Modulatory effects of melatonin on genotoxic ...

Mutation Research 417 Ž1998. 75–84

Modulatory effects of melatonin on genotoxic response of reference mutagens in the Ames test and the comet assay S.A. Musatov

a,b

, V.N. Anisimov b, V. Andre´ c , C. Vigreux c , T. Godard c , P. Gauduchon c , F. Sichel c,)

a

c

Department of Medical Genetics and Biology, I.P. PaÕloÕ St. Petersburg State Medical UniÕersity LeÕ Tolstoy St., 6 r 8, St. Petersburg 197022, Russian Federation b Laboratory of Experimental Tumors, N.N. PetroÕ Research Institute of Oncology, 68, Leningradskaya Str., Pesochny-2, St. Petersburg 189646, Russian Federation UPRES-EA1772 and CJF INSERM 96-03, UniÕersite´ de Caen, Esplanade de la Paix, B.P. 5186, 14032 Caen Cedex, France, and Laboratoire de Cancerologie Experimentale, Centre Franc¸ois Baclesse, Route de Lion-sur-Mer, 14076 Caen, Cedex 05, France ´ ´ Received 3 February 1998; revised 2 July 1998; accepted 3 July 1998

Abstract The effect of a potent endogenous antioxidant, the pineal gland indole melatonin ŽMLT. on the mutagenicity of twelve well-known mutagens and carcinogens has been investigated using two in vitro tests the Ames test and the single cell gel electrophoresis assay ŽSCGE assay or COMET assay.. The 12 mutagens used were 7,12-dimethylbenzŽ a.anthracene ŽDMBA., benzoŽ a.pyrene ŽBP., 2-aminofluorene ŽAF., 1,2-dimethylhydrazine ŽDMH., bleomycin, cyclophosphamide ŽCP., 4-nitroquinoline-N-oxide ŽNQO., 2,4,7-trinitro-9-fluorenone ŽTNF., 9-aminoacridine ŽAA., N-nitrosomethylurea ŽNMU., mitomycin C and sodium azide tested in the absence or in the presence of S9 mix. MLT alone turned out neither toxic nor mutagenic in the Ames test and revealed clastogenic activity at the highest concentration tested Ž100 mM. in the SCGE assay. In four Salmonella typhimurium tester strains TA 97, TA 98, TA 100 and TA 102 MLT significantly reduced the mutagenicity of chemicals which require S9 activation. In the SCGE assay performed on CHO cells, preincubation with MLT led to a strong inhibition of clastogenic activities of DMBA and CP, and in a lesser extent with BP and NMU. With mitomycin C, MLT exacerbated responses in both tests. The possible mechanisms of MLT’s inhibitory action are discussed. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Antioxidant; Melatonin; Ames test; DNA damage; SCGE assay; COMET assay; Chinese hamster ovary cell; 7,12-dimethylbenzŽ a.anthracene; BenzoŽ a.pyrene; 2-aminofluorene; 1,2-dimethylhydrazine; Bleomycin; Cyclophosphamide; 4-nitroquinoline-N-oxide; 2,4,7-trinitro-9-fluorenone; 9-aminoacridine; N-nitrosomethylurea; Mitomycin C; Sodium azide

1. Introduction Melatonin ŽMLT., the indole hormone of the pineal gland, is known to participate in the function ) Corresponding author. Tel.: q33-231-45-50-70; Fax: q33231-45-50-53; E-mail: [email protected]

of neuroendocrine system in mammals. Recently, however, it was shown to be a very efficient scavenger of free radicals and was found to be more effective than other well known antioxidants such as glutathione and vitamin E. Being both lipophilic and hydrophilic, it acts not only in every cell but also in every subcellular compartment. Beside its ability to

1383-5718r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 8 . 0 0 0 9 4 - 1

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S.A. MusatoÕ et al.r Mutation Research 417 (1998) 75–84

neutralize free radicals directly, MLT also stimulates the activity of the glutathione peroxidase enzyme, thereby reducing oxidative damage w1x. It is likely that antioxidative property of MLT is its phylogenetically primary one and, thus, it is probably produced by most organisms in the animal kingdom w2x. MLT has been found to inhibit X-ray induced mutagenesis in human lymphocytes in vitro w3x, to reduce cis-platinum-induced genetic damage in the bone marrow of mice w4x, to decrease hepatic DNA adduct formation caused by safrole in rats w2x and to protect rat hepatocytes from chromiumŽVI.-induced DNA single-strand breaks in vitro w5x. Recently, we studied the effect of MLT on the induction of chromosome aberrations and sperm head anomalies in mice treated with cyclophosphamide, 1,2-dimethylhydrazine and N-nitrosomethylurea and we found that MLT inhibited greatly the mutagenicity of these carcinogens w6x. This paper presents the results of a study on the effect of MLT on the genotoxicity of various wellknown mutagens and carcinogens with different mechanisms of action using two in vitro tests: the Ames test and the single cell gel electrophoresis assay ŽSCGE assay or COMET assay.. Since MLT has both direct and receptor mediated actions, it is of importance to compare the results of these tests performed with bacteria and mammalian cells, respectively.

2. Materials and methods 2.1. Chemicals

Colombes, France; bleomycin w9041-93-4x from Laboratoire Roger Bellon, Neuilly-sur-Seine, France; mitomycin C w50-07-7x from Choay, Paris, France. DMBA, BP, AF, NQO, TNF and AA were dissolved in DMSO, while CP, bleomycin, NMU, mitomycin C and sodium azide were dissolved in water Žin the Ames test. or in HAM F12 medium Žin the SCGE assay.. DMH was dissolved in 0.2 M phosphate buffer ŽpH 7.4. and MLT was dissolved in a 3% ethanol solution in water. S9 mix was prepared according to Maron and Ames Ž1984. from Aroclor 1254 induced rat liver S9 fraction ŽIFFA-CREDO, L’Arbresle, France.. 2.2. The Ames test Salmonella typhimurium tester strains TA 97, TA 98, TA 100 and TA 102 were kindly provided by Dr. B.N. Ames, Berkeley, CA, USA. The preincubation method was used according to DeMeo ´ et al. w7x with minor modifications. Strains were grown in Oxoid Nutrient Broth No. 2 with a 12-h shaking, except for testing DMH on TA 102 where a 5-h culture was used, according to Matsushita et al. w8x. About 100 ml of the culture of bacteria was mixed with 100 ml of the solution of MLT or solvent Ž3% ethanol., 10 ml of mutagens and 100 ml of 5% S9 mix Žwhen appropriate.. After 20 min incubation at 378C, molten agar Ž2 ml. was added to the tubes and poured on minimal glucose plates. All the manipulations were carried out in dim light because of the photosensitivity of MLT. After 48 h of incubation, the number of revertants was counted and the percentage of inhibition was calculated according to the formula: Rm y Rmt

The chemicals used were purchased from the following suppliers: MLT ŽCAS no. w73-31-4x., 7,12-dimethylbenzŽ a.anthracene w57-97-6x ŽDMBA., benzoŽ a.pyrene w50-32-8x ŽBP., 4-nitroquinoline-Noxide w56-57-5x ŽNQO., 9-aminoacridine w90-45-9x ŽAA., nitrosomethylurea w684-93-5x ŽNMU. and sodium azide w26628-22-8x from Sigma, Saint Quentin-Fallavier, France; 2-aminofluorene w153-786x ŽAF. and 2,4,7-trinitro-9-fluorenone w129-79-3x ŽTNF. from Aldrich, France; 1,2-dimethylhydrazine w306-37-6x ŽDMH. from Fluka; cyclophosphamide w6055-19-2 x ŽCP . from Laboratoire Lucien,

Rm

= 100

where Rm is the number of revertants in the presence of a mutagen and Rmt is the number of revertants in the presence of the mutagen and MLT, both minus the number of spontaneous revertants w9x. Since some mutagens can damage DNA through several mechanisms, these mutagens were tested in more than one strain which allows to detect different types of mutations. The concentrations of mutagens were chosen to produce the optimal mutagenic effect with minimal toxicity.


2.3. The SCGE assay The test was performed in Chinese Hamster Ovary cells ŽCHOK1. according to McKelvey-Martin et al. w10x with minor modifications. Briefly, cells seeded onto multiwell plates were cultivated overnight in HAM F12 medium supplemented with 10% fetal calf serum, penicillin Ž100 unitsrml. and streptomycin Ž100 mgrml. in a humidified incubator at 378C with 5% CO 2 . Cells were first treated with MLT Ž0.5 ml in 0.001% ethanol. in the absence of serum for 2 h, then solutions of mutagens were added Ž0.5 ml. and cells were further incubated for 1 h. DMBA, BP and CP were tested with S9 mix Ž1.7%.. After treatment, cells were washed with phosphate buffered saline ŽPBS. and trypsinized with 100 ml trypsin during 5 min. A 2 ml medium was added, cells were centrifuged Ž5 min, 800 = g ., the supernatant discarded and cells were resuspended in 70 ml low melting point agarose ŽLMA, 0.5% in PBS without calcium and magnesium. and plated on fully frosted slides ŽTouzard et Matignon, France. which were first covered with 80 ml of normal melting agarose Ž0.8% in PBS without calcium and magnesium.. The slides were kept on ice for 5 min. After solidification, a top layer of 80 ml LMA was added, again allowed to solidify for 5 min. Slides were then immersed in freshly prepared lysing solution Ž2.5 M NaCl, 0.1 M EDTA, 10 mM Tris pH 10, 10% DMSO and 1% Triton X-100. for 1 h. Then slides were left in electrophoresis buffer Ž0.3 M NaOH and 1 mM EDTA, pH 13.6. for 40 min and placed in a gel electrophoresis tank ŽHoeffer HE 99X@.. Elec-

77

trophoresis was conducted at room temperature for 24 min using 20 V and 300 mA current. After electrophoresis the excess alkali was neutralized in Tris buffer Ž0.4 M Tris, pH 7.5. two times for 5 min. Finally, the slides were stained with 50 ml ethidium bromide Ž2 mgrml. and examined at 250 = magnification using a Dialux fluorescence microscope ŽLeitz. equipped with a Ploemopak 2.3 Žexcitation filter: 515–560 nm; barrier filter: 580 nm.. Images of 30 randomly selected cells Ž15 cells from each of two duplicate slides. were analysed from each sample and tail moment was determined by means of an original software developed in our laboratory. The non-parametric Wilcoxon–Mann–Whitney test was performed to compare differences between the control and each dose tested.

3. Results In the Ames test, MLT itself was neither mutagenic nor toxic towards the four strains at the concentrations tested ŽTable 1.. The mutagenicity of DMBA, BP, AF and DMH was inhibited by MLT in a dose-dependent manner ŽTable 2.. The maximum inhibitions observed were: DMBA—84% and 76% for TA 97 and TA 98 respectively; BP—53%, 49% and 46% for TA 97, TA 98 and TA 100 respectively; AF—79%, 36% and 72% for TA 97, TA 98 and TA 100 respectively; DMH—67% for TA 102. Maximal inhibition of bleomycin mutagenicity Ž56%. was obtained at the lowest concentration of MLT. MLT did not affect the mutagenicity of direct-acting mutagens

Table 1 Effect of MLT on Hisq revertants in the Ames test MLT a mmolrplate

Net no. of revertantsrplate TA 97

0 0.25 0.5 1 2 a b

241 " 21.2 235 " 13.3 207 " 12.2 214 " 2.0 213 " 10.0

b

TA 98

TA 100

TA 102

35 " 5.1 33 " 9.1 31 " 2.3 31 " 6.7 35 " 3.6

140 " 5.7 161 " 2.1 143 " 29.7 141 " 9.9 144 " 9.2

516 " 16.3 533 " 4.2 546 " 59.4 505 " 12.7 480 " 25.5

Incubations performed in the presence of rat liver S9 microsomal mix. Mean of triplicate plates.


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Table 2 Effect of MLT on DMBA, BP, AF, DMH, bleomycin, NQO, TNF, AA, NMU, mitomycin C and sodium azide induced Hisq revertants in the Ames test Mutagen, mgrplate

DMBA 10 for TA 97 40 for TA 98

BP 1.0

AF 0.5 for TA 97 and TA 98, 1.0 for TA 100

S9 "

q

q

q

DMH 500

q

Bleomycin 10

q

NQO 0.2 for TA 97, 0.5 for TA 98, 0.1 for TA 100

_

TNF 0.05

_

AA 50

_

NMU 10 for TA 100, 3.0 for TA 102

_

Mitomycin C

_

MLT, mmolerplate

Net no. of revertantsrplate TA 97 TA 98

0 0.25 0.5 1 2 0 0.25 0.5 1 2 0 0.25 0.5 1 2 0 0.25 0.5 1 2 0 0.25 0.5 1 2 0 0.25 0.5 1 2 0 0.25 0.5 1 2 0 0.25 0.5 1 2 0 0.25 0.5 1 2 0 0.25 0.5

439 " 5.7 a Ž2.7. b 232 " 12.1 196 " 91.9 128 " 12.7 71 " 13.4 1021 " 42.4 Ž5.2. 869 " 11.3 774 " 18.4 539 " 19.8 479 " 22.6 375 " 62.2 Ž2.6. 264 " 35.4 192 " 12.7 159 " 59.4 79 " 11.3

45 " 9.2 Ž2.8. 11 " 0.7 15 " 3.5 22 " 0.7 18 " 7.1 453 " 2.8 Ž13.9. 381 " 33.9 362 " 51.6 248 " 21.2 230 " 17.0 738 " 9.9 Ž22.1. 655 " 76.4 558 " 9.9 487 " 0.7 470 " 4.2

TA 100

TA 102

c

591 " 1.4 Ž5.2. 551 " 72.1 552 " 69.3 539 " 49.5 269 " 7.1 304 " 14.1 Ž2.6. 174 " 0.7 148 " 5.7 114 " 11.3 84 " 14.1 527 " 12.7 Ž2.3. 468 " 14.1 423 " 43.8 248 " 39.6 175 " 21.2 932 " 141.4 Ž2.5. 410 " 82.0 644 " 158.4 780 " 17.0 882 " 93.3

492 " 15.6 Ž4.1. 436 " 66.5 531 " 82.0 373 " 65.1 440 " 24.0 341 " 45.3 Ž2.7. 303 " 2.8 320 " 24.0 259 " 17.0 220 " 32.5 591 " 28.3 Ž4.0. 553 " 84.9 549 " 90.5 645 " 152.7 572 " 1.4

426 " 31.1 Ž15.2. 520 " 5.7 483 " 15.6 441 " 38.2 436 " 67.9 472 " 35.4 Ž16.2. 490 " 52.3 510 " 77.8 575 " 22.6 475 " 263.0

1228 " 31.1 Ž13.0. 1040 " 132.9 1142 " 62.2 924 " 121.6 928 " 36.8

1585 " 613.8 Ž9.2. 1587 " 345.1 1202 " 547.3 1509 " 70.7 1647 " 59.4

774 " 203.6 Ž2.6. 616 " 31.1 529 " 63.6 956 " 2.8 840 " 31.1 1653 " 64.3 Ž3.5. 3315 " 240.4 4123 " 39.6


79

Table 2 Žcontinued. Mutagen, mgrplate

S9 "

0.02

Sodium azide 1.0

_

MLT, mmolerplate

Net no. of revertantsrplate TA 97 TA 98

1 2 0 0.25 0.5 1 2

TA 100

TA 102 4406 " 340.8 4434 " 60.1

1140 " 36.1 Ž6.9. 1077 " 70.7 1139 " 11.3 1173 " 59.4 1297 " 104.7

a

Mean of triplicate plates. Ratio between induced and spontaneous revertants. c No mutagenicity detected in the non-toxic range in the preincubation assay used. The spontaneous revertants for TA 97 qS9 were 180–191, TA 97 yS9 were 140–194, TA 98 qS9 were 21–41, TA 98 yS9 were 22–38, TA 100 qS9 were 136–190, TA 100 yS9 were 121–197, TA 102 qS9 were 520–610 and for TA 102 yS9 were 500–556. b

NQO, TNF, AA, NMU and sodium azide. In contrast, MLT enhanced mutagenicity of mitomycin C. The effect of MLT on the clastogenicity of four promutagens ŽDMBA, BP, CP and bleomycin. and two direct-acting mutagens ŽNMU, mitomycin C. in the SCGE assay has been studied. MLT itself produced an increase of DNA damage only at the

highest concentration tested Ž100 mM. Ž p - 0.001. ŽFig. 1.. In combination with DMBA, BP and CP, MLT was seen to have a significant protective effect ŽFig. 2A,B,C.. Slight but statistically significant and dose-related anticlastogenic action was observed towards NMU ŽFig. 2E.. With bleomycin ŽFig. 2D., the protective effect was very slight, and furthermore

Fig. 1. Effect of melatonin ŽMLT. on Chinese Hamster Ovary cells in the single cell gel electrophoresis assay ŽSCGE.. Cells were ranked according to increasing tail moment categories. HDC: highly damaged cells Žtail moment was not calculable.. Ž). Significantly different from control value Ž p - 0.001..

80



81

82


irrespective to the concentration of MLT. In contrast, the clastogenicity of mitomycin C was increased nearly 3 fold ŽFig. 2F..

4. Discussion Recently, the potent antioxidative properties of MLT were discovered. MLT was shown to be a very efficient scavenger of hydroxyl radicals and was found to be five times more effective in inactivating them than glutathione w11x. Likewise, MLT can stimulate glutathione peroxidase activity in different tissues thereby reducing generation of the OH . by neutralizing its precursor H 2 O 2 w12x. These properties allow MLT to preserve macromolecules including DNA, protein and lipid from oxidative damage resulted from ionizing radiation and chemical carcinogen exposure. Being both lipophilic and hydrophilic, MLT may prove to play an important role in the antioxidant defense system in all cells and tissues of the body w1x. Since MLT can protect cells directly as an antioxidant and indirectly through receptor-mediated activation of antioxidative enzymes we applied two different in vitro test systems: the Ames test and the SCGE assay. To date, bacteria have not been shown to have specific receptors to MLT. So we supposed that in the Ames test, we could study direct antioxidant properties of MLT. On the other hand, in the SCGE assay carried out with mammalian cells we could investigate both direct and indirect action of MLT. Some mutagens used are known to damage DNA through several mechanisms. In order to test these mutagens, we used the preincubation assay with four strains of S. typhimurium able to detect different mechanisms of mutagenicity. The strains TA 97 and TA 98 are sensitive to different frameshift mutagens. TA 100 detects G–A base-pair substitutions. TA 102 is sensitive to mutagens damaging A–T pairs, mainly through free radicals formation, and cross-linking

agents w13,14x. MLT being an antioxidant, we supposed that it could affect mutational events resulting from oxidative damage and so could inhibit revertant colonies formation in the stain TA 102. As oxidative mutagens we used DMH, bleomycin Žwith S9-mix. and mitomycin C Žwithout S9-mix. which are believed to generate oxygen radical species w15–17x. Furthermore, mitomycin C acts as a bifunctional alkylating agent w18x. Additionally, we tested eight other intercalating and alkylating agents both direct-acting and requiring metabolic activation. MLT inhibited mutagenic activity of promutagens DMBA, BP, AF, DMH and bleomycin in all strains tested. The mutagenicity of NQO, TNF, AA, NMU and sodium azide remained unaffected by MLT. It is to be noticed that MLT was effective as an antimutagen only at extremely high doses Ž0.25–2 mmolrplate.. The modifying activity of MLT was not related to the mechanism of action of the mutagens, i.e. frameshift mutations or base-pair substitutions. As MLT displayed its protective effect towards promutagens only we speculate that it can interact with the metabolic activation process, perhaps by inhibiting the cytochrome p450-dependant monooxygenase enzyme system of S9-mix. However, basing on the present data, we cannot suggest any precise mechanism of the interaction between MLT and metabolizing enzymes. S9 mediated effect of MLT could also be attributed to one of its metabolites such as 6-hydroxymelatonin, which was found able to inhibit the thiobarbituric-induced lipoperoxidation in mouse brain w19x. In the SCGE assay, MLT modulated the clastogenicity of DMBA, BP and CP. Compared to the data obtained in the Ames test, MLT inhibited the clastogenicity of the chemicals at lower concentrations Ž0.1–1 nM.. These findings reflect probably the fact that protective effect of MLT is receptor-mediated in this model. MLT has been shown to increase the level of glutathione in both the liver and the mammary gland and to stimulate glutathione peroxi-

Fig. 2. Effect of MLT on Chinese Hamster Ovary cells treated with ŽA. DMBA, ŽB. BP, ŽC. CP, ŽD. bleomycin, ŽE. NMU, and ŽF. mitomycin C in the SCGE assay. There are statistically significant protective Žor exacerbating in case of mitomycin C. effects due to the clastogens: ) p - 0.05; )) p - 0.005; ))) p - 0.001.


dase activity in these organs w12x and in the brain in rats w20x. In combination with mitomycin C, a significant dose-related exacerbating effect of MLT has been observed in both tests. We hypothesize that MLT could enhance the metabolic reduction of mitomycin C, which could follow two pathways: the first one involving one electron reductases such as cytochrome p450 reductase, xanthine oxydase or cytochrome b5 reductase, the second one involving two electrons pathway reductase such as DT-diaphorase w18x. Indole structure of melatonin could interact with one Žor more. of these enzymes, leading to raise the intracellular level of active reduced mitomycin C, thus increasing binding to DNA. With NMU, MLT has not affected the mutagenicity in the Ames test but has lowered clastogenicity in the SCGE assay. It could be due to different DNA damaging mechanisms in bacteria and mammals. NMU, an alkylating agent, is known to cause additional lesions in mammalian cells through free radicals formation by means of lipid peroxidation activation w21x. On the other hand MLT has been proved to be a very efficient neutralizer of the peroxyl radicals ŽROO . . generated during lipid peroxidation. MLT was thus found twice more effective than the wellknown membrane antioxidant vitamin E w22x. The present study shows that MLT may play an important role in defending cells from DNA damage induced not only by oxidative mutagens but also by different alkylating agents. Surprisingly, we found also that MLT can enhance the mutagenicity and clastogenicity of one compound, i.e., mitomycin C, which is known to be activated through a particular reductive process. This should urge caution in the use of MLT as an apparent anti-mutagen in human. Several explanations about the observed modulatory effect of MLT are possible. Further detailed investigations are to be carried out to assess the contribution of each mechanism into the MLT’s action.

Acknowledgements We thank Pr Jean-Franc¸ois Heron, Director of the Center F. Baclesse, for his constant support. The excellent technical assistance rendered by Edwige Lemoisson is gratefully acknowledged.

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