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The mutagenicity of N-nitrosopiperidines for Salmonella typhimurium
85
Mutation Research, 57 ( 1 9 7 8 ) 8 5 - - 8 9 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
Short Communication THE MUTAGENICITY OF N-NITROSOPIPERIDINES FOR SALMONELLA TYPHIMURIUM
E R R O L Z E I G E R * a n d A L B E R T T. S H E L D O N
Division of Toxicology, Food and Drug Administration, Washington, D.C. 20204 (U.S.A.) ( R e c e i v e d 18 J u l y 1 9 7 7 ) ( R e v i s i o n received 1 N o v e m b e r 1 9 7 7 ) (Accepted 9 November 1977)
Introduction NPIPD induces tumors in the nose and upper gastrointestinal tract of rats [3,8--10] and lung adenomas in mice [6]. It is also mutagenic for E. coli [4] and S. typhimurium in the presence of an in vitro metabolic activation system [2,7,11,15] and for S. typhimurium in the host-mediated assay (E. Zeiger, unpublished), and produces single-strand breaks in DNA of regenerating rat liver [13]. A number of methylated [8], oxygenated [9] and halogenated [10] nitrosopiperidines are also carcinogenic in rats. In this study, NPIPD, NPDO, DCNPD, NINA and NPIPA (Fig. 1) were tested for mutagenicity. Salmonella typhimurium TA-1535, which reverts to prototrophy by base-pair substitution, was used in both suspension and plate tests, and metabolic activation was provided by liver homogenates from rats and mice. Materials and methods
The test strain Salmonella typhimurium TA-1535, was obtained from Dr. Bruce Ames. It contains a missense mutation in the G gene of the histidine operon, a deletion covering the bio uvrB loci and an rfa mutation [1]. HA (ICR)f male mice were purchased from Sprague-Dawley, Madison, Wis: and were used when they weighed approximately 30 g. Sprague-Dawley derived Osborne-Mendel rats were obtained from the FDA colony and were used at approximately 300 g. All nitrosamines were supplied by Dr. William Lijinsky, Biology Division, DCNPD, N-nitroso-3,4-dichloropiperidine; DMSO, dimethylsulfoxide; NINA, Nnitrosoisonipecotic acid; NPDO, N-nitroso-4-pipcridone; NPIPA, N-nitrosopipecolic acid; NPIPD, N-nitrosopiperidine.
Abbreviations:.
* Present address: E n v i r o n m e n t a l Mutagenesis Branch, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, N.C., 27709 (U.S.A.)
86
C:: RI
N -- NO
N-NITROSOPIPERIDINE N-NITROSO-4-PIPERIDONE N-NITROSO-3 4-DICHLOROPIPERIDINE N-NITROSOI~ONIPECOTICACID N-NITROSOPIPECOLICACID
R1
R2
R3
-H =0 -CI -H -H
-H -H -H -H -CI -H -COOH-H -H -COOH
Fig. 1. Structures of N-nitrosopipendines.
Oak Ridge National Laboratory, Oak Ridge, Tenn. NADP and glucose 6-phosphate were purchased from Sigma Chemical Co., St. Louis, Mo. DMSO was purchased from Baker Chemical Co., Phillipsburg, N.J., and was used as a solvent for NPDO, DCNPD, NINA and NPIPA. The final DMSO concentrations never exceeded 3.3% in the suspension test or 2.5% in the 2 ml of top agar in the plate test. Livers were prepared and mutagenicity tests were performed as previously described [15]. Suspension test controls included the high dose of the test compound in the presence of heat-inactivated (100°C/10 min) homogenate. Plate test controls included the high dose in the absence of the supernatant of homogenate centrifuged at 9000 ×g (S-9). Results and discussion NPIPD, NPDO and DCNPD were mutagenic in both the plate and suspension tests with both mouse- and rat-liver preparations (Table 1). The mutagenicity of NPDO was significantly less than that of NPIPD; this finding was unexpected since the compounds were of equivalent carcinogenicity in rat feeding studies, producing tumors primarily in the nasal cavity, oral cavity and esophagus in male rats [9]. In addition, since NPIPD is metabolized to NPDO by rat-liver microsomes in vitro [12], the mutagenicity and carcinogenicity results would imply that NPDO is not an intermediate in the activation of NPIPD to its ultimate mutagenic form. The mutagenic response of NPDO shows that mammalian metabolism is required for its activity, although the rat non-activation controls (50.0 /~moles) showed mutagenic activity equivalent to that which would occur with between 1.0 and 5.0 pmoles of metabolized NPDO. DCNPD is the most potent of the carcinogenic heterocyclic nitrosamines in the rat, inducing tumors in the tongue, pharynx, esophagus and non-glandular stomach [10; W. Lijinsky, personal communication]. It gave a relatively low mutagenic response in the rat-liver activation plate and suspension tests (Table 1). The plate tests with rat-liver activation gave a positive result only in the 1.0--50.0 pmole dose range; however, the unmetabolized 50.0-pmole control showed that at this dose range DCNPD did not require metabolism for its in vitro mutagenicity. This could be the result of a spontaneous breakdown of
87 TABLE 1 RESPONSE OF SALMONELLA TYPHIMURIUM TO N-NITROSOPIPERIDINE, AND N-NITROSO-3,4-DICHLOROPIPERIDINE a
MUTAGENIC PIPERIDONE
Cornpound
Dose level (#moles)
Mouse-liver activation b
S u s p e n s i o n test
NPDO
0 0.01 0.05 0.10 0.50 1.00 1.00 c
Plate test
S u s p e n s i o n test
Plate test
Surrival
his +
his +
Stur-
his +
revertants/
revertants/
vival
108 cells
(%)
plate
108 cells
(%)
revertants/ plate
0.76 -7.71 9.45 25.30 31.64 _
100.0 -111.2 119.8 93.2 94.4 --
11.0 470.3 1256.3 2254.0 4324.3 7596.0 10.0
0.56 -2.56 4.98 15.22 16.13 0.93
+ 0.26 ± 0.75 ± 5.02 ± 1.22 _+ 0 . 1 2
100.0 -105.2 95.4 81.9 104.5 85.6
21.5 41.3 175.0 168.7 1248.0 1750.0 17.0
0 . 6 2 _+ 0 . 0 7
100.0
± 0.14 ,+ 0 . 5 8 ± 0.89 + 1.18 ± 4.07
0 0.01
0.59 ± 0.19 --
100.0 --
0.05
0.76 ± 0.25
119.0
0.I0
2.00 ± 0.25
62.5
0.50
5 . 6 3 _+ 0 . 4 3
104.6
128.7
1.00
9 . 4 2 ,+ 0 . 2 6
94.0
119.3
-+ 5.7 + 142.9 + 139.0 ± 537.1 ± 582.5 ,+ 1 5 1 1 . 5 ± 3.0
21.0 +
14.1
20.7 ±
7.8
--
39.0 ±
14.0 6.1
± ±
6 8 . 0 _+
,+ 0 . 1 7
-+ 7.8 _+ 8.1 ± 61.9 ± 21.8 ± 633.8 ± 322.8 -~ 1.4
12.3 +
--
--
--
--
--
--
--
19.0
--
--
4 0 . 3 -+
13.0
3.63 ± 0.65
94.1
64.3 ±
2.5
--
5.00
--
--
--
5.32 ± 0.92
107.4
218.0
±
4.5 11.6 19.1
10.00
--
--
--
8.54 ± 0.33
88.3
363.0
±
30.5
50.00
--
--
--
± 1.89
75.4
492.0
_+ 3 2 . 4
4.04 ± 0.36
64.2
93.5 ±
6.4
100.0
15.3 ± --
3.2
1.00 c 50.00 c
DCNPD
Rat-liver activation
revertants/
his +
NPIPD
N-NITROSO-4-
0.71 ± 0.08 _
~
0
0.52 ± 0.10
0.01
--
0.05 0.10
100.0 --
6.18 ± 1.50 10.46
126.2
12.25
1 4 . 7 _+
4.7
--
--
9.0 ±
2.6
0 . 5 7 _+ 0 . 1 3
6 0 . 3 -+
9.5
--
--
--
--
121.8
256.7
±
52.0
1 . 1 7 _+ 0 . 4 2
95.2
--
± 1.39
106.0
499.3
±
50.7
1.54 ± 0.07
106.4
--
0.50
14.84
± 6.56
118.8
1928.7
±
846.8
3.83 + 0.51
100.7
18.7 ±
1.00
22.15
± 1.24
84.3
1582.4
_+
172.5
6 . 6 7 ,+ 0 . 4 9
96.0
31.7 ±
5.00
.
.
.
.
.
10.00
.
.
.
.
.
50.00
.
.
.
.
1.00 e 50.00 e
0.53 ± 0.04 .
.
. 108.6 .
17.3 ± .
1.5
90.3 ± ± 301.5 ± 111.0
0 . 9 2 ,+ 0 . 3 6
95.3
.
4.5 8.7 41.5 4.0 30.4
-183.0
±
8.2
a V a l u e s are given as m e a n s + S D o f three replicates. b D o s e l e v e l s axe given as # m o l e s / m l for the s u s p e n s i o n test and p m o l e s / p l a t e for the plate test. c S u s p e n s i o n test, c o n t r o l w i t h b o i l e d h o m o g e n a t e ; plate test, c o n t r o l w i t h o u t S-9.
DCNPD, the ability of the bacteria to metabolize it to an active form, or the capacity of DCNPD to cause mutation without metabolism. Both NINA and NPIPA (Table 2) caused apparent mutagenic responses at the 50.0-~mole level in all test systems, with minimal or negative responses at the 10.0-~mole level. Neither compound induced tumors in rat feeding studies [5; W. Lijinsky, personal communication]. NINA and NPIPA were the only compounds of those reported here and of the 14 previously reported [15] that produced a toxic response in the suspension tests. This toxicity was seen both in the presence and absence of metabolic activation. As was the case with DCNPD, these compounds also had mutagenic activity at 50.0 pmoles in the
88 TABLE 2 MUTAGENIC RESPONSE OF SALMONELLA N-NITROSOPIPECOLIC ACID a
Cornpound
NINA
NIPA
Dose
Mouse-liver activation
level (pmoles) b
S u s p e n s i o n test
TYPHIMURIUM TO N-NITROSOISONIPECOTIC
ACID AND
Rat-liver activation Plate test
S u s p e n s i o n test
Plate test
his +
Sur-
his +
his*
Sur-
his +
revertants/
viral
revertants/
revertants/
viral
revertants/
108 cells
(%)
plate
108 cells
(%)
plate
100.0 -113.6 120.3 118.7 16.7 2.1
12.3 27.0 23.7 42.0 40.0 158.3 302.7
100.0 -95.3 101.5 112.3 74.6 1.7
23.3 23.7 23.3 40.7 33.3 162.7 302.7
100.0 -127.4 115.0 123.3 79.4 28.7
12.7 19.3 18.0 24.7 27.0 46.3 48.7
100.0 -138.7 116.8 123.5 77.2 49.7
19.3 17.7 14.7 23.7 20.3 45.7 36.7
0 0.50 1.00 5.00 10.00 50.00 50.00 e
1.15 -0.75 0.77 0.95 424.23 59.17
0 0.50 1.00 5.00 10.00 50.00 50.00 c
0.97 -0.97 0.78 0.72 1.64 2.71
+
0.97
+ 0.26 -+ 0 . 1 4 ± 0.11 + 86.48 ± 29.46 ±
0.04
± ± + ± _+
0.31 0.18 0.06 0.60 0.10
± 3.5 ± 7.0 ± 5.0 ± 0 ± 3.6 ± 19.2 ± 14.6 ± ± ± ± ± ± ±
6.5 4.7 6.2 4.7 9.5 6.8 3.2
0.51 -0.72 0.98 1.07 43.85 406.7 d 3.14 -2.07 2.83 2.50 3.54 5.46
+
0.01
± 0.48 ± 0.12 + 0.38 ± 11.76
±
0.55
± ± ± ± ±
0.43 0.15 0.37 0.43 1.09
-+ 2 . 5 ± 9.0 + 8.1 ± 2.1 -+ 2.9 -+ 8 . 7 ± 14.6 + ± ± ± ± + ±
0.6 4.0 0.6 7.5 6.8 3.5 8.4
a V a l u e s are given as m e a n s + S D o f three replicates. b D o s e l e v e l s are given as p m o l e s / m l for the s u s p e n s i o n test and p m o l e s / p l a t e for the plate test. c S u s p e n s i o n test, c o n t r o l with boiled h o m o g e n a t e ; plate test, c o n t r o l w i t h o u t S-9.
d Single d e t e r m i n a t i o n .
absence of metabolic activation, implying a spontaneous or bacteria-catalyzed activation to an ultimate mutagen or a direct action of the compounds to cause mutation. The three carcinogens -- NPIPD, NPDO, DCNPD -- were more mutagenic in the presence of mouse-liver homogenate than with rat-liver homogenate. DCNPD, unlike the other compounds, gave non-equivalent responses in the rat suspension and plate tests. The reason for this is unknown. Previously reported results with heterocyclic nitrosamines in vivo [ 14] and in vitro [ 4,15] showed no correlation between mutagenicity and carcinogenicity, in that noncarcinogenic nitrosamines were no less mutagenic than a number of carcinogens. The responses in this study, however, did distinguish the carcinogens (NPIPD, NPDO, DCNPD) from the noncarcinogens (NINA, NPIPA), although there was no correlation among the carcinogens with regard to relative mutagenic activity. Acknowledgement I thank Dr. William Lijinsky, whose present address is Frederick Cancer Research Center, Frederick Md., for supplying the nitrosamines and his unpublished observations, as well as for the many helpful discussions.
89 Note added in proof T.K. Rao et al. [Mutation Res., 56 (1977) 131--145] have recently reported testing a series of N-nitrosopiperidines for mutagenicity, using S. typhimurium TA-1535, and have concluded that there is a correlation between mutagenicity and carcinogenic potency of these compounds. References 1 A m e s , B . N . , F . D . 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 i a 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 c l a s s i f i c a t i o n o f m u t a g e n s a n d c a r c i n o g e n s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 0 ( 1 9 7 3 ) 7 8 2 - - 7 8 6 . 2 B a r t s c h , H . , A. C a m u s a n d C. Malaveflle, C o m p a r a t i v e m u t a g e n i c i t y o f N - n i t r o s a m i n e s in a s e m i - s o l i d a n d in a l i q u i d i n c u b a t i o n s y s t e m in t h e p r e s e n c e o f r a t o r h u m a n tissue f r a c t i o n s , M u t a t i o n R e s . , 3 7 (1976) 149--162. 3 D r u e k r e y , H . , R . P r e u s s m a n n , S. I v a n k o v i c a n d D. S c h m a h l , O r g a n o t r o p e c a r c i n o g e n e W i r k u n g e n bei 6 4 v e r s c h i e d e n e n N - N i t r o s o - V e r b i n d u n g e n a n B D - R a t t e n , Z. K r e b s f o r s c h . , 6 9 ( 1 9 6 7 ) 1 0 3 - - 2 0 1 . 4 E l e s p u r u , R . K . , a n d W. L i j i n s k y , M u t a g e n i c i t y o f c y c l i c n i t r o s a m i n e s in E s c h e r i c h i a coli f o l l o w i n g a c t i v a t i o n w i t h r a t liver m i c r o s o m e s , C a n c e r R e s . , 3 6 ( 1 9 7 6 ) 4 0 9 9 - - 4 1 0 1 . 5 G a r c i a , H . , a n d W. L i j i n s k y , S t u d i e s o f t h e t u m o r i g e n i c e f f e c t o f l o w d o s e s o f a m i n e s a n d n i t r i t e s t o r a t s , Z. K r e b s f o r s c h . , 7 9 ( 1 9 7 3 ) 1 4 1 - - 1 4 4 . 6 G r e e n b l a t t , M . , a n d W. L i j i n s k y , F a i l u r e t o i n d u c e t u m o r s in Swiss m i c e a f t e r c o n c u r r e n t a d m i n i s t r a tion of amino acids and sodium nitrite, J. Natl. Cancer Inst., 48 (1972) 1389--1392. 7 H s i e h , S.-T., P . L . K r a f t , M.C. A r c h e r , a n d S . R . T a n n e n b a u m , R e a c t i o n o f n i t r o s a r n i n e s i n t h e U d e n friend system: Principal products and biological activity, Mutation Res., 35 (1976) 23--28. 8 L i j i n s k y , W., a n d H . W . T a y l o r , C a r c i n o g e n i c i t y o f m e t h y l a t e d n i t r o s o p i p e r i d i n e s , I n t . J. C a n c e r , 16 (1975) 318--322. 9 L i j i n s k y , W., a n d H.W. T a y l o r , T u m o r i g e n e s i s b y o x y g e n a t e d n i t r o s o p i p e r i d i n e s in r a t s , J. N a t l . C a n c e r Inst., 55 (1975) 705---708. 1 0 L i j i n s k y , W., a n d H.W. T a y l o r , C a r c i n o g e n i c i t y o f N - n i t r o s o - 3 , 4 - d i c h l o r o - a n d N - n i t r o s o - 3 , 4 - d i b r o m o p i p e r i d i n e in r a t s , C a n c e r R e s . , 3 5 ( 1 9 7 5 ) 3 2 0 9 - - 3 2 1 1 . 11 M c C a n n , J . , E. C h o i , E. Y a m a s a k i a n d B . N . A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s i n t h e S a l m o n e l l a / m i c r o s o m e t e s t , A s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 5 1 3 5 - 5139. 1 2 R a y m a n , M . P . , B.C. Challis, P . J . C o x a n d M. J a r m a n , O x i d a t i o n o f N - n i t r o s o p i p e r i d i n e in t h e U d e n f r i e n d m o d e l s y s t e m a n d its m e t a b o l i s m b y r a t liver m i c r o s o m e s , B i o c h e m . P h a r m a c o l . , 2 4 ( 1 9 7 5 ) 621---626. 1 3 S t e w a r t , B.W., a n d E. F a r b e r , S t r a n d b r e a k a g e in r a t liver D N A a n d its r e p a i r f o l l o w i n g a d m i n i s t r a t i o n of cyclic nitrosamines, Cancer Res., 33 (1973) 3209--3215. 1 4 Z e i g e r , E., M.S. L e g a t o r a n d W. L i j i n s k y , M u t a g e n i c i t y o f N - n i t r o s o p i p e r a z i n e s f o r S a l m o n e l l a t y p h i m u r i u m in t h e h o s t - m e d i a t e d a s s a y , C a n c e r R e s . , 3 2 ( 1 9 7 2 ) 1 5 9 8 - - 1 5 9 9 . 1 5 Z e i g e r , E., a n d A . T . S h e l d o n , T h e m u t a g e n i c i t y o f h e t e r o c y c l l c N - n i t r o s a m i n e s f o r S a l m o n e l l a t y p h i r n u r i u m , M u t a t i o n R e s . , 57 ( 1 9 7 8 ) 1 - - 1 0 .
Mutation Research, 57 ( 1 9 7 8 ) 8 5 - - 8 9 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
Short Communication THE MUTAGENICITY OF N-NITROSOPIPERIDINES FOR SALMONELLA TYPHIMURIUM
E R R O L Z E I G E R * a n d A L B E R T T. S H E L D O N
Division of Toxicology, Food and Drug Administration, Washington, D.C. 20204 (U.S.A.) ( R e c e i v e d 18 J u l y 1 9 7 7 ) ( R e v i s i o n received 1 N o v e m b e r 1 9 7 7 ) (Accepted 9 November 1977)
Introduction NPIPD induces tumors in the nose and upper gastrointestinal tract of rats [3,8--10] and lung adenomas in mice [6]. It is also mutagenic for E. coli [4] and S. typhimurium in the presence of an in vitro metabolic activation system [2,7,11,15] and for S. typhimurium in the host-mediated assay (E. Zeiger, unpublished), and produces single-strand breaks in DNA of regenerating rat liver [13]. A number of methylated [8], oxygenated [9] and halogenated [10] nitrosopiperidines are also carcinogenic in rats. In this study, NPIPD, NPDO, DCNPD, NINA and NPIPA (Fig. 1) were tested for mutagenicity. Salmonella typhimurium TA-1535, which reverts to prototrophy by base-pair substitution, was used in both suspension and plate tests, and metabolic activation was provided by liver homogenates from rats and mice. Materials and methods
The test strain Salmonella typhimurium TA-1535, was obtained from Dr. Bruce Ames. It contains a missense mutation in the G gene of the histidine operon, a deletion covering the bio uvrB loci and an rfa mutation [1]. HA (ICR)f male mice were purchased from Sprague-Dawley, Madison, Wis: and were used when they weighed approximately 30 g. Sprague-Dawley derived Osborne-Mendel rats were obtained from the FDA colony and were used at approximately 300 g. All nitrosamines were supplied by Dr. William Lijinsky, Biology Division, DCNPD, N-nitroso-3,4-dichloropiperidine; DMSO, dimethylsulfoxide; NINA, Nnitrosoisonipecotic acid; NPDO, N-nitroso-4-pipcridone; NPIPA, N-nitrosopipecolic acid; NPIPD, N-nitrosopiperidine.
Abbreviations:.
* Present address: E n v i r o n m e n t a l Mutagenesis Branch, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, N.C., 27709 (U.S.A.)
86
C:: RI
N -- NO
N-NITROSOPIPERIDINE N-NITROSO-4-PIPERIDONE N-NITROSO-3 4-DICHLOROPIPERIDINE N-NITROSOI~ONIPECOTICACID N-NITROSOPIPECOLICACID
R1
R2
R3
-H =0 -CI -H -H
-H -H -H -H -CI -H -COOH-H -H -COOH
Fig. 1. Structures of N-nitrosopipendines.
Oak Ridge National Laboratory, Oak Ridge, Tenn. NADP and glucose 6-phosphate were purchased from Sigma Chemical Co., St. Louis, Mo. DMSO was purchased from Baker Chemical Co., Phillipsburg, N.J., and was used as a solvent for NPDO, DCNPD, NINA and NPIPA. The final DMSO concentrations never exceeded 3.3% in the suspension test or 2.5% in the 2 ml of top agar in the plate test. Livers were prepared and mutagenicity tests were performed as previously described [15]. Suspension test controls included the high dose of the test compound in the presence of heat-inactivated (100°C/10 min) homogenate. Plate test controls included the high dose in the absence of the supernatant of homogenate centrifuged at 9000 ×g (S-9). Results and discussion NPIPD, NPDO and DCNPD were mutagenic in both the plate and suspension tests with both mouse- and rat-liver preparations (Table 1). The mutagenicity of NPDO was significantly less than that of NPIPD; this finding was unexpected since the compounds were of equivalent carcinogenicity in rat feeding studies, producing tumors primarily in the nasal cavity, oral cavity and esophagus in male rats [9]. In addition, since NPIPD is metabolized to NPDO by rat-liver microsomes in vitro [12], the mutagenicity and carcinogenicity results would imply that NPDO is not an intermediate in the activation of NPIPD to its ultimate mutagenic form. The mutagenic response of NPDO shows that mammalian metabolism is required for its activity, although the rat non-activation controls (50.0 /~moles) showed mutagenic activity equivalent to that which would occur with between 1.0 and 5.0 pmoles of metabolized NPDO. DCNPD is the most potent of the carcinogenic heterocyclic nitrosamines in the rat, inducing tumors in the tongue, pharynx, esophagus and non-glandular stomach [10; W. Lijinsky, personal communication]. It gave a relatively low mutagenic response in the rat-liver activation plate and suspension tests (Table 1). The plate tests with rat-liver activation gave a positive result only in the 1.0--50.0 pmole dose range; however, the unmetabolized 50.0-pmole control showed that at this dose range DCNPD did not require metabolism for its in vitro mutagenicity. This could be the result of a spontaneous breakdown of
87 TABLE 1 RESPONSE OF SALMONELLA TYPHIMURIUM TO N-NITROSOPIPERIDINE, AND N-NITROSO-3,4-DICHLOROPIPERIDINE a
MUTAGENIC PIPERIDONE
Cornpound
Dose level (#moles)
Mouse-liver activation b
S u s p e n s i o n test
NPDO
0 0.01 0.05 0.10 0.50 1.00 1.00 c
Plate test
S u s p e n s i o n test
Plate test
Surrival
his +
his +
Stur-
his +
revertants/
revertants/
vival
108 cells
(%)
plate
108 cells
(%)
revertants/ plate
0.76 -7.71 9.45 25.30 31.64 _
100.0 -111.2 119.8 93.2 94.4 --
11.0 470.3 1256.3 2254.0 4324.3 7596.0 10.0
0.56 -2.56 4.98 15.22 16.13 0.93
+ 0.26 ± 0.75 ± 5.02 ± 1.22 _+ 0 . 1 2
100.0 -105.2 95.4 81.9 104.5 85.6
21.5 41.3 175.0 168.7 1248.0 1750.0 17.0
0 . 6 2 _+ 0 . 0 7
100.0
± 0.14 ,+ 0 . 5 8 ± 0.89 + 1.18 ± 4.07
0 0.01
0.59 ± 0.19 --
100.0 --
0.05
0.76 ± 0.25
119.0
0.I0
2.00 ± 0.25
62.5
0.50
5 . 6 3 _+ 0 . 4 3
104.6
128.7
1.00
9 . 4 2 ,+ 0 . 2 6
94.0
119.3
-+ 5.7 + 142.9 + 139.0 ± 537.1 ± 582.5 ,+ 1 5 1 1 . 5 ± 3.0
21.0 +
14.1
20.7 ±
7.8
--
39.0 ±
14.0 6.1
± ±
6 8 . 0 _+
,+ 0 . 1 7
-+ 7.8 _+ 8.1 ± 61.9 ± 21.8 ± 633.8 ± 322.8 -~ 1.4
12.3 +
--
--
--
--
--
--
--
19.0
--
--
4 0 . 3 -+
13.0
3.63 ± 0.65
94.1
64.3 ±
2.5
--
5.00
--
--
--
5.32 ± 0.92
107.4
218.0
±
4.5 11.6 19.1
10.00
--
--
--
8.54 ± 0.33
88.3
363.0
±
30.5
50.00
--
--
--
± 1.89
75.4
492.0
_+ 3 2 . 4
4.04 ± 0.36
64.2
93.5 ±
6.4
100.0
15.3 ± --
3.2
1.00 c 50.00 c
DCNPD
Rat-liver activation
revertants/
his +
NPIPD
N-NITROSO-4-
0.71 ± 0.08 _
~
0
0.52 ± 0.10
0.01
--
0.05 0.10
100.0 --
6.18 ± 1.50 10.46
126.2
12.25
1 4 . 7 _+
4.7
--
--
9.0 ±
2.6
0 . 5 7 _+ 0 . 1 3
6 0 . 3 -+
9.5
--
--
--
--
121.8
256.7
±
52.0
1 . 1 7 _+ 0 . 4 2
95.2
--
± 1.39
106.0
499.3
±
50.7
1.54 ± 0.07
106.4
--
0.50
14.84
± 6.56
118.8
1928.7
±
846.8
3.83 + 0.51
100.7
18.7 ±
1.00
22.15
± 1.24
84.3
1582.4
_+
172.5
6 . 6 7 ,+ 0 . 4 9
96.0
31.7 ±
5.00
.
.
.
.
.
10.00
.
.
.
.
.
50.00
.
.
.
.
1.00 e 50.00 e
0.53 ± 0.04 .
.
. 108.6 .
17.3 ± .
1.5
90.3 ± ± 301.5 ± 111.0
0 . 9 2 ,+ 0 . 3 6
95.3
.
4.5 8.7 41.5 4.0 30.4
-183.0
±
8.2
a V a l u e s are given as m e a n s + S D o f three replicates. b D o s e l e v e l s axe given as # m o l e s / m l for the s u s p e n s i o n test and p m o l e s / p l a t e for the plate test. c S u s p e n s i o n test, c o n t r o l w i t h b o i l e d h o m o g e n a t e ; plate test, c o n t r o l w i t h o u t S-9.
DCNPD, the ability of the bacteria to metabolize it to an active form, or the capacity of DCNPD to cause mutation without metabolism. Both NINA and NPIPA (Table 2) caused apparent mutagenic responses at the 50.0-~mole level in all test systems, with minimal or negative responses at the 10.0-~mole level. Neither compound induced tumors in rat feeding studies [5; W. Lijinsky, personal communication]. NINA and NPIPA were the only compounds of those reported here and of the 14 previously reported [15] that produced a toxic response in the suspension tests. This toxicity was seen both in the presence and absence of metabolic activation. As was the case with DCNPD, these compounds also had mutagenic activity at 50.0 pmoles in the
88 TABLE 2 MUTAGENIC RESPONSE OF SALMONELLA N-NITROSOPIPECOLIC ACID a
Cornpound
NINA
NIPA
Dose
Mouse-liver activation
level (pmoles) b
S u s p e n s i o n test
TYPHIMURIUM TO N-NITROSOISONIPECOTIC
ACID AND
Rat-liver activation Plate test
S u s p e n s i o n test
Plate test
his +
Sur-
his +
his*
Sur-
his +
revertants/
viral
revertants/
revertants/
viral
revertants/
108 cells
(%)
plate
108 cells
(%)
plate
100.0 -113.6 120.3 118.7 16.7 2.1
12.3 27.0 23.7 42.0 40.0 158.3 302.7
100.0 -95.3 101.5 112.3 74.6 1.7
23.3 23.7 23.3 40.7 33.3 162.7 302.7
100.0 -127.4 115.0 123.3 79.4 28.7
12.7 19.3 18.0 24.7 27.0 46.3 48.7
100.0 -138.7 116.8 123.5 77.2 49.7
19.3 17.7 14.7 23.7 20.3 45.7 36.7
0 0.50 1.00 5.00 10.00 50.00 50.00 e
1.15 -0.75 0.77 0.95 424.23 59.17
0 0.50 1.00 5.00 10.00 50.00 50.00 c
0.97 -0.97 0.78 0.72 1.64 2.71
+
0.97
+ 0.26 -+ 0 . 1 4 ± 0.11 + 86.48 ± 29.46 ±
0.04
± ± + ± _+
0.31 0.18 0.06 0.60 0.10
± 3.5 ± 7.0 ± 5.0 ± 0 ± 3.6 ± 19.2 ± 14.6 ± ± ± ± ± ± ±
6.5 4.7 6.2 4.7 9.5 6.8 3.2
0.51 -0.72 0.98 1.07 43.85 406.7 d 3.14 -2.07 2.83 2.50 3.54 5.46
+
0.01
± 0.48 ± 0.12 + 0.38 ± 11.76
±
0.55
± ± ± ± ±
0.43 0.15 0.37 0.43 1.09
-+ 2 . 5 ± 9.0 + 8.1 ± 2.1 -+ 2.9 -+ 8 . 7 ± 14.6 + ± ± ± ± + ±
0.6 4.0 0.6 7.5 6.8 3.5 8.4
a V a l u e s are given as m e a n s + S D o f three replicates. b D o s e l e v e l s are given as p m o l e s / m l for the s u s p e n s i o n test and p m o l e s / p l a t e for the plate test. c S u s p e n s i o n test, c o n t r o l with boiled h o m o g e n a t e ; plate test, c o n t r o l w i t h o u t S-9.
d Single d e t e r m i n a t i o n .
absence of metabolic activation, implying a spontaneous or bacteria-catalyzed activation to an ultimate mutagen or a direct action of the compounds to cause mutation. The three carcinogens -- NPIPD, NPDO, DCNPD -- were more mutagenic in the presence of mouse-liver homogenate than with rat-liver homogenate. DCNPD, unlike the other compounds, gave non-equivalent responses in the rat suspension and plate tests. The reason for this is unknown. Previously reported results with heterocyclic nitrosamines in vivo [ 14] and in vitro [ 4,15] showed no correlation between mutagenicity and carcinogenicity, in that noncarcinogenic nitrosamines were no less mutagenic than a number of carcinogens. The responses in this study, however, did distinguish the carcinogens (NPIPD, NPDO, DCNPD) from the noncarcinogens (NINA, NPIPA), although there was no correlation among the carcinogens with regard to relative mutagenic activity. Acknowledgement I thank Dr. William Lijinsky, whose present address is Frederick Cancer Research Center, Frederick Md., for supplying the nitrosamines and his unpublished observations, as well as for the many helpful discussions.
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