Fd Chem. Toxic. Vol. 20, pp, 461 to 465. 1982 0278-6915/82/040461.05~t03.00[0 Printed in Great Britain, All rights reserved Copyright O 1982 Pergamon Press Ltd

TARTRAZINE-INDUCED CHROMOSOMAL ABERRATIONS IN MAMMALIAN CELLS

R. M. PATTERSON and J. S. BUTLER Department of Biology, Atlanta University, Atlanta, GA 30314, USA

(Received 12 January 1982)

Abstract--Tartrazine (FD & C Yellow No.5) has been shown to induce chromosomal aberrations in fibroblast cells of Muntiacus ntuntjac in vitro. M. muntjac cells were exposed to various concentrations of tartrazine (in the 5-20/ag/ml range) and were evaluated for induced chromosomal aberrations after two different periods of culture. Total percentages of chromosomal aberrations were significantly increased above control levels in all experimental groups. The results suggest that further studies are needed to determine the potential mutagenic effects of tartrazine.

Introduction

Tartrazine (FD & C Yellow No.5) is a pyra- zoleaniline dye (5-oxo-l-(p-sulphophenyl)-4-(sulpho- phenylazo)-2-pyrazoline-3-carboxylic acid, trisodium salt), which is used to colour candy, soft drinks, drug capsules, hand lotions and many other foods, drugs and cosmetics. Rats fed tartrazine for prolonged periods did not reveal any evidence of toxic reactions (Mannell, Grice, Lu & Allmark, 1958). However, Ershoff (1977) reported that rats fed a 5~',; level of tartrazine in a purified diet showed marked retard- ation of growth and a 50To death rate during the 14-day experimental period. Tartrazine has been shown to induce allergic reactions in specific human populations (Neuman, Elian, Nahum et al. 1978; Ste- nius & Lemola, 1976). One third of an allergic popu- lation studied was shown to be clinically affected by tartrazine. A correlation has been demonstrated between tartrazine sensitivity and aspirin intolerance (Samter & Beers, 1968; Stenius & Lemola, 1976), up to 44~//, of aspirin-sensitive patients having been shown to react adversely to tartrazine. The apparent association between tartrazine sensitivity and aspirin intolerance is not yet understood,

In the investigations reported here, a study has been made of the effects of tartrazine on the chromo- somes of Muntiacus m,ntjac as reflected in induced percentage increases in chromosomal aberrations.

Experimental

Male M. muntjac fibroblast cells (American Type Culture Collection, Rockville, MD) were grown in Eagle's Basal Medium supplemented with 10To foetal calf serum and 100 units kanamycin/ml (GIBCO, Grand Island, NY). The fibroblast cells were treated with 3, 5, 10 or 20tlg tartrazine/ml (Warner-Jenkin- son Co., St Louis, MO) for 48 hr in short-term treat- ment studies. In a second series of investigations, fibroblast cells were exposed to 5 or 10/ag tartra- zine/ml for 3 days and were then cultured for an ad- ditional 13 days (a total of 16 days in culture).

After treatment with one of the specific concen- trations of tartrazine, the experimental fibroblast cells,

together with the control cells, were prepared for cytogenetic analysis as previously described (Patter- son & Petricciani, 1973). Briefly, the cells in culture were treated with 0'025 or 0'03 pg colcemld/ml for 15hr. The cells were then harvested, treated with hypotonic medium without serum, fixed in acetic acid-methanol (1:3, v/v) and stained in dilute Giemsa. Aberration counts were made on at least 100 recta- phase cells with well-spread chromosomes.

Results

M. muntjac fibroblasts treated with 3pg tartra- zine/ml for 48 hr yielded a 30~ incidence of aberra- tions, compared with 6-03~'0 in the control group (Table 1). In this experimental group, 15To of chromo- somes exhibited chromatid breaks (Fig. 1), and there were more chromatid gaps and dicentric chromo- somes than in control cells. With increasing concen- tration of tartrazine for the 48-hr treatment, there was a significant increase in chromosome damage; cells treated with 5#g tartrazine/ml showed a 46To inci- dence of aberrant chromosomes, and compared to the controls (Table 1) this group exhibited a high fre- quency of dicentric chromosomes (Fig. 2).

When the tartrazine concentration was increased to 10 ug/ml for 48 hr, there was an increase (to 53.45~) in the percentage of aberrant chromosomes, as well as an increase in the type of aberrations observed (Table 11. The highest percentage of aberrations occurred as breaks of chromosome No. 3 with and without displacement. Ring chromosomes (Fig. 3) were observed at a low frequency. Cells treated with 20/~g tartrazine/ml for 48 hr exhibited a 33"6~ inci- dence of chromosomal aberrations (Table 1). In this experimental group, 18-8To of the chromosomes were dicentrics; other aberrations included chromatid breaks (7.9~) and chromatid gaps (6.9~).

In M. muntjac fibroblast cells treated with 5 ltg tar- trazine/ml for 72 hr and allowed to remain in culture for an additional 13 days (a total of 16 days), 48~ of the chromosomes showed aberrations (Table 2). Chromatid separation was the most frequent type of aberration; breaks and gaps were also observed. Cells

461

462 R. M. PATTERSON and J. S. BUTLER

Table I. Incidence of chromosomal aberrations in Muntiacus muntjac cells exposed.for 48 hr to tartra- :ine in concentrations of 3-20 l~g/ml

Aberration

Incidence of aberration (o/~]

Experiment 1 Experiment 2 Tartrazine concn

(~ug/ml).., 0* 3 5 0* 10 20 No. of metaphases

examined ... 116 200 103 128 152 101

Chromatid breaks 0 15.0 10.6 0 19.7 7.9 Chromatid gaps 1.72 8.0 9.3 1.5 6.8 6.9 Dicentrics 0 5"0 16.0 0 18.4 18"8 Fusions 0 0 3.3 0 0 0 Rings 0 0 0 0 0.66 0 Polyploids 0 0 0 0 7.89 0 Chromatid separations 4.31 2.0 6.8 2.3 0 0

Total aberrations (°,) . . . 6.03 30'0 46.0 3-8 53.45 33"6

*Control values.

treated with 10 l 'g tartrazine/ml for the same culture times (Table 2) exhibited a 25°4, incidence of aberra- tions, including endoreduplications, breaks, gaps, dicentrics and chromatid separations.

Discussion

These investigations indicated that M. ,utntjac fibroblast cells exposed to 3, 5, l0 or 20/.tg tartra- zine/ml showed significant increases in chromosomal aberrations. The experimental cultures exposed to 3 pg/ml exhibited a significant increase in the percent- age of aberrations compared to the control group. With a tartrazine exposure of 5 ltg/ml for 48 hr, there was a corresponding increase in chromosome damage above that with the 3-1tg/ml exposure. Similar results were obtained with 10 Fg/ml, but in the 20-1~g/ml cul- tures the mitotic index was considerably lower than those for 3, 5 and 10/.tg tartrazine/ml, respectively. Lewis, Patterson & McBay (1981) also noted a de- crease in chromosomal-aberration percentages when M. muntjac fibroblast cells were exposed to the xan- thine dye Rhodamine B (D & C Red No. 19) at a level of 20/tg/ml. Although chromosomal aberration

studies with tartrazine have not previously been reported, Yahagi, Degawa, Seino et al. (1975) found several azo dyes and their derivatives to be mutage- nic/carcinogenic and suggested that these effects may involve modification of the DNA.

Cells exposed to 5 Fg tartrazine/ml for 3 days and allowed to remain in culture for an additional 13 days did not show a significant decrease in total chromoso- mal aberrations, but did show a significant decrease in breaks, gaps and dicentrics, compared to the short- term studies. Chromosomal structural changes that are due to lesions (breaks and/or gaps) are disconti- nuities that can occur as a result of chemical action (Evans, 1962). The vast majority (90-99.7°,,) of these lesions in a chromosome are rejoined in the original order by repair processes, and the chromosomes reveal no visible structural changes. In the 16-day study with 5/ag tartrazine/ml, a high percentage of chromatid separation accounted for most of the aber- rations observed. Cells treated with 10Fg tartrazi- ne/ml for 3 days showed a lower percentage of aberra- tions than did the 5-,ag/ml cultures. Although in this study, 10-~g tartrazine/ml did not increase chromoso- mal aberrations compared with the 5-/ag/ml exposure. as was the case in the short-term studies, it did induce

Table 2. Incidence of cllromosonxal aberrations in Muntiacus muntjac cells exposed.lor 3 days to tartra:ine in a concentration of 5 or 10 jtg/ml and then

cultured.lor a .lio'ther 13 days

Aberration

Incidence of aberration (%)

Tartrazine conch (#g/ml)... 0 5 10

No. of metaphases examined... 137 100 40

Chromatid breaks Chromatid gaps Dicentrics Chromatid separations Endoreduplications

Total aberrations (~'0)...

0 4.0 I0'0 0.7 5'0 2.5 0 0 7"5 2'9 39"0 2.5 0 0 2-5 3"6 48"0 25'0

Fig. 1. Chromatid break (arrowed) in chromosomes of a M. muntjac cell exposed to 3 ug tartrazine/ml for 48 hr. Giemsa x 1200.

k

Q Fig. 2. Dicentric chromosomes of M. muntjac cell exposed to 5 ug tartrazine/ml for 48 hr. Giemsa × 1200.

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Fig. 3. Ring chromosomes (arrowed)of M. muntjac cell exposed to 10.ug tartrazine/ml for 48hr. Giemsa x I200.

464

Cytogenetic effects of tartrazine 465

endoreduplicat ion, an aberrat ion not observed in the other experimental cultures.

Acknowledgements--This research was supported by NIH Grant RR 08806 and NSF Grant RIM 78-16355.

REFERENCES

Ershoff, B. H. (1977). Effects of diet on growth and survival of rats fed toxic levels of tartrazine (FD&C Yellow No. 5) and Sunset Yellow FCF (FD&C Yellow No. 6). J. Nutr. 107, 822.

Evans. H. J. (1962). Chromosome aberrations induced by ionizing radiations. Int. Rer. Cytol. 13, 221.

Lewis, I. L., Patterson. R. M. & McBay, H. C. 11981). The effects of Rhodamine B on the chromosomes of Mun- tiacus mun(jac. Mutation Res. 88, 21 I.

Mannell, W. A., Grice, H. C., Lu, F. C. & Allmark, M. G. (1958). Chronic toxicity studies on food colours. Part IV. Observations on the toxicity of tartrazine, amaranth and sunset yellow in rats. J. Pharm. Pharmac. I0, 625.

Neuman, l., Elian, R., Nahum, H., Shaked, P. & Creter, D. (1978). The danger of 'yellow dyes' (tartrazine) to allergic subjects. Clin. Allergy 8, 65.

Patterson, R. M. & Petricciani, J. C. (1973). A comparison of prophase and metaphase G-bands in the muntjac. J. Hered. 64, 80.

Samter, M. & Beers, R. F., Jr (1968). Intolerance to aspirin. Clinical studies and consideration of its pathogenesis. Ann. intern. Med. 68, 975.

Stenius, B. S. M. & Lemola, M. (1976). Hypersensitivity to acetylsalicylic acid (ASA) and tartrazine in patients with asthma. Clin. Alleryy 6, 119.

Yahagi, T., Degawa, M., Seino, Y., Matsushima, T., Nagao, M.. Sugimura T. & Hashimoto, Y. (1975). Mutagenicity of carcinogenic azo dyes and their derivatives. Cancer Lett. I, 91.