by Furthermore, caspase-3 activity was also markedly increased in a dose-dependent manner. experiment was performed. TUNEL analysis showed that Cd triggered liver cell apoptosis; caspase-3A activity was markedly increased; its proenzyme level was significantly decreased, and the levels of its cleaved forms were markedly increased. However, real-time quantitative PCR analysis revealed that the mRNA transcript level of caspase-3A was not significantly elevated. Immunoreactivities were observed in the cytoplasm of hepatocytes by immunohistochemical detection. The findings indicates that Cd can trigger liver cell apoptosis through the activation of caspase-3A. Caspase-3A may play an essential role in Cd-induced apoptosis. == Introduction == Apoptosis, or programmed cell death, plays a critical role in the survival of multicellular organisms by getting rid of damaged or infected cells that may interfere with normal function[1].It can be regulated by many modulators, including some ions (e.g. calcium), genes (e.g. c-myc, Bcl-2/Bax and Fas), proteins (e.g. p53, caspases, IAPs) and even organelles (e.g. mitochondria, endoplasmic reticulum)[2]. Among these regulators, members of the caspase family of aspartic acid-directed cysteine proteases, lead to the loss of cellular structure and function and eventually result in apoptotic cell death[3],[4]. In mammalian cells, the caspase family comprises at least 14 enzymes, which can be mainly divided into two categories, initiator caspases and executioner caspases depending on where they function in the apoptotic cascade[5]. Initiator caspases include caspase-2, -8, -9 and -10. Once activated, they cleave and activate the executioner caspases, which consist of caspases-3, to a lesser extent caspase-6 and -7. The activated executioner caspases then cleave their respective substrates to cause demolition of the cell[6]. Briefly, the caspase PF-4840154 activation is regulated through either extrinsic pathway (death receptor pathway) or intrinsic pathway (mitochondrial pathway)[7]. Both pathways converge on caspase-3 and subsequently on other proteases and nucleases that drive the terminal events of apoptosis[8],[9]. Apoptosis can be induced by many different stimuli such as ultraviolet (UV) irradiation[10], chemotherapy agents[11], infection by pathogens[12],[13], polychlorinated biphenyls (PCBs)[14], polycyclic aromatic hydrocarbons (PAHs)[15], insecticides[16]and heavy metals[17],[18]. Among these factors, heavy metals such as Cd play crucial roles in the process of apoptosis induction, and may thus tip the balance of cellular homeostasis towards an increased cellular PF-4840154 mortality[19]. As a widespread environmental pollutant, Cd has multiple effects on cells, affecting essential cellular processes such as cell division, proliferation, differentiation and apoptosis[20]. Cd triggers cell apoptosis, both in vitro[17],[21]and in vivo[22],[23]in several models, but the mechanisms remain controversial. Commonly, Cd can induce apoptosis via a caspase-dependent pathway or a caspase-independent pathway based on the different Cd exposure conditions[17],[24][26]. Similar to the findings reported for mammalian models, a few studies about fish tissues and cells have indicated Rabbit polyclonal to ARHGDIA the occurrence of apoptosis upon Cd exposure. In rainbow trout (Oncorhynchus mykiss) cell lines, Cd exposure promotes the elevation of caspase-3 activity[27]. In larval topsmelt (Atherinops affinis), Cd treatment increases apoptotic DNA fragmentation[28]. In zebrafish (Danio rerio), Cd contamination effects pro-apoptotic genes bax andc-junto be up-regulated[29]. Generally, Cd-induced apoptosis correlates with caspase-dependent pathway in fish. As an effector caspase, caspase-3 is situated at a pivotal junction in the apoptotic pathways triggered both by the mitochondrial and the death receptor pathways[30]. In fish, It has been mainly identified and characterized in the following fish species: cinnamon clownfish (Amphiprion melanopus)[31], rock bream (Oplegnathus fasciatus)[32], Western sea bass (Dicentrarchus labrax)[33], zebrafish[34], large yellow croaker (Pseudosciaena crocea)[35], Atlantic salmon (Salmo salar)[36], Nile tilapia (Oreochromis niloticus)[37]and Medaka (Oryzias latipes)[38]. Moreover, two isoforms of caspase-3 (A and B) have been recognized in Atlantic salmon[36]and Medaka[38]. These caspase-3 homologs display related and conserved immune-related functions between fish and mammals. They are involved in the process of apoptosis response to viral or bacterial challenge[32],[35]. Common carp is one of the major farmed freshwater varieties in the world[39]. It exhibits a strong tolerance to environmental stress and has become a widely used model varieties[40]. However, despite its great economic value and ecological importance in world aquaculture, innate immunological knowledge about common carp is definitely relatively poor compared with additional cultured fish such as rainbow trout, Atlantic salmon, Western sea bass and Medaka. To date, only a few apoptotic genes such as tumor necrosis element- (TNF-)[41]and recruitment website protein (Cards)[42]have been completely recognized and characterized. It is necessary to get a clear understanding of the PF-4840154 immunological mechanisms against stimuli such as infection and heavy metal toxicity in common carp. In this study, to explore whether caspase-3 is definitely involved in Cd-induced apoptosis in common carp, we isolated complementary DNA (cDNA) of caspase-3 from common carp and analyzed its tasks in liver.
