Mode of mutagenic action of 4-benzoylamido- and 4-acetamido-4-carboxamido-n(N-nitroso)-butylcyanamide

9

Mutation Research, 49 (1978) 9--18

© Elsevier/North-Holland Biomedical Press

MODE OF MUTAGENIC ACTION OF 4-BENZOYLAMIDO- AND 4-ACETAMIDO-4-CARBOXAMIDO-n(N-NITROSO)'BUTYLCYANAMIDE

NOZOMU OTSUJI a and HIDEYA ENDO b Department of Microbiology, a Faculty of Pharmaceutical Sciences and Cancer Research Institute, b Faculty of Medicine, Kyushu University, Fukuoka (Japan)

(Received 3 June 1977) (Revision received 23 August 1977) (Accepted 2 September 1977) Summary The mode of mutagenic action of 4-benzoylamido- and 4-acetamido- 4-carboxamido-n(N-nitroso)-butylcyanamide (BCNBC, ACNBC) was studied using Escherichia coli K12 strains. The strains carrying defects in DNA-repair mechanism, AB2463 (recA) and P3478 (polA) were more sensitive than their parent strains to both compounds, while AB1886 (uvrA) showed the same sensitivity as the parental strain. A b o u t 90% of tryptophan revertants from BE1043 (trpa~bPhOamb) b y both compounds were due to mutation in suppressor genes. Suppressor analysis by using BE1047 (trpambPhOoch) revealed that the most frequently occurring reversion was due to a mutation in suppressor gene, supE. This implies that these two alkylnitrosocyanamides predominantly induce GC -~ AT transition.

Introduction

With respect to a possible gastric carcinogenesis due to environmental factors, studies on the compounds structurally and functionally related to MNNG, a well known mutagen [1,15] and a gastric carcinogen [27--29], seem to be worthy of note. From this point of view we focused our first attention to the nitrosation products of various guanidine derivatives structurally similar to A part of this work was reported at the 2nd Annu a l Meeting of the Japanese E n v i r o n m e n t a l Mutagen Society, September 22nd 1973, Mishima (Abstract in Mutation Res. 26 (1974) 431).

Abbreviations: AAA, acetylarginineamide; BAA, benzoylarginineamide; ACNBC, 4-acetamido-4carboxamido-n(N-nitroso)butylcyanamide; BCNBC, 4-benzoylamido-4-carboxamido-n(N-nitroso)b u t y l c y a n a m i d e ; DCMTC, d e c a r b a m o y l m i t o m y c i n C; ETG, enriched TG m e d i u m ; MMS, m e t h y l m e t h a n e s u l f o n a t e ; MNC, m e t h y l n l t r o s o c y a n a m i d e ; MNG, N-methyl-NP-nltroguanidine; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; NB, n u t r i e n t b r o t h m e d i u m ; P me di um, p e p t o n e me di um; ST(}, semi-enriched TG m e d i u m ; Tris, t r i s ( h y d r o x y m e t h y l ) a m i n o m e t h a n e .

10 MNG which is converted b y nitrosation to MNNG [ 16]. During this study we found that two arginine derivatives, BAA and AAA, though not naturally-occurring guanidines, when nitrosated, showed a powerful mutagenic action for a strain of Salmonella typhimurium TA1535, a tester for base-substitution mutagens. The active principles responsible for the mutagenicity were identified as BCNBC and ACNBC respectively [5,8]. The mutagenic activity of the former was a b o u t 30 times as high as, and that of the latter was a b o u t the same as that of MNNG. The present study was thus made to explore the mode of their mutagenic action, in more detail, and the results of which will be described in this paper. Materials and methods

Bacterial and phage strains Bacterial strains of Escherichia coli K12 used in this experiment are listed in Table 1. Suppressor sensitive (sus) mutants of phage k; ksusG9, N53 and P3 were supplied by A. Campbell, and R 2 1 6 was obtained from R. Thomas.

Media TG medium contained 0.1 M tris(hydroxymethyl)aminomethane pH 7.2, 0.2% glucose, 8 X 10-2M NaC1, 2 × 10 -2 M KC1, 2 × 10 -2 M NH4C1, 3 × 10 -3 M Na2SO4, 10 -3 M MgC12,2 × 10 -4 CaC12, and 2 × 10-6M FeC13. As a "high phosphate" medium, 6.4 × 10 -4 M KH2PO4 was added; a "low phosphate" medium, 3.2 × 10-SM KH2PO4 was added. Tris buffer-salts was TG medium without glucose. ETG consisted of TG medium with high phosphate, 2.5 g casamino acids and 40 mg of L-tryptophan per liter of distilled water. STG agar was lowphosphate TG medium supplemented with 2.5% liquid Difco nutrient broth

TABLE 1 BACTERIAL STRAINS USED a Strains

Genotype

Source

BE1043 BEl115 BE1047 ABl157

F-, trP10242, p h o G $ , strr F-, trP10242, p h o o 5 , uvrA, strr F - , trp 1 0 2 4 2 , p h o u 2 8 , strr F - , thr-1, leu-6, p r o A - 2 , his-4, thi-1, a r g E - 3 , / a c Y - 1 , galK-2, a r a - 1 4 , xyl-5o mtl-1, tsx-33, s t r A - 3 1 , s u p - 3 7 ABl157 uvrA AB1157 fecAl3 thy W3110 thy polA1

This paper This paper This paper

AB1886 AB2463 W311Othy P3478

E.A. Adelberg P. H o w a r d - F l a n d e r s P. H o w s x d - F l a n d e r s

a T h e s y m b o l s arg, his, leu, pro, thi, thr. t h y a n d trp d e n o t e r e q u i r e m e n t s f o r a r g i n i n e , h i s t i d i n e , l e u c i n e , p r o l i n e , t h i a m i n e , t h r e o n i n e , t h y m i n e a n d t r y p t o p h a n , r e s p e c t i v e l y ; ara, gal, lac, rntl and x y ! d e n o t e t h e i n a b i l i t y t o utilize a r a b i n o s e , g a l a c t o s e , l a c t o s e , m a n n i t o l a n d x y l o s e , r e s p e c t i v e l y ; tsx and str d e n o t e response to T6 phage and to the antibiotics streptomycin (superscript r indicates resistance); pho d e n o t e s a l k a l i n e p h o s p h a t a s e ; sup d e n o t e s s u p p r e s s o r g e n e . uvr d e s i g n a t e s a g e n e a f f e c t i n g e x c i s i o n , h o s t - c e l l r e a c t i v a t i o n , a n d U V s e n s i t i v i t y ; rec d e n o t e s a g e n e a f f e c t i n g g e n e t i c r e c o m b i n a t i o n a n d U V s e n s i t i v i t y , t r P 1 0 2 4 2 a n d p h o G s are a m b e r m u t a t i o n s a n d p h o u 2 8 is a n o c h r e m u t a t i o n is r e s p e c t i v e genes; arab a n d och d e s i g n a t e a m b e r a n d o c h r e m u t a t i o n , r e s p e c t i v e l y ,

11 and solidified w i t h 1.5% agar. NB contained 5 g of meat extract and 10 g of polypeptone per liter of distilled water, and pH was adjusted to 7.2. P medium contained 10 g of polypeptone, 2.5 g of NaC1 per liter of distilled water and used for assaying k phage.

Determination of surviving cells To determine the survival of bacteria, cultures were first grown at 37 ° in ETG medium to a density of about 3 X l 0 s cells per ml. They were centrifuged, washed twice and resuspended in Tris buffer-salts. Then, they were exposed to various concentrations of the chemicals to be tested for 30 min at 37 ° . After dilution, samples were plated on NB plates and incubated about 20 h at 37 °.

Mutation induction Bacteria grown to log phase in TG medium with high phosphate and 20 pg per ml of t r y p t o p h a n were harvested by centrifugation, washed twice and resuspended in Tris buffer-salts. Cells at a concentration o f 2 X 109 per ml were exposed to various concentrations of chemicals at 37 ° for 30 min, and plated on STG agar which is sufficient to allow phenotypic expression of newly induced mutations. Viable cells were determined on the same plate after appropriate dilution. Colonies were counted after 2 days' incubation at 37 ° .

Test for presence and identification of suppressors Presence of suppressor was tested by the ability of Trp ÷ revertants to form alkaline phosphatase. Colonies grown on STG agar were sprayed with 2 mg per ml of ~-naphthylacid phosphate in 0.06% of borate and with 2% of o-dianisidine {Sigma). Strains carrying suppressors form alkaline phosphatase and turn to black after several minutes at room temperature. To identify suppressors, a strain BE1047 (trpambphOoch), which carries an amber mutation in trp gene and an ochre mutation in pho gene, was used. In each experiment, more than 50 Trp ÷ revertants were purified once by singlec o l o n y isolations on the selective STG agar plate. Each single colony was regrown at 37 ° for overnight in 1 ml of NB liquid medium as host cells for Xsus growth test and on STG agar for alkaline phosphatase test. To test ability to support Xsus phages, a portion of the culture grown in NB liquid was spread on P medium with melted soft agar, on which a drop of Xsus phages was spotted. After about 20 h, growth of the phages was assayed. Trp ÷ revertants were classified into 7 groups according to their ability to sup-

CONH2 i HC-(CH2)3-N-CN i

NH I

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Fig. 1.

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12 press alkaline phosphatase ochre mutation and to support various amber mutants of k phase [21]. These Xsus phages are already characterized to show suppression pattern different from each other for strains carrying supD, supE, supF, supC, sup(ochre) and sup ÷ of E. coli as shown in Table 2.

Chemicals BCNBC and ACNBC (Fig. 1) were synthesized in our laboratory [5,8]. DCMTC was kindly supplied from Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan. Remits

Survival of E. coli strains carrying defects in DNA-repair mechanism after treatment of BCNBC In most organisms damages on DNA induced by ultraviolet light or by some chemicals which bind to DNA bases are repaired by excision or by post-replication repair mechanisms [3,12]. Mutants of E. coli defective in such mechanisms are more sensitive to UV or to these chemicals than their parent wild-type strain. In order to see whether BCNBC causes chemical alteration in DNA molecule and to know how such change is repaired, we have tested bactericidal action of the drug on various strains of E. coli which have defects in repair mechanism of DNA damages. Bacterial strains, ABl157 (wild), AB1886 (uvrA), AB2463 (recA), W3110 thy (wild) and P3478 (polA), grown to log phase in ETG medium were exposed to various concentrations of BCNBC and plated on NB agar to determine surviving cells. As seen in Fig. 2, strains, AB2463 (recA) and P3478 (polA) were more sensitive to the drug than their parent strains, i

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0.13

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Fig. 2. Survivals o f U V sensitive strains o f E. coli a f t e r i n c u b a t i o n w i t h BCNBC. A f t e r i n c u b a t i o n o f E. coli strains w i t h v a r i o u s c o n c e n t r a t i o n s o f B C N B C for 30 rain, s a m p l e s w e r e p l a t e d o n N B agax t o c o u n t surviving ceils. S y m b o l s : o, A B l 1 5 7 (wild); ~, A B 1 8 8 6 ( u v r A ) ; o A B 2 4 6 3 ( r e c A ) ; e , W 3 1 1 0 t h y (wild); × , P 3 4 7 8 ( p o l A ) .

13 whereas a strain AB1886 (uvrA) is as sensitive as its parent strain. Since the strain AB1886 is defective in the process of incision of DNA strands nearby pyrimidine dimers on DNA molecule, and sensitive to UV [12], therefore, the results suggested that mechanism for repair of chemical alterations on DNA by BCNBC does not require the incision enzyme.

Induction of mutation by BCNBC and ACNBC In order to study frequency of induction of mutation by BCNBC and ACNBC, a strain BE1043 (trpambphOa~b) was exposed to various concentrations of the drugs and plated on STG agar to allow Trp ÷ revertant colonies to develop. Numbers of revertants due to suppressor mutation were determined by counting phosphatase-producing Cells by spraying a-naphthylacid phosphate on the selective agar. In Fig. 3, surviving cells and induced mutation frequency were presented as a function of concentration of BCNBC and ACNBC. The results on DCMTC was also included for comparison. The kinetics of mutation induction by BCNBC and ACNBC are very similar. A concentration of BCNBC and ACNBC which reduced cell survivals to 37% increased mutation frequency about 250- and 180-fold as that in the absence of the mutagens, respectively. To our surprise, more than 90% of the Trp ÷ revertants were due to suppressor mutation (dotted line). In contrast to m u t a t i o n by these two compounds, suppressor mutation induced by monofunctional mitomycin, DCMTC, was about 50% of the total Trp + revertants. In other experiments using strains with amber

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Fig. 3. I n d u c e d f r e q u e n c y o f m u t a t i o n f r o m trp t o T r p * ' i n a s t r a i n B E 1 0 4 3 ( t r P a r n b P h O a m b ) b y A C N B C , B C N B C a n d D C M T C . T h e b a c t e r i a g r o w n to l o g p h a s e w e r e t r e a t e d w i t h v a r i o u s c o n c e n t r a t i o n s o f c h e m icals a s d e s c r i b e d in Materials a n d m e t h o d s . A, s u r v i v i n g f r a c t i o n ; o, m u t a t i o n f r e q u e n c y o f T r p + r e v e r t a n t s ; • mutation frequency of suppressor mutation.

14

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ld'

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o.'3

o:,~

ACNBC (~rno|e/rnl)

Fig. 4. I n d u c e d f r e q u e n c y of m u t a t i o n f r o m trp to T r p + in a strain B E l l l 5 (trPambPhOarnbUVrA) b y ACNBC. ~, surviving f r a c t i o n ; o, m u t a t i o n f r e q u e n c y of T r p + r e v e r t a n t s ; o, m u t a t i o n f r e q u e n c y o f suppressor mutation.

mutation in lactose or arginine genes, 92 and 90% of the revertants by BCNBC were due to mutation in suppressor genes (data n o t presented). These results indicate that high specificity of the BCNBC and ACNBC for induction of suppressor mutation is not restricted only to amber mutation in trp cistron, but to general effects on amber codon in bacterial genes. In Fig. 4, survivals and m u t a t i o n frequency in a uvrA strain, B E l l l 5 , after treatment with ACNBC were presented. The strain was as sensitive to the drug in both survivals and mutation induction as its parent strain, BE1043. Induction of suppressor mutation was also high as was in its parent uvr ÷ strain.

Classification o f the revertants Since these two compounds caused mutation at high frequencies in suppressor gene, we next examined which suppressor gene is the most frequently modified. By classifying suppressor mutation, we could assess what type of base change is induced by these compounds [19,20,23], since it is known that amber and ochre suppressors result from change at the anticodons of tRNA molecules [2,11]. To classify the suppressors, a strain carrying an amber mutation in trp gene and an ochre mutation in p h o A , BE1047 (trpambphooch), was exposed to mutagens and Trp * revertants were selected. They were tested for synthesis of alkaline phosphatase and for supporting growth of amber m u t a n t of X phage, and classified into 7 groups according to the classification shown in Table 2. The compounds, BCNBC and ACNBC, induced suppressor mutation at high frequency, which agreed with the result obtained in the strain, BE1043. Furthermore, they produced a class 2 revertant predominantly, t h a t is, those induced by BCNBC and ACNBC were 76.6% and 79.9%, whereas the other

15 TABLE

2

RESPONSE

OF

AMBER

MUTANTS

OF

k PHAGE

TO

Trp + REVERTANTS

OF

A STRAIN

BE1047

( trP a m b P h Oo c h ) Class

Growth

of ksus

Suppressor

Alkaline

phosphatase G9

N53

P3

R216

1

+

+

+

--

--

amber

2

+

+

+

+

--

amber

3

+

+

--

--

--

4

+

+

--

--

+

5

+

+

_+

+_

+

ochre

(unknown)

6

--

--

--

+

+

ochre

(unknown)

7

.

+, clear plating

lysis

.

in t h e a r e a

. of

the

.

(supD) (supE) amber (supF) ochre (supC)

.

k sus s p o t ; - - , n o l y s i s ;

sup + ( s t r u c t u r e +, incomplete

lysis,

a

gene)

corresponding to a lower

efficiency,

a see f o o t n o t e

in T a b l e 3 .

treatments, DCMTC and UV, caused mutation in class 2 at frequencies o f 1.5% and 36%, respectively. A class 2 revertant in spontaneous Trp ÷ revertants was a b o u t 14%. These results indicate that specific ability of the compounds, BCNBC and ACNBC, to produce revertants in class 2 is n o t due to a specificity of an amber c o d o n in the trp gene, b u t is their specific mode of mutation induction. Discussion Our results indicate that BCNBC and ACNBC are mutagenic to E. coli at sublethal doses (Fig. 3). The responses of a strain deficient in excision repair (uvrA) and a wild-type strain to these c o m p o u n d s are qualitatively similar with regard to both survival (Fig. 2) and mutagenesis (Figs. 3 and 4). The irrelevance of the uvrA gene to bactericidal and mutagenic effects of ACNBC and BCNBC indicates that the lesions by these compounds responsible for the effects are n o t susceptible to repair enzyme for excision of pyrimidine dimers on DNA. However, the recA and polA mutants of E. coli are more sensitive to the compounds than wild-type strain, suggesting that they cause some alteration on DNA which are repaired by the mechanism involved by polA and recA genes. The response of these UV-sensitive mutants to ACNBC and BCNBC is very similar to the effect of monofunctional alkylating agent, MMS. It is reported that UV sensitive polA and recA mutants are sensitive to MMS, b u t UV-sensitive excision-repair negative mutants (uvrA, B, C) are insensitive to it [3,12, 24--26]. MMS reacts with DNA primarily by the addition of a methyl group to the purine residue, forming mainly 7-methylguanine [4]. We assume, therefore, that both BCNBC and ACNBC alkylate DNA bases which are repaired by similar molecular mechanisms responsible for the repair of MMS-induced DNA damage. Striking specificity in mutation induction by t w o compounds is of great interest. As seen in Fig. 3, more than 90% of the Trp ~ revertants by these c o m p o u n d s are due to mutation in suppressor gene. Furthermore, the most

16 TABLE 3 A N A L Y S I S O F M U T A G E N - P R O D U C E D T R Y P T O P H A N R E V E R T A N T S OF S T R A I N B E 1 0 4 7

( trPambPhO och ) Mutagen

BCNBC ACNBC DCMTC UV

Spontaneous

Total number tested

A m b e r suppressors class 1

class 2

class 3

class 4

class 5 *

class 6

128 74 260 39 94

0 1 47 0 4

98 59 4 14 13

0 1 5 2 15

2 5 12 0 16

10 1 24 1 11

0 3 3 3 3

(0) b (1.4) (18.1) (0) (4.3)

(76.6) (79.7) (1.5) (35.9) (13.8)

Ochre suppressors

(0) (1.4) (1.9) (5.1) (16.0)

(1.6) (6.8) (4.6) (0) (17.0)

(7.8) (1.4) (7.2) (2.6) (11.7)

Structural gene

(0) (4.1) (1.2) (7.7) (3.2)

18 4 165 17 32

(14) (5.4) (63.5) (43.6) (34.0)

Trl0 + r e v e r t a n t s w e r e p u r i f i e d b y single c o l o n y isolation, a n d classified into 7 g r o u p s b y t e s t i n g e a c h single c o l o n i e s f o r s y n t h e s i s o f alkaline p h o s p h a t a s e a n d f o r s u p p o r t i n g g r o w t h of k sus phages. a E x t r a c t s f r o m 3 r e v e r t a n t s b y BCNBC a n d t h o s e f r o m 5 b y D C M T C w e r e s u b j e c t e d t o starch-gel electrop h o r e s i s to see a n e l e e t r o p h o r e t i c m o b i l i t y of alkaline p h o s p h a t a s e a n d w e r e f o u n d to i n c o r p o r a t e basic a m i n o acid i n t o t h e p o s i t i o n o f p e p t i d e co~cesponding to a n o n s e n s e c o d o n in an alkaline p h o s p h a t a s e gene. Therefore, w e a s s u m e a s u p p r e s s o r in a class 5 i n c o r p o r a t e s lysine into n o n s e n s e c o d o n as supG does. b In p a r e n t h e s i s , % o f e a c h o f t h e r e v e r t a n t classes is s h o w n .

frequently occurring revertants belong to a class 2, which arises from mutation in supE gene (Table 3). A question arose whether this specificity is due to only the nonsense codon in trp gene. This is however not the case, since a majority of the revertants from strains carrying nonsense mutation in lactose or arginine gene by treatment with BCNBC and ACNBC were also shown to be due to mutation in supE gene (data not shown). Moreover, DCMTC was found to induce m u t a t i o n in structure gene in the present assay system at higher frequency than in suppressor genes. These results indicate that high specificity of BCNBC and ACNBC for induction of suppressor mutation is n o t restricted only to amber m u t a t i o n in trp cistron of this special strain, but to general effects on amber codon in bacterial genes. By classifying suppressor mutation, one can assess what type of base change is induced by mutagens [19,20,23]. Mutation to amber suppressor arises from change to CTA in the DNA coding for the anticodon of tRNA which is complementary to UAG. Similarly, the alteration leading to ochre suppressors is a change to TTA in the code for anticodon of tRNA in DNA molecule. The suppressor in a class 2 revertant inserts glutamine into the position of peptide corresponding to the nonsense codon and arise from mutation in DNA coding for glutamyl-tRNA. Folk and Yaniv [10] reported that the presumptive anticodons of glutamyl-tRNA of E. coli K12 were CUG and NUG where N is probably 2-thiouridine. These two anticodons can become complementary to UAG by a single base substitution, G to A. Therefore, a class 2 revertant is formed by GC-~ AT change in the DNA coding for an anticodon of glutamyl-tRNA. We could conclude that mutation of GC -~ AT transition was preferentially induced by BCNBC and ACNBC when the cells are exposed to these compounds. Using E. coli WWU strain, Osborn et al. [2] found t h a t EMS produced mutants of which 90% are class 2 revertants, and led to the similar conclusion that the EMS causes GC ~ AT transition. The present study prompted us to investigate nitrosation products of various naturally-occurring guanidine derivatives. Among them, MNC, a nitrosation

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