Melatonin and 6-hydroxymelatonin protect against iron-induced neurotoxicity

Authors


Address correspondence and reprint requests to S. Daya, PO Box 94, Faculty of Pharmacy, Rhodes University, Grahamstown, 6139, South Africa. E-mail: s.daya@ru.ac.za

Abstract

Oxidative damage of biological macromolecules is a hallmark of most neurodegenerative disorders such as Alzheimer, Parkinson and diffuse Lewy body diseases. Another important phenomenon involved in these disorders is the alteration of iron homeostasis, with an increase in iron levels. The present study investigated whether 6-hydroxymelatonin (6-OHM) can reduce Fe2+-induced lipid peroxidation and necrotic cell damage in the rat hippocampus in vivo. It was found that 6-OHM administration proved successful in reducing Fe2+-induced neurotoxicity in rat hippocampus. This study provides some evidence of the neuroprotective effects of 6-OHM.

Abbreviations used
DPX

Distrene Plasticiser in Xylene

6-OHM

6-hydroxymelatonin

MDA

Malondialdehyde

The activities of many enzymes depend on iron, but when this metal is unbound it can potentiate the formation of reactive oxygen species via the Fenton reaction. Iron-mediated oxidative stress has been implicated in a wide variety of neurological disorders, including hemorrhagic stroke (Koeppen et al. 1995), post-traumatic epilepsy (Willmore et al. 1978), Alzheimer's disease (Smith et al. 1997) and Parkinson's disease (Dexter et al. 1989). To reduce oxidative stress in these disorders, there has been interest in therapeutic approaches that either chelate iron, or increase the availability of antioxidants (Halliwell 1989; Simon and Standaert 1999) or agents that can reduce ferric ion (Fe3+) to ferrous ion (Fe2+), a more usable form (Maharaj et al. 2003a).

Melatonin and its primary hepatic metabolite, 6-hydroxymelatonin (6-OHM), have been shown to scavenge free radicals, inhibit lipid peroxidation (Pierrefiche et al. 1993; Reiter et al. 2000; Maharaj et al. 2003b), and directly bind transition metals such as iron (Limson et al. 1998; Maharaj et al. 2003a). Because melatonin and 6-OHM can cross the blood–brain barrier easily (Reiter et al. 2000), there is much interest in the therapeutic use of these agents in oxidative brain injury (Maharaj et al. 2003b). It has been shown previously in our laboratory that both melatonin and 6-OHM are able to bind to iron (Limson et al. 1998; Maharaj et al. 2003a), so in the present study we investigated the ability of 6-OHM to reduce iron-induced lipid peroxidation and necrotic cell damage in the hippocampus in vivo.

Experimental procedures

All experimental protocols were approved by the Rhodes University Animal Ethics Committee. Male Wistar rats weighing between 250 and 300 g were used, and supplied with food and water ad libitum. Rats were divided into four experimental groups containing 10 animals each and treated as shown in Table 1. To induce oxidative injury each rat was anesthetized with chloral hydrate (450 mg/kg i.p.; Sigma Chemical Co., St Louis, MO, USA) and placed in a stereotaxic apparatus (Stoelting, IL, USA). After skin incision and exposure of the parietal bones, holes were drilled above the cortical surface to allow an intrahippocampal unilateral infusion of 1 µL Kreb's Ringer solution containing ferrous citrate (iron, 3.4 mmol/L) in the Fe2+-only, melatonin and Fe2+, and 6-OHM and Fe2+ groups. The control group received an intrahippocampal infusion of 1 µL Kreb's Ringer solution only. The stereotaxic co-ordinates derived from the bregma (König and Klippel (1963) were 4.0 mm caudal to the bregma, 2.5 mm lateral to the saggital suture, and 3.2 mm ventral to the dura. The iron solution was infused at a rate of 0.2 µL/min through a 30-G stainless steel needle and the needle was held in place for an additional 5 min after the infusion.

Table 1.  Treatment regimen for each group of rats
Treatment groupIntrahippocampal injection (1 µL)Daily treatment (intraperitoneal injection)1
  • 1

    For 7 days after stereotaxic surgery.

ControlKreb's Ringer solution0.25 mL 2% ethanol and saline
Fe2+ alone3.4 mmol/L ferrous citrate0.25 mL 2% ethanol and saline
Fe2+ and melatonin3.4 mmol/L ferrous citrate0.25 mL 10 mg/kg/day melatonin
Fe2+ and 6-OHM3.4 mmol/L ferrous citrate0.25 mL 10 mg/kg/day 6-OHM

Melatonin (Sigma Chemical Co.) 10 mg/kg/day and Fe2+, and 6-OHM (Sigma Chemical Co.) 10 mg/kg/day and Fe2+ groups received intraperitoneal injections each day for 7 days after surgery. The control and Fe2+-only groups were injected in the same way but they received vehicle, i.e. 2% ethanol and water solution.

On the seventh day animals were killed by cervical dislocation. Brains were removed and the hippocampi of five rats were excised rapidly according to a modified method of Glowinski and Iversen (1966). The hippocampi were then assayed for lipid peroxidation levels using a modification of the method described by Anoopkumar-Dukie et al. (2001). Whole brains from the remaining five rats were fixed in 10% formaldehyde for 48 h before embedding in paraffin wax for histological analysis. The tissue was then processed and sectioned (10 µm thick) using a rotary microtome. The sections were Nissl stained using cresyl violet. The slides were then mounted with Distrene Plasticiser in Xylene (DPX; Philip Harris, Gauteng, South Africa) and viewed under a light microscope to detect necrotic cell death in the CA1 and CA3 regions of the hippocampus. The photomicrographs and cell counts of CA1 and CA3 regions were taken from every fifth section throughout the rostrocaudal extent for each animal and its control using an image analyzer. Care was taken to exclude neurons at and about 1 mm from the injection point to avoid any discrepancies in the results. To differentiate surviving from injured neurons, injured neuronal cells were determined according to the criteria of Eke and Conger (1989) and Eke et al. (1990). Neurons were examined at a magnification of × 1000. CA1 and CA3 regions of the hippocampus were examined and a damage score calculated; 0% represented the absence of detectable cell loss and 100% represented the absence of any normal pyramidal neurons in the field of view.

Results and discussion

The brain is known to be rich in iron, which plays a crucial role in initiating and propagating lipid peroxidation (Halliwell and Gutteridge 1989). The concentration of iron is known to increase with age and serves as a catalyst for in vivo lipid peroxidaiton (Floyd and Carney 1992; Minotti and Aust 1992). Triggs and Willmore (1984) demonstrated that intracerebral injection of Fe2+ particularly into the hippocampus significantly increases lipid peroxidation levels and induces neuronal damage (Sloot et al. 1994). The results of the present study are in accordance with this as intrahippocampal injection of Fe2+ resulted in a significant increase in lipid peroxidation levels in comparison to control levels (Fig. 1). Both melatonin and 6-OHM have been shown previously to significantly reduce Fe2+-induced lipid peroxidation in rat liver homogenate (Karbownik et al. 2000; Maharaj et al. 2003a). In addition, melatonin has been reported by Chen et al. (2003) to protect against Fe2+-induced lipid peroxidation in vivo. In the present study the level of lipid peroxidation in the melatonin and Fe2+-treated rats was reduced by ± 50% relative to levels measured in the hippocampus of rats that received Fe2+ only. Similarly, there was a 40% reduction in Malondialdehyde (MDA) in the Fe2+ + 6-OHM-treated rats relative to levels in rats treated with Fe2+ only (Fig. 1). Thus, the injection of melatonin or 6-OHM in combination with Fe2+ significantly inhibited lipid damage in the hippocampus induced by the neurotoxin. However, melatonin proved to be superior to 6-OHM as an antioxidant as it reduced the Fe2+-induced lipid peroxidation to a greater extent than 6-OHM.

Figure 1.

Effect of melatonin (MEL) and 6-OHM on Fe2+-induced lipid peroxidation in rat hippocampal homogenate in vivo. Values are mean ± SEM (n = 6). #p < 0.001 versus control (CON); *p < 0.01 versus Fe2+; @p < 0.05 versus Fe2+ + 6-OHM (Student–Newman–Keuls multiple range test).

Sections of the CA1 and CA3 regions from control animals (Fig. 2) showed optimally sized, pyramidal shaped and undamaged neuronal cells with a clearly observable cell nucleus and continuous cell membrane. However, neurons from the CA1 and CA3 regions (Fig. 2) of the Fe2+-treated animals showed extensive damage, which was reversed by treatment with melatonin or 6-OHM (Fig. 2). Figure 3 shows that treatment with Fe2+ only resulted in a significant increase in the proportion of damaged neurons in both CA1 and CA3 regions of the hippocampus. However, administration of 6-OHM or melatonin significantly reduced the number of damaged neurons compared with that in the Fe2+-only group.

Figure 2.

Fe2+ toxicity and the protective effects of melatonin and 6-OHM treatment on rat hippocampal neurons. Scale bar 10 µm.

Figure 3.

Effect of melatonin and 6-OHM on neuronal damage assessed in the CA1 and CA3 regions after Fe2+ injection. Values are mean ± SEM (n = 5 rats) *p < 0.05 versus Fe2+-only group. Statistical test is the Student Newman Keuls Multiple Range Test.

The above findings (Figs 1 and 2) are consistent with these results (Fig. 3) and further indicate that melatonin and 6-OHM are effective in partially reversing the damage caused by Fe2+ in the hippocampus. Melatonin has been shown previously to scavenge hydroxyl radicals effectively (Stasica et al. 1998) and one of the primary products formed is 6-OHM (Horstman et al. 2002). This implies that, even though melatonin interacts with Fenton-type hydroxyl radicals to form 6-OHM, 6-OHM retains significant ability to scavenge hydroxyl radicals and reduce lipid peroxidation induced by the Fenton reaction. However, melatonin remains superior in protecting the hippocampus against Fe2+-induced lipid peroxidation.

Both 6-OHM and melatonin are capable of rapidly crossing the blood–brain barrier and it has also been reported that melatonin accumulates in high concentrations in brain cells after entering the brain. Cabrera et al. (2000) and Menéndez-Peláez et al. (1993) found that 30 min after a subcutaneous injection of 0.5 mg/kg melatonin, the concentrations of melatonin in the cell nuclei in rat cerebral cortex and cerebellum were five times higher than those of control rats. This study provides evidence that 6-OHM, like melatonin, is able to protect the hippocampus against lipid peroxidation and neuronal damage caused by the neurotoxin Fe2+. Although the protection offered by 6-OHM is less than that offered by melatonin, 6-OHM is nonetheless an effective antioxidant.

Acknowledgements

The authors would like to thank the National Research Foundation and the Medical Research Council (MRC) for financial support. Deepa Sukhdev and Himant Maharaj would like to thank the MRC for their Postdoctoral Fellowships.

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