The mutagenic effect of amaranth (FD and C red no. 2) in bacteria and yeast

Environment International, Vol. 9, pp. 145-148, 1983

0160-4120/83 $3.00 + .00 Copyright © 1983 Pergamon Press Ltd.

Printed in the USA. All rights reserved.

THE MUTAGENIC EFFECT OF AMARANTH (FD AND C RED NO. 2) IN BACTERIA AND YEAST

M.A. JabarAI-Mossawi Environmental Sciences Department, Kuwait Institute for Scientific Research, P.O. Box 24885, Safat, Kuwait

(Received 7 September 1982; Accepted 13 December 1982) Although amaranth is consumed in large quantities as a food dye, its mutagenicityand/or carcinogenicity

is still controversial. Therefore, the investigationof the geneticactivityinduced by this dye might assist in the evaluation of its possible dangers to human health. Both bacteria and yeast strains were used for testing the mutagenic activity of amaranth. When the fluctuation test was used, amaranth was found to enhance both forward and reversemutations in bacterial strains that contain R-plasmids. Amaranth was not found to induce any genetic activity in yeast cells at 28 °C but a significant increase in mitotic gene conversionswas recorded at 37 °C in both acidic and neutral media. It has been postulated that amaranth has optimal action on microbial cells at 37 °C and should be considered a weak mutagen.

Introduction

of amaranth in over 60 countries has been estimated at 675,000 kg (Baigusheva, 1968).

Amaranth, originally synthesized by H. Baum in 1828 (Society o f Dyers and Colourists, 1971), is commercially synthesized by coupling diazotized naphthionic acid

Although amaranth is consumed in large quantities as a food dye, its carcinogenicity is still controversial. For instance, Boffey (1976), Baigusheva (1968), and Andrianova (1970) have reported the induction of different kinds o f tumors in rats fed amaranth. Others have claimed that amaranth is not carcinogenic when injected subcutaneously into Osborne Mendel rats (Nelson and Hogan, 1953) and that the reported carcinogenic effect might be due to the impurity o f the chemical used (Radomski, 1974). The working group of the International Agency for Research on Cancer (IARC) concluded that the carcinogenicity of amaranth in humans cannot be evaluated because o f the inadequacy o f the data available (IARC, 1975). Thus the investigation of the genetic activity of such a widely consumed food dye, using microbial assays, might help to predict its risk to humans, dependent upon the empirical relationship between the carcinogenicity and mutagenicity of chemical compounds (McCann et al., 1975).

(1 naphthylamine-4 sulphonic acid) with 2-naphthol-3, 6-disulphonic acid (Zuckerman, 1964)forming the following structure with an empirical formula of C2oH,,N2Na30,oS3 (molecular weight = 604.5):

HO

NaO3S~

.SO3Na

N=N

SOaNa

Amaranth is used as a coloring in a wide range of foods, including gelatine, maraschino cherries, sausage casings, frozen desserts, carbonated beverages, drink powders, sweets and confectionary products not containing oils and fats, bakery products and cereals, pudding, aqueous drug solutions, tablets, capsules, mouth washes, bath salts, hair rinses, and so on. (IARC, 1975; Society of Dyers and Colourists, 1971). The annual use

Materialsand Methods Chemicals The formula for Certicol amaranth S (a gift from William Hounslow Ltd., Middlesex, England) is tri145

146

M . A . J a b a r AI-Mossawi

sodium lene-3, (MMS) used as

3-hydroxy-4-(4-sulpho-l-napthylazo) naphtha7-disulphonate. Methyl methanesulphonate purchased from Eastman Kodak Co. (NY) was a positive control,

tryptophan or histidine, for measuring mitotic gene conversion (Parry et al., 1976). Amaranth lethality was measured by mixing different concentrations of amaranth with bacterial cells in nutrient broth (Difco) overlays and pouring them on nutrient-agar plates. The plates were then incubated overnight at 37 °C.

Tester strains The strains of bacteria used in this study included cultures of Salmonella typhimurium, auxotrophic for histidine (provided by Bruce Ames) and a variety of Escherichia coli strain (provided by G. Mohn, M. Green, and D. Tweats) and have all been described elsewhere (Parry et al., 1976; Parry and AI-Mossawi, 1979). The strains used were capable of detecting both frameshift and base substitution mutagens, The yeast strain JD1 was auxotrophic for both histidine and tryptophan and produces prototrophic colonies by the process of mitotic gene conversion, a process of genetic change that responds to treatment by mutagens and carcinogens in an essentially nonspecific manner. The use of this yeast strain has been described in detail elsewhere (Davies et al., 1975; Parry et aL, 1976).

Results Freshly prepared aqueous solutions of amaranth were screened for possible genetic activity. The bacterial fluctuation test and Ames test assays with and without liver microsomes were used, in addition to the measuremerit of induced mitotic gene conversion in the yeast Saccharomyces cerevisiae. All the assays involved the measurement of the frequencies of prototrophic cells produced in auxotrophic cultures. Figure 1 shows the induced mutation produced by the amaranth obtained from Williams Hounslow after exposure of both Escherichia coli and Salmonella typhimurium strains. In general, those bacterial strains that contain drug-resistant plasmids (the R-plasmids R46 and pKM101, in Escherichia coli and Salmonella typhimurium strains, respectively), were more sensitive to amaranth activity than strains lacking such plasmids. This activity can be clearly observed in Escherichia coli uvrA(R46) and in Salmonella typhimurium TA100, which are usually used for screening of base change mutagens (Green and Muriel, 1976; McCann et al.,

Mutagenicity assays The fluctuation test, as described by Green et al. (1976) and the Ames test (Ames et al., 1975), were used with and without liver microsomal activation. In addition, yeast cells were treated in liquid yeast complete (yc) media (pH 7.0 and 4.6) for 4 days at 37 °C, and then plated on yeast minimal plates containing either

2.o

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~.¢oli Wp2 uvrA(R46)

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E.coll 343/113(1846 ){y$60

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002

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Amaranth

20

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02

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Fig. 1. The mutagenic effect of a m a r a n t h on Eseherichia coil and Salmonella typhimurium as shown by fluctuation test without microsornal activation.

Mutagenic effects of amaranth

147

Table 1. Treatment of different bacterial strains with amaranth, with or without microsomal activation, using the Ames test. Mean Number of Prototrophs/Plate

Without microsomal activation

With microsomal activation

Strains

Locusa

Control

S. S. E. E.

typhimurium TA98 typhimuriurn TA100 coli uvrA (R46) coli 343/113 (R46)

his D3052 his C46 trp arg 56

29.0 44.7 4.5 75.2

S. S. S. E. E.

typhimuriurn TA98 typhimurium TAI00 typhimuriurn TA1538 coli WP2 uvrA (R46) coli 343/113 (R46)

his D3052 his G46 his D3052 trp arg 56

40.0 45.0 10.7 5.2 193.0

2 20 p.g/ml #g/ml 37.0 60.7 3.5 95.0

37.0 70.2 3.75 109.0

49.0 44.0 54.6 50.0 9.5 9.0 10.0 11.2 192.0 165.0

200 2000 v.g/ml tzg/ml MMS 31.0 57.2 69.5

1.6 36.2 -

34.2 319.5

35.5 50.0 9.2 7.2 145.2

2.8 28.6 7.0 -

79.2 406.2

ahis = histidine; trp = tryptophan; arg = arginine.

1975). T h e f o r w a r d m u t a t i o n s y s t e m g a l R % in E s c h e r i c h i a coli 343/113 (R46) strain, also c o n t a i n i n g R - p l a s m i d , was used to d e m o n s t r a t e the genetic activity of a m a r a n t h in the f l u c t u a t i o n test. A significant increase in the n u m b e r o f positive t u b e s was c o u n t e d , d e m o n s t r a t i n g the f o r w a r d m u t a t i o n i n d u c e d by a m a r a n t h . E s c h e r i c h i a coli 3 4 3 / 1 1 3 (R46) a n d S a l m o n e l l a t y p h i m u r i u m T A 9 8 were also used, a n d significant increases in the n u m b e r o f t u r b i d tubes were observed. These two strain can be reverted to prot o t r o p h y by frameshift m u t a g e n s ( M o h n et al., 1974; A m e s et al., 1973). T h e r e f o r e , the data presented i n F i g , 1 show that base change f o r w a r d a n d frameshift m u t a tions are i n d u c e d in bacterial strains b y low c o n c e n t r a tions o f a m a r a n t h . The effective c o n c e n t r a t i o n detected by the f l u c t u a t i o n test r a n g e d f r o m 0.02 to 0.2 # g / m l . O n the other h a n d , the e x p e r i m e n t i n v o l v i n g the use of the A m e s test failed to show a n y activity for a m a r a n t h , either with or w i t h o u t liver m i c r o s o m e activation, as presented in T a b l e 1. The results o b t a i n e d from the m e a s u r e m e n t o f induced m i t o t i c gene c o n v e r s i o n in the yeast strain JD1

using a m a r a n t h are s h o w n in T a b l e 2. These results were o b t a i n e d b y g r o w i n g the yeast cells in the presence of a m a r a n t h in liquid yc m e d i a at 37 ° C at n e u t r a l a n d acidic media. No genetic activity was observed when a m a r a n t h was tested at 28 °C on S a c c h a r o m y c e s cerevisiae ( P a r r y , 1977). A t 37 °C, a m a r a n t h shows a significant increase in the n u m b e r of revertants to trypt o p h a n a n d histidine p r o t o t r o p h s in b o t h n e u t r a l a n d acidic p H , with some v a r i a t i o n in the dose response. I n a n a t t e m p t to d e t e r m i n e the presence of toxic effects p r o d u c e d by a m a r a n t h for bacterial cells, cultures of the bacteria Escherichia coli a n d S a l m o n e l l a t y p h i m u r i u m were exposed to a m a r a n t h s o l u t i o n ( 0 . 0 2 - 2 0 0 / ~ g / m l ) a n d plated o n n u t r i e n t agar plates. It appears f r o m Fig. 2 that there was a toxic effect of a m a r a n t h u p o n bacterial cells a n d that S a l m o n e l l a strains are more susceptible to such toxicity. T h e lethal effects o f a m a r a n t h u p o n yeast cells was noticed in both acidic a n d n e u t r a l media; there is a significant difference in lethality response at the 2 0 0 0 / ~ g / m l dose between the two media; it seems more lethal at n e u t r a l p H (see T a b l e 2).

Table 2. Induction of mitotic gene conversion in yeast JDI by amaranth and methyl methanesulphonate (MMS) at 37 °C without liver activation, a pH 7 7 7 7 7 4.6 4.6 4.6 4.6 4.6

Treatment b Control 20/zg/ml A 200 ttg/ml A 2000 ttg/ml A MMS 0.2070 for 30 min Control 20 v.g/ml A 200 p.g/ml A 2000 p.g/ml A MMS 0.2070 for 30 min

ahis = histidine; trp = tryptophan. bm = amaranth.

Mean No. of His" Prototrophs/Plate 25.1 183.1 82.1 5.6 22.54 0.7 1.7 6.3 5.65 12.25

His" Prototrophs/ 106 Survival ±S.E. 47.0 404.0 194.5 37.3 102.4 11.1 35.4 141.5 131.3 284.8

± 1.06 ±24.0 ±39.0 +2.4 ±6.0 ±8.7 ±32.0 ±5.2

Mean No. of Trp" Prototrophs/Plate

Trp ~ Prototrophs/ l0 s Survival ±S.E.

Viability (°7o)

16.4 37.0 15.2 0.9 185.5 1.6 14.0 6.1 6.6 9.6

29.9 ±2.0 81.8 ± 10.3 36.0 ±3.7 71.3 ±0.63 840.9 25.3 ±1.7 291.6 ±23.0 135.5 ±23.0 155.3 ±6.5 224.4

100.0 82.8 77.0 21.0 39.0 100.0 76.1 70.9 68.2 68.2

148

M . A . Jabar AI-Mossawi

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with increase in temperature has been reported in plants also (Lewis, 1949). From these data one can conclude that amaranth is a weak mutagen, since its activity in bacteria and yeast has been demonstrated only by liquid treatment techniques. More investigation is needed to test such activity in other short-term and/or long-term systems to evaluate amaranth's effects on health. A c k n o w l e d g e m e n t - I would like to thank Dr. J. M. Parry for his encouragement to test this important compound and for providing the JD1 strain.

~

References Q=

o.,

Amaranth

"°

concentration

'* ( pg/ml )

~°0

Fig. 2. Survivals curve of Salmonella typhimurium and Escherichia coil uvrA (R46) after specimens were treated with amaranth and plated out the cells on nutrient agar plates.

Discussion Amaranth was found to enhance both forward and mutation in cultures of auxotrophs of

reverse

Escherichia coli and Salmonella typhimurium that con-

tain drug-resistant plasmids, when the fluctuation t e s t w a s used. The latter test is a liquid treatment procedure and is recommended for screening of weak mutagens at low doses (Green et al., 1976). When the Ames t e s t w a s used, amaranth was found not to be mutagenic in the absence or presence of liver microsomes in all the bacterial strains used. These observations support the data reported by Gatehouse (1978), who found that Azorubin (another commercial name for amaranth produced by Aldrich) was mutagenic to Escherichia coli WP2 and Salmonella typhimurium TA98 by his modified fluctuation test, but not by Ames test. The failure of the Ames test procedure in detecting the genetic activity of amaranth

might be due to the insensitivity of this procedure for detecting low levels of mutagens (Green et aL, 1 9 7 6 ) . The mutagenic effect of amaranth was generally detected in the strains containing drug-resistant plasmids. These plasmids (R46 and pKM101 in Escherichia coli and Salmonella typhimurium, respectively) may carry mutator genes that can enhance mutagenesis by both frameshift and basechange mutagens (McCann e t ak, 1975). The susceptibility of yeast Saccharomyces cerevisiae JD1 to the mutagenic effect of amaranth at 37 °C but not at 28 °C (Parry, 1977) suggests that amaranth has optimal action on yeast cells a t 37 °C. Lindgren (1972) postulated that some chemicals have optimal action a t certain temperatures, others have an increasing effect with augmented temperature, and still others a r e n o t affected by temperature. An increase in induced mutation

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289-296. Green, M. H. L. and Muriel, W. J. (1976): Mutagen testing using trp* reversion in Escherichia coli, Mutat. Res. 38, 3-32. Green, M. H. L., Muriel, W. J., and Bridges, B. A. (1976) Use of a simplified fluctuation test to detect low levels of mutagens, Mutat. Res. 38, 33-42.

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tion of carcinogensonmutagensin the Salmonella/microsome test assay: Assay of 300 chemicals, Proc. Nat. Acad. Sci. U.S. 72, 5135-5147. Mohn, G., Ellenberger, J., and McGregor, D. (1974)Development of mutagenicity tests using Escherichia coli K-12 as indicator organism, Mutat. Res. 25, 187-198. Nelson, A. A. and Hogan, E. C. (1953) Production of fibrosarcomas in rats at site of subcutaneous injection of various food dyes, Federation Proceedings, Federation Am. Soc. o f Exper. Biol. 12, 397. Parry, J. M., Tweats, D. J., and AI-Mossawi, M. A. J. 0976) Monitoring the marine environment for mutagens, Nature (Lon-

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genic chemicals in the tissue of mussel mytius edclis, Environ. Pollut. 19, 175-186. Radomski, J. L. (1974) Toxicology of food colours, Ann. Rev. Pharmacol. 14, 127-136. Society of Dyers and Colourists (1971) Colour Index, 3rd ed., Vol. 1, p. 1132. Deanhouse Picadilly, Bradford, Yorkshire, England. Zuckermann, S. (1964) Colors for food drugs and cosmetics, in Encyclopedia o f Chemical Technology, 2nd Ed., Vol. 5, p. 857. John Wiley and Sons, New York, NY.