Anita Krokosz

W niniejszej pracy oceniano uszkodzenia erytrocytów człowieka indukowane w obecności mannitolu lub etanolu przez radiacyjnie generowane RFT oraz podczas przechowywania (głodzenia) erytrocytów. Napromieniowane promieniowaniem X (400 Gy) lub nienapromieniowane zawiesiny erytrocytów (Ht 2%) w buforowanej fosforanami soli fizjologicznej traktowano mannitolem (do 100 mM) lub etanolem (do 60 mM) i przechowywano w 37C do 72 godz. Określano hemolizę, kształt i granularność erytrocytów, profil elektroforetyczny białek błonowych oraz poziom utleniania hemoglobiny i glutationu. Przeprowadzone badania wskazują, że zastosowane stężenia mannitolu hamują hemolizę erytrocytów, nie zapobiegając utlenianiu Hb i GSH. Mannitol zapobiegał nieznacznie pęcznieniu erytrocytów w stężeniach powyżej 20 mM. Etanol w stężeniach poniżej 60 mM nie wykazuje wpływu na poziom hemolizy erytrocytów, natomiast hamuje utlenianie Hb i GSH. Nie zaobserwowano znaczącego wpływu etanolu na wielkość erytrocytów. Jednak obser...

The mechanism of the interaction of highly hydroxylated fullerenol C60(OH)36 with erythrocyte membranes was studied by electron spin resonance spectroscopy (ESR) of stearic acid derivatives labeled with a nitroxyl radical at C-12 or C-16 and with a nitroxyl derivative of maleimide covalently attached to sulfhydryl groups of membrane proteins. A significant increase in membrane fluidity in the hydrophobic region of the lipid bilayer was observed for 12-doxylstearic acid at fullerenol concentrations of 100 mg/L or 150 mg/L, while for 16-doxylstearic acid significant increase in fluidity was only observed at 150 mg/L. Fullerenol at 100 mg/L or 150 mg/L caused conformational changes in membrane proteins, expressed as an increase in the hw/hs parameter, when fullerenol was added before the maleimide spin label (MSL) to the membrane suspension. The increase of the hw/hs parameter may be caused by changes in lipid-protein or protein-protein interactions which increase the mobility of the MSL label and as a result increase the membrane fluidity. Incubation of the membranes with the MSL before the addition of fullerenol blocked the available membrane protein –SH groups and minimized the interaction of fullerenol with them. This confirms that fullerenol interacts with erythrocyte membrane proteins via available protein –SH groups.

The effect of polyhydroxylated fullerene (fullerenol), C60(OH)36, on human peripheral blood mononuclear cells (PBMCs) exposed to X-rays was studied. PBMCs untreated and treated for 1 h with C60(OH)36 at the concentrations 75 and 150 mg/l were exposed to high doses of ionizing radiation (10, 30 and 50 Gy). After 24 and 48 h of post-irradiation incubation the viability and granularity of lymphocytes were determined applying the flow cytometry (FC) method. Moreover, after 24 h of incubation the membrane fluidity was investigated by measuring the fluorescence anisotropy of a 1,6-diphenyl-1,3,5-hexatriene (DPH) probe. Additionally, DNA damage of PBMCs after exposure to X-rays at the doses 0, 5, 10 and 15 Gy in the absence and presence of fullerenol (75 mg/l) was determined using the comet assay under alkaline conditions. Results show that the effects of fullerenol C60(OH)36 on X-irradiated human PBMCs are very small or inexistent. It was suggested that this action of C60(OH)36 may be related to its interactions with the surface of plasma membrane but not inside PBMCs.

Recently, much attention has been paid to the bioactive properties of water-soluble fullerene derivatives: fullerenols, with emphasis on their pro- and antioxidative properties. Due to their hydrophilic properties and the ability to scavenge free radicals, fullerenols may, in the future, provide a serious alternative to the currently used pharmacological methods in chemotherapy, treatment of neurodegenerative diseases, and radiobiology. Some of the most widely used drugs in chemotherapy are anthracycline antibiotics. Anthracycline therapy, in spite of its effective antitumor activity, induces systemic oxidative stress, which interferes with the effectiveness of the treatment and results in serious side effects. Fullerenols may counteract the harmful effects of anthracyclines by scavenging free radicals and thereby improve the effects of chemotherapy. Additionally, due to the hollow spherical shape, fullerenols may be used as drug carriers. Moreover, because of the existence of the currently ineffective ways for neurodegenerative diseases treatment, alternative compounds, which could prevent the negative effects of oxidative stress in the brain, are still sought. In the search of alternative methods of treatment and diagnosis, today’s science is increasingly reaching for tools in the field of nanomedicine, for example, fullerenes and their water-soluble derivatives, which is addressed in the present paper.

Fullerenols, the water-soluble derivatives of fullerenes, are currently being recently intensively studied in the context of the possibility of their application in the biomedicine. Due to their hydrophilic properties and the ability to eliminate free radicals, fullerenols may in the future provide a solid alternative to currently used pharmacological methods in chemotherapy, treatment of neurodegenerative diseases and radiobiology. Depending on the research protocol applied, fullerenols may also act as pro oxidants. The dualistic nature of fullerenols may contribute to finding new biomedical applications of these agents in the future, by exerting
a cytotoxic or protective effect respectively against cancer cells or healthy cells. Because of the encapsulated structure of fullerenols, there exists the possibility of their application in
medical diagnostics in the transfer of contrast agents or in the drug transport. During the planning of an experiment designed to investigate the effects of radiation in combination with derivatives of water-soluble fullerenes, the possibility of appearance of the
“dose-response effect” should be taken into consideration since it significantly contributes to one of the two possible effects: protection or sensitization. The same applies to the possibility
of using these compounds as potential neuroprotectors. Fullerenol may protect neurons in the particular areas of the brain but in the definedcertain doses it may also induce cell death. A giant leap in the field of nanotechnology not only leads scientists to search for new applications of nanomaterials such as fullerenols, but also raises the question about their harmful effect on the environment. High utilization of hardly biodegradable fullerenols increases the
likelihood of their accidental release into natural systems and their bioaccumulation. Despite convincing evidences about the potential applications of fullerenols in biomedicine, we still have insufficient knowledge about the mechanism of action of these molecules and their possible side effects.

"The present study was aimed at investigating the effect of fullerenol C60(OH)36 on chosen parameters of the human erythrocyte membrane and the preliminary estimation of the properties of fullerenol as a potential linking agent transferring the compounds (e.g., anticancer drugs) into the membrane of erythrocytes. The results obtained in this study confirm the impact of fullerenol on erythrocyte cytoskeletal transmembrane proteins, particularly on the band 3 protein. The presence of fullerenol in each of the concentrations used prevented degradation of the band 3 protein. The results show that changes in the morphology of red blood cells caused by high concentrations of fullerenol (up to 150 mg/L) did not lead to increased red blood cell hemolysis or the leakage of potassium. Moreover, fullerenol slightly prevented hemolysis and potassium efflux. The protective effect of fullerenol at the concentration of 150 mg/L was 20.3%, and similar results were obtained for the efflux of potassium. The study shows that fullerenol slightly changed the morphology of the cells and, therefore, altered the intracellular organization of erythrocytes through the association with cytoskeletal proteins.

Highlights
• Fullerenol C60(OH)36 can associate to the cytoskeletal proteins of erythrocyte membrane.
• Fullerenol binds mostly to the surface of the transmembrane proteins such as band 3 protein.
• Fullerenol decreases the level of the potassium ion leakage and autohemolysis.
• Fullerenol can moderately/slightly influence the morphology of erythrocytes by altering the inner-organization of the cells."""

Purpose: Melatonin (MEL) is an effective antioxidant in numerous experimental models, both in vitro and in vivo. However, it should be stressed that there are also papers reporting limited antioxidative activity of MEL or even giving evidence for its pro-oxidative properties. In the present paper we investigated the influence of MEL on the oxidative damage of human erythrocytes during prolonged incubation. Material/Methods: Human erythrocytes suspended in phosphate-buffered saline (PBS), pH 7.4 were incubated at 37ºC either in absence or presence of melatonin at concentration range 0.02 mM-3 mM for up to 96 hrs. The influence of MEL on erythrocyte damage was assessed on the basis of the intensity of intracellular oxidation processes (the oxidation of HbO2, GSH, fluorescent label DCFH2) as well as damage to the plasma membrane (lipid peroxidation, the potassium leakage) and the kinetics of hemolysis. Results: The prolonged incubation of erythrocytes induced a progressive destruction of erythrocytes. Melatonin prevented lipid peroxidation and hemolysis whereas the oxidation of HbO2 and DCFH2 was enhanced by melatonin at concentrations higher than 0.6 mM. In the case of erythrocytes incubated with 3 mM of MEL, the hemolysis rate constant (0.0498±0.0039 H%•h-1) was 50% lower than that of the control while the HbO2 oxidation rate constants were about 1.4 and 1.5 times higher for 1.5 and 3 mM of MEL, respectively. Melatonin had no influence on the oxidation of GSH and the potassium leakage. Conclusions: Probably, MEL can stabilize the erythrocyte membrane due to interaction with lipids, thus prolonging the existence of cells. On the contrary, in the presence of MEL the accelerated oxidation of HbO2 and generally, increased oxidative stress was observed in erythrocytes. Pro- and antioxidative properties of melatonin depend on the type of cells, redox state, as well as experimental conditions.

"The influence of fullerenol on the activities, of human erythrocyte membrane ATPases and the fluidity of the plasma membrane as well as the possibility of fullerenol incorporation into the plasma membrane, were investigated. Fullerenol at concentrations up to 150 µg/ml induced statistically significant decreases in the anisotropy of 1-anilino-8-naphthalene sulfonate (ANS) (14%), N,N,N-trimethyl-4-(6-phenyl-1,3,5,-hexatrien-1-yl)phenylammonium p-toluenesulfonate (TMA-DPH) (7.5%) and 1,6-diphenyl-1,3,5-hexatriene (DPH) (9.5%) after a 1-hour incubation at 37°C. The effect disappeared for ANS and TMA-DPH, but not for DPH, after washing out the fullerenol. Incubation of erythrocyte membranes with fullerenol led to decreases in the activities of Na+,K+-ATPase (to 23% of the control value), Ca2+-ATPase (to 16% of control) and Mg2+-ATPase (to 22% of control). Washing out the fullerenol lessened the inhibition of the Na+,K+-ATPase (37% of control) and Ca2+-ATPase (23.5% of control); however, it did not influence Mg2+-ATPase activity. Furthermore, fullerenol could associate with erythrocyte plasma membranes.
Our results suggest that fullerenol associates primarily with the surface of the plasma membrane; however, it can also migrate deeper inside the membrane. Moreover, fullerenol influences membrane ATPases so that it may modulate ion transport across membranes."

Fullerenes are chemical structures made of carbon atoms. The stable form is molecule composed of 60 carbon atoms arranged in a soccer ball-shaped structure. With respect to its electron donor and acceptor capability and photochemical behavior fullerenes can be effective antioxidants and radical scavengers or prooxidants and photosensitizers. These properties of fullerenes have paid attention on their possible biological applications. Results of previous studies point to the great dependance of fullerenes activity upon quality, quantity and geometry of substituents in fullerene derivatives. Some of fullerene derivatives show antiviral and antimicrobial activity, including anti-HIV properties. C60 and its derivatives are able to exhibit cytotoxic and enzyme-inhibiting abilities as well as radical-quenching and antioxidative abilities. Generation of reactive oxygen species under influence of visible light is another ability of fullerene derivetives desired in photodynamic therapy.

Molecule of fullerene, having a spherical or ellipsoidal shape, is made of rings consisting of five or six carbon atoms, combined with conjugated pi bonds. Delocalization of pi electrons in the molecule of fullerene makes it easy to scavenge free radicals. But, despite being the effective antioxidants and radical scavengers fullerenes may be prooxidants by reactive oxygen species generation. Mammalian cells consist mainly of water (about 70%). Thus, the radical and non-radical products of water radiolysis are the basic sources of radiation damage to biomolecules. Reactive oxygen species, such as hydroxyl (HO*) and superoxide (O2-*) radicals and hydrogen peroxide (H2O2), are responsible for radiation-induced damage in aerated systems. Free radical mechanism of radiation damage suggests that scavengers of free radicals should protect cellular structures against damage. Electron donor compounds should also exhibit protective properties towards oxidized functional groups by reducing them. However, the electron transfer from fullerene to oxygen may generate superoxide radical. The shape of fullerenes allows them to act as carriers of radioactive atoms of isotopes used in the therapy and medical diagnostics. Fullerenes and their derivatives due to its properties are new promising chemicals for application in radiobiology. Fullerenes may be radioprotectors, radiosensitizer or auxiliary compounds in diagnostic imaging. What they are depends on the experimental system used.

We studied the influence of hydroxyl radical scavengers mannitol and glucose on the level of lipid peroxidation in EG (erythrocyte ghosts). OH radicals were generated by gamma-radiation or by the Fenton or Fenton-like reaction. Glucose and mannitol protected EG from peroxidation caused by OH radicals generated by gamma-radiation and by the Fenton-likę reaction carried out in the presence of endogenous catalase and at a Fe(II)EDTA:H2O2 at ratio of 1:1. But when the ratio of a Fe(II)EDTA: H2O2 increased to 1:150, gIucose and mannitol radicals probably reacted with H2O2 undergoing degradation to products reacting with TBA.

Proteins are major targets for oxidative damage due to their abundance in cells and high reactivity with free radicals. In the present study we examined the influence of oxygen on radiation-induced inactivation and structural changes of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH). We chose these two enzymes because they occur at high concentrations and participate in the most important processes in organisms; furthermore, they show considerable similarity in their structure.

Protein solutions were irradiated with X-rays in doses ranging from 0.1 to 0.7 kGy, in air and N2O. The much higher radiation inactivation of GAPDH as compared to LDH is correlated with substantially greater structural changes in this protein, mainly involving the loss of free thiol groups (–SH). Of lesser importance in the differentiation of the radiosensitivity of the studied enzymes are tryptophan residues. Molecular oxygen, present during irradiation, increased to a significantly greater extent the inactivation and structural changes of GAPDH than that of LDH. The results suggest that the greater effect of oxygen on GAPDH is due to the higher efficiency of the superoxide radical, the higher amount of hydroperoxides generated, and the higher degree of unfolding of this protein.

Highlights
► Effect of irradiation on the activity and structure of GAPDH and LDH. ► GAPDH is considerably more radiosensitive than LDH. ► Oxygen during irradiation greatly enhances the radiosensitivity of GAPDH than LDH. ► The main role in radiosensitivity is played by — SH groups, Trp plays slight role. ► High radiosensitivity GAPDH is linked with higher level of hydroperoxides generated.

The erythrocyte suspensions in Na-phosphate buffered isotonic NaCl solution (PBS) or Na-phosphate isotonic buffer (PB) (hematocrit 1%) were irradiated with the dose of 400 Gy under N2O. Erythrocytes were incubated in the medium in which the cells were irradiated or in fresh PBS. The level of damage to cells was estimated on the basis of the course of post-radiation hemolysis and hemoglobin (Hb) oxidation. The medium in which the cells were irradiated and incubated influenced the course of the post-radiation hemolysis and Hb oxidation as well as some other parameters. We discussed the contribution of hydroxyl and chloride radicals in the initiation of erythrocyte damage and oxygen modification of these processes.

Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit of 2%) were irradiated with γ-rays at three dose-rates of 66.7, 36.7, 25 Gy min−1 in order to investigate the influence of the dose-rate on radiation-induced membrane damage, hemoglobin oxidation and loss of reduced glutathione.The obtained results showed that such processes as erythrocyte hemolysis, lipid and protein destruction depend on the radiation dose-rate. The parameter values describing these processes showed an inverse dose-rate effect.

The erythrocyte suspensions in PBS (Na-phosphate buffered isotonic NaCl solution) or PB (Na-phosphate isotonic buffer) (hematocrit 1%) were irradiated with the dose of 400 Gy in aerobic conditions. The level of damage to cells was estimated after incubation in different media. A higher level of destruction of cells irradiated in PBS than in PB was observed. The same level of MetHb and lipid peroxidation determined right after irradiation was detected. However, the loss of reduced glutathione was higher in PB than in PBS. We discussed the contribution of hydroxyl and chloride radicals in the initiation of erythrocyte damage.

Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit of 2%), were irradiated with γ-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.

The effect of phenolic compounds: phenol, 2,4–dichlorophenol (2,4-DCP), 2,4-dimethylphenol (2,4-DMP) and catechol on human erythrocytes was studied. The level of fluorescent label – 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) oxidation by phenolic compounds in erythrocytes as well as the carbonyl group content and hemoglobin denaturation were monitored. H2DCFDA has been utilized extensively as a marker for studies of oxidative stress at the cellular level.We noted that 2,4-DCP, 2,4-DMP and catechol induced an increase in the concentration- and time-dependent H2DCFDA oxidation. We also observed an increase in carbonyl group content and the changes in parameter T (denaturation of hemoglobin) in erythrocytes incubated with 2,4-DCP, catechol and 2,4-DMP. The highest level of H2DCFDA oxidation was provoked by 2,4-DCP. The biggest changes of proteins in erythrocytes measured as the carbonyl group content were induced by 2,4-DMP, but measured as parameter T they were induced by catechol. It was observed that phenol did not oxidize H2DCFDA up to the concentration of 2.5 mM after 3 h of incubation. Phenol did not affect the carbonyl group content but decreased parameter T (induced denaturation of hemoglobin).To sum up, the kind of the substituent in a phenolic ring determines the molecular mechanism of action of the individual compound and the capacity of reactive oxygen species generation and thus damages the specified structures in human erythrocytes.

Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit 2 %) were exposed under air to gamma radiation at a dose rates of 2.2 kGy·h-1 and 4.2 kGy·h-1. The dose-response curves for hemolysis of erythrocytes indicated that the process of hemolysis is inversely related to the dose-rate. At both dose-rates we observed a reduced level of hemolysis, when erythrocytes were irradiated with a split dose (0.4 kGy+2.3 kGy with an interval time between the subsequent exposures from 1 to 4 h) in comparison with the same single dose (2.7 kGy). The maximal effect of fractionation was observed when the interfraction time was equal to 3.5 h. The influence of the interfraction temperature on this effect was observed. The results obtained indicate that enucleated human erythrocytes under suitable radiation conditions are capable of repairing radiation damage which leads to hemolysis.

The effects of interaction of ethanol, l-butanol or t-butanol peroxyl radicals with oxyhemoglobin (HbO2) were estimated using absorption spectroscopy in the visible range and tryptophan fluorescence. Peroxyl radicals were generated by gamma-radiation. Alcohol peroxyl radicals showed the same effectiveness in oxidizing HbO2 iron as -OH radicals (GFe(III) = 1.8 for a dose 0.33 kGy). However, they degraded hemoglobin to hemichromes and cholehemichromes to a lesser degree than -OH radicals. In addition l- and t-butanol peroxyl radicals were less effective than peroxyl ethanol radicals. Alcohol peroxyl radicals caused unfolding of protein to a much lesser degree than -OH radicals. Their contribution to the destruction of tryptophan residues was nearly matched by those obtained with the -OH radicals.