Protection of rat chromosomes by melatonin against gamma radiation-induced damage

Mutation Research 677 (2009) 14–20

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Protection of rat chromosomes by melatonin against gamma radiation-induced damage Mohammad E. Assayed a,∗ , A.M. Abd El-Aty b a b

Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Menofiya University-Sadat City Branch, Egypt Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt

a r t i c l e

i n f o

Article history: Received 21 January 2009 Received in revised form 2 March 2009 Accepted 4 April 2009 Available online 22 May 2009 Keywords: Whole-body gamma irradiation Rat polychromatic erythrocytes Melatonin Micronucleus assay Mitotic index Chromosomal aberrations

a b s t r a c t The aim of this study was to investigate the protective effect of melatonin (2.5 mg/kg/day, given to rats five times by intra-peritoneal injection) against damage in bone-marrow chromosomes induced by a single dose of gamma radiation (4.0 Gy whole-body irradiation; WBI). Ninety-six male albino rats were divided into four equal groups of 24 rats each. They were designated as I—non-irradiated, non-treated control rats, II—non-irradiated rats treated with melatonin for five successive days, III—whole-body gamma-irradiated rats and IV—rats injected with melatonin daily for five successive days, then subjected to whole-body gamma irradiation 2 h after the final melatonin injection. Six rats from each group were sacrificed at days 1, 3, 7 and 10 following treatment and/or irradiation and their bone marrows were flushed out for micronuclei scoring and chromosomal analysis. WBI resulted in significant elevations in bone-marrow polychromatic erythrocytes containing micronuclei in their cytoplasm, and caused a significant decrease in the mitotic index of bone-marrow cells; in addition, there was a significant increase in the frequency of aberrant bone-marrow cells and in the different types of structural chromosomal aberration. Melatonin injection prior to WBI significantly reduced the mean frequencies of micro-nucleated polychromatic erythrocytes and of aberrant cells, as well as the incidence of structural chromosomal aberrations in bone-marrow cells; it also caused a highly significant elevation in the value of the mitotic index of bonemarrow cells. This investigation clarifies the protective and/or ameliorative role played by melatonin against deleterious effects of gamma radiation. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The increasing use of radiation in the modern world and recent incidents of massive radiation exposure dictate that certain basic elements of radiation toxicity should be investigated. Electromagnetic radiation is divided into non-ionizing and ionizing radiation according to the energy required to eject electrons from molecules [1]. Radiation-induced cellular changes may result in death of the organism, death of cells, modulation of physiological activity or cancer. Occupational exposure of employees at nuclear facilities, researchers using radioisotopes or of workers involved in the cleanup and/or storage of nuclear waste material, and exposure of patients during the therapeutic irradiation of cancer, would all pose a considerable biological hazard [2]. The deleterious effects of ionizing radiation in biological systems are mainly mediated through the generation of reactive oxygen

∗ Corresponding author. Tel.: +20 225190923; fax: +20 482603214. E-mail address: [email protected] (M.E. Assayed). 1383-5718/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2009.04.016

species (ROS) in cells as a result of water radiolysis [3]. Among them, particularly, the highly damaging hydroxyl radical (• OH) can cause injury by reacting with bio-molecules [4,5]. ROS and oxidative stress may contribute to radiation-induced cytotoxicity and to metabolic and morphologic changes in animals and humans during radiotherapy, experimentation [11], or even space flight [6]. In animal experiments, exposure to ionizing radiation leads to depletion of the endogenous antioxidants and ultimately to the development of systemic diseases [7,8]. Irradiation of rats with a total dose of 14.4 Gy decreased serotonin N-acetyltransferase activity and the concentration of pineal and serum melatonin 6 h and 3 days after the last radiation exposure [9]. In addition, total antioxidant capacity of plasma was reduced in patients exposed to whole-body irradiation for the purpose of reducing tumour growth. Consequently, the cellular antioxidant capacity is decreased and the organs become more susceptible to the deleterious effects of ROS [10]. The potential application of radio-protective chemicals in the event of planned exposures or radiation accidents/incidents has been investigated from the beginning of the nuclear era [11]. It has also been considered possible that radiation therapy for cancer patients could be improved by the use of radio-protectors to

M.E. Assayed, A.M. Abd El-Aty / Mutation Research 677 (2009) 14–20

protect normal tissue. Therefore, there is a constant need for the development of effective, non-toxic, radical scavengers that can protect humans against free-radical genetic damage induced by radiation and other agents. Unfortunately, most of the putative protective chemical substances would have to be used at relatively toxic doses in order to attain the desired protection, where their therapeutic index (toxic dose divided by protective dose) was small and this precluded their use in humans. The second drawback was that the duration of protection was short. The compound had to be injected about 10–30 min before irradiation. When the interval between administration and irradiation was extended beyond 1 h, protection became much less and disappeared completely [12]. Although biological and natural products are suitable tools for protection against radiation exposure, they are still receiving less attention [13]. Melatonin is a very potent and efficient endogenous radical scavenger. This pineal indolamine reacts with the highly toxic hydroxyl radical and provides on-site protection against oxidative damage to bio-molecules within every cellular compartment [14]. Melatonin acts as a primary nonenzymatic anti-oxidative defense against the devastating action of the extremely reactive hydroxyl radical. Melatonin and structurally related tryptophan metabolites are evolutionary conserved molecules principally involved in the prevention of oxidative stress in organisms as different as algae and rats [15]. Because radiation-induced cellular and sub-cellular injuries are attributed mainly to ROS, it is anticipated that melatonin treatment should delay or prevent the onset of radiation-induced oxidative stress and tissue injury [16]. Therefore, the present study was designed to investigate the possibly protective role of melatonin administration prior to radiation exposure against the clastogenic effect of whole-body gamma irradiation on rat chromosomes; to this end, we used the micronucleus assay and the analysis of bonemarrow metaphases for the presence of chromosomal aberrations.

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in DW. Freshly prepared, ice-chilled fixative consisted of absolute methyl alcohol: glacial acetic acid (3:1, v/v) (Nile Chemical Company, Cairo, Egypt). Diluted Giemsa Stain (1:10 in DW) and Wright stain (1.8 g/l in absolute methanol) were obtained from BDH Chemicals (AT, USA). 2.5. Melatonin A fresh solution of melatonin (N-acetyl-5-methoxytryptamine, Sigma–Aldrich Code M5250, purified crystals) in distilled water was made and adjusted to contain 0.25 mg melatonin per ml, corresponding to 2.5 mg/kg body weight. The latter dose was intra-peritoneally injected in rats of groups II and IV, daily for 5 consecutive days.

3. Cytogenetic investigations 3.1. Mitotic index The mitotic index (MI) was determined by examination of 1000 bone-marrow cells and by scoring the number of dividing cells per 1000 cells examined [17]. 3.2. Micronucleus assay The frequency of micro-nucleated polychromatic erythrocytes (PCEMN) in femoral bone-marrow preparations was scored and evaluated according to the previously published technique [18] with certain recommended modifications [19]. 3.3. Bone-marrow metaphase analysis for chromosomal aberrations Cytogenetic analysis of chromosomal aberrations (CA) of bonemarrow cells was carried out according to the CA technique [17] with further modifications [20].

2. Materials and methods

4. Statistical analysis

2.1. Radiation source

Data were expressed as arithmetical mean ± standard error of the mean (SEM). The Least Significance Difference test (LSD) was carried out and the Analyses of Variance (ANOVA) were conducted to obtain the significance of the treatments compared with the controls, using the statistical analysis methods described earlier [21].

The irradiation process was undertaken using a “Gamma Cell-40 unit”, manufactured by the Canadian Atomic Energy Authority and installed at the National Center for Radiation Research and Technology (NCRRT, Nasr City, Cairo, Egypt). The unit is a Cesium-137 type, which provides uniform gamma irradiation for small animals or biological samples, while providing complete protection for operating personnel. The unit gives a dose rate of 0.667 Gy/min. 2.2. Animals Ninety-six male albino Wistar rats of average body weight 75 g were used in the experimental work. Rats were obtained from the laboratory animal house of Ophthalmology Research Institute, Giza, Cairo. They were divided into four groups, each of twenty-four animals, as follows: Group I: Non-irradiated, non-treated normal control rats. Group II: Non-irradiated rats treated with melatonin. Group III: Whole-body gamma-irradiated rats (4 Gy). Group IV: Rats injected with melatonin on five consecutive days, then subjected to whole-body gamma irradiation (4 Gy), 2 h after the final melatonin injection. Six rats from each group were sacrificed at 1, 3, 7 and 10 days following treatment and/or irradiation and their bone marrows were flushed out for micronuclei scoring and cytogenetic analysis. 2.3. Irradiation dose Animals in groups III and IV received a whole-body gamma radiation dose of 4 Gy (estimated LD50/30, data not shown) at a dose rate of 0.667 Gy/min. 2.4. Chemicals Colchicine (4 mg/ml in distilled water, DW) (Sigma–Aldrich, St. Louis, MO, USA) was used as a mitotic inhibitor. Foetal calf serum (FCS) and absolute methyl alcohol (99.79%) were obtained from E. Merck (Darmstadt, Germany). Potassium chloride (Nile Chemical Company, Cairo, Egypt) was prepared as a hypotonic solution 0.56 g%

5. Results The mean values of PCEMN scored in 500 bone-marrow polychromatic erythrocytes are shown in Table 1. Data of scored PCEMN having only one MN, two MN, more than two MN as well as total PCEMN, showed that i.p. administration of melatonin produced no significant alterations of the mean values of such types of PCEMN during all sampling days of the experiment, compared with control values. In contrast, whole-body gamma irradiation (WBI) of rats induced a highly significant increase in the percentage of PCEMN containing one, two, or more than two MN and in the total number of PCEMN at all sampling days, compared with control values. Pre-treatment of the rats with i.p. injections of melatonin prior to their WBI highly significantly reduced the mean frequencies of PCEMN containing one, two, or more than two MN, as well as the total number of PCEMN at all sampling days of the experiment, as compared with the WBI-alone values. Regarding the mitotic index, data in Table 2 show that treatment of rats with melatonin by i.p. injection induced a significant decrease in the MI of bone-marrow cells at all sampling days, compared with control values. Furthermore, exposure of animals to WBI resulted in a highly significant decrease in the MI of bone-marrow cells on all the experimental sampling days, compared with control values.

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Table 1 Effect of whole-body gamma irradiation and melatonin pre-treatment on frequencies of micro-nucleated polychromatic erythrocytes in rat bone-marrow after different sampling times. Treatment

Frequency of micronuclei

Time after treatment

Control

1 MN 2 MN >2 MN Total PCEMN

5.5 1.35 0.66 7.51

± ± ± ±

0.84 0.42 0.03 1.14

6.0 1.32 0.64 7.96

± ± ± ±

0.88 0.35 0.01 1.12

5.6 1.34 0.66 7.60

± ± ± ±

0.82 0.42 0.03 1.14

6.2 1.33 0.68 8.21

± ± ± ±

0.86 0.40 0.02 1.24

Treatment with melatonin

1 MN 2 MN >2 MN Total PCEMN

4.33 1.66 0.50 6.50

± ± ± ±

0.91 0.61 0.34 1.83

4.83 1.83 0.66 7.33

± ± ± ±

0.7 0.31 0.91 1.11

4.16 1.33 0.66 6.16

± ± ± ±

1.07 0.42 0.33 1.6

4.83 2.00 1.00 7.83

± ± ± ±

0.79 0.57 0.25 1.3

WBI

1 MN 2 MN >2 MN Total PCEMN

55.5 13.83 9.3 78.66

± ± ± ±

3.18a 1.07a 0.66a 4.43a

55.16 15.00 7.5 77.66

± ± ± ±

3.5a 1.06a 0.42a 4.07a

50.5 11.83 5.5 67.83

± ± ± ±

2.95a 0.94a 0.70a 3.20a

52.5 11.83 6.0 70.30

± ± ± ±

1.89a 1.79a 0.57a 0.95a

Treatment with malatonin prior to WBI

1 MN 2 MN >2 MN Total PCEMN

25.66 3.5 2.5 31.66

± ± ± ±

1.42b 0.42b 0.56b 2.6b

30.83 2.83 2.0 35.66

± ± ± ±

1.19b 0.60b 0.36b 1.7b

19.5 2.0 0.66 22.16

± ± ± ±

1.38b 0.44b 0.21b 1.77b

41.16 6.5 3.83 51.0

± ± ± ±

1.99b 0.99b 0.6b 3.14b

Day 1

Day 3

Day 7

Day 10

Values expressed as mean ± SE, WBI: whole-body gamma irradiation. a Significantly different from control group (p < 0.01). b Significantly different from WBI group (p < 0.01).

Melatonin pre-treatment of rats, followed by WBI produced a highly significant elevation in the values of MI at all experimental sampling days, when compared with the WBI values. Such values of MI, although highly significantly elevated, are still significantly below the control values. Data in Table 3 show that no significant effect of melatonin injection was recorded on the numbers of either normal or aberrant cells of the bone marrow of rats, as compared with the corresponding control values. In contrast, exposure of rats to WBI produced highly significant decrease in the mean number of normal cells during all experimental sampling periods. At the same time, a highly significant increase was seen in the mean number of aberrant cells, comprising those with one aberration and more than one aberration, as well as in the total number of aberrant cells, compared with the corresponding control values. Treatment of rats with five daily i.p. doses of melatonin before WBI resulted in a highly significant increase in mean number of normal bone-marrow cells, associated with a highly significant decrease in the mean aberrant cell number, either of those containing one aberration, those having more than one aberration and hence the total aberrant cell mean numbers at all experimental sampling days, compared with the results without melatonin. Nonetheless, the normal cell mean values were still lower than those of the control group, while the aberrant cell values remain higher than their corresponding control values. Results concerning different types of CA showed that only structural aberrations, including chromatid-type and chromosome-type aberrations were recorded in bone-marrow cells of treated rats during the whole experiment. No numerical aberrations were found

during the cytogenetic examination of bone-marrow metaphase spreads. Table 4 shows the mean values of various scored chromatidtype aberrations, comprising chromatid gaps (cg), chromatid breaks (cb), chromatid deletions (cd) and acentric fragments (af) at different sampling days. It is obvious that rats injected with melatonin displayed non-significant alterations in the mean values of cg, cb, cd or af at different sampling days of the experiment, when compared with the equivalent control values. WBI of rats resulted in highly significant elevations in the mean values of all chromatidtype aberrations at all sampling days of the experiment compared with the equivalent control values of such aberrations. Melatonin injection followed by WBI produced a highly significant reduction in the mean values of cg at days 3, 7 and 10 of the experiment, while such treatment had a non-significant effect on cg at day 1. In addition, melatonin injection prior to WBI resulted in a highly significant reduction in the mean values of cb as well as af at all experimental sampling days compared with WBI mean values, while such treatment induced a significant reduction in the mean values of cd on days 1 and 3 of the experiment and a highly significant reduction on days 7 and 10, compared with the corresponding values without melatonin treatment. Table 5 shows the mean values of different chromosome-type aberrations: chromosome gaps (Sg), chromosome breaks (Sb), ring chromosomes (R) and dicentric chromosomes (DiC) at different sampling days. The results demonstrate that injection of rats with melatonin had no significant effect on the mean frequencies of any of these aberrations at all sampling days of the experiment, when compared with the equivalent control values.

Table 2 Effect of whole-body gamma irradiation and melatonin pre-treatment on the mitotic index of rat bone-marrow cells after different sampling times. Treatment

Mitotic index Day 1

Control Treatment with melatonin WBI Treatment with melatonin prior to WBI

65.66 60.33 26.33 41.66

Day 3 ± ± ± ±

1.29 1.35a 1.62a 0.98b

Values expressed as mean ± SE, WBI: whole-body gamma irradiation. a Significantly different from control group (p < 0.01). b Significantly different from WBI group (p < 0.01).

69.66 57.32 30.66 42.50

Day 7 ± ± ± ±

1.29 1.30a 0.91a 1.33b

66.61 55.42 33.50 47.16

Day 10 ± ± ± ±

1.19 1.32a 0.99a 0.87b

64.62 ± 1.19 50.53 ± 1.25a 41.16 ± 1.13a 51.33 ± 1.05b

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Table 3 Effect of whole-body gamma irradiation and melatonin pre-treatment on frequencies of aberrant cells in rat bone-marrow after different sampling times. Treatment

Frequency of cells

Time after treatment Day 7

Day 10

Control

Normal cells Cells with 1 aberr. Cells with >1 aberr. Total aberr. cells

43.83 5.166 1.00 6.16

± ± ± ±

1.62 1.53 0.25 1.6

44.83 5.132 1.06 6.192

± ± ± ±

1.66 1.55 0.26 1.12

44.46 ± 1.62 5.230 ± 1.42 1.06 ± 0.25 6.290 ± 1.14

43.88 5.133 1.00 6.133

± ± ± ±

1.64 1.53 0.22 1.24

Treatment with melatonin

Normal cells Cells with 1 aberr. Cells with >1 aberr. Total aberr. cells

44.33 5.16 0.50 5.66

± ± ± ±

0.49 0.31 0.34 0.49

44.00 5.66 0.33 6.0

± ± ± ±

0.86 0.80 0.21 0.85

45.66 4.00 0.33 4.33

± ± ± ±

0.75 0.57 0.21 0.71

44.33 5.33 0.33 5.66

± ± ± ±

1.08 0.98 0.21 1.08

WBI

Normal cells Cells with 1 aberr. Cells with >1 aberr. Total aberr. cells

12.0 20.81 17.16 38.00

± ± ± ±

0.96a 1.01a 1.53a 0.96a

12.16 25.83 12.0 37.83

± ± ± ±

0.87a 1.37a 1.23a 0.87a

20.83 21.83 7.33 29.16

± ± ± ±

1.16a 0.91a 0.84a 1.16a

13.83 28.66 7.5 36.16

± ± ± ±

1.24a 1.28a 0.76a 1.24a

Treatment with melatonin prior to WBI

Normal cells Cells with 1 aberr. Cells with >1 aberr. Total aberr. cells

32.83 13.0 4.16 17.16

± ± ± ±

1.3b 0.93b 0.6b 1.3b

34.5 12.33 3.16 15.50

± ± ± ±

0.76b 0.61b 0.47b 0.76b

40.33 8.0 1.66 9.66

± ± ± ±

0.84b 0.57b 0.42b 0.84b

37.00 9.5 3.5 13.0

± ± ± ±

1.71b 1.4b 0.72b 1.71b

Day 1

Day 3

Values expressed as mean ± SE, WBI: whole-body gamma irradiation. a Significantly different from control group (p < 0.01). b Significantly different from WBI group (p < 0.01).

WBI of rats induced highly significant elevations in the mean frequencies of Sg, Sb, and R at all sampling days of the experiment, while the same treatment produced a mildly significant increase in the frequencies of DiC at days 1 and 7, and a non-significant increase on days 3 and 10, compared with the equivalent control values. Melatonin i.p. injection to the rats followed by WBI resulted in a mildly significant reduction in the mean values of Sg at day 1, a highly significant reduction in Sg mean values at days 3 and 7, and a non-significant effect on Sg at day 10. Melatonin injection followed by WBI also caused a highly significant reduction in the mean values of Sb as well as R at all experimental sampling days. It was also shown that the same treatment induced a significant decrease in the mean values of DiC at day 7, while such treatment produced a non-significant reduction in the mean frequencies of DiC at days 1, 3 and 10, compared with the equivalent WBI mean values.

6. Discussion The aim of the present work was to investigate the extent to which the natural pineal mediator melatonin is able to control the damaging effect of gamma radiation on cell division and chromosomes. The results clearly show that WBI of rats at a dose of 4 Gy induced a highly significant increase in the percentages of PCEMN containing one, two, or more than two MN, as well as of the total PCEMN at all sampling days of the experiment. The micronucleus test is a simple method that assesses genotoxic damage in cells exposed to physical or chemical agents [22]. Micronuclei are formed of acentric fragments or whole chromosomes that have not been incorporated into daughter nuclei at mitosis [17,23]. Pre-treatment of the rats with i.p. injections of melatonin on five successive days prior to the WBI significantly reduced the mean frequencies of PCEMN containing one, two and more than two

Table 4 Effect of whole-body gamma irradiation and melatonin pre-treatment on the frequencies of structural aberrations in rat bone-marrow cells after different sampling times. Treatment

Frequency of cells

Time after treatment Day 1

Day 3

Day 7

Day 10

(A) Chromatid-type aberrations Control

Chromatid gaps Chromatid breaks Chromatid deletion Acentric fragments

3.66 0.66 0.16 4.16

± ± ± ±

0.66 0.42 0.01 1.32

3.61 0.63 0.16 4.46

± ± ± ±

0.66 0.39 0.01 1.46

3.55 0.66 0.16 4.16

± ± ± ±

0.61 0.42 0.01 1.32

3.66 0.63 0.16 4.56

± ± ± ±

0.66 0.39 0.01 1.48

Treatment with melatonin

Chromatid gaps Chromatid breaks Chromatid deletion Acentric fragments

2.83 0.16 0.16 1.66

± ± ± ±

0.31 0.01 0.01 0.61

4.16 0.5 0.5 1.50

± ± ± ±

0.31 0.34 0.05 0.67

2.83 0.00 0.16 1.50

± ± ± ±

0.54 0.00 0.01 0.36

2.50 0.66 0.16 3.16

± ± ± ±

1.08 0.33 0.01 0.70

WBI

Chromatid gaps Chromatid breaks Chromatid deletion Acentric fragments

9.83 9.83 4.50 15.83

± ± ± ±

1.63a 1.62a 0.62a 1.44a

11.66 9.00 3.66 14.00

± ± ± ±

0.88a 1.32a 0.42a 1.69a

10.00 8.66 4.83 10.50

± ± ± ±

0.83a 0.61a 0.47a 0.76a

11.66 9.66 3.66 14.33

± ± ± ±

0.88a 0.33a 0.42a 1.33a

Treatment with melatonin prior to WBI

Chromatid gaps Chromatid breaks Chromatid deletion Acentric fragments

8.66 2.00 2.16 6.00

± ± ± ±

0.84 0.57c 0.31b 0.73c

6.50 2.00 2.16 7.00

± ± ± ±

0.76c 0.36c 0.31b 0.36c

4.83 1.00 0.83 4.00

± ± ± ±

0.60c 0.36c 0.31c 0.51c

7.33 2.66 1.33 5.83

± ± ± ±

1.1c 0.55c 0.21c 0.11c

Values expressed as mean ± SE, WBI: whole-body gamma irradiation. a Highly significantly different from control group (p < 0.01). b Significantly different from WBI group (p < 0.01). c Highly significantly different from WBI group (p < 0.01).

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Table 5 Effect of whole body gamma irradiation and melatonin pretreatment on frequencies of structural aberrations in rat bone marrow cells after different sampling time. Treatment

Frequency of cells

Time post treatment Day 1

Day 3

Day 7

Day 10

(B) Chromosome-type aberrations Control

Chromosome gaps Chromosome breaks Ring Chromosomes Dicentric chromosome

0.33 0.00 0.00 0.00

± ± ± ±

0.21 0.00 0.00 0.00

0.33 0.00 0.00 0.00

± ± ± ±

0.21 0.00 0.00 0.00

0.33 0.00 0.00 0.00

± ± ± ±

0.21 0.00 0.00 0.00

0.33 0.00 0.00 0.00

± ± ± ±

0.21 0.00 0.00 0.00

Melatonin Treatment

Chromosome gaps Chromosome breaks Ring Chromosomes Dicentric chromosome

0.16 0.00 0.00 0.00

± ± ± ±

0.01 0.00 0.00 0.00

0.33 0.00 0.00 0.00

± ± ± ±

0.03 0.00 0.00 0.00

0.16 0.00 0.00 0.00

± ± ± ±

0.01 0.00 0.00 0.00

0.16 0.00 0.00 0.00

± ± ± ±

0.01 0.00 0.00 0.00

WBI

Chromosome gaps Chromosome breaks Ring Chromosomes Dicentric chromosome

6.33 4.16 4.16 0.83

± ± ± ±

1.33c 0.83c 0.83c 0.31a

6.33 3.00 5.00 0.66

± ± ± ±

0.84c 0.52c 0.51c 0.33

5.50 4.16 2.66 0.83

± ± ± ±

0.62c 0.47c 0.49c 0.31a

4.66 3.83 3.66 0.66

± ± ± ±

0.66c 0.47c 0.55c 0.33

Melatonin Treatment + WBI

Chromosome gaps Chromosome breaks Ring Chromosomes Dicentric chromosome

2.5 1.00 0.50 0.16

± ± ± ±

0.42b 0.36d 0.27d 0.01

3.16 0.66 0.16 0.16

± ± ± ±

0.47d 0.33d 0.01d 0.01

1.33 2.00 0.33 0.00

± ± ± ±

0.21d 0.12d 0.21d 0.00b

2.66 1.33 0.66 0.33

± ± ± ±

0.8 0.14d 0.33d 0.21

Values expressed as mean ± SE, WBI: whole-body gamma irradiation. a Significantly different from control group (p < 0.01). b Significantly different from WBI group (p < 0.01). c Highly significantly different from control group (p < 0.01). d Highly Significantly different from WBI group (p < 0.01).

MN as well as the total number of PCEMN, on all sampling days. Our results are in agreement with published data [23,24] showing that human peripheral blood lymphocytes pre-treated in vitro with melatonin and then exposed to in vitro gamma radiation, exhibited a significantly reduced incidence of micronuclei, as compared with similarly irradiated lymphocytes not pre-treated with melatonin. The present results revealed that the mitotic index (MI) was decreased below the control value in both groups II and III, while it was elevated over the control in group IV. The influence of melatonin on cell division and mitosis and its participation in the regulation of the growth of a variety of normal and cancer cells has been demonstrated in many investigations. Mice injected with 1, 10 and 100 ␮g of melatonin, and rat organ cultures treated in vitro with 5 × 10−7 M melatonin, were shown to exhibit a significantly decreased mitotic activity in thyroid follicular and adrenocortical cells [25], while pinealectomy in rats resulted in an increased proliferation of cells in the anterior pituitary gland, intestinal epithelium, liver and spleen [26]. Melatonin has also been shown to inhibit the growth of a variety of neoplasms, particularly mammary tumours, in vivo and in vitro [27]. It was also suggested that the influence of melatonin on cell growth can be considered in terms of its effect on (a) gene expression leading to changes in the process of cell division; (b) RNA processing to provoke alterations in cellular blast transformation; (c) posttranslational modifications to induce conformational changes in specific protein molecules involved in the regulation of cell growth; or (d) activation of specific receptor/acceptor molecules by classical ligand-transformation mechanisms [28]. The adverse effect of radiation on cell division has been well documented in the literature, and there are indications that the inhibition of nuclear DNA synthesis by irradiation is responsible for the longest delays in cell division [26,29,30]. Referring to the results obtained in the present study, exposure of rats to 4 Gy-WBI caused a highly significant decrease in normal cells during all experimental sampling periods, associated with a highly significant increase in all aberrant cell types. In addition, highly significant elevations were noted in the mean values of all chromatid-type and chromosome-type aberrations at all sampling days. It has long been known that the damaging effects of ionizing radiation on cellular DNA are brought about by both direct and indirect mechanisms. Direct action produces disruption

of chemical bonds in the molecular structure of DNA, while indirect effects result from highly reactive free radicals such as • OH, • H and e-aq produced during the radiolysis of water, and their subsequent interaction with cellular DNA [30]. Evaluation of chromosome aberrations is a fully accepted method for evaluation of genotoxic effects as it always indicative of real mutations [31]. The production of chromosomal aberrations (CA) is a complex cellular process. The mechanisms of chromosome breakage and re-joining are not yet completely understood. According to the prevailing theories, structural CA result from (i) direct DNA breakage, (ii) replication on a damaged DNA template, (iii) inhibition of DNA synthesis. Under in vivo conditions, assessment of genotoxicity and in particular the clastogenic potential of an agent is evaluated by use of the CA assay [20]. Melatonin, when given intra-peritoneally to rats prior to WBI caused protection of the bone-marrow cells and chromosomes of those rats – to a relatively high extent – against the detrimental effects of gamma irradiation at most sampling days of the experiment. Two hypotheses can be proposed for the observed protective effect of melatonin. First, melatonin may directly protect against chromosome damage by scavenging free radicals generated by ionizing radiation before they induce damage to the genetic material, i.e. the extent of primary damage in cellular DNA may be significantly reduced. Secondly, melatonin may indirectly alter the final level of chromosome damage by activating oxidative repair enzymes, so that the damaged DNA is repaired more rapidly in irradiated cells pre-treated with melatonin. In fact, the published literature on melatonin indicates that both of the proposed hypotheses may be valid [25]. The small size and high lipophilicity of the melatonin molecule permits its rapid diffusion through biomembranes. Melatonin crosses all morpho-physiological barriers, e.g., the blood–brain barrier and the placenta, and is distributed throughout the cell, reaching its highest concentrations in the nucleus, where it protects DNA from free-radical damage [32]. In mammalian tissues, evidence from both immuno-cytochemical studies and radio-immunoassays indicates that endogenously produced or exogenously administered melatonin is more highly concentrated in the nucleus than in the cytosol [33]. The tendency of melatonin to diffuse and accumulate within the nucleus along with its ability to scavenge free radicals would provide an effective

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and direct means of on-site protection of the cells against radiationinduced genetic damage [34]. On the other hand, melatonin also appears to activate certain cellular enzymatic antioxidant defense mechanisms, for instance glutathione peroxidase, which acts to decrease • OH formation by metabolizing its precursor, H2 O2 . Other enzymes involved in DNA repair may also be induced by melatonin, and such enzymes would facilitate quick repair of the damaged DNA [15,16]. Melatonin can also have a protective effect against oxidative DNA damage by chemical inactivation of a DNA-damaging agent as well as by stimulating DNA repair, but key factors in base-excision repair, like glycosylases and AP-endonucleases do not seem to be affected by melatonin [35]. In addition, melatonin may alter the activities of enzymes that improve the endogenous antioxidant defense capacity, including superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase [36]. Melatonin scavenges the • OH by donating an electron to the latter, resulting in the generation of the indolyl radical [37]. This radical product may interact with the superoxide anion radical to produce N-methyl-N-2-formyl5-methoxykynuramine, another biogenic amine and excellent scavenger [38,39]. The prophylactic action of N(1)-acetyl-N(2)formyl-5-methoxykynuramine (AFMK), an important melatonin metabolite, against X-irradiation-induced oxidative damage to bio-molecules (DNA, protein and lipid) in C57BL mice was demonstrated. Total antioxidant capacity of plasma was significantly reversed in AFMK-pretreated mice, and AFMK showed a very high level of in vitro hydroxyl radical-scavenging potential [40]. The same authors later [41] reported protective effects of AFMK against high energy charged particle radiation-induced oxidative damage to the brain, as it ameliorated the radiation-induced augmentation of protein carbonyls and 4-hydroxyalkenal plus malondialdehyde (HAE + MDA) in the brain and maintained the total antioxidant capacity of plasma and non-protein sulfhydryl content in brain tissue. A second product of melatonin’s scavenging of the • OH radical has been identified as cyclic 3-hydroxymelatonin, which is formed when melatonin scavenges two • OH. After its formation this product is secreted in the urine [42]. This provides direct evidence that melatonin under physiological conditions acts as a direct free-radical scavenger and detoxifies the highly cytotoxic • OH in vivo. Based on the results presented here it may be concluded that melatonin administration prior to irradiation decreases the oxidative stress and harmful effects of ionizing radiation on bonemarrow cells and stimulates the antioxidant enzyme activities. Hence melatonin could be of great benefit in alleviating radiation toxicity to the bone marrow in radiation-treated cancer patients. Moreover, melatonin may enable higher radiation dose rates to be applied in patients suffering from cancers. Since melatonin is not foreign to the human body, these results should provide an impetus to further research leading to the use of melatonin in the protection of the genome against effects of natural and other DNA-damaging agents. Conflict of interest The authors declare that there are no conflicts of interest. References [1] A.W. Hayes, Principles and Methods of Toxicology, Raven Press, New York, 2001, pp. 147–149. [2] J.D. Boice, Radiation carcinogenesis—human epidemiology, in: The Biological Basis of Radiation Protection Practice, Williams and Wilkins, Baltimore, 1992, pp. 89–120. [3] J.P. Kamat, K.K. Boloor, T.P.A. Devasagayam, S.R. Venkatachalam, Antioxidant properties of Asparagus racemosus against damage induced by gamma radiation in rat liver mitochondria, J. Ethnopharmacol. 71 (2000) 425–435.

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