Magnetite Is A Common Type Of Iron Oxide

Published Date: 02 Nov 2017

INTRODUCTION

Magnetite is a common type of iron oxide with the chemical formula Fe3O4 and is the only mineral that exhibits strong magnetism, whereas others, such as maghemite (γ-Fe2O3) and hematite (α-Fe2O3) have weak magnetic proprieties. Magnetite is widespread in the environment despite the fact that is thermodynamically unstable in the presence of oxygen. It occurs naturally in small grains in almost all types of igneous and metamorphic rocks and in many sedimentary rocks as well. The iron oxide Fe3O4 is also a major anthropogenic component of ambient particulate matter that is generated as emissions from traffic, industry and power stations but also synthesised chemically for a wide variety of applications (Karlsson H.L., 2008). These nanoparticles have attracted attention in biomedical applications due to their magnetic proprieties as MRI contrast agents, heating mediators for cancer therapy and for magnetically guided drug delivery.

The concerns about this magnetic nanoparticles is that they might have toxic effects on human body. There are three possibility of contact with nanoparticles via respiratory system, ingestion or through skin.

The principal way these particles are absorbed inside the human body is via the respiratory route, through inhalation. Inhaled nanoparticles can be deposited throughout the human respiratory system including pharyngeal, nasal, tracheobronchial and alveolar regions, depending on particle size (Price et al., 2002). After deposition in the respiratory tract, translocation of nanoparticles may potentially occur to the lung interstitium, the brain, liver, spleen and possibly to the foetus in pregnant females through the circulation system. (MacNee et al 2000, Oberdörster G et al 2000, 2002).

Results from toxicological research have shown that iron oxide nanoparticles have several mechanisms of adverse cellular effects, such as cytotoxicity through oxidative stress mechanisms, oxygen-free radical-generating activity, DNA oxidative damage, mutagenicity, and stimulation of proinflammatory factors. The most important mechanism that has been proposed to explain the toxicity of nanoparticle exposure is oxidative stress through the generation of ROS, although the extrapolation of this mechanism to all types of nanoparticles is not possible (Athanasios V. et al., 2008).

Recently, it was demonstrated that iron oxide (Fe3O4) deposotion in rat lungs cause pulmonary inflammation implied by goblet cell hyper- and/or metaplasia, intraepithelial eosinophilic globules in the nasal passages, increased lung and lung-associated-lymph node weights and inflammatory changes in the bronchiolo-alveolar region (Jürgen P., 2011). Another study suggest that ROS formation in human alveolar epithelial-like type-II cells exposed to magnetite nanoparticles may play an important role in genotoxicity but not in the activation of c-Jun N-terminal kinases that is ROS-independent (Mathias Könczöl et al., 2011).

Inside the cell, the iron oxide nanoparticles may presumably be degraded into iron ions within the lysosomes by hydrolysing enzymes effective at low pH. Free iron and can potentially cross the nuclear or mitochondrial membrane and in the latter case the free iron in the form of ferrous ions (Fe2+) can react with hydrogen peroxide and oxygen produced by the mitochondria to produce highly reactive hydroxyl radicals and ferric ions (Fe3+) via the Fenton reaction. Hydoxyl radicals (+OH) generated could indirectly damage DNA, proteins, polysaccharides and lipids in vivo (Halliwell B. et al., 2007). Also, it was demonstrated that high levels of free Fe ions in the exposed tissue can lead to an imbalance in its homeostasis and can cause deleterious cellular consequences including cytotoxicity, DNA damage, oxidative stress, epigenetic events, inflammatory processes (Veranth J. M. et al., 2007; Hafeli U.O. et al., 2009; Ankamwar B. et al., 2010) and eventually leading to cell death through alteration of the membrane potential, cytochrome c release, +O-2 production, and uncoupling of oxidative phosphorylation (Upadhyay D. et al., 2003).

Cell survival during stress requires induction of the heat shock response. The expression of heat shock genes provides an adaptive mechanism for stress tolerance. Therefore, production of high levels of heat shock proteins can be triggered by exposure to different kinds of environmental stress conditions, such as infection, inflammation, exposure of the cell to toxins (ethanol, arsenic, heavy metals, and ultraviolet light, among many others), starvation, hypoxia etc. (Santoro M. G., 2000). Another protein that becomes activated in response to different types of stress which include oxidative stress, DNA damage (induced by UV, IR or chemical agents), osmotic shock and deregulated oncogene expression is  the tumor suppressor protein - p53. This protein retain attention because modifications in p53 expression involves a number of disorders in human body.

To date, only limited nanoparticles compositions and structures have been tested, much work is required to identify the toxic potential and the mehanisms of action induced in human cells.

In the present study human lung fibroblasts (MRC-5) were exposed to Fe3O4 nanoparticles for analyzing their ability to generate reactive oxygen species,  the activities of antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase and glutathione-S-transferase), the content of total and reduced glutathione, lipid peroxidation levels, the changes in heat shock protein and gene expression and also modifications in p53 protein expression. For these investigations also determination of cellular content in iron ions was undertaken.

MATERIALS AND METHODS

Materials

The main materials used in this study were GIBCO® Modified Eagle’s Medium (MEM), fetal bovine serum, phosphate buffered saline (PBS) and Chromogenic Western Blot Immunodetection Kit anti-mouse purchased from Invitrogen (Carlsbad, California, USA). Nicotinamide adenine dinucleotide phosphate disodium salt (NADP+), nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt (NADPH) and malondialdehyde tetramethyl acetal were supplied by Merck (Darmstadt, Germany). Tetraethoxypropane (TEP) and thiobarbituric acid (TBA) were obtained from Fluka (Milwaukee, USA). The Detect X® Glutathione Colorimetric Detection Kit was purchased from Arbor Assay (Michigan, USA). Antibodies for heat shock proteins (anti-Hsp27, anti-Hsp60, anti-Hsp70, anti-Hsp90) and p53 were from Santa Cruz Biotechnology (Santa Cruz, CA). Other chemicals used were of analytical grade and were from Sigma (St. Louis, Missouri, USA).

Nanoparticles

Fe3O4 (magnetite) nanoparticles were purchased from Laser department, National Institute of Laser, Plasma and Radiation Physics, Bucharest-Magurele, Romania. They were obtained by laser reactive ablation and the size distribution estimated from transmission electron microscopy image (Philips CM120 model) statistics was a lognormal function, in the range 20 to180 nm, with the arithmetic mean value of the diameter of about 50 nm.

Cell culture conditions and treatment

MRC-5 (purchased from the American Type Culture Collection) is a fibroblast cell line derived from normal lung tissue of a 14-week-old male. This cell line is easy to maintain and is preferred in toxicological studies for better reproducibility of data. For this study the cells were cultured in MEM with 10% fetal bovine serum at 37°C in a 5% CO2 humidified atmosphere. They were seeded at a density of 7.5 × 105 cells/ 75cm2 flask and the growth medium was changed every 3 days. All the experiments were carried out between passage nos. 11 to 20. For the treatment the suspension of magnetite nanoparticles was sterilized and sonicated before use. The cells were incubated with nanoparticles at concentration of 12.5 μg/ml for 24, 48 and 72 hours. Controls without treatment were performed for each experiment.

Detection of intracellular iron

The reaction between ferrous ion and 1,10-phenanthroline to form a red complex serves as a sensitive method for determining iron at 520 nm. 100 µl cell lysate was mixted with HCl 2N and incubated for 10 min at room temperature. 100 µl TCA (trichloracetic acid) 20% was added and after 10 min incubation the mix was centrifuged at 5000 rpm for 10 min. 100 µl supernatant was mixed with 10 µl hydrochinone 4%, 5 µl o-phenanthroline 2% and 100 µl sodium acetat 35%. A stock solution of 10 mg% Mohr salt was used for standard curve. The results were expressed as µg% iron /mg protein.

Determination of intracellular ROS

For measurement of intracellular ROS level, after treatment with the indicated concentrations of Fe3O4 NPs, MRC-5 cells were immediately incubated with 10 µM 2’,7’- dichlorodihydrofluorescein diacetate (Sigma) at 37ºC in the dark for 30 min. Thereafter the cells were washed twice and resuspended in PBS; the fluorescence intensity was detected by Jasco Spectroflormeter FP-750 with Spectra Manager software at 488 nm excitation, 515 nm emission. The mean fluorescence intensity of Fe3O4-treated cells was normalized to that of untreated cells as control.

Preparation of cell extract and biochemical assays

For the preparation of cell extract, 75cm2 flasks containing 8 mL of growth medium were seeded with ca. 7.5×105 cells. After treatment with nanoparticles, the cells (about 80% confluent) were trypsinized and pelleted by centrifugation at 1500 rpm for 10 min. The cell pellet was washed with PBS, resuspended in 300 µL and lysed using ultrasonde sonicator. The lysate was centrifuged (5000 rpm, 10 min, 4ºC) and the supernatant (cell extract) was used in various biochemical assays. Protein concentration in the cell extract was estimated by Bradford (1976) method.

Catalase

The CAT (EC 1.11.1.6) activity was assayed by monitoring the disappearance of H2O2 at 240 nm, according to the Aebi method (1984). The CAT activity was calculated in terms of U/mg protein, where one unit is the amount of enzyme that catalyzed the conversion of one mole H2O2 in a minute.

Superoxide dismutase

The total SOD (EC 1.15.1.1) activity was measured according the spectrophotometric method of Paoletti (1986), based on NADPH oxidation. The method consists of a purely chemical reaction sequence which generates superoxide anion from molecular oxygen in the presence of EDTA, manganese (II) chloride and mercaptoethanol. The decrease in absorbance at 340 nm was followed for 10 min to allow NADPH oxidation. A control was run with each set of three duplicate samples and the percentage inhibition was calculated as (sample rate)/ (control rate) times100. One unit of activity was defined as the amount of enzyme required to inhibit the rate of NADPH oxidation of the control by 50%.

Glutathione peroxidase

The total GPX peroxidase (EC 1.11.1.9) was assayed by the Beutler (1971) method, using H2O2 and NADPH as substrates. The conversion of NADPH to NADP+ was followed by recording the changes in absorption intensity at 340 nm, and one unit was expressed as one mole of NADPH consumed per minute, using a molar extinction coefficient of 6.22x103 M-1 cm-1.

Glutathione-S-transferase

The glutathione S-transferase (GST) (EC 2.5.1.18) activity was assayed spectrophotometrically at 340 nm by measuring the rate of 1-chloro-2,4-dinitrobenzene (CDNB) conjugation with GSH, according to the Habig et al. method (1974). One unit of GST activity was defined as the formation of one mole of conjugated product per minute. The extinction coefficient 9.6 mM-1cm-1 of CDNB was used for the calculation.

Total glutathione (reduced) content

The cellular lysate, deproteinized with 5 % sulfosalicylic acid, was analyzed for total glutathione and oxidized glutathione (GSSG) using the Detect X® Glutathione colorimetric detection kit and following manufacturer’s instructions. GSH concentration is obtained by subtracting the GSSG level from the total glutathione. The total and GSH levels were calculated as nmoles/mg protein

Lipid peroxidation

The method described by Del Rio et al. (2003) was used to assess malondialdehyde (MDA), as a marker of lipid peroxidation. For 200 μL of sample with a protein concentration of 2 mg/ml, 700 μL 0.1 N HCl were added and the mixture was incubated for 20 minutes at room temperature. Then, 900 μL of 0.025 M thiobarbituric acid (TBA) were added and the total volume was incubated for 65 minutes at 37°C. Finally 400 μl of 0.1 M Tris-HCl, 5mM EDTA buffer, pH 7.4 was added. The fluorescence of MDA was recorded using a 520nm/549nm (excitation/emission) filter. A calibration curve with MDA tetramethyl acetal in the range 0.05-5 μM was used to calculate the MDA concentration. The results were expressed as nmoles of MDA/mg protein.

Western blot analysis

To determine heat shock protein expression of Hsp27, Hsp60, Hsp70, Hsp90 and p53, western blot analyses were performed with a Chromogenic Western Blot Immunodetection Kit anti-mouse and monoclonal antibodies against the respective proteins. Equal amounts of cell lysate were heated at 100°C for 5 min and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were electrotransferred to a PVDF membrane and probed with suitable antibodies. Secondary antibodie coupled with alkaline phosphatase is recognized by chromogen substrate that consists of 5-bromo-4-chloro-3-indolyl-1-phosphate (BCIP) and nitroblue tetrazolium (NBT) and form a very insoluble, purple formazan. β-actin protein (42 kDa) was use as standard.

Statistical analyses

All the experiments were carried out three times, independently. The data obtained were expressed in terms of "mean±standard deviation" values. The results were compared using Student’s t-test. Statistical significance was assigned if the probability value (p) was less than 0.05.

RESULTS

Cellular iron content

First, we examined whether the treatment with Fe3O4 nanoparticles had an influence in cellular iron content accumulation at different time intervals up to 72 hours. A significant increase in a time-dependent manner by 91% for 24 hours, by 200% for 48 hours and by 296% for 72 hours was observed.

Figure 1. The cellular iron content in untreated (control) and treated MRC-5 cells (with Fe3O4 for 24, 48 and 72 hours). Values are calculated as means ± SD (n = 5 in each group at each time point) and expressed as % from controls. *P\0.05, **P\0.01, ***P\0.001

ROS generation

The ability of Fe3O4 nanoparticles to generate ROS in MRC-5 cells was determined using 2,7 dichlorofluorescin diacetate (H2DCFDA). Exposure of pulmonary fibroblasts to nanoparticles resulted in a significant increase by 52% after 48 hours but the levels are high also at 24 (by 12%) and 72 hours (by 37%) when compared to cells not exposed to these particles (Figure 2).

Figure 2. ROS generated in MRC-5 cells exposed to Fe3O4 nanoparticles. Values are calculated as means ± SD (n = 5 in each group at each time point) and expressed as % from controls. *P\0.05, **P\0.01, ***P\0.001

Enzymes activity

Figure 3 shows the effects of Fe3O4 nanoparticles-exposure on enzyme activity, indicative of oxidative stress, in MRC-5 cells. The level of catalase in nanoparticles-exposed cells increase in a time-dependent manner by 4% after 24 hours and significantly by 6% after 48 hours and by 13% after 72 hours than control cells. In case of SOD activity, was also noticed a time-dependent increase by 8%, 11% and 38% after 24, 48 and 72 h, respectively but was not statistically significant. Changes observed in the levels of GST and GPx were also found to be statistically insignificant. An increase in GPx levels was observed after 24 and 72 hours by 2% and 22% respectively. In contradistinction, GST levels decrease by 8% and 5% for the same time points. After 48 hours of exposure to Fe3O4 nanoparticles, GPx activity in MRC-5 cells was found to decrease with 3% while GST activity was increased with 4%.

Figure 3. Levels of catalase, SOD, GPx and glutathione-S-transferase in untreated (control) and treated MRC-5 cells with Fe3O4 nanoparticles for 24, 48 and 72 hours. Values are calculated as means ± SD (n = 5 in each group at each time point) and expressed as % from controls. *P\0.05, **P\0.01, ***P\0.001

Lipid peroxidation and glutathione (total and reduced) content

Levels of total glutathione, GSH content and lipid peroxidation in MRC-5 cells are showed in Table 1. Lipid peroxidation levels in Fe3O4 nanoparticles-exposed cells were significantly higher than unexposed cells. In contrast, levels of GSH decreased significantly with 50% and 60% after 48 hours and respectively 72 hours of treatment with magnetite nanoparticles. The increase of lipid peroxidation was proportionally with treatment intervals, maximum level at 72 hours was with 680% higher than control cells.

Table 1. Levels of total glutathione, GSH content and lipid peroxidation in MRC-5 cells after treatment with magnetite nanoparticles. Values are means ± S.D. (n = 3)

Time (hours)

Sample

Total glutathione (nmoles/mg)

GSH (nmoles/mg)

Lipid peroxidation (nmoles MDA/mg)

24

Control

25.88±0.58

23.68±0.60

0.026±0.004

Fe3O4 nanoparticles

28.49±1.28

25.17±0.39*

0.062±0.001**

48

Control

35.42±0.85

31.78±0.95

0.014±0.004

Fe3O4 nanoparticles

21.37±1.11***

16.69±0.86***

0.128±0.039*

72

Control

31.29±1.10

26.16±0.85

0.016±0.002

Fe3O4 nanoparticles

15.59±0.62***

10.87±0.66***

0.155±0.033*

*p < 0.05; **p < 0.01; ***p<0.001.

Protein expression of heat shock and p53 proteins

In order to reveal the protein expression of the heat shock proteins (Hsp27, Hsp60, Hsp70, Hsp90) and tumor suppressor protein p53 the Western blot procedure was applied. As shown from figure 4, the levels of Hsp27 and Hsp60 proteins in MRC-5 treated cells were high down-regulated, almost inhibited at 24 and 48 hours comparative with control but at 72 hours a light band was observed. In case of Hsp70 protein protein expression was up-regulated at 48 and 72 hours after exposure. For Hsp90 protein an overexpression appeare at all time intervals of treatment but lower at 72 hours of nanoparticles treatment. Interestingly, p53 expression in MRC-5 treated cells was also down-regulated increasing in time but not over the control level.

Figure 4. Protein expression of Hsp27, Hsp60, Hsp70, Hsp90 and p53 in MRC-5 cells unexposed and exposed to magnetite nanoparticles. The Western Blot analysis, using antibodies against the respective proteins shows the levels of Hsp27, Hsp60, Hsp70, Hsp90, p53 in untreated cells (C) and treated cells (T) after 24, 48 and 72 hours (25 µg of protein loaded per lane). β-actin protein (42 kDa) was use as standard.

The bands corresponding for each sample were quantified with Image J program and the values were normalised to β-actin. In the table below are represented the relative values compared to control that was considered 100%.

Protein

Time interval

Hsp27

Hsp60

Hsp70

Hsp90

P53

24h

1.77%

12.80%

87.85%

57.22%

11.15%

48h

2.23%

2.15%

133.05%

46.85%

16.33%

72h

23.55%

6.86%

140.31%

34.77%

33.00%

Table 2. Relative values of Hsp27, Hsp60, Hsp70, Hsp90 and p53 expression after treatment with magnetite nanoparticles in MRC-5 cells. The value of control is considered 100% and the values of sample are obtained by ratio between the area of sample and coresponding control for each interval. All values of samples were refered to the respective β-actin values.

DISCUSSIONS

When cells are exposed to Fe3O4 nanoparticles, most nanoparticles probably were first adhered to the surface, internalized to the cells by endocytosis, and accumulated in lysosomes (Tomitaka A. et al. 2009; Wei Kai et al., 2011). Inside these digestive vacuoles iron oxide nanoparticles may be degraded into iron ions that can pottentially cross the nuclear or mitochondrial membrane and cause severe cellular damage. As we have seen, in our study, the Fe3O4 nanoparticles were accumulated in high levels in MRC-5 cells in a time-dependent manner. Studies have shown that exposure of cells to metal compounds, can generate excessive ROS in cells by Fenton-type reaction, Haber–Weiss reaction, or by reacting directly with cellular molecules (Gunnar F. et al., 2007). In addition to the these reaction, structural damage to ATP-generating mitochondria by iron oxide nanoparticles localisation or iron overload, could potentially result in anomalous functioning of the mitochondria such as altered membrane potential, cytochrome c release, +O-2 production, and uncoupling of oxidative phosphorylation (Mahmoudi M. et al., 2010), which may also contribute to the underlying mechanisms associated with cytotoxicity. Therefore, iron overload as a result of iron oxide nanoparticles-exposure could potentially result in deleterious cellular consequences eventually leading to cell death (Neenu Singh et al., 2010).

The level of ROS in MRC-5 cells treated with Fe3O4 nanoparticles increase up to 48 hours of exposure by 54%. At high concentrations, ROS can be important mediators of damage to cell structures, including lipids and membranes, proteins and nucleic acids and cause oxidative stress (G. Poli et al., 2004).

In order to counteract intracellular damage by ROS, cells have developed a intracellular antioxidant system with antioxidant scavengers (such as glutathione) and antioxidant enzymes (such as superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase) but they can adapt only to low physiological concentration of ROS (Gunnar F. et al., 2007). At high levels of oxidative stress, the antioxidant defenses are overwhelmed and cytotoxic effects ensue.

After 72 hours of nanoparticles exposure we observed a decrease of ROS level indicating the action of antioxidant system in protection of the MRC-5 cells. The levels of catalase and SOD activities increase in a time dependent-manner suggesting that MRC-5 cells respond to the nanoparticles toxic effects in attempt to protect the cells. Statistically insignificant changes observed in the levels of GPx and GST after treatment with Fe3O4 nanoparticles suggest a less pronounced response by these cellular defense mechanisms as compared to the other enzymes. The increase of GPx level by 22% after 72 hours can be correlated with the decrease of ROS level at the same time interval of treatment. As a marker of lipid peroxidation elicited by ROS, time-dependent increased MDA production was also observed in our study. This increase is correlated with the depletion of GSH content with up to 60% after 72 hours of exposure to nanoparticles. Glutathione is a cofactor for several detoxifying enzymes against oxidative stress such as GPx and GST, thus the decrease in the level of GSH may be explained also by its use as a substrate. We also found that MDA levels were highly correlated with the CAT and SOD activities.

Heat shock proteins (HSP) are important protective mechanisms involved in the folding and unfolding of other proteins. These heat shock proteins can inhibit caspase activation and thus prevent apoptosis (Garrido C. et al., 2001; Ito A. et al., 2006). HSPs 70, 90, and 27 can also promote cell survival mechanisms (Beere HM, 2005).

We chosed four heat shock proteins for study: Hsp27, Hsp60, Hsp70 and Hsp90. The expression of Hsp27 and Hsp60 was similiar, both showed a pronounced down-regulation., From other studies, we know that expression of these proteins is up-regulated in stress conditions. In our case the inhibition of Hsp27 and Hsp60 expression could be due from a denaturation of proteins caused by oxidative stress or another possibility can be the post-translational modifications. Hsp70 expression was decreased after the first 24 hours possible by ROS production effects and after 48 and 72 hours was elevated by 33% and 40% respectively as protective response to oxidative stress. Instead, the Hsp90, one of the highest proteins expressed in the cell, had low levels in Fe3O4 nanoparticles treated-cells compared to control for all time intervals. Several studies shows that hydrogen peroxide (Pantano C et al., 2003; Panopoulos A et al., 2005) or other reactive oxygen species (Shen SC et al., 2008) can promote Hsp90 cleavage resulting fragments with a small molecular weight, thus inactivating its function.

P53 protein is implicated in an array of functions, including modulation of the cell cycle, response to DNA damage, cellular differentiation, signal transduction, and control of gene expression. Treatment with magnetite nanoparticles reduced the expression of p53 protein in MRC-5 cells. The Hsp90 protein plays a number of important roles, which include assisting folding, intracellular transport, maintenance and degradation of proteins as well as cell signaling and tumor repression. This protein acts as a general protective chaperone for a wide range of „client" or „substrate" proteins and for the less stable proteins implicated in signaling cascades of cancerous cells (Wiech H. et al., 1992). The Hsp90 protein help the repress of cancerous tumours protecting the tumour suppressor protein p53 and enabling it to function by stabilizing the folded shape (Sung JP et al., 2011). Recent studies (Raphael B. et al., 2009) shows that Hsp90 cleavege an oxidative stress leads to its client proteins degradation and that explain the low level of p53 expression.

Overall, the treatment with Fe3O4 nanoparticles on MRC-5 cells resulted in time-dependent accumulation of Fe ions inside the cells which led to generation of ROS. The increased CAT and SOD activity, GSH depletion and MDA generation reflected the oxidative stress in the MRC-5 cells. The protein expression of Hsp27 and Hsp60 proteins was almost inhibated by nanoparticles treatment but not protein expression of Hsp70 that increased under stress. Oxidative stress promote Hsp90 cleavage losing, thus, the function to maintain the stability of its client protein p53.

Acknowledgement

This study was financially supported by Grant POSDRU 8/1.5/S/61150/2010 co-financed from European Social Fund by the Sectorial Operational Program for Development of Human Resources 2007-2013.