Effect of pH and temperature on nitrosamide-induced mutation in Escherichia coli

Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands

155

E F F E C T OF pH AND T E M P E R A T U R E ON N I T R O S A M I D E - I N D U C E D MUTATION IN E S C H E R I C H I A COLI

S. N E A L E

Courta~ld Institute of Biochemistry, 3Iiddlesex Hospital 34edicaI School, London, IVzP 5PR (Great Britain) (t~eceived August 23rd, 197 I)

SUMMARY

N-Nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) induced reversions in four mutant auxotropic strains of E. coll. Among other nitroso compounds tested only N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (MNG) was an active mutagen in the system used. The decay rate of the nitrosamides decreased with decreasing pH but the number of revertants induced was relatively constant between pH 7.9 and 6.o. The number of reversions induced was proportional to nitrosamide concentration and, at lower concentrations only, increased with length of exposure over a period of 2 h. Comparison of the reversion rates induced in three mutant strains carrying previously deduced altered nucleotide sequences suggested that nitrosamides induce both transitions and transversions. The nitrosamides had a lower lethal effect than MNG when inducing comparable reversion rates and it is suggested that they could form useful mutagenic agents.

INTRODUCTION

Nitrosamides and nitrosamines are among the most effective carcinogens reported is and have been shown to be powerful mutagens in m a n y species including Drosophila6,18, higher plantsS,~e, 27, fungi16,17,29, bacteria3,7,1~,lg,23, 25 and bacteriophages2,14. Both the carcinogenic and mutagenic effects of these compounds have been considered to be due not to the compound per se but to decomposition products (refs. 17,29), in particular to the possibility of diazoalkane formation3,5,~7,23,. Attemps to correlate the carcinogenicity of these alkylating agents with the degree of alkylation found in the nucleic acids have been discussed by other workers la, 25. Further, both LOVELESS13 and VELEMfNSK~" et al. 27 have discussed the possibility of a correlation between mutagenicity and 0-6 alkylation of guanine. The nitrosamides are unstable in aqueous solution and it is well-known that at Abbreviations: DEN, N,N'-diethyl-N-nitrosamine; DMN, N,N'-dimethyl-N-nitrosamine; MNG, N-methyl-N'-nitro-N-nitrosoguanidine; NEU, N-nitroso-N-ethylurea; NMU, N-nitrosoN-methylurea.

M~*lation Res., 14 (i972) 155 164

156

S. NEALE

alkaline pH the respective diazoalkane is one of the breakdown products formed while at acid pH it is probable that the nitroso group is lost as nitrous acid ~. The rate of decomposition is further affected by temperature. In the present work the effect of these factors on mutation rate was studied in E. coli in an attempt to obtain further information on the mechanism of mutagenicity of these compounds. Results with the unstable nitrosamides were compared with those for nitrosamines and nitrosomorpholine which are relatively stable in aqueous solution. Both DMN and DEN are known to break down in mammals under the conditions in which induction of tumors occurs. It was thought unlikely that the cyclic compound nitrosomorpholine, a potent carcinogen, could undergo decomposition by enzymic hydroxylation and that the compound per se might be the active carcinogen (see review by SWANN ANI) MAGEE22). Recently, nitrosomorpholine administration has been shown to result in alkylation of rat nucleic acid in vivo 1~ and the mode of action of this compound is under further investigation in our laboratory. MATERIALS AND METHODS

Bacterial strain, s. The following strains of Escheriehia coli were used: C6oo, lhr leu-B1, obtained from Dr. R. ROWBURY; trpA23 cys-his , lrpA58 and trpA78 originally isolated by Dr. C. YANOFSKV from Ymel strain of E. coli K I 2 and supplied by Drs. W. J. BRAMMAR,J. GUEST and C. YANOFSKY. Strains were maintained at 4 °, as stabs, on medium containing 1.25°/~, (w/v) Oxoid nutrient broth and 0.75% (w/v) Bacto agar. Chemicals and analytical methods. N-Methylurea was supplied by Kocb Laboratories Ltd. NMU, NEU and nitrosomorpholine were gifts from Dr. P. I:. SWAXN. The MNG was supplied by Aldrich Chemical Co., and DMN and DEN, which were redistilled before use, were supplied by British Drug Houses Ltd. and Eastman Chemical Co., respectively. Nitroso compounds were dissolved in twice glass-distilled water (pH 5) in the dark and either used immediately or stored in small portions at 2o °, and only thawed once immediately before use. Under tile storage conditions described no decay of nitrosamide was detected by polarograpby over a 3-month period. Nitroso compounds were determined polarographically on 0.2 ml samples in IO ml 2.5°/~ (w/v) sulphosalicylic acid stored at 20 ° in the dark until assayed. Growth media and exposure of cells to potential mulagens. The basal mineral salts medium (232) already described 24 was supplemented with 0.5% (w/v) glucose (232G) and further supplemented, as indicated. Cells for exposure to potential nmtagens were obtained by the following method. An overnight culture in 232G supplemented with o.5~/0 (w/v) Bacto peptone (232G / PEP) was subcultured into 232G supplemented witl~ the required amino acids (all at 3 °/~g/ml). The medium was further supplemented with thiamine (IO/~g/ml) for growth of C6oo or with I°/~1 (w/v) Oxoid nutrient broth for growth of tryptophan-dependent strains. The cultures were incubated at 37 ° in shake flasks through at least four generations and harvested by centrifugation while still in tbe exponential phase. The cell pellets were resuspended aseptically in o.o 4 M NaC1. Cells, at a final population density of 3" IO9 cells/ml, suspended in o.15 M buffer (see below) and 232 mineral salts (onesixth normal strength) adjusted to the required pH, were exposed in opaque tubes to Mulatio~z Rcs., 14 (i072 ) 155 164

157

NITROSAMIDE-INDUCED MUTATION IN E . coli

nitroso compounds at the concentrations indicated. Buffers used were tris (hydroxymethyl)aminomethane-hydrochloride (Tris HC1), pH 7.9; potassium phosphate, pH 7.0 and 6.0; acetate, pH 5.0. The pH value remained constant throughout each cell treatment. A control tube with water replacing the nitroso compound was included in every experiment for determination of the spontaneous reversion rate. The reaction tubes were incubated at 37 or 15 ° and, whenever nitroso compounds were present, o.2-ml samples were withdrawn at intervals for determination of residual nitroso compound. At suitable intervals samples of the treated cell suspensions were centrifuged, the cells washed once with o.16 M NaC1, pH 7.0, finally resuspended in o.16 M NaC1, pH 7.0, and serially diluted in saline for estimation of numbers of surviving cells and revertants. Total survivors were estimated by plating o.I ml of washed cell suspension on 232G medium supplemented with o.o1% (w/v) Difco nutrient broth 4 and 2% (w/v) Bacto agar and further supplemented with L-leucine, L-threonine and thiamine (for experiments with C6oo), or with L-tryptophan, L-cysteine and L-histidine (for A23, A58 and A78 ). Revertants to leucine independence (C6oo) or tryptophan independence (A23, A58, A78 ) were counted by plating o.I ml of washed cell suspensions containing 3 ' lO6, 3 lO7 and 3" lO8 cells on the above media from which either leucine or tryptophan was omitted. Colonies were counted after 2 days incubation at 37 ° after which time no further survivor colonies appeared. Revertant colonies were counted again on the 5th day of incubation by which time additional colonies of slow-growing revertants had appeared. Slow-growing colonies were divided arbitrarily on the basis of colony "FABLE I EFFECT O F p H ON N I T R O S A M I D E DECAY RATF AND NITROSAMIDE INDUCTION OF RFVERTANTS IN E. coli C6oo AND A58 Cells, 3" lO9 per nil, were e x p o s e d to 5 m M fresh or d e c a y e d n i t r o s a n l i d e s o l u t i o n in t h e presence of 232 p lus o . i 5 M buffer s o l u t i o n a t v a r i o u s p H v a l u e s (see ~ETHODS). S a m p l e s were w i t h d r a w n a t s u i t a b l e i n t e r v a l s for d e t e r u l i n a t i o n of t h e r e s i d u a l n i t r o s a n l i d e and, a f t e r 2 h, for d e t e r m i n a t i o n of t h e r e v e r s i o n rate. The s p o n t a n e o u s r e v e r s i o n rates, d e t e r m i n e d c o n c u r r e n t l y in t u b e s cont a i n i n g w a t e r in place of n i t r o s a m i d e , were c o n s t a n t o v e r t h e p H r a n g e used. All t h e v a l u e s were d e t e r m i n e d a t 37 ° e x c e p t for t h e figures in p a r e n t h e s e s w h i c h were o b t a i n e d a t 15". I n all t r e a t m e n t s b e t w e e n 95 a n d lOO% of the cells r e m a i n e d viable. THE

Treatment

p H of e#,l system

Number reversions/;o 8 viable cells 5 mM NMU 5 rnM NE U C6oo A58 A58

7.9

36o0

Nil Nil

7.0 6.0

4000 4200

Nil 15 rain p H 7.9 lOO% d e c a y a t p H 6.0 5 o% d e c a y a t p H 5.o lOO% d e c a y in light, no buffer

5.° 7.9 6.0 5.o 6.o

116 2 2 5°

Nitrosamide halJ2life (rain) NMU NEU

( A ) 5 m M nitrosamide Nitrosamide pretreatment Nil

(B) Spontaneous reversion

4oo

2oi 3

(520) 255 300

3.1

2.6

(~56) II7O 603

23.0 243.0

(50)

(~)a

IO

1638

14.o 364.0 15oo

6

2

2

0. 5

0. 5

a No d e c a y d e t e c t a b l e a f t e r 24o rain a t 15 °.

3lutation Res., 14 (1972) 155-164

I58

s. NEaLV;

size into p r o b a b l e p a r t i a l r e v e r t a n t s a n d suppressed m u t a n t s "s. Results are expressed as t o t a l n u m b e r of r e v e r t a n t s per I o 8 survivors (discounting p r o b a b l e suppressed mutants). W h e n the frequency of n m t a t i o n was low the n u m b e r of plates scored was such t h a t a m i n i m u m of 5 ' Io9 cells were screened for reversion. RESULTS

The m u t a g e n i c effects of several related c o m p o u n d s were s t u d i e d using all four E. coli strains. A m a r k e d increase over the s p o n t a n e o u s reversion r a t e was o b t a i n e d in the strains t e s t e d with NMU, N E U or MNG. W h e n cells of E. coli C6oo were exposed to 5 m M NMU at 37 ° the n u m b e r of reversions to leucine p r o t o t r o p h y which were i n d u c e d in 2 h showed little v a r i a t i o n between p H 6.o a n d 7.9 (Table I). A t p H 7.(3 NMU h a d a half-life of only 3 min, whereas at p H 6.o the half-life was a b o u t 4 h. However, at p H 5.o, where only 5 % of the N M U d e c a y e d during the 2-h exposure period, the n u m b e r of reversions decreased b y a factor of a b o u t 35. No increase in reversion r a t e over the s p o n t a n e o u s level was o b t a i n e d if NMU was allowed to d e c a y at p H 7.9 for 15 or 12o min before a d d i t i o n of cells to an otherwise complete reaction m i x t u r e (Table I). A similar failure to induce reversions in strain C6oo was observed when 5 m M NMU solutions were first allowed to d e c a y at p H 6.o or b y exposure to light. E x p o s u r e of 5 m M N M U to a c e t a t e buffer p H 5.o until 5o% of the n i t r o s a m i d e had decayed, a b o u t 26 h, resulted in a r e d u c e d r a t e of reversion c o m p a r e d with tile values o b s e r v e d with fresh NMU solution at the same p H (Table I). I n d u c t i o n of reversions in C6oo b y 5 n l M NMU at 37 ° was followed, at various p H values, over a period of 3 h. At this concentration the m a x i m u m n u m b e r of m u t a tions were induced within 15 rain at p H 7-9 and in 6o min at p H 6.o (Vig. Ia). The kinetics of induction of reversion can be c o m p a r e d with the time courses for NMU decay. The rate of NMU d e c a y at each p H was logarithmic (Fig. Ib) a n d there a p p e a r e d to be no direct correlation between d e c a y rate a n d induction of reversions. The effect of N M U c o n c e n t r a t i o n on induction of reversions was followed in E. coli A58 at p H 6.0 and 37 ° (Fig. 2) ; the cells were exposed to N M U for 7 ° rain during which time the original c o n c e n t r a t i o n of N M U decreased b y a b o u t lO%. The n u m b e r of r e v e r t a n t s to t r y p t o p h a n independence recovered following t r e a t m e n t with o.375 m M N M U was 4 per IO ~ viable cells above the s p o n t a n e o u s level, whereas 3.75 m M NMU induced an increase to 185 r e v e r t a n t s per IO 8 viable cells. These figures represent the n u m b e r of reversions induced in 7 ° min at c o n s t a n t p H a n d hence each cell sample h a d been exposed to concentrations both of m u t a g e n a n d b r e a k d o w n product(s) which could be d i r e c t l y r e l a t e d to the initial NMU concentration. A l t h o u g h at 5 m M N M U (pH 6.0) the t o t a l n u m b e r of r e v e r t a n t s per IO 8 surviw)rs was c o n s t a n t after 6o rain, at 1.25 m M the n u m b e r of r e v e r t a n t s per IOs survivors was still increasing at 12o min (Fig. 3). The n u m b e r of r e v e r t a n t s induced b y 1.25 m M NMU, p H 6.o, increased almost linearly with time over tim first 12o min and did not show the e x p o n e n t i a l relationship with time which was t y p i c a l for NMU decay. The r a t e of reversion was also m e a s u r e d at 15 ° in the presence of 5 m M NMU at p H 6.0. A t this t e m p e r a t u r e and pH NMU d e c a y was less t h a n 4 % in 5 h, lower t h a n t h a t observed in p H 5.o buffer at 37 °. The n u m b e r of reversions induced b y 5 m M NMU at p H 6.o, 15 °, was v e r y small c o m p a r e d with t h a t observed at 37 ° and, in the first 45 rain, b a r e l y exceeded the spontaneous level. A l t h o u g h not s t u d i e d in Mutation Res., 14 ( i 9 7 2 ) 15.5 164 .

159

NITROSAMIDE-INDUCED MUTATION IN E. coli

14°ll \ g

2 Time

(h)

3

o

1

2 Time

(h)

Fig. I. The effect of p H on reversion of E. coli C6oo to leucine independence during exposure to an initial concentration of 5 m M NMU. Cells, 3" IO9 per m], were exposed to 5 m M N M U at 37 °

in t h e presence of 232 a n d o.15 M buffer at v a r i o u s p H v a l u e s (see METHODS). S a m p l e s were rem o v e d a t s u i t a b l e i n t e r v a l s a n d a s s a y e d for: (a) r e v e r s i o n to leucine i n d e p e n d e n c e , e x p r e s s e d as t h e n u m b e r of reversions i n d u c e d p e r ios s u r v i v o r s after s u b t r a c t i o n of t h e n u m b e r of s p o n t a n e o u s r e v e r t a n t s ; (b) c o n c e n t r a t i o n of residual N M U , e x p r e s s e d as a p e r c e n t a g e of t h e initial value. A, Tris HC1 buffer (pH 7-9) ; A, p o t a s s i u m p h o s p h a t e buffer (pH 7.o) ; ©, p o t a s s i u m p h o s p h a t e buffer (pH 6.0) ; O, s o d i u m a c e t a t e buffer (pH 5.0).

detail, the number of reversion induced at 15 ° increased slowly with time (Fig. 3) and after both 45 rain and 4 h the number of reversions per lO 8 viable cells was consistently higher than that found at comparable times using pH 5.0 buffer and 37 °. The mutagenic effect of NEU was also studied in some detail. At 5 mM, NEU was found to be a more potent mutagen than NMU when tested with any of the three strains A23, A58 or A78 (Table II) or when tested with A58 at various pH values from 5.0 to 7-9 (Table I). The effect of NEU concentration, at pH 6.0, on induction of reversions in strain A58 is shown in Fig. 2. At 3-75 mM the number of revertants induced by NEU exceeded by 50% the number of revertants induced by the same concentration of NMU and at lower nitrosamide concentrations this effect was even more marked. The effects of 5 m M NMU or NEU on reversion of each of the three tryptophan auxotrophs were investigated at pH 7-9 (Table II). Both compounds increased the number of reversions to tryptophan independence in each of the three strains; however, they were more effective with strain A58 than with strains A23 or A78 and in each case induction of the same number of reversion required a higher concentration of NMU than NEU. The possible mutagenic activity of several other related compounds was tested on all four E. coli strains. Following exposure to IO mM DMN, DEM, N-nitrosomorpholine or N-methylurea for 4 h at pH 7.0 and 37 °, the number of revertants observed did not exceed the number of spontaneous reversions in parallel untreated suspensions. Mutation Res., 14 (1972) 155-164

160

S. NEALE

@

40C

_m 40C u

30O o

g >

%

300

~2oo o .o

200 /

L_

/

L 100 100

zx

E

2

2

~ Initiol

~

~

o

concn(MxlO -3)

Time

( lq )

Fig. 2. The effect of n i t r o s a m i d e c o n c e n t r a t i o n on i n d u c t i o n of r e v e r s i o n to t r y p t o p h a n i n d e p e n den ce in E. coli A58 a t p H 6.o. Cells, 3" IO9 per ml, in t h e pre s e nc e of 232 a n d o.15 M p o t a s s i u m p h o s p h a t e buffer p H 6.o were e x p o s e d to v a r i o u s i n i t i a l c o n c e n t r a t i o n s of N MU or N E U . A ft e r 7 ° m m a t 37" s a m p l e s were w i t h d r a w n for e s t i m a t i o n of t he n u m b e r of r e v e r s i o n s i n d u c e d (see METHODS). The n u m b e r s of r e v e r t a n t s are e x p r e s s e d per lO 8 s u r v i v o r s a f t e r s u b t r a c t i o n of t h e n u m b e r of s p o n t a n e o u s r e v e r t a n t s . <,, N M U ; O, N E U . Fig. 3. The effect of t i m e of e x p o s u r e to NMU on i n d u c t i o n of re ve rs i on to t r y p t o p h a n i n d e p e n den ce in E. coli A58 a t p H 6.o. Cells, 3" IO9 per ml, in t h e pre s e nc e of 232 a nd o.15 M p o t a s s i u m p h o s p h a t e buffer p H 6.o were e x p o s e d to 5 or 1.25 m M N MU a t 37 or 15 ° a n d s a m p l e s w i t h d r a w n a t s u i t a b l e i n t e r v a l s for e s t i m a t i o n of t h e n u m b e r of r e v e r t a n t s i n d u c e d (see METHODS). The n u m b e r s of r e v e r t a n t s are e x p r e s s e d per lO 8 s u r v i v o r s a f t e r s u b t r a c t i o n of t h e n u m b e r of splmt a n e o u s r e v e r t a n t s , c , 5 m M NMU, 37"; O, 1.25 m M NMU, 37); / , 5 m 3 I NM1T, I5'.

TABIA'2 11 FREQUENCY OF NITROSAMIDE-INDUCED REVERSIONS IN J2. CODON REVERTANT TYPES

Foil STRAINS

A 2 3 , A 5 8 AND A 7 8 AND THE POSSII3I

Cells of E. coli A23, A58 or A78 (3" IO~ per ml) were e x p o s e d t o a n i n i t i a l c o n c e n t r a t i o n of 5 h i M NMU or m M N E U in 232 p lus o.15 zTI T n s HCI buffer, p H 7.9. Af t e r 7 ° rain e x p o s u r e s a m p l e s were w i t h d r a w n f, d e t e r m i n a t i o n of t h e n u m b e r of r e v e r t a n t s . The s p o n t a n e o u s r e v e r s i o n r a t e s were o b t a i n e d from s i m i l a r t u b in w h i c h th e n i t r o s a m i d e was replaced b y water. I n all t r e a t m e n t s b e t w e e n 95 a n d 1oo% of t h e cells r e n i a i m viable. I n f o r n l a t i o n for the a s s i g n m e n t of codons was o b t a i n e d from YANOFSKY el al.~< r

3lutant slrain Number reversions per 1o 8 viable cells .Spontaneous 5 m M N M U 5 mM NEU p H 7.9 p H 7.9

Codons H"ild-type

Mutant

Possible codons in reverlants with funclional protein

:\23

404

GGA

AGA

(]Gi I

(arg) U GA C (asp} UGU2

GGA AGC,

2o 13

(gly) (;(; tT

(gly)

(cys)

A 58

I .o

0.5

272

4oo

(

(gly~ A78

I.O

65

3lutalion Res., 14 (I972) 1 5 5 - I 6 4

520

A UA ACA

A(; l,"

NITROSAMIDE-INDUCEI)

M U T A T I O N IN E . coli

IO;_

MNG (0.3 mM) caused an increase in the number of revertants of A58 but not of A78. In both strains there was an increase in the number of small slow-growing colonies, assumed to be suppressor mutations. The number of A58 revertants per IO8 survivors induced by o.3 mM MNG or 2.5 mM NEU was approximately equal. However, at these concentrations exposure to MNG had a pronounced lethal effect and after 7 ° min only about 33% of the cells survived whereas there was no significant decrease in viability among the cells treated with NEU for a similar period of time. DISCUSSION

Among the compounds tested for mutagenicity only NMU, NEU or MNG resulted in increased numbers of revertants to amino acid independence in the E. coli strains used. The other, related, compounds tested, namely DMN, DEN, N-nitrosomorpholine and methylurea, have been shown to be carcinogenic or to induce morphological changes in animalsl~,2°, 22. The failure of these compounds, even at high concentration, to induce reversions in the E. coli system used could be due to inability of E. coli to degrade the compounds to an active mutagen. Although POGODINA19 obtained a mutagenic effect when exposing E. coli for 23 h to I M DEN, other workers failed to induce mutation of E. coli with DMN or DENT, 23. Evidence that DMN degradation was a prerequisite for the induction of mutations in Neurospora has been obtained 16. In the present work no breakdown of DMN or DEN was detected throughout a 4-h experimental period. MNG has been widely used as a nmtagen and its possible mode of action has already been the subject of detailed experiments by other workers2,a,~,9, ~2. MNG was shown here to induce reversions in A58 but not in A78. Although only o.3 mM MNG was required to induce the same percentage of revertants as was obtained with 2.5 m M NEU, at these concentrations MNG had a pronounced lethal effect not apparent with NEU; NEU may therefore be a more convenient nmtagenic tool but should be used with great care since it has been shown to be a most potent carcinogen in animals~.~. It has been suggested that the mutagenic action of nitroso compounds is dependent on degradation to an active mutagen, and could be due to diazoalkane formationa, ~7. CERDA-OLMEDOAND HANAWALT a, during a detailed study of MNG mutagenesis in E. coli, concluded that diazomethane, probably acting through alkylation of the DNA, was the active mutagen at pH values above 5. More recently however it has been shown that diazomethane is at least not involved in the methylation of the N- 7 of guanine in nucleic acids either by MNG in an in vitro system 9 or by MNG or DMN in vivo in E. coli 1~ or rat n systems. This implies the existence of alternative pathways of breakdown for nitroso compounds. Both NMU and NEU are unstable in aqueous solution with half-lives which differ markedly with pH. Above pH 5 the respective diazoalkane is one of the decay products formed 2~. Consequently, if diazoalkane formation was a prerequisite for mutation, the rate of induction of mutation might be expected to vary with changes in the decay rate resulting from alterations of pH. Both NMU and NEU were powerful mutagens for all four E. coli strains used but the total number of revertants induced by a given nitrosamide concentration was approximately constant between pH 6.o and 7.9 though the nitrosamide half-life fell from about 25o min to 3 min (Table I, Fig. I). Although presumably exposed to the same 31utation Res., 14 ( i 9 7 2 ) I55--I04

102

S. NEALE

concentration of decay products, treatment of cells with o.8 mM NMU at pH 7.9 yielded a greatly reduced number of revertants compared to cells treated with 5 mM NMU at ptt 6.o. Consideration of these results suggests it was probable that the number of nitrosamide-induced mutations was independent of the decay rate of the compound between pH 7.9 and 6.o. The induction of mutations in strain A58 was, however, proportional to initial NMU concentration between o.3 and 5.0 mM at pH 6.o (Fig. 2). At pH 5.0 the half-life of NMU increased to 27 11 and the number of mutations to prototrophy was markedly reduced. This observation could indicate that at pH 5.o ttle nature of the decay product(s), the degree of celt permeability or the DNA conformation was less favourable for induction of nmtations. Tile decrease in the number of mutations induced at pH 5.o could not be ascribed entirely to the low rate of nitrosamide decay since at pH 6.o and 15 ° the decay was even slower while the number of mutations induced was higher. No mutations were induced if, before exposing strain C6oo to NMU, the carcinogen was first destroyed completely either in the presence of pH 6.o or pH 7.9 buffers in the dark or by exposure of an aqueous solution to light. Pretreatment of NMU with pH 5.o buffer for a period of one half-life resulted in induction of only 5o°,~ of the number of mutations induced by fresh nitrosamide solution. These results indicate that the final product of NMU decay was not the active mutagen but do not preclude the possibility of the active mutagen being a transitory intermediate in the decay process. The effectiveness of alkylating agents as mutagens has been shown to be related to a low Swain-Scott substrate constant (s) both in E. coli where a group of alkane sulphonates was studied "5 and in Arabidopsis using NMU and NEU2E Using isopropylmethane sulphonate (s = o.29), TURT6CZKYAND EHRENBERG2a obtained a linear dose response curve for induction of mutations in E. coli but with compounds with s > o.55 the dose-response curve was exponential. The s values for NMU and NEU are o.42 and o.26 respectively 27 and, as has already been noted, yielded approximately linear dose response curves for induction of reversions in E. c01iA58. Thus the dose response curves of nmtagenesis and the s values of NMU and NEU resemble closelv those of isopropylmethane sulphonate. In Arabidopsis NMU was found to be 3-fold more effective than NEU as a mutagen 27 but with coliphage T2 the ethyl homologue was marginally more efficient 14. In the present work NEU, with a lower s value than NMU, was more effective than NMU in inducing reversions in the tryptophan auxotrophs. The ratio of the number of reversions induced by NEU to the number induced by the same concentration of NMU varied with pH and the bacterial strain used (Fig. 2, Table II). I n vitro exposure of salmon sperm DNA to NEU did not result in the high level of alkvlation of the N- 7 position of guanine obtained with NMU x4. However NEU was 3 4 times more effective than NMU in alkylation of the 0-6 position of guanine when added to an aqueous solution of deoxyguanosinel:L The nucleophilic strength of guanine-O-6 is low and other possible sites for alkylation which also have low n-values include adenine-N- 3 and phosphate-O-. The original base changes resulting in the production of tryptophan-dependent mutants A23, A58 and A78 can be postulated from the known changes in amino acid residues in the tryptophan synthetase A protein (Table II) 28. The amino acid changes recorded as reversions can only represent those resulting in partial or full restoration of the tryptophan synthetase A protein activity. In the absence of amino acid analyses 2~lutation Res., 14 (1972 ) 155-164

N1TROSAMIDE-INDUCED MUTATION IN

E. coli

~63

of the tryptophan synthetase. A protein in nitrosamide-induced revertants it was assumed that only those changes recorded by YANOFSKV et al. 28 in tryptophan-independent revertants were likely to be found. YANOFSKY et al. recorded only one reversion type for A78, in which an arginine residue of A78 was replaced by glycine, and which would require a change of A ~ C in the DNA strand transcribed. Since NMU and NEU treatments both increased the number of reversions in A78 it is probable that these compounds can induce the transversion A ~ C . MNG is believed to cause predominantly transitions G-+A and C->T' and failed to induce reversions in strain A78. I t should be noted that YANOFSKY el al. ''s also failed to induce A78 reversions with the alkylating agent ethyl methanesulphonate (s -- o.69). Mutant A58 carries a mutation in the same base codon as A78, and reversions which require either T-+C or T ~ G changes in the transcribed DNA strand have been reported °-s. The relatively large number of reversions induced in A58 by both nitrosamides suggests that either both the possible base changes, a transition and a transversion, occur or the frequency of one of the possible changes was much greater than the frequency of the change A ~ C in A78. Four different amino acids were reported to yield enzymatically active protein when replacing arginine at amino acid residue 21o in the tryptophan synthetase A protein of strain A23 (ref. 28). The base changes required in the DNA strand transcribed were C ~ A , C-+G, T--~A, T-+G or T ~ C . Amino acid analyses of the tryptophan A gene protein in nitrosamide-indueed revertants of A23 and A58 are necessary to determine which base changes are probably induced by these compounds and whether the nitrosamides are capable of inducing transitions in addition to the transversion probably induced in revertants of A78. ACKNOWLEDGEMENT

This work was supported by a research grant from the Cancer Research Campaign. The author wishes to thank Drs. R. J. ROWBURY, W. J. BRAMMAn, J. R. GUEST and C. YANOFSKY for the bacterial strains used, Mrs. H. SLADE and Mr. J. HOLSMAN for their excellent technical assistance and Prof. P. N. MAGEE for valuable discussion.

1~I~I:I';t¢.ENC|';S i ABELSON, J. N., M. L. GEFTER, g. I~ARNETT, A. LANDY, t{. L. RUSSELL AND J. D. •MITH, M u t a n t tyrosine transfer ribonueleie acids, J. 1Viol. Biol., 47 (I97 °) 15 28. 2 BAKER, i{., AND [. TESSMAN, Different mutagenic specificities in phages SI 3 and T4; in vivo t r e a t m e n t with N-methyl-N'-nitro-N-nitrosoguanidine, .[. Mol. Biol., 35 (1968) 439 448. 3 CERDf~-OLMEDO, E., AND P. C. HANA~,VALT, D i a z o m e t h a n e as the active agent in nitrosoguanidine mutagenesis and lethality, 3Iol. Gen. Genet., i o i (I968) I9I-2O2. .4 DEMEREC, M., AND E. CAHN, Studies of m u t a b i l i t y in nutritionally deficient strains of Escherichia coli, 1. Genetic analysis of five a u x o t r o p h i c strains, J. t3acteriol., 65 (1953) 27 36. 5 DRUCKREY, [-[., R. PREUSSMANN, D. SCHMAHL AND M. MULLER, E r z e u g u n g yon Magenkrebs dureh Nitrosamide an Ratten, Nat~trwissenschaften, 48 (I96I) 165. 6 FAHMY, O. G., M. J. FAHMY, J. MASSASSO AND M. ONDRI~J, Differential m u t a g e n i c i t y of the amine and amide derivatives of nitroso c o m p o u n d s in Drosophila *nelanogas~er, 3lutation Res., 3 (I966) 2oi-217. 7 (;EISSLER, E., l~Jbar die W i r k u n g von N i t r o s a m i n e n auf Mikroorganismen, Naturwissenschaften, 48 (I962) 38o-38I. 8 GICHNER, T., AND J. VELEM[NSK~, The mutagenie activity of I - a l k y l - I - n i t r o s o u r e a s and Ialkyl-3-nitro-I-nitrosoguanidines, Mutation t?es., 4 (1967) 2o7-212.

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9 HAERLIN, R., R. S/]ISSMUTH AND F. LIXGENS, M e c h a n i s m of m u t a g e n c s i s by N - n l e t h y l - N ' -

n i t r o - N - n i t r o s o g u a n i d m e {MNNG), V. M e t h y l a t i o n of D N A by N - t r i d c u t e r i o m e t h y l - N ' n i t r o - N - n i t r o s o g u a n i d i n e (Da-MNN(;), FEI3S ],etters, 9 (I97O) I75 I76. IO LEE, I~. Y., AND ~\'. LIJINSKY, A l k y l a t i o n of r a t liver R N A b y cyclic N - n i t r o s a m i n e s in viv,, ,1. Natl. Cancer I~st., ,37 (1966) 4oI-4O7 II IAJINSKY, \\;., J. LOO AND A. E. R o s s , Mechanisnl of a l k y l a t i o n of nucleic aids l)y nitrosodinaethylamine, Nature, 218 (1968) 1174-1175. 12 LINGENS, F., R. HAERLIN AND R. ~t'SSMUTI], M e c h a n i s m of n l u t a g e n e s i s b y N - m e t h y l - N ' n i t r o - N - n i t r o s o g u a n i d i n e (MNNG), VI. M e t h y l a t i o n of nucleic acids by N-tridenteriomethyl-N'n i t r o - N - n i t r o s o g u a n i d i n e (D:3-MNNG) in t h e presence of c v s t e i n e a n d in cells of Escherichia coil, I,'EBS Letters, 13 11971) 241-242. 13 LOVELESS, A., Possible relevance of 0 - 6 a l k y l a t i o n of d e o x y g u a n o s i n e to t h e n l u t a g e n i c i t y a n d c a r c i n o g e n i c i t y of n i t r o s a m i n e s a n d nitrosanlides, Nature, 223 119691 2o6-2o7. 14 LOVELESS, A., AND C. g. HAMPTON, I n a c t i v a t i o n a n d m u t a t i o n of coliphage T2 by N - m e t h y l a n d N - e t h y l - N - n i t r o s o u r e a , Mutation Res., 7 11969) 1-12. 15 MAGEE, P. N., A l k y l a t i o n of nucleic acids a n d carcinogenesis, in ±7. Colloquium der (;csellschc~/i jiir Physiologische Chemie, 1966, pp. 79 95. i6 MALLIXCL H. V., M u t a g e n i c i t y of two p o t e n t carcinogens, d i n l e t h y h a i t r o s a m i n c a n d diethyln i t r o s a m i n e , in Neurospora erassa, Mutation Res., 3 11966) 537-54 °. 17 MARQUARDT, H., F. K. ZIMMERIXIANN AND R. SCHWAIER, Die \ ¥ i r k u n g K r c b s a u s l 6 s e n d e r N i t r o s a i n i n e u n d N i t r o s a m i d e a u f das a d e n i n e - 6 - 4 5 - R i i c k n - m t a t i o n s s y s t e m yon Saccharomyccs cvrevisiac, Z. l'ererbztngsl., 95 (I064) 82 96 . 18 PASTERNAK, L., U n t e r s u c h u n g e n fiber die n l u t a g e n e W i r k u n g v e r s c h i e d e n e r Nitrosanain- unll N i t r o s a m i d - V e r b i n d u n g e n , Arzneimitte!-Forsch., 14 (1964) 8o2 8o 4. ~9 POGODINA, (). N., On t h e nautagenic action of s o m e carcinogens belonging to t h e g r o u p of nitrosanlines, Tsitologlia, 8 11969) 5o3 5o9. 20 SANDERS, 1~'. ][{., AN[) B. O. BURFORD, Morphological conversion of cells in vitro b y N nitros o n l e t h y l u r e a , Nature, 213 (I967) 1171 1173. 21 SMITH, g . l., Aliphatic
31utatiotz Res., 14 (1972 ) 155 164