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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