Melatonin protects against copper-mediated free radical damage
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Transcript of Melatonin protects against copper-mediated free radical damage
Melatonin protects against copper-mediatedfree radical damage
Introduction
Copper is involved in the neuropathology of variousneurodegenerative disorders including Wilson’s, Menke’s,
Parkinson’s, Alzheimer’s, amyotrophic lateral sclerosis, andPrion diseases [1, 2]. Copper overload results in manypathological conditions that are consistent with oxidative
damage to membranes or molecules. Cu1+ is known togenerate free radicals by a Fenton type reaction. Copperions are able to catalyze the formation of hydroxyl radicalsvia the Haber-Weiss reaction (in vitro):
O�2 � þCu2þ ! O2 þ Cu1þ ð1Þ
Cu1þ þ H2O2 ! Cu2þ þ OH� þ �OH ð2Þ
Cupric chloride can also react with hydrogen peroxide toyield Cu1+ and the superoxide radical (O2
)Æ). Cu1+ formed
can then react with hydrogen peroxide (H2O2) according toEquation (2), increasing the potential for free radicaldamage.
Lipid peroxidation in biological membranes causes
impairment of membrane functioning, decreased fluidity,inactivation of membrane-bound receptors and enzymes,and an increased non-specific permeability to ions such as
Ca2+. Free, ligand-bound and vesicular copper is presentthroughout the brain. Copper is a rapidly acting neurotoxinat physiological concentrations (10–100 lM) [3]. Upon
exposure to iron or copper ions, lipid hydroperoxidesdecompose, resulting in hydrocarbon gases, for example,ethane and pentane, radicals that can abstract further
hydrogen atoms from fatty acid side chains and cytotoxiccarbonyl compounds [4].
Lipid peroxidation is often a late event, accompanyingrather than causing final cell death [5]. Thus, a failure toprotect against tissue damage by chain-breaking antioxid-
ant inhibitors of lipid peroxidation does not rule out freeradical damage as an injury mechanism [6].
Transition metals promote lipid peroxidation in two
ways (1): by catalyzing the formation of oxygen free radicalspecies capable of initiating lipid peroxidation and (2) bycatalyzing the decomposition of preformed lipid peroxidesto propagate lipid peroxidation [7]. Elevated copper levels
induce a variety of changes in tissues. In the liver, copperoverload results in hypertrophy of hepatocytes, hepatitis,hepatocellular necrosis and eventually hepatocellular death
[8].Melatonin, the principle secretory product of the pineal
gland is a potent free radical scavenger and general
antioxidant [9]. It efficiently scavenges the hydroxyl radical(ÆOH) [10] and possibly the peroxyl radical (LOOÆ) [11].Utilizing adsorptive cathodic stripping voltammetry, anelectrochemical method which has been used with success
to examine metal–ligand complex formation at an elec-trode, Limson et al. reported that melatonin binds heavymetals including Fe3+, Cu2+, Pb2+, Cd2+ and Zn2+ [12].
This implies that melatonin, besides acting as an antioxid-ant, may bind these metals and prevent them frompartaking in free radical generation.
In vivo studies have shown that melatonin offers neuro-protection against mercury [Hg(II)] toxicity in rats [13].
Parmar P, Limson J, Nyokong T, Daya S. Melatonin protects against copper-
mediated free radical damage. J. Pineal Res. 2002; 32:237–242. � Blackwell
Munksgaard, 2002
Abstract: Copper is an essential trace element which forms an integral
component of many enzymes. While trace amounts of copper are needed to
sustain life, excess copper is extremely toxic. Copper has been implicated in
various neurodegenerative disorders, such as Wilson’s and Alzheimer’s
diseases. Previous studies showed that melatonin, the principle secretory
product of the pineal gland, binds Cupric chloride (Cu2+) and that this may
have implications in copper-induced neurodegenerative diseases. In the
present study, in vitro copper-mediated lipid peroxidation was induced.
Melatonin (5 mM) protected against copper-mediated lipid peroxidation in
liver homogenates. Electron micrographs of in vivo administered Cu2+ and
melatonin show that melatonin affords some protection to rat hepatocytes in
the presence of copper. Electrochemical studies performed show that
melatonin, in addition to binding Cu2+, may provide protection against
copper-mediated free radical damage by binding Cu1+. The findings of these
studies provide further evidence for the neuroprotective role of melatonin.
Paresh Parmar1, Janice Limson1,Tebello Nyokong2 and SantyDaya1
1Faculty of Pharmacy and 2Department of
Chemistry, Rhodes University, Box 94,
Grahamstown, South Africa
Keywords: copper, lipid peroxidation,
melatonin, voltammetry
Address reprint requests to Santy Daya,
Faculty of Pharmacy, Rhodes University, Box
94, Grahamstown, 6140 South Africa.
E-mail: [email protected]
Received August 1, 2001;
accepted October 24, 2001.
J. Pineal Res. 2002; 32:237–242 Copyright � Blackwell Munksgaard, 2002
Journal of Pineal ResearchISSN 0742-3098
237
Melatonin may offer this protection by forming a metal–ligand complex with mercury. Further electrochemicalstudies have also shown an interaction of lithium and
aluminium with melatonin [14]. These findings suggest anew role for melatonin in sequestering and removing toxicmetals from the central nervous system. Since melatonin issuggested to bind Fe3+, melatonin may function as an
antioxidant by binding this metal ion. In the present study,electrochemical methods were employed to gauge theaffinity of melatonin for the reduced form of copper,
Cu1+. As glutathione is known to prevent copper toxicityby binding Cu1+, adsorptive stripping experiments werealso conducted with glutathione.
Materials and methods
Chemicals and reagents
Hexafluorophosphoric acid (HPF6), butylated hydroxy-
toluene (BHT), melatonin, bovine serum albumin (BSA)and 2-thiobarbituric acid (TBA) were purchased fromSigma Chemical Co., St Louis, MO, USA. Cupric chloride
(Cu2+), cuprous oxide (Cu1+), glutaraldehyde, sodiumphosphate, osmium tetroxide, trichloroacetic acid (TCA),Folin and Ciocalteu’s reagent and copper chloride (CuCl2)
were purchased from Saarchem (Pty) Ltd, Krugersdorp,South Africa. Tetrakis (acetonitrile) copper (I) hexafluoro-phosphate [Cu(CH3CN)4]PF6 was synthesized from Cu2O
and HPF6 [15]. 1.1.3.3-Tetramethoxypropane (MDA) waspurchased from Fluka AG, Switzerland. Cu(I) solution(1 mM) was prepared by dissolving [Cu(CH3CN)4]PF6 in5 mL 0.2% HNO3 before being made to volume with
water. Melatonin (Sigma) was dissolved in 95% ethanol.Glutathione (Sigma) solutions were prepared daily withwater. All aqueous solutions were prepared from Millipore
water. Sodium acetate buffer (0.2 M) of pH 4.4 wasprepared from stock solutions of 0.2 M acetic acid and0.2 M sodium acetate and used as the electrolyte solution
for adsorptive stripping voltammograms. 0.2 M Tris-HClbuffer (pH 7.3) was used as the electrolyte in all cyclicvoltammetry experiments. All other reagents were obtainedfrom local sources and were of the highest purity available.
In vitro lipid peroxidation
Male Wistar rats weighing 250–300 g were used in theexperiments. The rats were killed by cervical dislocationand rapidly decapitated. The liver was removed and a 10%
w/v homogenate was made with 0.1 M phosphate bufferedsaline (PBS) pH 7.4. The experiment included three groups,viz. a control group, a copper group and a copper/
melatonin group. The control group consisted of 0.9 mLhomogenate and 0.1 mL of drug vehicle (50:50 eth-anol:water). A stock solution of copper chloride (2 mM)was prepared. In the copper group, 0.1 mL of the copper
stock solution was added, plus 0.1 mL distilled water to0.8 mL homogenate to give a final copper concentration of1 mM. The copper/melatonin group consisted of 0.8 mL
homogenate, plus 0.1 mL copper of the various copperstock solutions and 0.1 mL melatonin (10 mM). This gave afinal melatonin concentration of 5 mM. Melatonin was
dissolved in 50:50 ethanol:water.
The lipid peroxidation assay was performed according tothe modified method of Anoopkumar-Dukie et al. [16].Aliquots of 1 mL of the homogenate were incubated at
37�C for 60 min. 0.5 mL BHT (0.5 mg/mL in methanol)and 1 mL TCA (15% w/v in water) was added to themixture. The tubes were sealed and heated for 10 min in aboiling water bath to release protein-bound MDA. The
samples were then cooled to avoid adsorption of MDAonto insoluble proteins and then centrifuged at 2000 g for15 min. Following centrifugation, 1 mL of this protein-free
supernatant was removed from each tube and a 1-mLaliquot of TBA (0.33% w/v in water) was added to thisfraction. The tubes were sealed and incubated in a boiling
water bath for 60 min. After incubation, the mixture wascooled for 10 min on ice. The TBA-MDA complex wasseparated from other possible interfering thiobarbituricacid-reactive substances (TBARS) using an IsoluteTM C18
solid phase extraction (SPE) column. The SPE column wasprewashed with 2 mL of methanol followed by 2 mL ofdistilled water. One milliliter of the sample was loaded onto
the column, which was subsequently washed with 1 mL ofdistilled water. The TBA–MDA complex was eluted with1 mL methanol. The absorbance was measured at 532 nm
with an UV-visible spectrophotometer (Shimadzu). Meth-anol was used as a blank. An MDA standard curve wasgenerated using 1,1,3,3-tetramethoxypropane as described
above. Standards were dissolved in 1 mL of distilled water,and a series (0–20 nmoles/mL) were prepared. Final resultswere expressed as nmoles/mg protein.
Protein estimation in the liver homogenates was deter-
mined using described methods [17].
Transmission electron microscopy
Male Wistar rats weighing 250–300 g were used in theexperiments. The rats were randomly assigned to one of the
three groups of five rats each, and were housed in acontrolled environment with a 12-hr light:dark cycle; theywere given access to standard laboratory chow and waterad libitum. The control group received the drug vehicle, i.e.
ethanol: 0.9% saline (40:60) for 2 wk. The copper treatedgroup received 2 mg/kg Cu2+ (copper chloride – CuCl2)for 2 wk. The copper/melatonin treated groups received
Cu2+ (2 mg/kg) and melatonin (12 mg/kg) for 2 wk. Therats were injected daily, with either the vehicle, Cu2+ orCu2+/melatonin. Copper/melatonin groups were injected
on different intraperitoneal sites to prevent any interactionof the copper with the melatonin. Protocols for theexperiments were approved by the Rhodes University
Ethics Committee. The rats were killed and tissue samplesof liver were placed in liquid nitrogen and stored in a )70�Cfreezer until used. The tissue pieces were cut using a scalpelinto pieces approximately 2 mm3 and placed in buffered
glutaraldehyde (2.5% glutaraldehyde in 0.1 M sodiumphosphate buffer) for 12–24 hr at 4�C. The glutaraldehydewas replaced with cold buffer and allowed to wash for
10 min. This was repeated twice. The final buffer wash wasfollowed by fixation in a 1% solution of osmium tetroxidein 0.1 M sodium phosphate buffer. After 60–90 min, the
tissue pieces were washed again in two changes of buffer(10 min each). The buffer washes were followed by
Parmar et al.
238
dehydration carried out by immersing the tissue pieces inascending concentrations of ethanol (30, 50, 70, 80, 90 and100% ethanol) for 3–5 min in each solution. A second
change of 100% ethanol was replaced by propylene oxide, atransitional solvent, and allowed to infiltrate for 15 min,after which the propylene oxide was changed. After afurther 15 min, resin infiltration was carried out by
replacing the propylene oxide by solutions containingincreasing concentrations of resin (TAAB 812 and Aralditemixture) propylene oxide. Three mixtures were used (25:75,
50:50, 75:25) and the infiltration carried out for 60–90 minin each mixture. The final mixture was replaced by pureresin and further infiltration allowed to take place over-
night. The tissue pieces were then transferred to mouldscontaining pure resin and allowed to polymerize at 60�C for36 hr. The solidified samples were removed and placed inlabeled tubes until use. The sample blocks were trimmed
and sectioned with an RMC MT-7 ultramicrotome forexamination. The sections were viewed using a Joel JEM1210 transmission electron microscope.
Electrochemistry
Cyclic and stripping voltammograms were obtained withthe Bio Analytical Systems (BAS) CV-50 W voltammetricanalyzer using a BAS C2 cell stand to maintain constant
atmosphere. An undivided cell was employed in all elect-rochemical experiments. A 3-mm diameter glassy carbonelectrode (GCE) was employed as a working electrode forcyclic voltammograms. A hanging mercury drop electrode
(HMDE) was employed as the working electrode in allcathodic adsorptive stripping voltammograms. A silver/silver chloride ([KCl ¼ 3 mol dm)3]) and a platinum wire
were employed as reference and auxiliary electrodes,respectively, in all electrochemical work. Prior to use,GCE was cleaned by polishing with alumina on a Buehler
pad, followed by washing in nitric acid and rinsing in waterand the buffer solution. Between scans, the GCE wascleaned by immersion in a dilute acid solution and rinsedwith water.
For cyclic voltammetric experiments, appropriate con-centrations of Cu1+ and melatonin in pH 7.3 Tris-HClbuffer were introduced into the glass cell, degassed for
5 min with nitrogen before scanning a potential window.For adsorptive stripping experiments, sodium acetate
buffer (pH ¼ 4.4) and appropriate concentrations of Cu1+
and of the ligands (melatonin or glutathione) were intro-duced into an electrochemical cell. The solution was thendeaerated with nitrogen for 5 min, after which a flow of
nitrogen was maintained over the solution throughout themeasurement. The metal complexes of melatonin or gluta-thione are expected to have formed at this stage. Anoptimum deposition potential of 150 mV versus Ag/AgCl
was applied for 60 s to effect the formation and adsorptionof the metal complex onto the HMDE. The voltammogramswere then scanned in the negative direction from 150 mV
versus Ag/AgCl to)300 mV versus Ag/AgCl at the scan rateof 100 mV/s to strip the adsorbed metal–ligand complexfrom the electrode. During the stripping step, current
response caused by the reduction of the metal complexesof melatonin or glutathione, were measured as a function of
potential. All potential values quoted are referenced againstthe silver/silver chloride reference electrode.
Results
As shown in Fig. 1, copper chloride (1 mM) produces asignificant increase in lipid peroxidation in the liver
(P < 0.001), as compared with the control liver group.The copper and copper/melatonin liver groups are signifi-cantly different (P < 0.001), indicating that melatonin
reduces the degree of copper-induced lipid peroxidation.The 2 wk experiment with 2 mg/kg Cu showed extensive
hepatocyte damage. Fig. 2A–C illustrate the control,
copper and copper/melatonin samples, respectively. InFig. 2A the nucleus is spherical, with normal mitochondriaand extensive glycogen. The sample from the copper-treated animals (Fig. 2B), exhibits damaged mitochondria
and a creatinated nucleus. The chromatin in the nucleus iscondensed, and there is less noticeable glycogen. Fig. 2Cshows a copper/melatonin treated sample. Here less dam-
aged mitochondria are noticeable when compared with thecopper sample (Fig. 2B). The nucleus is spherical and issimilar in appearance to the control nucleus (Fig. 2A).
The CV of melatonin in the absence of Cu1+ shows apeak as a result of the oxidation of melatonin at 0.73 Vversus Ag/AgCl (Fig. 3A). Upon several successive scans, a
quasi-reversible couple appears, which may be related tothe adsorption of melatonin at the electrode surface.
The CV of Cu1+ in the absence of melatonin shows anirreversible oxidation peak at 0.24 V versus Ag/AgCl.
Upon several successive scans, this peak does not showany significant increases indicating that it is not beingelectrodeposited at the electrode.
Cyclic voltammetry is an electrochemical method forcharacterizing species in solution. Species in solution tendto have characteristic oxidation/reduction patterns. Chan-
ges in the redox potential of these species indicate that thespecies has been altered, for example, by forming a complexwith another species introduced to the solution. Fig. 3Ashows the CV obtained for a solution of melatonin alone.
Upon addition of Cu1+, the melatonin peak shifts from0.73 to 0.87 V without much change in current intensity(Fig. 3B). The potential shift from 0.73 to 0.87 V for
Fig. 1. In vitro liver copper (1 mM) and melatonin (5 mM)experiments. *The control group (A) versus copper (B) is statisti-cally significant at P < 0.001. *The copper group (B) versus cop-per-melatonin group (C) is statistically significant at P < 0.001.
Melatonin protects against copper-mediated free radical damage
239
melatonin indicates that melatonin has formed a newspecies with copper. As seen in Fig. 3C, increasing theCu1+ concentration resulted in an increase in currentresponse of the melatonin peak and an enhancement of the
return peak. The weak peak observed for Cu1+ in solutionis absent in the presence of melatonin.
Upon several successive scans of a solution of melatoninand Cu1+ we observe the appearance of a new quasi-
reversible couple at 0.34 V versus Ag/AgCl (Fig. 4). Withincreasing scan number these peaks increase in strength,while the peak caused by melatonin oxidation at 0.87 V
versus Ag/AgCl decreases. This indicates the formation of anew compound electrodeposited at the electrode. Thedecrease in melatonin current response and disappearance
of the peak observed for Cu1+ alone and the increase incurrent strength of the couple at 0.34 V versus Ag/AgCl,indicates that both melatonin and Cu1+ are being used to
produce a new product. This electrode was rinsed in Tris-HCl buffer and placed in a fresh Tris-HCl buffer. No peak
Fig. 2. (A) A control rat hepatocyte. The nucleus is spherical andappears normal. There is extensive glycogen (G) present. Themitochondria (M) appear normal. (B) Hepatocyte of a rat treatedwith copper (2 mg/kg daily for 2 weeks). Extensive damage is seenin the mitochondria (M) and the nucleus is creatinated (�). (C) Ahepatocyte of a copper/melatonin treated rat in the 2 weekexperiment. There are some damaged (fi) pleomorphic mito-chondria (M), with a normal appearing nucleus. Lysosomes (�)are irregularly shaped and dense. Glycogen is present, ·5000.
Fig. 3. Cyclic voltammograms of (A) melatonin (1 · 10)5M) in
buffer in the absence of Cu1+; (B) solution of melatonin(1 · 10)5
M) and Cu1+ (1 · 10)5M) showing a shift in oxidation
potential for melatonin from 0.73 to 0.87 V versus Ag/AgCl in thepresence of Cu1+; (C) further increases in Cu1+ (3 · 10)5
M)results in a decrease in melatonin current.
Fig. 4. Cyclic voltammogram of a solution of Cu1+ (1 · 10)3M)
and melatonin (4 · 10)3M). A new reversible couple is observed at
0.34 V versus Ag/AgCl.
Parmar et al.
240
caused by the oxidation of free melatonin in solution isobserved at 0.87 V versus Ag/AgCl. The redox coupleobserved at 0.071 V versus Ag/AgCl is stable upon multiple
scans of the electrode. The electrode was allowed to dry anda stable blue-purple film was observed at the electrode.
Adsorptive stripping voltammetry has been used withsuccess to examine metal–ligand complex formation at an
electrode. This technique relies on the natural tendency ofanalytes to preconcentrate at the surface of a workingelectrode. Theoretically, the introduction of a successful
ligand to a metal solution will increase preconcentration ofthe metal at the electrode if it forms a metal–ligandcomplex. The increase in preconcentration of the metal as a
metal–ligand complex results in an increase in the currentresponse due to the metal reduction. A shift in thereduction potential further indicates that a new species isbeing reduced. Fig. 5A shows the cathodic adsorptive
stripping voltammogram (CSV) of Cu1+ in the absence ofmelatonin. A peak caused by the reduction of Cu1+ isobserved at 0.02 V versus Ag/AgCl. Fig. 5B shows the CSV
obtained for Cu1+ and melatonin. In the presence ofmelatonin, an increase in the Cu1+ reduction peak and anegative potential shift is observed. Increases in melatonin
concentration increased the current response caused byCu1+ (Figs 5B–E). For 0.04 mM melatonin, a shift to)0.78 V versus Ag/AgCl is observed. The increase in
current strength as well as the potential shift indicates theformation of a melatonin-Cu1+ complex.
As glutathione is known to bind Cu1+, we ran a similarset of experiments for Cu1+ with glutathione. As before
with melatonin, the presence of glutathione resulted in anincrease in current strength for Cu1+ reduction accompan-ied by a strong negative potential shift to )0.91 V versus
Ag/AgCl. An increase in glutathione concentration alsoincreased the observed current response.
Both cyclic voltammetric and adsorptive stripping vol-tammetric experiments indicate strongly that melatoninforms a complex with Cu1+. From these experiments it
appears that the bond is fairly strong but it is not possibleto conclude whether it is simple ligation or whether it ischelation. Cyclic voltammetric experiments indicate that anew product is being formed.
Discussion
Copper is bound in lysosomes within the cell to limit thedamage caused by free copper ions within the cell. Seques-tration of copper into lysosomes is a cytoplasmic adaptive
process, which protects the cell from free, toxic copper [18].Copper within the nucleus interacts with DNA and
results in cellular damage. Creatinated nuclei are a directresult of copper loading. Chromatin condensation occurs as
early as week 2 of copper loading [19]. Both creatinatednuclei and chromatin condensation evident in Fig. 2B arethus consistent with copper overloading.
Rats are rather tolerant to copper loading. Initially, therats receiving copper exhibit all the signs of acute coppertoxicity, but as time progresses, metallothionein (MT) and
ceruloplasmin (Cp) are induced. This aids in intercellularcopper chelation, and decreases the potential of furthercopper toxicity. Liver, kidney and intestines are able to
form MT when faced with initial copper loading [18, 20].On continuous copper exposure, additional protectivemechanisms are activated. The copper-MT complexes areincorporated into lysosomes, which then release the com-
plex into the biliary ducts for excretion. Other chelatingmolecules such as glutathione can remove the copper andprevent its destructive cellular effects [18]. Melatonin may
provide protection against copper toxicity in a similar wayto glutathione. Melatonin, which has been shown tointeract with Cu2+ in vitro [12], possibly interacts with
copper in vivo as well.The in vitro experiments were performed with the
addition of copper and copper/melatonin to liver homo-genate. When liver homogenate was incubated with copper
(Fig. 1) an increase in lipid peroxidation in the copper andcopper/melatonin groups was seen. Copper alone inducedmore lipid peroxidation (P < 0.001), indicating that mela-
tonin affords a degree of protection against copper-inducedlipid peroxidation, at a copper concentration of 1 mM. It islikely that the free radical induced by copper in these
experiments is the hydroxyl radical (via the Fenton reac-tion), as this is sufficiently reactive to initiate lipid perox-idation. Further studies are required to determine the
optimum concentration of melatonin needed to preventcopper-induced lipid peroxidation.
Previous experiments [12] provide evidence that melato-nin forms a metal–ligand bond with Cu2+. In these studies,
electrochemical studies of Cu1+ indicate strongly thatmelatonin interacts with Cu1+ and that a new productmay be formed from this interaction. In-depth studies are in
progress to determine the nature of this new compoundsuggested. In conclusion, it is suggested that melatonin mayprotect against copper-induced free radical damage by
forming a metal–ligand bond with free copper of bothoxidation states.
Fig. 5. Cathodic adsorptive stripping voltammograms of Cu1+
and melatonin. (A) Cu1+ (4 · 10)5M) in the absence of melatonin;
(B) Cu1+ (4 · 10)5M) in the presence of melatonin (1 · 10)5
M);(C–E), cathodic adsorptive stripping voltammograms of increasingmelatonin concentrations (2 · 10)5 to 4 · 10)5
M, respectively) inthe presence of Cu1+ (4 · 10)5
M).
Melatonin protects against copper-mediated free radical damage
241
Acknowledgements
Janice Limson thanks the Medical Research Council (South
Africa) for postdoctoral fellowship.
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