215
Mutation Research, 38 (1976) 215--224 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands
GENETIC ACTIVITY OF THE ANTIMICROBIAL FOOD ADDITIVES AF-2 AND H-193 IN SACCHAROMYCES CEREVI$IAE
MAJDI M. SHAHIN and R.C. VON BORSTEL
Department of Genetics, University of Alberta Edmonton, Alberta T6G 2E9 (Canada) (Received October 1st, 1975) (Revision received January 26th, 1976) (Accepted January 27th, 1975)
Summary The genetic activity of the antimicrobial food additives AF-2 and H-193 has been investigated in Saccharomyces cerevisiae. The strains chosen for the present studies were D5 for the induction of mitotic recombinational events and XV185-14C for the induction of reversion of the mutants lysl-1, his1-7 and hom3-7. When three concentrations (25, 50 and 100 pg/ml) of AF-2 were used in the reversion system of strain XV185-14C, there was an increase in the frequency of hom÷ and his* revertants as a function of incubation time, while the lysine m u t a n t exhibited a very low frequency of induced reversion. When AF-2 and H-193 were compared at the same concentration and exposure time, AF-2 exhibited a higher genetic activity in both systems than H-193. However, H-193 was genetically more active in inducing revertants than AF-2, when the comparison was made at the same survival level. Cells of both haploid and diploid strains were found to be more sensitive to inactivation by AF-2 than by H-193. It should be pointed out that the solubility of H-193 was lower (about 4 pg/ml saturation) than the solubility of AF-2 (120 #g/ml saturation). T h e h a p l o i d strain was more sensitive to both compounds than the diploid strain.
Introduction AF-2 (furylfuramide) and H-193 (Fig. 1) are two antimicrobial food additives. AF-2 induces mutations in bacteria [5--8], chromosome aberrations in human lymphocytes in vitro [24,25] and rat bone marrow cells in vivo [20], mutations in Neurospora and mitotic recombination in yeast [11]. It has also been shown to induce mutations in the silkworm using egg color mutants as markers [22]. Soares and Sheridan [19] have presented data indicating t h a t AF-2 is relatively non-toxic in unstarved mice and does not induce d o m i n a n t lethals in the strains of mice tested.
216 Studies on the mutagenic effect of the antimicrobial food additive H-193 have not been reported. The experiments reported herein were undertaken (1) to identify and investigate the genetic activity of H-193 in yeast, (2) to investigate the genetic activity of AF-2 using the gene mutation system of Saccharomyces cerevisiae where the reversion at lysl-1, his1-7 and horn3-10 could be studied, and (3) to compare the gene mutation and the mitotic recombination systems in their responses to AF-2 and H-193. Materials and Methods The strain used for mitotic recombination studies The diploid strain of S. cerevisiae (D5) used in the present study for the induction of mitotic recombination was developed b y Zimmermann [27]. The two haploid strains of which D5 is composed have the following genotypes: AZ--5A: a, trpl, ade2-40, M A L l
/
4--12D:
J
a, leul, ade2-119, MAL4
D5
Strain D5 carries two phenotypically distinguishable ade2 alleles. The strain homozygous for the ade2-40 allele forms red colonies. The other allele, ade2119, is leaky; therefore, the homozygotes form pink colonies. The two alleles complement fully so that the heterozygous diploid (D5) forms white colonies and does not require adenine for growth. In strain D5, genetic activity is indicated by the appearance of six phenotypically distinguishable colony types. The appearance of pink and red-sectored colonies (twin spots) provides evidence for mitotic crossing over. All red or all pink colonies can be due to mitotic crossing over, mutation, mitotic gene conversion, or aneuploidy. In addition to pink and red twin spots, sectored colony types include pink and white, red and white, and red, pink and white. Preparation o f cell samples, treatment conditions and plating Cells from stock culture were inoculated into liquid YPG medium (0.15% KH2PO4, 0.01% MgSO4, 0.45% (NH4)2SO4, 0.35% Difco proteose peptone, 0.5% Difco yeast extract and 2% glucose) and incubated at 30°C in a shaking water bath until they reached stationary phase. The cells were than harvested by centrifugation and washed twice in phosphate buffer (pH 7). Cell concentrations were adjusted to 1 × 10 s cells/ml. At zero time, the compound(s) to be tested was added to the cell suspension to give the desired final chemical concentration. Control and treated cell suspensions in Pyrex, low actinic, red erlenmeyer flasks were continually agitated at 30°C. At appropriate intervals, cell samples were withdrawn, centrifuged, and washed twice in phosphate buffer. The cell samples were then diluted and plated to give a b o u t 200--300 colonies per plate. The plates were incubated for 5 days at 30°C, and colony-forming ability was used as the criterion for survival. The strain used for gene mutation studies The haploid strain XV185-14C used for the induction of gene mutation was
217
developed by Quah and Von Borstel [13]. The genotype of XV185-14C is as follows: a, ade2-1, arg4-17, lysl-1, trp5-48, his1-7, hom3-10. Although strain XV185-14C has six markers, it requires six amino acids and one base for growth, because the homoserine marker blocks the synthetic pathway of threonine and methionine. Reversion at the lysl, his1 or horn3 loci produces cells capable of growth on medium deficient in either lysine, histidine or threonine.
Determination of spontaneous and induced reversion frequency For the determination of spontaneous or induced reversion frequency, undiluted samples (1 × 108 cells/ml) were plated (0.2 ml/plate) on medium containing Difco yeast nitrogen base without amino acids plus 2% glucose plus 2% agar plus all required supplements except lysine, histidine or threonine. For survival estimation, treated and control samples were diluted and plated on the same medium plus all required substances to give a b o u t 200--300 colonies per plate. The plates were incubated for six days at 300(2, and colony-forming ability was used as the criterion for survival. Preparation of cell samples, treatment conditions and plating are the same as described for strain D5. Results
Cell inactivation The cell inactivation effect of AF-2 on the haploid strain XV185-14C and on the diploid strain D5 is shown in Tables I and II. The decrease in the percentage of survival is dependent on the concentration used and time of exposure. Cells of b o t h strains were found to be m o r e sensitive to inactivation by AF-2 than H-193 (Tables I--IV). It should, however, be pointed o u t that the solubility of H-193 was lower in the 2% dimethyl sulfoxide phosphate buffer solution
TABLE I R E V E R S I O N F R E Q U E N C I E S OF S T R A I N XV185-14C A F T E R C O N C E N T R A T I O N S OF AF-2
TREATMENT
WITH DIFFERENT
N u m b e r s in p a r e n t h e s e s are t h e n u m b e r s of r e v e r t a n t c o l o n i e s c o u n t e d p e r 3 p l a t e s ( h i s t i d i n e ) or p e r 6 plates (homoserine, Iysine).
Dose (/~g/ml)
0 0 25 25 25 25 50 50 50 100 100 100 100
Treatm e n t (h)
0 24 2 4 8 24 2 8 24 2 4 ~ 8 24
R e v e r t a n t s p e r 107 survivors
Survival (%)
horn3-10
hisl-7
lys i-1
0.53 (8) 0.66 (10) 2.80 (38) 3.40 (28) 4.50 (40) 6.60 (40) 3.00 (36) 6.00 (18) 26.60 (16) 4.40 (36) 6.50 (22) 11.60 (10) 4 4 . 4 0 (2)
25 (198) 22 (168) 88 (592) 144 (666) 160 (699) 215 (644) 133 (799) 168 (253) 296 (89) 160 (648) 170 (286) 223 (100) 3 5 5 (8)
15 (228) 16 (244) 19 (250) 24 (214) 21 (188) 25 (152) 24 ( 2 8 2 ) 22 ( 6 6 ) 17 ( 1 0 ) 28 ( 1 2 6 ) 28 (94) 22 (20) - - (0)
I00 108 90 60 58 44 80 20 4 54 22 6 0.3
90
25
23
50 pg AF-2/ml 4
24
28
1.6
1.0
0.13
28
48
62
24
200 pg AF-2/ml 4
0.4
4.6
28
48
6.5
85
24
100 pg AF-2/ml 4
3.4
91
48
48
97
100
Control 0
24
% survival
OF ALTERED
Treatment (h)
FREQUENCY
TABLE II
789
1657
2524
3810
2767
2838
4044
5230
4959
3548
3876
5586
5625
5984
6189
scored
Number of colonies
COLONIES
29 0.761% 45 1.78% 31 1.87% 19 2.4%
32 0.612% 47 1.162% 33 1.163% 43 1.55%
7 0.i 25% 18 0.464% 20 0.563% 37 0.746%
6 0.157% 5 0.198% 4 0.241% 2 0.253%
9 0.172% 6 0.148% 7 0.247% 11 0.397%
5 0.089% 9 0.232% 8 0.225% 9 0.181%
9 0.236% II 0.436% 7 0.422% 3 0.380%
6 0.115% 6 0.148% 11 0.388% 7 0.253%
5 0.089% 7 0.180% 10 0.282% 17 0.343%
0.033% 1 0.018%
0.017% 1 0.018%
--
0.048% 2
1 0.016% 1
3
Pink
STRAIN
--
Red
CEREVISIAE
---
Red and pink (twin spots)
IN SACCHAROMYCES
3 0.079% 4 0.i 58% 5 0.302% 3 0.380%
7 0.134% 12 0.296% 6 0.211% 8 0.289%
3 0.054% 4 0.103% 5 0.141% 16 0.323%
--
0.017%
0.016% 1
1
Pink and white
D5 WITH AF-2
7 0.184% 5 0.198% 3 0.181% 2 0.253%
11 0.210% 3 0.074% 9 0.317% 5 0.181%
4 0.072% 5 0.129% 7 0.197% 11 0.22%
1 0.017% 1 0.018%
Red and white
7 0.184% 6 0.238% 7 0.422% 5 0.634%
6 0.115% 2 0.049% 9 0.317% 7 0.253%
4 0.072% 6 0.155% 5 0.141% 13 0.262%
2 0.032% 3 0.050% 4 0.071%
Hairline
61 1.60% 76 3.01% 57 3.43% 34 4.31%
71 1.36% 76 1.88% 75 2.64% 81 2.93%
28 0.50% 49 1.26% 55 1.55% 103 2.01%
7 0.113% 8 0.134% 7 0.124%
Total number and % of altered colonies
b~
219 T A B L E III REVERSION FREQUENCIES OF STRAIN CONCENTRATIONS OF H-193
XV185-14C
AFTER
TREATMENT
WITH DIFFERENT
N u m b e r s in p a r e n t h e s e s a r e t h e n u m b e r s o f r e v e r t a n t c o l o n i e s c o u n t e d p e r 3 p l a t e s ( h i s t i d i n e ) o r p e r 6 p l a t e s ( h o m o s e r i n e , lysine). Dose (~g/ml)
0 0 0 0 50 50 50 50 100 100 100 100 200 200 200 200
Treatm e n t (h)
0 24 28 48 4 24 28 48 4 24 28 48 4 24 28 48
Revertants per 10 ? survivors
Survival (%)
horn3-10
his l - 7
lysl -i
3.1 2.8 3.8 3.8 8.1 9.0 11.8 11.1 6.6 14.6 13.1 17.3 6.0 7.6 7.2 17.6
23 33 28 30 98 182 138 200 69 300 186 291 70 181 198 334
15 14 14 13 15 29 21 18 14 28 29 39 11 18 16 30
(42) (36) (50) (46) (106) (62) (74) (52) (86) (82) (66) (62) (80) (60) (46) (46)
(155) (214) (184) (178) (643) (637) (433) (468) (462) (842) (481) (521) (475) (713) (635) (455)
(202) (184) (182) (160) (200) (200) (134) (86) (184) (158) (150) (140) (148) (138) (104) (82)
I00 95 97 90 97 52 47 35 99 42 38 27 100 51 48 20
(about 4 pg/ml saturation) than the solubility of AF-2 in phosphate buffer (120 pg/ml saturation}. When a comparison of the haploid strain XV185-14C and the diploid strain D5 was made, the haploid strain (Tables I and III} was more sensitive to inactivation b y b o t h c o m p o u n d s than the diploid strain (Tables II and IV). For example, at a concentration of 100 pg AF-2/ml and an incubation time of 24 h, the surviving fraction increased from 0.3% (haploid strain XV18514C, Table I) to 6.5% (diploid strain D5, Table II) or increased from 42% (strain XV185-14C, Table III) to 83% (strain D5, Table IV) when 100 pg H193/ml was used.
Genetic activity of AF-2 and H-193 On the basis of earlier results, it was concluded that AF-2 is a genetically active c o m p o u n d [11,16]. Its ability to induce mitotic recombination in S. cerevisiae strain D5 is higher than that of the antischistosomal agents, SQ18, 506 or niridazole [16]. We have shown here that AF-2 also induces point mutations in yeast. It induces reversion at a relatively high frequency (Table I). When three concentrations (25, 50 and 100 pg/ml) of AF-2 were used, there was substantial increase in the frequency of hom÷ and his + revertants as a function of incubation time (Table I}. The lysl-1 mutant exhibited a very low frequency of induced reversion (Table I). By comparing AF-2 and H-193 (at the same concentration and exposure time) in the induction of hom3-10 and his1-7 revertants per 107 survivors, the mutation yields obtained b y AF-2 (Table I), were mostly higher than those produced b y H-193 (Table III}. This is n o t the case when the comparison was
103
97
96
93
96
92
91
87
4
24
28
48
50 ~ g H - 1 9 3 / m l 4
24
28
48
82
48
84
83
75
24
28
48
86
91
28
200 pg H-193/ml 4
83
24
89
100
Control O
100 #g H-193/ml 4
% survival
OF ALTERED
Treatment (h)
FREQUENCY
TABLE IV
4585
5082
5172
5290
5012
5555
5085
5442
5345
5585
5620
5862
5725
5852
5942
6315
6127
Number of colonies scored
COLONIES
1
10 0.189% 18 0.348% 23 0.453% 53 1.156%
0.12%
0.738% 15 0.284% 13 0.251% 19 0.374% 7 0.153%
10 0.184% 25 0.492% 23 0.414% 6
10 0.17% 25 0.445% 20 0.358% 23 0.43%
0.017% --
1
--
0.016% --
14 0.257% 17 0.334% 32 0.576% 37
5 0.085% 30 0.534% 25 0.448% 17 0.318%
--
--
--
--
--
Red
IN SACCHAROMYCES
Red and pink (twin spots)
INDUCED
9 0.174% 7 0.138% 2 0.044%
0.219%
6 0.11% 5 0.098% 4 0.072% 11
4 0.068% 7 0.125% 3 0.054% 3 0.056%
0.017% --
0.048% 5 0.084% 1
0.049% 3
3
Pink
CEREVISIAE
6 0.113% 7 0.135% 3 0.059% 5 0.109%
0.08%
4 0.074% 7 0.138% 5 0.09% 4
3 0.054% 5 0.094%
1 0.0175%
4 0.076% 13 0.251% 9 0.177% 18 0.393%
0.06%
5 0.092% 9 0.177% 8 0.144% 3
3 0.054% 9 0.168%
5 0.085%
2 0.032% 6 0.1%
Red and white
D5 WITH H-193
Pink and white
STRAIN
5 0.095% 6 0.116% 3 0.059% 13 0.284%
0.14%
3 0.055% 9 0.177% 12 0.216% 7
6 0.1% 7 0.125% 11 0.197% 9 0.168%
3 0.051% 5 0.087%
5 0.084%
3 0.049%
Hairline
40 0.756% 66 1.276% 64 1.259% 98 2.14%
1.357%
42 0.722% 72 1.416% 84 1.512% 68
30 0.512% 69 1.228% 65 1.164% 66 1.235%
5 0.085% 6 0.1%
7 0.114% 5 0.079% 16 0.269%
Total number and % of altered colonies
221
made at the same survival level. For example, at 20% survival (Tables I and III), the frequency of revertants per 107 survivors induced by AF-2 was 6 (homoserine), 168 (histidine) and 22 (lysine), whereas, the frequency of revertants per 107 survivors induced by H-193 was 17.6, 334 and 30 respectively. The AF-2 dose effects for the reversion of strain XV185-14C from hom- and histo hom÷ and his + are given in Table I. It can be seen that the frequency of induced revertants per 107 survivors is dependent on the concentration of AF-2 used and exposure time. This is not always the case when H-193 was employed (Table III). In addition, little increase was obtained in the frequency of homoserine and lysine revertants after treatment with various concentrations of H193 and incubation for various lengths of time (Table III). However, the frequency of his+ revertants is higher than the frequency of hom ÷ and lys ÷ reverrants (Table III). It should also be noted that a considerable variation for induction of revertants and recombinant colonies was always associated with the use of H-193. Studies with the mitotic recombination system support the observation made with the reversion system that AF-2 (Table I and II) is genetically more active than H-193 (Tables III and IV) when the comparison was made at the same concentration and exposure time per 107 survivors, but not when it was made at a similar survival level. The data show that the percentage of twin spots obtained with AF-2 (Table II, column 4) was higher in most cases than the percentage obtained with H193 (Table IV, column 4). On the other hand, H-193 was more effective in inducing red colonies (Table IV, column 5) than AF-2 (Table II, column 5). In strain D5, the appearance of twin spot colonies provides evidence for mitotic crossing over. Red colonies can be due to mitotic crossing over, mutation, mitotic gene conversion, or aneuploidy.
II II.
O21N-- C\O ~ C
= C--CONH2
AF -2 II I ~ CH= CH ~ O2N - C\ O
CONH2
H- 193 N~C-NH 2
,o SQ 18, 506
O2N- C\S ~ -
Nxc/NH II
©
Nir,dazole Fig. 1. C h e m i c a l s t r u c t u r e o f t h e c o m p o u n d s .
222 Discussion
Present and previous investigations [5--8,11,18,20,22,24,25] have shown that the nitrofuran derivative AF-2 is a potent mutagen. In the present two assay systems of yeast, the genetic activity of AF-2 is higher than that of the two nitrofuran derivatives SQ18,506 and H-193 at a given dose and exposure time (16,26 and present), and also higher than the genetic activity of the thiazole derivative, niridazole [16,26]. The opinion [2,10,12,14,15] is that the NO2 group is necessary for the activity of the molecule (Fig. 1). Bueding and his colleagues [3,4,9,14,15] suggest that the activity in vivo may depend upon the solution of the molecule to give a h y d r o x y l amino or a nitroso derivative. Asnis [2] and Paul et al. [12] found that the NO2 group of nitrofurazone is reduced by pyridine nucleotide-dependent enzymes both in animal tissues and in bacteria. McCalla [10] reported that in cells lacking reductase I, [C'4] nitrofurazone does not become bound to macromolecules nor is the DNA of such cells detectably damaged by nitrofurans. It is possible that yeast can reduce nitrofurans to the h y d r o x y l a m i n o or to the nitroso forms, because it is certain that nitrofuran derivatives are mutagenic in yeast [16,17]. However, it has not yet been demonstrated that yeast can reduce nitrofurans. Of course, there can be a number of reasons why the four compounds (Fig. 1) act differently in inducing mutation and recombination in the same systems and under the same experimental conditions. It is possible that the genetic activity of these compounds is related to their solubility in the reaction medium and to their penetration into the cells. It is also possible that some of these compounds at a certain concentration(s) reduce the activity of the enzyme(s) involved in the reduction process. For example, McCalla [10] found that NFT, 3-amino-6-[ 2-( 5-nitro-2-furyl)vinyl ]-1, 2,4-triazine, and FANFT, N- [ 4-( 5-nitro2-furyl)-2-thiazolyl] formamide produce more mutants at the lower than at the higher concentration. In our hands H-193 failed to show a clear dose effect (Table III) and in some instances the low concentration produced more revertants and recombinant colonies than the higher concentration. In addition, the physiological state of the cells under which treatment is taken place has to be considered. Our recent study on the mutagenicity of the nitrothiazole derivative niridazole and the antischistosomal agent h y c a n t h o n e have shown that niridazole is genetically active when the treatment of yeast cells is performed in a rich medium under growing conditions, but it is not active when treatment is carried out in a non-nutrient suspension (phosphate buffer). On the other hand, h y c a n t h o n e is genetically active on cells treated in phosphate buffer but inactive when cells are treated in rich medium [16,26]. Thus numerous factors probably influence the genetic activity of the compound, or as T o m o e d a et al. [23] noted, the unique biological activities of nitrofurans can be well correlated to their metabolic reduction potential. The observation that the lysl-1 m u t a n t had a low frequency of induced reversion may be due to the high sensitivity of the lysine revertants to killing by the compound, because the number of revertants decreases as a function of incubation time and with increasing concentration (cf. Ames et al. [1]; McCalla [10] and yon Borstel and Igali [26]). In the results, it is mentioned that H-193 is genetically more effective in in-
223 ducing revertants than AF-2 when both compounds are compared at the same survival level. We know that the solubility of H-193 (4 pg/ml) is lower than the solubility of AF-2 (120 pg/ml). This fact might be the reason that yeast cells are more sensitive to AF-2 at a given dose and exposure time than H-193. Presumably, the slow inactivation by H-193 enables the cells to repair over a longer period of time. We must assume thereby that the H-193 mutational damage is not as rapidly repaired as that of its lethal damage. This, of course, is based on the supposition that the initial killing and mutational damage is identical for both compounds. This assumption is further supported by the fact that an effect similar to the observations made for H-193 was observed when different concentrations of AF-2 were compared. For example, the frequency of revertants per 107 survivors induced by the lower concentration of AF-2 was found to be similar or higher when compared with the number of revertants induced by the higher concentration at the same survival level (Table I). The mutagenicity and mode of action of nitrofuran derivatives, including AF-2 has been reviewed recently by Tazima et al. [21]. The carcinogenicity of AF-2 to the mouse stomach was demonstrated in the National Institute of Hygienic Science, Tokyo, and the use of this compound in the foods was completely prohibited in Japan after October 1, 1974 [20]. Acknowledgement This research was supported by U.S. National Institute of Health contractNIH-74-C-798 and a grant to R.C. von Borstel from the National Research Council of Canada. References 1 A m e s , B . N . , W.E. D u r s t o n , E. Y a m a s a k i a n d F . D . Lee, C a r c i n o g e n s a r e m u t a g e n s ; a simple test system c o m b i n i n g liver h o m o g e n a t e s f o r a c t i v a t i o n a n d b a c t e r i a f o r d e t e c t i o n , P r o c . Natl. A e a d . Sci. U.S., 7 0 (1973) 2281--2285. 2 Asnis, R . E . , T h e r e d u c t i o n o f f u r a c i n b y cell-free e x t r a c t s o f f u r a c i n - r e s i s t a n t a n d parent-susceptible s t r a i n s o f Escherichia coli, A r c h . B i o e h e m . B i o p h y s . , 6 6 ( 1 9 5 7 ) 2 0 8 - - 2 1 6 . 3 B u e d i n g , E., C. N a q u i r a , S. B o u w m a n a n d G. R o s e , T h e a n t i s c h i s t o s o m a l a c t i v i t y o f a n i t r o v i n y l f u r a n d e r i v a t i v e ( S Q 1 8 , 5 0 6 ) in m i c e a n d h a m s t e r s , J. P h a r m a c o l . E x p . T h e r . , 1 7 8 ( 1 9 7 1 ) 4 0 2 - - 4 1 0 4 H u l b e r t , P.B., E. B u e d i n g a n d C.H. R o b i n s o n , S t r u c t u r e a n d a n t i s e h i s t s o m a l a c t i v i t y in t h e n i t r o f u r a n series. R e q u i r e m e n t f o r a 5 - n i t r o - 2 - f u r y l v i n y l m o i e t y b a s e d o n c o m p a r i s o n o f 3 - ( 5 - n i t r o ° 2 f u r y l ) - s u b s t i t u t e d p r o p i o n i c , a c r y l i c a n d p r o p i o l i c a c i d d e r i v a t i v e s , J. Med. C h e m . , 1 6 ( 1 9 7 3 ) 7 2 - - 7 8 . 5 K a d a , T., Escherichia coli m u t a g e n i c i t y o f f u r y l f u r a m i d e , J a p . J. G e n e t . , 4 8 ( 1 9 7 3 ) 3 0 1 - - 3 0 5 . 6 K a d a , T.~ Escherichia coli m u t a g e n i c i t y o f f u r y l f u r a m i d e , M u t a t i o n R e s . , 26 ( 1 9 7 4 ) 4 3 4 . 7 K o n d o , S. a n d H. I c h i k a w a - R y o , T e s t i n g a n d c l a s s i f i c a t i o n o f m u t a g e n i c i t y o f f u r y l f u r a m i d e in Escherichia coli, Jap. J. Genet., 4 8 ( 1 9 7 3 ) 2 8 3 - - 2 8 8 . 8 K o n d o , S. a n d H. I c h i k a w a - R y o , T e s t i n g a n d c l a s s i f i c a t i o n o f m u t a g e n i c i t y o f f u r y l f u r a m i d e in Escherichia coli, M u t a t i o n R e s . , 26 ( 1 9 7 4 ) 4 3 4 - - 4 3 5 . 9 L e n n o x , R.W. a n d E. B u e d i n g , T h e relative c h e m o t h e r a p e u t i c e f f i c a c y o f a n i t r o v i n y l f u r a n (SQ 1 8 , 5 0 6 ) a g a i n s t i m m a t u r e a n d m a t u r e s t a g e s o f S c h i s t o s o m a m a n z o n i , A m e r . J. T r o p . Med. H y g . , 21 (1972) 302--306. 10 McCalla, D . R . and D. V o u t s i n o s , On the m u t a g e n i c i t y o f n i t r o f u r a n s , M u t a t i o n R e s . , 2 6 ( 1 9 7 4 ) 3 - - 1 6 . 11 O n g , T o n g - m a n a n d M.M. S h a h i n , M u t a g e n i c a n d r e c o m b i n o g e n i c a c t i v i t i e s o f t h e f o o d a d d i t i v e f u r y l f u r a m i d e in e u k a r y o t e s , S c i e n c e , 1 8 4 ( 1 9 7 4 ) 1 0 8 6 - - 1 0 8 7 . 1 2 P a u l , H . E . , V . R . Ells, F. K o p k o a n d R . C . B e n d e r , M e t a b o l i c d e g r a d a t i o n o f t h e n i t r o f u r a n s , J. M e d . Phaxm. Chem., 2 (1960) 563--584. 1 3 Q u a h , S.K. a n d R . C . y o n B o r s t e l , u n p u b l i s h e d results. 1 4 R o b i n s o n , C . H . , E. B u e d i n g a n d J. Fisher, R e l a t i o n s h i p b e t w e e n s t r u c t u r e , c o n f o r m a t i o n , a n t i s c h i s t o s o m a l o f n i t r o b e t e r o c y c l i c c o m p o u n d s , MoL P h a r m a c o l . , 6 ( 1 9 7 0 ) 6 0 4 - - 6 1 6 .
224
15 R o b i n s o n , C.H., S. S p e n g e l a n d E. Bueding, S t r u c t u r e a n d a n t i s c h i s t o s o m a l a c t i v i t y in t h e n i t r o f u r a n series. R e q u i r e m e n t for a 5-nitro g r o u p in 3 - ( 5 - n i t r o - 2 - f u r y l ) a c r y l i c acid d e r i v a t i v e s , J. Med. C h e m . , 16 ( 1 9 7 3 ) 7 9 - - 8 0 . 16 S h a h i n , M.M., G e n e t i c a c t i v i t y o f n i r i d a z o l e in yeast, M u t a t i o n Res., 30 ( 1 9 7 5 ) 1 9 1 - - 1 9 8 . 17 Shahin, M.M. a n d B.J. K i l b e y , G e n e t i c a c t i v i t y o f t h e a n t i s c h i s t o s o m a l a g e n t SQ 1 8 , 5 0 6 in yeast, Mut a t i o n Res., 26 ( 1 9 7 4 ) 1 9 3 - - 1 9 8 . 18 Shahin, M.M. a n d R.C. yon Borstel, M u t a t i o n and r e c o m b i n a t i o n i n d u c t i o n b y AF-2 a n d H - 1 9 3 , t w o n i t r o f u r a n d e r i v a t i v e s , in yeast, M u t a t i o n Res., 31 ( 1 9 7 5 ) 308. 19 Soares, E.R. and W. S h e r i d a n , L a c k of i n d u c t i o n o f d o m i n a n t lethals in m i c e b y orally a d m i n i s t e r e d A F - 2 , M u t a t i o n Res., 31 ( 1 9 7 5 ) 2 3 5 - - 2 4 0 . 20 Sugiyarna, T., K. G o t o a n d H. U e n a k a , A c u t e c y t o g e n e t i c e f f e c t of 2 - ( 2 - f u r y l ) - 3 - ( 5 - n i t r o - f u r y l ) - a c r y l a r n i d e ( A F - 2 , a f o o d p r e s e r v a t i v e ) on rat b o n e m a r r o w cells in vivo, M u t a t i o n Res., 31 ( 1 9 7 5 ) 2 4 1 - 246. 21 T a z i m a , Y., T. K a d a a n d A. M u r a k a m i , M u t a g e n i c i t y of n i t r o f u r a n d e r i v a t i v e s , including f u r y l f u r a m i d e , a f o o d p r e s e r v a t i v e , M u t a t i o n Res., 23 ( 1 9 7 5 ) 5 5 - - 8 0 . 22 Tazirna, Y. and K. O n i r n a r u , R e s u l t s of r n u t a g e n i c i t y testing f o r s o m e n i t r o f u r a n d e r i v a t i v e s in a sensitive test s y s t e m w i t h s i l k w o r m o o c y t e s , M u t a t i o n Res., 26 ( 1 9 7 4 ) 440. 23 T o m o e d a , M., M. Y a r n a d a and J. Seto, T h e s t r u c t u r e a n d biological activities of n i t r o f u r a n s , M u t a t i o n Res., 26 ( 1 9 7 4 ) 440---441. 24 T o n o r n u r a , A. a n d M.S. Sasaki, C h r o m s o r n e a b e r r a t i o n s and D N A r e p a i r synthesis in c u l t u r e d h u m a n ceils e x p o s e d to n i t r o f u r a n s , .lap. J. G e n e t . , 48 ( 1 9 7 3 ) 2 9 1 - - 2 9 4 . 25 T o n o r n u r a , A. a n d M.S. Sasaki, T h e i n d u c t i o n o f c h r o m o s o m e a b e r r a t i o n s a n d D N A repair s y n t h e s i s in c u l t u r e d h u m a n cells b y s o m e n i t r o f u r a n s , M u t a t i o n Res., 26 ( 1 9 7 4 ) 441. 26 V o n Borstel, R.C., a n d S. Igali, M u t a g e n i c i t y testing of a n t i s c h i s t o s o m a l t h i o x a n t h c n o n e s and indazoles o n yeast, J. T o x i c o l . E n v i r o n m , H l t h , I ( 1 9 7 5 ) 2 8 1 - - 2 9 1 . 27 Z i m m e r r n a n n , F.K., A yeast strain for visual screening for the t w o r e c i p r o c a l p r o d u c t s o f m i t o t i c crossing o v e r , M u t a t i o n Res., 21 ( 1 9 7 3 ) 2 6 3 - - 2 6 9 .
Mutation Research, 38 (1976) 215--224 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands
GENETIC ACTIVITY OF THE ANTIMICROBIAL FOOD ADDITIVES AF-2 AND H-193 IN SACCHAROMYCES CEREVI$IAE
MAJDI M. SHAHIN and R.C. VON BORSTEL
Department of Genetics, University of Alberta Edmonton, Alberta T6G 2E9 (Canada) (Received October 1st, 1975) (Revision received January 26th, 1976) (Accepted January 27th, 1975)
Summary The genetic activity of the antimicrobial food additives AF-2 and H-193 has been investigated in Saccharomyces cerevisiae. The strains chosen for the present studies were D5 for the induction of mitotic recombinational events and XV185-14C for the induction of reversion of the mutants lysl-1, his1-7 and hom3-7. When three concentrations (25, 50 and 100 pg/ml) of AF-2 were used in the reversion system of strain XV185-14C, there was an increase in the frequency of hom÷ and his* revertants as a function of incubation time, while the lysine m u t a n t exhibited a very low frequency of induced reversion. When AF-2 and H-193 were compared at the same concentration and exposure time, AF-2 exhibited a higher genetic activity in both systems than H-193. However, H-193 was genetically more active in inducing revertants than AF-2, when the comparison was made at the same survival level. Cells of both haploid and diploid strains were found to be more sensitive to inactivation by AF-2 than by H-193. It should be pointed out that the solubility of H-193 was lower (about 4 pg/ml saturation) than the solubility of AF-2 (120 #g/ml saturation). T h e h a p l o i d strain was more sensitive to both compounds than the diploid strain.
Introduction AF-2 (furylfuramide) and H-193 (Fig. 1) are two antimicrobial food additives. AF-2 induces mutations in bacteria [5--8], chromosome aberrations in human lymphocytes in vitro [24,25] and rat bone marrow cells in vivo [20], mutations in Neurospora and mitotic recombination in yeast [11]. It has also been shown to induce mutations in the silkworm using egg color mutants as markers [22]. Soares and Sheridan [19] have presented data indicating t h a t AF-2 is relatively non-toxic in unstarved mice and does not induce d o m i n a n t lethals in the strains of mice tested.
216 Studies on the mutagenic effect of the antimicrobial food additive H-193 have not been reported. The experiments reported herein were undertaken (1) to identify and investigate the genetic activity of H-193 in yeast, (2) to investigate the genetic activity of AF-2 using the gene mutation system of Saccharomyces cerevisiae where the reversion at lysl-1, his1-7 and horn3-10 could be studied, and (3) to compare the gene mutation and the mitotic recombination systems in their responses to AF-2 and H-193. Materials and Methods The strain used for mitotic recombination studies The diploid strain of S. cerevisiae (D5) used in the present study for the induction of mitotic recombination was developed b y Zimmermann [27]. The two haploid strains of which D5 is composed have the following genotypes: AZ--5A: a, trpl, ade2-40, M A L l
/
4--12D:
J
a, leul, ade2-119, MAL4
D5
Strain D5 carries two phenotypically distinguishable ade2 alleles. The strain homozygous for the ade2-40 allele forms red colonies. The other allele, ade2119, is leaky; therefore, the homozygotes form pink colonies. The two alleles complement fully so that the heterozygous diploid (D5) forms white colonies and does not require adenine for growth. In strain D5, genetic activity is indicated by the appearance of six phenotypically distinguishable colony types. The appearance of pink and red-sectored colonies (twin spots) provides evidence for mitotic crossing over. All red or all pink colonies can be due to mitotic crossing over, mutation, mitotic gene conversion, or aneuploidy. In addition to pink and red twin spots, sectored colony types include pink and white, red and white, and red, pink and white. Preparation o f cell samples, treatment conditions and plating Cells from stock culture were inoculated into liquid YPG medium (0.15% KH2PO4, 0.01% MgSO4, 0.45% (NH4)2SO4, 0.35% Difco proteose peptone, 0.5% Difco yeast extract and 2% glucose) and incubated at 30°C in a shaking water bath until they reached stationary phase. The cells were than harvested by centrifugation and washed twice in phosphate buffer (pH 7). Cell concentrations were adjusted to 1 × 10 s cells/ml. At zero time, the compound(s) to be tested was added to the cell suspension to give the desired final chemical concentration. Control and treated cell suspensions in Pyrex, low actinic, red erlenmeyer flasks were continually agitated at 30°C. At appropriate intervals, cell samples were withdrawn, centrifuged, and washed twice in phosphate buffer. The cell samples were then diluted and plated to give a b o u t 200--300 colonies per plate. The plates were incubated for 5 days at 30°C, and colony-forming ability was used as the criterion for survival. The strain used for gene mutation studies The haploid strain XV185-14C used for the induction of gene mutation was
217
developed by Quah and Von Borstel [13]. The genotype of XV185-14C is as follows: a, ade2-1, arg4-17, lysl-1, trp5-48, his1-7, hom3-10. Although strain XV185-14C has six markers, it requires six amino acids and one base for growth, because the homoserine marker blocks the synthetic pathway of threonine and methionine. Reversion at the lysl, his1 or horn3 loci produces cells capable of growth on medium deficient in either lysine, histidine or threonine.
Determination of spontaneous and induced reversion frequency For the determination of spontaneous or induced reversion frequency, undiluted samples (1 × 108 cells/ml) were plated (0.2 ml/plate) on medium containing Difco yeast nitrogen base without amino acids plus 2% glucose plus 2% agar plus all required supplements except lysine, histidine or threonine. For survival estimation, treated and control samples were diluted and plated on the same medium plus all required substances to give a b o u t 200--300 colonies per plate. The plates were incubated for six days at 300(2, and colony-forming ability was used as the criterion for survival. Preparation of cell samples, treatment conditions and plating are the same as described for strain D5. Results
Cell inactivation The cell inactivation effect of AF-2 on the haploid strain XV185-14C and on the diploid strain D5 is shown in Tables I and II. The decrease in the percentage of survival is dependent on the concentration used and time of exposure. Cells of b o t h strains were found to be m o r e sensitive to inactivation by AF-2 than H-193 (Tables I--IV). It should, however, be pointed o u t that the solubility of H-193 was lower in the 2% dimethyl sulfoxide phosphate buffer solution
TABLE I R E V E R S I O N F R E Q U E N C I E S OF S T R A I N XV185-14C A F T E R C O N C E N T R A T I O N S OF AF-2
TREATMENT
WITH DIFFERENT
N u m b e r s in p a r e n t h e s e s are t h e n u m b e r s of r e v e r t a n t c o l o n i e s c o u n t e d p e r 3 p l a t e s ( h i s t i d i n e ) or p e r 6 plates (homoserine, Iysine).
Dose (/~g/ml)
0 0 25 25 25 25 50 50 50 100 100 100 100
Treatm e n t (h)
0 24 2 4 8 24 2 8 24 2 4 ~ 8 24
R e v e r t a n t s p e r 107 survivors
Survival (%)
horn3-10
hisl-7
lys i-1
0.53 (8) 0.66 (10) 2.80 (38) 3.40 (28) 4.50 (40) 6.60 (40) 3.00 (36) 6.00 (18) 26.60 (16) 4.40 (36) 6.50 (22) 11.60 (10) 4 4 . 4 0 (2)
25 (198) 22 (168) 88 (592) 144 (666) 160 (699) 215 (644) 133 (799) 168 (253) 296 (89) 160 (648) 170 (286) 223 (100) 3 5 5 (8)
15 (228) 16 (244) 19 (250) 24 (214) 21 (188) 25 (152) 24 ( 2 8 2 ) 22 ( 6 6 ) 17 ( 1 0 ) 28 ( 1 2 6 ) 28 (94) 22 (20) - - (0)
I00 108 90 60 58 44 80 20 4 54 22 6 0.3
90
25
23
50 pg AF-2/ml 4
24
28
1.6
1.0
0.13
28
48
62
24
200 pg AF-2/ml 4
0.4
4.6
28
48
6.5
85
24
100 pg AF-2/ml 4
3.4
91
48
48
97
100
Control 0
24
% survival
OF ALTERED
Treatment (h)
FREQUENCY
TABLE II
789
1657
2524
3810
2767
2838
4044
5230
4959
3548
3876
5586
5625
5984
6189
scored
Number of colonies
COLONIES
29 0.761% 45 1.78% 31 1.87% 19 2.4%
32 0.612% 47 1.162% 33 1.163% 43 1.55%
7 0.i 25% 18 0.464% 20 0.563% 37 0.746%
6 0.157% 5 0.198% 4 0.241% 2 0.253%
9 0.172% 6 0.148% 7 0.247% 11 0.397%
5 0.089% 9 0.232% 8 0.225% 9 0.181%
9 0.236% II 0.436% 7 0.422% 3 0.380%
6 0.115% 6 0.148% 11 0.388% 7 0.253%
5 0.089% 7 0.180% 10 0.282% 17 0.343%
0.033% 1 0.018%
0.017% 1 0.018%
--
0.048% 2
1 0.016% 1
3
Pink
STRAIN
--
Red
CEREVISIAE
---
Red and pink (twin spots)
IN SACCHAROMYCES
3 0.079% 4 0.i 58% 5 0.302% 3 0.380%
7 0.134% 12 0.296% 6 0.211% 8 0.289%
3 0.054% 4 0.103% 5 0.141% 16 0.323%
--
0.017%
0.016% 1
1
Pink and white
D5 WITH AF-2
7 0.184% 5 0.198% 3 0.181% 2 0.253%
11 0.210% 3 0.074% 9 0.317% 5 0.181%
4 0.072% 5 0.129% 7 0.197% 11 0.22%
1 0.017% 1 0.018%
Red and white
7 0.184% 6 0.238% 7 0.422% 5 0.634%
6 0.115% 2 0.049% 9 0.317% 7 0.253%
4 0.072% 6 0.155% 5 0.141% 13 0.262%
2 0.032% 3 0.050% 4 0.071%
Hairline
61 1.60% 76 3.01% 57 3.43% 34 4.31%
71 1.36% 76 1.88% 75 2.64% 81 2.93%
28 0.50% 49 1.26% 55 1.55% 103 2.01%
7 0.113% 8 0.134% 7 0.124%
Total number and % of altered colonies
b~
219 T A B L E III REVERSION FREQUENCIES OF STRAIN CONCENTRATIONS OF H-193
XV185-14C
AFTER
TREATMENT
WITH DIFFERENT
N u m b e r s in p a r e n t h e s e s a r e t h e n u m b e r s o f r e v e r t a n t c o l o n i e s c o u n t e d p e r 3 p l a t e s ( h i s t i d i n e ) o r p e r 6 p l a t e s ( h o m o s e r i n e , lysine). Dose (~g/ml)
0 0 0 0 50 50 50 50 100 100 100 100 200 200 200 200
Treatm e n t (h)
0 24 28 48 4 24 28 48 4 24 28 48 4 24 28 48
Revertants per 10 ? survivors
Survival (%)
horn3-10
his l - 7
lysl -i
3.1 2.8 3.8 3.8 8.1 9.0 11.8 11.1 6.6 14.6 13.1 17.3 6.0 7.6 7.2 17.6
23 33 28 30 98 182 138 200 69 300 186 291 70 181 198 334
15 14 14 13 15 29 21 18 14 28 29 39 11 18 16 30
(42) (36) (50) (46) (106) (62) (74) (52) (86) (82) (66) (62) (80) (60) (46) (46)
(155) (214) (184) (178) (643) (637) (433) (468) (462) (842) (481) (521) (475) (713) (635) (455)
(202) (184) (182) (160) (200) (200) (134) (86) (184) (158) (150) (140) (148) (138) (104) (82)
I00 95 97 90 97 52 47 35 99 42 38 27 100 51 48 20
(about 4 pg/ml saturation) than the solubility of AF-2 in phosphate buffer (120 pg/ml saturation}. When a comparison of the haploid strain XV185-14C and the diploid strain D5 was made, the haploid strain (Tables I and III} was more sensitive to inactivation b y b o t h c o m p o u n d s than the diploid strain (Tables II and IV). For example, at a concentration of 100 pg AF-2/ml and an incubation time of 24 h, the surviving fraction increased from 0.3% (haploid strain XV18514C, Table I) to 6.5% (diploid strain D5, Table II) or increased from 42% (strain XV185-14C, Table III) to 83% (strain D5, Table IV) when 100 pg H193/ml was used.
Genetic activity of AF-2 and H-193 On the basis of earlier results, it was concluded that AF-2 is a genetically active c o m p o u n d [11,16]. Its ability to induce mitotic recombination in S. cerevisiae strain D5 is higher than that of the antischistosomal agents, SQ18, 506 or niridazole [16]. We have shown here that AF-2 also induces point mutations in yeast. It induces reversion at a relatively high frequency (Table I). When three concentrations (25, 50 and 100 pg/ml) of AF-2 were used, there was substantial increase in the frequency of hom÷ and his + revertants as a function of incubation time (Table I}. The lysl-1 mutant exhibited a very low frequency of induced reversion (Table I). By comparing AF-2 and H-193 (at the same concentration and exposure time) in the induction of hom3-10 and his1-7 revertants per 107 survivors, the mutation yields obtained b y AF-2 (Table I), were mostly higher than those produced b y H-193 (Table III}. This is n o t the case when the comparison was
103
97
96
93
96
92
91
87
4
24
28
48
50 ~ g H - 1 9 3 / m l 4
24
28
48
82
48
84
83
75
24
28
48
86
91
28
200 pg H-193/ml 4
83
24
89
100
Control O
100 #g H-193/ml 4
% survival
OF ALTERED
Treatment (h)
FREQUENCY
TABLE IV
4585
5082
5172
5290
5012
5555
5085
5442
5345
5585
5620
5862
5725
5852
5942
6315
6127
Number of colonies scored
COLONIES
1
10 0.189% 18 0.348% 23 0.453% 53 1.156%
0.12%
0.738% 15 0.284% 13 0.251% 19 0.374% 7 0.153%
10 0.184% 25 0.492% 23 0.414% 6
10 0.17% 25 0.445% 20 0.358% 23 0.43%
0.017% --
1
--
0.016% --
14 0.257% 17 0.334% 32 0.576% 37
5 0.085% 30 0.534% 25 0.448% 17 0.318%
--
--
--
--
--
Red
IN SACCHAROMYCES
Red and pink (twin spots)
INDUCED
9 0.174% 7 0.138% 2 0.044%
0.219%
6 0.11% 5 0.098% 4 0.072% 11
4 0.068% 7 0.125% 3 0.054% 3 0.056%
0.017% --
0.048% 5 0.084% 1
0.049% 3
3
Pink
CEREVISIAE
6 0.113% 7 0.135% 3 0.059% 5 0.109%
0.08%
4 0.074% 7 0.138% 5 0.09% 4
3 0.054% 5 0.094%
1 0.0175%
4 0.076% 13 0.251% 9 0.177% 18 0.393%
0.06%
5 0.092% 9 0.177% 8 0.144% 3
3 0.054% 9 0.168%
5 0.085%
2 0.032% 6 0.1%
Red and white
D5 WITH H-193
Pink and white
STRAIN
5 0.095% 6 0.116% 3 0.059% 13 0.284%
0.14%
3 0.055% 9 0.177% 12 0.216% 7
6 0.1% 7 0.125% 11 0.197% 9 0.168%
3 0.051% 5 0.087%
5 0.084%
3 0.049%
Hairline
40 0.756% 66 1.276% 64 1.259% 98 2.14%
1.357%
42 0.722% 72 1.416% 84 1.512% 68
30 0.512% 69 1.228% 65 1.164% 66 1.235%
5 0.085% 6 0.1%
7 0.114% 5 0.079% 16 0.269%
Total number and % of altered colonies
221
made at the same survival level. For example, at 20% survival (Tables I and III), the frequency of revertants per 107 survivors induced by AF-2 was 6 (homoserine), 168 (histidine) and 22 (lysine), whereas, the frequency of revertants per 107 survivors induced by H-193 was 17.6, 334 and 30 respectively. The AF-2 dose effects for the reversion of strain XV185-14C from hom- and histo hom÷ and his + are given in Table I. It can be seen that the frequency of induced revertants per 107 survivors is dependent on the concentration of AF-2 used and exposure time. This is not always the case when H-193 was employed (Table III). In addition, little increase was obtained in the frequency of homoserine and lysine revertants after treatment with various concentrations of H193 and incubation for various lengths of time (Table III). However, the frequency of his+ revertants is higher than the frequency of hom ÷ and lys ÷ reverrants (Table III). It should also be noted that a considerable variation for induction of revertants and recombinant colonies was always associated with the use of H-193. Studies with the mitotic recombination system support the observation made with the reversion system that AF-2 (Table I and II) is genetically more active than H-193 (Tables III and IV) when the comparison was made at the same concentration and exposure time per 107 survivors, but not when it was made at a similar survival level. The data show that the percentage of twin spots obtained with AF-2 (Table II, column 4) was higher in most cases than the percentage obtained with H193 (Table IV, column 4). On the other hand, H-193 was more effective in inducing red colonies (Table IV, column 5) than AF-2 (Table II, column 5). In strain D5, the appearance of twin spot colonies provides evidence for mitotic crossing over. Red colonies can be due to mitotic crossing over, mutation, mitotic gene conversion, or aneuploidy.
II II.
O21N-- C\O ~ C
= C--CONH2
AF -2 II I ~ CH= CH ~ O2N - C\ O
CONH2
H- 193 N~C-NH 2
,o SQ 18, 506
O2N- C\S ~ -
Nxc/NH II
©
Nir,dazole Fig. 1. C h e m i c a l s t r u c t u r e o f t h e c o m p o u n d s .
222 Discussion
Present and previous investigations [5--8,11,18,20,22,24,25] have shown that the nitrofuran derivative AF-2 is a potent mutagen. In the present two assay systems of yeast, the genetic activity of AF-2 is higher than that of the two nitrofuran derivatives SQ18,506 and H-193 at a given dose and exposure time (16,26 and present), and also higher than the genetic activity of the thiazole derivative, niridazole [16,26]. The opinion [2,10,12,14,15] is that the NO2 group is necessary for the activity of the molecule (Fig. 1). Bueding and his colleagues [3,4,9,14,15] suggest that the activity in vivo may depend upon the solution of the molecule to give a h y d r o x y l amino or a nitroso derivative. Asnis [2] and Paul et al. [12] found that the NO2 group of nitrofurazone is reduced by pyridine nucleotide-dependent enzymes both in animal tissues and in bacteria. McCalla [10] reported that in cells lacking reductase I, [C'4] nitrofurazone does not become bound to macromolecules nor is the DNA of such cells detectably damaged by nitrofurans. It is possible that yeast can reduce nitrofurans to the h y d r o x y l a m i n o or to the nitroso forms, because it is certain that nitrofuran derivatives are mutagenic in yeast [16,17]. However, it has not yet been demonstrated that yeast can reduce nitrofurans. Of course, there can be a number of reasons why the four compounds (Fig. 1) act differently in inducing mutation and recombination in the same systems and under the same experimental conditions. It is possible that the genetic activity of these compounds is related to their solubility in the reaction medium and to their penetration into the cells. It is also possible that some of these compounds at a certain concentration(s) reduce the activity of the enzyme(s) involved in the reduction process. For example, McCalla [10] found that NFT, 3-amino-6-[ 2-( 5-nitro-2-furyl)vinyl ]-1, 2,4-triazine, and FANFT, N- [ 4-( 5-nitro2-furyl)-2-thiazolyl] formamide produce more mutants at the lower than at the higher concentration. In our hands H-193 failed to show a clear dose effect (Table III) and in some instances the low concentration produced more revertants and recombinant colonies than the higher concentration. In addition, the physiological state of the cells under which treatment is taken place has to be considered. Our recent study on the mutagenicity of the nitrothiazole derivative niridazole and the antischistosomal agent h y c a n t h o n e have shown that niridazole is genetically active when the treatment of yeast cells is performed in a rich medium under growing conditions, but it is not active when treatment is carried out in a non-nutrient suspension (phosphate buffer). On the other hand, h y c a n t h o n e is genetically active on cells treated in phosphate buffer but inactive when cells are treated in rich medium [16,26]. Thus numerous factors probably influence the genetic activity of the compound, or as T o m o e d a et al. [23] noted, the unique biological activities of nitrofurans can be well correlated to their metabolic reduction potential. The observation that the lysl-1 m u t a n t had a low frequency of induced reversion may be due to the high sensitivity of the lysine revertants to killing by the compound, because the number of revertants decreases as a function of incubation time and with increasing concentration (cf. Ames et al. [1]; McCalla [10] and yon Borstel and Igali [26]). In the results, it is mentioned that H-193 is genetically more effective in in-
223 ducing revertants than AF-2 when both compounds are compared at the same survival level. We know that the solubility of H-193 (4 pg/ml) is lower than the solubility of AF-2 (120 pg/ml). This fact might be the reason that yeast cells are more sensitive to AF-2 at a given dose and exposure time than H-193. Presumably, the slow inactivation by H-193 enables the cells to repair over a longer period of time. We must assume thereby that the H-193 mutational damage is not as rapidly repaired as that of its lethal damage. This, of course, is based on the supposition that the initial killing and mutational damage is identical for both compounds. This assumption is further supported by the fact that an effect similar to the observations made for H-193 was observed when different concentrations of AF-2 were compared. 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