CryoproteCtive agents affeCt amino aCids inCorporation into total proteins in Cells of lymphoid organs and liver of experimental animals

The effect of penetrating (glycerol, DMSO) and poorly penetrating (PEG-400) cryoprotective agents on labeled amino acids incorporation into de novo synthesized proteins in the cells of mice thymus, lymph nodes, spleen and cell-free rat liver extract was studied. cryoprotective agents within the range of concentrations which provide a cryoprotective effect were found to inhibit significantly protein synthesis in cell-free systems under investigation. The most effective inhibition was exerted by polymeric cryoprotective agent PEG-400. Cryoprotective agents more efficiently inhibited protein synthesis at a cell level as compared with that in cell-free system that was likely associated with their effect on amino acids transport system. An inhibitory effect of cryoprotective agents on the protein synthesizing apparatus of cells was determined to be Mg2+-dependent and reversible.

T he substances capable of protecting the living tissues against damaging effects of low temperatures, i.e. cryoprotective agents (CPAs), were reported to pseudotoxically affect thecryopreservedcellsandtissues.Thisinfluence consists in a reversible inhibition of their functions and metabolism [1][2][3][4]. As well according to the publisheddata [5][6][7],atoxiceffectofCPAisowingto their interaction with membranes, that disorders the transport properties of plasma membranes and mitochondria, leading to a change in ionic homeostasis and metabolic state of cells. The binding of CPAs to macromolecules and their complexes causes their partial dehydration, resulting in an alteration of spatial structure of macromolecules, their reversible denaturation and impaired enzymatic activity [7].
Modern data show that even in low concen-trationstheCPAsaffectsomaticandreproductive cells [8][9][10]. In this regard, the question of the CPAs influen ceonmetabolicprocessesincellsandtissues is getting relevant, especially in view of the development of such areas in medicine as cell and tissue therapy as well as assisted reproductive technologies [8][9][10]. Moreover, recently many authors have demanded the re-considering of using the CPAs in reproductive technologies, as modern experimental results demonstrate that CPAs within a wide range ofconcentrationshaveatoxiceffectatthegenome level, which leads to irreversible impairments in structure and functions of cells [8][9][10]. Earlier, we have found that CPAs inhibit the transport of amino acids into Ehrlich ascites carcinoma cells, as well as the aminoacylation [11,12]; in this regard, we could assumethattheyalsoinfluenceproteinbiosynthesis at the translation level.
This research aim was to investigate in vitro the effectofpenetrating(glycerol,DMSO)andpoorly penetrating (PEG-400) CPAs on incorporation of la-beled amino acids into de novo synthesized proteins in the cells of thymus, lymph nodes, spleen, and rat liver cell-free extract.

materials and methods
Isolation of lymphoid cells.Suspensionsofthymus, spleen and lymph nodes of male outbred mice wereobtainedwithEagle'smedium,bufferedwith 10 mM Tris-HCL, pH 7.4. Lymphoid organs were crushed with scissors, passed through several layers of nylon tissue and subjected to easy homogenization. Cell suspensions were removed to tubes and centrifuged for 10 min at 2500 g. The cell pellet was re-suspended in a portion of fresh medium. Cell viability using eosin staining was 89-94%.
Measurement of incorporation of 14 c amino acids in isolated cells of lymphoid organs. After ob-tainingthecellsweresuspendedinTris-salinebuffer (0.15 M NaCl, 10 mM Tris-HCl, pH 7.4) at the rate of50millioncellspermlofsolution,1μCi/mlof 14 C-amino acids of chlorella protein hydrolyzate was added(specificactivity10.4μCi/ml)andincubatedat 37°C. The incubation time when studying the incorporation of 14C-amino acids into total proteins was 15 min. To wash the amino acids not incorporated into proteins, the samples were cooled after incubation, 3 ml of cold 5% TCA were added, homogenized and heated at 90°C for 30 min to decompose the nucleic acid, after that the samples were cooled down to0°C,appliedtonitrocelluloseultrafilters(Whatman,USA)andsequentiallywashed,namely3times with 70% ethanol at 42°C, twice with a mixture of alcohol-chloroform (1:1) and twice with chloroform. Thefiltersweredriedandradioactivitywascounted in a standard toluene scintillator with an Intertech-nickSL-40scintillationcounter(France).
The incorporation of 14 C amino acids into proteins was evaluated according to the method described above.
Methods for isolating a cell-free protein synthesizing system (S 15 fraction) from rat liver and determining its activity are presented in the paper [12]. After decapitation and opening of the abdominal cavity, the liver through the inferior vena cava was perfused with medium comprising: 50 mM Tris HCl (pH 7.6), 25 mM KCl, 5 mM MgCl, 0.25 M sucrose. Then the liver was removed, crushed and homogenized in the same medium in a ratio of 1:2. The homogenate was centrifuged for 15 min at 15 000 g. Theupperpartofsupernatant(fractionS 15 ) was collected with removing the fat layer, if possible, and used in further experiments. All manipulations were carried out strictly in the cold.
The experiments were carried out in accordan-cewiththeLawofUkraine"OnAnimalsProtec-tionAgainstCruelty"(No.3447-IVofFebruary21 st, 2006, the rules of the Committee for Bioethics of the Institute for Problems of Cryobiology and Cryo-medicineoftheNationalAcademyofSciencesof Ukraine, provisions of the "European Convention for the Protection of Vertebrates Animals Used for Ex-perimentalandOtherScientificPurposes". We used glycerol and DMSO as penetrating CPAs, as well as non-penetrating CPA PEG-400 (MERCK, Germany). All of them were of chemically pure grade.
Statistical analysis of experimental data was carriedoutusingthesoftware"Statgraphicplusfor Windows"version2.1.byStudent'scriterion.The results are presented as a mean obtained in a series of similar experiments (at least 6) and the standard deviation.Thedifferenceswereconsideredsignificant at P < 0.05.

results and discussion
It is known that the cryoprotective concentrations of the most commonly used CPAs such as DMSOandglycerolare7.5-15%and20-40%,respectively [1,14], and for poorly penetrating CPA PEG-400 it makes 15-20% [1,14]. The cryopreserved cells are mainly pre-incubated with these CPAs at room temperature, that ensures a good permeability of cytoplasmic membranes and fast saturation of cells with CPAs [1,14].
Inthefirstseriesofexperiments,westudied theeffectofCPAsonproteinbiosynthesisincellsof lymphoid organs and liver within the concentration rangeprovidingacryoprotectiveeffect,aswellas at low concentrations, which can be considered as remaining after washing of cells [8]. Fig.1demonstratesthatafter15minofincubation of thymus cells in a medium containing 0.5% glycerol, there was a tendency to inhibit an incorporation of 14 C amino acids into total proteins.
A further increase of glycerol content in the incubation medium of thymocytes to 2.5% or more leads to reliable and progressive inhibition of the inclusion of amino acids in the de novo synthesized proteins. At cryoprotective concentrations of glycerol of 15 and 20% [1,14], the incorporation of labeled amino acids into total proteins of thymocytes was only 12% and 3% versus an initial level, respectively (Fig.1).
When studying the effect of DMSO on the ability of thymus cells to metabolize amino acids, it was found that the ability of cells to incorporate 14 C-amino acids into the acid-insoluble fraction sig-nificantlydecreasesat2.5-5%DMSOconcentration intheincubationmedium.At10%DMSOconcentration, which is most often used to cryopreserve the cells [1,14], the level of incorporation of labeled amino acids decreased down to 30.4% of the control, andat15%DMSOitreduceddownto14.5%versus thecontrol (Fig.1).
The incorporation of 14 C-amino acids into total proteins of thymocytes turned out to be extremely sensitivetotheactionofPEG-400,whichsignificantly suppressed this reaction even at 2.5% concentration and at 5% the inhibition of the process reached63.6%.Whencellswereincubatedinamedium containing 20% PEG-400, the level of incorporation of labeled amino acids into total thymocyte proteinsmadeonly1.2% (Fig.1).
Similarly,thestudiedCPAsaffectedthesynthesis of proteins in cells of lymph nodes. This is confirmedbyexperimentaldataontheincorporation of 14 C-amino acids into the acid-insoluble fraction (Fig.2).
The data presented show that PEG-400 inhibits the protein biosynthesis more effectively. This is evident when comparing the molar concentrations of the studied CPAs. The level of incorporation of 14 C-amino acids into total proteins of cells of lymph nodes at 15% concentration of glycerol in the incu-bationmediumwasreduceddownto22.6%.When thecontentofDMSOintheincubationmediumwas 15%, the inclusion of 14 C-amino acids in the total proteins of the cells of the lymph nodes was practically absent. At a 15% PEG-400 concentration in an incubation medium the test parameter decreased down to 15.9% if compared with the control.
The dynamics of incorporation of 14 C-amino acids into total splenocyte proteins during incubation in media containing CPAs was similar to the above results. In particular, a strong decrease in the studied parameter was observed at a 5% concentra-tionofglycerolandDMSOorhigher,aswellasfor PEG-400 at a concentration of 2.5% or higher.
With an increase in the concentration of glycerol , DMSO, or PEG-400 up to 15%, the incorporation of 14 C amino acids into total splenocyte proteins relative to the control was 19.9%, 5.1%, and 5.7%,respectively (Fig.3).
PEG-400 inhibits more markedly the protein biosynthesisinlivercells.Itsinhibitoryeffectwas manifested starting with a 5% concentration. At 10% PEG-400 in incubation medium, the incorporation of exogenous 14 C-amino acids into the total proteins of rat liver cells decreased down to 21.1%; at a con- centration of 20%, these values were reduced down to 3.6% versus the initial level. In this case, an increase in the level of incorporation of 14 C-amino acids into total proteins of rat liver sections was observed at low concentrations of PEG-400.
Whenstudyingthemechanismsoftheabove inhibitoryeffectofCPAs,inadditiontotheireffect on the transport of amino acids and aminoacylation [12],aninfluenceofpenetratingCPAsdirectlyon the structure and functions of ribosomes, including those in polyribosomes, as well as the components involved into aminoacylation, should not be excluded .
To verify our assumptions, we studied the effect of glycerol and PEG-400 on protein biosynthesis inaratlivercell-freesystem.Wheninvestigatingthe dynamics of functioning of the protein synthesizing apparatus of a cell and its elements, it is necessary to take into account the role of Mg 2+ ions, which cruciallyaffecttheproteinbiosynthesis.Mg 2+ is known to be a cofactor of nucleotide-binding enzymes and guarantees the tRNA tertiary structure [15]. Mg 2+ ions bind to each other subunits of ribosomes during protein biosynthesis [16]. Mg 2+ also has been recognized as activating many enzymes; it is part of kinases that transfer the phosphate group from the ATP molecule to various substrates [17].
Ourpreviousdatashowed [12]thatlowconcen-trationsofCPAsenhancedtheeffectofMg 2+ . Therefore, in this study, the effect of CPAs on protein synthesis in a cell-free system was examined using various concentrations of Mg 2+ ions (Fig.5,Table1).
AnalysisofthedatapresentedinFig.5reveals that the maximum incorporation of 14 C-amino acids into the proteins synthesized in a cell-free system was shifted toward lower concetrations of Mg 2+ with increasing concentration of PEG-400. In particular, if at an optimum concentration of Mg 2+ in the incubation medium (5 mM), PEG-400 at a concentration of 13.2% reduced the incorporation of 14 C-amino acids into the de novo synthesized proteins by about 20.7 times, then a decrease in the Mg 2+ content made it possible to almost completely restore this process (Fig.5).
This regularity was less pronounced in the presence of glycerol added to the incubation medium at 40% concentration (Table 1).
So,ifat5mMMg 2+ the protein biosynthesis in the postmitochondrial supernatant is inhibited by 67.2% of the initial level, then when the Mg 2+ content decreases to 2 mM, the process was inhibited by 45.4%. It should be noted that the level of 14 C-Leu incorporation into total proteins of the rat liver post-mitochondrialsupernatant(S 15 ) in 40% glycerol presence increased almost 2.5-fold with a decrease in the concentration of Mg 2+ in the medium down to 1 mM (Table 1).
When comparing an inhibitory effect of glycerol on protein biosynthesis and in a cell-free system this it can be found to be many times higher at a cell level. The fact discovered, most likely, indicates the role of amino acid transport in a mechanism of disruption of protein biosynthesis in cells underthisCPAinfluence.
It is important to note that the inhibition of protein biosynthesis by CPAs in a cell-free system is reversible, as indicated by restoration of the studied index after a decrease in CPA concentration in the incubation medium ( Table 2). The presented results are consistent with data demonstrating the reversibility of the CPA action in studying their effect on the respiratory chain parame ters [6].
Summarizingtheresultsofexperimentsonthe effectofCPAsonproteinbiosynthesisinacell-free system, we suppose that in the mechanisms of inhi- Thisassumptionisconfirmedwiththereports [18,19], which demonstrated that organic solvents with the properties of CPA, in particular methanol,ethanol,DMSO,etc.,contributedtotheMg 2+ -dependent stabilization of ribosomes in associated state, as well as altered activity of aminotransferases and other translation factors, which generally reduced the translation apparatus functional activity. The decrease in the translation apparatus functional activity can also be associated with the interaction of cryoprotective agents with nucleotide bases of various types of RNA, as was reported [20].
Considering the possible mechanisms of the observed phenomenon of inhibition of protein synthesis in cells and cell-free systems, we can assume that they are primarily based on a change in the physicochemical properties of water, in particular its structural state and dielectric constant [21][22][23], accompanied by strengthening of hydrogen bonds [21,22], which are important in interaction of metabolites with their carriers, as well as functioning of ribosomes and enzymes involved into the translation. Apparently, an increase in the concentration of electrolytes, including Mg 2+ , is also crucial, as a result of a decrease in the free water fraction due to its binding to cryoprotective agent molecules.
In addition to the certain interest for cryobiology, we believe that the performed studies out are of general biological fundamental value, since the data indicate the possibility of protein synthesis in mixed solvents under conditions of an altered structure of water.
Thus, the following conclusions can be drawn as a result of the experiments: 1.Glycerol,DMSOandPEG-400atcryoprotectiveconcentrationssignificantlyinhibitprotein biosynthesis in the thymus, spleen, lymph nodes and liver cells. PEG-400 polymeric CPA revealed the mosteffectiveinhibitoryeffect.
It is important to note that, in a cell-free system the PEG-400 CPA in low concentrations, probably remaining in cells and tissues after washing, contributes to an increase in protein synthesis intensity. Similar results were obtained in experiments on whole cells as part of liver slices.
2.Aninhibitoryeffectonproteinbiosynthesis in the studied cells was found to be due to a direct influenceoftheseCPAsonthetranslationapparatus.