127
Mutation Research, 57 (1978) 127-134 0 Elsevier/North-Holland Biomedical Press
MUTAGENICITY
OF N-NITROSOPIPERAZINE
SALMONELLA
T. KAMESWAR L. EPLER a
TYPHIMURIUM
RAO a, JENNIFER
DERIVATIVES
IN
*
A. YOUNG
a, WILLIAM
LIJINSKY
b and JAMES
a Biology Division, Oak Ridge National Laboratory, Post Office Box Y, Oak Ridge, Tenn. 37830 (U.S.A.) and b Frederick Cancer Research Center, Post Office Box B, Frederick, MD 21701 (U.S.A.) (Received 11 July 1977) (Revision received 15 December (Accepted 16 December 1977)
1977)
Summary
Mutagenicity of several nitroso derivatives of piperazine was assayed using typhimurium. Nitroso derivatives of histidine auxotrophic strains of Salmonellu piperazine required metabolic activation with preference to phenobarbital induced rat-liver microsomal enzymes. We observed a good correlation between a positive effect in the mutation assay and the carcinogenic potency of the compound. Even though our results are not in complete agreement with earlier published work using several microbial mutation assay systems, the differences we observed demonstrate the predictive value of an in vitro activation system to detect carcinogenic compounds as mutagens. using S. typhimurium Introduction
In order to investigate the correlation between mutagenicity and carcinogenicity and to aid in determining the structural relationships involved in activity, we tested a series of derivatives of nitrosopiperazines for mutagenicity using S. typhimurium/microsomal activation system [ 11. By acceptance ment to retain
this article, the a nonexclusive
publisher royalty-free
or recipient license
acknowledges in and to any
* This research was jointly sponsored by the Environmental the Energy Research and Development Administration poration.
the right copyright
Protection Agency under contract with
of the U.S. Governcovering the article. (IAG-D5-E681) Union Carbide
and Cor-
Abbreviations: Ar. Aroclor 1254 (injection) induced rat-liver microsomal preparation (S-9); DNHPZ. tiitrosohomopiperazine; DNPZ. dinitrosopiperazine: NPZ. N-nitrosopiperazine; QB. phenobarbital (in drinking water) induce rat-liver microsomal preparation; S-9. supernatant from 9000 X 8 of rat-liver homogenate; Ui. uninduced (control) rat-liver microsomal preparation.
128
Carcinogenicity of 1,4dinitrosopiperazine (DNPZ) and methylated DNPZs in rats was reported by Lijinsky and Taylor [8]. N-Nitrosopiperazine (NPZ) was reported to be weakly or non-carcinogenic by Love et al. [lo]; N-methyl NPZ was reported to be noncarcinogenic by Druckrey et al. [4] and Lijinsky and Taylor [9]. Mutagenicity assays with NPZ derivatives have been done in various microbial systems: Salmonella host-mediated assay [14] and Salmonella plate assay involving microsomal activation in both semisolid and liquid suspension tests [2]; in E. coli [12], including activation with rat-liver microsomes [ 51; and in Actinomyces rimosus [3]. This paper reports mutagenicity assays with NPZ derivatives and points out differences between our results and earlier reports. Materials and methods Chemicals Preparation
of NPZ and its derivatives
was previously
described
[8-lo].
Bat terial strains Histidine auxotrophic strains of S. typhimurium (TA-1535, TA-100, TA1537, TA-1538 and TA-98) were obtained through the courtesy of Dr. B.N. Ames. Genotypes and routine phenotype check of the strains used have been previously described [ll]. Cultures were grown in nutrient broth (Difco) and plated for mutant screening on minimal medium made up of 1.5% Bacto-Difcoagar in Vogel-Bonner medium E [13] with 2% glucose. Experimental details for mutagenicity analysis and rat-liver homogenate preparations were previously described [ 111. The test compound was mixed with bacteria and rat-liver homogenate (S-9) in agar (containing minimal amounts of histidine and biotin) and overlayered over minimal medium plates. Reversion at the histidine locus was measured after 48 h of incubation at 37°C. Results Mutagenicity of NPZ and its derivatives was assayed using various strains of S. typhimurium (TA-1535, TA-100, the base-pair substitution strains; TA-1537 and TA98, the frameshift strains). A clear specificity to revert the base-pair substitution strain (TA-1535) was noticed, and this strain was used in all the following studies. We reported earlier similar strain specificity with another family of cyclic nitrosamines, nitrosopiperidines [ 111. The structures and mutagenicity indexes of NPZ and its derivatives are given in Table I. Table IA lists the raw data for all NPZ derivatives in the presence and absence of metabolic activation. The mutation index was obtained from the slope value of the linear portion of a dose-response curve. The doseresponse curve was obtained from a plate assay using a wide dose range (65-625 pg/plate). Metabolic activation with rat-liver microsor& preparation (S-9) was required to convert various NPZ derivatives into active mutagens. Uninduced (Vi), Aroclor (Ar) and phenobarbital ($B) induced rat-liver microsomal enzymes were tested for their effectiveness in the mutation assay. $B induced S-9 gave maximum activity and was used in all the following studies. NPZ failed to revert the strain TA-1535 from histidine auxotrophy to proto-
129 TAaLE
I
MUTAGENICITY
OF y-NITROSOPIPERAZINE
(NPZ)
AND
ITS DERIVATIVES.
structure
Compwnd (CAS No.)~
ahis+ -59
N-NITROSOPIPERAZINE (5632-47-3)
REV/pm& +S9 (6B)
NM
NM
NM
6
C”3 ri N-METHYL NPZ (16339-07-4)
0 Y NO
‘iJo 0 N
DlNlTROSOPlPERAZlNE (140-79-s)
(DNPZ)
NM
35
NM
51
NM
26
NM
53
NM
NM
N
Y0 (1 N
P-METHYL
DNPZ
N
‘“3
1(10
NO 2,5-DIMETHYL (55556-88-2)
DNPZ
NO
2,6-DIMETHYL (55380-34-2)
DNPZ C”3 I NO NO I
2,3,5,&TETRAMETHYL
DNPZ
130
TABLE I (Can’t)
Q .+ hlr -59
structum
Compcund b (CAS No.)
REV/pm& +s9 (@a)
NO i.l DINITROSOHOMOPIPERAZINE (55557-00-l )
0
NM
76
NM
NM
N tJl0
$6 H5 F=O CH3 4-BENZOYL-3,5-DIMETHYL
NPZ I NO
‘Determined %AS NI4
numbers =
from the linear ore given
portion
whenever
of o dose-response
curve.
available.
Non-mutagenic.
trophy even after metabolic activation with rat liver S-9 (Vi, Ar, or $B). N-Methyl NPZ exhibited a slight mutagenic activity with 4B induced rat liver S-9. These results correlate with the carcinogenicity of these compounds which indicate them as noncarcinogenic or weakly carcinogenic [4,9,10]. DNPZ was found to be mutagenic after metabolic activation with r$B induced rat-liver S-9. DNPZ derivatives with methyl groups in the a-position (2-methyl DNPZ); 2,5 or 2,6 positions (2,5dimethyl or 2,6dimethyl DNPZ) were mutagenic, and all of them are carcinogenic [8]. However, a complete block in the positions alpha to the nitroso group, as in 2,3,5,6tetramethyl DNPZ, completely eliminated mutagenic potency as well as carcinogenic potency of this compound [9]. DNHPZ, which was found to be a stronger mutagen in the mutation assay than the other derivatives, was also a strong carcinogen [8]. 4-Benzoyl-3,5dimethyl DNPZ was nonmutagenic and carcinogenesis studies with this compound are still in progress. Thus, a qualitative correlation between mutagenicity in the Salmonella plate assay and carcinogenic potency of NPZ derivatives was observed. Discussion Various microbial genetic test systems are being extensively used for the detection of carcinogens as mutagens [ 71. These studies drastically cut down
10
625
ACTIVATION
10
375
27
22
30
250
375
625
47
38
43
--
24
14
24
17
11
11
17
14
N-methyl NPZ
reverta&/plote
or on overage
(oB, S-9)
was obkined
24
125
a Each number
15
10
0
62
MTH
13
10
125
14
250
14
0
ACTIVATION
62
WlTHOUT
I
NPZ
his’
TA-1535
Compound/dose (lJg/plote)
I
OF NITROSOPIPERAZINE
MUTAGENICITY
TABLE IA
of at least
13P
130
95
70
50
14
13
14
11
19
9
14
DNPZ
three
DERIVATIVES
independent
125
132
02
50
39
14
17
17
i
23
21
14
P-Methyl DNPZ
experiments.
73
60
53
39
27
14
3
11
13
12
8
14
2,5-Dimethyl DNPZ
159
131
97
57
37
14
20
14
16
15
10
14
2,6-Dimethyl DNPZ
11
6
11
15
2
15
10
10
a
7
9
14
2,3,5.6Tetromethyl DNPZ
113
107
85
61
40
14
9
7
20
21
10
14
DNHPZ
8
11
14
9
18
15
10
8
12
18
12
14
4-Benzoyl3,5-dimethyl NPZ
132
research time and expense while helping to set priorities for research activities in the determiriation of carcinogenic hazard due to environmental carcinogens. Ames et al. [l] have developed a simple, inexpensive, but very sensitive, bacterial assay using certain well characterized histidine auxotrophic strain of S. typhimurium. Our previous [ 111 and present work with a selected group of cyclic nitrosamines shows a substantial correlation between a positive result in the mutation assay and carcinogenicity. DNPZ, mono- or di-methylated DNPZs, and DNHPZ gave a positive response in the mutation assay (with #B S-9) and were shown to be potent carcinogens by Lijinsky and Taylor [8]. NPZ and 2,3,5,6-tetramethyl DNPZ [9,10], reported as weak or noncarcinogens, failed to exhibit mutagenic activity. N-Methyl NPZ, which was noncarcinogenic [9], showed a slight but definite positive effect in the mutation assay. This observation is in agreement with an earlier published report by Bartsch et al. [2] using strain TA-1530 of 5’. typhimurium (Table II). 4-Benzoyl-3,lidimethyl NPZ was not mutagenic and its carcinogenic potency is still under investigation (Lijinsky, personal communication). NPZ derivatives require metabolic activation with rat-liver microsomal preparation in order to convert them into active form. c$B induction was the choice of the induction systems suggesting [6] involvement of cytochrome P450 dependent oxidative enzymes to convert these compounds to active mu tagens. Lijinsky and Taylor [8] have proposed that the hydrogen attached to carbon atoms alpha to the nitroso group is important for carcinogenic activity. This hypothesis seems to apply for the mutagenic activity of NPZ derivatives. A complete block in the alpha positions, as in 2,3,5,6-tetramethyl DNPZ, eliminated both mutagenic and carcinogenic potency of that compound. However, a single methyl substitution or substitution by two methyl groups did not inhibit the mutagenic or carcinogenic potency of DNPZ. Table II illustrates a comparative analysis using several studies with S. typhimurium, E. coli and Actinomyces rimosus. Trams and Kuenkel [12] have reported DNPZ to be nonmutagenic in E. coli by using a forward mutation assay (without metabolic activation), selecting for biochemically deficient mutants. This observation supports our results that DNPZ is not a direct acting mutagen and requires metabolic activation in order to be an active mutagen. Results obtained by Elespuru and Lijinsky with E. coli incorporating metabolic activation show a similarity with the results we obtained using Salmonella. Both DNPZ and DNHPZ were identified as mutagenic in both systems. However, studies with E. coli [5] failed to identify 2,6dimethyl DNPZ as a mutagen, while carcinogenesis studies indicate it to be a strong carcinogen [ 8].2,6-Dimethyl DNPZ was a potent mutagen in our studies with Salmonella (Table I and II). Zeiger et al. [14] have reported NPZ, N-methyl NPZ, and DNPZ as mutagenic in host-mediated assay for the strain G46 of S. typhimurium. NPZ and N-methyl NPZ, both of them weak- or non-carcinogens, were identified as potent mutagens in that study. In our study we failed to detect mutagenic activity with NPZ and detected only slight mutagenic activity with N-methyl NPZ. Bartsch et al. [2] have reported a similar observation with N-methyl NPZ using the strain TA-1530 of S. typhimurium, which not only reconfirms our result but also indicates the usefulness and prediction
(DNHPZ)
NPZ
4-Benzoyl-3,5-dimethyl 0
+
0
+
DNPZ
DNPZ
+
DNPZ
Dinitrosohamapipemrine
2,3,5,6-Tetmmethyl
2,6-Dimethyl
2,5-Dimethyl
+
+
+
(DNPZ)
Dinitrosapipemrine
Z-Methyl
DNPZ
f
l
NPZ
z-methyl
0
Table
+
(NPZ)
2
1
WlTH
BACTERIAL
t
0
l
0
0
5
tyr reversion
E. coli
DERIVATIVES
Plate assay
OF NPZ
+
-N-nitrosopipemzine
Compound
14
Reference
array
typhimurium
CARCINOGENICITY
Host-mediated
AND
Assay
MUTAGENICITY
Salmonella
BETWEEN
System
A COMPARISON
TABLE II
12
forward
mutation
SYSTEMS
l
3
met reversion
Actinomyces rimorur
i-l-k
0
7
+4-a+
+I++
0 0
4-H
+t+t
8,
9,
COdWCJ~~&S
rats
10
134
value of in vitl;o activation system over host-mediated assays. Studies with A. rimosus [3] using reversion at methionine locus indicate a slight mutagenic activity with DNPZ. Failure to incorporate metabolic activation could be the reason for this effect. These differences from earlier published observations with other bacterial test systems demonstrate the use and predictive values of a simple, but very sensitive, Salmonella plate assay incorporating metabolic activation for the detection df these carcinogens as mutagens. Our previous report [ll] and this study illustrate a parallel relationship between mutagenicity of a group of cyclic nitrosamines (that might cause base-pair substitution) and their carcinogenicity . Acknowledgements This research was jointly sponsored by the Environmental Protection Agency (lAG-D5-E681) and the Energy Research and Development Administration under contract with Union Carbide Corporation. We thank Dr. B.N. Ames for providing the bacterial strains, Dr. P. Nettesheim for providing materials for rat-liver enzyme preparation, and D. Creasia and W. Winton for technical aid in making rat-liver enzyme preparation. We also express our appreciation to the staff of the Environmental Mutagen Information Center for their help in this project. References 1 Ames. B.N.. J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31 (1975) 347-364. 2 Bartsch, H.. A. Camus and C. Malaveille. Comparative mutagenicity of N-nitrossmines in a semisolid and in a liquid incubation system in the presence of rat or human tissue fractions, Mutation Res.. 37 (1976) 149-162. 3 Domracheva. A.G.. Comparation study on genetic activity of various N-nitroso compounds and ethylenimine on a biochemical mutant of Actinomyces rimosus. Sov. Genet.. 4 (1968) 40-46 4 Druckrey. H., R. Preussmann. S. Ivankovic and D. Schm$ Organotrope carcinogene Wirkungen bei 65 verschiedenen N-Nitroso-Verbindungen an BD-ratten. Z. Krebsforsch., 69 (1967) 103-121. 5 Elespuru. R.K. and W. Liiinsky. Mutagenicity of cvclic nitrosamines in Escherichia coli following activation with rat liver microsomes, Cancer Res.. 36 (1976) 4099-4101. 6 Guttenplan. J.B.. F. Huttever and A.J. Garro. Effects of cytochrome P-448 and P-450 inducers on microsomal dimethylnitrosamine dimethylase activity and the capacity of isolated microsomes to activate diiethylnitrosamine to a mutagen, Mutation Res.. 35 (1976) 415-422. 7 Lijinsky, W.. Carcinogenic and mutagenic N-nitroso compounds. in: A. Hollaender (Ed.), Chemical Mutagens, Vol. 4. Plenum, New York. 1976. pp. 193-217. 8 Lijinsky, W. and H.W. Taylor. Carcinogenicity of methylated dinitropiperazines in rats, Cancer Res.. 35 (1975) 1270-1273. 9 Lijinsky, W. and H.W. Taylor. Carcinogenesis tests of nitroso-N-methylpiperazine. 2,3.5,6-tetramethyldinitrosopiperazine, nitrosoisonipecotic acid, and nitrosomethoxymethylamine in rats, Z. Krebsforsch.. 89 (1977) 31-36. 10 Love, A.L., W. Lijinsky. L.F. Keefer and H. Garcia. Chronic oral administration of l-nitrosopiperazinc at high doses to MRC rats, Z. Krebsforsch.. 89 (1977) 69-73. 11 Rae. T.K.. A.A. Hardigree, J.A. Young. W. Lijinsky and J.L. Epler, Mutagenicity of N-nitrosopiperidines with SoZmoneZia typhimurium/microsomal activation system, Mutation Res., 56 (1977) 131-145. 12
Trams, zinc
13 14
A.
and
Kuenkel. Lack of mutagenicity of N-nitrosopiperidine and N-dinitrosopipera(German). Naturwissenchaften, 52 (1965) 650-651. and D.M. Banner, Acetylomithinase of E. coli: Partial purification and some properChem., 218 (1956) 97-106. M.S. Leg&or and W. Lijinsky, Mutagenicity of N-nitrosopiperazines for Salmonella typhithe host mediated assay. Cancer Res., 32 (1972) 1598-1599.
in Escherichia
Vogel. H.J. ties, J. Biol. Zeiger. E.. murium in
H.A.
coli
Mutation Research, 57 (1978) 127-134 0 Elsevier/North-Holland Biomedical Press
MUTAGENICITY
OF N-NITROSOPIPERAZINE
SALMONELLA
T. KAMESWAR L. EPLER a
TYPHIMURIUM
RAO a, JENNIFER
DERIVATIVES
IN
*
A. YOUNG
a, WILLIAM
LIJINSKY
b and JAMES
a Biology Division, Oak Ridge National Laboratory, Post Office Box Y, Oak Ridge, Tenn. 37830 (U.S.A.) and b Frederick Cancer Research Center, Post Office Box B, Frederick, MD 21701 (U.S.A.) (Received 11 July 1977) (Revision received 15 December (Accepted 16 December 1977)
1977)
Summary
Mutagenicity of several nitroso derivatives of piperazine was assayed using typhimurium. Nitroso derivatives of histidine auxotrophic strains of Salmonellu piperazine required metabolic activation with preference to phenobarbital induced rat-liver microsomal enzymes. We observed a good correlation between a positive effect in the mutation assay and the carcinogenic potency of the compound. Even though our results are not in complete agreement with earlier published work using several microbial mutation assay systems, the differences we observed demonstrate the predictive value of an in vitro activation system to detect carcinogenic compounds as mutagens. using S. typhimurium Introduction
In order to investigate the correlation between mutagenicity and carcinogenicity and to aid in determining the structural relationships involved in activity, we tested a series of derivatives of nitrosopiperazines for mutagenicity using S. typhimurium/microsomal activation system [ 11. By acceptance ment to retain
this article, the a nonexclusive
publisher royalty-free
or recipient license
acknowledges in and to any
* This research was jointly sponsored by the Environmental the Energy Research and Development Administration poration.
the right copyright
Protection Agency under contract with
of the U.S. Governcovering the article. (IAG-D5-E681) Union Carbide
and Cor-
Abbreviations: Ar. Aroclor 1254 (injection) induced rat-liver microsomal preparation (S-9); DNHPZ. tiitrosohomopiperazine; DNPZ. dinitrosopiperazine: NPZ. N-nitrosopiperazine; QB. phenobarbital (in drinking water) induce rat-liver microsomal preparation; S-9. supernatant from 9000 X 8 of rat-liver homogenate; Ui. uninduced (control) rat-liver microsomal preparation.
128
Carcinogenicity of 1,4dinitrosopiperazine (DNPZ) and methylated DNPZs in rats was reported by Lijinsky and Taylor [8]. N-Nitrosopiperazine (NPZ) was reported to be weakly or non-carcinogenic by Love et al. [lo]; N-methyl NPZ was reported to be noncarcinogenic by Druckrey et al. [4] and Lijinsky and Taylor [9]. Mutagenicity assays with NPZ derivatives have been done in various microbial systems: Salmonella host-mediated assay [14] and Salmonella plate assay involving microsomal activation in both semisolid and liquid suspension tests [2]; in E. coli [12], including activation with rat-liver microsomes [ 51; and in Actinomyces rimosus [3]. This paper reports mutagenicity assays with NPZ derivatives and points out differences between our results and earlier reports. Materials and methods Chemicals Preparation
of NPZ and its derivatives
was previously
described
[8-lo].
Bat terial strains Histidine auxotrophic strains of S. typhimurium (TA-1535, TA-100, TA1537, TA-1538 and TA-98) were obtained through the courtesy of Dr. B.N. Ames. Genotypes and routine phenotype check of the strains used have been previously described [ll]. Cultures were grown in nutrient broth (Difco) and plated for mutant screening on minimal medium made up of 1.5% Bacto-Difcoagar in Vogel-Bonner medium E [13] with 2% glucose. Experimental details for mutagenicity analysis and rat-liver homogenate preparations were previously described [ 111. The test compound was mixed with bacteria and rat-liver homogenate (S-9) in agar (containing minimal amounts of histidine and biotin) and overlayered over minimal medium plates. Reversion at the histidine locus was measured after 48 h of incubation at 37°C. Results Mutagenicity of NPZ and its derivatives was assayed using various strains of S. typhimurium (TA-1535, TA-100, the base-pair substitution strains; TA-1537 and TA98, the frameshift strains). A clear specificity to revert the base-pair substitution strain (TA-1535) was noticed, and this strain was used in all the following studies. We reported earlier similar strain specificity with another family of cyclic nitrosamines, nitrosopiperidines [ 111. The structures and mutagenicity indexes of NPZ and its derivatives are given in Table I. Table IA lists the raw data for all NPZ derivatives in the presence and absence of metabolic activation. The mutation index was obtained from the slope value of the linear portion of a dose-response curve. The doseresponse curve was obtained from a plate assay using a wide dose range (65-625 pg/plate). Metabolic activation with rat-liver microsor& preparation (S-9) was required to convert various NPZ derivatives into active mutagens. Uninduced (Vi), Aroclor (Ar) and phenobarbital ($B) induced rat-liver microsomal enzymes were tested for their effectiveness in the mutation assay. $B induced S-9 gave maximum activity and was used in all the following studies. NPZ failed to revert the strain TA-1535 from histidine auxotrophy to proto-
129 TAaLE
I
MUTAGENICITY
OF y-NITROSOPIPERAZINE
(NPZ)
AND
ITS DERIVATIVES.
structure
Compwnd (CAS No.)~
ahis+ -59
N-NITROSOPIPERAZINE (5632-47-3)
REV/pm& +S9 (6B)
NM
NM
NM
6
C”3 ri N-METHYL NPZ (16339-07-4)
0 Y NO
‘iJo 0 N
DlNlTROSOPlPERAZlNE (140-79-s)
(DNPZ)
NM
35
NM
51
NM
26
NM
53
NM
NM
N
Y0 (1 N
P-METHYL
DNPZ
N
‘“3
1(10
NO 2,5-DIMETHYL (55556-88-2)
DNPZ
NO
2,6-DIMETHYL (55380-34-2)
DNPZ C”3 I NO NO I
2,3,5,&TETRAMETHYL
DNPZ
130
TABLE I (Can’t)
Q .+ hlr -59
structum
Compcund b (CAS No.)
REV/pm& +s9 (@a)
NO i.l DINITROSOHOMOPIPERAZINE (55557-00-l )
0
NM
76
NM
NM
N tJl0
$6 H5 F=O CH3 4-BENZOYL-3,5-DIMETHYL
NPZ I NO
‘Determined %AS NI4
numbers =
from the linear ore given
portion
whenever
of o dose-response
curve.
available.
Non-mutagenic.
trophy even after metabolic activation with rat liver S-9 (Vi, Ar, or $B). N-Methyl NPZ exhibited a slight mutagenic activity with 4B induced rat liver S-9. These results correlate with the carcinogenicity of these compounds which indicate them as noncarcinogenic or weakly carcinogenic [4,9,10]. DNPZ was found to be mutagenic after metabolic activation with r$B induced rat-liver S-9. DNPZ derivatives with methyl groups in the a-position (2-methyl DNPZ); 2,5 or 2,6 positions (2,5dimethyl or 2,6dimethyl DNPZ) were mutagenic, and all of them are carcinogenic [8]. However, a complete block in the positions alpha to the nitroso group, as in 2,3,5,6tetramethyl DNPZ, completely eliminated mutagenic potency as well as carcinogenic potency of this compound [9]. DNHPZ, which was found to be a stronger mutagen in the mutation assay than the other derivatives, was also a strong carcinogen [8]. 4-Benzoyl-3,5dimethyl DNPZ was nonmutagenic and carcinogenesis studies with this compound are still in progress. Thus, a qualitative correlation between mutagenicity in the Salmonella plate assay and carcinogenic potency of NPZ derivatives was observed. Discussion Various microbial genetic test systems are being extensively used for the detection of carcinogens as mutagens [ 71. These studies drastically cut down
10
625
ACTIVATION
10
375
27
22
30
250
375
625
47
38
43
--
24
14
24
17
11
11
17
14
N-methyl NPZ
reverta&/plote
or on overage
(oB, S-9)
was obkined
24
125
a Each number
15
10
0
62
MTH
13
10
125
14
250
14
0
ACTIVATION
62
WlTHOUT
I
NPZ
his’
TA-1535
Compound/dose (lJg/plote)
I
OF NITROSOPIPERAZINE
MUTAGENICITY
TABLE IA
of at least
13P
130
95
70
50
14
13
14
11
19
9
14
DNPZ
three
DERIVATIVES
independent
125
132
02
50
39
14
17
17
i
23
21
14
P-Methyl DNPZ
experiments.
73
60
53
39
27
14
3
11
13
12
8
14
2,5-Dimethyl DNPZ
159
131
97
57
37
14
20
14
16
15
10
14
2,6-Dimethyl DNPZ
11
6
11
15
2
15
10
10
a
7
9
14
2,3,5.6Tetromethyl DNPZ
113
107
85
61
40
14
9
7
20
21
10
14
DNHPZ
8
11
14
9
18
15
10
8
12
18
12
14
4-Benzoyl3,5-dimethyl NPZ
132
research time and expense while helping to set priorities for research activities in the determiriation of carcinogenic hazard due to environmental carcinogens. Ames et al. [l] have developed a simple, inexpensive, but very sensitive, bacterial assay using certain well characterized histidine auxotrophic strain of S. typhimurium. Our previous [ 111 and present work with a selected group of cyclic nitrosamines shows a substantial correlation between a positive result in the mutation assay and carcinogenicity. DNPZ, mono- or di-methylated DNPZs, and DNHPZ gave a positive response in the mutation assay (with #B S-9) and were shown to be potent carcinogens by Lijinsky and Taylor [8]. NPZ and 2,3,5,6-tetramethyl DNPZ [9,10], reported as weak or noncarcinogens, failed to exhibit mutagenic activity. N-Methyl NPZ, which was noncarcinogenic [9], showed a slight but definite positive effect in the mutation assay. This observation is in agreement with an earlier published report by Bartsch et al. [2] using strain TA-1530 of 5’. typhimurium (Table II). 4-Benzoyl-3,lidimethyl NPZ was not mutagenic and its carcinogenic potency is still under investigation (Lijinsky, personal communication). NPZ derivatives require metabolic activation with rat-liver microsomal preparation in order to convert them into active form. c$B induction was the choice of the induction systems suggesting [6] involvement of cytochrome P450 dependent oxidative enzymes to convert these compounds to active mu tagens. Lijinsky and Taylor [8] have proposed that the hydrogen attached to carbon atoms alpha to the nitroso group is important for carcinogenic activity. This hypothesis seems to apply for the mutagenic activity of NPZ derivatives. A complete block in the alpha positions, as in 2,3,5,6-tetramethyl DNPZ, eliminated both mutagenic and carcinogenic potency of that compound. However, a single methyl substitution or substitution by two methyl groups did not inhibit the mutagenic or carcinogenic potency of DNPZ. Table II illustrates a comparative analysis using several studies with S. typhimurium, E. coli and Actinomyces rimosus. Trams and Kuenkel [12] have reported DNPZ to be nonmutagenic in E. coli by using a forward mutation assay (without metabolic activation), selecting for biochemically deficient mutants. This observation supports our results that DNPZ is not a direct acting mutagen and requires metabolic activation in order to be an active mutagen. Results obtained by Elespuru and Lijinsky with E. coli incorporating metabolic activation show a similarity with the results we obtained using Salmonella. Both DNPZ and DNHPZ were identified as mutagenic in both systems. However, studies with E. coli [5] failed to identify 2,6dimethyl DNPZ as a mutagen, while carcinogenesis studies indicate it to be a strong carcinogen [ 8].2,6-Dimethyl DNPZ was a potent mutagen in our studies with Salmonella (Table I and II). Zeiger et al. [14] have reported NPZ, N-methyl NPZ, and DNPZ as mutagenic in host-mediated assay for the strain G46 of S. typhimurium. NPZ and N-methyl NPZ, both of them weak- or non-carcinogens, were identified as potent mutagens in that study. In our study we failed to detect mutagenic activity with NPZ and detected only slight mutagenic activity with N-methyl NPZ. Bartsch et al. [2] have reported a similar observation with N-methyl NPZ using the strain TA-1530 of S. typhimurium, which not only reconfirms our result but also indicates the usefulness and prediction
(DNHPZ)
NPZ
4-Benzoyl-3,5-dimethyl 0
+
0
+
DNPZ
DNPZ
+
DNPZ
Dinitrosohamapipemrine
2,3,5,6-Tetmmethyl
2,6-Dimethyl
2,5-Dimethyl
+
+
+
(DNPZ)
Dinitrosapipemrine
Z-Methyl
DNPZ
f
l
NPZ
z-methyl
0
Table
+
(NPZ)
2
1
WlTH
BACTERIAL
t
0
l
0
0
5
tyr reversion
E. coli
DERIVATIVES
Plate assay
OF NPZ
+
-N-nitrosopipemzine
Compound
14
Reference
array
typhimurium
CARCINOGENICITY
Host-mediated
AND
Assay
MUTAGENICITY
Salmonella
BETWEEN
System
A COMPARISON
TABLE II
12
forward
mutation
SYSTEMS
l
3
met reversion
Actinomyces rimorur
i-l-k
0
7
+4-a+
+I++
0 0
4-H
+t+t
8,
9,
COdWCJ~~&S
rats
10
134
value of in vitl;o activation system over host-mediated assays. Studies with A. rimosus [3] using reversion at methionine locus indicate a slight mutagenic activity with DNPZ. Failure to incorporate metabolic activation could be the reason for this effect. These differences from earlier published observations with other bacterial test systems demonstrate the use and predictive values of a simple, but very sensitive, Salmonella plate assay incorporating metabolic activation for the detection df these carcinogens as mutagens. Our previous report [ll] and this study illustrate a parallel relationship between mutagenicity of a group of cyclic nitrosamines (that might cause base-pair substitution) and their carcinogenicity . Acknowledgements This research was jointly sponsored by the Environmental Protection Agency (lAG-D5-E681) and the Energy Research and Development Administration under contract with Union Carbide Corporation. We thank Dr. B.N. Ames for providing the bacterial strains, Dr. P. Nettesheim for providing materials for rat-liver enzyme preparation, and D. Creasia and W. Winton for technical aid in making rat-liver enzyme preparation. We also express our appreciation to the staff of the Environmental Mutagen Information Center for their help in this project. References 1 Ames. B.N.. J. McCann and E. Yamasaki, Methods for detecting carcinogens and mutagens with Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31 (1975) 347-364. 2 Bartsch, H.. A. Camus and C. Malaveille. Comparative mutagenicity of N-nitrossmines in a semisolid and in a liquid incubation system in the presence of rat or human tissue fractions, Mutation Res.. 37 (1976) 149-162. 3 Domracheva. A.G.. Comparation study on genetic activity of various N-nitroso compounds and ethylenimine on a biochemical mutant of Actinomyces rimosus. Sov. Genet.. 4 (1968) 40-46 4 Druckrey. H., R. Preussmann. S. Ivankovic and D. Schm$ Organotrope carcinogene Wirkungen bei 65 verschiedenen N-Nitroso-Verbindungen an BD-ratten. Z. Krebsforsch., 69 (1967) 103-121. 5 Elespuru. R.K. and W. Liiinsky. Mutagenicity of cvclic nitrosamines in Escherichia coli following activation with rat liver microsomes, Cancer Res.. 36 (1976) 4099-4101. 6 Guttenplan. J.B.. F. Huttever and A.J. Garro. Effects of cytochrome P-448 and P-450 inducers on microsomal dimethylnitrosamine dimethylase activity and the capacity of isolated microsomes to activate diiethylnitrosamine to a mutagen, Mutation Res.. 35 (1976) 415-422. 7 Lijinsky, W.. Carcinogenic and mutagenic N-nitroso compounds. in: A. Hollaender (Ed.), Chemical Mutagens, Vol. 4. Plenum, New York. 1976. pp. 193-217. 8 Lijinsky, W. and H.W. Taylor. Carcinogenicity of methylated dinitropiperazines in rats, Cancer Res.. 35 (1975) 1270-1273. 9 Lijinsky, W. and H.W. Taylor. Carcinogenesis tests of nitroso-N-methylpiperazine. 2,3.5,6-tetramethyldinitrosopiperazine, nitrosoisonipecotic acid, and nitrosomethoxymethylamine in rats, Z. Krebsforsch.. 89 (1977) 31-36. 10 Love, A.L., W. Lijinsky. L.F. Keefer and H. Garcia. Chronic oral administration of l-nitrosopiperazinc at high doses to MRC rats, Z. Krebsforsch.. 89 (1977) 69-73. 11 Rae. T.K.. A.A. Hardigree, J.A. Young. W. Lijinsky and J.L. Epler, Mutagenicity of N-nitrosopiperidines with SoZmoneZia typhimurium/microsomal activation system, Mutation Res., 56 (1977) 131-145. 12
Trams, zinc
13 14
A.
and
Kuenkel. Lack of mutagenicity of N-nitrosopiperidine and N-dinitrosopipera(German). Naturwissenchaften, 52 (1965) 650-651. and D.M. Banner, Acetylomithinase of E. coli: Partial purification and some properChem., 218 (1956) 97-106. M.S. Leg&or and W. Lijinsky, Mutagenicity of N-nitrosopiperazines for Salmonella typhithe host mediated assay. Cancer Res., 32 (1972) 1598-1599.
in Escherichia
Vogel. H.J. ties, J. Biol. Zeiger. E.. murium in
H.A.
coli
