Assessment of cytotoxic and apoptotic effects of benzaldehyde using different assays

Abstract

Benzaldehyde (BA) occurs naturally in a number of plants, including cherry, fig and peach fruit and carnation flowers at therapeutic doses. In addition, it is used in cosmetics, personal care products and food as a preservative. In this study, we aimed to determine the cytotoxic and apoptotic effects of different concentrations of BA on cultured human lymphocytes using lactate dehydrogenase assay, cell proliferation (water-soluble tetrazolium salts-1) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) test (apoptotic test) as a group of cytotoxicity tests at 6th and 24th h on human lymphocyte cell culture. The cytotoxicity increased when cells were treated with 10, 25 and 50 μg/mL concentrations of BA (p < 0.05). Moreover, treatment of the cells with the same concentrations significantly decreased the cell number at the 6th and 24th hours (p < 0.05). TUNEL assay results also show that the concentration of BA at 10, 25 and 50 μg/mL caused DNA damage significantly (p < 0.05). According to our results, the toxic and genotoxic effects of BA have to be further evaluated before using in cosmetic and food products.

Introduction

In many developed countries, food additives are used enormously for the last 30 years. Increased consumption of the food additives has increased health problems such as eczema, asthma, headaches, migraine, cancer, depression, behavioral problems, sleeping problems, allergic itching, urticaria, diarrhea (especially for kids) and hyperactivity. ¹ Benzaldehyde (BA) and its derivatives are used in the food and beverages as an additives and preservative, drugs, pharmaceutical, perfume, soap and as a solvent in oils, lacquers, inks, resin and plastics. ^1–4

Human exposure to BA (daily) is through foodstuffs. ⁵ Adult consumption of BA through foodstuff is approximately 48.2 mg/day. ⁶ Concentrations of BA when used directly range from 36 to 840 ppm as a flavoring agent in various food and beverage products, such as alcoholic and nonalcoholic beverages, ice cream, candy, gelatin, pudding and chewing gum. ^7–9 Feron et al. stated that BA also naturally occurs in alcoholic beverages (0.01–0.8 ppm), dairy products, meat (0.03–0.13 ppm), poultry, fruit (0.0003–8.9 ppm), vegetables (up to 1.2 ppm), coffee, tea, cocoa (0.7–7.4 ppm) and spices. ¹⁰ BA uptake is from cell membrane to cytoplasm where it can react with DNA, proteins and enzymes including glutathione peroxidase (GPx). ¹¹ Decreasing activity of GPx in turn can increase oxidative stress. BA is also metabolized to benzoic acid and benzyl alcohol by cytochrome P450 that can react with DNA. BA and its derivatives cause DNA damage by binding to DNA and increasing oxidative stress. ¹² (Figure 1)

Figure 1.

The effects of BA on antioxidant system, lipid peroxidation and DNA damage. BA: benzaldehyde.

BA is generally nongenotoxic. BA in different concentrations (from 21 to 3333 µg/plate) was not mutagenic in Salmonella gene mutation assays (Ames). ^8,13,14 Gee et al. reported that 50–1000 µg/mL concentrations of BA are not mutagenic in eight different Salmonella typhimurium strains and a mixture of the base-specific strains. ¹⁵ In addition, in Drosophila sex-linked recessive lethal assay, ¹⁶ sister chromatid exchange and chromosomal aberration test systems, it does not have any genotoxic and mutagenic effects. The using concentrations of BA did not increase chromosomal aberrations and sister chromatid exchange in Chinese hamster ovary (CHO) cells. ⁸ In another study, induction of chromosomal aberration and sister chromatid exchange by BA was found in CHO cells (in concentration 1600 µg/mL) ¹⁷ and human lymphocytes (in concentration 2 mM). ¹⁸ Recently Demir et al. reported that BA exposure to 10 mM (7.59) and 25 mM (12.57) concentrations in comet assay test system significantly increased tail moment, while it demonstrated positive results when exposed to 10 mM (14.61 ± 3.26), 25 mM (23.28) and 50 mM (10.14). ³ On the other hand, some researchers showed that BA and its derivatives showed slightly higher cytotoxicity against human tumor cell lines, when compared with normal cells. BA derivatives such as cyclodextrin BA inclusion compound has cytotoxic effects on different human cancer cell lines including human myelogenous leukemia (HL-60, ML-1 and KG-1), human oral squamous cell carcinoma (HSC-2, HSC-3 and HSC-4) and human glioblastoma (T98G, U87MG). ¹⁹ In another study, antiproliferative effect of BA and its derivative compound 5, 6-benzylidene-l-ascorbate (SBA) was investigated on oral squamous cell carcinoma, glioblastoma and myelogenous leukemia. BA showed much higher tumor-specific cytotoxicity than SBA. ²⁰

The aim of this study was to assess the possible genotoxicty and cytotoxicity of different concentrations of BA using current cell culture techniques (lactate dehydrogenase (LDH), water-soluble tetrazolium salts-1 (WST-1) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay) on cultured human lymphocytes.

Materials and methods

All products used for cell culture (Roswell Park Memorial Institute medium (RPMI), fetal bovine serum (FBS), ficoll, l-glutamine, penicillin and streptomycin) were purchased from Biochrom AG (Berlin, Germany) and Biological Industries (Kibbutz Beit-Haemek, Israel). WST-1, LDH and TUNEL kits were bought from Roche (Mannheim, Germany). Benzaldehyde (BA) was obtained from Sigma-Aldrich, Missouri, USA.

Isolation of PBMCs

The peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples by density gradient centrifugation with ficoll. For LDH and WST-1 assays, PBMCs were incubated in RPMI 1640 medium with 2 mmol/L l-glutamine, 10% FBS and antibiotics (penicillin and streptomycin) in the presence of 5% CO₂ at 37°C at 1.0 × 10⁶ cells/mL ²¹ with BA concentrations (1, 10, 25 and 50 µg/mL) for 24 and 48 h. All donors gave full written informed consent.

LDH assay

Isolated cells were exposed to the test compounds for the LDH assay, which was performed using Cytotoxicity Detection Kit LDH (Roche). For this assay, a positive control, leading to 100% cytotoxicity by lysing the cells completely was included. The positive control was 2% Triton X-100 solution in the assay medium, as proposed by the manufacturer. After the preincubation of cells, before addition of the test compounds, the growth medium was exchanged from medium containing 5% FBS to medium containing only 1% FBS. For testing the LDH activity, 100 μL of culture medium was transferred to a new 96-well plate at 24th and 48th hours. The reaction solution, 100 μL, from the kit, containing the detection dye and the catalyst were then added and the absorbance was measured after 30 min at 490 nm with 655 nm as reference wavelength in an enzyme-linked immunosorbent assay (ELISA) reader (BioTek-Power WaveS, Winooski, USA). Culture medium without cells was used as the blank. As for the other assays, background values from wells without cells were subtracted and average values are calculated. Mean ± SD values are presented in Figure 2. Cytotoxicity was then calculated according to the following equation:

$\begin{array}{rl}& \mathrm{C}\mathrm{y}\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{t}\mathrm{y}\left(\mathrm{\%}\right)\\ & =\left(\mathrm{e}\mathrm{x}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}-\mathrm{n}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)/\\ & \left(\mathrm{p}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{n}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)\times 100.\end{array}$

Proliferation assay (WST-1)

For WST-1 assay, cells were cultured in microplates (tissue culture grade, 96 wells and flat bottom) in a final volume of 100 μL/well culture medium in a humidified atmosphere (37°C, 5% CO₂). Cell proliferation reagent WST-1 was added in a 10 μL/well volume. Cells were incubated for 4 h in a humidified atmosphere (37°C, 5% CO₂). The absorbance of samples  was measured at 420 nm with ELISA reader (BioTek-PowerWaveS, USA). Mean ± SD values are presented in Figure 3. Culture medium without cells was used as the blank.

TUNEL assay

DNA fragmentation is an indicator for detecting the late apoptosis. TUNEL (Roche) is an assay that is composed of an enzyme solution and labeling solution, which detects the nicks (single strand breaks) in DNA and binds to free 3'–OH ends. Addition of dUTPs to 3'–OH ends by terminal deoxynucleotidyl transferase causes the labeling of DNA. Fluorescently labeled ends were detected using fluorescent microscopy. Blood samples were obtained by vein puncture from the nonsmoking, healthy volunteers. Lymphocyte cultures were set up by adding 0.5 mL of heparinized whole blood to RPMI 1640 chromosome medium supplemented with 15% heat-inactivated fetal calf serum, 100 IU/mL streptomycin, 100 IU/mL penicillin and 1% l-glutamine. Lymphocyte proliferation was induced by 1% phytohemagglutinin. Carbon tetra chloride as a positive control (in concentrations of 5 μM) and different concentrations (1, 10, 25 and 50 µg/mL) of BA were added to the cultures. The cultures were incubated at 37°C for 48 h. The cultures were centrifuged at 800 × g for 10 min. The following fixation protocols were applied: (1) 3:1 acetic acid–methanol for 10 min at 4°C 1200 r/min; (2) 2:1 acetic acid:methanol for 10 min at 4°C 1200 r/min and (3) 4% phosphate-buffered saline (PBS) (0.01 M and pH 7.4) for 5 min at room temperature followed by washing in PBS. Cells spread on the slides then ²² , DNA fragmentation of cells was determined with the In Situ Cell Death Detection Kit, Fluorescein (Roche), according to the manufacturer’s instructions. The results were analyzed by fluorescence microscopy.

Statistical analysis

The statistical analysis of LDH and WST-1 data was performed using Mann–Whitney U test. The statistical analysis of TUNEL was evaluated using χ ² test. A value of p < 0.05 was accepted as statistically significant. For these procedures, SPSS 15.0 version for Windows (SPSS Inc, Chicago, Illinois, USA) was used. Results were expressed as mean ± SD.

Results

LDH assay results

The cytotoxic effects of different doses of BA on human lymphocyte cells at the 6th and 24th hours were represented in Figures 2 and 4. All of BA concentrations caused releasing of LDH and cytotoxicity. Increases in the LDH level were observed after treatment with concentrations (1, 10, 25 and 50 µg/mL) of BA when compared with negative control groups. This increase was found to be statistically significant (p < 0.05). The highest cytotoxic effect of BA as shown in Figures 2 and 4 was in the 50 µg/mL concentration.

Figure 2.

The cytotoxic effects of different concentrations of BA on human lymphocyte culture at 6th and 24th hours. ^a p < 0.05 compare with control group, ^b p < 0.05 compare with BA-1 group, ^c p < 0.05 compare with BA-10 group, ^d p < 0.05 compare with BA-25 group at 6 h, ^e p < 0.05 compare with control group, ^f p < 0.05 compare with BA-1 group, ^g p < 0.05 compare with BA-10 group and ^h p < 0.05 compare with BA-25 group at 24 h. BA: benzaldehyde.

Cell proliferation assay results (WST-1)

Figures 3 and 4 show the results of the cell viability part of the present study, including the WST-1 assay in the control and experimental groups. Increases in the cell proliferation were observed after treatment with 1 µg/mL concentration of BA. Statistical analysis showed a significant decrease in cell viability between 10, 25 and 50 µg/mL concentrations of BA with control group at 6th hour (p < 0.05). In addition, different concentrations of BA (25 and 50 µg/mL) caused a decrease in cell proliferation at 24th hour (p < 0.05). BA at concentrations of 25 and 50 µg/mL decreased cell proliferation significantly at all time points during the 6th and 24th hours (p < 0.05) in comparison with the all concentrations and control. LDH release significantly correlated with decreased cell proliferation. BA at concentrations of 25 and 50 µg/mL increased LDH release significantly, and the increasing of cell viability was found to be at all time points during 6th and 24th hours (p < 0.05) as compared with control group.

Figure 3.

The cell proliferation effects of different concentrations of BA on human lymphocyte culture. ^a p < 0.05 compare with control group, ^b p < 0.05 compare with BA-1 group, ^c p < 0.05 compare with BA-10 group, ^d p < 0.05 compare with BA-25 group at 6 h, ^e p < 0.05 compare with control group, ^f p < 0.05 compare with BA-1 group, ^g p < 0.05 compare with BA-10 group and ^h p < 0.05 compare with BA-25 group at 24 h. BA: benzaldehyde.

Figure 4.

Correlation of cell viability (%) and cytotoxicity (%) treated with different concentrations of BA at 6th and 24th hours. BA: benzaldehyde.

TUNEL assay result

Figures 5 and 6 show the results of the apoptotic cells, including the TUNEL assay in the control and experimental groups. Significant inductions in the apoptotic cell percentage were found in all BA-treated groups except 1 µg/mL BA when compared with the control (p < 0.05). This increase in the percentage of apoptotic cells correlated with the increase in BA concentration. The most effective concentration of BA was 50 µg/mL and the lowest effective concentration of BA was 1 µg/mL (Figure 5).

Figure 5.

The apoptotic effects of different BA concentrations on the human lymphocyte cells at 48th hours. ^a p < 0.05 compare with control group, ^b p < 0.05 compare with CCI₄ group, ^c p < 0.05 compare with BA-1 group, ^d p < 0.05 compare with BA-10 group and ^e p < 0.05 compare with BA-25 group.

Figure 6.

Views of comparison effects of different concentrations of BA (50 µg/mL (a), 10 µg/mL (b and d) and 25 µg/mL (c)) observed fluorescence microscope on human lymphocyte culture cells (10× magnification). White arrows: DNA breaks, red arrow: human lymphocyte’s nucleus, blue arrow: a set of chromosome pair.

Discussion

There is no sufficient and reliable data in literature about the genotoxic and cytotoxic effects of different BA concentrations on cultured human cells and animal cells. Previous studies on the genotoxicty of these BA show quite different results depending on the use of model animals, cell lines or assay. Until now, the used test assays show high variation in sensitivity to assess the cytotoxic and genotoxic effects of BA on cells.

Many studies reported that only the high concentrations of BA are cytotoxic and genotoxic on animals and cell lines, whereas its lower concentrations are not. ⁹ In previous research conducted in Ames test ^8,13,14,23 and Drosophila sex-linked recessive lethal assay, ¹⁶ BA was not found mutagenic. Also, it has not exhibited genotoxic activity in unscheduled DNA synthesis assay ²³ and in assays for sister chromatid exchanges in both CHO cells ¹⁷ and human lymphocytes. ¹⁸

On the other hand, Demir et al. reported BA has genotoxic activity in two different test systems: Drosophila wing spot test and comet assay in human lymphocyte cells. ^3,24 In comet assay results, 10, 25 and 50 mM concentrations of BA significantly increased the percentage of  tail DNA positive results when exposed to 10 mM (14.61), 25 mM (23.28) and 50 mM (10.14). ³ The results obtained from balancer-heterozygous wings (multiple wing hairs (mwh)/TM3) exposed to 25 and 50 mM concentrations of the BA demonstrated clearly positive results for small single spots, total mwh spots and total spots. ²⁴

Another study reported BA exhibited genotoxic activity in the mouse lymphoma assay ^23,25 and chromosomal aberration in CHO cells. ²⁶ Heck et al. (1989) and McGregor et al. (1991) showed 600 and 800 µg/mL BA have genotoxic effect on mouse L5178Y lymphoma cells, respectively. ^23,25 Moreover, higher BA concentration (1200 µg/mL) was used; absolutely this concentration was mutagenic on chromosomal aberration test in CHO.

In our study, we showed that not only higher concentrations of BA but also its lower concentrations caused cytotoxic and genotoxic effects on cultured lymphocytes. BA concentration in 10, 25 and 50 µg/mL increased cytotoxicity and genotoxicity on cultured human lymphocyte cells. LDH level in culture medium was higher than control group significantly at 6th and 24th hours. As BA inhibits the GPx activity, oxidative stress and lipid peroxidation can increase in cells, therefore, LDH is released from cells to culture medium. ^11,12 Also, we presented that different concentrations of BA inhibited cell viability and increased apoptotic cells. BA and its metabolites such as benzoic acid and benzyl alcohol can bind  to DNA in cells; this can be the result of genotoxic and apoptotic effects in cells. ¹²

Conclusion

In conclusion, the study showed BA has cytotoxic and apoptotic effects. Chemicals used in cosmetic product and food preservatives are doubtful about the effects on human health. There are many controversial results in the toxic and genotoxic effects of BA and its derivatives. Test assays used to determine the harmful impact of the chemicals are different in sensitivity in assessing the cytotoxic and genotoxic effects on cells. Therefore, further studies should be carried out with different test systems.