Article Cite This: Chem. Res. Toxicol. 2018, 31, 876−884
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Low Doses of Polyethylene Glycol Coated Iron Oxide Nanoparticles Cause Significant Elemental Changes within Main Organs Agnieszka Skoczen,́ *,† Katarzyna Matusiak,† Zuzanna Setkowicz,‡ Aldona Kubala-Kukuś,§,⊥ Ilona Stabrawa,§,⊥ Małgorzata Ciarach,‡ Krzysztof Janeczko,‡ and Joanna Chwiej†
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†
Faculty of Physics and Applied Computer Science, Department of Medical Physics and Biophysics, AGH University of Science and Technology, 30-059 Krakow, Poland ‡ Institute of Zoology and Biomedical Research, Department of Neuroanatomy, Jagiellonian University, 31-007 Krakow, Poland § Institute of Physics, Jan Kochanowski University, 25-001 Kielce, Poland ⊥ Holy Cross Cancer Center, 25-734 Kielce, Poland S Supporting Information *
ABSTRACT: The main goal of this study was to evaluate the elemental changes occurring in the main rat organs (kidneys, spleen, heart, brain) as a result of polyethylene glycol-coated magnetic iron oxide nanoparticles (PEG-IONPs) administration. For this purpose, 24 animals were divided into four equinumerous groups, and the three of them were intravenously injected with PEG-IONPs dispersed in 15% solution of mannitol in dose of 0.03 mg of Fe per 1 kg of body weight. The organs were collected 2 h, 24 h and 7 days passing from NPs administration, respectively, for the 2H, 24H, and 7D experimental groups. The forth group of animals, namely control group, was injected with 1 mL of physiological saline solution. For the analysis of subtle elemental changes occurring in the organs after nanoparticles injection, highly sensitive method of total reflection X-ray fluorescence spectroscopy was used. Obtained results showed that administration of even such low doses of PEG-IONPs may lead to statistically significant changes in the accumulation of selected elements within kidneys and heart. Two hours and 7 days from NPs injection, the Fe level in kidneys was higher compared to that of control rats. Elevated levels of Cu, possibly associated with systemic action of ceruloplasmine enzyme, were found within kidneys in 24H and 7D groups, while in heart the similar observation was done only for 24H group. The levels of Ca and Zn increased in kidneys and heart during the first 2 h from the injection and were again elevated in these organs 7 days later. The abnormalities in Ca and Zn accumulations occurring exactly in the same manner may suggest that these elements may interplay either in the mechanisms responsible for the detoxification of the PEG-IONPs or pathological processes occurring as a result of their action.
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INTRODUCTION In the recent decades, great progress in the development of new nanomaterials (NMs) dedicated for various fields of life and science has been observed. Their precise definition was given by the European Commission in the recommendation from October 18, 2011. Accordingly, the term “nanomaterials” specifies any natural, incidental, or manufactured material containing particles, in an unbound, aggregated, or agglomerated state, where at least one of external dimensions, for 50% or more of the particles in the number size distribution, is in the size ranges 1−100 nm.1 In turn, nanoparticles (NPs) are most commonly defined as the objects with the diameter between 1 and 100 nm.2 NPs possess unique electrical, chemical, structural, and magnetic properties that makes them highly attractive especially for biomedical applications.3−5 The greatest potential to revolutionize clinical methods of diagnosis and therapy is attributed to magnetic nanoparticles (MNPs). The magnetic properties of these particular NPs allow them to be © 2018 American Chemical Society
used as a contrast agents for magnetic resonance imaging (MRI), and they can also serve as a targeted drug carriers, enabling monitoring of their therapeutics distribution. Furthermore, MNPs can induce local hyperthermia in response to an external magnetic field and therefore selectively destroy cancer cells.6−9 Because of their small sizes, comparable with cellular components and biological molecules, NPs can easily exceed the natural barriers protecting the organism and induce adverse health effects.10,11 Moreover, because of the complexity of in vivo systems, interaction of nanoparticles with the components such as cells or proteins may result in unique biodistribution, clearance, immune response, and metabolism.12 Therefore, increasing attention is paid to determine the potential risk of engineered NPs. In spite of a numerous publications concerning the subject of nanotoxicity, the health and safety Received: April 25, 2018 Published: August 2, 2018 876
DOI: 10.1021/acs.chemrestox.8b00110 Chem. Res. Toxicol. 2018, 31, 876−884
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central element of cardiovascular system, may be connected with its exposition to high blood flows.15,26 In this work, the temporary and long-term influences of PEG-IONPs on the contents of Fe and other important elements, including Cu, Ca, and Zn within selected organs, were examined. The elemental anomalies within kidneys, spleen, heart, and brain were assessed by comparative analysis of their concentrations quantified for animals subjected to NPs and controls. To achieve this goal, the modern and highly sensitive analytical technique of total reflection X-ray fluorescence (TXRF) spectroscopy was used. With low detection limits (ppm-ppb range), TXRF method enabled us to examine the changes in Fe, Cu, Ca, and Zn levels occurring in the mentioned organs after administration of relatively low dose of IONPs. By analyzing the tissues taken from animals at different times passing from NPs injection, we were able to follow the dynamics of changes in their organ accumulations.
aspects of nanotechnology are still far behind its development. In particular, the impact of NMs on biological systems and the relationship between their properties and toxic biological response need to be thoroughly investigated.10,12,13 In turn, evaluation of the biodistribution and pharmacokinetics of NPs after administration to the body is crucial to enhance their functionality.12−14 Currently, most studies concerning the toxic effects of nanoparticles utilize cell culture models. While in vitro studies have been widely performed, there is still a great need for research on NPs behavior and potential adverse effects under in vivo conditions,12,15 especially since the inconsistency between in vitro and in vivo effects of selected nanoparticles has recently been demonstrated.12,16 The essential elements of the in vivo studies are those with animal use, which allow researchers to evaluate the systemic impact of NPs on a living organism.12,15,17 Among different advantages of MNPs, predisposing them for the use in various medical applications, predominant attention is focused on biocompatible, biodegradable, and easy to synthesize iron oxide nanoparticles (IONPs).18,19 So far, the in vivo studies concerning IONPs toxicity have been focused on exposure to relatively high doses of NPs, which were equal or much higher than these used clinically in humans.6,18,20−22 In particular, Resovist, the drug clinically used in MRI diagnostics of liver, is administered at dose of 0.49 mg of Fe per kg of body mass.14,23 In turn, for Rienso, applied in anemia treatment, the injected dose is 12 mg of Fe per kg of body mass.24 To improve the nanoparticles biocompatibility and stability in the solution, it is necessary to apply appropriate coating. These properties are attributed to the commonly used compound, which is polyethylene glycol (PEG).27 Because of its hydrophobic properties, PEG prevents coated nano-objects from opsonization by proteins present within circulatory systems, leading to the inhibition of the immune response and capturing of the nanoparticles by the mononuclear phagocytic system present inter alia within spleen.28,29 Such property increases nanoparticles circulation time and internalization efficiency and thus makes PEG especially attractive coating for biomedical applications.27,30 In our last paper published in Nanotoxicology, the influence of much lower dose (0.03 mg Fe/kg of a body mass) of PEGcoated IONPs, suspended in 15% mannitol solution, on the elemental composition of rat liver was examined.25 The obtained results proved that even such small quantities of intravenously injected IONPs might induce changes in the systemic iron metabolism as well as in the liver contents of the elements such as Cu, Ca, and Zn.25 This paper is the continuation of the mentioned investigation and aims at evaluation of potential toxic effects of the same IONPs dose on the other rat organs, namely on kidneys, spleen, heart, and brain. Existing reports suggest that IONPs may accumulate within the aforementioned organs, causing some transient histopathological and functional changes.14,15,18 Apart from that, the choice of the tissues was dictated mainly by functions played by these organs in the organism. For example, the kidneys, as a crucial element of body clearance, are desirable pathway for NPs removal from the body.14,26 Spleen in turn, together with previously considered liver, is important for immune response and forming mononuclear phagocytic system (MPS), which is responsible for IONPs clearance from the bloodstream.14 The possible impact of intravenously injected IONPs on heart, the
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EXPERIMENTAL PROCEDURES
IONPs Solution. The mannitol (Baxter) solution of PEG-coated magnetic iron (II, III) oxide nanoparticles (Sigma-Aldrich 747408) was used in the experiment. The detailed protocol of solution preparation was placed in our previous publication.25 The hydrodynamic diameter and zeta potential of IONPs, evaluated with the dynamic light scattering (DLS) method at the wavelength of 633 nm, were equal to 35 nm and −98 mV, respectively. The final concentration of Fe in the NPs solution was determined using S2 PICOFOX spectrometer (Bruker) and was equal to 8.14 ppm (standard deviation of the concentration was 0.86 ppm). Experimental Animals and Sample Preparation. Adult male Wistar rats came from the animal colony of the Department of Neuroanatomy (Institute of Zoology and Biomedical Research, Jagiellonian University, Krakow). All procedures involving animals were approved by the Bioethical Commission of the Jagiellonian University, Krakow, Poland (agreement no. 121/2015) and were performed in accordance with international standards. On the 60th day of the postnatal life, 24 animals were divided into two groups. One of them included 18 rats, which were intravenously injected with 1 mL of the IONPs solution. To minimize the influence of the injection on the obtained results, the second control group (N) was treated with the same volume of physiological saline solution. After 2 h, 24 h, and 7 days, counting from IONPs administration, animals originating from groups 2H, 24H, and 7D (respectively) were weighted and perfused intracardially with 0.9% saline of high analytical purity to remove blood from their bodies. The kidneys, spleen, heart, and brain were taken from each rat and deeply frozen in liquid nitrogen. Afterward, the organs were separately packed and stored in the temperature of −80 °C until the microwave-assisted digestion. In the next step, the whole organs were subjected to the microwave assisted digestion process performed in the mineralizer SpeedWave 4 (Berghof). The 0.3 mL of Ga internal standard in the concentration of 10 ppm was added to 1 mL of each digested sample and the mixture was homogenized. Afterward, 2 μL of the solution was placed onto the special quartz glass carrier and dried on the heating plate. The detailed procedures of sample preparation were presented elsewhere.25 Apparatus and Measurement Conditions. The measurements were carried out in the Laboratory of X-ray Methods, Institute of Physics at The Jan Kochanowski University in Kielce. The multielemental analysis based on the TXRF method was performed with S2 PICOFOX automatic spectrometer. The spectrometer is equipped with metal-ceramic air-cooled X-ray tube with molybdenum anode and multilayer monochromator. The target was set at the angle of 6° and the focal spot size was 1.2 × 0.1 mm2. The energy of the exciting beam was 17.5 keV. Twenty-five quartz glass carriers with the samples were placed in the cassette and the measurement time of each sample was set to 20 min. 877
DOI: 10.1021/acs.chemrestox.8b00110 Chem. Res. Toxicol. 2018, 31, 876−884
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Figure 1. Comparison of the exemplary TXRF spectra obtained for examined organs from selected control rat. Emission K-α lines originating from the analyzed elements (Fe, Cu, Ca, Zn) and used for further quantifications were marked with the black squares. Ga was used as the internal standard.
Figure 2. Median, minimal, and maximal values as well as lower and upper quartiles range of Fe concentration in the examined rat organs obtained for N, 2H, 24H, and 7D groups. The p-values of Mann−Whitney U test for statistically significant differences between IONPs treated and control rats are placed on the charts. The statistically significant increases (p-value less than 5%) of Fe concentration with respect to N group are marked with regular up-arrows. Additionally, the dynamics of changes in Fe concentrations (statistically significant differences between subsequent moments after IONPs administration) is presented. Statistical Analysis. The Mann−Whitney U test was applied for statistical evaluation of changes in elemental composition of the examined organs after IONPs injection. The nonparametric statistical test was applied as our data might not meet the assumptions concerning the normality, homoscedasticity, and linearity (the population is too small to verify these assumptions), which are necessary for the use of parametric test. The differences were tested at the significance level of 5%. The statistical analysis was performed using the STATISTICA software (version 7.1). Elemental Analysis. Qualitative and quantitative elemental analysis was carried out with the use of TXRF spectroscopy. In Figure 1, the exemplary TXRF spectra recorded for kidneys, spleen, heart, and brain taken from selected control animal are presented. It is
necessary to mention that the spectral data acquisition as well as their fitting was performed with the Picofox Spectra 7 software. According to Figure 1, the following elements were detected within the all examined organs: P, S, Cl, K, Ca, Ti, Mn, Fe, Cu, Zn, Pb, Br, Se, and Rb. However, only Fe, Cu, Ca, and Zn were considered for further quantifications. It was due to the fact that light elements such as P, S, Cl, and K can be lost during wet digestion process while the concentrations of Mn, Pb, Br, Se, and Rb were below the detection limits for some of the examined samples. In turn, the Si and Ar lines in the spectra originated from sample carrier and air, respectively. Elemental Concentrations Calculation. The concentration Ci of the element i in the measured liquid sample was calculated as follows: 878
DOI: 10.1021/acs.chemrestox.8b00110 Chem. Res. Toxicol. 2018, 31, 876−884
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Figure 3. Median, minimal, and maximal values as well as lower and upper quartiles range of Cu concentration in rat organs taken from N, 2H, 24H, and 7D groups. The p-values of Mann−Whitney U test for statistically significant differences between IONPs treated and control rats and for trends are placed on the charts. The statistically significant increases (p-value less than 5%) of Cu concentration with respect to N group are marked with regular up-arrows while the upward trend (p-value between 5 and 10%) with dotted up-arrow. Additionally, the dynamics of changes in Cu accumulations is presented for selected organs and blood serum. Statistically significant difference between subsequent moments after IONPs administration is marked with regular sloping arrow, while trend with dotted sloping arrow.
Ci =
CIS × Ni NIS × si
ally, the dynamics of elemental changes occurring within examined organs are schematically presented there. It was done by the evaluation of the statistically significant differences in Fe, Cu, Ca, and Zn levels between subsequent moments after NPs injection. The arrows at the plots point at the direction of the observed changes (arrow sloping up, increase; arrow sloping down, decrease), while “+” or “−” mean the values higher or lower (respectively) comparing to controls. As one can see in Figures 2−5, PEG-IONPs administration to animals may lead to both temporary and long-term changes in the concentrations of the examined elements within the analyzed rat organs. Evaluation of Changes of Fe Content. As can be seen in Figure 2, kidneys were the only organ for which the statistically significant anomalies in Fe concentration were observed. Two hours after NPs injection, the accumulation of Fe in kidneys was significantly higher than in controls but in the next 22 h its content decreased to the normal level. However, on the seventh day from the injection, the level of Fe relevantly exceeded the normal concentration again. None statistically significant anomalies in Fe concentration was observed for brain, heart, and spleen. Nonetheless, as it can be seen from the plot showing the dynamics of changes in Fe levels, its concentration within spleen, similarly as in kidneys, tended to increase between first and seventh day from IONPs injection. Despite that, Fe content in 7D group did not exceed the normal level, which indicates the slower nature of the changes in this element concentration for spleen comparing to kidneys. Evaluation of Changes in Cu Accumulation. The differences in Cu concentrations between examined groups of rats are presented in the Figure 3. As one can see, 24 h and 7 days from IONPs administration, the levels of Cu in kidneys taken from NPs treated animals were significantly higher than in control rats. In case of heart, similar relation was found only for 24H group. However, it was not statistically relevant at the
(1)
where CIS is the concentration of the internal standard (Ga) in the liquid sample [ppm]; NIS is the net pulse number for the internal standard in the sample spectrum [a.u.]; Ni is the net pulse number for the element i in the sample spectrum [a.u.]; and si is the relative sensitivity for the element i. To calculate the final elemental concentration Cix within organ the sample dilution factor d and the liquid to organ mass conversion factor f x were considered, as it was described in our previous work:25
Ci x = Ci × d × fx
(2)
where Cix is the elemental concentration in the organ x [ppm]; Ci is the elemental concentration in the diluted sample [ppm]; d is the dilution coefficient (1.3 for each organ) [a.u.]; and f x is the liquid to organ mass conversion factor for organ x [a.u.].
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RESULTS Elemental Changes Occurring in Selected Rat Organs after IONPs Administration. The TXRF spectroscopy allowed us to evaluate concentrations of Fe, Cu, Ca, and Zn within the main organs taken from animals subjected to the PEG-IONPs and controls. To compare the data obtained for different experimental groups, the median contents of the elements in the examined tissues were calculated and are presented in Tables S1−S4. Additionally, in Figures 2−5, the median, minimal, and maximal concentrations of Fe, Cu, Ca, and Zn (respectively) together with the interquartile ranges recorded for 2H, 24H, and 7D animals as well as control rats are shown as the box-and-whiskers charts. The statistical evaluation of the differences in elemental concentrations between IONPs injected and control groups was performed with nonparametric Mann−Whitney U test. The statistically significant increases (none decreases was detected) in the levels of examined elements in animals treated with IONPs are marked in box-and-whiskers charts. Addition879
DOI: 10.1021/acs.chemrestox.8b00110 Chem. Res. Toxicol. 2018, 31, 876−884
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Figure 4. Median, minimal, and maximal values as well as lower and upper quartiles range of Ca concentration in rat organs obtained for N, 2H, 24H, and 7D groups. The p-values of Mann−Whitney U test for statistically significant differences between IONPs treated and control rats and for trends are placed on the charts. The statistically significant increases (significance level of 5%) of Ca concentration with respect to N group are marked with regular up-arrows while the upward trends (p-value between 5 and 10%) with dotted up-arrows. Additionally, the dynamics of changes in Ca concentrations is presented for selected organs. Statistically significant difference between subsequent moments after IONPs administration is marked with regular sloping arrow while trend with dotted sloping arrow.
Figure 5. Median, minimal, and maximal values as well as lower and upper quartiles range of Zn concentration in rat organs obtained for N, 2H, 24H, and 7D groups. The p-values of Mann−Whitney U test for statistically significant differences between IONPs treated and control rats are placed on the charts. The statistically significant increases (significance level of 5%) of Zn accumulation with respect to N group are marked with regular up-arrows. Additionally, the dynamics of changes in Zn concentrations (statistically significant differences between subsequent moments after IONPs administration) is presented.
assumed significance level of 5%. Neither statistically significant differences comparing to controls nor trends were noticed for brain and spleen of animals subjected to NPs action. The changes of Cu concentration in kidneys and heart may be a result of ceruloplasmin enzyme release from the liver to the bloodstream as it was described in our previous work.25
Accordingly, the elevated level of Cu was found within the blood serum 24 h and 7 days after administration of PEGIONPs. To make easier the interpretation of possible relations between the organ and serum levels of Cu, the dynamics of the changes in the serum concentration of the element is added in Figure 3. 880
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fluorescent radiation as well as signal-to-noise ratio (SNR) comparing to standard X-ray fluorescence (XRF).35 In the following study, the TXRF method was applied to evaluate the changes in Fe, Cu, Ca, and Zn concentrations occurring within the kidneys, spleen, heart, and brain of rats subjected to intravenous injection of the PEG-IONPs. Analysis of the tissues taken from animals at different time intervals from NPs injection allowed tracking the dynamics of elemental anomalies resulting from the nanoparticles systemic action. The core material of nanoparticles used in the present study is magnetite (Fe3O4). This particular iron (II, III) oxide is the mixture of ferrous Fe3+ and ferric Fe2+ ions, which may participate in the formation of reactive oxygen species (ROS).36,37 Fe2+ ions are able to catalyze the hydroxyl radical’s (OH•) formation process from hydrogen peroxide (H2O2) through the Fenton’s reaction. In turn, ferrous ions, in the presence of superoxide (O2•−) or other Fe3+ reducing agents, are reduced to Fe2+ form.38 This feature predisposes IONPs to induce cytotoxic effects such as an oxidative stress leading to damage within the organs in which they accumulate.39,40 The key role in the prevention from Fe induced oxidative stress plays ceruloplasmine enzyme, which as an antioxidant inhibits free radical formation through the catalysis of Fe2+ to Fe3+ oxidation reaction.36,37,41 Obtained results revealed elevated Fe level within the kidneys after 2 h and 7 days from IONPs injection. Rat kidneys are able to filter selectively the particles with the hydrodynamic diameter (dH) below 15 nm. The dH of the IONPs used in the presented study was equal to around 35 nm and they could not be removed from the body through the renal clearance but might accumulate in kidneys leading to the elevated Fe level.14,26,42 The decrease in the Fe concentration that was observed in the organs between second and 24th hour from the NPs administration may indicate at occurrence of nanoparticles redistribution from kidneys to the blood circulation.14,26,43 This conclusion seems to be confirmed by the gradual increase in Fe level within spleen between first and seventh day from subjection to NPs, which is probably connected with filtering of the redistributed IONPs from the bloodstream through the endocytosis in macrophages residing within the reticular meshwork of the organ.14,44 Nearly 95% of Cu within the serum is in the form associated with ceruloplasmin.36,45 As it was mentioned, this enzyme is essential for catalyzing Fe2+ oxidation to Fe3+, which enables Fe binding to the transferrin and its transport from the circulation to the cells via the transferrin receptor, thereby preventing from Fe overload in serum.36,37,41 It has also been shown that ceruloplasmin demonstrates antioxidant functions against a wide range of compounds. It is believed to be responsible for about 80% of plasma antioxidant properties.41 What is more, ceruloplasmin is classified to positive acute phase proteins group since its production increases due to infection and inflammation, which may be related to the response of the organism to the oxidative stress accompanying these processes.46,47 Other important enzyme of Cu-dependent activity is Cu,Zn-superoxide dismutase (Cu,Zn-SOD), which is the part of the cellular antioxidant protection network.48,49 Elevated concentration of Cu was observed within the kidneys after 24 h and 7 days from IONPs injection. Such effect the most probably results from the increase of Cu level in the serum as a consequence of ceruloplasmin release from the liver to the bloodstream what can be noticed from the dynamics chart in Figure 3. Observed changes in Cu
As one can notice from the plot showing the dynamics of changes in Cu levels, the concentrations of the element within heart and blood serum presented very similar patterns of variation. The content of Cu within the heart increased significantly between second and 24th hour from NPs injection, and this occurred in parallel with the elevation (trend with p-value between 5 and 10%) of Cu concentration in the serum. Both for heart and blood serum, Cu levels did not change during the first and the third observation periods. Although 24 h and 7 days from administration of IONPs the element level within kidneys was significantly higher in NPs injected animals comparing to control rats, the statistically relevant increase in Cu concentration did not occur during any of the three observation periods. Such result may suggest that changes in Cu level within the organ progressed much slower than within heart or blood serum. Furthermore, the accumulations of Cu within spleen and brain remained stable during all the observation periods. Evaluation of Ca and Zn Concentrations. As one can see from Figures 4 and 5, the differences in concentrations of Ca and Zn occurring between NPs treated and normal rats presented a very similar pattern. The levels of both elements were significantly elevated within kidney 2 h and 7 days from NPs injection. Similar relation was observed for heart but it was statistically significant only for Zn. In turn, Ca accumulation presented explicit upward trend both 2 h and 7 days after subjection to the NPs. Also the dynamics of changes in Ca and Zn contents exhibited very similar pattern. The increase of the concentrations of both elements within the organs occurred only during the first 2 h from NPs administration. No differences in Ca and Zn concentrations were observed for brain and spleen of rats subjected to NPs action comparing to controls. Furthermore, for both organs, the levels of the elements remained stable between the subsequent times passing from the IONPs injection.
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DISCUSSION In this paper, the elemental anomalies occurring in the main rat organs (kidneys, spleen, heart, brain) as a result of PEGcoated magnetic iron oxide nanoparticles administration were examined. To achieve this goal, 24 animals were divided into four equal groups, and three of them (2H, 24H, and 7D) were intravenously injected with PEG-IONPs dispersed in 15% solution of mannitol in dose of 0.03 mg of Fe per 1 kg of the body weight. The applied dose was about 16-times lower than diagnostic dose of Resovist and about 425-times lower than therapeutic doses of Rienso used clinically in humans.23,24,31 To evaluate the changes in elemental accumulation, a highly sensitive analytical technique of total reflection X-ray fluorescence spectroscopy was used. Among many advantages of the TXRF method, small amount of sample needed to spectral analysis and relatively low detection limits (ppm-ppb) make it especially suitable for detecting very subtle anomalies in elemental concentrations within biomedical samples.32−35 The high sensitivity of the TXRF technique results from the significant background reduction achieved by the measurement geometry of the method in which the primary beam does not penetrate the reflector but is almost entirely reflected from the sample surface. Hence, also, the sample is excited twice (once by primary beam and next by the beam reflected from the carrier), thereby enhancing the intensity of the detected 881
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core is dissolved in the acid environment of the lysosomes to free Fe ions.14,58 Thus, the occurrence of the oxidative stressmediated toxicity of the iron ions released from the IONP within heart tissue cannot be ruled out. However, to confirm such hypothesis, the assessment of the level of oxidative stress biomarkers such as lipid peroxidation level should be done.21,59,60 Hence, the challenge for future experiments is performing the biochemical analysis with use of Fourier transform infrared microspectroscopy, which will provide information on the content and structure of main biomolecules within the heart as well as the other organs.61 No statistically significant changes were observed within brain and spleen of rats subjected to NPs what may suggest the lack of PEG-IONPs influence on these organs. Such an observation is consistent with the existing literature, suggesting that IONPs accumulation occurs within the spleen when their injected dose is 20 mg per 1 kg of body weight or higher,62 while the dose administered to the animals in current study was three orders of magnitude lower. In turn, crossing the blood−brain barrier by IONPs is possible only after applying of appropriate coating consisting of chitosan-PEG copolymer, wherein the hydrodynamic diameter of such particles cannot exceed 30 nm,63,64 which may explain the absence of the IONPs within the brain tissue. However, it is also possible that the elemental anomalies occurring within brain or spleen were limited to small tissue areas and might not be detected with the use of bulk TXRF analysis showing the averaged elemental changes within the organ. Therefore, the next step of the study should be the topographic elemental analysis using more sensitive and highly spatially resolved method such as X-ray fluorescence microscopy.
concentration after 24 h from the NPs administration seem to follow organism reaction on enhanced iron metabolism triggered by IONPs appearance in bloodstream after redistribution from the kidneys. Moreover, elevated Cu level in both the serum and kidneys after 7 days from the treatment may confirm the occurrence of IONPs reaccumulation within the renal pathways. On the other hand, increased Cu concentration on seventh day from IONPs injection may also indicate on IONPs induced long-term adverse effects, for example, inflammation.46,47 Thus, the elevated level of the element may be connected with systemic response to the oxidative stress which is accompanied by enhanced activity of Cu,Zn-SOD or ceruloplasmin. Increasing trend was observed for Cu concentration in the heart after 24 h from subjection to the nanoparticles. The most possible reason for such anomaly is elevation of Cu level within the serum especially as the element contents both within the serum and the heart started to increase between second and 24th hour from IONPs treatment. Ca is the microelement with versatile biological functions, which controls multiple life processes.50−52 Ca has the largest gradient of any chemicals across the plasma membrane of all living cells: its extracellular concentration is 10 000-fold higher than the cytosol Ca content. Such disequilibrium is maintained by both the passive impermeability of the plasma membrane to Ca ions and by the active extrusion of Ca from the cell.53,54 Oxidative stress arising from, for example, exposure to oxidants like Fe2+ ions may simply alter Ca homeostasis leading to plasma membrane damage.52,53 Consequently, the large Ca gradient can no longer be maintained, which results in excess Ca ions accumulation in the injured cell.53,54 The occurrence of these phenomena may explain both the elevated concentrations of Ca within the kidneys and upward trends of the element level in heart, which were observed after 2 h and 7 days from IONPs injection. Zn, as an element responsible for proper folding and stability of Cu,Zn-SOD enzyme, is an important part of the antioxidant defense system against ROS.48,49 Cu,Zn-SOD, present in cytoplasm, nucleus, and plasma of all living cells, catalyzes the superoxide anion radical (O2•−) dismutation into hydrogen peroxide (H2O2).41,48,49,55,56 In case of ROS-induced oxidative stress, the enzyme activity rises, thus re-establishing redox homeostasis within the affected tissue.52 The data presented in the Figure 5 revealed significantly increased Zn concentration within the kidneys and heart after 2 h and 7 days from subjection to the NPs that may be explained by phenomenon of Cu,Zn-SOD activation resulting from IONPs-induced oxidative stress. What is more, the strong correlation between Zn and Ca levels as well as between the dynamics of their changes in animals treated with IONPs may further confirm the aforementioned explanation. Interestingly, the levels of Zn and Ca in the kidneys and heart seem to correlate also with Cu level in the serum. Furthermore, elevated concentration of Cu, probably connected with systemic releasing of ceruloplasmin being the positive acute phase protein, may additionally indicate at appearance of oxidative stress following inflammatory processes ongoing in the organism. It is also worth noticing that although the NPs accumulation was not observed in the heart, the adverse effects of their administration and redistribution also affected this organ. Myocardium is one of the most sensitive tissues to Fe.57 It is believed that mechanisms of IONPs degradation in the body are very similar to those associated with ferritin in which the IONP
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CONCLUSION Even low doses of intravenously injected IONPs may introduce significant elemental changes within kidneys and heart of treated animals. Although elevated Fe level, suggesting the presence of nanoparticles, was observed only within kidneys, the long-term elevation of Cu concentration accompanying enhanced iron metabolism was found not only in this organ, but also within the heart and serum. What is more, increased levels of Cu, Zn, and Ca in the kidneys and heart may indicate at progression of inflammation processes within the tissues. It may suggest that intravenously injected iron oxide nanoparticles could induce toxic effects within selected organs. Results obtained in frame of this paper allowed us to point out the target organs in which the NPs may accumulate as well as those in which elemental changes originating from IONPs systemic action can be observed. The presented data enabled us also to indicate the processes that may be responsible for the observed anomalies. Nevertheless, further research providing more detailed information about mechanisms underlying the presented adverse effects of subjection to the NPs and in particular relation between IONPs properties and systemic response to their intravenous administration is the challenging task for the future.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00110. 882
DOI: 10.1021/acs.chemrestox.8b00110 Chem. Res. Toxicol. 2018, 31, 876−884
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Median concentrations and LLD of Fe, Cu, Ca, and Zn for examined organs (DOCX)
AUTHOR INFORMATION
Corresponding Author
*E-mail: Agnieszka.Skoczen@fis.agh.edu.pl. ORCID
Agnieszka Skoczeń: 0000-0002-4604-4557 Funding
This work was partially financed by the Faculty of Physics and Applied Computer Science AGH UST statutory tasks as well as dean Grant No. 15.11.220.717/23 for PhD students and young researchers within subsidy of Polish Ministry of Science and Higher Education and the statutory research of the Institute of Zoology and Biomedical Research (Jagiellonian University) K/ZDS/007359. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Special thanks to PhD Eng. Przemyslaw Wachniew from Faculty of Physics and Applied Computer Science, University of Science and Technology in Krakow for providing access to chemical laboratory and sharing the mineralizer SpeedWave4. Special thanks to Prof. Piotr Warszynski and M.Sc. Aneta Kedra from The Jerzy Haber Institute of Catalysis and Surface Chemistry of the Polish Academy of Sciences in Krakow for DLS measurements with Nano ZS System.
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DOI: 10.1021/acs.chemrestox.8b00110 Chem. Res. Toxicol. 2018, 31, 876−884
