- Email: [email protected]
Comparisons of the promutagen-activating capacity of S9 liver preparations from mouse and chicken using in vitro tests with Salmonella and yeast
Mutation Research, 140 (1984) 75-79
75
Elsevier
MRLett 0568
Comparisons of the promutagen-activating capacity of $9 liver preparations from mouse and chicken using in vitro tests with Salmonella and yeast Y.I. Pavlov, N.N. K h r o m o v - B o r i s o v , L . P . Shevchenko, L . A , Alekseyevitch a n d S.G. Inge-Vechtomov Department of Genetics and Breeding, Leningrad State University, Leningrad, 199164 (U.S.S.R.) (Accepted 21 March 1984)
The methodology of in vitro activation is of considerable value in mutation research in such fundamental studies as carcinogen-mutagen correlations and genetic toxicology (Rinkus and Legator, 1979; Nagao et al., 1978). Rat and mouse liver microsomal preparations are most commonly used in these types of studies. However, the disadvantages of such preparations are obvious. First, it is necessary to use inducers of cytochrome P448/ P450 metabolism to obtain high activity in $9 microsomal preparations, and in some instances this procedure is potentially hazardous to laboratory workers (for example when one uses the carcinogenic Aroclor-1254; see Maron and Ames, 1983). Second, many animals are killed and incinerated, only the liver being excised and used in further procedures. Recently it was established that $9 preparations from chicken are efficient at metabolizing promutagens without requiring induction when used in both the Salmonella/ microsome test (see for example Tometsko et al., 1981; Curgeon et al., 1982) and in the yeast/ microsome test (Pavlov et al., 1980). There are at least 3 advantages to the use of chicken liver microsomes for mutagenicity testing: (a) No induction is needed. (b) They may show a wider metabolic profile. (c) Fewer animals are required. Data is presented here on the promutagenactivating capacity of chicken $9 in comparison with induced and uninduced mouse $9 using in vitro tests with both Salmonella and yeast. 0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
Materials and methods
We have used the Salmonella typhimurium tester strains TAI950, TA100 and TA98 (Ames et al., 1975) and Saccharomyces cerevisiae diploid tester strain P3288 of the following genotype: MATc~ ade2-916 lys2-A12 + MATa ade2-951 + metal This strain was constructed for the detection of heteroallelic mitotic recombination between the noncomplementing alleles ade2-916 and ade2-951 (Pavlov and Khromov-Borisov, 1981; Pavlov et al., 1981). Culture media for Salmonella and yeast were those described in Ames et al. (1975) and Pavlov et al. (1981), respectively. A leghorn chicken subline LR (low reactivity) was used (Alekseyevitch, 1976). Details concerning the chickens are in the legends to the figures. The male mice were F1 hybrids CBA x C57BL about 8 months old. According to the data of Mfiller et al. (1980), $9 microsomal preparations from mouse can be regarded as promutagen metabolizers as efficient as those from rat. We chose mouse because of the results of our preliminary experiments with Salmonella, where our $9 preparations from mouse livers were more active than $9 preparations from Wistar rat livers (2-aminofluorene and cyclophosphamide tested, data not presented). The standard methods of the $9 fraction
76
preparation and storage were used (Ames et al., 1975). The minor modification for chickens was that livers were prehomogenized for 1-2 min in a stainless steel tissue homogenizer at 2000 rev/min (Universal type, Poland) and only then homogenized in a Potter-type glass homogenizer with a teflon pestle. The $9 mix ÷ was a complete activation system (Ames et al., 1975) while in the $9 m i x - , NADP and glucose 6-phosphate were omitted. We used the $9 mix- for the nonactivation test conditions following the suggestion of Fonstein et al. (1978), because this appears to be the more appropriate control in the sense of an equal protein content of both the $9 mix ÷ and $9 m i x - . For example, yeast survival under our test conditions was similar in controls with the $9 mix + and the $9 m i x - , while survival was lower when yeasts were treated in buffer. 2-Aminofluorene (2-AF) was from 'Reanal' (Hungary), benzo[a]pyrene (BP) was kindly sent to us by Dr. D. Anderson (BIBRA, Great Britain), cyclophosphamide (CP) was from the Saransk
drug-producing factory (U.S.S.R.), sodium azide (NAN3) from 'BDH' (Great Britain), 2,7-diamino4,9-dihydroxy-5,10-dioxo-4,5,9,10-tetrahydro-4,9diazopyrene (DDDTDP) was a generous gift from Dr. L.M. Fonstein (U.S.S.R.). Experiments with Salmonella were performed according to Ames et al. (1975). Yeasts were treated at 36°C for 4 h as described earlier (Pavlov et al., 1981; Pavlov and Khromov-Borisov, 1983). Results and discussion
A summary of the results obtained with Salmonella strains are presented in Table 1. Three promutagens were tested: 2-AF, CP and BP. The results with the positive controls ensured that tester strains responded to the mutagens as predicted. TA1950 was reverted by NAN3, TA100 was reverted by both the base-pair substituting NaN3 and the frame-shifting DDDTDP (Abilev et al., 1979), while TA98 was reverted only by DDDTDP. It is important to note that the results presented for
TABLE 1 M U T A G E N I C ACTIVITY OF CP, BP A N D 2-AF METABOLIZED BY $9 P R E P A R A T I O N S FROM MOUSE AND CHICKEN. S A L M O N E L L A / M I C R O S O M E TEST Homo-
Variant
genatesa
Mean number of revertants per plate _+ S.E. in variants Strain TA1950 b Water
CP,500
Strain TAI00 c NAN3,10
DMSO
BP,10
Strain TA98 c NAN3,10
DDDTDP, 100
DMSO
2-AF,5
1
$9 mix + S9mix-
24+2 25+3
228+ 28 118+ 10
220± 16 224+65
298± 12 237±31
53+_ 5 31+_ 4
3124± 79 42+ 12
2
S9mix + S9mix-
24±4 25_+5
1094± 70 138_+ 8
224+10 267+_11
2743_+37 246_+11
42_+ 6 26+_ 4
4010_+143 76_+ 15
3
S9mix ÷ S9mix-
20_+3 14_+2
424_+ 58 120_+ 11
180±13 158_+20
1267_+38 165±20
44± 5 44± 6
7377± 87 50-+ 7
4
$9 mix + S9mix-
26-+4 21_+3
623-+112 146+ 8
241±42 283±12
1091_+23 207-+21
32± 4 25± 3
8687_+141 89_+ 23
5
S9mix + S9mix-
29_+4 30_+5
611-+ 79 180_+ 6
185±25 246± 16
901_+13 200+11
39+ 5 68± 15
5960_+133 42+ 5
none
-
19-+4
2859+53
202-+10
2439+50
859±30
33_+ 6
DDDTDP, 100
2106 ± 296
a (1) Livers pooled from 11 uninduced mice, (2) livers pooled from 16 Aroclor-1254 induced mice, (3,4,5) one liver from 2-year-old female chicken. b 300 /~1 of $9 per plate. c 150 td of $9 per plate. All mutagen concentrations are in/~g.
77
the promutagens are the maximal numbers of revertants in the linear part of the dose-response curves, which were determined for CP with varying concentrations, for 2-AF with the $9 concentration and for BP for both parameters. CP was slightly mutagenic without activation (variant $9 mix - ) or when uninduced mice were used. CP was moderately mutagenic when Aroclor-1254 induced mice or uninduced chickens were used for the $9 preparation. The former preparations were only 2 times more effective than the latter. The activity of the different $9 preparations were similar in the case o f BP. The $9 fractions from chicken were especially potent in converting 2-AF to mutagenic metabolites compared with the $9 preparations from uninduced and induced mice (Table 1). For yeast we utilized a liquid suspension test tor the in vitro metabolic activation experiments to
detect the induction of mitotic intragenic recombination (Pavlov and Khromov-Borisov, 1983). The same $9 preparations were used in experiments with yeast (Table 2) and with bacteria (Table 1). It is evident from the data that the $9 from chicken was far more effective in converting 2-AF, a known promutagen in yeast, to an active species than the $9 from uninduced mice. In order to examine further the parameters o f metabolic activation of 2-AF chicken liver we also used younger birds and compared males with females. The results are shown in Fig. 1. The $9 from chicken o f both sexes possessed a high activity. A direct comparison of the activity of older and younger chicken cannot be performed by comparing Table 2 and Fig. 1, because the spontaneous frequency of recombination was approximately 10 times higher in the experiments in Fig. 1. This dif-
TABLE 2 R E C O M B I N O G E N I C ACTIVITY OF 2-AF METABOLIZED BY $9 P R E P A R A T I O N S FROM MOUSE A N D CHICKEN. Y E A S T / M I C R O S O M E TEST Homogenate a Variant
Concentration Mean number of Survival (°70) Ozg/ml) recombinants per plate
1
S9mix +
0 5 10 20
19+ 2 133+13 130+_ 12 74-+ 8
100+19 69+__15 50-+ 15 29-+ 8
4.4+ 1.1 40.6_+ 6.0 53.8_+25.5 53.5+_17.9
2
S9mix +
0 2.5 5 10
20+_ 3 213_+11 508 _+ 13 513 + 19
89_+13 82_+11 67 ± 19 63 _+ 12
4.7+_ 1.1 54.3+_ 9.6 157.2 ± 55.2 170.1 _+30.6
3
S9mix +
0 2.5 5 10
20± 3 323 _+30 411 ± 12 370±11
93+19 80 + 20 63 ± 12 38-+ 8
4.4+_ 1.1 83.8 +_22.2 134.7 -+ 22.6 201.1 +_35.6
4
S9mix +
0 2.5 5 10
17_+ 3 667 ± 43 651 + 60 358 + 78
100+_15 83 + 16 77 + 18 38 _+ 15
3.5_+ 1.0 168.2 -+ 32.0 176.8 ± 46.0 195.5 ± 97.9
4
S9mix-
0 2.5 5 10
23+ 29± 26+24+
3 3 3 4
87+13 72±11 75+22 69+_16
Frequency of recombinants per 105 survivors
5.4± 8.4+7.1+_ 7.4-+
1.1 1.8 2.2 2.5
a Number of homogenates are as shown in Table 1. Test conditions were: 25% $9 mix in total 1 ml of incubation mixture was suspended in 0.1 M phosphate buffer (pH 7.4) yeast cells from logarithmic growth phase.
78
~00 o
o • L_ N .~
o
,p 8
"o
~0 •
o
o o
z~ A
N o I
q0~
0,~
I
I
I
~z5 ~
i
i
5 40
2-amJnofeuo~ne ( /.~ /m~) Fig. 1. Comparison of recombinogenic activity of 2-AF metabolized by $9 preparations from male and female chicken. O, female $9 mix ÷ ; e, male $9 mix ÷ ; ,a, female $9 mix- ; A, male $9 mix-. Test conditions: 1.5-month-old chickens were used; other conditions as in footnote to Table 2. ference was due mainly to the different numbers of cells plated on selective medium. It is known that when fe~ver cells o f the strain P3288 are plated the recombination frequency apparently increases (Pavlov and Khromov-Borisov, 1981). To summarize the data we can say that the $9 preparations f r o m chicken liver were more effective in the Salmonella/microsome test with all 3 promutagens, which have different activation pathways, than the analogous preparations f r o m uninduced mice. The activity o f chicken $9 was close to the activity o f the $9 f r o m Aroclor-1254 induced mice (in the case with 2-AF it was superior). The same high activity with respect to 2-AF was demonstrated for the yeast/chicken microsome test. The utilization of different species for in vitro metabolic activation is obviously o f great practical and theoretical interest. O f the 7 species studied by M/iller et al. (1980) the $9 preparations f r o m the livers o f b a b o o n and rhesus m o n k e y were the most effective in p r o m u t a g e n activation when uninduced animals were used. Mouse and dog were the most active a m o n g the same species induced with Aroclor. The main conclusion f r o m our study is
that the $9 preparations from uninduced chicken of either sex possess high promutagen activation capacity. Owing to the low cost of such preparations, which do not require enzyme induction, these preparations might be regarded as candidates to substitute or supplement the conventional activation systems for the in vitro mutagenicity testing of chemical compounds. It is known that birds possess an efficient xenobiotic metabolism. The $9 preparations from duck liver were far more active than m a m m a l i a n liver in activating aflatoxin B~ (Hsieh et al., 1977). Hepatic BP hydroxylase in pigeon was approximately 3 timers more active than that of rat or mouse (Hussain et al., 1981). Both male and female chickens develop a high level of xenobiotic metabolism at an early development stage (Rifkind et al., 1979; Bloom et al., 1982; Hamilton et al., 1983). Chicken $9 is a very active promutagen activation system in the Salmonella/microsome test (Tometsko et al., 1981; Curgeon et al., 1982; present data). In order to explain the high activity of the $9 preparations f r o m chicken liver one may propose that: (a) Chickens have an intrinsic high capacity to activate the promutagens we have studied or (b) The chickens chosen for these studies were already induced by some unidentified environmental factors. The same proposal was put forward by Hussain et al. (1981) to explain the different mixed function oxidation activity in wild and homing pigeon.
Acknowledgements We thank M.I. R a h m a n for suggestions on statistics and T.A. Kamilova for the help in figure preparation. We are grateful to the referees for the improvement o f the text. We thank Dr. S.K. Abilev for some references on use of chicken $9 preparations in promutagen activation and Dr. L.M. Fonstein for the gift of D D D T D P .
79
References Abilev, S.K., L.M. Fonstein, G.I. Migachev and A.M. Andrievsky (1979) On the mutagenic action of DDDTDP in bacteria, Genetika (U.S.S.R.), 15, 807-811. Alekseyevitch, L.A. (1976) Selection of chicken lines differing by reactivity, Research in Genetics, Published by Leningrad University, Vol. 6, pp. 123-128. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ microsome test, Mutation Res., 31, 347-364. Bloom, S.E., J.W. Hamilton and M.S. Denison (1982) Metabolic activation of promutagen-carcinogens in the early chick embryo, Genetics, 100, s36. Curgeon, R.D., R.G. Kmetz, A.S. Parmar and R.E. Nelson (1982) The use of S-9's prepared from livers of food production and laboratory animals in the Ames' Salmonella/ microsomal assay, Environ. Mutagen., 4, 329. Fonstein, L.M., S.K. Abilev and C.P. Akinshina (1978) A study of metabolic activation of chemicals using microorganisms, I. Effect of inducers of microsomal systems, Genetika (U.S.S.R.), 14, 1564-1570. Hamilton, J.W., M.S. Denison and S.E. Bloom (1983) Development of basal and induced aryl hydrocarbon benzo[a]pyrene hydroxylase activity in the chicken embryo in ovo, Proc. Natl. Acad. Sci. (U.S.A.), 80, 3372-3376. Hsieh, D.P.H., J.J. Wong, Z.A. Wong, C. Michas and B.H. Ruebner (1977) Hepatic transformation of aflatoxin and its carcinogenicity, in: Hiatt et al. (Eds.), Origins of Human Cancer, Cold Spring Harbor Laboratory, Book B, pp. 697-707. Hussain, M.M., A. Kumar, H. Mukhtar and C.R. Krishna Murti (1981) Xenobiotic biotransformation in wild birds: activity, induction, characterization and comparison with rat and mouse microsomal enzymes, Xenobiotica, 11, 785-793.
Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Miiller, D., J. Nelles, E. Deparade and P. Arni (1980) The activity of $9 liver fractions from seven species in the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 70, 279-300. Nagao, M., T. Sugimura and T. Matsushima (1978) Environmental mutagens and carcinogens, Annu. Rev. Genet., 12, 117-160. Pavlov, Y.I., and N.N. Khromov-Borisov (1981) Metabolic activation of promutagens in the yeast/microsome test, I. Development of tester strains and methods for their testing, Genetika (U.S.S.R.), 17, 1406-1412. Pavlov, Y.I., and N.N. Khromov-Borisov (1983) Metabolic activation of promutagens in the yeast/microsome test, II. Activation of cyclophosphamide and 2-aminofluorene, Genetika (U.S.S.R.), 19, 362-374. Pavlov, Y.I., N.N. Khromov-Borisov, L.A. Alekseyevitch and S.G. Inge-Vechtomov (1980) Metabolic activation of promutagens by microsomes from chicken, in: Genetic aspects of Environmental Pollution in Moldavia, Moldavian Acad. Sci., Kishinev, pp. 118-119. Pavlov, Y.I., N.N. Khromov-Borisov and S.G. IngeVechtomov (1981) 2-Fluorenamine, indirect recombinogen for Saccharomyces cerevisiae, Mutation Res., 91, 221-224. Rifkind, A.B., M. Troeger and T. Betschke (1979) Equality of the rates of mixed function oxidation in livers of male and female chick embryos, Biochem. Pharm., 28, 1681-1683. Rinkus, S.J., and M.S. Legator (1979) Chemical characterization of 465 known or suspected carcinogens and their correlation with mutagenic activity in the Salmonella typhimurium system, Cancer Res., 39, 3289-3318. Tometsko, A.M., K.M. Sheridan and M.C. DeTraglia (1981) Promutagen activation with mammalian and avian $9 liver microsomes, J. Appl. Toxicol., 1, 11-14.
75
Elsevier
MRLett 0568
Comparisons of the promutagen-activating capacity of $9 liver preparations from mouse and chicken using in vitro tests with Salmonella and yeast Y.I. Pavlov, N.N. K h r o m o v - B o r i s o v , L . P . Shevchenko, L . A , Alekseyevitch a n d S.G. Inge-Vechtomov Department of Genetics and Breeding, Leningrad State University, Leningrad, 199164 (U.S.S.R.) (Accepted 21 March 1984)
The methodology of in vitro activation is of considerable value in mutation research in such fundamental studies as carcinogen-mutagen correlations and genetic toxicology (Rinkus and Legator, 1979; Nagao et al., 1978). Rat and mouse liver microsomal preparations are most commonly used in these types of studies. However, the disadvantages of such preparations are obvious. First, it is necessary to use inducers of cytochrome P448/ P450 metabolism to obtain high activity in $9 microsomal preparations, and in some instances this procedure is potentially hazardous to laboratory workers (for example when one uses the carcinogenic Aroclor-1254; see Maron and Ames, 1983). Second, many animals are killed and incinerated, only the liver being excised and used in further procedures. Recently it was established that $9 preparations from chicken are efficient at metabolizing promutagens without requiring induction when used in both the Salmonella/ microsome test (see for example Tometsko et al., 1981; Curgeon et al., 1982) and in the yeast/ microsome test (Pavlov et al., 1980). There are at least 3 advantages to the use of chicken liver microsomes for mutagenicity testing: (a) No induction is needed. (b) They may show a wider metabolic profile. (c) Fewer animals are required. Data is presented here on the promutagenactivating capacity of chicken $9 in comparison with induced and uninduced mouse $9 using in vitro tests with both Salmonella and yeast. 0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
Materials and methods
We have used the Salmonella typhimurium tester strains TAI950, TA100 and TA98 (Ames et al., 1975) and Saccharomyces cerevisiae diploid tester strain P3288 of the following genotype: MATc~ ade2-916 lys2-A12 + MATa ade2-951 + metal This strain was constructed for the detection of heteroallelic mitotic recombination between the noncomplementing alleles ade2-916 and ade2-951 (Pavlov and Khromov-Borisov, 1981; Pavlov et al., 1981). Culture media for Salmonella and yeast were those described in Ames et al. (1975) and Pavlov et al. (1981), respectively. A leghorn chicken subline LR (low reactivity) was used (Alekseyevitch, 1976). Details concerning the chickens are in the legends to the figures. The male mice were F1 hybrids CBA x C57BL about 8 months old. According to the data of Mfiller et al. (1980), $9 microsomal preparations from mouse can be regarded as promutagen metabolizers as efficient as those from rat. We chose mouse because of the results of our preliminary experiments with Salmonella, where our $9 preparations from mouse livers were more active than $9 preparations from Wistar rat livers (2-aminofluorene and cyclophosphamide tested, data not presented). The standard methods of the $9 fraction
76
preparation and storage were used (Ames et al., 1975). The minor modification for chickens was that livers were prehomogenized for 1-2 min in a stainless steel tissue homogenizer at 2000 rev/min (Universal type, Poland) and only then homogenized in a Potter-type glass homogenizer with a teflon pestle. The $9 mix ÷ was a complete activation system (Ames et al., 1975) while in the $9 m i x - , NADP and glucose 6-phosphate were omitted. We used the $9 mix- for the nonactivation test conditions following the suggestion of Fonstein et al. (1978), because this appears to be the more appropriate control in the sense of an equal protein content of both the $9 mix ÷ and $9 m i x - . For example, yeast survival under our test conditions was similar in controls with the $9 mix + and the $9 m i x - , while survival was lower when yeasts were treated in buffer. 2-Aminofluorene (2-AF) was from 'Reanal' (Hungary), benzo[a]pyrene (BP) was kindly sent to us by Dr. D. Anderson (BIBRA, Great Britain), cyclophosphamide (CP) was from the Saransk
drug-producing factory (U.S.S.R.), sodium azide (NAN3) from 'BDH' (Great Britain), 2,7-diamino4,9-dihydroxy-5,10-dioxo-4,5,9,10-tetrahydro-4,9diazopyrene (DDDTDP) was a generous gift from Dr. L.M. Fonstein (U.S.S.R.). Experiments with Salmonella were performed according to Ames et al. (1975). Yeasts were treated at 36°C for 4 h as described earlier (Pavlov et al., 1981; Pavlov and Khromov-Borisov, 1983). Results and discussion
A summary of the results obtained with Salmonella strains are presented in Table 1. Three promutagens were tested: 2-AF, CP and BP. The results with the positive controls ensured that tester strains responded to the mutagens as predicted. TA1950 was reverted by NAN3, TA100 was reverted by both the base-pair substituting NaN3 and the frame-shifting DDDTDP (Abilev et al., 1979), while TA98 was reverted only by DDDTDP. It is important to note that the results presented for
TABLE 1 M U T A G E N I C ACTIVITY OF CP, BP A N D 2-AF METABOLIZED BY $9 P R E P A R A T I O N S FROM MOUSE AND CHICKEN. S A L M O N E L L A / M I C R O S O M E TEST Homo-
Variant
genatesa
Mean number of revertants per plate _+ S.E. in variants Strain TA1950 b Water
CP,500
Strain TAI00 c NAN3,10
DMSO
BP,10
Strain TA98 c NAN3,10
DDDTDP, 100
DMSO
2-AF,5
1
$9 mix + S9mix-
24+2 25+3
228+ 28 118+ 10
220± 16 224+65
298± 12 237±31
53+_ 5 31+_ 4
3124± 79 42+ 12
2
S9mix + S9mix-
24±4 25_+5
1094± 70 138_+ 8
224+10 267+_11
2743_+37 246_+11
42_+ 6 26+_ 4
4010_+143 76_+ 15
3
S9mix ÷ S9mix-
20_+3 14_+2
424_+ 58 120_+ 11
180±13 158_+20
1267_+38 165±20
44± 5 44± 6
7377± 87 50-+ 7
4
$9 mix + S9mix-
26-+4 21_+3
623-+112 146+ 8
241±42 283±12
1091_+23 207-+21
32± 4 25± 3
8687_+141 89_+ 23
5
S9mix + S9mix-
29_+4 30_+5
611-+ 79 180_+ 6
185±25 246± 16
901_+13 200+11
39+ 5 68± 15
5960_+133 42+ 5
none
-
19-+4
2859+53
202-+10
2439+50
859±30
33_+ 6
DDDTDP, 100
2106 ± 296
a (1) Livers pooled from 11 uninduced mice, (2) livers pooled from 16 Aroclor-1254 induced mice, (3,4,5) one liver from 2-year-old female chicken. b 300 /~1 of $9 per plate. c 150 td of $9 per plate. All mutagen concentrations are in/~g.
77
the promutagens are the maximal numbers of revertants in the linear part of the dose-response curves, which were determined for CP with varying concentrations, for 2-AF with the $9 concentration and for BP for both parameters. CP was slightly mutagenic without activation (variant $9 mix - ) or when uninduced mice were used. CP was moderately mutagenic when Aroclor-1254 induced mice or uninduced chickens were used for the $9 preparation. The former preparations were only 2 times more effective than the latter. The activity of the different $9 preparations were similar in the case o f BP. The $9 fractions from chicken were especially potent in converting 2-AF to mutagenic metabolites compared with the $9 preparations from uninduced and induced mice (Table 1). For yeast we utilized a liquid suspension test tor the in vitro metabolic activation experiments to
detect the induction of mitotic intragenic recombination (Pavlov and Khromov-Borisov, 1983). The same $9 preparations were used in experiments with yeast (Table 2) and with bacteria (Table 1). It is evident from the data that the $9 from chicken was far more effective in converting 2-AF, a known promutagen in yeast, to an active species than the $9 from uninduced mice. In order to examine further the parameters o f metabolic activation of 2-AF chicken liver we also used younger birds and compared males with females. The results are shown in Fig. 1. The $9 from chicken o f both sexes possessed a high activity. A direct comparison of the activity of older and younger chicken cannot be performed by comparing Table 2 and Fig. 1, because the spontaneous frequency of recombination was approximately 10 times higher in the experiments in Fig. 1. This dif-
TABLE 2 R E C O M B I N O G E N I C ACTIVITY OF 2-AF METABOLIZED BY $9 P R E P A R A T I O N S FROM MOUSE A N D CHICKEN. Y E A S T / M I C R O S O M E TEST Homogenate a Variant
Concentration Mean number of Survival (°70) Ozg/ml) recombinants per plate
1
S9mix +
0 5 10 20
19+ 2 133+13 130+_ 12 74-+ 8
100+19 69+__15 50-+ 15 29-+ 8
4.4+ 1.1 40.6_+ 6.0 53.8_+25.5 53.5+_17.9
2
S9mix +
0 2.5 5 10
20+_ 3 213_+11 508 _+ 13 513 + 19
89_+13 82_+11 67 ± 19 63 _+ 12
4.7+_ 1.1 54.3+_ 9.6 157.2 ± 55.2 170.1 _+30.6
3
S9mix +
0 2.5 5 10
20± 3 323 _+30 411 ± 12 370±11
93+19 80 + 20 63 ± 12 38-+ 8
4.4+_ 1.1 83.8 +_22.2 134.7 -+ 22.6 201.1 +_35.6
4
S9mix +
0 2.5 5 10
17_+ 3 667 ± 43 651 + 60 358 + 78
100+_15 83 + 16 77 + 18 38 _+ 15
3.5_+ 1.0 168.2 -+ 32.0 176.8 ± 46.0 195.5 ± 97.9
4
S9mix-
0 2.5 5 10
23+ 29± 26+24+
3 3 3 4
87+13 72±11 75+22 69+_16
Frequency of recombinants per 105 survivors
5.4± 8.4+7.1+_ 7.4-+
1.1 1.8 2.2 2.5
a Number of homogenates are as shown in Table 1. Test conditions were: 25% $9 mix in total 1 ml of incubation mixture was suspended in 0.1 M phosphate buffer (pH 7.4) yeast cells from logarithmic growth phase.
78
~00 o
o • L_ N .~
o
,p 8
"o
~0 •
o
o o
z~ A
N o I
q0~
0,~
I
I
I
~z5 ~
i
i
5 40
2-amJnofeuo~ne ( /.~ /m~) Fig. 1. Comparison of recombinogenic activity of 2-AF metabolized by $9 preparations from male and female chicken. O, female $9 mix ÷ ; e, male $9 mix ÷ ; ,a, female $9 mix- ; A, male $9 mix-. Test conditions: 1.5-month-old chickens were used; other conditions as in footnote to Table 2. ference was due mainly to the different numbers of cells plated on selective medium. It is known that when fe~ver cells o f the strain P3288 are plated the recombination frequency apparently increases (Pavlov and Khromov-Borisov, 1981). To summarize the data we can say that the $9 preparations f r o m chicken liver were more effective in the Salmonella/microsome test with all 3 promutagens, which have different activation pathways, than the analogous preparations f r o m uninduced mice. The activity o f chicken $9 was close to the activity o f the $9 f r o m Aroclor-1254 induced mice (in the case with 2-AF it was superior). The same high activity with respect to 2-AF was demonstrated for the yeast/chicken microsome test. The utilization of different species for in vitro metabolic activation is obviously o f great practical and theoretical interest. O f the 7 species studied by M/iller et al. (1980) the $9 preparations f r o m the livers o f b a b o o n and rhesus m o n k e y were the most effective in p r o m u t a g e n activation when uninduced animals were used. Mouse and dog were the most active a m o n g the same species induced with Aroclor. The main conclusion f r o m our study is
that the $9 preparations from uninduced chicken of either sex possess high promutagen activation capacity. Owing to the low cost of such preparations, which do not require enzyme induction, these preparations might be regarded as candidates to substitute or supplement the conventional activation systems for the in vitro mutagenicity testing of chemical compounds. It is known that birds possess an efficient xenobiotic metabolism. The $9 preparations from duck liver were far more active than m a m m a l i a n liver in activating aflatoxin B~ (Hsieh et al., 1977). Hepatic BP hydroxylase in pigeon was approximately 3 timers more active than that of rat or mouse (Hussain et al., 1981). Both male and female chickens develop a high level of xenobiotic metabolism at an early development stage (Rifkind et al., 1979; Bloom et al., 1982; Hamilton et al., 1983). Chicken $9 is a very active promutagen activation system in the Salmonella/microsome test (Tometsko et al., 1981; Curgeon et al., 1982; present data). In order to explain the high activity of the $9 preparations f r o m chicken liver one may propose that: (a) Chickens have an intrinsic high capacity to activate the promutagens we have studied or (b) The chickens chosen for these studies were already induced by some unidentified environmental factors. The same proposal was put forward by Hussain et al. (1981) to explain the different mixed function oxidation activity in wild and homing pigeon.
Acknowledgements We thank M.I. R a h m a n for suggestions on statistics and T.A. Kamilova for the help in figure preparation. We are grateful to the referees for the improvement o f the text. We thank Dr. S.K. Abilev for some references on use of chicken $9 preparations in promutagen activation and Dr. L.M. Fonstein for the gift of D D D T D P .
79
References Abilev, S.K., L.M. Fonstein, G.I. Migachev and A.M. Andrievsky (1979) On the mutagenic action of DDDTDP in bacteria, Genetika (U.S.S.R.), 15, 807-811. Alekseyevitch, L.A. (1976) Selection of chicken lines differing by reactivity, Research in Genetics, Published by Leningrad University, Vol. 6, pp. 123-128. Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ microsome test, Mutation Res., 31, 347-364. Bloom, S.E., J.W. Hamilton and M.S. Denison (1982) Metabolic activation of promutagen-carcinogens in the early chick embryo, Genetics, 100, s36. Curgeon, R.D., R.G. Kmetz, A.S. Parmar and R.E. Nelson (1982) The use of S-9's prepared from livers of food production and laboratory animals in the Ames' Salmonella/ microsomal assay, Environ. Mutagen., 4, 329. Fonstein, L.M., S.K. Abilev and C.P. Akinshina (1978) A study of metabolic activation of chemicals using microorganisms, I. Effect of inducers of microsomal systems, Genetika (U.S.S.R.), 14, 1564-1570. Hamilton, J.W., M.S. Denison and S.E. Bloom (1983) Development of basal and induced aryl hydrocarbon benzo[a]pyrene hydroxylase activity in the chicken embryo in ovo, Proc. Natl. Acad. Sci. (U.S.A.), 80, 3372-3376. Hsieh, D.P.H., J.J. Wong, Z.A. Wong, C. Michas and B.H. Ruebner (1977) Hepatic transformation of aflatoxin and its carcinogenicity, in: Hiatt et al. (Eds.), Origins of Human Cancer, Cold Spring Harbor Laboratory, Book B, pp. 697-707. Hussain, M.M., A. Kumar, H. Mukhtar and C.R. Krishna Murti (1981) Xenobiotic biotransformation in wild birds: activity, induction, characterization and comparison with rat and mouse microsomal enzymes, Xenobiotica, 11, 785-793.
Maron, D.M., and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Miiller, D., J. Nelles, E. Deparade and P. Arni (1980) The activity of $9 liver fractions from seven species in the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 70, 279-300. Nagao, M., T. Sugimura and T. Matsushima (1978) Environmental mutagens and carcinogens, Annu. Rev. Genet., 12, 117-160. Pavlov, Y.I., and N.N. Khromov-Borisov (1981) Metabolic activation of promutagens in the yeast/microsome test, I. Development of tester strains and methods for their testing, Genetika (U.S.S.R.), 17, 1406-1412. Pavlov, Y.I., and N.N. Khromov-Borisov (1983) Metabolic activation of promutagens in the yeast/microsome test, II. Activation of cyclophosphamide and 2-aminofluorene, Genetika (U.S.S.R.), 19, 362-374. Pavlov, Y.I., N.N. Khromov-Borisov, L.A. Alekseyevitch and S.G. Inge-Vechtomov (1980) Metabolic activation of promutagens by microsomes from chicken, in: Genetic aspects of Environmental Pollution in Moldavia, Moldavian Acad. Sci., Kishinev, pp. 118-119. Pavlov, Y.I., N.N. Khromov-Borisov and S.G. IngeVechtomov (1981) 2-Fluorenamine, indirect recombinogen for Saccharomyces cerevisiae, Mutation Res., 91, 221-224. Rifkind, A.B., M. Troeger and T. Betschke (1979) Equality of the rates of mixed function oxidation in livers of male and female chick embryos, Biochem. Pharm., 28, 1681-1683. Rinkus, S.J., and M.S. Legator (1979) Chemical characterization of 465 known or suspected carcinogens and their correlation with mutagenic activity in the Salmonella typhimurium system, Cancer Res., 39, 3289-3318. Tometsko, A.M., K.M. Sheridan and M.C. DeTraglia (1981) Promutagen activation with mammalian and avian $9 liver microsomes, J. Appl. Toxicol., 1, 11-14.