Extraction of ultrafine carbon nanoparticles from samooli Bread and evaluation of their in vitro cytotoxicity in human mesenchymal stem cells

Accepted Manuscript Title: Extraction of Ultrafine Carbon Nanoparticles from Samooli Bread and Evaluation of Their In Vitro Cytotoxicity in Human Mesenchymal Stem Cells Author: Ahmed M. Al-Hadi Vaiyapuri Subbarayan Periasamy Jegan Athinarayanan Abdulrahman Saleh Al-Khalifa Ali A. Alshatwi PII: DOI: Reference:

S1359-5113(16)30681-X http://dx.doi.org/doi:10.1016/j.procbio.2016.10.018 PRBI 10840

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

28-6-2016 29-9-2016 19-10-2016

Please cite this article as: Al-Hadi Ahmed M, Periasamy Vaiyapuri Subbarayan, Athinarayanan Jegan, Al-Khalifa Abdulrahman Saleh, Alshatwi Ali A.Extraction of Ultrafine Carbon Nanoparticles from Samooli Bread and Evaluation of Their In Vitro Cytotoxicity in Human Mesenchymal Stem Cells.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.10.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Extraction of Ultrafine Carbon Nanoparticles from Samooli Bread and Evaluation of Their In Vitro Cytotoxicity in Human Mesenchymal Stem Cells

Ahmed

M.

Al-Hadi,

Vaiyapuri

Subbarayan

Periasamy,

Jegan

Athinarayanan,

Abdulrahman Saleh Al-Khalifa and Ali A. Alshatwi*

Nanobiotechnology and Molecular Biology Research Lab, Department of Food Science and Nutrition, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia.

*Corresponding author Dr. Ali A. Alshatwi Professor Department of Food Science and Nutrition College of Food Sciences and Agriculture King Saud University, P.O. Box 2460 Riyadh 11451, Kingdom of Saudi Arabia Tel: +996 1 467 7122; Fax: +996 1 467 8394. E-mail: [email protected]

Graphical abstract

Highlights: 

2-10 nm ultrafine carbon nanoparticles isolated



Ultrafine carbon nanoparticles induced cell death at high concentration



Ultrafine carbon nanoparticles alters GPX1, Bcl2, CAT and SOD genes expression

Abstract Modern technological strategies of food manufacturing may inevitably produce an unknown quantity of nanoscale ingredients or impurities in many processed or packaged foods. The indirect formation of these nanoscale contaminants, which are predicted to be emulsifier-like complex lipidaceous materials that form during heat-induced food processing, has not been studied. Moreover, the effects of chronic exposure to nanoscale contaminants on human health

and the environment are also unknown. In this present study, we identify and extract emulsifierlike ultrafine carbon nanostructures (UFCNs) from bread using a facile method. The physicochemical properties of the isolated particles were analyzed using UV-Vis-NIR spectroscopy, Xray diffraction (XRD) and transmission electron microscopy (TEM). The isolated nanoparticles exhibited fluorescence and a crystalline nature. TEM images revealed that carbon particles are 210 nm in diameter. An in vitro toxicological assessment was carried out in human mesenchymal stem cells (hMSCs) as a cellular model. Acute dose-response relationships were analyzed using an MTT assay, cell morphological assessments, and assessments of intra-cellular stress, i.e., ROS and mitochondrial trans-membrane potential and analysis cell cycle progression. The expression of oxidative stress-related genes was analyzed using real-time qPCR. Cell viability and morphology analysis results indicated that UFCNs slightly reduced cell viability and induced cellular morphological changes. UFCNs induced acute mitochondrial membrane damage at 400 µg/mL and generated ROS in hMSCs in a dose-dependent manner. The expression of GPX1, Bcl2, CAT and SOD genes showed dose- and time-dependent relationships in UFCN-treated cells. Our findings suggest that UFCNs induced cytotoxicity at 400 µg/mL in hMSCs.

Keywords: Nanoparticles, Food process, Oxidative stress, Cell viability, Cellular uptake

1.

Introduction Nanotechnology is a research field that has grown exponentially over the past few

decades, with a quickly expanding global market expected to be worth 3 trillion dollars in 2020 [1]. Nanotechnology has been applied in various sectors, including the biomedical, agricultural, food, textile, electronic and automobile sectors. Nanoparticles are more chemically and biologically active than bulk materials. Owing to their unique physico-chemical properties, nanoparticles are used as nutritional supplements, flavoring and coloring agents and antibacterial food packaging materials in the food industry [2, 3]. Recently, nanoscale ingredients have been found in processed food products, food packaging and food contact materials [4-10]. Several direct and indirect opportunities exist for the formation or introduction of nanoscale materials in food products. Synthetic food additives are used directly in many food products to improve their taste, color and texture and to enhance nutrition [11]. Our recent studies have suggested that 50% of E171 and E551 food additives comprise nanoscale ingredients [10]. Nanoscale TiO2 (E171) and SiO2 (E551) were identified in commercially available food products due to the introduction of food additives [6, 7, 9, 10]. Ahamed et al. found carbon nanostructures in bakery products and suggested that these nanostructures are formed during food processing [12]. Palashuddin et al. demonstrated that carbon nanoparticles are detected in commercial food products, such as bread, jaggery and sugar caramels [5]. These indirect unknown nanoscale contaminations can be formed during the synthesis and manufacture of food additives or the preparation, processing or packaging of food products [5, 12,13]. In addition, Metak et al. reported that silver nanoparticles migrate from nanosilver-impregnated food packaging materials to food products [8].

Modern techniques, including microwave heating, radiating, milling, grinding, fermentation and the addition of chemical agents, are used in food production [14-16]. The above-mentioned physical methods and fermentation processes have also been used for commercial or standard methods for nanoparticle synthesis. For instance, microwave irradiation, radiation and microbes are used to easily reduce metal ions to metal nanoparticles [17-19]. Therefore, modern food production methods, such as exposure to high temperatures and microwaving, may trigger nanoparticle formation in food. Principally, undefined carbonaceous particulates were found to be formed due to spontaneous reactions during high temperaturemediated food processing and preparation [5,12,13]. Samooli, a type of bread, is a stable food source eaten regularly by major populations around the world. Most bread products are produced on a large scale using modern industrial practices to reduce time, labor and manufacturing costs [20]. Large-scale manufacturing companies have introduced a variety of synthetic or modified additives, which are mostly lipidaceous materials. The three main sources of lipids in a typical bread formula are wheat flour, shortening and surfactants/emulsifiers. Apart from natural surfactants, more than 40 different emulsifiers/surfactants are applied in the food industry. Generally, surfactants are used in bread making as crumb softeners, dough strengtheners, or anti-firming agents at levels ranging from 0.3% to 1.0%. Frequently used surfactants, such as esters of citric acid esters of mono- and diglycerides (E472c), mono- and diglycerides (E471), polyglycerol esters of fatty acids (E475), diacetyl tartaric acid esters of mono- and diglycerides (E472e), sucrose esters of mono- and diglycerides (E473) and sorbitan esters of fatty acids (E491-496), are used in bakery products [21]. Remarkably, food emulsifier-based pharmaceutical formulations have been used to enhance the intestinal absorption of poorly soluble drug materials.

These types of synthetic/modified food additives have been used to accelerate bread production [22-26]. Large-scale manufacturing of emulsifiers or lipid ingredients may have indirect or direct contaminants. For example, thermally mediated fat-refinery processes generate complexations in lipid molecules, producing several hazardous compounds, such as hydroperoxides, trans-fatty acids and aldehydes [22, 27-32]. Moreover, during bread preparation, thermally mediated chemical reactions may occur, resulting in deteriorative changes or the formation of carbonaceous materials or partially combusted proteins, lipids, other food macromolecules and resultant substances that may be allergenic or toxic to humans [21,22]. Chemically, amphiphilic and surface-active materials may act as GI-permeation enhancers of poorly absorbed UFCNs in the gastrointestinal environment. During baking, predominantly in hot surfaces or environments, bread may undergo many changes on its surface, particularly gelatinization, the Maillard reaction, and caramelization (non-enzymatic browning), resulting in the formation of crust. At 100-200°C, oxygen enhances lipid oxidation, which produces nonvolatile and volatile substances. There have been no reports thus far identifying thermally induced lipidaceous materials (emulsifier-like) in bread samples. In this study, we attempted to identify non-biological exogenous lipidaceous particulates from bread samples. Earlier studies examining the formation of ultrafine particulates with lipid products and their effects on biological systems have shown that they induce metabolic stress, are cytotoxic and mutagenic and are implicated in the onset of coronary heart disease and arteriosclerosis in humans [33-35]. There is clear evidence of ultrafine particles in cooking exhaust; these are mostly insoluble/metabolically active organic particulates bound with toxic aldehydes, alcohols, ketones, alkanes, polycyclic aromatic hydrocarbons and aromatic amines [36-38]. The toxicity of nanoparticles varies based on their shape, size, surface area, chemical composition, dispersion

and aggregation [39-40]. A variety of engineered surfactant-based nano-emulsion/nanoparticles used for drug delivery have exhibited toxic effects in biological systems through inflammasomemediated oxidative stress pathways, inflammatory responses, DNA mutation, nuclear and mitochondrial damage and cell death [41-42]. Because several other indirect food contaminants, such as oxidized foodborne chemicals, enterotoxins, carcinogenic food substances, etc., are hydrophilic or hydrophobic in nature, the interfacial behavior of these lipidaceous materials may increase their intracellular permeability and contribute to oxidative stress, which would in turn lead to metabolism-related diseases. Chronic or acute intake of these ultrafine carbonaceous particulates (UFCPs) or surfactant-like emulsified compounds may contribute to the generation of free radicals (ROS) and lead to acute and chronic changes at the cellular and molecular levels. Here, we used a stem cell model to access basic acute changes after exposure. Adult stem cells can serve as in vitro model for studying cellular and molecular toxicological profiles because stem cells have the potential to differentiate into various lineages, such as adipocytes, chondrocytes, osteoblasts, nerve cells and muscle cells [43-44]. There is a substantial lack of information concerning the impact of unwanted acute or chronic intake of these foodborne lipidaceous materials on biological systems. Foodborne nanosized particulate identification and physico-chemical characterization are challenging and emerging areas of study. Moreover, the physico-chemical nature and toxicological properties of foodborne nanoparticles remain unknown. Thus, we have isolated ultrafine carbon nanostructures (UFCNs) with lipidaceous properties (such as surfactant properties) from bread and analyzed their morphology and size. Furthermore, we have assessed their in vitro cytotoxicity in human mesenchymal stem cells serving as a cellular model.

2.

Materials and methods

2.1.

Materials 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT),

5,5',6,6'-tetra

chloro-1,1',3,3'-tetra ethyl benz-imidazolyl-carbocyanine iodide (JC-1), dimethyl sulfoxide (DMSO),

acridine

orange/ethidium

bromide

(AO/EB)

and

DCFH-DA

(2',7'-

dichlorodihydrofluorescein diacetate) assay kits were procured from Invitrogen (Carlsbad, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA). Eagle's minimum essential medium (EMEM), trypsin-EDTA solution (0.25%), fetal bovine serum (FBS) and 100 U/mL penicillin/100 μg/mL streptomycin were purchased from ATCC (USA). The QuantiTect Primer Assay, FastLane Cell cDNA Kit and QuantiFast SYBR Green PCR Kit were obtained from Qiagen (Hilden, Germany). 2.2.

Extraction of ultrafine carbon nanostructures The samooli bread sample was collected from a local supermarket in Riyadh, Saudi

Arabia. The foodborne carbon nanostructure-associated surfactant was separated using a previously described method, with a few modifications [4]. One gram of crust or charred brown samooli bread was carefully removed and mixed with 20 mL of methanol. The mixture was sonicated using an ultrasonicator at 35 kHz for 60 min at 15 min intervals to reduce the temperature. Consequently, the large sediment particles were removed from the mixture using centrifugation at 12000 rpm for 10 min. After further purification, the obtained brown solution was filtered by a 0.22 µm pore size membrane filter. An additional liquid-liquid extraction method was used and validated based on the phase behavior of a ternary system of water and oil. Amphiphilic behaviors were favored to separate simultaneously as the water-soluble (polar) and

oil-soluble (non-polar) fractions. After final purification steps, the fractions were used for further analysis. 2.3.

Physico-chemical characterization of ultrafine carbon nanostructures The optical properties of isolated ultrafine carbon nanostructures were assessed using

UV-Vis-NIR spectroscopy and fluorescence spectroscopy. The isolated carbon nanostructure crystalline nature was investigated using powder XRD (PANalytical X’Pert X-ray diffractometer equipped with Cu Kα (λ=1.54056 Å)). To verify the size and shape of the carbon nanostructures using transmission electron microscopy (TEM). 2.4.

Cell culture Human mesenchymal stem cells (hMSCs) were provided as a gift by the stem cell unit,

King Khalid Hospital, Riyadh, Saudi Arabia. The cells were grown in EMEM with 10% FBS and antibiotics in T25 cm2 and T75 cm2 flasks and in 6-, 12-, 24- and 96-well culture plates at 37°C in 5% CO2. All experiments were performed using cells from passage 15 or less. The ultrafine carbon nanostructures were sonicated with cell culture medium for in vitro studies. 2.5.

Cell viability assay hMSC viability was assessed as previously described [45]. Briefly, cells were seeded in

200 μL of medium at a density of 1 x 104 cells per well in 96-well plate. After overnight incubation, hMSCs were exposed to different concentrations of ultrafine carbon nanostructures (0, 25, 50, 100, 200 and 400 μg/mL) for 24, 48 and 72 h. After exposure, 20 μL of MTT solution (5 mg/mL in phosphate-buffered saline (PBS)) was added to each well. The plates was covered with aluminum foil and incubated for 6 h at 37°C. After incubation, the supernatant was discarded, and 100 μL of DMSO was added to each well. The absorbance was monitored at 570 nm (measurement) and 630 nm (reference) using a 96-well plate reader (Bio-Rad, CA, USA).

Data were collected for triplicates each and used to calculate the mean. The percentage of cell viability was calculated using the formula:

Mean OD of untreated cells (control)-Mean OD of treated cells X 100

Cell viability (%) = Mean OD of untreated cells (control)

2.6.

Analysis of cellular morphology The effect of ultrafine carbon nanostructures on hMSCs morphology was assessed using

fluorescent microscopy. Approximately 1 × 105 hMSCs were seeded in each well of a 6-well plate. After overnight growth, the hMSCs were exposed to different concentrations (control, 50, 200 and 400 μg/mL) of UFCNs for 24 h. The supernatant medium was aspirated and rinsed with phosphate buffered saline (PBS). The hMSCs were stained using AO/EB (Acridine orange/Ethidium bromide) for 5 min at 37°C in the dark. The stained cells were examined under a fluorescence microscope (Carl Zeiss, Jena, Germany). The results are representative of a series of three independent experiments. 2.7.

Assessment of mitochondrial membrane potential Mitochondrial membrane potential was assessed using the fluorescent probe JC-1, and

cells were observed by fluorescence microscopy. JC-1 is a fluorescent dye that is able to selectively enter mitochondria. At low mitochondrial membrane potential, the dye emits green fluorescence, whereas at higher membrane potential, it forms red-orange fluorescent ‘J’ aggregates. The hMSCs were seeded at 5 x 104 cells per well in 12-well plates. After incubation, the cells were treated with UFCNs (Control, 50, 200 and 400 μg/mL) for 24 h. After 24 h of exposure, the hMSCs were stained with JC-1 (2 μg/mL) for 30 min. Stained cells were then washed with PBS thrice for fluorescence microscopy analysis (Carl Zeiss USA).

2.8.

Intracellular reactive oxygen species analysis DCFH-DA was used to analyze intracellular reactive oxygen species (ROS), which can

easily diffuse through the cell membrane. DCFH-DA is enzymatically hydrolyzed by intracellular esterases to form non-fluorescent DCFH, which is rapidly oxidized to highly fluorescent DCF by ROS. DCF fluorescence intensity is indicative of the amount of ROS formed intracellularly. The hMSCs were grown in 12-well plates, and the cells were treated with UFCNs (Control, 50, 200 and 400 μg/mL) for 24 h. After treatment, the cells were trypsinized and treated with 10 µM DCFH-DA probe. Intracellular fluorescence intensity was observed using flow cytometry (BD FACSCantoTM II, San Jose, CA, USA). 2.9.

Cell cycle analysis hMSCs were plated at 1×105 cells per well in six-well plates. After overnight incubation,

the cells were exposed to different concentrations of UFCNs for 24 h. Cell cycle analysis was performed as previously described by Alshatwi et al. [46]. Treated and untreated cells were trypsinized and fixed in 70% cold ethanol for 16 h at 4˚C. The fixed cells were washed with PBS thrice with centrifugation at 1000×g for 10 min. Consequently, the pellet was resuspended in 1 mL of propidium iodide (PI) staining solution for 15 min at 37°C. The cell cycle stage was observed using flow cytometry (BD FACSCantoTM II, San Jose, CA, USA), with an excitation wavelength of 488 nm and an emission at 670 nm. The data presented are representative of those obtained from at least three independent experiments conducted in triplicate. 2.10.

Quantitative real-time PCR analysis Gene expression was analyzed using reverse transcription-PCR (RT-PCR; Applied

Biosystems 7500 Fast, Foster City, CA) with the real-time SYBR Green/ROX gene expression assay kit. cDNA was synthesized from UFCN-treated and untreated hMSCs using a Fastlane®

Cell cDNA Kit. The mRNA levels of glutathione peroxidase (GPX), tumor suppressor protein (p53), cytochrome P450 1A (CYP1A), glutathione S-transferase A4 (GSTA4), B-Cell CLL/lymphoma 2 (Bcl2), glutathione S-transferase M3 (GSTM3), glutathione reductase 1 (GSR1), catalase (CAT), superoxide dismutase (SOD) and the reference gene glyceraldehyde 3phosphate dehydrogenase (GAPDH), were assayed using gene-specific SYBR Green-based QuantiTect® Primer Assays (Table 1). According to manufacturer’s instructions, a 25 μL reaction volume was used in each well of the PCR plates. Briefly, 12.5 μL of master mix, 2 μL of assay primer (10×) and 10 μL of template cDNA (500 pg) were added to each well. After centrifugation, the PCR plate was subjected to 40 cycles under the following conditions: (i) PCR activation at 95°C for 5 minutes, (ii) denaturation at 95°C for 5 seconds and (iii) annealing/extension at 60°C for 30 seconds. Quantitative RT-PCR data were analyzed using the comparative threshold (Ct) method, and fold inductions of samples were compared with the control. GAPDH was used as an internal reference gene to normalize the expression of the oxidative stress related genes. The Ct method was used to determine the expression level in untreated and UFCN-treated hMSCs for 24 and 48 h. Gene expression level was calculated as previously described by Al-Hadi et al. (2016) [12]. The results are expressed as the ratio of the reference gene to the target gene using the following formula: ΔCt = Ct (target genes) - Ct (GAPDH). To determine relative expression levels, the following formula was used: ΔΔCt = ΔCt (Treated) - ΔCt (untreated control). Thus, expression levels are expressed as n-fold differences relative to the reference gene. The value was used to plot the expression of oxidative stress related genes using the expression of 2-ΔΔCt. The results were obtained from three independent experiments.

2.11.

Statistical analysis The statistical parameters (i.e., mean + SD, student t-test) of all experimental data were

analyzed for three independent experiments using SPSS (IBM Corporation, USA) and Microsoft Excel software (Microsoft Corp., KY, USA). For all comparisons, differences were considered statistically significant at p < 0.05. 3.

Results

3.1.

Physico-chemical analysis Figure 1B and C shows the ultrafine carbon nanostructures under visible and UV light

irradiation respectively. The UFCNs exhibited a brown color and transparent nature under visible light and emitted a strong blue fluorescence under UV light irradiation (λex=365 nm). Isolated UFCNs were characterized using UV-Vis spectroscopy, XRD and TEM. Figure 2A shows the UV–Vis spectra of isolated UFCNs that exhibited strong absorbance between 260 nm to 290 nm, which confirms n-π* and π-π* transitions corresponding to C=O and C=C functional groups, respectively. Figure 2B shows the XRD pattern of UFCNs, which shows a broad single peak with 2θ value of 22.7°. Our result indicates that UFCNs have highly disordered carbon with an amorphous nature. The morphology and particle size of UFCNs were analyzed using TEM, and resulting images are shown in Figure 2C-D. The TEM images indicate a cluster of UFCNs with a 5-20 nm diameter. These results revealed that the surface of samooli bread contains ultra-fine carbon nanostructures. 3.2.

Cytotoxicity of UFCNs on hMSCs After 24, 48 and 72 h of exposure of UFCNs to hMSCs, cytotoxicity was measured using

the MTT assay (Figure 3). No significant differences were observed at low UFCN concentrations

in the exposed cells compared with the control cells. However, a 15-25% decrease in cell viability was observed at high UFCN concentrations in treated cells. Our results suggested that UFCNs induced acute toxicity in hMSCs. Moreover, UFCNs exhibited dose- and time-dependent cytotoxic effects on hMSCs after 24 and 48 h of exposure. However, after 72 h of UFCN exposure, cells showed increased viability due to particle uptake tolerance. In order analyze acute UFCN toxicity, we chose different concentrations such as control, 50, 200 and 400 µg/mL for further analysis. 3.3.

Morphological assessment by fluorescent microscopy The effect of UFCNs on hMSC morphology was measured using AO/EB staining under

fluorescent microscopy. AO/EB staining is used to observe nuclear changes and the formation of apoptotic bodies in cells under fluorescence microscopy. Acridine orange is a cell-permeable dye that can stain both live and dead cells, whereas ethidium bromide can stain only cells that have lost their membrane integrity. hMSCs were exposed to different concentrations of UFCNs for 24 h (Figure 4). Few necrotic cells were observed at high concentrations of UFCN-exposed hMSCs. Moreover, dense granularity in the cytoplasmic region, but not in the nuclear region, was observed. Neither individual or monolayer colony cells show significant intracellular structural changes or nuclear fragmentation. Our results clearly indicate that UFCNs did not induce significant cell death, which is an indication of necrosis rather than apoptosis. However, UFCNs were able to be taken up and induce necrotic cell death in hMSCs at high doses through acute oxidative stress. 3.4.

Assessment of mitochondrial membrane potential (∆ψM) ∆ψM is an important factor for cellular processes, such as the induction of apoptosis and

programmed cell death and cellular calcium and redox homeostasis. We analyzed whether

UFCNs induced the disintegration of mitochondrial membrane potential. We assessed ∆ψM using JC-1 dye, which emits green fluorescence when the ∆ψM has collapsed (Figure 5). As shown in Figure 5, after hMSCs were exposed to UFCNs (control, 50, 200 and 400 µg/mL) for 24 h, the number of green cells increased as UFCN concentration increased. Moreover, 12% and 25% of green cells were observed after exposure to 200 and 400 µg/mL UFCNs, respectively. Our results clearly suggest that UFCNs induced acute mitochondrial membrane damage. 3.5.

Intracellular ROS level measurement After hMSCs were exposed to different concentrations of UFCNs for 24 h, intracellular

ROS production was assessed using DCFH-DA. Figure 6 illustrates intracellular ROS generation using a flow cytometric assay. These results indicate that green fluorescence intensity was significantly increased in UFCN-exposed cells compared with control cells. Moreover, UFCNs exhibited dose-dependent ROS generation in hMSCs. Notably, oxidative stress may contribute to disease pathogenesis. 3.6.

Cell cycle analysis The cell cycle progression of UFCN-treated hMSCs was analyzed using flow cytometry

after PI staining. Figure 7 illustrates that UFCNs affect cell cycle distribution at the sub-G0, G0/G1, S and G2/M phases. These results show no significant (p<0.05) changes in cell cycle phase distribution or arrest in the G0/G1, S and G2/M phases. However, we observed a slight increase in the percentage of cells in the sub-G0 phase accompanied by a decrease in the proportion of cells in the other phases of the cell cycle at later time points. 3.7.

Quantitative real-time PCR analysis of gene expression To analyze the expression level of selected redox system-based genes in UFCN-treated

hMSCs, the relative mRNA levels of GPX1, p53, CYP1A, GSTA4, Bcl2, GSTM3, GSR1, CAT

and SOD were quantified using a SYBR green-based assay on a 7500 Fast Real-Time System. Figure 8 shows the expression of GPX1, p53, CYP1A, GSTA4, Bcl2, GSTM3, GSR1, CAT and SOD genes in UFCN-treated hMSCs, with results expressed as mean fold changes over the control. The expressions of GPX1, Bcl2, CAT and SOD genes were dose- and time-dependent in UFCN-treated cells. The expression level of the GPX1 gene was decreased by dose and increased by time. Bcl2 and SOD gene expression levels increased significantly in high concentration of UFCN-exposed hMSCs. Moreover, the levels of CAT gene expression decreased in high-dose UFCN-treated cells compared with low-dose UFCN-treated cells. When low-dose and high-dose UFCN-treated cells were compared, the gene expression levels of GSTM3 and p53 were significantly higher at 24 h. GSTA4 gene expression decreased after 24 h and increased after 48 h of exposure to UFCNs in a dose-dependent manner. Our results clearly indicate that UFCNs may induce metabolic stress by altering redox homeostasis at the intracellular level through alterations in Bcl2, CAT and SOD gene expression. 4.

Discussion Nanotechnology-based food industrial applications are expanding exponentially. With

this comes the increased probability of human exposure to foodborne nano- and microstructures, which have outstretched health safety concerns for regulatory authorities [2-4]. Regulations for the usage of nanomaterials in food products are currently undeveloped for research communities and remain debatable public health issues. Exposure of biological systems to foodborne nanoand microstructures occurs via two major routes: (i) intentional (food additives, supplements) and (ii) unintentional (food production methods and migration from environment, food packaging, transfer surface coatings of food equipment to food products) [47]. However, the degree to which food contains nanoscale ingredients and contaminants, the physiochemical

properties of these contaminants and the role they play in biological systems remain unknown and thus are challenging issues for researchers. Several previous studies have demonstrated that a variety of engineered nanoparticles have toxic effects on mammalian cell lines. For instance, engineered nanoparticles (silver, silica, zinc oxide, copper oxide and gold nanoparticles) exhibited size-, chemical composition-, surface area- and shape-dependent cytotoxicity [48, 49]. A few studies demonstrated that nanoscaled ingredients from food products showed cytotoxicity in human cell lines. Our previous studies suggested that the nanoscale ingredients SiO2, TiO2 and carbon were present in commercial food products and that they induced cell death via metabolic stress in lung epithelial and adult mesenchymal stem cell lines [6, 7, 9, 10, 12]. In particular, several synthetic/modified emulsifiers have been used in food industry. Apart from intentional route of contaminations, unintentional sources of contamination (i.e., lipid derived) may be the main route for micro- and nanomaterials in bakery products. Emulsifiers (E471, E472c, E472e, E475, E491-496 and E473) are widely used synthetic/semi-synthetic amphiphilic lipid-derived molecules in the food industry. The US-FDA (United States Food and Drug Administration) suggests using meals high in fat for food-effect studies because such fatty meals affect GI physiology and maximize drug transfer into the systemic circulation [50]. Several lipid-based (such as triglycerides, polar and non-polar surfactants) pharmaceutical formulations have been used clinically to increase absorption from the gastrointestinal tract. These lipid materials usually accelerate the release of cellular drugs to the systemic circulation via the intestinal lymphatic system [51]. In this study, we identified ultrafine lipidaceous carbon nanostructures from bread surfaces, which were 5-20 nm in diameter. The present study results are consistent with our previous study [12]. Isolated UFCNs induced acute intake by hMSCs in a dose- and time-

dependent manner for 24 and 48 h. However, UFCNs did not cause significant cell death at low concentrations. Similar results have been reported previously [6, 7]. These emulsifier-like materials may have unique interfacial hydrophobic attraction and repulsion features that aid in their uptake by cell membranes. They may have also have the ability to solubilize ingredients and thus easily facilitate rapid absorption of carbohydrates, proteins and lipids and other food ingredients, such as minerals, vitamins, endotoxins, allergens, or particulates into cells. AO/EB staining microscopic images showed few late apoptotic and more necrotic cells, clearly indicating that UFCNs induced necrotic cell death in hMSCs. Excessive uptake of UFCNs may trigger free radical generation, leading to morphological changes, such as cell aggregation and cytoplasmic vesicle formation, both of which are well-characterized features caused by oxidative stress. An earlier study by Wei et al. suggested that cationic nanocarriers triggered necrosis based on their positive surface charge. Moreover, necrosis is an abnormal type of cell death that can result in several diseases [52]. Mitochondrial membrane potential plays a vital role in cell life and death, and its depletion can lead to various diseases, including diabetes and cardiovascular and neurologic diseases [53]. Our fluorescence microscopic images of a mitochondrial membrane potential assay show a high concentration of green fluorescent cells after treatment with UFCNs, which indicates that UFCNs disrupt the mitochondrial membrane via changes in the membrane and oxidation–reduction potentials of the mitochondria. Mitochondrial membrane potential collapses due to increased ROS production, leading to cellular necrosis. Previous studies asserted that nanoparticles can induce chronic changes at the intracellular level by the depletion of mitochondrial membrane potential and intracellular ROS generation [6, 7, 9, 10, 45, 54]. Reactive oxygen species (ROS) are important factors in the cellular redox homeostasis

mechanism and mitochondrial membrane potential disruption [12]. Our present study results indicate that intracellular ROS levels increased with mitochondrial membrane damage in cells exposed to 200 or 400 µg/mL UFCNs. Oxidative stress can damage hMSCs by inducing genetic damage in proliferating cells and inducing cell differentiation (i.e., induced adipogenesis). For instance, free radical generation cause changes in intracellular signaling in various types of cells. Moreover, ROS play an important role in cell signaling by producing crucial epigenomic changes and mRNA transcription profiles. This abnormal regulation may affect tissue homeostatic control of tissue development, leading to various stem cell-dependent diseases (irregular adipogenesis-obesity, diabetes and cancer) [43-44]. Moreover, the proportions of cells in the sub-G0, G0/G1, S, G2/M phases were not significantly affected compared with the proportion of cells in the other cell cycle phases (p<0.05). These results clearly indicate that UFCPs have demonstrated self-uptake properties intracellularly (Figure 7). These types of physiological changes may have also occured for the rapid or easy absorption or high uptake of unwanted lipidaceous substances (hydrophilic and hydrophobic forms) in intestinal cells. Gene expression results revealed that GPX1, Bcl2, CAT and SOD show dose- and time-dependent expression in UFCN-treated cells. In particular, Bcl2 and SOD gene expression levels were significantly upregulated in UFCN-exposed hMSCs. The expression of the antioxidant defense enzyme SOD gene was elevated due to increased protection against ROS. Previous studies have similarly demonstrated that increased SOD expression for antioxidant defense mechanisms adapts to changes in oxidative stress [55]. Bcl2 is an anti-apoptotic gene whose upregulation indicates the prevention of cells from undergoing apoptosis by inhibiting the activation of mitochondria [56]. Moreover, CAT and GPX1 gene

expression was downregulated by UFCN treatment due to suppression of antioxidant enzymes and excessive increases in ROS. Our findings suggested that UFCNs induce oxidative stress in a chronic manner through the SOD-, CAT- and GPX1-mediated redox pathway. Most importantly, chronic exposure to these unwanted lipidaceous substances may be key factor underlying the increased risk of many cardiovascular diseases and cancers. Acknowledgments We gratefully acknowledge financial support from Research Center, Deanship of Scientific Research, College of Food and Agriculture Science, King Saud University, Riyadh, Saudi Arabia and King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia.

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Figure Captions Figure 1:

A photograph of (A) samooli bread, (B) ultrafine carbon nanostructures under daylight and (C) UV light at 365 nm wavelength.

Figure 2: Physico-chemical analysis of UFCNs isolated from samooli bread (A) UV-Vis spectra, (B) XRD spectra and (C-D) Field emission transmission electron microscope images of ultrafine carbon nanostructures. Figure 3: Cell viability assay of hMSCs exposed to UFCNs Cells were treated with different concentrations of UFCNs for 24, 48 and 72 h, and cell viability was then determined using the MTT assay. The data are presented as the mean ± SD of three determinations, each performed in triplicate. Figure 4: Cellular morphological analysis of hMSCs treated with UFCNs Fluorescent microscopic images of untreated (A) Control hMSCs and hMSCs treated with different concentrations of UFCNs for 24 h: (B) 50 µg/mL, (C) 200 µg/mL and (D) 400 µg/mL. The experiments have been repeated at least in triplicate with similar results as those shown. Figure 5: Fluorescence microscopic images of mitochondrial membrane potential detection The mitochondrial membrane potential of hMSCs treated with UFCNs for 24 h: (A) Control, (B) 50 µg/mL, (C) 200 µg/mL and (D) 400 µg/mL. The experiments have been repeated at least in triplicate with similar results as those shown. Figure 6: Intracellular ROS analysis

Intracellular ROS generation in hMSCs is dependent on the incorporation of UFCNs. Total ROS of untreated (A) Control and different concentrations of UFCN treatment of hMSCs for 24 h: (B) 50 µg/mL, (C) 200 µg/mL and (D) 400 µg/mL. Figure 7: Cell cycle analysis using flow cytometry Histogram of (A) Control and UFCN-treated (B) 50 µg/mL (C) 200 µg/mL hMSCs for 24 h cell cycle distribution. (D) Graphical representation of cell cycle distribution. Figure 8: Quantitative RT-PCR analysis of redox response genes in hMSC cells Comparison of the change in expression level, expressed as the fold change (i.e., the ratio of the target gene to the reference gene [GAPDH]) in hMSCs after exposure to UFCNs for 24 and 48 h (200 µg/mL and 400 µg/mL). The data represent the mean ± SD of three determinations, each performed in triplicate (p<0.05).

Figure 1

Figure 2

24 hr

Cell viability (%)

120

48 hr 72hr

100 80 60 40 20 0 Control

25

50

100

Doses (µg/mL) Figure 3

200

400

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Tables Table 1: QuantiTect primer assays

Accession Gene

Number

UniGene

GPX1(Glutathione peroxidase 1)

NM_000581

Hs.76686

P53 (Tumor protein p53)

NM_000546

Hs.437460

CYP1A (Cytochrome P450, family 1, subfamily A, NM_000499

Hs.72912

polypeptide 1) GSTA4 (Glutathione S-transferase alpha 4)

NM_001512

Hs.485557

BCL2(BCL2-like 1)

NM_138578

Hs.516966

GSTM3 (Glutathione S-transferase M3)

NM_000849

Hs.2006

GSR1 (Glutathione reductase)

NM_000637

Hs.271510

CAT (Catalase)

NM_001752

Hs.502302

SOD (Superoxide dismutase )

NM_000454

Hs.443914