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Cell Biology International (2009) 33, 12801286 (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 24
Keywords: Apoptosis, Caspases, HepG2 cells, N-Nitrosamines, Reactive oxygen species.
*Corresponding author. Tel.: +34 91 394 37 47; fax: +34 91 394 37 43.
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
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 (H
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, 50
2.3 Morphological evaluation of cell death
HepG2 cells (1
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 45
2.5 Western blot
After incubation of cells with NPIP (10, 25 and 45
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 H
2.8 Statistical analyses
The Student's t-test was used for statistical comparison and differences were considered significant at P
3.1 Analysis of morphological changes induced by NPIP and NDBA
The effect of NPIP (1–45
Morphological changes of nuclear chromatin in HepG2 cells treated with N-Nitrosamines. Cells were plated in the absence (A) or the presence of 45
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 (10
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. C
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 (116
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 72
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 (10
Effect of specific caspase inhibitor on apoptosis induced by 10
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 1
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 ** p
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 1
Effect of NAC on ROS production (A) and apoptosis (B) induced by NPIP. C
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 1
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 85
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 24
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 2009doi:10.1016/j.cellbi.2009.08.015