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THE USE OF MELATONIN AND CO-TREATMENT WITH AUTOLOGOUS OR ALLOGENEIC CELLS AS A MODEL FOR CONTROL OF MALIGNANT p-CELL LEUKEMIA

1 Hebrew University Faculty of Medicine

2 Hadassah University Hospital Jerusalem, Israel

The purpose of the present study was to evaluate the possible effects of pharmacological doses of melatonin upon the survival of leukemic mice using a murine model of originally spontaneous, subsequently transplantable and non-immunogenic P-cell leukemia (BCL1). (BALB/C x C57BL/6) F1 mice inoculated with BCL1 cells were conditioned by total irradiation (750cGy) and reconstituted with either bone marrow (BM) cells, a syngeneic transplantation, as a model of autologous transplantation, or BMC 57BL/6(C57) allogeneic transplantation (1). The leukemia produced is believed to exert damaging effects on cellular DNA, causing disruption of chemical bonds in the molecular structure of DNA, the destruction by the active free radicals interacting with cellular DNA. Melatonin, the compound tested, is an antioxidant believed to detoxify the devastating OH radicals, thus providing protection against the destructive biological effect and genetic damage of malignancy.

Our aim was to establish whether protection is provided by melatonin against the destructive effect of leukemia, and if so, whether it is due to its antioxidative capacity. Four groups of 12 mice each (2-3months old, purchased from Harlem, Jerusalem) were irradiated according to the outlined procedure and on the following day injected with BCL1 cells and reconstituted with either BMC57 or BMF1 cells. Melatonin was given by S.C. injection 10mg/kg for 4 consecutive days and the animals left for observation until the development of leukemia or death. Leukemia and consequently death developed twice as quickly in the mice reconstituted with syngeneic cells [Figure 1

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Figure 1. Development of leukemia (mean number of days) in mice receiving syngeneic cells (Gr1) syngeneic cells and melatonin (Gr2), allogeneic cells (Gr3), or allogeneic cells and melatonin (Gr4). Groups 5 and 6 equal Groups 3 and 4 respectively with some variation in the mode of application of melatonin. Groups 2-6 are statistically different from group 1 (p < 0.05).

Gr 1

Gr 2

Gr 3

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Figure 1. Development of leukemia (mean number of days) in mice receiving syngeneic cells (Gr1) syngeneic cells and melatonin (Gr2), allogeneic cells (Gr3), or allogeneic cells and melatonin (Gr4). Groups 5 and 6 equal Groups 3 and 4 respectively with some variation in the mode of application of melatonin. Groups 2-6 are statistically different from group 1 (p < 0.05).

(Gr1)] compared with those reconstituted with allogeneic cells (Gr. 3). The simultaneous application of melatonin, however, could reduce the destruction in animals given the syngeneic cells to the same level as those of the mice reconstituted with allogeneic cells [Figure 1 (Gr2)]. No difference was found in the pace of development of leukemia in the mice with allogeneic cells, irrespective of whether or not the animals received melatonin [Figure 1 (Gr3 & 4)].

This finding indicates the absence of a leukemia defensive factor in the syngeneic cells such as is found in allogeneic cells. Melatonin administration to the syngeneic cell recipients raised the defensive function to that recorded in the mice reconstituted with allogeneic cells. The question arising, furthermore, was whether the additional protection against leukemic malignancy provided by melatonin in the presence of syngeneic cells is due to an antioxidative effect against free radicals. This possibility was tested by determining the antioxidative protective capacity and actual erythrocyte sensitivity of the various groups of experimental animals. The erythrocyte sensitivity and oxida-tive stress was determined with 22 azobis-amidinopropane (AAPH), a radical inhibitor which brings about membrane alteration and hemolysis (2). The toxicity related to oxygen free radicals appeared not to differ in the plasma and erythrocytes of the animals under the various experimental conditions, indicating a similar deficit of protection against radicals in the various instances (Figure 2). In vitro melatonin significantly inhibited hemolysis of red blood corpuscles, thus indicating a protective capacity against oxygen free radicals. Furthermore, an additional screening test to establish the anti-radical elements present in the plasma of the variously treated mice was undertaken using a cyclic voltametry procedure to determine low molecular weight antioxi-dative capacity (3). The major contributors of the antioxidative capacity of biological fluids and tissues were established to be ascorbic-dehydro ascorbic acid and uric acid, acting as direct chemical scavengers. Here too, the values determined appear to be similar in the animals under the various experimental conditions.

It may be concluded, based on the results recorded in our leukemic model of mice, that melatonin could, under certain experimental conditions, bring about a restraint

AAPH mVI

Figure 2. The antioxidative capacity of the plasma obtained from the six variously treated groups of mice (a,b,c,d,e,f) as indicated from the degree of hemolysis determined in vitro (inhibitory 50% plasma volume). There was no statistical difference between any of the groups determined. (a,b,c,d,e,f correspond to the groups 1,2,3,4,5,6 described in Figure 1).

in oncogenicity, this effect not being dependent on its antioxidative status. Moreover, the positive effect of melatonin cotreatment with syngeneic cells established in BCL1 leukemic sygeneic mice, if found applicable for human bone marrow transplantation, would be of prime importance in clinical practice, allowing use of cells taken from the patient himself instead of the accepted use of cells from donors.

1. Weiss, L., Reich, S., and Slavin, S. Use of recombinant human interleukin 2 in conjunction with bone marrow transplantation as a model for control of minimal residual disease in malignant hematological disorders: I. Treatment of murine leukemia in conjunction with allogeneic bone marrow transplantation and IL-2 activated cell-mediated immunotherapy. Cancer Investigation, 10, 19-26

2. Abella, A,, Messaoudi, C., Laurent, D., Marot, D., Chalas, J., Breux, J., Claise, C., and Lindenbaum, A. A method for simulataneous determination of plasma and erythrocyte antioxidant status. Evaluation of the antioxidant activity of vitamin E in healthy volunteers. BrJClin. Pharmacol, 42, 737-741 (1996).

3. Chevion, S., Berry, E.M., Ktrowssky, N., and Kohen, R. Evaluation of plasma low molecular weight antioxidants by voltametry. FreeRad. Biol. Med., 22, 411-471 (1997).

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