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Effects of SILICON DIOXIDE ON Lung cells using High Content Microscopy

Abstract

The aim of the experiment was to find out the effect of Silicon iv Oxide nanoparticles on the cancerous alveoli epithelial cells of the cell line H441.  Such parameters as cell area, permeability intensity, mitochondrial intensity, cell diameter, cell heterogeneity, cell circularity, cell roundness, cell clumpiness, vacuole area and segmented mitochondrial activity using high content microscopy. To determine the effect of the Nano-SiO2 standards were used such as FCCP, DSMO and water as the standards Data analysis was carried out using the combined standard deviation and standard errors of the mean of the five 96-well plates and graphs drawn of the standard error of the mean and combined standard deviation.  From the graphs, using the standard as the reference point, the study findings showed that Nano-SiO2 increases membrane permeability and it has an effect on the mitochondrial activity.

 

Introduction

Background

There is a growing demand for synthetic silicon dioxide nanoparticles in both the food and construction industry as they are used in coatings, paints, adhesives, composites and food additives (Athinarayanan et al., 2015; Younes et al., 2018). There is evidence that some cosmetic products contain silicon dioxide. Additionally, silicon dioxide nanoparticles have found use in the pharmaceutical industry in drug delivery systems (Wiemann et al., 2018).  However, there is evidence of adverse effects of silicon dioxide on the lungs in the sense it leads to silicosis which is a progressive and irreversible lung disease, lung cancer and COPD (Forbes et al., 2014; Wiemann et al., 2016; Younes et al., 2018). There is a need to find out the effect of the nanoparticles of silicon dioxide on the lungs’ alveolar epithelial cells.

Literature Review and Objectives

The effect of silicon dioxide nanoparticles on animal cells is well documented in various studies. Breznan et al. (2017) conducted a study to find out the biological potency of amorphous silicon nanoparticles (SiNPs) with similar molecular size in a wide of cell lines to determine their physicochemical and biological factors on their toxicity. The findings of this study showed that mice macrophages had the highest SiNP exposure. The cytotoxicity of the individual cell lines was correlated with  the cytokine responses differed which was evidenced through type-specific differences in inflammation-associated pathways. In another study, the ex vivo cardiac function and mitochondria were investigated after exposure to SiO2 nanoparticles (Lozano et al., 2020).  The findings indicated that exposure to nanoparticles of SiO2 compromised cardiac function through mitochondrial dysfunction through the opening of the mitochondrial permeability transition pore (Lozano et al., 2020). Therefore, the current study focuses on the effect of SiO2 nanoparticles on the alveolar epithelial cells cell line.

Various studies have been conducted to determine the toxicity of SI02 nanoparticles on the alveolar epithelial cells using a wide range of concentrations. For example, in a study conducted by Okoturo-Evans et al.(2013) to find out the effects of SiO2 nanoparticles on  A549 lung epithelial cells, the scholars used SiO2 nanoparticles at the concentration of 100ug/ml in the presence of 1.25nserum. The results showed that there was a rapid deterioration of cells in the presence of Si02 nanoparticles.  There is evidence of  SiO2 nanoparticles toxicity on the epithelial human cell line NCI-H441 through the production of proinflammatory cytokines and oxidative stress (Farcal et al., 2013). The findings of the study indicate the possibility of using vitro models to study pulmonary toxicity as long as the models closely mimic the complexity of the in-vivo situation. In another study, human lung epithelial cells, BEAS-2B were continuously exposed to amorphous silica nanoparticles (SiNP) at a concentration of  5ug/ml for 40 passages (Guo et al., 2017). It was evident from the study that prolonged exposure of the silica nanoparticles to amorphous SiNPs induced malignant transformation as indicated by aggressive cellular proliferation and increased migration of the cells (Guo et al., 2017).  A similar study has supported the potential SiO2 toxicity on nanoparticles that was conducted by Li et al. (2019). The study aimed at exploring the role and mechanism of SiNPs in the tumorigenesis of lung cancer in Xuanwei human mononuclear cells (THP-1) as well as the human bronchial epithelial cells (BEAS-2B). The findings of the study suggested that SiNPs enhanced the proliferation and Epithelial-Mesenchymal Transition of BEAS-2B cells by inducing the releases of TGF-α from THP-1 cells (Y. Li et al., 2019).

There is a need to understand alveolar epithelial cells’ response mechanistically and come up with a method of detecting nanoparticles that are toxic and discriminating them from those that have a non-toxic epithelial response (Wiemann et al., 2018). Validated tools with this capacity will ensure an efficient process of investigating the safety of nanoparticles.  The same scientific, evidenced-based approach will reduce the burden on animal studies by eliminating the need to do or repeat in vivo testing especially if there is an in vitro method.

The safety of inhaled compounds is investigated in vivo using rats. The studies are usually 30-to-90-day studies which require sacrificing a minimum of six rats at a single point over a wide range of doses meaning that a total of 180 rats are needed (Forbes et al., 2014; Hoffman et al., 2017, 2020).  The end-point of assessment of inflammatory changes heavily depends on histopathologic examination of lung slices that are assigned a qualitative description of inflammation or pathological manifestations (Hoffman et al., 2017).  There is no standardized procedure for the same in vivo assessments besides the fact that they are time-consuming.  On top of that, there is no clear-cut correlation between immune responses in rats and healthy human lungs. Rats are obligate breathers with significant differences in their lung anatomy, physiology and biology meaning that they are more sensitive to inflammation-based responses than human beings (Wiemann et al., 2018). It is an uphill task to conduct alveolar epithelial cells studies using cells derived from animals because of the inaccessibility of the cells. Besides, the alveolar epithelial population from the bronchoalveolar lavage fluid is small and the lavage procedure can damage both the health and phenotype of macrophages.

Hoffman et al. (2017) developed a high content image analysis assay and employed it to offer a detailed morphological characterization of rate and human alveolar-like macrophages coupled with their response to a phospholipidosis-activating agent.  High-Content Imaging/Analysis commonly referred to as high content screening (HCS) is one of the most powerful tools to be invented which facilitates assessing molecular, cellular and tissue-based toxicity, more specifically in the field of predictive toxicology. Studies have been conducted using high content imaging to investigate alveolar macrophage response to novel inhaled compounds.  Hoffman et al. (2020), conducted a study to find out if high content image analysis can distinguish between a wide range of drug-induced foamy macrophages phenotypes as well as to find out the level of reversibility of the foamy phenotypes through assessment of changes in morphology over time. The cell morphometric properties were determined by employing the high content image analysis.  The findings of the study showed that high content analysis can differentiate between phenotypes of foamy macrophages.

Therefore, this study aims to find out the deleterious effect of silicon dioxide on the alveolar epithelial cells through consideration of such morphometric parameters as cell diameter, mitochondrial activity, cell clumpiness, circularity, cell permeability and intensity and heterogeneity using high content microscopy analysis.  This study will contribute toward informing policy on the use of silicon dioxide on various human consumed products to avert potential health hazards.

Materials and Methods

NCI-H441 (H441) is the cell line that was used in this experiment that was first isolated from the pericardial fluid of a male patient with papillary adenocarcinoma of the lung.  The cells were exposed to different nanoparticles in the 96-well plate.  These lines of cells were grown in RPMI-1640 medium which was having 10%v/v FBS supplemented with 100IU/ml penicillin-100 ug/ml streptomycin solution and 2Mm L-glutamine in a flask and after getting enough of them, they were placed in the 96 well plates. The cells were allowed to grow for 24 hours before getting exposed to nanoparticles which is a set-up that simulates nanoparticles toxicity on the lungs.

Cells were incubated with either negative and positive controls, that is, 2Mm L-glutamine having DMSO 1% as the vehicle or dilution control, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) for mitochondrial activity control, 0.5% v/v Triton X (TX) for cell wall permeability control and 20% water for dilution control at a concentration of 100-0.4 µg/ml in a full cell culture medium supplemented with supplemented with 100IU/ml penicillin-100 ug/ml streptomycin solution or silicon iv oxide at various concentrations. The compound exposure studies were carried out 24 hours after seeding.  It should be noted that for epithelial cells, they were exposed to the nanoparticles 2 hours after isolation as soon as the cells had attached to the 96-well plate.  For studying the cell health and morphology, the cells were stained with a cocktail of dyes, that is, Hoechst 33342 10 μg/mL, MitoTracker Red 300 nM and Image-It Dead Green 25 nM (Invitrogen, Renfrewshire, UK) for 30 min.  The cells were then washed once with 100 μL PBS before getting centrifuged at 380g for fixation using 3.7% w/v paraformaldehyde for 15 min.  The fixed cells were then stained overnight using Blue Nuclear stain diluted 1:1000 based on the protocol of the manufacturer.  Cells were washed once using PBS before imaging.  It is essential to bear in mind that cells are fixed on the nanoparticles, that is killed but preserved in their original formation with nanoparticles (Silicon dioxide).  Cells were stored at 4°C. The EVOS M7O0 Imaging System was employed to capture images using a 40x objective employing a standard 2D imaging mode with an exposure time of 0.1s

Image analysis was carried out using the Celeste Image Analysis Software.  For cell health and morphology analysis, Hoechst 33.342 cell nuclear was employed to identify nucleated cells.  Cell Mask Deep Red dye is one of the most established cell delineation tools that have found use in cellular imaging and contributed towards highlighting the cytoplasmic regions in the cells that were identified.  On the other hand, vacuoles within the cells were identified because of negative staining with Cell Mask Deep Red.  MitoTracker Red is known to detect changes in the mitochondrial membrane potential and it accumulates in the mitochondria.  Image-It Green Dead cannot penetrate health cells and does so when the plasma membrane of the cells is compromised.  The two health stains were reported as fluorescence intensity values.

From the analysis, quantitative measurements such as cell area, nuclear area, mitochondrial activity, cell permeability, number of vacuoles per cell, cell diameter, cell circularity, cell roundness, cell clumpiness, segmented mitochondrial activity, permeability intensity and vacuole area in each cell.  The experiment was carried out in a total of 5 96-well plates at varying concentrations of the nanoparticles.

The data were normalized based on the control and the maximum of the nanoparticles within the cell. The normalized data was then used to calculate the mean and standard deviation of each well in all the 96-well plates. Data was then summarized using the combined mean and standard deviation before presentation in a line graph with errors bars as the standard deviations.

 

Results

The results of the experiment were summarized using line graphs of the concentration of the various treatments against the various cell characteristics that were being measured.  The data presented here is for only silicon oxide’s effect on the alveolar epithelial cells NCl-H441 cells.  Only three graphs were selected which exhibited significant difference between the treatments.  The three characteristics were selected since they had complete data in sharp contrast to the rests of the graph.

Cell Mean Diameter

There was a significant difference in terms of cell mean diameter between silicon oxide at the concentration of 11.11ug/dl (Combined Mean=11.0585, Combined SD=3.19091) and Triton x at the concentration of 1% (Combined Mean =23.55914, Combined SD=0.780107) which was a positive control in the experiment.  The difference cell diameter mean was also evident for silicon at the concentration of 11.11ug/dl (Combined Mean=8.8654, Combined SD=0.049772) and water (Combined Mean=16.163, Combined SD=3.163896) which is essentially doubled. It should be noted that some of the treatments did not have data which explains the rationale behind their absence on the graphs.

Reducing the concentration of silicon led to an increase in the cell mean diameter as evidenced in the graph between silicon at the concentration of 11.11ug/dl (Combined Mean=8.8654, Combined SD=0.04977) and 1.235ug/dl (Combined Mean=16.1354, Combined SD=0.121818852). Similar findings were obtained for cell clumpiness and its graph is shown in the appendix.

Cell Circularity

The effect of silicon iv oxide on the cell circularity is illustrated in the graph shown below.

There was a significant difference between Triton x 1%w/v (Mean= 26.16311483, SD=3.247569957) and silicon iv oxide (mean= 8.5882, SD=1.6361) at the concentration of 3.704. There was a small margin between the negative control (Combined Mean= 4.7046, Combined SD=0.8229) and silicon iv oxide (Mean= 3.6212, SD= 0.587931934) at the concentration of 1.235ug/dl, as evidenced on the graph above.  Reducing the concentration of silicon iv oxide significantly reduced its effect on cell circularity as evidenced by the negative slope on the graph of treatment concentration vs cell circularity.

Mitochondrial Activity

The effect of silicon iv oxide on the mitochondrial activity is shown in the graph below.

There was no significant difference between silicon iv oxide (Combined Mean=5.0573, SD=0.6493) at the concentration of 3.704ug/dl and negative control (Mean=5.0204, SD=2.2809). However, there was a huge difference on the effect of silicon iv oxide (Combine mean= 5.0573, SD=0.649278) and water (Combined mean=26.91046, SD=3.02797) on the mitochondrial activity.

The effect on of silicon iv oxide on the other characteristics are on the appendix.

Discussion

The experiment shows that Nano-SiO2 particles have  an effect on epithelial alveolar cells of the cell line through modification of such parameters as cell area, nuclear area, mitochondrial activity, cell permeability, number of vacuoles per cell, cell diameter, cell circularity, cell roundness, cell clumpiness, segmented mitochondrial activity, permeability intensity and vacuole area in each cell.  The toxicity of Nano-SiO2 towards cancer cells is well established( Kong et al., 2015; Liu et al., 2021; Niu et al., 2019) . The cytotoxicity of nano-SiO2 in breast cancer lines occurs through a disturbance in cancer cells growth, viability, and sensitivity towards doxorubicin induced by nano-SiO2 increased apoptosis as well as changes in several markers of apoptosis (Jeon et al., 2017). In the experiment silicon iv oxide was observed to have a variation on its effects on the alveolar epithelial cells of the cell line NCI-H441 relative to the positive and negative controls in the experiment.

There is evidence that nanoparticles can cross cellular membranes through passive processes such as diffusion and adhesive interactions facilitated by thermal capillary waves and line tension. Triton X increases cell permeability by starting with inserting into the membrane and immediately equilibrates between both the monolayers leading to an increase in the vesicle surface area (Mattei et al., 2017). Solubilization starts as soon as the visible pores open making the membrane gradually and fully solubilized. Galabova et al. (1996)  have it in their study results that there is a positive effect of the non-ionic surfactant Triton X-100 on the permeability of the Yarrowia lipolytica cells. It should be noted in their study, permeability of the cells was achieved with as little as 0.1-0.2% of Triton X-100.  In terms of permeability intensity, the effects of silicon iv oxide nanoparticles were comparable to that of Triton x as shown in Figure A02. This means that silicon iv oxide might be having an effect on the membrane permeability of human cells. The results can be supported by a study conducted on the effect of the silicon iv oxide nanoparticles on a single living HeLa cells’ membrane permeability by Kong et al. (2015).  The findings of the study had it that SiO2 NPs have cytotoxicity, but the 50 and 100 nm SiO2 NPs can elevate the cell membrane permeability by 12.5% and 9% respectively. However, increasing the size of SiO2 to 200 nm did not yield an effect on the cell membrane permeability.  (Kong et al., 2015).  In another study done by C. Li and  Poznansky, (1990) the findings of the study pointed to the effect of the uncoupler of oxidative phosphorylation, FCCP (carbonylcyanide ptrifluoromethoxyphenylhydrazone) on the tight junctions of the canine kidney cells’ permeability as well as cellular distribution of the tight junction protein ZO-1 in the epithelial cells. Its mechanism is through breakdown of proton gradients and changing the intracellular pH.   However, fewer studies have been conducted on the effect of silicon iv oxide on the cell permeability on the NCI-H441, and therefore, more experiments are required to evaluate the effect of reducing or increasing concentration on the human alveolar epithelia cells. Additionally, the effect on increasing the concentration of silicon iv oxide has not explored widely.

Studies have been done on the effect of DMSO on cells. DMSO induces reduction in cellur adenosine triphosphate more specifically during the cleavage stage which leads to downstream effects that might disrupt cellular functions.  Additionally, DMSO has a significant inhibitory effect on the growth of cells as well as the viability of cancer cells. Baldelli et al. (2021) conducted a study to investigate the off-target effect of DMSO on the functional signalling networks, drug targets, as well as downstream substrates. The effects of DMSO were heterogenous across the cell lines and the variation was based on concentration, exposure time and cell line. Out of the 187 proteins which were measured, majority of them were statistically different in at least one comparison at the highest DMSO concentration, followed by 99.5% and 98.9% at the lower concentrations. The results of this experiments suggested that there was a difference on the effect of Silicon iv Oxide (Combined Mean= 139.8641, Combined SD= 7.384088) on the cell heterogeneity as compared to DMSO (Combined Mean=207.2392, Combined SD= 11.04648). Therefore, the silicon iv oxide might not be having an effect on cell heterogeneity since the results were not comparable.

FCCP is one the most potent uncouplers of mitochondrial oxidative phosphorylation. It is well known to disrupt ATP synthesis through transportation of protons across the inner membrane of mitochondria and thereby interferes with proton gradient (Demine et al., 2019).  DMSO also has known deleterious effects on the mitochondrial integrity through making it swell, impairing membrane potential and producing reactive oxygen species followed by cytochrome c release. Studies shows that DMSO at concentrations that are more than 5% will significantly inhibit cell variability and enhance apoptosis, followed by severe mitochondrial damage (Yuan et al., 2014). The findings of this study have it that mitochondrial impairment is the major effect of DMSO induced cytotoxicity.  However, in the current study, Silicon iv Oxide does not exhibit similar results to FCCP and DMSO as shown in Figure A01 in the appendices section. Therefore, its mechanism of cytotoxicity on the epithelial alveolar lung cells of the is not related that of both FCCP and DMSO.

The current study was not devoid of limitations.  It should be noted that the high content microscopy method that was used requires special skills in terms of sample preparation, image acquisition, data storage and handling, image analysis as well as data mining which is also time consuming.  It requires complex hardware and software that might not be available in all laboratories.

The results of this study suggested that Nano-SiO2 can potentially be used as an antitumor therapy agent on the alveoli. However, more data from animal experiments are required to make the same conclusion. Future studies should focus on findings out the effects of altering the size of SiO2 on the cytotoxicity towards the alveolar epithelial cells of the NCI-H441 cell line. For example, there is need to compare the phenotypic attributes of using Nano-SiO2 against the use of the microparticles of the SiO2. The same will elucidate the efforts that need to be made to avert possible toxicity exposed to human beings.

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