Modulation of Antioxidant Enzymatic Activities by Certain Antiepileptic Drugs (Valproic Acid, Oxcarbazepine, and Topiramate): Evidence in Humans and Experimental Models

It is estimated that at least 100 million people worldwide will suffer from epilepsy at some point in their lives. This neurological disorder induces brain death due to the excessive liberation of glutamate, which activates the postsynaptic N-methyl-D-aspartic acid (NMDA) receptors, which in turn cause the reuptake of intracellular calcium (excitotoxicity). This excitotoxicity elicits a series of events leading to nitric oxide synthase (NOS) activation and the generation of reactive oxygen species (ROS). Several studies in experimental models and in humans have demonstrated that certain antiepileptic drugs (AEDs) exhibit antioxidant effects by modulating the activity of various enzymes associated with this type of stress. Considering the above-mentioned data, we aimed to compile evidence elucidating how AEDs such as valproic acid (VPA), oxcarbazepine (OXC), and topiramate (TPM) modulate oxidative stress.


Introduction
Neurological diseases are a major cause of health concerns at different life stages and lead to considerable utilization of medical resources [1]. Epilepsy is one of the most common neurological disorders in both children and adults [2,3]. The term epilepsy describes a group of disorders characterized by the presence of chronic, recurrent, and paroxysmal alterations of the motor and sensory neurological functions secondary to a disorder in the electrical activity of a neuron population [4]. The term epileptic syndrome refers to various disorders characterized by a group of signs and symptoms that occur simultaneously. These signs include the type of crisis, causes, anatomic aspects, precipitating factors, age of onset, severity, prognostics, chronicity, and electroencephalographic activity, and the clinical characteristics are identified based on the patient's age [2,5].
Epileptic seizures and syndromes are classified according to the International League Against Epilepsy (ILAE), using genetic studies and electroclinical, neuropsychological, and neuroimaging research. Epilepsy can be divided, based on its etiology, into idiopathic disease or disease associated with a hereditary predisposition, as symptomatic or associated with any event that damages the brain, and as cryptogenic or of unknown cause [6,7].
Currently, the epilepsy prevalence is reported to be five to 10 cases per 1,000 individuals. It is estimated that at least 100 million people worldwide will present with epilepsy at a certain life stage [4,8]. The ILAE reports that the disease prevalence lies between four and 10 cases per 1,000 individuals, and the incidence lies between 20 and 70 cases per 100,000 individuals per year. The prevalence rate in Latin-American countries is the highest, in the range of 14 to 57 per 1,000 individuals [6,7].
Epilepsy control using antiepileptic drugs (AEDs) depends on several factors: efficacy, side effects of the hormonal alteration, teratogenicity, pharmacokinetics, interactions between AEDs or other drugs, serum levels, cost, and the neurologist's experience with AED use [9]. The patient may respond in three different manners: remitting seizures spontaneously (without AED use), responding adequately to AED administration, or presenting refractoriness to the treatment drug. The most commonly used AEDs are valproic acid (VPA), oxcarbazepine (OXC), and topiramate (TPM), which are considered the first-option treatments for the diverse manifestations of this pathology.
A wide variety of AEDs have been divided into generations according to their date of introduction to clinical use. These agents are categorized as first-(1857-1978), second-(1993-2009), and third-(2009 to date) generation AEDs. The second-and third-generation drugs are described in Table 1 [10, 11].

Overview of Valproic Acid, Oxcarbazepine, and Topiramate
VPA is a carboxylic acid composed of eight carbons and is used to treat several types of epilepsy due to its broad action spectrum and efficiency [12] (Figure 1(a)). The mechanism of action, similarly to that of other AEDs, is not fully known; however, it has been reviewed in various articles. These reports can be divided into two groups: studies suggesting that VPA increases gamma aminobutyric acid (GABA) transmission and research indicating that this AED may directly interact with the neuronal membrane. Löscher [12] studied VPA interference with GABAergic transmission in 1993. This report is based on the observation that VPA increases the levels of the inhibitory neurotransmitter GABA [12]. Other researchers have confirmed Löscher's studies [13][14][15]. This effect can be produced either by glutamate decarboxylase activation [16,17]; by the inhibition of GABA-degrading enzymes such as GABA aminotransferase [17], succinic semialdehyde dehydrogenase [18], aldehyde reductase [19], and -ketoglutarate dehydrogenase [17]; or by an increase in glutaminase activity [20]. Alternative mechanisms involve potentiation of the postsynaptic response of GABA, GABA A receptor modulation, and depolarization induced by Nmethyl-D-aspartic acid (NMDA) [21][22][23].
With respect to the mechanism of VPA interactions with the neuronal membrane, it has been reported that this AED decreases the excitatory synaptic potential necessary for the synchronization network and the neuronal firing in the substantia nigra [24][25][26]. VPA also activates conductance in the potassium channels and interferes with other biochemical pathways related to energy metabolism in the brain. However, to date, this activity has not been proven to be an additional mechanism of AED action [27]. OXC was developed as a carbamazepine analog (Figure 1(b)) [28], and its pharmaceutical activity occurs mainly through its active metabolite, 10,11-dihydro-10hydroxy-carbazepine ( Figure 2). The mechanisms of action have not been clarified, but it is reported that the principal mechanism involves a blockade of voltage-dependent sodium [29]. In vitro electrophysiological and animal studies have demonstrated that this AED activity is based on the interference with transmembranal sodium, calcium, and potassium (i.e., voltage-dependent) ionic currents. These agents also modify the release of certain neurotransmitters, such as glutamate [29,30].
TPM is a monosaccharide substituted with sulfate groups [2,3:4,5-bis-O-(1-methylethylidene)-beta-D-fructopyranose sulfamate] (Figure 1(c)). Five mechanisms that reportedly contribute to its antiepileptic action are as follows: blockage of the sodium channels, which reduces the duration and frequency of the action potentials [31]; a positive modulating effect of the GABA A receptors [32,33]; inhibition of the ionotropic glutamate receptors alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid receptor (AMPA)/kainate; a negative modulatory effect of the calcium channels activated by L-type voltage; and inhibition of carbonic anhydrase subtypes II and IV [32].

Oxidative Stress and Its Role in Epilepsy
In the last two decades, the study of ROS and reactive nitrogen species (RNS) has sparked great interest in clinical Oxidative Medicine and Cellular Longevity and experimental medicine. Both species are (a) generated during the irradiation of ultraviolet (UV) light, X-rays, and gamma rays, (b) products of reactions catalyzed by metals, (c) present in air pollutants; (d) produced by neutrophils and macrophages during inflammation, and (e) byproducts of reactions catalyzed by the electron carriers in the mitochondria [34]. ROS and RNS are known for their dual role in biological systems, as they can be beneficial or harmful. The beneficial effects of ROS can be observed in their physiological role in numerous cellular responses and cell signaling systems. By contrast, at high concentrations, ROS can be important mediators of cell damage to various structures such as lipids, proteins, and nucleic acids. The beneficial effects of ROS are supplemented by the action of nonenzymatic antioxidants and by the antioxidant enzyme system. Despite the presence of the antioxidant defense system to combat oxidative damage caused by ROS, this damage accumulates throughout life [34].
The imbalance between the ROS and RNS levels (such as superoxide, radical O 2 •− ; hydrogen peroxide, H 2 O 2 ; hydroxyl radical, HO • ; and nitric oxide, NO • ) and the cellular antioxidant defense system (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPx; glutathione reductase, GR; and glutathione-S-transferase, GST) is defined as "oxidative stress" [34]. Because this disequilibrium can appear at the cellular level (involving the mitochondria, cytochrome P450 system, peroxisomes, and activation of inflammatory cells [35]), it is involved in the development of several diseases such as cancer, atherosclerosis, and arthritis and in neurodegenerative disorders such as epilepsy [34,36].
The participation of oxidative stress in diseases of the central nervous system (CNS) is well established [37,38]. The brain is highly sensitive to oxidative damage because this organ contains a large number of easily oxidized fatty acids (20 : 4 and 20 : 6) and a limited antioxidant system [37].
Oxidative stress is strongly implicated during seizures induced by excitotoxicity, due to mitochondrial ROS generation. Since the beginning of the 1990s, oxidative stress has been associated with neuronal hyperexcitation caused by CNS diseases [39]. Dalton, in 1995, was the first to identify brain damage induced by the presence of oxidative stress in an animal experimental model [40].
The presence of NO • is known to be a cause of seizures [41]. NO • is formed from high concentrations of inducible nitric oxide synthase (iNOS). The role of oxidative stress in pentylenetetrazole-induced epilepsy has been proven in rodents [42][43][44]. The increased activity of glutamatergic systems induces status epilepticus and causes an energy imbalance, increasing ROS formation [45]. Several studies have linked seizures and cell damage to the excitotoxicity induced by pentylenetetrazole [46]. Seizures are linked to the increased release of glutamate and NMDA receptor activation. In fact, during epileptic seizures induced by different models, there is an extracellular Ca 2+ concentration decrease and a cytosolic Ca 2+ concentration increase [47].
The effects mediated by Ca 2+ during excessive glutamate receptor activation (excitotoxicity) lead to neuronal degeneration and give rise to oxidative stress. The phospholipase A 2 -dependent activity of Ca 2+ mediated by glutamatergic receptors liberates arachidonic acid (AA), which generates O 2 •− through its metabolism by lipoxygenases and cyclooxygenases for eicosanoid formation [48].
The constant formation of NO • by the glia is neurotoxic because it increases the neuronal sensitivity to this reactive species. The neurotoxic action of NO • is likely caused by the formation of peroxynitrite (ONOO − ), which is rapidly formed by the reaction of NO • with O 2 •− . Under conditions of energy deficit and elevated intracellular Ca 2+ concentration, xanthine oxidase generates O 2 •− . In this environment, lactic acid is generated, which promotes the release of Fe 2+ , the Haber-Weiss reaction, and the production of HO • [49]. Furthermore, ONOO − can react with tyrosine in proteins to form 3-nitrotyrosine (3-NT) [50].  With combined drugs, they were no significant differences in the SOD activity and lipid peroxidation levels. In patients treated with combination therapy, the SOD activity was lower, whereas the activity of GPx and GR was higher. In addition, lower lipid peroxidation levels were displayed. [60] Oxidative Medicine and Cellular Longevity 5  [65]

Antioxidant Enzymes Induced by VPA, OXC, and TPM: Evidence in Humans and Experimental Models
It is established that VPA, OXC, and TPM are capable of modulating the oxidant-antioxidant system. Particularly, enzyme antioxidant activity studies have demonstrated that the administration of certain AEDs causes decrease in enzyme activity, increase in the antioxidant system, or even the absence of an effect upon the antioxidant system. VPA can modulate negatively or positively the enzymatic activity. Chaudhary and Parvez, in 2012, found a significant reduction of GST, GR, GPx, SOD, and CAT activity. They also observed significantly increased xanthine oxidase activity and lipid peroxidation levels in the cerebellum and cerebral cortex of rats [66]. Zhang  With respect to the positive regulatory effect of TPM on the antioxidant system, a significant increase in SOD, CAT, GPx, and neuronal nitric oxide synthase (nNOS) activities, as well as in GSH levels, has been observed. Significantly decreased lipid peroxidation levels have also been observed in experimental epilepsy models [43,[73][74][75][76]. Our research group has demonstrated for the first time that TPM has direct antioxidant activity in vitro against O 2 •− , H 2 O 2 , HO • , and hypochlorous acid (HOCl) in a concentration-dependent manner. In this study, we demonstrated that the scavenging activity of TPM might explain its neuroprotective properties [77]. Human studies have evaluated the effects of AEDs on the antioxidant system; see Table 2. Although there is evidence in humans and experimental models that these AEDs (VPA, OXC, and TPM) modulate the activity of antioxidant enzymes, this evidence is not conclusive. It is necessary to study further how AEDs induce effects in antioxidant capacities in different types of epilepsy. In particular, our research group is interested in determining the activity of the antioxidant enzymes and their modulation by AEDs in epileptic pediatric patients.

Therapeutic Relevance
To date, the mechanisms involved in the etiopathogenesis of epilepsy remain unclear; however, the evidence showed that oxidative stress is involved in the epilepsy development. Therefore, the knowledge that current AEDs can modulate other systems opens a new therapeutic window for the population suffering from epilepsy and other chronic and degenerative CNS diseases, in which oxidative stress is one of the mechanisms underlying these pathologies.

Conflict of Interests
The authors of this work have no conflict of interests to declare.