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Testing of some permitted food colours for the induction of gene conversion in diploid yeast
309
Mutation Research, 67 (1979) 309--314 © Elsevier/North-Holland Biomedical Press
T E S T I N G OF SOME P E R M I T T E D FOOD COLOURS F O R T H E INDUCTION OF GENE CONVERSION IN DIPLOID YEAST
N. SANKARANARAYANAN and M.S.S. MURTHY Division of Radiological Protection, Bhabha Atomic Research Centre, Trombay, Bombay400085 (India)
(Received 9 August 1978) (Revision received 8 March 1979) (Accepted 9 March 1979)
Summary 12 p er mitted f o od colours in use were screened for geno-toxicity. Mitotic gene conversion in S a c c h a r o m y c e s cerevisiae was used as the end-point. Each food colour was tested in stationary-phase as well as log-phase cells but w i t h o u t microsomal activation. These f o o d colours did n o t cause any increase in mitotic gene conversion in diploid yeast BZ 34.
A m o n g the m a n y f ood additives currently in use, colouring agents form an i m p o r t a n t group. Most of these agents have been tested for acute and chronic t o x i c i t y in mammals [3,13]. However, n o t much i nform at i on is available on the geno-toxicity of these c om pounds . Wherever available, such tests are cond u c t e d mostly with microorganisms. When microorganisms are used to test for geno-toxicity, it becomes necessary to c o n d u c t the tests on as m a n y different systems and end-points as possible to rule out organism specificities, false positives and false negatives. In this paper, 12 per mi t t ed food colours used in India and elsewhere were tested for the induction of mitotic gene conversion in diploid yeast S a c c h a r o m y c e s cerevisiae. Mitotic gene conversion results in the transfer o f some genetic i nf or m a t i on between non-sister chromatids of homologous chromosomes. Its relevance in screening of environmental chemicals has been discussed by Z i m m e r m a nn [17,18] and Murthy [7]. Materials and m e t h o d s Y e a s t strain The diploid yeast BZ 34 was kindly d o n a t e d by Dr. R o b e r t Mortimer of the
310 D o n n e r L a b o r a t o r y , Berkeley. The g e n o t y p e of BZ 34 is as follows: a
+ arg4-4 + thrl + trp 5-48 + ura3his5-21ysl-lade2-1 pet + arg 4-17 + leu 1-12 trp 5-48 m e t 1 + his 5-2 lys l - l a d e 2-1
It has two n o n - c o m p l e m e n t i n g m u t a n t alleles (4-4 and 4-17) in the arginosuccinase locus and hence requires arginine in the growth medium. The spontaneous f r e q u e n c y of mitotic gene conversion to arginine independence is very low. However, DNA-damaging chemical and physical agents can induce mitotic gene conversion at a high rate resulting in arginine p r o t o t r o p h y [11,10]. This strain also carries mutations at o t h e r loci, such as ade, his, trp, lys making it auxotrophic to all these amino-acid requirements. However, these loci were rather insensitive to the induction of back-mutations. Induction of mitotic gene conversions in this strain can be d e t e c t e d by plating treated cell suspensions on m e d i u m from which arginine is omitted. T o optimize the yield of arginine p r o t o t r o p h s , omission medium was supplemented with trace quantities of arginine [11]. These will be designated as Arg- plates. The ability of BZ 34 to screen DNA-damaging agents is comparable to those of strain D-4 and D-7 developed by Dr. Zimmermann. This test system was successfully used for estimating the Radiation Equivalent Chemical (REC) value of ethyl methanesulp h o n a t e [9] and AF-2 [8]. Testing chemicals The f o o d colours Amaranth, Sunset Yellow, Erythrosine, Carmoisine, Tartrazine, Fast Red E, Scarlet-F, Indigo Carmine (Indigotine) and Ponceau-4R were obtained f r o m Hickson and Dadajee, Bombay. These materials c o n f o r m to the specifications laid down by the Indian Standards Institution (ISI) for food colours. Wool Green and Fast Green were obtained from the local market. For turmeric, an ex tr ac t was prepared by refluxing pow dered turmeric in 70% ethanol for 1 h at 90 ° C. When the supernatant was diluted with water, a yellow precipitate soluble in di m e t hyl s ul phoxi de (DMSO) was formed. This was used for testing purposes. Gene c o n v e r s i o n test There are man y examples of chemicals that show geno-toxicity in stationary phase cells but n o t when treated under growth conditions and vice versa. This is attributed mainly to the metabolic activation or inactivation of these compounds in growing cultures [14]. Hence, each food colour was tested for induction of gene conversion under log phase as well as stationary phase. The stationary phase tests were carried out in potassium phosphate buffer (0.1 M, pH 7). In a typical t r e a t m e n t a cell suspension of about 10 s cells/ml was prepared from a culture with a low background conversion frequency. 1 ml of this was added to 9 ml o f phosphate buffer which contained the colour at 5 mg/ml. The t r e a t m e n t was carried o u t at 30°C in a shaker water-bath in the dark. After a lapse of 4 h, suitable dilutions were made and plated on yeast extract, pept one, dextrose (YEPD) and Arg- plates for scoring survival and gene conversion respectively. T r e a t m e n t under growth conditions was done in YEPD broth. 10 ml of brot h were inoculated with 1000 cells and placed on a shaker water-bath at 30°C.
311 After 24 h o f growth a given f ood colour was added (5 mg/ml) and the treatm e n t c o n t i n u e d in the dark. At 72 h (i.e. 48 h after addition of the colour) the t r e a t m e n t was terminated. The pH at this stage was a b o u t 6. Cell counts were made with a h a e m o c y t o m e t e r to see w he t he r the food colours inhibited cell division. Suitable dilutions were made and plated on YEPD as well as Argplates. After 3 days the colonies were c o u n t e d and the n u m b e r of convertants per million survivors was calculated. Control samples w i t h o u t the colours were treated identically. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) served as a positive control. Tr eat m e nt s were repeated 2--3 times for each colour. Results and discussion Results summarized in Tables I and 2 show that the 12 perm i t t ed colours tested in this investigation did n o t induce mitotic gene conversion in yeast when treated either in stationary-phase or log-phase culture. Under these t r e a t m e n t conditions neither significant cell killing nor inhibition of cell division was observed. T ha t the absence of lethal and genetic effects may be due to the lack o f penetration of the dye into the cell can be ruled out by the following observations. When the cells were plated after t r e a t m e n t w i t h o u t further dilution on Arg- plates, the colonies picked up the colour of the dye in question. This suggested t ha t the dye could indeed enter the cells. This was further c o n f i r med in some cases by s p e c t r o p h o t o m e t r i c measurements. The absorbance o f the s u p er n atan t after t r e a t m e n t was less than that before treatment. However, the d y e uptake was n o t enough to be seen on microscopic examination of invidual cells.
TABLE
1
TREATMENT OF DIPLOID YEAST IN STATIONARY TION OF MITOTIC GENE CONVERSION
PHASE
WITH
FOOD
Concentration of colour: 5 mg/ml. Numbers in paxentheses are the numbers Conversion frequencies shown are the means of 2 experiments, Food
colour
Colour index
Arginine convertants survivors Control
COLOURS
of colonies scored on 4 plates.
per 106
Percentage survival
Treated
Amaranth Indigo Carmine Carmoisine
16 185 73 015 14 720
21 24 22
(854) (665) (854)
29 ( 2 8 7 ) 26 (584) 26 (275)
100 103 100
Ponceau-4R Fast Red E Scarlet F
16 255 37 035 16 155
26 (1101) 22 (854) 28 (330)
27 ( 2 7 8 ) 35 (272) 33 (342)
100 100 100
Fast Green FCF Wool Green BS Tartrazine
42 053 44 090 19 1 4 0
33 33 38
38 (384) 30 (336) 40 (220)
100 100 98
Erythrosine Sunset Yellow FCF Turmeric MNNG (0.02 raM)
45 430 15 985 75 300 positive control
25 (1101) 20 (535) 41 ( 2 0 4 ) 16 (191)
(232) (232) (701)
25 23 49 2026
FOR INDUC-
(524) (620) (254) (547)
100 100 97 64
312
TABLE 2 TREATMENT OF DIPLOID YEAST MITOTIC GENE CONVERSION
IN LOG PHASE WITH FOOD
COLOURS
FOR
INDUCTION
OF
C o n c e n t r a t i o n o f c o l o u r : 5 m g / m l . N u m b e r s i n p a r e n t h e s e s are t h e n u m b e r s o f c o l o n i e s s c o r e d o n 4 p l a t e s . C o n v e r s i o n f r e q u e n c i e s s h o w n are t h e a v e r a g e s o f 2 e x p e r i m e n t s . Food colour
Colour index
Arginine convertants per 106 survivors Control
Percentage survival
Treated
Amaranth Indigo Carmine Carmoisine
16 1 8 5 73 015 14 7 2 0
52 (1075) 37 (242) 37 ( 2 4 2 )
56 30 30
(993) (292) (286)
98 101 100
Ponceau-4R Fast Red E Scarlet F
16 2 5 5 37 0 3 5 16 1 5 5
37 45 73
(242) (990) (492)
36 (269) 46 (1018) 83 (851)
100 100 100
Fast Green FCF W o o l G r e e n BS Tartrazine
42 053 44 090 19 1 4 0
81 ( 1 0 6 2 ) 43 (1308) 66 (1117)
93 (982) 44 (724) 60 (1195)
100 100 100
Erythrosine Sunset Yellow FCF Turmeric MNNG (0.05raM)
45 430 15 985 75 300 positive control
37 37 33 75
(242) (242) (393) (710)
24 33 36 2600
(484) (346) (986) (515)
100 100 103 63
Kada et al. [6] conducted a "rec" assay on Amaranth, Fast Green, Erythrosine, Indigo Carmine, Sunset Yellow and Tartrazine; but all were negative. Luck et al. [3] have reported that Erythrosine was slightly mutagenic in E. coli. This colour also exhibited very slight mutagenic activity in E. coli [3]. However, it TABLE 3 SUMMARY OF DIFFERENT TYPES OF GENOTOXICITY MICROORGANISMS AND MAMMALIAN CELL SYSTEMS Mutation
(E. coli)
TESTS WITH SOME FOOD
Mutation ( A m e s test), a
Chromosomal aberrations and micronucleus test [5,15,161
negative [2,4] negative [4] negative [2,4]
-+ negative --
negative negative --
negative negative negative
positive . .
--
negative negative negative
[3]
" R e c " assay [6]
COLOURS
Gene conversion in yeast ( p r e s e n t investigation)
Amaranth Indigo Carmine Carmoisine
negative negative --
Scarlet-F Poneeau-4R Fast Red E
. negative . .
Wool Green F.S. Fast Green FCF Tartrazine
---
negative I1] negative [4,2] n e g a t i v e [ 1]
--positive
-negative negative
negative negative negative
Erythrosine Sunset Yellow FCF Tu r m e r i c
weak -.
negative [ 4,2] -. .
±
negative negative
negative negative negative
.
.
.
-.
.
.
--, M e a n s d a t a n o t a v a i l a b l e . a N u m b e r s i n b r a c k e t s are r e f e r e n c e n u m b e r s .
.
.
IN
313
failed to induce gene conversion in yeast strain BZ 34. Amaranth,when tested through the fluctuation test by Parry, was negative [12]. Amaranth, Tartrazine, Erythrosine, Indigotine, Fast Green, and Sunset Yellow were also negative in the Ames test with microsomal activation [2,1,4]. Ishidate and Odashima screened Sunset Yellow and Amaranth for chromosomal aberration on Chinese hamster cells in culture [ 5]. Whereas Sunset Yellow was negative, Amaranth was classified as "suspicious" in this system. Zhurkov et al. [16] tested Indigo Carmine, Sunset Yellow, Amaranth and Tartrazine in a human lymphocyte culture. Only Tartrazine showed a weak cytogenetic effect. Amaranth, Ponceau4R, Sunset Yellow and Tartrazine were investigated for mutagenicity in human leukocyte and mouse micronucleus test systems [15]. All 4 food colours were reported to be positive in both systems at low concentrations. Table 3 shows a summary, from published literature, of the different types of geno-toxicity tests conducted on these food colours in both microorganisms and cultured mammalian cell systems. In view of the conflicting results with some compounds and the large population exposed, it is necessary to carry out further tests on them. Acknowledgements We thank Dr. K.G. Vohra for his encouragement, Dr. U. Madhvanath for useful suggestions and U.R. Kini for spectrophotometric measurements. References 1 A u l e t t a , A.E., J.M. K u z a v a a n d A.S. P a r m a r , L a c k of m u t a g e n i c i t y of a series of f o o d d y e s for Salmonella t y p h i m u r i u m , M u t a t i o n Res., 56 ( 1 9 7 7 ) 2 0 3 - - 2 0 7 . 2 B r o w n , J.P., W.R. G e r a l d a n d R.J. B r o w n , M u t a g e n i c i t y t e s t i n g o f f o o d c o l o u r s a n d r e l a t e d azo, xant h e n e a n d t r i p h e n y l m e t h a n e d y e s w i t h t h e S a l m o n e l l a / m i c r o s o m e s y s t e m , M u t a t i o n Res., 56 ( 1 9 7 8 ) 249--271. 3 F . A . O . N u t r i t i o n Meetings R e p o r t Series No. 4 6 A , T o x i c o l o g i c a l E v a l u a t i o n of s o m e f o o d colours, emulsifiers, a n t i - c a k i n g a g e n t s a n d c e r t a i n o t h e r s u b s t a n c e s . W H O / F O O D - A D D ] 7 0 , pp. 2 3 - - 5 7 . 4 G a r n e r , C.R., a n d C.A. N u t m a n , T e s t i n g o f s o m e a z o - d y e s a n d t h e i r r e d u c t i o n p r o d u c t s for m u t a g e n i c i t y using S a l m o n e l l a t y p h i m u r i u m T A 1 5 3 8 , M u t a t i o n Res., 44 ( 1 9 7 7 ) 9 - - 1 9 . 5 I s h i d a t e Jr., M., a n d S. O d a s h i m a , C h r o m o s o m e tests w i t h 1 3 4 c o m p o u n d s o n Chinese h a m s t e r cells in vitro: A s c r e e n i n g for c h e m i c a l c a r c i n o g e n s , M u t a t i o n Res., 48 ( 1 9 7 7 ) 3 3 7 - - 3 5 4 . 6 K a d a , T., K. T u f i k a w a a n d Y. Sadaie, In v i t r o a n d h o s t - m e d i a t e d " r e c - a s s a y " p r o c e d u r e s f o r s c r e e n i n g c h e m i c a l m u t a g e n s a n n p h l o x i n e , a r e d d y e d e t e c t e d , M u t a t i o n Res., 16 ( 1 9 7 2 ) 1 6 5 - - 1 7 4 . 7 M u r t h y , M.S.S., I n d u c t i o n of gene c o n v e r s i o n in d i p l o i d y e a s t b y c h e m i c a l s : C o r r e l a t i o n w i t h m u t a genie a c t i o n a n d its r e l e v a n c e in g e n o t o x i c i t y screening, M u t a t i o n Res., 64 ( 1 9 7 9 ) 1 - - 1 7 . 8 M u r t h y , M.S.S., a n d N. S a n k a r a n a x a y a n a n , I n d u c t i o n o f ' ~ e n e c o n v e r s i o n in S a c c h a r o m y c e s cerevisiae b y t h e n i t r o f u r a n d e r i v a t i v e f u r y l f u r a m i d e ( A F - 2 ) , M u t a t i o n Res., 58 ( 1 9 7 8 ) 9 9 - - 1 0 1 . 9 M u r t h y , M.S.S., and N. S a n k a r a n a r a y a n a n , D e t e r m i n a t i o n o f r a d i a t i o n e q u i v a l e n c e of e t h y l m e t h a n e s u l p h o n a t e ( E M S ) for i n d u c t i o n of gene c o n v e r s i o n in diploid y e a s t , M u t a t i o n Res., 51 ( 1 9 7 8 ) 1 8 1 - 188. 10 M u r t h y , M.S.S., B.S. R a o , N.M.S. R e d d y , P. S u b r a h m a n y a n a n d U. M a d h v a n a t h , N o n - e q u i v a l e n c e of Y E P D a n d s y n t h e t i c c o m p l e t e m e d i a in y e a s t r e v e r s i o n studies, M u t a t i o n Res., 27 ( 1 9 7 5 ) 2 1 9 - - 2 2 3 . 11 M u r t h y , M.S.S., B.S. R a o a n d V.V. D e o r u k h a k a r , D e p e n d e n c e of e x p r e s s i o n of the r a d i a t i o n - i n d u c e d gene c o n v e r s i o n to a r g i n i n e i n d e p e n d e n c e in d i p l o i d y e a s t o n t h e a m i n o acid c o n c e n t r a t i o n : E f f e c t on allelic m a p p i n g , M u t a t i o n Res., 35 ( 1 9 7 6 ) 2 0 7 - - 2 1 2 . 12 P a r r y , J.M., Use of y e a s t c u l t u r e s for t h e d e t e c t i o n of e n v i r o n m e n t a l m u t a g e n e s i s using a f l u c t u a t i o n test, M u t a t i o n Res., 46 ( 1 9 7 7 ) 1 6 5 - - 1 7 6 . 13 R a d o m s k i , J . L . , T o x i c o l o g y of f o o d c o l o u r s , A n n u . Rev. P h a r m a c o l . , 14 ( 1 9 7 4 ) 1 2 7 - - 1 3 7 . 14 S h a h i n , M.M., G e n e t i c a c t i v i t y of n i r i d a z o l e in y e a s t , M u t a t i o n Res., 30 ( 1 9 7 5 ) 1 9 1 - - 1 9 8 . 15 V a i d y a V., a n d N.N. G o d b o l e , M u t a g e n i c i t y studies w i t h f o u r f o o d e o l o u r s using h u m a n l e u c o c y t e a n d m o u s e m i c r o n u e l e u s test s y s t e m s , in: Proc. I n t . S y m p . E n v i r o n . A g e n t s a n d t h e i r Biological Effects, Osmania University, Hyderabad-7, India, 1978.
314
16 Z h u r k o v , V.S., a n d K.N. Y a k o v e n k o , T h e c u l t u r e of h u m a n l y m p h o c y t e s as a test s u b j e c t for evaluation of m u t a g e n i c a c t i v i t y o f c h e m i c a l s , M u t a t i o n Res., 41 ( 1 9 7 6 ) 1 0 7 - - 1 1 2 . 17 Z i m m e r m a n n , F.K., D e t e c t i o n o f g e n e t i c a l l y a c t i v e c h e m i c a l s using v a r i o u s y e a s t s y s t e m s , in: A. Holl a e n d e r ( E d . ) , C h e m i c a l M u t a g e n s : Principles a n d M e t h o d s for t h e i r D e t e c t i o n , Vol. 3, P l e n u m , N e w Y o r k , 1 9 7 3 , pp. 2 0 9 - - 2 3 9 . 18 Z i m m e r m a n n , F.K., I n d u c t i o n of m i t o t i c gene c o n v e r s i o n b y m u t a g e n s , M u t a t i o n Rcs., 11 ( 1 9 7 1 ) 321--337.
Mutation Research, 67 (1979) 309--314 © Elsevier/North-Holland Biomedical Press
T E S T I N G OF SOME P E R M I T T E D FOOD COLOURS F O R T H E INDUCTION OF GENE CONVERSION IN DIPLOID YEAST
N. SANKARANARAYANAN and M.S.S. MURTHY Division of Radiological Protection, Bhabha Atomic Research Centre, Trombay, Bombay400085 (India)
(Received 9 August 1978) (Revision received 8 March 1979) (Accepted 9 March 1979)
Summary 12 p er mitted f o od colours in use were screened for geno-toxicity. Mitotic gene conversion in S a c c h a r o m y c e s cerevisiae was used as the end-point. Each food colour was tested in stationary-phase as well as log-phase cells but w i t h o u t microsomal activation. These f o o d colours did n o t cause any increase in mitotic gene conversion in diploid yeast BZ 34.
A m o n g the m a n y f ood additives currently in use, colouring agents form an i m p o r t a n t group. Most of these agents have been tested for acute and chronic t o x i c i t y in mammals [3,13]. However, n o t much i nform at i on is available on the geno-toxicity of these c om pounds . Wherever available, such tests are cond u c t e d mostly with microorganisms. When microorganisms are used to test for geno-toxicity, it becomes necessary to c o n d u c t the tests on as m a n y different systems and end-points as possible to rule out organism specificities, false positives and false negatives. In this paper, 12 per mi t t ed food colours used in India and elsewhere were tested for the induction of mitotic gene conversion in diploid yeast S a c c h a r o m y c e s cerevisiae. Mitotic gene conversion results in the transfer o f some genetic i nf or m a t i on between non-sister chromatids of homologous chromosomes. Its relevance in screening of environmental chemicals has been discussed by Z i m m e r m a nn [17,18] and Murthy [7]. Materials and m e t h o d s Y e a s t strain The diploid yeast BZ 34 was kindly d o n a t e d by Dr. R o b e r t Mortimer of the
310 D o n n e r L a b o r a t o r y , Berkeley. The g e n o t y p e of BZ 34 is as follows: a
+ arg4-4 + thrl + trp 5-48 + ura3his5-21ysl-lade2-1 pet + arg 4-17 + leu 1-12 trp 5-48 m e t 1 + his 5-2 lys l - l a d e 2-1
It has two n o n - c o m p l e m e n t i n g m u t a n t alleles (4-4 and 4-17) in the arginosuccinase locus and hence requires arginine in the growth medium. The spontaneous f r e q u e n c y of mitotic gene conversion to arginine independence is very low. However, DNA-damaging chemical and physical agents can induce mitotic gene conversion at a high rate resulting in arginine p r o t o t r o p h y [11,10]. This strain also carries mutations at o t h e r loci, such as ade, his, trp, lys making it auxotrophic to all these amino-acid requirements. However, these loci were rather insensitive to the induction of back-mutations. Induction of mitotic gene conversions in this strain can be d e t e c t e d by plating treated cell suspensions on m e d i u m from which arginine is omitted. T o optimize the yield of arginine p r o t o t r o p h s , omission medium was supplemented with trace quantities of arginine [11]. These will be designated as Arg- plates. The ability of BZ 34 to screen DNA-damaging agents is comparable to those of strain D-4 and D-7 developed by Dr. Zimmermann. This test system was successfully used for estimating the Radiation Equivalent Chemical (REC) value of ethyl methanesulp h o n a t e [9] and AF-2 [8]. Testing chemicals The f o o d colours Amaranth, Sunset Yellow, Erythrosine, Carmoisine, Tartrazine, Fast Red E, Scarlet-F, Indigo Carmine (Indigotine) and Ponceau-4R were obtained f r o m Hickson and Dadajee, Bombay. These materials c o n f o r m to the specifications laid down by the Indian Standards Institution (ISI) for food colours. Wool Green and Fast Green were obtained from the local market. For turmeric, an ex tr ac t was prepared by refluxing pow dered turmeric in 70% ethanol for 1 h at 90 ° C. When the supernatant was diluted with water, a yellow precipitate soluble in di m e t hyl s ul phoxi de (DMSO) was formed. This was used for testing purposes. Gene c o n v e r s i o n test There are man y examples of chemicals that show geno-toxicity in stationary phase cells but n o t when treated under growth conditions and vice versa. This is attributed mainly to the metabolic activation or inactivation of these compounds in growing cultures [14]. Hence, each food colour was tested for induction of gene conversion under log phase as well as stationary phase. The stationary phase tests were carried out in potassium phosphate buffer (0.1 M, pH 7). In a typical t r e a t m e n t a cell suspension of about 10 s cells/ml was prepared from a culture with a low background conversion frequency. 1 ml of this was added to 9 ml o f phosphate buffer which contained the colour at 5 mg/ml. The t r e a t m e n t was carried o u t at 30°C in a shaker water-bath in the dark. After a lapse of 4 h, suitable dilutions were made and plated on yeast extract, pept one, dextrose (YEPD) and Arg- plates for scoring survival and gene conversion respectively. T r e a t m e n t under growth conditions was done in YEPD broth. 10 ml of brot h were inoculated with 1000 cells and placed on a shaker water-bath at 30°C.
311 After 24 h o f growth a given f ood colour was added (5 mg/ml) and the treatm e n t c o n t i n u e d in the dark. At 72 h (i.e. 48 h after addition of the colour) the t r e a t m e n t was terminated. The pH at this stage was a b o u t 6. Cell counts were made with a h a e m o c y t o m e t e r to see w he t he r the food colours inhibited cell division. Suitable dilutions were made and plated on YEPD as well as Argplates. After 3 days the colonies were c o u n t e d and the n u m b e r of convertants per million survivors was calculated. Control samples w i t h o u t the colours were treated identically. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) served as a positive control. Tr eat m e nt s were repeated 2--3 times for each colour. Results and discussion Results summarized in Tables I and 2 show that the 12 perm i t t ed colours tested in this investigation did n o t induce mitotic gene conversion in yeast when treated either in stationary-phase or log-phase culture. Under these t r e a t m e n t conditions neither significant cell killing nor inhibition of cell division was observed. T ha t the absence of lethal and genetic effects may be due to the lack o f penetration of the dye into the cell can be ruled out by the following observations. When the cells were plated after t r e a t m e n t w i t h o u t further dilution on Arg- plates, the colonies picked up the colour of the dye in question. This suggested t ha t the dye could indeed enter the cells. This was further c o n f i r med in some cases by s p e c t r o p h o t o m e t r i c measurements. The absorbance o f the s u p er n atan t after t r e a t m e n t was less than that before treatment. However, the d y e uptake was n o t enough to be seen on microscopic examination of invidual cells.
TABLE
1
TREATMENT OF DIPLOID YEAST IN STATIONARY TION OF MITOTIC GENE CONVERSION
PHASE
WITH
FOOD
Concentration of colour: 5 mg/ml. Numbers in paxentheses are the numbers Conversion frequencies shown are the means of 2 experiments, Food
colour
Colour index
Arginine convertants survivors Control
COLOURS
of colonies scored on 4 plates.
per 106
Percentage survival
Treated
Amaranth Indigo Carmine Carmoisine
16 185 73 015 14 720
21 24 22
(854) (665) (854)
29 ( 2 8 7 ) 26 (584) 26 (275)
100 103 100
Ponceau-4R Fast Red E Scarlet F
16 255 37 035 16 155
26 (1101) 22 (854) 28 (330)
27 ( 2 7 8 ) 35 (272) 33 (342)
100 100 100
Fast Green FCF Wool Green BS Tartrazine
42 053 44 090 19 1 4 0
33 33 38
38 (384) 30 (336) 40 (220)
100 100 98
Erythrosine Sunset Yellow FCF Turmeric MNNG (0.02 raM)
45 430 15 985 75 300 positive control
25 (1101) 20 (535) 41 ( 2 0 4 ) 16 (191)
(232) (232) (701)
25 23 49 2026
FOR INDUC-
(524) (620) (254) (547)
100 100 97 64
312
TABLE 2 TREATMENT OF DIPLOID YEAST MITOTIC GENE CONVERSION
IN LOG PHASE WITH FOOD
COLOURS
FOR
INDUCTION
OF
C o n c e n t r a t i o n o f c o l o u r : 5 m g / m l . N u m b e r s i n p a r e n t h e s e s are t h e n u m b e r s o f c o l o n i e s s c o r e d o n 4 p l a t e s . C o n v e r s i o n f r e q u e n c i e s s h o w n are t h e a v e r a g e s o f 2 e x p e r i m e n t s . Food colour
Colour index
Arginine convertants per 106 survivors Control
Percentage survival
Treated
Amaranth Indigo Carmine Carmoisine
16 1 8 5 73 015 14 7 2 0
52 (1075) 37 (242) 37 ( 2 4 2 )
56 30 30
(993) (292) (286)
98 101 100
Ponceau-4R Fast Red E Scarlet F
16 2 5 5 37 0 3 5 16 1 5 5
37 45 73
(242) (990) (492)
36 (269) 46 (1018) 83 (851)
100 100 100
Fast Green FCF W o o l G r e e n BS Tartrazine
42 053 44 090 19 1 4 0
81 ( 1 0 6 2 ) 43 (1308) 66 (1117)
93 (982) 44 (724) 60 (1195)
100 100 100
Erythrosine Sunset Yellow FCF Turmeric MNNG (0.05raM)
45 430 15 985 75 300 positive control
37 37 33 75
(242) (242) (393) (710)
24 33 36 2600
(484) (346) (986) (515)
100 100 103 63
Kada et al. [6] conducted a "rec" assay on Amaranth, Fast Green, Erythrosine, Indigo Carmine, Sunset Yellow and Tartrazine; but all were negative. Luck et al. [3] have reported that Erythrosine was slightly mutagenic in E. coli. This colour also exhibited very slight mutagenic activity in E. coli [3]. However, it TABLE 3 SUMMARY OF DIFFERENT TYPES OF GENOTOXICITY MICROORGANISMS AND MAMMALIAN CELL SYSTEMS Mutation
(E. coli)
TESTS WITH SOME FOOD
Mutation ( A m e s test), a
Chromosomal aberrations and micronucleus test [5,15,161
negative [2,4] negative [4] negative [2,4]
-+ negative --
negative negative --
negative negative negative
positive . .
--
negative negative negative
[3]
" R e c " assay [6]
COLOURS
Gene conversion in yeast ( p r e s e n t investigation)
Amaranth Indigo Carmine Carmoisine
negative negative --
Scarlet-F Poneeau-4R Fast Red E
. negative . .
Wool Green F.S. Fast Green FCF Tartrazine
---
negative I1] negative [4,2] n e g a t i v e [ 1]
--positive
-negative negative
negative negative negative
Erythrosine Sunset Yellow FCF Tu r m e r i c
weak -.
negative [ 4,2] -. .
±
negative negative
negative negative negative
.
.
.
-.
.
.
--, M e a n s d a t a n o t a v a i l a b l e . a N u m b e r s i n b r a c k e t s are r e f e r e n c e n u m b e r s .
.
.
IN
313
failed to induce gene conversion in yeast strain BZ 34. Amaranth,when tested through the fluctuation test by Parry, was negative [12]. Amaranth, Tartrazine, Erythrosine, Indigotine, Fast Green, and Sunset Yellow were also negative in the Ames test with microsomal activation [2,1,4]. Ishidate and Odashima screened Sunset Yellow and Amaranth for chromosomal aberration on Chinese hamster cells in culture [ 5]. Whereas Sunset Yellow was negative, Amaranth was classified as "suspicious" in this system. Zhurkov et al. [16] tested Indigo Carmine, Sunset Yellow, Amaranth and Tartrazine in a human lymphocyte culture. Only Tartrazine showed a weak cytogenetic effect. Amaranth, Ponceau4R, Sunset Yellow and Tartrazine were investigated for mutagenicity in human leukocyte and mouse micronucleus test systems [15]. All 4 food colours were reported to be positive in both systems at low concentrations. Table 3 shows a summary, from published literature, of the different types of geno-toxicity tests conducted on these food colours in both microorganisms and cultured mammalian cell systems. In view of the conflicting results with some compounds and the large population exposed, it is necessary to carry out further tests on them. Acknowledgements We thank Dr. K.G. Vohra for his encouragement, Dr. U. Madhvanath for useful suggestions and U.R. Kini for spectrophotometric measurements. References 1 A u l e t t a , A.E., J.M. K u z a v a a n d A.S. P a r m a r , L a c k of m u t a g e n i c i t y of a series of f o o d d y e s for Salmonella t y p h i m u r i u m , M u t a t i o n Res., 56 ( 1 9 7 7 ) 2 0 3 - - 2 0 7 . 2 B r o w n , J.P., W.R. G e r a l d a n d R.J. B r o w n , M u t a g e n i c i t y t e s t i n g o f f o o d c o l o u r s a n d r e l a t e d azo, xant h e n e a n d t r i p h e n y l m e t h a n e d y e s w i t h t h e S a l m o n e l l a / m i c r o s o m e s y s t e m , M u t a t i o n Res., 56 ( 1 9 7 8 ) 249--271. 3 F . A . O . N u t r i t i o n Meetings R e p o r t Series No. 4 6 A , T o x i c o l o g i c a l E v a l u a t i o n of s o m e f o o d colours, emulsifiers, a n t i - c a k i n g a g e n t s a n d c e r t a i n o t h e r s u b s t a n c e s . W H O / F O O D - A D D ] 7 0 , pp. 2 3 - - 5 7 . 4 G a r n e r , C.R., a n d C.A. N u t m a n , T e s t i n g o f s o m e a z o - d y e s a n d t h e i r r e d u c t i o n p r o d u c t s for m u t a g e n i c i t y using S a l m o n e l l a t y p h i m u r i u m T A 1 5 3 8 , M u t a t i o n Res., 44 ( 1 9 7 7 ) 9 - - 1 9 . 5 I s h i d a t e Jr., M., a n d S. 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