Study On The Pathways Of Lead Acetate-induced Apoptosis In PC 12 Cells

Lead is a common kind of heavy metal, and it is widely used in industry and daily lives because of its malleability, low melting point and resistance to corrosion. As an unbiodegradable material, lead exists persistently in all phases of environment and biological systems, therefore causes global contaminations of air, water and soil, and presents a great threat to human health. Lead exposure can induce a wide range of physiological, biochemical and behavioral dysfunctions in human beings. A variety of researches showed that lead could cause toxic effects to almost all human systems, including nervous system, hematological system, cardiovascular system, urinary system, reproductive system, skeletal system, endocrine system, and immune system. Due to the severe damage to the mental development of preschool children, neurotoxicity of lead gained even more attention.The molecular mechanisms of lead toxicity have been well eclucidated in many previous studies, and it is presumed that lead may exert toxicity through its affinity with certain proteins and its ability to mimic calcium. Recent studies also found that lead could induce apoptosis in rat brain, testis, fibroblasts, lung, and retinal rod cells, which indicated that apoptosis might also play an important role in the lead toxicity. Apoptosis is a highly controlled process in which cell death is executed to maintain the steady state under physiological conditions and respond to various stimuli. The regulations of apoptosis are complex, involving three important pathways: death receptor pathway, mitochondrial pathway, and endoplasmic reticulum pathway. Hundreds of genes and proteins are involed in these pathways and accordingly form a network to regulate apoptosis precisely. In lead-induced apoptosis, it is widely accepted that the mitochondria are most pertinent in mediating apoptosis. However, it is still unclear how lead induces mitochondrial dysfunction and which factors are involved in the upstream of mitochondrial pathway. Furthermore, the fact that lead can disturb intracellular Ca²⁺ homeostasis also leads us to investigate the involvement of endoplasmic reticulum pathway in lead-induced apoptosis.In summary, the present study used PC 12 cell line, a regular model for neuron study, to confirm whether lead could induce apotosis. Moreover, according to the known apoptotic mechanisms gained from model organisms, we speculated the possible participants of upstream mitochondrial pathway (such as DNA damage, p53, Bcl-2 family, cytochrome c and caspase-3, etc.) and tried to identify them through a series of experiments. Therefore we might draw a relatively complete picture of the mitochondrion-mediated apoptotic pathway. Besides, the endoplasmic reticulum events (such as the activations of caspase-12 and calpain, and overexpressions of CHOP and GRP 78) were measured to explore whether endoplasmic reticulum played an important role in this process, which could give us a more comprehensive understanding of apoptotic mechanism induced by lead.The main results: 1. Lead acetate decreased the proliferation of PC 12 cells.2. Under light microscope, lead acetate had no effect on the cell morphology and cytoskeleton.3. Under electron microscope, lead acetate induced nuclear pyknosis, mitochondrial swelling and vacuolization, cell blebbing. But the structure of endoplasmic reticulum remained unchanged.4. Lead acetate induced DNA damage with a concentration-dependent manner in PC 12 cells.5. Lead acetate induced apoptosis with a concentration-dependent manner in PC 12 cells.6. Lead acetate increased the activation level of caspase-3 with a concentration-dependent manner in PC 12 cells.7. Lead acetate had no effect on the activity of calpain in PC 12 cells.8. Lead acetate increased cytosolic cytochrome c level and the expressions of Bax and p53. The expression of Bcl-2 decreased, and the ratio of Bax/Bcl-2 increased significantly.9. Lead acetate increased the expression of GRP78, but the expressions of CHOP and procaspase-12 remained unchanged. Furthermore, caspase-12 wasn't activated.Conclusions:1. The possible mitochondrial pathway of lead-induced apoptosis: Lead can induce DNA damage, which may activate p53. After the ratio of Bax/Bcl-2 increases, the apoptotic factors (such as cytochrome c) are released from mitochondria, which induce caspases cascade activation and apoptosis.2. The morphiological and biochemical results indicate that under the conditions of this research, lead may induce apoptosis through mitochondrial pathway, but not endoplasmic reticulum pathway.3. Accompanied with the lead-induced apoptotic biochemical alterations, the morphology of organelle also changes.4. Apoptosis may play an important role in lead toxicity.