HEMOLYTIC ACTIVITY OF SKIN SECRETIONS OF AMPHIBIANS THAT INHABIT THE UKRAINE TERRITORY

Secretions derived from amphibian skin glands serve as a potential reservoir of various valuable active molecules. Currently, the multiple substances with diverse therapeutic activities among the components of glandular secretions of different species of amphibians have been found. It has been proven that they have antibacterial, antifungal, antiprotozoal, antidiabetic, antineoplastic, analgesic, and sleep-inducing properties. Taking this into consideration, to get the basic knowledge about the properties of the components of skin secretions of some Anura species that inhabit the territory of Ukraine is crucial for further investigation of the most potential ones. The red blood cell hemolysis assay is a prevalent test to study the cytotoxicity of studied samples. The aim of the present study was to analyze the hemolytic activity of skin secretions of Bombina bombina, Bombina variegata, Bufotes viridis, Rana temporaria, Pelophylax ridibundus, and Pelobates fuscus, and to obtain the primary data on the possible mechanism of their toxicological action on the blood cells membranes. The skin secretions of six amphibian species mentioned above were incubated with erythrocyte suspension in different concentrations. Eminently active B.variegata skin secretions, having the HD HD50 value at 0.5 μg/ml, were taken for the subsequent researches, where the effects of osmotic protectants, divalent cations, antioxidants, chelating agent, and serine protease inhibitor on the cell lysis ability of B. variegata skin secretions was studied. All studied cations inhibited the hemolytic activity of B. variegata secretions in a dose-depend manner. While the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), markedly decreased the hemolytic activity of studied skin secretions. We can assume that the bioactive peptides in these skin secretions have an enzymatic mechanism of action.


Introduction.
The Amphibians skin provides a rich source of bioactive molecules (peptides, proteins, steroids, alkaloids, opiods) that have some effective therapeutic potencies, such as: antibacterial, antifungal, antiprotozoal, antidiabetic, antineoplastic, analgesic and sleep-inducing properties [1]. Isolation and identification of novel metabolites from amphibian skin secretions could be a promising course to create efficient drugs with valuable therapeutic and pharmaceutical potential [2,3,4]. Nevertheless, the composition and the mechanism of action of biologically active compounds from amphibian skin secretions are not fully investigated by this time.
Toxicity of an active molecule is a key factor during drug design, and the hemolytic activity represents a useful starting point in this regard. It provides the primary information on the interaction between molecules and biological entities at cellular level. Hemolytic activity of any compounds is an indicator of general cytotoxicity towards normal healthy cells. On the other hand, some proteins that affect the biological membranes might induce the formation of pores or channels in natural and model bilayer lipid membranes [5,6]. Thus, hemolytic activity that is induced by these protein toxins could be used as a sensitive toxicological tool to investigate the targeting and attachment of proteins to the cell membranes.
Thus, the aim of this work was to study the hemolytic activity of skin secretions of amphibians prevalent on the territory of Ukraine, such as Bombinabombina, Bombinavariegata, Bufotesviridis, Rana temporaria, Pelophylaxridibundus, and Pelobatesfuscus, and to get the basic information on the possible mechanism of their toxicological action on the blood cells membranes.

Materials and Methods.
Adult specimens (both sexes) of B. bombina, B. variegata, B. viridis, R. temporaria, P. ridibundus and P. fuscus were collected and authenticated by the Department of Zoology and Ecology of Taras Shevchenko National University of Kyiv, Ukraine. All animal procedures followed the European Directive 2010/63/EU (EC, 2010) on protecting animals used for experimental and other scientific purposes. All manipulations were approved by the Ethical Committee of Educational and Scientific Centre "Institute of Biology and Medicine", Taras Shevchenko National University of Kyiv, Ukraine.
The crude skin secretions were collected after a shortterm mechanical irritation of amphibian skin. The secret was washed with a small amount of distilled water, filtrated, lyophilized (Testar Lyo Quest, Spain) and stored at 4 °C until use. Before each experiment dried material was dissolved in phosphate-buffered saline (PBS), pH 7.2, that contained 137 mM NaCl, 1.5 mM KH2PO4, 2.7 mM KCl and 8.1 mM Na2HPO4. Further, the suspension was centrifuged at 2500 g for 10 min and the supernatant was collected and used in the study. Bradford method [7] was used to measure the concentration of total protein in the samples.
Rabbit blood was collected from the ear artery in the tubes containing 3.8 % sodium citrate in ratio of 9:1 to prevent coagulation, and centrifuged at 500 g for 10 min at 4 °C. Plasma was removed carefully and the erythrocytes were washed for additional three times in 5 volumes of PBS, pH 7.2. Washed erythrocytes were stored at 4 °C and used within 6 h for the hemolysis assay. For the experiment 2 % (v/v) erythrocyte suspension was prepared by mixing 0.1 ml of packed red blood cells with 4.9 ml of PBS, pH 7.2.
For the preliminary study of hemolytic activity of the skin secretions of six studied amphibian species, erythrocyte suspension was incubated with various concentrations of these secretions (the final concentrations of total protein were 0.5, 5 and 50 µg per 1 ml of erythrocyte suspension) at 37 ºC for 30 min and then centrifuged at 2500 g for 6 min. The absorbance of supernatant was measured at 541 nm to establish the amount of hemoglobin released due to erythrocytes lysis. Two controls were prepared (both without frog secretions): negative control contained only PBS, and positive control -1 % Triton X-100 that was taken as 100 % cell lysis. The most active B. variegata skin secretion, which had HD50 value 0.5 µg/ml, was used for further research to establish basic information on its toxicological properties. For this purpose we investigated the effects of different factors on the skin secretion hemolytic activity, including osmotic protectants -25 mM D-glucose and 25 mM D-lactose; divalent cations, such as Mn 2+ , Mg 2+ , Ca 2+ , Zn 2+ in concentration range from 1 to 100 mM, Fe 2+ in concentration range from 10 to 50 µM, and Cu 2+ in concentration range from 1 to 100 µM; antioxidants -2 mM ascorbic acid and 2 mM cysteine; chelating agent -2 mM EDTA; serine protease inhibitor -2 mM PMSF.
Each of these was added to erythrocytes suspension followed by addition of B. variegata skin secretion (the final concentration of total protein in all experiments was 0.5 µg per 1 ml of erythrocytes). After incubation at 37 °C for 30 min, the hemolytic activity was determined as described above.
Data analysis was performed using Student's t test to evaluate the statistical significance of experimental groups. The results were recorded as means M±SD. p≤0.05 Results and Discussion. Granular glands (also called serous glands or poison glands) of amphibian skin produce a large variety of bioactive substances, including antimicrobial peptides [8], neurotoxic peptides [9], gastric disturbance peptides [10], and alkaloids [11]. In the middle of the grand the granules with active peptides are located. When the animal is injured or alarmed, the content is released through skin secretions [12]. Despite the immense richness of wild amphibians in Ukraine, current knowledge about the presence of bioactive molecules and mechanisms of their action is limited to only few species. The literature data on the biological activity of peptides isolated from amphibians indicates that skin secretions could be an attractive source of new therapeutic candidates. Although, the therapeutic potential of many active molecules from amphibian skin secretions is limited due to their high hemolytic activity against human erythrocytes.
Taking into account that skin secretions, which are used for isolation and purification of bioactive molecules, have to be evaluated for their potential hemolytic activity, in this study we examined the effect of skin secretions from six amphibian species widespread on Ukrainian territory -B. bombina, B. variegata, R. temporaria, B. viridis, P. fuscus and P. ridibundus on the red cell membrane integrity.
The hemolytic activity of the amphibian crud skin secretions was tested at three protein concentrations 0.5, 5 and 50 μg per 1 ml of erythrocytes suspension (Table 1). According to the obtained results, the skin secretions of R. temporaria and B. viridis had no effect on the erythrocytes integrity even at the highest level of tested concentrations (50 μg/mL), while skin secretions of other studied species with various intensity induced the erythrocyte lysis. As follows, the skin secretion of P. ridibundus showed significant hemolytic values (76 %) only at the concentration of 50 μg/mL, while at the lower concentration no negative influence on the red blood cells was observed. B. bombina, P. fuscus and B. variegata skin secretions showed strong hemolytic effect in a concentration-depend manner. As demonstrated in the Table 1, the most harmful effect on the erythrocytes was observed while studying the B. variegata skin secretion, which caused 50 % of cell lysis even at the lowest concentration -0.5 μg/ml. The skin secretion of this species therefore has been chosen for further experiment to investigate the possible mechanism of its action. To evaluate the HD50 value -the concentration of secretion causing 50 % hemolysis of red blood cellserythrocyte suspension was incubated with various concentrations of B.variegata skin secretion (the final concentrations of total protein were 0.25, 0.5, 1, 2.5, 5, 10, 25 µg per 1 ml). The HD50 value of B. variegata skin secretion was found to be 0.5 μg/mL (Fig. 1). Thus, the degree of hemolysis caused by this concentration of B. variegata skin secretions was used as the reference value in our following experiments, which were carried out to investigate the effects of different factors, such as osmotic protectants, divalent cations, antioxidants, EDTA, and serine protease inhibitor (PMSF) on skin secretion hemolytic activity. The influence of cations on the hemolytic activity of B. variegata skin secretion was determined using divalent cations Mn 2+ , Mg 2+ , Ca 2+ , Zn 2+ , Cu 2+ , Fe 2+ at different concentrations. Cations were incubated separately with erythrocyte suspension and skin secretion at the final concentration of total protein of 0.5 μg/mL for 30 minutes, centrifuged and the absorbance of supernatant was measured at 541 nm.

T a b l e 1. The degree of erythrocyte lysis (%) caused by skin secretions of studied amphibian species
As shown in Fig. 2, all cations decreased the hemolytic activity of B. variegata skin secretion in dose-depend manner, but with different intensity. The lowest effect had Mg 2+ which at its highest concentration decreased the hemolytic effect only to 29±2 %. Ca 2+ at low concentrations (1-10 mM) enhanced the cell lysis to 69±7 %, but inhibited it at highest studied concentration (100 mM) to 26±3 %. Mn 2+ showed full inhibition effect on hemolytic activity at concentration of 50 mM, while Cu 2+ and Fe 2+ had the same effect at concentration of 50 μM. Zn 2+ at concentration ranging from 1 to 100 mM completely inhibited the hemolytic activity of B. variegata skin secretion (Fig. 2).  Table 2. The degree of erythrocyte lysis (50±3 %) caused by B. variegata skin secretion at the concentration of 0.5 µg/mL was taken as the reference value for this experiment (control). The percentage of inhibition was calculated having regard to the control (without agent) which was defined as "100 %".
Glucose and lactose had no significant effect on hemolytic activity of B. variegata skin secretion. The antioxidants ascorbic acid and cysteine, as well as EDTA, also showed no remarkable outcome. In contrast, a serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), substantially inhibited hemolytic activity of frog's skin secretion (Table 2).

Agent
Hemolysis (%) Inhibition (%) control (without agent) 50 ± 3 no effect 25mM D-glucose 49 ± 3 no effect 25mM D-lactose 58 ± 5 no effect 2mM ascorbic acid 51 ± 4 no effect 2mM cysteine 51 ± 2 no effect 2mM EDTA 50 ± 3 no effect 2mM PMSF 3 ± 2* ~ 95 *р ≤ 0.05 the difference is comparable to the basal level which mean the degree of erythrocyte lysis (%) caused by B. variegata skin secretion at the concentration of 0.5 µg/mL ISSN 1728-2748 БІОЛОГІЯ. 1(80)/2020 ~ 9 ~ Based on our results, as the hemolytic activity of B.variegata secretions was inhibited by PMSF, we can suggest, that the mode of action of bioactive peptides in this secret was enzymatic. Our results are proved by other literature data. Thuswise, in the late 1980s and early 1990s, certain studies described the peptidases discovered in the cutaneous secretion of Xenopus laevis [13,14]. The recent study showed, that the skin secretions of several amphibians contained a wide variety of proteins (molecular weight from 8 to 150 kDa), which exhibited proteolytic activity with selective effects on different substrates [15]. In addition, Jilek et al. found, that skin secretion of B. variegata contains the protein, which catalyzes the conversion of L-Ile to D-aIle at position 2 of a model peptide with the N-terminal sequence of bombinin H. It approves the fact, that amphibian skin secretions have various enzymes in their content [16]. However, further investigations are needed to better characterize the active peptides of this secretion and to clarify their biochemical functions.
Conclusion. In this study, we examined the effects of osmotic protectants, cations, and antioxidants on erythrocyte hemolysis stimulated by the skin secretions from the frog B. variegata. We observed that serine protease inhibitor -PMSF -completely inhibited the hemolytic activity of studied secterion. Consequently, an assumption was made, that this skin secretion induced erythrocytes lysis by catalytic way. This hypothesis should be explored in more depth in future studies.