Mutagenesis, Vol. 14, No. 1, 107-112,
January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Inhibitory action of melatonin on H2O2- and cyclophosphamide-induced DNA damage
1 Centro di Genetica Evoluzionistica, CNR, c/o Università `La Sapienza', Via degli Apuli 4, 00185 Roma and 2 Dipartimento di Biologia, Università degli Studi `Roma TRE`, Roma, Italy
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Abstract |
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Melatonin, the pineal gland hormone known for its ability to modulate circadian rhythm, has recently been studied in its several functions. It is believed to inhibit cancer growth, to stimulate the immune system and to act as an antioxidant. In particular, this latter activity is ascribed to two different mechanisms: stimulation of radical detoxifying enzymes and scavenging of free radicals. We used this compound in mammalian cells in vitro to investigate its mechanism of action in modulating DNA damage. Cytogenetic and cytofluorimetric analyses were performed. We show that melatonin is able to modulate chromosome damage (chromosomal aberrations and sister chromatid exchanges) induced by cyclophosphamide. Conversely, its involvment in modulating oxidative processes, thereby reducing DNA damage, is less clear. In particular, melatonin is able to decrease H2O2-induced chromosomal aberrations but not sister chromatid exchanges and has been found to induce oxygen species in a cytofluorimetric test (DCFH assay).
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Introduction |
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All organisms are able to produce oxygen radicals during aerobic metabolism, the functions of which have been extensively studied (Pryor, 1976
; Clayson, 1994). On the other hand many enzymatic mechanisms within the organism have evolved to reduce/minimize their negative effects (cell toxicity, mutagenicity, ageing). Epidemiological studies and experimental analyses have addressed the identification of exogenous elements which exhibit scavenging or chelating activity against oxidant molecules. Among these putative agents, vitamins and phenols have been shown to exert a modulating effect both in vivo and in vitro (Sies et al., 1992). In recent years particular attention has been devoted in studying melatonin, the pineal gland hormone, produced by almost all animals and best known for its ability to modulate circadian rhythm (for a review see Brzezinski, 1997). There are numerous reports, dealing with the various effects of melatonin, which include inhibition of cancer growth (Blask, 1993), stimulation of the immune system (Maestroni, 1993) and, above all, free radical scavenging activity (Ianas, 1991; Marshall, 1996). In particular, the antioxidant activity of melatonin is ascribed to two different mechanisms: (i) melatonin stimulates radical detoxifying enzymes, such as glutathione peroxidase (Pablos et al., 1995); (ii) melatonin directly scavenges OH· radicals with an efficacy greater than vitamin E or mannitol (Tan et al., 1993). These data, coupled with those relative to its inhibitory effects on cytochrome P-450 (Kothari and Subramanian, 1992), actually render melatonin one of the most studied agents in the field of antimutagenesis/anticarcinogenesis research.
We used this compound, with other alleged antioxidants, in a project aimed at investigating the mechanism of action of such agents in mammalian cells in vitro and their ability to modulate DNA damage. We recently postulated that ellagic acid, a naturally occurring phenolic lactone, ascorbic acid and ß-carotene act differently in modulating oxidative damage (Cozzi et al., 1995, 1997).
In the present work melatonin was analysed for its protective activities both as an antioxidant agent and as a cytochrome P-450 inhibitor. For this purpose CHO cells were treated in vitro with H2O2, a well-known generator of oxygen reactive species, or cyclophosphamide (CP), which is a well-known alkylating agent activated in one or more metabolic steps. The initial step in bioactivation involves cytochrome P-450, followed by decomposition to the ultimate mutagens, acrolein and phosphamide mustard (Le Blanc and Waxman, 1990). In our experimental protocols the cells were also treated with melatonin and cytofluorimetric and cytogenetic analyses were performed. The results provide evidence of melatonin's ability to inhibit the metabolism of CP, thus reducing chromosome damage; on the other hand, we found conflicting results concerning melatonin's scavenging activity against oxygen species.
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Materials and methods |
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Chemicals
The chemicals were freshly prepared immediately before use. Melatonin (Sigma) was dissolved in DMSO and dispensed to the cultures at the final concentrations required by the various protocols. DMSO in the culture media never exceeded 1%. CP (Endoxan) was dissolved in distilled sterile water and added to the cultures at the final concentrations shown in the Tables. H2O2 was dissolved in NaCl (0.9%) from a 30% stock solution and added to the cultures at a final concentration of 10-4 M. 2'-7'-Dichlorofluorescein diacetate (DCFH-DA) was dissolved in ethanol and sodium azide (NaN3) in distilled sterile water. Bromodeoxyuridine (BrdUrd) (Sigma) (for sister chromatid differentiation) was added to the cultures at a final concentration of 5x10-6 M from a stock solution stored at -20°C. BrdUrd for cell cycle cytofluorimetric analysis was added to the cultures at a final concentration of 45 µM. Anti-BrdUrd monoclonal antibody was obtained from Becton Dickinson. Secondary antibody-FITC (fluorescein thiocyanate) conjugate was obtained from Vector Laboratories. Propidium iodide (Sigma) for DNA staining was used at a final concentration of 20 µg/ml. For metabolic activation, rat liver S9 homogenate and S9 mix were used, as previously described (Palitti et al., 1985
). The S9 homogenate was prepared from the livers of young male Sprague-Dawley/CD rats (weight 200-250 g; Charles River), whose hepatic enzymes had been induced by mixed induction with phenobarbital and ß-naphthoflavone. The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows: 3 ml S9 fraction, 1 ml 40 mM NADP, 1 ml 50 mM glucose-6-phosphate, 2 ml 20 mM HEPES buffer, 1 ml 40 mM MgCl2, 1 ml 330 mM KCl and 1 ml distilled water (total 10 ml).
Cell cultures and experimental protocols
CHO cells are routinely cultured in our laboratory in Ham's F-10 medium (Flow) supplemented with 10% fetal calf serum (FCS), 1% L-glutamine, 2% penicillin (5000 U/ml) and streptomycin (5000 µg/ml). Under these conditions, the average cell cycle lasts 12 h. In all experiments, cells were seeded at a density of 1x106/5 ml flask. After 4 h, cells were treated according to the various protocols.
For sister chromatid exchange (SCE) and chromosomal aberrations (ChAb) analysis, cultures were treated with the appropriate concentrations of melatonin in the presence or not of rat S9 preparation. CP was added in the combined treatment to cultures in complete medium plus S9 fraction, for 3 h; treatment with H2O2 was performed in NaCl for 30 min at 37°C. The cultures were then incubated in fresh complete medium containing BrdUrd. (For concentrations of reagents see Figures and Tables.) The cells were fixed after 26 h for SCE analysis and 18 h for ChAb analysis. Colchicine (5x10-7 M) was always added 2 h before the cells were fixed. The normal Giemsa-Hoechst technique, as previously described (Cozzi et al., 1989) with slight modifications, was used for differential staining of sister chromatids.
For SCE analysis and ChAb analysis, 40 second mitoses (M2) and 100 first mitoses (M1), respectively, were scored from coded slides for each point in each experiment. All experiments were repeated three times. For each experimental point, 1000 cells were scored for mitotic index (MI) and 100 metaphases were analysed for first (M1), second (M2) and successive mitosis determination (proliferation index).
For cytofluorimetric analysis, cells were seeded for 15 min in phosphate-buffered saline (PBS) lacking Ca2+ and Mg2+ and containing DCFH-DA (5 µM), as well as sodium azide (5 mM), which was added to inhibit cellular catalase. The medium was then removed and the cells seeded in NaCl (0.9%) containing melatonin and H2O2, in the combined treatment, for 30 min. The cells were then trypsinized and analysed in a FACSTAR cytometer (Becton Dickinson) equipped with a 5 W argon laser (coherent, 488 nm emission). Ten thousand cells were analysed for each sample.
Cell cycle cytofluorimetric analysis
Exponentially growing cells were treated for 3 h with melatonin and for the last 15 min of the treatment pulsed with 45 µM BrdUrd. At the end of treatment (t = 0) the cells (previously washed with PBS without Ca2+ and Mg2+) were harvested and fixed in a 1:1 methanol/PBS mixture. Successive times of fixing (15 and 24 h) were used to reveal a treatment effect of delay. The fixed cells were then processed for flow cytometric analysis of DNA content. Immunostaining with BrdUrd monoclonal antibody involved DNA denaturation by incubating a cell suspension in 1 ml 3 N HCl buffered with 1 ml 0.1 M sodium tetraborate for 45 min at room temperature after washing in PBS. BrdUrd detection was carried out by incubating in 0.5% Tween 20, 0.1 mg primary antibody anti-BrdUrd and 5 µl goat serum for 30 min at room temperature. Cell suspensions were then washed twice with PBS, 0.5% Tween 20, pelleted and resuspended in fluorescein-conjugated antibody directed against mouse anti-BrdUrd. After 75 min at 4°C, 20 µl goat serum were added. The cell suspensions were then washed and resuspended in 1 ml PBS containing 20 µg/ml propidium iodide for DNA staining. Flow cytometric analysis was carried out using dual fluorescence excitation at 488 nm. Red fluorescence (DNA content) was detected with a 600 nm long pass filter and green fluorescence (BrdUrd content) was measured at 510 nm. Ten thousand events were collected for each sample and biparametric analysis of total DNA content and BrdUrd content was performed.
Statistical analysis
For the SCE analysis, means and standard errors were determined. For SCE and ChAb analysis, mutagen-treated cultures and mutagen + melatonin-treated cultures were compared by Student's t-test. For DCFH the potentiation factor (PF) was used as defined by Sturelid and Kihlman (1978). Cytofluorimetric data on the cell cycle were quantitatively analysed by the program Winmdi v.2.5, wich measures the percentage of cell cycle phases on the basis of biparametric representation (green fluorescence versus red fluorescence). The DCFH assay data were also analysed with Winmdi. The computer analysis was performed by determining the channel of green fluorescence for each of the 10 000 cells analysed. The media of green fluorescence intensity were calculated and the t-test was used to verify the significance of the fluorescence increase in the positive fraction of cells induced by chemical compounds.
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Results |
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Melatonin was not able to induce SCEs or ChAb under our experimental conditions at the tested doses in CHO cells, with or without metabolic activation (S9) (Table I
). Furthermore, the agent did not appreciably modify mitotic and proliferation indices.
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When we performed experimental protocols aimed at investigating the antioxidant activity of melatonin, we observed an evident reduction in ChAb induced by H2O2. In fact H2O2 induced 62% of abnormal cells; in combined treatment with melatonin this percentage was reduced to 35 and 31% (Table IIa), depending on the melatonin doses used. In particular, we found a drastic reduction in chromosome exchanges, which were reduced from 39 to 7 out of 100 cells scored at the highest dose of melatonin. Conversely, we observed an increase in mitotic index from 3.8% in H2O2-treated cells to 6.7 and 7.2% in combined treatment with melatonin.
Table IIb shows the effect of melatonin on SCEs induced by H2O2. H2O2 induced a slight increase in SCEs which was significantly augmented by the two melatonin doses used (from 13.7 to 21.0 and 25.9). Furthermore, we observed a recovery of mitotic and proliferation indices. In order to elucidate these contradictory results, we performed a quantitative assay using cytofluorimetric analysis to measure oxidative products, i.e. oxygen and radical species (Cozzi et al., 1997).
Figure 1 shows the fluorescence distribution of cells previously treated with DCFH-DA. The histograms represent the mean fluorescence intensity in arbitrary units (a.u.) (ordinate) in different experimental treatments. Melatonin showed an ability to induce oxidative species by itself, in a dose-related manner. Furthermore, cells treated with H2O2 and melatonin showed a higher mean fluorescence intensity than those treated with a single agent (H2O2 or melatonin), exceeding an additive effect.
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We used the potentiation factor (PF) defined by Sturelid and Kihlman (1978) as the ratio of the effect of the combined treatment (Ei + p - Ec, Ec = control) to the sum of the effect of the two agents (H2O2 and melatonin) applied separately (Ei + Ep) - 2Ec, i.e. Pf = Ei + p - Ec/(Ei + Ep) - 2Ec. A potentiation factor
1 corresponds to no enhancement of the induced effect. Our data thus analysed showed a value of PF always >1 when comparing all combined treatments with single ones. Melatonin proved capable of boosting the oxidative stress induced by H2O2, at least under our experimental conditions and using our system. In addition, we performed experimental protocols with CHO cells incubated in the presence of a metabolic activation system (S9) and treated with CP or melatonin. The results are shown in Table IIIa and b. Melatonin caused a drastic reduction in both CP-induced ChAb and SCEs. In fact the percentage of abnormal cells decreased from 60% (CP treatment alone) to 44 and 26% in the combined treatment at the two melatonin doses used (Table IIIa) and the total aberrations decreased from 105 to 57 and 27. This reduction may be ascribed in particular to chromatid exchanges, with a reduction from 45, out of 100 cells scored, to 5 at the highest dose of melatonin.
In SCE experiments we used four different doses of CP. At the highest doses (4.2 and 5.6 µg/ml) we failed to detect differentiated metaphases (second and successive) owing to the drastic reduction in the proliferation index induced by CP. When melatonin was present in the combined treatment, we obtained an evident reduction (~20 and 50%) in SCEs induced by the two lowest doses of CP (0.8 and 1.6 µg/ml). Furthermore, we noted an increase in mitotic and proliferation indices (Table IIIb). When the highest doses of CP were used in combination with melatonin, we observed the appearance of second mitoses, showing a recovery of proliferation index. These second metaphases, even when badly damaged, showed a level of SCEs that decreased with decreasing melatonin dose: at the highest dose (3 mM) we found 36.1 versus 61.5 (1 mM) or 44.6 versus 77.0 SCEs (1 mM).
As some reports in the literature describe melatonin's ability to affect cell cycle progression, we performed an experimental protocol of CHO cells treated with melatonin and analysed by cytofluorimeter. In Table IV we show the results expressed as percentage of cells in G1, S and G2 phases in cultures analysed at different times after treatment. We observed no melatonin effect on our cell cultures and the percentage of the cells in the three different phases remained more or less the same for all recovery times.
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Discussion |
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Our experiments demonstrate the inhibitory activity of melatonin on DNA damage induced by CP in CHO cells. This effect appeared as decreases in both ChAb and SCEs induced. Furthermore, we underline a strong recovery of mitotic and proliferation indices, particularly in the case of the highest doses of CP used. It is well known that metabolic activation of alkylating agents such as CP may be realized principally by cytochrome P-450 (Sanderson and Shield, 1996
). The mutagenicity of CP in particular is related to formation of the ultimate cytotoxic metabolite phosphoramide mustard through the intermediate agents hydroxycyclophosphamide and deschloroethylcyclophosphamide (Le Blanc and Waxman,1990; Ren et al., 1997), which is capable of inducing DNA crosslinks and strand lesions (Hengstler et al., 1997). In our experimental conditions we supplied rat S9 preparation to cell cultures in order to obtain activation of the promutagen CP. Co-incubation of CP with melatonin may affect CP metabolism, thereby reducing chromosomal damage and cytotoxicity. In fact, as reported by Kothari and Subramanian (1992) and Praast et al. (1995), melatonin influences phase I and II drug metabolizing enzymes. This action may have critical importance for the inhibitory behaviour of melatonin on the growth of some types of tumour, both in vivo and in vitro. Furthermore, Anisimov et al. (1997) have demonstrated the capacity of melatonin to reduce the development of intestinal tumours induced by dimethylhydrazine in rats, ascribing it in part to the interaction of melatonin with the metabolites of dimethylhydrazine.
On the other hand, Tan et al. (1994) reported that melatonin suppresses DNA adduct formation by safrole, a naturally occurring carcinogen, in rat livers because of its ability to inhibit cytochrome P-450 mixed function oxidases. Consequentely our data are interpretable in the light of the ability of melatonin both to modify the activity of cytochrome P-450 and to trap the CP electophilic metabolites produced.
Melatonin exerts a wide range of actions inside organisms; one of the most studied is its ability to reduce oxidative stress (for review see Reiter et al., 1997). We tested this activity both as modulation of chromosome damage and as direct scavenging of radical species.
Oya et al. (1986) and Rueff et al. (1993) claim that H2O2 is an inducer of DNA damage (single- and double-strand breaks, base destruction and crosslinking) by itself and/or through highly reactive oxygen and radical species. These lesions may result in chromosome damage. We obtained strong induction of ChAbs which was significantly reduced in cell cultures co-treated with melatonin in a dose-related manner and recovery of mitotic index which was severely delayed by H2O2. These data are in agreement with those presented by Vijayalaxmi et al. (1995, 1996), who showed a decrease in ChAbs and micronuclei induced by 
-radiation in human lymphocytes, which has been ascribed to the melatonin-scavenging activity of free radicals.
When we tested melatonin modulation activity on SCEs induced by H2O2, surprisingly we obtained a significant dose-related increase and recovery of proliferation index. This may be accounted for by the possibility that melatonin is a scavenger only of hydroxyl radicals, which induce ChAb, thus decreasing this type of damage. In fact, Ianas et al. (1991) and Tan et al. (1993) reported that melatonin neutralizes OHo generated in a cell-free system. On the other hand, Marshall et al. (1996) showed that melatonin does not scavenge superoxide radicals, which probably cause SCE lesions in vitro.
In order to gain further insight into these contradictory results, we then performed a DCFH test. As previously shown (Cozzi et al., 1995, 1997), this assay probably reveals superoxide but not hydroxyl radicals. We obtained an increase in mean fluorescence intensity of cells treated with melatonin alone with a dose-effect relation and a synergistic effect when cells were treated with H2O2 in combination with melatonin. In our opinion these data confirm the SCE results regarding the increase in H2O2-induced damage caused by melatonin. Furthermore, the presence of oxidation revealed by the DCFH test in cells treated with melatonin alone suggests that this compound per se can have a pro-oxidative effect, as stated by Ianas et al. (1991).
Finally, we performed an experimental protocol to examine the effect of melatonin on cell cycle progression. Cos et al. (1991, 1996) have in fact demonstrated an antiproliferative action of melatonin in human breast cancer cells. In particular, they found a delay in transition from G1 to S phase. Similar results were obtained by Hill and Blask (1988), who demonstrated an antiproliferative effect of physiological concentrations of melatonin in the same cells, but not at supraphysiological concentrations and in estrogen receptor-negative cancer cells in culture. Furthemore, Persengiev and Kyurkchiev (1993) obtained inhibition of S phase by melatonin in normal mouse and human lymphocytes, as well as in T lymphoblastoid cell lines.
On the whole, these data seem to indicate that melatonin has an antitumour effect which is exerted through specific cell cycle phase delay. On the other hand, we failed to find any significant effect in our cell cultures, even if CHO cells stably express melatonin receptor (Witt-Enderby and Dubocovich, 1996).
On the basis of these data we have to conclude that the mechanism of action of melatonin is still unclear and further thorough investigation is needed. We demonstrate a reduction in DNA damage induced by one promutagen, CP, probably ascribed to melatonin modulating the activity of P-450 and/or to its ability to interact with CP electrophilic metabolites. On the other hand, its involvment in oxidative processes is less well defined and probably ranges from an antioxidative to a pro-oxidative effect, as in the case of many other antioxidant compounds (ascorbic acid and ß-carotene) (Shamberger, 1984)
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Acknowledgments |
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We gratefully acknowledge our colleague Carmen Cassero for helpful suggestions. This work was partially supported by a grant from the Consiglio Nazionale delle Ricerche, Italy, no. 97.04641.CT13
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Notes |
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3 To whom correspondence should be addressed. Tel: +39 6 4457527; Fax:+39 6 4457529; Email: desalvia{at}axcasp.caspur.it
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References |
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Received on May 15, 1998; accepted on August 7, 1998.
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