Behavioral effects of manganese injected in the rat substantia nigra are potentiated by dicumarol, a DT-diaphorase inhibitor

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Abstract

The purpose of this study was to evaluate the contribution of DT-diaphorase inhibition to in vivo neurodegenerative effects of dopamine (DA) oxidation to the corresponding o-quinones. The neurotoxicity to nigrostriatal DA neurons was induced by injection of manganese pyrophosphate (Mn3+) complex as a prooxidizing agent alone or together with the DT-diaphorase inhibitor dicumarol into the right rat substantia nigra. The behavioral effects were compared with those induced after selective lesions of dopaminergic neurons with 6-hydroxydopamine (6-OHDA). Intranigral injection of Mn3+ and Mn3+ plus dicumarol produced significant impairment in motor behavior compared with control animals. However, the effect seen in the Mn3+ plus dicumarol injected group was significantly more severe than that observed in the Mn3+ alone injected group. In motor activity and rearing behavior, the simultaneous injection of Mn3+ plus dicumarol produced a 6-OHDA-like impairment. Similar effects were observed in the acquisition of a conditioned avoidance response (CAR). Dicumarol significantly impaired avoidance conditioning although without affecting the motor behavior. The behavioral effects were correlated to the extent of striatal tyrosine hydroxylase (TH)-positive fiber loss. Rats receiving unilateral intranigral Mn3+ and Mn3+ plus dicumarol injections exhibited a significant reduction in nigrostriatal TH-positive fiber density in medial forebrain bundle compared with the contralateral noninjected side. In conclusion, this study provides evidence that the neurotoxicity of Mn3+ in vivo is potentiated by DT-diaphorase inhibition, suggesting that this enzyme could play a neuroprotective role in the nigrostriatal DA systems.

Introduction

Although it is generally accepted that free radicals are involved in the neurodegeneration of the nigrostriatal dopamine (DA) system observed in Parkinson's disease, the exact mechanism of neurodegeneration in vivo is still unknown. Much attention has focused on the fact that oxidation of DA results in the formation of cytotoxic compounds, which can cause massive brain damage if they are allowed to accumulate. However, the rate at which oxygen oxidizes DA at neutral pH is very low (Graham et al., 1978) and probably does not produce any significant amount of reactive oxygen intermediates. Manganese in the Mn3+ state is a potent oxidizing agent and can accelerate the oxidation of DA to its o-quinone Barbeau, 1984, Archibald and Tyree, 1987, which instantaneously cyclizes to form aminochrome Segura-Aguilar and Lind, 1989, Shen and Dryhurst, 1998, Brenneman et al., 1999, Dorman et al., 2000, Lee, 2000. This observation may explain the drastic decrease in the level of DA because this reaction appears to be irreversible (Segura-Aguilar and Lind, 1989). Previously, it was demonstrated that aminochrome resulting from oxidation by DA with Mn3+ was toxic in a mouse-derived neuronal cell line (CNh) (Arriagada et al., 2000) and also induces a significant behavioral impairment in vivo (Dı́az-Véliz et al., 2002). The one-electron reduction of aminochrome to leukoaminochrome o-semiquinone radicals can be one possible source of reactive species Stokes et al., 1999, Segura-Aguilar et al., 2001, Smythies, 2002. Leukoaminochrome o-semiquinone radical is a very reactive metabolite (Segura-Aguilar et al., 1998) that autoxidizes in the presence of oxygen or transition metal ions like manganese, copper or iron Segura-Aguilar and Lind, 1989, Shen and Dryhurst, 1998, Paris et al., 2001, initiating a redox cycling process (Baez et al., 1995). This aberrant one-electron metabolism of aminochrome can be prevented by a two-electron reduction of aminochrome to leukoaminochrome, catalyzed by DT-diaphorase Segura-Aguilar and Lind, 1989, Segura-Aguilar et al., 1998, Segura-Aguilar et al., 2001. There is evidence that DT-diaphorase, an enzyme that in rat substantia nigra constitutes the 98% of the total quinone reductase activity (Schultzberg et al., 1988), prevents aminochrome one-electron reduction by reducing aminochrome with two electrons to leukoaminochrome (Segura-Aguilar and Lind, 1989). The selective inhibition of this enzyme leads to an autoxidative cascade due to the ability of leukoaminochrome o-semiquinone to induce redox cycling (Baez et al., 1995). Thus, very low concentrations of aminochrome can produce a large amount of reactive oxygen species (Segura-Aguilar et al., 2001). In vitro studies have demonstrated that DT-diaphorase is inhibited by dicumarol Schultzberg et al., 1988, Segura-Aguilar and Lind, 1989, Paris et al., 2001. Recently, we demonstrated that inhibition of DT-diaphorase is a requirement for Mn3+ to produce a 6-hydroxydopamine (6-OHDA)-like rotational behavior in rats (Segura-Aguilar et al., 2002).

To evaluate the contribution of DT-diaphorase inhibition to in vivo neurodegenerative effects of oxidation products of DA, we injected into substantia nigra (1) Mn3+ as a general prooxidizing agent to accelerate the oxidation of endogenous DA and (2) dicumarol as a selective inhibitor of DT-diaphorase. We evaluated the degeneration of the nigrostriatal pathway through the expression of spontaneous motor activity and avoidance conditioning, considering the influence of integrity of DA systems on these behaviors.

Section snippets

Animals

Fifty adult, male Sprague–Dawley rats, weighing 180–220 g, were housed six per cage in a temperature-controlled vivarium under a 12:12 light/dark cycle (lights on from 08:00 to 20:00 h) with free access to food and water. The experimental protocols followed the Guide for Care and Use of Laboratory Animals and were approved by the Faculty of Medicine Committee. The rats were assigned to five experimental groups injected with (1) Tris-HCl vehicle, (2) dicumarol, (3) Mn3+, (4) dicumarol plus Mn3+

Spontaneous motor activity

Fig. 1 shows the time course of intranigral injection on spontaneous motor activity. Two-way ANOVA revealed a significant effect of Treatment [F(4,135)=30.15, P<.0001] and Time [F(2,135)=117.78, P<.0001]. The interaction between Treatment and Time was also significant [F(8,135)=2.47, P<.05], suggesting that the effect of treatment on motor activity was dependent on the time of observation. Subsequent multiple comparison tests indicated that motor activity was not affected as a result of

Discussion

The results of the current study support the in vivo neurotoxicity of Mn3+ and the contribution of DT-diaphorase inhibition by dicumarol to the behavioral consequences of the nigrostriatal pathway degeneration. In fact, Mn3+ injected into the rat substantia nigra decreased spontaneous motor activity, rearing behavior and acquisition of an avoidance response. These effects were potentiated by the concomitant administration of dicumarol; in this condition, they were not significantly different to

Acknowledgments

This work was supported by grant 1020672 from Fondecyt, Chile.

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