Cancer Letters, (1986) 299-303 299

Scientific Publishers Ltd.

SISTER CHROMATID EXCHANGE INDUCED BY METANIL YELLOW

AND NITRITE SINGLY AND IN COMBINATION IN VIVO ON MICE

ASHOK KUMAR GIRI, GEETA TALUKDER and ARCHANA SHARMA

Centre for Advanced Study in Cell and Chromosome Research, Department of Botany,
University of Calcutta, 35, Ballygunge Circular Road, Calcutta-700 019 (India)

(Received 25 September 1985)

(Revised version received 12 March 1986)
(Accepted 25 March 1986)

SUMMARY

In vivo sister chromatid exchanges (SCEs) induced by metanil yellow (a

dye containing secondary amino group), sodium nitrite, and dye in combi-
nation with nitrite following treatment with acute doses were studied on mice.
The incidence of SCEs was significantly high in both dye- and nitrite-treated
series. However, a combination of half the concentrations of dye and nitrite,
when used together, gave a frequency of SCE higher than that of either
chemical, when given in full dose, indicating the stronger clastogenicity of
the nitrosamine formed.

INTRODUCTION

The N-nitrosoderivatives of secondary amines are known to be highly

carcinogenic [4,11,12] and are produced in the human stomach by acid
catalysed reaction between nitrites present in food [ 13-161. Both nitrate
and nitrite may be synthesized in the body from nitrogenous components
of the diet [8,18] and human saliva also contains a significant amount of
nitrite [20]. Several vegetables also concentrate nitrates [l]. Interaction
between certain dyes and nitrite in the laboratory has shown the presence of
nitrosamines [ 21. Apparently, the use of dyes having nitrosable groups may
lead to the production of nitrosamines in the nitrite- or nitrate-containing
food or in the stomach itself. Metanil yellow, not permissible as a dye, is
yet often used to colour sweets and soft drinks in India [9]. It contains a
secondary amino group.
The clastogenic and mutagenic effects of this dye have not been worked
out in detail [3], though it has been shown to induce chromosomal ab-
normalities in bone marrow cells of mice [ 5,6] . In the present investigation,

0304-3835/86/$03.50 0 1986 Elsevier Scientific Publishers Ireland Ltd

Published and Printed in Ireland
300

marrow chromosomes

albino male mice (Mus musculus)

90-100 days old were used for this experiment.
housed in different

tablet of 5-bromodeoxyuridine
(BrDU) [lo].
Metanii yellow and sodium nitrite were dissolved in distilled water and were
given separately as a single i.p. injection to 2 sets of 7 mice each at the rate
of 2.5, 5, 10, 20, 40, 100 and 200 mg/kg body wt immediately after tablet
implantation. Similarly metanil yellow and sodium nitrite were given simul-
taneously (equal amounts) as a single i.p. injection to 7 mice each at the rate
of 2.5, 5,10, 20, 40, 100 and 200 mg/kg body wt. For controls, a single i.p.
injection of 0.05 ml distilled water was given to each of 7 animals in each
of 3 sets immediately after subcutaneous implantation of BrDU tablets. All
mice received an i.p. injection of colchicine (5 mg/kg) 21-22 h after sub-
cutaneous implantation of BrDU. Two hours later the animals were killed by
cervical dislocation and bone marrow chromosomes were prepared following
the method of King et al. [lo].
Differential staining of the sister chromatids was carried out by a modifi-
cation of the fluorescence-plus-Giemsa (FPG) technique [ 171. Slides, aged
for 4-5 days, were stained for 10 min in Hoechst 33258 dissolved in NaCl/
KC1 solution; rinsed and mounted with M/15 Sorensens phosphate buffer
(pH 6.8). Then slides were irradiated with a 254-nm UV mineralogic lamp
for 30 min. Slides were incubated in BXSSC (0.3 M NaCl, 0.03 M trisodium
citrate) solution for 90 min at 59C immediately after irradiation. Slides
were then rinsed thoroughly in distilled water and stained with 7% phosphate-
buffer-Giemsa solution for 10 min, then mounted as usual. About 150
metaphase cells from 7 animals in each concentration in all sets were scanned
for SCEs. Due to the scarcity of exchange metaphase cells in 200 mg/kg
treated series only 100 plates were scanned in this concentration in all sets.

RESULTS AND DISCUSSION

Figure 1 shows the results on the induction of SCEs exposed to metanil

yellow, sodium nitrite and metanil yellow in combination with sodium
nitrite for 24 h. Marked increase in the frequency of SCEs was observed in
all the treated series except 2.5 mg/kg cont. when compared with distilled
water controls. There was a significant increase in SCEs in dye with nitrite
treated series except a few concentrations when compared with dye and
nitrite treated series individually. There were no significant differences
 301

16
q Metani I yellow
q Sodium nitrite
1 m Metoni\ yel\ow p\us sodium nitrite
1L 1 Standard deviation
1

T
zz
12-
+
*

:
lo-
T 1
:
CL
Lu
a-
=:

0
ki 6-
J3 T

0 2.5 5 10 20 LO 100 200

Concentration (mg /kg body weight j
Fig. 1. In vivo SCE induced by metanil yellow and nitrite singly and in combination on
bone marrow cells of mice. *P < 0.02 significant in comparison to nitrite alone, **P <
0.001 significant in comparison to both dye and nitrite aione.

between dye, nitrite, and dye plus nitrite treated series in the concentration
2.5 and 200 mg/kg treated series. When given the highest concentration, i.e.
200 mg/kg, 2 mice died in each of the dye and dye plus nitrite groups and
one in the nitrite group. The number of exchange metaphase plates were
very few in all 3 sets using this concentration and hence, only 100 metaphase
plates could be scanned. This concentration was apparently too toxic for the
cells.
The overall data indicate an additive effect when dye is given in com-
bination with nitrite. It is also interesting to note that this effect could not
be observed with the initial and last 2 concentrations. Although the highest
concentration was too toxic, yet the relatively higher doses showed a reduc-
tion of this additive effect. The maximum additive effect was with the 10
mg/kg dose, above and below which the effects were decreased (see Fig. 1).
In these experiments, with all 3 treatments, there is a progressive increase in
the SCE until a maximum is reached after which the effect declines. It is
possible that after the maximum effect, lethality ensues, decreasing the
302

number of viable exchange metaphase plates. The synergistic effects of

certain dyes in combination with nitrite have been worked out in vivo on
bone marrow cells of mice [7]. In the present study it appears that metanil
yellow, which showed a high level of cytotoxicity [7] and clastogenicity in
human leucocyte cultures [ 191, has also a strong effect on the induction of
SCEs when used in combination with nitrite. A higher incidence of SCEs
induced by metanil yellow in combination with nitrite may be due to the
simple chemical structure of nitrosamine formed from it, which can easily
penetrate through cell membranes. So the present investigation shows a
possible carcinogenic risk to human beings exposed to high amounts of dye
and nitrite through various sources.

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