25
Mutation Research, 60 (1979) 25--32
© Elsevier/North-Holland Biomedical Press
A R Y L - M O N O A L K Y L AND CYCLIC TRIAZENES: DIRECT-ACTING MUTAGENS
HARVEY F. THOMAS * 1, D I A N E L. BROWN 1, PHILIP E. HARTMAN ..1, EMIL H. WHITE 2 and ZLATA HARTMAN 1 Departments of 1 Biology and 2 Chemistry, The Johns Hopkins University, Baltimore, MD 21218 (U.S.A.) (Received 25 July 1978)
(Accepted 30 October 1978)
Summary An aryl-monoalkyl triazene, methyl-p-tolyl triazene (MTT) and a cyclic triazene (A2-triazoline) are direct-acting mutagens for Salmonella t y p h i m u r i u m bacteria and for cell-free Hemophilus influenzae DNA. MTT causes reversion of the hisG46 base-substitution mutation, b u t no reversion of the hisD3052 frameshift mutation. Induced mutation frequency is n o t strongly influenced by modifications in the genetic background of the S. t y p h i m u r i u m Ames tester strains, but is mildly enhanced by the addition of a pool of amino acids to the plating medium and is strongly enhanced by liquid preincubation before plating.
Linear aryl--dialkyl triazenes are strong carcinogens and teratogens [19,20]. In various test systems many of these dialkyl triazenes induce mitotic gene conversion [4,5,11] and mutations [5,11,14--16,26,27]. The linear aryl--dialkyl triazenes are indirect-acting agents and require for genetic activity either nonenzymatic cleavage of the diazoamino side-chain or microsomal activation and subsequent heterolysis [5,11,12,19,20]. Alkylation of DNA occurs in vivo [13]. Linear aryl--monoalkyl triazenes [30--32] are carcinogenic, in which case tumors occur mainly at the site of application (i.e., "proximal" carcinogens)
o f Medical Genetics, U n i v e r s i t y o f W i s c o n s i n , M a d i s o n , WI 5 3 7 0 6 (U.S.A.) ** F r o m w h o m reprints should be requested. Contribution No. 978 of the Department of Biology, The Johns Hopkins University. Abbreviations: Ery R, erythromycin resistance; M T T , methyl-p-tolyl triazene; StrR~ streptomycin resistance. * Present address: Department
26
[ 17 ]. A monoalkyl triazene is mutagenic for Neurospora, in which case activity is higher than with the dialkyl homologue [16]. Predominantly base-pair substitutions are induced [16], in keeping with observations of alkylation of guanine residues in RNA and DNA treated in vitro [18]. We report here on the mutagenicity of a linear aryl--monoalkyl triazene and a cyclic triazene for Salmonella typhimurium bacteria and for Hemophilus influenzae cell-free DNA. We conclude that these two agents are potent directacting mutagens which predominantly induce base-substitution mutations. Conditions of liquid preincubation are essential for demonstration of optimal genetic activity. Materials and methods
Hemophilus influenzae strain Rd [10] was a gift of Dr. Roger M. Herriott, and Salmonella typhimurium strains were from our collection (hisG46) or, in the case of the TA strains, gifts of Dr. Bruce N. Ames [1]. Mutation hisG46, also contained in strains T A 1 5 3 0 and T A 1 0 0 [1], was originally used in an analysis of the mutagenicity of cycasin aglycone [22]. Mutation hisD3052, contained in strains T A 1 5 3 8 and T A 9 8 [1], was specially chosen from a large collection of histidine-requiring mutants [8] for its particular sensitivity to mutagenesis by hycanthone and certain other frameshift mutagens [9]. Purification and treatment of cell-free H. influenzae transforming D N A from TABLE
1
SPECIFICITY
FOR
INDUCTION
Mutagen
OF
BASE-SUBSTITUTION
MUTATIONS
BY MTT
hlsG46
TA1530
TA100
TA1538
TA98
Pre a
hisG46 AuvrB
hisG46 AuvrB rfa / p K M l O!
hisD3052 AuvrB rfa
hisD3052 AuvrB rfa / p K M l O1
Pre
Ames
(nmoles/Plate) Ames
a
Pre 0
6
3
41 b
__
Ames
Pre
Ames
I0
12
81
196
16
12
14
--
512
--
--
--
--
2172
--
--
--
MTT 5 25
2092
125
--
--
200
12
4096
500
--
6
.
--
--
2811
-0
ICR-191
1620
255
--
193
.
.
.
--
--
304
0
0
--
--
c
20
0
--
0
--
1911
--
2005
--
N-Methyl-N-nitrosoacetamide
e
2
2656
--
4800
--
4216
--
0
--
--
750
--
773
--
0
--
N-ChloroethylN-nitrosoacetamide
c
2 a Pre,
-preincubation
test [1]. b Experimental
values
in liquid are
medium
average
before
number
plating
of mutant
ground (0 mutagen) subtracted. c Tests performed in a parallel set of experiments
that
(see
Materials
colonies
per
and plate
serve as additional
Methods); with
Ames,
standard
Ames
spontaneous
mutant
back-
controls
for tests
on MTT.
~1- e m a
-4712
-489
_
3500 ~4500
521 1534
93
0.5
em + amino acids
0
196 . ~4500
.
.
1.5
. 5080
. 644
.
em + 2.5 mM caffeine
TREATMENT
5640
549
1.5
em + 5 mM caffeine
of mutant
colonies per plate with spontaneous
(0 mutagen)
controls subtracted.
2058 ~4500
142 670
27
13
em + adenosine
2051 ~4500
134 662
34
6
em+ methionine
3500 ~4500
574 1326
69
0.5
adenosine
em + amino acids +
OFhisG46 WITH MTT BY THEPREINCUBATION
L-amino acids at 20 pg/ml and lacking L-histidine and L-methionine.
.
.
.
caffeine
~ml + 2 5 mM
AFTER
em, 1.25% broth.
. ~4500
. 193
.
3
caffeine
2.5 mM
~1- e m +
OF HIS+MUTATIONS
broth supplement;
0.5
acids b
amino
~em a
1
ON RECOVERY
values are average number
acids, pool of 18 common
c Experimental
b Amino
1744 ~4500
115 502
20 c
8
em a
MEDIA
0.625% (V/V) liquid Difco-nutrient
-~4500
20 40
1 a ~em,
-405
2.5
5 10
6
--
0
plate)
OFPLATING
EFFECTS
MTT (nmoles/
TABLE2
3500 ~4500
719 1588
101
0
methionine
em + amino acids +
METHOD
b~
28 Str R bacteria and selection of mutations to Ery a followed procedures outlined by Herriott [10] with the modifications noted elsewhere [25]. Briefly, native DNA was extracted and purified by the chloroform--alcohol procedures described by Herriott [10] except that octanol replaced isoamyl alcohol, as recommended by Herriott (personal communication). For mutagenesis tests, 10 pg/ml DNA was suspended in 150 mM NaC1, 200 mM phosphate buffer, pH 7.4, 37°C, in the presence of varying concentrations of triazene. After 30 min at 37°C, samples were diluted 100-fold into ice-cold 150 mM NaC1, 100 mM phosphate buffer, pH 7.61, then 4-fold into HI broth [10] containing 5 ?( 10a/ml competent H. influenzae bacteria. The transformation mixtures were incubated for 30 min at 37°C and 2 ml distributed on 4 plates of selective medium for scoring of antibiotic resistant mutants as described elsewhere [25]. S. typhimurium strains grown overnight in Difco-nutrient broth were exposed to triazene under two sets of conditions in parallel experiments performed simultaneously by two different investigators. In one series, the directplating assay of Ames et al. [1] was used. In the second series, a preincubation test was used [cf. 33]. 1 part of cold triazene solution was added to 9 parts of bacterial suspension at 37°C. After 2 rain incubation, samples of 0.1 ml were spread directly onto each of duplicate or triplicate plates of minimal E medium [28] containing 0.2% glucose and supplemented with 1.25% (V/V) liquid Difco-nutrient broth (em plates) and, when necessary, 4 ~g/ml D-biotin. Other supplements used in one series of tests are described in the legend to Table 2. Plates were scored for His* revertants after 48 h incubation or, when amino acid pool was used (Table 2), after 20 h incubation at 37°C. 2 min was used for the preincubation period since there was no increase in the number of revertant colonies with longer incubation and, in fact, some decrease after about 5--6 min of triazene--ethanol exposure. A similar level of mutation induction was noted for bacterial cultures exposed to triazene directly in nutrient broth as for cultures previously sedimented, washed, and resuspended in 0.1 M phosphate buffer, pH 7.6. The cyclic triazene (A2-triazoline) was a gift of Dr. C. Ticozzi (Politecnico di Milano, Instituto di Chimica, Milano, Italy) and was synthesized by previously described methods [6,7]. Methyl-p-tolyl triazene (MTT)was synthesized as described by White et al. [29]. Immediately before use, each triazene was dissolved and serially diluted in 95% ethanol at 4°C. Operations were performed in subdued light as MTT was found to be slightly photosensitive (at 37°C). A repeated set of mutagenicity tests performed at the end of the experiment (with strain hisG46) ensured that no significant inactivation of triazene had occurred during the course of the experimental period. Results Data in Table 1 indicate that mutagenicity by methyl-p-tolyl triazene (MTT) is highly selective for reversion of S. typhimurium strains carrying the hisG46 base-substitution mutation. No mutagenicity is detected for frameshift mutation hisD3052. The strains tested carrying the hisG46 mutation appear approximately equally sensitive to mutagenesis, independent of genetic background.
29
8000}-
fx
4000-
his
G46
"
x/
"
t
hi I-.J QI--
Z
2000
--
1000 800
-
H
•
CH3@NfN_~_CH HE MOPHI LUS
~DNA~I I //
600
40o-
3
PREINCUBATION
/
/
ZOO -
AMES
II
O0 - -
/// i/ &/ /
80
60 40o
20
~/
I
//
x~~O ~
¢
/ / c.3c.2-~.--,c.2 /
I
N
~"N'CH2
C
C,
/ I0
I
~
2
-
4
-
8 I0 20 nmoles
40
80 000
TRIAZENE
200
400
800 iO0O 2000 4000
/ PLATE
Fig. 1. D o s e - - - r e s p o n s e r e l a t i o n s h i p s for t w o t r i a z e n e s t h a t axe d i r e c t - a c t i n g m u t a g e n s f o r S. t ~ p h i m u r i u m strain h i s G 4 6 a n d for native H. i n f l u e n z a e cell-free D N A . R e s p o n s e s t o M T T ( f o r m u l a at t o p , c e n t e r ) are s h o w n f o r i n d u c t i o n o f h i s G 4 6 + m u t a t i o n s b y t h e p r e i n c u b a t i o n m e t h o d ( . ) a n d b y the s t a n d a r d A m e s p r o c e d u r e ( X ) a n d for i n d u c t i o n o f E r y R m u t a t i o n in H. i n f l u e n z a e DNA (A). S i m i l a r l y , r e s p o n s e s t o t h e c y c l i c t r i a z e n e ( f o r m u l a at b o t t o m , c e n t e r ) are s h o w n for t h e p r e i n c u b a t i o n m e t h o d ( o ) a n d for t h e s t a n d a r d A m e s p r o c e d u r e (•) u s i n g h i s G 4 6 as w e l l as for H. i n f l u e n z a e DNA (.).
Dose--response curves for mutagenesis of S. typhimurium strain hisG46 by MTT (formula at top, center of Fig. 1) are shown for the preincubation method ( . ) and for the standard Ames assay where mutagen is incorporated directly into top agar (×). The preincubation method is roughly 50-fold more sensitive for detection of MTT mutagenicity. Analogous data are shown for the cyclic triazene (formula at bottom, center of Fig. 1) by the preincubation (©) and standard Ames ((}) tests. Mutant yields for induction of Ery R mutations in H. influenzae native D N A are depicted for MTT (A) and for cyclic triazene (m). No microsomal extract was added in the above experiments (Table 1, Fig. 1). In fact, addition of S-9 mix [1] lowered mutant yield slightly when the standard Ames procedure was used. We conclude that the triazenes tested are direct-acting mutagens with ability to mutate native, purified cell-free D N A as well as readily penetrate and cause base-substitution mutations in Salmonella bacteria. A variety of c o m p o u n d s which are not mutagenic themselves have been found to raise or to decrease induced mutant yield in bacteria [2]. Data in Table 2 indicate that the presence of 2.5 or 5 mM caffeine in the plating medium may decrease mutant yield and that a pool of c o m m o n L-amino acids (lacking histidine) increases mutant yield. Addition of the amino acid pool also
30
allows scoring of plates after overnight incubation tion customary in the standard Ames test.
instead
of the 48 h incuba-
Discussion Our data indicate that a linear aryl-monoalkyl triazene is a direct-acting mutagen for S. typhimurium bacteria and for H. influenzae cell-free DNA. Base-substitution mutations are induced, in keeping with observations on N. crassa [ 161. These results are in accord with the mechanisms by which linear aryl-dialkyl triazenes are considered to exhibit genetic activity (see Introduction) and the observation of nucleic acid alkylation in vitro by an aryl-monoalkyl triazene [ 181. Mutation induction by MTT shares two features that we also have found for the direct-acting base-substitution mutagen N-methyl-N-nitrosoacetamide (to be submitted). There is little effect on mutation frequency when u&3 or rfa mutations or when plasmid pKMlO1 is present in the test strain (Table 1). In addition, the mutation frequency is proportional to dose, but the slope of the response curve is significantly greater than 1 (Fig. 1). Departure from strict proportionality is not merely due to cell penetration, for tests on treated cellfree DNA also depart from unity (Fig. 1). Two attractive and mutually compatible explanations for these phenomena are: (a) bacteria contain an extremely active system, independent of the uurAB system, that elicits effective error-free repair of MTT-induced genetic lesions, perhaps a certain class of alkylation damage, and (b) multiple lesions are required for mutagenesis. Further
N”2 N
N’
so
‘I? @ -so /
NITROSAMINE
\
0 NH2
N=N
HONO
H-
N
Nk POLYAMINE
H-
-7
‘R
DIAZONIUM ION
-H20
N \‘N NCR TRIAZENE
I 0 + N2 c
N,H R
CARBONIUM ION Fig. 2. Speculative reaction mechanisms for cyclic triazene formation from naturally occurring PdYamines and synthetic primary diamines during treatment with nitrous acid (BONO). R. -H in simple diamines while R can have varying structures in spermidine. spermine and other polyamines carrying secondary amino groups; the upper pathway of cyclic triazene formation would apply only to this latter class of comPounds.
31
work will be necessary to elucidate the operative mechanism(s). In any event, departure of certain dose--response curves from strict proportionality poses problems for the calculation of comparative mutagenicity of various chemicals and, perhaps, for the organ-specificity of carcinogens. The current report is the first to demonstrate mutagenicity by a cyclic triazene. Our interest in the cyclic triazene was elicited by our observation of the production of unstable, direct-acting mutagens after nitrosation of various diamines and polyamines [25] and the consideration that formation of cyclic triazenes might allow DNA alkylation (Fig. 2) [24]. Tannenbaum et al. [23] have observed a product formed by nitrosation of n-butylamine whose mass spectrum is suggestive of a "linear N-nitrosotriazene structure", and Endo et al. [3] have observed the formation of a diphenyltriazene derivative from a sulfonamide and nitrite under gastric conditions. In these latter cases, triazene formation would be second-order with respect to substrate, whereas in the case of a diamine the reaction might not be so limited with respect to substrate since the attacking group would be located on the same molecule. However, to date we have been unable to provide direct demonstration of triazene formation in diamine + nitrous acid reaction mixtures, and an alternate explanation namely C- and/or O-nitrosation of deamination products, has been proposed for the mutagenicity detected [25]. Acknowledgements We are indebted to Drs. Calimero Ticozzi and Hugh J. Creech for supplying the cyclic triazene and ICR-191, respectively. This investigation was supported by Public Health Service Research Grants AI-01650 from the National Institute of Allergy and Infectious Diseases (to P.E.H.) and CA-18387 from the National Cancer Institute {to E.H.W.). References 1 A m e s , B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s for d e t e c t i n g c a r c i n o g e n s and m u t a g e n s with the S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 Clarke, C.H. a n d D.M. S h a n k e l , A n t i m u t a g e n e s i s in m i c r o b i a l s y s t e m s , Bacteriol. R e v . , 39 ( 1 9 7 5 ) 33--53. 3 E n d o , H., H. N o d a , N. K i n o s h i t a , N. I n u i , M. U m e d a a n d T. Ochi, F o r m a t i o n o f d i p h e n y l t r i a z e n e derivatives f r o m a s u l f o n a m i d e a n d n i t r i t e u n d e r gastric c o n d i t i o n s , s u b m i t t e d f o r p u b l i c a t i o n . 4 Fahrig, R., M e t a b o l i c a c t i v a t i o n of a x y l d i a l k y l t r i a z e n e s in the m o u s e : i n d u c t i o n of m i t o t i c gene c o n v e r s i o n in S a c c h a r o m y c e s cerevisiae in the h o s t - m e d i a t e d assay, M u t a t i o n Res., 13 ( 1 9 7 1 ) 4 3 6 - - 4 3 9 . 5 F a h r i g , R., D e v e l o p m e n t of h o s t - m e d i a t e d m u t a g e n e c i t y tests, 1. D i f f e r e n t i a l r e s p o n s e o f y e a s t cells i n j e c t e d into t e s t e s o f rats a n d p e r i t o n e u m o f m i c e a n d rats to m u t a g c n s , M u t a t i o n Res., 26 ( 1 9 7 4 ) 29--36. 6 G a u d i a n o , G., C. T i c o z z i , A. U m a n i - R o n c h i a n d P. B r a v o , Sulla r e a z i o n e t r a a z i d i e ilidi dello z o l f o : u n a n u o v a sintesi di kx2-1,2,3-triazoline, Gaz. C h i m . Ital., 97 ( 1 9 6 7 ) 1 4 1 1 - - 1 4 2 2 . 7 G u a d i a n o , G., A. U m a n i - R o n c h i , P. B r a v o a n d M. A c a m p o r a , D i m e t h y l o x o s u l p h o n i n m m e t h y l i d e : A tool for the s y n t h e s i s of f i v e - m e m b e r e d h e t e r o c y c l e s , T e t r a h e d r o n L e t t . , ( 1 9 6 7 ) 1 0 7 - - 1 1 1 . 8 H a r t m a n , P.E., Z. H a r t m a n , R.C. Stahl and B.N. A m e s , Classification and m a p p i n g of s p o n t a n e o u s a n d i n d u c e d m u t a t i o n s in t h e histidine o p e r o n of S a l m o n e l l a , Adv. G e n e t . , 16 ( 1 9 7 1 ) 1--34. 9 H a r t m a n , P.E., K. L e v i n e , Z. H a r t m a n and H. Berger, H y c a n t h o n e : A f r a m e s h i f t m u t a g e n , Science, 172 (1971) 1058--1060. 10 H e r r i o t t , R.M., E f f e c t s o n D N A : T r a n s f o r m i n g principle, in: A. H o l l a e n d e r ( E d . ) , C h e m i c a l M u t a g e n s , Principles a n d M e t h o d s f o r T h e i r D e t e c t i o n , Vol. 1, P l e n u m , N e w Y o r k , 1 9 7 1 , pp. 1 7 5 - - 2 1 7 . 11 K o l a r , G . F . , R. F a h r i g a n d E. Vogel, S t r u c t u r e - - a c t i v i t y d e p e n d e n c e in s o m e n o v e l r i n g - s u b s t i t u t e d
32
12 13
14
15 16
17
18
19
20 21
22 23
24 25 26
27
28 29 30 31 32 33
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I v a n k o v i c a n d A. y o n H o d e n b e r g , C h e m i c a l s t r u c t u r e a n d c a r c i n o g e n icity of aliphatic h y d r a z o , azo, a n d a z o x y c o m p o u n d s and of triazenes, p o t e n t i a l in vivo a l k y l a t i n g agents, A n n . N.Y. A c a d . Sci., 163 ( 1 9 6 9 ) 6 9 7 - - 7 1 4 . P r e u s s m a n n , R., a n d A. v o n H o d e n b e r g , M e c h a n i s m of c a r c i n o g e n e s i s with 1-aryl-3,3-dialkyltriazenes, II. In v i t r o - a l k y l a t i o n of g u a n o s i n e , R N A a n d D N A w i t h a r y l - r n o n o a l k y l t r i a z e n e s to f o r m 7-alkylg u a n i n e , B i o c h e m . P h a r m a c o l . , 19 ( 1 9 7 0 ) 1 5 0 5 - - 1 5 0 8 . P r e u s s m a n n , R . A . yon H o d e n b e r g a n d H. H e n g y , M e c h a n i s m of c a r c i n o g e n e s i s w i t h 1-aryl-3,3-dialkyltriazenes, E n z y m a t i c d e a l k y l a t i o n by r a t liver m i c r o s o m a l f r a c t i o n in v i t r o , B i o c h e m . Pharrnacol., 18 ( 1 9 6 9 ) 1--13. P r e u s s m a n n , R., S. I v a n k o v i c , C. L a n d s c h f i t z , J. G i m m y , E. F l o h r and U. G r i e s b a c h , C a r c i n o g e n e W i r k u n g y o n 13 A r y l d i a l k y l t r i a z e n e n an B D - R a t t e n , Z. K r e b s f o r s c h . , 81 ( 1 9 7 4 ) 2 8 5 - - 3 1 0 . Siebert, D. a n d G.F. K o l a r , I n d u c t i o n of m i t o t i c gene c o n v e r s i o n by 3 , 3 - d i m e t h y l - l - p h e n y l t r i a z e n e , 1 - ( 3 - h y d r o x y p h e n y l ) - 3 , 3 - d i m e t h y l t r i a z e n e a n d by 1 - ( 4 - h y d r o x y p h e n y l ) - 3 , 3 - d i r n e t h y l t r i a z e n e in S a c c h a r o r n y c e s cerevisiae, M u t a t i o n Res., 18 ( 1 9 7 3 ) 2 6 7 - - 2 7 4 . S m i t h , D.W.E., M u t a g e n i e i t y of c y c a s i n a g l y c o n e ( m e t h y l a z o x y m e t h a n o l ) , a n a t u r a l l y o c c u r r i n g c a r c i n o g e n , Science, 152 ( 1 9 6 6 ) 1 2 7 3 - - 1 2 7 4 . T a n n e n b a u m , S.R., J.S. Wishnok, J.S. ttovis a n d W.W. Bishop, N - N i t r o s o c o m p o u n d s f r o m the reaction of p r i m a r y a m i n e s w i t h nitrite a n d t h i o c y a n a t e , I n t e r n a t i o n a l A g e n c y for R e s e a r c h on Cancer, L y o n , F r a n c e , 1 9 7 8 , in press. T h o m a s , H . F . , P.E. H a r t m a n a n d D.L. B r o w n , D i r e c t - a c t i n g m u t a g e n s resulting f r o m n i t r o s a t i o n of p o l y a r n i n e s , lysine, s y n t h e t i c d i a m i n e s , a n d N - m o n o a c e t y l p u t r e s c i n e , E n v i r o n . Mut., 1 ( 1 9 7 9 ) in press. T h o m a s , H . F . , P.E. H a r t m a n , M. M u d r y j a n d D.L. B r o w n , N i t r o u s acid m u t a g e n e s i s of d u p l e x D N A as a t h r e e - c o m p o n e n t s y s t e m , M u t a t i o n Res., s u b m i t t e d . Vogel, E., C h e m i s c h e K o n s t i t u t i o n and m u t a g e n e Wirkung, VI. I n d u k t i o n d o m i n a n t e r u n d rezessivg e s c h l e c h t s g e b u n d e n e r L e t a l m u t a t i o n e n d u t c h A r y l d i a l k y l t r i a z e n e bei Drosophila melanogaster, Mutation Res., 11 ( 1 9 7 1 ) 3 9 7 - - 4 1 0 . Vogel, E., R. F a h r i g a n d G. Obe, T r i a z e n e s , a n e w g r o u p of i n d i r e c t m u t a g e n s , C o m p a r a t i v e investigations of the genetic e f f e c t s of d i f f e r e n t a x y l d i a l k y l t r i a z e n e s using S a e c h a r o m y e e s eerevisiae, the hostm e d i a t e d assay, Drosophila rnelanogaster, a n d h u m a n c h r o m o s o m e s in v i t r o , M u t a t i o n Res., 21 ( 1 9 7 3 ) 123--136. Vogel, H.J., a n d D.M, B o n n e r , A c e t y l o r n i t h i n a s e of Escherichia toll: partial p u r i f i c a t i o n a n d s o m e p r o p e r t i e s , J. Biol. C h e m . , 2 1 8 ( 1 9 5 6 ) 9 7 - - 1 0 6 . White, E.H., A . A . B a u m and D.E. Eitel, 1 - M e t h y l - 3 - p - t o l y l t r i a z e n e a n d its use in the e s t e r i f i c a t i o n of acids, O r g a n i c S y n t h . , 48 ( 1 9 6 8 ) 1 0 2 - - 1 0 5 . White, E.H., H. Maskill, D.J. W o o d c o c k a n d M.A. S c h r o e d e r , C a r b o n i u m ions f o r m e d in the triazene m e t h o d of d e a m i n a t i o n , T e t r a h e d r o n L e t t . , ( 1 9 6 9 ) 1 7 1 3 - - 1 7 1 6 . White, E . H . , and H. S c h e r r e r , T h e t r i a z e n e m e t h o d for the d e a m i n a t i o n of aliphatic a m i n e s , T e t r a hedron Lett., (1961) 758--762, White, E.H., a n d D.J. W o o d c o c k , Cleavage of the c a r b o n - - n i t r o g e n b o n d , in: S. Patai ( E d . ) , T h e C h e m i s t r y of the A m i n o G r o u p , Wiley, N e w Y o r k , 1 9 6 8 , pp. 4 0 7 - - 4 9 7 . Yahagi, T., M. N a g a o , Y. Seino, T. M a t s u s h i m a , T. S u g i r n u r a a n d M. O k a d a , M u t a g e n i c i t i e s of N-nitros a m i n e s on S a l m o n e l l a , M u t a t i o n Res., 48 ( 1 9 7 7 ) 1 2 1 - - 1 3 0 .
Mutation Research, 60 (1979) 25--32
© Elsevier/North-Holland Biomedical Press
A R Y L - M O N O A L K Y L AND CYCLIC TRIAZENES: DIRECT-ACTING MUTAGENS
HARVEY F. THOMAS * 1, D I A N E L. BROWN 1, PHILIP E. HARTMAN ..1, EMIL H. WHITE 2 and ZLATA HARTMAN 1 Departments of 1 Biology and 2 Chemistry, The Johns Hopkins University, Baltimore, MD 21218 (U.S.A.) (Received 25 July 1978)
(Accepted 30 October 1978)
Summary An aryl-monoalkyl triazene, methyl-p-tolyl triazene (MTT) and a cyclic triazene (A2-triazoline) are direct-acting mutagens for Salmonella t y p h i m u r i u m bacteria and for cell-free Hemophilus influenzae DNA. MTT causes reversion of the hisG46 base-substitution mutation, b u t no reversion of the hisD3052 frameshift mutation. Induced mutation frequency is n o t strongly influenced by modifications in the genetic background of the S. t y p h i m u r i u m Ames tester strains, but is mildly enhanced by the addition of a pool of amino acids to the plating medium and is strongly enhanced by liquid preincubation before plating.
Linear aryl--dialkyl triazenes are strong carcinogens and teratogens [19,20]. In various test systems many of these dialkyl triazenes induce mitotic gene conversion [4,5,11] and mutations [5,11,14--16,26,27]. The linear aryl--dialkyl triazenes are indirect-acting agents and require for genetic activity either nonenzymatic cleavage of the diazoamino side-chain or microsomal activation and subsequent heterolysis [5,11,12,19,20]. Alkylation of DNA occurs in vivo [13]. Linear aryl--monoalkyl triazenes [30--32] are carcinogenic, in which case tumors occur mainly at the site of application (i.e., "proximal" carcinogens)
o f Medical Genetics, U n i v e r s i t y o f W i s c o n s i n , M a d i s o n , WI 5 3 7 0 6 (U.S.A.) ** F r o m w h o m reprints should be requested. Contribution No. 978 of the Department of Biology, The Johns Hopkins University. Abbreviations: Ery R, erythromycin resistance; M T T , methyl-p-tolyl triazene; StrR~ streptomycin resistance. * Present address: Department
26
[ 17 ]. A monoalkyl triazene is mutagenic for Neurospora, in which case activity is higher than with the dialkyl homologue [16]. Predominantly base-pair substitutions are induced [16], in keeping with observations of alkylation of guanine residues in RNA and DNA treated in vitro [18]. We report here on the mutagenicity of a linear aryl--monoalkyl triazene and a cyclic triazene for Salmonella typhimurium bacteria and for Hemophilus influenzae cell-free DNA. We conclude that these two agents are potent directacting mutagens which predominantly induce base-substitution mutations. Conditions of liquid preincubation are essential for demonstration of optimal genetic activity. Materials and methods
Hemophilus influenzae strain Rd [10] was a gift of Dr. Roger M. Herriott, and Salmonella typhimurium strains were from our collection (hisG46) or, in the case of the TA strains, gifts of Dr. Bruce N. Ames [1]. Mutation hisG46, also contained in strains T A 1 5 3 0 and T A 1 0 0 [1], was originally used in an analysis of the mutagenicity of cycasin aglycone [22]. Mutation hisD3052, contained in strains T A 1 5 3 8 and T A 9 8 [1], was specially chosen from a large collection of histidine-requiring mutants [8] for its particular sensitivity to mutagenesis by hycanthone and certain other frameshift mutagens [9]. Purification and treatment of cell-free H. influenzae transforming D N A from TABLE
1
SPECIFICITY
FOR
INDUCTION
Mutagen
OF
BASE-SUBSTITUTION
MUTATIONS
BY MTT
hlsG46
TA1530
TA100
TA1538
TA98
Pre a
hisG46 AuvrB
hisG46 AuvrB rfa / p K M l O!
hisD3052 AuvrB rfa
hisD3052 AuvrB rfa / p K M l O1
Pre
Ames
(nmoles/Plate) Ames
a
Pre 0
6
3
41 b
__
Ames
Pre
Ames
I0
12
81
196
16
12
14
--
512
--
--
--
--
2172
--
--
--
MTT 5 25
2092
125
--
--
200
12
4096
500
--
6
.
--
--
2811
-0
ICR-191
1620
255
--
193
.
.
.
--
--
304
0
0
--
--
c
20
0
--
0
--
1911
--
2005
--
N-Methyl-N-nitrosoacetamide
e
2
2656
--
4800
--
4216
--
0
--
--
750
--
773
--
0
--
N-ChloroethylN-nitrosoacetamide
c
2 a Pre,
-preincubation
test [1]. b Experimental
values
in liquid are
medium
average
before
number
plating
of mutant
ground (0 mutagen) subtracted. c Tests performed in a parallel set of experiments
that
(see
Materials
colonies
per
and plate
serve as additional
Methods); with
Ames,
standard
Ames
spontaneous
mutant
back-
controls
for tests
on MTT.
~1- e m a
-4712
-489
_
3500 ~4500
521 1534
93
0.5
em + amino acids
0
196 . ~4500
.
.
1.5
. 5080
. 644
.
em + 2.5 mM caffeine
TREATMENT
5640
549
1.5
em + 5 mM caffeine
of mutant
colonies per plate with spontaneous
(0 mutagen)
controls subtracted.
2058 ~4500
142 670
27
13
em + adenosine
2051 ~4500
134 662
34
6
em+ methionine
3500 ~4500
574 1326
69
0.5
adenosine
em + amino acids +
OFhisG46 WITH MTT BY THEPREINCUBATION
L-amino acids at 20 pg/ml and lacking L-histidine and L-methionine.
.
.
.
caffeine
~ml + 2 5 mM
AFTER
em, 1.25% broth.
. ~4500
. 193
.
3
caffeine
2.5 mM
~1- e m +
OF HIS+MUTATIONS
broth supplement;
0.5
acids b
amino
~em a
1
ON RECOVERY
values are average number
acids, pool of 18 common
c Experimental
b Amino
1744 ~4500
115 502
20 c
8
em a
MEDIA
0.625% (V/V) liquid Difco-nutrient
-~4500
20 40
1 a ~em,
-405
2.5
5 10
6
--
0
plate)
OFPLATING
EFFECTS
MTT (nmoles/
TABLE2
3500 ~4500
719 1588
101
0
methionine
em + amino acids +
METHOD
b~
28 Str R bacteria and selection of mutations to Ery a followed procedures outlined by Herriott [10] with the modifications noted elsewhere [25]. Briefly, native DNA was extracted and purified by the chloroform--alcohol procedures described by Herriott [10] except that octanol replaced isoamyl alcohol, as recommended by Herriott (personal communication). For mutagenesis tests, 10 pg/ml DNA was suspended in 150 mM NaC1, 200 mM phosphate buffer, pH 7.4, 37°C, in the presence of varying concentrations of triazene. After 30 min at 37°C, samples were diluted 100-fold into ice-cold 150 mM NaC1, 100 mM phosphate buffer, pH 7.61, then 4-fold into HI broth [10] containing 5 ?( 10a/ml competent H. influenzae bacteria. The transformation mixtures were incubated for 30 min at 37°C and 2 ml distributed on 4 plates of selective medium for scoring of antibiotic resistant mutants as described elsewhere [25]. S. typhimurium strains grown overnight in Difco-nutrient broth were exposed to triazene under two sets of conditions in parallel experiments performed simultaneously by two different investigators. In one series, the directplating assay of Ames et al. [1] was used. In the second series, a preincubation test was used [cf. 33]. 1 part of cold triazene solution was added to 9 parts of bacterial suspension at 37°C. After 2 rain incubation, samples of 0.1 ml were spread directly onto each of duplicate or triplicate plates of minimal E medium [28] containing 0.2% glucose and supplemented with 1.25% (V/V) liquid Difco-nutrient broth (em plates) and, when necessary, 4 ~g/ml D-biotin. Other supplements used in one series of tests are described in the legend to Table 2. Plates were scored for His* revertants after 48 h incubation or, when amino acid pool was used (Table 2), after 20 h incubation at 37°C. 2 min was used for the preincubation period since there was no increase in the number of revertant colonies with longer incubation and, in fact, some decrease after about 5--6 min of triazene--ethanol exposure. A similar level of mutation induction was noted for bacterial cultures exposed to triazene directly in nutrient broth as for cultures previously sedimented, washed, and resuspended in 0.1 M phosphate buffer, pH 7.6. The cyclic triazene (A2-triazoline) was a gift of Dr. C. Ticozzi (Politecnico di Milano, Instituto di Chimica, Milano, Italy) and was synthesized by previously described methods [6,7]. Methyl-p-tolyl triazene (MTT)was synthesized as described by White et al. [29]. Immediately before use, each triazene was dissolved and serially diluted in 95% ethanol at 4°C. Operations were performed in subdued light as MTT was found to be slightly photosensitive (at 37°C). A repeated set of mutagenicity tests performed at the end of the experiment (with strain hisG46) ensured that no significant inactivation of triazene had occurred during the course of the experimental period. Results Data in Table 1 indicate that mutagenicity by methyl-p-tolyl triazene (MTT) is highly selective for reversion of S. typhimurium strains carrying the hisG46 base-substitution mutation. No mutagenicity is detected for frameshift mutation hisD3052. The strains tested carrying the hisG46 mutation appear approximately equally sensitive to mutagenesis, independent of genetic background.
29
8000}-
fx
4000-
his
G46
"
x/
"
t
hi I-.J QI--
Z
2000
--
1000 800
-
H
•
CH3@NfN_~_CH HE MOPHI LUS
~DNA~I I //
600
40o-
3
PREINCUBATION
/
/
ZOO -
AMES
II
O0 - -
/// i/ &/ /
80
60 40o
20
~/
I
//
x~~O ~
¢
/ / c.3c.2-~.--,c.2 /
I
N
~"N'CH2
C
C,
/ I0
I
~
2
-
4
-
8 I0 20 nmoles
40
80 000
TRIAZENE
200
400
800 iO0O 2000 4000
/ PLATE
Fig. 1. D o s e - - - r e s p o n s e r e l a t i o n s h i p s for t w o t r i a z e n e s t h a t axe d i r e c t - a c t i n g m u t a g e n s f o r S. t ~ p h i m u r i u m strain h i s G 4 6 a n d for native H. i n f l u e n z a e cell-free D N A . R e s p o n s e s t o M T T ( f o r m u l a at t o p , c e n t e r ) are s h o w n f o r i n d u c t i o n o f h i s G 4 6 + m u t a t i o n s b y t h e p r e i n c u b a t i o n m e t h o d ( . ) a n d b y the s t a n d a r d A m e s p r o c e d u r e ( X ) a n d for i n d u c t i o n o f E r y R m u t a t i o n in H. i n f l u e n z a e DNA (A). S i m i l a r l y , r e s p o n s e s t o t h e c y c l i c t r i a z e n e ( f o r m u l a at b o t t o m , c e n t e r ) are s h o w n for t h e p r e i n c u b a t i o n m e t h o d ( o ) a n d for t h e s t a n d a r d A m e s p r o c e d u r e (•) u s i n g h i s G 4 6 as w e l l as for H. i n f l u e n z a e DNA (.).
Dose--response curves for mutagenesis of S. typhimurium strain hisG46 by MTT (formula at top, center of Fig. 1) are shown for the preincubation method ( . ) and for the standard Ames assay where mutagen is incorporated directly into top agar (×). The preincubation method is roughly 50-fold more sensitive for detection of MTT mutagenicity. Analogous data are shown for the cyclic triazene (formula at bottom, center of Fig. 1) by the preincubation (©) and standard Ames ((}) tests. Mutant yields for induction of Ery R mutations in H. influenzae native D N A are depicted for MTT (A) and for cyclic triazene (m). No microsomal extract was added in the above experiments (Table 1, Fig. 1). In fact, addition of S-9 mix [1] lowered mutant yield slightly when the standard Ames procedure was used. We conclude that the triazenes tested are direct-acting mutagens with ability to mutate native, purified cell-free D N A as well as readily penetrate and cause base-substitution mutations in Salmonella bacteria. A variety of c o m p o u n d s which are not mutagenic themselves have been found to raise or to decrease induced mutant yield in bacteria [2]. Data in Table 2 indicate that the presence of 2.5 or 5 mM caffeine in the plating medium may decrease mutant yield and that a pool of c o m m o n L-amino acids (lacking histidine) increases mutant yield. Addition of the amino acid pool also
30
allows scoring of plates after overnight incubation tion customary in the standard Ames test.
instead
of the 48 h incuba-
Discussion Our data indicate that a linear aryl-monoalkyl triazene is a direct-acting mutagen for S. typhimurium bacteria and for H. influenzae cell-free DNA. Base-substitution mutations are induced, in keeping with observations on N. crassa [ 161. These results are in accord with the mechanisms by which linear aryl-dialkyl triazenes are considered to exhibit genetic activity (see Introduction) and the observation of nucleic acid alkylation in vitro by an aryl-monoalkyl triazene [ 181. Mutation induction by MTT shares two features that we also have found for the direct-acting base-substitution mutagen N-methyl-N-nitrosoacetamide (to be submitted). There is little effect on mutation frequency when u&3 or rfa mutations or when plasmid pKMlO1 is present in the test strain (Table 1). In addition, the mutation frequency is proportional to dose, but the slope of the response curve is significantly greater than 1 (Fig. 1). Departure from strict proportionality is not merely due to cell penetration, for tests on treated cellfree DNA also depart from unity (Fig. 1). Two attractive and mutually compatible explanations for these phenomena are: (a) bacteria contain an extremely active system, independent of the uurAB system, that elicits effective error-free repair of MTT-induced genetic lesions, perhaps a certain class of alkylation damage, and (b) multiple lesions are required for mutagenesis. Further
N”2 N
N’
so
‘I? @ -so /
NITROSAMINE
\
0 NH2
N=N
HONO
H-
N
Nk POLYAMINE
H-
-7
‘R
DIAZONIUM ION
-H20
N \‘N NCR TRIAZENE
I 0 + N2 c
N,H R
CARBONIUM ION Fig. 2. Speculative reaction mechanisms for cyclic triazene formation from naturally occurring PdYamines and synthetic primary diamines during treatment with nitrous acid (BONO). R. -H in simple diamines while R can have varying structures in spermidine. spermine and other polyamines carrying secondary amino groups; the upper pathway of cyclic triazene formation would apply only to this latter class of comPounds.
31
work will be necessary to elucidate the operative mechanism(s). In any event, departure of certain dose--response curves from strict proportionality poses problems for the calculation of comparative mutagenicity of various chemicals and, perhaps, for the organ-specificity of carcinogens. The current report is the first to demonstrate mutagenicity by a cyclic triazene. Our interest in the cyclic triazene was elicited by our observation of the production of unstable, direct-acting mutagens after nitrosation of various diamines and polyamines [25] and the consideration that formation of cyclic triazenes might allow DNA alkylation (Fig. 2) [24]. Tannenbaum et al. [23] have observed a product formed by nitrosation of n-butylamine whose mass spectrum is suggestive of a "linear N-nitrosotriazene structure", and Endo et al. [3] have observed the formation of a diphenyltriazene derivative from a sulfonamide and nitrite under gastric conditions. In these latter cases, triazene formation would be second-order with respect to substrate, whereas in the case of a diamine the reaction might not be so limited with respect to substrate since the attacking group would be located on the same molecule. However, to date we have been unable to provide direct demonstration of triazene formation in diamine + nitrous acid reaction mixtures, and an alternate explanation namely C- and/or O-nitrosation of deamination products, has been proposed for the mutagenicity detected [25]. Acknowledgements We are indebted to Drs. Calimero Ticozzi and Hugh J. Creech for supplying the cyclic triazene and ICR-191, respectively. This investigation was supported by Public Health Service Research Grants AI-01650 from the National Institute of Allergy and Infectious Diseases (to P.E.H.) and CA-18387 from the National Cancer Institute {to E.H.W.). References 1 A m e s , B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s for d e t e c t i n g c a r c i n o g e n s and m u t a g e n s with the S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 Clarke, C.H. a n d D.M. S h a n k e l , A n t i m u t a g e n e s i s in m i c r o b i a l s y s t e m s , Bacteriol. R e v . , 39 ( 1 9 7 5 ) 33--53. 3 E n d o , H., H. N o d a , N. K i n o s h i t a , N. I n u i , M. U m e d a a n d T. Ochi, F o r m a t i o n o f d i p h e n y l t r i a z e n e derivatives f r o m a s u l f o n a m i d e a n d n i t r i t e u n d e r gastric c o n d i t i o n s , s u b m i t t e d f o r p u b l i c a t i o n . 4 Fahrig, R., M e t a b o l i c a c t i v a t i o n of a x y l d i a l k y l t r i a z e n e s in the m o u s e : i n d u c t i o n of m i t o t i c gene c o n v e r s i o n in S a c c h a r o m y c e s cerevisiae in the h o s t - m e d i a t e d assay, M u t a t i o n Res., 13 ( 1 9 7 1 ) 4 3 6 - - 4 3 9 . 5 F a h r i g , R., D e v e l o p m e n t of h o s t - m e d i a t e d m u t a g e n e c i t y tests, 1. D i f f e r e n t i a l r e s p o n s e o f y e a s t cells i n j e c t e d into t e s t e s o f rats a n d p e r i t o n e u m o f m i c e a n d rats to m u t a g c n s , M u t a t i o n Res., 26 ( 1 9 7 4 ) 29--36. 6 G a u d i a n o , G., C. T i c o z z i , A. U m a n i - R o n c h i a n d P. B r a v o , Sulla r e a z i o n e t r a a z i d i e ilidi dello z o l f o : u n a n u o v a sintesi di kx2-1,2,3-triazoline, Gaz. C h i m . Ital., 97 ( 1 9 6 7 ) 1 4 1 1 - - 1 4 2 2 . 7 G u a d i a n o , G., A. U m a n i - R o n c h i , P. B r a v o a n d M. A c a m p o r a , D i m e t h y l o x o s u l p h o n i n m m e t h y l i d e : A tool for the s y n t h e s i s of f i v e - m e m b e r e d h e t e r o c y c l e s , T e t r a h e d r o n L e t t . , ( 1 9 6 7 ) 1 0 7 - - 1 1 1 . 8 H a r t m a n , P.E., Z. H a r t m a n , R.C. Stahl and B.N. A m e s , Classification and m a p p i n g of s p o n t a n e o u s a n d i n d u c e d m u t a t i o n s in t h e histidine o p e r o n of S a l m o n e l l a , Adv. G e n e t . , 16 ( 1 9 7 1 ) 1--34. 9 H a r t m a n , P.E., K. L e v i n e , Z. H a r t m a n and H. Berger, H y c a n t h o n e : A f r a m e s h i f t m u t a g e n , Science, 172 (1971) 1058--1060. 10 H e r r i o t t , R.M., E f f e c t s o n D N A : T r a n s f o r m i n g principle, in: A. H o l l a e n d e r ( E d . ) , C h e m i c a l M u t a g e n s , Principles a n d M e t h o d s f o r T h e i r D e t e c t i o n , Vol. 1, P l e n u m , N e w Y o r k , 1 9 7 1 , pp. 1 7 5 - - 2 1 7 . 11 K o l a r , G . F . , R. F a h r i g a n d E. Vogel, S t r u c t u r e - - a c t i v i t y d e p e n d e n c e in s o m e n o v e l r i n g - s u b s t i t u t e d
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