Food and Chemical Toxicology 49 (2011) 763–769

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

The evaluation of the genotoxicity of two food preservatives: Sodium benzoate

and potassium benzoate
N. Zengin a, D. Yüzbasßıoğlu a,⇑, F. Ünal a, S. Yılmaz b, H. Aksoy c
a
Genetic Toxicology Laboratory, Department of Biology, Gazi University, Ankara, Turkey
b
Faculty of Health Sciences, Ankara University, 06190 Altındağ Ankara, Turkey
c
Department of Biology, Faculty of Art and Science, Sakarya University, Sakarya, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: In this study, the genotoxic effects of sodium benzoate (SB) and potassium benzoate (PB) were investi-
Received 12 August 2010 gated in cultured human peripheral lymphocytes using chromosomal aberrations (CA), sister chromatid
Accepted 29 November 2010 exchange (SCE), and micronuclei (MN). The level of nuclear DNA damage of SB and PB were also evaluated
Available online 3 December 2010
using the comet assay. The lymphocytes were incubated with different concentrations of SB (6.25, 12.5,
25, 50, and 100 lg/ml) and PB (62.5, 125, 250, 500, and 1000 lg/ml). A significant increase was observed
Keywords: in CA, SCE, and MN, in almost all treatments compared to negative controls. SB and PB significantly
Genotoxicity
decreased the mitotic index (MI) in all the treatments, compared to the negative controls. However, nei-
Food additive
Sodium benzoate
ther of the additives affected the replication index (RI). Although SB significantly increased DNA damage,
Potassium benzoate PB did not cause a significant increase in DNA damage. The present results indicate that SB and PB are
Human lymphocytes clastogenic, mutagenic and cytotoxic to human lymphocytes in vitro.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction 75% of the Western diet is made up of various processed foods,

With the increase in the production of processed and conve-
nience foods, food additives have become an increasingly impor-
tant practice in modern food technology (Saad et al., 2005).
Additives are widely used for various purposes, including preserva-
tion, coloring, and sweetening. The preservatives are added to stop
or delay nutritional losses due to microbiological, enzymatic or
chemical changes of foods and to prolong the shelf life and quality
of foods. SB and PB, also known salts of benzoic acid, are food pre-
servatives that inhibit the growth of mold, yeast, and some bacte-
ria. The Joint FAO/WHO Committee on Food Additives (JECFA) has
allocated an acceptable daily intake (ADI) of sodium benzoate and
potassium benzoate at 0–5 mg/kg body weight. These preserva-
tives are commonly used as antimicrobial substances in many
kinds of foods, such as, marinated fish, fruit-based fillings, jam,
salad cream, soft drinks, and beer. They have been found to pro-
voke urticaria, angioedema, and asthma (Michaelsson and Juhlin,
1973; Food Intolerance and Food Aversion, 1984; Miller and
Millstone, 1987; Tuormaa, 1994; Doğruyol, 2006). Furthermore,
they have also been directly linked with childhood hyperactivity
(Egger et al., 1985).
The use of food additives has increased enormously in the last
few decades. As a result, it has been estimated that today about
⇑ Corresponding author. Tel.: +90 312 202 1205; fax: +90 312 212 2279.
E-mail address: deniz@gazi.edu.tr (D. Yüzbasßıoğlu).
each person consuming an average of 8–10 lbs of food additives
per year, with some possibly eating even more. The following
adverse effects have been attributed to the consumption of food
additives: eczema, urticaria, angioedema, exfoliative dermatitis,
irritable bowel syndrome, nausea, vomiting, diarrhea, rhinitis,
bronchospasm, migraine, anaphylaxis, hyperactivity, and other
behavioral disorders (Feingold, 1973; Smith, 1991; Tuormaa,
1994; Doğruyol, 2006). It was reported that certain food preserva-
tives, especially antimicrobial agents, were genotoxic in different
test systems (Türkoğlu, 2007; Yavuz-Kocaman et al., 2008;
Mpountoukas et al., 2008; Yılmaz et al., 2009; Mamur et al., 2010).
The genotoxic activity of SB revealed highly contradictory re-
sults. Negative results have been obtained by using the Ames test,
with different Salmonella typhimuirum strains (Ishidate et al.,
1984), S. typhimuirum assay, and the tryptophan reversion assay
with Escherichia coli strain WP2 (Prival et al., 1991) and the comet
assay with different mouse organs (Sasaki et al., 2002). In contrast,
positive results have been obtained in Vicia faba (Xing and Zhang,
1990), Allium cepa (Türkoğlu, 2007), Chinese hamster fibroblast cell
line (Abe and Sasaki, 1977; Ishidate et al., 1984), and in the human
lymphocyte culture (Mpountoukas et al., 2008). On the other hand,
genotoxicity studies related to PB are limited. The studies are asso-
ciated with potassium benzoate salts. Tamaro et al. (1986) showed
the mutagenic activity of p-(3,3-dimethyl-1-triazeno) benzoic acid
potassium salts in E. coli B strains. However, the last study
indicated that p-(3,3-dimethyl-1-triazeno) has a low mutagenic

0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.11.040
764 N. Zengin et al. / Food and Chemical Toxicology 49 (2011) 763–769
activity in S. typhimurium strains. Vernole et al. (1987) studied the
cytogenetic effects of 1-p-(3-methyltriazeno) benzoic acid potas-
sium salts in human lymphocytes. They reported that this chemical
caused a dose-dependent increase in the frequency of chromo-
somal breaks. Vernole et al. (1988) also reported the clastogenic
potential of 1-p-(3-methyltriazeno) benzoic acid potassium salts
via sister-chromatid exchange in human lymphocytes.
To evaluate the genotoxic effects induced by physical and
chemical agents, various test systems have been described in bac-
teria, in mammalian cells, and in plants (Prival et al., 1991; Mac-
ioszek and Kononowicz, 2004; Yılmaz et al., 2008a; Arslan et al.,
2008; Mamur et al., 2010). Some of these test systems that are
widely used are chromosomal aberrations (CAs), sister-chromatid
exchanges (SCEs), and micronuclei (MN) tests in cultured human
peripheral lymphocytes, and recently the comet assay, in isolated
lymphocytes (Rencüzoğulları et al., 2001; Macioszek and Kon-
onowicz, 2004; Yüzbasßıoğlu et al., 2006; Yılmaz et al., 2008b; Ma-
mur et al., 2010).
The purpose of this study was to evaluate the potential geno-
toxic effects of food preservatives SB and PB, using in vitro CA,
SCE, and MN tests on human peripheral lymphocytes and the co-
met assay on isolated human lymphocytes. With the great increase
in the use of food additives this study obtained more data about
screening the potential effects.
2. Materials and methods
In the current study, human peripheral lymphocytes were used as the test sys-
tem. The test substance SB (Cas. No: 532-32-1) was obtained from Merck and PB
(Cas. No: 582-25-2) was obtained from Emerald. The chemical structure, molecular
formula and molecular weight of SB and PB are shown in Fig. 1. These chemicals
were dissolved in distilled water. The other chemicals cytochalasin-B (Cas. No:
14930-96-2), mitomycin C (Cas. No: 50-07-7), bromodeoxyuridine (Cas. No: 59-
14-3), NaCl (Cas. No: 7647-14-5), colchicine (Cas. No: 64-86-8) were obtained from
Sigma. DMSO (Cas. No: 67-68-5), NaOH (Cas. No: 1310-73-2), Tris (Cas. No: 77-86-
1), EDTA (Cas. No: 6381-92-6), Triton X-100 (Cas. No: 9002-93-1), Low Melting Aga-
rose (Cas. No: 9012-36-6), Normal Melting Agarose (Cas. No: 9012-36-6), EtBr (Cas.
No: 1239-45-8), H2O2 (Cas. No: 7722-84-1) were obtained from Applichem.
The study was carried out using blood samples from two healthy donors (non-
smokers, of age 25 years), with no medication for at least 3 weeks prior, and not
having had a radiological examination within the prior 3 months.
For the SCE and CA studies, whole blood 0.2 ml was added to 2.5 ml chromo-
some medium B (containing fetal bovine serum, heparin, antibiotics, phytohemag-
glutinin) supplemented with 10 lg/mL bromodeoxyuridine. The cultures were
incubated at 37 °C for 72 h. SB and PB did not change the pH of the culture medium.
Human lymphocytes were treated with different concentrations of SB (6.25, 12.5,
25, 50, and 100 lg/mL) and PB (62.5, 125, 250, 500, and 1000 lg/mL) for 24 and
48 h. In addition, negative and positive controls (mitomycin-C = MMC, 0.20 lg/
mL) were also maintained in all experiments. The methods of Evans (1984) and
Perry and Thompson (1984) were followed in the preparation of CA and SCE tests
with minor modifications (Yüzbasßıoğlu et al., 2006). Chromosomal abnormalities
were scored from 100 well-spread metaphases per donor (totally 200 metaphases
per dose level). The mitotic index (MI) was determined by scoring 1000 cells from
each donor.
For the SCE assay, the slides were stained with Giemsa, according to Speit and
Houpter’s (1985) method, with some modifications (Mamur et al., 2010). The num-
ber of SCEs (25 cells from each donor, a total of 50 cells per concentration) under the
second metaphases was scored. In addition, 100 cells from each donor were scored
Fig. 1. Chemical structures of sodium benzoate and potassium benzoate.
for determination of the replication index (RI). The RI was calculated according to
the following formula; (1  M1) + (2  M2) + (3  M3)/N (N = number of observed
cells) where M1, M2, and M3 represented the number of cells undergoing the first,
second, and third mitosis, respectively (Schneider et al., 1981).
Micronuclei preparation was performed according to the procedures of Fenech
(2000) and Palus et al. (2003). The human lymphocyte cultures were incubated at
37 °C for 72 h and treated with five different concentrations of SB and PB during
the last 48 h. Forty-four hours after the start of the culture, cytochalasin B (Cyt-B)
was added at a final concentration of 5.2 lg/mL, to arrest cytokinesis. SB and PB
did not alter the pH of the culture medium. Micronuclei were scored from 1000
binucleated cells (BN) per donor (totally 2000 binucleated cells per concentration).
Cell proliferation was evaluated using the cytokinesis-block proliferation index
(CBPI), which indicated the average number of cell cycles a given cell had under-
gone (Scarfi et al., 1997). Five hundred lymphocytes were scored from per donor
(totally 1000 lymphocytes) to evaluate the percentage of cells with 1–4 nuclei. CBPI
was calculated according to Surrales et al. (1995), as follows; (1  N1) + (2  N2) +
(3  (N3 + N4)/N, where N1–N4 represent the number of cells with 1–4 nuclei,
respectively, and N is the total number of cells scored. MN were accepted only when
(i) they were separated from the main nuclei, but included within the correspond-
ing cytoplasm, (ii) they had a chromatin material similar to that of the main nuclei,
(iii) they were coplanar to the main nuclei (Çelik, 2006).
Primary DNA damaging effects caused by the SB and PB were determined using
the comet assay according to Singh et al.’s (1988) method, with some modifications.
The methods followed for the comet assay in human lymphocytes was given in de-
tail in the study of Mamur et al. (2010). The lymphocytes were isolated using the
Biocoll separating solution. To detect the viability of cells, the trypan blue exclusion
test was used. Cell viability was >98%. Isolated human lymphocytes were incubated
with concentrations of SB (6.25, 12.5, 25, 50, and 100 lg/mL) and PB (62.5, 125, 250,
500, and 1000 lg/mL) for 1 h at 37 °C. Negative and positive controls (100 mM
H2O2, 0.30 lg/mL) were also included. To detect DNA damaging capacity of the
SB and PB, DNA was electrophoresed at 25 V, 300 mA for 20 min.
Image analysis and comet scoring were examined using the fluorescent micro-
scope (Olympus BX51) equipped with an excitation filter of 546 nm and a barrier
filter of 590 nm, at 400 magnification. A slide was prepared for each dose of SB
and PB. The tail moment (%) of 100 comets on the slide (totally 200 comets per con-
centration) were determined using a specialized image analyzes system (Comet As-
say IV, Perceptive Instruments Ltd., UK).
For the statistical analysis of the results, the z-test was applied for the percent-
age of abnormal cells with CA, CA/cell, RI, CBPI, MI, MN, and the t-test for SCEs and
the comet assay. Dose–response relationships were determined from the correla-
tion and regression coefficients for the percentage of abnormal cells, CA/cell, SCE,
MN, and the mean comet tail moment.
3. Results
3.1. Chromosomal aberrations assay
Sodium benzoate and PB induced a significant increase in the
frequency of CAs and CA/cell in all concentrations and treatment
periods as compared to the negative control (Table 1). These in-
creases in the frequency of CAs and CA/cell were dose-dependent,
both in the 24- and 48-h treatments (for SB, r = 0.99 and 0.97 and
for PB, r = 0.97 and 0.97 at 24- and 48-h, respectively). Both the
additives caused six types of structural aberrations (chromatid
and chromosome breaks, chromatid exchanges, fragments, sister-
chromatid unions, and dicentric chromosomes) and a numerical
aberration (polyploidy). Chromatid (SB = 73.98%; PB = 58.64%)
and chromosome breaks (SB = 16.47%; PB = 29.51%) were the most
frequent aberrations in all the experimental groups.
3.2. Sister-chromatid exchanges, cell cycle and mitotic index
The results of the SCE analysis are shown in Table 2. SB and
PB increased the frequency of SCEs/cell. This increase was signifi-
cant in almost all concentrations and treatment times (except
62.5 lg/mL PB at 24 h). The increase in SCEs was concentration-
dependent at both treatment periods (for SB r = 0.97, 0.89; for PB
r = 0.98, 0.99, at 24 and 48 h, respectively).
3.3. Micronucleus assay
Table 3 shows that SB and PB increased the frequency of
lymphocytes with micronuclei. Micronuclei frequency significantly
 N. Zengin et al. / Food and Chemical Toxicology 49 (2011) 763–769 765

Table 1
Chromosome aberrations in cultured human lymphocytes treated with sodium benzoate and potassium benzoate.

Test substance Treatment Aberrations Abnormal cell ± SH CA/cell ± SH

(%)
Structural Numerical
Period Concentration (lg/ ctb csb cte f scu dic p
(h) ml)
Control 24 0.00 4 – – – – – – 2.00 ± 0.98 0020 ± 0009
MMC 24 0.20 44 14 7 2 1 5 – 32.00 ± 3.29 0365 ± 0034
S. benzoate 24 6.25 11 1 1 – – 1 – 7.00 ± 1.80* 0070 ± 0018*
12.5 13 4 1 – – 1 – 9.50 ± 2.07** 0095 ± 0020**
25 19 2 1 – – 1 – 11.50 ± 2.25*** 0115 ± 0022***
50 19 4 – 1 2 2 – 12.00 ± 2.29*** 0140 ± 0024***
100 28 10 – 1 3 3 – 21.50 ± 2.90*** 0225 ± 0029***
MMC 48 0.20 90 36 20 2 2 7 – 57.50 ± 3.49 0785 ± 0029
S. benzoate 48 6.25 17 3 – 2 – – 1 10.50 ± 2.16*** 0115 ± 0022***
12.5 19 2 – – – – – 10.00 ± 2.12*** 0105 ± 0021***
25 22 4 – – 1 2 – 13.00 ± 2.37*** 0145 ± 0024***
50 48 9 – – 2 2 – 27.00 ± 3.13*** 0305 ± 0032***
100 60 18 – – 1 4 – 36.50 ± 3.40 *** 0415 ± 0034***
Frequency of abnormalities 73.98 16.47 0.86 1.15 2.60 4.62 0.28
(%)
Control 24 0.00 2 4 – – – – – 3.00 ± 1.20 0030 ± 0012
MMC 24 0.20 52 29 8 2 2 5 – 40.00 ± 3.46 0490 ± 0035
P. benzoate 24 62.5 11 4 – – – 1 – 6.50 ± 1.74 0080 ± 0019*
125 15 4 – – 1 2 1 11.00 ± 2.21** 0115 ± 0022***
250 21 9 – – – 4 – 16.00 ± 2.59*** 0170 ± 0026***
500 29 19 1 2 2 3 – 24.00 ± 3.01*** 0280 ± 0031***
1000 44 25 – 1 1 5 – 30.50 ± 3.25*** 0380 ± 0034***
MMC 48 0.20 100 28 22 4 1 4 – 60.50 ± 3.45 0795 ± 0028
P. benzoate 48 62.5 16 7 1 1 – 1 – 12.00 ± 2.29 *** 0130 ± 0023***
125 24 13 – – 1 1 – 16.00 ± 2.59 *** 0195 ± 0028***
250 33 14 – 1 – 7 – 20.50 ± 2.85 *** 0275 ± 0031***
500 50 25 – 3 1 3 – 34.00 ± 3.34 *** 0410 ± 0034***
1000 59 32 1 2 4 10 – 36.50 ± 3.40 *** 0540 ± 0035***
Frequency of abnormalities 58.64 29.51 0.58 1.94 1.94 7.18 0.19
(%)

ctb: Chromatid break, csb: chromosome break, cte: chromatid exchange, f: fragment, scu: sister chromatid union, dic: dicentric chromosome, p: polyploidy.
Totally 200 cells were scored for each treatments.
MMC: mitomycin-C.
*
Significant from the control P < 0.05 (z test).
**
Significant from the control P < 0.01 (z test).
***
Significant from the control P < 0.001 (z test).

increased at 25, 50, and 100 lg/mL concentrations of SB and 125,
250, 500, and 1000 lg/mL concentrations of PB. These increases
were dose-dependent (SB r = 0.97, PB r = 0.91).
3.4. Mitotic, replication and nuclear division indices
Sodium benzoate significantly decreased the frequency of the
mitotic index at concentrations of 12.5, 25, 50, and 100 lg/mL at
the 24-h treatment and all concentrations at the 48-h treatment.
The other additive PB significantly decreased the frequency of
the mitotic index at concentrations of 250, 500, and 1000 lg/mL
at the 24-h treatment and all concentrations at the 48-h treatment.
Significant concentration response correlations were observed in
the MI at both treatment periods (for SB r = 0.97, 0.89, for PB
r = 0.93, 0.95 at 24 and 48 h, respectively) (Table 4). On the
other hand neither had an additive effect in RI, compared to the
negative controls (Table 4). The CBPI value was not affected by
the treatments of these additives.
3.5. Comet assay
According to the comet assay results, the comet tail moment
significantly increased in all the concentrations of sodium benzo-
ate, but this increase was not concentration-dependent. On the
other hand, potassium benzoate did not significantly increase the
comet tail moment in any concentration (Table 5).
4. Discussion
In this study, the genotoxic potential of SB and PB was investi-
gated, with chromosomal aberrations, sister-chromatid exchanges
and micronucleus analysis in cultured human peripheral lympho-
cytes, as well as, single cell gel electrophoresis (SCGE) or ‘comet as-
say’ in isolated lymphocytes, which are used as the most rapid,
sensitive, and useful assays and have been proved to be good indi-
cators of DNA damage (Macioszek and Kononowicz, 2004; Mpoun-
toukas et al., 2008; Mamur et al., 2010). In vitro genotoxicity tests
detected compounds that induced genetic damage, directly or indi-
rectly, by different mechanisms. They are considered to be the
markers of early biological effects of carcinogen exposure (Liou
et al., 2002). In all these test systems, data showed that both addi-
tives induced genotoxicity at almost all concentrations and treat-
ment times and also decreased the mitotic index.
Both food additives significantly increased the frequency of CAs
and CA/cell in all treatments groups when compared with their
negative controls. These additives induced seven types of chromo-
somal aberrations, which indicated their clastogenic effects. In this
study chromatid and chromosome breaks had been observed as the
most common aberrations. Chromosomal breakage could result in
a number of different structural rearrangements, some of which
gave rise to abnormalities of chromosomal segregation at mitosis
(Gisselsson, 2001). Increased levels of CA have been associated
with increased cancer risk (Hagmar et al., 1994).
766 N. Zengin et al. / Food and Chemical Toxicology 49 (2011) 763–769

Table 2 damage in blood lymphocytes of individuals exposed to genotoxic

Frequency of the sister-chromatid exchanges in cultured human lymphocytes treated carcinogens (Yager et al., 1993). High levels of SCE frequency has
with sodium benzoate and potassium benzoate.
been observed in persons at higher cancer risk, due to occupational
Test substance Treatment Min–max SCE SCE/cell ± SE or environmental exposure to a wide variety of carcinogens (Sinues
Period (h) Concentration et al., 1991; Fucic et al., 2000; Bolognesi, 2003).
(lg/ml) The comet or the single cell gel electrophoresis assay is a sensi-
Control 24 0.00 0–6 3.28 ± 0.27 tive, reliable, and rapid method for assessing DNA damage in indi-
MMC 24 0.20 10–75 34.42 ± 2.67 vidual eukaryotic cells. Comet assays are among the relatively new
S. benzoate 24 6.25 2–8 4.34 ± 0.22* assays recommended for use in investigating potentially genotoxic
12.5 2–12 5.58 ± 0.28*
substances, including food additives (Collins et al., 1997). In this
25 1–13 5.78 ± 0.33*
50 2–14 7.34 ± 0.38* study, the alkaline version of the comet assay has been used. It is
100 3–16 8.46 ± 0.40* capable of detecting single and double strand breaks, alkali labile
MMC 48 0.20 48–115 84.02 ± 2.37 sites in nuclear DNA, and delayed or incomplete excision repair
S. benzoate 48 6.25 2–10 5.50 ± 0.32*
sites in the DNA of individual cells (Rojas et al., 1999). Further-
12.5 2–12 6.10 ± 0.33*
25 2–14 6.94 ± 0.39*
more, under certain circumstances, the comet assay can also detect
50 3–16 8.26 ± 0.47* DNA–DNA and DNA–protein cross-linking, which appears as a rel-
100 3–17 8.96 ± 0.53* atively reduced distance of DNA migration when compared with
Control 24 0.00 0–8 3.42 ± 0.25 the concurrent controls (Hartmann et al., 2003).
MMC 24 0.20 10–75 37.16 ± 2.58 Sodium benzoate and PB also increase the MN frequency in a
P. benzoate 24 62.5 1–11 4.18 ± 0.29 dose-dependent manner. MN assay detects both clastogenicity
125 1–10 4.98 ± 0.29*
(chromosome breakage) and aneugenicity (chromosome lagging
250 2–10 5.22 ± 0.25*
500 2–10 6.04 ± 0.27* due to dysfunction of the mitotic apparatus). MN may reflect geno-
1000 4–12 7.46 ± 0.29* mic instability (Inoue et al., 1997; Albertini et al., 2000).
MMC 48 0.20 24–115 65.76 ± 4.47 In the results of our experiments, SB and PB decreased the mi-
P. benzoate 48 62.5 2–11 5.70 ± 0.31* totic index. Decreasing of the MI could be due to blocking of G2,
125 3–12 6.70 ± 0.30*
250 3–12 7.62 ± 0.30*
preventing the cell from entering mitosis or decreasing the ATP le-
500 3–18 9.62 ± 0.49* vel and the pressure from the energy production center. Inhibition
1000 5–25 12.56 ± 0.63* of certain cell cycle-specific enzymes, such as DNA polymerase,
Totally 50 s mitosis were scored for SCEs/cell. MMC: mitomycin-C.
which was necessary for the synthesis of DNA precursors as well
*
Significant from the control P < 0.05 (t test). as other enzymes, more directly involved with spindle production,
assembly or orientation, may also explain the reported antimitotic
effects as well as changes in the frequencies of different cell stages
Table 3 (Jain and Andsorbhoy, 1988; Hidalgo et al., 1989; Yılmaz et al.,
Micronuclei in cultured human lymphocytes treated with sodium benzoate and 2008b). Replication and nuclear division indices were not effected
potassium benzoate.
by both of the additives.
Test Treatment Distribution MN (%) Genotoxicity of SB was investigated in some test systems. SB
substance of BN cells was found to be negative in different S. typhimurium strains
according to
(3 mg/plate) in the presence or absence of metabolic activation
the no. of MN
(Ishidate et al., 1984). In another study with the S. typhimurium as-
Period Concentration (1) (2) (3)
say and the tryptophan reversion assay in the E. coli strain WP2
(h) (lg/ml)
(0.033–10 mg/plate) this preservative showed no evidence of car-
Control 48 0.00 1 – – 0.05 ± 0.04
cinogenicity (Prival et al., 1991). Sasaki et al. (2002) reported that
MMC 48 0.20 76 4 2 4.50 ± 0.46
S. benzoate 48 6.25 2 – – 0.10 ± 0.07
SB did not yield a statistically significant increase (2000 mg/kg) in
12.5 6 – – 0.30 ± 0.12 DNA damage in any of the mouse organs studied. On the other
25 8 – – 0.40 ± 0.14* hand, the SCE test carried out on the V. faba root tip cells and hu-
50 8 1 – 0.50 ± 0.15** man lymphocytes, at a dose level of 10 2 M, gave a significant in-
100 17 2 1 1.20 ± 0.24***
crease of the mean number of SCEs/cell in comparison with the
Control 48 0.00 4 – – 0.20 ± 0.09
MMC 48 0.20 81 4 1 4.60 ± 0.46 control (Xing and Zhang, 1990). Studies denominate that SB inhib-
P. benzoate 48 62.5 7 2 – 0.55 ± 0.16 its DNA synthesis, induces anaphase bridges, and causes premature
125 10 2 – 0.70 ± 0.18* chromosome condensation, leading to picnotic nuclei and chroma-
250 12 3 – 0.90 ± 0.21**
tin erosion in the interphase nuclei, in the roots of V. faba (Njagi
500 17 1 – 0.95 ± 0.21**
1000 17 3 – 1.15 ± 0.24***
and Gopalan, 1982). Türkoğlu (2007) showed that SB increased
chromosomal aberrations and decreased the mitotic index in A.
Totally 2000 binucleated cells were scored for each treatments. cepa. In Chinese hamster, the fibroblast cell line, chromosome
MMC: mitomycin-C.
*
Significant from the control P < 0.05 (z test).
aberration test (2 mg/ml), and SCE test (P2 mM) were also posi-
**
Significant from the control P < 0.01 (z test). tive (Abe and Sasaki, 1977; Ishidate et al., 1984). In a recent study
***
Significant from the control P < 0.001 (z test). SB at 8 mM showed an increase in SCE/cell (1.4 times over control)
in human lymphocytes (Mpountoukas et al., 2008).
Studies on the point of the genotoxicity of potassium benzoate
These additives also significantly increased the frequency of are limited. There are only some researches about certain salts of
SCEs/cell in all treatments groups as compared to their negative PB. The mutagenic activity of the p-(3,3-dimethyl-1-triazeno) ben-
controls. The ready quantifiable nature of SCEs with high sensitiv- zoic acid potassium salt has been studied in bacterial cells by
ity for revealing toxicant–DNA interaction and the demonstrated Tamaro et al. (1986). The results indicate that the p-(3,3-di-
ability of genotoxic chemicals to induce a significant increase in methyl-1-triazeno) benzoic acid potassium salt has a very low
SCEs in cultured cells and in cells sampled from treated animals, mutagenic activity on the S. typhimurium strains tested, while it
has resulted in this endpoint being used as an indicator of DNA is more effective in inducing trp + revertants in E. coli B strains
 N. Zengin et al. / Food and Chemical Toxicology 49 (2011) 763–769 767

Table 4
Frequency of the replication, mitotic and cytokinesis-block proliferation indices in cultured human lymphocytes treated with sodium benzoate and potassium benzoate.

Test substance Treatment M1 M2 M3 RI ± SE MI ± SE CBPI ± SE

Period (h) Concentration (lg/ml)
Control 24 0.00 43 54 103 2.30 ± 0.05 7.55 ± 0.59 –
MMC 24 0.20 44 70 86 2.21 ± 0.05 4.35 ± 0.45 –
S. benzoate 24 6.25 35 60 105 2.35 ± 0.05 6.20 ± 0.53 –
12.5 40 48 112 2.36 ± 0.05 5.95 ± 0.52* –
25 41 48 111 2.35 ± 0.05 5.80 ± 0.52* –
50 39 58 103 2.32 ± 0.05 5.70 ± 0.51* –
100 37 54 109 2.36 ± 0.05 5.00 ± 0.48 *** –
Control 48 0.00 43 54 103 2.30 ± 0.05 7.55 ± 0.59 1.40 ± 0.37
MMC 48 0.20 74 45 81 2.03 ± 0.05 3.75 ± 0.42 1.17 ± 0.34
S. benzoate 48 6.25 33 53 114 2.40 ± 0.05 5.45 ± 0.50** 1.27 ± 0.35
12.5 43 47 110 2.33 ± 0.05 5.00 ± 0.48*** 1.38 ± 0.36
25 35 46 119 2.42 ± 0.05 4.80 ± 0.47*** 1.36 ± 0.36
50 43 57 100 2.28 ± 0.05 4.60 ± 0.46*** 1.41 ± 0.37
100 54 67 79 2.12 ± 0.05 4.30 ± 0.45*** 1.33 ± 0.36
Control 24 0.00 41 64 95 2.27 ± 0.05 7.55 ± 0.59 –
MMC 24 0.20 51 77 72 2.10 ± 0.05 3.75 ± 0.42 –
P. benzoate 24 62.5 30 60 110 2.40 ± 0.05 6.90 ± 0.56 –
125 52 47 101 2.24 ± 0.05 6.35 ± 0.54 –
250 38 58 104 2.33 ± 0.05 5.65 ± 0.51* –
500 43 56 101 2.29 ± 0.05 4.85 ± 0.48*** –
1000 54 71 75 2.10 ± 0.05 4.30 ± 0.45*** –
Control 48 0.00 41 64 95 2.27 ± 0.05 7.55 ± 0.59 1.62 ± 0.40
MMC 48 0.20 62 66 72 2.05 ± 0.05 3.60 ± 0.41 1.27 ± 0.35
P. benzoate 48 62.5 44 52 104 2.30 ± 0.05 5.80 ± 0.52* 1.65 ± 0.40
125 55 65 80 2.12 ± 0.05 5.30 ± 0.50** 1.48 ± 0.38
250 57 81 62 2.02 ± 0.05 5.00 ± 0.48*** 1.36 ± 0.36
500 75 86 39 1.82 ± 0.05 4.30 ± 0.45*** 1.25 ± 0.35
1000 98 68 34 1.68 ± 0.05 3.75 ± 0.42*** 1.24 ± 0.35

Totally 200 cells scored for RI.

Totally 2000 cells were scored for MI.
Totally 1000 cells were scored for CBPI.
MMC: mitomycin-C.
*
Significant from the control P < 0.05 (z test).
**
Significant from the control P < 0.01 (z test).
***
Significant from the control P < 0.001 (z test).

stimulated by phytohemagglutinin. SCE values increase signifi-

Table 5 cantly in a dose-dependent manner up to 200 lg/mL. However,
DNA damaging activity of sodium benzoate and potassium benzoate in isolated SCE frequencies, as well as chromosome breaks, do not increase
human lymphocytes. dramatically (Vernole et al., 1988). Benzoic acid is commonly used
Test substance Concentration (lg/ml) Tail moment (%) as an antimicrobial substance in many food products. Sarıkaya and
Solak (2003) have published that benzoic acid significantly de-
Control 0.00 0.35 ± 0.05
H2O2 0.30 4.27 ± 0.91 creases the life period and increases somatic mutation and recom-
Sodium benzoate 6.25 1.53 ± 0.33* bination (SMART) in Drosophila melanogaster. Yılmaz et al. (2008a)
12.5 1.77 ± 0.39* has reported that benzoic acid significantly increases the chromo-
25 3.03 ± 0.65* somal aberrations and decreases the mitotic index in the Allium sat-
50 1.18 ± 0.30*
100 1.61 ± 035*
ivum root tips. In another study, Yılmaz et al. (2009) reports that
benzoic acid significantly increases chromosomal aberrations, sis-
Control 0.00 0.35 ± 0.05
H2O2 0.30 4.27 ± 0.91
ter-chromatid exchanges, and micronucleus frequency in human
Potassium benzoate 62.5 0.62 ± 0.20 lymphocytes. They indicate that benzoic acid is a weak genotoxic
125 1.05 ± 0.36 agent, especially in lower doses, in human lymphocyte cultures.
250 0.35 ± 059 The mechanism operating in SB- and PB-mediated genotoxicity
500 1.20 ± 061
in human lymphocytes is currently unknown. However, damages
1000 0.63 ± 0.24
may be mediated by the inhibition of the activation of XRCC1,
Totally 200 comets were scored. PARP-1, and DNA LIG3 proteins, which are responsible for DNA re-
H2O2: hydrogen peroxide.
*
pair (Wang et al., 2005). The XRCC1 protein plays an important role
Significant from the control P < 0.05 (t test).
in base excision repair; after excision of a damaged base, it stimu-
lates an endonuclease action and acts as a scaffold in the subse-
(Tamaro et al., 1986). Vernole et al. (1987) has shown the in vitro quent restoration of the site (Vidal et al., 2001; Goode et al.,
cytogenetic effects of 1-p-(3-methyltriazeno) benzoic acid potas- 2002). Although DNA ligase III is predominantly considered as a
sium salt on human lymphocytes. It has been tested at different component of a single-strand break and base-excision repair
culture times in a range of concentrations from 2 to 500 lg/mL. where it interacts with XRCC1 and PARP-1, other observations hint
This chemical causes a dose-dependent increase in the frequency at a potential role of DNA double strand breaks rejoining as well
of chromosomal breaks. Vernole et al. (1988) has reported the (Caldecott et al., 1994; Wang et al., 2005).
clastogenic potential of 1-p-(3-methyltriazeno) benzoic acid potas- In conclusion SB and PB are genotoxic additives in human lym-
sium salt on human lymphocytes. They assess the SCE frequencies phocytes in vitro. However, the mechanisms of the damaging ef-
induced by benzoic acid potassium salt on human lymphocytes fects of these substances need to be clarified by more detailed
768 N. Zengin et al. / Food and Chemical Toxicology 49 (2011) 763–769
studies. All food additives must be kept under continuous observa-
tion and must be re-evaluated whenever necessary, in the light of
changing conditions of use and new scientific information (Council
directive 89/107/EEC). Further studies on the genotoxic properties
of sodium benzoate and potassium benzoate, with the help of other
tests for genotoxicity, should be conducted.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgement
This study was partially supported by Gazi University Research
Fund under Project No. 05/2008-54.
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