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Mutagenicity of 4-nitropyridine-1-oxide for Salmonella typhimurium
371
Mutatton Research, 58 ( 1 9 7 8 ) 3 7 1 - - 3 7 4 © E l s e v i e r / N o r t h - H o l l a n d B m m e d l c a l Press
Short Communication
MUTAGENICITY OF 4-NITROPYRIDINE-1-OXIDE F O R Salmonella typbimurium *
A N G E L A E. A U L E T T A a n d P A U L J P R I C E
JRB Assoctates, Inc , McLean, Va 22101, and Mwrobmlogwal Assoctates, Torrey Pines Research Center, La JoUa, Cahf 92037 (U.S A.) ( R e c e i v e d 31 J a n u a r y 1 9 7 8 ) ( R e w s m n received 11 May 1 9 7 8 ) ( A c c e p t e d 22 May 1 9 7 8 )
4-Nltropyndme-l-oxide (4-NPO) is a known carcinogen [3] which has prevlsouly been reported to induce DNA repair synthesis in human cells in culture [7], to preferentially inhibit the growth of polA- strains of Eschenchia coli [5] and to exert a cocarcinogenic effect in the transformation of cells in culture [6]. It has n o t previously been reported to be mutagenm in bacteria, however. Accordingly, 4-NPO was screened for mutagenic activity in Salmonella typhim u r m m and for its ability to inhibit repair-deficient strains of this organism. Mutagemcity was determmed by the method of Ames et al. [2] with S. typhimurium stran TA100. This strain is a histidine auxotroph whmh can be reverted to p r o t o t r o p h y by agents which cause either base-pair or frameshlft mutations. Strain TA100 carries an R factor plasmld, p k m l 0 1 , which confers resistance to ampmillin and concomitantly enhances sensitivity to mutation [4]. In subsequent studies, 4-NPO failed to reduce mutation with any of the other Salmonella tester strains in the present series (TA1535, TA1537, TA1538, or TA98). Enzyme activation with liver microsomes was n o t performed because 4-NPO proved to be a direct acting mutagen for S. typhtmurlum For the spot test, 0.1 ml of bacterial culture was added to 2 ml of soft agar overlay supplemented with trace amounts of histidine and biotin. The agar was mixed and poured over a base plate of Spizzen's minimal m e d m m [8] and 25 pg of 4-NPO in DMSO was added to the center of the plate. The plate was scored for the presence of m u t a n t colomes after 48 h at 37°C. For the pour plate assay, 0.1 ml of 4-NPO at the appropriate concentrahon was incorporated into the top agar along with the bacteria. Plates were poured as noted * Supported m part by Contract N01-CP-43240, section B, from the National Cancer Institute, NIH, DHEW, Bethesda, Md. (U.S.A)
Abbrevmt~ons MMS, methyl methanesulfonate, MNNG, N-methyl-N'onltro-Nonltrosoguamdme, 4-NPO, 4-mtropyndme-l-oxlde, 4-NQO, 4-mtroqmnohne-N-oxlde.
372
above. Revertant colonies were counted after 48 h at 37 ° C. The repair test with S. typhzmurium strains TA1538 and TA1978 was performed according to the m e t h o d of Ames et al. [1]. Strain TA1538 has a deletion through the uvrB region of the chromosome which has eliminated excision repair m this organism. Strain TA1978 has an excision repair system. Both strmns contain the h~sD3052 m u t a t i o n and an additional rfa m u t a t i o n which has removed the lipopolysaccharide coat and made the bacteria permeable to large agents [ 1 ]. For the assay, plates were poured as noted for the spot test, except the top agar contmned an excess of histidlne to allow for full growth of the culture. 44-NPO was loaded onto 7-mm discs in the center of the plate, plates were incubated overnight at 37°C and the zone of inhibition for each strain measured and compared. The results of the spot test for mutageniclty are shown in Fig. 1; the results
Fig. I Spot test for mutagemelty of 4 - N P O wlth strata T A 1 0 0 . To]) spontaneous revertants. B o t t o m left 10 #g 4 - N Q O B o t t o m rzght 25 #g 4 - N P O
373 400
S typhlmurlum [TA 100]
L.
300 n"
~- 200 -r
E ~ I00
;'2
2',
32
IJg NPO per Plate Flg 2. D o s e - - r e s p o n s e c u r v e of 4 - N P O - m d u c e d m u t a n t s w i t h S. tyhp~rnurzum s t r a t a T A 1 0 0 .
o f the quantitative pour plate assay are shown in Fig. 2. The n u m b e r of spontaneous reversions occurred in the u n t r e a t e d cont rol plates was substracted f r o m the n u m b e r of reduced m ut a nt s before the graph in Fig. 2 was plotted. In Fig. 1, 10 #g per plate 4-mtroquinoline-N-oxide (4-NQO) is shown on the b o t t o m left and 25 #g 4-NPO is shown on the b o t t o m right. T r e a t m e n t with 4-NPO resulted in fewer induced mutants and a smaller zone of inhibition than t r e a t m e n t with 4-NQO, a know n p o t e n t carcinogen and mutagen. In the quantitative pour plate assay, t r e a t m e n t with 4-NPO p r o d u c e d a typical dose--response curve of mutagen-induced revertant colonies. The results o f the repair test with strains T A 1 9 7 8 and T A 1 5 3 8 are shown m Table 1. There was no appreciable difference in the zone of inhibition o f either strain with 4-NPO. This is m essential agreement with the results o f Nagao and Sugimura [5]. These authors f o u n d t hat 4-NPO preferentially inhibited the growth o f polA- strains of E. coh over the parent wild-type strains but had no differenhal effect on cell kill between parent and UV-sensihve mutants of B. subtilis and S. cerevzsiae. 4-NQO preferentially inhibited the m u t a n t strains over t h e parents in all instances. The authors postulated that there may be differences m the mechanisms o f repair of DNA damage by agents such as 4-NQO on the one hand and 4-NPO on the other. T he UV-sensltive strains of B subtzhs and S. cereviszae were also f o u n d to be insensitive to m e t h y l m et hanesul fonat e
TABLE 1 I N H I B I T I O N OF S A L M O N E L L A Concentration (#g/plate)
50 25 10 2
T Y P H I M U R I U M M U T A N T S BY 4-NPO
D i a m e t e r o f z o n e of m h z b l t l o n ( r a m ) T A 1 9 7 8 ( r e p a i r +)
TA1538 (repaxr--)
9 5 8 5 5.7 0
10 8.5 5 2 0
374
(MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), thus suggesting a c o m m o n factor in the mechanism of repmr of these agents and 4-NPO. Strain T A 1 5 3 8 is a uvrB mutant whmh is sensitive to both UV and 4-NQO but is insensitive to MMS and MNNG. In this study, there was no preferential lnhibltmn o f strain T A 1 5 3 8 over strain T A 1 9 7 8 by 4-NPO giving added evidence that the repmr mechanism for agents such as 4-NQO and UV. There is presently much interest in use of short-term tests as prescreens for potentmlly n o x i o u s agents. The results reported here, although limited m scope, support the use of the Salmonella/microsomal assay as a prescreen for potential carcinogens. The results of the repmr test suggest that the polAmutants of E. colt whmh appear to be sensitive to a broader range o f comp o u n d s than UV mutants of other organisms are the organisms of chome for use m such an assay system. However, since the total number of agents tested in these systems is still rather small, they should be used in c o n j u n c t i o n with other short-term assays until the data base on whmh to vahdate their performance has been broadened. References I A m e s , B N , F.C L e e a n d W.E. D u r s t o n , A n i m p r o v e d b a c t e r m l t e s t s y s t e m f o r t h e d e t e c t i o n a n d classif i c a t i o n of m u t a g e n s a n d c a r c i n o g e n s , P r o c Natl. A c a d Scl ( U . S . A . ) , 70 ( 1 9 7 3 ) 7 8 2 - - 7 8 6 . 2 A m e s , B.N., J M c C a n n a n d E Y a m a s a k l , 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 a n d m u t a g e n s w i t h t h e salm o n e l l a / m a m m a h a n - m m r o s o m e m u t a g e m c l t y test, M u t a h o n R e s , 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 K a w a z o e , Y , a n d M. Aralcl, C h e m m a l p r o b l e m s m 4 N Q O c a r c i n o g e n e s i s , m W N a k a h a r a ( E d . ) C h e m m a l T u m o r P r o b l e m s , J a p a n e s e S o c i e t y f o r t h e A d v a n c e m e n t o f Science, T o k y o , 1 9 7 0 , p p 4 5 - 104. 4 M c C a n n , J , N E S p m g a r n , J K o b o r l a n d B N A m e s , D e t e c h o n of c a r c i n o g e n s as m u t a g e n s B a c t e r m l t e s t e r s t r a t u s w i t h R f a c t o r p l a s m l d s , Proc N a t l . A c a d Scl (U S.A ), 72 ( 1 9 7 5 ) 9 7 9 - - 9 8 3 5 N a g a o , M , a n d T. S u g n n u r a , S e n s l h v l t y o f r e p a l r - d e f m m n t m u t a n t s a n d similar m u t a n t s t o 4 - m t r o q u m o h n e 1 - o x i d e , 4 - n l t r o p y r l d m e 1 - o x i d e , a n d t h e i r d e r i v a t i v e s , C a n c e r Res , 32 ( 1 9 7 2 ) 2 3 6 9 - - 2 3 7 4 6 Price, P J , A E A u l e t t a , M P K i n g , P M H u g m m n a n d R J H e u b n e r , T h e c o - c a r c m o g e m c a c h v l t y of 4 - m t r o p y n d m e - l - o x l d e ( 4 - N P O ) a n d p r e v e n t i o n of t r a n s f o r m a t i o n b y t y p e - s p e c i f i c a n h - v l r a l antib o d i e s , I n v i t r o , 12 ( 1 9 7 6 ) 5 9 5 - - 5 9 8 . 7 Stlch, H . F , R H C San a n d Y. K a w a z o e , D N A r e p a i r s y n t h e s i s m m a m m a h a n cells e x p o s e d to a s e r i e s of o n c o g e m c a n d n o n - o n c o g e m c d e r i v a t i v e s o f 4 - n l t r o q u m o h n e 1-oxide, N a t u r e ( L o n d o n ) , 2 2 9 ( 1 9 7 1 ) 416--419 8 S p l z z l z e n , J , T r a n s f o r m a h o n of b l o c h e m m a U y d e h c l e n t stratus of Baczllus subt~lls b y d e o x y n b o n u c l e a t e , Proc Natl A c a d Scl ( U S A ), 44 ( 1 9 5 8 ) 1 0 7 2 - - 1 0 7 8
Mutatton Research, 58 ( 1 9 7 8 ) 3 7 1 - - 3 7 4 © E l s e v i e r / N o r t h - H o l l a n d B m m e d l c a l Press
Short Communication
MUTAGENICITY OF 4-NITROPYRIDINE-1-OXIDE F O R Salmonella typbimurium *
A N G E L A E. A U L E T T A a n d P A U L J P R I C E
JRB Assoctates, Inc , McLean, Va 22101, and Mwrobmlogwal Assoctates, Torrey Pines Research Center, La JoUa, Cahf 92037 (U.S A.) ( R e c e i v e d 31 J a n u a r y 1 9 7 8 ) ( R e w s m n received 11 May 1 9 7 8 ) ( A c c e p t e d 22 May 1 9 7 8 )
4-Nltropyndme-l-oxide (4-NPO) is a known carcinogen [3] which has prevlsouly been reported to induce DNA repair synthesis in human cells in culture [7], to preferentially inhibit the growth of polA- strains of Eschenchia coli [5] and to exert a cocarcinogenic effect in the transformation of cells in culture [6]. It has n o t previously been reported to be mutagenm in bacteria, however. Accordingly, 4-NPO was screened for mutagenic activity in Salmonella typhim u r m m and for its ability to inhibit repair-deficient strains of this organism. Mutagemcity was determmed by the method of Ames et al. [2] with S. typhimurium stran TA100. This strain is a histidine auxotroph whmh can be reverted to p r o t o t r o p h y by agents which cause either base-pair or frameshlft mutations. Strain TA100 carries an R factor plasmld, p k m l 0 1 , which confers resistance to ampmillin and concomitantly enhances sensitivity to mutation [4]. In subsequent studies, 4-NPO failed to reduce mutation with any of the other Salmonella tester strains in the present series (TA1535, TA1537, TA1538, or TA98). Enzyme activation with liver microsomes was n o t performed because 4-NPO proved to be a direct acting mutagen for S. typhtmurlum For the spot test, 0.1 ml of bacterial culture was added to 2 ml of soft agar overlay supplemented with trace amounts of histidine and biotin. The agar was mixed and poured over a base plate of Spizzen's minimal m e d m m [8] and 25 pg of 4-NPO in DMSO was added to the center of the plate. The plate was scored for the presence of m u t a n t colomes after 48 h at 37°C. For the pour plate assay, 0.1 ml of 4-NPO at the appropriate concentrahon was incorporated into the top agar along with the bacteria. Plates were poured as noted * Supported m part by Contract N01-CP-43240, section B, from the National Cancer Institute, NIH, DHEW, Bethesda, Md. (U.S.A)
Abbrevmt~ons MMS, methyl methanesulfonate, MNNG, N-methyl-N'onltro-Nonltrosoguamdme, 4-NPO, 4-mtropyndme-l-oxlde, 4-NQO, 4-mtroqmnohne-N-oxlde.
372
above. Revertant colonies were counted after 48 h at 37 ° C. The repair test with S. typhzmurium strains TA1538 and TA1978 was performed according to the m e t h o d of Ames et al. [1]. Strain TA1538 has a deletion through the uvrB region of the chromosome which has eliminated excision repair m this organism. Strain TA1978 has an excision repair system. Both strmns contain the h~sD3052 m u t a t i o n and an additional rfa m u t a t i o n which has removed the lipopolysaccharide coat and made the bacteria permeable to large agents [ 1 ]. For the assay, plates were poured as noted for the spot test, except the top agar contmned an excess of histidlne to allow for full growth of the culture. 44-NPO was loaded onto 7-mm discs in the center of the plate, plates were incubated overnight at 37°C and the zone of inhibition for each strain measured and compared. The results of the spot test for mutageniclty are shown in Fig. 1; the results
Fig. I Spot test for mutagemelty of 4 - N P O wlth strata T A 1 0 0 . To]) spontaneous revertants. B o t t o m left 10 #g 4 - N Q O B o t t o m rzght 25 #g 4 - N P O
373 400
S typhlmurlum [TA 100]
L.
300 n"
~- 200 -r
E ~ I00
;'2
2',
32
IJg NPO per Plate Flg 2. D o s e - - r e s p o n s e c u r v e of 4 - N P O - m d u c e d m u t a n t s w i t h S. tyhp~rnurzum s t r a t a T A 1 0 0 .
o f the quantitative pour plate assay are shown in Fig. 2. The n u m b e r of spontaneous reversions occurred in the u n t r e a t e d cont rol plates was substracted f r o m the n u m b e r of reduced m ut a nt s before the graph in Fig. 2 was plotted. In Fig. 1, 10 #g per plate 4-mtroquinoline-N-oxide (4-NQO) is shown on the b o t t o m left and 25 #g 4-NPO is shown on the b o t t o m right. T r e a t m e n t with 4-NPO resulted in fewer induced mutants and a smaller zone of inhibition than t r e a t m e n t with 4-NQO, a know n p o t e n t carcinogen and mutagen. In the quantitative pour plate assay, t r e a t m e n t with 4-NPO p r o d u c e d a typical dose--response curve of mutagen-induced revertant colonies. The results o f the repair test with strains T A 1 9 7 8 and T A 1 5 3 8 are shown m Table 1. There was no appreciable difference in the zone of inhibition o f either strain with 4-NPO. This is m essential agreement with the results o f Nagao and Sugimura [5]. These authors f o u n d t hat 4-NPO preferentially inhibited the growth o f polA- strains of E. coh over the parent wild-type strains but had no differenhal effect on cell kill between parent and UV-sensihve mutants of B. subtilis and S. cerevzsiae. 4-NQO preferentially inhibited the m u t a n t strains over t h e parents in all instances. The authors postulated that there may be differences m the mechanisms o f repair of DNA damage by agents such as 4-NQO on the one hand and 4-NPO on the other. T he UV-sensltive strains of B subtzhs and S. cereviszae were also f o u n d to be insensitive to m e t h y l m et hanesul fonat e
TABLE 1 I N H I B I T I O N OF S A L M O N E L L A Concentration (#g/plate)
50 25 10 2
T Y P H I M U R I U M M U T A N T S BY 4-NPO
D i a m e t e r o f z o n e of m h z b l t l o n ( r a m ) T A 1 9 7 8 ( r e p a i r +)
TA1538 (repaxr--)
9 5 8 5 5.7 0
10 8.5 5 2 0
374
(MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), thus suggesting a c o m m o n factor in the mechanism of repmr of these agents and 4-NPO. Strain T A 1 5 3 8 is a uvrB mutant whmh is sensitive to both UV and 4-NQO but is insensitive to MMS and MNNG. In this study, there was no preferential lnhibltmn o f strain T A 1 5 3 8 over strain T A 1 9 7 8 by 4-NPO giving added evidence that the repmr mechanism for agents such as 4-NQO and UV. There is presently much interest in use of short-term tests as prescreens for potentmlly n o x i o u s agents. The results reported here, although limited m scope, support the use of the Salmonella/microsomal assay as a prescreen for potential carcinogens. The results of the repmr test suggest that the polAmutants of E. colt whmh appear to be sensitive to a broader range o f comp o u n d s than UV mutants of other organisms are the organisms of chome for use m such an assay system. However, since the total number of agents tested in these systems is still rather small, they should be used in c o n j u n c t i o n with other short-term assays until the data base on whmh to vahdate their performance has been broadened. References I A m e s , B N , F.C L e e a n d W.E. D u r s t o n , A n i m p r o v e d b a c t e r m l t e s t s y s t e m f o r t h e d e t e c t i o n a n d classif i c a t i o n of m u t a g e n s a n d c a r c i n o g e n s , P r o c Natl. A c a d Scl ( U . S . A . ) , 70 ( 1 9 7 3 ) 7 8 2 - - 7 8 6 . 2 A m e s , B.N., J M c C a n n a n d E Y a m a s a k l , 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 a n d m u t a g e n s w i t h t h e salm o n e l l a / m a m m a h a n - m m r o s o m e m u t a g e m c l t y test, M u t a h o n R e s , 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 K a w a z o e , Y , a n d M. Aralcl, C h e m m a l p r o b l e m s m 4 N Q O c a r c i n o g e n e s i s , m W N a k a h a r a ( E d . ) C h e m m a l T u m o r P r o b l e m s , J a p a n e s e S o c i e t y f o r t h e A d v a n c e m e n t o f Science, T o k y o , 1 9 7 0 , p p 4 5 - 104. 4 M c C a n n , J , N E S p m g a r n , J K o b o r l a n d B N A m e s , D e t e c h o n of c a r c i n o g e n s as m u t a g e n s B a c t e r m l t e s t e r s t r a t u s w i t h R f a c t o r p l a s m l d s , Proc N a t l . A c a d Scl (U S.A ), 72 ( 1 9 7 5 ) 9 7 9 - - 9 8 3 5 N a g a o , M , a n d T. S u g n n u r a , S e n s l h v l t y o f r e p a l r - d e f m m n t m u t a n t s a n d similar m u t a n t s t o 4 - m t r o q u m o h n e 1 - o x i d e , 4 - n l t r o p y r l d m e 1 - o x i d e , a n d t h e i r d e r i v a t i v e s , C a n c e r Res , 32 ( 1 9 7 2 ) 2 3 6 9 - - 2 3 7 4 6 Price, P J , A E A u l e t t a , M P K i n g , P M H u g m m n a n d R J H e u b n e r , T h e c o - c a r c m o g e m c a c h v l t y of 4 - m t r o p y n d m e - l - o x l d e ( 4 - N P O ) a n d p r e v e n t i o n of t r a n s f o r m a t i o n b y t y p e - s p e c i f i c a n h - v l r a l antib o d i e s , I n v i t r o , 12 ( 1 9 7 6 ) 5 9 5 - - 5 9 8 . 7 Stlch, H . F , R H C San a n d Y. K a w a z o e , D N A r e p a i r s y n t h e s i s m m a m m a h a n cells e x p o s e d to a s e r i e s of o n c o g e m c a n d n o n - o n c o g e m c d e r i v a t i v e s o f 4 - n l t r o q u m o h n e 1-oxide, N a t u r e ( L o n d o n ) , 2 2 9 ( 1 9 7 1 ) 416--419 8 S p l z z l z e n , J , T r a n s f o r m a h o n of b l o c h e m m a U y d e h c l e n t stratus of Baczllus subt~lls b y d e o x y n b o n u c l e a t e , Proc Natl A c a d Scl ( U S A ), 44 ( 1 9 5 8 ) 1 0 7 2 - - 1 0 7 8