MARKERI OKSIDATIVNOG STRESA U HRONIČNOJ LIMFOCITNOJ LEUKEMIJI

Transcription

1 Original article Medicus 2006; 7(2): UDK Originalni nauèni rad OXIDATIVE STRESS MARKERS IN CHRONIC LYMPHOCYTIC LEUKEMIA Predrag Djurdjević, Ivanka Zelen 2, Petar Ristić 5, Ivan Jovanović 3, Vladimir Jakovljević 4, Dejan Baskić 3, Suzana Popović 3 and Nebojša Arsenijević 3 Department of Pathophysiology, Faculty of Medicine, University of Kragujevac, 2 Department of Biochemistry, Faculty of Medicine, University of Kragujevac, 3 Department of Microbiology and Immunology, Faculty of Medicine, University of Kragujevac, 4 Department of Physiology, Faculty of Medicine, University of Kragujevac, 5 Public Health Institute, Kragujevac MARKERI OKSIDATIVNOG STRESA U HRONIČNOJ LIMFOCITNOJ LEUKEMIJI Predrag Djurdjević, Ivanka Zelen 2, Petar Ristić 5, Ivan Jovanović 3, Vladimir Jakovljević 4, Dejan Baskić 3, Suzana Popović 3 i Nebojša Arsenijević Katedra za patofiziologiju, Medicinski fakultet, Univerzitet u Kragujevcu, 2 Katedra za biohemiju, Medicinski fakultet, Univerzitet u Kragujevcu, 3 Katedra za mikrobiologiju i imunologiju, Medicinski fakultet, Univerzitet u Kragujevcu, 4 Katedra za fiziologiju, Medicinski fakultet, Univerzitet u Kragujevcu, 5 Institut za zaštitu zdravlja, Kragujevac Received/Primljen: Accepted/Prihvaćen: ABSTRACT Chronic lymphocytic leukemia is characterized by the progressive accumulation of small immature lymphocytes which do not proliferate and that remain predominantly (more than 95%) in the G0 phase of the cell cycle. Expansion of malignant cell clone appears to be due to an underlying defect in its ability to undergo apoptosis. One of the potential mechanisms of defective apoptosis could be irregular oxidative stress. The goal of our investigation was to determine the plasma level of nitric oxide, superoxide anion and malondialdehyde in patients with chronic lymphocytic leukemia as markers of oxidative stress. Thirty patients with untreated chronic lymphocytic leukemia in the A stage of the disease classified by Binet and thirty healthy volunteers were examined. Nitric oxide (its stable metabolites, nitrite (NO 2-2 ) and nitrate (NO 3-2 ), superoxide anion and malondialdehyde were measured by spectrophotometry in plasma of both investigated groups which were obtained from heparinized whole blood after centrifugation. Our results showed that the plasma levels of nitrate/nitrite (32.2 ± 7.25 vs ± 7.27, p<0.0), superoxide anion (0.34 ± 9.40 vs 8.52 ± 6.29, p<0.0) and malondialdehyde (2.76 ±.6 vs.37 ± 0.90, p<0.00) were increased in patients with chronic lymphocytic leukemia than in the group of healthy volunteers. These data suggest that there is more intensive oxidative stress in patients with chronic lymphocytic leukemia. It could be one of the potential mechanisms in the pathogenesis of irregular apoptosis of malignant lymphocytes and it could take part in the genesis of chronic lymphocytic leukemia. Key words: chronic lymphocytic leukemia, nitric oxide, superoxide anion, malondialdehyde. Abbreviations: CLL chronic lymphocytic leukemia, NO - nitric oxide, MDA malondialdehyde, ROS reactive oxygen species, IL-4 interleukin 4, CD cluster differentiation molecule, SOD superoxide dismutase INTRODUCTION Chronic lymphocytic leukemia (CLL) is predominantly clonal B-cell neoplasm of small, resting, long-lived B- cells. Despite recent advances in understanding of genetics (), biology (2), clinical behavior (3) and treatment (4), CLL has not been cured yet and its progression and outcome are highly unpredictable. CLL cells co-express low levels of surface membrane molecules CD5, CD23 and weak CD22 and surface membrane immunoglobulin (5). Expansion of these malignant cells leads to the accumulation in the peripheral blood, bone marrow and many tissues. These cells are functionally defective and immunologically distinct from normal B cells (6). The clinical course of is highly heterogenous, ranging from less than two years in symptomatic patients with the advanced disease to more than twenty years for patients with an early stage and non-progressive SAŽETAK Hronična limfocitna leukemija je progresivna maligna bolest koja nastaje proliferacijom i akumulacijom klona imunski nekompetentnih limfocita u kostnoj srži, limfnim žlezdama, slezini i drugim organima. Smatra se da je osnovni mehanizam nastanka bolesti smanjene apoptoze limfocita in vivo. Uzroci ovog poremećaja nisu potpuno istraženi, ali se smatra se da jedan od mogućih mehanizama može biti i oksidativni stres. Cilj ovog istraživanja bio je određivanje plazmatskih koncentracija azotmonoksida, superoksid anjona i malonildialdehida kao markera oksidativnog stresa. Ispitivanu grupu sačinjavalo je 30 obolelih od hronične limfocitne leukemije koji nisu lečeni antineoplastičnom terapijom i koji su bili u A stadijumu bolesti po Binet-u, dok je kontrolnu grupu sačinjavalo 30 zdravih ispitanika slične polne i starosne strukture. Posle centrifugiranja heparinizovane venske krvi svih ispitanika izdvajana je plazma u kojoj su određivane koncentracije markera oksidativnog stresa. Koncentracije azotmonoksida (32.2 ± 7.25 ), superoksid anjona (0.34 ± 9.40 ) i malonilaldehida (2.76 ±.6 ) u plazmi bolesnika obolelih od hronične limfocitne leukemije visoko statistički značajno veće od koncentracija istih molekula u plazmi ispitanika kontrolne grupe (NO ± 7.27 ; superoksid anjon 8.52 ± 6.29 ; malonildialdehid.37 ± 0.90 ). Ovi podaci ukazuju da je oksidativni stres bolesnika znatno veći u poređenju sa kontrolnom grupom i da bi smanjenje apoptoze limfocita obolelih od hronične limfocitne leukemije moglo biti povezano sa modifikovanim oksidativnim stresom. Ključne reči: hronična limfocitna leukemija, azotmonoksid, superoksid anjon, malonilaldehid, oksidativni stres. Skraćenice: CLL chronic lymphocytic leukemia, NO - nitric oxide, MDA malondialdehyde, ROS reactive oxygen species, IL-4 interleukin 4, CD cluster differentiation molecule, SOD superoxide dismutase disease (7). Although the pathogenesis of has not been fully elucidated, the progressive increase of lymphocyte count coupled with the very low proportion of proliferating cells has led to the notion that may be determined by defective apoptosis (8). Precise mechanisms underlying apoptosis have still remained unknown. Dysregulation of the p-53, c-myc and bcl-2 oncogenes can be the cause of defective apoptosis in B- CLL and even though the cell molecular alterations involving different oncogenes and tumor supressor genes have been established, the role of oxidative stress in the pathogenesis of this disease is poorly understood and it is a matter of interest (9). Oxidative stress is a well-known phenomenon in the body which plays an important role in the pathogenesis of various diseases and syndromes (0). Reactive oxygen species (ROS) consist of superoxide anion (O 2 ), Correspondence: Predrag M Djurdjević Department of Pathophysiology, Faculty of Medicine, University of Kragujevac Svetozara Markovića 69, Kragujevac Phone: , Mob phone: zekapeka@ptt.yu

2 hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (OH.) which are generated by cells in some physiological and pathological conditions. Any dearrangement between pro-oxidants and antioxidants in which pro-oxidants prevail, is known as oxidative stress. ROS can react with all macromolecules, particularly with polyunsaturated fatty acid on the cell membrane and this process is named lipid peroxidation. The secondary product of this process is malondialdehyde which is a useful indicator for this reaction (). After the beginning of an initial reaction with ROS a continuing chain reaction starts followed by cell injury (2). One of the most abundant free radicals in the body is nitric oxide (NO) and its excess production can cause inhibition of mitochondrial respiratory enzymes (3). All these radicals can interact with the nucleic acid, proteins and lipids causing cellular dysfunction and even death. Interaction with the nucleic acids can lead to genetic disturbances which may cause irregular B lymphocyte apoptosis and that can be a critical event in the pathogenesis of. According to these facts, the aim of our investigation was to determine the plasma level of nitric oxide species (nitrates and nitrites), superoxide anion, hydrogen peroxide and malondialdehyde in CLL patients as parameters of oxidative stress. MATERIAL AND METHODS Patients and s Thirty patients (3 females and 7 males, median age 67.8 years, range years) and thirty healthy subjects (5 females and 5 males, median age 63.8 years, range years) were included in the study. was diagnosed according to standard clinical and laboratory criteria. Only patients with the stage A, according to Binet s clinical classification of the disease were studied. None of the patients had been treated with any kind of cytoreductive therapy at least 6 months at the time samples were obtained. Patients with infectious diseases or other conditions which could alter results and consequently influence the outcomes of our study were excluded. The s were healthy volunteers without known acute and chronic diseases. Exlusion criteria were positive parameters of the systemic inflammation (high erythrocyte sedimentation rate, high serum fibrinogen level, high serum C-reactive protein level) due to other etiologies or positive anamnestic data for other illnesses (eg autoimmune diseases, acute and chronic infections, systemic and local inflammations etc) that might have influenced the investigated parameters. All subjects (patients and s) were non-smokers, none of whom received any systemic and topical treatment such as corticosteroids, cyclosporine A and similar drugs which might affect oxidative stress parameters within six months prior to initiation of the investigation. None of the patients and s had alcohol abuse problems and none of them performed regular exercise other than daily activities. The local Ethics Committee approved the study and prior to initiation, the written informed consent was obtained from all subjects according to the Declaration of Helsinki. Methods All blood samples were obtained after 2 hours of fasting, in the morning, at the same time and were collected in polystyrene tubes. Nitric oxide, superoxide anion and malondialdehyde were investigated in the plasma samples. The plasma samples of both groups were stored at -20 C until assayed. NO determination Before testing, the plasma samples were deproteinazed by using acid solution. In 500μL tubes, 00 μl of 3M perchloric acid, 400 μl of 20mM EDTA and 200 μl of serum were added. Extracts were incubated in ice for 20 minutes with occasional mixing and then centrifugated at 5000rpm for 5 minutes. The supernatants were removed into other tubes and 20μL 2M potassium-carbonate was added to neutralize the extracts. The neutralized extracts were stored at -20 C until testing. Just before the use, extracts were defrosted and centrifuged in order to reduce presence of potassiumperchlorate particles. NO values in plasma were measured in the form of nitrites (NO 2 ) and nitrates (NO 3 ) as final stable products of NO metabolism. The method for detection the plasma nitrate and nitrite levels was based on the Griess reaction modified by Baskic et al (4). Griss reagent was prepared ex tempore just before the experiment by mixing equal amounts of stocks: 2% (w/v) sulfanil-amide dissolved in 5% HCl and 0.% (w/v) aqueous solution of N-- naphtyl-ethylene-diamine-dihydrochloride (N-NEDA). Nitrate and nitrite solutions in H 2 O (0mM) were prepared fresh, daily. Nitrate standard solution was serially diluted (final concentration after adding other reagents μm) in a 96-well, flat-bottom polystirene microtiter plate in final volume of 00μL. The destilated aqua (diluting medium) was used as the standard blank. After loading the plate with samples (00μl), addition of VCl3 (00μl) to each well was rapidly followed by addition of the Griess reagents (00μl). Blank consisted of the diluting medium and Griess reagent. Nitrite was measured in a similar manner except samples and nitrite standards that were exposed only to the Griess reagent. In either case the absorbance was measured at 540nm (Multiplate Reader 230S, Organon) following 30 minutes of incubation. Concentration of NOx (NO NO 2 2 ) was determined by using Xia software for data analysing based on the standard curve which was obtained by linear regression of absorbance values for each standard reduced for blank values. Results were expressed as nanomoles per mililiter (nmol/ml). Superoxide anion determination Superoxide anion values in plasma were measured by using the method of Auclair et Voisin (5). Actually, determination was performed by spectrophotometric method

3 based on the reaction of superoxide anion with Nitro Blue Tetrazolium (NBT) which formed nitrophormazan blue. As a standard solution, we used adequate amount of Krebbs-Hensenleite solution. Absorbance was registered at 550nm with appliance of spectrophotometer Multiplate Reader 230S, Organon. Concentration of superoxide anion was calculated by using mathematical formula which was noted in the method. Results were expressed as nanomoles per mililiter (nmol/ml). MDA determination Lipid peroxidation product malonildialdehyde (MDA) concentration in plasma was determined by thiobarbituric acid assay according to the protocol of Ohkawa (6). At high temperature and low ph value, malondialdehyde reacts with 2-thiobarbituric acid via nucleophylic addition. The product of this reaction is a colored substance and concentration of MDA correlates with the color intensity of this mixture. Extracts of lipid peroxides were obtained from plasma by using 0.5 vol 28% TCA (three chlor acetate). Interval of extraction was 30 minutes in ice container with sporadic stirring. Prior to performing the tests, TCA treated exctracts were stored at the temperature -20 C. Defrosted extracts were centrifuged at 5000 rpm (Eppendorf 545 D) for 4 minutes. TBA was added to translucent supernatants thus creating mixture with volumetric ratio 4: (exctract:tba). For this purpose we applied 0.% TBA dissolved in 0.05 M NaOH. These mixtures were then incubated for 5 minutes at 95 C. Color intensity was determined by using spectrophotometric method (LKB Biochrom Ultrospec 4050). Absorbance was measured at 532 nm. Molar absorbance coeffitient for MDA:TBA complex is.56x0 5 mol L - cm-. For apparatus calibration, the blank sample was used. It consisted of all required reagents except the plasma sample which was replaced by destilated water. Concentration of thiobarbituric acid reactans (TBARs) was calculated according to formula: CMDA = [ΔA x TV E x D x V an ] x R x F (nmol/ml) ΔA absorbance of serum sample excluding absorbance of blank sample E molar absorbance coefficient for MDA:TBA complex.56x0 5 mol L x cm D optical path length TV amount of incubated mixture V an amount of plasma sample R dispersion of plasma sample F multiplying factor (x0 6 ) for conversion from mol/l to nmol/ml. Statistical analysis All values are expressed as mean ± standard deviation (SD). Commercial SPSS (Statistical Package for the Social Sciences) version.0. for Windows was used for the statistical analysis. Statistical evaluation was performed by Student s T test for paired observations, one-factorial and two-factorial analysis of variance. The differences were considered to be significant when p value was less than 0.05 and highly significant when p value was less than 0.0. RESULTS We found that NO plasma values were higher in CLL patients than in the group. Average NO value in patients with was 32.2 ± 7.26 and in the subjects, it was ± 7.27 (p<0.0) (Figure ). Figure. NO plasma level in patients and in the group Superoxide anion plasma level in patients was higher than in the subjects (Figure 2). Difference in superoxide anion concentration between the tested groups was highly statistically significant (0.34 ± 9.40 vs 8.52 ± 6.29 ; p<0.0) p<0.0 Figure 2. Superoxide anion plasma level in patients and in the group. We found similar results in concentration of MDA (figure 3). MDA plasma level in patients was higher than in the group and the observed difference was highly statistically significant (2,76 ±.6 vs.37 ± 0.90, p<0.00). 3 2,5 2,5 0,5 0 p<0.0 p<0.00 Figure 3. MDA concentrationin patients and in healthy volonteers.

4 DISCUSSION For organisms living in an aerobic environment, exposure to ROS is continuous and unavoidable. ROS are potentially toxic products of cellular metabolism that are generated during the production of adenosine triphosphate by aerobic metabolism in the mitochondria where electrons escaping from the respiratory complexes I and III can react with molecular oxygen to produce oxygen radicals (7). According to their reactive chemical properties, ROS may cause various types of tissue injury. Overproduction of ROS above capability of naturally produced antioxidants may represent a molecular basis for initiation and promotion of multistage carcinogenesis. Highly reactive ROS may interact with DNA inducing a multitude of oxidative modifications and they are implicated in the mutagenesis by protooncogene activation and tumor supressor gene inhibition (8). Our results indicate that oxidative stress and lipid peroxidation are accelerated in patients with. We demonstrated that, although the plasma levels were heterogenous, there was a significant increase in the plasma NO value in patients. In the study of Bakan et al (9) similar results were demonstrated as well as there was no significant difference in NO level on the basic disease stages. NO exerts different effects on cell death depending on conditions (20). Effects on apoptosis depend on NO concentration, flux and cell type (2). In some situation NO activates the transduction pathways which leads to apoptosis whereas in other cases NO protects cells against spontaneous or induced apoptosis (20, 2). Many mechanisms for inhibition of apoptosis have not been known yet. NO inactivates caspases (such as caspase-3) through oxidation and nitrosylation of the active cystein (20), stimulates cgmp-dependent protein kinase, modulates the member bcl-2/bax family oncogenes, induces synthesis of heat shock protein 70 and takes part in the ceramide pathways (2). Otherwise NO is synthesized from L-arginine by three isoforms of NO synthase (NOS), two of which (endothelial and neuronal NOS) are constitutively expressed and are regulated by calcium/calmodulin and phosphorylation while the third (inducible NOS) synthase is induced during different pathological events and it produces higher level of NO (22). Inducible isoform of NOS is spontaneously expressed in cells (23, 24) which is the reason for increased generation of NO by. This spontaneous expression of inos is caused by cytokines IL-4 and interferon-gamma which prevent spontaneous in vitro apoptosis of cells (25). Flavones and polyphenols can down-regulate inos and consequtively decrease the endogenous NO production which induces apoptosis of cells in vitro (26). Inhibition of the inos pathway leads to the increased apoptosis of tumor cells in vitro indicating that endogenous production of NO increases malignant cell resistance to the normal apoptotic process All factors which induce high expression of inos in vivo in the cells have not been identified yet.. Engagement of CD23 stimulates inos activation in malignant cells suggesting that in vivo interaction of CD23 with one of its ligands may contribute to inos induction. Interaction of CD40-CD40 ligand may also be involved in the inhibition of apoptosis via the inhibition of caspase activities (24). Mitochondrial respiration is the major biochemical pathway for production of superoxide anion in cells. We showed that the plasma level of O 2 was significantly higher in patients than in the group. Recent studies have demonstrated similar results.the accumulation of O 2 leads to the free-radical-mediated damage of mitochondrial membranes and activation of apoptotic cascade (27). CLL cells generate increased levels of O 2 which is associated with mitochondrial DNA mutations (28). Accumulation of this radical in cells was associated with morphological and biochemical changes typical of apoptosis (29). Leukemia cells from different patients have different rates of O 2 generation (29). Inhibition of superoxide dismutase (SOD) led to the accumulation of O 2 (27, 29). Cell contents of O 2 depend on previously used therapeutic agents. Actually, the O 2 level was significantly higher in CLL cells isolated from previously treated patients with various treatment than the CLL cells from previously untreated patients (29). CLL cells at different stages of the disease may have different metabolic activities and thus produce various level of O 2 depending on the energy requirement by leukemia cells (29). Using agents for ROS production and SOD inhibition in CLL cells may be a new strategy to enhance therapeutic activity (30). The process of lipid peroxidation is one of oxidative conversions of polyunsaturated fatty acids which are important for normal function of most mammalian cells. MDA is one of end-products of lipid peroxidation induced by ROS and well-correlated with the degree of lipid peroxidation (3). Lipid peroxidation of the cellular structrure may play an important role in the pathogenesis of many pathological processes such as carcinogenesis. In our study, significantly higher MDA plasma level in patients than in the group indicates the intensive lipid peroxidation process which can be the pathogenetic basis of the disease. There are different results of serum MDA level in patients in literature. Devi et all (32) showed that plasma lipid peroxidation products in untreated leukemia patients (such as ) were in normal range. Our results are in accordance with some other studies (9, 33). There is also extensive lipid peroxidation in malignant B-cells as indicated by an increased MDA value in these cells which is in accordance with different forms of DNA bases lesions (33). MDA level and degree of DNA bases damages in transformed lymphocytes are positively correlated with the duration of disease (33). Our findings suggest that extensive oxidative stress caused by ROS may be related to the pathogenesis of CLL. The identification of adequate oxidative markers for tumor cell metabolism may be useful for early diagnosis and assessment of tumor progression. Understanding of endogenous mechanisms of carcinogenesis by serious

5 oxidative stress and molecular action of carcinogens must be further elucidated. REFERENCES. Döhner H, Stilgenbauer S, Benner A et al. Genomic aberration and survival in chronic lymphocytic leukemia. N Engl J Med 2000; 343: Meinhardt G, Wendtner C, Hallek M. Molecular pathogenesis of chronic lymphocytic leukemia: factors and signaling pathways regulating cell growth and survival. J Mol Med 999; 77: Rai K, Peterson B, appelbaum F et al. Fludarabin compared with chlorambucil as primary therapy for chronic lymphocytic leukemia. N. Engl J Med 2000; 343: Kipps T. Chronic lymphocytic leukemia. Curr Opin Hematol 2000; 7: Moreau EJ, Matutes E, A Hern RP et al. Improvement of the chronic lymphocytic leukemia scoring system with the monoclonal antibody SN8 (CD79b). Am J Clin Pathol 997; 08: Mainou-Fowler T, Miller s, Proctor SJ, Dickinson AM. The levels of TNFα, IL4 and IL0 production by T-cells in B-cell chronic lymphocytic leukemia (). Leukemia Res 200; 25: Eisterer W, Bechter O, Söderberg O et al. Elevated levels of soluble CD44 are associated with advanced disease and in vitro proliferation of neoplastic lymphocytes in B-cell chronic lymphocytic leukemia. Leukemia Res 2004; 28: Jones DT, Geneshaguru K, Anderson RJ et al. Albumin activates the AKT signaling pathway and protects B-chronic lymphocytic leukemia cells from chlorambucil- and radiation-induced apoptosis. Blood 2003; 0: Dierlamm J, Michaux L, Criel a et al. Genetic abnormalities in chronic lymphocytic leukemia and their critical and prognostic implications. Cancer Gen Cytogenet 997; 94: Ben-Menachem E, Kyllerman M, Marklund S. Superoxide dismutase and gluthatione peroxidase function in progressive myoclonus epilepsies. Epilepsy Res 2000; 40: De Zwart LL, Meerman JHN, Commandeur JNM, Vermeuoen MPE. Biomarkers of free radical damage: Application in experimental animals and in humans. Free Radic Biol Med 999; 26: Kannan K, Jain SK. Oxidative stress and apoptosis. Pathophysiology 2000; 7: Sozmen EY, Kerry Z, Uysal F et al. Antioxidant enzyme activities and total nitrite/nitrate levels in the collar model. Effect of nicardipine. Clin Chem lab Med 2000; 38: Baskić D, Jovanović I, Ristić P, Jakovljević V, Delibašić Đ, Arsenijević N. Spectrophotometric method for simultaneous detection of nitrate and nitrite. Medicus 2005; 6(2): Auclair C, Voisin E. Nitroblue tetrazolium reduction. In: Greenwald RA (ed): Handbook of methods for oxygen radical research, CRC Press, Inc, Boka Raton 985, pp Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxide s in animal tissue by thiobarbithuric acid reaction. Anal Biochem 979; 95: Sankarapandi S, Zweier JL. Evidence against the generation of free hydroxyl radicals from the interaction of copper, zinc su- Acknowledgements: This study was supported by grant 637 from the Ministry of Science and Enviromental Protection, Serbia peroxide dismutase and hydrogen peroxide. J Biol Chem 999; 274: Du MQ, Carmichael PL, Phillips DH. Induction of activating mutations in the human C-Ha-ras- protooncogenen by oxygen free radicals. Mol Carcinog 994; : Bakan N, Taysi S, Yilmaz O et al. Glutathione peoxidase, glutathione reductase, Cu-Zn superoxide dismutase activities, glutathione, nitric oxide and malondialdehide concentrations in serum of patients with chronic lymphocytic leukemia. Clin Chim Acta 2003; 338(-2): Brown GC, Borutaite V. Nitric oxide inhibition of mitochondrial respiration and its role in cell death. Free Radic Biol Med 2002; 33: Kolb JP. Mechanisms involved in the pro- and antiapoptotic role of NO in human leukemia. Leukemia 2000; 4(9): Ignarro JL. Nitric oxide: biology and pathobiology. Ed. San Diego: Academic Press, Billard C, Kern C, Tang R, Ajchenbaum Cymbalista F. Kolb JP. Flavopiridol downregulates the expression of both the inducible NO synthase and p27(kip) in malignant cells from B-cell chronic lymphocytic leukemia. Leukemia 2004; 8(2): Kolb JP, Roman V, Mentz F et al. Contribution of nitric oxide to the apoptotic process in human B cell chronic lymphocytic leukemia. Leuk Lymphoma 200, 40(3-4): Levesque MC, Misukonis MA, O Loughlin CW et al. IL-4 and interferon gamma regulate expression of inducible nitric oxide synthase in chronic lymphocytic leukemia cells. Leukemia 2003; 7(2): Quiney C, Dauzonne D, Kern C et al. Flavones and polyphenols inhibit the NO pathway during apoptosis of leukemia B-cells. Leuk Res 2004; 28(8): Huang P, Feng L, Hileman EO, Keating MJ, Plunkett W. Superoxide dismutase as a target for the selective killing of cancer cells. Nature 2000; 407: Carew JS, Nawrocki ST, Xu RH et al. Increased mitochondrial biogenesis in primary leukemia cells: the role of endogenous nitric oxide and impact on sensitivity to fludarabin. Leukemia 2004; 8(2): Zhou Y, Hileman EO, Plunkett W, Keating MJ Huang P. Free radical stres sin chronic lymphocytic leukemia cells and its role in cellular sensitivity to ROS-generating anticancer drugs. Blood 2003; 0: Miller WH Jr. Moleculare targets of arsenic trioxide in malignant cells. Oncologist 2002; 7(suppl ): 4-9. Latha B, Babu M. The involvement of free radicals in burn injury: a review. Burns 200; 27: Devi GS, Prasad MH, Sarawathi I, Raghu D, Rao DN, Reddy PP. Free radicals antioxidant enzymes and lipid peroxidation in different types of leukemias. Clin Chim Acta 2000; 293(-2): Oltra AM, Carbonell F, Tormos C, Iradi A, Saez GT. Antioxidant enzyme activities and the production of MDA and 8-OXO-DG in chronic lymphocytic leukemia. Free Radic Biol Med 200; 30: