O. Voronina1, D. Golyshkin2, S. Beregovyi1, T. Beregovа1, L. Ostapchenko1
1ESC «Institute of Biology and Medicine» Taras Shevchenko National University of Kyiv, Ukraine
2«Yuria Pham», Kyiv, Ukraine
Abstract. Sub-acute toxicity of a natural pigment melanin, produced by Antarctic yeast-like fungi Pseudonadsoniella brunnea was studied in rats. Morphological and biochemical changes in liver of rats following 14-day intragastric treatment with melanin in doses 50,100, or 200 mg/kg bw were evaluated. It was shown that melanin injections did not cause significant pathological changes in the rats liver. This presumes melanin to be safety used for long-term treatments.
Key words: melanin, subacute toxicity, morpho-functionalstate of the liver.
Introduction. Studies on the effects of polyphenols on human health have increased significantly over the past 10 years. It is associated with the role of polyphenols in the prevention of degenerative diseases of the gastrointestinal tract, cardiovascular system, reproductive system, etc. as well as the development of inflammation and carcinogenesis. The antioxidant properties of polyphenols have long been studied, but recently there are data on other mechanisms of their projective action, in particular, the effect on the signalling pathways of the cell, on the potential of the mitochondrial membrane, decrease of caspase-3 activity [1]; in tumour cells — inhibition of metalloproteinases and proteins that regulate DNA replication and transformation [2].
Among the variety of polyphenolic compounds, pigment melanin, present in almost all living organisms has a special place. Its photoprotective [3], radioprotective [4, 5], stress-protective [6], neuroprotective [7], hepatoprotective [8], gastroprotective [9], antitumour [10] actions have been proved.
Consequently, the creation of new agents based on the natural pigment melanin is justified and far-reaching. One of the first stages of such development is the study of melanin toxicity.
The aim of the study was to evaluate the subacute toxicity of melanin in terms of biochemical and pathomorphological changes in the liver of rats.
Object and methods of study. The study was carried out on 80 Wistar white laboratory rats weighing 180–260 g. In accordance with the method of administration practised in the clinic, an intragastric route via an elastic gastric catheter was used for administration of Melanin test sample. The study of subacute toxicity lasted 14 days.
The melanin producer is yeast-like fungi Pseudonadsoniella brunnea (formerly Nadsoniella nigra, strain X-1) from the Antarctic, sown in the Educational and Scientific Centre “Institute of Biology and Medicine”, Taras Shevchenko National University of Kyiv from samples of vertical rocks of Galindez Island of the Argentine Archipelago (The Vernadsky Research Base, Ukrainian Antarctic Station) [11].
In order to determine the dosage regimen of the product, the individual body mass of rats was recorded in the required time frames. The study doses were determined based on the maximum tolerated dose of 200 mg/kg body weight with single administration. Dosages ¼, ½, of DL0, corresponding to doses of 50 and 100 mg/kg, were also studied.
The rats were randomly divided into 4 groups: the first — control — the animals received physical solution for 14 days; the second group received melanin at a dose of 50 mg/kg body weight; the third group — 100 mg/kg of body weight; and the fourth group — 200 mg/kg of body weight (the maximum single dose that can be administered). Each group included 20 rats — 10 females and 10 males.
The experiments were carried out in compliance with the international principles of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes [12], in accordance with Law of Ukraine of February 21, 2006 No. 3447-IV “On the Protection of Animals from Cruelty” [13] and in accordance with the ethical norms and rules of work with laboratory animals (Guide for the Care and Use of Laboratory Animals, National Academy Press, Washington DC, 1996) [14].
During 14 days, we observed physiological changes and body weight changes in the experimental animals. After completion of the experiment, all animals were weighed, followed by instantaneous decapitation.
For histological examination, the liver was fixed in 10 % neutral formalin solution, then poured with paraffin under the standard histological technique. Histological sections with the thickness of 7 μm were made on a rotary microtome and stained with Bomer’s haematoxylin and eosin. Coloured micrographs were obtained using the Olympus C-5050 Zoom digital camera (Olympus Europe GmbH, Japan) and the Olympus BX-41 microscope (Olympus Europe GmbH, Japan).
Biochemical studies included an analysis of serum parameters characterizing the functional state of the liver, namely: alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, bilirubin, gamma-glutamyl transferase, total protein, and cholesterol, using the Global Scientific test kits (USA).
Comparison of the difference between control and experimental data was performed using Student’s t-test for independent samples.
Study results and their discussion. Studying the mortality rate of rats shows that subacute administration of melanin did not lead to death of rats, survival was 100 % regardless of the dose of the test sample. Immediately after administration of melanin, and throughout the observation period, no changes in the functional state of the animals were observed. The general condition of the animals is satisfactory: They were fed, the fur was smooth and silky, the skin was intact, the mucous was clean.
The autopsy of all the animals demonstrates that the internal organs had the correct anatomical location. The size and form of the liver in all groups of animals were within the normal range. The liver was divided into 4 parts, the lobe edges were not rounded. The surface of the body was smooth, without tumours and nodes. The liver capsule was thin, clear, not tense. The parenchyma of the organ had a reddish-brown colour and moderately dense consistency.
During the experiment, the weight of internal organs and their relative weight were also determined (Table 1). In rats, melanin at all doses reduced the relative weight of the liver. Thus, in the group of males which received a test sample at a dose of 200 mg/kg, the relative weight of the liver decreased by 17.8 % (p ≤ 0.01) compared to the control, in females — 19.8 % (p ≤ 0.01). Melanin at a dose of 100 mg/kg reduced the weight of the liver in males and females by 15.2 % (p ≤ 0.01) and 25.6 % (p ≤ 0.001) respectively. Administration of the product at a dose of 50 mg/kg reduced the relative weight of the liver in males by 25.2 % (p ≤ 0.001), in females — 19 % (p ≤ 0.01) compared to the control. Also, liver enlargement was observed in males which received melanin at a dose of 100 mg/kg, 13.4 % (p ≤ 0.05) compared to males which received a dose of 50 mg/kg.
Table 1. Relative weight of the liver of rats in the study of subacute toxicity of Melanin test sample
* - p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 — relative to the animals of the control group;
● - p ≤ 0.05 — relative to the animals receiving melanin at a dose of 50 mg/kg.
The morphological structure of the liver of rats in the control group is normal, there is a radial orientation of hepatic trabeculae, the borders of liver lobes are clear, the central vein is filled with blood (Fig. 1). Hepatocytes have a polygonal form, containing clear 1–2 nuclei with nucleoli, light pink cytoplasm. The degree of anisonucleosis severity is negligible, the number of binuclear cells is moderate. In the microcirculatory system, no abnormalities were observed. The morphofunctional analysis indicates a moderate functional state of the liver.
When melanin is administered at a dose of 50 mg/kg, no morphological changes in the liver were observed, trabeculae have clear borders. The organ blood filling is normal. Hepatocytes have a polygonal form, there is increased basophilia in their cytoplasm. The cells contain 1–2 nuclei in the centre, in which the nucleoli, eu- and heterochromatin are clearly differentiated. In general, the structure of the liver corresponds to the physiological norm (Fig. 2).
Fig. 1. Micrograph of the liver of the rat in the control group (staining with haematoxylin-eosin. Magn. 400).
Fig. 2. Micrograph of the liver of the rat receiving melanin at a dose of 50 mg/kg (staining with haematoxylin-eosin Magn. 400).
When the product is administered at a dose of 100 mg/kg, the structure of hepatic trabeculae is preserved, but the intertrabecular sheets are slightly narrowed. Oedema is observed in hepatocytes, in some cells there is a marked vacuolation of the cytoplasm, indicating the development of granular degeneration. Cell nuclei are hyperchromatic. Consequently, the first signs of protein degeneration are visible in the liver, but it is known that early granular degenerative changes are reversible and do not entail an organ physiological failure (Fig. 3).
With an increase in the dose to 200 mg/kg hepatocytes and Kupffer cells are somewhat swollen, hypertrophied, the trabecular pattern is fuzzy, there are marked focal enlargement of the intertrabecular sheets. The interlobar arteries are filled with blood. However, the nuclei of hepatocytes remain weakly basophilic, with euchromatin domination; 1–2 nucleoli are clearly visible. Hepatocyte cytoplasm contains small and large drops of fat and protein, indicating signs of early dystrophic changes. Signs of inflammation or necrosis were not detected (Fig. 4). Consequently, in the parenchyma of the body, there are signs of steatosis and granular degeneration (most often reversible when eliminating the pathological factor), indicating impaired protein-lipid metabolism, possibly due to intoxication.
Fig. 3. Micrograph of the liver of the rat receiving melanin at a dose of 100 mg/kg (staining with haematoxylin-eosin Magn. 400).
Fig. 4. Micrograph of the liver of the rat receiving melanin at a dose of 200 mg/kg (staining with haematoxylin-eosin Magn. 400).
Consequently, there were no significant morphological changes in the liver when melanin was administered. However, it should be noted that when studying the toxic effect of melanin at doses of 100 and 200 mg/kg, dose-dependent signs of steatosis and proteinosis were observed, although it is known that such changes are reversible if the pathological factor is eliminated [15, 16].
Biochemical changes in blood parameters show that melanin at the dose of 50 and 100 mg/kg in females did not significantly affect the contents of both total protein and albumin in the blood of animals, which values were at the level of the control ones. Administration of melanin 100 mg/kg in males and 200 mg/kg in animals of both sexes did not lead to a statistically significant change in total protein and serum albumin in relation to control. This indicates that protein metabolism did not change in rats. The activity of gamma-glutamyl transpeptidase (GGT), an enzyme that is involved in the exchange of amino acids, was not significantly changed.
Particular attention should be paid to the content of bilirubin, cholesterol and alkaline phosphatase in serum since they reflect the state of the liver of experimental animals and development of cytolytic processes under the influence of the study drug. Increased bilirubin is a symptom of acute and chronic liver disease; toxic and drug liver poisoning with further disorders in the activity of the whole body. In the experiment, melanin did not significantly affect the content of bilirubin and cholesterol in serum in all dose groups of animals of both sexes (Table 2).
The level of cholesterol and alkaline phosphatase at high doses of melanin (200 mg/kg) did not increase when compared to the control. Consequently, the functional state of the liver was not impaired.
Studies of the level of total protein, alanine aminotransferase (ALT), aspartate aminotransferase (AST) demonstrate that these parameters upon the action of melanin did not significantly change. These results also confirm that the liver remains within the physiological norm.
Table 2. Blood biochemistry of rats, which characterizes the functional state of the liver in the study of subacute toxicity in the setting of administration of melanin
▲ — p ≤ 0.05 difference between sexes; ALT — alanine aminotransferase; AST — aspartate aminotransferase; GGT — gamma-glutamyl transferase; AP — alkaline phosphatase.
Consequently, as a result of the 14-day administration of melanin, no significant morphological and functional changes in the liver were observed. However, it should be noted that during the study of the toxic effects of melanin at doses of 100 and 200 mg/kg, dose-dependent signs of steatosis and proteinosis were observed, although it is known that such changes are reversible if the pathological factor is eliminated.
Conclusions. The study of subacute toxicity of melanin in rats has shown that when melanin was administered at doses of 50, 100 and 200 mg/kg, it did not cause significant pathological changes in the morphofunctional status of the liver of rats, which characterizes it as a very safe agent for long-term use.
References
1. Therapeutic properties of green tea against environmental insults / L. Chen, H. Mo, L. Zhao [et al.] // J. Nutr. Biochem. – 2017. – № 40. – Р. 1–13.
2. Ping Dou Q. Molecular Mechanisms of Green Tea Polyphenols / Q. Ping Dou // Nutr. Cancer. – 2009. – № 61(6). – Р. 827– 835.
3. Solano F. Melanins: Skin Pigments and Much More — Types, Structural Models, Biological Functions, and Formation Route / F. Solano // New Journal of Science Volume. – 2014. – Р. 1–28.
4. Melanin, a promising radioprotector: mechanisms of actions in a mice model / A. Kunwar, B. Adhikary, S. Jayakumar [et al.] // Toxicol Appl Pharmacol. – 2012. – №264(2). – Р. 202–211.
5. Melanin is effective in protecting fast and slow growing fungi from various types of ionizing radiation / C. Pacelli, R. Bryan, S. Onofri [et al.] // Environ Microbiol. – 2017. – №19(4). – Р. 1612–1624.
6. The influence of melanin on the gastric mucosa and hypothalamic-pituitary-adrenocortical axis under acute stress conditions / D. Golyshkin, T. Falalyeyeva M, K. Neporada [et al.] // Fiziol Zh. – 2015. – №61(2). – 65–72.
7. Brenner S. Parkinson's disease may be due to failure of melanin in the Substantia Nigra to produce molecular hydrogen from dissociation of water, to protect the brain from oxidative stress / S.Brenner // Med Hypotheses. – 2014. – № 82(4). – Р. 503–508.
8. Effects of polyphenol compounds melanin on NAFLD/NASH prevention / N. Belemets, N. Kobyliak, O. Virchenko [et al.] // Biomed Pharmacother. – 2017. – №88. – Р. 267–276.
9. Farzaei M.H. Role of dietary polyphenols in the management of peptic ulcer / M. H. Farzaei // World journal of gastroenterology: WJG. – 2015. – Vol. 21, No. 21. – P. 6499–6517
10. El-Naggar N. Bioproduction, characterization, anticancer and antioxidant activities of extracellular melanin pigment produced by newly isolated microbial cell factories Streptomyces glaucescens NEAE-H / N. El-Naggar, S. El-Ewasy // Sci Rep. – 2017. – №7. – Р. 4212–4223.
11. Confirmation of taxonomic status of black yeast-like fungus by three gene phylogeny / Т.О. Kondratiuk, S.Y. Kondratyuk, M.V. Khimich [et al.] // Acta Botanica Hungarica. – 2016. – V. 59, Issue 3-4, Р. 287-302.
12. Rozemond H. Laboratory animal protection: the European Convention and the Dutch Act. / H. Rozemond // Vet. Q. – 1986. – V. 8. – P. 346–349.
13. Zakon Ukrainy vid 21.02.2006 № 3447-IV «Pro zakhyst tvaryn vid zhorstokoho povodzhennia»: 2006.
14. Life Sciences C.I. Guide for the Care and Use of Laboratory Animals // Laboratory Animals. – 1996. – V. 66, № 4. – 248 p.
15. Strukov A. I. Patologicheskaya anatomiya. / A. I. Strukov, V.V.Serov. – M.: GOЄTAR-Media. 2015. – 880s.
16. Physiological and biochemical basis of clinical liver function tests: a review / L.T. Hoekstra, W. de Graaf, G.A. Nibourg [et al.] // Ann. Surg. – 2013. – Vol. 257, №1. – P. 27–36.
Надійшла до редакції 19.06.2017