cm), one can derive Equation B to calculate the retention volume of approximately 55 ml. The residue from the [NaMDA]. lyophilization was dissolved in a minimal volume of H2O, and [NaMDA]= 1.09 (29 A267—8.2A245) (B) acetone was added to the cloud point. On standing overnight at —15°,fine white needles of NaMDA formed (260 mg, 40% The [BMA] is then calculated from Equation C. yield). Combustion analysis indicates that these crystals con tam 2 to 3 water molecules/molecule of NaMDA. [BMA] •=55A245—O.55[NaMDA] (C) BEA. A modificationof the procedure of Rüeggeta!. (19) was used. Toluenesulfonic acid (54.3 mg; 0.3 mmol) and H2O A267and A245are the measured absorbances at 267 and 245 (1.8 g; 100 mmol) were added to TEP (22 g; 100 mmol) and nm, respectively. The concentrations of NaMDA and BMA stirred at 80°for 2 hr. The reflux condenser was replaced with determined from Equations B and C are in @tMandrepresent a short-path distillation apparatus, and ethanol (4.6 g; 100 the concentrations in the solutions diluted for spectrophoto mmol) distilled off. NaHCO, (0.5 g) was added, and the mixture metric analysis. The concentrations in the stock solutions are was stirred at room temperature for 1 hr. Ethanol was removed determined by multiplying by the appropriate dilution factor. by distillation at reduced pressure (41 to 45°at 140 torr). Analysis of TEP, BEA, and NaMDA in Hydrolyzed TEP Crude BEA distilled from 130 to 153°(140 torn). Further Solutions. The hydrolyzed TEP solutions were neutralized (pH purification was affected by preparative GC on a 6-ft column of 7) and extracted with an equal volume of CCL. An aliquot of 10% OV-17 on Chromosorb W-HP (35 ml helium per mm at the organic extract was injected onto a 6-ft column of 10% OV 1100). The isolated material exhibited a single peak when 1 7 on Chromosorb W-HP (2 mm i.d.) at 70°. Isothermal elution reanalyzed under these conditions. NMR (CCL): @1.4t, J = 7 was continued for 3 mm after which the temperature was Hz (3 H); 4.1 q, J = 7 Hz (2 H); 5.6 dd, J = 7, 13 Hz (1 H); 7.4 programmed to 170°at 10°/mm(30 ml helium per mm). The d, J = 13 Hz (1 H); and 9.3 d, J = 7 Hz (1 H). Mass spectrum ‘amountsofBEA (8 mm) and TEP (11 mm) eluting from the (70 eV), m/e (intensity): 100 (24%) and 71 (100%). column were quantitated by electronic integration. DMP and BMA. The above procedure was used for the The concentration of NaMDA in the aqueous phase was hydrolysis of TMP (16.4 g; 100 mmol). Following the removal determined from the absorbance at 267 nm. of methanol by distillation, a fraction which contained both UV spectra were recorded on a Perkin-Elmer 124 spectro DMP and BMA was collected at 110 to 1200 (145 torr).