D.R WADE, S C AIRY and J.E. SINSHEIMER *. College of Pharmacy, Unzverszty of Mzchzgan, Ann Arbor, MI 48109 (U S.A ). (Received 18 Apml 1978).
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Mutatton Research, 58 (1978) 217--223 © Elsevmr/North-Holland Blomedmal Press
MUTAGENICITY OF ALIPHATIC EPOXIDES
D.R WADE, S C AIRY and J.E. SINSHEIMER *
College of Pharmacy, Unzverszty of Mzchzgan, Ann Arbor, MI 48109 (U S.A ) (Received 18 Apml 1978) (Revision received 11 July 1978) (Accepted 20 July 1978)
Summary The mutagenicity of 17 ahphatic epoxides was determined using the specially constructed mutants of Salmonella typhimurium developed by Ames. The activity of these epoxides together with those reported in the literature as mutagens in strains T A 1 0 0 and TA1535 depended on the degree of substitution around the oxirane ring. Monosubstituted oxiranes were the most p o t e n t mutagens in both strains. 1,1-Disubstltution resulted m the complete loss or reduction of mutagenicity, trans-l,2-Disubstituted, and tetrasubstituted oxiranes all lacked mutagenicity, while the cis-l,2-disubstituted oxiranes tested were weakly mutagenic in strain TA100 only. For the monosubstituted compounds the ,rresence of electron-withdrawing substituents increased mutagenicity.
Introduction
Parallel to the biotransformation of arene oxides from aromatic compounds [5], it is assumed that ahphatic epoxides (alkene oxides} are active intermediates in the metabolism of alkene compounds [15]. The concept that arene oxides, through their binding to biopolymers such as DNA, R N A and protein, are responsible for the toxic, carcmogenic, and mutagemc effects of aromatic c o m p o u n d s is well estabhshed [5,9]. However, as pointed o u t by Oesch [13], it does n o t follow that the adverse properties of some arene oxides can be extrapolated to epoxides in general. This is the case m the Ames test for mutagenicity of alkene oxides. Thus, it has been reported that 2,3-epoxypropionaldehyde, 2,3-epoxypropanol, styrene oxide, 1,2-epoxybutane, 1,2,3,4-dmpoxybutane, and 1,2,7,8-dmpoxyoctane [11] as well as chloroethylene oxide [10] are mutagenic. While c~s- and trans-4* To whom correspondence should be
addressed
218 acetylaminostilbene~fl-oxide [6] as well as carbamazeplne-10,11-oxlde, cyporheptadine-10,11-oxide, and cyclobenzaprine-10,11-oxide are not mutagenic [7]. It is the purpose of this investigation to extend mutagemclty testing of alkene oxides m the Ames assay so as to better predict the toxicity of such c o m p o u n d s per se and as an mitml step m evaluating their possible role m the toxicity of alkene compounds subsequent to blotransformatmn. Materials and methods The followmg epoxldes were obtmned from the sources indicated in the purest grade available and their structures were confirmed by n.m.r, spectrometry. 3,3,3-Trichloropropylene oxide (purity 98%), 1-bromo-2,3~epoxypropane (b.p. 134--136°C), 1-chloro-2,3-epoxypropane (purity 99+%), 1,2-epoxy3-fluoropropane (purity 98%), propylene oxide (purity 99%), styrene oxide ( p u n t y 97%), trans-stllbene oxide, cyclohexene oxide (purity 98%), and glycldol (b.p. 160--161 °C) were obtained from Aldrich Chemmal Co. 2,3-Epoxybutane, a-methylstyrene oxide and 1,2-epoxyisobutane were obtained from ICN Life Sciences Group. a-Phenylstyrene oxide (m.p. 55--57°C; Ref. [4], 5 4 - - 5 6 ° C ) a n d 2-methyl3,3,3-trichloropropylene oxide (b.p. 57°C/0.6 mm) were synthesized from benzophenone and 1,1,1-trichloroacetone by the general procedure described by Corey and Chaykovsky [4]. cis-Stilbene oxide was prepared b y dehydrohalogenation of threo-2-chloro1,2-dlphenylethanol with 1.0 N NaOH. The threo isomer was synthesized by the procedure described by Bert1 et al. [3]. trans-Dmthylstilbestrol (DES) oxide (m.p. 142--143°; Ref. [16], 145°C) was prepared from trans-DES by reaction with peracetic acid in a modified procedure of Wessely et al. [16] in which the oxide was isolated only after the removal of acids to prevent the plnacolone rearrangement noted m the original procedure. czs-DES oxide (m.p. 132--133 °) was prepared by peracetm acid oxidation of cls-DES m dlmethylsulfoxlde. The cts-DES was obtained by lsomenzatlon of the trans-lsomer m dunethylsulfoxide by the procedure of White and Ludwig [17]. Mutagenesls assays were performed as described by Ames [2]. Bacteria used in the assay we,'e hlstldme-dependent mutants of Salmonella typhtmurzum, strains TA1537, TA1535, TA100 and TA98 supplied by Professor B.N. Ames, Berkeley, CA. The cells were grown overnight m nutrient broth (Dffco) at 37°C. Test compounds dissolved m 0.1--0.5 ml dlmethylsulfoxlde were added to 2 ml of top agar [1] along with 0.1 ml of the bacterial suspensmn (2--4 × 108 cells) and plated m duphcate on petri dishes with semi-enriched agar (Vogel and Bonner minimal medium E) as described [2]. Control plates were treated in the same manner, b u t received only dlmethylsulfoxide m place of the test c o m p o u n d . The revertant colonies were counted after incubation for 2 days at 37°C and a dose--response curve established for all positive compounds. Dose-response curves were confirmed by repeated determlnatmns. Benzo[a]pyrene, /3-naphthylamme and methyl methanesulfonate were rou-
219
TABLE 1 M U T A G E N I C I T Y A N D S T R U C T U R E OF A L I P H A T I C EPOXIDES Monosubshtuted No
Structure
Mutagemclty a
Ref
TA100
TA1535
1
C13C
/1
2355/500
131/500
A b
2
E~rCH2
//dO
1502/500
456•500
A
3
C1CH2
//10
674•500
402/500
A
4
FCFI2
,~10
766/500
265/500
A
897/500
34/500
A
1120/200
622/200
6
HOCH2 ,,,//'10 CH3
7
//dO
166/1500
8
HC
5260•20
9
CI
NR d
C~H5 - - ~ 0
10
11
0~C4H8
,~
22/1000
+ c
61/0 4 e
A/ll
A
11
10
333/4200
+
11
690/900
+
11
NR
71/50
11
1,1-Dlsubshtuted No
Structure
Mutagenlmty TA100
13
C13C~ ( ~
560/500
Ref. TA1535
nm-1000 f
A
14
~ 0
nm-5000
nm-5000
A
15
CH3 -~-~0
nm-5000
nm-5000
A
nm-2000
nm-2000
A
1o
220
TABLE 1 (continued) 1,2-Dlsubshtuted No.
Structure
Mutagenlclty TA100
%
17
79/100 g
Ref TA1535
nm-100
A
18
100/1000
nm-1500
A
19
nm-100
nm-100
A
rim-500
nm-5000
A
nm-100
nm-100
6
nm-100
nm-100
6
23
NR
nm-500
7
24
NR
rim-100
7
25
NR
nm-500
7
H3C.,-'~CH3
20
21
AcFIN-~
22 AcHN
CNCH2CH2N(H3) C2 Tetrasubstztuted No
26
Stmc~re
Ref
Mutagemclty
H O ~
27
OH
ON
TA100
TA1535
rim-100
nm-100
A h
nm-100
nm-lO0
A h
221 tinely included as positive controls. Benzo[a]pyrene and ~-naphthylamine required the addition of a rat-liver homogenate and a NADPH-generatlng system prepared as described b y Ames [2]. Bactenal survival was determined b y plating bacterial dilutions on nutrient agar (Difco) with the test compound. With the exception of czs-stilbene oxide no reduction of bacterial growth was observed in the linear portion of the dose--response curve. Results
Table 1 summarizes the mutagenlclty results m strains TA1535 and TA100 for c o m p o u n d s investigated m the present study and those previously reported m the literature. All the compounds in our study, with the exception of 2-methyl-3,3,3-trichloropropylene oxide (13), cis-stilbene oxide (17) and cyclohexene oxide (18) that were mutagenic in strain TA100 were also mutagenic, b u t - w i t h reduced sensitivity, m the second strmn, TA1535. Both of these strains detect base-pair mutagens. However, none of the epoxides in the present study were found to be mutagenic in strains TA1537 and TA98 which detect frame-shift mutagens. All the monosubstituted epoxldes tested in the present study were mutagenic (1--7). Other monosubstituted epoxides previously reported to be mutagenic by the Ames assay include c o m p o u n d s 8--12. The only disubstituted epoxldes found to be mutagenlc were 2-methyl-3,3,3-trichloropropylene oxide (13), cts-stilbene oxide (17) and cyclohexene oxide (18). Mutagenicity for these three c o m p o u n d s was restricted to strain TA100. The other 1,1~lisubstlt u t e d and trans-l,2-dlsubstituted epoxides tested (14--16, 19, 20) as well as reported (22) were found to be nonmutagenic. Neither of the tetrasubstituted epoxides (26, 27) were found to be mutagenlc. In the monosubstituted series, those c o m p o u n d s with electron-withdrawing groups on the oxirane ring proved to be the more p o t e n t mutagens. Discussion
The results indicate that the monosubstituted epoxldes are the most potent mutagens and that even the addition of a single methyl group to the oxirane
a M u t a g e m c l t y 1S r e p o r t e d a s r e v e r t a n t s m i n u s s p o n t a n e o u s r e v e r t a n t s p e r m i c r o g r a m s o f c o m p o u n d u n l e s s otherwise stated The mmrogram quantity is taken from the hnear portion of the dose--response curve A v e r a g e s p o n t a n e o u s r e v e r t a n t s w e r e 1 8 0 f o r staLn T A 1 0 0 , 2 0 f o r T A 1 5 3 5 , 3 9 f o r T A 9 8 a n d 7 f o r TA1537 Pomtlve controls used included 5 pg benzo[a]pyrene (131 revertants/TA1537 and 425 revertants/TA98), 0 2 ill m e t h y l m e t h a n e s u l f o n a t e (2150 revertants/TA100) and 100 pg ~-naphthylamme (500 revertants/TA1535). Benzo[a]pyrene and ~-naphthylarnlne required, for mutagenlc actlwty, the adchtlon of a rat-hvcr homogenate and a NADPH-generatmg s y s t e m p r e p a r e d a s d e s c r i b e d m R e f 2. b A = this study c R e p o r t e d as m u t a g e n i c m R e f 1 1 , b u t n o a c c o m p a n y i n g d a t a d Not reported. e R e p o r t e d i n R e f 1 0 as 6 1 r e v e r t a n t s p e r p l a t e a t a d o s e l e v e l o f 0 4 p m o l e s / m l o f s o f t a g a r l a y e r f Nonmutagenic at the highest dose tested in/~g g Results are expressed in terms of the highest concentration with minimal (18%) toxicity h Dlethylshlbestrol-~fl-oxlde of unspecified stereochemlstry has been reported as being nonmutagemc in Ref 12
222 ring can reduce or eliminate mutagenicity. A reduction in mutagenicity in strain T A 1 0 0 is demonstrated in the comparison of c o m p o u n d 13 to the strong mutagen 3,3,3-trichloropropylene oxide (1). The ehmination of mutagemcity with the addition of a methyl substituent is demonstrated in both strains with a comparison of compounds 15 and 20 to propylene oxide (7) as well as in the comparison of 14 to styrene oxide (5). For the monosubstituted compounds, mutagenicity appears to be related to the electrophilicity of the epoxide. Electron-withdrawing groups on the epoxlde would create a stronger electrophile with increased reactivity to blonucleo. phfles such as DNA. The electron-withdrawing groups present m compounds 1 through 6 as well as 8 and 9 do result in an increase in mutagemcity for these c o m p o u n d s compared to propylene oxide (7) in both TA100 and TA1535. C o m p o u n d 10 in comparison to 7 can be considered to have an electron-donating group present and on a per microgram basis has decreased mutagemclty. However, the strength of the elctron-wlthdrawing group alone cannot always be correlated to the extent of mutagemcity. For example, while trmhloropropylene oxide (1) is more mutagenic than the monochloro c o m p o u n d 3 in strain TA100, the reverse is true in TA1535. It is of interest that the only 1,2-disubstltuted epoxldes that were found to be mutagenm were c~s-stflbene omde (17) and cyclohexene oxide (18) with mutagenicity m both cases being restricted to strata TA100. The number of revertant colonies for both c o m p o u n d s was low and for czs-stllbene oxide (17) the results are further comphcated by the inablhty to establish a meaningful dose-response range due to cell toxmlty. Compounds 21, 23--25 have a similar cis arrangement of substituents on the oxirane ring as do c o m p o u n d s 17 and 18 yet have been reported as being nonmutagemc m the Ames assay [6,7]. More c o m p o u n d s of the c l s stereochemlstry must be tested to better define the mu: tageniclty of these dlsubstituted compounds. The structure--activity relationships indicated in our results are similar in several respects to those reported by Oesch [14] in his study of the specificity of epoxldes as substrates and inhibitors of epoxide hydratase. In both ours and Oesch's studies, reactivity was most pronounced for the monosubstltuted, oxides, and for the 1,2-dlsubstituted compounds only the czs but not the t r a n s c o m p o u n d s were active. In both studies, tetrasubstituted compounds were inactive. The two studies differed, however, in that there was a lipophilic substituen~ effect for compounds most effective m the hydratase study that is n o t evident in the mutagemcity response. Also, 1,1-dlsubstituted c o m p o u n d s are active in the hydratase investigation but, except for c o m p o u n d 13 in strain TA100, are inactive as mutagens. Some of the same alkene oxides and related compounds have been tested as substrates for glutathione-S-epoxlde transferase [8]. The relationships noted for their substrate--transferase activity do n o t always parallel the structure-mutagenicity results. For example n o t all the monosubstltuted epoxides, including the relatively strong mutagenic compounds 6 and 8, were substrates for glutathione transferase, while also m contrast to the mutagenicity series, most of the multisubstituted compounds tested were active substrates for the transferase.
223
Thus, it is of interest that structure--activity for neither of the two principal epoxide detoxlfication mechamsms completely parallel toxicity as measured by the Ames mutagenicity test. A knowledge of all three systems is required to make predictions concerning the in vivo toxicity of a given alkene oxide. Such informatmn would be Important for the proper handling as well as for the design of useful alkene oxides and their parent alkene compounds of reduced toxicity. Acknowledgements The authors wish to express appreciation to Professor B.N. Ames, Berkeley, CA for supplying the Salmonella mutants. We are grateful to Kathleen Dnnan for technical assistance. This work was supported in part by Grant GM 19815 from the National Institutes of Health, Bethesda, MD. 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