Elsevier

Toxicology Letters

Volume 137, Issue 3, 3 February 2003, Pages 169-174
Toxicology Letters

Short communication
Melatonin protects the central nervous system of rats against toluene-containing thinner intoxication by reducing reactive gliosis

https://doi.org/10.1016/S0378-4274(02)00400-9Get rights and content

Abstract

Neuroprotective effects of melatonin against free radical damage have been studied extensively. Thinner containing 60–70% toluene is a neurotoxic mixture which is widely used as an aromatic industrial solvent. This product has been shown to cause functional and structural changes in the central nervous system. Toluene generates reactive oxygen species (ROS) and the toxic effects relating to these reactants. In the present study we investigated glial reactivity in hippocampus, cortex and cerebellum and the expression of glial fibrillary acidic protein (GFAP) after exposure of rats to toluene vapor (3000 ppm) for 45 days. We also examined the protective effects of melatonin against gliosis. Western blots demonstrated a marked elevation in total GFAP, a specific marker for astrocytes, by thinner fume inhalation in the hippocampus (P<0.001), cortex (P<0.01) and cerebellum (P<0.05) of rats. Melatonin administration prevented the increase of total GFAP induced by thinner fume inhalation. Thinner exposure caused a significant increase of lipid peroxidation products (malondialdehyde and 4-hydroxyalkenals) in all brain regions (P<0.01 for each region), and this elevation was also was inhibited by melatonin. Furthermore, melatonin augmented glutathione levels in all brain regions (P<0.05 for each region) investigated. In conclusion, melatonin treatment may provide neuroprotection against toluene neurotoxicity by increasing the survival of glial cells possibly by directly scavenging ROS and by indirectly augmenting their antioxidant capacity.

Introduction

Industrial thinners are chemical mixtures frequently containing toluene, benzene, acetone, methanol, hexane, and other substances. They are widely used in textiles, paints and solvent-based cleaning fluids. The main agent is the neurotoxin toluene, which is approximately 60–70% of most thinners. Thinners are abused substances and well known as neurotoxic agents (Escobar and Aruffo, 1980). The chronic abuse of solvents results in structural and functional impairment of a variety of organs. Thinner fume inhalation is an important cause of encephalopathy and may lead to irreversible brain damage. Particularly, toluene abuse has been shown to cause permanent changes in brain structures which correlate with neural dysfunction (Baelum, 1991, Ladefoged et al., 1991, Hass et al., 1999).

Acute and chronic effects of toluene on neurons have been well documented (Burmistrov et al., 2001). In addition, toluene inhalation was found to enhance astrocyte activation (Gotohda et al., 2000). Astrocytes are known to play an important role in survival of neurons in the brain and they have been implicated in the regulation of ionic environments which are required for proper physiological function of neurons (Moonen et al., 1990). Glial cells exhibit early cellular responses following a variety of insults to the central nervous system (CNS) (Papadopoulos et al., 1999). Reactive gliosis is a reaction of astrocytes to neuronal damage resulting from physical or chemical insults. It is characterized by an over expression of glial fibrillary acidic protein (GFAP), a specific marker for astrocytes (Janeczko, 1989, Janeczko, 1993).

Melatonin, a pineal derived product, is a potent free radical scavenger (Donder et al., 1999a, Reiter et al., 2001a, Tan et al., 2002) which is known to reduce oxidation-based neurotoxicity (Reiter, 1998). Melatonin pharmacologically prevents oxidative damage to macromolecules including lipids, proteins and nucleic acids (Donder et al., 1999b, Reiter et al., 2001a, Okatani et al., 2002) and preserves membrane fluidity. As a free radical scavenger, it reportedly quenches the hydroxyl radical, superoxide anion radical, singlet oxygen, peroxyl radical, nitric oxide, peroxynitrite anion and peroxynitrous acid (Reiter et al., 2001b, Tan et al., 2002). In addition, melatonin stimulates several antioxidant enzymes including superoxide dismutase and glutathione peroxidase (GSH-Px) (Baydas et al., 2001, Reiter et al., 2001a). GSH-Px is particularly important antioxidative enzyme in the protection of the brain from damaging effects of reactive oxygen species (ROS).

Effects of toluene inhalation on the induction of oxidative stress have been, in part, defined (Burmistrov et al., 2001). However, to date the potential protective actions of melatonin against the toxic effects of thinner and/or toluene exposure on glial and neuronal cell functions have yet to be investigated. Glial cells contain augmented levels of glutathione (GSH) as well as elevated activities of the enzymes involved in GSH metabolism and higher superoxide dismutase activity than neurons (Savolainen, 1978, Raps et al., 1989). Possibly one of the most protective actions of melatonin, which is attributed to its indirect antioxidant activity, in CNS is to elevate GSH content and increase antioxidant enzyme activities such as GSH-Px (Baydas et al., 2002a, Baydas et al., 2002b). Studies to date have shown that melatonin is generally highly protective of neurons and presumably glial cells against chemical insults.

In the present study we investigated the effects of melatonin on the glial reactivity induced by the exposure of rats to the fumes of thinner, which due to the presence of toluene, are known to be neurotoxic. Endpoints in these studies included an estimation of GFAP expression, levels of lipid peroxidation products and glutathione levels in various brain areas.

Section snippets

Material and methods

Forty-five adult male Wistar rats (weighing 200–250 g), obtained from Biomedical Unit of Firat University, were randomly divided into three groups. The experimental protocols were approved by the appropriate animal care committee of Firat University. The rats were placed 5/cage in a room under controlled photoperiod conditions (12 h light/12 h dark cycles; lights off was 1900 h). Food and water were provided ad libitum. Two groups (15/group) of rats were exposed to 3000 ppm thinner fumes

Results

After thinner fume inhalation, levels of lipid peroxidation products, i.e. MDA and 4-HDA, in hippocampus, cortex and cerebellum were significantly increased compared to those of control rats (P<0.01). Melatonin administration to thinner-exposed animals significantly reduced the levels of MDA+4-HDA in these brain regions compared levels in the brain of exposed rats not given melatonin (Fig. 1; P<0.05 for hippocampus and cerebellum; P<0.01 for cortex).

There was no significant effect of thinner

Discussion

Astrocytes exhibit the earliest cellular responses following an insult to the CNS. This response is characterized by an over expression of GFAP (Janeczko, 1989, Janeczko, 1993). In the present study, we show that thinner inhalation promotes GFAP expression and its degradation in several brain regions. Enhanced GFAP expression, reflecting reactive gliosis, has been recently described in rat brain after toluene fume inhalation (Gotohda et al., 2000). In that study, toluene exposure augmented the

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