Potential Protective Effects of Melatonin on Bone Marrow of Rats Exposed to Cytotoxic Drugs

Comp. Biochem. Physiol. Vol. 119A, No. 2, pp. 493–501, 1998 Copyright  1998 Elsevier Science Inc. All rights reserved.

ISSN 1095-6433/98/$19.00 PII S1095-6433(97)00456-X

Potential Protective Effects of Melatonin on Bone Marrow of Rats Exposed to Cytotoxic Drugs Mamdouh M. Anwar,1 Hussein A. Mahfouz,2 and Arafat S. Sayed 3 1

Department of Physiology, 2 Radiation Oncology, Faculty of Medicine, and 3 Medicine and Clinical Laboratory Diagnosis Department, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt ABSTRACT. Myelosuppression is the most serious, dose limiting, toxicity of cytotoxic drugs. Efforts to protect the bone marrow have been only variably successful, and no agreement exists on how to approach this problem. Melatonin, the major hormonal product of the pineal gland, is supposed to have both chemoprotective and myelostimulatory effects. This experimental study was carried out to test these two effects on the bone marrow of rats, daily intraperitoneally injected with 100 µg melatonin. Injection of 10 mg aracytin for 10 days produced a significant (P , 0.01) decrease in red blood cells count (RBCs), total leucocytic count, as well as platelets count. When melatonin was injected along with aracytin, it would significantly increase (P , 0.05) RBC count and (P , 0.01) blood platelet count. Injection of melatonin after aracytin treatment would significantly increase (P , 0.01) RBC, total leucocytic and platelet counts in comparison with rats treated with aracytin only. The effects of melatonin were more clear in rats treated with it after aracytin injection than those treated with melatonin and aracytin at the same time. Furthermore, it was found that aracytin produced a significant (P , 0.01) decrease in serum total proteins, albumin, and significantly increased the (P , 0.01) albumin/globulin ratio. Melatonin injection would significantly increase (P , 0.01) total protein, globulin, and significantly decrease (P , 0.01) the albumin/glubulin ratio when injected either with aracytin or after aracytin treatment. These results indicate that melatonin protects bone marrow, lymphoid tissues from damaging effect of cytotoxic drugs, as well as stimulating the suppressed bone marrow. comp biochem physiol 119A;2:493–501, 1998.  1998 Elsevier Science Inc. KEY WORDS. Melatonin, pineal gland, aracytin, bone marrow, white blood cells, red blood cells, hemoglobin, cancer

INTRODUCTION Melatonin, nacetyl-5-methoxytryptamine, is a hormonal product of the pineal gland. Its synthesis was higher at night than during the day in all vertebrates including man (20). This hormone has many functions in the body and may protect the body from many diseases, including control of fertility (1,17), anticancer (2,18), antioxidant (20), and finally immunostimulant (13). Patients with cancer are usually treated with cytotoxic drugs, which produce variable degree of myelosuppression expressed on leucopenia, thrombocytopenia, and anemia. One of the potent myelosuppressive drugs is cytosine arabinosite (Aracytin) (7). Moreover, myelosuppression is a dose-limiting toxicity that prevents administration of intensive doses of the cytotoxic drugs. The aim of this work is to study the protective and stimulatory effects of melatonin on bone marrow of rats exposed to a potent myelosuppresAddress reprint requests to: Mamdouh M. Anwar, Dept. of Physiology, Faculty of Medicine, Assiut University, Assiut, Egypt. Fax 088-332278. Received 25 January 1996; accepted 23 May 1996.

sive cytotoxic drug (aracytin), and to study the best effect of melatonin administration is given with or after aracytin administration. MATERIALS AND METHODS Materials ANIMALS. Fifty adult male albino rats (Sprague-Dawley strain) were used in this study. The animals were kept in the physiology department laboratory, Assiut University, for 10 days to exclude the diseased rats. CHEMICALS. Melatonin (Sigma Chemical Co.) was dissolved in a few drops of ethanol then distilled water where 0.5 ml contained 100 µg melatonin. This is a dose dependant according to Lang et al. (11) who reported that the most effective dose of melatonin in rats was 100 µg/day. Aracytin [cytarabine (1-β-d-arabinofuranosyl cytosine), (cytosine arabinoside)] was a synthetic nucleoside (Upjohn Co.). It was dissolved in benzyl alcohol 0.990 w/w, where each 0.25 ml contained 10 mg aracytin.

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TABLE 1. The RBCs count, Hb concentration, and PCV% in control, aracytin, and melatonin treated groups

Control group Aracytin-treated group Melatonin-treated group N.S.

RBCs (106 /mm3)

Hb (g%)

PCV (%)

7.59 6 0.58 4.42 6 1.47 b 7.80 6 0.74 N.S.

14.55 6 0.93 10.75 6 1.36 b 14.91 6 0.86 N.S.

42.6 6 1.65 31.7 6 3.16 b 44 6 3.43 N.S.

Non-significant; a significant at P , 0.05; and b significant at P , 0.01.

Methods The rats divided into 5 groups (10 rats each): 1. The first group (group I) was injected intraperitoneally (i.p.) with the solvent of aracytin and melatonin for 10 successive days and kept as a control. 2. The second group (group II) was injected i.p. with 10 mg of aracytin daily for 10 days. 3. The third group (group III) was injected i.p. with 100 µg melatonin for 10 successive days 2 hr prior to sunset. 4. The fourth group (group IV) was injected with 10 mg aracytin and 100 µg melatonin daily for 10 days. 5. The fifth group (group V) was injected with 10 mg aracytin for 10 days, then injected for another 10 days with 100 µg melatonin daily. Two blood samples were collected from each rat in clean, sterile centrifuge tubes, by puncture of the retro-orbital sinus. One blood sample was tested with anticoagulant (EDTA) for determination of packet cell volume (PCV%), total erythrocytic count (10 6 /mm 3 ), hemoglobin (Hbg/100 ml), total leucocytic count (10 3 /mm 3 ), hemogram (10 3 / mm 3 ), platelets count (10 3 /mm 3 ), corpuscular volume (MCV-F/L), mean corpuscular hemoglobin (MCH-Pg), and mean corpuscular hemoglobin concentration (MCHCg/100 ml), according to Schalm et al. (22). Another blood sample was tested without anticoagulant for obtaining blood serum for determination of corticosterone hormone thiobarbituric acid reactive substances (TBARS) acid phosphatase, alkaline phosphatase, total protein, and albumin. Total protein, albumin, according to King and Wootton (9), acid phosphatase, according to Moss (14), and alkaline phosphatase, according to Varley (24), were determined by using test kits supplied from Bio Meriex and spectrophotometer (ultraspect, pharmacia). TBARS was determined in accordance to the technique described by El-Saadani (5). Corti-

costerone hormone was determined in serum samples by using a RIA kit and Gama counter. Statistical analyses were done by one-way analysis of variance using a PC-state computer program (16). RESULTS Hematological investigations of blood samples including RBCs count (10 6 /mm 3 ), Hb (g%) PCV (%), MCV (f/L) MCH (Pg), MCHC (g/dL, total leucocytic count (10 3 / mm 3 ), hemogram, (10 3 /mm 3 ), and platelets count (10 3 / mm 3 ) were shown in Tables 1–6 and in Figs. 1–5. Biochemical analyses of blood serum for determination of corticosterone hermone (ng/ml) TBARS (mmol Eq./L), acid phosphatase (U/L), alkaline phosphatase (U/L), total protein (g/100 ml), albumin (g/100 ml), globulin (g/100), and albumin globulin ratio are shown in Tables 7–10 and in Figs. 6–10. DISCUSSION The potent biological active substance produced by the pineal gland, melatonin, participate in many important physiological functions, including the control of seasonal reproduction, as well as influence of the immune system (8,19). In this study, the effect of a 100 µg-injection melatonin on bone marrow exposed to cytotoxic drug aracytin was investigated. A daily injection of 10 mg aracytin in the rats produced a marked bone marrow suppression, which lead to a reduction in the process of hemopiosis, and it was represented by a significant decrease (P , 0.01) in RBC count, Hb concentration, PCV (%), MCV, MCH, total leucocytic, neutrophil, lymphocyte, and platelet counts. These results were in agreement with Herzig et al. (7), who reported that

TABLE 2. The RBCs count, Hb concentration, PCV% in aracytin, aracytin with melatonin, and aracytin then melatonin-

treated groups

Aracytin-treated group Aracytin with melatonin-treated group Aracytin then melatonin-treated group N.S.

Non-significant. Significant at P , 0.05. Significant at P , 0.01.

a

b

RBCs (106 /mm3)

Hb (g%)

PCV (%)

4.42 6 1.47 5.66 6 1.15 a 7.71 6 1.88 b

10.75 6 1.36 11.20 6 1.45 N.S. 13.03 6 2.07 b

31.7 6 3.16 31.6 6 2.27 N.S. 36.4 6 3.76 a

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TABLE 3. The MCV, MCH, and MCHC in control-, aracytin-, and melatonin-treated groups

MCV (f/L) Control group Aracytin-treated group Melatonin-treated group

56.44 6 5.10 83.63 6 41.59 b 56.62 6 5.09 N.S.

MCH (Pg) 19.25 6 1.69 27.98 6 12.88 b 19.49 6 2.07 N.S.

MCHC (g/dL) 34.23 6 2.93 34.08 6 4.77 N.S. 34.13 6 3.86 N.S.

N.S.

Non-significant. Significant at P , 0.05. b Significant at P , 0.01. a

aracytin was primarily a potent myelosuppressive agent capable of producing severe leucopenia, thrombocytopenia, and anemia. Calabresi and Chabner (3) attributed the destructive effects of aracytin on bone marrow to its inhibitory effect on β-DNA polymerase, an enzyme involved in DNA repair. It appeared that MCV and MCH were reduced, while MCHc was not altered; this effect may be attributed to increase the demand of O 2 and, therefore, MCV and MCH increased as a compensatory mechanism to the reduction of total RBC count. Daily i.p. injection of 100 µg melatonin alone produced an increase in RBC count, Hb concentration, PCV%, MCV, MCH, and MCHc, but without statistical significant value when compared with control. It caused a significant increase (P , 0.01) in total leucocytic, neutrophils, lymphocytes, eosinophils, and basophils counts, while it led to a significant (P , 0.05) increase in the platelet count. When melatonin was injected with the cytotoxic drug, it caused a significant increase (P , 0.05) in the RBC count and MCH, and it caused a significant (P , 0.01) increase in MCV and platelet count. Injection of melatonin after complete treatment by aracytin produced a marked stimulatory effect on suppressed bone marrow. It caused a significant increase (P , 0.01) in the RBC count, Hb concentration, MCV, MCH, total leucocytic, neutrophil, lymphocyte, monocyte, eosinophils, and platelet counts. Also, it led to a significant increase (P , 0.05) in PCV% and basophil count. These results indicated that melatonin had a protective and stimulatory effect on bone marrow exposed to damage by chemical substances. The effects of melatonin on bone marrow cells were cleared in lymphocytes, RBCs, as well as blood platelets. Kuci et al. (10) reported that pinealectomy lowered the number of progenitor cells for granulocytes and monocytes

in bone marrow. Moreover, they suggested that injection of P-chlorophenylalanine, which produced pharmacological pinealectomy, would be reduced the night peak of antibody production. On blood platelets, melatonin stimulated the platelets’ production. Rossi and Bella (21) reported that melatonin promotes the protrusion formation by megakaryocytes, thus facilitating the engagement of megakaryocytes into sinusoides. Moreover, melatonin spreads rapidly as far as the megakaryocytes nucleus, where it stimulates tubuloflamentous system to increase the platelets production. Melatonin stimulates some cytokins (IL-2), which have a powerful stimulatory effect on bone marrow cells (12). Moreover, Tan et al. (23) suggested that melatonin protects cellular DNA from damage by chemical substances and stimulates DNA polymerase enzymes, which are responsible for DNA repair. Biochemical investigation of serum levels of corticosterone, TBARs, acid and alkaline phosphatase, total protein, albumin and globulin were done. Serum corticosterone level was significantly elevated (P , 0.01) after aracytin injection, which attributed to a stress condition with cellular damage by aracytin. A daily i.p. injection of melatonin in rats produced a significant decrease (P , 0.01) in the serum corticosterone level. Injection of melatonin with aracytin did not alter serum corticosterone from the aracytin treated group, while it caused a marked significant decrease (P , 0.01) in rats treated with melatonin after aracytin. These results indicated that melatonin interacts with stress by reduction of corticosterone to relieve stress effects. Weidenfeld et al. (25) reported that melatonin had an antistress effect and it lowered corticosterone level in rats expossed to stress. Furthermore, the reduction in serum corticosterone might help stimulate both cellular and humoral immunity.

TABLE 4. The MCV, MCH, and MCHC in aracytin, aracytin with melatonin, and aracytin then melatonin-treated groups

Aracytin-treated group Aracytin with melatonin-treated group Aracytin then melatonin-treated group N.S.

Non-significant. Significant at P , 0.05. Significant at P , 0.01.

a

b

MCV (f/L)

MCH (Pg)

MCHC (g/dL)

83.63 6 41.59 57.89 6 12.57 b 49.04 6 9.49 b

27.88 6 12.38 20.38 6 4.13 a 17.50 6 3.99 b

34.08 6 4.77 34.70 6 4.42 N.S. 35.83 6 5.07 N.S.

3.39 6 0.56 0.89 6 0.49 b 2.37 6 0.47 b

8.54 6 0.98 4.59 6 1.82 b 10.92 6 0.91 b

4.64 6 0.77 3.3 6 1.33 b 7.76 6 0.78 b

Lymphocytes count (103 /mm3) 0.33 6 0.05 0.25 6 0.09 N.S. 0.30 6 0.15 N.S.

Monocytes count (103 /mm3) 0.06 6 0.09 0.04 6 0.02N.S. 0.35 6 0.06b

Eosinophils count (103 /mm3) 0.02 6 0.05 0.003 6 0.009N.S. 0.14 6 0.11 b

Basophil count (103 /mm3)

832.2 6 48.30 482 6 210.28 a 955.5 6 38.11 a

Platelets count (103 /mm3)

a

Non-significant. Significant at P , 0.05. b Significant at P , 0.01.

N.S.

Aracytin-treated group Aracytin with melatonin-treated group Aracytin then melatonin-treated group

0.89 6 0.49 1.13 6 0.41N.S. 1.79 6 0.34 b

5.13 6 2.03 N.S. 8.84 6 1.24 b

Neotrophils count (103 /mm3)

4.59 6 1.82

Total leucocytes (103 /mm3)

then melatonin-treated groups

6.05 6 0.77 b

3.68 6 1.52 N.S.

3.3 6 1.33

Lymphocytes count (103 /mm3)

0.63 6 0.12 b

0.25 6 0.17 N.S.

0.25 6 0.09

Monocytes count (103 /mm3)

0.32 6 0.15b

0.08 6 0.06 N.S.

0.04 6 0.02

Eosinophils count (103 /mm3)

0.07 6 0.13 a

0.0 6 0.0 N.S.

0.003 6 0.009

Basophil count (103 /mm3)

876 6 77.49 b

662 6 107.99 b

482 6 210.28

Platelets count (103 /mm3)

TABLE 6. The total leucocytic, neutrophil, monocyte, eosinophil, basophil, lymphocyte, and platelet counts in aracytin, aracytin with melatonin and aracytin,

a

Non-significant. Significant at P , 0.05. b Significant at P , 0.01.

N.S.

Control group Aracytin-treated group Melatonin-treated group

Neotrophils count (103 /mm3)

Total leucocytes (103 /mm3)

TABLE 5. The total leucocytic, neutrophil, monocyte, eosinophil, basophil, lymphocyte, and platelet counts in control, aracytin, and melatonin-treated groups

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FIG. 3. PCV (%) in control, aracytin, melatonin, aracytin FIG. 1. RBCs count (10/mm) in control, aracytin, melato-

with melatonin and aracytin then melatonin treated groups.

nin, aracytin with melatonin and aracytin then melatonin treated groups.

Malondialdehyde (MDA), as a marker of lipid peroxidation, proved to be sensitive. It was measured by using the thiobarbituric acid reaction. It was observed that MDA Eq⋅mmol/L elevated significantly (P , 0.01) in rats treated with aracytin. The elevation of oxygen-free radicals in these rats indicated that cellular damage was severe and cleared. On the other hand, TBARs was reduced significantly (P , 0.01) in rats treated with melatonin with and after aracytin treatment. Lipid peroxidation was an indicator and a measurement for free radicals in biological systems (6). The reduction of free radical by melatonin would protect the cellular DNA from damage by aracytin. Reiter (20) suggested that melatonin was a powerful scavenger and protects

FIG. 2. Hemoglobin (g%) in control, aracytin, melatonin,

aracytin with melatonin and aracytin then melatonin treated groups.

DAN from oxidative damage in comparison with the well known ideal scavengers, glutathion and mannitol. Moreover, Reiter (20) stated that melatonin stimulates the endogenous antioxidants glutathion, glutathion peroxidase, and superoxided dismutase. Serum acid and alkaline phosphatases were measured in our study. It was found that aracytin treatment elevated both acid and alkaline phosphatases significantly (P , 0.01). These elevations might be due to mass cellular damage produced by aracytin treatment. Moss and Henderson (15) reported that phosphatases enzymes increased during cellular damage, especially bone marrow damage. When melatonin was injected alone it caused significant elevation (P , 0.01) in acid phosphatase. Injection of melatonin with aracytin reduced acid phosphatase enzyme, but without sig-

FIG. 4. Total and differential leucocytic counts (10/mm) in control, aracytin, melatonin, aracytin with melatonin and aracytin then melatonin treated groups.

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FIG. 5. Platelets count (10/mm) in control, aracytin, melato-

nin, aracytin with melatonin and aracytin then melatonin treated groups.

nificant value and alkaline phosphatase decreased significantly (P , 0.01). On the other hand, melatonin injection after complete aracytin injection produced a significant decrease (P , 0.05) in alkaline phosphatase, while serum acid phosphatase was not altered. The acid and alkaline phosphatases could be used as an indicator for cellular damage, as well as repairing bone marrow. For investigating the effect of aracytin and melatonin on liver and humoral immunity serum total protein, albumin, globulin, as well as albumin/globulin ratio, were measured. It was observed that serum total protein and albumin significantly decreased (P , 0.01) after aracytin treatment, and albumin/globulin ratio increased significantly (P , 0.01) when compared with control. These results attributed to the destructive effect of aracytin on liver and lymphoid tissues. Herzig et al. (7) suggested that aracytin leads to hepatic dysfunction. Calabresi and Chabner (3) also stated that aracytin inhibits liver DNA synthesis and repair. Daily i.p. injection of 100 µg melatonin alone produced

TABLE 7. The concentration of corticosterone, TBARS, acid phosphatase, and alkaline phosphatase in control-, aracytin-,

and melatonin-treated groups

Control group Aracytin-treated group Melatonin-treated group N.S.

Corticosterone ng/ml

TBARS mmolEq./L

Acid phosphatase U/L

Alkaline phosphatase U/L

34.63 6 7.42 68.27 6 2.63 b 15.73 6 2.55 b

5.65 6 1.61 11.79 6 4.10 b 4.25 6 2.04 N.S.

41.86 6 3.04 54.04 6 14.03 b 57.9 6 6.85 b

158.91 6 18.31 254.03 6 64 b 159 6 52.91 N.S.

Non-significant. Significant at P , 0.05. Significant at P , 0.01.

a

b

TABLE 8. The concentrations of corticosterone, TBARS, acid phosphatase and alkaline phosphatase in aracytin, aracytin with

melatonin, and aracytin then melatonin-treated groups Corticosterone ng/ml Aracytin-treated group Aracytin with melatonintreated group Aracytin then melatonintreated group

68.27 6 2.63

TBARS mmolEq./L

Acid phosphatase U/L

Alkaline phosphatase U/L

11.79 6 4.10

54.04 6 14.03

254.03 6 64

65.82 6 20.43 N.S.

7.42 6 2.64 b

46.23 6 8.25 N.S.

160.36 6 35.31 b

40.63 6 11.15 b

7.08 6 3.09 b

48.35 6 8.65 N.S.

203.18 6 48.77 a

N.S.

Non-significant. Significant at P , 0.05. b Significant at P , 0.01. a

TABLE 9. Serum total protein, albumin, globulin, and albumin globulin ratio in control, aracytin, and melatonin-treated groups

Control group Aracytin-treated group Melatonin-treated group N.S.

Non-significant. Significant at P , 0.05. b Significant at P , 0.01. a

Total protein g/100 ml

Albumin g/100 ml

Globulin g/100 ml

Albumin/ globulin ratio

2.91 6 0.13 2.26 6 0.22 b 3.01 6 0.29 N.S.

2.34 6 0.25 1.90 6 0.26 b 1.99 6 0.16 b

0.35 6 0.11 0.24 6 0.13 N.S. 1.02 6 0.33 b

4.98 6 1.30 6.46 6 1.18 b 1.73 6 0.89 b

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TABLE 10. Serum total protein, albumin, globulin, and albumin globulin ratio in aracytin, aracytin with melatonin, and ara-

cytin then melatonin-treated groups Total protein g/100 ml Aractytin-treated group Aracytin with melatonintreated group Aracytin then melatonintreated group

Albumin g/100 ml

Globulin g/100 ml

Albumin/ globulin ratio

2.26 6 0.22

1.90 6 0.26

0.24 6 0.13

6.46 6 1.18

2.79 6 0.12 b

2.03 6 0.29 N.S.

0.67 6 0.34 b

2.45 6 0.99 b

2.86 6 0.31 b

1.99 6 0.28 N.S.

0.92 6 0.30 b

2.03 6 0.59 b

N.S.

Non-significant. Significant at P , 0.05. b Significant at P , 0.01. a

FIG. 6. Corticosteron level (ng/ml) in control, aracytin, mel-

atonin, aracytin with melatonin and aracytin then melatonin treated groups.

FIG. 7. TBARS (MDA Eq⋅mmol/L) in control, aracytin, mel-

atonin, aracytin with melatonin and aracytin then melatonin treated groups.

a significant increase (P , 0.01) in serum globulin and a significant decrease (P , 0.01) in the albumin globulin ratio, which led to a significant increase (P , 0.05) in serum albumin. When melatonin was injected with aracytin or after arcytin treatment, it significantly increased (P , 0.05 and P , 0.01) serum levels of total protein and globulin respectively. Also, it decreased the albumin/globulin ratio significantly (P , 0.01). These results prove that aracytin damage lymphoid tissues and melatonin protect and stimulate DNA repair in these tissues. Moreover, melatonin could be considered as humoral immune stimulating agent. There are several roles for possible mechanism of melatonin actions. The first possible mechanism may be a direct action on bone marrow, as reported by Kuci et al. (10). Also, Maestroni and Conti (13) reported that melatonin stimulates cellular proliferation in lymphatic tissues. Lissoni et al. (12) reported that melatonin acts as a growth factor, especially for granulocytes on bone marrow, and suggested that melatonin stimulates the release of IL-2, which stimulates bone marrow cells. The second possible mechanism may be an indirect action through reduction of corticosterone hor-

FIG. 8. Acid and alkaline phosphatase (U/L) in control, aracytin, melatonin, aracytin with melatonin and aracytin then melatonin treated groups.

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may be able to repair damaged bone marrow cells. It also appeared that the repairing effect of melatonin is more clear than its preventing effect. Moreover, melatonin protects liver and lymphoid cellular DNA from damage by chemical substances. It could also be considered as an immunostimulant agent for both cellular and humoral immunity. The multiple disclosed effects of melatonin on the bone marrow sufficiently justify its clinical employment both in chemotherapy by cytotoxic drugs and in cancer. References

FIG. 9. Total protein, albumin and globulin in control, aracytin, melatonin, aracytin with melatonin and aracytin then melatonin treated groups.

mone, which was reflected by the elevation of the leucocytic count. The third possible mechanism may be through the antioxidant effect of melatonin, which prevents further damage of bone marrow and stimulate cellular proliferation, according to Tan et al. (23), who reported that melatonin protect liver tissue from damage-induced substances. The fourth possible mechanism, as described by Conti et al. (4), indicates that melatonin accelerated leucogenesis and this action of melatonin was blocked by naltrexone, resulting in the involvement of melatonin-induced-immuno-opioids in the leucogenesis. From this study, it could be concluded that melatonin prevents bone marrow damage by cytotoxic drugs and

FIG. 10. Albumin/globulin ratio in control, aracytin, melatonin, aracytin with melatonin, and aracytin then melatonin treated groups.

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