A rapid and transient ROS generation by cadmium triggers apoptosis via caspase-dependent pathway in HepG2 cells and this is inhibited through N-acetylcysteine-mediated catalase upregulation
Introduction
Cd is a widespread environmental and industrial pollutant which has been classified as a type I carcinogen by the International Agency for Cancer Research (IARC, 1993). Although the general level of Cd exposure is normally quite low, Cd has a long biological half-life of over 20 years, which classifies it as a cumulative toxin (Sugita and Tsuchiya, 1995). The chronic intake of Cd, through both occupational and nonoccupational exposure, can result in inflammation and fibrosis, and can eventually cause organ dysfunction (Falck et al., 1983, Driscoll et al., 1992). Cd has also been implicated in the development of cancers, including cancers of the lung, prostate, kidney, liver and hemapoietic system (Waalkes, 2000, Elinder et al., 1985). However, the mechanism by which Cd-induced cytotoxicity or carcinogenesis occurs remains fairly unclear.
Apoptosis not only plays a crucial role in tissue development and homeostasis, but is also involved in a wide range of pathological conditions (Thompson, 1995, Fadeel et al., 1999). Apoptotic cell death is accompanied by a series of complex biochemical events and definite morphologic changes, which include cell shrinkage, chromatin condensation, DNA fragmentation, membrane budding and the appearance of membrane-associated apoptotic bodies (Wylli et al., 1981). Apoptosis can be induced through two distinct pathways; one involves the ligation of the TNF/Fas-receptor with its ligand and then this is followed by caspase-8 activation. This in turn either directly activates caspase-3, or causes it to merge with the mitochondrial pathway via cleavage of the Bcl-2 family member, Bid. The other pathway is the mitochondria-mediated caspase-9 activation pathway. Both pathways converge in caspase-3, culminating in cell death.
Cd, however, is considered to be a multitarget toxicant, and it accumulates principally in the liver and kidney after absorption (Dudley et al., 1982, Rikans and Yamano, 2000, Habeebu et al., 1998). The liver, in particular, constitutes the target organ of both chronic and acute exposure to Cd. In vivo studies of mouse and rat liver have shown that apoptosis plays a primary role in Cd-induced hepatotoxicity (Habeebu et al., 1998, Harstad et al., 1999, Yu et al., 2001, Tzirogiannis et al., 2003). Several in vitro examinations have also demonstrated that Cd-induced cytotoxicity is associated with apoptotic cell death in several cell lines including the liver (Hart et al., 1999, Li et al., 2000, Oh et al., 2004, Kondoh et al., 2002, Aydin et al., 2003). However, the above studies indicate that apoptosis may play an important role in the Cd-induced cytotoxicity, the particular molecular events which result in cell death remain fairly unclear.
ROS are generated in the mitochondria and from other sources, and inflict serious damage to lipids, proteins and DNA (Orrenius, 1993). Cd-induced cytotoxicity has been implicated in ROS generation due to the depletion of glutathione and protein-bound sulfhydryl groups (Rikans and Yamano, 2000, Hart et al., 1999, Stohs et al., 2000), demonstrated in experiments involving several antioxidants and antioxidant enzymes (Shih et al., 2004, Chiba et al., 1996, Déas et al., 1997, Poliandri et al., 2003). ROS has also been demonstrated to perform certain functions in the early stages of apoptosis, and to induce the depolarization of the mitochondrial membrane. This eventually results in an increase in the level of ROS along with the level of other proapoptotic molecules in the cytosol (Shih et al., 2004, Buttke and Sandstorm, 1994, Pourahmad and O'Brien, 2000). Although these results indicate that ROS may constitute a direct cause of mitochondrial dysfunction, it remains controversial as to how ROS actually functions during apoptosis. Additionally, the role of ROS in Fas-mediated apoptosis has been demonstrated in a variety of cell types (Um et al., 1996, Denning et al., 2002, Bauer et al., 1998, Jayanthi et al., 1999, Castaneda and Kinne, 2001). In these investigations, ROS was derived from mitochondria as the result of Fas activation. Denning et al. (2002) found that ROS, in the form of H2O2, was capable of inducing the expressions of both Fas and FasL mRNA and protein during apoptosis in intestinal epithelial cells, indicating that ROS might be functioning upstream of the mitochondria. Additionally, a recent study has demonstrated that Fas-mediated apoptosis was potentiated by attenuated ROS generation in human Jurkat T cells (Aronis et al., 2003). Taken together, the above all data indicate that the molecular events associated with ROS in the cytotoxic mechanism have yet to be clarified.
Although the above studies cited were performed in order to elucidate the mechanisms of underlying Cd-induced cytotoxicity, no clear results have not yet been reported. One of the reasons for this involves the use of different experimental parameters, including drug's concentrations and incubation times. Thus, in the present study, the cellular transduction signaling pathway at the early stage of Cd-induced cytotoxicity in HepG2 cells was investigated in both concentration- and time-dependent manner.
Section snippets
Reagents
Cadmium acetate [Cd(Ac)2], cychloheximide, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide, N-acetylcysteine, superoxide dismutase, glutathione and cyclosporin A were purchased from the Sigma-Aldrich Co. (St. Louis, MO, USA). Cytochrome c oxidase subunit IV (Cox 4), dichlorodihydrofluorescein diacetate and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide were obtained from Molecular Probes (Eugene, OR, USA). The inhibitors of
Cd induces apoptosis in HepG2 cells
In order to evaluate the ability of Cd to inhibit the growth of the HepG2 cells, the cells were exposed to different Cd concentrations for 12 and 24 h. Cell growth was inhibited in a concentration- and time-dependent manner (Fig. 1A), and the IC50 dose of Cd for 24 h was determined to be 15 μM. DNA fragmentation, a feature characteristic of apoptosis, was found to be induced by Cd in the HepG2 cells in a concentration- and time-dependent manner. As shown in Fig. 1B, after the cells had been
Discussion
Although previous reports have demonstrated that Cd-associated cytotoxicity is intimately related to apoptosis, investigations into the signaling mechanisms of this process have yielded different results. Consistent with this, we previously determined that Cd-cytotoxicity occurred via apoptosis in lung epithelial fibroblast WI 38 cells, and that the apoptotic pathway demonstrated an extrinsic as well as an intrinsic signaling pathway (Oh et al., 2004). In this study, we attempted to
Acknowledgments
This work was supported by a grant (R13-2003-009) from the Ministry of Science and Technology, Korea, and by the Korea Science and Engineering Foundation through the Research Center for Resistant Cells. The authors would also like to thank Dr. H.J. Song of the Jeil Sungmo Hospital for his helpful advice.
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