307
Mutation Research, 48 (1977) 307--312 © Elsevier/North-Holland Biomedical Press
MUTAGENICITY TESTS ON ANTHELMINTICS: MICROSOMAL ACTIVATION O F VIPRYNIUM EMBONATE TO A MUTAGEN
D.G. MACPHEE and D.M. PODGER Department of Genetics, La Trobe University, Bundoora, Victoria, 3083 (Australia) (Received November 1st, 1976) (Revision received February 1st, 1977) (Accepted March 1st, 1977)
Summary Eight anthelmintic preparations readily available in Australia were tested for mutagenicity in the Salmonella typhimurium test system. A slightly modified version of the procedure r e c o m m e n d e d b y Ames et al. [2] was adopted, in that the test samples were simply placed in "wells" cut o u t of the agar of a plate previously seeded with the appropriate tester strain. Addition of a mixture of rat liver microsomal enzymes and appropriate co-factors ("S-9 mix") to one of the t w o wells on a single plate allowed a possible requirement for metabolic activation to be recognised. Using this procedure, viprynium embonate was found to be non-mutagenic. It was, however, activated by the rat liver microsome preparation to a mutagen capable of causing both base-pair substitution (detected with strain TA100) and frameshift (detected with strain TA98) mutations. The other seven c o m p o u n d s tested all gave negative results in this system.
Introduction Ames et al. [2] have developed a m e t h o d of detecting a high proportion of those chemicals in our environment which are potentially hazardous as mutagens, carcinogens, or (as is more usual) both. The basic method involves incorporating the test chemical in an agar overlay containing a small inoculum of one of several carefully chosen auxotrophic mutant strains of the bacterium Salmonella typhimurium, and then scoring the numbers of back mutants to p r o t o t r o p h y which appear on the appropriate selective medium. It is recommended that this test be carried o u t both with and without addition to the overlays of a mixture of rat liver microsomal enzymes and certain co-factors. This allows one to include an important aspect of mammalian metabolism in the test system, which in turn allows one to detect many possible effects of mammalian metabolism on test chemicals.
308 Because it utilizes bacteria and relatively simple media and chemicals, this test is both rapid and extremely cheap by comparison with other methods of mutagenicity and carcinogenicity testing [2]. However, when carried out as described above, it does require a considerable number of plates for each test compound -- plates with and without liver homogenate, for example, and also plates containing several concentrations of the test compound. Faced with requests to test a large number of compounds together with a shortage of funds for the purpose, we modified one version of the test system so that only one plate is required per test strain per compound. The present paper reports the results of testing eight anthelmintic preparations in this modified system. Materials and methods Bacterial strains The strains used were TA98 and TA100, which carry the R plasmid pKM101 and have been described in detail by Ames and his colleagues [2,8]. Other strains used were TA1535 and TA1538, which lack the R plasmid; TA1975, TA1976, TA1977 and TA1978, which are excision-proficient, deep rough, tester strains [ 1]; DG1339 (TA1975 carrying pKM101), DG1340 (TA1976 carrying pKM101), DG1341 (TA1977 carrying pKM101), and DG1342 (TA1978 carrying pKM101). The latter four strains were made by transferring the R plasmid from a derivative of strain DG2009 [6] carrying pKM101 to the TA1975TA1978 series, selection being made for ampicillin resistance. Media All media used were as previously described [4]. Mutagenesis tests Samples (0.1 ml) of stationary phase cultures of the appropriate tester strains were spread over the surface of plates of the selective medium (minimal medium E supplemented with biotin and a trace of either histidine or, in some cases, 2.5% v/v broth) and allowed to dry. Using a sterile cork-borer attached to a vacuum line, plugs of agar 1.2 cm in diameter were removed from each plate and trapped in a water trap which could later be autoclaved. The bottom of each well was then sealed with 0.1 ml of molten soft agar. Samples (50 pl) of the test compound, dissolved in either dimethyl sulfoxide or (in some cases) water, were then placed in each of two wells per plate. One of these wells was used to determine whether the rat liver microsome system had any effect on mutagenicity. This was done by adding 50/~l of S-9 mix, which contained per ml: 0.3 ml of S-9 fraction 8 pmol MgC12, 33 ~umol KC1, 5 #mol glucose-6-phosphate, 4 pmol NADP, and 100 #mol sodium phosphate (pH 7.4). The S-9 fraction was obtained as described by Ames et al. [2] from rats induced with Aroclor 1254, a mixture of polychlorinated biphenyls. The plates were incubated at 37°C for 2--3 days prior to inspection. Test chemicals At least one representative of each type of anthelmintic listed in the Australian Drug Compendium was obtained in the form recommended for use. Corn-
309 pounds were dissolved in dimethyl sulfoxide (DMSO, Ajax Chemical Company, Melbourne) for initial testing at concentrations of either 5000 pg/ml or 50,000 pg/ml. The preparations used, and the ingredients specified by the manufacturers, are shown in Table I. Results and discussion The results of testing eight anthelmintics for mutagenicity in S. typhimurium are summarized in Table I. Preliminary experiments carried out with strains TA98 and TA100 alone indicated that none of the eight preparations were mutagenic by themselves. However, one preparation (containing viprynium embonate; see Fig. 1 for structure) gave clear positive results with both strains when the rat liver microsomes/co-factor mixture was present in the wells. Typical results for this preparation, Vanquin, are shown in Fig. 2. All other preparations gave negative results even when the opportunity for metabolic activation was provided. Controls with DMSO alone and S-9 mix alone also gave negative results. To obtain some idea of the sensitivity of the method, all eight preparations were tested on strains TA1535 and TA1538, which are less sensitive to many mutagens because they lack the R plasmid pKM101 [8]. Vanquin could still be detected as a mutagen when activated by the S-9 mix in strain TA1538, but not in strain TA1535 (the R- equivalent of TA100). These results suggest that the well method of testing may be at its most useful only with strains carrying an R plasmid. Ames et al. [2] discussed the merits and demerits of spot tests for mutagenicity (carried out by placing a drop or a few crystals of the test chemical directly on the agar surface) in some detail. Most of their discussion is also relevant to testing by the well method as described here, in particular their point that as the test chemical diffuses out from the spot (well) a range of
TABLE
I
MUTAGENICITY Anthelmintic
TESTS
ON
EIGHT
Active
ANTHELMINTIC Tester
PREPARATIONS
DISSOLVED
IN DMSO
strain
ingredient(s) TA9S
TA100
--$9 Antepar
Piperazine
Combantrin
Pyrantel
Divermex
PiPerazine
Hetrazan Mintezol Pripsen
+$9
TA1538
--$9
+$9
--$9
TA1535 +$9
citrate
.
.
.
.
.
.
.
embonate
.
.
.
.
.
.
.
.
adipate
.
.
.
.
.
.
.
.
Diethylcaxbamazine
.
.
.
.
.
.
.
.
Thiabendazole
.
.
.
.
.
.
.
.
Piperazine
.
.
.
.
.
.
.
.
.
.
phosphate
--$9
+$9
--
--
.
+ senna Vanquin Yomesan
a
Viprynium Niclosamide
embonate
-.
+ .
-.
+
-.
+ .
.
a Although it is sparingly soluble in water, Vanquin also gave positive results with strains T A 9 8 and T A I 0 0 w h e n aqueous preparations m a d e up at 5 m g / m l were added to wells containing the microsomal activation system.
310 C6H5 I
YY
H3C
~H3 N÷
CH
CH
N
CH
~
COOOH CH2
( C H 3 ) 2 N ~ CO0Fig. 1. S t r u c t u r e o f v i p r y n i u m e m b o n a t e .
concentrations are tested simultaneously. This means of course that fewer plates are required per test chemical. Also, we have found that good results are obtained by the well method using only about one tenth of the amount per plate of the (relatively) expensive S-9 mix usually recommended for use in overlays. To some extent, testing by the well method would appear to combine the advantages of incubation of metabolic enzymes and the test chemical in liquid suspension (necessary for the detection of some promutagens [7]) with the advantages associated with stabilization of metabolic enzymes in agar [2]. This is so because the liquid suspension originally placed in the well dries in to the agar plug and the surrounding agar 1--2 h after being added. One other advantage of the well method is that it is very much harder to mis.
Fig. 2. R e s u l t s o f m u t a g e n i c i t y t e s t s w i t h V a n q u i n . T h e l e f t hand plate is T A 1 0 0 , t h e right hand plate T A 9 8 . The w e l l s o n t h e l e f t o f e a c h plate c o n t a i n 2 5 0 / ~ g V a n q u i n a l o n e , w h i l e t h o s e on the right c o n t a i n 2 5 0 / J g V a n q u i n plus " S 9 m i x "
311
interpret than the overlay method. This may be of particular significance when the Salmonella test system is first being used by inexperienced operators. We are aware of a number of people who became needlessly disenchanted with the Salmonella test because they misinterpreted as positives the results of overlay experiments which were in fact negatives. Such problems seem to arise mainly when the test chemical causes significant lethality, and occur for reasons which are fully discussed b y Green and Muriel (appendix A to ref. [3] ). Certain of the chemical structures used in this study had at least some features which suggested they might be capable of cross-linking the t w o strands of a DNA molecule. Some agents which do cause the formation of cross-links {e.g. mitomycin C) can be detected as mutagens only if the test bacteria are capable of excision repair, apparently because excision of at least one arm of a crosslink is a necessary prelude to this type of mutagenesis. We therefore tested all eight preparations on the excision-proficient (but deep rough) strains TA1975, TA1976, TA1977 and TA1978, together with derivatives of these strains given the R plasmid pKM101 by conjugation. No positive results were obtained for any preparation other than viprynium embonate, which gave only a very weak positive result when tested with (but n o t without) metabolic activation on strain TA1978 (pKM101). The far greater sensitivity of excision-deficient strains such as TA98, TA100, and TA1538 to the effects of the mutagenic metabolite of viprynium embonate provides evidence that this metabolite interacts with DNA in such a way that it produces damage which can be excised but if n o t excised may lead to fixation of a mutation. The present results show that when one of eight anthelmintic preparations tested is incubated with a rat liver extract/co-factor mixture, it becomes capable of causing both base-pair substitution and frameshift mutations in S. typhimurium. We conclude that this preparation, which contains "viprynium as the e m b o n a t e " , contains a promutagen capable of being converted by mammalian metabolic processes to a mutagenic metabolite (or metabolites). Further evaluation of the safety of this preparation would appear to be warranted, particularly since it is widely used b y children and often seems to be recommended for use b y individuals who may n o t even be worm-infested (e.g. the whole family of an affected individual). Acknowledgements We are grateful to Dr. B.N. Ames for kindly providing most of the strains used in this study, and to Mrs. M. Shallies for capable technical assistance. 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 class i f i c a t i o n o f m n 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 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 f o r 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 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 R e s . , 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 G r e e n , M . H . L . a n d W . J . M u r i e l , M u t a g e n t e s t i n g u s i n g T R P + r e v e r s i o n in Escherichia coli, M u t a t i o n Res., 38 (1976) 3 --32. 4 M a c P h e e , D . G . , E f f e c t s o f a n R f a c t o r a n d caffein o n u l t r a v i o l e t m u t a b i l i t y in Salmonella typhimurium, Mutation Res., 18 (1973) 367--370. 5 M a c P h e e , D . G . , Salmonella t y p h i m u r i u m hisG46 ( R - U t r e c h t ) : p o s s i b l e use in s c r e e n i n g r n u t a g e n s a n d carcinogens, Applied Microbiol., 26 (1973) 1004--1005.
312 6 M a c P h e e , D . G . a n d M . R . Beazer, M u t a n t s o f Salmonella typhimurium d e f i c i e n t in D N A p o l y m e r a s e I: d e t e c t i o n b y t h e i r f a i l u r e t o p r o d u c e c o l i c i n E l , Molec. Gen. G e n e t . , 1 2 7 ( 1 9 7 3 ) 2 2 9 - - 2 4 0 . 7 Malling, H . V . , D i m e t h y i n i t r o s a m i n e : f o r m a t i o n o f m u t a g e n i c c o m p o u n d s b y i n t e r a c t i o n w i t h m o u s e liver m i c r o s o m e s , M u t a t i o n R e s . , 1 3 ( 1 9 7 1 ) 4 2 5 - - 4 2 9 . 8 M c C a n n , J . , N . E . S p i n g a r n , J. K o b o r 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 : b a c t e r i a l t e s t e r s t r a i n s w i t h R f a c t o r p l a s m i d s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 9 7 9 - - 9 8 3 .
Mutation Research, 48 (1977) 307--312 © Elsevier/North-Holland Biomedical Press
MUTAGENICITY TESTS ON ANTHELMINTICS: MICROSOMAL ACTIVATION O F VIPRYNIUM EMBONATE TO A MUTAGEN
D.G. MACPHEE and D.M. PODGER Department of Genetics, La Trobe University, Bundoora, Victoria, 3083 (Australia) (Received November 1st, 1976) (Revision received February 1st, 1977) (Accepted March 1st, 1977)
Summary Eight anthelmintic preparations readily available in Australia were tested for mutagenicity in the Salmonella typhimurium test system. A slightly modified version of the procedure r e c o m m e n d e d b y Ames et al. [2] was adopted, in that the test samples were simply placed in "wells" cut o u t of the agar of a plate previously seeded with the appropriate tester strain. Addition of a mixture of rat liver microsomal enzymes and appropriate co-factors ("S-9 mix") to one of the t w o wells on a single plate allowed a possible requirement for metabolic activation to be recognised. Using this procedure, viprynium embonate was found to be non-mutagenic. It was, however, activated by the rat liver microsome preparation to a mutagen capable of causing both base-pair substitution (detected with strain TA100) and frameshift (detected with strain TA98) mutations. The other seven c o m p o u n d s tested all gave negative results in this system.
Introduction Ames et al. [2] have developed a m e t h o d of detecting a high proportion of those chemicals in our environment which are potentially hazardous as mutagens, carcinogens, or (as is more usual) both. The basic method involves incorporating the test chemical in an agar overlay containing a small inoculum of one of several carefully chosen auxotrophic mutant strains of the bacterium Salmonella typhimurium, and then scoring the numbers of back mutants to p r o t o t r o p h y which appear on the appropriate selective medium. It is recommended that this test be carried o u t both with and without addition to the overlays of a mixture of rat liver microsomal enzymes and certain co-factors. This allows one to include an important aspect of mammalian metabolism in the test system, which in turn allows one to detect many possible effects of mammalian metabolism on test chemicals.
308 Because it utilizes bacteria and relatively simple media and chemicals, this test is both rapid and extremely cheap by comparison with other methods of mutagenicity and carcinogenicity testing [2]. However, when carried out as described above, it does require a considerable number of plates for each test compound -- plates with and without liver homogenate, for example, and also plates containing several concentrations of the test compound. Faced with requests to test a large number of compounds together with a shortage of funds for the purpose, we modified one version of the test system so that only one plate is required per test strain per compound. The present paper reports the results of testing eight anthelmintic preparations in this modified system. Materials and methods Bacterial strains The strains used were TA98 and TA100, which carry the R plasmid pKM101 and have been described in detail by Ames and his colleagues [2,8]. Other strains used were TA1535 and TA1538, which lack the R plasmid; TA1975, TA1976, TA1977 and TA1978, which are excision-proficient, deep rough, tester strains [ 1]; DG1339 (TA1975 carrying pKM101), DG1340 (TA1976 carrying pKM101), DG1341 (TA1977 carrying pKM101), and DG1342 (TA1978 carrying pKM101). The latter four strains were made by transferring the R plasmid from a derivative of strain DG2009 [6] carrying pKM101 to the TA1975TA1978 series, selection being made for ampicillin resistance. Media All media used were as previously described [4]. Mutagenesis tests Samples (0.1 ml) of stationary phase cultures of the appropriate tester strains were spread over the surface of plates of the selective medium (minimal medium E supplemented with biotin and a trace of either histidine or, in some cases, 2.5% v/v broth) and allowed to dry. Using a sterile cork-borer attached to a vacuum line, plugs of agar 1.2 cm in diameter were removed from each plate and trapped in a water trap which could later be autoclaved. The bottom of each well was then sealed with 0.1 ml of molten soft agar. Samples (50 pl) of the test compound, dissolved in either dimethyl sulfoxide or (in some cases) water, were then placed in each of two wells per plate. One of these wells was used to determine whether the rat liver microsome system had any effect on mutagenicity. This was done by adding 50/~l of S-9 mix, which contained per ml: 0.3 ml of S-9 fraction 8 pmol MgC12, 33 ~umol KC1, 5 #mol glucose-6-phosphate, 4 pmol NADP, and 100 #mol sodium phosphate (pH 7.4). The S-9 fraction was obtained as described by Ames et al. [2] from rats induced with Aroclor 1254, a mixture of polychlorinated biphenyls. The plates were incubated at 37°C for 2--3 days prior to inspection. Test chemicals At least one representative of each type of anthelmintic listed in the Australian Drug Compendium was obtained in the form recommended for use. Corn-
309 pounds were dissolved in dimethyl sulfoxide (DMSO, Ajax Chemical Company, Melbourne) for initial testing at concentrations of either 5000 pg/ml or 50,000 pg/ml. The preparations used, and the ingredients specified by the manufacturers, are shown in Table I. Results and discussion The results of testing eight anthelmintics for mutagenicity in S. typhimurium are summarized in Table I. Preliminary experiments carried out with strains TA98 and TA100 alone indicated that none of the eight preparations were mutagenic by themselves. However, one preparation (containing viprynium embonate; see Fig. 1 for structure) gave clear positive results with both strains when the rat liver microsomes/co-factor mixture was present in the wells. Typical results for this preparation, Vanquin, are shown in Fig. 2. All other preparations gave negative results even when the opportunity for metabolic activation was provided. Controls with DMSO alone and S-9 mix alone also gave negative results. To obtain some idea of the sensitivity of the method, all eight preparations were tested on strains TA1535 and TA1538, which are less sensitive to many mutagens because they lack the R plasmid pKM101 [8]. Vanquin could still be detected as a mutagen when activated by the S-9 mix in strain TA1538, but not in strain TA1535 (the R- equivalent of TA100). These results suggest that the well method of testing may be at its most useful only with strains carrying an R plasmid. Ames et al. [2] discussed the merits and demerits of spot tests for mutagenicity (carried out by placing a drop or a few crystals of the test chemical directly on the agar surface) in some detail. Most of their discussion is also relevant to testing by the well method as described here, in particular their point that as the test chemical diffuses out from the spot (well) a range of
TABLE
I
MUTAGENICITY Anthelmintic
TESTS
ON
EIGHT
Active
ANTHELMINTIC Tester
PREPARATIONS
DISSOLVED
IN DMSO
strain
ingredient(s) TA9S
TA100
--$9 Antepar
Piperazine
Combantrin
Pyrantel
Divermex
PiPerazine
Hetrazan Mintezol Pripsen
+$9
TA1538
--$9
+$9
--$9
TA1535 +$9
citrate
.
.
.
.
.
.
.
embonate
.
.
.
.
.
.
.
.
adipate
.
.
.
.
.
.
.
.
Diethylcaxbamazine
.
.
.
.
.
.
.
.
Thiabendazole
.
.
.
.
.
.
.
.
Piperazine
.
.
.
.
.
.
.
.
.
.
phosphate
--$9
+$9
--
--
.
+ senna Vanquin Yomesan
a
Viprynium Niclosamide
embonate
-.
+ .
-.
+
-.
+ .
.
a Although it is sparingly soluble in water, Vanquin also gave positive results with strains T A 9 8 and T A I 0 0 w h e n aqueous preparations m a d e up at 5 m g / m l were added to wells containing the microsomal activation system.
310 C6H5 I
YY
H3C
~H3 N÷
CH
CH
N
CH
~
COOOH CH2
( C H 3 ) 2 N ~ CO0Fig. 1. S t r u c t u r e o f v i p r y n i u m e m b o n a t e .
concentrations are tested simultaneously. This means of course that fewer plates are required per test chemical. Also, we have found that good results are obtained by the well method using only about one tenth of the amount per plate of the (relatively) expensive S-9 mix usually recommended for use in overlays. To some extent, testing by the well method would appear to combine the advantages of incubation of metabolic enzymes and the test chemical in liquid suspension (necessary for the detection of some promutagens [7]) with the advantages associated with stabilization of metabolic enzymes in agar [2]. This is so because the liquid suspension originally placed in the well dries in to the agar plug and the surrounding agar 1--2 h after being added. One other advantage of the well method is that it is very much harder to mis.
Fig. 2. R e s u l t s o f m u t a g e n i c i t y t e s t s w i t h V a n q u i n . T h e l e f t hand plate is T A 1 0 0 , t h e right hand plate T A 9 8 . The w e l l s o n t h e l e f t o f e a c h plate c o n t a i n 2 5 0 / ~ g V a n q u i n a l o n e , w h i l e t h o s e on the right c o n t a i n 2 5 0 / J g V a n q u i n plus " S 9 m i x "
311
interpret than the overlay method. This may be of particular significance when the Salmonella test system is first being used by inexperienced operators. We are aware of a number of people who became needlessly disenchanted with the Salmonella test because they misinterpreted as positives the results of overlay experiments which were in fact negatives. Such problems seem to arise mainly when the test chemical causes significant lethality, and occur for reasons which are fully discussed b y Green and Muriel (appendix A to ref. [3] ). Certain of the chemical structures used in this study had at least some features which suggested they might be capable of cross-linking the t w o strands of a DNA molecule. Some agents which do cause the formation of cross-links {e.g. mitomycin C) can be detected as mutagens only if the test bacteria are capable of excision repair, apparently because excision of at least one arm of a crosslink is a necessary prelude to this type of mutagenesis. We therefore tested all eight preparations on the excision-proficient (but deep rough) strains TA1975, TA1976, TA1977 and TA1978, together with derivatives of these strains given the R plasmid pKM101 by conjugation. No positive results were obtained for any preparation other than viprynium embonate, which gave only a very weak positive result when tested with (but n o t without) metabolic activation on strain TA1978 (pKM101). The far greater sensitivity of excision-deficient strains such as TA98, TA100, and TA1538 to the effects of the mutagenic metabolite of viprynium embonate provides evidence that this metabolite interacts with DNA in such a way that it produces damage which can be excised but if n o t excised may lead to fixation of a mutation. The present results show that when one of eight anthelmintic preparations tested is incubated with a rat liver extract/co-factor mixture, it becomes capable of causing both base-pair substitution and frameshift mutations in S. typhimurium. We conclude that this preparation, which contains "viprynium as the e m b o n a t e " , contains a promutagen capable of being converted by mammalian metabolic processes to a mutagenic metabolite (or metabolites). Further evaluation of the safety of this preparation would appear to be warranted, particularly since it is widely used b y children and often seems to be recommended for use b y individuals who may n o t even be worm-infested (e.g. the whole family of an affected individual). Acknowledgements We are grateful to Dr. B.N. Ames for kindly providing most of the strains used in this study, and to Mrs. M. Shallies for capable technical assistance. 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 class i f i c a t i o n o f m n 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 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 f o r 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 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 R e s . , 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 G r e e n , M . H . L . a n d W . J . M u r i e l , M u t a g e n t e s t i n g u s i n g T R P + r e v e r s i o n in Escherichia coli, M u t a t i o n Res., 38 (1976) 3 --32. 4 M a c P h e e , D . G . , E f f e c t s o f a n R f a c t o r a n d caffein o n u l t r a v i o l e t m u t a b i l i t y in Salmonella typhimurium, Mutation Res., 18 (1973) 367--370. 5 M a c P h e e , D . G . , Salmonella t y p h i m u r i u m hisG46 ( R - U t r e c h t ) : p o s s i b l e use in s c r e e n i n g r n u t a g e n s a n d carcinogens, Applied Microbiol., 26 (1973) 1004--1005.
312 6 M a c P h e e , D . G . a n d M . R . Beazer, M u t a n t s o f Salmonella typhimurium d e f i c i e n t in D N A p o l y m e r a s e I: d e t e c t i o n b y t h e i r f a i l u r e t o p r o d u c e c o l i c i n E l , Molec. Gen. G e n e t . , 1 2 7 ( 1 9 7 3 ) 2 2 9 - - 2 4 0 . 7 Malling, H . V . , D i m e t h y i n i t r o s a m i n e : f o r m a t i o n o f m u t a g e n i c c o m p o u n d s b y i n t e r a c t i o n w i t h m o u s e liver m i c r o s o m e s , M u t a t i o n R e s . , 1 3 ( 1 9 7 1 ) 4 2 5 - - 4 2 9 . 8 M c C a n n , J . , N . E . S p i n g a r n , J. K o b o r 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 : b a c t e r i a l t e s t e r s t r a i n s w i t h R f a c t o r p l a s m i d s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 9 7 9 - - 9 8 3 .
