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. Author manuscript; available in PMC: 2023 Sep 11.
Published in final edited form as: Brain Res Bull. 2021 Feb 2;170:246–253. doi: 10.1016/j.brainresbull.2021.01.021

Haloperidol elicits oxidative damage in the brain of rats submitted to the ketamine-induced model of schizophrenia

Samira S Valvassori a,*, José H Cararo a, Samira Menegas a, Taise Possamai-Della a, Jorge M Aguiar-Geraldo a, Simone Lespinasse Araujo a, Gustavo Antunes Mastella a, João Quevedo a,b,c,d, Alexandra I Zugno a
PMCID: PMC10494233  NIHMSID: NIHMS1925120  PMID: 33545309

Abstract

The present study aims to evaluate the effects of haloperidol, an important first-generation antipsychotic, on the antioxidant system parameters in the brain of animals subjected to a model of schizophrenia induced by ketamine. Adult rats intraperitoneally received saline (1 mL/kg) or ketamine (25 mg/kg body weight) for 15 days, and saline or haloperidol (0.1 mg/kg body weight) via gavage once a day, between the 9th and 14th days. In the frontal cortex, hippocampus, and striatum, assessments of lipid (4-hydroxy-2-nonenal and 8-isoprostane levels) and protein (protein carbonyl content) oxidative damage were conducted. It was also measured the glutathione peroxidase and glutathione reductase activities in the same cerebral structures. Increases in the 4-hydroxy-2-nonenal and 8-isoprostane levels were detected in rats receiving haloperidol and ketamine. An increase in the carbonyl content was also observed in animals receiving ketamine, haloperidol, or a combination thereof. In animals receiving the antipsychotic, there was a decrease in the activity of the enzymes. Therefore, both ketamine and haloperidol induced oxidative damage. A possible energy dysfunction or a haloperidol effect targeting the glutathione enzymes, and then disrupting the redox homeostasis in neurons, could not be ruled out, although further studies are required to confirm or refute a direct interaction.

Keywords: Lipid peroxidation, Protein carbonylation, Redox imbalance, Side effects, Typical antipsychotics

1. Introduction

Schizophrenia (SZ) is a lifelong mental disorder characterized by positive and negative symptoms, as well as cognitive impairment (Krzystanek and Pałasz, 2019). The SZ prevalence is 0.3–1% worldwide (Cattane et al., 2018; World Health Organization (WHO), 2018). SZ pathophysiology remains poorly understood, while the alterations observed in several brain structures and the lack of responsiveness of negative and cognitive symptoms to antipsychotics are still a challenge (Dean, 2017; Rodrigues-Amorim et al., 2017). Nevertheless, it was hypothesized that an excess or depletion of dopamine, serotonin, glutamate, and γ-aminobutyric acid (GABA) collaborates for the pathophysiology of the disorder (Patel et al., 2014). There is evidence of immune system dysfunction in schizophrenic patients, which could lead to neuroinflammation (Miller et al., 2011). Moreover, studies have indicated that an impairment in the antioxidant defenses is also detectable in patients (Pavlović et al., 2002; Gysin et al., 2007; Dadheech et al., 2008).

In the present study, an SZ model was induced using ketamine (Ket), a well-known anesthetic drug (Canever et al., 2010). Ket administration is regarded as a suitable SZ animal model since it mimics the pathogen mechanisms associated with the symptoms of the disorder. Indeed, Ket administration enables the study of positive, negative, and cognitive alterations observed in SZ, which contributes to providing face validity to this model (Chatterjee et al., 2011; Frohlich and Van Horn, 2014). Neurochemical effects induced by Ket include oxidative stress and blockade of N-methyl-D-aspartic acid (NMDA) receptors, preventing the influx of calcium in neurons, which induces behavioral changes similar to that observed in the patients, collaborating to provide construct validity to the model (Chatterjee et al., 2011). Additional evidence about how to induce the Ket model and how it works regarding the behavioral analysis is showed in recent studies (Zugno et al., 2016; Damazio et al., 2017; Canever et al., 2018; Supp et al., 2020). Data from these papers enable the reproduction of the model to assess potential neurochemical alterations similar to that observed in schizophrenic patients using haloperidol or other drugs.

The therapy of SZ and other psychotic disorders can be carried out by first-generation antipsychotics. These drugs primarily act by inhibiting dopaminergic receptors (Dean, 2017). Among the drugs of this class, haloperidol (Hal) is recommended for acute episodes of positive symptoms and as maintenance therapy for SZ (Haddad and Correll, 2018; Chokhawala and Stevens, 2019). Although the antipsychotics have persisted as the standard therapy for SZ and are effective in treating the positive symptoms, these drugs may induce several side effects, including tardive dyskinesia, muscle rigidity, and tremors (Buchanan et al., 2007; Chatterjee et al., 2011; Dean, 2017). At least in part, oxidative stress seems to be involved in such effects.

In this regard, Hal (2 mg kg—1 day—1) was found to impair the activity of enzymes such as catalase and superoxide dismutase as well as elicit an increase in the levels of the lipid peroxidation by-product hydroxyalkenal in rat brain (Parikh et al., 2003). Besides, Hal (1.5 mg kg—1 day—1) induced significant increases in the content of thiobarbituric acid-reactive species (TBARS) in the striatum as well as in the protein carbonyl content in hippocampus of rats (Reinke et al., 2004). Hal was also linked to increased serum TBARS in SZ patients, in comparison with olanzapine (Singh et al., 2008). In the Raudenska and coworkers’ review (2013) is outlined a more detailed picture of the mechanisms involved in the cytotoxicity of Hal-induced oxidative stress.

Other antipsychotics with a possible role in oxidative stress include clozapine, chlorpromazine, risperidone, and ziprasidone (Martins et al., 2008; Elmorsy et al., 2017; Dietrich-Muszalska and Kolińska-Łukaszuk, 2018). The first-generation drugs are more prone to induce lipid peroxidation than the most recent ones (Kropp et al., 2005). Despite the repertory of alterations mediated by reactive oxygen species described to date, the precise mechanisms underlying the toxicity of antipsychotics are still incomplete, even at the lowest doses of the drugs. In this scenario, the present study aimed to evaluate the effects of a low dose of Hal on oxidative stress parameters in the brain of rats submitted to an animal model of SZ induced by Ket.

2. Materials and methods

2.1. Animals

Twenty adult male Wistar rats (Rattus norvegicus; 250–350 g body weight; heterogenic strain) acquired from the Central Animal House at the University of Southern Santa Catarina were kept as 5 per cage and received ad libitum chow and drinking water. The cages were maintained in a room with a 12 h-light/dark cycle (lights on at 7:00 a.m.) at 22 ± 1 °C. All procedures were conducted following the National Institutes of Health (US) “Guide for the Care and Use of Laboratory Animals” (National Research Council (US), 2011) and the Brazilian Society for Neuroscience and Behavior. Experiments started after approval by the Local Ethics Committee for Animal Use (record no. 49/2012) and were performed in the morning, to avoid circadian variations. All efforts were undertaken to reduce the number and the suffering of the animals in the procedures.

2.2. Drugs and pharmacological procedures

Animals received saline (Sal, NaCl 0.9 %) or Ket (25 mg/kg body weight) by intraperitoneal (i.p.) injection for 15 days. Rats also received Sal (1 mL/kg) or Hal (0.1 mg/kg body weight) once a day, via gavage, between the 9th and 14th days of the experiment. Therefore, the procedures were performed with four groups (n = 5, per group), as follows: 1) Sal + Sal (control); 2) Sal + Hal; 3) Ket + Sal; and 4) Ket + Hal. Animals were randomly assigned to the experimental groups. The volume proportion of the drugs was 1 ml per kg body weight. Ket dose (25 mg/kg) was based on the SZ rat model developed by Canever and coworkers (2010). Thirty minutes after the last injection of Ket or Sal, the animals were submitted to euthanasia by decapitation without anesthesia (to avoid a potential bias on the biochemical analysis).

It is worth to note that the Hal dose used here is lower than the standard dose (2 mg kg−1 day−1) orally administered in rats aiming to reach plasma concentrations close to the therapeutic range of adult patients using Hal (Terry et al., 2010). However, the rationale of the present study was to investigate if Hal can affect oxidative stress parameters even as a sub dose (since studies in similar conditions to the present paper but using higher doses have already been performed), which provides novel data on the safety profile of the drug.

2.3. Brain samples

It was excised the rat brains in order to obtain the frontal cortex, hippocampus, and striatum. These cerebral structures were dissected on a cleaned, cold surface and then immersed in liquid nitrogen. The samples were stored at −80 °C until the biochemical analysis was carried out.

2.4. Protein quantification

All biochemical analysis data (Section 2.5) were normalized according to the total protein level in each sample. The protein content was measured through the Lowry and coworkers’ procedure (1951), using bovine serum albumin as a standard.

2.5. Biochemical analysis

2.5.1. Oxidative damage to lipids and proteins

Lipid oxidative damage was estimated based on the 4-hydroxy-2-nonenal (4-HNE) content by using a Cell Biolabs’ assay kit (Cell Biolabs, Inc.; San Diego, CA, USA; catalog no. STA-338). Content of 4-HNE-protein adducts (expressed as mg/mg protein) generated by modifications in lysine, histidine, and cysteine residues was measured using the Kimura and colleagues’ immunoassay (2005).

Another significant parameter of lipid damage is 8-isoprostane (8-ISO), whose content in the samples (expressed as μg/mg protein) was measured using a competitive ELISA assay kit (Cayman Chemical Company, Ann Arbor, MI, USA; catalog no. 516351). Briefly, the principle of the method relies on the competition between 8-ISO (analyte) and 8-ISO-acetylcholinesterase conjugate (labeled analyte) for several boundary sites (antiserum specific for 8-ISO).

It was conducted an assessment on the oxidative damage in proteins, through the carbonylation level on these molecules. Carbonyl group content (expressed as nmol/mg protein) was measured using a specific kit purchased from Cell Biolabs (OxiSelect™ Protein Carbonyl ELISA Kit; catalog no. STA-310).

2.5.2. The activity of antioxidant enzymes

The glutathione peroxidase (GPx; EC 1.11.1.9) activity was measured according to the assay method described by Wendel (1981). Briefly, oxidized glutathione is released, through the hydrogen peroxide reduction catalyzed by GPx; glutathione then is recycled to its reduced state by the glutathione reductase (GR; EC 1.8.1.7) activity; this process is dependent on the oxidized nicotinamide adenine dinucleotide phosphate (NADP+) coenzyme. One unit of GPx is equivalent to the amount of enzyme that will lead to the oxidation of 1.0 nmol NADPH to NADP+ per minute at 25 °C. Therefore, the method relies on NADPH consumption, which is spectrophotometrically followed (340 nm).

By using the Carlberg and Mannervik assay (1985), it was possible to evaluate the GR activity. The method relies on the rate of NADPH oxidation to NADP+, followed by a decrease in absorbance at 340 nm. One unit of GR is the amount of enzyme that leads to oxidation 1.0 nmol NADPH to NADP+ per minute at 25 °C. Data on the GPx and GR activity were expressed as nmol min−1 mg protein−1.

2.6. Statistical analysis

The procedures were conducted in duplicate, including appropriate positive and negative controls. The analysis of variables ran through Shapiro Wilk’s test for normality, according to their distribution. Data are mean ± standard error of the mean. The approach for evaluation of differences between groups was the two-way analysis of variance (ANOVA) and Tukey post hoc test – when F value was significant. Comparisons of means ran using Statistica 7 (StatSoft, Inc., Tulsa, OK, USA). Differences were rated as statistically significant when p < 0.05. The GraphPad Prism software (version 5.00 for Windows; GraphPad, San Diego, CA, USA) was used to generate the figure graphics.

3. Results

The present study was designed to investigate the effects of the Hal administration in an animal model of SZ induced by Ket. First, it was evaluated the oxidative damage to lipids in the cerebral structures, in terms of 4-HNE and 8-ISO contents. The administration of Ket elicited an increase in the 4-HNE levels in the frontal cortex, hippocampus, and striatum, as compared to the control group. Interestingly, Hal did not alter the increase in the 4-HNE levels induced by Ket in any brain structure evaluated. Besides, the administration of Hal per se increased the levels of 4-HNE in all brain structures. Regarding the 8-ISO content, the administration of Ket increased this parameter in the striatum and hippocampus, while Hal did not change this damage. In the frontal cortex, the administration of Ket did not alter the levels of 8-ISO, and the treatment with Hal in animals previously receiving Ket increased 8-ISO in this brain structure. Hal per se increased the levels of 8-ISO in all brain structures evaluated (Fig. 1).

Fig. 1.

Fig. 1.

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Effect of haloperidol (Hal), ketamine (Ket) or the combined drugs on the levels of 4-hydroxy-2-nonenal (4-HNE: 1A–1C) and 8-isoprostane (8-ISO: 1D–1F) in rats submitted to the experimental model of schizophrenia induced by Ket. Values represent mean (bars) ± standard error (vertical lines above the bars). Data are expressed as mg/mg protein or μg/mg protein. *p < 0.05, as compared to Saline (Sal) + Sal group. #p < 0.05, as compared to Ket + Sal group. Two-way analysis of variance (ANOVA).

Data from two-way ANOVA revealed significant effects of Ket administration [4-HNE: Frontal cortex: F(1,16) = 33.249, p < 0.05; Striatum: F(1,16) = 40.174, p < 0.05; Hippocampus: F(1,16) = 61.446, p < 0.05; 8-ISO: Frontal cortex: F(1,16) = 10.0219, p < 0.05; Striatum: F(1,16) = 22.365, p < 0.05; Hippocampus: F(1,16) = 33.252, p < 0.05], treatment [4-HNE: Frontal cortex: F(1,16) = 22.623, p < 0.05; Striatum: F(1,16) = 24.724, p < 0.05; Hippocampus: F(1,16) = 50.377, p < 0.05; 8-ISO: Frontal cortex: F(1,16) = 30.0728, p < 0.05; Striatum: F(1,16) = 8.2036, p < 0.05; Hippocampus: F(1,16) = 45.274, p < 0.05], and significant Ket administration × Hal interactions [4-HNE: Frontal cortex: F(1,16) = 1.762, p = 0.2029; Striatum: F(1,16) = 6.697, p < 0.05; Hippocampus: F(1,16) = 0.132, p = 0.7209; 8-ISO: Frontal cortex: F(1,16) = 0.0536, p = 0.819; Striatum: F(1,16) = 19.761, p < 0.05; Hippocampus: F(1,16) = 0.841, p = 0.373].

The second step was to estimate the protein oxidative damage, expressed as carbonyl content. The administration of Ket elicited a significant increase in the carbonyl levels in frontal cortex, striatum, and hippocampus, while Hal treatment was not able to counteract this effect. Hal itself induced damage in all structures evaluated (Fig. 2).

Fig. 2.

Fig. 2.

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Effect of haloperidol (Hal), ketamine (Ket) or the combined drugs on the carbonyl content in the frontal cortex (A), hippocampus (B), and striatum (C) of rats submitted to the experimental model of schizophrenia induced by Ket. Data are expressed as nmol/mg protein. Values represent mean (bars) ± standard error (vertical lines above the bars). *p < 0.05, as compared to Saline (Sal) + Sal group. Two-way analysis of variance (ANOVA).

Data from ANOVA (carbonyl) indicated significant effects of Ket administration [Frontal cortex: F(1,16) = 43.411, p < 0.05; Striatum: F(1,16) = 25.295, p < 0.05; Hippocampus: F(1,16) = 56.3043, p < 0.05], treatment [Frontal cortex: F(1,16) = 12.853, p < 0.05; Striatum: F(1,16) = 14.588, p < 0.05; Hippocampus: F(1,16) = 27.00569, p < 0.05], and a significant Ket administration × Hal interaction [Frontal cortex: F(1,16) = 12.5044, p < 0.05; Striatum: F(1,16) = 34.0715, p < 0.05; Hippocampus: F(1,16) = 13.0141, p < 0.05].

Finally, it was evaluated the activities of glutathione antioxidant enzymes in the samples. A significant decrease in the GPx activity, induced by Ket, Hal or Ket + Hal was detected in striatum and hippocampus. In frontal cortex, Hal per se or combined with Ket decreased GPx activity, while Ket itself induced an increase in this enzyme activity. Additionally, a significant decrease in GR activity elicited by the administration of Hal and Ket (isolated or in combination) was detectable in all brain structures evaluated (Fig. 3).

Fig. 3.

Fig. 3.

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Effect of haloperidol (Hal), ketamine (Ket) or the combined drugs on the activity of glutathione peroxidase (GPx: 3A–3C) and glutathione reductase (GR: 3D–3F) in cerebral structures of rats submitted to the experimental model of schizophrenia induced by Ket. Data are expressed as nmol min−1 mg protein−1. Values represent mean (bars) ± standard error (vertical lines above the bars). *p < 0.05, as compared to Saline (Sal) + Sal group. #p < 0.05, as compared to Ket + Sal group. Two-way analysis of variance (ANOVA).

Data from ANOVA showed significant effects of Ket administration [GPx: Frontal cortex: F(1,16) = 25.659, p < 0.05; Striatum: F(1,16) = 35.248, p < 0.05; Hippocampus: F(1,16) = 30.799, p < 0.05; GR: Frontal cortex: F(1,16) = 22.411, p < 0.05; Striatum: F(1,16) = 0.368, p = 0.553; Hippocampus: F(1,16) = 57.243, p < 0.05], treatment [GPx: Frontal cortex: F(1,16) = 69.003075, p < 0.05; Striatum: F(1,16) = 31.185, p < 0.05; Hippocampus: F(1,16) = 26.455, p < 0.05; GR: Frontal cortex: F(1,16) = 21.276, p < 0.05; Striatum: F(1,16) = 0.344, p = 0.566; Hippocampus: F(1,16) = 47.227, p < 0.05], and significant Ket administration × Hal interactions [GPx: Frontal cortex: F(1,16) = 1.899, p = 0.187; Striatum: F(1,16) = 43.00149, p < 0.05; Hippocampus: F(1,16) = 33.821, p < 0.05; GR: Frontal cortex: F(1,16) = 17.367, p < 0.05; Striatum: F(1,16) = 8.659, p < 0.05; Hippocampus: F(1,16) = 50.928, p < 0.05].

4. Discussion

In the present study, a significant increase in the lipid peroxidation parameters was found in the brain structures in rats receiving Hal itself or combined with Ket. The evaluation of protein carbonylation in the samples was also carried out and revealed that all groups receiving Hal, Ket, or a combination thereof presented an increase in this parameter in the three cerebral structures analyzed. Finally, the evaluation of the activity of GPx and GR demonstrated a decrease in these parameters in the experimental groups receiving Hal, regardless of brain structure. To date, this is the first study to show alterations induced by Hal in the glutathione metabolism enzymes, lipid peroxidation markers and protein oxidation in the SZ animal model induced by Ket administration.

The SZ positive and negative symptoms, as well as the cognitive impairment induced by Ket, are partly assigned to the blockade in NMDA receptors (Balla et al., 2009; Chatterjee et al., 2011). Inhibition of these receptors is associated with an increase in the release of dopamine in the mesolimbic system and upregulation of other glutamatergic receptors (Kokkinou et al., 2018). Ket can also induce oxidative stress in the brain of animals (de Oliveira et al., 2011), as detected in rats receiving Ket in the present paper. This effect was stable and not reversible by Hal. Additionally, Hal was also associated with oxidative stress in the brain structures. This data is partly in accordance with the paper of El-Awdan and coworkers (2015), describing the pro-oxidant effect of the antipsychotic. Therefore, Hal therapeutic activity is independent on the antioxidant mechanisms since the drug can exert the opposite effect on oxidative stress parameters.

Inhibition of dopaminergic neurotransmission through the blockade of D2 dopamine receptors, preventing dopamine reuptake in the brain, is involved in the action mechanism of Hal. This drug may also lead to blocking action on noradrenergic, cholinergic, and histaminergic pathways (Chokhawala and Stevens, 2019). Moreover, Hal can be neurotoxic, since it was associated with neuronal death at different doses (Nasrallah and Chen, 2017). Hal administration alone or combined with Ket elicited oxidative damage to lipids and proteins in the rats, a finding that seems to be intrinsic to the antipsychotic. At least in part, data from the present study corroborate the hypothesis that Hal increases the production of reactive oxygen species, leading to oxidative stress (Nasrallah and Chen, 2017).

As the first step of the present study, it was carried out measurements on lipid peroxidation markers, which were significantly increased in rats receiving Hal alone or combined with Ket. In this context, Kamyar et al. (2016) detected increased levels of a lipid peroxidation by-product called malondialdehyde (MDA) in the cortex, hippocampus, and striatum of rats receiving Hal (1 mg/kg body weight, i.p.), although their experiment lasted 21 days. Similar data is provided by de Araújo et al. (2017), which demonstrated that MDA levels increased in brain structures of rats receiving Hal for 31 days. In striatum of rats receiving Hal (0.2 mg kg−1 day−1) for five weeks, Samad and Haleem (2017) detected increased MDA content following an increase in the H2O2 levels. Besides, Hal treatment (1 mg/kg body weight, for 21 days) seems also involved in an increase in MDA and a decrease in glutathione levels in rat forebrain (Dhingra et al., 2018). Indeed, studies have described significant lipid peroxidation in the striatum of rodents, but the damage is preventable or even reversible with antioxidant supplementation (Chen et al., 2018; Sonego et al., 2018; Tsai et al., 2019). A comprehensive picture of the putative mechanism involved in these alterations in striatum remains incomplete. However, it is well-known that Hal plays roles in striatum dopaminergic neurons, whereas Hal and its metabolites likely elicit neurotoxicity in nucleus basalis (Castagnoli et al., 1999). Hal also presents basal cytotoxicity higher than other antipsychotics; this may be in part because it may increase 4-HNE and protein carbonyl contents, as demonstrated in the present study, while the drug is not useful to scavenge reactive species as other antipsychotics (Yang et al., 2014).

The evaluation of protein carbonylation in the samples was the second step of the present research, a parameter significantly increased in animals receiving Hal, Ket, or a combination of both. In this context, Martins et al. (2008) reported an increase in the carbonyl content in the hippocampus of rats receiving Hal (1.5 mg/kg for 28 days), which was followed by an increase in the MDA levels in the striatum of these animals. In rats receiving a Hal solution (12 mg kg−1 mL−1) subchronically, it was showed a significant increase in the levels of protein carbonyl and reactive species in the cerebral cortex, striatum, and substantia nigra, which was followed by critical impairments in locomotion (Kronbauer et al., 2017). Moreover, the intramuscular administration of the Hal-decanoate ester (4.2–50 mg, once a week) was positively associated with an increase in plasma protein carbonyl levels in patients (Bošković et al., 2013). Collectively, the findings and the presented literature indicate that Hal could trigger the protein carbonylation, in a way that seems independent on the dose or route of administration.

The final step of the present paper was the evaluation of the activity of GPx and GR – antioxidant enzymes crucial for life. Hal administration was associated to a decrease in these parameters, independent on the brain structure. In rats receiving Hal (0.2 mg kg−1 day−1) for five weeks, it was detected several oxidative modifications in the striatum, including a decrease in GPx activity (Samad and Haleem, 2017). On the other hand, Pillai et al. (2007) conducted a study showing no significant alterations in GPx activity in the brain samples from rats receiving the drug for a long term (2 mg kg−1 day−1). In other protocol, Hal (2 mg/kg for 21 days) induced a decrease in the GPx activity in rat brain; this effect was significantly prevented by supplementation with the lipoic acid antioxidant (Perera et al., 2011). Interestingly, according to Fendri et al. (2006), a decrease in GPx activity is associated with negative SZ symptoms. This finding collaborates to understand why Hal and other typical antipsychotics are few effective in treating these symptoms. The GR activity is probably one of the most sensitive to the deleterious effects assigned to Hal. In guinea pigs who received this drug (42, 000 μg/kg body mass), the animals with the higher Hal levels exhibited an increase in the content of reactive species and activity of antioxidant enzymes. GR was the protein that most collaborated for the observed effects on the enzymes (Gumulec et al., 2013). In contrast, a 10-weeks Hal administration (1.5 mg kg−1 day−1) had an association with significant decreases in the activity of GR, GPx, and other enzymes in rat brain (Vairetti et al., 2004).

Indeed, a glutathione deficiency in the posterior medial frontal cortex is associated with the severity of negative symptoms in schizophrenic patients (Matsuzawa et al., 2008). It is worthy to note that glutathione deficiency in mice markedly altered tissue levels of dopamine and 5-hydroxytryptamine and their metabolites in a region-specific manner (Jacobsen et al., 2005). More specifically, the levels of homovanillic acid (frontal cortex and hippocampus) and 5-hydroxyindoleacetic acid (nucleus accumbens), two metabolites from 5-hydroxytryptamine, were elevated by the glutathione deficiency itself (Jacobsen et al., 2005). Therefore, altered glutathione metabolism can lead to and sustain oxidative stress, which may alter the availability of relevant neurotransmitters. GPx and GR activities seem relevant targets of the oxidative imbalance observed in schizophrenia and increased by Hal, which renders the supplementation with antioxidants an important way to bypass these effects.

The precise mechanisms by which Hal induces oxidative disturbances are not fully understood, but it is well-known that Hal has a pyridinium metabolite (HPP+), whose synthesis is catalyzed by the P4503A cytochrome enzymes. HPP+ is detectable in the urine of SZ patients in therapy with Hal, and the presence of this metabolite with certain organic acids and acylcarnitine in urine suggested that HPP+ could impair a step of energy metabolism (Castagnoli et al., 1999). However, incubation of sections of mouse brain with Hal (10 nM) showed that the drug inhibits the mitochondrial electron transport chain complex I, in a time and dose-dependent manner. No significant inhibitory activity of HPP+ was detected at this same concentration in the medium, indicating that Hal can be most deleterious than HPP+ and could induce oxidative stress secondary to an energy dysfunction. Besides, this activity of Hal is higher than that of other antipsychotics (Balijepalli et al., 1999). Another possibility is that antipsychotics as Hal can induce an increase in the intracellular levels of the β-catenin protein, whereas certain antioxidant compounds counterbalance this effect via activation of the mammalian target of rapamycin pathway (Deslauriers et al., 2013).

4.1. Conclusions

In summary, the present study reports the effects of Hal administration on the neurochemical parameters linked to oxidative stress in rats subjected to an experimental model of SZ. Both Hal and Ket triggered remarkable oxidative damage. These findings corroborate previous evidence that Hal, in different doses and duration from that used here, induces significant lipid peroxidation and glutathione depletion in the chronic Ket model (Onaolapo et al., 2017; Abdel-Salam et al., 2018). An energy dysfunction or a primary effect of the drug on the GPx and GR enzymes, disturbing the redox homeostasis in neurons, can be implicated in the alterations reported above. At present, this is the first study to reveal alterations elicited by Hal in the glutathione-recycling enzymes, parameters of lipid peroxidation and protein carbonylation in the SZ animal model induced by Ket. Further neurochemical and behavioral research is required to afford a more comprehensive picture of the factors involved in all these alterations, as well as the impact of supplementation with antioxidants in these experimental circumstances.

The limitations of the present study include the relatively small size of the sample, leading to the need for replication by additional research. Furthermore, it was used only one Hal dose (0.1 mg/kg), while the data detailing its involvement in oxidative stress comprise several doses, which collaborates to hamper comparisons between the findings of the present study and literature. The SZ model induced by subchronic Ket has certain limitations, and additional research using other models mimicking the episodes of the mental disorder could circumvent these drawbacks. Moreover, it was chosen to evaluate only one first-generation antipsychotic. Although Hal was discovered more than 60 years ago and is still in use today, the inclusion of second- and third-generation antipsychotics in the study could reflect a more comprehensive therapeutic significance. Nevertheless, Hal was chosen due to several misunderstood effects regarding its pharmacological activity, including oxidative stress-mediated cytotoxicity.

Acknowledgments

Translational Psychiatry Program (USA) is supported by the Department of Psychiatry and Behavioral Sciences, McGovern Medical School, The University of Texas Health Science Center at Houston (UTHealth). Translational Psychiatry Laboratory (Brazil) is one of the centers of the National Institute for Molecular Medicine (INCT-MM) and one of the members of the Center of Excellence in Applied Neurosciences of Santa Catarina (NENASC).

Funding sources

Translational Psychiatry Program (USA) is funded by a grant from the National Institute of Health/National Institute of Mental Health (1R21MH117636-01A1, to JQ). Center of Excellence on Mood Disorders (USA) is funded by the Pat Rutherford Jr. Chair in Psychiatry, John S. Dunn Foundation and Anne and Don Fizer Foundation Endowment for Depression Research. Translational Psychiatry Laboratory (Brazil) is funded by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC), and Instituto Cérebro e Mente. JQ is a 1A CNPq Research Fellow.

Footnotes

CRediT authorship contribution statement

Samira S. Valvassori: Conceptualization, Methodology, Supervision, Project administration. José H. Cararo: Writing - original draft, Visualization. Samira Menegas: Writing - original draft, Visualization. Taise Possamai-Della: Investigation, Validation. Jorge M. Aguiar-Geraldo: Investigation, Validation. Simone Lespinasse Araujo: Investigation. Gustavo Antunes Mastella: Investigation. João Quevedo: Writing - review & editing, Funding acquisition. Alexandra I. Zugno: Writing - review & editing, Resources.

Declaration of Competing Interest

JQ has the following declarations of interest: Clinical research support: Janssen Pharmaceutical (Clinical Trial), Allergan (Clinical Trial); Advisory boards, speaker bureaus, expert witness, or consultant: Daiichi Sankyo (Speaker Bureau); Patent, equity, or royalty: Instituto de Neurociências Dr. João Quevedo (Stockholder); Other: Artmed Editora (Copyright), Artmed Panamericana (Copyright). All the other authors have no conflicts of interest.

References

  1. Abdel-Salam OM, El-Shamarka ME-S, Omara EA, 2018. Brain oxidative stress and neurodegeneration in the ketamine model of schizophrenia during antipsychotic treatment: effects of N-acetylcysteine treatment. ROS 6, 253–266. [Google Scholar]
  2. Balijepalli S, Boyd MR, Ravindranath V, 1999. Inhibition of mitochondrial complex I by haloperidol: the role of thiol oxidation. Neuropharmacology 38, 567–577. [DOI] [PubMed] [Google Scholar]
  3. Balla A, Nattini ME, Sershen H, Lajtha A, Dunlop DS, Javitt DC, 2009. GABAB/NMDA receptor interaction in the regulation of extracellular dopamine levels in rodent prefrontal cortex and striatum. Neuropharmacology 56, 915–921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bošković M, Grabnar I, Terzič T, Kores Plesničar B, Vovk T, 2013. Oxidative stress in schizophrenia patients treated with long-acting haloperidol decanoate. Psychiatry Res 210, 761–768. [DOI] [PubMed] [Google Scholar]
  5. Canever L, Oliveira L, D’Altoé de Luca R, Correa PT, de B Fraga D, Matos MP, Scaini G, Quevedo J, Streck EL, Zugno AI, 2010. A rodent model of schizophrenia reveals increase in creatine kinase activity with associated behavior changes. Oxid. Med. Cell. Longev 3, 421–427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Canever L, Freire TG, Mastella GA, Damázio L, Gomes S, Fachim I, Michels C, Carvalho G, Godói AK, Peterle BR, Gava FF, Valvassori SS, Budni J, Quevedo J, Zugno AI, 2018. Changes in behavioural parameters, oxidative stress and neurotrophins in the brain of adult offspring induced to an animal model of schizophrenia: the effects of FA deficient or FA supplemented diet during the neurodevelopmental phase. Prog. Neuropsychopharmacol. Biol. Psychiatry 86, 52–64. [DOI] [PubMed] [Google Scholar]
  7. Castagnoli N Jr., Castagnoli KP, Van der Schyf CJ, Usuki E, Igarashi K, Steyn SJ, Riker RR, 1999. Enzyme-catalyzed bioactivation of cyclic tertiary amines to form potential neurotoxins. Pol. J. Pharmacol 51, 31–38. [PubMed] [Google Scholar]
  8. Cattane N, Richetto J, Cattaneo A, 2018. Prenatal exposure to environmental insults and enhanced risk of developing Schizophrenia and Autism Spectrum disorder: focus on biological pathways and epigenetic mechanisms. Neurosci. Biobehav. Rev S0149-7634(17)30972-30977. [DOI] [PubMed]
  9. Chatterjee M, Ganguly S, Srivastava M, Palit G, 2011. Effect of’ chronic’ versus’ acute’ ketamine administration and its’ withdrawal’ effect on behavioural alterations in mice: implications for experimental psychosis. Behav. Brain Res 216, 247–254. [DOI] [PubMed] [Google Scholar]
  10. Chen CN, Chang KC, Wang MH, Tseng HC, Soung HS, 2018. Protective effect of L-theanine on haloperidol-induced orofacial. Chin. J. Physiol 61, 35–41. [DOI] [PubMed] [Google Scholar]
  11. Chokhawala K, Stevens L, 2019. Antipsychotic Medications (Accessed 5 September, 2019). https://www.ncbi.nlm.nih.gov/books/NBK519503/. [PubMed]
  12. Damazio LS, Silveira FR, Canever L, Castro AA, Estrela JM, Budni J, Zugno AI, 2017. The preventive effects of ascorbic acid supplementation on locomotor and acetylcholinesterase activity in an animal model of schizophrenia induced by ketamine. An. Acad. Bras. Cienc 89, 1133–1141. [DOI] [PubMed] [Google Scholar]
  13. de Araújo DP, Camboim TGM, Silva APM, Silva CDF, de Sousa RC, Barbosa MDA, Oliveira LC, Cavalcanti JRLP, Lucena EES, Guzen FP, 2017. Behavioral and neurochemical effects of alpha lipoic acid associated with omega-3 in tardive dyskinesia induced by chronic haloperidol in rats. Can. J. Physiol. Pharmacol 95, 837–843. [DOI] [PubMed] [Google Scholar]
  14. de Oliveira L, Fraga DB, De Luca RD, Canever L, Ghedim FV, Matos MP, Streck EL, Quevedo J, Zugno AI, 2011. Behavioral changes and mitochondrial dysfunction in a rat model of schizophrenia induced by ketamine. Metab. Brain Dis 26, 69–77. [DOI] [PubMed] [Google Scholar]
  15. Dean L, 2017. Thioridazine therapy and CYP2D6 genotypes. In: Pratt V, McLeod H, Dean L (Eds.), Medical Genetics Summaries National Center for Biotechnology Information, Bethesda (MD) (Accessed 5 September, 2019). https://www.ncbi.nlm.nih.gov/books/NBK424018/. [Google Scholar]
  16. Deslauriers J, Desmarais C, Sarret P, Grignon S, 2013. α-Lipoic acid interaction with dopamine D2 receptor-dependent activation of the Akt/GSK-3β signaling pathway induced by antipsychotics: Potential relevance for the treatment of schizophrenia. J. Mol. Neurosci 50, 134–145. [DOI] [PubMed] [Google Scholar]
  17. Dhingra D, Goswami S, Gahalain N, 2018. Protective effect of hesperetin against haloperidol-induced orofacial dyskinesia and catalepsy in rats. Nutr. Neurosci 21, 667–675. [DOI] [PubMed] [Google Scholar]
  18. Dietrich-Muszalska A, Kolińska-Łukaszuk J, 2018. Comparative effects of aripiprazole and selected antipsychotic drugs on lipid peroxidation in plasma. Psychiatry Clin. Neurosci 72, 329–336. [DOI] [PubMed] [Google Scholar]
  19. El-Awdan SA, Abdel Jaleel GA, Saleh DO, 2015. Alleviation of haloperidol induced oxidative stress in rats: effects of sucrose vs grape seed extract. Bull. Fac. Pharm. Cairo Univ 53, 29–35. [Google Scholar]
  20. Elmorsy E, Al-Ghafari A, Aggour AM, Khan R, Amer S, 2017. The role of oxidative stress in antipsychotics induced ovarian toxicity. Toxicol. In Vitro 44, 190–195. [DOI] [PubMed] [Google Scholar]
  21. Fendri C, Mechri A, Khiari G, Othman A, Kerkeni A, Gaha L, 2006. Oxidative stress involvement in schizophrenia pathophysiology: a review. Encephale 32, 244–252. [DOI] [PubMed] [Google Scholar]
  22. Gumulec J, et al. , 2013. Determination of oxidative stress and activities of antioxidant enzymes in guinea pigs treated with haloperidol. Exp. Ther. Med 5, 479–484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Haddad PM, Correll CU, 2018. The acute efficacy of antipsychotics in schizophrenia: a review of recent meta-analyses. Ther. Adv. Psychopharmacol 8, 303–318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jacobsen JP, Rodriguiz RM, Mørk A, Wetsel WC, 2005. Monoaminergic dysregulation in glutathione-deficient mice: possible relevance to schizophrenia? Neuroscience 132, 1055–1072. [DOI] [PubMed] [Google Scholar]
  25. Kamyar M, Razavi BM, Hasani FV, Mehri S, Foroutanfar A, Hosseinzadeh H, 2016. Crocin prevents haloperidol-induced orofacial dyskinesia: possible an antioxidant mechanism. Iran. J. Basic Med. Sci 19, 1070–1079. [PMC free article] [PubMed] [Google Scholar]
  26. Kokkinou M, Ashok AH, Howes OD, 2018. The effects of ketamine on dopaminergic function: meta-analysis and review of the implications for neuropsychiatric disorders. Mol. Psychiatry 23, 59–69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kronbauer M, Metz VG, Roversi K, Dias VT, de David Antoniazzi CT, da Silva Barcelos RC, Burger ME, 2017. Influence of magnesium supplementation on movement side effects related to typical antipsychotic treatment in rats. Behav. Brain Res 320, 400–411. [DOI] [PubMed] [Google Scholar]
  28. Kropp S, Kern V, Lange K, Degner D, Hajak G, Kornhuber J, Rüther E, Emrich HM, Schneider U, Bleich S, 2005. Oxidative stress during treatment with first- and second-generation antipsychotics. J. Neuropsychiatry Clin. Neurosci 17, 227–231. [DOI] [PubMed] [Google Scholar]
  29. Krzystanek M, Pałasz A, 2019. NMDA receptor model of antipsychotic drug-induced hypofrontality. Int. J. Mol. Sci 20, E1442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Martins MR, Petronilho FC, Gomes KM, Dal-Pizzol F, Streck EL, Quevedo J, 2008. Antipsychotic-induced oxidative stress in rat brain. Neurotox. Res 13, 63–69. [DOI] [PubMed] [Google Scholar]
  31. Matsuzawa D, Obata T, Shirayama Y, Nonaka H, Kanazawa Y, Yoshitome E, Takanashi J, Matsuda T, Shimizu E, Ikehira H, Iyo M, Hashimoto K, 2008. Negative correlation between brain glutathione level and negative symptoms in schizophrenia: a 3T 1H-MRS study. PLoS One 3, e1944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nasrallah HA, Chen AT, 2017. Multiple neurotoxic effects of haloperidol resulting in neuronal death. Ann. Clin. Psychiatry 29, 195–202. [PubMed] [Google Scholar]
  33. National Research Council (US), 2011. Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 8th edition. Guide for the Care and Use of Laboratory Animals; (Accessed 5 September, 2019). https://www.ncbi.nlm.nih.gov/books/NBK54050/. [Google Scholar]
  34. Onaolapo AY, Aina OA, Onaolapo OJ, 2017. Melatonin attenuates behavioural deficits and reduces brain oxidative stress in a rodent model of schizophrenia. Biomed. Pharmacother 92, 373–383. [DOI] [PubMed] [Google Scholar]
  35. Parikh V, Khan MM, Mahadik SP, 2003. Differential effects of antipsychotics on expression of antioxidant enzymes and membrane lipid peroxidation in rat brain. J. Psychiatr. Res 37, 43–51. [DOI] [PubMed] [Google Scholar]
  36. Perera J, Tan JH, Jeevathayaparan S, Chakravarthi S, Haleagrahara N, 2011. Neuroprotective effects of alpha lipoic acid on haloperidol-induced oxidative stress in the rat brain. Cell Biosci 1, 12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pillai A, Parikh V, Terry AV Jr., Mahadik SP, 2007. Long-term antipsychotic treatments and crossover studies in rats: differential effects of typical and atypical agents on the expression of antioxidant enzymes and membrane lipid peroxidation in rat brain. J. Psychiatr. Res 41, 372–386. [DOI] [PubMed] [Google Scholar]
  38. Raudenska M, Gumulec J, Babula P, Stracina T, Sztalmachova M, Polanska H, Adam V, Kizek R, Novakova M, Masarik M, 2013. Haloperidol cytotoxicity and its relation to oxidative stress. Mini Rev. Med. Chem 13, 1993–1998. [DOI] [PubMed] [Google Scholar]
  39. Reinke A, Martins MR, Lima MS, Moreira JC, Dal-Pizzol F, Quevedo J, 2004. Haloperidol and clozapine, but not olanzapine, induces oxidative stress in rat brain. Neurosci. Lett 372, 157–160. [DOI] [PubMed] [Google Scholar]
  40. Rodrigues-Amorim D, Rivera-Baltanás T, López M, Spuch C, Olivares JM, Agís-Balboa RC, 2017. Schizophrenia: a review of potential biomarkers. J. Psychiatr. Res 93, 37–49. [DOI] [PubMed] [Google Scholar]
  41. Samad N, Haleem DJ, 2017. Antioxidant effects of rice bran oil mitigate repeated haloperidol-induced tardive dyskinesia in male rats. Metab. Brain Dis 32, 1099–1107. [DOI] [PubMed] [Google Scholar]
  42. Singh OP, Chakraborty I, Dasgupta A, Datta S, 2008. A comparative study of oxidative stress and interrelationship of important antioxidants in haloperidol and olanzapine treated patients suffering from schizophrenia. Indian J. Psychiatry 50, 171–176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sonego AB, Prado DS, Vale GT, Sepulveda-Diaz JE, Cunha TM, Tirapelli CR, Del Bel EA, Raisman-Vozari R, Guimarães FS, 2018. Cannabidiol prevents haloperidol-induced vacuos chewing movements and inflammatory changes in mice via PPARγ receptors. Brain Behav. Immun 74, 241–251. [DOI] [PubMed] [Google Scholar]
  44. Supp AD, Avila S Jr., Mastella GA, Damásio L, de Oliveira IH, Godoi AK, Michels A, Schuck PF, Zugno AI, 2020. Ascorbic acid supplementation attenuates schizophrenia-like symptoms in an animal model induced by ketamine. Int. J. Dev. Neurosci 10.1002/jdn.10058. Online ahead of print. [DOI] [PubMed]
  45. Terry AV Jr, Gearhart DA, Pillai A, Zhang G, Bartlett MG, 2010. Chronic antipsychotic treatment: protracted decreases in phospho-TrkA levels in the rat hippocampus. Int. J. Neuropsychopharmacol 13, 799–805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tsai CC, Wang MH, Chang KC, Soung HS, Yang CC, Tseng HC, 2019. Possible nitric oxide mechanism involved in the protective effect of L-theanine on haloperidol-induced orofacial dyskinesia. Chin. J. Physiol 62, 17–26. [DOI] [PubMed] [Google Scholar]
  47. Vairetti M, Ferrigno A, Canonico PL, Battaglia A, Bertè F, Richelmi P, 2004. Nicergoline reverts haloperidol-induced loss of detoxifying-enzyme activity. Eur. J. Pharmacol 505, 121–125. [DOI] [PubMed] [Google Scholar]
  48. World Health Organization, WHO, 2018. Schizophrenia (Accessed 5 September 2019). https://www.who.int/news-room/fact-sheets/detail/schizophrenia.
  49. Yang MC, Chen KP, Lung FW, 2014. Generalized estimating equation model and long-term exposure effect of antipsychotics on SH-SY5Y cells against oxidative stressors. Eur. J. Pharmacol 740, 697–702. [DOI] [PubMed] [Google Scholar]
  50. Zugno AI, Canever L, Heylmann AS, Wessler PG, Steckert A, Mastella GA, de Oliveira MB, Damázio LS, Pacheco FD, Calixto OP, Pereira FP, Macan TP, Pedro TH, Schuck PF, Quevedo J, Budni J, 2016. Effect of folic acid on oxidative stress and behavioral changes in the animal model of schizophrenia induced by ketamine. J. Psychiatr. Res 81, 23–35. [DOI] [PubMed] [Google Scholar]

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