CARYOLOGIA Vol. 60, no.

4: 309-314, 2007

Impact of Food Additives on Mitotic Chromosomes of Vicia

faba L.
Pandey* Ram Milan and Santosh Upadhyay
Cytogenetics Laboratory, National Botanical Research Institute, Lucknow-226001 (India; Ph. 0522-2205831-35/
extn. 307, Fax. 0522-2205836.

Abstract — Present study was conducted to study the impact of food additives like boric acid, citric acid and
sodium metabisulphite individually, in different concentrations, on root tips of Vicia faba L. Cytological studies
revealed significant decrease in mitotic index with an increase in concentration of the food additives. Most frequent
cytological abnormalities observed are fragments, disturbed metaphase, C-mitosis, laggards, bridges, stickiness,
precocious movement of chromosomes, unequal and late separation of chromosomes. Bridges and fragments were
more frequent at anaphase. The percentage of chromosomal aberrations at mitosis increased with an increase in
concentration of the food additives.
Key words: chromosome aberration, food additives, genotoxicity, mitotic index, Vicia faba.

INTRODUCTION medicinal and nonmedicinal purposes (HEINDEL

In this era of increasing world population, it
is necessary to investigate new food sources and
preserve them for a long period without affect-
ing their quality. Several methods have been in-
vestigated for food storage and many chemical
substances are being used for food preservation
as antimicrobial agent. These antimicrobial agents
were reported as mitotoxic and genotoxic in sev-
eral test systems (NAGAO et al. 1977; ISHIDATE et
al. 1977; MATSUOKA et al. 1979; LUCA et al. 1987;
MUKHERJEE et al. 1988). Sodium metabisulphite
induced mitotic abnormalities and decreased the
mitotic index in Allium cepa (RENCUZOGULLARI et
al. 2001a) and also induced the chromosomal ab-
errations in cultured human lymphocytes (RENCU-
ZOGULLARI et al. 2001b). The Cytogenetic effects
of food additives sodium benzoate and sodium
sulphite, which inhibit the DNA synthesis and
induce the formation of anaphase bridges, prema-
ture chromosome condensation and chromatin
erosion in root meristems of V. faba,was reported
by NJAGI and GOPALAN (1982).
Boric acid is used as a food preservative (4gm/
lit) in food product like caviar (The Ministry of
Agriculture of Turkey 1997) and used for both
* Corresponding author: e-mail: rmp12@rediffmail.com
et al. 1997), while citric acid is used as a strong
acid, flavoring and chelating agents in ice cream,
sherbet, fruit drink, candy, carbonated beverages,
instant potatoes and other foods. The use of so-
dium metabisulphite has been reported in typical
products as beer, soft drinks, dried fruit, juices,
cordials, wine, vinegar and potato products. Pres-
ently no published data is reported on the effect
of citric acid as well as comparative effects of bo-
ric acid, citric acid and sodium metabisulphite on
mitosis. Vicia faba is an excellent model for the
assay of chromosomal aberration after chemi-
cal treatment (MA 1982). Therefore, the present
study has been undertaken to investigate the im-
pact of food additives i.e. boric acid, citric acid
and sodium metabisulphite on somatic cells of V.
faba L.
MATERIAL AND METHODS
Several concentrations i.e. 1, 2 and 4gm/l of
food additives namely boric acid, citric acid and
sodium metabisulphite solutions were prepared in
distilled water. Seeds were directly placed in test
solutions and in distilled water as control. The
seeds were treated with test solution for twelve
hours and then allowed a recovery period for two
hours in distilled water. The treated seeds were
placed on wet filter paper in petridishes and left
310 pandey and upadhyay

Table 1 — Effect of food additives on mitotic index in Vicia faba L.

Number of cells Number of actively
Treatments Concentration (gm/l) Mitotic Index (MI) %
observed dividing cells
Control 1418 304 21.44
Boric acid 1 1571* 335** 21.32**
2 1304* 227** 17.41**
4 1478* 164** 11.10**
1451 + 78.34 242 + 49.99 16.61 + 2.97

Citric acid 1 1652* 353** 21.37**

2 1647* 296** 17.97**
4 1460* 173** 11.85**
1586.33 + 63.25 274 + 53.17 17.06 + 2.78

Sodium meta 1576* 337** 21.38**

bisulphite 1 1506* 271** 17.99**
2 1492* 182** 12.20**
4 1524.66 + 26.01 263.33 + 44.96 17.19 + 2.68

Mean 1520.66 + 511.30 259.77 + 25.16 16.95 + 1.41

CD at:
5% 1179.05 58.02 3.25
1% 1715.41 84.41 4.73

*, ** significant at 5% and 1% level of probability, respectively

in dark at 22-25oC. Root tips were obtained after
germination. Both experimental and control root
meristems were excised from the seeds and fixed
in acetic alcohol solution (1 acetic acid: 3 alco-
hol). For each variable, ten root tip squashes were
prepared and minimum 150 mitotic cells were
counted from each slide. Several chromosome ab-
errations scored were fragments, disturbed met-
aphase, C- mitosis, laggards, chromatid bridge,
stickiness, precocious movement, unequal sepa-
ration and late separation (Table 4). The micro-
photographs of desired cells were taken using Tri-
nacular microscope attached with PM-6 camera
and the chromosome were enlarged X = 500.
tion of food additives in both the stages in actively
dividing cells (Table 3). Several chromosomal ab-
normalities viz. fragments, disturbed metaphase,
C- mitosis, laggards, chromatid bridge, sticki-
ness, precocious movement, unequal separation
and late separation were observed (Figs. 1-9), in
which C-mitosis was more frequent. Disturbed
metaphase, precocious movement, laggards and
unequal separation were also found in high per-
centage, but fragments, chromatid bridge, sticki-
ness and late separation were in lower percentage
(Table 4). The most interesting investigation in all
results was the significant increase in abnormality
percentage, which is considered important for the
use of food additives.

RESULTS
Mitotic index significantly decreased with in-
creasing concentration of food additives, but bo-
ric acid was more effective at higher concentration
(4gm/l) in comparison to citric acid and sodium
metabisulphite (Table 1). Significant increase in
abnormality percentage was also observed with
increasing concentration of food additives, in
which boric acid was more prevalent (Table 2). A
significant increase in metaphase percentage and
abnormality percentage and decrease in anaphase
percentage was noticed with increasing concentra-
DISCUSSION
The impact of food additives like boric acid,
citric acid and sodium metabisulphite has been
studied on somatic cells of Vicia faba L. and a sig-
nificant decrease in mitotic index and an increase
in the abnormality percentage with increasing
concentrations were noticed. RENCUZOGULLARI et
al. (2001 a; b) have reported decrease in mitotic
index in Allium cepa and human lymphocytes, due
to the effect of sodium metabisulphite. Its effect
on human lymphocytes was also investigated by
impact of food additives on mitotic chromosomes of vicia faba l. 311

Table 2 — Effect of food additives on abnormality (%) in mitotic cells of Vicia faba L.
Number of Number of ab- Abnormality (%) Abnormality
Concentration Number of
Treatments actively normally dividing in actively (%) in observed
(gm/l) observed cells
dividing cells cells dividing cells cells
Control 1418 304 - - -
Boric acid 1 1571* 335** 9 2.69 0.57
2 1304* 227** 50** 22.03** 3.83**
4 1478* 164** 70** 42.68** 4.74**
1451 + 78.34 242 + 49.99 43 + 31.09 22.46 + 11.55 3.04 + 1.26

Citric acid 1 1652* 353** 9 2.55 0.54

2 1647* 296** 61** 20.61** 3.70**
4 1460* 173** 73** 42.20** 5.00**
1586.33 + 63.25 274 + 53.17 47.66 + 19.66 21.78 + 11.47 3.08 + 1.32

Sodium 1 1576* 337** 9 2.67 0.57

meta 2 1506* 271** 54** 19.93** 3.59**
bisulphate 4 1492* 182** 76** 41.76** 5.09**
1524.66 + 26.01 263.33 + 44.96 46.33 + 19.74 21.45 + 11.32 3.08 + 1.33

Mean 1520.66 + 511.30 259.77 + 25.16 45.66 + 9.58 21.90 + 5.72 3.07 + 0.65
CD at:
5% 1179.05 58.02 22.09 13.19 1.49
1% 1715.41 84.41 32.14 19.19 2.18

Table 3 — Effect of food additives on metaphase (%) and anaphase (%) in mitotic cells of Vicia faba L.
Number Number of Metaphase (%) in actively dividing cells Anaphase (%) in actively dividing cells
Concentra-
Treatments of observed actively dividing
tion gm/l) Normal Abnormal Total Normal Abnormal Total
cells cells
Control 1418 304 54.16 - 54.16 45.84 - 45.84
Boric acid 1 1571 335 54.62 1.62 56.24 42.69* 1.07 43.76
2 1304 227 51.06 11.05 62.11 26.91 10.98 37.89
4 1478 164 46.42 23.46** 69.88 10.90 19.22** 30.12
1451 + 78.34 242 + 49.98 50.7 + 2.37 12.04 + 6.33 62.74 + 1.73 26.83 + 34.25 10.42 + 5.25 37.25 + 3.95

Citric acid 1 1652* 353 53.44 1.52 54.96 44.01* 1.03 45.04*
2 1647* 296 48.74 11.52 60.26 30.66 9.10 39.76
4 1460 173 44.70 23.22** 67.92 13.11 18.97** 32.08
1586.33 + 63.25 274 + 53.17 48.96 + 2.52 12.08 + 6.27 61.04 + 3.76 29.26 + 8.95 9.7 + 5.19 38.96 + 3.76

Sodium meta 1 1576 337 54.11 1.37 55.48 43.22* 1.30 44.52*
bisulphite 2 1506 271 50.27 10.86 61.13 29.80 9.07 38.87
4 1492 182 46.81 23.27** 70.08 11.43 18.49** 29.92
1524.66 + 26.01 263.33 + 44.96 50.39 + 3.70 11.83 + 0.05 62.23 + 44.14 28.15 + 9.22 9.62 + 4.97 37.77 + 4.25

Mean 1520.66 + 761.08 259.77 + 451.36 50.01+ 30.71 11.98 + 5.99 62.00 + 31.00 28.08 + 14.05 9.91 +4.96 37.99 + 19.00
CD at:
5% 1755.05 1040.83 70.81 13.81 71.48 32.39 11.44 43.81
1% 2553.42 1514.31 103.03 20.09 104.00 47.14 16.64 63.74

MENG and ZHANG (1992). Several food preserva-
tives viz. sodium benzoate and sodium sulphite
inhibit the DNA synthesis as reported by NJAGI
and GOPALAN (1982) in V. faba root meristems.
The inhibition of DNA synthesis and decrease in
mitotic index might be caused due to decrease in
ATP level and the pressure from the functioning
of energy producing centre (EPEL 1963; JAIN and
ANDSORBHOY 1988). It was interesting to note that
food additives i.e. boric acid, citric acid and so-
dium metabisulphite had increased the percent-
age of metaphase in actively dividing cells with in-
creasing concentrations, while gesagard increased
the percentage of prophase in barley (TOPAKTAS
312 pandey and upadhyay

Figs. 1-9 — Showing cytological effects of food additives in Vicia faba L., 1. Normal metaphase, 2. Normal ana-
phase, 3. Disturbed metaphase, 4. C-mitosis, 5. Late separation at anaphase, 6. Unequal separation, 7. Fragmenta-
tion at anaphase, 8. Chromatid bridge, 9. Stickiness and Laggard at anaphase.

et al. 1991). It was also reported in V. faba and
A. cepa with aventox by BADR (1983), in A. cepa
with garlon by EL-KHODARY et al. (1989) and with
sodium metabisulphite by RENCUZOGULLARI et al.
(2001a). It is evident from the present study that
increasing concentrations of food additives de-
crease the mitotic index.
A strong genotoxicity was also reported by
food additives, which increased the percentage of
chromosomal abnormalities with increasing con-
centrations and also produced strong chromotox-
ic effects in stored solution as compared to fresh
solution (MUNZER et. al. 1990). Boric acid showed
synergistic effect in the induction of mutagenecity
when used in mixture with gamma rays and fast
neutron (KINASHI et al. 1997). Boric acid, citric ac-
id and sodium metabisulphite induced mitotic ab-
normalities and decreased the mitotic index in the
present case and the most frequent abnormality
was C-mitosis, while disturbed metaphase, preco-
cious movement, laggards and unequal separation
of chromosomes (Fig.s 1-9) were also observed
in high percentage. fragments, bridges, sticki-
ness and late separation were in lower percentage,
which suggests an eugenic effect of food additives
by inhibiting spindle formation, causes C-mitosis
impact of food additives on mitotic chromosomes of vicia faba l. 313

Table 4 — Effect of food additives on chromosomal aberrations (%) in mitotic cells of Vicia faba L.
Metaphase abnormalities (%) in actively
Anaphase abnormalities (%) in actively dividing cells
dividing cells
Concen-
Treat-
tration Precocious Late Unequal
ments
(gm/l) Disturbed movement C- Stickiness Fragments Chromatid separation separation
Total Laggards Total
metaphase of chromo- mitosis bridges of chromo- of chromo-
somes somes somes
Control - - - - - - - - - - -
Boric acid 1 0.53 0.47 0.62 1.62 0.47 - 0.43 - - 0.11 1.07
2 3.80 3.02** 4.23 11.05 2.12 1.20 1.23 1.03 1.20 2.12 10.98
4 6.67** 6.33** 10.46** 23.46** 4.48* 2.35* 3.36** 2.43 2.41* 4.21** 19.22**
3.66+1.77 3.27+1.69 5.10+5.88 12.04+15.65 2.35+2.34 1.77+0.57 1.67+0.87 1.73+0.70 1.80+0.6 2.14+1.18 10.42+5.24

Citric acid 1 0.52 0.46 0.54 1.52 0.46 - 0.41 - - 0.16 1.03
2 3.99 2.99** 4.52 11.52 2.07 1.13 1.37 1.14 1.33 2.06 9.10
4 6.50** 6.50** 10.22** 23.22** 4.20* 2.39* 3.08** 3.02* 2.20* 4.08** 18.97**
3.67+1.73 3.31+3.07 5.09+2.81 12.08+10.61 2.24+1.02 1.76+0.63 1.62+0.78 2.08+1.06 1.76+0.43 2.1+1.12 9.7+5.19

Sodium- 1 0.43 0.39 0.55 1.37 0.48 - 0.42 - - 0.40 1.30

meta- 2 3.82 3.18** 3.86 10.86 2.05 1.12 1.14 1.33 1.36 2.07 9.07
bisulphite 4 6.59** 6.41** 10.27** 23.27** 4.12* 2.20* 2.24* 3.43* 2.47* 4.03** 18.49**

3.61+1.78 3.32+1.73 4.89+2.85 11.83+14.51 2.21+1.89 1.66+0.54 1.26+0.52 2.38+1.05 1.91+0.55 2.16+1.05 9.53+9.09

Mean 3.65+1.82 3.30+0.86 5.03+2.51 11.98+5.99 2.27+1.39 1.73+0.77 1.52+0.76 2.06+1.31 1.82+0.81 2.13+1.06 9.88+4.94
CD at:
5% 4.19 1.98 5.78 13.81 3.20 1.77 1.75 3.02 1.86 2.44 11.39
1% 6.10 2.88 8.42 20.09 4.66 2.58 2.56 4.39 2.71 3.55 16.57

*, ** significant at 5% and 1% levels, respectively

CD = Critical difference ; *, ** – significant at 5% and 1% levels respectively

and also effects the chromosomal protein which
induces stickiness.
It was concluded from the present investiga-
tion that the food additives used in the experiment
showed mitotoxicity and genotoxicity. Hence, this
would yield detailed information on the relevance
of genotoxic effects of food additives and their
potential risks for human health.
Acknowledgement — The authors are thankful to
Director, N. B. R. I., Lucknow for providing the neces-
sary facilities to carryout the present investigation.
REFERENCE
BADR A., 1983 — Mitodepressive and chromotoxic ac-
tivities of two herbicides in A. Cepa. Cytologia, 48:
451-457.
EL-KHODARY S., HABIB A. and HALIEM A., 1989 — Cy-
tological effect of herbicide garlon-4 on root mitosis
of A. cepa. Cytologia, 54: 465-472.
EPEL D., 1963 — The effects of carbon mono oxide in-
hibition of ATP level and the date of mitosis in Sea
Urchin egg. J. Cell. Biol., 17: 315-319.
HEINDEL J., FAIL P., GEORGE J. and GRIZZLE T., 1997
— Reproduction toxicology of boric acid. Environ.
Health Perspect., 105: 275-276.
ISHIDATE M.JR. and ODASHIMA S., 1977 — Chromosome
test with 134 compounds of Chinese hamster cells
in vitro-screening for chemical carcinogens. Mutat.
Res., 48: 337-353.
JAIN A.K. and ANDSORBHOY R.K., 1988 — Cytogenetical
studies on the effects of some chlorinated pesticide
III. Concluding Remarks. Cytologia, 53: 427-436.
KINASHI Y., MASUNAGA S., TAKAGAKI M. and ONO K.,
1997 — Mutagenic effects at HPGRT locus induced
in Chinese hamster Ovary cells by thermal neutron
with or without boron compound. Mutat. Res., 377:
211-215.
LUCA D., LUCA V., COTOR F. and RAILEANU L., 1987
— In vivo and in vitro Cytogenetic damage induced
by sodium nitrite. Mutat. Res., 189: 333-339.
MA T.H., 1982 — Vicia faba Cytogenetic test for envi-
ronmental mutagens. A report of the US environ-
mental protection agenc. Gene-tox Program, Mut.,
USA.
MATSUOKA A., HAYASHI M. and ISHIDATE M.JR., 1979
— Chromosomal aberration test on 29 chemicals
combined with S9 mix in vitro. Mutat. Res., 66: 277-
290.
314
MENG Z. and ZHANG L., 1992 — Cytogenetic damage
induced in human lymphocytes by sodium bi sul-
phite. Mutat. Res., 298: 63-69.
MUKHARJEE A., GIRI A.K., TALUKDER G. and SHARMA
A., 1988 — Sister chromatid exchange and micronu-
clei formation induced by sorbic acid and sorbic acid-
nitrite in vivo in mice. Toxicol. Let., 42: 47-53.
MUNZER M., GUIGAS C. and RENNER H.W., 1990 — Re-
examination of potassium sorbate and sodium sorb-
ate for possible genotoxic potential. Food. Chem.
Toxicol., 28: 397-401.
NAGAO M., HONDA M., SEINO Y., YAHAGI T., and KA-
WACHI T., 1977 — Mutagenecity of protein pyrolates.
Cancer Let., 2: 335-339.
NJAGI G.D. and GOPALAN H.N., 1982 — Cytogenec ef-
fects of the food preservatives- sodium benzoate and
sodium sulphite on Vicia faba root meristems. Mutat.
Res., 102: 213-219
pandey and upadhyay
RENCUZOGULLARI E., KAYRALDIZ A., Ila H.B., CAKMAC
T. and TOPAKTAS M., 2001a — The Cytogenetic ef-
fect of sodium metabisulphite, a food preservative in
root tip cells of Allium cepa L. Turkish J. Biol., 25:
361-371.
RENCUZOGULLARI E., ILA H.B., KAYRALDIZ A. and TOPA-
KTAS M., 2001b -Chromosome aberrations and sister
chromatid exchange in cultured human lymphocytes
treated with sodium metabisulphite, a food preserva-
tive. Mutat. Res., 490: 107-112.
THE MINISTRY OF AGRICULTURE OF TURKEY., 1997 —
Food Codex Instructions. Dunya Publication. 197
pp.
TOPAKTAS M. and RENCUZOGULLARI E., 1991 — Cytoge-
netic effects of gesagard and igran in barley. Cytolo-
gia, 56: 419-424.
Received August 28th 2006; accepted December 14th 2006