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Environmental enrichment restores oxidative balance in animals chronically exposed to toluene: Comparison with melatonin
Accepted Manuscript Title: Environmental enrichment restores oxidative balance in animals chronically exposed to toluene: Comparison with melatonin Authors: Sergio Montes, Yepci Yee-Rios, Nayeli P´aez-Mart´ınez PII: DOI: Reference:
S0361-9230(18)30041-8 https://doi.org/10.1016/j.brainresbull.2018.11.007 BRB 9544
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
20 January 2018 28 August 2018 14 November 2018
Please cite this article as: Montes S, Yee-Rios Y, P´aez-Mart´ınez N, Environmental enrichment restores oxidative balance in animals chronically exposed to toluene: Comparison with melatonin, Brain Research Bulletin (2018), https://doi.org/10.1016/j.brainresbull.2018.11.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Environmental enrichment restores oxidative balance in animals chronically exposed to toluene: comparison with melatonin
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Sergio Montesa, Yepci Yee-Riosb, Nayeli Páez-Martínezb,c*
Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía
Manuel Velasco Suarez. Avenida Insurgentes Sur No. 3877. Ciudad de México. C.P. 14269. México. b
Sección de Posgrado e Investigación, Escuela Superior de Medicina, Instituto
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Politécnico Nacional. Plan de San Luis y Díaz Mirón. Ciudad de México. C.P.
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Laboratorio Integrativo para el Estudio de Sustancias Inhalables Adictivas.
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c
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11340. México.
Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría
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C.P. 14370. México.
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Ramón de la Fuente Muñiz. Calzada México Xochimilco 101. Ciudad de México.
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*Corresponding author: Dr. Nayeli Páez Martínez.
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Sección de Posgrado e Investigación. Escuela Superior de Medicina, Instituto Politécnico Nacional. Plan de San Luis y Díaz Mirón, Col. Santo Tomás, Ciudad de México. C.P. 11340. México. Tel.: +52 (55) 4160-5116 Fax: +52 (55) 5655-9980 E-mail: [email protected]
HIGHLIGHTS
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Chronic toluene exposure induced an oxidative unbalance in the hippocampus and prefrontal cortex. EE restored oxidative balance in animals previously exposed to toluene. Effects of EE were similar to those obtained with melatonin. Abstinence did not spontaneously repair the alterations in oxidative stress induced by toluene.
ABSTRACT
Inhalants are widely used as recreational drugs, and toluene is the main chemical
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compound present in most inhalants used for these purposes. Previous studies have
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shown that repeated toluene exposure produces cellular death and memory impairment,
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while environmental enrichment (EE) rescues from those effects. However, the
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mechanisms involved in those responses are unclear. Previous studies have shown that toluene induces a redox imbalance at the neuronal level; although, details on the
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mechanism of action of environmental enrichment enhancing antioxidant capacity remain
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to be explored. It is also unexplored whether this putative antioxidant capacity is similar to
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that produced by pharmacological antioxidants. To study this hypothesis, Swiss-Webster male mice were chronically exposed to toluene (0 or 4000 ppm, 30 min/day/4 weeks).
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Subsequently, neurochemical tests were conducted to measure biomarkers of oxidative stress (ROS, NO, GSH/GSSG ratio and SOD activity) in the hippocampus and the
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prefrontal cortex. In the second part of the study, we evaluated the putative antioxidant capacity of environmental enrichment and compared it to that of melatonin, a known free radical scavenger and inductor of antioxidant defences. The results showed that chronic toluene exposure increased the levels of pro-oxidative molecules and decreased the
antioxidant markers. Conversely, environmental enrichment restored oxidative balance in animals previously exposed to toluene. Furthermore, the effects of EE were similar to those obtained with melatonin. Altogether, alterations in oxidative balance could represent an
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intermediate signalling pathway in the cascade of effects induced by toluene, while EE and melatonin appear to have the ability to rescue those effects.
Keywords: environmental enrichment, melatonin, oxidative stress, toluene
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1. Introduction
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Deliberate consumption of inhalants for intoxication purposes is practised worldwide, and
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its use has emerged as a relatively common problem among children and adolescents
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(Lubman et al., 2008). Toluene-based products (paint thinner, glues, and “Activo” [a toluene-enriched formulation distributed by drug dealers]) are inhalants most commonly
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used in Mexico (Villatoro et al., 2011). Despite their important deleterious health
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consequences, inhalants continue to be one of the least studied psychoactive substances. Therefore, treatment and prevention research has only recently emerged (Howard et al.,
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2011). In previous studies from our lab, it was observed that environmental enrichment
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(EE) reversed memory impairment and reduced neuronal death in animals previously exposed to toluene (Montes et al., 2017; Paez-Martinez et al., 2013); however, the
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mechanisms involved are still unclear.
It has been shown that NMDA receptors (channels permeable to Ca2+) are molecular targets of toluene (Cruz et al., 1998). Furthermore, chronic toluene exposure enhanced NMDA-mediated currents and increased the expression of NR2A and NR2B NMDA
receptor subunits in neuronal cell cultures (Bale et al., 2005). It is well known that NMDA receptors play a key role in long-term potentiation, even though overstimulation of NMDA receptors causes excitotoxicity. Pathological increases of Ca2+ ions into the postsynaptic
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neurons activate the Ca2+-dependent pathways (Feldman et al., 1997; Marks et al., 2009). On one hand, this pathway leads to metabolic changes such as arachidonic acid metabolism and generating reactive oxygen species (ROS). On the other hand, Ca2+ activates neuronal NO synthase, which synthesises nitric oxide (NO), while NO, as a free radical, is transformed to a highly reactive product, peroxynitrite, upon reaction with ROS
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(Feldman et al., 1997; Marks et al., 2009). Altogether, these reactions may result in
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oxidative stress that has been observed to result in apoptosis.
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Oxidative stress is defined as the imbalance between cellular pro-oxidants and antioxidants in favour of oxidant activity that potentially leads to tissue damage (Uttara et al., 2009).
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There is some evidence in the literature showing that toluene exerts part of its toxicity via
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oxidative stress. For instance, toluene administration (0.5, 1 and 1.5 g/kg, i.p.) induced
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ROS production in synaptosomal fractions from the striatum, hippocampus and cerebellum. Additionally, 1 g/kg toluene increased lipid peroxidation in the hippocampus (Mattia et al.,
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1993). Similarly, paint thinner vapours (3000 ppm/1 h/day for 45 days) enhanced lipid
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peroxidation in the hippocampus, cortex and cerebellum (Baydas et al., 2003).
In contrast, EE is a housing condition that provides social, cognitive and physical stimulation (Nithianantharajah and Hannan, 2006). Several studies have shown that EE reduces the levels of oxidative biomarkers (such as lipid peroxidation, protein oxidation, and free radical levels) while enhancing values of antioxidant parameters (such as catalase
and SOD activity), when compared with standard housing rodents (Cechetti et al., 2012; Herring et al., 2010; Marmol et al., 2015). Furthermore, the expression of the SOD enzyme
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is increased in mice housed in EE conditions (Herring et al., 2010).
On the other hand, melatonin is an endogenous mitochondria-targeted molecule with high efficacy in reducing oxidative damage. This molecule possesses a variety of mechanisms by which it reaches its antioxidant effects. Melatonin and its metabolites are radical scavengers of reactive oxygen and reactive nitrogen species and they have the ability to
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bind heavy metals, which enhance melatonin’s capacity to quench oxidative damage
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(Reiter et al., 2016). Melatonin also interacts with membrane receptors, which indirectly
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stimulates antioxidant enzymes, including glutathione peroxidase and glutathione
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reductase, superoxide dismutase, while suppressing the activity of pro-oxidant enzymes
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(Reiter et al., 2000; Reiter et al., 2018).
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Altogether, the main aims of the present study were to evaluate oxidative stress induced by
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chronic toluene exposure and to analyse whether EE restores the imbalance in oxidative stress in mice with a history of toluene exposure. Finally, as melatonin is a compound with
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potent antioxidant properties (Reiter et al., 2000), it was of our interest to compare the effects induced by EE with those produced by melatonin in terms of antioxidant mechanism
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of action.
2. Material and methods 2.1 Animals
Swiss-Webster male mice (postnatal day 35-40) were used in the present study. This stage was selected because mice at this age exhibit behavioral, neurochemical and endocrine patterns of human adolescent subjects (Abreu-Villaca et al., 2008; Burns and Proctor,
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2013; Laviola et al., 2003) and because this age extrapolates to the age of onset of inhalant abuse in humans (Dell et al., 2011). All animals were maintained under inverted controlled light-dark cycle conditions [12 h:12 h; lights on at 19:00 h, zeitgeber time = 0 (ZT0)] at 22 to 26°C and had access to food and water ad libitum. All experimental procedures followed the regulations of the Mexican Official Norm NOM-062-ZOO-1999 for
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the use and care of laboratory animals (DOF, 2001) and had the approval of the Ethics
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Committee for Animal Experimentation of the “Instituto Nacional de Psiquiatría Ramón de
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la Fuente Muñiz, Mexico” (IACUC: CEI/C/059/2016).
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2.2 Toluene exposure
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The exposure was conducted in a 27-litre glass cylindrical inhalation chamber. The
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chamber was covered with a polycarbonate lid that was fitted with an injection port and a fan that projected into the chamber above a stainless-steel mesh platform. At the beginning
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of exposure, two mice were placed in the bottom of the chamber and the lid was set. A predetermined volume of toluene (purity 99.9%; Sigma-Aldrich, Toluca, Mexico) was
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delivered through an injection port, via a microsyringe, onto a filter paper located on a stainless-steel mesh (Nelson, 1971). After removing the needle, the fan was turned on to distribute the solvent into the glass chamber. Animals were exposed either to 0 ppm (air or control group) or 4000 ppm toluene vapours (Sigma-Aldrich, Toluca Mexico). The exposure was conducted thirty minutes a day, for four weeks. After two hours of the last toluene
administration, animals were euthanized, their brains were removed and hippocampus and prefrontal cortex were quickly dissected out and stored at −70°C until biochemical assays. These brain regions were selected because they are involved in processes impaired by
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toluene administration, such as memory in the object recognition test, neurogenesis and neuronal death.
2.3 Environmental enrichment
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Independent groups of animals were exposed to toluene (4000 ppm, 30 min/day for 4
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weeks); afterwards, animals were housed with EE or standard housing conditions. A
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control group consisted of mice exposed to air and then housed in standard conditions. EE
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animals were maintained in larges home cages of 34 x 44 x 20 cm, in groups of 5. The environment included a series of 5 objects of different shapes, sizes and textures. Objects
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included toys and tunnels; these objects were changed twice a week to stimulate animals´
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curiosity. In addition, a wheel was included so that the animals had the opportunity to
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perform voluntary exercise. Environmental enrichment was carried out 24 hours a day, for 4 weeks. Standard housing groups (n= 5) were placed in cages (18 ×28 × 15 cm) without
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any stimulation during 4 weeks. At the end of the housing period, animals were challenged with a single dose of toluene (4000 ppm/30 min). The intention of this administration was to
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prime a new oxidative stress state and to evaluate the ability of EE to buffer this potential oxidative damage. Two hours after challenge, animals were euthanized, their brains were removed and the hippocampus and prefrontal cortex were obtained. Samples were stored at −70°C until biochemical assays.
2.4 Melatonin administration Similar to the previous protocol, mice were exposed to toluene for 4 weeks and
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subsequently administered melatonin daily (Sigma-Aldrich, Toluca, México) for 4 more weeks (10 mg/kg, i.p.) (Chabra et al., 2014; El-Sokkary et al., 2002; Solis-Munoz et al., 2011). Length of treatment was stablished (four weeks) to have the possibility to compare melatonin effect with that of EE; however it is important to mention that the dose of
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melatonin selected considered the animal species utilized and the chronicity of treatment.
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Melatonin was administered at zeitgeber time 11 (ZT11), which represent one hour before
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endogenous indolamine starts to rise; at this point the levels of melatonin are low (Estrada-
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Reyes et al., 2018). The vehicle consisted of 1% ethanol in 0.9% saline solution. Control animals (named the saline solution group) were injected with the vehicle during the same
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schedule of administration as that of melatonin group. Two hours after toluene challenge,
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animals were euthanized, their brains were removed and the hippocampus and prefrontal
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cortex were obtained. Samples were stored at −70°C until biochemical assays.
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2.5 Oxidative stress balance
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To analyse the changes in pro-oxidant molecules, we evaluated ROS and nitrites plus nitrates (an indirect estimation of NO) levels. On the other hand, partial antioxidant capacity was evaluated through SOD activity and the glutathione reduced/glutathione oxidized ratio (GSH/GSSG ratio).
2.5.1 Reactive oxygen species (ROS) This parameter was evaluated via oxidation and deacetylation of 2´7´-dichlorofluorescein diacetate
(DCFH-DA),
which
is converted
to
the fluorescent
compound,
2´7´-
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dichlorofluorescein (DCF), by free radicals. The determination was conducted with homogenates of hippocampus and prefrontal cortex with 40 nM pH 7.4 TRIS-HEPES buffer (18:1). A standard curve was prepared using increasing concentrations of DCF with TRISHEPES buffer (9:1). For the test, we used a microplate reader Biotek instrument. Fluorescence signals were recorded at 488 nm excitation and 525 nm emission for 1 h at
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37°C with constant stirring. Total protein was analysed by the Bicinchoninic acid (BCA)
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method. The results were expressed as pmol of 2´7´-dichlorofluorescein formed per
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2.5.2 Nitric oxide (NO)
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milligram of protein/min.
Nitric oxide production was estimated by measuring nitrates and nitrites, the oxidation
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products of nitric oxide. Nitrites were measured with the Griess reagent. The procedure consisted of homogenizing the tissues with 1 mL of distilled water in a Teflon-glass system
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on ice. The samples were then centrifuged at 12 500 rpm for half an hour. Then, 500 μl of the supernatant was collected and transferred to Eppendorf tubes with Millipore filtration
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system for 3 kDa and centrifuged again at 7 500 rpm for 15 min. A 100 μL aliquot was transferred to a 96-well plate and the following reagents were added: 50 mU Nitrate Reductase and 25 μM β-NADPH. This mixture was incubated for 30 minutes at room temperature, and then 100 mU of glutamic dehydrogenase, 100 mM ammonium chloride,
and 4 mM α-ketoglutaric acid were added. The mixture was incubated for 30 minutes at room temperature and Griess's reagent was then added. The plate was placed in a Biotek Eon plate reader for UV-vis at 548 nm absorbance. A calibration curve of nitrites was
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performed along with the test. The final result was expressed as μMol of nitrites / g wet tissue.
2.5.3 Glutathione determination
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Glutathione (GSH), in its reduced form, is one the most important endogenous antioxidant
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molecule in cells. Under oxidative stress, reduced glutathione is utilized to inactivate
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hydrogen peroxide, and at the same time, oxidized glutathione (GSSG) is produced;
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therefore, by measuring the GSH/GSSG ratio we obtained a close estimate of tissue oxidative status. For GSH quantification, the tissue was homogenized in 250 µl PB-EDTA
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buffer solution (0.1 M-0.005 M, pH 8) in a Teflon-glass system, on ice. The samples were
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then centrifuged at 12 500 rpm for 30 minutes at 4°C. An aliquot was taken and diluted with
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PB-EDTA to be mixed with 100 μL of OPA reagent and then incubated for 15 minutes at room temperature. Finally, fluorescence was determined at 350 nm excitation wavelength
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and 420 nm emission (Perkin Elmer LS-50). For the quantification of GSSG, a 250 μL aliquot of the original homogenate was
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transferred to a clean tube and 100 μL of N-ethyl-maleimide 0.4 mM (NEM) reagent was added; the mix was incubated at room temperature for 30 min. Subsequently, the sample was diluted with NaOH 0.1 M and then 100 μL of OPA was added and incubated for 15 min
at room temperature. Fluorescence was determined for GSH as well. All samples were run in duplicate. The results are expressed as μmol/g wet tissue.
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2.5.4 Superoxide dismutase (SOD) activity
The SOD enzymatic activity was measured through the reduction of cytochrome C by superoxide generated from the Xanthine-Xanthine oxidase system.
The tissue samples were homogenized with a buffer solution of 20 mM sodium
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Bicarbonate- 0.02% Triton X-100 at pH 10.2 and then centrifuged at 5 000 g, and
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supernatants were stored for SOD activity. A reaction mixture (substrate) was prepared
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previously with 10 μM sodium azide, 100 μM Xanthine, 10 μM reduced cytochrome C and
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1 mM EDTA in the bicarbonate buffer. The solution was stored in a water bath at 37°C
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throughout the test.
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The assay started with the addition of 0.08 U Xanthine oxidase to 2.9 ml of the reaction mixture and 50 µl of sample supernatant. Changes in the absorbance at 560 nm were
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recorded for 3 minutes, every 30 seconds (Lambda 25 spectrophotometer, Perkin Elmer). Mn-SOD activity was calculated as the total activity minus the activity inhibited by 1 mM
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KCN in the reaction mixture, as this substance directly inhibits the Cu/Zn-SOD isoform. The
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results were reported as international units, SOD / g wet tissue / min.
2.6 Experimental design 2.6.1 Chronic exposure to toluene
In the first set of experiments, we explored the effect of chronic toluene exposure (for four weeks) on free radicals-related variables, in hippocampus and prefrontal cortex from mice. At the beginning of the experiments, 35-40 days-old Swiss-Webster mice were allocated
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into two groups:, 1) a non-exposed control, and 2) a solvent exposed group (4 000 ppm airborne inhalation toluene, 30 min/day for 4 weeks). Control animals were submitted to inhalation chamber 30 min daily, in the same way as exposed rodents. After this period, animals were euthanized and oxidative stress markers were determined, (ROS, GSH/GSSG, nitrites, and SOD activity) in hippocampus and frontal cortex, as described
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above.
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2.6.2 Treatments
To explore the possibility to reverse the oxidative damage caused by toluene exposure, we
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used two experimental approaches, EE and melatonin. The effect of these treatments was
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compared. For this purpose, we used another set of experiment that consisted in: a non-
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exposed air-treated group (n=5) and a group of rodents exposed to 4000 ppm toluene (n=20). After 4 weeks of exposure, mice were divided into 4 groups (n=5 each). 1) A
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subgroup that was allocated in standard vivarium conditions during 4 weeks (Std), 2) a subgroup of mice housed in enriched environment (EE) for 4 weeks, 3) a control subgroup
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receiving vehicle solution consisting in ethanol 1% in saline solution (SS) (daily, during 4 weeks), and 4) a melatonin-treated (10 mg/Kg, i.p. daily/ 4 weeks) subgroup (Mel). At the end of all treatments, animals were challenge with a single exposure of 4000 ppm toluene (during 30 min), afterwards mice were euthanized and their hippocampus and frontal cortex were obtained. Subsequently, biochemical analyses were conducted to assay ROS,
GSH/GSSG, nitrites, and SOD activity. It is important to note that due to the long-term exposure of both, toluene and EE, the evaluation of the biochemical studies were carried
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out at the adulthood of mice.
Statistical analysis
All data were expressed as the mean ± SE. To analyse the effects of chronic toluene exposure on oxidative markers, a Student’s t-test was conducted. To evaluate the action of
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EE or melatonin on oxidative stress, in animals previously exposed to toluene, one-way ANOVAs followed by Student- Newman-Keuls Test were performed. In all cases, p-values
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≤0.05 were considered statistically significant. All statistical analyses were carried out using
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the Sigma-Stat program (version 3.5, Jandel Scientific).
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3. Results
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3.1 Chronic toluene exposure
The effects of toluene on the production of pro-oxidant molecules are shown in Fig. 1A-D.
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2A-F.
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The GSH/GSSG ratio is depicted in Fig. 1 E-F. SOD isoform activities are shown in Fig.
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Mice repeatedly exposed to toluene shown significantly enhanced ROS levels in the hippocampus (p = 0.003; Fig. 1A), while the levels had a tendency to increase in the prefrontal cortex (p = 0.141; Fig. 1B). On the other hand, nitrite levels were significantly
increased in both brain areas evaluated (p = 0.036 hippocampus, Fig. 1C and p =.035 prefrontal cortex, Fig. 1 D).
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On the other hand, toluene produced a significant reduction of the GSH/GSSG ratio, both in the hippocampus (p = 0.044; Fig. 1E) and in the prefrontal cortex (p = 0.005; Fig. 1F). Total SOD activity was significantly reduced in the hippocampus from toluene-exposed rodents (p = 0.004; Fig. 2A), and the same effect was observed with Cu/Zn-SOD activity in
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this brain area (p = 0.003; Fig. 2E). Mn-SOD activity was unaltered after toluene
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administration in the hippocampus (p = 0.0334; Fig. 2C). As to the prefrontal cortex, none
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of the SOD isoform enzymes nor the SOD total showed a significant change in activity after
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repeated toluene exposure (p = 0.582 SOD total, p = 0.690 Mn-SOD, p = 0.878 Cu/ZnSOD; Fig. 2B, 2D and 2F). Altogether, these results suggest that toluene modified the
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redox balance, enhancing the pro-oxidant biomarkers and reducing antioxidant capacity;
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this effect was more pronounced in the hippocampus than in the prefrontal cortex.
3.2 Environmental enrichment and melatonin
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The effect of EE and melatonin on the parameters of oxidative stress is shown in Fig. 3 and
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4. The data showed that repeated toluene exposure followed by standard housing (toluene + Std) significantly enhanced the ROS levels in the hippocampus and the prefrontal cortex (second bar, Fig. 3A and B), when compared with the air + Std (first bar, Fig. 3A and B). Similarly, toluene + SS group significantly increased the ROS levels in the hippocampus (fourth bar, Fig. 3A), and the tendency to increase was observed in the prefrontal cortex
(fourth bar, Fig. 3B). In contrast, repeated toluene exposure and EE housing (toluene + EE) resulted in a significant reduction of the ROS levels in the hippocampus (Fig. 3A), while no statistically significant changes were observed in the prefrontal cortex (Fig. 3B). A
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similar pattern in the reduction of ROS was obtained with repeated toluene exposure and melatonin (toluene + Mel), compared with the toluene + SS group, in the hippocampus and prefrontal cortex (Fig. 3A and B). Both, EE and melatonin comparatively reduced ROS levels, with respect to their control groups [hippocampus (F4,20 = 87.002; p < 0.001);
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prefrontal cortex (F4,20 = 12.637; p < 0.001)].
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A similar pattern of effects, as those observed with ROS, was obtained in the nitrites
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evaluation in the hippocampus (F4,20 = 10.677; p < 0.001), except that the toluene + SS group did not show significant changes compared with the air + Std group (Fig. 3C, first
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versus fourth bar). On the other hand, no significant changes were obtained in the
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prefrontal cortex in any of the groups evaluated (F4,20 = 1.631; p = 0.206) (Fig. 3D).
In animals exposed to toluene and afterwards treated with either EE or melatonin, the
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GSH/GSSG ratio in the hippocampus was increased, when compared with its respective control group (F4,20 = 39.550; p < 0.001) (Fig. 3E, second versus third bar, and fourth
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versus fifth bar). In the prefrontal cortex, the toluene + EE group showed a significant increase in the GSH/GSSG ratio compared with air + Std, while the GSH/GSSG ratio was not changed in animals with a history of toluene exposure and treated with melatonin (F4,20 = 21.747; p < 0.001).
The total SOD analysis showed that EE and melatonin treatments produced a significant change when compared with their control treatments (standard housing or saline solution)
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in the hippocampus (F5,25 = 9.223; p < 0.001); although the effects of EE or melatonin were comparable with those of the air + Std group. In the prefrontal cortex, all treatments resulted in SOD total activity below that obtained in the air + Std group. However, the effect of EE and melatonin was significantly higher than those obtained with their control groups (F4,20 = 52.448; p < 0.001). In the case of Mn-SOD activity, all groups showed a response
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below to that produced by the air + Std group, although the effect was most evident in the
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prefrontal cortex. Notwithstanding, only melatonin (but not EE) treatment showed a
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significant change compared with its control group [hippocampus (F 4,20 = 30.361; p <
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0.001); prefrontal cortex (F4,20 = 27.333; p < 0.001)] (Fig. 4C and D). Finally, the activity of
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Cu/Zn-SOD was significantly increased in both treatments (EE or melatonin) in the
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hippocampus when compared with their control groups (F4,20 = 8.787; p < 0.001) (Fig. 4E). Similarly, in the prefrontal cortex, EE and melatonin significantly increased Cu/Zn-SOD
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activity compared with toluene + Std or toluene + SS, respectively; although the effects were below that obtained with the air + Std group (F4,20 = 23.497; p < 0.001) (Fig. 4EF).
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Altogether, these results suggest that EE has the ability to rescue the redox imbalance, buffering pro-oxidant activity and triggering antioxidant defences. Moreover, the effects
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observed with EE were comparable with those induced by melatonin treatment.
4. Discussion
The main results from the present study showed that chronic toluene exposure in mice (4000 ppm/30 min day/ 4 weeks) resulted in increased pro-oxidant biomarkers (ROS, nitrites), in both the hippocampus and prefrontal cortex, while decreased markers related to
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antioxidant capacity (GSH/GSSG ratio and SOD activity), mainly in the hippocampus. Conversely, EE was able to rescue from the effects produced by toluene, as this decreased ROS and nitrites, while it enhanced the GSH/GSSG ratio and SOD activity. The comparison between treatments showed that EE produced similar effects than those of melatonin, in the pro-oxidant and the antioxidant parameters; the only differences were
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where EE produced a larger effect than melatonin.
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found in the GSH/GSSG ratio in the prefrontal cortex and Mn-SOD in the hippocampus,
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In agreement with our results, there is some evidence showing that different patterns of
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toluene administration produce oxidative stress. Mattia and colleagues (1993) described
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that a single toluene administration in rats (0.5, 1.0, and 1.5 g/kg, i.p.) increased ROS in the hippocampus, cerebellum and striatum, along with enhanced lipid peroxidation at the
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hippocampus. Similarly, 3000 ppm paint thinner chronic administration (toluene 60–70%; 1 h/day for 45 days) increased lipid peroxidation in the hippocampus, cortex and cerebellum
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(Baydas et al., 2003). To our knowledge, this is the first report showing the effects of toluene on NO production; although, the data also support the idea of enhancement in pro-
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oxidative molecules.
The evaluation of partial brain antioxidant capacity was conducted through two biomarkers, the GSH/GSSG ratio and SOD (total and two isoforms). The results showed that toluene
reduced brain antioxidant defences by decreasing the GSH/GSSG ratio, and the SOD activity in the hippocampus. In contrast to the present results, a previous study showed that rats exposed to paint thinner for 45 days did not show significant changes in reduced
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glutathione (Baydas et al., 2003). The difference may be related to the nature of the substance analysed (paint thinner versus toluene), the pattern of administration (3000 ppm/1 h a day/45 days vs 4000 ppm/30 min a day/4 weeks in present study) or the animal species studied (rats vs mice). As to the SOD activity, to our knowledge, there are no studies in the literature analysing this parameter after toluene exposure. The present
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results showed a reduction in the activity of SOD in the hippocampus but not in the
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prefrontal cortex. Thus, the hippocampus appears to be more sensitive to the effects of
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toluene, at least after the present pattern of administration studied. Furthermore, the
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deleterious action of toluene on total SOD may involve Cu/Zn-SOD, as this compound did not modify the activity of Mn-SOD. Since the Cu/Zn-SOD enzyme is located in the cytosol,
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while the Mn-SOD enzyme is found in the mitochondria, the alterations in antioxidant
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capacity of this enzyme after toluene exposure appears to be confined to the cytosol.
Altogether, the evidence suggests that toluene administration may lead to redox imbalance
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in favour of oxidant activity that potentially will lead to apoptosis, as it has been observed in previous studies (Paez-Martinez et al., 2013). If this assumption is correct, possible
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neuronal death in the hippocampus could explain the alterations in memory observed in chronically exposed toluene animals (Montes et al., 2017). The mechanisms behind these effects may involve NMDA receptors. In vitro studies have shown that acute toluene administration inhibits NMDA receptor currents (Cruz et al., 1998); however, repeated administration induced the opposite effects and enhanced calcium currents carried by
NMDA receptors (Bale et al., 2005). The enhancement of the activity in NMDA may cause a dangerous triggering of Ca2+-dependent pathways. One important approach of the present study was that some of the pathological consequences of toluene exposure may
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be mediated through ROS and NO. The present results seem to support this idea, as ROS and NO production were elevated after repeated toluene administration.
After treatment with EE or melatonin, animals were challenged with a dose of toluene, with the aim to induce a new oxidative stress state and to evaluate the capability of those
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treatments to buffer it. The treatment of EE in animals with a previous history with toluene
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dampened the production of the assayed pro-oxidant molecules (ROS and NO) in the
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hippocampus (Fig. 3A and C). In contrast, the same challenging dose in animals with
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standard housing maintained the high levels of ROS and NO in the hippocampus. A similar effect was observed in the prefrontal cortex regarding the production of ROS, whereas no
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changes in nitrites were observed in this brain area. These results suggest once again that
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the hippocampus appears to be more sensitive to the pro-oxidative effects induced by
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toluene than the prefrontal cortex. On the other hand, EE appears to enhance the antioxidant defences in the hippocampus, as the challenge with toluene enhanced the
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GSH/GSSG ratio, as well as the activity of total SOD and Cu/Zn-SOD, in which such an effect was not observed in the toluene + Std group. These results suggest that without
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environmental stimulation, hippocampal cells could still be vulnerable to oxidative stress and that toluene abstinence by itself does not produce spontaneous recovery in the redox balance. Similar to the chronic toluene exposure, the challenge with toluene activates Cu/Zn-SOD; this result suggests that the SOD cytosolic activity has an important role in toluene-induced injury. In the prefrontal cortex, SOD enzymes also respond to the
challenge with toluene, but at a lower proportion than hippocampal neurons. The present findings are in agreement with previous studies showing that EE reduced oxidative stress, evaluated through different biomarkers (i.e., lipid peroxidation levels and superoxide anion
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activity) in brain areas such as the hippocampus, the prefrontal cortex and striatum, or in the whole brain (Cechetti et al., 2012; Herring et al., 2010; Marmol et al., 2015). In parallel, EE housing has also been associated with increased antioxidant defences, enhancing SOD activity or even upregulating SOD genetic expression (Cechetti et al., 2012; Herring
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et al., 2010; Marmol et al., 2015).
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Melatonin, a pineal-derived hormone, is known to reduce oxidative stress-based
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neurotoxicity (Reiter et al., 2016; Srinivasan, 2002). Thus, the aim to evaluate this
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substance in the present study was to compare its effect with that of EE on oxidative stress induced by toluene. In all cases, 10 mg/kg daily melatonin, dosed for four weeks, produced
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similar antioxidant effects as those resulting from housing animals with an enrichment
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environment for 4 weeks. The only differences between treatments were found in the
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GSH/GSSG ratio in the prefrontal cortex and Mn-SOD in the hippocampus; in both cases, EE produced larger effects than melatonin. The present results are in line with the study of
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Baydas and colleagues (2003), where the administration of melatonin after paint thinner exposure (3000 ppm 1 h/day for 45 days) reduced lipid peroxidation levels and increased
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GSH levels after inhalation. It is important to mention that the ability of melatonin to rescue from toluene oxidative unbalance could be explained due to its properties as a radical scavenger, of both oxygen and nitrogen reactive species (Reiter et al., 2016); also to its action to stimulate antioxidant defences, including glutathione peroxidase, glutathione reductase and superoxide dismutase (Reiter et al., 2000; Reiter et al., 2018). Moreover, in
the present study the evaluation of melatonin´s effect was conducted four hours after last administration, thus we suggest that responses observed are the consequence of
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cumulative dosage, more likely than the result of the last single administration.
Altogether, present findings suggest that EE conferred the ability to rescue from oxidative stress induced by toluene exposure, and such effects are comparable with melatonin, a compound with potent antioxidant properties. Although translation from preclinical to clinical settings is still in his infancy (McDonald et al., 2018), some approaches have shown
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that enriched environment, or some of its components, have been used in human patients
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aimed to provide protection from neurodegeneration (Tanaka et al., 2009), for a faster
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recovery from stroke (McDonald et al., 2018) and ameliorating some of the symptoms of
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autism (Woo and Leon, 2013). Since results observed in humans have been promising, we believe that mechanistic basic studies, as the present, contribute and support to the use of
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environmental enrichment as an excellent alternative for treating patients dependent to
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inhalants.
5. Conclusion
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The present study confirmed that toluene induces an oxidative imbalance in brain areas, such as the hippocampus and prefrontal cortex. This oxidative stress could be, in turn,
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related to some other effects induced by toluene, such as the apoptotic process and memory impairment. While toluene abstinence by itself was not enough to spontaneously repair alterations in oxidative stress induced by toluene; EE or melatonin had the ability to restore redox imbalance after toluene exposure. Beneficial effects of environmental
enrichment and melatonin set them as possible therapeutic alternatives to counteract deleterious effects induced by toluene.
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Acknowledgements This paper includes data from the Master's dissertation by Yepci Guadalupe Yee Ríos. This study was supported by projects SIP-Instituto Politécnico Nacional and Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz NC103380.2.
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Declarations of interest: none.
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Figure captions Fig. 1. Effect of repeated toluene administration (4000 ppm/30 min a day/4 weeks) on the
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levels of pro-oxidant biomarkers ROS (A, B), nitrites (C, D) and the antioxidant levels of the GSH/GSSG ratio (E, F) in the hippocampus (left panels) and prefrontal cortex (right panels). Data are expressed as the mean ± SEM, n=5. *p<0.05, Student t-test.
Fig. 2. Activity of total SOD (A, B), Mn-SOD (C, D) and Cu/Zn-SOD (E, F) in the
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hippocampus and prefrontal cortex of mice exposed chronically to toluene. Data are
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expressed as the mean ± SEM, n=5. *p<0.05, Student t-test.
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Fig. 3. Effects of environmental enrichment or melatonin on biomarkers of oxidative stress in animals previously exposed to toluene. The pro-oxidant markers, ROS and NO
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(indirectly evaluated as nitrite formation), as well as the GSH/GSSG ratio, were studied in
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the hippocampus and prefrontal cortex. Std = standard housing, EE = environmental
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enrichment, SS = saline solution group, Mel = melatonin administration. Data are expressed as the mean ± SEM of group values, n=5. * p ≤ 0.05, Student-Newman-Keuls
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test.
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Fig. 4. SOD activity in mice chronically exposed to toluene and later housed in EE or administered with melatonin. Upper panels represent the effects of treatments on total SOD activity in the hippocampus and prefrontal cortex. Middle panels show the effects of EE or melatonin on Mn-SOD activity and low panels represent the effects of the treatments on Cu/Zn-SOD activity. Std = standard housing, EE = environmental enrichment, SS =
saline solution group, Mel = melatonin administration. Data are expressed as the mean ±
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SEM of group values, n=5. * p ≤ 0.05, Student-Newman-Keuls test.
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S0361-9230(18)30041-8 https://doi.org/10.1016/j.brainresbull.2018.11.007 BRB 9544
To appear in:
Brain Research Bulletin
Received date: Revised date: Accepted date:
20 January 2018 28 August 2018 14 November 2018
Please cite this article as: Montes S, Yee-Rios Y, P´aez-Mart´ınez N, Environmental enrichment restores oxidative balance in animals chronically exposed to toluene: Comparison with melatonin, Brain Research Bulletin (2018), https://doi.org/10.1016/j.brainresbull.2018.11.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Environmental enrichment restores oxidative balance in animals chronically exposed to toluene: comparison with melatonin
a
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Sergio Montesa, Yepci Yee-Riosb, Nayeli Páez-Martínezb,c*
Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía
Manuel Velasco Suarez. Avenida Insurgentes Sur No. 3877. Ciudad de México. C.P. 14269. México. b
Sección de Posgrado e Investigación, Escuela Superior de Medicina, Instituto
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Politécnico Nacional. Plan de San Luis y Díaz Mirón. Ciudad de México. C.P.
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Laboratorio Integrativo para el Estudio de Sustancias Inhalables Adictivas.
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c
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11340. México.
Dirección de Investigaciones en Neurociencias, Instituto Nacional de Psiquiatría
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C.P. 14370. México.
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Ramón de la Fuente Muñiz. Calzada México Xochimilco 101. Ciudad de México.
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*Corresponding author: Dr. Nayeli Páez Martínez.
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Sección de Posgrado e Investigación. Escuela Superior de Medicina, Instituto Politécnico Nacional. Plan de San Luis y Díaz Mirón, Col. Santo Tomás, Ciudad de México. C.P. 11340. México. Tel.: +52 (55) 4160-5116 Fax: +52 (55) 5655-9980 E-mail: [email protected]
HIGHLIGHTS
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Chronic toluene exposure induced an oxidative unbalance in the hippocampus and prefrontal cortex. EE restored oxidative balance in animals previously exposed to toluene. Effects of EE were similar to those obtained with melatonin. Abstinence did not spontaneously repair the alterations in oxidative stress induced by toluene.
ABSTRACT
Inhalants are widely used as recreational drugs, and toluene is the main chemical
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compound present in most inhalants used for these purposes. Previous studies have
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shown that repeated toluene exposure produces cellular death and memory impairment,
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while environmental enrichment (EE) rescues from those effects. However, the
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mechanisms involved in those responses are unclear. Previous studies have shown that toluene induces a redox imbalance at the neuronal level; although, details on the
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mechanism of action of environmental enrichment enhancing antioxidant capacity remain
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to be explored. It is also unexplored whether this putative antioxidant capacity is similar to
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that produced by pharmacological antioxidants. To study this hypothesis, Swiss-Webster male mice were chronically exposed to toluene (0 or 4000 ppm, 30 min/day/4 weeks).
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Subsequently, neurochemical tests were conducted to measure biomarkers of oxidative stress (ROS, NO, GSH/GSSG ratio and SOD activity) in the hippocampus and the
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prefrontal cortex. In the second part of the study, we evaluated the putative antioxidant capacity of environmental enrichment and compared it to that of melatonin, a known free radical scavenger and inductor of antioxidant defences. The results showed that chronic toluene exposure increased the levels of pro-oxidative molecules and decreased the
antioxidant markers. Conversely, environmental enrichment restored oxidative balance in animals previously exposed to toluene. Furthermore, the effects of EE were similar to those obtained with melatonin. Altogether, alterations in oxidative balance could represent an
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intermediate signalling pathway in the cascade of effects induced by toluene, while EE and melatonin appear to have the ability to rescue those effects.
Keywords: environmental enrichment, melatonin, oxidative stress, toluene
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1. Introduction
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Deliberate consumption of inhalants for intoxication purposes is practised worldwide, and
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its use has emerged as a relatively common problem among children and adolescents
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(Lubman et al., 2008). Toluene-based products (paint thinner, glues, and “Activo” [a toluene-enriched formulation distributed by drug dealers]) are inhalants most commonly
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used in Mexico (Villatoro et al., 2011). Despite their important deleterious health
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consequences, inhalants continue to be one of the least studied psychoactive substances. Therefore, treatment and prevention research has only recently emerged (Howard et al.,
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2011). In previous studies from our lab, it was observed that environmental enrichment
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(EE) reversed memory impairment and reduced neuronal death in animals previously exposed to toluene (Montes et al., 2017; Paez-Martinez et al., 2013); however, the
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mechanisms involved are still unclear.
It has been shown that NMDA receptors (channels permeable to Ca2+) are molecular targets of toluene (Cruz et al., 1998). Furthermore, chronic toluene exposure enhanced NMDA-mediated currents and increased the expression of NR2A and NR2B NMDA
receptor subunits in neuronal cell cultures (Bale et al., 2005). It is well known that NMDA receptors play a key role in long-term potentiation, even though overstimulation of NMDA receptors causes excitotoxicity. Pathological increases of Ca2+ ions into the postsynaptic
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neurons activate the Ca2+-dependent pathways (Feldman et al., 1997; Marks et al., 2009). On one hand, this pathway leads to metabolic changes such as arachidonic acid metabolism and generating reactive oxygen species (ROS). On the other hand, Ca2+ activates neuronal NO synthase, which synthesises nitric oxide (NO), while NO, as a free radical, is transformed to a highly reactive product, peroxynitrite, upon reaction with ROS
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(Feldman et al., 1997; Marks et al., 2009). Altogether, these reactions may result in
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oxidative stress that has been observed to result in apoptosis.
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Oxidative stress is defined as the imbalance between cellular pro-oxidants and antioxidants in favour of oxidant activity that potentially leads to tissue damage (Uttara et al., 2009).
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There is some evidence in the literature showing that toluene exerts part of its toxicity via
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oxidative stress. For instance, toluene administration (0.5, 1 and 1.5 g/kg, i.p.) induced
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ROS production in synaptosomal fractions from the striatum, hippocampus and cerebellum. Additionally, 1 g/kg toluene increased lipid peroxidation in the hippocampus (Mattia et al.,
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1993). Similarly, paint thinner vapours (3000 ppm/1 h/day for 45 days) enhanced lipid
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peroxidation in the hippocampus, cortex and cerebellum (Baydas et al., 2003).
In contrast, EE is a housing condition that provides social, cognitive and physical stimulation (Nithianantharajah and Hannan, 2006). Several studies have shown that EE reduces the levels of oxidative biomarkers (such as lipid peroxidation, protein oxidation, and free radical levels) while enhancing values of antioxidant parameters (such as catalase
and SOD activity), when compared with standard housing rodents (Cechetti et al., 2012; Herring et al., 2010; Marmol et al., 2015). Furthermore, the expression of the SOD enzyme
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is increased in mice housed in EE conditions (Herring et al., 2010).
On the other hand, melatonin is an endogenous mitochondria-targeted molecule with high efficacy in reducing oxidative damage. This molecule possesses a variety of mechanisms by which it reaches its antioxidant effects. Melatonin and its metabolites are radical scavengers of reactive oxygen and reactive nitrogen species and they have the ability to
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bind heavy metals, which enhance melatonin’s capacity to quench oxidative damage
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(Reiter et al., 2016). Melatonin also interacts with membrane receptors, which indirectly
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stimulates antioxidant enzymes, including glutathione peroxidase and glutathione
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reductase, superoxide dismutase, while suppressing the activity of pro-oxidant enzymes
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(Reiter et al., 2000; Reiter et al., 2018).
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Altogether, the main aims of the present study were to evaluate oxidative stress induced by
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chronic toluene exposure and to analyse whether EE restores the imbalance in oxidative stress in mice with a history of toluene exposure. Finally, as melatonin is a compound with
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potent antioxidant properties (Reiter et al., 2000), it was of our interest to compare the effects induced by EE with those produced by melatonin in terms of antioxidant mechanism
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of action.
2. Material and methods 2.1 Animals
Swiss-Webster male mice (postnatal day 35-40) were used in the present study. This stage was selected because mice at this age exhibit behavioral, neurochemical and endocrine patterns of human adolescent subjects (Abreu-Villaca et al., 2008; Burns and Proctor,
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2013; Laviola et al., 2003) and because this age extrapolates to the age of onset of inhalant abuse in humans (Dell et al., 2011). All animals were maintained under inverted controlled light-dark cycle conditions [12 h:12 h; lights on at 19:00 h, zeitgeber time = 0 (ZT0)] at 22 to 26°C and had access to food and water ad libitum. All experimental procedures followed the regulations of the Mexican Official Norm NOM-062-ZOO-1999 for
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the use and care of laboratory animals (DOF, 2001) and had the approval of the Ethics
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Committee for Animal Experimentation of the “Instituto Nacional de Psiquiatría Ramón de
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la Fuente Muñiz, Mexico” (IACUC: CEI/C/059/2016).
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2.2 Toluene exposure
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The exposure was conducted in a 27-litre glass cylindrical inhalation chamber. The
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chamber was covered with a polycarbonate lid that was fitted with an injection port and a fan that projected into the chamber above a stainless-steel mesh platform. At the beginning
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of exposure, two mice were placed in the bottom of the chamber and the lid was set. A predetermined volume of toluene (purity 99.9%; Sigma-Aldrich, Toluca, Mexico) was
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delivered through an injection port, via a microsyringe, onto a filter paper located on a stainless-steel mesh (Nelson, 1971). After removing the needle, the fan was turned on to distribute the solvent into the glass chamber. Animals were exposed either to 0 ppm (air or control group) or 4000 ppm toluene vapours (Sigma-Aldrich, Toluca Mexico). The exposure was conducted thirty minutes a day, for four weeks. After two hours of the last toluene
administration, animals were euthanized, their brains were removed and hippocampus and prefrontal cortex were quickly dissected out and stored at −70°C until biochemical assays. These brain regions were selected because they are involved in processes impaired by
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toluene administration, such as memory in the object recognition test, neurogenesis and neuronal death.
2.3 Environmental enrichment
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Independent groups of animals were exposed to toluene (4000 ppm, 30 min/day for 4
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weeks); afterwards, animals were housed with EE or standard housing conditions. A
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control group consisted of mice exposed to air and then housed in standard conditions. EE
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animals were maintained in larges home cages of 34 x 44 x 20 cm, in groups of 5. The environment included a series of 5 objects of different shapes, sizes and textures. Objects
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included toys and tunnels; these objects were changed twice a week to stimulate animals´
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curiosity. In addition, a wheel was included so that the animals had the opportunity to
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perform voluntary exercise. Environmental enrichment was carried out 24 hours a day, for 4 weeks. Standard housing groups (n= 5) were placed in cages (18 ×28 × 15 cm) without
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any stimulation during 4 weeks. At the end of the housing period, animals were challenged with a single dose of toluene (4000 ppm/30 min). The intention of this administration was to
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prime a new oxidative stress state and to evaluate the ability of EE to buffer this potential oxidative damage. Two hours after challenge, animals were euthanized, their brains were removed and the hippocampus and prefrontal cortex were obtained. Samples were stored at −70°C until biochemical assays.
2.4 Melatonin administration Similar to the previous protocol, mice were exposed to toluene for 4 weeks and
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subsequently administered melatonin daily (Sigma-Aldrich, Toluca, México) for 4 more weeks (10 mg/kg, i.p.) (Chabra et al., 2014; El-Sokkary et al., 2002; Solis-Munoz et al., 2011). Length of treatment was stablished (four weeks) to have the possibility to compare melatonin effect with that of EE; however it is important to mention that the dose of
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melatonin selected considered the animal species utilized and the chronicity of treatment.
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Melatonin was administered at zeitgeber time 11 (ZT11), which represent one hour before
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endogenous indolamine starts to rise; at this point the levels of melatonin are low (Estrada-
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Reyes et al., 2018). The vehicle consisted of 1% ethanol in 0.9% saline solution. Control animals (named the saline solution group) were injected with the vehicle during the same
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schedule of administration as that of melatonin group. Two hours after toluene challenge,
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animals were euthanized, their brains were removed and the hippocampus and prefrontal
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cortex were obtained. Samples were stored at −70°C until biochemical assays.
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2.5 Oxidative stress balance
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To analyse the changes in pro-oxidant molecules, we evaluated ROS and nitrites plus nitrates (an indirect estimation of NO) levels. On the other hand, partial antioxidant capacity was evaluated through SOD activity and the glutathione reduced/glutathione oxidized ratio (GSH/GSSG ratio).
2.5.1 Reactive oxygen species (ROS) This parameter was evaluated via oxidation and deacetylation of 2´7´-dichlorofluorescein diacetate
(DCFH-DA),
which
is converted
to
the fluorescent
compound,
2´7´-
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dichlorofluorescein (DCF), by free radicals. The determination was conducted with homogenates of hippocampus and prefrontal cortex with 40 nM pH 7.4 TRIS-HEPES buffer (18:1). A standard curve was prepared using increasing concentrations of DCF with TRISHEPES buffer (9:1). For the test, we used a microplate reader Biotek instrument. Fluorescence signals were recorded at 488 nm excitation and 525 nm emission for 1 h at
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37°C with constant stirring. Total protein was analysed by the Bicinchoninic acid (BCA)
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method. The results were expressed as pmol of 2´7´-dichlorofluorescein formed per
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2.5.2 Nitric oxide (NO)
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milligram of protein/min.
Nitric oxide production was estimated by measuring nitrates and nitrites, the oxidation
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products of nitric oxide. Nitrites were measured with the Griess reagent. The procedure consisted of homogenizing the tissues with 1 mL of distilled water in a Teflon-glass system
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on ice. The samples were then centrifuged at 12 500 rpm for half an hour. Then, 500 μl of the supernatant was collected and transferred to Eppendorf tubes with Millipore filtration
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system for 3 kDa and centrifuged again at 7 500 rpm for 15 min. A 100 μL aliquot was transferred to a 96-well plate and the following reagents were added: 50 mU Nitrate Reductase and 25 μM β-NADPH. This mixture was incubated for 30 minutes at room temperature, and then 100 mU of glutamic dehydrogenase, 100 mM ammonium chloride,
and 4 mM α-ketoglutaric acid were added. The mixture was incubated for 30 minutes at room temperature and Griess's reagent was then added. The plate was placed in a Biotek Eon plate reader for UV-vis at 548 nm absorbance. A calibration curve of nitrites was
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performed along with the test. The final result was expressed as μMol of nitrites / g wet tissue.
2.5.3 Glutathione determination
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Glutathione (GSH), in its reduced form, is one the most important endogenous antioxidant
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molecule in cells. Under oxidative stress, reduced glutathione is utilized to inactivate
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hydrogen peroxide, and at the same time, oxidized glutathione (GSSG) is produced;
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therefore, by measuring the GSH/GSSG ratio we obtained a close estimate of tissue oxidative status. For GSH quantification, the tissue was homogenized in 250 µl PB-EDTA
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buffer solution (0.1 M-0.005 M, pH 8) in a Teflon-glass system, on ice. The samples were
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then centrifuged at 12 500 rpm for 30 minutes at 4°C. An aliquot was taken and diluted with
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PB-EDTA to be mixed with 100 μL of OPA reagent and then incubated for 15 minutes at room temperature. Finally, fluorescence was determined at 350 nm excitation wavelength
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and 420 nm emission (Perkin Elmer LS-50). For the quantification of GSSG, a 250 μL aliquot of the original homogenate was
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transferred to a clean tube and 100 μL of N-ethyl-maleimide 0.4 mM (NEM) reagent was added; the mix was incubated at room temperature for 30 min. Subsequently, the sample was diluted with NaOH 0.1 M and then 100 μL of OPA was added and incubated for 15 min
at room temperature. Fluorescence was determined for GSH as well. All samples were run in duplicate. The results are expressed as μmol/g wet tissue.
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2.5.4 Superoxide dismutase (SOD) activity
The SOD enzymatic activity was measured through the reduction of cytochrome C by superoxide generated from the Xanthine-Xanthine oxidase system.
The tissue samples were homogenized with a buffer solution of 20 mM sodium
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Bicarbonate- 0.02% Triton X-100 at pH 10.2 and then centrifuged at 5 000 g, and
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supernatants were stored for SOD activity. A reaction mixture (substrate) was prepared
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previously with 10 μM sodium azide, 100 μM Xanthine, 10 μM reduced cytochrome C and
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1 mM EDTA in the bicarbonate buffer. The solution was stored in a water bath at 37°C
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throughout the test.
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The assay started with the addition of 0.08 U Xanthine oxidase to 2.9 ml of the reaction mixture and 50 µl of sample supernatant. Changes in the absorbance at 560 nm were
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recorded for 3 minutes, every 30 seconds (Lambda 25 spectrophotometer, Perkin Elmer). Mn-SOD activity was calculated as the total activity minus the activity inhibited by 1 mM
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KCN in the reaction mixture, as this substance directly inhibits the Cu/Zn-SOD isoform. The
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results were reported as international units, SOD / g wet tissue / min.
2.6 Experimental design 2.6.1 Chronic exposure to toluene
In the first set of experiments, we explored the effect of chronic toluene exposure (for four weeks) on free radicals-related variables, in hippocampus and prefrontal cortex from mice. At the beginning of the experiments, 35-40 days-old Swiss-Webster mice were allocated
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into two groups:, 1) a non-exposed control, and 2) a solvent exposed group (4 000 ppm airborne inhalation toluene, 30 min/day for 4 weeks). Control animals were submitted to inhalation chamber 30 min daily, in the same way as exposed rodents. After this period, animals were euthanized and oxidative stress markers were determined, (ROS, GSH/GSSG, nitrites, and SOD activity) in hippocampus and frontal cortex, as described
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above.
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2.6.2 Treatments
To explore the possibility to reverse the oxidative damage caused by toluene exposure, we
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used two experimental approaches, EE and melatonin. The effect of these treatments was
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compared. For this purpose, we used another set of experiment that consisted in: a non-
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exposed air-treated group (n=5) and a group of rodents exposed to 4000 ppm toluene (n=20). After 4 weeks of exposure, mice were divided into 4 groups (n=5 each). 1) A
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subgroup that was allocated in standard vivarium conditions during 4 weeks (Std), 2) a subgroup of mice housed in enriched environment (EE) for 4 weeks, 3) a control subgroup
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receiving vehicle solution consisting in ethanol 1% in saline solution (SS) (daily, during 4 weeks), and 4) a melatonin-treated (10 mg/Kg, i.p. daily/ 4 weeks) subgroup (Mel). At the end of all treatments, animals were challenge with a single exposure of 4000 ppm toluene (during 30 min), afterwards mice were euthanized and their hippocampus and frontal cortex were obtained. Subsequently, biochemical analyses were conducted to assay ROS,
GSH/GSSG, nitrites, and SOD activity. It is important to note that due to the long-term exposure of both, toluene and EE, the evaluation of the biochemical studies were carried
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out at the adulthood of mice.
Statistical analysis
All data were expressed as the mean ± SE. To analyse the effects of chronic toluene exposure on oxidative markers, a Student’s t-test was conducted. To evaluate the action of
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EE or melatonin on oxidative stress, in animals previously exposed to toluene, one-way ANOVAs followed by Student- Newman-Keuls Test were performed. In all cases, p-values
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≤0.05 were considered statistically significant. All statistical analyses were carried out using
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the Sigma-Stat program (version 3.5, Jandel Scientific).
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3. Results
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3.1 Chronic toluene exposure
The effects of toluene on the production of pro-oxidant molecules are shown in Fig. 1A-D.
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2A-F.
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The GSH/GSSG ratio is depicted in Fig. 1 E-F. SOD isoform activities are shown in Fig.
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Mice repeatedly exposed to toluene shown significantly enhanced ROS levels in the hippocampus (p = 0.003; Fig. 1A), while the levels had a tendency to increase in the prefrontal cortex (p = 0.141; Fig. 1B). On the other hand, nitrite levels were significantly
increased in both brain areas evaluated (p = 0.036 hippocampus, Fig. 1C and p =.035 prefrontal cortex, Fig. 1 D).
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On the other hand, toluene produced a significant reduction of the GSH/GSSG ratio, both in the hippocampus (p = 0.044; Fig. 1E) and in the prefrontal cortex (p = 0.005; Fig. 1F). Total SOD activity was significantly reduced in the hippocampus from toluene-exposed rodents (p = 0.004; Fig. 2A), and the same effect was observed with Cu/Zn-SOD activity in
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this brain area (p = 0.003; Fig. 2E). Mn-SOD activity was unaltered after toluene
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administration in the hippocampus (p = 0.0334; Fig. 2C). As to the prefrontal cortex, none
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of the SOD isoform enzymes nor the SOD total showed a significant change in activity after
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repeated toluene exposure (p = 0.582 SOD total, p = 0.690 Mn-SOD, p = 0.878 Cu/ZnSOD; Fig. 2B, 2D and 2F). Altogether, these results suggest that toluene modified the
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redox balance, enhancing the pro-oxidant biomarkers and reducing antioxidant capacity;
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this effect was more pronounced in the hippocampus than in the prefrontal cortex.
3.2 Environmental enrichment and melatonin
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The effect of EE and melatonin on the parameters of oxidative stress is shown in Fig. 3 and
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4. The data showed that repeated toluene exposure followed by standard housing (toluene + Std) significantly enhanced the ROS levels in the hippocampus and the prefrontal cortex (second bar, Fig. 3A and B), when compared with the air + Std (first bar, Fig. 3A and B). Similarly, toluene + SS group significantly increased the ROS levels in the hippocampus (fourth bar, Fig. 3A), and the tendency to increase was observed in the prefrontal cortex
(fourth bar, Fig. 3B). In contrast, repeated toluene exposure and EE housing (toluene + EE) resulted in a significant reduction of the ROS levels in the hippocampus (Fig. 3A), while no statistically significant changes were observed in the prefrontal cortex (Fig. 3B). A
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similar pattern in the reduction of ROS was obtained with repeated toluene exposure and melatonin (toluene + Mel), compared with the toluene + SS group, in the hippocampus and prefrontal cortex (Fig. 3A and B). Both, EE and melatonin comparatively reduced ROS levels, with respect to their control groups [hippocampus (F4,20 = 87.002; p < 0.001);
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prefrontal cortex (F4,20 = 12.637; p < 0.001)].
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A similar pattern of effects, as those observed with ROS, was obtained in the nitrites
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evaluation in the hippocampus (F4,20 = 10.677; p < 0.001), except that the toluene + SS group did not show significant changes compared with the air + Std group (Fig. 3C, first
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versus fourth bar). On the other hand, no significant changes were obtained in the
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prefrontal cortex in any of the groups evaluated (F4,20 = 1.631; p = 0.206) (Fig. 3D).
In animals exposed to toluene and afterwards treated with either EE or melatonin, the
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GSH/GSSG ratio in the hippocampus was increased, when compared with its respective control group (F4,20 = 39.550; p < 0.001) (Fig. 3E, second versus third bar, and fourth
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versus fifth bar). In the prefrontal cortex, the toluene + EE group showed a significant increase in the GSH/GSSG ratio compared with air + Std, while the GSH/GSSG ratio was not changed in animals with a history of toluene exposure and treated with melatonin (F4,20 = 21.747; p < 0.001).
The total SOD analysis showed that EE and melatonin treatments produced a significant change when compared with their control treatments (standard housing or saline solution)
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in the hippocampus (F5,25 = 9.223; p < 0.001); although the effects of EE or melatonin were comparable with those of the air + Std group. In the prefrontal cortex, all treatments resulted in SOD total activity below that obtained in the air + Std group. However, the effect of EE and melatonin was significantly higher than those obtained with their control groups (F4,20 = 52.448; p < 0.001). In the case of Mn-SOD activity, all groups showed a response
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below to that produced by the air + Std group, although the effect was most evident in the
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prefrontal cortex. Notwithstanding, only melatonin (but not EE) treatment showed a
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significant change compared with its control group [hippocampus (F 4,20 = 30.361; p <
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0.001); prefrontal cortex (F4,20 = 27.333; p < 0.001)] (Fig. 4C and D). Finally, the activity of
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Cu/Zn-SOD was significantly increased in both treatments (EE or melatonin) in the
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hippocampus when compared with their control groups (F4,20 = 8.787; p < 0.001) (Fig. 4E). Similarly, in the prefrontal cortex, EE and melatonin significantly increased Cu/Zn-SOD
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activity compared with toluene + Std or toluene + SS, respectively; although the effects were below that obtained with the air + Std group (F4,20 = 23.497; p < 0.001) (Fig. 4EF).
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Altogether, these results suggest that EE has the ability to rescue the redox imbalance, buffering pro-oxidant activity and triggering antioxidant defences. Moreover, the effects
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observed with EE were comparable with those induced by melatonin treatment.
4. Discussion
The main results from the present study showed that chronic toluene exposure in mice (4000 ppm/30 min day/ 4 weeks) resulted in increased pro-oxidant biomarkers (ROS, nitrites), in both the hippocampus and prefrontal cortex, while decreased markers related to
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antioxidant capacity (GSH/GSSG ratio and SOD activity), mainly in the hippocampus. Conversely, EE was able to rescue from the effects produced by toluene, as this decreased ROS and nitrites, while it enhanced the GSH/GSSG ratio and SOD activity. The comparison between treatments showed that EE produced similar effects than those of melatonin, in the pro-oxidant and the antioxidant parameters; the only differences were
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where EE produced a larger effect than melatonin.
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found in the GSH/GSSG ratio in the prefrontal cortex and Mn-SOD in the hippocampus,
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In agreement with our results, there is some evidence showing that different patterns of
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toluene administration produce oxidative stress. Mattia and colleagues (1993) described
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that a single toluene administration in rats (0.5, 1.0, and 1.5 g/kg, i.p.) increased ROS in the hippocampus, cerebellum and striatum, along with enhanced lipid peroxidation at the
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hippocampus. Similarly, 3000 ppm paint thinner chronic administration (toluene 60–70%; 1 h/day for 45 days) increased lipid peroxidation in the hippocampus, cortex and cerebellum
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(Baydas et al., 2003). To our knowledge, this is the first report showing the effects of toluene on NO production; although, the data also support the idea of enhancement in pro-
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oxidative molecules.
The evaluation of partial brain antioxidant capacity was conducted through two biomarkers, the GSH/GSSG ratio and SOD (total and two isoforms). The results showed that toluene
reduced brain antioxidant defences by decreasing the GSH/GSSG ratio, and the SOD activity in the hippocampus. In contrast to the present results, a previous study showed that rats exposed to paint thinner for 45 days did not show significant changes in reduced
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glutathione (Baydas et al., 2003). The difference may be related to the nature of the substance analysed (paint thinner versus toluene), the pattern of administration (3000 ppm/1 h a day/45 days vs 4000 ppm/30 min a day/4 weeks in present study) or the animal species studied (rats vs mice). As to the SOD activity, to our knowledge, there are no studies in the literature analysing this parameter after toluene exposure. The present
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results showed a reduction in the activity of SOD in the hippocampus but not in the
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prefrontal cortex. Thus, the hippocampus appears to be more sensitive to the effects of
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toluene, at least after the present pattern of administration studied. Furthermore, the
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deleterious action of toluene on total SOD may involve Cu/Zn-SOD, as this compound did not modify the activity of Mn-SOD. Since the Cu/Zn-SOD enzyme is located in the cytosol,
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while the Mn-SOD enzyme is found in the mitochondria, the alterations in antioxidant
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capacity of this enzyme after toluene exposure appears to be confined to the cytosol.
Altogether, the evidence suggests that toluene administration may lead to redox imbalance
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in favour of oxidant activity that potentially will lead to apoptosis, as it has been observed in previous studies (Paez-Martinez et al., 2013). If this assumption is correct, possible
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neuronal death in the hippocampus could explain the alterations in memory observed in chronically exposed toluene animals (Montes et al., 2017). The mechanisms behind these effects may involve NMDA receptors. In vitro studies have shown that acute toluene administration inhibits NMDA receptor currents (Cruz et al., 1998); however, repeated administration induced the opposite effects and enhanced calcium currents carried by
NMDA receptors (Bale et al., 2005). The enhancement of the activity in NMDA may cause a dangerous triggering of Ca2+-dependent pathways. One important approach of the present study was that some of the pathological consequences of toluene exposure may
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be mediated through ROS and NO. The present results seem to support this idea, as ROS and NO production were elevated after repeated toluene administration.
After treatment with EE or melatonin, animals were challenged with a dose of toluene, with the aim to induce a new oxidative stress state and to evaluate the capability of those
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treatments to buffer it. The treatment of EE in animals with a previous history with toluene
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dampened the production of the assayed pro-oxidant molecules (ROS and NO) in the
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hippocampus (Fig. 3A and C). In contrast, the same challenging dose in animals with
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standard housing maintained the high levels of ROS and NO in the hippocampus. A similar effect was observed in the prefrontal cortex regarding the production of ROS, whereas no
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changes in nitrites were observed in this brain area. These results suggest once again that
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the hippocampus appears to be more sensitive to the pro-oxidative effects induced by
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toluene than the prefrontal cortex. On the other hand, EE appears to enhance the antioxidant defences in the hippocampus, as the challenge with toluene enhanced the
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GSH/GSSG ratio, as well as the activity of total SOD and Cu/Zn-SOD, in which such an effect was not observed in the toluene + Std group. These results suggest that without
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environmental stimulation, hippocampal cells could still be vulnerable to oxidative stress and that toluene abstinence by itself does not produce spontaneous recovery in the redox balance. Similar to the chronic toluene exposure, the challenge with toluene activates Cu/Zn-SOD; this result suggests that the SOD cytosolic activity has an important role in toluene-induced injury. In the prefrontal cortex, SOD enzymes also respond to the
challenge with toluene, but at a lower proportion than hippocampal neurons. The present findings are in agreement with previous studies showing that EE reduced oxidative stress, evaluated through different biomarkers (i.e., lipid peroxidation levels and superoxide anion
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activity) in brain areas such as the hippocampus, the prefrontal cortex and striatum, or in the whole brain (Cechetti et al., 2012; Herring et al., 2010; Marmol et al., 2015). In parallel, EE housing has also been associated with increased antioxidant defences, enhancing SOD activity or even upregulating SOD genetic expression (Cechetti et al., 2012; Herring
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et al., 2010; Marmol et al., 2015).
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Melatonin, a pineal-derived hormone, is known to reduce oxidative stress-based
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neurotoxicity (Reiter et al., 2016; Srinivasan, 2002). Thus, the aim to evaluate this
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substance in the present study was to compare its effect with that of EE on oxidative stress induced by toluene. In all cases, 10 mg/kg daily melatonin, dosed for four weeks, produced
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similar antioxidant effects as those resulting from housing animals with an enrichment
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environment for 4 weeks. The only differences between treatments were found in the
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GSH/GSSG ratio in the prefrontal cortex and Mn-SOD in the hippocampus; in both cases, EE produced larger effects than melatonin. The present results are in line with the study of
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Baydas and colleagues (2003), where the administration of melatonin after paint thinner exposure (3000 ppm 1 h/day for 45 days) reduced lipid peroxidation levels and increased
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GSH levels after inhalation. It is important to mention that the ability of melatonin to rescue from toluene oxidative unbalance could be explained due to its properties as a radical scavenger, of both oxygen and nitrogen reactive species (Reiter et al., 2016); also to its action to stimulate antioxidant defences, including glutathione peroxidase, glutathione reductase and superoxide dismutase (Reiter et al., 2000; Reiter et al., 2018). Moreover, in
the present study the evaluation of melatonin´s effect was conducted four hours after last administration, thus we suggest that responses observed are the consequence of
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cumulative dosage, more likely than the result of the last single administration.
Altogether, present findings suggest that EE conferred the ability to rescue from oxidative stress induced by toluene exposure, and such effects are comparable with melatonin, a compound with potent antioxidant properties. Although translation from preclinical to clinical settings is still in his infancy (McDonald et al., 2018), some approaches have shown
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that enriched environment, or some of its components, have been used in human patients
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aimed to provide protection from neurodegeneration (Tanaka et al., 2009), for a faster
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recovery from stroke (McDonald et al., 2018) and ameliorating some of the symptoms of
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autism (Woo and Leon, 2013). Since results observed in humans have been promising, we believe that mechanistic basic studies, as the present, contribute and support to the use of
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environmental enrichment as an excellent alternative for treating patients dependent to
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inhalants.
5. Conclusion
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The present study confirmed that toluene induces an oxidative imbalance in brain areas, such as the hippocampus and prefrontal cortex. This oxidative stress could be, in turn,
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related to some other effects induced by toluene, such as the apoptotic process and memory impairment. While toluene abstinence by itself was not enough to spontaneously repair alterations in oxidative stress induced by toluene; EE or melatonin had the ability to restore redox imbalance after toluene exposure. Beneficial effects of environmental
enrichment and melatonin set them as possible therapeutic alternatives to counteract deleterious effects induced by toluene.
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Acknowledgements This paper includes data from the Master's dissertation by Yepci Guadalupe Yee Ríos. This study was supported by projects SIP-Instituto Politécnico Nacional and Instituto Nacional de Psiquiatría Ramón de la Fuente Muñiz NC103380.2.
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Declarations of interest: none.
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Figure captions Fig. 1. Effect of repeated toluene administration (4000 ppm/30 min a day/4 weeks) on the
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levels of pro-oxidant biomarkers ROS (A, B), nitrites (C, D) and the antioxidant levels of the GSH/GSSG ratio (E, F) in the hippocampus (left panels) and prefrontal cortex (right panels). Data are expressed as the mean ± SEM, n=5. *p<0.05, Student t-test.
Fig. 2. Activity of total SOD (A, B), Mn-SOD (C, D) and Cu/Zn-SOD (E, F) in the
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hippocampus and prefrontal cortex of mice exposed chronically to toluene. Data are
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expressed as the mean ± SEM, n=5. *p<0.05, Student t-test.
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Fig. 3. Effects of environmental enrichment or melatonin on biomarkers of oxidative stress in animals previously exposed to toluene. The pro-oxidant markers, ROS and NO
D
(indirectly evaluated as nitrite formation), as well as the GSH/GSSG ratio, were studied in
TE
the hippocampus and prefrontal cortex. Std = standard housing, EE = environmental
EP
enrichment, SS = saline solution group, Mel = melatonin administration. Data are expressed as the mean ± SEM of group values, n=5. * p ≤ 0.05, Student-Newman-Keuls
CC
test.
A
Fig. 4. SOD activity in mice chronically exposed to toluene and later housed in EE or administered with melatonin. Upper panels represent the effects of treatments on total SOD activity in the hippocampus and prefrontal cortex. Middle panels show the effects of EE or melatonin on Mn-SOD activity and low panels represent the effects of the treatments on Cu/Zn-SOD activity. Std = standard housing, EE = environmental enrichment, SS =
saline solution group, Mel = melatonin administration. Data are expressed as the mean ±
A
CC
EP
TE
D
M
A
N
U
SC RI PT
SEM of group values, n=5. * p ≤ 0.05, Student-Newman-Keuls test.
D
TE
EP
CC
A
SC RI PT
U
N
A
M
D
TE
EP
CC
A
SC RI PT
U
N
A
M
D
TE
EP
CC
A
SC RI PT
U
N
A
M