sodium benzoate-mediated cytotoxicity in mammalian cells

jfbc_432 1034..1046

SODIUM BENZOATE-MEDIATED CYTOTOXICITY INMAMMALIAN CELLS

HYE-WON PARK1,2, EDWIN H. PARK3, HYUNG-MUN YUN1,2 andHYEWHON RHIM1,4

1Life Sciences DivisionKorea Institute of Science and Technology

Seoul 136-791, Korea

2Graduate School of BiotechnologyKorea University

Seoul, Korea

3Seoul International School (SIS)Seongnam, Gyeonggi-do, Korea

Accepted for Publication September 18, 2009

ABSTRACT

Sodium benzoate (SB) is widely used in food chemistry, cosmetics and thepharmaceutical industry. Previous studies suggest possible toxic effects of SBwhen used as a food preservative. However, these studies were confined tomicrobiological systems. Therefore, we investigated the cytotoxic effects of SBin rat-cultured cortical neurons and in human clonal epithelial cells (HeLacells) using cell death assays and the fluorescent measurements of intracellu-lar Ca2+concentration ([Ca2+]i) and mitochondrial transmembrane potential(Dym). We found that SB induced cell death in a dose-dependent manner. Even0.1% (w/v) SB, a concentration typically added to soft drinks, produced asignificant decrease in cell viability in rat cortical and HeLa cells. We alsofound that SB caused [Ca2+]i and Dym increases in a dose-dependent manner.Based on our results, more caution and further cellular studies are requiredwith respect to the use of SB as a food preservative.

PRACTICAL APPLICATIONS

Our research is mainly focused on the toxic effects of sodium benzoate(SB), a common preservative used in foods, in mammalian cell systems.

4 Corresponding author. TEL: +82-2-958-5923; FAX: +82-2-958-5909; EMAIL: [email protected]

DOI: 10.1111/j.1745-4514.2010.00432.x

Journal of Food Biochemistry 35 (2011) 1034–1046.© 2011 Wiley Periodicals, Inc.1034

Recently, there are many mass media reports noticing the damaging effects ofSB on vital parts of mitochondrial DNAs, which based on a study, can causemany diseases including Parkinson’s and other neurodegenerative disorders.In addition, there is a human study that reports that consumption of certainmixtures of artificial food colors and SB preservative are associated withincreases in hyperactive behavior in children. Therefore, our research suggeststhe necessity of further caution and cellular studies before the continued use ofSB as a food preservative can be considered free of risk to human health.

INTRODUCTION

Sodium benzoate (SB), the sodium salt of benzoic acid, is a commonpreservative used in beverages, jams and condiments (Nair 2001; Tsay et al.2007). SB can inhibit bacterial and fungal growth under acidic conditions, inwhich the benzoate moiety is uncharged and therefore readily diffuses acrosscell membranes (Peter 1999). SB is “Generally Recognized as Safe” in foods;however, the allowed concentration of this preservative is limited by the U.S.Food and Drug Administration to 0.1% by weight (Pylypiw and Grether 2000;Nair 2001). Nonetheless, there have been several reports related to toxicity ofSB in foods which suggest the need for limitations on the uses of SB as a foodpreservative. In combination with ascorbic acid (vitamin C) which is containedin many soft drinks, SB may form benzene, a known carcinogenic agent.However, previous studies on the potential carcinogenicity of SB reportednegative results (Nair 2001). Interestingly, it has been reported that SB leads todysfunction of the mitochondrial respiratory chain through mitochondrialDNA damage in yeast (Peter 1999).

Reactive oxygen species (ROS) lead to oxidative damage, and mito-chondria are particularly susceptible to damage induced by ROS, which aregenerated continuously by the mitochondrial respiratory chain (Turrens 1997;Kowaltowski and Vercesi 1999). A number of recent studies have shown thatmitochondrial dysfunction caused by ROS is associated with cell necrosisand apoptosis (Zamzami et al. 1997; Kowaltowski and Vercesi 1999). Weakorganic acid food preservatives, such as sorbic acid and benzoic acids, areknown to function as both prooxidants and mutagens for mitochondrial DNAin yeast (Peter 1999). The presumptive factors causing oxidative stress underaerobic conditions are high superoxide free-radicals (O2

-) and highly reactivehydroxyl radicals (-OH) (Kowaltowski and Vercesi 1999). The production ofhydroxyl radicals is catalyzed by Fe2+ released from ferritin, an intracellulariron storage protein that plays a role in iron-induced lipid peroxidation inmitochondrial membranes (Minotti and Aust 1987; Hermes-Lima et al.1995). Based on results using yeast, Peter (1999) suggested that SB may also

1035SODIUM BENZOATE-MEDIATED CYTOTOXICITY

cause oxidative stress in human cells. Because yeast and human cells sharesignificant sequence homology in many genes associated with human dis-eases, yeast is frequently used for human drug screening (Schwimmer et al.2006; Sturgeon et al. 2006). In recent decades, only a few SB-related studieshave been conducted using human cells. In one such study, treatment oflymphocytes with SB resulted in significant changes in morphology andmembrane ultrastructure compared with untreated lymphocytes (Hu et al.2008). According to Mpountoukas et al. (2008), weak cytostaticity and geno-toxicity, but no cytotoxicity, were observed after exposure of human lympho-cytes to 0.12% SB.

In the presence work, we examined metabolic inhibition- or mito-chondrial dysfunction-induced cell death caused by SB in two differentmammalian cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay (Mosmann 1983). We also tested SB-mediatedchanges in intracellular concentration of Ca2+ ([Ca2+]i) and mitochondrialtransmembrane potential (Dym) as possible causes of SB-mediatedtoxicity.

MATERIALS AND METHODS

Materials

Sodium benzoate and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis,MO). Fura-2-acetoxymethyl ester (Fura-2/AM) and rhodamine-123 were pur-chased from Molecular Probes (Eugene, OR).

Cell Culture

Rat cortical neurons were prepared following procedures described byKim et al. (2008). Briefly, cerebral cortices were isolated from 18-day-oldfetal Sprague-Dawley rats and were incubated with 0.25% trypsin in Hank’sBalanced Salt Solution for 20–30 min at 37C. Cells were then mechanicallydissociated by trituration with fire-polished Pasteur pipettes and plated onpoly-L-lysine-coated coverslips or culture dishes. Cultures were maintained inNeurobasal/B27 medium containing 0.5 mM L-glutamine, 100 units and100 mg/mL penicillin/streptomycin under a humidified atmosphere of 95% airand 5% CO2 at 37C. Experiments were carried out on rat cortical neurons after7 days in culture. Henrietta Lacks (HeLa) cells were grown in Dulbecco’sModified Eagle’s Medium supplemented with 10% fetal bovine serum andpenicillin/streptomycin (100 units and 100 mg/mL) at 37C in a humidifiedatmosphere of 5% CO2 and 95% air.

1036 H-W. PARK ET AL.

Cell Death Assay

Cell viability was measured via detection of dehydrogenase activity inliving cells using a MTT assay. Cells were washed with phosphate bufferedsaline (PBS) and were then incubated with various concentrations of SBin serum-free medium. SB was applied for 24 h using either single or mul-tiple applications. For multiple applications, each indicated concentration(0.01–1%) of SB was applied five times at 3-h intervals as a cumulativeapplication without washout. The final doses of SB were therefore 0.05–5.0% at the end of the 24-h treatment. Optical densities (OD) were measuredwith an automated spectrophotometric multi-well plate reader at a wave-length of 575 nm.

Measurements of Intracellular Ca2+ Concentration and MitochondrialTransmembrane Potential

Fura-2/AM and rhodamine-123 were used as fluorescent Ca2+ in-dicator and mitochondrial transmembrane potential (Dym)-sensitive dye,respectively. Cells were incubated with 5-mM Fura-2/AM for 30–40 minat room temperature or with 5-mM rhodamine-123 for 15–30 min at 37C ina 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-bufferedsolution composed of (in mM): 150 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10HEPES, 10 glucose, pH adjusted to 7.4 with NaOH. The cells were washedtwice with HEPES-buffered solution and placed on an inverted microscope(Olympus, Tokyo, Japan). Cells were illuminated using a xenon arc lamp,and required excitation wavelengths (340/380 nm or 495 nm) were selectedusing a computer-controlled filter wheel (Sutter Instruments Corp., Novato,CA). Emitter fluorescence light was reflected through a 515 or 537 nm long-pass filter to a frame transfer cooled charge-coupled device camera. Allimaging data were collected and analyzed using MetaFluor Imaging Soft-ware (Molecular Devices Corp., Downingtown, PA).

To examine the effect of long-term application of SB on Dym, HeLa cellswere incubated with increasing concentrations of SB in serum-free mediumfor 24 h. After incubation with 5-mM rhodamine-123 for 15–30 min, the cellswere washed three times with PBS and fixed with 4% paraformaldehyde inPBS for 20 min at room temperature. Next, the cells were washed again threetimes with PBS, mounted on glass slides and viewed on a confocal microscope(Olympus).

Data Analysis

All numerical values were analyzed using the GraphPad Prism Version 4program (GraphPad Software Inc., San Diego, CA) and are represented as


means � SE. For all concentration-response curves, best-fit lines wereobtained using the logistical equation y/ymax = 1/(1 + (k1/2/[A])nH) where ymax isthe maximum response, k1/2 is the concentration for half-maximum response(IC50), [A] is the concentration of drug, and nH is the Hill coefficient. Statisticalsignificance was determined using an unpaired Student’s t-test. A P value of<0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Sodium Benzoate-Induced Mammalian Cell Death

To examine mitochondrial dysfunction-induced cell death resulting fromexposure to SB, cultured rat cortical neurons were incubated with variousconcentrations of SB, and the cell viability was then quantified by measure-ment of MTT-reducing activity. Application of SB for 24 h significantlyreduced cell viability as shown in Fig. 1A. SB at concentrations of 0.01–1.0%(w/v) induced rat cortical neuronal cell death in a concentration-dependentmanner with a half-maximal inhibitory concentration (IC50 value) of 0.29%.We also performed SB-induced cell death experiments using human clonalHeLa epithelial cells. Application of SB for 24 h reduced HeLa cell viabilitygradually, but significantly, as shown in Fig. 1B. SB at concentrations of0.01–1.0% (w/v) induced HeLa cell death in a concentration-dependentmanner, with an IC50 value of 0.35%. The toxic effect of 1% SB on HeLa cellswas significantly higher (5.0 � 0.4% cell viability, n = 4, Fig. 1B) than that ofcortical neurons (51.4 � 1.4% cell viability, n = 4, Fig. 1A), suggesting thathuman HeLa epithelia cells are more vulnerable to SB than are cultured ratcortical neurons.

We next examined whether repeated application of SB affected cellviability in HeLa cells. As shown in Fig. 1C, five treatments with SB at 3-hintervals during a 24 h period resulted in increased cell death at all doses tested(0.01–1.0%). The dosages used in this experiment are comparable to levels ofbenzoic acid and benzoates commonly used to preserve acidic foods andbeverages; the typical levels of use for benzoic acid as a preservative in foodare between 0.05 and 0.1%. Even at 0.01% concentration of SB, a lowerconcentration than typically used, five treatments with 0.01% SB resulted in agreater decrease in cell viability (63.2 � 1.0% of control, n = 4) than did asingle application of 0.05% SB (87.4 � 3.7%, n = 4), suggesting accumulativetoxic effects of SB at the mammalian cellular level.

SB-Induced Changes in [Ca2+]i and Dym in Mammalian Cells

Alteration of cellular Ca2+ homeostasis followed by excessive Ca2+

cycling and collapse of mitochondrial transmembrane potential (Dym) are very


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FIG. 1. EFFECTS OF SB-INDUCED CELL DEATH IN MAMMALIAN CELLS(A) Bar graph illustrating effects of single sodium benzoate (SB) application on cell viability in rat

cortical neurons. The cells were treated with 0.01–1.0% (w/v) of SB for 24 h. (B) Bar graphillustrating effects of single SB application on cell viability in human HeLa cells. (C) Bar graph

illustrating effects of multiple SB applications on cell viability in human HeLa cells. The cells weretreated five times with the indicated concentrations of SB at 3 h intervals during a 24-h period,

which are represented using gray bars. Cell viability was measured using an MTT assay. *P < 0.05,**P < 0.01, ***P < 0.001 compared with the buffer-treated cells (CTL, black bar).


critical causes of oxidative stress- and neurotoxin-induced cell death. Wetherefore examined SB-mediated changes in [Ca2+]i and Dym as possiblecauses of SB-mediated toxicity using digital imaging techniques. We firstexamined the effects of SB in [Ca2+]i and Dym in rat-cultured cortical neurons.When [Ca2+]i was measured using a fluorescent Ca2+ indicator, Fura/2 AM,acute application (15 s) of SB (0.1–1.0%) induced a small but notable increasein [Ca2+]i (Fig. 2A). When we monitored Dym in rat cortical neurons using theDym-sensitive dye rhodamine-123, we found that acute application (15 s) of0.1% to 1.0% SB induced a small increase in Dym (Fig. 2B). Normal mito-chondrial transmembrane potential in these cells was confirmed using a potentuncoupling agent, carbonylcyanide p-trifluoromethoxyphenylhydrazone(FCCP) in cultured control neurons.

We further examined whether similar small SB-induced changes in[Ca2+]i and Dym are reproducibly observed in human HeLa cells. As shown inFig. 3A, acute application (15 s) of 0.1% to 1.0% SB also induced a small, butnotable, increase in [Ca2+]i in HeLa cells. The SB-induced increase of [Ca2+]i

in Hela cells was more clearly observed after long-term application (5 min) ofSB, following such application, [Ca2+]i remained at a stable plateau afterwashout of the drug (Fig. 3B). The mean peak 340/380 ratios of [Ca2+]i were0.64 � 0.01 (n = 27), 0.69 � 0.01 (n = 24) and 0.73 � 0.01 (n = 8) after5-min application of 0.1, 0.5 and 1.0% SB, respectively. When we monitoredDym in HeLa cells, we also found that acute application (15 s) of SB (0.1–1.0%) induced a small increase of Dym (Fig. 3C) in cells in which the normalfunction of the mitochondrial transmembrane potential was confirmed usingFCCP. To examine whether long-term SB treatment of HeLa cells was asso-ciated with collapse of Dym, a consequence of severe mitochondrial dysfunc-tion, we assessed Dym using rhodamine-123 fluorescence as previouslyreported (Johnson et al. 1981; Rao and Norenberg 2004). As shown in Fig. 3D,HeLa cells exposed to 0.01–1.0% SB showed a progressive dissipation of Dym

as demonstrated by decreased rhodamine-123 fluorescence. The SB-induceddecrease in rhodamine-123 fluorescent intensity occurred in a dose-dependentmanner; it was notably observed in cells treated with 0.05% SB, rhodamine-123 fluorescence almost disappeared in cells treated with 1.0% SB, and nochange in rhodamine-123 fluorescence was observed in control or 0.01% SBtreated cells. These results suggest that severe impairment of mitochondrialmembrane function occurred following 24-h treatment of human HeLa epi-thelial cells with SB.

This study demonstrates that exposure of mammalian cell cultures toSB results in significant cell death and produces increases in [Ca2+]i as wellas final dissipation of Dym in a time- and dose-dependent manner. TheSB-mediated increase in [Ca2+]i and dissipation of Dym may be the maincauses for the SB-induced cell death assessed using the MTT assay,


suggesting the potential involvement of oxidative stress in SB-mediatedmammalian cell death. A noteworthy observation is that the cultured rat cor-tical neurons were far less affected by treatment with a high dose (1.0%) ofSB than were HeLa cells. The reason for the selective vulnerability of HeLa

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FIG. 2. EFFECTS OF SB-INDUCED [CA2+]I INCREASE AND DyM CHANGE IN PRIMARYCULTURED RAT CORTICAL NEURONS

The acute application of sodium benzoate (SB) (0.1–1.0%, a 15 s duration, ) produced [Ca2+]i

increase (A, n = 10) and transient increase of Dym (B, n = 5) in cultured rat cortical neurons. Cellswere incubated with 5 mM Fura-2/AM for 30–40 min at room temperature or with 5 mM

rhodamine-123 for 15–30 min at 37C in a HEPES-buffered solution. SB was applied to the cells for15 s with a fast perfusion system by a computer-controlled timer. FCCP (1 mM) was used as a

potent uncoupling agent of mitochondria transmembrane potential.


cells to SB is not clearly elucidated in this study. However, mitochondrialheterogeneity between different cell types may explain the phenomenon.According to Huang et al. (2004), primary cultured neurons are known tohave highly localized synaptic signaling mechanisms, which demand highenergy production and utilization. Thus, neurons have a higher sum of Dym/cell area compared with skin fibroblasts, showing that mitochondria mayspecifically adapt to meet the unique energy demands of cell process and cellsurvival. In addition, Fiskum (2000) demonstrated that neuronal mitochon-dria show greater resistance to the effects of Ca2+ overload on the inductionof oxidative stress and subsequent opening of the mitochondrial permeabilitytransition pore than do astrocytic mitochondria.

As an important stimulator of mitochondrial ROS generation, increasedlevels of intracellular Ca2+ ions promote a nonspecific inner membrane

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FIG. 3. EFFECTS OF SB-INDUCED [CA2+]I AND DyM CHANGES IN HUMAN HELA CELLS(A and B) Effect of sodium benzoate (SB)-induced [Ca2+]i increase using Ca2+ imaging techniques.

(A) The acute application of SB (0.1–1.0%, 15 s duration, , n = 13) and (B) the long-termapplication of SB (5 min duration, �: control [n = 5], �: 0.1% SB [n = 27], �: 0.5% SB [n = 24],�: 1.0% SB [n = 8]) produced [Ca2+]i increase in a dose-dependent manner. (C and D) Effects ofSB-induced Dym alteration. (C) The acute application of SB (0.1–1.0%, n = 5) induced transientincrease in Dym. The SB-induced Rh123 fluorescence was normalized by 1 mM FCCP-induced

fluorescence. (D) Rhodamine-123 fluorescent intensity (green) after long-term application of SB(0.01–1.0%, 24 h treatment). These experiments were repeated three times using different

cell seeding.


permeabilization referred to as mitochondrial permeability transition (MPT),which can lead to collapse of mitochondrial transmembrane potential (Dym).The rapid changes in permeability occurring during MPT lead to mitochon-drial swelling, rupture of the mitochondrial outer membrane and the release ofcytochrome c and other proapoptotic proteins (Richter 1993; Lemasters andNieminen 1997; Green and Reed 1998; Kowaltowski and Vercesi 1999). If SBproduces dysfunction of the mitochondrial respiratory chain through mito-chondria DNA damage in the mammalian system as Peter (1999) reported inyeast, our data showing the effect of SB in [Ca2+]i increase suggest an essentialand additive pathway of SB-mediated toxicity in mammalian cells. SB has alsobeen shown to produce toxic effects on marine bacteria and to affect thesurvival rate of zebrafish larvae (Xu et al. 2005; Tsay et al. 2007). The con-centrations of SB required for half-maximal toxic response were 0.06% and0.15% for marine bacteria and zebrafish larvae, respectively. Although thesevalues are much lower than the IC50 values of 0.29–0.35% that we found in thepresent study, it is difficult to compare or discuss the relative effective dosevalues in terms of mitochondrial heterogeneity or oxidative stress becausecomparative data have been obtained only in nonmammalian systems.

The maximum permitted concentration of SB for soft drinks, 0.1%,produced a significant decrease in cell viability in cortical and HeLa cells(86% and 84% of the control, respectively). Furthermore, repeated treatmentswith SB within 24 h produced a greater toxic effect than did a comparativesingle dose as shown in Fig. 1B,C. Although it is necessary to compare in vitroand in vivo concentrations of SB by means of bioavailability studies, theconcentrations (0.01–1.0%) of SB employed in the present study are in therange of the typical usage levels of SB added to foods as a preservative. SB israpidly absorbed from the gastrointestinal tract and is subsequently conjugatedwith glycine in the liver (Bridges et al. 1970). Hippuric acid (N-benzoylglycine), the metabolized product of SB formed by conjugation withglycine, is excreted via the urine. Although the accumulation of benzoic acidor SB in the body is not expected under this process, continuous and excessintake of beverages containing SB should be cautioned against because ofpotential damaging effects of SB in the gut or liver. In rats, treatment with1,090 mg/kg/day SB resulted in lesions of several organs including intestineand liver (Nair 2001; Williams and Lock 2005). Higher dose (3,750 mg/kg/day) of SB led to diarrhea and intestinal hemorrhage in rats (Nair 2001).Although detailed data for tissue distribution of SB in animals is not availableyet, animal studies suggest that i.p. injected SB may reach the brain andkidney. SB may cross the blood brain barrier (Kido et al. 2000; Tamai andTsuji 2000) and modulate kidney enzyme activities (Williams and Lock 2005).Quantitation of SB levels in the kidney showed a dose- and time-relatedpresence of SB, with the maximum concentration being observed 1 h


postdosing; when 1,000 mg/kg of SB administered, 3.24–3.45 mmol/g of SBwas detected within 1/2–1 h in the kidney. These results document a certaindegree of bioavailability of SB and underscore the possibility that SB mayexert potentially damaging effects in the brain and kidney. In summary, ourresults suggest the possibility that the continuous infusion of concentrated SBmay produce accumulative cytotoxicity through an increase in [Ca2+]i andmitochondrial dysfunction in mammalian cells. Taken together with previousreports of mitochondrial damage resulting from exposure to SB, furthercaution and cellular studies are required before the continued use of SB as afood preservative can be considered free of risk to human health.

ACKNOWLEDGMENTS

This work was supported by KIST Core-Competence program, KOSEFGrant (No. 20090079000), the Pioneer Grant and Brain Research Center of the21st Century Frontier Research Program (No. 2009K001265) funded by theKorea government (MEST).

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sodium benzoate-mediated cytotoxicity in mammalian cells

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