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Induction of transient radioresistance in human erythrocytes
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Radiation Physics and Chemistry 75 (2006) 967–976 www.elsevier.com/locate/radphyschem
Induction of transient radioresistance in human erythrocytes Anita Krokosz, Zofia Szweda-Lewandowska Department of Molecular Biophysics, University of Lodz, 90-237 Lodz, Poland Received 28 December 2005; accepted 10 February 2006
Abstract Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit of 2%), were irradiated with g-rays with single and split doses under air or N2O in order to determine the physicochemical changes caused by the dose inducing an increase in resistance to radiation-induced hemolysis. The obtained results showed that under the applied irradiation conditions, the dose of 0.4 kGy induced changes in erythrocytes, which were responsible for temporary resistance of erythrocytes to hemolysis. We concluded that the observed resistance is caused mainly by the structural changes in proteins. r 2006 Elsevier Ltd. All rights reserved. Keywords: Gamma radiation; Radioresistance; Human erythrocytes
1. Introduction Numerous studies have shown that small doses of ionizing radiation of low linear energy transfer (LET) induce various stimulating effects on living systems uch as radio-adaptive response, an increase in reproductive ability, stimulatory effects on immune systems and an extended life span (Luckey, 1991). It is commonly known that many cells exposed to small doses of ionizing radiation show reduced cytogenetic damage as well as higher survival rate after a subsequent higher dose given a short time later. These doses are called ‘‘priming’’ or ‘‘conditioning’’ and ‘‘challenging’’ doses. Induced radioresistance or adaptive response to the first dose has been proposed as an explanation of this effect (Joiner, 1994a, b). The adaptive response was reported in various types of cellular systems including human lymphocytes, Chinese hamster cells, human fibroblasts, bacteria, protozoa, algae, lepidopCorresponding author. Tel.: +48 42 6354480; fax: +48 42 6354473. E-mail address: [email protected] (A. Krokosz).
tera insects and higher plants (Joiner, 1994a, b and references cited therein; UNSCEAR, 1994). The experiments on tissue cultures of human lymphocytes (Wolff et al., 1988), Chinese hamster V79 cells (Ikushima, 1989) and human 41-Mel cells (Meyers et al., 1992; Boothman et al., 1993) have shown that new proteins are induced after exposure to low doses of radiation, presumably by gene activation. Especially poly (ADP-ribose) polymerase, an enzyme which is activated by radiation-induced DNA breaks, plays an important role in DNA repair and has been implicated in the adaptive response in human lymphocytes (Wiencke et al., 1986). The experiments on protein induction with low doses showed the appearance of protein characteristic of X-irradiation (not induced by heat shock, hypoxia or alkylating agents). It should be stressed that previous studies of radioresistance mechanisms were related mainly to the damage and repair of DNA in dividing cells without taking into consideration other cellular structures, particularly membranes. The evidence is that membrane-associated damage may be important in radiation-induced interphase cell death (Wolter and Konings, 1985).
0969-806X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2006.02.004
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Mature human erythrocytes are highly specialized cells, which have no nucleus or any other cellular organelles. This is why they are a suitable model for studying the influence of various factors on the plasma membrane, which stays in its natural environment without isolation. For the first time, Lee and Ducoff (1994) have shown that fractionation of a radiation dose causes a decrease in hemolysis of nucleated avian erythrocytes. In our previous papers (Zaborowski and SzwedaLewandowska, 1997; Koziczak et al., 1999, 2003), we showed that the enucleated human erythrocytes irradiated under air with split doses, with a time interval between subsequent expositions, hemolyzed slower in comparison with cells irradiated with the same single doses. Simultaneously, we observed a lower degree of lipid peroxidation, hemoglobin (Hb) oxidation and smaller changes in the fluidity of lipid bilayer. The effect of the decrease in hemolysis of enucleated human erythrocytes was closely dependent on the dividing conditions. The critical factors limiting this effect were the magnitude of the first radiation dose and the interval between fractions. To continue the above experiments, in this paper we focused on the effects of split doses on human erythrocytes irradiated under air or N2O. The aim of this work was to determine the physicochemical changes taking place in the human erythrocytes caused by the dose inducing an increase in resistance to radiationinduced hemolysis.
2. Materials and methods 2.1. Chemicals a-Cellulose, 5-, 12-, 16-doxylstearic acids, 2,2,6,6tetramethyl-4 maleimidopiperidine-N-oxyl (MSL), ATP, ouabain, NADPH, EGTA, glutathione reductase, t-butyl hydroperoxide, thiobarbituric acid, and phenylmethylsulfonyl fluoride were purchased from Sigma Chemical Co., Poole Dorset, UK. Other chemicals were obtained from POCh (Gliwice, Poland) (all of analytical grade). All solutions were made with water purified by the Milli-Q system. 2.2. Preparation of erythrocyte suspension Blood samples from healthy adult donors were provided by the Central Blood Bank in Lodz. Erythrocytes were separated from blood plasma and leucocytes by centrifugation. In order to remove residual leucocytes, erythrocytes were passed through an a-microcrystalline cellulose column, washed with 0.1 M Na-phosphate buffer (pH 7.4) and resuspended in the same buffer to obtain a hematocrit of 2%.
2.3. Irradiation conditions Erythrocyte suspensions in a Na-phosphate buffer were irradiated with a 60-Co g-rays at room temperature in the atmosphere of air or N2O. The dose rate estimated with modified Fricke dosimeter was 4 kGy h1. During irradiation, the erythrocyte suspensions were stirred with a magnetic bar. In the experiments with a split dose, the first dose was equal to 0.4 kGy and the second dose was equal to 2.3 kGy when preparations were irradiated under air and 5.6 kGy when preparations were irradiated under N2O. The total dose received by cells was 2.7 and 6 kGy, respectively. The time interval between subsequent doses was 3.5 h. Irradiation of erythrocytes under N2O was performed in a glass tonometer, preflushed with N2O for 1–1.5 h, with gentle mixing. Irradiation was performed in the Institute of Applied Radiation Chemistry, the Technical University of Lodz. 2.4. Measurement of hemolysis Hemolysis of erythrocytes was determined on the basis of the ratio of Hb released from cells to the total cellular Hb content. After irradiation, erythrocyte suspensions were centrifuged, the supernatant was carefully collected and the absorbance at 630 nm was measured after oxidation of iron with K3[Fe(CN)6]. The residual erythrocytes were hemolyzed with distilled water and the absorbance of the solution was measured as described above. The percentage of hemolysis was calculated as described in the earlier paper (Koziczak et al., 1999). Post-radiation kinetics of hemolysis was determined for erythrocytes irradiated with the dose of 0.4 kGy after indicated time intervals. After irradiation, 0.5% of penicillin and 0.1% of streptomycin were added to stop bacterial growth. 2.5. Determination of osmotic resistance Osmotic fragility curves were drawn on the basis of the percentage of erythrocyte hemolysis in solutions of various NaCl concentrations buffered with 5 mM Naphosphate (pH 7.4). Suspensions of erythrocytes were diluted to a hematocrit of 0.2%, and the degree of hemolysis was estimated after 10 min.The samples were centrifuged at 10,000 rpm for 3 min.The absorbance of the supernatants was assessed at 570 nm.The degree of hemolysis was calculated on the assumption that the absorbance of a sample hemolyzed with distilled water equaled 100%. 2.6. Determination of lipid peroxidation Lipid peroxidation was quantified spectrophotometrically at 532 nm by measuring the formation of
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thiobarbituric acid reactive substances (TBARS) (Sandhu et al., 1992). The main terminal product of lipid peroxidation reacting with TBA is malondialdehyde (MDA). However, besides MDA other aldehydes formed during the decomposition of lipid peroxides might give colored complex with TBA (Hageman et al., 1992). Lipid peroxidation was expressed in absorbance units. 2.7. Measurement of methemoglobin (MetHb) in erythrocytes The percentage of MetHb content was measured in the residual erythrocytes after their hemolysis. The absorbance of the solutions was measured at 630 nm before the oxidation of iron and after its total oxidation with K3[Fe(CN)6]. The content of Fe(III) was calculated according to Puchala et al. (2004). 2.8. Measurement of glutathione peroxidase (GPx) (EC.1.11.1.9) activity GPx activity was determined by following NADPH oxidation using t-butyl-hydroperoxide as a substrate. The decrease in absorbance was measured at 340 nm for 3 min. Activity was expressed as IU/g Hb indicating such an amount of enzyme, which was equivalent to the oxidation of 0.5 mmol NADPH in 1 min at 37 1C. The molar absorption coefficient of NADPH at 340 nm is 6220 mol1 dm3 cm1 (Rice-Evans et al., 1991). 2.9. Determination of catalase (EC.1.11.1.6) activity in erythrocytes Catalase activity was determined according to Aebi (1984). The method is based on spectrophotometrically monitoring the rate of decomposition of H2O2 at 240 nm at room temperature. One IU represents the amount of enzyme necessary to breakdown 1 mmol H2O2 in 1 min.The molar absorption coefficient of H2O2 at 240 nm is 43.6 mol1 dm3 cm1. The activity was expressed in IU/mg Hb. 2.10. Determination of Na,K-ATPase activity (EC 3.1 6.37) Na,K-ATPase activity was determined by measuring the difference in the level of liberation of inorganic phosphate (Pi) from ATP in the absence and in the presence of 0.1 mM ouabain during a 30-min incubation of the membrane preparations at 37 1C. The amount of released Pi was determined spectrophotometrically at 610 nm as a complex with heptamolybdate and malachite green (Baykov et al., 1988). Pi concentration was taken from a calibration curve made for 2–40 mM of KH2PO4 as a standard. Na,K-ATPase activity was
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calculated by subtracting the activity assessed in the presence of ouabain from the activity in the absence of ouabain (total ATPase activity). The enzyme activity 1 was expressed in nmol Pi mg1 prot h . 2.11. Preparation of erythrocyte membranes Erythrocyte membranes were prepared according to the method of Dodge et al. (1963) with some modifications. Hemolysis was carried out at 4 1C with 20 volumes of 20 mM Na-phosphate buffer (pH 7.4), containing 1 mM ethylenediaminetetraacetate (EDTA) and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) as protease inhibitors. The membrane ghosts were washed successively with 20, 10 and 5 mM ice-cold Na-phosphate buffer (pH 7.4) until the ghosts were free of residual hemoglobin. Protein concentration in the membrane preparations was determined by the method of Lowry et al. (1951). 2.12. Spin labeling of erythrocytes and erythrocyte membranes The electron spin resonance spectra of erythrocytes labeled with 5-doxylstearic acid (5-DSA), 12-doxylstearic acid (12-DSA) and 16-doxylstearic acid (16-DSA) were used to monitor the fluidity of membrane lipids (Koziczak et al., 2003). From the ESR spectra of 5-DSA, an order parameter (S) was derived by measuring the outer and inner hyperfine splitting, 2TII and 2T?, using the following formula: S¼
T II ðT ? þ CÞ 1:723, T II þ 2ðT ? þ CÞ
where C ¼ 1.40.0053(TIIT?). The order parameter is a degree of the distribution of molecular orientations with respect to a reference axis, which is the normal to the membrane surface. An increase in the order parameter reflects a decrease in segmental flexibility of the spin label (Schreier et al., 1978) For the 12-DSA spectra, the ratio of the low-field peak height to the midfield peak height, A/h0, was calculated, characterizing a mobility of the spin label in the environment of double bonds of unsaturated fatty acids. From the 16-DSA spectra, the difference between the rotational correlation time, tC, and tB was calculated using the formula Dt ¼ tC tB , where hqffiffiffiffiffiffi qffiffiffiffiffiffi i h0 h0 tC ¼ 12 3:25 1010 DW hþ1 þ h1 2 , tB ¼ 12ð3:25 1010 ÞDW
hqffiffiffiffiffiffi h0 hþ1
qffiffiffiffiffiffii h0 h1
,
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and DW the midfield peak width; h0 the midfield peak height; h+1 the low-field peak height; h1 the high-field peak height. Rotational correlation times reflecting the spin label motion in the directions perpendicular and parallel to the long axis of a lipid molecule. Changes in protein conformation were estimated according to Barber et al. (1983) by spin labeling of the erythrocyte membranes with 1 mg of MSL per 25 mg of protein and incubation at 4 1C for 30 min.Then the excess of the label was removed by washing it with PBS several times. The spectra of MSL attached to the membrane proteins were analyzed by measuring the ratio of weakly (hW) and strongly (hS) immobilized components in the low-field peak (hW/hS). All ESR spectra were measured at ambient temperature using a Bruker 300 E ESR spectrometer. 2.13. Determination of reduced glutathione (GSH) concentration in erythrocytes The content of GSH was determined fluorimetrically as described by Akerboom and Sies (1981), with oftalaldehyde. The fluorescence was measured at an excitation wavelength of 320 nm and an emission wavelength of 420 nm using Perkin-Elmer fluorometer. GSH concentration in the studied samples was taken from a calibration curve made for 0.2–5 mM concentration of GSH.
3. Results 3.1. The influence of a split dose on human erythrocytes irradiated in the atmosphere of air and N2O In this work, the influence of a split dose on erythrocytes was presented on the example of two chosen doses for which irradiation conditions were previously found by a trial and error method (Zaborowski and Szweda-Lewandowska, 1997; Koziczak et al., 1999). Fig. 1 shows the level of hemolysis of erythrocytes irradiated under air and N2O with a single dose and with the same dose split into two fractions with a break of 3.5 h between subsequent exposures. Hemolysis of erythrocytes was measured 18 h after the end of exposure. Apart from hemolysis, the following parameters were determined in the irradiated erythrocytes: the intensity of lipid peroxidation and the MetHb level. The intensity of lipid peroxidation was estimated on the basis of the presence of TBARS. The level of hemolysis in suspensions of erythrocytes irradiated under air with a split dose was 2-fold lower than in erythrocytes irradiated with a single dose (Fig. 1). However, the level of lipid peroxidation was
Fig. 1. Hemolysis of erythrocytes irradiated under air and N2O with a single or split dose of g-radiation. The break between two fractions of split dose was 3.5 h. Irradiation and incubation during the break took place in the atmosphere of air or N2O as mentioned. After irradiation, erythrocyte suspensions were incubated at room temperature under air and analyzed 18 h after the end of the exposure.
approximately the same for both doses and equaled 0.050470.0043 for the split dose (0.052370.0035 for the single dose). Also, the MetHb level was the same in both cases and equaled (12.070.7)%. The effect of dose splitting in erythrocytes irradiated in the atmosphere of N2O was examined at significantly higher dose than under air (Fig. 1). The division of the dose caused a decrease in hemolysis by 36.3% and in the level of lipid peroxidation by 31.2%. However, the process of dose splitting did not influence the level of hemoglobin oxidation. Thus, the level of MetHb in erythrocytes irradiated with single and split doses was approximately the same and equaled about 15%. In further studies, the changes in erythrocytes irradiated in the atmosphere of air or N2O with the dose of 0.4 kGy were determined, but we concentrated mainly on the erythrocytes irradiated under air.
3.2. Characterization of erythrocytes irradiated in the atmosphere of air or N2O with the dose of 0.4 kGy 3.2.1. The post-radiation kinetics of hemolysis of erythrocytes irradiated under air The post-radiation kinetics of hemolysis of erythrocytes irradiated under air with 0.4 and 1 kGy and the auto-hemolysis of non-irradiated erythrocytes (control) were examined. The obtained results are presented in Fig. 2. The erythrocytes irradiated with 0.4 kGy hemolyzed considerably slower than the control. The biggest difference in the hemolysis of the control erythrocytes and those irradiated with 0.4 kGy appeared in 94th hour after irradiation. The erythrocytes irradiated with the
ARTICLE IN PRESS A. Krokosz, Z. Szweda-Lewandowska / Radiation Physics and Chemistry 75 (2006) 967–976
Fig. 2. Post-radiation kinetics of hemolysis of erythrocytes irradiated under air with the dose of 0.4, 1 kGy and unirradiated erythrocytes.
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Fig. 3. Influence of 5 mM glucose on post-radiation hemolysis of erythrocytes. Glucose was added to erythrocyte suspension immediately after the end of irradiation. Hemolysis was assessed 48 hrs after the end of irradiation.
dose of 1 kGy for up to 96 h of incubation hemolyzed slightly faster than the control erythrocytes. After that time, the level of hemolysis was rapidly growing. It should be stressed that in the described experiments, erythrocytes were suspended in a phosphate buffer without glucose. However, when glucose was added to erythrocyte suspensions immediately after irradiation up to the final concentration of 5 mM, the same level of hemolysis was found in both experimental systems after 48 h of incubation at room temperature (Fig. 3). 3.2.2. Physicochemical state of a plasmatic membrane of erythrocytes irradiated with the dose of 0.4 kGy Fig. 4 shows the osmotic resistance curves of control erythrocytes and those irradiated with the dose of 0.4 kGy under air or N2O determined 2 h after irradiation. The erythrocytes irradiated under air showed the same osmotic resistance as the control erythrocytes. However, saturation of the erythrocyte suspension with N2O caused a slight decrease in their osmotic resistance, which increased insignificantly after irradiation with the dose of 0.4 kGy. In the erythrocytes irradiated with the dose of 0.4 kGy both under air or N2O, no lipid peroxidation products were found (results not shown). The spin label evaluation of the fluidity of membrane lipids indicated an increase in the fluidity of the lipid bilayer of erythrocytes irradiated under air (Table 1). The order parameter (S) for 5-DSA showed a slight but statistically significant increase in the lipid fluidity on the surface of the membrane close to the polar heads of lipids. The values of the ratio A/h0 characterizing the lipid fluidity at the depth of 12-carbon of hydrocarbon chains of lipids were higher in irradiated preparations than in the control. The highest value of the lipid fluidity parameter was observed for the dose of 0.4 kGy. The increase in the A/h0 ratio indicated lipid fluidization in
Fig. 4. Osmotic fragility test for unirradiated erythrocytes and irradiated with the dose of 0.4 kGy under air and N2O.
the membrane hydrophobic regions. The rotational correlation time for 16-DSA was also determined but remained on the level of control, indicating a lack of changes in lipid fluidity in these membrane regions. Changes occurring in membrane proteins of irradiated erythrocytes were examined using a maleimide spin label. The analysis of the maleimide spectrum, attached to the ghost membrane proteins was performed on the basis of the value of the hW/hS ratio as outlined in Methods. Values of the hW/hS ratios for erythrocytes irradiated under air as well as under N2O are presented in Table 2. A decrease in the hW/hS ratio in irradiated erythrocytes indicated conformational changes in the membrane proteins connected with the reduction of accessibility of the protein –SH groups for the label. In general, a decrease in the hW/hS ratio of MSL bound to erythrocyte membrane implies decreased segmental motion or an increased protein–protein interaction of
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Table 1 Changes in order parameter S, A/h0 parameter and correlation time t of the lipid spin label, respectively, 5-doxylstearic acid, 12doxylstearic acid and 16-doxylstearic acid in membrane of human erythrocytes irradiated with g-rays under air Dose
Order parameter S value7SD
A/h0 parameter value7SD
Correlation time t value (ns)7SD
Unirradiated (control) 0.4 kGy 0.8 kGy
0.748170.0034 0.737470.0009 0.739770.0068
0.208870.0091 0.272370.0200 0.239570.0161
0.215870.0171 0.192570.0078 0.204070.0035
Table 2 Changes of hW/hS ratio in ESR spectra of MSL attached to membrane proteins for human erythrocytes irradiated with g-rays under air and N2O Dose
Unirradiated (control) 0.4 kGy 0.8 kGy
W/S parameter value7SD Air
N2O
2.09170.011 1.65670.058 1.23370.027
2.18370.087 1.93270.040 1.72870.077
spin-labeled domains of the proteins (Palmieri and Butterfield, 1990). The decrease in the hW/hS ratio was observed at the dose of 0.4 kGy. It was higher in the erythrocytes irradiated under air than those irradiated under N2O. Fig. 5 presents the activity of Na,K-ATPase in the membranes of control erythrocytes and those irradiated under air and N2O with the dose of 0.4 kGy and under air with the dose of 1 kGy. There is an increase in Na,KATPase activity in the erythrocytes irradiated with 0.4 kGy under air in comparison with the control. Further increase in the dose up to 1 kGy caused a contrary effect i.e. a decrease in the activity. However, there were no changes observed in Na,K-ATPase activity in the erythrocytes irradiated with the dose of 0.4 kGy under N2O.
3.2.3. Changes in intracellular compounds of erythrocytes irradiated with the dose of 0.4 kGy The erythrocytes irradiated with 0.4 kGy both under air and N2O did not indicate an increase in the MetHb level above the control. Nevertheless, in the irradiated erythrocytes, a rapid decrease in the amount of reduced glutathione was observed (Fig. 6). The GSH level was decreasing considerably faster in the erythrocytes irradiated under air than N2O. Irradiation of the erythrocytes under air with the dose of 0.4 kGy led to a decrease in the GSH level by 65%, while in the erythrocytes irradiated with the same dose under N2O, the GSH level decreased by 31%.
Fig. 5. Na,K-ATPase activity in the membranes of erythrocytes irradiated under air and N2O. Each bar represents the mean of 3–5 separate experiments7SD.
Fig. 6. Glutathione level in erythrocytes irradiated under air and N2O determined 3.5 h after the end of irradiation. Each points represents the mean of 3–7 separate experiments7SD.
In the preparations irradiated with the dose of 0.4 kGy and higher doses, the activity of catalase and GPx was determined. As it is visible in Fig. 7, the irradiation of erythrocytes under air with the doses of 0.4 and 0.8 kGy did not cause any changes in the activity
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Fig. 7. Catalase (A) and glutathione peroxidase (B) activity in erythrocytes irradiated under air and N2O determined 3.5 h after the end of irradiation. Each bar represents the mean of 3–5 separate experiments7SD.
of both enzymes. On the contrary, in the erythrocytes irradiated under N2O, a statistically significant increase in the activity of catalase was found at the doses of 0.4 and 0.8 kGy. Higher doses led to a decrease in the activity of this enzyme. However, the GPx activity remained at the level of control.
4. Discussion The obtained results show that under the conditions of irradiation, the dose of 0.4 kGy induced a specific response in human erythrocytes to a high dose applied subsequently. Hemolysis was used as the end-point and on its basis erythrocyte response was assessed. The induced radioresistance was observed during erythrocyte irradiation under air and N2O. It is known that g-radiation generates active oxygen species resulting in the exposure of the irradiated systems to oxidative stress (Riley, 1986). In both experimental systems studied in this work the observed effects were initiated mainly by dOH radicals. Their radiation yield G is equal to 2.7 under air and 5.4 under N2O (Sontag, 1987). Apart from the fact that yield of dOH radicals under N2O is twice higher than under air, the effectiveness of d OH radicals in the induction of hemolysis under N2O is smaller than under air, as we presented in the previous paper (Szweda-Lewandowska et al., 2003). That is why the applied dose, which ‘‘challenged’’ hemolysis after 18 h was considerably higher under N2O than under air.
Erythrocytes contain very efficient antioxidant systems consisting of small antioxidant compounds such as glutathione (GSH), vitamin E, vitamin C and enzymes such as catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). These compounds can scavenge free radicals and also take part in the repair processes of target molecules by adding hydrogen to organic radicals (Rd) induced by dOH radicals on target biomolecules (Kollmann et al., 1969; Petkau, 1986; Chiu et al., 1989). Under anaerobic conditions, there is a possibility of repair of damaged proteins and lipids by means of chemical and enzymatic defense systems. The molecular oxygen present during exposition of erythrocytes to dOH radicals modifies the reactions of secondary radicals in the membrane and accelerates the process of hemolysis. These secondary reactions taking place in both experimental systems caused the erythrocytes irradiated with the dose of 0.4 kGy to indicate significant differences in their physicochemical properties. As it was shown in our previous paper (Zaborowski and Szweda-Lewandowska, 1997), the maximal effect of decreased hemolysis appeared after a three-and-a-half-hour break. Longer or shorter breaks between subsequent expositions caused a decrease of this effect. Thus the reactions initiated in erythrocytes with the dose of 0.4 kGy and post-radiation processes occurring during the break led to a temporary resistance of erythrocytes to the hemolysis induced by a higher subsequent dose of radiation and even hypochlorite (Krokosz, 2003). Erythrocytes irradiated with an ‘‘inducing’’ dose and incubated in a phosphate buffer at ambient temperature
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hemolyzed considerably slower than the unirradiated erythrocytes but only for 100 h. After that time, there was a rapid increase in hemolysis, which after 120 h leveled to the control. Under the conditions of incubation, erythrocytes were devoid of the source of energy. It is known that the lack of ATP turns off the activity of the ion-depending ATPases, which leads to the disturbance of ion balance and to the osmotic hemolysis. However, determining the hemolysis of erythrocytes irradiated with the dose of 0.4 kGy and unirradiated erythrocytes incubated for 48 h in a buffer with the addition of glucose showed a significant decrease of hemolysis to the same level in spite of the fact that the initial state of erythrocytes was different in both cases. In case of unirradiated erythrocytes, hemolysis was probably caused by the changes in the membrane resulted from malnutrition. However, in the case of irradiated erythrocytes the obtained results suggested that the reactions initiated by d OH radicals in the presence of air caused temporary membrane rearrangement, which lasted for a short time after irradiation and led to a delay in hemolysis. It may be a pre-hemolytic phase of the changed membrane, which, with the lack of energy, finally leads to hemolysis. Nevertheless, in the presence of glucose, some elements of the membrane may be regenerated. Some papers provide a proof of the occurrence of reparation processes in irradiated mammalian erythrocytes. These processes concerned the reduction of radiation-induced disulfide groups in human erythrocyte membranes as well as the recovery of radiation-induced potassium lost in human and rat erythrocytes after the incubation of damaged cells at 36 1C (Myers and Bide, 1966; Sutherland and Pihl, 1968; Brugnara and Churchill, 1992). In addition, literature provides data showing temporary early membrane alterations of human thymocytes induced by ionizing radiation in vitro (Kubasova et al., 1993). In erythrocytes irradiated with the dose of 0.4 kGy there were changes in parameters describing the physical state of the membrane and the elements of cytoplasma. In case of a plasmatic membrane, any products of lipid peroxidation were not found. However, the fluidity parameters of the lipid bilayer in preparations irradiated under air indicated changes in the order of lipids on the surface of hydrophilic areas and on the depth of existence of unsaturated bonds. In both cases, the decrease of microviscosity was noticed. Analogical measures for erythrocytes irradiated under N2O with the dose of 0.4 kGy were not performed. Such parameters described in our previous paper (SzwedaLewandowska et al., 2003) for the dose of 6 kGy indicated the lack of changes in microviscosity. The increase in fluidity of the unsaturated area of the lipid bilayer was observed at the dose preceding hemolysis, i.e., 9 kGy. Taking the above into consideration, it
cannot be unequivocally claimed that there were no such changes at the dose of 0.4 kGy (Krokosz and SzwedaLewandowska, 2005). It seems that the changes in the lipid bilayer described above as well as the lack of terminal products of lipid peroxidation suggest the alteration of protein–lipid relations caused by the increase in protein–protein interactions described by the hW/hS parameter. The decrease of hW/hS parameter was higher under air than under N2O. In our previous paper (Szweda-Lewandowska et al., 2003), the aggregation of proteins was observed in the membranes of erythrocytes irradiated under N2O with the dose of 0.4 kGy. The aggregates were formed by –S–S– bridges. Under air, molecular oxygen suppressed crosslinking. It is probable that in the absence of oxygen, OH radicals attack the –SH groups of membrane proteins, the tiyl radicals appear and they react with each other before they are regenerated by GSH. Such course of action is compatible with the fact that in erythrocytes irradiated under N2O at the dose of 0.4 kGy, the level of GSH decreased only by 33%. Whereas irradiation under air at the same dose caused decrease of the GSH level by 65%. Apart from scavenging radicals, such a significant decrease in GSH level may result from its participation in reaction with protein peroxide radicals created under air, because in such conditions no protein aggregates were found. Guille et al. (1987) suggested that the alteration in motion of spin labels observed in the lipid bilayer of erythrocyte membrane after irradiation were most likely to be due to changes in the structure of proteins, which then affected membrane lipids. Cantafora et al. (1987) observed changes in 31P NMR spectra correlated with changes in MSL motion, which revealed the formation of the cytoskeletal proteins crosslinking and changes in the interactions between proteins and lipids in erythrocyte membrane under the influence of irradiation. Yonei et al. (1984) suggested that reactions of radiation-generated radicals are directed at –SH groups including membrane proteins. Changes in the protein conformation cause changes in the protein–lipid interactions, which impose changes in the order of lipids. Simultaneously, the observed decrease in GSH level, active GPx and catalase indicate the activity of intracellular defense systems against oxygen radicals. In case of erythrocytes irradiated under N2O, the catalase activity is even higher after the dose of 0.4 and 0.8 kGy than in the control erythrocytes. It suggests structural changes in this protein. In the erythrocytes irradiated under air, the dose of 0.4 kGy stimulated the Na,K-ATPase activity, whereas under N2O the activity of this enzyme remained at the control level. As it was shown earlier (Krokosz, 2000), the increase in the Na,K-ATPase activity did not prevent the loss of K+ from the erythrocytes irradiated with the dose of 0.4 kGy and incubated at room temperature without exogenic glucose. In such
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conditions, the ATP level decreased gradually, which led to the inhibition of processes requiring energy. The increase in ATPase activity determined in isolated membranes in a proper medium indicated protein conformation changes, which correlated with the decrease in hW/hS parameter. Generally, the results concerning conformational changes in proteins indicate that they are more sensitive to the modifications than lipids, which is in accordance with our previous papers as well as suggestions of other authors (Guille et al., 1987; Verma and Rastogi, 1990). It may suggest that the structural changes in proteins are responsible for temporary resistance to hemolysis. Thus, d OH radicals at an approximately low concentration may be the factors modifying the structures of biological compounds leading to the transient increase in their resistance.
References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Akerboom, T.P., Sies, H., 1981. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol. 77, 373–382. Barber, M.J., Rosen, G.M., Rauckman, L.J., 1983. Studies on the mobility of maleimide spin labels within the erythrocyte membrane. Biochim. Biophys. Acta 732, 126–132. Baykov, A.A., Evtushenko, O.A., Avaeva, S.M., 1988. A malachite green procedure for orthophosphate determination and its use inalkaline phosphatase-based enzyme immunoassay. Anal. Biochem. 171, 266–270. Boothman, D.A., Meyers, M., Fukunaga, N., Lee, S.W., 1993. Isolation of X-ray inducible transcripts from radioresistant human melanoma cells. Proc. Natl. Acad. Sci. USA 90, 7200–7204. Brugnara, C., Churchill, W.H., 1992. Effect of irradiation on red cell cation content and transport. Transfusion 32, 246–252. Cantafora, A., Ceccarini, M., Guidoni, L., Ianzini, F., Minetti, M., Viti, V., 1987. Effects of gamma-irradiation on the erythrocyte membrane: ESR, NMR and biochemical studies. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 51, 59–69. Chiu, D., Kuypers, F., Lubin, B., 1989. Lipid peroxidation in human red cells. Semin. Hematol. 26, 257–276. Dodge, J.T., Mitchell, C., Hanahan, D.J., 1963. The preparation and chemical characteristic of hemoglobin free ghosts of human erythrocytes. Arch. Biochem. Biophys. 100, 119–130. Guille, J., Raison, J.K., Gebicki, J., 1987. Radiation induced lipid peroxidation and fluidity of erythrocyte membrane lipids. Free Radical Biol. Med. 3, 147–152. Hageman, J.J., Bast, A., Vermeulen, N.P.E., 1992. Monitoring of oxidative free radical damage in vivo: analytical aspects. Chem. Biol. Interact. 82, 243–293. Ikushima, T., 1989. Radio-adaptive response. Characterization of a cytogenetic repair induced by low-level ionizing
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radiation in cultured Chinese hamster cells. Mutat. Res. 227, 241–246. Joiner, M.C., 1994a. Induced radioresistance: an overview and historical perspective. Int. J. Radiat. Biol. 65, 79–84. Joiner, M.C., 1994b. Evidence for induced radioresistance from survival and other points: an introduction. Radiat. Res. 138, 55–58. Kollmann, G., Shapiro, B., Martin, D., 1969. The mechanism of radiation hemolysis in human erythrocytes. Radiat. Res. 37, 551–566. Koziczak, R., Krokosz, A., Szweda-Lewandowska, Z., 1999. Effect of dose-rate and dose fractionation on radiationinduced hemolysis of human erythrocytes. Biochem. Mol. Biol. Int. 47, 865–872. Koziczak, R., Gonciarz, M., Krokosz, A., Szweda-Lewandowska, Z., 2003. The influence of split doses of gammaradiation on human erythrocytes, J. Radiat. Res. 44, 217–222. Krokosz, A., 2000. Structural changes of human erythrocyte membranes after low doses of gamma radiation. Ph.D. Thesis, Lodz. Krokosz, A., 2003. The effect of hypochlorite on human erythrocytes pretreated with X-radiation. Cell. Mol. Biol. Lett. 8, 215–219. Krokosz, A., Szweda-Lewandowska, Z., 2005. Changes in the activity of acetylcholinesterase and Na,K-ATPase in human erythrocytes irradiated with X-rays. Cell. Mol. Biol. Lett. 10, 471–478. Kubasova, T., Chukhlovin, A.B., Somosy, Z., Ivanov, S.D., Koteles, G.J., Zherbin, E.A., Hanson, K.P., 1993. Detection of early membrane and nuclear alterations of thymocytes upon in vitro ionizing irradiation. Acta Physiol. Hung. 81, 277–288. Lee, S.W., Ducoff, H.S., 1994. The effect of ionizing radiation on avian erythrocytes. Radiat. Res. 137, 104–110. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, J.R., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Luckey, T.D., 1991. Radiation Hormesis, CRC Press, Boca Raton Ann Arbor, Florida. Meyers, M., Schea, R., Seabury, H., Petrovski, A., McLaughlin, W.P., Lee, J., Boothman, D.A., 1992. In: Sugahara, T., Sagan, L.A., Aoyama, T., (Eds.), Low Dose Irradiation and Biological Defense Mechanisms, Elsevier, Amsterdam pp. 263–266. Myers, D.K., Bide, R.W., 1966. Biochemical effects of X-radiation on erythrocytes. Radiat. Res. 27, 250–263. Palmieri, D.A., Butterfield, D.A., 1990. Structure-activity investigation of the alteration of the physical state of the skeletal network of proteins in human erythrocyte membranes induced by 9-amino-1,2,3,4-tetrahydroacridine. Biochim. Biophys. Acta 1024, 285–288. Petkau, A., 1986. Protection and repair of irradiated membranes. In: Free Radicals, Aging and Degenerative Diseases. Allan R. Liss Inc., New York, pp. 481–508. Pucha"a, M., Szweda-Lewandowska, Z., Kiefer, J., 2004. The influence of radiation quality on radiation-induced hemolysis and hemoglobin oxidation of human erythrocytes. J. Radiat. Res. 45, 1–5. Rice-Evans, C., Diplock, A.T., Symons, M.C., 1991. Techniques in Free Radical Research. Elsevier, Amsterdam.
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Riley, P.A., 1986. Free radicals in biology: oxidative stress and the effects of ionising radiation. Int. J. Radiat. Biol. 65, 27–33. Sandhu, J.S., Ware, K., Grisham, M.B., 1992. Peroxyl radicalmediated hemolysis: role of lipid, protein and sulfhydryl oxidation. Free Radical Res. Commun. 16, 111–122. Schreier, S., Polnaszek, C., Smith, J., 1978. Spin labels in membranes. Problems in practice. Biochim. Biophys. Acta 515, 375–436. von Sontag, C., 1987. The chemical basis of radiation biology. Taylor and Francis, London, New York, Philadelphia, pp. 31–36. Sutherland, R.M., Pihl, A., 1968. Repair of radiation damage to erythrocyte membranes. The reduction of radiationinduced disulfide groups. Radiat. Res. 34, 300–314. Szweda-Lewandowska, Z., Krokosz, A., Gonciarz, M., Zajeczkowska, W., Puchala, M., 2003. Damage to human erythrocytes by radiation-generated HO* radicals: molecular changes in erythrocyte membranes. Free Radical Res. 37, 1137–1143. UNSCEAR, 1994. Sources and effects of ionizing radiation, United Nations Scientific Committee on the effects of atomic radiation. Report to the General Assembly with Scientific Annexes, United Nations, New York, 1994.
Verma, S.P., Rastogi, A., 1990. Role of proteins in protection against radiation-induced damage in membranes. Radiat. Res. 122, 130–136. Wiencke, J.K., Afzal, V., Olivieri, G., Wolff, S., 1986. Evidence that the [H-3] thymidine-induced adaptive response of human lymphocytes to subsequent doses of X-rays involves the induction of chromosomal repair mechanism. Mutagenesis 1, 375–380. Wolff, S., Afzal, V., Wiencke, J.K., Olivieri, G., Michaeli, A., 1988. Human lymphocytes exposed to low doses of ionizing radiation become refractory to high dose of radiation as well as chemical mutagenesis that induce double-strand breaks in DNA. Int. J. Radiat. Biol. 53, 39–48. Wolter, H., Konings, A.W.T., 1985. Membrane radiosensitivity of fatty acid supplemented fibroblast as assayed by loss of intracellular potassium. Int. J. Radiat. Biol. 48, 963–973. Yonei, S., Akasaka, S., Kato, M., 1984. Studies on the mechanism of radiation-induced structural disorganization of human erythrocyte membranes. Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 46, 463–471. Zaborowski, A., Szweda-Lewandowska, Z., 1997. The influence of dose fractionation on radiation-induced hemolysis of human erythrocytes. Cell Biol. Int. 21, 559–563.
Radiation Physics and Chemistry 75 (2006) 967–976 www.elsevier.com/locate/radphyschem
Induction of transient radioresistance in human erythrocytes Anita Krokosz, Zofia Szweda-Lewandowska Department of Molecular Biophysics, University of Lodz, 90-237 Lodz, Poland Received 28 December 2005; accepted 10 February 2006
Abstract Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit of 2%), were irradiated with g-rays with single and split doses under air or N2O in order to determine the physicochemical changes caused by the dose inducing an increase in resistance to radiation-induced hemolysis. The obtained results showed that under the applied irradiation conditions, the dose of 0.4 kGy induced changes in erythrocytes, which were responsible for temporary resistance of erythrocytes to hemolysis. We concluded that the observed resistance is caused mainly by the structural changes in proteins. r 2006 Elsevier Ltd. All rights reserved. Keywords: Gamma radiation; Radioresistance; Human erythrocytes
1. Introduction Numerous studies have shown that small doses of ionizing radiation of low linear energy transfer (LET) induce various stimulating effects on living systems uch as radio-adaptive response, an increase in reproductive ability, stimulatory effects on immune systems and an extended life span (Luckey, 1991). It is commonly known that many cells exposed to small doses of ionizing radiation show reduced cytogenetic damage as well as higher survival rate after a subsequent higher dose given a short time later. These doses are called ‘‘priming’’ or ‘‘conditioning’’ and ‘‘challenging’’ doses. Induced radioresistance or adaptive response to the first dose has been proposed as an explanation of this effect (Joiner, 1994a, b). The adaptive response was reported in various types of cellular systems including human lymphocytes, Chinese hamster cells, human fibroblasts, bacteria, protozoa, algae, lepidopCorresponding author. Tel.: +48 42 6354480; fax: +48 42 6354473. E-mail address: [email protected] (A. Krokosz).
tera insects and higher plants (Joiner, 1994a, b and references cited therein; UNSCEAR, 1994). The experiments on tissue cultures of human lymphocytes (Wolff et al., 1988), Chinese hamster V79 cells (Ikushima, 1989) and human 41-Mel cells (Meyers et al., 1992; Boothman et al., 1993) have shown that new proteins are induced after exposure to low doses of radiation, presumably by gene activation. Especially poly (ADP-ribose) polymerase, an enzyme which is activated by radiation-induced DNA breaks, plays an important role in DNA repair and has been implicated in the adaptive response in human lymphocytes (Wiencke et al., 1986). The experiments on protein induction with low doses showed the appearance of protein characteristic of X-irradiation (not induced by heat shock, hypoxia or alkylating agents). It should be stressed that previous studies of radioresistance mechanisms were related mainly to the damage and repair of DNA in dividing cells without taking into consideration other cellular structures, particularly membranes. The evidence is that membrane-associated damage may be important in radiation-induced interphase cell death (Wolter and Konings, 1985).
0969-806X/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.radphyschem.2006.02.004
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Mature human erythrocytes are highly specialized cells, which have no nucleus or any other cellular organelles. This is why they are a suitable model for studying the influence of various factors on the plasma membrane, which stays in its natural environment without isolation. For the first time, Lee and Ducoff (1994) have shown that fractionation of a radiation dose causes a decrease in hemolysis of nucleated avian erythrocytes. In our previous papers (Zaborowski and SzwedaLewandowska, 1997; Koziczak et al., 1999, 2003), we showed that the enucleated human erythrocytes irradiated under air with split doses, with a time interval between subsequent expositions, hemolyzed slower in comparison with cells irradiated with the same single doses. Simultaneously, we observed a lower degree of lipid peroxidation, hemoglobin (Hb) oxidation and smaller changes in the fluidity of lipid bilayer. The effect of the decrease in hemolysis of enucleated human erythrocytes was closely dependent on the dividing conditions. The critical factors limiting this effect were the magnitude of the first radiation dose and the interval between fractions. To continue the above experiments, in this paper we focused on the effects of split doses on human erythrocytes irradiated under air or N2O. The aim of this work was to determine the physicochemical changes taking place in the human erythrocytes caused by the dose inducing an increase in resistance to radiationinduced hemolysis.
2. Materials and methods 2.1. Chemicals a-Cellulose, 5-, 12-, 16-doxylstearic acids, 2,2,6,6tetramethyl-4 maleimidopiperidine-N-oxyl (MSL), ATP, ouabain, NADPH, EGTA, glutathione reductase, t-butyl hydroperoxide, thiobarbituric acid, and phenylmethylsulfonyl fluoride were purchased from Sigma Chemical Co., Poole Dorset, UK. Other chemicals were obtained from POCh (Gliwice, Poland) (all of analytical grade). All solutions were made with water purified by the Milli-Q system. 2.2. Preparation of erythrocyte suspension Blood samples from healthy adult donors were provided by the Central Blood Bank in Lodz. Erythrocytes were separated from blood plasma and leucocytes by centrifugation. In order to remove residual leucocytes, erythrocytes were passed through an a-microcrystalline cellulose column, washed with 0.1 M Na-phosphate buffer (pH 7.4) and resuspended in the same buffer to obtain a hematocrit of 2%.
2.3. Irradiation conditions Erythrocyte suspensions in a Na-phosphate buffer were irradiated with a 60-Co g-rays at room temperature in the atmosphere of air or N2O. The dose rate estimated with modified Fricke dosimeter was 4 kGy h1. During irradiation, the erythrocyte suspensions were stirred with a magnetic bar. In the experiments with a split dose, the first dose was equal to 0.4 kGy and the second dose was equal to 2.3 kGy when preparations were irradiated under air and 5.6 kGy when preparations were irradiated under N2O. The total dose received by cells was 2.7 and 6 kGy, respectively. The time interval between subsequent doses was 3.5 h. Irradiation of erythrocytes under N2O was performed in a glass tonometer, preflushed with N2O for 1–1.5 h, with gentle mixing. Irradiation was performed in the Institute of Applied Radiation Chemistry, the Technical University of Lodz. 2.4. Measurement of hemolysis Hemolysis of erythrocytes was determined on the basis of the ratio of Hb released from cells to the total cellular Hb content. After irradiation, erythrocyte suspensions were centrifuged, the supernatant was carefully collected and the absorbance at 630 nm was measured after oxidation of iron with K3[Fe(CN)6]. The residual erythrocytes were hemolyzed with distilled water and the absorbance of the solution was measured as described above. The percentage of hemolysis was calculated as described in the earlier paper (Koziczak et al., 1999). Post-radiation kinetics of hemolysis was determined for erythrocytes irradiated with the dose of 0.4 kGy after indicated time intervals. After irradiation, 0.5% of penicillin and 0.1% of streptomycin were added to stop bacterial growth. 2.5. Determination of osmotic resistance Osmotic fragility curves were drawn on the basis of the percentage of erythrocyte hemolysis in solutions of various NaCl concentrations buffered with 5 mM Naphosphate (pH 7.4). Suspensions of erythrocytes were diluted to a hematocrit of 0.2%, and the degree of hemolysis was estimated after 10 min.The samples were centrifuged at 10,000 rpm for 3 min.The absorbance of the supernatants was assessed at 570 nm.The degree of hemolysis was calculated on the assumption that the absorbance of a sample hemolyzed with distilled water equaled 100%. 2.6. Determination of lipid peroxidation Lipid peroxidation was quantified spectrophotometrically at 532 nm by measuring the formation of
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thiobarbituric acid reactive substances (TBARS) (Sandhu et al., 1992). The main terminal product of lipid peroxidation reacting with TBA is malondialdehyde (MDA). However, besides MDA other aldehydes formed during the decomposition of lipid peroxides might give colored complex with TBA (Hageman et al., 1992). Lipid peroxidation was expressed in absorbance units. 2.7. Measurement of methemoglobin (MetHb) in erythrocytes The percentage of MetHb content was measured in the residual erythrocytes after their hemolysis. The absorbance of the solutions was measured at 630 nm before the oxidation of iron and after its total oxidation with K3[Fe(CN)6]. The content of Fe(III) was calculated according to Puchala et al. (2004). 2.8. Measurement of glutathione peroxidase (GPx) (EC.1.11.1.9) activity GPx activity was determined by following NADPH oxidation using t-butyl-hydroperoxide as a substrate. The decrease in absorbance was measured at 340 nm for 3 min. Activity was expressed as IU/g Hb indicating such an amount of enzyme, which was equivalent to the oxidation of 0.5 mmol NADPH in 1 min at 37 1C. The molar absorption coefficient of NADPH at 340 nm is 6220 mol1 dm3 cm1 (Rice-Evans et al., 1991). 2.9. Determination of catalase (EC.1.11.1.6) activity in erythrocytes Catalase activity was determined according to Aebi (1984). The method is based on spectrophotometrically monitoring the rate of decomposition of H2O2 at 240 nm at room temperature. One IU represents the amount of enzyme necessary to breakdown 1 mmol H2O2 in 1 min.The molar absorption coefficient of H2O2 at 240 nm is 43.6 mol1 dm3 cm1. The activity was expressed in IU/mg Hb. 2.10. Determination of Na,K-ATPase activity (EC 3.1 6.37) Na,K-ATPase activity was determined by measuring the difference in the level of liberation of inorganic phosphate (Pi) from ATP in the absence and in the presence of 0.1 mM ouabain during a 30-min incubation of the membrane preparations at 37 1C. The amount of released Pi was determined spectrophotometrically at 610 nm as a complex with heptamolybdate and malachite green (Baykov et al., 1988). Pi concentration was taken from a calibration curve made for 2–40 mM of KH2PO4 as a standard. Na,K-ATPase activity was
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calculated by subtracting the activity assessed in the presence of ouabain from the activity in the absence of ouabain (total ATPase activity). The enzyme activity 1 was expressed in nmol Pi mg1 prot h . 2.11. Preparation of erythrocyte membranes Erythrocyte membranes were prepared according to the method of Dodge et al. (1963) with some modifications. Hemolysis was carried out at 4 1C with 20 volumes of 20 mM Na-phosphate buffer (pH 7.4), containing 1 mM ethylenediaminetetraacetate (EDTA) and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) as protease inhibitors. The membrane ghosts were washed successively with 20, 10 and 5 mM ice-cold Na-phosphate buffer (pH 7.4) until the ghosts were free of residual hemoglobin. Protein concentration in the membrane preparations was determined by the method of Lowry et al. (1951). 2.12. Spin labeling of erythrocytes and erythrocyte membranes The electron spin resonance spectra of erythrocytes labeled with 5-doxylstearic acid (5-DSA), 12-doxylstearic acid (12-DSA) and 16-doxylstearic acid (16-DSA) were used to monitor the fluidity of membrane lipids (Koziczak et al., 2003). From the ESR spectra of 5-DSA, an order parameter (S) was derived by measuring the outer and inner hyperfine splitting, 2TII and 2T?, using the following formula: S¼
T II ðT ? þ CÞ 1:723, T II þ 2ðT ? þ CÞ
where C ¼ 1.40.0053(TIIT?). The order parameter is a degree of the distribution of molecular orientations with respect to a reference axis, which is the normal to the membrane surface. An increase in the order parameter reflects a decrease in segmental flexibility of the spin label (Schreier et al., 1978) For the 12-DSA spectra, the ratio of the low-field peak height to the midfield peak height, A/h0, was calculated, characterizing a mobility of the spin label in the environment of double bonds of unsaturated fatty acids. From the 16-DSA spectra, the difference between the rotational correlation time, tC, and tB was calculated using the formula Dt ¼ tC tB , where hqffiffiffiffiffiffi qffiffiffiffiffiffi i h0 h0 tC ¼ 12 3:25 1010 DW hþ1 þ h1 2 , tB ¼ 12ð3:25 1010 ÞDW
hqffiffiffiffiffiffi h0 hþ1
qffiffiffiffiffiffii h0 h1
,
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and DW the midfield peak width; h0 the midfield peak height; h+1 the low-field peak height; h1 the high-field peak height. Rotational correlation times reflecting the spin label motion in the directions perpendicular and parallel to the long axis of a lipid molecule. Changes in protein conformation were estimated according to Barber et al. (1983) by spin labeling of the erythrocyte membranes with 1 mg of MSL per 25 mg of protein and incubation at 4 1C for 30 min.Then the excess of the label was removed by washing it with PBS several times. The spectra of MSL attached to the membrane proteins were analyzed by measuring the ratio of weakly (hW) and strongly (hS) immobilized components in the low-field peak (hW/hS). All ESR spectra were measured at ambient temperature using a Bruker 300 E ESR spectrometer. 2.13. Determination of reduced glutathione (GSH) concentration in erythrocytes The content of GSH was determined fluorimetrically as described by Akerboom and Sies (1981), with oftalaldehyde. The fluorescence was measured at an excitation wavelength of 320 nm and an emission wavelength of 420 nm using Perkin-Elmer fluorometer. GSH concentration in the studied samples was taken from a calibration curve made for 0.2–5 mM concentration of GSH.
3. Results 3.1. The influence of a split dose on human erythrocytes irradiated in the atmosphere of air and N2O In this work, the influence of a split dose on erythrocytes was presented on the example of two chosen doses for which irradiation conditions were previously found by a trial and error method (Zaborowski and Szweda-Lewandowska, 1997; Koziczak et al., 1999). Fig. 1 shows the level of hemolysis of erythrocytes irradiated under air and N2O with a single dose and with the same dose split into two fractions with a break of 3.5 h between subsequent exposures. Hemolysis of erythrocytes was measured 18 h after the end of exposure. Apart from hemolysis, the following parameters were determined in the irradiated erythrocytes: the intensity of lipid peroxidation and the MetHb level. The intensity of lipid peroxidation was estimated on the basis of the presence of TBARS. The level of hemolysis in suspensions of erythrocytes irradiated under air with a split dose was 2-fold lower than in erythrocytes irradiated with a single dose (Fig. 1). However, the level of lipid peroxidation was
Fig. 1. Hemolysis of erythrocytes irradiated under air and N2O with a single or split dose of g-radiation. The break between two fractions of split dose was 3.5 h. Irradiation and incubation during the break took place in the atmosphere of air or N2O as mentioned. After irradiation, erythrocyte suspensions were incubated at room temperature under air and analyzed 18 h after the end of the exposure.
approximately the same for both doses and equaled 0.050470.0043 for the split dose (0.052370.0035 for the single dose). Also, the MetHb level was the same in both cases and equaled (12.070.7)%. The effect of dose splitting in erythrocytes irradiated in the atmosphere of N2O was examined at significantly higher dose than under air (Fig. 1). The division of the dose caused a decrease in hemolysis by 36.3% and in the level of lipid peroxidation by 31.2%. However, the process of dose splitting did not influence the level of hemoglobin oxidation. Thus, the level of MetHb in erythrocytes irradiated with single and split doses was approximately the same and equaled about 15%. In further studies, the changes in erythrocytes irradiated in the atmosphere of air or N2O with the dose of 0.4 kGy were determined, but we concentrated mainly on the erythrocytes irradiated under air.
3.2. Characterization of erythrocytes irradiated in the atmosphere of air or N2O with the dose of 0.4 kGy 3.2.1. The post-radiation kinetics of hemolysis of erythrocytes irradiated under air The post-radiation kinetics of hemolysis of erythrocytes irradiated under air with 0.4 and 1 kGy and the auto-hemolysis of non-irradiated erythrocytes (control) were examined. The obtained results are presented in Fig. 2. The erythrocytes irradiated with 0.4 kGy hemolyzed considerably slower than the control. The biggest difference in the hemolysis of the control erythrocytes and those irradiated with 0.4 kGy appeared in 94th hour after irradiation. The erythrocytes irradiated with the
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Fig. 2. Post-radiation kinetics of hemolysis of erythrocytes irradiated under air with the dose of 0.4, 1 kGy and unirradiated erythrocytes.
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Fig. 3. Influence of 5 mM glucose on post-radiation hemolysis of erythrocytes. Glucose was added to erythrocyte suspension immediately after the end of irradiation. Hemolysis was assessed 48 hrs after the end of irradiation.
dose of 1 kGy for up to 96 h of incubation hemolyzed slightly faster than the control erythrocytes. After that time, the level of hemolysis was rapidly growing. It should be stressed that in the described experiments, erythrocytes were suspended in a phosphate buffer without glucose. However, when glucose was added to erythrocyte suspensions immediately after irradiation up to the final concentration of 5 mM, the same level of hemolysis was found in both experimental systems after 48 h of incubation at room temperature (Fig. 3). 3.2.2. Physicochemical state of a plasmatic membrane of erythrocytes irradiated with the dose of 0.4 kGy Fig. 4 shows the osmotic resistance curves of control erythrocytes and those irradiated with the dose of 0.4 kGy under air or N2O determined 2 h after irradiation. The erythrocytes irradiated under air showed the same osmotic resistance as the control erythrocytes. However, saturation of the erythrocyte suspension with N2O caused a slight decrease in their osmotic resistance, which increased insignificantly after irradiation with the dose of 0.4 kGy. In the erythrocytes irradiated with the dose of 0.4 kGy both under air or N2O, no lipid peroxidation products were found (results not shown). The spin label evaluation of the fluidity of membrane lipids indicated an increase in the fluidity of the lipid bilayer of erythrocytes irradiated under air (Table 1). The order parameter (S) for 5-DSA showed a slight but statistically significant increase in the lipid fluidity on the surface of the membrane close to the polar heads of lipids. The values of the ratio A/h0 characterizing the lipid fluidity at the depth of 12-carbon of hydrocarbon chains of lipids were higher in irradiated preparations than in the control. The highest value of the lipid fluidity parameter was observed for the dose of 0.4 kGy. The increase in the A/h0 ratio indicated lipid fluidization in
Fig. 4. Osmotic fragility test for unirradiated erythrocytes and irradiated with the dose of 0.4 kGy under air and N2O.
the membrane hydrophobic regions. The rotational correlation time for 16-DSA was also determined but remained on the level of control, indicating a lack of changes in lipid fluidity in these membrane regions. Changes occurring in membrane proteins of irradiated erythrocytes were examined using a maleimide spin label. The analysis of the maleimide spectrum, attached to the ghost membrane proteins was performed on the basis of the value of the hW/hS ratio as outlined in Methods. Values of the hW/hS ratios for erythrocytes irradiated under air as well as under N2O are presented in Table 2. A decrease in the hW/hS ratio in irradiated erythrocytes indicated conformational changes in the membrane proteins connected with the reduction of accessibility of the protein –SH groups for the label. In general, a decrease in the hW/hS ratio of MSL bound to erythrocyte membrane implies decreased segmental motion or an increased protein–protein interaction of
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Table 1 Changes in order parameter S, A/h0 parameter and correlation time t of the lipid spin label, respectively, 5-doxylstearic acid, 12doxylstearic acid and 16-doxylstearic acid in membrane of human erythrocytes irradiated with g-rays under air Dose
Order parameter S value7SD
A/h0 parameter value7SD
Correlation time t value (ns)7SD
Unirradiated (control) 0.4 kGy 0.8 kGy
0.748170.0034 0.737470.0009 0.739770.0068
0.208870.0091 0.272370.0200 0.239570.0161
0.215870.0171 0.192570.0078 0.204070.0035
Table 2 Changes of hW/hS ratio in ESR spectra of MSL attached to membrane proteins for human erythrocytes irradiated with g-rays under air and N2O Dose
Unirradiated (control) 0.4 kGy 0.8 kGy
W/S parameter value7SD Air
N2O
2.09170.011 1.65670.058 1.23370.027
2.18370.087 1.93270.040 1.72870.077
spin-labeled domains of the proteins (Palmieri and Butterfield, 1990). The decrease in the hW/hS ratio was observed at the dose of 0.4 kGy. It was higher in the erythrocytes irradiated under air than those irradiated under N2O. Fig. 5 presents the activity of Na,K-ATPase in the membranes of control erythrocytes and those irradiated under air and N2O with the dose of 0.4 kGy and under air with the dose of 1 kGy. There is an increase in Na,KATPase activity in the erythrocytes irradiated with 0.4 kGy under air in comparison with the control. Further increase in the dose up to 1 kGy caused a contrary effect i.e. a decrease in the activity. However, there were no changes observed in Na,K-ATPase activity in the erythrocytes irradiated with the dose of 0.4 kGy under N2O.
3.2.3. Changes in intracellular compounds of erythrocytes irradiated with the dose of 0.4 kGy The erythrocytes irradiated with 0.4 kGy both under air and N2O did not indicate an increase in the MetHb level above the control. Nevertheless, in the irradiated erythrocytes, a rapid decrease in the amount of reduced glutathione was observed (Fig. 6). The GSH level was decreasing considerably faster in the erythrocytes irradiated under air than N2O. Irradiation of the erythrocytes under air with the dose of 0.4 kGy led to a decrease in the GSH level by 65%, while in the erythrocytes irradiated with the same dose under N2O, the GSH level decreased by 31%.
Fig. 5. Na,K-ATPase activity in the membranes of erythrocytes irradiated under air and N2O. Each bar represents the mean of 3–5 separate experiments7SD.
Fig. 6. Glutathione level in erythrocytes irradiated under air and N2O determined 3.5 h after the end of irradiation. Each points represents the mean of 3–7 separate experiments7SD.
In the preparations irradiated with the dose of 0.4 kGy and higher doses, the activity of catalase and GPx was determined. As it is visible in Fig. 7, the irradiation of erythrocytes under air with the doses of 0.4 and 0.8 kGy did not cause any changes in the activity
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Fig. 7. Catalase (A) and glutathione peroxidase (B) activity in erythrocytes irradiated under air and N2O determined 3.5 h after the end of irradiation. Each bar represents the mean of 3–5 separate experiments7SD.
of both enzymes. On the contrary, in the erythrocytes irradiated under N2O, a statistically significant increase in the activity of catalase was found at the doses of 0.4 and 0.8 kGy. Higher doses led to a decrease in the activity of this enzyme. However, the GPx activity remained at the level of control.
4. Discussion The obtained results show that under the conditions of irradiation, the dose of 0.4 kGy induced a specific response in human erythrocytes to a high dose applied subsequently. Hemolysis was used as the end-point and on its basis erythrocyte response was assessed. The induced radioresistance was observed during erythrocyte irradiation under air and N2O. It is known that g-radiation generates active oxygen species resulting in the exposure of the irradiated systems to oxidative stress (Riley, 1986). In both experimental systems studied in this work the observed effects were initiated mainly by dOH radicals. Their radiation yield G is equal to 2.7 under air and 5.4 under N2O (Sontag, 1987). Apart from the fact that yield of dOH radicals under N2O is twice higher than under air, the effectiveness of d OH radicals in the induction of hemolysis under N2O is smaller than under air, as we presented in the previous paper (Szweda-Lewandowska et al., 2003). That is why the applied dose, which ‘‘challenged’’ hemolysis after 18 h was considerably higher under N2O than under air.
Erythrocytes contain very efficient antioxidant systems consisting of small antioxidant compounds such as glutathione (GSH), vitamin E, vitamin C and enzymes such as catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). These compounds can scavenge free radicals and also take part in the repair processes of target molecules by adding hydrogen to organic radicals (Rd) induced by dOH radicals on target biomolecules (Kollmann et al., 1969; Petkau, 1986; Chiu et al., 1989). Under anaerobic conditions, there is a possibility of repair of damaged proteins and lipids by means of chemical and enzymatic defense systems. The molecular oxygen present during exposition of erythrocytes to dOH radicals modifies the reactions of secondary radicals in the membrane and accelerates the process of hemolysis. These secondary reactions taking place in both experimental systems caused the erythrocytes irradiated with the dose of 0.4 kGy to indicate significant differences in their physicochemical properties. As it was shown in our previous paper (Zaborowski and Szweda-Lewandowska, 1997), the maximal effect of decreased hemolysis appeared after a three-and-a-half-hour break. Longer or shorter breaks between subsequent expositions caused a decrease of this effect. Thus the reactions initiated in erythrocytes with the dose of 0.4 kGy and post-radiation processes occurring during the break led to a temporary resistance of erythrocytes to the hemolysis induced by a higher subsequent dose of radiation and even hypochlorite (Krokosz, 2003). Erythrocytes irradiated with an ‘‘inducing’’ dose and incubated in a phosphate buffer at ambient temperature
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hemolyzed considerably slower than the unirradiated erythrocytes but only for 100 h. After that time, there was a rapid increase in hemolysis, which after 120 h leveled to the control. Under the conditions of incubation, erythrocytes were devoid of the source of energy. It is known that the lack of ATP turns off the activity of the ion-depending ATPases, which leads to the disturbance of ion balance and to the osmotic hemolysis. However, determining the hemolysis of erythrocytes irradiated with the dose of 0.4 kGy and unirradiated erythrocytes incubated for 48 h in a buffer with the addition of glucose showed a significant decrease of hemolysis to the same level in spite of the fact that the initial state of erythrocytes was different in both cases. In case of unirradiated erythrocytes, hemolysis was probably caused by the changes in the membrane resulted from malnutrition. However, in the case of irradiated erythrocytes the obtained results suggested that the reactions initiated by d OH radicals in the presence of air caused temporary membrane rearrangement, which lasted for a short time after irradiation and led to a delay in hemolysis. It may be a pre-hemolytic phase of the changed membrane, which, with the lack of energy, finally leads to hemolysis. Nevertheless, in the presence of glucose, some elements of the membrane may be regenerated. Some papers provide a proof of the occurrence of reparation processes in irradiated mammalian erythrocytes. These processes concerned the reduction of radiation-induced disulfide groups in human erythrocyte membranes as well as the recovery of radiation-induced potassium lost in human and rat erythrocytes after the incubation of damaged cells at 36 1C (Myers and Bide, 1966; Sutherland and Pihl, 1968; Brugnara and Churchill, 1992). In addition, literature provides data showing temporary early membrane alterations of human thymocytes induced by ionizing radiation in vitro (Kubasova et al., 1993). In erythrocytes irradiated with the dose of 0.4 kGy there were changes in parameters describing the physical state of the membrane and the elements of cytoplasma. In case of a plasmatic membrane, any products of lipid peroxidation were not found. However, the fluidity parameters of the lipid bilayer in preparations irradiated under air indicated changes in the order of lipids on the surface of hydrophilic areas and on the depth of existence of unsaturated bonds. In both cases, the decrease of microviscosity was noticed. Analogical measures for erythrocytes irradiated under N2O with the dose of 0.4 kGy were not performed. Such parameters described in our previous paper (SzwedaLewandowska et al., 2003) for the dose of 6 kGy indicated the lack of changes in microviscosity. The increase in fluidity of the unsaturated area of the lipid bilayer was observed at the dose preceding hemolysis, i.e., 9 kGy. Taking the above into consideration, it
cannot be unequivocally claimed that there were no such changes at the dose of 0.4 kGy (Krokosz and SzwedaLewandowska, 2005). It seems that the changes in the lipid bilayer described above as well as the lack of terminal products of lipid peroxidation suggest the alteration of protein–lipid relations caused by the increase in protein–protein interactions described by the hW/hS parameter. The decrease of hW/hS parameter was higher under air than under N2O. In our previous paper (Szweda-Lewandowska et al., 2003), the aggregation of proteins was observed in the membranes of erythrocytes irradiated under N2O with the dose of 0.4 kGy. The aggregates were formed by –S–S– bridges. Under air, molecular oxygen suppressed crosslinking. It is probable that in the absence of oxygen, OH radicals attack the –SH groups of membrane proteins, the tiyl radicals appear and they react with each other before they are regenerated by GSH. Such course of action is compatible with the fact that in erythrocytes irradiated under N2O at the dose of 0.4 kGy, the level of GSH decreased only by 33%. Whereas irradiation under air at the same dose caused decrease of the GSH level by 65%. Apart from scavenging radicals, such a significant decrease in GSH level may result from its participation in reaction with protein peroxide radicals created under air, because in such conditions no protein aggregates were found. Guille et al. (1987) suggested that the alteration in motion of spin labels observed in the lipid bilayer of erythrocyte membrane after irradiation were most likely to be due to changes in the structure of proteins, which then affected membrane lipids. Cantafora et al. (1987) observed changes in 31P NMR spectra correlated with changes in MSL motion, which revealed the formation of the cytoskeletal proteins crosslinking and changes in the interactions between proteins and lipids in erythrocyte membrane under the influence of irradiation. Yonei et al. (1984) suggested that reactions of radiation-generated radicals are directed at –SH groups including membrane proteins. Changes in the protein conformation cause changes in the protein–lipid interactions, which impose changes in the order of lipids. Simultaneously, the observed decrease in GSH level, active GPx and catalase indicate the activity of intracellular defense systems against oxygen radicals. In case of erythrocytes irradiated under N2O, the catalase activity is even higher after the dose of 0.4 and 0.8 kGy than in the control erythrocytes. It suggests structural changes in this protein. In the erythrocytes irradiated under air, the dose of 0.4 kGy stimulated the Na,K-ATPase activity, whereas under N2O the activity of this enzyme remained at the control level. As it was shown earlier (Krokosz, 2000), the increase in the Na,K-ATPase activity did not prevent the loss of K+ from the erythrocytes irradiated with the dose of 0.4 kGy and incubated at room temperature without exogenic glucose. In such
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conditions, the ATP level decreased gradually, which led to the inhibition of processes requiring energy. The increase in ATPase activity determined in isolated membranes in a proper medium indicated protein conformation changes, which correlated with the decrease in hW/hS parameter. Generally, the results concerning conformational changes in proteins indicate that they are more sensitive to the modifications than lipids, which is in accordance with our previous papers as well as suggestions of other authors (Guille et al., 1987; Verma and Rastogi, 1990). It may suggest that the structural changes in proteins are responsible for temporary resistance to hemolysis. Thus, d OH radicals at an approximately low concentration may be the factors modifying the structures of biological compounds leading to the transient increase in their resistance.
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