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Cell Biology International (2009) 33, 1280–1286 (Printed in Great Britain)
N-Nitrosopiperidine and N-Nitrosodibutylamine induce apoptosis in HepG2 cells via the caspase dependent pathway
Almudena Garcíaa, Paloma Moralesa, Joseph Rafterb and Ana I. Hazaa*
aDepartamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain
bDepartment of Biosciences and Nutrition, Karolinska Institutet, Huddinge University Hospital, NOVUM, S-141 86, Huddinge, Sweden


The human hepatoma cell line (HepG2) exhibited a dose and time-dependent apoptotic response following treatment with N-Nitrosopiperidine (NPIP) and N-Nitrosodibutylamine (NDBA), two recognized human carcinogens. Our results showed a significant apoptotic cell death (95%) after 24h treatment with NDBA (3.5mM), whereas it was necessary to use high doses of NPIP (45mM) to obtain a similar percentage of apoptotic cells (86%). In addition, both extrinsic (caspase-8) and intrinsic pathway (caspase-9) could be implicated in the N-Nitrosamines-induced apoptosis. This study also addresses the role of reactive oxygen species (ROS) as intermediates for apoptosis signaling. A significant increase in ROS levels was observed after NPIP treatment, whereas NDBA did not induce ROS. However, N-acetylcysteine (NAC) did not block NPIP-induced apoptosis. All these findings suggest that NPIP and NDBA induce apoptosis in HepG2 cells via a pathway that involves caspases but not ROS.

Keywords: Apoptosis, Caspases, HepG2 cells, N-Nitrosamines, Reactive oxygen species.

*Corresponding author. Tel.: +34 91 394 37 47; fax: +34 91 394 37 43.

1 Introduction

Exposure to N-Nitroso compounds (NOC), which are potential carcinogens, can occur through either ingestion or inhalation of preformed N-Nitrosamines or by ingestion of their precursors (Lijinsky, 1999). Significantly higher amounts of N-Nitrosopiperidine (NPIP) may be formed by nitrosation of piperidine, main principle of pepper, by the nitrite added to the spice mixture (Shenoy and Choughuley, 1992), whereas N-Nitrosodibutylamine (NDBA) is a contaminant in industrial rubber products and rubber toys (Spiegelhalder and Preussmann, 1983). Both NPIP and NDBA are carcinogens in laboratory animals (Gray et al., 1991; Magee and Barnes, 1967) and possible causative agents in human cancer (IARC, 1978).

Apoptosis is characterized by membrane blebbing, cytoplasmic shrinkage and reduction of cellular volume, condensation of the chromatin, and fragmentation of the nucleus, all of which ultimately lead the formation of apoptotic bodies, a prominent morphological feature of apoptotic cell death (Kroemer et al., 2005). The caspases, a family of cysteine proteases, play a central role in most apoptotic processes constructing the protease cascade including the initiator caspases (caspase-8 and -9) and the effector caspases (caspase-3, -6 and -7) (Taylor et al., 2008). It has been also highlighted the correlation between the chemical potential for the induction of apoptosis and carcinogenesis (Holme et al., 2007). The fate of cells with DNA damage either to undergo apoptosis or to survive seems to be dependent on the intensity of DNA damage. When weak DNA damage was induced, the cellular response allows repair of the damage. However, if the damage failed to be repaired, mutagenic lesions could be propagated and might lead to carcinogenesis.

Numerous studies have demonstrated that food mutagens (Hashimoto et al., 2001, 2004; Salas and Burchiel, 1998; Shiotani and Ashida, 2004) and tobacco specific N-Nitrosamine (Tithof et al., 2001) induce apoptosis. Our previous work also reported that NPIP and NDBA-induced apoptosis in human leukemia HL-60 cell line (García et al., 2008). However, the liver is its major target for carcinogenesis, since alkylating species is produced in hepatocytes (Mirvish, 1995). Numerous in vitro studies have employed human hepatoma HepG2 cells to characterize the apoptotic programmed cell death (Kim et al., 2006; Matsuda et al., 2002), becoming a very useful tool for the study of the apoptotic effect of several hepatocarcinogens (Chen et al., 2003; Panaretakis et al., 2001). Thus, the aim was to investigate the induction of apoptosis by NPIP and NDBA in the human hepatoma cell line (HepG2).

As well as DNA damage constitutes the primary signal for the induction of apoptosis, others mechanisms such as oxidative stress may play an important role during apoptosis induction (Chandra et al., 2000). N-Nitrosamines may cause the generation of reactive oxygen species (ROS) resulting in oxidative stress and cellular injury (Bansal et al., 2005; Yeh et al., 2006). For that reason, we also asked whether the induction of apoptosis in HepG2 cells by NPIP and NDBA is mediated by a ROS-dependent cell death pathway.

2 Material and methods

2.1 Chemicals

N-Nitrosopiperidine (NPIP), N-Nitrosodibutylamine (NDBA), Dimethyl sulfoxide (DMSO), Etoposide, N-Acetyl-L-cysteine (NAC) and Acridine orange (AO) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO). Culture medium and supplements were purchased from Gibco Laboratories (Life Technologies, Inc., Gaithersburg, MD 20884-9980). 2′, 7′-dichlorodihydroflourescein diacetate (H2DCFDA) was obtained from Molecular Probes (Eugene, Oregon, USA). The caspase inhibitors, Z-DEVD-FMK (caspase-3 inhibitor), Z-VEID-FMK (caspase-6 inhibitor), Z-IETD-FMK (caspase-8 inhibitor) and Z-LEHD-FMK (caspase-9 inhibitor) were purchased from BD Pharmigen (USA) and dissolved at 10mM in DMSO (0.1%). All other chemicals and solvents were of the highest grade commercially available.

2.2 HepG2 cells

Human hepatoma cells (HepG2) were obtained from the Biology Investigation Center Collection (BIC, Madrid) and maintained in Dulbeccós Modified Eaglés Medium supplemented with 10% v/v heat-inactivated foetal calf serum, 50μg/ml streptomycin, 50UI/ml penicillin and 1% v/v L-Glutamine at 37°C humidified atmosphere containing with 5% CO2. Controls included a medium control without N-Nitrosamines as negative control. Etoposide has been extensively studied (Custódio et al., 2002) and was used here as a positive control (100μM) of apoptosis.

2.3 Morphological evaluation of cell death

HepG2 cells (1×106/ml) were treated with NPIP (10–45mM) or NDBA (1–3.5mM) at different incubation times. After treatments, cells were stained with acridine orange (5μg/ml) for 10min and observed by fluorescence microscopy (Axiostar plus microscope, Zeiss) as described by Gregory et al. (1991). A total of 200 cells were counted in multiple randomly selected fields, and the percentage of apoptotic cells was calculated.

2.4 TdT-dUTP Terminal Nick-End Labeling (TUNEL) assay

Apoptotic cell death was also measured by the In Situ Cell Death Detection Kit, Fluorescein according to the manufacturer's protocol (Roche, Indianapolis, USA). HepG2 cells were treated with NPIP (10, 25 and 45mM) or NDBA (1, 2.5 and 3.5mM) for 24, 48 and 72h. When NAC was used, cells were pre-incubated with 20mM NAC for 1h and exposed to N-Nitrosamines. Briefly, 3×106 cells were washed with PBS and fixed in 2% formaldehyde in PBS (1ml) for 1h at room temperature. The cells were permeabilized with 0.1% triton X-100 in 0.1% sodium citrate for 2min on ice and incubated with the TUNEL reaction mixture [50μl of enzyme solution (TdT) and 450μl of label solution (fluorescein-dUTP)] for 1h at 37°C in the dark in a humidified atmosphere. Finally, the percentage of apoptotic cells was measured by FACS Calibur flow cytometer (Becton & Dickinson) and the CellQuest software. For each experiment 104 cells were analysed.

2.5 Western blot

After incubation of cells with NPIP (10, 25 and 45mM) for 24 and 72h or NDBA (1, 2.5 and 3.5mM) for 3 and 6h, protein extracts were obtained with Nucbuster Protein Extraction Kit Novagen (Darmstadt, Germany). Samples containing 30μg of protein, measured by the Non-Interfering Protein Assay Kit (Calbiochem, San Diego, CA) were resolved on a 10% SDS-PAGE and electroblotted onto an immune-blot PVDF membrane (Bio-Rad Laboratories). The membranes were blocked overnight in milk block buffer (PBS, 0.2% Tween, 10% non fat dry milk) and then incubated for 1h with polyclonal poly (ADP-ribose) polymerase (PARP) antibody (Alexis Biochemicals, Lausen, Switzerland). The blots were further incubated for 1h with goat anti-rabbit peroxidase conjugated (Chemicon, Temecula, CA). Bound antibodies were detected by the super signal substrate (Pierce, Rockford, IL) using Bio-Rad Fluor S instrument and analysed used the Bio-Rad quantity one software package.

2.6 Caspase activity

To address the significance of caspases activation in NPIP/NDBA-induced apoptosis in HepG2 cells, we used permeable, specific and potent caspase inhibitors, Z-DEVD-FMK, Z-VEID-FMK, Z-IETD-FMK and Z-LEHD-FMK. After incubation of HepG2 cells with N-Nitrosamines in the presence or absence of caspase inhibitors, the percentage of apoptotic cells was determined by TUNEL assay and flow cytometry.

2.7 Measurement of ROS

ROS production was determined using H2DCFDA, which diffuses through the cell membrane and is hydrolyzed by intracellular esterases to non-fluorescent dichlorofluorescein (DCFH). In the presence of ROS, this compound is oxidized to highly fluorescent dichlorofluorescein (DCF). HepG2 cells were treated with different concentrations of NPIP (10, 25 and 45mM) or NDBA (1, 2.5 and 3.5mM) for different time intervals (0.25–24h). To study the role of antioxidants, NAC (20mM) was added 1h before the addition of N-Nitrosamine. Then 2.5×105 cells were washed with PBS loaded for 30min with H2DCFDA (10μM) and incubated in a waterbath (37°C). The cells were kept on ice and fluorescence intensity was read immediately with a FACS Calibur flow cytometer (Becton & Dickinson) and the CellQuest software. For each experiment, 104 cells were analysed.

2.8 Statistical analyses

The Student's t-test was used for statistical comparison and differences were considered significant at P0.05. Descriptive and graphical methods were used to characterize the data. All tests were performed with the software package Statgraphics Plus 5.0.

3 Results

3.1 Analysis of morphological changes induced by NPIP and NDBA

The effect of NPIP (1–45mM) and NDBA (1–45mM) on HepG2 cell viability was previously determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay). A moderate inhibition of HepG2 cell viability (20%) was found at 24h treatment with 10–45mM NPIP and 1–3.5mM NDBA (data not shown). Subsequently, we analysed morphological changes to clarify whether NPIP and NDBA-induced cytotoxicity against HepG2 cells was due to the induction of apoptosis (Fig. 1). The percentage of apoptotic cells was >30% after treatment with 10 and 25mM NPIP at 24, 48 and 72h. The highest dose of NPIP (45mM) induced a percentage of apoptosis around 50% after 24h treatment, reaching 91% of apoptotic cells at 72h. NDBA also induced a concentration (1–3.5mM) and time (1–6h) dependent increase in the percentage of apoptotic cells. One hour treatment at 3.5mM NDBA induced 52% of apoptosis and at 6h apoptotic bodies were abundant.

Fig. 1

Morphological changes of nuclear chromatin in HepG2 cells treated with N-Nitrosamines. Cells were plated in the absence (A) or the presence of 45mM NPIP for 48h (B) and 3.5mM NDBA for 3h (C).

3.2 TUNEL assay

The TUNEL assay is a common method for detecting DNA strand breaks that result from the apoptotic signaling cascades (Frohlich and Madeo, 2000). TUNEL analysis showed that NPIP and NDBA-induced apoptosis in a concentration and time dependent-manner (Fig. 2). The lowest dose of NPIP (10mM) induced 6% of apoptotic cells at 24h, whereas a markedly higher percentage of apoptotic cells (23 and 86%) was noted at higher concentrations of NPIP (25 and 45mM, respectively) (Fig. 2A). An increase in the number of apoptotic cells was apparent after 72h incubation with 1 and 2.5mM NDBA (27 and 51%, respectively; Fig. 2B). Finally, the results indicate that the percentage of apoptotic HepG2 cells obtained with the highest concentration of NPIP (45mM; 87%) or NDBA (3.5mM; 94%) at all incubation times was similar to the percentage obtained with etoposide for 72h (100μM, 90%).

Fig. 2

Flow cytometric analysis using TUNEL assay of HepG2 cells treated with different concentrations of NPIP (A) and NDBA (B) for 24 (□), 48 (&z.sqshd;) and 72 (■) h. C0, untreated cells; C1, cells treated with etoposide (100μM). Asterisks indicate significant difference from control C0 *** p0.001, ** p0.01 and * p0.05.

3.3 Western blot

It was of interest to identify by Western blot the cleavage of Poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme (116kDa) degraded by caspase-3 into 85 and 24kDa fragments during the execution of apoptosis (Chiarugi, 2002). Protein extracts from HepG2 cells (untreated and treated with NPIP, NDBA or etoposide) were electroblotted and probed against a PARP polyclonal antibody that recognizes the 116kDa intact PARP as well as an 85kDa cleaved product (Fig. 3). Untreated HepG2 cells showed only intact PARP at 116kDa (Fig. 3A and B, lane 1). All the PARP present in the 100μM etoposide treated cells had not been cleaved (Fig. 3A and B, lane 2), whereas the concomitant disappearance of the original 116kDa PARP fragment at long time incubations was seen (Fig. 3A, lane 2). Similarly, the 116kDa band disappeared in 25 and 45mM NPIP treated cells after 72 and 24–72h treatment, respectively (Fig. 3A, lanes 6, 7 and 8). NDBA caused PARP cleavage as reflected by the intensity of the 85kDa PARP fragments which were visible after 6h incubation with 2.5mM (Fig. 3B, lane 6) and after 3 and 6h incubation with 3.5mM (Fig. 3A, lanes 7 and 8). Quantification of PARP cleavage was determining by densitometry of the intensity of full-length protein signal visualized by polyclonal anti-PARP antibody.

Fig. 3

Western blot of PARP cleavage in HepG2 cells treated with NPIP (A) and NDBA (B). Lane 1 represents untreated cells (A and B) and lane 2 represents etoposide treated cells for 72h (A) and 24h (B). (A) Lanes 3 and 4 represent cells treated with 10mM NPIP for 24 and 72h, respectively. Lanes 5 and 6 represent cells treated with 25mM for 24 and 72h, respectively. Lanes 7 and 8 represent cells treated with 45mM for 24 and 72h, respectively. (B) Lanes 3 and 4 represent cells treated with 1mM NDBA for 3 and 6h, respectively. Lanes 5 and 6 represent cells treated with 2.5mM NDBA for 3 and 6h, respectively. Lanes 7 and 8 represent cells treated with 3.5mM NDBA for 3 and 6h, respectively.

3.4 Effects of NPIP and NDBA on the caspase pathway in HepG2 cells

Since the caspases are considered universal effectors of apoptosis (Hashimoto et al., 2001), we evaluated the ability of NPIP (10mM) and NDBA (2.5mM) to induce apoptosis in HepG2 cells in the presence or absence of different caspase inhibitors (100μM) for 72h. Z-DEVD-FMK (caspase-3 inhibitor) inhibited 73% both N-Nitrosamines and Z-VEID-FMK (caspase-6 inhibitor) reduced the apoptotic effect of NPIP and NDBA in 74–80%, respectively (Fig. 4). The addition of Z-IETD-FMK (caspase-8 inhibitor) diminished NPIP and NDBA-induced apoptosis in 69–74%, respectively. The blockage of apoptosis by Z-LEHD-FMK (caspase-9 inhibitor) caused an inhibition of both N-Nitrosamines of 65%.

Fig. 4

Effect of specific caspase inhibitor on apoptosis induced by 10mM NPIP or 2.5mM NDBA (72h) in HepG2 cells, using TUNEL assay and flow cytometry. C (■), HepG2 cells treated with N-Nitrosamines and without caspase inhibitor. (&z.sqshd;) HepG2 cells treated with N-Nitrosamines and specific caspase inhibitor. Asterisks indicate significant difference from control *** p0.001.

3.5 ROS production

After treatment of HepG2 cells, DCF fluorescence was measured by flow cytometry and expressed as percentage of control. A significant time and dose-dependent increase of ROS levels was observed in NPIP treated cells, reaching the maximum signal after 1h treatment with the highest dose (45mM) (Fig. 5A). A slight increase of ROS levels was only found in 1mM NDBA treated cells for 0.5, 1 and 3h compared with the untreated cells, and it was reduced after 24h As (Fig. 5B).

Fig. 5

Time-course of ROS production in untreated HepG2 cells (◇) and treated with NPIP (A) at 10 (■), 25 (▲) and 45 (●) mM and NDBA (B) at 1 (■), 2.5 (▲) and 3.5 (●) mM. Asterisks indicate significant difference from control ** p0.01 and * p0.05.

3.6 Effect of NAC on ROS production and apoptosis induced by NPIP

We tested whether NAC, a recognized radical scavenger and antioxidant (Zafarullah et al., 2003), could affect ROS production in NPIP treated cells. Since the experiments revealed that ROS production was maximal at 1h of NPIP treatment (Fig. 5A), this time-point was chosen. The increased ROS levels caused by exposure of HepG2 cells to NPIP for 1h were suppressed to the control levels by pretreatment with 20mM NAC for 1h (Fig. 6A). Moreover, to determine the involvement of ROS in NPIP-induced apoptosis, we performed experiments confirming the effects of NAC on NPIP-mediated apoptosis. We selected 10mM NPIP for 72h incubation time because it enhanced the percentage of apoptotic cells above 40%. However, pretreatment with NAC at 20mM for 1h caused a significant increase in the percentage of apoptotic cells (30%), and therefore the percentage of apoptosis was not reduced in the subsequent combined treatment with NPIP (10mM for 72h) (Fig. 6B).

Fig. 6

Effect of NAC on ROS production (A) and apoptosis (B) induced by NPIP. C0, untreated HepG2 cells. (A) Flow cytometric analysis using H2DCFDA of HepG2 cells pretreated with (■) or without (□) NAC at 20mM for 1h and then incubated in the presence of NPIP. (B) Flow cytometric analysis using TUNEL assay of HepG2 cells pretreated with or without NAC at 20mM for 1h and then incubated in the presence of NPIP (10mM for 72h). Asterisks indicate significant difference from control *** p0.001, ** p0.01 and * p0.05.

4 Discussion

It is widely accepted that N-Nitrosamines require metabolic activation by cytochrome P-450 to become carcinogenic (Fujita and Kamataki, 2001). The activated N-Nitrosamine attacks and covalently binds to DNA, forming DNA adducts. DNA damage induces the production of p53 protein, the activation of protease, and the subsequent activation of endonucleases to catalyze DNA fragmentation, leading to apoptosis (Roos et al., 2004). In the present study, a variety of methods have been employed to detect and quantify apoptosis, since the utilisation of two or more different techniques may be convenient to avoid errors (Baskic et al., 2006; Gómez-Lechón et al., 2002). Our results demonstrated that NPIP and NDBA-induced apoptosis in HepG2 cells in a concentration and time dependent-manner, as judged by fluorescence microscopy and TUNEL assay. The chromatin condensation could be visualized in HepG2 cells by fluorescence microscopy after 1h treatment with NDBA, whereas NPIP was effective after 24h (Fig. 1). However, DNA strand breaks, as detected by the TUNEL assay, were not found until 24h after treatment with both N-Nitrosamines (Fig. 2). There is no reason to assume that nuclear morphological changes and detectable DNA strand breaks occur at the same time, in many cases, detection of DNA strand breaks occurs in the lytic stage of apoptosis (Willingham, 1999).

The proteolytic cleavage of PARP was used as a third marker for NPIP and NDBA-induced apoptosis. While PARP cleavage was evident in NDBA treated cells (Fig. 3B), the 85kDa PARP fragment was absent in etoposide and NPIP treated cells (Fig. 3A). However, inhibition of the PARP expression occurred after treatment with high doses of NPIP (25 and 45mM) or at long incubation times with etoposide (72h) (Fig. 3A). These results are in agreement with DiBartolomeis and Moné (2003), who assumed that PARP cleavage was based on the disappearance of the 116kDa fragment in Jurkat cells treated with 500μM etoposide.

To determine whether the caspases were involved in NPIP and NDBA-induced cell death, we also analysed the effects of the specific inhibitors of caspase activity. The two major apoptotic pathways described in eukaryotic cells are extrinsic and intrinsic, whose initiator caspases are caspase-8 and -9, respectively. A signal transmitted from activated caspase-8 is bifurcated into two pathways: direct activation of caspase-3 (Hirata et al., 1998) and the mitochondria-mediated caspase cascade (Wolf and Eastman, 1999). Thus, the caspase-9 activated will function downstream from caspase-8 and upstream from caspase-3. Furthermore, caspase-3 activates caspase-6 (Hirata et al., 1998), which in turn causes nuclear shrinkage and fragmentation (Takahashi et al., 1996). Both the intrinsic and extrinsic pathways are similarly involved in the NPIP and NDBA-induced apoptosis in HepG2 cells (Fig. 4). These findings agree with those of Hashimoto et al. (2004), who reported that the 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces the activation of both caspase-8 and caspase-9 in rat splenocytes.

In comparison with our previous studies (Arranz et al., 2008), NDBA was the most potent N-Nitrosamine analysed in HepG2 cell line. Thus, after 24h incubation with NDBA at 3.5mM, the percentage of apoptotic cells reached 95%, whereas it was necessary to use doses of 45mM NPIP (86%), 50mM NPYR (68%) and 68mM NDMA (54%) and longer incubations times (72h) to obtain >50% of apoptotic cells by the TUNEL assay. The fact that the percentage of apoptotic cells varied with the type of N-Nitrosamine suggests that the apoptotic effect depended on the chemical structure of N-Nitrosamine. In the metabolic activation of these compounds, the number of carbon atoms of the chains bound to the nitroso group of N-Nitrosamines is one of the determinants of a certain CYP(s) responsible (Fujita and Kamataki, 2001). Similar findings have also reported in the leukemia HL-60 cell line (Arranz et al., 2008; García et al., 2008), HepG2 cells being more resistant to treatment with NPIP and NDBA. Likewise, higher concentrations of etoposide in HepG2 (100μM) than HL-60 (5μM) cells were needed to induce a similar percentage of apoptosis. Thus, a possible explanation of the variation in the percentage of apoptosis induced by N-Nitrosamines could be attributed to the differences in the levels of enzymatic activities in HepG2 and HL-60 cells.

The role of ROS as intermediates for apoptosis signaling is well recognized because of various antioxidants such as NAC can block apoptosis (Kannan and Jain, 2000). NPIP treated HepG2 cells showed a dose-dependent increase of ROS production, but not with NDBA (Fig. 5). This finding suggests that the initial toxic insults in response to NDBA in HepG2 cells are not related to ROS. However, we previously had found that NDBA induced a slight dose and time dependent increase of ROS production in HL-60 cells (García et al., 2008). Holme et al. (2007) reported specific differences in the ROS production between two cell lines treated with benzo(a)pyrene, detecting a significant increase of ROS levels in F258 cells, while no such increase was observed in Hepa1c1c7 cells. NAC decreased the ROS production induced by NPIP to control levels (Fig. 6A), whereas the exposure of cells to NAC did not confer protection from NPIP-induced apoptosis (Fig. 6B). These results agree with our recent work that demonstrated that NPIP and NDBA-induced apoptosis in leukemia HL-60 cells via a ROS-independent cell death pathway (García et al., 2008). Moreover, other studies suggest that NAC does not confer protection from apoptosis, and therefore ROS do not contribute to the regulation of apoptosis (Kinoshita et al., 2007; Lin et al., 2003). In conclusion, the present study proves that NPIP and NDBA induce apoptosis in HepG2 cells via a pathway that involves caspases but not ROS.


This work has been supported by Grant ALI2002-01033 from the Ministerio de Ciencia y Tecnología (Spain) and by Grant 910177 from the Comunidad de Madrid and the Universidad Complutense (UCM). A. García is a recipient of Fellowships from the Universidad Complutense. This work was also partly supported by ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: ‘Food Quality and Safety’ (contract no. 513943).


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Received 19 May 2009/2 July 2009; accepted 27 August 2009


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