Determination of sodium in the liver and changes of the biochemical indicators of the ratches blood syrups for the introduction of Fe2O3 koloid solutions with different nanoparticle sizes

  • Authors: L.V. Bakalo, N.M. Dmytrukha, I.M. Andrusyshina, I.P. Lubyanova, L.A. Klymenko
Download attachments:

Abstract. Introduction. The use of nanomaterials, especially nanoparticles, is one of the most promising directions in the development of science and technology of the twentieth century. An important place among them belongs to the iron oxide nanoparticles. Using them in medicine and pharmacy, the limited amount of data that assess the impact on the human body requires careful medical and biological research.
The Aim of the Study. The aim was to determine the iron content in the liver and the biochemical parameters of blood in rats, which characterize its functional state after the introduction of colloidal solutions of Fe2O3 with particles of different sizes.
Materials and methods of research. In an experiment on male Wistar male rats, the effect of Fe2O3 solutions with particles 19, 75 and 400 nm was investigated. Chemically-analytical, biochemical researches are carried out.
Results. The article presents data on the study of a violation of the functional activity of the liver as a result of the accumulation of iron after prolonged flow into the body of rats colloidal solutions of Fe2O3. The established increase in the activativity of the enzymes AST, ALT, an increase in the factor de Rithis indicate the damaging effect of LF. Fe2O3, especially 19 nm on liver cells, their necrosis. An increase in the content of uric acid may indicate that the mechanism of cytotoxic action of Fe2O3 is oxidative stress with the formation of reactive oxygen compounds.
Conclusions. The experiment found that the accumulation of iron in the liver, the hepatotoxic effect of the LF Fe2O3 depends on the size of the particles, the dose and the time of exposure. The established violations of biochemical parameters for the influence of colloidal solutions of Fe2O3 can be used in substantiating preventive measures.
Key words: Iron oxide nanoparticles, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, uric acid.

Introduction. The most common nanomaterials with unique properties today are nanoparticles (LF) of metals and their oxides, in particular, of iron, gold, silver, copper, and others. Particularly noteworthy is magnetoactive iron oxide nanoparticles (Fe2O3, Fe3O4) that are used in medicine and biology for the treatment and diagnosis of oncological diseases [1,2].

Due to the ultra-small size (<100 nm) iron oxide NPs can penetrate through cell membranes, overcome biological barriers, initiate the formation of reactive oxygen compounds (oxidative stress) and inflammation, damage organelles and DNA involved in the process of their division, lead to apoptosis and cell necrosis [3–5]. Such properties of magnetic NPs not only have a detrimental effect on tumour cells, but may also damage the cells of normal tissues and organs.

The studies [6, 7] on the effect of iron NPs on cellular metabolism have shown that in general, standardized biocompatible forms of magnetite NPs (Micromagia-B, MUS-B, ICNB) exhibit a non-specific modulating effect on metabolic processes.

The experiment has proved that, as a result of ultra-structural studies of the reticulo-endothelial system organs (liver, lungs, kidneys), intravenous administration of biocompatible forms of magnetite NPs causes non-specific activation of metabolic processes, increasing adaptation-adaptive mechanisms and potential cell capabilities, accelerating reparative processes at the membrane level, and macromolecules [8].

The main body of the depot, which can store and mobilize iron in the body depending on the systemic needs, is the liver. Liver cells (hepatocytes) produce hepcidin, a circulating factor regulating iron homeostasis. Accumulation of iron in the liver leads to cell degeneration, damage and violation of the regulation of its function, development of pathology [9].

Recent publications have clearly shown that the accumulation of iron in the liver parenchyma, found in a number of infectious and non-infectious diseases, is accompanied by further complications, in particular cirrhosis and hepatocellular cancer [10].

In view of the above, it can be assumed that with excessive supply of iron oxide to the body, there may be accumulation of iron in the liver with further development of the pathological process in it, which determines the necessity and urgency of the in-depth study.

The purpose of the work was to determine iron content in the liver and blood biochemical parameters in rats that characterize its functional state following administration of colloidal solutions of iron oxide with particles of different sizes to them.

Materials and methods. The study was carried out on male Wistar rats weighing 150–180 g. The control and experimental animals were kept in a vivarium on a standardized diet with free access to drinking water. Colloidal solutions with iron oxide nanoparticles (Fe2O3 NPs) of 19 nm and 75 nm in size were obtained at the Department of Photochemistry of the L. V. Pisarzhevskyi Institute of Physical Chemistry of Sciences of Ukraine (Kyiv).

In studies, the rats were divided into two series, four groups (20 animals per group). The study drugs were administered intraperitoneally at a dose of 1 mL per 100 g of body weight (iron content of 0.156 mg/mL) 5 times a week. The first group of experimental rats received a colloidal solution of Fe2O3 with a particle size of 19 nm; the second group of experimental rats also received a colloidal solution of Fe2O3 with a particle size of 75 nm; the third group of experimental rats received a colloidal solution of Fe2O3 powder with a particle size of 400 nm; the fourth group (control) received in a similar manner a 0.1% solution of gelatin, which was used as a stabilizer of nanoparticles. The studies were carried out immediately after 30 injections of Fe2O3 solutions (Series I) and 30 days after the end of administration — the post-exposure period (Series II). The studies were conducted in accordance with the requirements of the European Convention for the Protection of Vertebrate Animals, Used for Experimental and Other Scientific Purposes (Strasbourg, 1986) [11].

Iron content in blood and organs of experimental animals was determined using the method of inductively coupled plasma-atomic emission spectroscopy (Optima 2100 DV by Perkin Elmer [USA]) according to methodological recommendations (2003) [12].

During the study in the control and experimental animals after 30 injections of colloidal solutions of Fe2O3 iron oxide with different particle sizes, the registration of weak magnetic fields in the liver was performed using a SQUID device (Superconducting Quantum Interference Sensor) at V. M. Hlushkov Institute of the National Academy of Sciences of Ukraine [13]. These data are an integral parameter of iron accumulation in the liver.

At the end of the experiment, the animals were decapitated under mild aether anaesthesia, blood and internal organs were taken for further study. Among the biochemical parameters, the activity of the enzymes characterizing liver damage was determined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP) and uric acid in serum using a biochemical auto analyser VITLAB FLEXOR E (the Netherlands) using standard test kits EliteTech [14].

Processing of the obtained results involved the methods of variation statistics using Microsoft Office Exсel 2003 programs of statistical analysis [15].

Study results and their discussion. The study has shown that 30 injections of colloidal Fe2O3 solutions over 30 days with different particle sizes resulted in an increase in iron content in the whole blood and internal organs of the experimental rats (liver, kidney) preserved in the post-exposure period. An especially pronounced effect of iron accumulation in the body was observed in animals of the 1st experimental group, which received Fe2O3 NPs of 19 nm in size, in particular, in the whole blood there was an increase by 24.9 %, and in the liver — by 57.6 %. In the group of rats, which received Fe2O3 NPs of 75 nm in size, iron content was increased only in the liver (by 25.8 %). In rats of the third experimental group, which received Fe2O3 of 400 nm in size, there was an increase in iron content in the blood (by 19.8 %), especially in the liver (2.55 times), whereas in the kidneys on the contrary there was its decrease (1.7 times) (Table 1).

It should be noted that after 30 days of the post-exposure period, iron content in the blood of experimental rats, which received Fe2O3 NPs of both sizes, increased even more (in the 1st group — by 3.5 times, in the 2nd group — by 2.5 times), while in the liver (3.0 and 3.1 times, respectively). The level of iron content was particular in the liver of animals, which received Fe2O3 of 400 nm in size (4.5 times).  In parallel, iron content in the kidneys of rats exposed to Fe2O3 NPs of 19 nm and 400 nm was reduced (by 21.7 % and 32.5 %) (Table 1). The latter may indicate an impaired utilization of iron due to changes in an excretory function of the kidneys.
Fig. 1 shows the results of measurement of weak magnetic fields over the liver of control and experimental rats using SQUID magnetometry. The obtained data indicate that the magnetic signal was increased in the experimental rats, which received iron nanoparticles, compared to control animals, and was larger after administration of Fe2O3 NPs of 19 nm in size. This is confirmed by the data obtained by atomic emission spectroscopy to determine iron content in the liver.

Table 1

Concentration of iron in the whole blood and internal organs of control and experimental rats, given solutions of Fe2O3 with different particle sizes

Notes: * — p < 0.05 in comparison with the control; ** — p < 0.05 compared to the values after 30 injections.

Fig. 1.  Characteristic curve of the parameters of the magnetic signal evaluation for the group of rats, given colloidal solutions of Fe2O3 NPs.
The figure shows: 1 — magnetic noise; 2 —control group No. 1; 3 — control group No. 2; 4 — Fe2O3 NPs of 19 nm; 5 — Fe2O3 NPs of 75 nm; 6 — Fe2O3 NPs of 400 nm).

Thus, the study using SQUID magnetometry confirms the fact that upon entering the body, Fe2O3 NPs enter the bloodstream in the liver and accumulate in it (Table 1).

The conducted biochemical studies have shown that changes in the parameters characterizing the functional state of the liver were detected in the blood serum of rats following administration of colloidal solutions of Fe2O3 iron oxide with different particle sizes (Table 2). After 30 days of intraperitoneal administration of colloidal Fe2O3solutions with a particle size of 19 nm and 75 nm to experimental rats, there was a significant increase in the activity of the enzymes AST and ALT, in the 1st group (3.9 and 1.9 times), and in the 2nd group (3.4 and 1.8 times) compared to the parameters in the control group (p < 0.05) (Table 2).

Table 2

Activity of blood serum enzymes characterizing the functional state of the liver in rats after administration of colloidal solutions of Fe2O3 iron oxide

Notes: * — p < 0.05 in comparison with the control; ** — p < 0.05 compared to the values after 30 injections.

Increased activity of AST and ALT was also determined in blood serum of rats after 30 injections of Fe2O3 solution with particles of 400 nm (2.5 and 1.7 times, p < 0.05). After the post-exposure period in the groups of rats, which received Fe2O3 NPs of both sizes, AST activity remained increased compared to the control value p < 0.05 (Table 2). ALT increased activity in the blood that was observed during the experiment in rats exposed to Fe2O3 of both sizes was more pronounced in animals following administration of Fe2O3 NPs of 19 nm in size.

To assess the severity of liver damage, de Ritis ratio was used, which is determined by AST to ALT activity ratio. It is known that in inflammatory processes in the liver, there is an increase in the activity of ALT and a decrease in de Ritis ratio, while in hepatocyte necrosis the activity of AST increases significantly, which leads to an increase in the ratio.

De Ritis ratio calculated by us in the control group of rats was 2.17 ± 0.03, and in rats of the experimental groups, which received Fe2O3 NPs with different particle sizes, the value of this ratio was significantly higher (in the 1st group by 2.2 times, in the 2nd — by 1.9 times, in the 3rd — by 1.6 times), which may indicate necrosis of hepatocytes (Table 3).

It should be noted that after the post-exposure period, de Ritis ratio in the experimental groups of rats slightly decreased, which may indicate a cessation of the destructive process in the liver and hepatocyte recovery.

Table 3

De Ritis value in the control and experimental groups of rats, given solutions of iron oxide with different particle sizes

Notes: * — p < 0.05 in comparison with the control; ** — p < 0.05 compared to the values after 30 injections.

From the data presented in Table 2, it is evident that the activity of alkaline phosphatase (AP) in blood serum of rats of all experimental groups was significantly increased (in the 1st — by 2.3 times, in the 2nd — by 2.7 times and in the 3rd group of rats — 2.6 times, p < 0.05 compared to the values in the control). After 30 days of the post-exposure period, AP activity in the experimental rats also remained increased compared to the control (by 2.0, 2.7 and 2.5 times, respectively, p < 0.05).  Increased AP activity indicates the presence of a pathological process associated with impaired excretion of this enzyme by the liver cells in the gall bladder capillaries.

During the experiment, in all experimental groups in rats, severe unidirectional changes in uric acid levels were found after 30 injections of colloidal Fe2O3 solutions and after 30 days of the post-exposure period. A significant increase in uric acid content was observed in blood serum of rats, which received Fe2O3 NPs of 19 nm in size (by 3.0 times) and 75 nm (by 2.8 times), as well as Fe2O3 of 400 nm (by 1.6 times) compared to the control animals (p < 0.05). After the post-exposure period, increased levels of uric acid in the experimental groups of rats compared to control were even more severe: In the 1st group (by 4.0 times), in the 2nd group (by 4.3 times), and in the 3rd group (by 2.7 times), p < 0.05 (Table 2).

As it is known, uric acid is the product of exchange of purine bases that are part of nucleoproteids and is excreted by the kidneys. It is considered to be the main endogenous antioxidant in response to generation of reactive oxygen species by iron ions. Mainous AG et al. (2011) suggest using parameters of uric acid content as a marker for increasing iron content in the body [16]. Consequently, a significant increase in the content of uric acid in the serum may be associated with the above-mentioned processes of inflammation due to the accumulation of iron in the organs and tissues.

The obtained data allow us to conclude the following.

Conclusions

1. Administration of colloidal Fe2O3 solutions resulted in an increase in iron content in the whole blood and liver of the experimental rats, especially injection of Fe2O3 of 19 nm, which was preserved in the post-exposure period. Iron accumulation in the liver is evidenced by the increased magnetic signal in the group of experimental rats, especially after administration of Fe2O3 of 19 nm, compared to the animals of the control group.

2. Administration of colloidal Fe2O3 NPs solutions caused an increase in the activity of the enzymes AST and ALT, which persisted in the post-exposure period, indicating damage to the liver cells.  An increase in de Ritis ratio may indicate hepatocyte necrosis, and its decrease in the post-exposure period — recovery of liver cells.

3. Increased levels of uric acid in blood serum of experimental rats given colloidal Fe2O3 NPs solutions may indicate the activation of oxidative stress in the body.

4. These violations of biochemical parameters under the influence of colloidal solutions of Fe2O3 of 19 and 75 nm are signs of their negative effect on the body of rats and can be used in substantiating preventive measures.

 

REFERENCES

1. Nanosized ferromagnetic powders obtained by the thermal process and possible pathways for biomedical use. / Kuschevskaya N.F. // Powdery Metal. 2006. —№ 7/8. P. 116–121.

2.Nanomaterials of Biology. / Borisevich V.B., Kaplunenko V.G. // Fundamentals of Nanotechnology. —Kiev. —Avicena. —2010. —415 р.

3. Nanoрharmacology: Experimental-Clinical Aspect. / Chekman I.S. Medical case. 2008. № 3–4. —Р. 104–109.

4. The present and future of nanotechnology in human health care / Sahoo S.K., Parveen S., Panda J.J. Nanomedicine, —2007. —N 3(1). —P. 20–31.

5. Nanotechnology in modern pharmacology. / Shimanovsky N. // International Medical Journal. —2009. —№ 1. —Р. 131–135.

6. Converging technologics, shifting boundaries. / Swierstra T., Boenink M., Wolhout B. [et al.] // Nano-ethics, 2009. —N 3. —Р. 213–216.

7. Cancer nanotechnology opportunities and challenges. / Ferrari M. // Nat. Rev. Cancer. 2005. —N 3. —Р. 161–171.

8. Ultrastructure of the liver cells after administration of magnetite./ Belousov A.N., Nevzorov V.P. // International collection of scientific papers 4 scientific and practical conference on the creation and testing of new drugs. —Moscow. —1997. —T. 4. — Р. 71–77.

9. Hepcidin: from discovery to differencial diagnosis./ Kemna E.H. [et al.] // Haematologica. —2008. —Jan; 93(1). —P. 90–97.

10. Iron, hemochromatosis, and hepatocellular carcinoma / Kowdley K.V. // Gastroenterology. —2004. —V. 127. —P. 79–86.

11. Scientific and practical recommendations on keeping and working with laboratory animals./ Kozhemyakin Yu.M., Khromov O.S., Filonenko M.A. [et al.] —Kiev. —«Avicenna». —2002. —156 р.

12. Methodical instructions 4.1.1482-03 "Determination of chemical elements in biological media and preparations by the methods of atomic-emission spectrometry with inductively coupled plasma and mass spectrometry with inductively coupled plasma". —Moscow: Ministry of Health of Russia, 2003. —16 p.

13. Noninvasive method for the determination of iron accumulation in the liver of rats with lead intoxication. / Lubyanova I.P., Krasnokutskaya L.M., Dmytrukha N.M., Legkostup L.A. and etc. // Ukrainian Journal of Medical Problems. 2011. —№ 3(27). —Р. 43–47.

14. Medical laboratory technology. Volume 2. / Karpishchenko A.I. // St. Petersburg «Intermedica». —2002.

15. Mathematical processing and analysis of medical and biological data. Antonomov M.Yu. / Kiev. —2006. —558 р.

16. Uric acid as a potential cue to screen for iron overload./ Mainous A.G.// Am. Board Fam. Med. —2011. —Jul-Aug; —24(4): —415–21.

 

Надійшла до редакції 20.07.2017 р.