6-Hydroxymelatonin protects against cyanide induced oxidative stress in rat brain
homogenates
Deepa S. Maharaj, Roderick B. Walker, Beverley D. Glass and Santy Daya
Abstract
Both 6-hydroxymelatonin and N-acetyl-N-formyl-5-methoxykynurenamine are
photodegradants and enzymatic metabolites of melatonin and are known to retain
equipotent activity against potassium cyanide-induced superoxide generation compared
to melatonin. It is not clear whether one or both of these metabolites is responsible for
this effect. The present study therefore investigates the possible manner in which 6-
hydroxymelatonin protects against oxidative stress induced by cyanide in rat brain
homogenates. We examined the ability of 6-hydroxymelatonin to scavenge KCN-induced
superoxide anion generation as well as lipid peroxidation. In addition, we also examined
the effect of this indole on lactate dehydrogenase activity (LDH) as well as mitochondrial
electron transport using dichlorophenol–indophenol as an electron acceptor. The results
of this study show that 6-hydroxymelatonin significantly reduces KCN-induced
superoxide anion generation, which is accompanied by a commensurate reduction in lipid
peroxidation. Partial reversal of the KCN-induced reduction in mitochondrial electron
transport is accompanied by a similar reversal of mitochondrial LDH activity blunted by
KCN. It can thus be proposed that 6-hydroxymelatonin is potentially neuroprotective
against KCN-induced neurotoxicity.
1. Introduction
The brain is the primary target organ for cyanide toxicity ([Gunesekar et al., 1996]).
Acute cyanide neurotoxicity has been attributed to production of cellular anoxia in the
brain ( [Ballantyne, 1987] [Yamamato and Tang, 1996]) and produces tonic and clonic
seizures, convulsions ( [Way, 1985]) while in some individuals a Parkinson-like
condition may develop as a post toxicity sequel ( [Utti et al., 1985]). Cyanide also
produces dopaminergic toxicity accompanied by impaired motor function ( [Gunesekar et
al., 1996]). Due to a number of antioxidant enzymes being inhibited by cyanide, it is also
believed that oxidative stress plays an important role in cyanide induced neurotoxicity (
[Ardelt et al., 1989]). [Johnson et al., 1987] proposed that increased intracellular calcium
after cyanide treatment generates reactive oxygen species leading to peroxidation of
lipids and subsequent neuronal damage.
The primary function of the mitochondria is to generate ATP, the energy currency of the
cell. Since ATP is an ubiquitous store of energy that is needed for transport across
membranes for all synthetic processes and for the mechanical work involved in motor
activities of the cell ([Hammans and Tipton, 1994]), energetically compromised
mitochondria may have detrimental effects on the survival of the cell, leading to potential
apoptosis. Mitochondrial respiratory chain defects have been implicated in the
pathogenesis of Alzheimer's disease ( [Grunewald and Beal, 1999]) and mitochondrial
dysfunction has been associated with the neurodegeneration of Parkinson's disease (
[Berman and Hastings, 1999]). Cyanide, implicated as a mitochondrial electron transport
inhibitor, is also an inhibitor of complex IV and causes severe depletion of cellular ATP (
[Ottino and Duncan, 1997]). Cyanide causes a rapid and severe depletion of cellular ATP
and cell death that is dependent on cellular energy impairment but not lipid peroxidation.
The final transport of electrons across the inner mitochondrial membrane is inhibited with
cyanide by inhibition of cytochrome a1a3 which reduces the number and rate of electron
production by mitochondrial metabolism ([Plummer, 1971]).
It has been shown that 6-hydroxymelatonin and N-acetyl-N-formyl-5-
methoxykynurenamine, both known enzymatic metabolites of melatonin in the body
([Garfinkel et al., 1995] [Maharaj et al., 2002]) can be generated by UV-irradiation of
melatonin ( [Anoopkumar-Dukie et al., 2000].
[Pierrefiche et al., 1993] have attributed the antioxidant activity of melatonin, both in
vivo and in vitro, to the indole structure of the molecule, still present in the principal
hepatic metabolite, 6-hydroxymelatonin. Thus, 6-hydroxymelatonin is considered to be
an ideal candidate to be evaluated as a neuroprotectant against oxidative stress. In the
present study, we thus examined the potential protective effects of 6-hydroxymelatonin
using cyanide-induced free radical damage to rat brain homogenate in vitro, using the
nitroblue tetrazolium assay (NBT), biological oxidation assay, lactate dehydrogenase
assay (LDH) with samples analyzed by ultraviolet/visible (UV/Vis) spectroscopy and the
lipid peroxidation assay with samples analyzed by high performance liquid
chromatography (HPLC).
2. Materials and methods
2.1. Chemicals and reagents
All reagents were at least analytical grade. 6-Hydroxymelatonin, KCN, 2-thiobarbituric
acid (TBA), 1,1,3,3-tetramethoxypropane (98%), and butylated hydroxytoluene (BHT),
NBT, nitroblue diformazan (NBD), nicotinamide adenine dinucleotide (NAD), 2,6-
dichlorophenolindophenol (DPI), pyruvic acid and 3-[N-morpholino]propanesulfonic
acid (MOPS) were purchased from Sigma Chemical Corporation, St. Louis, MO, USA.
Trichloroacetic acid (TCA), glacial acetic acid, and sucrose were purchased from
Saarchem (PTY) Ltd., Krugersdorp, South Africa. -Malate was purchased from Eastman
Organic Chemicals and NAD, reduced form NADH from Boehringer Mannheim,
Germany.
2.2. Animals
Adult male albino rats of the Wistar strain, weighing between 250 and 300 g used were
housed in a controlled environment with a 12 h light:dark cycle, and were given access to
standard laboratory food and water ad libitum. The experiments were approved by the
Rhodes University Animal Ethics Committee.
2.3. Homogenate preparation
The rats were killed by neck fracture, the heads decapitated and the brains rapidly excised
and chilled on crushed ice and thereafter these were rinsed in ice-cold saline [0.9% (w/v)
NaCl]. The whole rat brain was homogenised with 0.1 M phosphate buffer saline (PBS)
at pH 7.4, to give a final concentration of 10% w/v for the NBT and lipid peroxidation
assays. This is necessary to prevent lysosomal damage of the tissue. Rat brain
mitochondrial suspensions were used for the biological oxidation assay of the electron
transport chain. The whole brains were homogenized in 0.32 M sucrose+1 M MOPS
buffer at pH 7.4 in a manual glass–teflon homogeniser on ice to yield a 10% w/v
homogenate. Mitochondrial suspensions were prepared by differential centrifugation to
obtain relatively pure suspensions of intact mitochondria.
2.4. Instrumentation
Samples for NBT, biological oxidation assay and LDH assay were analyzed using a Cary
500 UV/Vis/NIR spectrophotometer.
Samples for lipid peroxidation analysis were analyzed on a modular, isocratic HPLC
system, consisting of a Spectraphysics Iso Chrom LC Pump (Spectraphysics, USA), a
linear UV/Vis 200 detector and a Rikadenki chart recorder (Tokyo, Japan). Samples were
introduced onto the chromatographic system using a Rheodyne Model 7125 20 µl fixed
loop injector. An N-EVAP analytical evaporator was used to evaporate the methanol
using nitrogen.
2.5. Chromatographic conditions
The HPLC separation was achieved with a Spherisorb (250×4.6 mm i.d. 5 µm, C18)
column (Waters, Masachussets, USA), fitted with an in-line pre-column filter. The
mobile phase for the lipid peroxidation analysis was 14% methanol in Milli-Q water,
degassed and filtered prior to use. The mobile phase flow rate was set at 1.2 ml/min, the
detection wavelength 532 nm, the response time at 0.1 s, the detector sensitivity at 0.1-
absorbance units full scale (AUFS) and the data were recorded at a chart speed of 5
mm/min. Resorcinol (0.1 mg/ml in water) was used as an external standard.
2.6. Lipid peroxidation assay
The method used in this experiment is described by [Anoopkumar-Dukie et al., 2001].
Briefly, homogenate (1 ml) containing varying concentrations (0, 0.25, 0.5, 1 mM) of
KCN in the absence and presence of 6-hydroxymelatonin (0, 0.25, 0.5, 1 mM) was
incubated in an oscillating water bath for 1 h at 37±2 °C. At the end of the incubation
period, 0.5 ml BHT (0.5 mg/ml in methanol) and 1 ml TCA (15% v/v in water) were
added to the mixture. The tubes were sealed and heated for 15 min in a boiling water bath
to release protein-bound MDA with subsequent cooling and centrifugation.
TBA–MDA was determined by HPLC analysis and the final results expressed as
nmoles/mg tissue.
2.7. Nitroblue tetrazolium assay (NBT)
This method is generally accepted as a simple and reliable method for assaying the
superoxide free radical ([Ottino and Duncan, 1997]). A modification of the assay used by
[Ottino and Duncan, 1997] was used in this set of experiments.
Homogenate (1 ml) containing varying concentrations of KCN (0, 0.25, 0.5, 1 mM) alone
or in combination with 6-hydroxymelatonin (0, 0.25, 0.50, 0.1 mM) was incubated with
0.4 ml 0.1% NBT in an oscillating water bath for 1 h at 37±2 °C. Termination of the
assay and extraction of reduced NBT was carried out by centrifugation of the samples at
2000×g and the resuspension of the pellet with 2 ml glacial acetic acid. The absorbance
of the glacial acetic acid fraction was measured at 560 nm and converted to µmoles
diformazan using a standard curve generated from NBD with final results expressed as
µmoles/mg protein.
2.8. Biological oxidation assay
A modified method described by [Plummer, 1971] was used. This spectrophotometric
technique was employed to determine the ‘activity’ of the inner mitochondrial membrane
electron transport chain of the mitochondrial suspension. The latter was determined by
the rate of reduction of the synthetic electron acceptor dye 2,6-dichlorophenol–
indophenol (DPI; 50 µM) in the presence of -malate. The substrate and NAD+ were
present in saturating final concentrations of 15 and 0.0899 mM, respectively. Potassium
phosphate buffer pH 7.4 buffer (1.5 ml) containing 300 µl of homogenate, 100 µl of 1
mM KCN in combination with 6-hydroxymelatonin (0.25, 0.5 and 1 mM) were placed in
a 37±2 °C water bath for varying preincubation times (0, 60 min). Following incubation
500 µl of substrate/buffer (control), 500 µl NAD and 100 µl DPI was added in that order.
This was inverted once to mix solutions and the decrease in absorbance at 600 nm was
read over a 5 min period at 30 s intervals. Data are expressed as ∆Abs600nm/min and
corrected for appropriate controls.
2.9. Lactate dehydrogenase assay (LDH)
LDH activity of the ‘whole brain’ mitochondrial suspension was determined
spectrophotometrically as the rate of NADH utilization ([Plummer, 1971]). The latter was
measured as ∆Abs340nm/min and the data converted to µmoles/min ( NADH=6.22×103
l/mol). Saturating final concentrations of NADH (0.2 mM) and pyruvate (0.76 mM) were
used as substrates. MOPS (1.0 M, pH 7.4) was used as the assay buffer. All reagents were
prepared in 0.1 M potassium phosphate buffer pH 7.4 with appropriate controls being
run. The assay mixture contained 1 ml buffer, 100 µl NADH, 100 µl pyruvate, 1.5 ml
water, 100 µl (0.25, 0.5, and 1 mM) KCN, 100 µl 6-hydroxymelatonin (0.25, 0.5 and 1
mM) and 0.1 ml homogenate. The reaction was started on addition of NADH following
60 min preincubation with the homogenate. The absorbance was read at 340 nm for 5 min
at 30 s intervals.
2.10. Protein assay
Protein estimation was performed using the method described by [Lowry et al., 1951].
2.11. Statistical analysis
The results were analyzed using a one-way analysis of variance (ANOVA) followed by
the Student–Newman–Keuls Multiple Range Test. The level of significance was accepted
at P<0.05 ([Zar, 1974]).
3. Results
As seen in Fig. 1, co-treatment of the rat brain homogenate with KCN and increasing
concentrations of 6-hydroxymelatonin (0.5, 1 mM) resulted in an overall decline in MDA
production. At a concentration of 1 mM, 6-hydroxymelatonin reduced the MDA formed
by ±1.94 nmoles/mg tissue.
Fig. 1. The effect of varying concentrations of 6-hydroxymelatonin on KCN (1 mM)-induced lipid peroxidation in whole rat brain homogenate.
Each bar represents the mean±SEM; n=5; *P<0.05 in comparison to 1 mM KCN alone (Student–Newman–Keuls Multiple Range Test).
As evident in Fig. 2, the 0.5 and 1 mM 6-hydroxymelatonin significantly reduced KCN-
induced superoxide generation.
Fig. 2. The effect of varying concentrations of 6-hydroxymelatonin on KCN (1 mM)-induced superoxide anion generation on whole rat brain homogenate.
Each bar represents the mean±SEM; n=5. *P<0.05 in comparison to 1 mM KCN alone (Student–Newman–Keuls Multiple Range Test).
As shown in Fig. 3, the mitochondrial electron transport was dose and time dependently reduced by KCN with malate as the substrate. This reduction in the mitochondrial electron transport caused by 1 mM cyanide was reversed by the addition of increasing concentrations of 6-hydroxymelatonin, with the greatest effect observed with 1 mM 6-hydroxymelatonin.
Fig. 3. The effect of KCN (1 mM) alone or in combination with 6-hydroxymelatonin (0, 0.25, 0.5, 1 mM) on rat brain mitochondrial electron transport utilizing -malate as a substrate, at time 0 and 60 min. [Data represents mean±SEM (n=5) #(P<0.001) 1 mM KCN alone vs. control, at time 5 and 60 min; *P<0.05].
Fig. 4, shows that cyanide dose-dependently reduced NADH-dependent LDH activity, with almost complete inhibition apparent at 1 mM. The use of increasing concentrations of 6-hydroxymelatonin resulted in reversal of this inhibition, causing an induction of LDH, with greatest induction evident with 1 mM 6-hydroxymelatonin.
Fig. 4. The effect of cyanide (1 mM) in combination with varying concentrations of 6-hydroxymelatonin (0, 0.25, 0.5, 1 mM) on LDH activity of rat brain mitochondrial preparation following 60 min preincubation with cyanide. [Data represents mean±SEM (n=5); for all data, #(P<0.001) a vs. b; *(P<0.05) c, d and e vs. b].
4. Discussion
It has been known for a number of years that indoles possess antioxidant properties and
are able to provide tissue protection ([Wattenberg, 1983]). The ability of indoles to do
this has been ascribed to their ability to scavenge free radicals. The indole 6-
hydroxymelatonin has been shown to operate both as a hydroxyl radical generation
promoter as well as a hydroxyl radical scavenger ( [Matuszak et al., 1997]).
At concentrations of 0.5 and 1 mM, 6-hydroxymelatonin significantly reduces KCN-
induced lipid peroxidation (Fig. 1), whilst concentrations of 6-hydroxymelatonin ranging
from 0.5 to 1 mM were found to be effective in scavenging superoxide anions formed by
1 mM KCN ( Fig. 2). It is well known that cyanide induces neurotoxicity due to oxidative
damage resulting in extensive lipid peroxidation of neuronal membranes. Cyanide-
induced oxidative stress is believed to result from inhibition of antioxidant enzymes (
[Ardelt et al., 1989]). Such toxicity induced by cyanide is known to result in a variety of
CNS disorders, ranging from tonic and clonic seizures ( [Way, 1985]) to a Parkinson-like
syndrome. Free radical scavengers have become increasingly popular as a means of
reducing or preventing the hazardous effects of free radicals and their inducers. In the
present study, we have also shown that 6-hydroxymelatonin not only scavenges the
hydroxyl radical but also superoxide radicals that are generated by potassium cyanide.
Furthermore, 6-hydroxymelatonin is able to partially reverse the KCN-induced inhibition
of mitochondrial electron transport. As shown in Fig. 3, this reversal is more apparent at
5 min than at 60 min. Similarly, the KCN-induced drastic decrease in NADH utilization
by the cytosolic enzyme, lactate dehydrogenase, is partially reversed by 6-
hydroxymelatonin. The exact mechanism by which 6-hydroxymelatonin reverses the
effects of KCN is not known but it is possible that 6-hydroxymelatonin could bind to
cyanide thus removing its inhibitory effect or preventing cyanide from binding to
cytochrome a1a3. This however requires further investigation. Thus, the findings imply
that 6-hydroxymelatonin is potentially neuroprotective in that it partly reverses the
deleterious effects of KCN.
Acknowledgements
This work was supported by a grant from the South African Medical Research Council to
Professor S. Daya. The authors would like to thank Sally and Dave Morley for their
technical assistance.
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