The mutagenic activity of ethyl N-hydroxycarbamate and its related compounds in Salmonella typhimurium

145

Mutation Research, 78 (1980) 145--150 © Elsevier/North-HollandBiomedicalPress

THE MUTAGENIC ACTIVITY OF ETHYL N-HYDROXYCARBAMATE AND ITS RELATED COMPOUNDS IN Salmonella typbimurium

HIDEO KOGA, YUICHI KAWAZOE,KIYOSHI TATSUMIand TADAO HORIUCHI Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812 (Japan)

(Received 14 September 1979) (Revision received 18 December 1979) (Accepted 2 January 1980)

Summary Alkyl N-hydroxycarbamates exhibited weak but significant mutagenic activity for Salmonella typhimurium TA100. The mutagenic potencies of these N-hydroxycarbamates were ranked thus: ethyl N-hydroxycarbamate ~ propyl N-hydroxycarbamate ~ methyl N-hydroxycarbamate. Acylation of ethyl N-hydroxycarbamate markedly enhanced its mutagenic activity for TA100. The highest mutagenic activity was observed with ethyl N-benzoyloxycarbamate among these acyl derivatives. Almost all the compounds were mutagenic to all the strains TA1535, TA100, TA1538 and TA98, especially to TA100.

Ethyl N-hydroxycarbamate is nearly as active a carcinogen to mice as ethyl carbamate (Berenblum et al., 1959; Miller et al., 1960). The compound also resembles ethyl carbamate in causing chromosome damage to rat tissue in vivo (Boyland and Nery, 1965; Philips et al., 1964), but is m u c h the more active compound as a chromosome-damaging agent for root tips of Vicia faba (Boyland et al.,1963), tissue cultures of rodent cells(Borenfreund et al.,1964) and transforming D N A of Bacillus subtilis (Freese, 1965). Furthermore, the acyl derivatives of ethyl N-hydroxycarbamate are chemically and biologicallyinteresting compounds, because the acyl~derivativesof aromatic hydroxylamines are "ultimate" carcinogens which react readily with guanyl moieties in D N A (Miller and Miller,1966). To date, the A m e s system with Salmonella typhimurium series has demonstrated, for a variety of chemicals, a strong correlation between mutagenicity and carcinogenicity (McCann et al., 1975). However, there is virtually no information available about the mutation test of ethyl N-hydroxycarbamate and itsrelated compound in this system~ In the present work, we have used 4 strains of Salmonella typhimurium to evaluate the mutagenic properties of alkyl N-hydroxycarbamates and acyl

146 derivatives of ethyl N-hydroxycarbamate. The study has provided evidence that all the compounds tested, unlike the corresponding alkyl carbamates, were mutagenic in the above tester strains. Materials and methods Chemicals. Methyl N-hydroxycarbamate (m.p. 51°), ethyl N-hydroxycarbamate (b.p. 86--88 ° at 0.6 mm Hg), propyl N-hydroxycarbamate (b.p. 90-92 ° at 0.6 mm Hg), ethyl N-acetoxycarbamate (b.p. 73 ° at 3 mm Hg) and ethyl N-acetoxy-N-acetylcarbamate (b.p. 86 ° at 2 mm Hg) were prepared by the method of Boyland and Nery (1964). Ethyl N-benzoyloxycarbamate (b.p. 129 ° at 5 m m Hg) and ethyl N-benzoyloxy-N-benzoylcarbamate (m.p. 73--75 °) were prepared by the method of Jones and Oesper (1914). Ethyl N-benzoy!oxy-Nacetylcarbamate (b.p. 111 ° at 2 mm Hg) was synthesized by acetylation of ethyl N-benzoyloxycarbamate with acetic anhydride in pyridine. The data of elementary analysis a r e as follows. Found: C, 57.14; H, 5.11; N, 5.58%. C12H13OsN requires C, 57.37; H, 5.22; N, 5.58%. Ethyl N-acetoxy-N-benzoylcarbamate (m.p. 65.5--66.5 °) was synthesized by benzoylation of ethyl N-acetoxycarbamate with benzoylchloride in pyridine. The data of elementary analysis are as follows. Found: C, 57.22; H, 5.19; N, 5.61%. C12H,3OsN requires C, 57.37; H, 5.22; N, 5.58%. The purity of the chemicals was checked by thin-layer chromatography. When trace amounts of impurities were noted, the compounds were purified by recrystaUization or by distillation. Growth media. TA1535, TA1538, TA100 and TA98 were grown in L-broth (Lennox, 1955) consisting of 10 g polypeptone, 5 g yeast extract, 5 g NaC1, 1 g glucose and 1 1 water, adjusted to pH 7.2 with NaOH. For mutation assay, two kinds of agar medium were used: 1.2% agar in Vogel--Bonnet minimal medium E (pH 7.2) (Vogel and Bonnet, 1956) consisting of 0.2 g MgSO4 • 7H20, 2 g citric acid, 10 g K2HPO4, 3.5 g NaNH4HPO4 • 4H20, 20 g glucose and 1 1 water; and 0.5% soft agar consisting of 6 g NaC1, 9.58 mg (final concentration, 50 laM) L-histidine • HC1, 12.22 mg (final concentration, 50 #M) biotin and 1 1 water. The trace o f histidine in the soft agar allows all the bacteria on the plate to undergo several cell divisions, and this growth is usually necessary for mutagenesis to occur. Mutation assay. The assay was carried out as described by Ames et al. (1973) with a slight modification. Chemicals dissolved in 0.1 ml of dimethyl sulfoxide were pre-incubated for 20 min at 37 ° with 0.5 ml of 0.1 M sodium phosphate buffer (pH 7.4) and 0.1 ml of overnight culture of a tester strain. 2 ml of soft agar molten at 48 ° were added and mixed, poured immediately over 1.2% agar in Vogel--Bonnet minimal medium E, and the plate was left to harden for several minutes. After incubation for 2 days at 37 °, the numbers of colonies that were revertants to histidine prototroph were counted in both test and control plates. Results are expressed as numbers of revertant colonies per test plate after subtraction of those per control plate.

147

Results and discussion

As shown in Fig. 1, methyl, ethyl and propyl N-hydroxycarbamates exhibited weak but significant mutagenic activity for TA100. On the other hand, none of the corresponding alkyl carbamates was mutagenic in the same tester strain (data not shown), in agreement with a previous report (McCann et al., 1975). This indicates that the hydroxylamino group of alkyl N-hydroxycarbamates is the essential part of the molecule for their mutagenic activity in TA100, although the mutagenic potency is dependent on the chain length of the "alkyl group as described below. The activity of these N-hydroxycarbamates was in the order: ethyl N-hydroxycarbamate >> propyl N-hydroxycarbamate > methyl N-hydroxycarbamate (Table 1). As regards the carcinogenic activity of these alkyl N-hydroxycarbamates, methyl and propyl N-hydroxycarbamates were not carcinogenic in mice whereas the ethyl derivative was carcinogenic in mice (Mirvish, 1968). These findings suggest that there is some correlation between the mutagenic activity and the carcinogenic activity of alkyl N-hydroxycarbamates. It is known that ethyl N-hydroxycarbamate is partly metabolized to its acetyl derivative in rats and rabbits (Boyland and Nery, 1965), and to its glucuronide in rats (Mirvish, 1966). On the other hand, the N-acyloxy derivatives of the carcinogenic aromatic amines are more biologically active than the corresponding N-hydroxy compounds (Miller and Miller, 1966). It has been suggested that the O-acyl derivatives or O-glucuronide of ethylN-hydroxycarbamate might react by decomposing to give the very active nitrene (Rose, 1967). Therefore, it would be interesting to determine whether the acyl derivatives of ethyl N-hydroxycarbamate are, generally, more effective than the parent N-hydroxy com-

Ethyl N-hydroxycarbamate

~'200/ r~

Methyl N-hydroxycarbamate

0 20 40

6D 8D jurnotes/plate

Fig. 1. D o s e - - r e s p o n s e c u r v e s of the mutagenie effects of alkyl N - h y d z o x y e a r b a m a t e s typhlmurlum TAt00. Spontaneous zevertants, about 140--170, have been subtracted.

o n 8almonelhz

148 TABLE 1 MUTAGENIC POTENCY OF ETHYL N - H Y D R O X Y C A R B A M A T E AND ITS R E L A T E D COMPOUNDS ON Salmonella typhimurium TA100 Compound

Revertants/nmole

Compound

Revertants/nmole

Methyl N - h y d r o x y c a r b a m a t e

0.001

Ethyl N-benzoyloxycarbamate

5.2

Ethyl N-hydroxycsxbamate

0.020

Ethyl N-acetoxycarbamate

1.6

Propyl N - h y d r o x y c a r b a m a t e

0.004

E t h y l N-benzoyloxy-Nacetylearbamate E t h y l N-acetoxy-Nbenzoylcarbamate E t h y l N-acetoxy-Nacetylcarbama t e E t h y l N-benzoyloxy-Nbenzoylcarbamate

1.7 1.6 1.5 1.4

The n u m b e r s are results from linear-response curves (Figs. 1 and 2).

pound with respect to the mutagenic activity in a bacterial system. Fig. 2 shows the mutagenic activity of several kinds of acyl derivatives of ethyl N-hydroxycarbamate in TA100. From the data shown in Figs. 1 and 2, the revertants per. nanomole of a chemical per plate were calculated (Table 1). The highest mutagenic activity was observed with ethyl N-benzoyloxycarbamate. The further N-acylation of this compound or ethyl N-acetoxycarbamate did not result in an increase in their mutagenic activity. Until now, the carcinogenic activity of acyl

1000. 800 ~c~'600, ÷.~ 400 201

0 a()40.:l

Q2 ,umoles/plate 014

Fig. 2. Dose--response curves of the mutagenie effects of aeyl derivatives of e t h y l N - h y d r o x y e a r b a m a t e on Salmonella typhimurium TA100. S p o n t a n e o u s revertants, a b o u t 140--170, have be e n subtracted. E t h y l N - b e n z o y l o x y c a r b a m a t e (X), e t h y l N - a c e t o x y c a r b a m a t e (e), e t h y l N -be nz oyl oxy-N -a c e t yl c a rba ma t e (4), e t h y l N-acetoxy-N-benzoylearbamate (o), e t h y l N-aeetoxy-N-aeetylcarbamate (z~), e t h y l N-benzoyloxy-Nb e n z o y l c a r b a m a t e (v).

149 TABLE 2 R E S U L T S O F M U T A T I O N T E S T S ON A C Y L D E R I V A T I V E S O F E T H Y L N - H Y D R O X Y C A R B A M A T E IN Salmonella typhimurtum Compound

His+ r e v e r t a n t s / p l a t e ~mole

TA1535

TA100

TA1538

TA98

Ethyl N-benzoyloxycarbamate

0.1 1.0

4 56

522 644

28 21

43 133

Ethyl N-aeetoxycarbamate

0.1 1.0

0 19

219 537

6 23

13 79

E t h y l N - b e n z o y l o xy-N-ac e t y l c a ~ b a m a t e

0.1 1.0

17 47

228 567

II 47

22 118

E t h y l N-acetoxy-N-benzoylearbamate

0.I 1.0

7 30

232 572

4 55

31 124

E t h y l N-ace t o x y - N - a e e t y l e a r b a m a t e

0.1 1.0

0 2

124 752

6 40

0 49

Ethyl N-benzoyloxy-N-benzoylcarbamate

0.1 1.0

0 48

202 404

18 55

18 99

T h e assays w e r e p e r f o r m e d as d e s c ~ b e d i n m u t a t i o n assay. S p o n t a n e o u s r e v e r t a n t s h a v e b e e n s u b t r a c t e d : II(TA1535), 170(TAI00), i2(TAI53S), 26(TA9S).

derivatives of ethyl N-hydroxycarbamate remains unknown. Based on the present study, however, we strongly suggest that these acyl derivatives should be more potentially carcinogenic than ethyl N-hydroxycarbamate. We have also tested the acyl derivatives of ethyl N-hydroxycarbamate for mutagenic activity with strains TA1535 and TA100 which contain a base-pair substitution mutation, and with strains TA1538 and TA98 which contain a frameshift mutation. Ethyl N-acetoxy-N-acetylcarbamate was active for TA100, TA98 and TA1538, but not for TA1535. However, 5 other acyl derivatives were mutagenic to all the strains tested, expecially to TA100 (Table 2). Acknowledgement This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education of Japan, Science and Culture to which the authors are greatly indebted. References A m e s , B.N., F.D. Lee a n d W.E. D u r s t o n ( 1 9 7 3 ) A n i m p r o v e d b a c t e r i a l t e s t s y s t e m for t h e d e t e c t i o n a n d c l a s s i f i c a t i o n o f m u t a g e n s a n d c a r c i n o g e n s , Proc. Natl. A e a d . Sci. (U.S.A.), 70, 7 8 2 - - 7 8 6 . B e r e n b l u m , I., D. Ben-Ishal, N. H a r a n - G h e r a , A. L a p i d o t , E. S i m o n a n d N. T r a l n i n ( 1 9 5 9 ) S k i n initiating a c t i o n a n d l u n g c a r c i n o g e n e s i s b y derivatives o f u r e t h a n e ( e t h y l e a r b a m a t e ) a n d r e l a t e d c o m p o u n d s , B i o e h e m . P h a n n a c o l . , 2, 1 6 8 - - 1 7 6 . B o r e n f r e u n d , E., M. K r i m a n d A. B e n d i c h ( 1 9 6 4 ) C h r o m o s o m a l a b e r r a t i o n s i n d u c e d b y h y p o n i t r i t e a n d h y d z o x y | a r n t n e derivatives, J. Natl. C a n c e r Inst., 3 2 , 6 6 7 - - 6 7 9 . B o y l a n d , E., a n d R . N e r y ( 1 9 6 4 ) The e o l o r i m e t r i e d e t e r m i n a t i o n o f N - h y d ~ o x y t t r e t h a n e a n d r e l a t e d c o m p o u n d s , Analyst, 89, 520---528. Boy]and, E., and R. Nery (1965) The metabolism of urethane and related compounds, Bioehem. J., 94, 198--208.

150 Boyland, E., R. Nery, K.S. Peggie and K. Williams (1963) The m e t a b o l i s m of possible mode of a c t i on of urethane, Biochem. J., 89, 113P--114P. Freese, E.B. (1965) The effects of urethane and h y d r o x y u r e t h a n e on transforming DNA, Genetics, 51, 953--960. Jones, L.W., and R. Oesper (1914) Some new h y d r o x y u r e t h a n e s and c hromoi s ome ri c silver salts of their aeyl derivatives, J. Am. Chem. Soc., 36, 2208--2223. Lennox, E.S. (1955) T ransduction of Hnked genetic characters of the hos t by bacteriophage P1, Virology, 1, 190--206. McCann, J., E. Choi, E. Yamasaki and B.N. Ames (1975) Dete c t i on of carcinogens as mut a ge ns in the S a l m o n e l l a / m i c r o s o m e test: Assay of 300 chemicals, Proc. Natl. Aead. Sei. (U.S.A.), 72, 5135--5139. Miller, E.C., and J.A. Miller (1966) Mechanism of chemical carcinogenesis: Nature of p r o x i m a t e carcinogens and i n t e r a c t i o n s with macromoleeules, Pharmaeol. Rev., 18,805---838. Miller, J.A., J.W. Cramer and E.C. Miller (1960) The N- and r i n g - h y d r o x y l a t i o n of 2-acetylaminofluorene during carcinogenesis in the rat, Cancer Res., 20, 950--962. Mirvish, S.S. (1 966) The m e t a b o l i s m of N - h y d r o x y u r e t h a n e in relation t o its carcinogenic action: Conversion into ureth ane and an N - h y d r o x y u r e t h a n e glucuronlde, Biochim. Biophys. Aeta, 117, 1--12. Mirvish, S.S. (1968) The carcinogenic action and m e t a b o l i s m of ure t ha ne and N - h y d r o x y u r e t h a n e , Adv. Cancer Res., 11, 1--42. Philips, F.S., S.S. Sternberg, A.P. Cronin and P.M. Vidal (1964) C y t o t o x i c i t y and disposition of h y d r o x ylurea and o t h e r h y d r o x y l a m i n e s in rat, Proc. Am. Assoc. Cancer Res., 5, 50. Rose, F.L. (1967) Possible c y t o t o x i c role of nitrenes, Nature (Lo ndon), 215, 1492. Vogel, H.J., and D.M. B o n n e t (1956) A c e t y l o r n i t h i n a s e of E. coli: Partial puri fi c a t i on and some properties, J. Biol. Chem., 218, 97--106.