Major biological activities and protein profiles of skin secretions of Lissotriton vulgaris and Triturus ivanbureschi Lissotriton vulgaris ve Triturus ivanbureschi deri başlıca biyolojik aktiviteleri ve protein

Objective: The aim of this study was to determine the total protein amounts, protein profiles, in vitro cytotoxicities, antimicrobial activities and hemolytic effects of skin secretions of the Lissotriton vulgaris and Triturus ivanbureschi . Methods: Skin secretions were obtained, clarified, supernatants snap-frozen then lyophilized. Total protein amounts were determined by BCA assay kit. Protein profiles were revealed by the SDS-PAGE. The cytotoxicity and antimicrobial activity were determined by using MTT assay and minimum inhibitory concentration (MIC) method. Hemolytic effects were measured on rabbit red blood cells. Results: Lissotriton vulgaris and T. ivanbureschi skin secretions have totally 18 and 20 protein fractions. IC 50 values were detected between 1.40 and 40.28 μ g/mL. The MIC results were found between 7.8 and 250 μ g/mL. Lissotriton vulgaris skin secretion showed low hemolytic effect while T. ivanbureschi skin secretion showed high hemolytic effect. Conclusion: This study is the first report showing the potential of L. vulgaris and T. ivanbureschi skin secretions for cytotoxicity, antimicrobial and hemolytic activity as an alternative therapeutic approach for traditional uses. active components from these skin secretions and mode of action on cancer cell lines and microorganisms as anti-agents.


Introduction
Skin secretions from many species of amphibians contain a wide range of compounds with biological activity in very high concentration that has excited interest because of their potential for novel drug development [1]. Also researches have shown that skin secretions from many species of Anura are containing high concentrations of peptides with cytotoxic activities against prokaryotic and eukaryotic cells [2]. Amphibian skin/parotoid gland secretions are rich sources of antimicrobial activity against strains of antibiotic-resistant microorganisms such as bacteria and fungi, also highly cytotoxic effects on cancer cell lines [3]. Besides, it is known that dermal glands indicate physiological functions on skin and play several roles of protection from predators [4,5].
The secreted materials of amphibian skin have been used extensively by several cultures. Chansu and Cinobufacini (Huachansu) are traditional Chinese medicines that have been used for centuries as therapy for inflammation, anesthesia and infections in China and other Asian countries [6]. Studies on bufadienolidic steroids led to isolation of marinobufagin, bufalin, telocinobufagin, and cinobufagin. These bufadienolidic steroids have shown efficiency against cancer cells in animal models and in migration of colon cancer cells (Colon 26-L5) [7,8], human leukemia cells (K562, U937, ML1 and HL-60) [9] and breast tumor cells (MCF-7) [10]. In Telocinobufagin studies, bufadienolid was found active against Staphylococcus aureus and Escherichia coli [11]. Three components of Chansu (Bufalin, Telocinobufagin and Cinobufagin) demonstrate a collection of biological activities, including cardiotonic, anesthetic, antineoplastic activity and blood pressure stimulation [10,12].
Natural products of amphibian skin and their proficiency are not limited with bufodienolidic steroids. Amphibian skin secretion studies have showed that Anura families such as Ranidae, Hylidae, Bombinatoridae, Pipidae, Alytidae, Ascaphidae and Leptodactylidae have various therapeutic potentials as anti-diabetic agents, immunomodulatory agents, anti-viral and anti-cancer agents [13]. Approximately 30 years have passed since the discovery of the Magainins in the skin of the African clawed frog, Xenopus laevis in the family Pipidae [14]. Magainin from X. laevis show potential as anti-cancer agents displaying tumoricidal activity against human small cell lung cancer cell lines [15], the RT4, 647V, and 486P bladder cancer cell lines [16], and against suspension cultures of a wide range of hematopoietic cell lines [17]. Another biologically active compound of amphibian skin is steroidal samandarine from salamanders. Studies show that samandarine have high toxicity is likely due to its potent local anesthetic activity [18].
The smooth newt, Lissotriton vulgaris and Balkan-Anatolian crested newt, Triturus ivanbureschi that are studied in this survey have similar distribution range in western and European part of Turkey. The main purpose of this study was to investigate cytotoxic, antimicrobial and hemolytic effects of L. vulgaris and T. ivanbureschi skin secretions on various cancer and non-cancerous cells, microorganisms and rabbit red blood cells to form an estimate of their potential use in medicine as therapeutic agents for the first time.

Field studies
Smooth newt -Lissotriton vulgaris specimens (five males, five females) were collected during the field excursion in Urla/Izmir province, western Turkey in March 2016. Balkan-Anatolian crested newt -Triturus ivanbureschi specimens (five males, five females) were collected during the field excursion in Uzunköprü/Edirne province, Turkish Thrace in April 2013. These two species can be easily distinguished from each other. Lissotriton vulgaris is an obviously smaller newt species than T. ivanbureschi. Besides, male specimens of L. vulgaris have continuous dorsal fin in reproduction period, while dorsal fins of the male specimens of T. ivanbureschi have an interruption at the base of the tail.

Collection of skin secretions
Skin secretions obtained by mild electrical stimulation (5-10 V) by stimulator (C.F. Palmer, London) according to Tyler et al. [24]. Each individual was rinsed with ultra-pure water into the tubes. Skin secretions clarified by centrifugation (5635 × g for 10 min.), supernatants were snapfrozen by liquid nitrogen then lyophilized and stored at +4°C until the bioactivity assays are set up. Secretion harvesting was performed in the field, and then newts were released to their natural habitats, unharmed.

Determination of the protein profiles by the SDS-PAGE and protein concentration
Protein content was assayed triplicate for each diluted skin secretion (2 mg/mL) samples in ultra-pure water, using bovine serum albumin as a standard by BCA assay kit (Thermo Scientific, MA, USA). The protein content was calculated with using a UV/Vis spectrophotometer (Thermo Multiskan Spectrum, Bremen, Germany) at 562 nm.
Electrophoretic processes were performed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a discontinuous buffer system according to Laemmli [25]. The running process was executed on Bio-Rad Mini Protein Tetra Cell electrophoresis device. TGX Stain Free (Bio-Rad, CA, USA) gel was used which does not need any staining processes. The Thermo PageRuler Plus Protein Standard (10,15,25,35,55,70, 100, 130 and 250 kD) was used as a marker. In protein profile studies, samples were repeated three times on gel. Approximate molecular weights of the protein fractions were calculated and photographed with using a software program (Image Lab, Bio-Rad, CA, USA) of the gel imaging system ChemiDoc (Bio-Rad, CA, USA).
Cytotoxicity of crude skin secretions were determined by colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay [26]. The optical density (OD) was measured at 570 nm (with a reference wavelength 630 nm) by UV/Vis spectrophotometry (Thermo Multiskan Spectrum, Bremen, Germany). Cytotoxicity was assayed triplicate. All cell lines were cultivated in 96-well microplates for 24 h with an initial concentration of 1 × 10 5 cells/mL. Subsequently, the cultured cells were treated with different doses of skin-secretions and incubated at 37°C for 48 h. The plant-derived sesquiterpene-lactone (parthenolide) was used as positive cytotoxic control agent. The viability (%) was determined by the following formula: In cell culture studies for untreated cell lines (negative controls) cytotoxicity was set to 0%. The IC 50 values were calculated by fitting the data to a sigmoidal curve and using a four parameter logistic model and presented as an average of three independent measurements. The IC 50 values were reported at 95% confidence interval and calculations were performed using Prism 5 software (Graph-Pad5, San Diego, CA, USA). The values of the blank wells were subtracted from each well of treated and control cells and half maximal inhibition of growth (IC 50 ) were calculated in comparison to untreated controls. Antimicrobial effects of skin secretions were determined by broth micro-dilution method. Test microorganisms were grown in MH broth for 5 h (exponential phase) and adjusted to 0.5 McFarland turbidity standard (A 600 = 1.0), corresponding to 1.5 × 10 6 colony forming unit (CFU)/mL. MICs were determined according to the Clinical and Laboratory Standard Institute [27]. Serial dilutions of skin secretions (0.9-500 μg/mL) were prepared in 96-well microtiter trays, at a final volume of 80 μL. Then, 20 μL of the adjusted bacterial and fungal inocula (1.5 × 10 5 CFU/ mL) were added to each well and incubated at 37°C for 24 h. Inhibition of microorganisms' growth was determined by visual observation. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of skin secretions required to inhibit microbial growth. Each dilution series included control wells, which consisted of 80 μL of it and 80 μL of Mueller Hinton broth. Ampicillin and flucytosine were used as standard drugs for comparison. All assays were studied three replicates.

Hemolytic activity assay
The hemolytic effect of crude L. vulgaris and T. ivanbureschi skin secretions were measured according to the modified method of [28]. Red blood cells were obtained from healthy New Zealand rabbit (Bornova Veterinary Control and Research Institute, Izmir, Turkey). Blood was collected with BD Vacutainer TM (NH 143 I.U., Belliver Industrial Estate, Plymouth, UK). Aliquots of 7 mL of blood were washed three times with sterile saline solution (0.89%, w/v NaCl, pyrogen free) by centrifugation at 2000 × g for 5 min. The cell suspension was prepared by finally diluting the pellet to 0.5% in saline solution. A volume of 0.05 mL of the cell suspension was mixed in U bottom 96-well microplate with 0.05 mL diluents containing 50, 5 and 0.5 μg/mL concentration of crude L. vulgaris and T. ivanbureschi skin secretions in saline solutions. The mixtures were incubated for 30 min at 37°C and centrifuged at 800 × g for 10 min. The free hemoglobin in the supernatants was measured spectrophotometrically at 412 nm. Saline and distilled water were included as minimal and maximal hemolytic controls. The hemolytic percent developed by the saline control was subtracted from all groups. Each experiment included triplicates at each concentration.

Protein contents and electrophoretic protein profiles
The total protein and peptide concentrations were determined by BCA assay for L. vulgaris and T. ivanbureschi as 1775 and 1470 μg/mL, respectively.
The electrophoretic profiles of the skin secretions revealed numerous proteins with a wide spectrum of molecular masses (Figure 1)

Cytotoxicity screening
The cytotoxicity of crude skin secretions is measured against the following cell lines: HeLa, A549, Caco-2,  Table 1.
Crude L. vulgaris and T. ivanbureschi skin secretions were showed high cytotoxic effects on all cancer and non-cancerous cell lines with IC 50 values varying between 1.58-26.15 and 1.40-40.28 μg/mL, respectively. Additionally, we found the highest anticancer activity for both secretion samples on MDA-MB-231 cell line (IC 50 : 1.58 and 1.40 μg/mL). These results are higher than plantderived sesquiterpene lactone parthenolide, remarkably. The lowest cytotoxic effect observed on human cervix adenocarcimoma (HeLa) by T. ivanbureschi crude skin secretion (IC 50 : 40.28 μg/mL). Morphological changes were observed after 48 h exposure to different doses of skin secretions. Increasing concentrations resulted in increased number of rounded cells, growth inhibition and the rate of various morphological abnormalities with larger areas devoid of cells when compared with the untreated control cells.

Antimicrobial activities
MIC was determined using broth dilution method (0.9-500 μg/mL). The MIC values of skin secretions against various Gram-negative and Gram-positive bacterial strains and yeasts are presented in Table 2.
The values indicate that skin secretion have MIC values varying from 3.9 to 250 μg/mL.

Hemolytic effects
The hemolytic effects of crude L. vulgaris and T. ivanbureschi skin secretions on rabbit red blood cells were shown in Table 3. Hemolytic activities were observed at Cell viability was determined by MTT assay, control was exposed to vehicle only which was taken as 100% viability. 1: Lissotriton vulgaris, 2: Triturus ivanbureschi. concentrations between 0.5 and 50 μg/mL. Hemolytic effect of L. vulgaris skin secretion was found only in the highest concentration (50 μg/mL) with 13.6% hemolytic percent. No hemolytic activity was observed in other concentrations of the L. vulgaris skin secretion. Hemolytic activities were seen at all concentrations of the T. ivanbureschi skin secretions in dose dependent manner. It showed very high hemolytic activity at 50 and 5 μg/mL concentrations which were more than the positive control distilled water (hemolytic percents: 115.1% and 103.7%).

Discussion
The skin secretions of amphibians show differentiation across different species with biologically active compounds such as peptides/proteins, steroids, alkaloids, biogenic amines and enzymes [29]. They might be useful as pharmacological implements in drug research and potential drug design models. In this study, cytotoxic, antimicrobial and hemolytic properties of crude L. vulgaris and T. ivanbureschi skin secretions were determined. Also, protein profiles of these crude skin secretions were revealed. The antimicrobial and cytotoxic activities are results of the active peptide inducing alterations in the hydrophobic-hydrophilic seal of the cell membrane, effecting lysis of the bacterial or cancer cells [30].
Salamanders and newts are known to secrete toxic and noxious compounds, such as neurotoxic tetrodotoxin [31]. Nevertheless, the exact mode of action of the salamander alkaloids is almost unknown. They have local anesthetic effects by nerve-blocking activity. Respiratory paralysis was found as the reason of death according to in vivo studies [32]. Moreover, salamander alkaloids, especially samandarone, exhibit distinct antimicrobial activities, although being less potent than most antibiotics [22,33]. Also, several peptides such as corticotropin-releasing peptides and vasoactive intestinal peptides were isolated from the newt, Cynops pyrrhogaster by Teranishi et al. [34]. Ampicillin and flucytosine were used as positive controls. -, Not detected. Hemolytic percent of saline and distilled water were included as minimal and maximal hemolytic control, respectively. All values represent the mean ± SD (n = 3 test). -, Not detected.
Protein profiles of crude skin secretions of salamanders and newts are almost unknown. Up to now, only crude skin secretion of A. davidianus is determined. The molecular masses of the proteins in the skin secretions of the smooth newt and Balkan-Anatolian crested newt are distributed over a wide range between 9 and 272 kDa, while those of the A. davidianus skin secretion are above 66 kDa and between 14 and 66 kDa [19]. There is no further study on bioactivity of skin secretions of newts.
In conclusion, biological activities and protein profiles of skin secretions of the smooth newt, L. vulgaris and Balkan-Anatolian crested newt, T. ivanbureschi were revealed for the first time. Besides, the results indicated that these crude skin secretions have a high inhibition on cancer cell lines and microorganisms with relatively high hemolytic activity and wide range molecular masses of proteins. Further studies need to focus on purification of the active components from these skin secretions and mode of action on cancer cell lines and microorganisms as anti-agents.