in vitro studies with human volunteers

Genetic Toxicology

ELSEVIER

Mutation Research 371 (1996) 221-228

Melatonin and radioprotection from genetic damage: In vivo/in vitro studies with human volunteers Vijayalaxmi a,*,

Russel J. Reiter b, Terence S. Herman a, Martin L. Meltz a

Department of Radiology/Division of Radiation Oncology, The Unieersity of Texas Health Science Center, 7703 Floyd Curl Drip,e, San Antonio, TX 78284, USA b Department of Cellular and Structural Biology, The UnicersiO' of Texas Health Science Center, 7703 Floyd Curl DriL,e, San Antonio, TX 78284, USA

Received 21 May 1996;revised 13 August 1996; accepted 13 August 1996

Abstract Peripheral blood samples were collected from human volunteers at 0 ( 5 - l 0 min before), and at 1 and 2 h after a single oral dose of 300 mg of melatonin. At each time point, (i) the concentration of melatonin in the serum and in the leukocytes was determined, and (ii) the whole blood was exposed in vitro to 150 cGy of ~37Cs gamma radiation, and the lymphocytes were cultured with mitogenic stimulation to determine the extent of radiation-induced genetic damage, viz., chromosome aberrations and micronuclei. For each volunteer, the results showed a significant increase in the concentration of melatonin in the serum and in the leukocytes at 1 h after the oral dose of melatonin, as compared to the sample collected at 0 h. The lymphocytes in the blood samples collected at 1 and 2 h after melatonin ingestion and exposed in vitro to 150 cGy gamma radiation exhibited a significant decrease in the incidence of chromosome aberrations and micronuclei, as compared with similarly irradiated lymphocytes from the blood sample collected at 0 h; the frequencies observed in the cells sampled at 2 h after the ingestion of melatonin were consistently lower when compared with those collected at 1 h. The data may have important implications for the protection of human lymphocytes from the genetic damage induced by free radical-producing mutagens and carcinogens. Keywords: Melatonin;Gamma radiation;Chromosome aberrations;Micronuclei;Radioprotection

1. Introduction In humans, melatonin (N-acetyl-5methoxytryptamine) is synthesized mainly by the pineal gland in the brain, where it exhibits a circadian rhythm with maximum production occurring

* Corresponding author. Tel.: (210) 567-5576; Fax: (210) 5673446; E-mail: [email protected]

during the night [ 1,2]. It participates in the regulation of a number of important physiological and pathological processes, and reportedly has receptor- and non-receptor-mediated effects [3-7]. Of particular interest is the demonstration that melatonin scavenges hydroxyl radicals ( . O H ) generated in vitro when hydrogen peroxide is exposed to ultraviolet light [8]. In in vivo studies, melatonin has been shown to inhibit the formation of DNA adducts in the liver of rats treated with safrole, a chemical

0165-1218/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S01 65- 12 18(96)00099-7

222

Vijayalaxmi et al. / Mutation Research 371 (1996) 221-228

carcinogen which acts in part due to its ability to generate reactive oxygen species [9,10], and also has been reported to prolong the survival of irradiated mice [11]. Recently, human peripheral blood lymphocytes which were pretreated in vitro with 0.5, 1.0 and 2.0 mM melatonin for 20 min, washed, and then exposed in vitro to ionizing radiation were reported to exhibit a significant and melatonin concentrationdependent decrease in the extent of genetic damage, as compared with similarly irradiated lymphocytes which were not pretreated with melatonin [12,13]. The damaging effects of ionizing radiation on cellular DNA have long been known to be due to both direct and indirect mechanisms. A number of investigators, using radical scavenging compounds, have shown that a significant proportion of radiation-induced biological effects are attributable to indirect action, and that • OH radicals are primarily responsible [14-16]. The study described here utilized human volunteers to test the hypothesis that oral administration of a single pharmacological dose of 300 mg melatonin would increase its concentration in serum, and that the increased level would be sufficient to protect the blood lymphocytes (obtained at the times of the increased levels of melatonin) from in vitro gamma radiation-induced genetic damage, viz., chromosome aberrations and micronuclei. To our knowledge, this is the first study to demonstrate in vivo protective ability of melatonin on in vitro radiation-induced genetic damage in human blood lymphocytes.

2. Materials and methods 2.1. Blood collection

A protocol approved by the Institutional Review Board was followed. Informed consent was obtained from four healthy, nonsmoking human volunteers, two males and two females aged between 30-53 years. Powdered melatonin (Regis Chemical Co., Morton Grove, IL, 99.9% purity) was accurately weighed and placed in a gelatin capsule using sterile procedures. After overnight fasting, each volunteer was given a single oral dose of 300 mg of melatonin (Regis Chemical Co., Morton Grove, IL) at 8:45 a.m. Blood samples were collected in sterile vacutainer tubes (without any preservative to separate the

serum, and with heparin as an anticoagulant for the separation of leukocytes and for in vitro exposure to gamma radiation) 5 - 1 0 min before, and at 1 and 2 h (_+ 5 min) following the ingestion of melatonin. 2.2. Melatonin concentration in the serum and leukocytes

At each of the collection times, for each volunteer, (i) serum was separated from the blood in the vacutainer tube without any preservative, and (ii) 5 ml of heparinized whole blood was used to separate the leukocytes by carefully layering it over 10 ml of ficoll-hypaque gradient in a centrifuge tube (25 ml capacity). All tubes were centrifuged for 15 min at 800 × g. The interface layer containing the leukocytes was separated, washed twice with phosphate buffered saline and homogenized in ice cold 10 mM phosphate buffer, pH 7.8. Since the extent of radiation-induced genetic damage is assessed in the lymphocytes, the study included the determination of the concentration of melatonin in leukocytes (melatonin concentration in the serum represents the circulating levels in the body). All serum and leukocyte samples were coded before determining the concentration of melatonin using a highly specific antibody (Guildhay Stockgrand Antisera, Guilford, UK) in a direct radioimmunoassay [17]. One ml of each sample was extracted 1 : 1 with chloroform in ice cold water for 15 min, and evaporated overnight in the dark at room temperature. Each dried residue was dissolved in radioimmunoassay buffer. Serial dilutions were mixed with antiserum solution (diluted 1:5000) and [3H]melatonin. After incubating the mixtures for 18 h at 34 _+ I°C, the amount of radioactivity was measured in a liquid scintillation counter. The concentration of melatonin in each sample was calculated. The recovery of exogenous standard melatonin determined in the serum and leukocyte samples was 81% and 78%, respectively. 2.3. Gamma radiation and culture set up to determine genetic damage

At each of the collection times, for each volunteer, duplicate aliquots of heparinized whole blood were immediately exposed in vitro to 150 cGy gamma radiation (137Cs source, GammaCell-40 Irradiator, Atomic Energy of Canada Ltd.; dose rate of 1.087

223

Vijayalaxmi et al. / Mutation Research 371 (1996) 221-228

G y / m i n ) . Duplicate aliquots of sham irradiated blood samples were used as a control. Immediately after irradiation, from each aliquot of the blood sample, separate cultures were set up for the chromosome aberration and for micronuclei assays, each using 0.5 ml of whole blood mixed with 4.5 ml of RPMI 1640 culture medium (Mediatech, Washington, DC) containing 15% fetal bovine serum (JRH Biosciences, Lenexa, KS), 1% phytohemagglutinin (Gibco, Grand Island, NY), 50 U / m l penicillin, 50 i~g/ml streptomycin, 2 mM glutamine (Mediatech) and 25 IxM bromodeoxyuridine (Sigma, St. Louis, MO). 2.4. C h r o m o s o m e aberration analysis

All cultures were incubated at 37___ I°C in a humidified atmosphere of 5% C O 2 / 9 5 % air for 48 h. During the last 2 h of incubation, colcemid solution (Gibco) was added to all cultures at a final concentration of 0.1 i x g / m l and the incubation continued. At the end of 48 h of incubation, the cells were collected by centrifugation and resuspended in 8 ml of potassium chloride for 8 min; they were then fixed in three changes of 3 : 1 methanol/acetic acid mixture. Fixed cells were dropped onto clean microscopic slides, air-dried and stained with the standard fluorescence-plus-Giemsa technique [18]. All slides were coded by an individual other than the scorer, and evaluated for chromosome aberrations. At each blood collection time, for each volunteer, from irradiated and control cultures, a total of 200 cells (100 cells each from duplicate cultures) in their first mitotic division, as defined by the absence of harlequin staining, were examined. Gaps and achromatic lesions less than the width of a chromatid were not included in the scoring. The number of abnormal cells showing chromosome aberrations, acentric fragments and exchange aberrations (dicentric, tricentric, ring, and tri/quadriradial chromosomes) were tabulated. Appropriate numbers of accompanying acentric fragments were assigned to each inter-chromosomal or inter-arm exchange chromosome; all excess acentric fragments were recorded separately [121. 2.5. M i c r o n u c l e i

All cultures were incubated at 37_+ I°C in a humidified atmosphere of 5% C O 2 / 9 5 % air for 72

h. Cytochalasin B (Sigma, St. Louis, MO) was added to all cultures (4 txg/ml) at 44 h to block the dividing cells in cytokinesis, and the incubation continued. At the end of the 72 h incubation, cells were collected, treated with 0.8% sodium citrate for 3 to 5 min and fixed in 5 : 1 methanol/acetic acid mixture. Fixed cells were dropped gently onto clean microscope slides, air dried and stained with 4% Giemsa (Sigma) using standard procedures. All slides were coded by an individual other than the scorer, and evaluated at 1000 × magnification for the frequency of micronuclei in cytokinesis blocked binucleate cells with well-preserved cytoplasm. The protocol described earlier for the identification of binucleate cells and micronuclei was followed [13]. At each blood collection time, for each volunteer, from irradiated and control cultures, a total of 2000 binucleate cells (1000 cells each from duplicate cultures) were examined to record the frequency of cells with one (C1MN), two (C2MN) or three (C3MN) micronuclei. The total number of micronuclei observed were derived from (1 × C1MN) + (2 × C2MN) + (3 × C3MN). The number of binucleate cells containing micronuclei was assessed as (C1MN) + (C2MN) + (C3MN).

Table 1 The concentration of melatonin in the blood samples collected from human volunteers, at 0 (5-10 min before), and at 1 and 2 h (+ 5 min) after a single oral ingestion of 300 mg melatonin Volunteer Time Serum Leukocytes (h) (ng/ml) (ng/mg protein) 1

0 1 2

0.04 0.96 83.13

0.01 0.44 0.07

0 1 2

0.04 1.37 0.86

0.01 29.69 33.55

0 1 2

0.04 80.95 1.70

0.01 30.45 1.27

0 1 2

0.02 85.41 1.38

* Leukocyte separation from the whole blood failed.

Vijayalaxmi et al./ Mutation Research 371 (1996) 221 228

224

2.6. S t a t i s t i c a l a n a l y s i s

the values obtained

in c o r r e s p o n d i n g

control

lym-

phocytes from those recorded for irradiated lymphoF o r e a c h v o l u n t e e r , at e a c h b l o o d c o l l e c t i o n t i m e , the incidence of radiation-induced chromosome

c y t e s . F o r e a c h v o l u n t e e r , t h e d i f f e r e n c e in t h e i n c i -

aber-

dence of radiation-induced

rations and micronuclei was computed by subtracting

and

micronuclei

between

chromosome 0, a n d

aberrations

1 and

2 h after

Table 2 The incidence of cells with chromosome damage, and the numbers of exchange aberrations and acentric fragments induced in vitro by 150 cGy gamma radiation in cultured blood lymphocytes from human volunteers examined at 0 (5-10 min before), and at 1 and 2 hours ( + 5 minutes) after a single oral ingestion of 300 mg melatonin Cells with Ch' damage Observed Volunteer 1 * 0 hr-control 0 hr-150 cGy 1 hr- control 1 hr-150 cGy 2 hr-control 2 hr-150 cGy

1 63 l 30 2 28

Volunteer 2 * 0 hr-control 0 hr-150 cGy 1 hr-control 1 hr-150 cGy 2 hr-control 2 hr-150 cGy

2 72 l 35 1 31

Volunteer 3 * 0 hr-control 0 hr-150 cGy 1 hr-control 1 hr-150 cGy 2 hr- Control 2 hr-150 cGy

1 63 2 30 2 26

Volunteer 4 * 0 hr-control 0 hr- 150 cGy 1 hr-control 1 hr-150 cGy 2 hr-control 2 hr- 150 cGy

2 69 2 32 2 27

Percent decrease

Total exchange Abr' p value

53.2

3.9

58.1

4.3

51,4

4.1

57.1

4.6

54.8

4.0

61.3

4.6

55.2

4.2

62.7

4.9

Total for 4 volunteers * * 0 hr-Control 6 0 hr-150 cGy 267 1 hr-control 6 1 hr-150 cGy 127 53.6 2 hr-Control 7 2 hr-150 cGy 112 59.8

8.1 9.2

Observed 0 51 0 22 0 20

0 57 0 24 0 22

0 59 0 28 0 22

0 64 0 26 0 25

0 231 0 100 0 89

* 200 metaphases were examined in each culture. * * A total of 800 metaphases were examined in each culture. p value: Difference between O and 1 or 2 h < 0.001.

Percent decrease

Total Acentric Frag' p value

56.9

3.8

60.8

4.1

57.9

4.2

61.4

4.5

52.5

3.8

62.7

4.7

59.4

4.7

60.9

4.8

56.7

8.3

61.5

9.1

Observed 1 31 1 14 2 12

2 36 1 15 1 ll

1 30 2 13 2 10

2 41 2 20 2 15

6 138 6 62 7 48

Percent decrease

p value

56.7

2.6

66.7

3.1

58.8

2.9

70.6

3.7

62.1

2.8

72.4

3.4

53.8

2.8

66.7

3.7

57.6

5.6

68.9

6.9

Vijayalaxmi et al./Mutation Research 371 (1996) 221-228

225

Table 3 The percentages of binucleate cells (BN), the incidence of BN cells with 1 (CIBN), 2 (C2MN) or 3 (C3MN) micronuclei, and the total micronuclei (MN) induced in vitro by 150 cGy gamma radiation in cultured blood lymphocytes from human volunteers examined at 0 (5-10 min before), and at 1 and 2 h (___5 min) after a single oral ingestion of 300 mg melatonin % BN cells

BN cells with

BN cells with MN d

1 MN

2 MN

3 MN

(CIMN)

(C2MN)

(C3MN)

Observed

Volunteer 1 * 0 hr-Control 0 hr-150 cGy 1 hr-Control 1 hr-150 cGy 2 hr-control 2 hr-150 cGy

50 48 49 40 47 43

20 312 21 138 22 136

0 18 0 15 0 7

0 6 0 4 0 2

20 336 21 157 22 145

Volunteer 2 0 hr-Control 0 hr- 150 cGy 1 hr-Control 1 hr-150 cGy 2 hr-Control 2 hr- 150 cGy

55 52 52 48 42 50

23 305 26 141 24 124

0 11 0 9 0 6

0 4 0 2 0 2

23 320 26 152 24 132

Volunteer 3 * 0 hr-Control 0 hr-150 cGy 1 hr-Control 1 hr-150 cGy 2 hr-Control 2 hr-150 cGy

52 47 53 52 54 54

25 237 23 104 26 97

0 28 0 16 0 13

0 7 0 4 0 2

25 272 23 124 26 112

Volunteer 4 * 0 hr-Control 0 hr-150 cGy 1 hr-Control 1 hr-150 cGy 2 hr-Control 2 hr-150 cGy

53 50 50 53 54 55

26 317 23 140 21 13t

0 22 0 13 0 6

0 6 0 3 0 3

26 345 23 156 21 140

94 1171 93 523 93 488

0 79 0 53 0 32

0 23 0 13 0 9

Total for 4 Volunteers * * 0 hr-Control 53 0 hr-150 cGy 49 1 hr-Control 51 1 hr-150 cGy 48 2 hr-Control 49 2 hr-150 cGy 51

d (CIMN) + (C2MN) + (C3MN). t, (1 × CIMN) + (2 × C2MN) + (3 × C3MN) * 2000 BN cells were examined in each culture. * * A total of 8000 BN cells were examined in each culture. p value: Difference between 0 and 1 or 2 h < 0.001.

94 1273 93 589 93 529

Percent decrease

Total MN b p value

57.0

8.3

61.1

9.0

57.6

7.9

63.6

9.0

59.1

7.3

65.2

8.1

58.3

8.6

62.7

9.4

57.9

19.0

63.0

18.2

Observed

20 366 21 180 22 156

23 339 26 165 24 142

25 314 23 148 26 129

26 379 23 175 21 152

94 1398 93 668 93 579

Percent decrease

p value

54.0

8.4

61.3

9.6

56.0

8.0

62.7

9.2

56.7

7.7

64.4

8.8

56.9

8.9

62.9

10.1

55.9

19.6

62.7

19.3

226

Vijayalaxmi et al. / Mutation Research 371 (1996) 221-228

melatonin ingestion, was verified by statistical analysis using a one-tailed t-test [19].

3. Results

None of the volunteers reported any obvious side effects from the single oral dose of 300 mg melatonin. The data on the concentration of melatonin in the serum and in leukocytes are presented in Table 1. For each volunteer, the values of melatonin in blood samples collected at 1 and 2 h after a single oral ingestion were significantly higher than in those sampled at 0 h. The data thus confirms reports that orally administered melatonin is rapidly absorbed and distributed through blood circulation [20-22]. However, a considerable individual- and time-related variation was obvious among the four volunteers; this may be due to differences between individual rates of absorption and/or clearance of melatonin from blood circulation. The genotoxic responses of the lymphocytes from all four volunteers to in vitro gamma radiation exposure at 0 (before melatonin ingestion), and at 1 and 2 h after the ingestion of melatonin were similar, although the absolute values were slightly different. The data presented in Tables 2 and 3 indicate a significant increase in the incidence of chromosome aberrations (exchange aberrations and acentric fragments) and micronuclei in irradiated lymphocytes as compared with those in corresponding control cells. Lymphocytes in the blood samples collected at 1 and 2 h after the oral ingestion of 300 mg melatonin and exposed in vitro to 150 cGy gamma radiation exhibited a significant and time-dependent decrease in the incidence of chromosome aberrations and micronuclei as compared with similarly irradiated lymphocytes in the blood collected at 0 h ( p < 0.001); the indices were usually lower at 2 h as compared with those at 1 h after the oral dose of melatonin. The average decreases in the lymphocytes examined at 1 and 2 h after melatonin ingestion were 56.7% and 61.5% for exchange type of aberrations, 57.6% and 68.9% for acentric fragments, and 55.9% and 62.7% for micronuclei, respectively. In all volunteers, the incidence of abnormal cells showing chromosome damage and numbers of binucleate cells with micronuclei were significantly de-

creased at 1 and 2 h after the ingestion of melatonin (as compared with the lymphocytes collected before the ingestion of melatonin); the average decreases were 53.6% and 59.8% for chromosome damage and 57.9% and 63.0% for micronuclei, at 1 and 2 h after the oral ingestion of melatonin, respectively (Tables 2 and 3). Similarly, the frequency distribution of binucleate cells with 1, 2 or 3 micronuclei was significantly decreased at 1 and 2 h after the ingestion of melatonin. For each volunteer, at the end of 72 h culture period, the percentages of lymphocytes that were binucleate ranged from 40 to 55% (scored in 2000 nucleated cells in each culture) (Table 3). The numbers of cells with 1 (mononucleate) and > 4 nuclei (multinucleate) were similar; these data indicate that there was no significant alteration in cell cycle kinetics.

4. Discussion

In mammalian tissues, exogenously administered or endogenously synthesized melatonin has been shown to be concentrated more in the nucleus than in the cytosol [23]. The tendency of melatonin to accumulate in the nucleus, coupled with its ability to scavenge .OH radicals, could provide an effective and direct means of protecting the lymphocytes against radiation-induced genetic damage, and this could be the mechanism for the radioprotective effect observed here. It is also possible that melatonin might have activated cellular DNA repair enzymes, facilitating a rapid repair of the damaged DNA, or altered some other biochemical process whereby the initial/primary genetic damage is significantly reduced. The observations made in three of the four volunteers in this study clearly demonstrate the need for further investigation; they showed very low melatonin concentrations in serum and leukocytes at 2 h following the oral ingestion of melatonin (as compared with the values at 1 h after oral melatonin), and yet there was an additional reduction in the incidence of chromosome aberrations and micronuclei in their lymphocytes. These data suggest that while melatonin acts effectively as a -OH radical scavenger, this is not the only mechanism of action, since the radioprotection by radical scavenging would be expected to be concentration dependent. Also, the

Vijayalaxmi et al./ Mutation Research 371 (1996) 221-228

close similarity in radioprotection f r o m genetic d a m age o b s e r v e d in four volunteers, despite the variation in serum and l e u k o c y t e concentration of m e l a t o n i n is interesting. Perhaps there is a m a x i m u m l e u k o c y t e radioprotection affordable after oral administration, and this c o u l d be i n d e p e n d e n t o f m e l a t o n i n concentration in serum and leukocytes. It is important to investigate w h e t h e r l o w e r or higher levels o f radioprotection can be o b s e r v e d with a smaller or larger oral dose(s) of melatonin. Acute and chronic doses o f melatonin ranging f r o m 1 - 3 0 0 m g have been used in humans to alleviate the s y m p t o m s o f jet lag, for the induction o f sleep, for the treatment of patients with locally adv a n c e d metastatic solid neoplasms, and as a contraceptive; no untoward side effects have been reported [20,21,24-29]. This in v i v o / i n vitro study in blood l y m p h o c y t e s o f h u m a n volunteers is the first to demonstrate a significant decrease in the extent o f genetic d a m a g e induced in vitro by g a m m a radiation, with this protection occurring within 1 h after the ingestion o f a n o n - t o x i c dose of 300 m g melatonin. Further investigations are necessary to d e t e r m i n e the lowest dose o f melatonin which can offer significant radioprotection f r o m genetic d a m a g e in h u m a n b l o o d l y m p h o c y t e s , and to determine w h e t h e r similar radioprotection occurs in other normal cell types. In the context o f the present study, melatonin can potentially be useful in occupational and medical settings where ionizing radiation is used a n d / o r repeated e x p o s u r e occurs.

Acknowledgements This research was supported by the D i v i s i o n of Radiation O n c o l o g y , The U n i v e r s i t y of Texas Health Science Center, and grants f r o m U n i t e d States Air F o r c e O f f i c e o f Scientific R e s e a r c h (F49620-95-10337) and National Institutes of Health (ES07132).

References [1] Waldhauer, F. and M. Dietzel (1985) Daily and annual rhythms in human melatonin secretion: Role in puberty control. Ann. NY Acad. Sci., 453, 205-214.

227

[2] Reiter, R.J. (1991) Melatonin: The chemical expression of darkness. Mol. Cell Endocrinol., 79, c153-159. [3] Reiter, R.J. (1980) The pineal gland and its hormones in the control of reproduction in mammals. Endocrine Rev., 1, 109-131. [4] Reiter, R.J. (1991) Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocrine Rev., 12, 151-180. [5] Reiter, R.J. (1992) The ageing pineal gland and its physiological consequences. BioEssays, 14, 169-175. [6] Reppert, S.M., D.R. Weaver and T. Ebisawa (1994) Cloning and characterization of a mammalian melatonin receptor that mediates reproductive and circadian rhythms. Neuron, 13,

1177-1185. [7] Carlberg, C. and I. Wiesenberg (1995) The orphan receptor family RZR/ROR, melatonin and 5-1ipoxygenase: an unexpected relationships. J. Pineal Res., 18, 171-178. [8] Tan, D.-X., L.-D. Chen, B. Poeggeler, L.C. Manchester and R.J. Reiter (1993) Melatonin: a potent, endogenous hydroxyl radical scavenger. Endocr. J., 1, 57-60. [9] Tan, D.-X., B. Poeggeler, R.J. Reiter, L.-D. Chen, S. Chen, L.C. Manchester and L.R. Barlow-Walden (1993) The pineal hormone melatonin inhibits DNA-adduct formation induced by the chemical carcinogen safrole in vivo. Cancer Lett., 70, 65-71. [10] Tan, D.-X., R.J. Reiter, L.-D. Chen, B. Poeggeler, L.C. Manchester and L.R. Barlow-Walden (1994) Both physiological and pharmacological levels of melatonin reduce DNA adduct formation induced by the carcinogen safrole. Carcinogenesis, 15, 215-218. [11] Blickenstaff, R.T., S.M. Brandstadter, S. Reddy and R. Witt (1994) Potential radioprotective agents. 1. Homologs of melatonin. J. Pharm. Sci., 83, 216-218. [12] Vijayalaxmi, R.J. Reiter and M.L. Meltz (1995) Melatonin protects human blood lymphocytes from radiation-induced chromosome damage. Mutation Res., 346, 23-31. [13] Vijayalaxmi, R.J. Reiter, E. Sewerynek, B. Poeggeler, B.Z. Leal and M.L. Meltz (1995) Marked reduction of radiationinduced micronuclei in human blood lymphocytes pre-treated with melatonin. Radiat. Res., 143, 102-106. [14] Roots, R. and S. Okada (1972) Protection of DNA molecules of cultured mammalian cells from radiation-induced singlestrand scissions by various alcohols and SH compounds. Int. J. Radiat. Biol., 21, 329-342. [15] Okada, S., N. Nakamura and K. Sasaki (1983) Radioprotection of intracellular genetic material. In: O.F. Nygaard and M.G. Simic (Eds.), Radioprotectors and Anticarcinogens. Academic Press, New York. pp. 339-356. [16] Littlefield, L.G., E.E. Joiner, S.P. Colyer, A.M. Sayer and E.L. Frome (1988) Modulation of radiation-induced chromosome aberrations by DMSO, an OH radical scavenger. 1: Dose-response studies in human lymphocytes exposed to 220 kV X-rays. Int. J. Radiat. Biol., 53, 875-890. [17] Fraser, S., P. Cowen, M. Franklin, C. Franey and J. Arendt (1983) Direct radioimmonuassay for melatonin in plasma. Clin. Chem., 29, 396-397.

228

Vijayalaxmi et a l . / Mutation Research 371 (1996) 221-228

[18] Perry, P. and S. Wolff (1974) New Giemsa method for the differential staining of sister chromatids. Nature (Lond.). 251, 156-158. [19] Vijayalaxmi and W. Burkart (1989) Resistance and cross-resistance to chromosome damage in human blood lymphocytes adapted to bleomycin. Mutation Res., 211, 1-5. [20] Lieberman, H.R., F. Waldhauser, G. Garfield, H.J. Lynch and R.J. Wurtman (1984) Effects of melatonin on mood and performance. Brain Res., 323, 201-207. [21] Voordouw, B.C., R. Euser, R.E. Verdonk, B.T. Alberda+ F.H. de Jong, A.C. Drogendijk, B.C. Fauser and M. Cohen (1992) Melatonin and melatonin-progestin combinations alter pituitary-ovarian function and can inhibit ovulation. J. Clin. Endocrinol. Metab., 74, 108-117. [22] Dollins, A.B., I.V. Zbdanova, R.J. Wurtman, H.J. Lynch and M.H. Deng (1994) Effect of inducing nocturnal serum melatonin concentrations in daytime on sleep, mood, body temperature, and performance. Proc. Natl. Acad. Sci. USA. 91, 1824-1828. [23] Menendez-Pelaez, A. and R.J. Reiter (1993) Distribution of melatonin in mammalian tissues: the relative importance of nuclear versus cytosolic localization. J. Pineal Res., 15, 59-69. [24] Arendt, M., M. Aldhous and V. Marks (1986) Alleviation of

[25]

[26]

[27]

[28]

[29]

jet lag by by melatonin: priliminary results of controlled double blind trial. Br. Med. J. Clin. Res. Ed., 292, 1170. Lissoni, P., S. Barni, S. Crispino, G. Tancini and F. Fraschini (1989) Endocrine and immune effects of melatonin therapy in metastatic cancer patients. Eur. J. Cancer Clin. Oncol.+ 25, 789-795. Lissoni, P., S. Barni, F. Rovelli, F. Brivio, A. Adrizzoia, G. Tancini, A. Conti and G.J. Maestroni (1993) Neuroimmunotherapy of advanced solid neoplasm with single evening subcutaneous injection of low-dose interleukin-2 and melatonin: Preliminary results. Eur. J. Cancer. 29, 185-189. James, S.P.+ D.A. Sack, N.E. Rosenthal and W.B. Mendelson (1990) Melatonin administration in insomnia. Neuropsychopharmacology, 3, 19-23. Sarnel, A+, A. M. Wegmann, M. Vejvoda, H. Maass, A. Gundel and M. Schutz (1991) Influence of melatonin treatment on human circadian rhythimicity before and after a simulated 9 hour time shift. J. Biol. Rhythms., 6, 235-248. Neri, B., C. Fiorelli, F. Moroni, G. Nicita, M.C. Paoletti, R. Ponchietti, A. Raugei and G. Santoni (1994) A. Trippitelli and G. Grechi. Modulation of human lymphoblastoid interferon activity by melatonin in metastatic renal cell carcinoma. A phase II study. Cancer, 73, 3015-3019.