Riboflavin and pyridoxine restore dopamine levels and reduce oxidative stress in brain of rats

The main incident in neurological disorders is excitotoxicity which entails an extensive upsurge in the concentrations of intracellular Ca²⁺ and the production of reactive species such as ROS and RNS by lethal pathways [1]. In addition, there are alterations of the mitochondrial ultrastructure and DNA damage caused by nitric oxide- (NO-) dependent oxidative stress [2]. This damage is the primary event in 3-nitropropionic acid (3-NPA) toxicity.

In Wistar rats, it has been reported that 3-nitropropionic acid leads to neurodegeneration and that the intravenous administration in rats provides valuable insight of Huntington’s disease model [3]. There is evidence that metabolism of transmitter dopamine by monoamine oxidase enzyme may promote striatal damage in mitochondrial toxin induced models of Huntington’s disease (HD) [4], and that HD is a devastating neurodegenerative disorder that reflect neuronal dysfunction and ultimately death in selected brain regions with striatum and cerebral cortex being the principal targets [5]. Some nutrients are known to act as antioxidants; and although neglected as an antioxidant, riboflavin is one of such nutrients that independently or as a component of the glutathione redox cycle has an important antioxidant action [6]. On the other hand, pyridoxine alters serotonin metabolism [7]. Both compounds are water-soluble vitamins that possess antioxidant activity [8].

Neuromodulation activity of the NO- is a documented fact, however, an excess amount of it can produce oxidative damage or nitroso-glutathione (NOGSH) inside the cell, thus; causing the damage of the cell [9]. It is known that free radicals (FR) induce damage to the components of the cell [10]. Such FR-induced damage particularly affects the plasma membrane lipids [11]. In addition, the cells of the central nervous system are extremely susceptible to these unpaired electron molecular species. Nitric oxide (NO) acts on hypothalamic neurocircuits and on higher brain circuits, e.g. dopaminergic system to regulate energy and glucose homeostasis [12]. Therefore, the presence of FR may upset this regulatory function of NO. The structural proteins in the lipid bilayer are contiguous with brain plasma membrane phospholipids [13] and the ionic inflow and outflow through the lipid bilayer is maintained by Na⁺, K⁺ ATPase enzyme which stimulates Na⁺ and K⁺ flows [14]. It is know that when the activity of the enzyme Na⁺, K⁺ ATPase is inhibited, it triggers the release of excitatory amino acid in the CNS [15].

In view of all the aforementioned, the objective of the present work was to make a comparative analysis of the protective effects derivable from riboflavin (B₂) and pyridoxine (B₆) on 5-HIAA and dopamine levels, as well as on selected markers of oxidative stress in rats’ brain regions after an induction of Huntington’s disease (HD). Literature reports suggest that these substances may participate in the neutralization of excess free radicals in oxidation mechanisms. The production of free radicals, a usual biological phenomenon, is regulated by different metabolic routes, which constitute the first line of defence in the human body.

The aim of this work was to determine if riboflavin (B₂) and pyridoxine (B₆) provide protection to the brain against free radicals generated by 3-nitropropionic acid (3-NPA) by measuring the levels of dopamine (DA) and selected oxidative stress markers.

Thirty-six male Wistar rats (250 g) were procured from Bioterium of Metropolitan University of Mexico City and housed in clean plastic cages, separated into 6 groups and treated as follows: group 1, control (NaCl 0.9%); group 2, 3-NPA (20 mg/kg); group 3, B₂ (10 mg/kg); group 4, B₂ (10 mg/kg) + 3-NPA (20 mg/kg); group 5, B₆ (10 mg/kg); group 6, B₆ (10 mg/kg) + 3-NPA (20 mg/kg), each group N = 6. The administration of treatments was by i.p. The animals received the drugs every 24 h during 3 days. At the end of the treatment period and 30 min after the last drug administration, the animals were sacrificed with guillotine without anaesthetic procedure. The animal brains were immediately extirpated and put in saline solution (NaCl 0.9%) at 4 °C. Tissues were immediately dissected in regions and used to evaluate reduced glutathione (GSH), H₂O₂, lipid peroxidation, ATPase, and the levels of DA and 5-hydroxyindolacetic acid (5-HIAA).

The rats or breeds employed in the study were subjected to a selection process based on phenotypic variety; genetic, environmental and compartmental factors. Longitudinal weight curves, weight and physical exploration were the means employed to select a breed for inclusion or exclusion in the study. Also, to select a breed, it is fundamental that inbreeding is non-existence, thus having a hereditary control of traits with continuous variation.

The selected animals were kept in cool and dry place at a temperature of 15–16 °C and with air filter and humidity of between 50 and 60. The place was maintained clean and was continuously sterilized to avoid bacterial and fungal growth.

The breeds were fed with standardized diet based on 3800 kcal/kg, proteins 12%, fat 5%, vitamins and minerals. The selected animals were 3 months old male Wistar of approximately 250 g weight.

In cortex, the administration of 3-NPA produced a significant increase in the levels of dopamine as well as a significant decrease in the levels of 5-HIAA (Fig. 1). GSH decreased in all animals groups that received the treatments however, the decrease of GSH had statistical significant difference only in those treated with Rivoflavin (B₂), Rivoflavin (B₂) + 3-NPA and Pyridoxine (B₆) + 3-NPA when compared with the control group (Fig. 2). Significant lipid peroxidation was appreciated in animals treated with Rivoflavin in comparison with control, B₂ + 3-NPA and B₆ + 3-NPA groups (Fig. 3). Calcium Magnesium dependent ATPase activity increased significantly in the group of animals that received 3-NPA when compared with other treatments (Fig. 4). In this region, the changes in H₂O₂ levels did not have statistical differences among treated and control groups (Fig. 5).

In striatum similar results as those observed in cortex were found. 3-NPA decreased 5-HIAA concentration (Fig. 1). Rivoflavin administration produced an increase in lipid peroxidation and 3-NPA treated animals showed significant increase in ATPase activity (Fig. 4).

In the cerebellum/Medulla oblongata of the animals that received 3-NPA alone, Dopamine, H₂O₂ and ATPase activity increased while 5-HIAA levels were found to. In this region significant lipid peroxidation increment was observed in Rivoflavin treated group when compared with the rest of the treated animals decrease (Figs. 1, 4, 5, 6).

Evidence has shown that Huntington’s disease (HD) can be caused by mitochondrial toxin produced as a result of striatal damaged that is provoked when the transmitter dopamine is metabolized by monoamine oxidase enzymes [4]. Also, HD is a destructive neurodegenerative disorder and indicates dysfunction in the neurons that may eventually lead to death of selected brain regions; especially, the striatum and cerebral cortex [5]. In recent studies, the dendritic spine density of striatal projection neurons was reported to be seriously decreased after 3-nitropropionic acid treatment [23]. This finding is in conformity with the results of the present study where dopamine levels increased in cortex, striatum and cerebellum/medulla oblongata regions of animals that received 3-nitropropionic acid treatment. Moreover, it is possible that the increase in dopamine turnover produces an increase in oxygen radical by monoamine oxidase activity.

H₂O₂ concentration increased in cerebellum/medulla oblongata regions in animals treated with 3-NPA. This demonstrates that reactive oxygen species is the primary event in 3-NPA toxicity. In the same brain regions, lipid peroxidation increased in animals treated with riboflavin. Such increase may be due to the association of this substance with increased mitochondrial energy metabolism that is probably responsible for the high rate of oxidative metabolic activity in the brain which gives rise to intense production of reactive oxygen metabolite and subsequently to the generation of free radicals implicated in the pathogenesis of neurological disorders. These results may have relation with the reports of Kumar et al. [24] and Kaur et al. [25], which suggest that 3-NPA depleted GSH in cortex, although the present study was made with young animal models.

Calcium and magnesium-dependent ATPase activity increased in cortex, striatum and cerebellum/medulla oblongata regions of the animals that received 3-NPA alone. This could be attributed to changes in the affinity of the enzyme [26]. These results may have relation with the reports of Naziroğlu et al. [27], which suggest that increase in Ca²⁺-ATPase activities may have protective effects against substances that induce brain injury by inhibiting free radical production, regulating calcium-dependent processes and supporting the antioxidant redox system.

The results of the present study suggest that the partial increase in antioxidant capacity in brain due to B₂ and B₆ vitamins promotes an effect in dopamine or serotonin metabolisms when we consider 3-NPA capacity to generate oxidative stress.

We recommend that further studies be carried out to investigate the possible relationship between B₂ and B₆ vitamins on dopamine and serotonin levels in different animal models. Undoubtedly, we believe that this would help in the study of neurodegenerative diseases.