| Innovation of materials technology is promoted by the rapid development of industrialization. Traditional and emerging industries make people’s lives more convenient but bringing risks to environment and human health because of the constant exposure to raw materials and industrial products. In this study, we explored the toxic effects of traditional industrial raw and novel materials (cadmium and silica nanoparticles, Cd2+ and SiO2-NPs) on organisms and the underlying mechanisms. So far, there have been numerous studies on the mechanisms of Cd2+-mediated cytotoxicity, but the investigations of effective prevention and cure of Cd2+ poisoning are still limited. Based on the physical and chemical properties of Cd+, we used epigallocatechin-3-gallate (EGCG) to protect cells from Cd2+-induced damage, and elucidated its protective mechanism. In addition, SiO2-NPs are widely used as an essential new type of NPs, which possess more advantages than traditional raw materials. However, despite a potentially increasing exposure of the gastrointestinal tract (GIT) to NPs, the toxicological effects of SiO2-NPs in the GIT are unclear. Therefore, we also explored the potential toxic effects of SiO2-NPs on epithelial cells of the intestine and the underlying mechanisms. Ultimately, we seek to characterize the unique properties of SiO2-NPs through which it exerts its cytotoxicity.Part One:The antagonistic effect of EGCG on Cd2+-induced apoptosisIn the present study, human liver cells (HL-7702) were treated with CdCl2 for 21 h and then co-treated with EGCG for 3 h. Cell viability and apoptosis were detected to explore whether EGCG was capable of inhibiting Cd2+-induced cytotoxicity. The study on its mechanisms was conducted from the perspective of EGCG working as an effective anti-oxidative agent as well as a metal chelating agent. Then we tried to find a new and effective therapeutic pathway to reduce Cd2+-induced toxicity through this study.The results were shown as follows:1. Treatments with different concentrations of Cd2+ for 24 h resulted in significant inhibition of cell viability (p< 0.01). EGCG reduced Cd2+-induced cytotoxicity (p< 0.05) except for the group exposed to the highest dose of 80 μM Cd2+.2. Treatment with Cd2+ significantly caused some typical features of apoptosis such as nuclear condensation (p< 0.01). However, treatment with EGCG suppressed the induction of apoptosis in response to Cd2+ exposure (p< 0.05). These data suggested that EGCG was able to relieve HL-7702 cells from Cd2+-induced apoptosis.3. As shown in present study, the ROS and MDA levels of Cd2+ treated cells were much higher than that of untreated cells by 2.4-fold and 1.78-fold respectively (p< 0.01). Addition of EGCG significantly impaired Cd2+-induced ROS and MDA production in HL-7702 cells (p< 0.05), indicating that EGCG was able to reduce Cd2+-induced oxidative stress by acting as a potent ROS scavenger in HL-7702 cells.4. As the main cellular source of ROS, mitochondria are the primary targets of ROS burst inevitably. The mitochondrial membrane potential (MMP) could be disrupted, and then it would enhance apoptosis in a vicious circle. Following the exposure of cells to Cd+ for 24 h, the MMP dropped to 88.56 ± 0.69% compared with the control (p< 0.01). In contrast, post-treatment with EGCG could recover MMP to 94.34 ± 0.75% in HL-7702 cells (p< 0.01).5. Caspase-3 has been confirmed to play a pivotal role in the execution phase of mitochondria mediated apoptotic pathway. Caspase-3 activity was significantly increased in HL-7702 cells treated with Cd2+ for 24 h (p< 0.01), while EGCG significantly reversed the Cd2+-induced up-regulation of caspase-3 activity (p< 0.01). The increase of caspase-3 activity was accompanied by the collapse of MMP. These results implied that Cd2+-induced apoptosis occurs through the activation of common executors of the mitochondrial signaling pathway such as caspase-3. EGCG appeared to participate in preventing Cd2+-induced apoptosis via suppressing caspase-3 activity.6. UV-Vis spectroscopic analysis showed the absorption peak of EGCG was slightly reduced when Cd2+ was added. Besides, no new absorption peak was observed in spectral region. Meanwhile, the results of ’H-NMR were consistent with that of the UV-Vis analysis, implying that there was no formation of Cd2+-EGCG complex at neutral condition.In conclusion, all the results suggested Cd2+ considerably decreased the cell viability and induced apoptosis of HL-7702 cells. Conversely, EGCG co-treatment resulted in significant inhibition of Cd2+ -induced reduction of cell viability and apoptosis, implying a rescue effect of EGCG against Cd2+ poisoning. The rescue effect most likely arose from scavenging ROS and maintaining redox homeostasis, as the generation of intracellular ROS and MDA was significantly reduced by EGCG, which further prevented MMP collapse and suppresses caspase-3 activity. However, no evidence about the chelation of EGCG with Cd2+ under neutral condition was observed. Therefore, a clear conclusion from this work can be drawn that EGCG could inhibit Cd2+-induced apoptosis by acting as a ROS scavenger rather than a metal chelating agent.Part two:The toxicity of silica-nanoparticles in HCT 116 cellsThe aim of the present study was to answer the question that the potential cell biological effects of SiO2-NPs on human colon carcinoma (HCT 116) cells and the toxic mechanisms. Silica submicron particles (SiO2-SMPs) were introduced in the study as a blank control for nanoparticles (NPs) in order to identify the effects which are only observed for SiO2-NPs. In summary, we hope to provide the evidence of toxicological study on SiO2-NPs exposure to GIT and possible novel mechanisms.Results and conclusions:1. In serum-free medium, HCT 116 wild type cells (HCT 116 wt) were exposed to 12 rnm SiO2-NPs at various concentrations (10,25,50 and 100 ug/mL). The total numbers of cells were significantly decreased and accompanied with phase changes of morphogenesis. The results indicated that cell growth and proliferation were inhibited and the cells showed typical apoptotic morphological features. The activity of succinate dehydrogenase (SDH) was lowered, which suggested the cell viability was decreased. Meanwhile, cytotoxicity was determined by measuring lactic dehydrogenase (LDH) activity released into the extracellular space, which also indicated that the cell membrane integrity was lost. These results implied that SiO2-NPs could induce cell damage in a time- and dose-dependent manner.2. In serum-free medium, HCT 116 wt cells were exposed to SiO2-NPs and SiO2-SMPs of different sizes for 24 h in concentrations of 10 and 50 μg/mL. The results showed that the corresponding cytotoxic effects were decreased with the increase of SiO2 particle sizes. The significantly different toxic effects of SiO2-NPs and SiO2-SMPs indicated that when the sizes of substance were reduced to the nanometer scale, the biological activity and toxicity of the material probably be changed a lot. Therefore, the nanometer scale materials may have potential and severe toxic effects on organisms.3. In serum-free medium, HCT 116 wt cells were exposed to non-modified 70 nm SiO2-NPs and chemically modified group 70 nm H2N-SiO2-NPs and 70 nm HOOC-SiO2-NPs for 24 h in concentrations of 10 and 50 μg/mL respectively. The results demonstrated that the cytotoxicities of modified SiO2-NPs were lower than non-modified 70 nm SiO2-NPs, and the toxic effects of two chemical-modified SiO2-NPs (-NH2 and -COOH) showed minor differences. It is indicated that individual chemical modification may reduce SiO2-NPs toxic effects to cells. Consequently, we can artificially modify the NPs via different strategies to reduce the risk of bio-safety without changing the properties of nanomaterials.4. In the medium with the presence of proteins (FCS, BSA, Mucin), HCT 116 wt cells were exposed to 10 μg/mL,12 nm SiO2-NPs for 24 h. The results showed FCS and BSA can notably inhibit the SiO2-NPs cytotoxic effect in a dose-dependent manner, while the effect of Mucin protein was not potent. Therefore, different proteins have different effects on the biological effects of SiO2-NPs. This can be explained by the various affinities between proteins and SiO2-NPs which contributes the formation of various corona.5. HCT 116 wt and HCT p53 knockout (HCT 116 p53-/-) cell lines were exposed to different concentrations of SiO2-NPs for 24 h. The results indicated that there was no significant difference in cellular damage between the two cell lines. Similarly, there was also not much difference could be observed in HCT 116 Bax control (HCT 116 Bax+/-) and HCT 116 Bax knockout (HCT 116 Bax-/-) cell lines. It implied that p53 and Bax were not the major regulation factors in HCT 116 apoptosis induced by SiO2-NPs.6. The broad-spectrum caspase inhibitor Z-VAD-FMK and Q-VD-OPH were used here to intervene the process of 12 nm SiO2-NP-induced HCT 116 wt apoptosis. The results showed that Z-VAD-FMK and Q-VD-OPH were able to inhibit 12 nm SiO2-NPs-induced apoptosis and prevent the chromatin fragmentation. Western blot results demonstrated the activated protein caspase-3,-8,-9 in HCT 116 wt cells were also up regulated after the SiO2-NPs treatment. These results implicate that caspase family members play an important role in apoptosis caused by SiO2-NPs in HCT 116 wt cells.In conclusion, the potential toxic effects of SiO2-NPs on HCT 116 cells have been confirmed, which depend on the sizes of NPs, chemical modifications, the bio-macromolecules like proteins and other factors. The mechanism of the toxic effects of SiO2-NPs was not significantly correlated with p53 and Bax, but mainly with the regulation of caspase cascade in HCT 116 wt cells. |
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