Benzoato de sodio y benzoato de potasio.docx

7/29/2019 Benzoato de sodio y benzoato de potasio.docx

1/7

Introduction

With the increase in the production of processed and convenience foods, food additiveshave become an increasingly important practice in modern food technology (Saad et al.,2005). Additives are widely used for various purposes, including preservation, 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 andquality of foods. SB and PB, also known salts of benzoic acid, are food preservatives thatinhibit the growth of mold, yeast, and some bacteria. The Joint FAO/WHO Committee onFood Additives (JECFA) has allocated an acceptable daily intake (ADI) of sodiumbenzoate and potassium benzoate at 05 mg/kg body weight. These preservatives arecommonly used as antimicrobial substances in many kinds of foods, such as, marinatedfish, fruit-based fillings, jam, salad cream, soft drinks, and beer. They have been found toprovoke urticaria, angioedema, and asthma (Michaelsson and Juhlin, 1973; FoodIntolerance and Food Aversion, 1984; Miller and Millstone, 1987; Tuormaa, 1994;Dogruyol, 2006). Furthermore, they have also been directly linked with childhoodhyperactivity (Egger et al., 1985). The use of food additives has increased enormously inthe last few decades. As a result, it has been estimated that today about 75% of the

Western diet is made up of various processed foods, each person consuming an averageof 810 lbs of food additives per year, with some possibly eating even more. The followingadverse effects have been attributed to the consumption of foodadditives: 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; Dogruyol,2006). It was reported that certain food preservatives, especially antimicrobial agents,were genotoxic in different test systems (Turkoglu, 2007; Yavuz-Kocaman et al.,2008;Mpountoukas et al., 2008; Ylmaz et al., 2009; Mamur et al., 2010 ). The genotoxicactivity of SB revealed highly contradictory results. Negative results have been obtained byusing 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 (Turkoglu, 2007), Chinese hamster fibroblast cell line (Abe and Sasaki, 1977;Ishidate et al., 1984), and in the human lymphocyte culture (Mpountoukas et al., 2008). Onthe other hand, genotoxicity studies related to PB are limited. The studies are associatedwith 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, thelast study indicated that p-(3,3-dimethyl-1-triazeno) has a low mutagenic. activity in S.typhimurium strains. Vernole et al. (1987) studied the cytogenetic effects of 1-p-(3-methyltriazeno) benzoic acid potassium salts in human lymphocytes. They reported thatthis chemical caused a dose-dependent increase in the frequency of chromosomal 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. Toevaluate the genotoxic effects induced by physical and chemical agents, various testsystems have been described in bacteria, in mammalian cells, and in plants ( Prival et al.,1991; Macioszekand Kononowicz, 2004; Ylmaz et al., 2008a; Arslan et al., 2008; Mamuret al., 2010). Some of these test systems that are widely used are chromosomalaberrations (CAs), sister-chromatid exchanges (SCEs), and micronuclei (MN) tests incultured human peripheral lymphocytes, and recently the comet assay, in isolatedlymphocytes (Rencuzogullar et al., 2001; Macioszek and Kononowicz, 2004;Yuzbas_oglu et al., 2006; Ylmaz et al., 2008b; Mamuret al., 2010). The purpose of this


2/7

study was to evaluate the potential genotoxic effects of food preservatives SB and PB,using in vitro CA, SCE, and MN tests on human peripheral lymphocytes and the cometassay on isolated human lymphocytes. With the great increase in the use of food additivesthis study obtained more data about screening the potential effects.

Materials and methods

In the current study, human peripheral lymphocytes were used as the test system. The testsubstance 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 molecularweight of SB and PB are shown in Fig. 1. These chemicals were dissolved in distilledwater. 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: 9002-931), Low Melting Agarose (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 obtainedfrom Applichem. The study was carried out using blood samples from two healthy donors

(nonsmokers, of age 25 years), with no medication for at least 3 weeks prior, and nothaving had a radiological examination within the prior 3 months. For the SCE and CAstudies, whole blood 0.2 ml was added to 2.5 ml chromosome medium B (containing fetalbovine serum, heparin, antibiotics, phytohemagglutinin) supplemented with 10 lg/mLbromodeoxyuridine. The cultures were incubated at 37 _C for 72 h. SB and PB did notchange 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 inall experiments. The methods of Evans (1984) and Perry and Thompson (1984) werefollowed in the preparation of CA and SCE tests with minor modifications (Yuzbas_ogluet al., 2006). Chromosomal abnormalities were scored from 100 well-spread metaphases

per donor (totally 200 metaphases per dose level). The mitotic index (MI) was determinedby scoring 1000 cells from each donor. For the SCE assay, the slides were stained withGiemsa, according to Speit and Houpters (1985) method, with some modifications(Mamur et al., 2010). The number of SCEs (25 cells from each donor, a total of 50 cellsper concentration) under the second metaphases was scored. In addition, 100 cells fromeach donor were scored for determination of the replication index (RI). The RI wascalculated 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 cellsundergoing the first, second, and third mitosis, respectively (Schneider et al., 1981).Micronuclei preparation was performed according to the procedures ofFenech(2000) andPalus et al. (2003). The human lymphocyte cultures were incubated at 37 _C for 72 h andtreated 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 finalconcentration of 5.2 lg/mL, to arrest cytokinesis. SB and PB did not alter the pH of theculture medium. Micronuclei were scored from 1000 binucleated cells (BN) per donor(totally 2000 binucleated cells per concentration). Cell proliferation was evaluated usingthe cytokinesis-block proliferation index (CBPI), which indicated the average number ofcell cycles a given cell had undergone (Scarfi et al., 1997). Five hundred lymphocyteswere scored from per donor (totally 1000 lymphocytes) to evaluate the percentage of cellswith 14 nuclei. CBPI was calculated according to Surrales et al. (1995), as follows; (1 _N1) + (2 _ N2) + (3 _ (N3 + N4)/N, where N1N4 represent the number of cells with 14


3/7

nuclei, respectively, and N is the total number of cells scored. MN were accepted onlywhen (i) they were separated from the main nuclei, but included within the correspondingcytoplasm, (ii) they had a chromatin material similar to that of the main nuclei, (iii) theywere coplanar to the main nuclei (Celik, 2006). Primary DNA damaging effects caused bythe 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 detail in the study ofMamur et al. (2010). The lymphocytes wereisolated using the Biocoll separating solution. To detect the viability of cells, the trypanblue exclusin test was used. Cell viability was >98%. Isolated human lymphocytes wereincubated 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, DNAwas electrophoresed at 25 V, 300 mA for 20 min. Image analysis and comet scoring wereexamined using the fluorescent microscope (Olympus BX51) equipped with an excitationfilter of 546 nm and a barrier filter of 590 nm, at 400_ magnification. A slide was preparedfor each dose of SB and PB. The tail moment (%) of 100 comets on the slide (totally 200comets per concentration) were determined using a specialized image analyzes system(Comet Assay IV, Perceptive Instruments Ltd., UK). For the statistical analysis of the

results, the z-test was applied for the percentage of abnormal cells with CA, CA/cell, RI,CBPI, MI, MN, and the t-test for SCEs and the comet assay. Doseresponse relationshipswere determined from the correlation and regression coefficients for the percentage ofabnormal cells, CA/cell, SCE, MN, and the mean comet tail moment.

Results

Chromosomal aberrations assay

Sodium benzoate and PB induced a significant increase in the frequency of CAs andCA/cell in all concentrations and treatment periods as compared to the negative control(Table 1). These increases 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 and0.97 at 24- and 48-h, respectively). Both the additives caused six types of structuralaberrations (chromatid and chromosome breaks, chromatid exchanges, fragments,sisterchromatid 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.

Sister-chromatid exchanges, cell cycle and mitotic index

The results of the SCE analysis are shown in Table 2. SB and PB increased the frequencyof SCEs/cell. This increase was significant in almost all concentrations and treatmenttimes (except 62.5 lg/mL PB at 24 h). The increase in SCEs was concentrationdependent

at both treatment periods (for SB r = 0.97, 0.89; for PB r = 0.98, 0.99, at 24 and 48 h,respectively).


4/7

Micronucleus assay

Table 3shows that SB and PB increased the frequency of lymphocytes with micronuclei.

Micronuclei frequency significantly 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).

Mitotic, replication and nuclear division ndices

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 affectedby the treatments of these additives.

Comet assay

According to the comet assay results, the comet tail moment significantly increased in all

the concentrations of sodium benzoate, 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).

Discussion

In this study, the genotoxic potential of SB and PB was investigated, with chromosomal

aberrations, sister-chromatid exchanges and micronucleus analysis in cultured human

peripheral lymphocytes, as well as, single cell gel electrophoresis (SCGE) or comet

assay in isolated lymphocytes, which are used as the most rapid, sensitive, and useful

assays and have been proved to be good indicators of DNA damage (Macioszek and

Kononowicz, 2004; Mpountoukas et al., 2008; Mamur et al., 2010). In vitro genotoxicity

tests detected compounds that induced genetic damage, directly or indirectly, 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

additives induced genotoxicity at almost all concentrations and treatment times and alsodecreased 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 chromosomal 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


5/7

chromosomal segregation at mitosis (Gisselsson, 2001). Increased levels of CA have been

associated with increased cancer risk (Hagmar et al., 1994).

These additives also significantly increased the frequency of SCEs/cell in all treatments

groups as compared to their negative controls. The ready quantifiable nature of SCEs with

high sensitivity for revealing toxicantDNA interaction and the demonstrated ability of

genotoxic chemicals to induce a significant increase in SCEs in cultured cells and in cells

sampled from treated animals, has resulted in this endpoint being used as an indicator of

DNA damage in blood lymphocytes of individuals exposed to genotoxic carcinogens

(Yager et al., 1993). High levels of SCE frequency has been observed in persons at higher

cancer risk, due to occupational or environmental exposure to a wide variety of

carcinogens (Sinues et al., 1991; Fucic et al., 2000; Bolognesi, 2003). The comet or the

single cell gel electrophoresis assay is a sensitive, reliable, and rapid method for

assessing DNA damage in individual eukaryotic cells. Comet assays are among the

relatively new assays recommended for use in investigating potentially genotoxic

substances, including food additives (Collins et al., 1997). In this study, the alkaline

version of the comet assay has been used. It is capable of detecting single and doubl

strand breaks, alkali labile sites in nuclear DNA, and delayed or incomplete excision repair

sites in the DNA of individual cells (Rojas et al., 1999). Furthermore, under certain

circumstances, the comet assay can also detect DNADNA and DNAprotein cross

linking, which appears as a relatively reduced distance of DNA migration when compared

with the concurrent controls (Hartmann et al., 2003). Sodium benzoate and PB also

increase the MN frequency in a dose-dependent manner. MN assay detects both

clastogenicity (chromosome breakage) and aneugenicity (chromosome lagging due to

dysfunction of the mitotic apparatus). MN may reflect genomic instability (Inoue et al.,

1997; Albertini et al., 2000). In the results of our experiments, SB and PB decreased the

mitotic index. Decreasing of the MI could be due to blocking of G2, preventing the cell fromentering mitosis or decreasing the ATP level and the pressure from the energy production

center. Inhibition of certain cell cycle-specific enzymes, such as DNA polymerase, which

was necessary for the synthesis of DNA precursors as well 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

(Jain and Andsorbhoy, 1988; Hidalgo et al., 1989; Ylmaz et al., 2008b). Replication and

nuclear division indices were not effected by both of the additives. Genotoxicity of SB was

investigated in some test systems. SB was found to be negative in different S. typhimurium

strains (3 mg/plate) in the presence or absence of metabolic activation (Ishidate et al.,

1984). In another study with the S. typhimurium assay and the tryptophan reversion assay

in the E. coli strain WP2 (0.03310 mg/plate) this preservative showed no evidence of

carcinogenicity (Prival et al., 1991). Sasaki et al. (2002) reported that SB did not yield a

statistically significant increase (2000 mg/kg) in DNA damage in any of the mouse organs

studied. On the other hand, the SCE test carried out on the V. faba root tip cells and

human lymphocytes, at a dose level of 10_2 M, gave a significant increase of the mean

number of SCEs/cell in comparison with the control (Xing and Zhang, 1990). Studies

denominate that SB inhibits DNA synthesis, induces anaphase bridges, and causes


6/7

premature chromosome condensation, leading to picnotic nuclei and chromatin erosion in

the interphase nuclei, in the roots of V. faba (Njagi and Gopalan, 1982). Turkoglu (2007)

showed that SB increased chromosomal aberrations and decreased the mitotic index in A.

cepa. In Chinese hamster, the fibroblast cell line, chromosome aberration test (2 mg/ml),

and SCE test (P2 mM) were also positive (Abe and Sasaki, 1977; Ishidate et al., 1984). In

a recent study SB at 8 mM showed an increase in SCE/cell (1.4 times over control) inhuman lymphocytes (Mpountoukas et al., 2008). Studies on the point of the genotoxicity of

potassium benzoate are limited. There are only some researches about certain salts of

PB. The mutagenic activity of the p-(3,3-dimethyl-1-triazeno) benzoic acid potassium salt

has been studied in bacterial cells by Tamaro et al. (1986). The results indicate that the p-

(3,3-dimethyl- 1-triazeno) benzoic acid potassium salt has a very low mutagenic activity on

the S. typhimurium strains tested, while it is more effective in inducing trp + revertants in E.

coli B strains (Tamaro et al., 1986). Vernole et al. (1987) has shown the in vitro

cytogenetic effects of 1-p-(3-methyltriazeno) benzoic acid potassium salt on human

lymphocytes. It has been tested at different culture times in a range of concentrations from

2 to 500 lg/mL. This chemical causes a dose-dependent increase in the frequency of

chromosomal breaks. Vernole et al. (1988) has reported the clastogenic potential of 1-p-

(3-methyltriazeno) benzoic acid potassium salt on human lymphocytes. They assess the

SCE frequencies induced by benzoic acid potassium salt on human lymphocytes

stimulated by phytohemagglutinin. SCE values increase significantly in a dose-dependent

manner up to 200 lg/mL. However, SCE frequencies, as well as chromosome breaks, do

not increase dramatically (Vernole et al., 1988). Benzoic acid is commonly used as an

antimicrobial substance in many food products. Sarkaya and Solak (2003) have published

that benzoic acid significantly decreases the life period and increases somatic mutation

and recombination (SMART) in Drosophila melanogaster. Ylmaz et al. (2008a) has

reported that benzoic acid significantly increases the chromosomal aberrations and

decreases the mitotic index in the Allium sativum root tips. In another study, Ylmaz et al.(2009) reports that benzoic acid significantly increases chromosomal aberrations, sister-

chromatid exchanges, and micronucleus frequency in human lymphocytes. They indicate

that benzoic acid is a weak genotoxic agent, especially in lower doses, in human

lymphocyte cultures. The mechanism operating in SB- and PB-mediated genotoxicity in

human lymphocytes is currently unknown. However, damages may be mediated by the

inhibition of the activation of XRCC1, PARP-1, and DNA LIG3 proteins, which are

responsible for DNA repair (Wang et al., 2005). The XRCC1 protein plays an important

role in base excision repair; after excision of a damaged base, it stimulates an

endonuclease action and acts as a scaffold in the subsequent restoration of the site (Vidal

et al., 2001; Goode et al., 2002). Although DNA ligase III is predominantly considered as a

component of a single-strand break and base-excision repair where it interacts with

XRCC1 and PARP-1, other observations hint at a potential role of DNA double strand

breaks rejoining as well (Caldecott et al., 1994; Wang et al., 2005). In conclusion SB and

PB are genotoxic additives in human lymphocytes in vitro. However, the mechanisms of

the damaging effects of these substances need to be clarified by more detailed studies.


7/7

All food additives must be kept under continuous observation 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.

Benzoato de sodio y benzoato de potasio.docx

Documents

Transcript of Benzoato de sodio y benzoato de potasio.docx