Protective Effects of Garlic Sulfur Compounds

Protective effects of garlic sulfur compounds

PROTECTIVE EFFECTS OF GARLIC SULFUR COMPOUNDS AGAINST DNA DAMAGE INDUCED BY DIRECT- AND INDIRECT-ACTING GENOTOXIC AGENTS IN HEPG2 CELLS.[1]

C. Belloir, V. Singh, C. Daurat, M.H. Siess and A.M. Le Bon*

Institut National de la Recherche Agronomique, Unité Mixte de Recherche de Toxicologie Alimentaire, BP 86510, 17 rue Sully, 21065 Dijon Cedex, France

* Corresponding author : Anne-Marie Le Bon,

e-mail: ; Tel: +33 380693215; Fax: +33 380693225

Running title : Protective effects of garlic sulfur compounds

Key-words: garlic; organosulfur compounds; HepG2 cells; DNA damage; comet assay.

Abstract

The aim of this study was to assess the antigenotoxic activity of several garlic organosulfur compounds (OSC) in the human hepatoma cell line HepG2, using comet assay. The OSC selected were allicin (DADSO), diallyl sulfide (DAS), diallyl disulfide (DADS), S-allyl cysteine (SAC) and allyl mercaptan (AM). To explore their potential mechanisms of action, two approaches were performed : (i) a pre-treatment protocol which allowed study of the possible modulation of drug metabolism enzymes by OSC before treatment of the cells with the genotoxic agent; (ii) a co-treatment protocol by which the ability of OSC to scavenge direct-acting compounds was assessed. Preliminary studies showed that, over the concentration range tested (5-100 µM), the studied OSC neither affected cell viability nor induced DNA damage by themselves. In the pre-treatment protocol, aflatoxin B1 genotoxicity was significantly reduced by all the OSC tested except AM. DADS was the most efficient OSC in reducing benzo(a)pyrene genotoxicity. SAC and AM significantly decreased DNA breaks in HepG2 cells treated with dimethylnitrosamine. Additionally, all the OSC studied were shown to decrease the genotoxicity of the direct-acting compounds, hydrogen peroxide and methyl methanesulfonate. This study demonstrated that garlic OSC displayed antigenotoxic activity in human metabolically competent cells. This mechanism could be part of the cancer preventive of garlic in man.

1. Introduction

It has been well established that a diet rich in vegetables and fruit is associated with cancer risk reduction (WCRF and AICR, 1997). As such, Allium vegetables have particularly attracted attention as potential chemopreventive vegetables. Available epidemiologic evidence is consistent in showing a correlation between consumption of high amounts of garlic and a reduced risk of cancer at most sites, particularly cancers of the stomach and colon (Fleischauer and Arab, 2001). Protective effects of garlic are attributed to organosulfur compounds (OSC) found in these vegetables. Numerous in vitro and in vivo experimental studies have shown that OSC exhibit a wide range of biological activities, including antimutagenic, antioxidant, antiproliferative and apoptotic effects which would protect against critical events that are involved in cancer process (Le Bon and Siess, 2000; Thomson and Ali, 2003).

Genetic damage represents a crucial initiating event in carcinogenesis. A number of studies have been carried out aimed at identifying OSC which inhibit the genotoxic activities of chemical carcinogens but in most cases, the evidence for their protective effects was demonstrated using the Ames test (Le Bon and Siess, 2000). However the relevance of this test to the human situation is restricted by the experimental conditions employing mutant bacterial cells and an exogeneous metabolic activation mixture from rat liver. Comparatively, less studies have reported antimutagenic activities of OSC in mammalian cellular models and investigations in human metabolically competent cell lines are scarce (Le Bon and Siess, 2000).

We therefore investigated the protective effect of several garlic OSC towards DNA damage induced by carcinogens in the human hepatoma cell line HepG2. HepG2 cells have retained many of the functions of normal liver cells (Knowles et al., 1980) and express different inducible xenobiotic metabolising enzymes (Doostdar et al., 1993; Knasmuller et al., 1998). These cells have been extensively used in metabolism studies and are recommended for the detection/screening of mutagens (Natarajan and Darroudi, 1991; Knasmuller et al., 1998; Valentin-Severin et al., 2003). To evaluate the effects of OSC on DNA damage, we used the single cell gel electrophoresis (comet) assay which is a rapid and sensitive technique to analyse DNA damage at the individual cell level (Fairbairn et al., 1995; Singh et al., 1988). Recent studies have shown that the comet assay can successfully be used to demonstrate the antigenotoxic effects of vegetable constituents in HepG2 cells (Aherne and O'Brien, 1999; Laky et al., 2002; Dauer et al., 2003).

Five OSC were selected for this study (figure 1): allicin (DADSO), diallyl sulfide (DAS), diallyl disulfide (DADS), S-allyl cysteine (SAC) and allyl mercaptan (AM). DADSO is the main thiosulfinate identified in crushed raw garlic (Lawson, 1996). DAS and DADS are typical lipophilic sulfur compounds found in crushed garlic and garlic oils; they result from the transformation of allicin (Lawson, 1996). SAC is a hydrophilic compound found in high amount in aged garlic extract (Lawson, 1996). Conversely, AM has been identified as an in vivo metabolite of DADS (Germain et al., 2002) and is assumed to be also a metabolite of DADSO (Lawson, 1998).

To explore their potential mechanisms of action, two approaches were performed : (i) a pre-treatment protocol which allowed study of the possible modulation of drug metabolism enzymes by OSC before treatment of the cells with the genotoxic agent: in this case, aflatoxin B1 (AFB1), benzo(a)pyrene (BaP) and N-nitrosodimethylamine (DMN) were chosen since they are metabolised by different cytochrome P450 (CYP) and phase 2 enzymes; (ii) a co-treatment protocol by which the ability of OSC to scavenge reactive species was assessed: direct-acting compounds such as hydrogen peroxide (H2O2) and methylmethane sulfonate (MMS) were selected for this study.

2. Materials and methods

2.1. Chemicals

Collagen IV, dimethylsulfoxide (DMSO), propidium iodide, low melting-point agarose (LMP), AFB1 (CAS n° 1162-65-8), BaP (CAS n° 50-32-8), DMN (CAS n° 62-75-9), H2O2 (CAS n° 7722-84-1), MMS (CAS n° 60-27-3), DAS (purity>97%, CAS n° 592-88-1), DADS (purity>80%, CAS n° 2179-57-9) and AM (purity>80%, CAS n° 870-23-5) were purchased from Sigma-Aldrich (La Verpillère, France). Dulbecco's Modified Eagle's Medium (DMEM) with glutamax, phosphate-buffered saline without calcium and magnesium (PBS), heat-inactivated fetal bovine serum (FBS) and trypsin (0,05%) - EDTA (0,02 %) solution were obtained from Invitrogen (Cergy Pontoise, France). Normal melting point agarose was from Coger Promega (Charbonnières les Bains, France). DADSO and SAC were synthesised by Pr. J. Auger (Université F. Rabelais, Tours, France).

Stock solutions of DAS, DADS, DADSO, SAC and AM were prepared in sterile water and diluted (1/100) in DMEM supplemented with 2.5% FBS just before use. AFB1 and BaP were initially dissolved in DMSO then diluted in the culture medium so that the final concentration of DMSO was 0.1%. DMN, H2O2 and MMS were dissolved in the culture medium just before use.

2.2. HepG2 cell culture conditions

HepG2 cells were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, Wiltshire, UK) at passage 97. Cell stocks (aliquots of 1x106 viable cells in DMEM supplemented with 20% (v/v) FBS and 5% (v/v) DMSO) were stored at passage 103 in liquid nitrogen. After thawing, cells were routinely grown in DMEM supplemented with 10% FBS in T75 flasks (Falcon, Merck Eurolab, Lyon, France) coated with a thin layer of collagen. Cells were subcultured at 5 days intervals using an initial inoculum of 3x106 cells per flask and medium was changed every 2 days. Cells were incubated at 37°C in a humidified atmosphere containing 10.2% CO2 and were cultured in the absence of antibiotics. Treatments were performed on HepG2 cells at passages between 106 and 110.

2.3. Cell treatments

2.3.1. Cytotoxicity studies

By using an electronic multichannel pipet (Biohit Proline, Merck Eurolab), cells were seeded on special black collagen-precoated 96-well tissue cultureplates (Falcon Optilux, Merck Eurolab) at a density of 25000 cells per well and incubated in 0.2 ml DMEM supplemented with 2.5% FBS. In these culture conditions, confluence was reached after 72 hours. Separate experiments were performed to evaluate the cytotoxicity of OSC and genotoxic compounds. Thirty-two hours or 68 hours after seeding, cells were exposed to increasing concentrations of OSC (5-100 µM) for 20 hours or 4 hours, respectively. Concerning genotoxic compounds, cells were treated by direct-acting or indirect-acting agents 4 hours or 20 hours before harvesting, respectively. At the end of treatment, cell viability was assessed using the neutral red assay as described below.

2.3.2. Genotoxicity studies

Individual wells of classic 96-well tissue culture plates (Falcon, Merck Eurolab) coated with collagen were inoculated with 0.2 ml of the culture medium containing 1.25x105 cells/ml in DMEM supplemented with 2.5% FBS. To check the absence of genotoxicity of OSC, cells were challenged for 20 hours with increasing concentrations of OSC (5-100 µM) 32 hours after seeding. After the treatment, cells were washed with PBS, then harvested and processed for comet assay.

2.3.3. Anti-genotoxicity studies

Anti-genotoxicity studies were carried out using classic 96-well tissue culture plates (Falcon, Merck Eurolab) coated with collagen and inoculated with 0.2 ml of DMEM supplemented with 2.5% FBS and containing 1.25x105 cells/ml.

The experimental design of these studies is presented in figure 2. In the pre-treatment study (protocol A), cells were exposed 32 hours after seeding to increasing concentrations of DAS, DADS, DADSO, SAC and AM (5 to 100 µM) for 20 hours. Then cells were washed twice with 0.2 ml PBS and treated for 20 hours with the indirect-acting genotoxic compound (AFB1 (25 µM) or BaP (25 µM) or DMN (75 mM)). In the co-treatment study (protocol B), cells were simultaneously treated with the garlic compound (5 to 100 µM) and the direct-acting genotoxic compound (H2O2 (0.75 mM) or MMS (75 µM)) for 4 hours before confluence. After the treatment, cells were washed with PBS, then harvested and processed for comet assay.

2.4. Cytotoxicity assays

2.4.1. Neutral red assay

In the preliminary cytotoxicity studies, cell viability was determined by the neutral red assay using the fluorimetric method described by Rat et al. (Rat et al., 1994). Briefly, after treatments, cells were washed twice with PBS (0.2 ml per well). The medium was replaced with 0.2 ml DMEM per well containing 50 µg/ml neutral red and cells were incubated for 3 hours at 37°C. Thereafter, the cells were washed twice with 0.2 ml PBS to eliminate extracellular neutral red. The incorporated dye was eluted from the cells by adding 0.2 ml elution medium (50% ethanol supplemented with 1% acetic acid, v/v) into each well followed by gentle shaking of the microplate for 1 hour (Titramax, Merck Eurolab, France). Fluorescence was measured using a microplate spectrofluorometer (Dynex Fluorolite 1000, Dynatech laboratories, Chantilly, VA, USA) with excitation and emission wavelengths fixed at 535 nm and 600 nm respectively. Results were expressed as percentage of neutral red fluorescence in untreated cells.

2.4.2. Trypan blue exclusion assay

Throughout the antigenotoxicity studies, the cell viability was routinely checked using the trypan blue exclusion assay. Cell suspensions were stained with the trypan blue (0.4%) and the percentage of viable (uncolored) and dead (colored) cells was scored. The cell viability was greater than 75% in all the experiments.

2.5 Comet assay

2.5.1. Cell recovery

At the end of treatments, cells were washed twice with 0.2ml PBS and incubated with trypsin-EDTA (50µl per well) for 1 min. Excess of trypsin-EDTA was removed by turning over the plate. Cells were then incubated at 37°C for 10 min before adding DMEM supplemented with 10% FBS (50 µl per well). Cells were gently detached from wells by pipetting the medium using a micropipette equipped with a 0.2 ml tip. Cells from 5 wells treated in the same way were pooled. The cell suspensions were kept on ice and assayed immediately.

2.5.2. Experimental procedure

The alkaline version of the comet assay was carried out according to the procedure of Singh et al. (Singh et al., 1988), with slight modifications. Microscope slides (Kimble Kontes, Baillet-en-France, France) were pre-coated by dipping them in 1% normal melting point agarose and allowed to dry overnight. After treatment, 20 µl of cell suspension (around 10000 cells) was mixed with 75 µl of 0.5% LMP agarose maintained at 37°C. This solution was rapidly pipetted over the pre-coated slide and covered with a coverslip to allow homogeneous spreading of agarose. Solidification of the agarose layer was performed on an ice-cold tray for 5 min. After gently removing the coverslip, 75 µl of 0.5% LMP agarose was layered, covered as described before, then allowed to solidify for 5 min over ice. After removal of the coverslip, the slides were immersed into the lysing solution (1% sodium sarcosinate, 2.5M NaCl, 100 mM Na2-EDTA, 10mM Tris-HCl, pH10, 1% TritonX-100 and 10% DMSO) for 60 min at 4°C. Then the slides were placed in a 1-l horizontal electrophoretic tank (Maxigel, Apelex, Paris, France). The reservoir was filled with freshly prepared electrophoresis buffer (1mM Na2-EDTA, 300mM NaOH, pH13). DNA unwinding was allowed to proceed for 40 min at room temperature. Electrophoresis was conducted for next 20 min at 25V (300 mA) using a compact power supply (ST600, Apelex). All the steps were carried out under yellow light to prevent additional DNA damage. After electrophoresis, the slides were rinsed three times with 0.4M Tris, pH 7.5. For preservation, slides were fixed by dipping them 1 min in 96% ethanol and were allowed to dry at air temperature until staining.

2.5.3. Quantification

Each slide was stained by rehydrating it with 75 µl propidium iodide (2µg/ml water) and was covered with a coverslip. Slides were examined at x200 magnification using a fluorescence Nikon E600 microscope (excitation filter 515 nm, barrier filter 560 nm). Image analysis was performed using the software Komet IV (Kinetic Imaging, Liverpool, UK) on 100 randomly selected cells (50 cells from each of two replicate slides). The extent of DNA damage was quantified by the Olive Tail Moment (OTM) which is defined as the product of the mean distance of DNA migration in the tail and the percentage of DNA in the comet tail (Olive et al., 1990).

2.6. Statistical analysis

The statistical analyses were carried out using the Stat Box Pro software (Addinsoft, version 5). Since the OTM data do not follow a normal distribution, the non-parametric Mann-Whitney-U test was used to perform pairwise comparisons between OSC-treated groups and genotoxic-treated groups. Results were considered to be significant if a P-value  0.05 was recorded. The data are presented as Box-Wisker plots which represent the range (minimum and maximum), medians and the 25th and 75th percentile of the OTM of 3 to 5 experiments.

3. Results

3.1. Cytotoxicity studies

Cytotoxicity studies (neutral red assay) were performed prior to antigenotoxicity studies in order to select non-toxic concentrations of chemicals. Over the concentration range tested (5-100 µM), none of the OSC was found to affect cell viability (data not shown). This concentration range was therefore used in subsequent studies. Selected doses of genotoxic compounds also did not affect the viability of cells (neutral red assay). Viability was always above 75% of control viability (data not shown). This viability level avoids false positive responses due to cytotoxicity in the comet assay (Henderson et al., 1998).

3.2. Genotoxicity studies

The potential of OSC to induce DNA damage was assessed using the comet assay. HepG2 cells were incubated with increasing concentrations (5 to 100 µM) of DAS, DADS, DADSO, SAC or AM for 20 hours. None of the five sulfur compounds investigated increased the formation of DNA single-strand breaks compared with non supplemented cells (data not shown).

3.3. Antigenotoxicity studies

3.3.1. Pre-treatment of HepG2 cells with OSC

Pretreatment of cells with DAS, DADS, DADSO and SAC significantly decreased AFB1-induced DNA damage at all concentrations tested (Figure 3). At 100 µM, these OSC reduced AFB1 genotoxicity by 34-47%. AM also induced a protective effect but this was only observed at the highest concentration tested (22 % decrease at 100 µM).

The DNA damage provoked by BaP was prevented by all the tested OSC except DADSO (Figure 4). The most protective effect was obtained with DADS which reduced BaP-induced DNA damage by 20% at 5 µM and by 40% at 100 µM. AM also inhibited BaP genotoxicity but no dose-response relationship could be discerned. DAS and SAC only offered protection against BaP at the highest concentrations employed (50 and 100 µM).

SAC and AM significantly decreased DNA damage in HepG2 cells treated with DMN (Figure 5). AM induced a dose-dependent protective effect : at 5 µM, DMN genotoxicity was reduced by 17% and the reduction reached 30% at 100µM. DADS inhibited DMN genotoxicity at low concentrations (5-25 µM). DAS and DADSO showed poorly preventive effect on DMN genotoxicity.

3.3.2. Co-treatment of HepG2 cells with OSC and genotoxic compounds

All the OSC tested produced a significant reduction in DNA damage induced by H2O2 (Figure 6). SAC and AM showed an inhibitory effect at all concentrations but only protection afforded by SAC occurred in a dose-dependent manner : H2O2 genotoxicity was reduced by 24, 49, 54 % at 25, 50, 100 µM SAC, respectively.

The DNA damage provoked by MMS in HepG2 cells was significantly prevented by DAS, DADS, DADSO and SAC from 5µM (Figure 7). DADSO and SAC showed the most protective effect (40-44% decrease at 100 µM). AM afforded significant protection against MMS at the highest dose employed (26% reduction at 100 µM).

4. Discussion

In the present study, we investigated the antigenotoxic properties of OSC towards a variety of genotoxic agents in HepG2 cells. We showed that OSC displayed antigenotoxic activity when administered before the genotoxic agents, suggesting an effect of the OSC on the metabolic pathways of the genotoxic agents.

Most of the OSC studied were shown to modify the DMN genotoxicity in HepG2 cells. The genotoxic effects of DMN depend upon metabolic activation to reactive intermediates catalyzed by CYP2E1 (Yamazaki et al., 1992). The genotoxicity of DMN in HepG2 cells was significantly inhibited by AM, SAC and to a lesser extent by DADS. These results are consistent with previous studies that have shown an inhibitory effect of these sulfur compounds on CYP2E1 activity and expression (Kwak et al., 1994; Wu et al., 2002). Surprisingly, DAS was not found to reduce DMN genotoxicity in HepG2 cells. This finding is not in agreement with the preventive properties of DAS towards nitrosamine toxicity observed in a number of experimental studies (Surh et al., 1995; Haber-Mignard et al., 1996; Le Bon et al., 1997) and the decrease in CYP2E1 activity that this compound induces in vivo and in vitro(Brady et al., 1991a; Kwak et al., 1994; Wu et al., 2002). These effects are attributed to diallyl sulfone, a metabolite of DAS identified in tissues of DAS-treated rats (Brady et al., 1991b). The fact that DAS showed no effect towards DMN genotoxicity in HepG2 cells may be the consequence of differences in metabolism of this OSC between experimental systems.