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Changes in lymphocyte properties after employment of the combination of carbamylation and oxidative stress, an in vitro study
Toxicology in Vitro 34 (2016) 105–112
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Changes in lymphocyte properties after employment of the combination of carbamylation and oxidative stress, an in vitro study Anna Pieniazek a,⁎, Krzysztof Gwozdzinski b a b
Department of Medical Biophysics, University of Lodz, 90-2367 Lodz, Poland Department of Molecular Biophysics, University of Lodz, 90-2367 Lodz, Poland
a r t i c l e
i n f o
Article history: Received 30 September 2015 Received in revised form 18 March 2016 Accepted 29 March 2016 Available online 2 April 2016 Keywords: Carbamylation Oxidative stress Lymphocytes Chronic renal failure
a b s t r a c t It is well known that oxidative stress and carbamylation alter macromolecule properties and functions. We evaluated the influence of sodium cyanate (NaOCN) and the combination of cyanate and hydrogen peroxide (H2O2) on nonenzymatic antioxidant capacity (NEAC), total thiols, reduced glutathione (GSH) and hydroperoxide level in mononuclear blood cells (MNCs). We also examined plasma membrane properties of MNCs using the spin labeling method in EPR spectroscopy (electron paramagnetic resonance spectroscopy). We showed that MNCs are resistant to cyanate treatment up to a concentration of 2 mM (survival test). On the other hand, a significant loss of antioxidant defense of cells, e.g. NEAC upon NaOCN, H2O2 and the combination of cyanate and hydrogen peroxide was observed. Carbamylation slightly decreased GSH and the free thiol level, but H2O2 and its combination with NaOCN lead to a decrease in their amounts. A markedly higher level of hydroperoxides was only observed in the cells treated with H2O2. We found a significant decrease in lipid membrane fluidity at the depth of 12th and 16th carbon atoms of fatty acids in lymphocytes treated with cyanate or H2O2. The combination of both substances acted synergistically and induced profound changes in comparison to cyanate and hydrogen peroxide used alone. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Carbamylation of proteins and oxidation of cellular components during oxidative stress occur in pathology of many diseases [e.g., diabetes mellitus, atherosclerosis, chronic renal failure (CRF)] (Jaisson and Gillery, 2010; Jaisson et al., 2011). Carbamylation is the posttranslational modification of proteins resulting from the nonenzymatic reaction between isocyanic acid and specific free functional groups, especially amino groups. Isocyanic acid is a very reactive form of cyanate derived from the spontaneous decomposition of urea ( Wynckler et al., 2000; Jaisson et al., 2011). In the body, an aqueous environment, urea and cyanate occur in equilibrium (99:1) (Wynckler et al., 2000). It has been demonstrated that in patients with chronic renal failure (CRF) the concentration of urea is 5–7 times higher than in healthy individuals (Malyszko et al., 2006; Selvaraj et al., 2002; Wyncler et al., 2000; Trepanier et al., 1996). Moreover, it has been demonstrated that in CRF hemodialysis (HD) patients the concentration of cyanate before HD increases up to 150 nM and in comparison to healthy volunteers is about three times greater (Nilsson et al., 1996).
⁎ Corresponding author at: Department of Thermobiology, University of Lodz, ul. Pomorska 141/143, 90-236 Lodz, Poland. E-mail address: [email protected] (A. Pieniazek).
http://dx.doi.org/10.1016/j.tiv.2016.03.017 0887-2333/© 2016 Elsevier Ltd. All rights reserved.
Another serious problem occurring in CRF patients is oxidative stress and microinflammation (Jaber et al., 2001). These problems may contribute to progression of renal disease, and can be associated with higher mortality of patients. It has been suggested that two main reasons of an increase of oxidative stress in hemodialysis patients exist. The first relates to increased production of reactive oxygen species as a result of activation of immune cells in response to a small biocompatibility of the dialysis membrane (Hörl, 2002). The second reason is the low selectivity of the dialysis membrane which may result in the removal of uremic toxins and low molecular weight antioxidants from the blood of uremic patients. It has been observed that in hemodialyzed patients, the antioxidant capacity of plasma before HD is higher than in healthy volunteers but after a dialysis session it drops significantly below the level of normal value (Pieniazek et al., 2002a, 2002b). Several studies have demonstrated synergism between oxidative stress and inflammatory response. Hydrogen peroxide as a metabolic oxidant can activate the NF-kB pathway, leading to synthesis of proinflammatory cytokines and resulting in amplification of the inflammatory cascade (Libetta et al., 2011). A small number of works associated with the combined action of carbamylation and oxidative stress available in the literature prompted us to study the effect of both factors on the properties of MNCs. The aim of this study was to evaluate the in vitro influence of carbamylation and the combination of carbamylation and oxidative stress on isolated human peripheral blood mononuclear cells. We determined the effect
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of the combination of carbamylation and oxidation on oxidative stress parameters such as nonenzymatic antioxidant capacity, total thiols, reduced glutathione and peroxide level. Additionally, we examined also the plasma membrane after incubation of cells with cyanate, hydrogen peroxide and their combination by measuring lipid membrane fluidity using EPR spectroscopy. 2. Material and methods
2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5carboxyanilide inner salt (XTT) was used to quantify the metabolically active living cells. The mitochondrial dehydrogenases of viable cells reduce the tetrazolium ring of XTT, yielding an orange formazan derivative. The obtained values of absorbance of the reaction product were presented as a percentage, taking the value of absorbance of the control as 100%.
2.1. Chemicals
2.4. Nonenzymatic antioxidant capacity
2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5carboxyanilide inner salt (XTT), Phenazine methosulfate (PMS), 2,4,6tripyridyl-s-triazine (TPTZ), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 5doxyl-stearic acid (5-DS), 12-doxyl-stearic acid (12-DS), 16-doxylstearic acid (16-DS), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), ophthalaldehyde (OPA), and N-ethylmaleimide (NEM) were obtained from the Sigma-Aldrich company. The RPMI 1640 bicarbonate medium, fetal bovine serum (FBS), penicillin and streptomycin were from PAA (Germany). All other chemicals were purchased from POCH S.A. (Gliwice, Poland) if not otherwise indicated. All dishes necessary for cell culture were obtained from the NUNC company.
The nonenzymatic antioxidant capacity (NEAC) of MNCs was measured using two different methods: TPTZ and DPPH. The spectrophotometric method is based on reduction of the ferric tripyridyltriazine [Fe(III)-TPTZ] complex to ferrous [Fe(II)] at a low pH. The iron complex [Fe(III)-TPTZ] reduced by antioxidants has an intense blue color with absorption maximum at 593 nm (Benzie and Strain, 1996). The samples of lysed cells (0.05 ml) were mixed with a working reagent (300 mM acetate buffer (pH 3.6); 10 mM TPTZ in 40 mM HCl; 20 mM FeCl3 freshly prepared in a volume ratio (10:1:1)) (0.1 ml) and incubated at room temperature for 30 min. At the end of incubation the absorbance was measured. The electron paramagnetic resonance (EPR) method is based on reduction of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. In the presence of antioxidants, loss of EPR signal of 1,1-diphenyl-2picrylhydrazyl (DPPH) is observed. The loss of EPR signal is inversely proportional to the concentration of antioxidants (Pieniazek et al., 2002a, 2002b). The samples of lysed cells (0.01 ml) were then mixed with DPPH (300 μM in methanol) (0.04 ml) and incubated for 30 min at room temperature. At the end of incubation the samples were centrifuged and the intensity of EPR signal in the supernatant was measured. In both methods the antioxidant activity of MNCs was expressed as Trolox equivalents calculated on the basis of the calibration curve (in nmol/mg protein). EPR spectra were performed on the Bruker ESP 300 E spectrometer at room temperature (22 + 2 °C), operating at a microwave frequency of 9.73 GHz. The instrumental settings were as follows: center field set at 3480 G, range of 80 G, with a 100 kHz modulation frequency and modulation amplitude of 1.01.
2.2. Peripheral blood mononuclear cells (MNCs) All experiments were carried out on human normal peripheral blood mononuclear cells (MNCs) isolated from the blood obtained from the Blood Bank in Lodz, Poland (each time, from leukocyte layers yielded separately from blood of different healthy volunteers). Therefore, every single experiment was performed on cells from one donor — nnumbers represent cells from different individuals. Cell isolation was performed by centrifugation of blood in the density gradient of Histopaque 1077 (300 g for 30 min at 22 °C). The cells were washed two times with phosphate buffered saline (PBS). The MNCs were grown in the RPMI medium supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) in a 37 °C humidified 5% CO2 incubator. Isolated cells were plated on a sterile dish (2 × 106 cells/ml) (5 ml each sample) then sodium cyanate (NaOCN) to the final concentration of 1 mM or 2 mM was added and incubated in a 37 °C humidified 5% CO2 incubator for 23 h. At the end of this period, hydrogen peroxide was added to a final concentration of 100 μM and left for 1 h of incubation. The collected cells were washed with phosphate buffered saline (pH 7.4) (PBS) and used for membrane lipid fluidity determination. For measurements of reduced glutathione and thiol group concentration as well as antioxidant activity, the cells were lysed in a buffer containing 10 mM Tris, 1 mM EDTA, 0.1 M NaCl and 0.01% Triton X-100 (about 1 × 107 cells in 0.1 ml buffer). 2.3. Cell survival The survival of blood mononuclear cells after treatment with sodium cyanate was estimated by the standard microplate XTT colorimetric method. Isolated blood mononuclear cells (15 × 104 cells/well) were seeded on 96-well microplates in the growth medium with a wide range of sodium cyanate concentrations (0–10,000 μM) for 24 h and incubated at 37 °C in a humidified 5% CO2 incubator. At the end of the exposure time the medium with NaOCN was replaced with a fresh one and the cells were grown for additional 24 h. At this time point the XTT test was performed. Freshly prepared XTT (0.05 ml, to the final concentration of 1 mg/ml) with PMS (to the final concentration of 0.017 mg/ml) was added to each well of the microplates. Subsequently, the microplates were incubated for 4 h. Absorbance was measured at 495 nm using a microplate reader (BioTek).
2.5. Free thiol group concentration The quantification of free thiol groups was based on reaction with Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid); DTNB). Upon reaction with free thiol groups 2-nitro-5-thiobenzoate (NTB), optically active at 412 nm, is released (Ellman, 1959). The samples of lysed cells (0.015 ml) were then mixed with 10 mM phosphate buffer (pH 8.0) containing 0.5% SDS (0.235 ml) and the absorbance at 412 nm was measured (A0). Then the DTNB reagent was added to the samples (final concentration of 0.1 mM) (0.025 ml) and they were incubated for 1 h at 37 °C. After this time the absorbance of the samples was again measured A1. ΔA = A1 − A0 for calculation of the thiol group concentration was used. The concentration of thiol groups was calculated based on the calibration curve for different concentrations of reduced glutathione and expressed as nmol/mg protein. 2.6. Glutathione The reduced glutathione (GSH) concentration was determined using the fluorimetric method with o-phthalaldehyde (OPA) (Senft et al., 2000). The reaction product of OPA and GSH has high fluorescence quantum yields. The mononuclear cell lysate (0.01 ml) was mixed with redox quenching buffer with trichloroacetic acid (RQB-TCA (20 mM HCl, 5 mM DTPA, 10 mM ascorbic acid); 5% TCA)
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(0.005 ml),7.5 mM N-ethylmaleimide (NEM) in RQB (0.002 ml), and 1.0 M potassium phosphate buffer (KP) (pH 7.0) (0.025 ml) and incubated at room temperature for 5 min. At the end of incubation the 0.1 M KP buffer (pH 6.9) (0.1 ml) and OPA (5 mg/mL in methanol) (0.015 ml) were added and incubated in the dark at room temperature for 30 min. The OPA-derived fluorescence was measured at 365 nm excitation and 430 nm emission. The glutathione concentration was calculated using the calibration curve for different concentrations of reduced glutathione, as a standard, and expressed as nmol/mg protein.
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with one-way ANOVA for repeated measures and the post-hoc multiple comparisons Tukey's test. In addition, due to relatively small sample sizes and low statistical power of numerous estimated inferences, we employed the resampling bootstrap technique (10,000 iterations) to reason on how likely the revealed differences could be observed by a pure chance. The power of the used test was checked for each analysis and always was more than 80%. Statistical analysis was performed using Statistica v. 12.5 and Resampling Stats Add-in for Excel v.4.
2.7. Hydroperoxide 3. Results For determination of MNC hydroperoxides, the spectrophotometric method with xylenol orange was employed. The reaction was based on rapid oxidation of Fe(II) to Fe(III) in the presence of peroxides (Gay et al., 1999; Gay and Gebicki, 2000). 25 mM ammonium iron(II) sulfate in 2.5 M H2SO4 and 125 μM xylenol orange (1:100) were used to prepare a working solution. The samples of lysed cells (0.01 ml) were then mixed with a working reagent (0.1 ml) and incubated for 30 min in the dark. The reaction of Fe(III) with xylenol orange yielded a violet-colored complex, which was quantified spectrophotometrically at 560 nm. The concentration of peroxides was expressed as μmol/mg protein. 2.8. Protein concentration The protein concentration in MNC lysates was determined using the method of Lowry et al. (1951). 2.9. MNC lipid membrane fluidity The lipid membrane fluidity of MNCs was estimated using three different spin labels (5-DS, 12-DS and 16-DS) in the EPR technique. The paramagnetic group of doxyl stearic acid is located on different carbon atoms in the hydrocarbon chain, and provides information on phospholipid fatty acyl mobility at different depths of lipid bilayer. The spin label, in ethanol solution was added into cell suspension (5 × 107 cells/ml) to the final concentration of 0.1 μM and incubated for 30 min at room temperature. The final ethanol concentration in cell suspension was less than 0.05% (v/v). Data obtained from the spectra was used to calculate the rotational correlation time (τc), according to formula (1) (Schreier et al., 1978):
τc ¼
1 kw0 2
sffiffiffiffiffiffiffiffi sffiffiffiffiffiffiffiffiffi ! h0 h0 −2 þ hþ1 h−1
ð1Þ
where: time when the spin label undergoes full rotation τc k constant equal to 1.19 × 10−9 s width of the mid-line of spectrum w0 height of the mid-line of spectrum h0 height of the high-field line of spectrum. h−1 EPR spectra were performed on the Bruker ESP 300 E under the same conditions as described above. 2.10. Statistical analysis All data were expressed as mean ± standard deviation. The normality of data was tested using the Shapiro–Wilk test and variance homogeneity was verified with the Brown Fosythe test. Mauchly's test was used to test for data sphericity. Sets of data with confirmed normal distributions and homoscedasticity/sphericity were tested with the use of parametric tests, while non-parametric tests were used for those without. The significance of differences between the groups was estimated
Isolated blood mononuclear cells were exposed to a wide range of sodium cyanate concentrations (0–10,000 μM) for 24 h. The cytotoxicity of sodium cyanate on MNCs was compared to untreated (control) cells, which survival was arbitrarily taken as 100%. Obtained results demonstrated that sodium cyanate did not change MNC survival at concentrations below 2 mM in a logarithmic scale on the abscissa axis (Fig. 1). A significant decrease in survival was observed for mononuclear blood cells after their exposure to cyanate at concentrations higher than 2 mM. The value of IC50 for mononuclear blood cells after treatment with sodium cyanate was about 9 mM. For further studies, two high sodium cyanate concentrations (1 mM and 2 mM), which did not affect the cell viability were selected, as seen in Fig. 1. For determination of nonenzymatic antioxidant capacity the MNCs were treated with sodium cyanate and hydrogen peroxide as well as with the combination of both substances using two independent methods. The changes in NEAC in cells measured with DPPH showed a significant decrease of this parameter in all selected groups in comparison to control (Fig. 2). The highest decrease in antioxidant capacity in the cells was noted after treatment with hydrogen peroxide as well as with the combination of cyanate at two concentrations with hydrogen peroxide. We found also a significant difference in antioxidant capacity after treatment of cells with the combination of NaOCN and H2O2 in comparison to cells treated with cyanate alone. Results similar to DPPH in antioxidant capacity were observed in the experiment using the TPTZ method. A significant decrease in antioxidant capacity in MNCs after treatment with hydrogen peroxide and the combination of NaOCN and H2O2 at two concentrations in comparison to control was found (Fig. 3). However, there were no differences between cells treated with cyanate alone in comparison to combinations of cyanate and hydrogen peroxide. As seen in Fig. 4, in the MNCs treated with hydrogen peroxide and the combination of NaOCN and H2O2, the level of free thiol groups significantly decreased in comparison to control. The sodium cyanate added to the cells, at two different concentrations led to a slight, not significant, decrease of free thiol concentration. However, the combination of NaOCN and H2O2 resulted in a significant loss of free thiol groups in comparison to cells treated with cyanate only. Simultaneously, we observed a significantly decreased concentration of reduced glutathione in cells after treatment with hydrogen peroxide and the combination of NaOCN and H2O2 (Fig. 5). On the other hand, in cells treated with sodium cyanate the concentration of GSH did not significantly decrease. Nevertheless, a significant decrease of reduced glutathione concentration in MNCs after treatment with the combination of sodium cyanate and hydrogen peroxide in comparison to cells treated with sodium cyanate alone was noted. The level of hydroperoxides in cells treated with sodium cyanate, hydrogen peroxide and their combinations with two concentrations of cyanate was determined. A statistically significant increase of hydroperoxides in cells treated with hydrogen peroxide in comparison to control cells was observed (Fig. 6). On the other hand, we did not find any differences in MNCs treated with both concentrations of sodium cyanate in comparison to control. However, in cells treated with the
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Fig 1. Survival of human mononuclear blood cells after 24 h of treatment with sodium cyanate (concentration range: 0–10,000 μM). Each circle represents cells from 8 different individuals (n = 8).
combination of NaOCN and H2O2 a slight, not significant, increase of hydroperoxides versus the control level was found. We also examined the plasma membrane fluidity of MNCs upon treatment with cyanate, hydrogen peroxide and combinations of two concentrations of NaOCN with H2O2 using the spin labeling method. For the determination of spin labeled fatty acid mobility after incorporation into the lipid milieu the relative rotational correlation time was calculated. In the polar region of the external lipid layer of MNCs, treated with sodium cyanate, hydrogen peroxide and their combination, there
Fig. 2. Nonenzymatic antioxidant activity of MNCs treated with sodium cyanate, hydrogen peroxide and their combinations using two concentrations of NAOCN with H2O2 measured by reduction of DPPH radical in EPR technique. Each bar represents cells from 6 different individuals (n = 6). Statistical significances: control vs. H2O2, P b 0.0002; control vs. cyanate (1 mM), P b 0.01; control vs. cyanate (2 mM), P b 0.0005; control vs. cyanate (1 mM) + H2O2, P b 0.0002; control vs. cyanate (2 mM) + H2O2, P b 0.0002; cyanate (1 mM) vs. cyanate (1 mM) + H2O2, P b 0.02; cyanate (2 mM) vs. cyanate (2 mM) + H2O2, P b 0.005.
were no significant differences in the rotational correlation time of the 5-DS spin label (data not shown). In the deeper region of the lipid bilayer, at the depth of the 12th carbon atom of fatty acid the changes of lipid membrane fluidity in MNCs after treatment with sodium cyanate, hydrogen peroxide, and their combinations were observed (Fig. 7). A statistically significant increase of the rotational correlation time of the 12-DS spin label in cells treated with hydrogen peroxide and two concentrations of cyanate in comparison to control cells was noted. A large increase in the rotational correlation time was found in cells exposed to a combination of sodium cyanate and hydrogen peroxide compared to control and compared to cells treated with sodium cyanate alone (Fig. 7).
Fig. 3. Antioxidant activity of MNCs treated with sodium cyanate, hydrogen peroxide and their combinations with two concentrations of NAOCN with H2O2 measured by the spectrophotometric method with TPTZ. Each bar represents cells from 6 different individuals (n = 6). Statistical significances: control vs. H2O2, P b 0.0002; control vs. cyanate (1 mM) + H2O2, P b 0.001; control vs. cyanate (2 mM) + H2O2, P b 0.001.
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Fig. 4. The level of free thiol groups in MNCs treated with sodium cyanate, hydrogen peroxide and their combinations with two concentrations of cyanate. Each bar represents cells from 6 different individuals (n = 6). Statistical significances: control vs. H2O2, P b 0.0005; control vs. cyanate (1 mM) + H2O2, P b 0.0002; control vs. cyanate (2 mM) + H2O2, P b 0.0002; cyanate (1 mM) vs. cyanate (1 mM) + H2O2, P b 0.01; cyanate (2 mM) vs. cyanate (2 mM) + H2O2, P b 0.02.
Results of spin label mobility similar to 12-DS were also obtained for the 16-DS spin label incorporated into the mononuclear blood cell membrane. There was a statistically significant increase in the rotational correlation time of 16-DS after incubation of cells with the higher
Fig. 5. The level of reduced glutathione in MNCs treated with sodium cyanate, hydrogen peroxide and their combination. Each bar represents cells from 6 different individuals (n = 6). Statistical significances: control vs. H2O2, P b 0.0005; control vs. cyanate (1 mM) + H2O2, P b 0.0002; control vs. cyanate (2 mM) + H2O2, P b 0.0002; cyanate (1 mM) vs. cyanate (1 mM) + H2O2, P b 0.002; cyanate (2 mM) vs. cyanate (2 mM) + H2O2, P b 0.0005.
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Fig. 6. The level of peroxides in MNCs treated with sodium cyanate, hydrogen peroxide and combinations of H2O2 with two concentrations of cyanate (n = 6). Each bar represents cells from 6 different individuals (n = 6). Statistical significance: control vs. H2O2, P b 0.05.
concentration of cyanate and with the combinations of the two concentrations of cyanate with hydrogen peroxide in comparison to control cells (Fig. 8). A significant increase of this parameter after treatment of
Fig. 7. Relative rotational correlation time of 12-DS spin label incorporated into the MNCs treated with sodium cyanate, hydrogen peroxide and their combinations. Each bar represents cells from 15 different individuals (n = 15). Statistical significances: control vs. H2O2, P b 0.01; control vs. cyanate (1 mM), P b 0.02; control vs. cyanate (1 mM) + H2O2, P b 0.00001; control vs. cyanate (2 mM) + H2O2, P b 0.00001; cyanate (1 mM) vs. cyanate (1 mM) + H2O2, P b 0.05; cyanate (2 mM) vs. cyanate (2 mM) + H2O2, P b 0.00002.
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Fig. 8. Relative rotational correlation time of 16-DS spin label incorporated into the MNCs treated with sodium cyanate, hydrogen peroxide and their combinations. Each bar represents cells from 15 different individuals (n = 15). Statistical significances: control vs. cyanate (2 mM), P b 0.01; control vs. cyanate (1 mM) + H2O2, P b 0.0002; control vs. cyanate (2 mM) + H2O2, P b 0.0002; cyanate (1 mM) vs. cyanate (1 mM) + H2O2, P b 0.05.
cells with the combination of NaOCN and H2O2 in comparison to the cells treated with cyanate only was also observed. 4. Discussion Carbamylation of proteins and amino acids leads to changes in their structure and function (Trepanier et al., 1996; Wynckel et al., 2000; Jaisson and Gillery, 2010; Jaisson et al., 2011). Similarly, oxidative stress also changes their structure and function during the oxidation process (Pieniazek et al., 2002a, 2002b; Fisher-Wellman and Bloomer, 2009). Both of these processes are observed in many diseases for example arteriosclerosis and chronic renal failure (Himmelfarb et al., 2000; Pieniazek et al., 2009; Jaisson et al., 2011). It seems that a study of carbamylation and oxidative stress induced in vitro may be useful in explaining changes in peripheral blood mononuclear cells of patients undergoing chronic hemodialysis. In the beginning of our study we evaluated the cytotoxicity of sodium cyanate on mononuclear blood cells. We found that this compound did not have influence on cell survival up to the concentration of 2 mM. Considering that the concentration in physiologically healthy subjects is 50 nM an elevated level was observed in patients with chronic renal failure before hemodialysis, approx. 150 nM (Nilsson et al., 1996). In our experiments the concentration of sodium cyanate (2 mM) was approx. 50,000 times higher than the physiological concentration. Based on our studies it can be concluded that the in vivo cyanate has no impact on the survival of MNCs. However, higher concentrations of NaOCN lead to a decrease in cell survival. Recently, it has been shown that application of 1 mM cyanate did not influence on endothelial cell (HCAEC) viability (El-Gamal et al., 2014). This result is in line with our finding. The physiological concentration of cyanate was too low to evoke any effect. Generally, we were interested on what is the combined effect of carbamylation and oxidative stress on MNCs. It is noteworthy that in the blood of CRF patients about 100 other toxic substances are present and their range of concentrations is 2–200 higher than in healthy subjects (Vanholder et al., 2003). Moreover, interactions between them are also possible.
Additionally, in our previous work, using the EPR method, we showed that in the 20th minute of hemodialysis the respiratory burst releases hydroxyl and superoxide radicals (Gwozdzinski and Janicka, 1995; Gwozdzinski et al., 1997). Both of them can oxidize biological material and induce damage including whole cells. Additionally during recombination/dismutation of both radicals hydrogen peroxide is produced. It has been well documented that oxidative stress induced by hydrogen peroxide can disrupt normal functions of cells by oxidation of proteins, lipids and nucleic acids (Pieniazek et al., 2002a). Additionally, it has been shown that hemodialyzed patients have elevated genomic damage and oxidized DNA in peripheral blood lymphocytes (Stopper et al., 2001; Tarng et al., 2004; Moffitt et al., 2014). Oxidative genetic damage can be a consequence of an increase of ROS formation as well as a decrease of antioxidant defense. In our studies we observed a loss of antioxidant defense of cells e.g. a decrease of nonenzymatic antioxidant capacity and a decrease of reduced glutathione and the free thiol group level. Additionally, we found a decrease in lipid membrane fluidity at the depth of the 12th carbon atom of fatty acids in lymphocytes treated with hydrogen peroxide. Both carbamylation and oxidation lead to a decrease of nonenzymatic antioxidant capacity of cells indicated by TPTZ and DPPH methods. The antioxidant capacity of the cells determined using both methods allows the assessment of the presence of low molecular weight antioxidants, including reduced glutathione (Benzie and Strain, 1996; Kedare and Singh, 2011). It can be assumed that the decline in antioxidant activity may be an effect, among others, of the loss of part of reduced glutathione, while thiol groups could be oxidized or carbamylated. It has been reported that thiol and phenol groups exhibit the highest reactivity with isocyanic acid (Stark, 1964; Jaisson et al., 2011; Praschberger et al., 2013). It is interesting that the use of the combination of cyanate and hydrogen peroxide, resulted in a greater loss of antioxidant activity of the cells than after application of the cyanate alone, but these changes were not as large as in the case of hydrogen peroxide alone. These results showed that carbamylated low molecular weight antioxidants are more resistant to oxidation than those unmodified. In patients with CRF undergoing hemodialysis it has been observed that the antioxidant activity of plasma was much higher than in normal subjects (Pieniazek et al., 2002b; Clermont et al., 2000). Nevertheless, in the plasma of patients with CRF a large amount of uric acid responsible for the increase in the antioxidant activity of plasma is accumulated. Analysis of total thiols and glutathione revealed a slight decrease in their amounts after carbamylation. The reaction of isocyanic acid with thiol groups is reversible, but nevertheless reproduced isocyanic acid may again cause carbamylation of proteins and amino acids (Stark, 1964). It has been reported that in the plasma of CRF patients the thiol level was lower in comparison to healthy subjects (Clermont et al., 2000). Application of hydrogen peroxide as an oxidant leads to the loss of antioxidant defense of cells e.g. a decrease of nonenzymatic antioxidant capacity and a decrease in the free thiol level and of reduced glutathione. The changes in these parameters were higher than after the carbamylation process. A similar effect was obtained when the combination of carbamylation and oxidative stress was applied. Oxidation of both proteins and lipids can lead, inter alia, to the formation of their peroxides. We observed a significant increase in this parameter in cells treated with hydrogen peroxide. Interestingly, the cells exposed to the process of carbamylation were slightly less sensitive to oxidation. In this study we showed that oxidation has a greater potential for damaging biological material than carbamylation. We also examined the effect of carbamylation, oxidation and both processes combined on the properties of the plasma membrane of MNCs. Three fatty acid spin labels were applied to determine lipid plasma fluidity. We did not find any changes in the polar region of the lipid layer after carbamylation, oxidation and the combination of both processes. However, the changes in lipid fluidity were monitored with spin labels located in the deeper region of the cell membrane indicated
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by 12-DS and 16-DS. Interestingly, carbamylation significantly declined membrane fluidity. It is possible that changes in membrane lipid fluidity were caused by carbamylation of amino groups of phospholipids (phosphatidylserine and phosphatidylethanolamine) and proteins as well as interaction of modified lipids and proteins. Changes in membrane fluidity have also influence on: lipid phase transition (the gel–liquid crystalline transition), changes in lipid–protein interactions, transport across the membrane, alterations in cell deformability, disturbances in intracellular signaling, changes in cell interactions, etc. Most research suggests that carbamylation by isocyanic acid refers to proteins and free amino acids. However, the possibility that carbamylation may extend beyond the level of protein to other nonprotein primary amino group-containing compounds has been suggested (Trepanier and Thibert, 1996). One of such potential targets is the plasma membrane aminophospholipids such as phosphatidylserine (PS) and phosphatidylethanolamine (PE). Both of these phospholipids contain a primary amino group in their polar region, which is potentially accessible to bind with exogenous amino-specific chemical compounds. Externalization of phosphatidylserine (PS) from the internal monolayer of lipids in the apoptosis pathway is also possible. Development of apoptosis in mononuclear cells in hemodialyzed patients has been reported (Jaber et al., 2001). We found also that oxidation and the combination of carbamylation and oxidation lead to a significant decrease in membrane fluidity. We showed that oxidation of MNCs leads to peroxide formation, but carbamylation attenuated this process. Unexpectedly, we observed a synergistic effect of hydrogen peroxide and cyanate which leads to a major change in fluidity unlike each of them separately. The changes in this parameter can be a result of lipid oxidation (fragmentation or/ and aggregation), lipid crosslinking, and changes in lipid–protein interactions. Carbamylation and oxidation have an impact on the function and structure of proteins and lipids. Oxidation of both proteins and lipids can lead, inter alia, to the formation of their peroxides. Interaction of isocyanic acid with proteins and amino phospholipids may form carbonyl groups on them. In our work, we observed a significant increase in the level of peroxides in lymphocytes incubated with hydrogen peroxide. Slight increases in peroxides were also observed in samples of cells treated with a combination of sodium cyanate and hydrogen peroxide. Because the cells were first treated with sodium cyanate and hydrogen peroxide, the slight increase of peroxides in these samples may be due to a previous interaction of sodium cyanate with proteins and phospholipids and blocking the possibility of oxidation and thus the formation of peroxides. 5. Conclusion Although mechanisms of action of cyanate and hydrogen peroxide are different, sometimes we observed a similar action of both reagents. Generally, carbamylation of proteins indicates a weaker effect on investigated parameters than oxidative stress. However, when used together, carbamylation “protected” against oxidative stress. On the other hand, in the case of membrane fluidity the synergistic effect of both substances was observed. These studies showed that carbamylation has rather small participation in MNC damage in hemodialyzed patients. It seems that oxidative stress dominated in cell injury in CRF patients. Transparency document The Transparency document associated with this article can be found, in the online version. Acknowledgment The authors thank Dr. Jan Czepas for language corrections and Prof. Cezary Watala for help in statistical analysis.
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Changes in lymphocyte properties after employment of the combination of carbamylation and oxidative stress, an in vitro study Anna Pieniazek a,⁎, Krzysztof Gwozdzinski b a b
Department of Medical Biophysics, University of Lodz, 90-2367 Lodz, Poland Department of Molecular Biophysics, University of Lodz, 90-2367 Lodz, Poland
a r t i c l e
i n f o
Article history: Received 30 September 2015 Received in revised form 18 March 2016 Accepted 29 March 2016 Available online 2 April 2016 Keywords: Carbamylation Oxidative stress Lymphocytes Chronic renal failure
a b s t r a c t It is well known that oxidative stress and carbamylation alter macromolecule properties and functions. We evaluated the influence of sodium cyanate (NaOCN) and the combination of cyanate and hydrogen peroxide (H2O2) on nonenzymatic antioxidant capacity (NEAC), total thiols, reduced glutathione (GSH) and hydroperoxide level in mononuclear blood cells (MNCs). We also examined plasma membrane properties of MNCs using the spin labeling method in EPR spectroscopy (electron paramagnetic resonance spectroscopy). We showed that MNCs are resistant to cyanate treatment up to a concentration of 2 mM (survival test). On the other hand, a significant loss of antioxidant defense of cells, e.g. NEAC upon NaOCN, H2O2 and the combination of cyanate and hydrogen peroxide was observed. Carbamylation slightly decreased GSH and the free thiol level, but H2O2 and its combination with NaOCN lead to a decrease in their amounts. A markedly higher level of hydroperoxides was only observed in the cells treated with H2O2. We found a significant decrease in lipid membrane fluidity at the depth of 12th and 16th carbon atoms of fatty acids in lymphocytes treated with cyanate or H2O2. The combination of both substances acted synergistically and induced profound changes in comparison to cyanate and hydrogen peroxide used alone. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Carbamylation of proteins and oxidation of cellular components during oxidative stress occur in pathology of many diseases [e.g., diabetes mellitus, atherosclerosis, chronic renal failure (CRF)] (Jaisson and Gillery, 2010; Jaisson et al., 2011). Carbamylation is the posttranslational modification of proteins resulting from the nonenzymatic reaction between isocyanic acid and specific free functional groups, especially amino groups. Isocyanic acid is a very reactive form of cyanate derived from the spontaneous decomposition of urea ( Wynckler et al., 2000; Jaisson et al., 2011). In the body, an aqueous environment, urea and cyanate occur in equilibrium (99:1) (Wynckler et al., 2000). It has been demonstrated that in patients with chronic renal failure (CRF) the concentration of urea is 5–7 times higher than in healthy individuals (Malyszko et al., 2006; Selvaraj et al., 2002; Wyncler et al., 2000; Trepanier et al., 1996). Moreover, it has been demonstrated that in CRF hemodialysis (HD) patients the concentration of cyanate before HD increases up to 150 nM and in comparison to healthy volunteers is about three times greater (Nilsson et al., 1996).
⁎ Corresponding author at: Department of Thermobiology, University of Lodz, ul. Pomorska 141/143, 90-236 Lodz, Poland. E-mail address: [email protected] (A. Pieniazek).
http://dx.doi.org/10.1016/j.tiv.2016.03.017 0887-2333/© 2016 Elsevier Ltd. All rights reserved.
Another serious problem occurring in CRF patients is oxidative stress and microinflammation (Jaber et al., 2001). These problems may contribute to progression of renal disease, and can be associated with higher mortality of patients. It has been suggested that two main reasons of an increase of oxidative stress in hemodialysis patients exist. The first relates to increased production of reactive oxygen species as a result of activation of immune cells in response to a small biocompatibility of the dialysis membrane (Hörl, 2002). The second reason is the low selectivity of the dialysis membrane which may result in the removal of uremic toxins and low molecular weight antioxidants from the blood of uremic patients. It has been observed that in hemodialyzed patients, the antioxidant capacity of plasma before HD is higher than in healthy volunteers but after a dialysis session it drops significantly below the level of normal value (Pieniazek et al., 2002a, 2002b). Several studies have demonstrated synergism between oxidative stress and inflammatory response. Hydrogen peroxide as a metabolic oxidant can activate the NF-kB pathway, leading to synthesis of proinflammatory cytokines and resulting in amplification of the inflammatory cascade (Libetta et al., 2011). A small number of works associated with the combined action of carbamylation and oxidative stress available in the literature prompted us to study the effect of both factors on the properties of MNCs. The aim of this study was to evaluate the in vitro influence of carbamylation and the combination of carbamylation and oxidative stress on isolated human peripheral blood mononuclear cells. We determined the effect
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of the combination of carbamylation and oxidation on oxidative stress parameters such as nonenzymatic antioxidant capacity, total thiols, reduced glutathione and peroxide level. Additionally, we examined also the plasma membrane after incubation of cells with cyanate, hydrogen peroxide and their combination by measuring lipid membrane fluidity using EPR spectroscopy. 2. Material and methods
2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5carboxyanilide inner salt (XTT) was used to quantify the metabolically active living cells. The mitochondrial dehydrogenases of viable cells reduce the tetrazolium ring of XTT, yielding an orange formazan derivative. The obtained values of absorbance of the reaction product were presented as a percentage, taking the value of absorbance of the control as 100%.
2.1. Chemicals
2.4. Nonenzymatic antioxidant capacity
2,3-bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5carboxyanilide inner salt (XTT), Phenazine methosulfate (PMS), 2,4,6tripyridyl-s-triazine (TPTZ), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 5doxyl-stearic acid (5-DS), 12-doxyl-stearic acid (12-DS), 16-doxylstearic acid (16-DS), 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), ophthalaldehyde (OPA), and N-ethylmaleimide (NEM) were obtained from the Sigma-Aldrich company. The RPMI 1640 bicarbonate medium, fetal bovine serum (FBS), penicillin and streptomycin were from PAA (Germany). All other chemicals were purchased from POCH S.A. (Gliwice, Poland) if not otherwise indicated. All dishes necessary for cell culture were obtained from the NUNC company.
The nonenzymatic antioxidant capacity (NEAC) of MNCs was measured using two different methods: TPTZ and DPPH. The spectrophotometric method is based on reduction of the ferric tripyridyltriazine [Fe(III)-TPTZ] complex to ferrous [Fe(II)] at a low pH. The iron complex [Fe(III)-TPTZ] reduced by antioxidants has an intense blue color with absorption maximum at 593 nm (Benzie and Strain, 1996). The samples of lysed cells (0.05 ml) were mixed with a working reagent (300 mM acetate buffer (pH 3.6); 10 mM TPTZ in 40 mM HCl; 20 mM FeCl3 freshly prepared in a volume ratio (10:1:1)) (0.1 ml) and incubated at room temperature for 30 min. At the end of incubation the absorbance was measured. The electron paramagnetic resonance (EPR) method is based on reduction of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. In the presence of antioxidants, loss of EPR signal of 1,1-diphenyl-2picrylhydrazyl (DPPH) is observed. The loss of EPR signal is inversely proportional to the concentration of antioxidants (Pieniazek et al., 2002a, 2002b). The samples of lysed cells (0.01 ml) were then mixed with DPPH (300 μM in methanol) (0.04 ml) and incubated for 30 min at room temperature. At the end of incubation the samples were centrifuged and the intensity of EPR signal in the supernatant was measured. In both methods the antioxidant activity of MNCs was expressed as Trolox equivalents calculated on the basis of the calibration curve (in nmol/mg protein). EPR spectra were performed on the Bruker ESP 300 E spectrometer at room temperature (22 + 2 °C), operating at a microwave frequency of 9.73 GHz. The instrumental settings were as follows: center field set at 3480 G, range of 80 G, with a 100 kHz modulation frequency and modulation amplitude of 1.01.
2.2. Peripheral blood mononuclear cells (MNCs) All experiments were carried out on human normal peripheral blood mononuclear cells (MNCs) isolated from the blood obtained from the Blood Bank in Lodz, Poland (each time, from leukocyte layers yielded separately from blood of different healthy volunteers). Therefore, every single experiment was performed on cells from one donor — nnumbers represent cells from different individuals. Cell isolation was performed by centrifugation of blood in the density gradient of Histopaque 1077 (300 g for 30 min at 22 °C). The cells were washed two times with phosphate buffered saline (PBS). The MNCs were grown in the RPMI medium supplemented with 10% inactivated fetal bovine serum (FBS), penicillin (100 U/ml), and streptomycin (100 μg/ml) in a 37 °C humidified 5% CO2 incubator. Isolated cells were plated on a sterile dish (2 × 106 cells/ml) (5 ml each sample) then sodium cyanate (NaOCN) to the final concentration of 1 mM or 2 mM was added and incubated in a 37 °C humidified 5% CO2 incubator for 23 h. At the end of this period, hydrogen peroxide was added to a final concentration of 100 μM and left for 1 h of incubation. The collected cells were washed with phosphate buffered saline (pH 7.4) (PBS) and used for membrane lipid fluidity determination. For measurements of reduced glutathione and thiol group concentration as well as antioxidant activity, the cells were lysed in a buffer containing 10 mM Tris, 1 mM EDTA, 0.1 M NaCl and 0.01% Triton X-100 (about 1 × 107 cells in 0.1 ml buffer). 2.3. Cell survival The survival of blood mononuclear cells after treatment with sodium cyanate was estimated by the standard microplate XTT colorimetric method. Isolated blood mononuclear cells (15 × 104 cells/well) were seeded on 96-well microplates in the growth medium with a wide range of sodium cyanate concentrations (0–10,000 μM) for 24 h and incubated at 37 °C in a humidified 5% CO2 incubator. At the end of the exposure time the medium with NaOCN was replaced with a fresh one and the cells were grown for additional 24 h. At this time point the XTT test was performed. Freshly prepared XTT (0.05 ml, to the final concentration of 1 mg/ml) with PMS (to the final concentration of 0.017 mg/ml) was added to each well of the microplates. Subsequently, the microplates were incubated for 4 h. Absorbance was measured at 495 nm using a microplate reader (BioTek).
2.5. Free thiol group concentration The quantification of free thiol groups was based on reaction with Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid); DTNB). Upon reaction with free thiol groups 2-nitro-5-thiobenzoate (NTB), optically active at 412 nm, is released (Ellman, 1959). The samples of lysed cells (0.015 ml) were then mixed with 10 mM phosphate buffer (pH 8.0) containing 0.5% SDS (0.235 ml) and the absorbance at 412 nm was measured (A0). Then the DTNB reagent was added to the samples (final concentration of 0.1 mM) (0.025 ml) and they were incubated for 1 h at 37 °C. After this time the absorbance of the samples was again measured A1. ΔA = A1 − A0 for calculation of the thiol group concentration was used. The concentration of thiol groups was calculated based on the calibration curve for different concentrations of reduced glutathione and expressed as nmol/mg protein. 2.6. Glutathione The reduced glutathione (GSH) concentration was determined using the fluorimetric method with o-phthalaldehyde (OPA) (Senft et al., 2000). The reaction product of OPA and GSH has high fluorescence quantum yields. The mononuclear cell lysate (0.01 ml) was mixed with redox quenching buffer with trichloroacetic acid (RQB-TCA (20 mM HCl, 5 mM DTPA, 10 mM ascorbic acid); 5% TCA)
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(0.005 ml),7.5 mM N-ethylmaleimide (NEM) in RQB (0.002 ml), and 1.0 M potassium phosphate buffer (KP) (pH 7.0) (0.025 ml) and incubated at room temperature for 5 min. At the end of incubation the 0.1 M KP buffer (pH 6.9) (0.1 ml) and OPA (5 mg/mL in methanol) (0.015 ml) were added and incubated in the dark at room temperature for 30 min. The OPA-derived fluorescence was measured at 365 nm excitation and 430 nm emission. The glutathione concentration was calculated using the calibration curve for different concentrations of reduced glutathione, as a standard, and expressed as nmol/mg protein.
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with one-way ANOVA for repeated measures and the post-hoc multiple comparisons Tukey's test. In addition, due to relatively small sample sizes and low statistical power of numerous estimated inferences, we employed the resampling bootstrap technique (10,000 iterations) to reason on how likely the revealed differences could be observed by a pure chance. The power of the used test was checked for each analysis and always was more than 80%. Statistical analysis was performed using Statistica v. 12.5 and Resampling Stats Add-in for Excel v.4.
2.7. Hydroperoxide 3. Results For determination of MNC hydroperoxides, the spectrophotometric method with xylenol orange was employed. The reaction was based on rapid oxidation of Fe(II) to Fe(III) in the presence of peroxides (Gay et al., 1999; Gay and Gebicki, 2000). 25 mM ammonium iron(II) sulfate in 2.5 M H2SO4 and 125 μM xylenol orange (1:100) were used to prepare a working solution. The samples of lysed cells (0.01 ml) were then mixed with a working reagent (0.1 ml) and incubated for 30 min in the dark. The reaction of Fe(III) with xylenol orange yielded a violet-colored complex, which was quantified spectrophotometrically at 560 nm. The concentration of peroxides was expressed as μmol/mg protein. 2.8. Protein concentration The protein concentration in MNC lysates was determined using the method of Lowry et al. (1951). 2.9. MNC lipid membrane fluidity The lipid membrane fluidity of MNCs was estimated using three different spin labels (5-DS, 12-DS and 16-DS) in the EPR technique. The paramagnetic group of doxyl stearic acid is located on different carbon atoms in the hydrocarbon chain, and provides information on phospholipid fatty acyl mobility at different depths of lipid bilayer. The spin label, in ethanol solution was added into cell suspension (5 × 107 cells/ml) to the final concentration of 0.1 μM and incubated for 30 min at room temperature. The final ethanol concentration in cell suspension was less than 0.05% (v/v). Data obtained from the spectra was used to calculate the rotational correlation time (τc), according to formula (1) (Schreier et al., 1978):
τc ¼
1 kw0 2
sffiffiffiffiffiffiffiffi sffiffiffiffiffiffiffiffiffi ! h0 h0 −2 þ hþ1 h−1
ð1Þ
where: time when the spin label undergoes full rotation τc k constant equal to 1.19 × 10−9 s width of the mid-line of spectrum w0 height of the mid-line of spectrum h0 height of the high-field line of spectrum. h−1 EPR spectra were performed on the Bruker ESP 300 E under the same conditions as described above. 2.10. Statistical analysis All data were expressed as mean ± standard deviation. The normality of data was tested using the Shapiro–Wilk test and variance homogeneity was verified with the Brown Fosythe test. Mauchly's test was used to test for data sphericity. Sets of data with confirmed normal distributions and homoscedasticity/sphericity were tested with the use of parametric tests, while non-parametric tests were used for those without. The significance of differences between the groups was estimated
Isolated blood mononuclear cells were exposed to a wide range of sodium cyanate concentrations (0–10,000 μM) for 24 h. The cytotoxicity of sodium cyanate on MNCs was compared to untreated (control) cells, which survival was arbitrarily taken as 100%. Obtained results demonstrated that sodium cyanate did not change MNC survival at concentrations below 2 mM in a logarithmic scale on the abscissa axis (Fig. 1). A significant decrease in survival was observed for mononuclear blood cells after their exposure to cyanate at concentrations higher than 2 mM. The value of IC50 for mononuclear blood cells after treatment with sodium cyanate was about 9 mM. For further studies, two high sodium cyanate concentrations (1 mM and 2 mM), which did not affect the cell viability were selected, as seen in Fig. 1. For determination of nonenzymatic antioxidant capacity the MNCs were treated with sodium cyanate and hydrogen peroxide as well as with the combination of both substances using two independent methods. The changes in NEAC in cells measured with DPPH showed a significant decrease of this parameter in all selected groups in comparison to control (Fig. 2). The highest decrease in antioxidant capacity in the cells was noted after treatment with hydrogen peroxide as well as with the combination of cyanate at two concentrations with hydrogen peroxide. We found also a significant difference in antioxidant capacity after treatment of cells with the combination of NaOCN and H2O2 in comparison to cells treated with cyanate alone. Results similar to DPPH in antioxidant capacity were observed in the experiment using the TPTZ method. A significant decrease in antioxidant capacity in MNCs after treatment with hydrogen peroxide and the combination of NaOCN and H2O2 at two concentrations in comparison to control was found (Fig. 3). However, there were no differences between cells treated with cyanate alone in comparison to combinations of cyanate and hydrogen peroxide. As seen in Fig. 4, in the MNCs treated with hydrogen peroxide and the combination of NaOCN and H2O2, the level of free thiol groups significantly decreased in comparison to control. The sodium cyanate added to the cells, at two different concentrations led to a slight, not significant, decrease of free thiol concentration. However, the combination of NaOCN and H2O2 resulted in a significant loss of free thiol groups in comparison to cells treated with cyanate only. Simultaneously, we observed a significantly decreased concentration of reduced glutathione in cells after treatment with hydrogen peroxide and the combination of NaOCN and H2O2 (Fig. 5). On the other hand, in cells treated with sodium cyanate the concentration of GSH did not significantly decrease. Nevertheless, a significant decrease of reduced glutathione concentration in MNCs after treatment with the combination of sodium cyanate and hydrogen peroxide in comparison to cells treated with sodium cyanate alone was noted. The level of hydroperoxides in cells treated with sodium cyanate, hydrogen peroxide and their combinations with two concentrations of cyanate was determined. A statistically significant increase of hydroperoxides in cells treated with hydrogen peroxide in comparison to control cells was observed (Fig. 6). On the other hand, we did not find any differences in MNCs treated with both concentrations of sodium cyanate in comparison to control. However, in cells treated with the
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Fig 1. Survival of human mononuclear blood cells after 24 h of treatment with sodium cyanate (concentration range: 0–10,000 μM). Each circle represents cells from 8 different individuals (n = 8).
combination of NaOCN and H2O2 a slight, not significant, increase of hydroperoxides versus the control level was found. We also examined the plasma membrane fluidity of MNCs upon treatment with cyanate, hydrogen peroxide and combinations of two concentrations of NaOCN with H2O2 using the spin labeling method. For the determination of spin labeled fatty acid mobility after incorporation into the lipid milieu the relative rotational correlation time was calculated. In the polar region of the external lipid layer of MNCs, treated with sodium cyanate, hydrogen peroxide and their combination, there
Fig. 2. Nonenzymatic antioxidant activity of MNCs treated with sodium cyanate, hydrogen peroxide and their combinations using two concentrations of NAOCN with H2O2 measured by reduction of DPPH radical in EPR technique. Each bar represents cells from 6 different individuals (n = 6). Statistical significances: control vs. H2O2, P b 0.0002; control vs. cyanate (1 mM), P b 0.01; control vs. cyanate (2 mM), P b 0.0005; control vs. cyanate (1 mM) + H2O2, P b 0.0002; control vs. cyanate (2 mM) + H2O2, P b 0.0002; cyanate (1 mM) vs. cyanate (1 mM) + H2O2, P b 0.02; cyanate (2 mM) vs. cyanate (2 mM) + H2O2, P b 0.005.
were no significant differences in the rotational correlation time of the 5-DS spin label (data not shown). In the deeper region of the lipid bilayer, at the depth of the 12th carbon atom of fatty acid the changes of lipid membrane fluidity in MNCs after treatment with sodium cyanate, hydrogen peroxide, and their combinations were observed (Fig. 7). A statistically significant increase of the rotational correlation time of the 12-DS spin label in cells treated with hydrogen peroxide and two concentrations of cyanate in comparison to control cells was noted. A large increase in the rotational correlation time was found in cells exposed to a combination of sodium cyanate and hydrogen peroxide compared to control and compared to cells treated with sodium cyanate alone (Fig. 7).
Fig. 3. Antioxidant activity of MNCs treated with sodium cyanate, hydrogen peroxide and their combinations with two concentrations of NAOCN with H2O2 measured by the spectrophotometric method with TPTZ. Each bar represents cells from 6 different individuals (n = 6). Statistical significances: control vs. H2O2, P b 0.0002; control vs. cyanate (1 mM) + H2O2, P b 0.001; control vs. cyanate (2 mM) + H2O2, P b 0.001.
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Fig. 4. The level of free thiol groups in MNCs treated with sodium cyanate, hydrogen peroxide and their combinations with two concentrations of cyanate. Each bar represents cells from 6 different individuals (n = 6). Statistical significances: control vs. H2O2, P b 0.0005; control vs. cyanate (1 mM) + H2O2, P b 0.0002; control vs. cyanate (2 mM) + H2O2, P b 0.0002; cyanate (1 mM) vs. cyanate (1 mM) + H2O2, P b 0.01; cyanate (2 mM) vs. cyanate (2 mM) + H2O2, P b 0.02.
Results of spin label mobility similar to 12-DS were also obtained for the 16-DS spin label incorporated into the mononuclear blood cell membrane. There was a statistically significant increase in the rotational correlation time of 16-DS after incubation of cells with the higher
Fig. 5. The level of reduced glutathione in MNCs treated with sodium cyanate, hydrogen peroxide and their combination. Each bar represents cells from 6 different individuals (n = 6). Statistical significances: control vs. H2O2, P b 0.0005; control vs. cyanate (1 mM) + H2O2, P b 0.0002; control vs. cyanate (2 mM) + H2O2, P b 0.0002; cyanate (1 mM) vs. cyanate (1 mM) + H2O2, P b 0.002; cyanate (2 mM) vs. cyanate (2 mM) + H2O2, P b 0.0005.
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Fig. 6. The level of peroxides in MNCs treated with sodium cyanate, hydrogen peroxide and combinations of H2O2 with two concentrations of cyanate (n = 6). Each bar represents cells from 6 different individuals (n = 6). Statistical significance: control vs. H2O2, P b 0.05.
concentration of cyanate and with the combinations of the two concentrations of cyanate with hydrogen peroxide in comparison to control cells (Fig. 8). A significant increase of this parameter after treatment of
Fig. 7. Relative rotational correlation time of 12-DS spin label incorporated into the MNCs treated with sodium cyanate, hydrogen peroxide and their combinations. Each bar represents cells from 15 different individuals (n = 15). Statistical significances: control vs. H2O2, P b 0.01; control vs. cyanate (1 mM), P b 0.02; control vs. cyanate (1 mM) + H2O2, P b 0.00001; control vs. cyanate (2 mM) + H2O2, P b 0.00001; cyanate (1 mM) vs. cyanate (1 mM) + H2O2, P b 0.05; cyanate (2 mM) vs. cyanate (2 mM) + H2O2, P b 0.00002.
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Fig. 8. Relative rotational correlation time of 16-DS spin label incorporated into the MNCs treated with sodium cyanate, hydrogen peroxide and their combinations. Each bar represents cells from 15 different individuals (n = 15). Statistical significances: control vs. cyanate (2 mM), P b 0.01; control vs. cyanate (1 mM) + H2O2, P b 0.0002; control vs. cyanate (2 mM) + H2O2, P b 0.0002; cyanate (1 mM) vs. cyanate (1 mM) + H2O2, P b 0.05.
cells with the combination of NaOCN and H2O2 in comparison to the cells treated with cyanate only was also observed. 4. Discussion Carbamylation of proteins and amino acids leads to changes in their structure and function (Trepanier et al., 1996; Wynckel et al., 2000; Jaisson and Gillery, 2010; Jaisson et al., 2011). Similarly, oxidative stress also changes their structure and function during the oxidation process (Pieniazek et al., 2002a, 2002b; Fisher-Wellman and Bloomer, 2009). Both of these processes are observed in many diseases for example arteriosclerosis and chronic renal failure (Himmelfarb et al., 2000; Pieniazek et al., 2009; Jaisson et al., 2011). It seems that a study of carbamylation and oxidative stress induced in vitro may be useful in explaining changes in peripheral blood mononuclear cells of patients undergoing chronic hemodialysis. In the beginning of our study we evaluated the cytotoxicity of sodium cyanate on mononuclear blood cells. We found that this compound did not have influence on cell survival up to the concentration of 2 mM. Considering that the concentration in physiologically healthy subjects is 50 nM an elevated level was observed in patients with chronic renal failure before hemodialysis, approx. 150 nM (Nilsson et al., 1996). In our experiments the concentration of sodium cyanate (2 mM) was approx. 50,000 times higher than the physiological concentration. Based on our studies it can be concluded that the in vivo cyanate has no impact on the survival of MNCs. However, higher concentrations of NaOCN lead to a decrease in cell survival. Recently, it has been shown that application of 1 mM cyanate did not influence on endothelial cell (HCAEC) viability (El-Gamal et al., 2014). This result is in line with our finding. The physiological concentration of cyanate was too low to evoke any effect. Generally, we were interested on what is the combined effect of carbamylation and oxidative stress on MNCs. It is noteworthy that in the blood of CRF patients about 100 other toxic substances are present and their range of concentrations is 2–200 higher than in healthy subjects (Vanholder et al., 2003). Moreover, interactions between them are also possible.
Additionally, in our previous work, using the EPR method, we showed that in the 20th minute of hemodialysis the respiratory burst releases hydroxyl and superoxide radicals (Gwozdzinski and Janicka, 1995; Gwozdzinski et al., 1997). Both of them can oxidize biological material and induce damage including whole cells. Additionally during recombination/dismutation of both radicals hydrogen peroxide is produced. It has been well documented that oxidative stress induced by hydrogen peroxide can disrupt normal functions of cells by oxidation of proteins, lipids and nucleic acids (Pieniazek et al., 2002a). Additionally, it has been shown that hemodialyzed patients have elevated genomic damage and oxidized DNA in peripheral blood lymphocytes (Stopper et al., 2001; Tarng et al., 2004; Moffitt et al., 2014). Oxidative genetic damage can be a consequence of an increase of ROS formation as well as a decrease of antioxidant defense. In our studies we observed a loss of antioxidant defense of cells e.g. a decrease of nonenzymatic antioxidant capacity and a decrease of reduced glutathione and the free thiol group level. Additionally, we found a decrease in lipid membrane fluidity at the depth of the 12th carbon atom of fatty acids in lymphocytes treated with hydrogen peroxide. Both carbamylation and oxidation lead to a decrease of nonenzymatic antioxidant capacity of cells indicated by TPTZ and DPPH methods. The antioxidant capacity of the cells determined using both methods allows the assessment of the presence of low molecular weight antioxidants, including reduced glutathione (Benzie and Strain, 1996; Kedare and Singh, 2011). It can be assumed that the decline in antioxidant activity may be an effect, among others, of the loss of part of reduced glutathione, while thiol groups could be oxidized or carbamylated. It has been reported that thiol and phenol groups exhibit the highest reactivity with isocyanic acid (Stark, 1964; Jaisson et al., 2011; Praschberger et al., 2013). It is interesting that the use of the combination of cyanate and hydrogen peroxide, resulted in a greater loss of antioxidant activity of the cells than after application of the cyanate alone, but these changes were not as large as in the case of hydrogen peroxide alone. These results showed that carbamylated low molecular weight antioxidants are more resistant to oxidation than those unmodified. In patients with CRF undergoing hemodialysis it has been observed that the antioxidant activity of plasma was much higher than in normal subjects (Pieniazek et al., 2002b; Clermont et al., 2000). Nevertheless, in the plasma of patients with CRF a large amount of uric acid responsible for the increase in the antioxidant activity of plasma is accumulated. Analysis of total thiols and glutathione revealed a slight decrease in their amounts after carbamylation. The reaction of isocyanic acid with thiol groups is reversible, but nevertheless reproduced isocyanic acid may again cause carbamylation of proteins and amino acids (Stark, 1964). It has been reported that in the plasma of CRF patients the thiol level was lower in comparison to healthy subjects (Clermont et al., 2000). Application of hydrogen peroxide as an oxidant leads to the loss of antioxidant defense of cells e.g. a decrease of nonenzymatic antioxidant capacity and a decrease in the free thiol level and of reduced glutathione. The changes in these parameters were higher than after the carbamylation process. A similar effect was obtained when the combination of carbamylation and oxidative stress was applied. Oxidation of both proteins and lipids can lead, inter alia, to the formation of their peroxides. We observed a significant increase in this parameter in cells treated with hydrogen peroxide. Interestingly, the cells exposed to the process of carbamylation were slightly less sensitive to oxidation. In this study we showed that oxidation has a greater potential for damaging biological material than carbamylation. We also examined the effect of carbamylation, oxidation and both processes combined on the properties of the plasma membrane of MNCs. Three fatty acid spin labels were applied to determine lipid plasma fluidity. We did not find any changes in the polar region of the lipid layer after carbamylation, oxidation and the combination of both processes. However, the changes in lipid fluidity were monitored with spin labels located in the deeper region of the cell membrane indicated
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by 12-DS and 16-DS. Interestingly, carbamylation significantly declined membrane fluidity. It is possible that changes in membrane lipid fluidity were caused by carbamylation of amino groups of phospholipids (phosphatidylserine and phosphatidylethanolamine) and proteins as well as interaction of modified lipids and proteins. Changes in membrane fluidity have also influence on: lipid phase transition (the gel–liquid crystalline transition), changes in lipid–protein interactions, transport across the membrane, alterations in cell deformability, disturbances in intracellular signaling, changes in cell interactions, etc. Most research suggests that carbamylation by isocyanic acid refers to proteins and free amino acids. However, the possibility that carbamylation may extend beyond the level of protein to other nonprotein primary amino group-containing compounds has been suggested (Trepanier and Thibert, 1996). One of such potential targets is the plasma membrane aminophospholipids such as phosphatidylserine (PS) and phosphatidylethanolamine (PE). Both of these phospholipids contain a primary amino group in their polar region, which is potentially accessible to bind with exogenous amino-specific chemical compounds. Externalization of phosphatidylserine (PS) from the internal monolayer of lipids in the apoptosis pathway is also possible. Development of apoptosis in mononuclear cells in hemodialyzed patients has been reported (Jaber et al., 2001). We found also that oxidation and the combination of carbamylation and oxidation lead to a significant decrease in membrane fluidity. We showed that oxidation of MNCs leads to peroxide formation, but carbamylation attenuated this process. Unexpectedly, we observed a synergistic effect of hydrogen peroxide and cyanate which leads to a major change in fluidity unlike each of them separately. The changes in this parameter can be a result of lipid oxidation (fragmentation or/ and aggregation), lipid crosslinking, and changes in lipid–protein interactions. Carbamylation and oxidation have an impact on the function and structure of proteins and lipids. Oxidation of both proteins and lipids can lead, inter alia, to the formation of their peroxides. Interaction of isocyanic acid with proteins and amino phospholipids may form carbonyl groups on them. In our work, we observed a significant increase in the level of peroxides in lymphocytes incubated with hydrogen peroxide. Slight increases in peroxides were also observed in samples of cells treated with a combination of sodium cyanate and hydrogen peroxide. Because the cells were first treated with sodium cyanate and hydrogen peroxide, the slight increase of peroxides in these samples may be due to a previous interaction of sodium cyanate with proteins and phospholipids and blocking the possibility of oxidation and thus the formation of peroxides. 5. Conclusion Although mechanisms of action of cyanate and hydrogen peroxide are different, sometimes we observed a similar action of both reagents. Generally, carbamylation of proteins indicates a weaker effect on investigated parameters than oxidative stress. However, when used together, carbamylation “protected” against oxidative stress. On the other hand, in the case of membrane fluidity the synergistic effect of both substances was observed. These studies showed that carbamylation has rather small participation in MNC damage in hemodialyzed patients. It seems that oxidative stress dominated in cell injury in CRF patients. Transparency document The Transparency document associated with this article can be found, in the online version. Acknowledgment The authors thank Dr. Jan Czepas for language corrections and Prof. Cezary Watala for help in statistical analysis.
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