Mutagenicity of chloroalkene epoxides in bacterial systems

Mutation Research, 101 (1982) 115-125 Elsevier Biomedical Press

115

Mutagenicity of chloroalkene epoxides in bacterial systems S t a n l e y A. K l i n e t, E l e n a C. M c C o y 3, H e r b e r t S. R o s e n k r a n z a n d B e n j a m i n L. V a n D u u r e n 2 / t:)lzo Bio¢'hem, Inc., 325 Itu~,(~'on St., New York, N Y 10013: ' l+ahoratol T ~/ Orgam~ ('/icml~trl a~M ('arcinogenesis, htstitute of l'nt~ironnlental Medicine. New York Unit~er.~i(l' Medical ('enter. 550 Ftl:~t 4 t+e.+ New York, N Y 1 (7016: and + Center for Enetronmental llealth Selettcey. Sch¢ud ¢~/ Medicine. ( 'a~'e [lk~ter~ Reverve Unit'ersi(v, Clet~eland, 0 1 t 441(76 (U.S.A.)

(Received 8 May 1981) (Revision received 5 October 19811 (Accepted 21 October 1981)

Summa~ 6 a-chloroepoxides have been tested for in vitro activity in a variety of systems. The epoxides were cis- and trans-l-chloropropene oxide, cis- and trans-l,3dichloropropene oxide, trichloroethylene oxide and tetrachloroethylene oxide. The epoxides were assayed for mutagenicity in the absence of metabolic activation in S. typhimurium TA1535 and E. colt WP2 uvrA and for preferential inhibition of growth of DNA-repair-deficient E. colt. All 6 epoxides possessed DNA-modifying activity as evidenced by their ability to preferentially inhibit DNA polymerase-deficient E. colt. All of the test chemicals except trichloroethylene oxide were mutagenic for S. typhimurium and all except trichloroethylene oxide and tetrachloroethylene oxide were mutagenic for E. colt WP2 uvrA. Cis- and trans-l,3-dichloropropene oxide were the most potent mutagens and DNA modifiers. For all cases, the cis isomers were more active than the corresponding trans isomers, a-Chloroepoxides are considered likely to be the active intermediates of the carcinogenic parent halo-olefins. These mutagenicity studies are considered relevant in assessing the carcinogenicity of the parent hydrocarbons.

O1efinic halogenated hydrocarbons are widely used in agriculture and industry. Since the initial discovery of the carcinogenicity of vinyl chloride (Viola et al., 1971; Maltoni and Lefemine, 1974) there have been numerous studies concerning the carcinogenicity and mode of action of these compounds. It has been suggested (Van Duuren, 1975) that vinyl chloride might be metabolized in the liver via an epoxide intermediate which could mediate its carcinogenic effects. While this unstable intermediate has not been directly detected as a metabolite of vinyl chloride, its presence (or that of its isomeric rearrangement product, chloroacetaldehyde) has been inferred from trapping experiments in the presence of an in vitro rat-liver 0165-1218/82/0000-0000/$02.75 .c:~Elsevier Biomedical Press

116 microsomal system (G/Sthe et al., 1974) and by identification of alkylated RNA adducts in vitro and in vivo (Laib and Bolt, 1977). Vinyl chloride epoxide itself is a direct-acting mutagen for bacteria (Malaveille et al., 1975; Rannug et al., 1976) and yeast (Loprieno et al., 1977); this is in contrast to vinyl chloride which requires conversion by microsomes to exhibit mutagenicity. Vinyl chloride epoxide is carcinogenic in mouse skin (Zajdela et al., 1980). Epoxides have been implicated in the metabolism of other chlorinated olefins (Powell, 1945; Bonse et al., 1975; Uehleke et al., 1977; Banerjee and Van Duuren, 1978). Greim et al. (1975) examined the microsome-mediated mutagenicity of a series of chlorinated ethylenes and concluded that their mutagenicity could be correlated with the reactivity of the postulated epoxide intermediates. We had previously synthesized and characterized epoxides of 6 widely used chloro-olefins (Kline and Van Duuren, 1977; Kline et al., 1978) including cis- and trans-lchloropropene oxide (1 and 2), cis- and trans-l,3-dichloropropene oxide (3 and 4), trichloroethylene oxide (5) and tetrachloroethylene oxide (6) (see Fig. 1). The biological activities of these chloroalkene epoxides, which are potential activated carcinogenic intermediates of their respective parent olefins, were thus of interest. We have examined their mutagenicity in S. typhimurium TA1535 and in E. coli WP2 uvrA and their genotoxicity in E. coli pol A / p o l A + DNA-repair assay in the absence of metabolic activation. The ability of these epoxides to transform Syrian hamster embryo cells in culture (Kline et al., 1980) and their tumorigenicity in I C R / H a Swiss mice have also been studied and will be reported elsewhere (Van Duuren et al. submitted).

Compound

Struclure

Half Life (min.)°

Products

H~--~-~H~o /

11.0

2-chloropropanal

H CI C~/N--------~Hs\o/ H

4.5

2-chloroproponol

CH s

ci._ss-l-Chloropropeneo x i d e Z trans-l-Chloropropene oxide 2

CICH2

cis-l,3-Dichloropropene oxide trans-l,3-Dichloropropeneoxide 4__ Trichloroethylene oxide --5 Tetrachloroethylene oxide __6

Cl

CI

~------~H H H CI C~/%-~HH2~O / Cl CI CI H~N-----~C~O /

2880

2-chloroacrolein

300.0

2-chloroocrolein

1.5

dichloroaceticacid, formic acid, CO

11.5

trichloroaceticacid, CO, COz

I

CI CI XO/~-~-~C I C I

a- In aqueous solution; pH 7.4; 37 o

Fig. I. Structure and stabilityof c~-chloroalkeneoxides.

117 Materials and methods

Chemicals Cis- and trans-l-chloropropene oxide (1 and 2) were synthesized by rnchloroperbenzoic acid (m-CPBA) oxidation of cis- and trans-l-chloropropene as previously described (Kline et al., 1978) except that tetrachloroethylene rather than methylene chloride was used as the reaction solvent. Yields were 70% and the chemicals were stored at - 7 8 ° C as 10% (w/v) solutions in acetone. Cis- and trans-l,3-dichloropropene oxide (3 and 4) were synthesized from the corresponding alkenes by m-CPBA oxidation as previously described (Kline et al., 1978) except that reactions were carried out in refluxing methylene chloride for 40 h. The dichlorobenzene impurity present in the original preparations (Kline et al., 1978) was not present under these conditions. Yields were - 3 0 % . Trichloroethylene oxide and tetrachloroe'~hylene oxide (5 and 6) were synthesized by the autooxidation of tri- and tetrachloroethylene as previously described (Kline and Van Duuren, 1977; Kline et al., 1978) except that the dichloroacetyl chloride and trichloroacetyl chloride byproducts were removed by extraction with 0.5 N sodium hydroxide.

Aqueous decomposition of trans-l,3-dichloropropene oxide 4 2/zl 4 (0.017 mmole) was dissolved in 8.5 ml 0.2 M potassium phosphate, pH 7.4 and 1.5 ml acetone. The mixture was stirred in the dark for 30 h (6 half-lives) and tested in the subsequent mutagenicity assay.

Bacterial mutagenesis assays The tester strains were grown overnight in trypticase soy broth (BBL). l0 /~l dilutions of the test chemical in acetone were assayed using the preincubation procedure of Yahagi et al. (1975). A preincubation assay (Yahagi et al., 1975) rather than the standard plate-incorporation assay (Ames et al., 1975) was used due to the volatility and hydrolytic lability of the chemicals (McCoy et al., 1978).

Mutagenicity of 4 and aqueous decomposition product of 4 in TA1535 by liquid assay In order to ascertain that the mutagenicity of 3 and 4 for Salmonella was due to the respective epoxides rather than their common aqueous decomposition product, 2-chloroacrolein (Kline et al., 1978), trans-l,3-dichloropropene oxide was tested concurrently with a decomposed sample using a liquid assay. In this assay, chemicals were incubated with bacteria in suspension for 90 min (less that 1/3 half-life of 4) and the compounds were removed immediately following exposure. Specifically, overnight cultures of S. typhimurium TA1535 in trypticase soy broth were grown to the exponential growth phase, washed with PBS and resuspended to a cell density of 3 × 10 9 bacteria/ml. Portions of this bacterial suspension (0.5 ml) were added to 2.5 ml of Davis-Mingiuli minimal medium (Davis and Mingiuli, 1950) together with 100-/~1 dilutions of chemical in either acetone in the case of 4 or phosphate buffer, pH 7.4, in the case of decomposed 4. Suspensions were incubated for 90 min at 37°C whereupon 1-ml portions were centrifuged and washed. These were then assayed for survivors and for revertants to histidine independence.

TABLE l M U T A G E N I C I T Y O F a - C H L O R O E P O X I D E S IN S. l.vphimurium T A I 5 3 5 A N D E. colt WP2 u~rA IN THE ABSENCE OF METABOLIC ACTIVATION Compound

None Acetone AF-2 ~ NaN 3 I

2

3

4

5

6

Concentration (raM) b

R e v e r t a n t colonies T A _._~/p ~te c 4 13

(/.()()l 2 0.15 I1 5.5 2.25 1.13 0.55 55 27.5 11 5.5 2.25 1.13 0.55 5 2.5 0.5 0.25 0.05 0.O25 0.013 5 2.5 0.5 0.25 O.05 O.025 0.013 5 2.5 1.3 0.5 0.25 25 5 2.5 1.3 (1.5

R c v c r t a n t colonies W P ~_ u',rA, plate ~ I l) 15 I O3 I

656 19 (3) 124(17) 304 (34) 208 (31) 118 I l l

149 (l) 95 121h 42 (7) 16 (3) 538 (135) 14~ 163)

17 51 28 23 16

13) 16) 10) (3) (4)

52 ?2

(g) (4)

9

(I)

(I 720 16) 472 (31) 121 (17) 76 (I0) 40 (7)

47 85 56 14

(3) (4) (6) (I)

() 350 24O 67 39 17

(37) (42) (7) (I) (3)

0 0 ,~ (6) 11 (3) 0 78 54

125 fl7) 62 (7) 36 (6) 13 (3)

() 13 (7) 13 i l l ) 16 (3~ II 11 ()

(4) (4)

II II X II

11 II It 1)

Bacteria were grown overnight in nutrient media. Test chemical was added to aliquots c o n t a i n i n g a p p r o x i m a t e l y 1() ~ bacteria and these were i n c u b a t e d in screw cap vials for 20 min at _ 7 ( . After a d d i t i o n of soft agar, the m i x t u r e was poured o n t o either m i n i m a l histidine ( T A I 5 3 5 ) or mininlal tr?eptophane ( W P 2 uvrA) plates and i n c u b a t e d in the d a r k at 37°C for 2 days after which cohmies ,acre counted. a 2_(2_Furyl)_3_(5_nitro_2_ f u w 1 ) a e w l a m i d e b , a m o l e / m l of bacterial suspension. • M e a n average of 3 plates ( s t a n d a r d deviation).

119

E. coli pol A 1 assay This assay was performed as previously described (Hyman et al., 1980). 100-txl portions of bacterial cultures containing approximately 1500-2000 bacteria/ml received 10-/~1 aliquots of the test chemicals diluted in acetone and, following a 90-min incubation at 37°C in screw cap tubes, soft agar was added and the mixture poured onto agar plates which upon solidification of the soft agar were incubated at 37°C for 2 days and survivors counted.

Results

Mutagenicity in S. typhimurium TA1535 and E. coil WP2 uvrA Compounds 1 6 were tested for mutagenicity in the absence of metabolic activation. Results are summarized in Table 1. Compounds generally exhibited toxicity, as evidenced by a decrease in the spontaneous frequency of the revertants a n d / o r by an inhibition of the growth of the bacterial lawn, at the higher doses. However, at lower levels, concentration-dependent mutagenic responses in Salmonella TA 1535 were obtained for all chemicals except for trichloroethylene oxide, 5. Similar results were obtained with E. coli WP2 uvrA except that neither 5 nor tetrachloroethylene oxide 6 were mutagenic. Cis- and trans-l,3-dichloropropene oxide, 3 and 4, showed the highest specific mutagenicities. Mutagenicity in Salmonella of 4 vs. hydrolyzed 4 trans-l,3-Dichloropropene oxide, 4, was assayed before appreciable aqueous decomposition could occur. In Table 2, the results are compared with those from a sample of 4 which had been allowed to decompose in aqueous solution. It was thus shown that both trans-l,3-dichloropropene oxide and its aqueous decomposition product exhibited a dose-dependent mutagenicity. The unhydrolyzed oxide, however, was 2-3 times more potent at a given dose than the hydrolyzed compound. Genotoxicity Genotoxicity was assayed by the differential growth inhibition of a DNA polymerase-deficient E. coli mutant, pol A~, as compared to its polymerase-proficient pol A~ parent (Slater et al., 1971). This assay magnifies, in effect, a chemical's DNA-modifying ability. Results are expressed as a survival index, i.e.: % survival pol A ~ / % survival pol A +. Values below 0.85 are taken to indicate preferential inhibition of pol A (Hyman et al., 1980). All test chemicals preferentially inhibited growth of pol A~ (Table 3). Compounds 3 and 4 were again significantly more active per/~mole than the other compounds. Results of these assays are summarized in Table 4. Cis- and trans-1-chloropropene oxide (1 and 2) and cis- and trans-l,3-dichloropropene oxide (3 and 4) were mutagenic for S. typhirnurium TA1535 and for E. coli WP2 uvrA and differentially inhibited the DNA E. coli repair-deficient strain. Trichloroethylene oxide 5 was not mutagenic for Salmonella and E. coli WP2 uvrA, but exhibited DNA-modifying

120 TABLE 2 MUTAGENICITY OF HYDROLYZED vs, U N H Y D R O L Y Z E D trans-I,3-DICHLOROPROPENE O X I D E IN S. (vphimurtum T A I 5 3 5 IN T H E A B S E N C E O F M E T A B O L I C A C T I V A T I O N Compound

Noi1c Acetone NaN+

Concentration (raM)

Revertant colonies / pl a t e ~'

Survivors > 10 ')~

Rex crtant.,,/1() ~ sur', ivors

3.0 2.8 3.2

7 15 188

20/*g/nil

20 42 600

ram.s-1,3-Diehloropropene oxide hydrolyzed 6

0.5 0.05 0.025 0.0025

3N0 87 27 33

(10) (6) (6) (11)

2.g (0.3) 3.3 ((I.3) 1.8(0.1) 3.6 (0.2)

132 24 15 15

1,3-I)ichloropropene oxide unhydrolyzed

0.5

780 (60)

1.8 (0.2)

433

0.05 0.025 0.0025

143 (35) 127 (45) 40 (22)

2.9 ((t.2) 2.7 (0.2) 3.0 (0.3)

49 47 [3

tram'-

Bacteria were grown overnight in nutrient media. Test chemicals ,acre added to aliquots containin~ a p p r o x i m a t e l y 1 . 5 > l0 '~ bacteria and incubated 90 min at 3 7 0 ( ". Bacteria ',',ere centrifuged, v, ashed. rcpelleted and resuspended in PBS. Aliquots were ~,erially diluted v, ith PBS and, after addition of soft agar, plated on either m i n i m a l histidine to detcrminc revertant colonic.,, or complete media to determine surxivors. Plates were incubated in thc dark at 370( ` for 2 days after which colonies were c o u n t e d " Average of 3 plates (standard dcviation). b 0.2 M potassium phosphate: pit 7.4.

activity as evidenced by its ability to preferentially inhibit E. coli pol A 1 . Tetrachloroethylene oxide 6, while non-mutagenic for E. coli WP2 uvrA, was mutagenic for Salmonella, and positive in the E. coli pol A I assay. Compounds 3 and 4 exhibited highest potencies in all the systems studied.

Discussion The biological potency of direct-acting alkylating agents such as ~-chloroepoxides are expected to be related to 4 factors: (a) chemical reactivity; (b) stability; (c) ability to interact with critical target molecules; and (d) nature of the lesion (i.e. ability to be repaired or mispaired, ability to induce mispairing, frameshift mutations, etc.). Compounds 1 - 6 are labile under physiological conditions (Fig. 1) and their decomposition products must. be considered in a discussion of biological activity. Chloropropanal, the decomposition product of 1 and 2 was inactive for S. typhirnurium (Rosen et al., 1980). 2-Chloroacrolein, the common aqueous decomposition product of 3 and 4 was recently shown to be a potent direct mutagen for Salmonella TA100 (Rosen et al., 1980). In order to ascertain that 4 was itself active, the mutagenicities of trans-l,3-dichloropropene oxide, and decomposed trans-l,3-

0.11 0.08 0.06 0.01 0.006

0.44 0.09 0.04

$

6

182 178 179

104 163 178 186 170

4 22 132 134

66

106 103 104

61 95 103 108 99

2 12 77 78

0 18 52 65

103 99

22 56 56

29 44 68 69 74

0 0 6 23

o 0 0 2

38 74

0

29 72

I

84 6

Colonies/plate ~ Pol A i strain

26 66 66

34 52 80 82 88

0 0 7 27

0 0 0 I

45 88

0

20 85

I

100 5

Survival pol A~ (% of control)

0.25 0.64 0.64

0.56 0.55 0.78 0.76 0.89

0 0 0.09 0.35

0 0 0 0.02

0.44 0.89

0

0.01 0.40 0.94

1.00 0.07

Survival index h)

100-/H aliqouts of overnight cultures of E. coil pol A~- or pol A [ strains (containing 150-200 bacteria) were incubated with the test chemical in screw cap vials for 20 rain at 37°C. Soft agar was added and the mixtures were poured onto nutrient agar plates. Surviving colonies were counted after 2 days incubation in the dark. a Average of 2 plates. b % Survival pol A ] - / % survival pol A~. c Ethyl methanesulfonate.

0.05 0.005 0.001 0.0005

4

0 32 89 I 12

178 171

0.05 0.005 0.001 0.0005

114

I. I

0.54 0.11

3

2

59 73 90

102 126 156

1.1

0.54 0.1 I

I

Survival pol A ~ (% of control) 100 84

Colonies/plate" pol A{ strain 172 145

Concentration (/~M/ml)

Acetone EMS ~

Compound

GENOTOXICITY OF a-CHLOROEPOXIDES IN E. coli (pol A I / p o l A~ ) IN THE ABSENCE OF METABOLIC ACTIVATION

TABLE 3

122 TABI,E

4

SUMMARY

OF BIOLOGICAL

('Olllpottrld ~half-lifc, rain)

I

ACTIVITIES

.~'. (l'p]llmtlrzt#~l TA1535

OF a-CHLOROEPOXIDES 1:'. col* WP2 uxrA

Di f f c r c n i i a l toxicit', to I:'.
(ll)

2 (4.3) 3 (2,'48) 4 (300) 5 (I.5) 6 Ill5

t ! ÷ # ~ +

, ; . .

. t .

. t

dichloropropene oxide were compared in a suspension system. Exposure time of the bacteria was 90 rain (less 1/3 half-life of 4). It was thus found (Table 2) that 4 was, in fact, 2 3 times more mutagenic than its decomposition product which is also a direct-acting mutagen. Compounds 3 and 4 were the most active in all the assays indicating that stability rather than reactivity was the predominant factor. Whereas the half-lives of 3 and 4 are 30 times greater at physiological temperature and pH than the next most stable compound in the series (Fig. 1), alkylating activity, [as measured by rate of reaction with p-nitrobenzylpyridine (data not shown)], was lowest in these and greatest in 5. Only 3 and 4 would survive the 20-30-rain incubation period of assays without appreciable decreases in effective concentration. These results must be contrasted with those obtained with more stable epoxides. In those a direct correlation between alkylating reactivity and mutagenicity was observed (Hemminki and Falck, 1979; Sugiura et al., 1978). The lack of activity of 5 in S. typhimurium and E. coli WP2 uvrA is of interest. To be sure, this compound was the least stable in aqueous solution of all the epoxides tested. On the other hand, vinyl chloride epoxide, which has a half-life in aqueous solution almost identical to that of 5 (Barbin et al., 1975), was mutagenic for S. typhimurium TA1535 under conditions similar to those described herein (Malaveille et al., 1975). That 5 was positive in the E. coli pol A assay, more so even than 1 and 2, indicates that it can react with the bacterial genome. In this DNA-repair assay any potentially lethal but repairable lesion in DNA, regardless of type or genomic location will be detected. The inactivity of 5 in Salmonella TA1535 and in E. co//WP2 uvrA may reflect a preference for reaction at a site other than the critical target molecule, presumably a guanine (McCann and Ames, 1977). Alternatively, the lack of activity of 5 compared to the other epoxides may reflect differences in the chemical structure of the adducts. 5 reacts with model nucleophiles to yield substituted chloroacetic acid adducts (Kline and Van Duuren, 1977). Vinyl chloride epoxide alkylates adenine and cytosine forming the imidazole derivatives 1,N6-ethenoadenine and 3, Na-ethenocytosine (Secrist et ai., 1972). 1 and 2 react with guanine at the 2-amino position to form the substituted chloropropene,

123 l-(2-N-guaninyl)-2-chloropropene (Goldschmidt et al., 1979). A comparison of the activities of the cis compounds 1 and 3 with their respective trans isomers 2 and 4 reveals that in each assay the cis isomer was the more active. The potency of 1 relative to 2 may, in part, be simply ascribed to the stability of the cis isomer. This explanation is insufficient, however, for 3 and 4 where the trans isomer (4) is slightly more stable. Rather, one may hypothesize that the steric interactions between the CH2CI and C1 moieties in the cis compound 3, which are relieved by epoxide opening, would render the cis compound more prone to nucleophilic attack. Parent halo-olefins of the mutagenic epoxides 1-4 were themselves mutagenic in Salmonella (Neudecker et al., 1977; Rosen et al., 1980). 1-Chloropropene (isomer not specified) required metabolic activation to express activity, whereas, cis- and trans-l,3-dichloropropene did not. The direct activity of the latter compounds has been ascribed to the allyl chloride moiety (Neudecker et al., 1980). The potency of 3 and 4, on the other hand (about 20 times more active per ~mole in Salmonella TA1535 than 1,3-dichloropropene), indicates that these may contribute to biological activity in vivo. Trichloroethylene was marginally active in Salmonella TA100 and tetrachloroethylene was inactive (Bartsch et al., 1979). No c~-chloroepoxides have been detected directly as metabolic intermediates of their parent olefins, undoubtedly due to their reactivity. However, there is indirect evidence to suggest their transient presence. These include trapping experiments (GOthe et al., 1974) and identification of alkylated RNA adducts (Laib and Bolt, 1977) in the case of vinyl chloride and in vitro DNA-binding experiments (Banerjee and Van Duuren, 1978) as well as spectral evidence (Uehleke et al., 1977) in the case of trichloroethylene. Trichloroethylene oxide intermediacy may also be inferred from the traces of dichloroacetic acid present in the urine of mice fed trichloroethylene (Hathway, 1980) since 5 decomposes predominantly to dichloroacetic acid. The major urinary metabolites of trichloroethylene in dogs, rats and man, however, are derived from trichloroacetaldehyde (Waters et al., 1977, and references therein). It has been suggested (Henschler et al., 1979) that 5 is formed in the environment of the cytochrome P-450 monooxygenase a n d i t s subsequent decomposition to trichloroacetaldehyde is immediately catalyzed by the trivalent iron in the heme moiety of this enzyme. 5 does, in fact, decompose to trichloroacetaldehyde in the presence of Lewis acids such as FeCI 3 (Henschler, 1977). In conclusion, we have demonstrated that a-chloroepoxides of biologically active halo-olefins are themselves biologically active in the absence of metabolic activation. Mutagenic activity in S. typhimurium and E. coli WP2 uvrA was to a large part dependent on the stability of the epoxides in aqueous solutions. The cis isomers were in both cases more active than the corresponding trans compounds. These compounds should, therefore, be considered likely candidates for reactive metabolic intermediates of their parent olefins.

124

Acknowledgements This work was supported by U.S.P.H.S. grants ES-01150, CA-13343 and ES-00260 (B.L.V.D. and S.A.K.) and U.S.P.H.S. contract 1-CP-65855 (H.S.R. and E.C.M.).

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