Toxicology and Applied Pharmacology 239 (2009) 37–45

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Toxicology and Applied Pharmacology

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Rapid and transient stimulation of intracellular reactive oxygen species by melatoninin normal and tumor leukocytes

Flavia Radogna a, Laura Paternoster a,c, Milena De Nicola a, Claudia Cerella a, Sergio Ammendola d,Annalida Bedini b, Giorgio Tarzia b, Katia Aquilano a, Maria Ciriolo a, Lina Ghibelli a,⁎a Dipartimento di Biologia, Universita' di Roma Tor Vergata, via Ricerca Scientifica, 1, 00133 Roma, Italyb Istituto di Chimica Farmaceutica, Universita' di Urbino Carlo Bo, Italyc Istitututo di Chimica Biologica, Universita' di Urbino Carlo Bo, Italyd Ambiotec, Italy

⁎ Corresponding author. Fax: +39 06 2023500.E-mail address: ghibelli@uniroma2.it (L. Ghibelli).

0041-008X/$ – see front matter © 2009 Elsevier Inc. Adoi:10.1016/j.taap.2009.05.012

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 November 2008Revised 21 April 2009Accepted 12 May 2009Available online 19 May 2009

Keywords:DichlorofluoresceinU937LuzindoleMelatonin analoguesOxidative stress

Melatonin is a modified tryptophan with potent biological activity, exerted by stimulation of specific plasmamembrane (MT1/MT2) receptors, by lower affinity intracellular enzymatic targets (quinone reductase,calmodulin), or through its strong anti-oxidant ability. Scattered studies also report a perplexing pro-oxidantactivity, showing that melatonin is able to stimulate production of intracellular reactive oxygen species(ROS). Here we show that on U937 human monocytes melatonin promotes intracellular ROS in a fast(b1 min) and transient (up to 5–6 h) way. Melatonin equally elicits its pro-radical effect on a set of normal ortumor leukocytes; intriguingly, ROS production does not lead to oxidative stress, as shown by absence ofprotein carbonylation, maintenance of free thiols, preservation of viability and regular proliferation rate. ROSproduction is independent from MT1/MT2 receptor interaction, since a) requires micromolar (as opposed tonanomolar) doses of melatonin; b) is not contrasted by the specific MT1/MT2 antagonist luzindole; c) is notmimicked by a set of MT1/MT2 high affinity melatonin analogues. Instead, chlorpromazine, the calmodulininhibitor shown to prevent melatonin–calmodulin interaction, also prevents melatonin pro-radical effect,suggesting that the low affinity binding to calmodulin (in the micromolar range) may promote ROSproduction.

© 2009 Elsevier Inc. All rights reserved.

Introduction

Melatonin is a hormone principally produced and released by thepineal gland under the influence of the environmental light/darkcycle (Wurtman et al., 1964). It participates in several importantphysiological functions, including biological regulation of circadianrhythms, sleep, mood, reproduction and neuro-immuno-modulation.Exogenous melatonin has been used for treating a variety of circadianrhythm disorders, including jet-lag and insomnia. Melatonin appearsto evoke its effects in humans through two subtypes of high affinity, 7-pass G protein-coupled plasma membrane receptors: MT1 (preferen-tially expressed in the brain), andMT2 (preferentially expressed in theretina) (Reppert et al., 1994; Lai et al., 2002). The two receptors arealso widely expressed in non-neuroendocrine tissue (Carrillo-Vico etal., 2004; Yuan et al., 2002). MT1/MT2 engagement triggers G proteinmediated signal transduction that can imply either the phospholipaseC (Brydon et al., 1999) or the adenylate cyclase (Vanecek, 1998)pathways; the study of the different roles of the two receptor subtypes

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has been impeded so far by the lack of selective melatonin receptoragonists and antagonists. Melatonin also possesses high affinitybinding for nuclear receptors ROR/RZR, which act as transcriptionalactivators (Wiesenberg et al., 1998); however, their role in melatoninsignalling is still debated. In addition to regular receptors, melatoninbinds at lower affinities two cytosolic enzymes, namely quinonereductase 2 (that coincides with the binding site formerly known asMT3 (Tan et al., 2007), and calmodulin (Benítez-King et al., 1993). It ispresently unknown whether these binding may exert any physiolo-gical functions, since they require higher melatonin concentrationsthan those present in human blood. Melatonin is a powerful anti-oxidant, acting both as a direct radical scavenger (Reiter et al., 2007)and by stimulating production/activity of intracellular anti-oxidantenzymes (Rodriguez et al., 2004); melatonin contrasts oxidative stressin tissues (Baydas et al., 2007) as well as within cells (Bongiovanni etal., 2007; Rubio et al., 2007), ameliorating tissue homeostasis inoxidative related pathologies (Clapp-Lilly et al., 2001). The mechan-ism through which melatonin stimulates anti-oxidant enzymes isunclear: generally speaking, the increase of anti-oxidant defense is acell response to oxidative stress, and the ability of an anti-oxidant suchas melatonin to promote anti-oxidant defense remains an intriguingissue (Rodriguez et al., 2004).

Table 1

Numbers Compounds pKi MT1 pKi MT2 Intrinsicactivity

pKi references

1 Melatonin 9.5 9.4 A2 UCM412 10.5 9.9 A (Mor et al., 2001)3 UCM 608 10.7 10.4 A (Mor et al., 2001)4 UCM 245 8.6 8.6 A (Spadoni et al. 1998)5 UCM92 8.8 9.2 A (Tarzia et al., 1997)6 Luzindole 6.8 8.0 ANT (Dubocovich, 1988)7 UCM 765 8.4 10.2 PA (Rivara et al., 2007)8 UCM 231 5.9 6.2 PA (Mor et al., 1998)9 UCM 454 5.9 8.1 ANT (Spadoni et al., 2001)10 UCM 353 4.8 5.0 ANT (Spadoni et al., 2001)

A: Agonist; PA: Partial Agonist; ANT: Antagonist.

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Possibly, the phenomenon may be related to the surprising recentfindings indicating that melatonin is able to produce intracellularreactive oxygen species (ROS) (Albertini et al., 2006; Büyükavci et al.,2006; Medina-Navarro et al., 1999), detected as the increase influorescence of oxidation-sensitive intracellular probes (namely dihy-drorhodamine (Pieri et al., 1998), dichlorofluorescein (Osseni et al.,2000), dihydroethidium (D'agostino et al., 2007). The pro-radicalactivity of melatonin has been reported on a set of cells, most of whichof tumor origin, which proved positive with very few exceptions(Albertini et al., 2006; Büyükavci et al., 2006; Medina-Navarro et al.,1999; Wölfler et al., 2001). Intriguingly, even though intracellular freethiols are decreased bymelatonin (Eskiocak et al., 2007), frank oxidativestress such as lipid peroxidation seems not to occur (Büyükavci et al.,2006). The origin of melatonin-produced ROS is still ignored. A directmolecular pro-oxidant effect can be excluded on the basis of the knownchemical properties of the molecule. Since the pro-radical effect wasreported only as an intracellular phenomenon, we explored whether itmay be the consequence of a signal transduction originated bymelatonin binding to any of its cellular targets.

In this study, we show that it is not due to the interaction withMT1/MT2 receptor, providing instead evidence that it may result frombinding to the lower affinity intracellular target calmodulin.

Methods

Cell culture. U937 are human tumor monocytes stabilized from ahistiocytic lymphoma; Jurkat are a human leukemic T-cell line; E2R arean Epstein–Barr virus (EBV) positive B-cell line obtained fromBurkitt'slymphoma. Cells were cultured in RPMI medium supplemented with10% FCS. Cells were routinely checked for absence of mycoplasm byusing a mycoplasm detection kit (Mycoalert TM., Cambrex Bio ScienceMilano, Italy). The experiments were performed on cells in thelogarithmic phase of growth under condition of ≥98% viability, asassessed by trypan blue exclusion. Peripheral blood mononuclearleukocytes (PBML) were isolated from heparinized blood samples ofhealthy individuals by collectionwith Ficoll–Hypaque (Sigma-Aldrich)density gradient centrifugation. Red cells were removed by hypotoniclysis. PBML were then plated in RPMI 1640 plus 10% human AB serumin culture flasks (pre-treated with the same human AB serum) topromote monocytes adhesion. After 2 h, floating cells (lymphocytes)were washed out, spun down and separately plated in RPMI 1640 plus10% hi-FCS. Adherent monocytes were washed 3 times with RPMI toremove residual lymphocytes, detached by cell scraping, and re-suspended in RPMI 1640plus 10%hi-FCS. Treatments (see below)wereperformed at 20–24 h post-separation. Cell viability in both fractionswas N98% as assessed by trypan blue exclusion test. Purity of theenriched fractions was controlled by labelling (20 min at roomtemperature) with cell-type specific antibodies: FITC-conjugated-anti-CD45 (from Becton-Dickinson) for lymphocytes; and PE-conjugated-anti-CD14 (from Becton-Dickinson) for monocytes. Cellswere then washed with PBS and labelling evaluated. Both monocyteand lymphocyte fractions were N95% pure.

Analysis of ROS. Cells were loaded with 10 μM dichloro-dihydrofluorescein diacetate (DCFH-DA, Molecular Probes), 2 μMdihydrorhodamine (DHR, Molecular Probes) or 5 μMdihydroethidium(DHE, Molecular Probes) by incubation at 37° for 30 min aftermelatonin treatments. These probes are non-fluorescent cell-permeable compounds; once inside the cell, DCFH-DA is de-esterified(DCFH) and turn fluorescent upon oxidation (DCF, Rh, Et,respectively), fluorescence being proportional to ROS production.Analyses were performed by flow cytometry using FACScanBecton&Dickinson.

Evaluation of cell viability and cell number. Apoptosis and necrosiswere evaluated as previously described (Colussi et al., 2000). Cells

were stained with a mixture of 10 μg/ml Hoechst 33342 (for nuclearmorphology) and 5 μg/ml propidium iodide (PI, for dye exclusion) inculture medium for 15 min at RT. Cells were analyzed using afluorescence microscope; cells with nuclear apoptotic morphology, ornon-excluding PI, were counted (at least 300 cells in at least 3independent fields randomly selected to avoid biases) and the fractionof apoptotic or necrotic cells among total cells was evaluated. Hoechst33342 and PI were purchased from Calbiochem (San Diego, CA, USA).

For evaluation of cell proliferation, cells were incubated for 5 dayswith 1 mM melatonin and cell number was quantified every 24 h for5 days in a haemocytometer in the presence of PI. The values arerelated to PI-excluding cells.

Cell cycle analysis. U937 cells (2×106) were washed with 1% PBSand fixed in 65% ice cold ethanol for 15 min. Cells were then stainedwith 5 μg/ml PI in RNase solution for 15 min at RT. Samples wereprocessed for flow cytometry in a FACScan Becton&Dickinson. Cellcycle was analyzed with Cylchred software.

Determination of glutathione. Intracellular glutathione was assayedupon formation of S-carboxymethyl derivatives of free thiols withiodoacetic acid, followed by the conversion of free amino groups to2,4-dinitrophenyl derivatives by the reaction with 1-fluoro-2,4-dinitrobenzene as previously described (Circolo et al., 2001). Thederivatized small thiols are run on HPLC column, and the elution timeof GSH and GSSG is calculated on the basis of the elution times of thestandards. Data are expressed as nmoles of GSH or GSSG/mg protein.

Determination of protein oxidation. Carbonylated proteins weredetected using the Oxyblot kit (Cayman) as previously described(Aquilano et al., 2003). Briefly, 20 μg of proteins were reacted withDNP for 15 min at 25 °C. Samples were resolved on 12% SDS-polyacrylamide gels, and DNP-derivatized proteins were identified byimmunoblot using an anti-DNP antibody.

Melatonin and analogues. Melatonin was purchased from SigmaChemical Co (St. Louis, MO, USA), and used at the concentration of1 mM unless otherwise specified. For the experiments, melatoninwasadded 1 h prior to apoptosis induction. Compounds UCM412,UCM608, UCM245, UCM92, UCM765, UCM231; UCM454, UCM353,(see Table 1) were synthesized according to procedures described inDuranti et al. (1992), Spadoni et al. (1993, 1998, 2001), Tarzia et al.(1997), Rivara et al. (2007), and Mor et al. (1998). The set of ninecompounds was constructed by selecting known derivativescharacterized by differing affinity and intrinsic activity for melatoninmembrane receptors. High affinity receptor agonists (see Table 1,compounds # 1–5, 7,) were chosen together with derivatives endowedwith lower affinity (see Table 1, compounds # 8–10) and differentagonist potency at membrane receptors.

Other treatments. Trolox was used at 1 mM and added 30 beforetreatments. o-phenanthroline (Sigma Chemical Co, St. Louis, M, USA)

Fig. 1. Immediate and transient stimulation of intracellular reactive oxygen species. (A) Flow cytometric analysis of ROS measured with DCFH-DA for 24 h of treatment with 1 mMmelatonin; the insert magnifies the first 15 min. Values indicate fold-increase (ratio between treated sample and the control values) with respect to control posed=1; one of 3independent experiments with similar results is shown. Panel B shows a flow cytometric analysis of ROS measured with DCFH-DA after melatonin±1 mM trolox C or 5 μM o-phenanthroline. Results indicate fold-increase (ratio between treated sample and the control values) with respect to control posed=1 and are the average of 3 independentexperiment±S.D. Identical symbols mark homogeneous subgroups (treatments). ROS increase by melatonin over control is highly significant (p=b0.01); ROS scavenging by Troloxor o-phenanthroline over melatonin alone is significant (pb0.05). In (C) it is shown the flow cytometric analysis of ROS measured with three different redox-sensitive probes, DHR,DCFH-DA or DHE after 3 h of 1 mMmelatonin treatments. Results indicate fold-increase (ratio between treated sample and the control values) with respect to control posed=1 andare the average of 3 independent experiment±SD. ROS increase measured with DCFH-DA is highly significant (pb0.01)⁎⁎; by DHR and DHE is significant (pb0.05)⁎. The effect ofPEG-catalase on each of the three probes is also shown; 100 U/ml of PEG-catalase reduce the melatonin-induced increment for DCF and rhodamine signal (both are significant,pb0.05), whereas the ethidium signal is unaffected. Panel D shows a flow cytometric analysis of ROS measured with DCFH-DA after 3 h of increasing doses of melatonin.Identical symbols mark homogeneous subgroups (treatments).

Fig. 2. ROS production does not involve mitochondria disturbance. Cells were pre-treated with the mitochondria complex I inhibitor rotenone; ROS were measured byflow cytometric analysis with DCFH-DA after 3 h of 1 mM melatonin. Results indicatefold-increase (ratio between treated sample and the control values) with respect tocontrol posed=1 and are the average of 3 independent experiment±S.D. Identicalsymbols mark homogeneous subgroups (two ways ANOVA with interaction).

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was used at the concentration of 5 μM and pre-incubated for 20 min.PEG-catalase (Sigma Chemical Co, St. Louis, M, USA) was used at 100 U/ml and was pre-incubated for 2 h. For PLC inhibition, U73122 (SigmaChemical Co, St. Louis, M, USA) was used at 10 μM and added 30 minbefore treatments. Extracellular Ca2+ influxwas inhibited by incubatingthe cells with ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetra-acetic acid (EGTA, 650 μM) for 15min before other treatments. G proteininhibition was achieved by pertussis toxin (PTX, 200 nM), added 24 hbefore the other treatments. Melatonin action on MT1/MT2 receptorswas antagonized with 50 μM 2-benzyl-N-acetyltryptamine (luzindole)(Sigma), a concentration proved able to antagonize 1 mMmelatonin inprevious studies (Spadoni et al., 1998), which was added 30 min beforethe other treatments. For mitochondrial respiration inhibition a specificinhibitor of complex I (rotenone, 5 μM) was added 30 min beforetreatments. For calmodulin inhibition, chlorpromazine (Sigma) orcalmidazolium (Sigma) respectively inhibiting or non affectingmelatonin-specific binding site to calmodulin, were used at 0.5 μM.BSO was used at the concentration of 1 mM and maintained for 24 hbefore medium change and melatonin addition (BSO was re-addedduring the experiment). At this time, no GSH is detectable (see Ghibelliet al., 1998).

Statistical analyses. The results are presented as means+−SD.Statistical evaluation was conducted by a one-way ANOVA, followedby the Student–Newman–Keuls multiple comparisons. Significancelevel was fixed at alpha=0.05. With respect to the data depicted inFig. 2 independent factors were rotenone (0 or 1 μm) and melatonin(0 or 10 μm). Association of DCF fold-increase with factors was testedusing two ways analysis of variance (ANOVA) with interaction.

Results

Immediate and transient stimulation of intracellular reactive oxygenspecies by melatonin

Melatonin promotes reactive oxygen species (ROS) formation onU937 tumormonocytic cells in a potent and rapidway: ROS are detectedby theoxidation-sensitive probe dichlorofluorescein (DCFH-DA) as earlyas after 1min of 1mMmelatonin challenge, followed bya sharp increasethat peaks at2–3h, as shownby the timecourse in Fig.1A. TheDCF signal

40 F. Radogna et al. / Toxicology and Applied Pharmacology 239 (2009) 37–45

then decreases slowly, to reach control values at 5–6 h. The signal isefficiently scavenged by the vitamin E analogue Trolox and by the ironchelator (inhibitor of the Fenton reaction) o-phenanthroline (panel B).

To evaluate if the DCF signal was really to attribute to melatonin-produced ROS, two other oxidation-sensitive probes such as dihy-drorhodamine (DHR) and dihydroethidium (DHE) were used. The 3probes recognize a wide spectrum of ROS, DCFDA being consideredvery general, DHR more sensitive to H2O2 and OHU, and DHE to anionsuperoxide. Melatonin stimulates oxidation of these additionalprobes, thus showing that a frank oxidation is produced by melatonin(panel C). The extent of stimulation greatly varies, DCF being the mostaltered signal, suggesting that most of the produced ROS might be

Fig. 3. MT1/MT2 receptor-induced signal transduction is not involved in melatonin pro-oxid(PTX), or the MT1/MT2 antagonist luzindole, or the extracellular Ca2+ chelator EGTA, or theROS were evaluated with DCFH-DA by flow cytometry at 3 h of 1 mMmelatonin treatment. Rrespect to control posed=1 and are the average of 3 independent experiment±S.D. For(pb0.001) Identical symbols mark homogeneous subgroups (treatments). (B) Chemical strumeasured with DCFH-DA 3 h after addition of 1 mM melatonin or its synthetic analogues (sevalues) with respect to control posed=1 and are the average of 3 independent experimen

different from H2O2 or superoxide (see also Radogna et al., in press).To evaluate the production of H2O2, we reinforced intracellularcatalase levels by supplementing the cell-permeable PEG-catalaseconjugate. This reduced by about 30% the extent of rhodamine (Rh)increment due to melatonin, by about 7% the DCF increment, leavingunaltered the ethidium (Et) (panel C), indicating that H2O2 is indeedproduced (though at a low level) as a response to melatonin addition.The different responses are conceivably due to the different affinitiesof the three probes for H2O2, DHR being the most sensitive.

A dose effect was then investigated, as shown in panel D, where thefluorescence values measured at 3 h are presented; an increase overbasal levels starts at 10 μM, but is significant only at 100 μM.

ant activity. (A) U937 cells were pre-treated with the G protein inhibitor pertussis toxinPLC inhibitor U73122, as indicated in Methods; then 1 mMmelatonin (mel) was added.esults indicate fold-increase (ratio between treated sample and the control values) witheach treatment, melatonin produces a significant increase with respect to its controlctures of the melatonin analogues listed in Table 1. (C) Flow cytometric analysis of ROSe Table 1). Results indicate fold-increase (ratio between treated sample and the controlt±S.D. Identical symbols mark homogeneous subgroups (treatments).

Fig. 5. The pro-radical effect of melatonin is general among leukocytes. (A) Flowcytometric analysis of ROS measured with DCFH-DA after 3 h of 1 mM melatonintreatment in normal or tumor haematopoietic cells. Results are the average of 3independent experiment±S.D. ROS generation in all cell types examined is significant(pb0.05).

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MT1/MT2 receptor engagement is not involved in ROS production

In order to explore the origin of melatonin-induced ROS, weevaluated whether melatonin, which is known to interact withmitochondria and their functioning, might produce ROS by disturbingthe respiratory chain. However we found that rotenone, an uncouplerof mitochondrial respiratory chain, does not impair melatonin abilityof producing ROS (Fig. 2).

Then, we evaluated the role of the intracellular melatonin bindingsites, namely MT1/MT2 plasma membrane receptors (which areengaged at nM doses) and calmodulin (bound at high μM doses).

The requirement of μM melatonin doses would suggest indepen-dence of ROS production from MT1/MT2 receptors, engaged at muchlower, i.e., nM concentrations. To actually investigate the role of MT1/MT2, wemeasured DCF fluorescence in cells at 3 h of 1 mMmelatoninin the presence of: luzindole, a specific MT1/MT2 antagonist;pertussis toxin, which inhibits the G protein responsible for thepropagation of the signal; the PLC inhibitor U73122, and theextracellular Ca2+ chelator EGTA. These compounds are reported tototally (luzindole) or partially (the others) inhibit melatonin-elicitedintracellular signal transductions (Radogna et al., 2007). None of thesetreatments impairs the ROS-promoting ability ofmelatonin, indicatingthat it occurs independently of MT1/MT2 interaction (Fig. 3A). Tofurther study the involvement of MT1/MT2, we also analyzed theeventual pro-radical effect of a set of melatonin analogues interactingwithMT1/MT2with different affinities (see Table 1 and Fig. 3B): noneof these exerted any pro-radical activity, confirming the independenceof melatonin ROS production from MT1/MT2 interaction (Fig. 3C).

Possible involvement of calmodulin binding in melatonin-induced ROSproduction

The doses required for melatonin pro-radical effect would beinstead compatiblewith binding to calmodulin (63 μM). Calmodulin isa cytosolic enzyme with a Ca2+ binding site and a domain ofinteraction with many different protein partners. The use of inhibitorsacting on the one or the other domain, namely calmidazolium andchlorpromazine, respectively, allowed establishing that melatonininteracts with the chlorpromazine-sensitive domain, being insensitiveto calmidazolium (Romero et al., 1998). To explore whether the pro-radical effect of melatonin may derive from calmodulin binding, weprobed the same couple of calmodulin inhibitors on melatonin-induced ROS production. As shown in Fig. 4, calmidazolium wasineffective, whereas chlorpromazine efficiently prevented melatonin-

Fig. 4. Possible involvement of calmodulin binding in melatonin-induced ROSproduction. U937 cells were pre-treated for 30 min with two calmodulin inhibitors,which inhibit (chlorpromazine) or not inhibit (calmidazolium) melatonin binding tocalmodulin; 1 mMmelatoninwas then added. ROSwere evaluated with DCFH-DA at 3 hof melatonin. Results indicate fold-increase (ratio between treated sample and thecontrol values) with respect to control posed=1 and are the average of 3 independentexperiment±S.D. Identical symbols mark homogeneous subgroups (treatments).

pro-radical effect. This strongly suggests that the pro-radical effect ofmelatonin may be the consequence of calmodulin binding.

The pro-radical effect of melatonin is general among leukocytes

To understand if the pro-radical effect of melatonin is a generalphenomenon among leukocytes, we examined a panel of cells ofhaematopoietic origin, i.e., lymphocytes and monocytes freshlyexplanted from healthy donors, and the human tumor T lymphocyticJurkat cell line. 1 mM melatonin was able to produce a DCF signal onall of these cells, independently of they being tumor or normal cells(Fig. 5).

Melatonin treatment does not alter cell viability or proliferation

Then we analyzed which consequences for the cells may derivefrom melatonin-induced oxidative status. First of all, we analyzedwhether 1 mMmelatonin may impair cell viability. Fig. 6A shows thatmelatonin does not elicit cell death for up to one week in the form ofapoptosis/necrosis; to ascertain that scattered events of cells deathmay occur un-noticed, control and melatonin-treated cells werecounted every 24 h; Fig. 6B shows that the proliferation rate ofmelatonin-treated cells perfectly overlap that of control cells. We alsoanalyzed cell cycle in the first 24 h of melatonin treatment, but again,no substantial changes with respect to control were observed, asshown in Fig. 6C. These results indicate that no toxicity can beattributed to melatonin, in spite of its pro-radical effect. Lack oftoxicity/apoptosis was also found in all leukocytes examined (notshown).

The pro-radical effect of melatonin does not imply oxidative stress

To give a rationale to the lack of toxic effect of melatonin in spite ofits pro-radical effect, we analyzed whether melatonin-produced ROSreally provoke an oxidative stress. We considered three intracellularmarkers of oxidative stress, i.e., protein carbonylation and the level ofreduced (GSH) and oxidized (GSSG) glutathione, by evaluating theseparameters at different time points after 1 mM melatonin treatment.Fig. 7A shows that no protein carbonylation is induced by melatonin,its level being in fact even decreased with time. GSH is substantiallyunaffected by melatonin (Fig. 7B); interestingly, no significantoxidation of glutathione was found.

We also analyzed whether depletion of GSH by BSO would affectcell response to melatonin in terms of ROS production or toxicity/apoptosis. Fig. 7C shows that cells maintain normal viability after1 mM BSO, independently of melatonin challenge. In terms of ROSproduction, BSO by itself slightly increases DCF signal (see alsoCristofanon et al., 2009), but does not sensitize to melatonin-inducedROS, since the two effects appear merely additive (Fig. 7D).

Fig. 6.Melatonin does not affect cell viability or proliferation. (A) U937 cells were incubated with melatonin 1 mM and apoptosis was quantified every 24 h for 5 days. (B) U937 cellswere incubatedwith 1mMmelatonin and cell number was quantified every 24 h for 5 days. One of 3 independent experiments with similar results is shown. (C) U937 cells cycle wasanalyzed as described in Methods at different times of 1 mM melatonin treatment. One of 3 independent experiments with similar results is shown.

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These results indicate that the pro-radical effect of melatonin is nottranslated within the cell into a proper oxidative stress.

Discussion

Promotion of free radical by melatonin within cells, thoughreported by several studies in the past years (Albertini et al., 2006;Büyükavci et al., 2006; Medina-Navarro et al., 1999; Wölfler et al.,2001), is not an openly accepted notion. We report here that the pro-radical activity is transient, peaking at 2-3 h after melatonin addition,to disappear after 6 h, and that it is elicited at melatoninconcentrations ≥10 μM, 4 orders of magnitude higher than thoseconsidered as “physiological”, i.e., necessary for receptor stimulation.This may explain why many studies, examining later time pointsand/or lower concentrations, failed to reveal it. We have shown thatboth normal and tumor white blood cells react to melatoninproducing free radicals: thus, at least for leukocytes, the paradigmthat melatonin only elicits a pro-radical effect on tumor cells is notapplicable.

We began to assess the nature of melatonin-produced ROS byusing different types of probes and scavengers. As far as the latter areconcerned, we used the soluble vitamin E derivative trolox, which is

active against a wide spectrum of ROS; the iron chelator o-phenanthroline, which inhibits the Fenton reaction; and PEG-catalase,a cell-permeable conjugate of catalase, the enzyme that decomposesH2O2 into H2O and oxygen. The strong response to o-phenanthroline(indeed similar in extent to trolox) shows that the ROS produced bymelatonin derive in large part from a Fenton reaction. The lowerresponse to PEG-catalase may be explained by considering that U937cells are very rich in endogenous catalase, so that the addition of theexogenous enzyme poorly affects the total response. In addition, thismay suggest that the half-life of H2O2 may be very short in the radicalchain created by melatonin addition. As far as the different probes areconcerned, we used DHR, DHE, and DCFH-DA; DHR and DHE areespecially sensitive to H2O2 and superoxide, respectively, whereasDCFH is considered very general, recognizing the above mentionedROS plus reactive nitrogen species and being also a substrate forlipoxygenase (Pufahl et al., 2007). The different responses with theseprobes support the notion that H2O2 may not be the main ROSproduced by melatonin stimulation, recommending exploring thepossible involvement of the pro-radical enzyme lipoxygenase(Radogna et al., in press). This might help in resolving the (apparent)contradiction, which still needs to be explained, that a well-knownanti-oxidant such as melatonin may behave as a pro-oxidant. The

Fig. 7. The pro-radical effect of melatonin does not imply oxidative stress. U937 cells were incubated with melatonin (Mel) 1 mM. For A, at the indicated time points, cells were lysedand proteins extracts were incubated with dinitrophenylhydrazine (DNP). After derivatization, proteins (20 μg) were loaded onto 10% SDS polyacrilamide gel and carbonyls residuesdetected by Western blot using a rabbit anti-DNP antibody. Alpha-tubulin was used as loading control. (B) At the indicated time points, cells were prepared for GSH/GSSG assay byHPLC as described under Methods; the elution time ensures the recognition of GSH or GSSG over other small thiols according to the elution times of the standards. Data are expressedas nmol of GSH or GSSG/mg total protein and reported as means±S.D. (n=4). Identical symbols mark homogeneous subgroups (treatments). (C) U937 cells were pre-treated withBSO as described in Methods; cells were then analyzed for apoptosis in the presence/absence of 1 mMmelatonin (4 h) as described for Fig. 6A. No significant toxicity was detectablein any condition; data aremean of 3 experiments±SD. Identical symbols mark homogeneous subgroups (treatments). (D) BSO pre-treated U937were analyzed for DCH signal in thepresence/absence of 1 mMmelatonin (2 h); the two pro-radical effects are additive. The data are the mean of 3 experiments±SD. Identical symbols mark homogeneous subgroups(treatments).

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possible activation of lipoxygenase as the major source of melatonin-produced ROS would imply that the pro-radical effect is not a directchemical pro-oxidant ability (which in fact was never demonstratedin vitro), but a cellular reaction. Alternatively, we can consider thatanti-oxidants often exert pro-oxidant activities according to theirconcentrations and/or the surrounding milieau, as shown for manyendogenous or exogenous compounds, such as tocopherol orascorbate (Maellaro et al., 1996); this is probably related with thereactivity of each redox couple. We can conceivably exclude insteadthat the pro-oxidant effect could be attributed to an exhaustion of cellanti-oxidant defenses, because it is too rapid, being in fact detectableas early as after 1 min of treatment; moreover, in our system the anti-oxidant defenses are unaffected in the first 24 h of melatonintreatment in terms of anti-oxidant enzyme activities (Albertini et al.,2006) or of glutathione concentration or oxidative status (see Fig. 7).

We show that ROS production is independent of stimulation of theplasma membrane MT1/MT2 receptors, since a), it is unaffected bythe MT1/MT2 antagonist luzindole; b) requires doses 4 orders ofmagnitude higher than those required for MT1/MT2 binding; c) is notelicited by melatonin analogues with high affinity for the MT1/MT2receptors. Instead, we present evidence that the pro-radical activitymay derive from the binding to calmodulin, by showing that the effectis sensitive to the same calmodulin inhibitor (chlorpromazine) whichwas shown to prevent melatonin binding to calmodulin, being insteadinsensitive to calmidazolium, which did not prevent the interaction.Melatonin, as an amphipathic molecule, may freely cross the plasmamembrane, thus it may rapidly accumulating within cells and reactwith cytosolic target.

An open, intriguing question that arises from this study is how cancalmodulin binding elicit a pro-oxidant effect. Calmodulin is animportant signalling enzyme; it is activated by transient increases incytosolic Ca2+, which binds to calmodulin inducing an allostericchange that alters the propensity of calmodulin to bind to an abundantset of target proteins, altering their activity (Benítez-King et al., 1993;Colomer and Means, 2007). In this guise, calmodulin controls theimmediate activation/silencing of many key cytosolic proteins, actingas a pivot of intracellular signalling (Shigeri et al., 2008). Thus, it isconceivable that calmodulin might trigger a signal that leads to theimmediate (i.e., b1 min) activation of a pro-oxidant enzyme. We havedelineated an intracellular signalling pathway leading from calmodu-lin binding to the activation of the pro-oxidant enzyme lipoxygenase(Radogna et al., in press), thus providing a direct evidence of thenature of melatonin-produced ROS.

The binding of calmodulin to melatonin was demonstrated fromthe structural point of view, but a possible physiological significance ofsuch an interaction was not provided or hypothesized. To ourknowledge, the pro-radical effect reported here is the first hypothesisof a functional role of melatonin binding to calmodulin, in spite of therelatively long time elapsed since the publication of the study(Romero et al., 1998). A possible reason for this is that the doses ofmelatonin necessary for this binding exceed by far the physiologicalblood concentration, thus possibly rendering the phenomenon acuriosity rather than a biomedical issue. However, it has been reportedrecently that bonemarrow concentrations of melatonin are 2 orders ofmagnitude higher than the blood ones. Interestingly, we report thepro-radical effect of melatonin in cells of bone marrow origin;

44 F. Radogna et al. / Toxicology and Applied Pharmacology 239 (2009) 37–45

possibly, micro-environments with even higher melatonin levelsmight be recognized by more focused analyses, thus providing thesecondary targets of melatonin, i.e., calmodulin and quinone reduc-tase, with a more straightforward biological significance.

The involvement of an intracellular signal as possible target ofmelatonin for the pro-radical effect of melatonin may give a logicalframework to explain how two opposite phenomena, radical scaven-ging and radical production, may co-exist. As an example, we reportthat two markers of oxidation are differently affected by melatonin:protein carbonylation is decreased (anti-oxidant effect), whereas freethiols (glutathione) are unaffected. This may be explained by thenotion that the scavenging efficiency of melatonin depends on thetype of radical involved (Clapp-Lilly et al., 2001); thus, the chain ofoxidations primed by melatonin may be differently scavenged bymelatonin itself, thus allowing only part of radical signalling toactually take place. The potent anti-oxidant ability of melatonin mayalso explain why melatonin-produced ROS do not lead to an openoxidative stress; indeed, melatonin has often been demonstrated toprotect against cellular oxidative stress (e.g., see Kimball et al., 2008).

As far as the lack of toxicity instead, it is not obvious that this can beascribed to the anti-oxidant properties of melatonin. Melatonin is arecognized anti-apoptotic agent, preventing apoptosis induced by avariety of stimuli in many cell systems (Andrabi et al., 2004; Radognaet al., 2008); it is often given for granted that this anti-apoptotic effectis due to melatonin anti-radical activity, since very often apoptosis istriggered by, or at least implies, an oxidative stress (D'Alessio et al.,2003; Bauer et al., 1998). However, amechanistic analysis showed thatthe anti-apoptotic effect of melatonin on U937 cells requires bindingto MT1/MT2 receptors and the downstream signal transduction(Radogna et al., 2007); this signal leads to the translocation of theanti-apoptotic protein Bcl-2 to mitochondria, where it prevents thedimerization and activation of the cognate pro-apoptotic protein Bax(Radogna et al., 2008); down-regulation of Bcl-2 by RNA interferencenot only abrogates melatonin anti-apoptotic effect, but also turnmelatonin into a pro-apoptotic agent (Radogna et al., 2008), thussuggesting that this Bcl-2 pathway may be required also to maintainviability in melatonin-treated cells. Other studies showed that Bcl-2levels can be up-regulated by melatonin (Ling et al., 1999). Interest-ingly, Bcl-2 is especially famous for behaving as an intracellular anti-oxidant, in some instances even compensating for glutathione(D'Alessio et al., 2004): the ability of melatonin to enhance Bcl-2levels/functions may be an alternative way of melatonin to behave asa biological (as opposed to chemical) anti-oxidant.

Acknowledgments

We wish to thank Prof. Piero Sestili, Prof. Gilberto Spadoni,Universita' di Urbino Carlo Bo, and Prof. Giampiero Gualandi,Universita' della Tuscia, for valuable and constructive support. Weare grateful to Prof. Marco Rocchi, Universita' di Urbino Carlo Bo, forvaluable help with the statistical analysis.

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