Mutagenic potency of haloacroleins and related compounds

113

Mutation Research, 78 (1980) 113--119 © Elsevier/North-HollandBiomedicalPress

MUTAGENIC POTENCY OF HALOACROLEINS AND RELATED COMPOUNDS *

JOSEPH D. ROSEN **, YOFFI SEGALL *** and JOHN E. CASIDA Pesticide Chemistry and Toxicology Laboratory, Department of Entomological Sciences, University of California, Berkeley, CA 94720 (U.S.A.)

(Received 8 October 1979) (Revision received 2 January 1980) (Accepted 4 January 1980)

Summary 2-Chloroacrolein, the ultimate mutagen formed on metabolism of the carcinogenic herbicides diallate and sulfallate, and its 2-bromo-, 2,3~lichloro-and 2,3,3-trichloro- analogs are much more potent mutagens in the Ames Salmonella typhimurium strain TA100 assay than any other aldehydes examined previously or in this study. Polymer formation on reaction of deoxyadenosine with the difunctional 2~hloroacrolein probably involves crosslinking via Schiff base formation at the carbonyl group and Michael ad.dition at the double bond.

Mono-, di- and tri-chloroallyl substituents are present in sulfaUate, diallate and triallate, respectively, 3 thiocarbamate herbicides that are potent mutagens in the Salmonella typhimurium test (Ames assay) with the TA100 strain and metabolic activation [4,5,27]. The mutagenic metabolite in the first 2 cases is 2~hloroacrolein formed from diallate through a series of reactions initiated by sulfoxidation [18,19,22] and from sulfallate probably by hydroxylation at the 8-methylene group [15]. The high mutagenic potency of 2-chloroacrolein [22] relative to other aldehydes previously examined prompted a study of related compounds to define structural features critical for high potency.

* Supported b y NIH grant 5 PO1 E800049 (to J.E.C.) and t he Embassy of Israel in WH ht ngt on (to Y.S.). ** Perman ent address: D e p a r t m e n t of Food Science, Cook College, Rutgers University, New Brunswick, NJ 08903 (U.S.A.) *** P e r m a n e n t address: Israel I n s t i t u t e for Biological Research, Ness-Ziona, P.O.B. 19 (Israel). R e p r i n t request address: Joseph D. Rosen, D e p a r t m e n t of F ood Science, C o o k College, Rutgers Uzi/versity, New Brunswick, NJ 08903 (U.S.A.).

114

Materials and methods

Mutagenesis assays The Ames assay was performed in the usual manner with and without 20 #l of $9 fraction from the liver of Aroclor 1254-induced rats and using dimethylsulfoxide (DMSO) as the carrier solvent [1]. Correction is made for the background revertants which varied between 110 and 153. Values for the revertants/nmole were obtained from the linear portion of the dose--response curves and are the average of 2 or more assays. Mutagenic activity is tabulated as 0 when the number of revertants at any dose tested never exceeded twice the background level.

Chemicals Chemicals were from commercial sources or earlier syntheses of Schupan [19--21] or analogous preparation procedures except as indicated below. 1H and 13C nuclear magnetic resonance (NMR) spectra were obtained with 90 and 45.3 MHz spectrometers, resp. Mass spectroscopy (MS) was performed in the chemical ionization (CI) mode using isobutane as the reagent gas or by the field desorption technique. 2-Chloropropanal was synthesized according to Dick [6]. 3-Chloropropanal was prepared by the method of Witzemann [28]. NMR (CDC13, ppm downfield from tetramethylsilane): 9.75 (1H, t, J = 1 Hz), 3.79 (2H, t, J = 6.4 Hz), 2.90 (2H, double t, J l = 6.4 Hz, J2 = 1 Hz). The product readily polymerizes. 2,2,3Trichloropropanal was prepared according to the general procedure of Shostakovskii et al. [26]. On addition of gaseous chlorine to neat 2-chloroacrolein the temperature rose spontaneously to 55°C. After cooling, the product was distilled at 53°C/20 mm Hg (95%). NMR (CDC13): 9.28 (1H, s), 4.16 (2H, s). (DMSO-d6): 9.48 (1H, s), 4.57 (2H, s). CI-MS: 161 [M + 1] +. 2,3-Dibromo. propanal. Bromine was added dropwise to an equimolar amount of acrolein with care to maintain the temperature below 10 ° C. The product was distilled at 74--75°C/16 mm Hg (82%). NMR (CDC13): 9.39 (1H, d, J = 2.6 Hz), 4.56 (1H, asymmetric heptet), 3.82 (2H, double doublet, Jl = 6.3 Hz, J2 = 3.6 Hz). (DMSO-d6): 9.41 (1H, d, J = 2.2 Hz), 5.03 (1H, double triplet, J = 2.2 Hz, J = 6.8 Hz), 4.01 (2H, d, J = 6.8 Hz). CI-MS: 215 [M + 1] ÷, 135 [M + 1 -- HBr]*. 2-Bromoacrolein was made by dissolving 2,3~libromopropanal in excess diethylaniline below 10°C. Following filtration to remove diethylaniline hydrobromide, the product was distilled at 39°C/15 mm Hg (85%). NMR (CDC13): 9.27 (1H, s), 6.93 (2H, s). (DMSO~I6): 9.34 (1H, s), 7.25 (1H, d, J = 2.5 Hz), 7.13 (1H, d, J ffi 2.5 Hz). CI-MS: 135 [M + 1] ÷. 2,3-Dichloroacrolein and 2,3,3-trichloroacrolein. 2,3-Dichloroallyl alcohol or 2,3,3-trichloroallyl alcohol (7.8 mmole), N-bromosuccinimide (7.8 mmole) and benzoylperoxide (10 mg) were warmed gently in carbon tetrachloride (60 ml). A spontaneous reaction occurred at 45°C resulting in immediate elevation of the temperature to reflux with evolution of hydrogen bromide. After the spontaneous reflux ceased, the mixture was refiuxed for 1 h, cooled, and treated with solid Na2CO3 (1 g). After filtering off the succinimide and drying with MgSO4, dichloroacrolein was distilled at 55--61°C/15 mm Hg (85%) and trichloroacrolein at 68°C/15 mm Hg (100%). 2,3-Dichloroacrolein NMR

115 (CDC13, cis/trans mixture): 9.45 (1H, s), 7.90, 7.57 (1H, s, cis,trans). 2,4-Dinitrophenylhydrazone derivative, m.p. 244°C (dec.) and appropriate NMR and CI-MS: 305 [M + 1] ÷. 2,3,3-Trichloroacrolein NMR (CDC13): 10.00 (s). 2,4-Dinitrophenylhydrazone derivative, m.p. 239°C, with appropriate NMR and CI-MS: 339 [M + 1] ÷. Methyl 2.chloroacrylate was synthesized by methylation of 2-chloroacrylic acid with diazomethane in ether. NMR (CDC13): 6.53 (1H, d, J = 1.4 Hz), 6.02 (1H, d, J = 1.4 Hz), 3.85 (3H, s). The compound undergoes rapid polymerization. Stability of halopropanals. All halopropanals considered in this study, except 3-chloropropanal, are stable at --18°C for at least 2 months based on NMR monitoring. 2-Chloro- and 2,2,3-trichloro-propanals are stable as 40% solutions in DMSO for at least 24 h at 37°C. In contrast, 2,3~lichloro- and 2,3-dibromopropanals in DMSO under the same conditions dehydrohalogenate to 2-chloroand 2-bromo-acroleins, with half-life times of 5--7 and 29--31 min, resp. 2-Chloroacrolein thus generated in DMSO (with 1 : 1 HC1) has only 2 signals in the NMR spectrum (9.63 and 6.89 ppm), but'when the product is distilled and dissolved in CDC13 it shows the appropriate spectrum of 2-chloroacrolein [19]. Reaction of 2-chloroacrolein and deoxyadenosine. The reaction of deoxyadenosine (as the monohydrate) with excess 2~hloroacrolein was studied in DMSO-de at 37°C and in aqueous medium at 25°C. NMR monitoring revealed that the reaction of 0.3 M deoxyadenosine with 1.0 M 2~hloroacrolein in DMSO-d6 proceeded to a significant extent in 1 h and was complete by 16 h. Excess 2-chloroacrolein was removed and the product was isolated either by precipitation on addition of acetone or by evaporation of the DMSO under vacuum below 40°C. Alternatively, a solution of deoxyadenosine (2.5 mmole) in 0.1 M Na phosphate pH 6.3 buffer (55 ml) was treated with 2-chloroacrolein (5 mmoles at each of 0 and 3 h and 10 mmoles at 18 h). After 6 days incubation and evaporation to dryness at reduced pressure, the product was chomatographed on cellulose thin-layer chromatoplates using methanol--ammonium hydroxide (99 : 1). The material moving free from the origin was recovered by methanol extraction and fractionated into acetone~oluble product (~ 80%) and then into ether~oluble product (~30% of the portion soluble in acetone). Essentially identical IH NMR spectra were obtained for each of these fractions (regardless of their solubility characteristics) as for the product from the DMSO~le reaction. Results

Mutagenic potency of haloacroleins and chloroallyl alcohols. 2-Chloroacrolein, cis/trans-2,3-dichloroacrolein, 2,3,3-trichloroacrolein and particularly 2-bromoacrolein are very p o t e n t mutagens (Table 1). The activity of 2-chloroacrolein is reduced by the $9 mix possibly due to oxidation to the acid. Glutathione also reduces the activity, by 43% at 50 nmoles and 92% at 1000 nmoles, suggesting rapid reaction of 2-chloroacrolein with this nucleophile. The chloroallyl alcohols are less than 0.1% as active as the corresponding chloroacroleins. However, the potency of these alcohols is greatly increased with the $9 mix indicating oxidation to the corresponding acrolein. Acrolein and

116 TABLE 1 MUTAGENIC POTENCY OF HALOACROLEINS

A N D C H L O R O A L L Y L A L C O H O L S IN S. typhirnu-

rium S T R A I N T A 1 0 0 Compound

CH2=CHR CH2=C(CI)R CH2=C(Br)R CICHffiC(C1)R CI2CffiC(C1)R

Revertants/nmole a R = CHO

R = CH2OH

0 (0) b 113 (74) 700 104 224

0 (0) 0.03 (0.11) 0.07 (0.29) 0.03 (~1.6)

a Results w i t h $ 9 are given in p a r e n t h e s e s . b A n o t h e z s t u d y indicates acrolein is inactive in T A 9 8 a n d T A 1 0 0 [ 1 6 ] .

allyl alcohol (with and without $9 mix) are not mutagenic but they are highly bactericidal at 2000 nmoles/plate; even at 20 nmoles/plate, acrolein consistently gives less revertant colonies than background. Effect of changing aldehyde substituent on mutagenic activity. 2-Chloroacrolein is more potent than any monochloro compound tested without the aldehyde group (Tables 1 and 2). Reduction to 2-chloroallyl alcohol greatly reduces the potency and oxidation to the acid completely abolishes activity even at 9400 nmoles/plate. 2-Chloroacrylyl chloride is slightly mutagenic and would probably be much more active except for its rapid hydrolysis to the inactive acid. Methyl 2-chloroacrylate shows activity that is difficult to quantitate because this compound polymerizes rapidly. Activity is abolished (even at 5600 nmoles/plate) on replacing the aldehyde by sodium methanesulfonate in mono-, di- and tri-chloroacroleins. Replacing the aldehyde substituent by a cyano or a chloromethyl group greatly reduces but does not destroy activity. Interrelationships of mutagenic activities of halopropanals and haloacroleins. The monohalopropanals are devoid of mutagenic activity, the 2,3-dihalopropanals are very potent mutagens and 2,3,3-trichloropropanal is moderately active (Table 3). 2-Chloropropanal is inactive at levels up to 5920 nmoles/plate TABLE 2 MUTAGENIC POTENCY OF 2-CHLOROACROLEIN ANALOGS WITH REPLACEMENTS FOR THE A L D E H Y D E S U B S T I T U E N T I N S. t y p h i m u r i u m S T R A I N T A 1 0 0 R s u b s t i t u e n t o f CH2=C(CI)R C(O)OH C(O)CI

C(O)OCH3 a CH2SO3Na b CN CH2C1

Revertants/nmole 0 0.02 ~1

0 0.01 0.10 (0.07) c

a M o n o m e r c o n t e n t at the t i m e o f adding to t h e a s s a y plates w a s e s t i m a t e d b y quantitative N M R m o n i t o r ing o f s a m p l e s h e l d u n d e r c o m p a r a b l e c o n d i t i o n s . b T h e 2 , 3 - d i c h l o r o a n d 2 , 3 , 3 o t r l c h l o r o analogs are also inactive. c Results w i t h S9 are given in parenthesis.

117

TABLE 3 MUTAGENIC POTENCY OF HALOPROPANALS Compound

Revertants/nmole

CH 3 CH(CI)CHO CICH 2 CH 2 CHO CICH2CH(CI)CHO BrCH2 CH(Br)CHO C1CH2C(CI 2 ) C H O

0 0 91 a 770 a 3.3

IN S. typhtmurium S T R A I N T A 1 0 0

a T h i s a c t i v i t y is d u e t o e s s e n t i a l l y c o m p l e t e c o n v e ~ d o n t o t h e c o r r e s p o n d i n g 2 - h s l o a c r o l e i n .

and 3-chloropropanai at the non-lethal level of 1400 nmoles/plate; however, 3~hloropropanal undergoes rapid polymerization. The mutagenic activities of the 2,3~lihalopropanals are similar to those of the corresponding 2-haloacroleins (Tables I and 3) due to their rapid dehydrohalogenation to the potent mutagens. 2,2,3-Trichloropropanal is very resistant to dehydrochlorination so its mutagenic activity, in contrast to that of 2,3~lichloropropanal, may be attributable to the parent compound rather than the dehydrochlorination product. Reaction of 2-chloroacrolein and deoxyadenosine. There are 3 types of evidence that the reactions in DMSO~I6 and aqueous medium yield polymeric materials. The products contained chlorine equivalent to 2--3 molecules of chloroacrolein per molecule of deoxyadenosine but they gave no chlorine-containing fragments up to 1500 mass units on field desorption MS. Major linebroadening occurred in the 13C NMR spectrum for resonances in the base region ( ~ 1 2 0 - 1 6 0 ppm) and peak-broadening was prominent throughout the IH NMR spectrum. The solubility properties in acetone, acetone--DMSO and ether were appropriate for a mixture of polymers. Polymer formation results from reactions in the base moiety only. Thus, 13C NMR established that the reaction does not remove or modify the sugar, open the rings or destroy the aromaticity of the base. IH NMR (DMSO~i6) established complete loss of the resonance peaks associated with the NH2 at 7.33 ppm and the H-8 and H-2 protons at 8.20 and 8.39 ppm. The IH N-MR spectrum is further characterized as follows: appearance of 2 new bands in the 8.8 and 5.5 ppm regions, the former appropriate for the signal of a - C H = N-- proton; appearance of water released during the reaction; multiplication of the resonance lines at 6.4 ppm corresponding to the I'-H of deoxyribose. This spectral evidence suggests Schiff-base formation on reaction of th e aldehyde and amino group plus other reactions that probably contribute to polymer formation. Discussion

It was surprising to find that the haioacroleins are highly potent mutagens in the Ames assay with S. typhimurium strain TA100 since only a few other aldehydes s h o w even a low order of activity with this strain or TA98 [16] (Table 4). Malonaldehyde, inactive in TA98 and 100 [16], exhibits low mutagenic activity in other strains. Such active compounds as glyoxal, mono- and di-chlo-

118 TABLE 4 R E L A T I O N OF MUTAGENIC POTENCY A N D R E A C T I V I T Y OF V A R I O U S A L D E H Y D E S Compound

Revertants/nmole [and ref.]

Fused ring [and ref.] Guanosine [25]

OHCCHO

0.2, T A 1 0 0 [2]

OHCCH2CHO

0, T A 1 0 0 / T A 9 8 [16] 0.02, his D 3052 [24] 0.03, T A 1 9 7 5 [24]

C1CH2CHO

0.6, T A 1 0 0 [10,12]

C12CHCHO

0.06, T A 1 0 0 [ I 0 ]

CH 3 CH(CI)CHO

0, T A 1 0 0 (this study)

Adenosine [ 23 ], cytidine [ 23 ], guanosine [17]. 9-methyladenine [9], 1-methylcyto~flne [9]

0CH2(~HCHO

19, T A 1 0 0 [11]

Guanosfine, deoxyguanosine [7]

CH2fC(CI)CHO

113, T A 1 0 0 (this study)

Cross-linking deoxyadenosine (this s t u d y )

roacetaldehyde and glycidylaldehyde have reported values of 0.06--19 revertants/nmole as compared with the value of 113 for 2-chloroacrolein. Several aldehydes considered above give fused-ring compGunds on reaction with purines, pyrimidines or their nucleosides (i.e. glyoxal, chloroacetaidehyde and glycidylaidehyde) (Table 4). 2-Chloropropanal, lacking mutagenic activity with TA100, reacts in DMSO with the guanine amino group but does n o t give a fused ring c o m p o u n d [8]. Certain malonaldehyde derivatives also form fusedring compounds with guanine [13]. Both glyoxal [3] and malonaldehyde [3, 14] cross-link DNA. These reactions have been cited as possible reasons for the mutagenic activity of these compounds. Bromoacrolein is a more p o t e n t mutagen than the chloroacroleins and particularly than acrolein itself. This may be associated with enhanced reactivity since both the aldehyde and double-bond functions of bromoacrolein are more polarized than the corresponding positions of acrolein and the chloroacroleins. 2-Chloroacrolein reacts rapidly with deoxyadenosine to give polymeric material, reflecting its difunctional reaction properties. We envision polymer formation as most likely occurring via 2 distinct types of reactions, i.e. Schiff-base formation at the aldehyde carbon and Michael addition at the ~--7 position. The analogous reaction with DNA and limiting amounts of 2-chloroacrolein would probably lead to cross-linking. The structure--mutagenic activity relationships in the present study for compounds related to 2~hloroacrolein appear to be consistent with the need for 2 reactive groups of distinct types to facilitate cross-linking.

Acknowledgments We thank B.N. Ames for stimulating discussions, generous aid and supplying the tester strain. Technical advice and assistance were provided by I. Schuphan, E.C. Kimmel and L.O. Ruzo. J.E. Hearst and H. Rapaport provided useful comments. The field desorption MS analyses were made by courtesy of A.L. Burlingame as supported by NIH grant RR00719.

119

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