Role of Free Radical Reactions in Myelodysplastic Syndrome. Hitoshi IMANISHI,1,* Shinichi MISAWA,1 Tatsuro and Tatsuo ABE 2

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1 J. Clin. Biochem. Nutr., 5, 75-79, 1988 Role of Free Radical Reactions in Myelodysplastic Syndrome Hitoshi IMANISHI,1,* Shinichi MISAWA,1 Tatsuro and Tatsuo ABE 2 TAKINO,1 1Third Department of Internal Medicine and 2Department of Hygiene, Kyoto Prefectural University of Medicine, Kyoto 602, Japan (Received December 28, 1987) Summary Activities of glutathione linked enzymes in red blood cells and concentrations of other free radical scavengers in red blood cells and in plasma were assayed in patients with myelodysplastic syndromes (MDS). Glutathione peroxidase (GSH-Px) activity, concentrations of both reduced glutathione and oxidized glutathione in red blood cells, and lipid peroxide levels in plasma were all significantly increased in MDS patients. Levels of other free radical scavengers in plasma (total vitamin B2, FAD, and coenzyme Q10), however, were decreased. The data suggest that MDS patients may have been exposed to free radicals, and may be hypersensitive to the oxygen intoxication. Key Words: free radical reactions, myelodysplastic syndrome, oxidoreductive system The myelodysplastic syndromes (MDS) are considered to be preleukemic states that are indicated by abnormalities in more than two cell lines in the bone marrow, and are characterized by one or more cytopenias in the peripheral blood. The French-American-British (FAB) Co-operative group proposed a classification of MDS based on morphological criteria [1]. MDS may precede most cases of acute leukemia. Some enzymatic and biochemical investigations in dyserythropoiesis have been reported recently. Enzyme deficiencies of glycolytic enzymes including pyruvate kinase, changes in antigen (A, B, H, and I), and increased hemoglobin F levels have been observed in the peripheral red blood cells of patients with dyserythropoiesis [2-4]. It was also reported that in most cases of MDS, hematological stem cells are hypersensitive to oxygen intoxication [5]. In relation to this, we previously found that the activities of the free radical-scavenging enzymes (glutathione linked enzymes) and the reduced glutathione (GSH) levels in red blood cells decrease with their aging [6, 7]. *To whom correspondence should be addressed. 75

2 76 H. IMANISHI et al. In the present study, changes in the oxidoreductive systems in peripheral red blood cells and in plasma were investigated in MDS patients. MATERIALS AND METHODS 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB), N-ethyl-maleimide, flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NADP), reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), glutathione reductase (GR) (Type III), and t-butyl hydroperoxide were purchased from Sigma Chemical Co. Ltd. (St. Louis, MO.). Other chemical reagents were of reagent grade. Human blood from 8 healthy male donors and that from 14 untreated patients with MDS were collected from the brachial vein with a heparinized tube syringe. The blood cells were washed with ice-cold physiological saline and centrifuged at 1,350 x g for 7 min three times after removal of the buffy coat. Plasma was stored at - 70 C until estimations for riboflavin and its derivatives, co-enzyme Q10 (CoQ1o) and lipid peroxide level. The classifications and numbers of the patients with MDS were as follows : refractory anemia (RA), 3 ; RA with ring sideroblasts (RARE), 2 ; RA with excess of blasts (RAEB), 6; RAEB in transformation (RAEB-T), 2; and idiopathic macrocytosis, 1. The concentrations of GSH and those of oxidized glutathione (GSSG), and activities of glutathione peroxidase (GSH-Px) and GR in washed red blood cells were assayed according to Beutler's method [8]. Enzymatic reactions were recorded at 340 nm at 37 C in the Hitachi Model recording spectrophotometer. Enzyme activities were expressed as pmol of NADP reduced or NADPH oxidized per min per g of hemoglobin (Hb). The plasma levels of riboflavin and its derivatives, and those of CoQ10 were estimated by high-performance liquid chromatography methods [9, 10]. The values of lipid peroxides in plasma were obtained by the method of Ohishi et al. [11]. Statistical method : Mean values and standard errors (SE) were calculated and were analyzed statistically by Student's t-test; values of p <0.05 were considered significant. RESULTS GSH-Px and GR activities of red blood cells were measured in normal human donors and patients with MDS. The activity of GSH-Px was significantly elevated in MDS patients. That of GR both with and without FAD supplementation was not significant. The activity coefficient (AC = GR activity with FAD/GR activity without FAD), however, increased in MDS patients, but was not significant (Table 1). The concentrations (pmol/g Hb) of both GSH and GSSG in red blood cells were significantly elevated in the patients compared with the levels in the controls (Table 1). The values of the other free radical scavengers; total vitamin B2, J. Gun. Biochem. Nutr.

3 . FREE RADICAL REACTIONS IN MDS 77 Table 1. Activities of glutathione peroxidase and glutathione reductase and concentrations of reduced and oxidized glutathione in red blood cells. Values are means +SE of 8 normal control, and 14 MDS patients for GSH-Px and GR and 13 MDS patients for GSH and GSSG. *p<0.05; **p<0025. Table 2. Levels of riboflavin and its derivatives and CoQ10 in plasma. Values are means +SE of 8 normal control, and 13 MDS patients for riboflavin and its derivatives and 9 MDS patients for CoQ10. *p< especially FAD, and CoQlo in plasma were significantly decreased in MDS patients (Table 2). On the other hand, lipid peroxide level as an index of the oxidation stress was significantly elevated in the plasma of MDS patients compared with that of controls (1.6 times, p <0.025). DISCUSSION MDS are heterogenous disorders that commonly show chromosomal abnormalities, but have many morphological and metabolic similarities from case to case. In some cases of dyserythropoiesis including MDS, decreased enzyme activity of pyruvate kinase with a concomitant increase in other enzyme activities and increase in red blood cell GSH, which were not related to reticulocytosis have been documented [3, 4]. Recently, it has been postulated that the cause of drug-induced hemolytic anemia, aging, carcinogenesis etc. may be due to the action of free radicals and other activated oxygens [12-17]. Life itself, however, is protected against these oxidant stresses by reducing enzymes such as superoxide dismutase, catalase, GSH-Px, etc., and other free radical scavengers, like vitamins A, B2, C, E (tocopherol), and ubiquinone (CoQ). Vol. 5, No. 1, 1988

4 78 H. IMANISHI et al. As mentioned above, enzyme deficiency of pyruvate kinase with a concomitant increase in other enzyme activities and in the GSH level in red blood cells may occur as a result of the free radical reactions and the concomitant defense mechanisms against oxidation stresses. In this study, the elevated activity of GSH-Px and the increase in concentrations of both GSH and GSSG in red blood cells, which are age-dependent [6, 7], were not related to reticulocytosis. In some cases, the activity of GR was restored to its normal level by supplementation with FAD. This was shown by the elevated activation coefficient (AC) of GR and deficiencies of total vitamin B2 and FAD in plasma. Another free radical scavenger, CoQlo, also decreased in plasma concomitantly with an increase in lipid peroxides. Thus, these data suggest that MDS patients have been exposed to free radicals and they may be hypersensitive to the oxygen intoxication. Further cytochemical or biochemical examinations of hematological cells in MDS patients are, however, necessary. REFERENCES 1. Bennet, J.M., Catovsky, D., Daniel, M.T., Flandrin, G., Galton, D.A.G., Gralnic, H.R., and Sultan, C. (1982) : Proposals for the classification of the myelodysplastic syndromes. Br. J. Haematol., 51, Dreyfus, B., Sultan, C., Rochant, H., Salmon, C.H., Mannoni, P., Carton, J.P., Boivin, P., and Galand, C. (1969) : Anomalies of blood group antigens and erythrocyte enzymes in two types of chronic refractory anaemia. Br. J. Haematol., 16, Valentine, W.N., Konrad, P.N., and Paglia, D.E. (1973) : Dyserythropoiesis, refractory anemia, and "preleukemia" : Metabolic features of the erythrocytes. Blood, 41, Arnold, H., Blume, K.G., Lohr, G.'W., Boulard, M., and Najean, Y. (1974) : "Acquired" red cell enzyme defects in hematological disease. Clin. Chim. Acta, 57, Yamada, H., and Maeda, H. (1986) : The erect of oxygen tension on the growth of human hemopoietic stem cells. Jpn. J. Clin. Hemat., 27, (in Japanese). 6. Imanishi, H., Nakai, T., Abe, T., and Takino, T. (1985) : Glutathione metabolism in red cell aging. Mech. Ageing Dev., 32, Imanishi, H., Nakai, T., Abe, T., and Takino, T. (1986) : Glutathione-linked enzyme activities in red cell aging. Clin. Chim. Acta, 159, Beutler, E. (1975) : Red Cell Metabolism (2nd ed.), Grune and Stratton, New York. 9. Ohkawa, H., Ohishi, N., and Yagi, K. (1982) : A simple method for micro-determination of flavins in human serum and whole blood by high-performance liquid chromatography. Biochem. Int., 4, Abe, K., Ishibashi, K., Ohmae, M., Kawabe, K., and Katsui, G. (1978) : Determination of ubiquinone in serum and liver by high-speed liquid chromatography. J. Nutr. Sci. Vitaminol., 24, Ohishi, N., Ohkawa, H., Miike, A., Tatano, T., and Yagi, K. (1985) : A new assay method for lipid peroxides using a methylene blue derivative. Biochem. Int., 10, Itano, H.A., Hirota, K., and Vedvick, T.S. (1977) : Ligands and oxidants in ferrihemochrome formation and oxidative hemolysis. Proc. Natl. Acad. Sci. USA., 74, Imanishi, H., Nakai, T., Abe, T., and Takino, T. (1985) : Role of free radical scavengers on phenylhydrazine induced hemolysis. Acta Vitaminol. Enzymol., 7, Imanishi, H., Hosokawa, K., and Itano, H.A. (1981) : Induction of Heinz body formation by sodium dithionite. Hemoglobin, 5, J. Clin. Biochem. Nutr.

5 FREE RADICAL REACTIONS IN MDS Harman, D. (1956): Aging: Theory based on free radical and radiation chemistry. J. Gerontol., 11, Goldstein, I.M. (1978) : Mechanism of Tumor Promotion and Carcinogenesis, Raven Press, New York. 17. Kensler, T.W., and Trush, M.A. (1981): Inhibition of phorbol ester-stimulated chemiluminescence in human polymorphonuclear leukocytes by retinoic acid and 5,6-epoxyretinoic acid. Cancer Res., 41, Vol. 5, No. 1, 1988