Effects of food dyes on Paramecium caudatum: Toxicity and inhibitory effects on leucine aminopeptidase and acid phosphatase activity

rOXICOLOGY

Toxicity

AND

APPLIEDPHARMACOLOGY 39, 111-l 17 (1977)

Effects of Food Dyes on Paramecium cwdatum: and Inhibitory Effects on Leucine Aminopeptidase Acid Phosphatase Activity

and

FUMIYO SAKO,~NAOYUKI

TANIGUCHI,~ NORIKOKOBAYASHI,AND EIMATSUTAKAKUWA

Department of Home Economics, Hokkaidb University of Education, and Department of Hygiene and Preventive Medicine, Hokkaido University School of Medicine, Sapporo, Japan Received June 4,1976 accepted August 24,1976

Effects of Food Dyes on Paramecium caudatum: Toxicity and Inhibitory Effects on Leucine Aminopeptidase and Acid PhosphataseActivity. SAKO,F., TANIGUCHI,N., KOBAYASHI,N., AND TAKAKUWA, E. (1977). Toxicol. Appl. Pharmacol. 39,ll l-l 17.The toxic effect of 14food dyeswas studiedin Paramecium caudatum. It wasfound that xanthenedyescontaining halogenatomsin their moleculesweremore toxic than other groupsof food dyes. Phloxin and rose bengal containing chlorine were especially toxic. The effect of food dyeson leucineaminopeptidase,acid phosphatase, and y-glutamyl transpeptidaseactivity in P. caudafum wasstudiedin order to investigatethe mechanismof toxicity. Phloxin and rosebengalinhibited leucineaminopeptidaseremarkably. The inhibitory effect of food dyeson leucine aminopeptidasein vitro is consistentwith the toxic effect of the dyes on the survival time of P. caudatum. A possiblecorrelation between toxicity and inhibition of the activity of enzymesinvolved in the digestive processis discussed. The death of Paramecium caudatum (PC), a unicellular animal, can be observed more readily and in far less time than that of small animals. In our previous report it was shown that PC can serve as an indicator for the toxicity of insecticides (Takakuwa et al., 1971).

In a bioassay system, a problematic point is whether the reaction of agents on PC, which is a lower animal in the evolutionary scale than the rat and mouse, can be applied to higher animals. It can be postulated, however, that an agent that is toxic on PC would have some abnormal action on the tissues of higher animals. Little work has been done on the effect of toxicity of food additives to PC, however some reports are available in the literature on the sensitivity of PC to caffeine (Joan, 1974), sodium 2,4-D (Kokia, 1971), detergent enzyme (Cairns et al., 1971), aflatoxin B (Reiss, 1971), and insecticides (Takakuwa et al., 1971). Recently, Tanaka and Okahara (1973) reported that erythrosine, phloxin, rose

bengal, and acid violet showed an extreme inhibitory

effect on amino acid transport

1Departmentof HomeEconomics, HokkaidoUniversityof Education,Sapporo,Japan. z Reprintsshouldbe requestedfrom Dr. N. Taniguchi,Departmentof Hygieneand Preventive Medicine,HokkaidoUniversitySchoolof Medicine,Sapporo,Japan. 111 Copyright Q 1977 by Academic Press, Inc. All rights of reproduction Printed in Great Britain

in any form

reserved.

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ET AL.

and leucine aminopeptidase (LAP) activity of rat small intestine. These authors proposed that the dyes have some effect on amino acid transport or the digestive process of the small intestine. Esteve (1970) found that acid phosphatase existed in food vacuoles, autophagic vesicles, and dense bodies, and was related to the digestive processes of PC. It is known that y-glutamyl transpeptidase plays an important role in the amino acid transport of mammalian tissues such as kidney (Orlowski and Meister, 1970) or tumor tissue (Taniguchi et al., 1975). This report describes the acute toxicity of 14 food dyes, using PC as an indicator. Also, the effects of food dyes on LAP, acid phosphatase, and y-glutamyl transpeptidase activities were assayed in vitro in order to investigate the toxic mechanisms for food dye interactions with PC.

METHODS

Chemicals L-Leucine p-nitroanilide, L-y-glutamyl y-nitroanilide, and p-nitrophenyl phosphate were obtained from C. F. Boehringer, Mannheim, Yamanouchi, Japan. All other reagents used were of analytical grade. Food dyes tested were as follows : azo dyes : Ponceau 3R, amaranth, Ponceau R, new coccine, tartrazine, sunset yellow; xanthene dyes : erythrosine, eosine, phloxin, rose bengal, acid red; triphenylmethane dyes: brilliant blue FCF, acid violet, fast green FCF. Cultures qf Pc PC was maintained aerogenes.

at 22°C on 0.15 % dried lettuce infusion and fed with Aerobacter

Determination of Survival Time and Death Rate Food dyes of various concentrations were put in a hollow slide glass, and an equal volume of 0.04 M phosphate buffer, pH 7.0, was added. After 5 to 10 PC were added, their survival times were measured microscopically. Thirty to forty PC for each concentration were tested by the same method, and the mean survival time and the death rate were calculated. The survival time was defined as the time required until death was observed for each concentration. Death was assumed to have occurred when there was no movement. The death rate was defined as the percentage of deaths observed during 20 min. Preparation of Enzyme Extracts One week after inoculation, PC culture was filtered through two thicknesses of muslin. This was centrifuged at 8000 g for 10 min and the packed cells were then washed in a solution of 0.12 M NaCl and resedimented by centrifugation at 8000 g for 10 min. The pellet of cells was then sonicated in 0.1 M sodium phosphate buffer, pH 7.2, for 20 sec. The resulting sonicate was centrifuged at 8000 g for 10 min, and the supernatant fraction was used as the crude enzyme extract. The centrifugation and the subsequent procedures were carried out at 0-4”C.

FOOD

DYES

Assay of Leucine Aminopeptidase, Activity

AND

PARAMECIUM

ENZYMES

Acid Phosphatase, and y-Glutamyl

113 Transpeptidase

Leucine aminopeptidase activity in the PC extract was assayed according to the method of Tuppy et al. (1962). The reaction mixture was composed of 0.03 ml of 0.025 M L-leucine p-nitroanilide, 0.67 ml of 0.1 M phosphate buffer of pH 7.2, and 0.2 ml of the crude enzyme extract as mentioned above. Acid phosphatase activity was assayed according to the method of Alvarez (1962). The reaction mixture was composed of 0.02 ml of 0.6 M p-nitrophenyl phosphate, 0.4 ml of 0.1 M citrate buffer of pH 5.6, and 0.1 ml of the crude enzyme extract. y-Glutamyl transpeptidase activity was assayed according to the method of Orlowski and Meister (1965). The reaction mixture was composed of 0.05 ml of 0.04 M L-y-glutamyl p-nitroanilide, 0.16 ml of 1 M Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) buffer of pH 9.0, 0.04 ml of 0.1 M MgC12, 0.04 ml of 0.1 M glycylglycine, and 0.075 ml of the crude enzyme extract. The amount of p-nitroaniline and p-nitrophenol formed from the substrate by the enzyme was determined calorimetrically at 405 and 412 nm, respectively, on a Hitachi 624 type recording spectrophotometer. Inhibition Study Enzyme inhibition experiments by food dyes were carried out by adding the solution of food dyes to the standard reaction mixture. Each food dye was diluted with the same buffer used in the enzyme assay.

RESULTS

Eflect of Food Dyes on the Mean Survival Time of Pc As shown in Table 1, the effect of food dyes on the survival time of PCwas measured microscopically for 20 min. The concentration of food dyes in the bathing fluid was 0.1 or 1.O%. The mean survival time of PC in 0.1% concentration varied with the chemical structure of the dye. Xanthene dyes (erythrosine, eosine, phloxin, and rose bengal) containing halogen atoms in their molecules showed smaller survival times, and thus these dyes were considered to be more toxic than other groups of dyes. The death rate of PC in 0.1% solution of these dyes was 100%. Phloxin and rose bengal containing chlorine were especially toxic ascompared to erythrosine and eosinewhich contained iodine and bromine, respectively. Toxic effects of chloramine T which contains chlorine were next to those of rose bengal in magnitude (data not shown). It was assumed,therefore, that chlorine played an important role in the lethal action to PC. Among xanthene dyes, only acid red was shown to have a low toxicity. Acid red contains no fluorescein in its molecule, and therefore this fact may suggestthat the structure of fluorescein also may have somerelation to the lethal action. On the other hand, there was no difference between azo dyes (Ponceau 3R, amaranth, Ponceau R, new coccine, tartrazine, sunset yellow) and triphenylmethane dyes (brilliant blue FCF, acid violet and fast green FCF) in toxicity. A mark of a representing mean survival time in 0.1% solution, as shown in Table 1, indicates that almost all PCsurvived even after a lapse of 20 min, and therefore for thesedyes, 1.O:;i solutions were tested.

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ET AL.

The effect of food dyes on the cumulative mortality of PC was studied. The lag phase of the death rate for each food dye was observed as shown in Fig. I. Following the lag phase the death rate increased exponentially, and the rate of death was almost linear when the numbers of deaths were plotted against time semilogarithmically. After the logarithmic phase, the death rate reached a stationary phase. It seems possible that the duration of the lag phase and the slope of the death rate in the logarithmic phase could be used as indicators of toxicity. In the cases of phloxin, rose bengal and erythrosine, the lag phase was quite short and the slope of the logarithmic phase was steep. On the TABLE EFFECT

OF FOOD

DYES ON THE MEAN

1

SURVIVAL

TIME

AND DEATH

RATE OF

Paramecium caudatum Dye concentration 1.0%

Food dyes Ponceau 3R Amaranth Ponceau R New coccine Tartrazine Sunset yellow Erythrosine Eosine Phloxin Rose bengal Acid red Brilliant blue FCF Acid violet Fast green FCF u No deaths * Impossible

0.1%

Mean survival time (set)

Death rate (%I

860 695 640 690 914 850

93.3

879 881 298

-*

were observed for 20 min. to observe microscopically

77.4 100 90.3

Mean survival time (set) -a -Q -a 1034

100

--a

83.3

1049 219 457 47 7

96.7 63.3

loo -*

because

-0 -0 865 1037

Death rate (%) 3.3 3.3 12.5 63.3 6.7 30.0 100

100 100 loo 12.5 7.7 83.3 36.7

of high concentration.

other hand, in the casesof acid violet and new coccine, the lag phasewas long and the slope in the logarithmic phasewas not steep. From these results, the toxicity of food dyes on PC was found to be in the order of rose bengal > phloxin > erythrosine > eosine > acid violet > new coccine > fast green FCF > sunset yellow > Ponceau R > acid red > brilliant blue FCF > tartrazine > Ponceau 3R and amaranth. Efect of Food Dyes on LAP, Acid Phosphatase,and y-Glutamyl TranspeptidaseActivity The eflrect of food dyes on these enzymes activities was assayedin vitro, in order to investigate the toxic mechanism of food dyes. The results are shown in Table 2. Rose

FOOD

DYES AND

PARAMECIUM

115

ENZYMES

Phloxin RoseBengal

0

200

400 600 800 Time(seconds)

10001200

FIG. 1. Typical cumulative mortality of Paramecium caudatum. PC was exposed to solutions of food dyes. The concentration of food dyes employed was 0.1 %, except for the concentration of rose bengal which was 0.001%. The death rate of PC in these dyes was found to be above 50%.

bengal inhibited LAP activity almost completely. Phloxin and acid violet also inhibited the enzyme activity by 70 and 900?, respectively. Amaranth and new coccine, however, had no inhibitory effect. The order of inhibitory effects was consistent with those of the death rate of PC described in Table 1. The correlation coefficient between the inhibitory TABLE EFFECT OF Foot DYES ON LEUCINE y-GLUTAMYLTRANSPEPTIDASEACTIVITY

2

ACIDPHOSPHATASE AND IN&ZY.~~~C~U~~~~~~~~~EXTRACTS~

AMINOPEPTIDASE,

Relative enzymeactivity (%) Food dyes Amaranth New coccine Phloxin Rosebengal Acid red Brilliant blue FCF Acid violet

Leucine aminopeptidase 100

Acid phosphatase 80

y-Glutamyl transpeptidase

94

104 67 65 121

93 113 98 93 113

71 10

106 125

80 107

99 33

0

0 This experiment was carried out by adding a solution of food dye (lOA M) to the standard reaction mixture. b Each value represents the average of closely agreeing duplicate analyses.

116

SAKO ET AL.

activity of these food dyes on LAP and the death rate showed r values of 0.7952 (p < 0.05). Acid phosphatase activity was also inhibited by phloxin, rose bengal, and slightly by amaranth. But acid red and acid violet activated this enzyme. The mechanism of the activation is not yet clear. y-Glutamyl transpeptidase activity was not inhibited by any food dye except brilliant blue FCF. The Type qf Inhibition of LAP by RoseBengal

The type of inhibition of LAP by rose bengal was studied. A double reciprocal plot showed an uncompetitive type of inhibition as shown in Fig. 2.

A 5-

0,0 1d’M RoseBengal

P4

c 3-.,/o’ yzS 1 -fl’O 0

/o I 5

None I 10 (l/S)

FIG. 2. Inhibition pattern of leucine aminopeptidase by rose bengal. The reaction mixture contained 0.312 ml of 0.1 M phosphate buffer, pH 7.2, 0.038 ml of 10 mM L-leucine p-nitroanilide, enzyme, and 10V5 M rose bengal in a total volume of 0.45 ml. Where Vis the velocity of the enzyme reaction expressed as optical density per minute, and S is the molar concentration of the substrate.

DISCUSSION Although the LD50 values for small animals have been generally accepted as indicators of the toxicity of food additives, use of such animals seems both time consuming and uneconomical, judging from the fact that there has been a rapid increase in the number of various chemical compounds, including food additives, that requires testing. Chlorella, Saccharomycesceretiisiaevar. sake (Yamamoto et al., 1967) bacteria and Tetrahymena have been used as indicators for these purposes. In contrast, the present study describes the toxicity of food dyes using PC, a unicellular animal, as an indicator. The results suggest that xanthene dyes such as phloxin and rose bengal, not permitted to be used in Europe and United States, but permitted in Japan, are most toxic. On the other hand, the inhibitory action of food dyes to LAP and acid phosphatase, which may play an important role in the digestive process of PC, was in good agreement with the toxic effects on PC. y-Glutamyl transpeptidase activity was not affected by these dyes. These facts suggest that the inhibition of LAP and acid phosphatase activity by food dyes may have some relation to the digestive process of PC rather than to the amino acid transport of the PC tissues. It seems to be one of the lethal factors that PC takes food dyes into food vacuoles, after which these enzymes are inactivated. It is possible

FOOD DYES AND PARAMEClUM

ENZYMES

117

that the rate of inhibition of these enzyme activities can be used as an indicator of toxicity of food dyes or other chemical substances. Inada and Miyazawa (1969) reported that the lethal action of xanthene dyes on some bacteria is explained by photodynamic action. It is possible that inactivation of LAP and acid phosphatase by xanthese dyes is brought about by chlorine that is released from xanthene dyes by visible light. This consideration would be supported by the fact that chloramine T is toxic to PC. The authors are now continuing the study along these lines.

ACKNOWLEDGMENT The PC specimens were supplied

by Professor S. Hayashi, Fuji Women’s

College, Sapporo.

REFERENCES E. F. (1962). The kinetics and mechanism of the hydrolysis of phosphoric acid esters by potato acid phosphatase. Biochim. Biophys. Acta 59, 663-672. CAIRNS, J., BEAMER, T., CHURCHILL, S., AND RUTHVEN, J. (1971). Response of protozoans to detergent-enzyme. Hydrobiologia 38, 193-205. ESTEVE, J. C. (1970). Distribution of acid phosphatase in Paramecium caudatum. J. Protozool. 17, 24-35. INADA, K., AND MIYAZAWA, F. (1960). Photodynamic action of food dye on bacteria. J. Food Hyg. Sot. 10,344-347. JOAN, S. S. (1974). Age-correlated effects of caffeine on nonirradiated and UV-irradiated Paramecium aarefia. J. Gerontol. 29,256-260. KOKIA, I. Y. (1971). Sensitivity of Paramecium caudatum to the action of lethal concentrations of sodium 2,4-D. Eksp. Vod. Toksikol. 2,4348. ORLOWSKI, M., AND MEISTER, A. (1965). Isolation of y-glutamyl transpeptidase from hog kidney. J. Biol. Chem. 240,338-347. ORLOWSKI, M., AND MEISTER, A. (1970). The gamma-glutamyl cycle: A possible transport system for amino acid. Proc. Nat. Acad. Sci. USA 67, 1248-1255. REISS, J. (1971). Der einflul3 von Aflatoxin B, auf Paramecium caudatum und Paramecium bursaria. Arch. Hyg. 154, 533-536. TAKAKUWA, E., SAITO, K., KAMORI, K., YATAKE, M., HONMA, H., AND SAITO, M. (1971). Studies on the determination of drug toxicity. Jap. J. Pub. Health. 18,459-461. TANAKA, K., AND OKAHARA, K. (1973). Effects of food additives on alimentary tract. J. Food Hyg. Sot. 14,234-238. TANIGUCHI, N., SAITO, K., AND TAKAKUWA, E. (1975). y-Glutamyl transferase from azo dye induced hepatoma and fetal rat liver. Biochim. Biophys. Acta 391,265-271. TUPPY, H., WIESBAUER, V., AND WINTERSBERGER, E. (1962). Amino acid-p-nitroanilide as a substrate for aminopeptidases and other proteolytic enzymes. Z. Physiol. Chem. 329, 278288. YAMAMOTO, S., ITO, Y., AND NODA, S. (1967). Toxicity test of food additives by the disturbance of life cycles of Chlorella and Saccharomyces cerevisiae var. sake. J. Food Hyg. Sot. 8, 396407. ALVAREZ,