Aldehydes pArticipAtion in oxidAtive stress in rAt thymocytes in vitro

a variety of lipid radicals are formed under oxidative stress development. The further oxidation of these radicals leads to formation of numerous aldehydes. They can form postsynthetic modifications in proteins and nucleic acids that disrupt their functions. in the present study aldehydes role in the formation of oxidative stress parameters in rat thymocytes was investigated. Two models were used: iron-stimulated oxidative stress and exogenous aldehydes exposure to thymocytes. For oxidative stress induction, thymocytes (2×106 cells/ml hBss, рН 7.2) were exposed to different concentrations of Feso4 (20, 30, 40 μМ) and ascorbic acid (100 μМ) for 6 h. it resulted in increase of levels of aldehydes 29 times (90 ± 6 nmol/107 cells), these changes led to increase of TBars levels 4.4 times; the levels of protein co groups 10 times, cell mitochondrial activity and low-molecular weight sh groups were decreased 1.5 and 2.3 times, respectively. Treatment with aldehydes acceptor dimedone (200 μМ) significantly decreased the levels of aldehydes 3.7 times, TBars 1.6 times and protein co groups 5 times. it was shown that the levels of cell mitochondrial activity increase 1.4 times and the levels of sh groups 1.8 times. To compare the effects of aldehydes in induction of oxidative stress, thymocytes (2×106 cells/ml hBss, рН 7.2) were exposed to 50-600 μМ formaldehyde (Fa), 50-600 μМ glyoxal (GL), 50-600 μМ methylglyoxal (MGL), 1-15 μМ acrolein (Аcr) for 6 h. TBars levels were increased for Fa 1.3 times and for other aldehydes about 5-7 times. The levels of protein co groups were increase for Fa 3.7 times, for MGL 7 times, for GL 13 times, for acr 22 times. Levels of sh groups were decreased for Fa 1.5 times, for MGL 2.6 times, for GL 3 times, for acr 9 times. a decrease of cell mitochondrial activity 1.5 times observe for all aldehydes. obtained results prove the aldehydes participation in the formation of oxidative stress parameters and their capability to oxidative stress induction in the rat thymocytes.

a variety of lipid radicals are formed under oxidative stress development. The further oxidation of these radicals leads to formation of numerous aldehydes. They can form postsynthetic modifications in proteins and nucleic acids that disrupt their functions. in the present study aldehydes role in the formation of oxidative stress parameters in rat thymocytes was investigated. Two models were used: iron-stimulated oxidative stress and exogenous aldehydes exposure to thymocytes.
For oxidative stress induction, thymocytes (2×10 6 cells/ml hBss, рН 7.2) were exposed to different concentrations of Feso 4 (20, 30, 40 μМ) and ascorbic acid (100 μМ) for 6 h. it resulted in increase of levels of aldehydes 29 times (90 ± 6 nmol/10 7 cells), these changes led to increase of TBars levels 4.4 times; the levels of protein co groups 10 times, cell mitochondrial activity and low-molecular weight sh groups were decreased 1.5 and 2.3 times, respectively. Treatment with aldehydes acceptor dimedone (200 μМ) significantly decreased the levels of aldehydes 3.7 times, TBars 1.6 times and protein co groups 5 times. it was shown that the levels of cell mitochondrial activity increase 1.4 times and the levels of sh groups 1.8 times.
To compare the effects of aldehydes in induction of oxidative stress, thymocytes (2×10 6 cells/ml hBss, рН 7.2) were exposed to 50-600 μМ formaldehyde (Fa), 50-600 μМ glyoxal (GL), 50-600 μМ methylglyoxal (MGL), 1-15 μМ acrolein (Аcr) for 6 h. TBars levels were increased for Fa 1.3 times and for other aldehydes about 5-7 times. The levels of protein co groups were increase for Fa 3.7 times, for MGL 7 times, for GL 13 times, for acr 22 times. Levels of sh groups were decreased for Fa 1.5 times, for MGL 2.6 times, for GL 3 times, for acr 9 times. a decrease of cell mitochondrial activity 1.5 times observe for all aldehydes. obtained results prove the aldehydes participation in the formation of oxidative stress parameters and their capability to oxidative stress induction in the rat thymocytes.
K e y w o r d s: oxidative stress, aldehydes, dimedone, thymocytes. E ndogenous aldehydes are formed in the organism as by-products under lipid peroxidation (POL) or non-enzymatic glycosylation of proteins. POL, a complex process that occurs in all cellular membranes and involves the interaction of oxygen-derived free radicals with polyunsaturated fatty acids, finally results in a variety of reactive aldehydes. Glycation is a complex series of parallel and sequential reactions, in which reducing free carbonyl groups of carbohydrates react with the nucleophilic amino groups of biomolecules, producing a large number of various aldehydes. Reactive aldehydes formed during these ways are, for example, saturated aldehydes (ethanal [1], propanal), non-saturated ones (acrolein (ACR) [2], 4-hydroxynonenal [3]) and dicarbonyls (glyoxal (GL), methylglyoxal (MGL) [4]). Formaldehyde (FA) is an intermediate product of amino acid catabolism.
Aldehydes can alkylate amino groups of amino acids and basics of nucleic acids; react with proteins and peptides with further creation of stable intermediate products such as Schiff bases or N-hydroxymethyl adducts. In addition, these carbonyl compounds are able to form methylene bridges between close parts in proteins, that causes defection in their activity [5,6].
Due to formation of end products of glycosylation and POL, aldehydes participate in pathogenesis of diseases related to oxidative stress, such as diabetic nephropathy, Parkinson's, Alzheimer's and other diseases that Fig. 1 illustrated. [7,8,28].
Liesivuori and Savolainen illustrated that aldehydes addition in vitro leads to the increase of reactive oxygen species (ROS) and POL products, and decrease of the level of glutathion [9]. For example, the most reactive among non-saturated aldehydes ACR is formed endogenously under POL and polyamines metabolism [10]. ACR may induce oxidative stress [11], react with proteins, phospholipids and DNA accompanied by formation of Michael adducts [12]. Mechanisms of the ACR action are not fully established, but recent researches demonstrate that this carbonyl compound is able to connect and cleavage cell nucleophiles, such as reduced glutathione, lipoic acid and thioredoxin [10]. It can attack free thiol groups of cysteine and γ-amino groups of lysine and histidine with ACR-amino acid adducts and carbonyl groups of proteins [13] formation. These conversions lead to disturbance of protein functions.
GL and MGL are well-researched glycation agents, which throughout modification of arginine residues form cross-links in proteins and cause their dysfunction [14]. The ability of MGL to induce oxidative stress [15] was demonstrated by Desai, Chang et al. on the different types of cells. These results indicate that aldehydes, are formed on the crossing of different metabolic ways, are very important for research of these processes.

Fig. 1. Formation of aldehydes and advanced glycation and lipooxidation end products (aGes and aLes) in oxidative and monosaccharide stresses
Oxidative stress is one of the cell death inductors. An increase of ROS production by cell causes potential threat of oxidative damage to biomolecules. Suspension of isolated thymocytes (non-fully differentiated T-cells) is a useful model for research of oxidative stress development. Maturation of T-cells in thymus is a highly-organized process. Any deviation in it causes immune deficiency, autoimmune reactions or cancer. These cells, in comparison to other types of cells, are easily isolated, morphologically homogeneous and more sensitive to different damaging influences of chemical or physical factors. They are suitable for flow cytometry because of their spherical shape and size.
Oxidative stress is implicated in the pathogenesis of many chronic and degenerative diseases. Carbonyl compounds increase or carbonyl stress is a consequence of oxidative stress. Nowadays, the research of aldehydes role during oxidative stress and development of treatment aimed at carbonyl stress decrease under pathologies related to oxidative and carbon stresses, are important. екСпериментальні рОБОти The aim of this research is the investigation of oxidative stress parameters formation and aldehydes possibility to oxidative stress induction in rat's thymocytes. Two models were used: iron-induced oxidative stress and exogenous aldehydes such as FA, GL, MGL and ACR exposure to thymocytes.

materials and methods
Experiments were performed on thymocytes obtained from Wistar rats of 80-100 g weight which have been used for research. All manipulations with animals followed the Guide for the Care and Use of Laboratory Animals (1996) and followed Law of Ukraine on Protection of Animals from Cruel Treatment (published by Supreme Council of Ukraine, 2006, No 27, paper 230). Thymus glands were gently triturated in chilled Hanks' balanced salt solution (HBSS, Sigma, USA) supplemented with 10 mм тris-нСl (рн 7.2) to dissociate single cells. This solution with cells was passed through a nylon mesh and suspended for two times in HBSS centrifuged at 1000 g for 5 min. Cell counting was performed with 0.4% trypan blue (Sigma, USA) in Neubauer hemocytometer.
Aldehydes concentrations in cell lysates were determined by Purpald (Sigma, USA). The optical density was measured spectrophotometrically by μQuant (Biotek, USA) at 550 nm. Different-concentrated formaldehyde solutions were used for calibration [19].
Protein carbonyl groups were determined by modified spectrophotometric method [20].
The levels of low-molecular weight SH-groups were determined in lysates with ο-phthalaldehyde (Fluka, аustria) assay by M. L. Hu [17]. Excitation wavelength for ο-phthalaldehyde was 360 nm and emission was detected at 420 nm by FL800 (Biotek, USA). Reduced glutation was used as standard (Sigma, USA).
Cell mitochondrial activity was determined by Alamar Blue (AB, Sigma, USA) reduction. аB is a redox indicator, which determine the live cell metabolic activity. The system includes redox nonfluorescent indicator (resazurin), which is converted in fluorescent form (rezorufin) as a result of chemical reduction by living cells [21]. AB final concentration in a sample was 5 μм. Cells were incubated at 37 °С for 2 h. Fluorescence is monitored at 560 nm excitation wavelength and 600 nm emission wavelength by FL800 (Biotek, USA). Cell viability was expressed as a percentage of the AB reduction in control (100%).
Statistical analyses has been done with using Student's test (р < 0.05) and MS Excel 2007.

results and discussion
In this paper, iron-induced oxidative stress in thymocytes was used to research the aldehydes involvement in development of oxidative stress. Tran-sition metals act as catalysts during oxidative damage of biological macromolecules. Number of metals such as iron, copper, cadmium, chrome, nickel and vanadium are able to create active forms of oxygen that lead to POL increase, DNA damage, sulfhydryl group depletion and calcium homeostasis disturbance. It is well documented that iron, the most abundant transition metal, can induce the generation of free radicals, which attack important biomolecules including proteins, deoxynucleic acids, and lipid membranes and thus cause oxidative stress. H 2 O 2 with iron can produce reactive HO • radicals or ferryl intermediate by the Fenton reaction.
It was demonstrated that exposure of cells to 20, 30 and 40 μM FeSO 4 for 6 h resulted in significant dose-dependent decrease of cell viability: 1.7 times, 2.2 times and 3.5 times, respectively, compared with control. Obtained results were demonstrated in Table 1.
Total levels of aldehydes increase at 20 μM 13.5 times, at 30 μM 23 times and at 40 μM 29 times compared with control were demonstrated. This changes of levels of aldehydes led to dosedependent lipid peroxidation in thymocytes that was indicated by increase in TBARS levels about 3-4 times. Levels of protein СО groups were increased 5 times, 8.3 times and 10 times, respectively, at 20, 30 and 40 μм FeSO 4 compared with control. Lowmolecu lar weight SH groups and mitochondrial activity were decreased about 2 times and 1.5 times compared with control. тhus, the obtained results was proved oxidative stress development which was T a b l e 1. Biochemical parameters of oxidative stress development in thymocytes under iron-induced oxidative stress and dimedone addition, (n = 6) accompanied by formation of active carbonyl compounds such as aldehydes.
Dimedone was used to separate the aldehydes participation in formation of oxidative stress parame ters in rat's thymocytes. Dimedone is used as acceptor of aldehydes through complex al-dimedone formation. In such way there occurred a decrease of aldehydes concentration. Fig. 2 demonstrates the mechanism of interaction between dimedone and aldehydes.
After dimedone addition a decrease of the total levels of aldehydes 3.7 times and 1.2 times at 20 and 30 μм FeSO 4 , respectively, were observed. At 40 μм FeSO 4 aldehydes level was not changed due to high concentration of iron but there was a visib le tendency to a decrease that proved the effective acceptance of it. A decrease of other parameters were observed. TBARS levels were decreased 1.6 times, 1.2 times and 1.3 times, protein CO groups levels 2 times, 5 times and 3 times compared with 20, 30 and 40 μм FeSO 4 without dimedone addition. SH groups levels were increased 1.5 and 1.8 times at 30 μM and 40 μM FeSO 4 . Mitochondrial activity increase and improvement of cell viability after dimedone addition were observed.
ACR is highly reactive air pollutant, a metabolic product of the anti-cancer drug and by-product of lipid peroxidation. It is known that ACR is estimated to reach concentrations up to 80 μм in respiratory tract lining fluids as a result of smoking (Eiserich et al., 1995) [22]. in vivo ACR concentrations in Alzhheimer' disease brain are 2. Exposure of various concentrations of ACR to thymocytes was resulted in a dose-dependent decrease in cell viability. After 6h exposure with 15 μм ACR, the cell death level increased 4.5 times compared with control.
These results demonstrated the strongest toxic effect of ACR compared to the other investigated aldehydes to thymocytes even in low concentration. Based on these results, 1, 5, 10, 15 μм аCR were used in research.
Exact concentrations of endogenous FA are quite hard to determine, because it metabolises very fast and non-metabolized one connects with tissue proteins. Nakao, Umebaysh et al. defined that concentration of FA in blood is about 100 μм [24]. 50, 200, 600 μм FA were used in the investigation. The obtained data demonstrated that FA has the lowest cytotoxic effect to thymocytes compared with other aldehydes, because the percentage of dead cells at maximal concentration of FA (600 μM) decreased 4 times compared with control.
Recent estimates of the concentrations of MGL and GL in human blood plasma are in the range of 100-120 nм [25] and cellular concentrations of MGL 1-5 μм and GL 0,1-1 μм [26]. It is known that MGL (> 10 mM) inhibits glycolytic enzymes (Leonici et al., 1989). DNA crosslinking was observed at 1.5 mM MGL in Chinese hamster ovary cells [27]. The IC 50 for MGL is 8.6 mM (for s. cerevisiae strain BY4743), approximately 5-fold less than that for glyoxal (IC 50 of 45.5 mM) (Hoon et al., 2011). Thornalley proposed that the effects of GL and MGL on cultured cells and tissues using at concentration > 10-fold higher than this are likely to be only of rele vance for acute intoxication and cytotoxicity [28].
Thornalley in their experiments with e. coli have used 0.2-1 mм MGL concentrations [27]. MGL and GL in the amount of 50, 200, 600 μM were used in our research. It has been demonstrated that after exposure to 600 μM MGL and GL the level of dead cells increased 4.4 and 4.8 times, respectively. These results indicated the similarity of their cytotoxic effect.
The ability of investigated aldehydes to induce oxidative stress development was determined by change of such oxidative stress parameters as TBARS, protein CO groups, low-molecular weight SH groups, cell mitochondrial activity by AB reduction.
As shown in Table 2, TBARS level for FA was increased only 1.3 times while for GL about 5-6 times, for MGL 13 times and for ACR 30 times compared with control. The level of protein CO groups in cell lysates was increased in the same way. The lowest level of protein CO groups (about 3-4 times increase) was observed after exposure to FA. Depending on concentration this parameter was increase for GL 9-13 times, for MGL 5-7 times, for ACR 22 times compared with control. Level of lowmolecular weight SH groups was decreased for FA about 1.5 times, for MGL up to 2 times, for ACR up to 9 times compared with control. Depending on concentration of aldehydes, a decrease of mitochondrial activity was observed for FA up to 1.3 times, for GL 1.3 times and for MGL 1.2 times, for ACR 1.7 times compared with control, which was accepted as 100%.
The obtained results demonstrated that ACR is the most toxic aldehyde from investigated ones to thymocytes because it is a by-product of lipid peroxidation. FA demonstrates the least toxic effect, because it does not belong to aldehydes which generated under oxidation processes development.