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THE USE OF THE SALMONELLA/MICROSO%MA% ASSAY TO DETERMINE MUTAGENICITY IN PAIRED CHEMICAL MIXTURES M. F. SALAMONE, .?. A. HEDDLEA N D M. KATZ Ceraare ,for Research
or1
Environmental Quality, York University, 4700 Keele Srrert, Llownsvicw (Toronro). Ontario M3J ZW3 C'ma(ia
The rnutagenicities of two sets of chemicals acting singly and in p a i r ~ i s ecornbinatiox~s %ere deterininecl by use of the Suln~onelEn/microsomalassay. The first set consisted of the promutagens of benzo(a)pyrene and benzo(rst)pentraphene. The second set contained the direct-acting mutagens methyl-nitro-nitroso-guanidine and ethyl methane sulfonate. In the tests with the promutagens, the quantities of S-') mix were varied over the range of 0.05 ml to 1 . 0 mi with increasing quantities of each chemical. The mutagenic respoalses or production of revertant colonies of the promutagens, acting singly and in pairwise cornbinations failed to show an additive effect. Excess quantities of §-9 mix appeared to inhibit partially OH totally the rnutageslic activity of each chemical, although for each particular dose there was an optima! quantity of S-9 mix to induce maximum activity. However, the direct-acting mutagens produced, individually, almost linear dose responses with increasing concentrations. In pairwise combinations, these chemicals also showed linear responses that closely approximated the theoretical additivity indicating that the mutagenicity of the mixtures was the sum of the activities of each component. La mutagenicit6 de deux skies de composCs chimiques agissant seuls en paires fut determinee par I'utillisation de Ba technique de Salmonelin sur microsomes. La premikre sCrie consistait des prornutagknes de benzcs(a)pyrknc et de henzo(rst)pentaph;tne. La seconde sCrie contenait les mutag6nes d'action disecte wmkthpl-nitro-nitroso guanidine et sulfonate de mithane Cthylique. Dans les tests avec les prc~mutagewes,les quantitks du milange S9 variaient entre 0.85 nl! et 1.0 ml avcc des quantitCs croissantes de chaque composk chimique. Les sCponses ou %aproduction mutagknique de colonies de retour agissant seules et en paires n'ont pas riussi $ dernontrer un effet additif. Des quantitis excessives du tnkiange S-9 ant semblk inhiber partiellernent ou totalernent B'activitC rnutagknique de chaque compost5 chirnique. quoique pour chaqale dose particulikre il y avait une quantitC c~ptimaledc melange S-9 pour induire une acaiviti maximale. Cependant, Bes mutagenes d'action tlirecte ont produit individuelBement des r6gonses aux doses presque linkaires avec i'augmentation des concentrations. Dans les conabinaisons cn paires, ces composes chimiques oilt aussi tl6montri des reponses lineaires qui approchaient de prks l'additivit6 thCorique. ceci indiquant que la mutagCnicit6 des mklanges etait ia somrne des activites [Traduit par le journal] de chaque constituant.
Iwts~duction The Sa&rzac~nella/microsoma1 assay developed by Ames and his collaborators (Arnes (PI., 1973, 197%)has proved to be a rapid Bow cost screen far chemical mutagens. One important component of the Scal~rzsrzellaassay is the exogenous microsomal enzyme activation system. This system is used to convert promutagens, chemicals which produce a mutagenic response only after they are metabolized or "activrated9', to their mutagenic form. Appropriate microsomal enzymes can be obtained in sufficient quantities from a number of animal tissue sources. However, recent reports indicate that the action of the microsomal enzymes from genetically similar or different sources may differ (Ashky and Styles, f978a; McGregor, 197%; Tang and Friedman, 1977). This suggests inherent variability in the type or quantity of microsomal enzymes occurring in animal tissue. In this paper two-component mixtures of direct acting mutagens and promutagens were used, as models s f more complex mixtures, to study the influence of the microsoma1 astivation system on the additive response of chemicals in cornbination. Manuscript received July 3 1 , 1978.
Can. $. Genet. d'ytd. 21: POP-l07,P979.
102
M . F . SALAMONE ETAL.
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Materials and Methods The chemicals used were benzo(a)pyrene (B(a)P), benzo(rsr)pentaphene (B(rst)P), n-nethylnitro-a~itrosoguanidine (MNWG) and ethyl methane sulfonate (EEa.IS). B(a)P and B(rst)P both require metabolic activation in order to produce a mutagenic response. while MNNG and EMS arc direct-acting mutagens. A 10 pl/ml base solution of EMS was prepared using water. M N N G was freshly prepared kg: dissolving I mg MNNG in 10 rnl diinethylsulfoxide (DMSO). B(a)P and B(rst)P were dissolved in DMSO to an initial concentration of 10 mg/ml. A11 solutions were serially diluted to more usable concentrations as follows: EMS w ~ t hwater, MNNG and BQa)P with DMSO, B(rst)P with a 1O:I water-DMS0 mixture. The aqueous dilutions served to reduce the potential deleterious effects of DMSO to the bacteria (Annes er a ! ., 1975; Salarnone er a / . , 1938). AII assays were carried out as dcssribed by Ames cb a!. (1933, 8975), using testes strains TA9X and TAiOO. The activation system contained aroslor 11254 (Monsanto) induced S-9 homogenate. This differed slightly frorn that described by Aines in that the final S-9 mixture contained !BB% instead of 4% S-9 homogenate. Mowekcr, the ratio of buffer and NADPH remained as suggested. In the coinbination study with 1 pg B(a)P and 2 p g B(rst)P. each chemical was first tested individually over a range of S-9 mix quantities. Then both chemicals were added together, in the same tube. and again tested using various quantities of S-9 lanix. Tester strain TA98 was the strain of choice for this phase of the study as both B(2a)P and B(rst)P respond hvcdrably with it. B(a)P produces a slightly stronger mutages-eic rcsponsc with TA $00but the response of B(rst)P is weak. No S-9 mix was used in the EMS-MNNC combinatioa~study. Initially, a dose-response was determined for each chemical using five concentrations which produced revertant counts with strain TA B 00 of between $00-500/plate. Then a combination dose-response was tested by adding together the appropriate quantity of each respective chemical over the five-concentration range. Hn both combination studies the individual and combination experiments were performed sirmultaneously. In addition. the volu~nesof the final top agar solutions in the individual and combination tests were equaiized by the addition of sterile water.
ResuIts The mutagenic response (production of revertant colonies) of B(a)P relative to various quantities of S-9 mix is presented in Table I . At any one chemical dose. there is a quantity of S-9 mix which prc~ducesa maximum mutagenic response. For the most part. the quantity of %-9 mix, which induces the greatest number of revertant colonies, increases with an increase in dose of chemical. A graph of the data i n Table I (Fig. 1) plc~ttingrevertantslplate versus pg B(a)B/plate for each crf the S-9 anix quantities further illustrates the responses obtained by varying the quantity of the activation complex. At the lowest S-9 mix quantities (0.05, 0. 1 and 0.2 ml) the curves reach a plateau as the B(a)P concentration is increased. As the
Relationchip between quantity of S-9 mixturc and ability of B(a)P to revert tester strain 'I'A98. All numbers for revcraant colonies given are the averages of 3 replicate place\ minus the average nunmbes (33) of spontaneous TCVCFlaflBS --
Pamount
----
--
-
Quantity of S-9 mixture per plate b~nl)
per plate i ~ g ?
I5
12
18
18 45 131
17 100
143 340
85 220
23 88 52 42
24
lh8
10 18 'PO 40
33 84
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MUTAGENICITY OF PAIRWISE MIXTURES
Fig. I . Mutagenic response of increasing doses of B(a)P to different quantities (ml) of total S-9 mix. The plotted data are taken from Table I. The number near the end of each curve signifies the quantity (ml)of S-9 mix and the symbol at the end of the curve designates the syrnbots for that curve.
quantity of S-9 increases the curves show less s f a tendency to plateau. However, there is a ~narlced decrease in the slope of the curves as the S-9 mix quantities increase from 0.4 to 8.0 mi. Similar results to the above were also obtained In tests with B(rst)P, and increasing quantities of S-9 mix. The data of the combination study with B(a)P and B(rst)P are depicted in Fig. 2. The maximum number of revertant colonies induced by 2 p g of B(rst)P was 245 and this required 0.2 rnl S-9 mix. For B(a)P, the maximum colony production (333) occurred at 0.3 ml S-9 mix. Therefore, the total number of revertants expected in (rst)P combination study, using 8.5 ml of S-9 mix, might be the sum of the maximum mutagenic responses of each chemical or, in this case, 578 revertant colonies. This prediction depends upon the total S-9 quantity producing arm additive mutagenic effect. However, the actual value obtained for the combination study at 0.5 ml of S-9 mix is 317 5 26. suggesting a nonadditive effect of these two chemicals.
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104
ELI. F. SAEAMQNE ETA&.
To determine if there would be any additive effect of direct-acting mutagens in a two component mixture, tests were made with EMS and MNNG. These data (Fig. 3) indicated that both EMS and MNNG individually produced approximately linear dose reponses. The response of the combination test is also Hinear and the curve closely approximated the theoretical additivity curve, indicating that the mutagenicity s f the mixture is the sum s f the activities of each component.
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MUTAGENICITY OF PAIRWISE MIXTURES
105
Fig. 3 . Dose response of MNNG (open circles) and EMS (triangles) singly and i n pairwise combination (closed circles uppermost line). The predicted, con~binedresponse of the two individual curves is represented by the curve depicted with squares.
Discussion The data in Table I on B(a)P and our findings with B(rst)P suggest that for each particular dose of chemical, there is an optimal quantity of S-9 mix which will induce a maximum activity. Quantities sf S-9 mix less than c~ptimaHappear insufficient to activate maxirnaIIy the chemical and, in turn, induce the maximum number of revertant colonies. Excess quantities sf S-9 mix appear to inhibit partially or totally the mutagenic activity of the chemical. Similar results have been found with
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benzo(ghi)perylene, benzo(e)pyrene, coronene arad diarninoanisole using strain TA98 (Salamone and Katz. in prepration). Ashby and Styles (1978a) have indicated that enzymes from naouse and rat Iiver (S-9 mix) induced by aroclor 1254, caused a decrease in the observed mutagenic potency of B(a)B in the Ser&monuklcs/microssmaIassay, as compared with uninduced S-9. They demonstrated the variable effect of the exogenous activation system and pointed out the difficulties of using such activation systems in quantitative studies. In other studies, De Flora (19'78) noted that the addition of an activation mixture containing aroclor 1254 induced liver komogenate. reduced or "deactivated" the activity of direct-acting mutagens. This illustrates further the potential inhibiting effect of the rnicrosornal activation cc~anplex. Ames and Hooper (1998) cite an experiment with B(a)P in which the use of a series of increasing enzyme concentrations caused the mutagenic activity first to increase and then to diminish when more than the maximum recommended amount of enzyme was added per plate. This finding is in agreement with our results on the activity of B(a)P and other promutagens cited here. A decrease in mutagenic response by an excess concentration of S-9 mix is probably due to the presence of enzymes possessing oxidation. reduction and transferase properties. At the higher levels of S-9, competitive reactions between these enzymes and the chemicals may favor partial deactivation by conversion to less toxic or nontoxic products. Because of this inhibition effect, there is an inherent danger in using a single S-9 quantity over a small dose range of chemical, as the mutagenic response may be missed. In the combination studies it seems apparent that with mixtures of direct-acting mutagens the mutagenic activities of the cornponents are additive. The mutagenic response of EMS-MNNG combination was equal to the sum of their individual activities. On the other hand, with promutagens the mutagenicity of the mixture is not the sum of the mutagenicities of the con~ponentsas illustrated in the B(a)PB(rst)P results. These findings seem to implicate some action of the S-9 mix in the lack of additivity in pairwise promutagen cormabinations. Other experimental data support these findings. Using disect-acting mutagens, whose mutagenic response is only slightly affected by S-9 activation, in combination studies with and without S9 activation, we have found that the presence of the S-9 mix interferes with the predicted additive respolase (Salarnsne and Katz, in preparation). This lack cmf additivity in the pairwise coanbination studies with promartagens is not surprising, since B(a)P and B(rst)P individually showed an increasing but not additive response to increasing doses of these chemicals at the higher S-9 mix quantities. This nonadditivity could be related to competition for activation enzymes. Such competition may or may not occur in vivo, but some evidence for competition does exist. Brunette and Katz (1975) were able to show that perylene and, to a lesser extent. chrysene, may interfere with the uptake sf B(a)P by Chinese hamster ovary cells. However, none of these data conclusively implicates enzyme competition and many alternate hypotheses can be formulated to explain these findings. It could be the mutagenic potential c~fthe cells that is limiting. "Mutagenic pathways" or '"error-prone repair" might be saturated. suggesting that the lack of additivity demonstrates that both compounds act via the same mechanism which is saturated already by one compound. The presence of an exogenous activation system in the Sadmoszslla assay may lead to misinterpretation of data. S-9 hornogenates from different species (Bartsch k t a l . , 1975; McGregor, t 975) or individuals s f the same species (Garner et ak., 1972; Tang and Friedman, 1977) may differ in the levels of constituent (or induced enzymes due to genetic or dietary differences. These differences may produce results
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MUTAGENICITY OF PAHRWISE MIXTURES
107
i n the SaClnonella assay which vary significantly with regard to the mutagenic pc~tentialof a chemical. It is clear that a variation in the test conditions established for the Salmos~eHHu assay could change the observed potency of a mutagen (Amaes and Hooper, 1978; Ashby and Styles, 1978b). However, even if the standard conditions are not optimal for two or more mutagens in a mixture, one might expect to find an additive response. Our findings indicate that this is not the case for the promutagens tested. This lack of additivity appears to be an artifact of the exogenous activation system. The present study illustrates further the difficulty of quantifying ijs v i ~ r oresults where an exogenous activator is presetat. Acknowledgments This work was carried out with the technical assistance of Earl Stuart arad John Gingerich. Thanks are due to Drs. Earle Nestman and George Douglas, Health Protection Branch, National Health and Welfare for their review of the manuscript. This research was supported in part by Contract Grant 03 1-530-0927 of the Associate Committee on Scientific Criteria for Environmental Quality, National Research @ o m cil of Canada. References .4mes, B . N . , Da~rston, W . E . , Yarnasaki, E. and Lee, F. I). 1973. CarcEnogeins arc nnutagens. .4 sirnplz tzst system combining liver hornogenatrs for activation and bacteria for detection. $roc. Natl. Acad. Sci.. U.S.A. 70: 2288-2285. Ara-ees. B . X . , McCann, J . and 'famasaki, E. 1975. Methods for detecting carcinogens and mutagens with the S~lmonellu/manimalianmicrosome mutagenicity Best. Mutat. Res. 31: 347-364. Ames, B . N , and Hooper, K . 1978. Does carcinogenic potency correlate with mutapcnic potency in the: Ames assay'! Nature (London), 274: 19-20. Ashby, J . and Styles, J . A. I978a. Does carcinogenic potsncy correlate with mutagenic potency in the Amss Assay'? Nature (London), 271: 452-455. Ashby, J . and Styles, J . A . I998b. Factors inftucncing mutagenic potency irr r.itt.o. Nature (Laondun), 2'74: 20-22. Bartsch. H . . Zalalavcille, C . and Montcsano. K. 1975. Human, rat and niouse liver - rnediatzd mutagenicity of vinyl chloride in .F. fpkiniuriutn strains. Int. J . Cancer, 15: 429-437. Brunette, D. M. and Katz, M . 1975. The interaction of bcnzo(a)pyrene with cc1I rnernhranes: Uptake into Chinese hamster ovary cells and fluorescent stuefics with isolated meinhranes. Chem.-Biol. Interact. 11: 1-14. De Flora. S. 1978. Metabolic deactivation of niutagens in the .Fultnonclla-niicrosonsr Bsst. Nature (LoncBon), 271: 455-456. Garner, R . C.. Miller, E;.. C, and Miller, J . A . 1972.' Liver ~nicrosornaI metabolism of atlatoxin B , to a reactive derivative toxic to Salrnonclla ryphit?~uriumTA l 5 30. Cancer Wes. 32:24358-2066. McGregor, D. 8975. The relationship of 2-aceiamidofluorcne mutagenicity in plate tests with its in vivo liver cell component distribution and its carcinogenic potentias. Rlutat. Res. 30: 305-3 i 5 . Salamone, M . F., Heddle, J . A. and Katz. M. 1978. The rnutagenic activity of thirty polycyclic arornatic hydrocarbons anti oxides iu urban airborne particulates. Int. J . Environ. Stud. (in press). Tang, T . and Friedman, M. A . 1977. Carcinogen activation by huinan liver enzyme in the Alnes mutagenicity test. Mutat. Res. 46: 387-394.
THE USE OF THE SALMONELLA/MICROSO%MA% ASSAY TO DETERMINE MUTAGENICITY IN PAIRED CHEMICAL MIXTURES M. F. SALAMONE, .?. A. HEDDLEA N D M. KATZ Ceraare ,for Research
or1
Environmental Quality, York University, 4700 Keele Srrert, Llownsvicw (Toronro). Ontario M3J ZW3 C'ma(ia
The rnutagenicities of two sets of chemicals acting singly and in p a i r ~ i s ecornbinatiox~s %ere deterininecl by use of the Suln~onelEn/microsomalassay. The first set consisted of the promutagens of benzo(a)pyrene and benzo(rst)pentraphene. The second set contained the direct-acting mutagens methyl-nitro-nitroso-guanidine and ethyl methane sulfonate. In the tests with the promutagens, the quantities of S-') mix were varied over the range of 0.05 ml to 1 . 0 mi with increasing quantities of each chemical. The mutagenic respoalses or production of revertant colonies of the promutagens, acting singly and in pairwise cornbinations failed to show an additive effect. Excess quantities of §-9 mix appeared to inhibit partially OH totally the rnutageslic activity of each chemical, although for each particular dose there was an optima! quantity of S-9 mix to induce maximum activity. However, the direct-acting mutagens produced, individually, almost linear dose responses with increasing concentrations. In pairwise combinations, these chemicals also showed linear responses that closely approximated the theoretical additivity indicating that the mutagenicity of the mixtures was the sum of the activities of each component. La mutagenicit6 de deux skies de composCs chimiques agissant seuls en paires fut determinee par I'utillisation de Ba technique de Salmonelin sur microsomes. La premikre sCrie consistait des prornutagknes de benzcs(a)pyrknc et de henzo(rst)pentaph;tne. La seconde sCrie contenait les mutag6nes d'action disecte wmkthpl-nitro-nitroso guanidine et sulfonate de mithane Cthylique. Dans les tests avec les prc~mutagewes,les quantitks du milange S9 variaient entre 0.85 nl! et 1.0 ml avcc des quantitCs croissantes de chaque composk chimique. Les sCponses ou %aproduction mutagknique de colonies de retour agissant seules et en paires n'ont pas riussi $ dernontrer un effet additif. Des quantitis excessives du tnkiange S-9 ant semblk inhiber partiellernent ou totalernent B'activitC rnutagknique de chaque compost5 chirnique. quoique pour chaqale dose particulikre il y avait une quantitC c~ptimaledc melange S-9 pour induire une acaiviti maximale. Cependant, Bes mutagenes d'action tlirecte ont produit individuelBement des r6gonses aux doses presque linkaires avec i'augmentation des concentrations. Dans les conabinaisons cn paires, ces composes chimiques oilt aussi tl6montri des reponses lineaires qui approchaient de prks l'additivit6 thCorique. ceci indiquant que la mutagCnicit6 des mklanges etait ia somrne des activites [Traduit par le journal] de chaque constituant.
Iwts~duction The Sa&rzac~nella/microsoma1 assay developed by Ames and his collaborators (Arnes (PI., 1973, 197%)has proved to be a rapid Bow cost screen far chemical mutagens. One important component of the Scal~rzsrzellaassay is the exogenous microsomal enzyme activation system. This system is used to convert promutagens, chemicals which produce a mutagenic response only after they are metabolized or "activrated9', to their mutagenic form. Appropriate microsomal enzymes can be obtained in sufficient quantities from a number of animal tissue sources. However, recent reports indicate that the action of the microsomal enzymes from genetically similar or different sources may differ (Ashky and Styles, f978a; McGregor, 197%; Tang and Friedman, 1977). This suggests inherent variability in the type or quantity of microsomal enzymes occurring in animal tissue. In this paper two-component mixtures of direct acting mutagens and promutagens were used, as models s f more complex mixtures, to study the influence of the microsoma1 astivation system on the additive response of chemicals in cornbination. Manuscript received July 3 1 , 1978.
Can. $. Genet. d'ytd. 21: POP-l07,P979.
102
M . F . SALAMONE ETAL.
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Materials and Methods The chemicals used were benzo(a)pyrene (B(a)P), benzo(rsr)pentaphene (B(rst)P), n-nethylnitro-a~itrosoguanidine (MNWG) and ethyl methane sulfonate (EEa.IS). B(a)P and B(rst)P both require metabolic activation in order to produce a mutagenic response. while MNNG and EMS arc direct-acting mutagens. A 10 pl/ml base solution of EMS was prepared using water. M N N G was freshly prepared kg: dissolving I mg MNNG in 10 rnl diinethylsulfoxide (DMSO). B(a)P and B(rst)P were dissolved in DMSO to an initial concentration of 10 mg/ml. A11 solutions were serially diluted to more usable concentrations as follows: EMS w ~ t hwater, MNNG and BQa)P with DMSO, B(rst)P with a 1O:I water-DMS0 mixture. The aqueous dilutions served to reduce the potential deleterious effects of DMSO to the bacteria (Annes er a ! ., 1975; Salarnone er a / . , 1938). AII assays were carried out as dcssribed by Ames cb a!. (1933, 8975), using testes strains TA9X and TAiOO. The activation system contained aroslor 11254 (Monsanto) induced S-9 homogenate. This differed slightly frorn that described by Aines in that the final S-9 mixture contained !BB% instead of 4% S-9 homogenate. Mowekcr, the ratio of buffer and NADPH remained as suggested. In the coinbination study with 1 pg B(a)P and 2 p g B(rst)P. each chemical was first tested individually over a range of S-9 mix quantities. Then both chemicals were added together, in the same tube. and again tested using various quantities of S-9 lanix. Tester strain TA98 was the strain of choice for this phase of the study as both B(2a)P and B(rst)P respond hvcdrably with it. B(a)P produces a slightly stronger mutages-eic rcsponsc with TA $00but the response of B(rst)P is weak. No S-9 mix was used in the EMS-MNNC combinatioa~study. Initially, a dose-response was determined for each chemical using five concentrations which produced revertant counts with strain TA B 00 of between $00-500/plate. Then a combination dose-response was tested by adding together the appropriate quantity of each respective chemical over the five-concentration range. Hn both combination studies the individual and combination experiments were performed sirmultaneously. In addition. the volu~nesof the final top agar solutions in the individual and combination tests were equaiized by the addition of sterile water.
ResuIts The mutagenic response (production of revertant colonies) of B(a)P relative to various quantities of S-9 mix is presented in Table I . At any one chemical dose. there is a quantity of S-9 mix which prc~ducesa maximum mutagenic response. For the most part. the quantity of %-9 mix, which induces the greatest number of revertant colonies, increases with an increase in dose of chemical. A graph of the data i n Table I (Fig. 1) plc~ttingrevertantslplate versus pg B(a)B/plate for each crf the S-9 anix quantities further illustrates the responses obtained by varying the quantity of the activation complex. At the lowest S-9 mix quantities (0.05, 0. 1 and 0.2 ml) the curves reach a plateau as the B(a)P concentration is increased. As the
Relationchip between quantity of S-9 mixturc and ability of B(a)P to revert tester strain 'I'A98. All numbers for revcraant colonies given are the averages of 3 replicate place\ minus the average nunmbes (33) of spontaneous TCVCFlaflBS --
Pamount
----
--
-
Quantity of S-9 mixture per plate b~nl)
per plate i ~ g ?
I5
12
18
18 45 131
17 100
143 340
85 220
23 88 52 42
24
lh8
10 18 'PO 40
33 84
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MUTAGENICITY OF PAIRWISE MIXTURES
Fig. I . Mutagenic response of increasing doses of B(a)P to different quantities (ml) of total S-9 mix. The plotted data are taken from Table I. The number near the end of each curve signifies the quantity (ml)of S-9 mix and the symbol at the end of the curve designates the syrnbots for that curve.
quantity of S-9 increases the curves show less s f a tendency to plateau. However, there is a ~narlced decrease in the slope of the curves as the S-9 mix quantities increase from 0.4 to 8.0 mi. Similar results to the above were also obtained In tests with B(rst)P, and increasing quantities of S-9 mix. The data of the combination study with B(a)P and B(rst)P are depicted in Fig. 2. The maximum number of revertant colonies induced by 2 p g of B(rst)P was 245 and this required 0.2 rnl S-9 mix. For B(a)P, the maximum colony production (333) occurred at 0.3 ml S-9 mix. Therefore, the total number of revertants expected in (rst)P combination study, using 8.5 ml of S-9 mix, might be the sum of the maximum mutagenic responses of each chemical or, in this case, 578 revertant colonies. This prediction depends upon the total S-9 quantity producing arm additive mutagenic effect. However, the actual value obtained for the combination study at 0.5 ml of S-9 mix is 317 5 26. suggesting a nonadditive effect of these two chemicals.
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104
ELI. F. SAEAMQNE ETA&.
To determine if there would be any additive effect of direct-acting mutagens in a two component mixture, tests were made with EMS and MNNG. These data (Fig. 3) indicated that both EMS and MNNG individually produced approximately linear dose reponses. The response of the combination test is also Hinear and the curve closely approximated the theoretical additivity curve, indicating that the mutagenicity s f the mixture is the sum s f the activities of each component.
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MUTAGENICITY OF PAIRWISE MIXTURES
105
Fig. 3 . Dose response of MNNG (open circles) and EMS (triangles) singly and i n pairwise combination (closed circles uppermost line). The predicted, con~binedresponse of the two individual curves is represented by the curve depicted with squares.
Discussion The data in Table I on B(a)P and our findings with B(rst)P suggest that for each particular dose of chemical, there is an optimal quantity of S-9 mix which will induce a maximum activity. Quantities sf S-9 mix less than c~ptimaHappear insufficient to activate maxirnaIIy the chemical and, in turn, induce the maximum number of revertant colonies. Excess quantities sf S-9 mix appear to inhibit partially or totally the mutagenic activity of the chemical. Similar results have been found with
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benzo(ghi)perylene, benzo(e)pyrene, coronene arad diarninoanisole using strain TA98 (Salamone and Katz. in prepration). Ashby and Styles (1978a) have indicated that enzymes from naouse and rat Iiver (S-9 mix) induced by aroclor 1254, caused a decrease in the observed mutagenic potency of B(a)B in the Ser&monuklcs/microssmaIassay, as compared with uninduced S-9. They demonstrated the variable effect of the exogenous activation system and pointed out the difficulties of using such activation systems in quantitative studies. In other studies, De Flora (19'78) noted that the addition of an activation mixture containing aroclor 1254 induced liver komogenate. reduced or "deactivated" the activity of direct-acting mutagens. This illustrates further the potential inhibiting effect of the rnicrosornal activation cc~anplex. Ames and Hooper (1998) cite an experiment with B(a)P in which the use of a series of increasing enzyme concentrations caused the mutagenic activity first to increase and then to diminish when more than the maximum recommended amount of enzyme was added per plate. This finding is in agreement with our results on the activity of B(a)P and other promutagens cited here. A decrease in mutagenic response by an excess concentration of S-9 mix is probably due to the presence of enzymes possessing oxidation. reduction and transferase properties. At the higher levels of S-9, competitive reactions between these enzymes and the chemicals may favor partial deactivation by conversion to less toxic or nontoxic products. Because of this inhibition effect, there is an inherent danger in using a single S-9 quantity over a small dose range of chemical, as the mutagenic response may be missed. In the combination studies it seems apparent that with mixtures of direct-acting mutagens the mutagenic activities of the cornponents are additive. The mutagenic response of EMS-MNNG combination was equal to the sum of their individual activities. On the other hand, with promutagens the mutagenicity of the mixture is not the sum of the mutagenicities of the con~ponentsas illustrated in the B(a)PB(rst)P results. These findings seem to implicate some action of the S-9 mix in the lack of additivity in pairwise promutagen cormabinations. Other experimental data support these findings. Using disect-acting mutagens, whose mutagenic response is only slightly affected by S-9 activation, in combination studies with and without S9 activation, we have found that the presence of the S-9 mix interferes with the predicted additive respolase (Salarnsne and Katz, in preparation). This lack cmf additivity in the pairwise coanbination studies with promartagens is not surprising, since B(a)P and B(rst)P individually showed an increasing but not additive response to increasing doses of these chemicals at the higher S-9 mix quantities. This nonadditivity could be related to competition for activation enzymes. Such competition may or may not occur in vivo, but some evidence for competition does exist. Brunette and Katz (1975) were able to show that perylene and, to a lesser extent. chrysene, may interfere with the uptake sf B(a)P by Chinese hamster ovary cells. However, none of these data conclusively implicates enzyme competition and many alternate hypotheses can be formulated to explain these findings. It could be the mutagenic potential c~fthe cells that is limiting. "Mutagenic pathways" or '"error-prone repair" might be saturated. suggesting that the lack of additivity demonstrates that both compounds act via the same mechanism which is saturated already by one compound. The presence of an exogenous activation system in the Sadmoszslla assay may lead to misinterpretation of data. S-9 hornogenates from different species (Bartsch k t a l . , 1975; McGregor, t 975) or individuals s f the same species (Garner et ak., 1972; Tang and Friedman, 1977) may differ in the levels of constituent (or induced enzymes due to genetic or dietary differences. These differences may produce results
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MUTAGENICITY OF PAHRWISE MIXTURES
107
i n the SaClnonella assay which vary significantly with regard to the mutagenic pc~tentialof a chemical. It is clear that a variation in the test conditions established for the Salmos~eHHu assay could change the observed potency of a mutagen (Amaes and Hooper, 1978; Ashby and Styles, 1978b). However, even if the standard conditions are not optimal for two or more mutagens in a mixture, one might expect to find an additive response. Our findings indicate that this is not the case for the promutagens tested. This lack of additivity appears to be an artifact of the exogenous activation system. The present study illustrates further the difficulty of quantifying ijs v i ~ r oresults where an exogenous activator is presetat. Acknowledgments This work was carried out with the technical assistance of Earl Stuart arad John Gingerich. Thanks are due to Drs. Earle Nestman and George Douglas, Health Protection Branch, National Health and Welfare for their review of the manuscript. This research was supported in part by Contract Grant 03 1-530-0927 of the Associate Committee on Scientific Criteria for Environmental Quality, National Research @ o m cil of Canada. References .4mes, B . N . , Da~rston, W . E . , Yarnasaki, E. and Lee, F. I). 1973. CarcEnogeins arc nnutagens. .4 sirnplz tzst system combining liver hornogenatrs for activation and bacteria for detection. $roc. Natl. Acad. Sci.. U.S.A. 70: 2288-2285. Ara-ees. B . X . , McCann, J . and 'famasaki, E. 1975. Methods for detecting carcinogens and mutagens with the S~lmonellu/manimalianmicrosome mutagenicity Best. Mutat. Res. 31: 347-364. Ames, B . N , and Hooper, K . 1978. Does carcinogenic potency correlate with mutapcnic potency in the: Ames assay'! Nature (London), 274: 19-20. Ashby, J . and Styles, J . A. I978a. Does carcinogenic potsncy correlate with mutagenic potency in the Amss Assay'? Nature (London), 271: 452-455. Ashby, J . and Styles, J . A . I998b. Factors inftucncing mutagenic potency irr r.itt.o. Nature (Laondun), 2'74: 20-22. Bartsch. H . . Zalalavcille, C . and Montcsano. K. 1975. Human, rat and niouse liver - rnediatzd mutagenicity of vinyl chloride in .F. fpkiniuriutn strains. Int. J . Cancer, 15: 429-437. Brunette, D. M. and Katz, M . 1975. The interaction of bcnzo(a)pyrene with cc1I rnernhranes: Uptake into Chinese hamster ovary cells and fluorescent stuefics with isolated meinhranes. Chem.-Biol. Interact. 11: 1-14. De Flora. S. 1978. Metabolic deactivation of niutagens in the .Fultnonclla-niicrosonsr Bsst. Nature (LoncBon), 271: 455-456. Garner, R . C.. Miller, E;.. C, and Miller, J . A . 1972.' Liver ~nicrosornaI metabolism of atlatoxin B , to a reactive derivative toxic to Salrnonclla ryphit?~uriumTA l 5 30. Cancer Wes. 32:24358-2066. McGregor, D. 8975. The relationship of 2-aceiamidofluorcne mutagenicity in plate tests with its in vivo liver cell component distribution and its carcinogenic potentias. Rlutat. Res. 30: 305-3 i 5 . Salamone, M . F., Heddle, J . A. and Katz. M. 1978. The rnutagenic activity of thirty polycyclic arornatic hydrocarbons anti oxides iu urban airborne particulates. Int. J . Environ. Stud. (in press). Tang, T . and Friedman, M. A . 1977. Carcinogen activation by huinan liver enzyme in the Alnes mutagenicity test. Mutat. Res. 46: 387-394.
