Nowadays, nanoparticles are widely used in different industries and businesses such as textiles, staining, medical imaging, and disease diagnosis. Therefore, global investment in nanotechnology research and development is increased significantly [1, 2]. This potential is due to unique physiochemical characteristics of materials such as large surface, different electronic properties, and reactivity level [3, 4]. Currently, NPs are used in different industrial processes and a vast spectrum of products and systems of daily life (e.g. food, drinks, and sunscreen creams). Moreover, nanotechnology has many promising applications in the food industry, such as improvement of taste and texture and increasedproduct shelf life [5, 6].
One of the useful nanoparticles in industry and medicine is MgO NPs . MgO NPs as an important alkali metal oxide is used in catalytic reactions and wastewater treatment. MgO NPs show high capacity in absorption and analysis of pollutants and fabrication of insulation and resistant materials against high temperature . The results of different studies show that MgO NPs alone or with other antimicrobial groups are used as an effective antibacterial agentto increase the health of food products . High stability, biocompatibility, and low toxicity are among the important properties of magnesium oxide .
SiO2 NPs is a non-metallic oxide and has different applications in chemical industries and is used as an additive in foodstuff, drugs, cosmetics, etc. This NP is used in biotechnology and medicine, too . The usage of SiO2 NPs as an anticaking agent in powder and flavoring products is taken into consideration.[11, 12].Nowadays, SiO2 NPs is one of the components used in foodstuff and is confirmed as an additive (E551). Therefore, finding information about the toxicity and safety of SiO2 NPs as a food additive is urgently need. So far, different studies are conducted on cytotoxicity of NPs and shows that size, structure, and surface properties are important factors for their toxicities [14, 15]. It has been indicated that the toxicity of NPs is dependent on their concentration . Determination of suitable dose to evaluate cell toxicity is a key component to understand toxic effects of NPs under real physiological conditions . Kyung et al. (2009) investigated SiO2 NPs toxicity in rat keratinocyte cells (HEL-30) and showed that SiO2NPs toxicity is dependent on exposure and size of NPs. LDH leakage was only observed in cells exposed to NPs with the diameters of 30 and 40 nm and NPs with the diameters of 118 and 535 nm did not show any leakage. The results of MTT assay showed a certain degree of toxicity for 30-40 nm particles at high concentrations (100 µg) compared with 118 and 535 nm . NPs due to their small size can pass through physiological barriers and enter the host circulation system and cause disorders in cell process when they reach the tissues and organs and this leads to disease . Different studies have been conducted on the effect of NPs toxicity and toxicities of titanium oxide, zinc oxide, silver, and gold on the different cells, such as skin, macrophage, heart, liver, and kidneys . The distribution of NPs in tissues and organs due to their small size and creation of structural and histopathological changes and also tissue necrosis and dystrophy can be caused concerns . NPs toxicity is mainly dependent on the action of NPs and biomolecules. Oxidative stress inductionresulting reactive oxygen species (ROS) generationis one of the main toxicity mechanisms of NPs. High production of ROS leads to DNA damage, releases intracellular calcium and disorders in mitochondrial function followed by cell death [21, 22]. Recently, the effect of MgO NPs toxicity in human cardiovascular endothelial cells has been reported that is dependent on exposure duration, size, and NPs concentration [23, 24].
Modern nanotechnology is developing rapidly and the production of NPs as the main component of this science is increased. Therefore, human exposure to NMs increasesconsequently.Increased use of different nanoparticles that most of them are related to biological systems necessitates careful examination of the side effects. This research aims to investigate the cytotoxic effects of MgO and SiO2NPs on human colon adenocarcinoma cells (HT-29) after 24 hours of exposure.
SiO2NPs and MgO NPs were synthesized according to the methods proposed by Ding et al. (2016) and Parvajanti et al. (2015), respectively [25, 26].Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and X-ray diffraction (XRD) have been used in the characterization of NPs to acquire the size, shape, and crystalline character(Fig. 1and 2).
SiO2 NPs and MgO NPs sample preparation
To prepare different concentrations of MgO (50 nm) and SiO2 (90-110 nm) NPs suspension, MgO and SiO2 NPs were suspended in deionized water and various concentrations of these NPs (25, 50, 100, and 200 µg/ml) were prepared. The suspension was homogenized with an ultrasonic bath (Sonica MH S3, Soltec, Italy) and sterilized using a syringe filter.
HT-29 cellswere obtained from Cancer Research Center, Shiraz University of Medical Sciences. This cell line category belongs to human colon adenocarcinoma. The cells were cultured in RPMI1640 containing glutamate (Sell Max Company, Iran)supplemented with10% fetal bovine serum (Gibco Company, USA), antibiotics penicillin-streptomycin 100 units/ml (Bio Idea Company, Iran), and amphotericin B 2.5mg/ml (Sigma Company, USA).Cells were maintained in a humidified incubator at37 C in a 5% CO2 atmosphere. The supernatant of cells was changed every 48 hours and at 80-90% confluence, cells were detached by using 0.25% trypsin and were sub-cultured into 25 cm2 flasks.
The Cytotoxic effects of the MgOand SiO2NPs were evaluated by MTT assay, which is based on the reduction of MTT to insoluble purple formazan crystals by mitochondrial dehydrogenases.A mitochondrial dehydrogenase activity is an important marker in cellviability.Briefly, for this assay, the cells were seeded (1× 104 cells per well) in a 96-well culture plate. After overnight incubation, the cells were exposed at various concentrations (25-200µg/ml) of MgO and SiO2NPs for 24h. The MTT assay was performed according to the manufacturer’s procedure(Pars Tous Company, Iran), following the treatment, 0.1 mg of MTT was added to each well, then incubation of the cell was performed at 37°C for 4h. After removing of culture media, 100 µl of DMSO was added to each well to dissolve the purple formazan crystals. Absorbance was read using a microplate reader at 570 nm (Tecan, Salzburg, Austria).All experiments were performed in triplicate.Cell viability was determined as a percent of the control culture value.
Fluorescence technique (Ethidium bromide/Acridine orange staining)
Fluorescence staining and cell counting under microscope is one of the simple methods to identify living and dead cells. In order to detect apoptotic or necrotic nuclei ethidium bromide and acridine orange was used. Acridine orange is a vital color that can be absorbed by living cells and penetrate into the cells’ DNA. In this condition, living cells’ chromatin under microscope appears uniformly green. Ethidium chromatin stains dead cells that have lost their membrane uniformity. Necrotic cells stained orange with no condensed chromatin.The late apoptotic cells appeared green with condensed and often fragmented nuclei. .
HT-29 Cells were grown in 6-well culture plate at a density of 5×105 cells/welland allowed to attach for 24 h. After 24 h of cell attachment, plates were washed with phosphate buffered saline (PBS) and then cells were incubated with various concentrations (25–200 μg/ml) of MgO and SiO2 NPs for 24 h. HT29 cells in culture medium without NPs were used as the control. After incubation, the supernatant medium was removed and the cells were washed with a sterile PBS. Then 200µl/ml of filtered EDTA (1mM) was used for each well to harvest the cells from 6 well culture plate. A centrifugation (200g,10min) was performed and the cells were collected and suspendedin 1 ml of fresh RPMI 1640 medium.10 ul of dye-cell suspension was placed on hemocytometer and cells are viewed under a fluorescence microscope and counted to quantify apoptosis.
Data analysis was performed with SPSS 16 and the results were reported as mean and standard deviation. In order to investigate the changes in different treatments, one-way ANOVA was used. In cases in which a significant difference existed between treatments, means were compared with Duncan test. Thelevel of P<0.05 were considered as significance.
In the current study, HT29 cells were exposed to MgO NPs (50nm) and SiO2 NPs (90-110nm)at 25-200 µg/ml for 24 h.The amounts of cytotoxicity created by MgO and SiO2 NPs were evaluated using the MTT assay and fluorescent(ethidium bromide/acridine orange) staining. As shown in Fig. 3, treating the HT29 cells with SiO2 NP and MgO NP increased cytotoxicity in a dose-dependent manner. The results indicated that the percentage of cytotoxicity increased when the concentrations of both types of NPs increased.
The results of fluorescence staining confirmed the results related to MTT. A significant increase in the cytotoxicity was observed by increasing the NPs dosage. As shown in Fig. 4,high dosage of MgO and SiO2 NPs showed a significant cytotoxicity. At 200 µg/ml dosage, the apoptosis percentages in the cells treated with SiO2 and MgO NPs were78/91 and 82/78, respectively. Also The results of Fluorescence staining by using ethidium bromide/acridine orange stain showed that the living cells appeared in green and dead cells appeared in orange or brown (Fig. 5).
DISCUSSION AND CONCLUSIONS
In recent years, the studies that have focused on the negative effects of NPs on human health are increasing significantly but still is a concern about the potential biological effects and toxicity of these particles. In the current study, the cytotoxicity of MgOand SiO2 NPs was investigated using MTT assay and fluorescence technique. In the current study, it was found that exposure to SiO2 and MgO nanoparticles at a dosage level of 25-200 µg/ml caused dose dependent cytotoxicity as revealed by MTT assay and fluorescence technique.These findings are in accordence with previous studies. Schneideret al. (2017) investigated the cytotoxic effects of metal nanoparticles and metal oxide (copper, silver, gold, zinc, and titanium oxide) on human colon adenocarcinoma cells using MTT assay and trypan blue staining. Their findings showed a significant decrease in mitochondrial activity, that this value in cells treated by high silver concentration was 40% and in cells treated with gold was 60% . These results were similar to our results. The results of previous studies proved that SiO2 NPs can intervene with molecular cells and influence the cell growth and also, they showed cytotoxic effects . In another study, Sergentet al. (2012) investigated the cytotoxic and genotoxic effect of SiO2 NPs with the diameters of 25 and 100 nm. Their results showed that the exposure of HT29 cells to SiO2 NPs induced slightly cytotoxicity effect. There is a concern about the distribution of nanoparticles in tissues and organs due to their small size and creating structural and hisopathologic transformation and also tissue necrosis and dystrophy. Among metal oxide nanomaterials, MgO NPs due to low cost and biocompatibility are very important. Also, studies showed that MgONPs hadlower toxicity compared with other NPs such as ZnO and TiO2. The toxic effect mechanism of MgONPs in HT-29 cell after 48 hours exposure was investigated by Alqahtaniet al.In theirstudycytotoxicity was evaluated by using MTT and neutral red uptake assays. The results showed that MgONPs reduced cell viability in concentration and time-dependent manner. MgO NPs caused oxidative stress by reducing glutathione (GSH) concentrations and enhancingreactive oxygen species (ROS) and lipid peroxidation levels . Jebali et al. (2013) reported the toxic effect of SiO2NPs in the form of wire, rod,and sphere with different sizes (20, 50 and 100 nm) on rat pulmonary cells after 6 and 24 hours of exposure, using MTT and MTS cytotoxicity tests. Their results showed that NPs with smaller size (20 nm) have higher toxicity in compared with larger sizes. Also, silica nanoparticles in spherical form had higher cytotoxic effects. Moreover, 24 hours of incubationed to higher cell death . Bohmertet al. (2012) in their study on NPs toxicity of silver coated with peptide, showed the strong cytotoxic effects of silver NPs on duplication and differentiation where these effects intensified with increased incubation time and dose and decreased particles size. Nanoparticles toxicity in the cell started with morphological transformation and decreased cell binding capacity and after 24-48 hours of exposure, cell death occurred .
In a recent study by Mai et al. (2012), MgO NPs showed considerable toxicity in AGS gastric cancer cells that was dose and time dependent. These findings are similar to the results of the present study. This study also showed structural and physiological transformations including chromatin compression and fragmentation .
In conclusion, our results showed that MgO and SiO2NPs can be toxic elements for HT-29 cells and this toxicity is dose-dependent. Therefore, it is better to avoid them in foodstuffs and packages that are directly connected to food.
We would like to thank University of Shahrekord, University of Shiraz, Miss.M Aghazi, Dr A Mohammadi, DrM Masoudian, Dr A Kawarizadeh,and M Sangabi for supporting and their technical assistance.
CONFLICT OF INTEREST
The authors did not report no conflict of interest.