The Effect of Particle Size on the Cytotoxicity of Amorphous Silicon Dioxide: An in Vitro Toxicological Study

The effect of UV/visible/NIR light (380/450/530/650/808/1064 nm) on ROS generation, mitochondrial activity and viability is experimentally compared in human neuroblastoma cancer cells. The absorption of photons by mitochondrial photoacceptors in Complexes I, III and IV is in detail investigated by sequential blocking with selective pharmaceutical blockers. Complex I absorbs UV/blue light by heme P450, resulting in a very high rate (14 times) of ROS generation leading to cell death. Complex III absorbs green light, by cytochromes b, c1 and c, and possesses less ability for ROS production (seven times), so that only irradiation lower than 10 mW cm-2 causes an increase in cell viability. Complex IV is well-known as the primary photoacceptor for red/NIR light. Light of 650/808 nm at 10-100 mW cm-2 generates a physiological ROS level about 20% of a basal concentration, which enhance mitochondrial activity and cell survival, while 1064 nm light does not show any distinguished effects. Further, ROS generation induced by low-intensity red/NIR light is compared in neurons, immune and cancer cells. Red light seems to more rapidly stimulate ROS production, mitochondrial activity and cell survival than 808 nm. At the same time, different cell lines demonstrate slightly various rates of ROS generation, peculiar to their cellular physiology.

Magnetite as iron oxide is widely used in various industries, in the pharmaceutical industry in particular where it is used for its magnetic properties. The environmental and occupational exposure to airborne nanoparticles and microparticles of iron oxide compounds have been reported. Since authors have reported contradictory results, the objective of this study was to investigate the effect of particles' size in their toxicities. The human cell line A549 was exposed with magnetite iron oxide in two size categories of micro (≥5 µm) and nano (<100 nm), with four concentrations of 10, 50, 100, and 250 µg/ml at two time periods of 24 and 72 h. The cell viability, reactive oxygen species (ROS), changes in mitochondrial membrane potential, and incidence of apoptosis were studied. Nano and micro magnetite particles demonstrated diverse toxicity effects on the A549 cell line at the 24- and 72-h exposure periods; however, the effects produced were time- and concentration-dependent. Nano magnetite particles produced greater cellular toxicities in forms of decreased viabilities at concentration exposures greater than 50 µg/ml (p < 0.05), along with increased ROS (p < 0.05), decreased cellular membrane potential (p < 0.05), and reduced rate of apoptosis (p < 0.05). The results of this study demonstrated that magnetite iron in nano-range sizes had a greater absorbability for the A549 cell line compared to micro sizes, and at the same time, nanoparticles were more toxic than microparticles, demonstrating higher production of ROS and decreased viabilities. Considering the greater toxicity of nanoparticles of magnetite iron in this study, thorough precautionary control measures must be taken before they can be used in various industries.

Airborne crystalline silica (SiO2) particles are one of the most common pollutants in stone industries. Limited studies have investigated the health effects of crystalline SiO2 nanoparticles. Hence, the objective of this study was to study the cytotoxicity of SiO2 in nano and micron sizes. A mineral quartz sample in the range of 0.2-0.8mm sizes was purchased. These particles were ground at about 5 and 0.1 microns. Human cell line A549 was exposed to micro and nanometer particles at concentrations of 10, 50, 100, and 250μg/ml for 24 and 72 h. Subsequently, the cytotoxicity of exposed cells was investigated by measuring cell survival, ROS generation, mitochondrial permeability, and intracellular glutathione content. The results showed that crystalline SiO2 nano and microparticles decreased cell survival, increased ROS generation, damaged the mitochondrial membrane, and lowered the antioxidant content of these cells in a concentration- and time-dependent manner. The toxicity of crystalline SiO2 microparticles at concentrations ≤50μg/mL was greater than for nanoparticles, which was the opposite at concentrations ≥100μg/mL. Exposure time and concentration were crucial factors for the cytotoxicity of exposed A549 cells to crystalline SiO2 particles, which can affect the severity of the effect of particle size. Due to the limitation of exposure concentration and test durations in this study, further studies on the parameters of nanoparticle toxicity and underlying mechanisms could advance our knowledge.

Size dependent cytotoxicity of ZnS nanoparticles (NPs) was investigated in Human embryonic kidney (HEK-293) cell lines by MTT assay. The cells were incubated with varying concentration of ZnS NPs of sizes 4 nm, 10 nm and 25 nm for 48 h under different (cell culture) media viscosity conditions. The results showed that the toxicity is decreased with the particle size while it is negatively correlated with the viscosity of the media. Theoretical calculations were performed, by assuming equivolume stress model and the same is explained with schematics. Similarly, the effect of particle size and shape on toxicity is explained based on the theoretical calculation of the stress. The calculations showed that out of the possible cellular entry mechanisms for the cubic or cage shaped NPs, the highest toxicity is predicted for the entry through the corners while the lowest toxicity is predicted for the entry through the faces. The experimental observations depicting the cytotoxicity as a function of the viscosity of cell culture media was also validated by stress calculations and are found to be consistent. Studies on size and shape dependence of semiconductor NPs like ZnS is rather scarce, while the role of viscosity of cell culture media on the cytotoxicity is being reported for the first time. In summary, the study indicates that the cytotoxicity is an integral function of size and shape of NPs, physical parameters of the cell culture media in addition to the post entry biochemical interactions with the host cell.

Effect of coal particle size on the coal hydrogenation reaction — In relation to the plasticity of coal

BackgroundCancer has been one of the leading causes of mortality in this era. Ruta angustifolia L. Pers has been traditionally used as an abortifacient, antihelmintic, emmenagogue and ophthalmic. In Malaysia and Singapore, the local Chinese community used it for the treatment of cancer.MethodsIn this study, the methanol and fractionated extracts (hexane, chloroform, ethyl acetate and water) of R. angustifolia were tested for its cytotoxicity using the sulforhodamide (SRB) cytotoxicity assay against HCT-116, A549, Ca Ski and MRC5 cell lines. Chemical isolation was carried out by using the high performance liquid chromatography (HPLC) and the isolated compounds were tested for its cytotoxicity against A549 cell line. Cellular and nuclear morphological changes were observed in the cells using phase contrast microscopy and Hoechst/PI fluorescent staining. The externalisation of phosphatidylserine was observed through FITC-labelling Annexin V/PI assay whilst DNA fragmentation was observed through the TUNEL assay. Other indication of apoptosis occuring through the mitochondrial pathway were the attenuation of mitochondrial membrane potential and increase in ROS production. Activation of caspase 9 and 3 were monitored. Western blot analysis was done to show the expression levels of apoptotic proteins.ResultsThe chloroform extract (without chlorophyll) exhibited the highest cytotoxic activity with IC50 of 10.1 ± 0.15 μg/ml against A549 cell line. Further chemical investigation was thus directed to this fraction which led to the isolation of 12 compounds identified as graveoline, psoralen, kokusaginine, methoxysalen, bergapten, arborinine, moskachan B, chalepin, moskachan D, chalepensin, rutamarin and neophytadiene. Among these compounds, chalepin exhibited excellent cytotoxicity against A549 cell line with an IC50 value of 8.69 ± 2.43 μg/ml (27.64 μM). In western blot analysis, expression of p53, truncated Bid, Bax and Bak while the anti-apoptotic proteins Bcl-2, survivin, XIAP, Bcl-XL,cFLIP decreased in a time-dependent manner when A549 cells were treated with 36 μg/ml of chalepin. In addition, the level of PARP was found to decrease.ConclusionHence these findings indicated that chalepin-induced cell death might involve the intrinsic mitochodrial pathway resulting in the upregulation of pro-apoptotic proteins and downregulation of anti-apoptotic proteins. Thus, chalepin could be an excellent candidate for the development of an anticancer agent.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1368-6) contains supplementary material, which is available to authorized users.

Results Aqueous extract and essential oil reduced the viability of A549 cancer cells in a concentration-dependent manner. The lowest inhibitory concentrations (IC50) for both samples of D. ammoniacum oleo-gum resin were 10 and 2.5 μg/ml for 24 hours in A549 cell line, respectively. After treatment with extract and essential oil of D. ammoniacum oleo-gum resin, ROS increased significantly compared to the control group. Although changes in caspase-3 did not show a significant increase in extract, the caspase-3 was found to be increased after exposure to essential oil and caspase-9 was downregulated after exposure to essential oil. Also, exposure to essential oil of D. ammoniacum caused a reduction in MMP level. Conclusion Based on results, the cytotoxic effect of essential oil of D. ammoniacum can induce apoptosis toward A549 cell line via induction of oxidative stress, MMP depletion, and caspase-3 activation, which is independent to mitochondrial cytochrome c release and caspase-9 function.

Two novel steroidal saponins, trilliumosides K (1) and L (2), were isolated from the rhizomes of Trillium govanianum led by bioactivity-guided phytochemical investigation along with seven known compounds: govanoside D (3), protodioscin (4), borassoside E (5), 20-hydroxyecdysone (6), 5,20-hydroxyecdysone (7), govanic acid (8), and diosgenin (9). The structure of novel compounds 1-2 was established using analysis of spectroscopic data including 1D and 2D nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HR-ESI-MS) data. All isolated compounds were evaluated for in vitro cytotoxic activity against a panel of human cancer cell lines. Compound 1 showed significant cytotoxic activity against the A-549 (Lung) and SW-620 (Colon) cancer cell lines with IC50 values of 1.83 and 1.85 µM, respectively whereas the IC50 value of Compound 2 against the A-549 cell line was found to be 1.79 µM. Among the previously known compounds 3, 5, and 9, the cytotoxic IC50 values were found to be in the range of 5–10 µM. Comprehensive anti-cancer investigation revealed that Compound 2 inhibited in vitro migration and colony-forming capability in the A-549 cell line. Additionally, the mechanistic analysis of Compound 2 on the A-549 cell line indicated distinctive alterations in nuclear morphology, increased reactive oxygen species (ROS) production, and decreased levels of mitochondrial membrane potential (MMP). By upregulating the pro-apoptotic protein BAX and downregulating the anti-apoptotic protein BCL-2, the aforementioned actions eventually cause apoptosis, a crucial hallmark in cancer research, which activates Caspase-3. To the best of our knowledge, this study reports the first mechanistic anti-cancer evaluation of the compounds isolated from the rhizomes of T. govanianum with remarkable cytotoxic activity in the desired micromolar range.

Effect of NaCl concentration and particle size on chalcopyrite leaching in cupric chloride solution

Oridonin, a diterpenoid compound, extracted and purified from Rabdosia rubescen has been reported to have cytotoxic effect on tumour cells through apoptosis, and tyrosine kinase pathways are involved in these processes. A specific epidermal growth factor receptor (EGFR) inhibitor AG1478 was used to examine the relationship between EGFR signal pathways and oridonin-induced apoptosis and autophagy in EGFR abundant human epidermoid carcinoma A431 cells. Inhibition of EGFRaugmented oridonin-induced A431 cell apoptosis, while the changes of expression of downstream proteins, Bcl-2, Bcl-xL, Bax, cytochrome c, pro-caspase-3, Fas, FADD and pro-caspase-8 suggested that both the intrinsic and extrinsic apoptotic pathways are involved in these processes. Pretreatment with AG1478 aggravated oridonin-induced loss of mitochondrial membrane potential (MMP) and increased ROS generation in A431 cells, while a ROS scavenger, N-acetylcysteine (NAC) completely reversed oridonin- and AG1478-induced ROS generation and apoptosis. Therefore, AG1478 augmented oridonin-induced apoptosis by enhancing oxidative stress. Pretreatment with AG1478 decreased the expression of downstream MAPK proteins ERK, JNK and P38 and their phosphorylated forms to varying degrees compared with oridonin alone treatment. Then after administration of ERK, JNK and P38 inhibitors, only JNK inhibitor SP600125 effectively augmented oridonin-induced apoptosis and ROS generation. Therefore, in EGFR downstream pathways, JNK played a major role in preventing oridonin-induced apoptosis. Autophagy antagonised apoptosis and exerted a protective effect in A431 cells, and both AG1478 and SP600125 decreased oridonin-induced autophagy. Inhibition of EGFR augmented oridonin-induced apoptosis and this was caused by enhanced oxidative stress, and JNK played a major protective role by increasing autophagy, leading to antagonising apoptosis and ROS generation.

The molecular basis by which dietary restricted feeding reduces mitochondrial reactive oxygen species generation

Introduction: BKCa channels are thought to provide protection during ischemic insults in the heart. Rottlerin, has been implicated as a potent BKCa activator, however this has not been firmly established. The purpose of this study was two fold: 1) assess the specificity of rottlerin for BKCa channels and 2) investigate the efficacy of BKCa channel activation as a cardioprotective agent during cardioplegia/reperfusion (CP/R). Methods: Wild type and BKCa knock-out mice with and without rottlerin (only administered in CP) were subjected to an isolated heart model of CP/R. Hearts were perfused for 30 min at 37C (baseline), followed by intermittent cold crystalloid cardioplegia (intermittent St Thomas II, 10C), and normothermic, normoxic, reperfusion for 30 min. In additional studies, mechanism of rottlerin-induced cardioprotection was investigated using H9c2 cells subjected to in vitro CP/reoxygenation and assessed for mitochondrial membrane potential (TMRE) and ROS (DCFDA) production. Results: CP and 30 min reperfusion decreased LVDP (58.7 +/- 5 % baseline, n=8 ), +/- dP/dt ( 71.1+/- 6.4, 59.2 +/-6.6, % baseline respectively) and coronary flow (CF) (66.8+/-7.4, %baseline) in wild type mice. Rottlerin, (100 (n=3), 500 nM (n=5)) delivered in the CP solution dose dependently increased the recovery of LV function and CF to near baseline levels (500 nM rottlerin, LVDP: 94.1+/- 2.3, +/-dPdt: 99.6 +/- 7.2, 111.0+/- 15.0, % baseline, and CF: 128.9 +/-30.0 %baseline ). BKCa KO hearts treated with (n=4)or without (n=3) 500nM rottlerin, were similar to wt CP hearts, showing no improved cardioprotection (BKCa KO + 500 nM rott: LVDP: 57.7+/- 4.0, +/-dP/dt 69.5 +/-12.0, 59.8+/- 4.9, and CF 73 +/- 6.7). H9c2 cells subjected to in vitro CP/R, displayed reduced mitochondrial membrane potential (39% +/-.09 % decrease relative to control, p&lt;.01) and increased ROS generation (50%+/-.04 increase, p&lt;.01), both of which were dose dependently normalized by rottlerin (100 nM-1 uM) (minimum n=8). Conclusions: Activation of BKCa channels profoundly rescues ischemic damage associated with CP, likely via mitochondrial effects on improved mitochondrial membrane potential and reduced ROS generation.

Introduction: Mitochondrial BKCa channels are thought to provide protection during ischemic insults in the heart. Rottlerin, has been implicated as a potent BKCa activator, however this has not been firmly established. The purpose of this study was twofold: 1) investigate the efficacy of BKCa channel activation as a cardioprotective agent during cardioplegia/reperfusion (CP/R) and 2) assess the specificity of rottlerin for BKCa channels. Methods: Wt and BKCa KO mice were subjected to an isolated heart model of CP/R. Hearts were perfused for 30 min at 37C (baseline), followed by intermittent cold hypoxic CP with or without rottlerin (St Thomas II, 10C), and normoxic 37C reperfusion for 30 min. Mechanism of rottlerin-induced cardioprotection was also investigated using H9c2 cells subjected to in vitro CP/reoxygenation and assessed for mitochondrial membrane potential (TMRE) and ROS (DCFDA) production. Results: CP/R decreased LVDP (61.6 +/- 6 % baseline, n=6 ), +/- dP/dt (76.6+/- 7.3, 63.4 +/-7.7, % baseline respectively) and coronary flow (CF) (69.6+/-6.0 % baseline) in wt mice. Rottlerin, (100 nM (n=3), 500 nM (n=6)) dose dependently increased the recovery of LV function and CF to near baseline levels (% baseline 500 nM rottlerin, LVDP: 87.6+/- 4.0, +/-dPdt: 97.6 +/- 5.9, 90.9+/- 6.6, and CF: 125.3 +/-22.8, all p&lt;.01 vs CP ). BKCa KO hearts treated with (n=7) or without (n=7) 500nM rottlerin, were similar to wt CP hearts (% baseline BKCa KO + 500 nM rott: LVDP: 66.9+/- 6.0, +/-dP/dt 73.0 +/-3.6, 65.2+/- 4.2, and CF: 94.1 +/- 9.0). H9c2 cells subjected to in vitro CP/R, displayed reduced mitochondrial membrane potential (39 +/-.09 % decrease) and increased ROS generation (50+/-.04% increase), both of which were significantly (p&lt;.01) normalized by rottlerin (100 nM-1 uM) (n=8). Conclusions: Activation of BKCa channels profoundly rescues ischemic damage associated with CP/R, likely via effects on improved mitochondrial membrane potential and reduced ROS generation.

The increasing trend of nanoparticle usage in science and technology has led to significant human exposure. Occupational exposure to iron oxides and silica dust has been reported in mining, manufacturing, construction, and pharmaceutical operations. The combined toxicological effects of nanoparticles and simultaneous exposure to other compounds have given rise to a new concern. Hence, the objective of this study was to investigate the toxicological effects of magnetite and polymorphous silicon dioxide nanoparticles in single and combined exposures. The polymorphous silicon dioxide nanoparticles were obtained from the milled quartz particles under 100 nm in diameter. The milled particles were purified through chloric and nitric acid wash processes. The toxic effects of the magnetite nanoparticles were investigated independently and in combination with quartz using the A549 cell line for durations of 24 and 72 h, and using diverse concentrations of 10, 50, 100, and 250 μg/mL. MTT, ROS, mitochondrial membrane potential, and cell glutathione content assays were used to evaluate the amount of cell damage in this study. The statistical significance level in one-way ANOVA and independent t test was considered to be at the 5% confidence level. The size and purity of polymorphous silicon dioxide nanoparticles were measured by TEM and ICP-OES analysis, respectively. The particles' diameters were under 100 nm and demonstrated a purity of higher than 99%. The toxicity results of this study showed a dependency on concentration and exposure duration in reducing the cell viability, cellular glutathione content, and mitochondrial membrane potential, as well as increasing the ROS generation in single and combined exposures with magnetite and polymorphous silicon dioxide nanoparticles. The toxic effects of combined exposure to these nanoparticles were less than the single exposures, and statistically significant antagonistic interactions were detected. Combined exposure to polymorphous silicon dioxide and magnetite nanoparticles, in comparison with their single exposures, could affect health in an antagonistic manner. Since this study has been the first of its kind, further studies investigating the health effects of single and combined exposures to these compounds are needed to verify our findings. Generally, studies such as this one could contribute to the field of combined toxicity effects.

HO-1 Regulated Autophagy and Apoptosis In CML Cells Through Mitochondrial-beclin1 Pathway