Accelerated Recovery of 5-Fluorouracil-Damaged Bone Marrow after Oxytocin Treatment

 

Shadan Muslim Aljanabi1*, Shaza Al-Laham1, Adnan M. Ekhtiar2

¹Pharmacology Department, Collage of Pharmacy, Damascus University, Syria

²Biotechnology Department, Syrian Atomic Energy Commission, Syria

*Corresponding Author E-mail:- shadan.m.aljanabi@gmail.com

 

Our preliminary data indicate that Oxytocin may be myeloprotective. We investigated whether it can modify bone marrow recovery. Oxytocin (150 µg/kg) accelerated the recovery of the number of medium size cells (MI%) and platelets (PLT) but did not affect the number of circulating red blood cells (RBC) and total count of white blood cells (WBC).Oxytocin injected subcutaneously for 5 consecutive days at doses of 150 µg/kg (twice a day) after 5-FU single dose. Glutathione GSH (anti-oxidant) has preventive effects against 5-FU-induced myelosuppression and can accelerate recovery of bone marrow. Oxytocin only increase GSH levels insignificantly (P > 0.05).These findings suggest that Oxytocinselectively enhances the recovery of the population of progenitor cells reduced by 5-FU in rats.

 

 

 

1. INTRODUCTION:

Cancer chemotherapy causes severe damage to haematopoietic stem cells. Meylotoxicity is a serious risk for chemotherapy, associated with leukopenia infectious complications necessitating the use of intravenous antibiotics and hospitalization, and may even result in death.(1)Severe anemia that is resistant to medical treatment is also observed in patients with malignancies and who are undergoing chemotherapy. The pathogenesis of this anemiais multifunctional. Low levels of circulating EPO, shortened survival time of circulating red blood cells (RBCs), and a decrease in the number of immature erythroid cells in bone marrow probably due to chemotherapy or cytokines such as tumor necrosis factor (TNF), have been demonstrated to be the causes.(2) The 5-fluorouracil (5-FU) is one of the most widely used agents in the treatment of advanced colorectal cancer. The primary target of this agent is thymidylate synthase (TS), which catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) by methylene tetrahydro folate(CH2H4PteGlu), to form thymidine monophosphate (TMP).

 

This reaction is the only de novo source of TMP, and is critical for proper DNA synthesis and cell proliferation. 5-Fluoro-20-deoxyuridylate (FdUMP), the active metabolite of 5-FU, inhibits TS via formation of a covalent ternary complex with CH2H4PteGlu and the enzyme(3)Various antioxidants and free radical scavengers have been suggested to be general cytoprotective agents of therapeutic benefit. In case of reduced or impaired defense mechanism and excess generation of free radicals that are not counterbalanced by endogenous antioxidant defense exogenously administered antioxidants have been proven useful to overcome oxidative damage.(4)

 

Oxytocin (OT), a thiol anti-oxidant agent is a non a peptide produced in the paraventricular and supraoptical nuclei in the hypothalamus, exerts a wide spectrum of central and peripheral effects. In addition to its reproduction-related classical functions, such as stimulation of uterine smooth muscle during labour and milk ejection during lactation, OT displays a potent antistress and wound healing effect that involves the suppression of hypothalamic pituitary- adrenal (HPA) axis (5) The cell defensive system consists of antioxidative free-radical scavenging molecules such as glutathione (GSH—a tripeptide consisting of glutamic acid-cysteine-glycine). GSH acts as the substrate for the enzyme glutathione peroxidase. As such it is an important component of intracellular antioxidant defense, protecting cytosolic organelles, in particular, from the damaging effects of hydroperoxides. In addition, GSH also acts synergistically with ascorbic acid and alphatocopherol to recycle these nutrient antioxidant vitamins to their reduced state after their interaction with reducing chemical species inside the cell (6)

 

Our new studies are designed to evaluate whether Oxytocinhas beneficial effects on bone marrow preservation when administered concurrently with 5-FU.

 

2. MATERIALS AND METHODS:

2.1. Animals care and handling:

The animal care and handling were done according to the guidelines set by the World Health Organization (WHO), Geneva, Switzerland and the INSA (Indian National Science Academy, New Delhi). Seven to eight weeks old male Wister Rats weighing 150g to 200g were maintained under controlled conditions of temperature (25± 2°C), humidity (40 ± 2%) and light (10 h and 14 h of light and dark, respectively). The animals were provided with clean food and water ad libitum. Five to six animals were housed in a polypropylene cage containing clean paddy husk (procured locally) as bedding throughout the experiment. An adaptation period to these conditions of at least 1 week was allowed before the experiment.

 

2.2 Study design:

A rat model of myelotoxic effects was made by single intraperitoneal injection of 5-FU at a dose of150 mg/kg. The first day of experiment was defined as day 0. Oxytocin (150 µg/kg, subcutaneous injection) was injected twice a day for 5 consecutive days after one hour of the 5-FU injection. The control group received a vehicle instead of test samples. Rats were scarified by an anesthetization with overdose of ether and then femurs and whole blood were collected at indicated time points.

 

2.3 Hematological analysis:

Blood sample (0.5–0.8 ml) was obtained every second day from anesthetized rats by cardiac puncture using an EDTA-coated syringe attached to a 21- guage needle. White blood cell (WBC), platelet (PLT) and red blood cell (RBC) count were performed using a Hematology System (Humacount).

 

2.4 Blood:

Rats were anesthetized with ether, and blood samples were collected by intraventricular exsanguinations on day 9 of 5-FU administration. Blood samples were centrifuged (2000g) for 5 minutes and the supernatant plasma samples were placed in microtubes and held in (-80°C) until further processing. Plasma (200 µL/tube) were deproteinized with the 5%5-Sulfosalicylic Acid Solution, centrifuged to remove the precipitated protein, and then assayed for glutathione (GSH).

 

2.5 Glutathione assay:

The measurement of GSH uses a kinetic assay in which catalytic amounts (n moles) of GSH cause a continuous reduction of 5,5`-dithiobis (2-nitrobenzoic acid) (DTNB)to TNB and the GSSG formed is recycled by glutathione reductase and NADPH. The GSSG present will also react to give a positive value in this reaction. Samples are prepared in 96-wellplates and absorbance at 415 nm with a reference wavelength of 595 is determined every 60 s for 5 min. The rate of increase in absorbance is directly proportional to the amount of glutathione reductase in the sample. Activity in an unknown sample is determined from a standard curve (7)

 

2.6 Statistical analysis:

The mean of the cell numbers and the glutathione concentration were considered as a single datapoint for analysis of results from at least three or four independent experiments. All data are expressed as mean ± S.E.M. Statistical significance was determined by Dunnett’s multiple test after one-way analysis of variance (ANOVA)with comparison to a control group, and the differences were considered significant if P<0.05.

 

3. RESULT:

After administration of 5-FU, the numbers of circulating RBC, WBC and PLT were reduced on day 9 by 26.6, 50.4 and 90%, respectively. The haemoglobin concentration were significantly decreased by 26% in 5-FU only group. In 24 hours of 5-FU administration the MI% (medium size cells percentage) was significantly decreased by 51%.The results are presented in Table 1. Subcutaneously injected Oxytocin at dose of 150µg/kg (twice a day) after 5-FU injection had no influence on the most observed parameters (P>0.05).However, there was a significant increase         (P< 0.05) of MI% in Oxytocin+ 5-FU group by 38%.The values of other haematological parameters (GRA%,HGB, LY% and RBC)were not influenced(P>0.05) after Oxytocin treatment. Administration of  (150µg/kg) Oxytocinafter 5-FU treatment increased the platelet count in blood by 66% (P> 0.05) at day 9.There was a good effect of Oxytocin (150µg/kg twice a day)  after-treatment on the  plasma GSH levels by 45% (P> 0.05).

 

Figure 1: Effect of Oxytocin on GSH levels in plasma Values represent mean ± S.E.M. of the data from three independent experiments. Results considered significantly different if P< 0.05

 

Table 1Effects of oxytocin (150µg/kg twice a day) on RBC, WBC and PLT numbers in peripheral blood after 5-FU administration

Day	treatment	RBC (x106 / mm3)	WBC (x103 /mm3)	LY%	GR%	MI%	PLT (x 103/ml)	HGB	n
	Normal	8.10 ± 0.27	6.48 ± 0.35	72 ± 1.9	20 ± 1.5	8 ± 0.93	780 ± 45	160 ± 2.7	10
1	5-FU alone	9.08 ± 0.24	4.67 ± 0.39	64 ± 3.6	32 ± 3.4	3.9 ± 0.37	554 ± 28	150 ± 1.4	10
	5-FU + Oxytocin	9.4 ± 0.23	5.4 ± 0.84	62 ± 7.7	32 ± 7.5	6.3 ± 0.71	560 ± 53	160 ± 3.4	10
	5-FU alone	9.03 ± 0.20	6.1 ± 1.3	67 ± 2.9	26 ± 2.5	7 ± 2.4	370 ± 60	150 ± 2.8	10
3	5-FU + Oxytocin	8.8 ± 0.58	3.27 ± 0.44	66 ± 2.0	24 ± 1.7	9.6 ± 2.8	370 ± 77	150 ± 8.5	10
	5-FU alone	7.77 ± 0.19	3.6 ± 0.46	90 ± 1.3	4 ± 0.38	6.1 ± 1.0	132 ± 12	130 ± 3.9	9
6	5-FU + Oxytocin	7.6 ± 0.6	4.17 ± 0.89	85 ± 2.7	7.6 ± 1.6	7.5 ± 2.0	150 ± 6.3	130 ± 7.5	10
	5-FU alone	6.35 ± 0.46	3.6 ± 0.45	77 ± 2.9	14 ± 3.2	8.2 ± 0.99	59 ± 12	110 ± 6.5	8
9	5-FU + Oxytocin	7.6 ± 0.60	6.41 ± 0.74	85 ± 2.7	7.6 ± 1.2	11 ± 3.7	157 ± 7	130 ± 7.5	9

Values represent mean ± S.E.M. of the data from three independent experiments.

 

 

4. DISCUSSION:

To examine further action of Oxytocin, we investigated the ability of Oxytocin to decrease the myelotoxic effect of 5-FU in this study. Oxytocin injected subcutaneously for 5 consecutive days at doses of 150 µg/kg (twice a day) after 5-FU single dose, improved the decrease in medium size cells caused by 5-FU. Petruška et al. found the similar results in study with of quercetin (flavonoid  with  antioxidant capacity) and acute dose of  T-2 toxin (an immune depressive) on selected haematological parameters of rabbit's blood. Authors observed significant increase of MI% after chronic application of quercetin(8). The increase in medium size cells (a subgroup of the WBC that consists primarily of monocytes and progenitor cells) could refer to an ability of Oxytocin to release bone marrow progenitor cells. The administration of chemotherapeutic agents alone or in combination with hematopoietic growth factorscan result in a transient expansion and mobilization of progenitor cells into the peripheral circulation. In addition, the increases in peripheral blood progenitor cells of CFU-GM and BFU-E progenitor cells occurs subsequent to recovery of bone marrow progenitor cells but precedes or occurs simultaneously with the recovery of peripheral blood cell numbers. Therefore, changes in the number of progenitor cells in the peripheral blood may reflect hematopoietic recovery and may also reflect the ability of cytokines to expand the progenitor cells pool size(9).

 

Oxytocin after-treatment also increased platelets count in blood by 66%, an effect may be due to Oxytocin ability to induce a significant increase in relative renin secretion (10). This stimulation of renin-agiotensin system directly induces erythropoietin which increases platelets production. (11).It was reported that glutathione showed preventive effects against 5-FU-induced leukopenia, thrombocytopenia and erythrocytopenia, and this anti-oxidant accelerated recovery from them after cessation of 5-FU treatment (12). But Oxytocin after-treatment only increased GSH levels insignificantly (Fig.1) which can refer to a different mechanism of Oxytocin to increase peripheral blood progenitor cells. Further studies can be done on Oxytocin meylosuppression protective effect after chemotherapy in different doses.

 

5. REFERENCES:

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9.     Laurenz JC, Hadjisavas M, Chovanic GW, Bazer FW. Myelosuppression in the Pig ( Susscrofa ): Uteroferrin Reduces the Myelosuppressive Effects of 5-Fluorouracil in Young Pigs. 1997;116(4):369–77.

10.   Jong WD, Helwig J, Imbs J, Barthelmebs M. Oxytocin-induced renin secretion by denervated kidney in anaesthetized rat. European journal of pharmacology. 2002;454:241–7.

11.   Haznedaroglu IC, Arici M, Buyukasik Y. A unifying hypothesis for the renin-angiotensin system and hematopoiesis: Sticking the pieces together with the JAK-STAT pathway. Medical Hypotheses. 2000;54:80–3.

12.   Kojima S, Takaba K, Kimoto N, Takeda T. Protective effects of glutathione on 5-fluorouracil-induced myelosuppression in mice. Archives of Toxicology. 2003;77(5):285–90.

 

 

 

Received on 01.03.2013          Modified on 30.03.2013

Accepted on 12.04.2013         © RJPT All right reserved