Neuroscience Letters 624 (2016) 1722

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Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet

Research paper

URB597 inhibits oxidative stress induced by alcohol binging in the

prefrontal cortex of adolescent rats
Renan Pelico a , Matheus C. Santos a , Leandro C. Freitas-Lima b , Silvana S. Meyrelles a ,
Elisardo C. Vasquez a , Ester M. Nakamura-Palacios a , Lvia C.M. Rodrigues a,
a
b

Department of Physiological Sciences, CCS/UFES, Vitria, ES, Brazil

Department of Physiology and Biophysics, ICB/UFMG, Belo Horizonte, MG, Brazil

h i g h l i g h t s
Acute and chronic binge ethanol induces oxidative stress in PFC of adolescent rats.
Previous administration of URB597 inhibits oxidative stress caused by binge alcohol.
Increased availability of anandamide is related to an antioxidant effect in PFC.

a r t i c l e

i n f o

Article history:
Received 23 February 2016
Received in revised form 9 April 2016
Accepted 30 April 2016
Available online 2 May 2016
Keywords:
Alcohol
Binge
Prefrontal cortex
Oxidative stress
URB597
Antioxidant effect

a b s t r a c t
Heavy episodic drinking (binging), which is highly prevalent among teenagers, results in oxidative damage. Because the prefrontal cortex (PFC) is not completely mature in adolescents, this brain region may
be more vulnerable to the effects of alcohol during adolescence. As endocannabinoids may protect the
immature PFC from the harmful effects of high doses of alcohol, this study investigated the effect of the
fatty acid amide hydrolase (FAAH) inhibitor URB597 on oxidative stress induced by acute or chronic binge
alcohol intake in adolescent rats. At 40 min after intraperitoneal pre-treatment with URB597 (0.3 mg/kg)
or vehicle (Veh), ethanol (EtOH; 3 or 6 g/kg, intragastrically) or distilled water (DW) was administered in 3
consecutive sessions (acute binging) or 3 consecutive sessions over 4 weeks (chronic binging). Oxidative
stress in PFC slices in situ was measured by dihydroethidium uorescence staining. At the higher EtOH
dose (6 g/kg), pre-treatment with URB597 signicantly reduced (p < 0.01) the production of superoxide
anions in the PFC after acute (42.8% decrease) and chronic binge EtOH consumption (44.9% decrease) compared with pre-treatment with Veh. As URB597 decreases anandamide metabolism, this evidence shows
an antioxidant effect of endocannabinoids to suppress acute and chronic binge alcohol intake-induced
oxidative stress in the PFC of adolescent rats.
2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Heavy episodic or binge drinking has been dened by the
National Institute on Alcohol Abuse and Alcoholism (NIAAA) as a
pattern of alcohol drinking that results in blood alcohol concentrations (BACs) of approximately 0.08 g-percent or above [1]. This
type of consumption, which is highly prevalent in adolescents [2,3],
can lead to alterations in brain structure, physiology and behavior
[46]. Binge drinking can lead to neurotoxic damage by inducing oxidative stress, a serious imbalance between reactive oxygen

Corresponding author at: Av. Marechal Campos, 1468, Marupe, Vitria, Esprito
Santo, 29043-900, Brazil.
E-mail address: livia.melo.rodrigues@gmail.com (L.C.M. Rodrigues).
http://dx.doi.org/10.1016/j.neulet.2016.04.068
0304-3940/ 2016 Elsevier Ireland Ltd. All rights reserved.

species and antioxidant mechanisms [7], in the central nervous

system [812]. Indeed, ethanol (EtOH) causes severe toxicity and
degeneration in different brain areas [13], notably the prefrontal
cortex (PFC) [1416].
The PFC is responsible for integrating cortical and subcortical
inputs to modulate essential cognitive functions, such as attention,
working memory, planning, response inhibition and decisionmaking [17]. It is also a key structure involved in the brain reward
system [18,19], which provides cognitive control over the functions
that are relevant to drug addiction [2022]. Thus, the neurotoxic
damage caused by EtOH abuse, specically its damage to the immature PFC of adolescents, may account for the observed decits in
behavior and cognition among young alcohol abusers [1,2325]
that may last in adulthood.

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R. Pelico et al. / Neuroscience Letters 624 (2016) 1722

The endocannabinoid system is a central regulatory system

that modulates a wide range of biological processes. Arachidonoylethanolamine (anandamideAEA), one of the most studied
endocannabinoids [26], is inactivated by fatty acid amide hydrolases (FAAH). AEA plays an important role in neuronal plasticity
and memory and exhibits protective properties against neuronal
injury [2729]. To the best of our knowledge, under heavy alcohol
dosage, the potential antioxidant effects of AEA on the immature
brain via selective inhibition of FAAH in have not been investigated.
This study examined the effect of an inhibitor of FAAH, URB597, on
brain oxidative stress induced by acute or chronic alcohol binging
in adolescent rats.
2. Materials and methods
2.1. Animals
Eighty-one male Wistar rats (30 days old at the beginning of the
experiments) obtained from our animal care facilities were used.
The animals were provided with free access to water and standard
commercial chow and were housed in groups of four per cage on
a 12:12 h light/dark cycle in a temperature-controlled (22 1 C)
room. The Guide for the Care and Use of Laboratory Animals [30]
and Directive 2010/63/EU were followed in all experiments. This
study was approved by the Local Ethics Committee on Animal Use
for Research.

The slices were incubated in a 2-M DHE-modied Krebs

solution containing 20 mM HEPES for 30 min in a light-protected
chamber at 37 C. DHE acts as a superoxide anion marker, as superoxide oxidizes DHE to form 2-OH-ethidium, which uoresces red
[32,33]. The uorescence intensity was proportional to the tissue
oxidative stress level and was quantied in photomicrographs captured with a microscope (Leica DM 2500) and a camera equipped
with an A4 lter to detect 528 nm light (Leica DFC310FX). The
images were analyzed using ImagePro Plus 4.5 software.
2.5. Experimental design
The animals received Veh or 0.3 mg/kg URB597 intraperitoneally, and 40 min later, the animals received distilled water or
3 or 6 g/kg EtOH intragastrically. The animals were divided into six
independent treatment groups: (1) Veh followed by DW, (2) Veh
followed by EtOH 3, (3) Veh followed by EtOH 6, (4) URB597 followed by DW, (5) URB597 followed by EtOH 3, and (6) URB597
followed by EtOH 6. Drug administration was performed for three
consecutive days (acute binge, n = 35) or for three consecutive
days over four weeks (chronic binge, n = 36), between 8:00 and
11:00 a.m. The animals were weighed daily to track weight gain.
Then, they were euthanized 24 h after the nal drug administration,
and their brains were removed for the superoxide anion production
assay.
2.6. Statistical analysis

2.2. Drugs
EtOH (Dinmica Qumica Contempornea, Diadema, SP, Brazil)
(99.5%) was diluted in distilled water to 10% or 20% (w/v).
EtOH was intragastrically administered at a dose of 3 (EtOH 3)
or 6 g/kg (EtOH 6). URB597 (3 -(aminocarbonyl)[1,1 -biphenyl]3-yl)-cyclohexyl carbamate (Cayman Chemicals Company, Ann
Arbor, MI, USA) was stored at 20 C until dilution to a concentration of 0.3 mg/ml in a vehicle (Veh) solution consisting of
phosphate-buffered saline (PBS): dimethyl sulfoxide (2:1) (pH 7.5)
immediately before intraperitoneal (IP) administration at a volume of 0.1 ml/100 g body weight. The dose of 0.3 mg/kg URB597
was based on its pharmacological prole as determined by Piomelli
et al. [31]. Veh (for IP administration) and distilled water (DW) (for
intragastric (IG) administration) were used as control solutions.
2.3. BAC measurement
To evaluate the BAC after IG administration, ten alcohol-naive
rats received a 3 g/kg (n = 5) or 6 g/kg (n = 5) dose of EtOH one hour
prior to euthanasia. The rats were decapitated, and trunk blood
samples were collected in sterile tubes containing EDTA and uorite to prevent coagulation. The blood samples were rapidly frozen
and maintained at 20 C until the time of the assay. The BACs
were determined via gas spectrometry coupled to a ame detector
(Varian 450-GC/FID) using headspace automatic injection.
2.4. Detection of superoxide anion production (dihydroethidium
(DHE) assay)
To examine in situ tissue oxidative stress, we adjusted the
assay for DHE (Sigma-Aldrich, MO, USA) to evaluate brain tissue. The animals were deeply anaesthetized with ketamine and
xylazine (75 mg/kg and 10 mg/kg, respectively) and were transcardially perfused with a Krebs-HEPES solution (pH 7.4) at a pressure
of 100 mmHg. The brains were removed and stored at 80 C. Then,
the brains were serially sectioned into slices of approximately
14 m using a cryostat (CM 1850, Leica, Nussloch, Germany) for
analysis of the PFC.

Data are presented as means SEM. Two-way analysis of variance (ANOVA) followed by the LSD post hoc test was used for the
DHE assay. For BACs, statistical analysis was performed using Students t-test for independent samples. A two-tailed level of 0.05
was considered signicant. SPSS 17.0 software was used for statistical analysis, and GraphPad Prism 5.0 (GraphPad Software Inc., San
Diego, CA, USA) was used for graphic presentations.
3. Results
IG administration of EtOH has been established to effectively
and proportionally yield high EtOH concentrations in blood. The
mean BACs measured 1 h after IG EtOH administration at 3 or 6 g/kg
(n = 5 per group) were 11.5 0.6 and 24.6 1.5 dg/L, respectively.
Fig. 1 (top panel) shows DHE uorescence in representative
PFC sections after acute EtOH binging; DHE uorescence was more
intense at the higher dose of EtOH (EtOH 6) (Fig. 1a). Notably, this
pattern was not observed when URB597 was administered before
EtOH binging at both EtOH doses (Fig. 1b). The average DHE uorescence values of the 6 groups are shown in Fig. 1. The uorescence
intensity produced by DHE oxidation in PFC slices from adolescent
rats was signicantly different [F(2,29) = 3.41, p = 0.047] between
the EtOH doses administered during acute binging. EtOH binging
for three consecutive days at 3 g/kg (p < 0.01) or 6 g/kg (p < 0.001)
increased the production of superoxide anion (by 41.8 and 74.1%,
respectively) compared with vehicle administration; these results
indicates the occurrence of oxidative stress resulting from EtOH
binging. There was also a signicant difference between EtOH
doses [F(1,29) = 9.19, p = 0.005]. Compared with the Veh group, pretreatment with the FAAH inhibitor URB597 signicantly decreased
(p < 0.05 for EtOH 3; p < 0.01 for EtOH 6) the production of superoxide anions induced by acute EtOH binging. The superoxide anion
level was reduced by approximately 27.4 and 42.8% in the EtOH 3
and EtOH 6 groups, respectively (Fig. 1).
Fig. 2 (top panel) shows DHE uorescence in PFC sections after
chronic EtOH binging with or without previous administration of
URB597. The intense uorescence observed after chronic binging
with the higher dose of EtOH (6 g/kg) was not observed in the

R. Pelico et al. / Neuroscience Letters 624 (2016) 1722

19

Fig. 1. Effect of URB597 on oxidative stress induced by acute alcohol binging in the prefrontal cortex of adolescent rats. A, representative longitudinal PFC tissue sections
(14 m thick) showing the characteristic red DHE uorescence produced by the tissue when producing reactive oxygen species, comparing URB597-pre-treated (b) with
vehicle-pre-treated rats (a). Magnication: 10. B, Average DHE uorescence intensity values after acute binging with or without URB597 pre-treatment. The values are
presented as the means SEM (n = 6 per group). *p < 0.05, **p < 0.01 and ***p < 0.001 (two-way ANOVA). (For interpretation of the references to colour in this gure legend,
the reader is referred to the web version of this article.)

PFC of animals pre-treated with URB597. Fig. 2 shows the DHE

uorescence intensity in all 6 groups. The uorescence intensity
produced by DHE oxidation in PFC slices of adolescent rats was
signicantly different between EtOH doses during chronic binging
[F(2.29) = 4.40, p = 0.021]. In the setting of chronic EtOH binging,
the superoxide anion levels in the PFC were signicantly (p < 0.05)
higher in the EtOH 6 group than in the Veh and EtOH 3 groups. This
increase was approximately 103.3% greater than the superoxide
anion level in the Veh group. There was also a difference between
the treatment conditions [F(1.29) = 5.25, p = 0.029]. Pre-treatment
with the FAAH inhibitor URB597 signicantly decreased (p < 0.001)
chronic 6 g/kg EtOH binging-induced superoxide anion production
by 44.9% compared with pre-treatment with the control (Veh).
4. Discussion
Oxidative stress occurs when oxidative events and endogenous
antioxidant mechanisms become imbalanced [34]. As expected,
acute and chronic EtOH binging induced oxidative stress in the
PFC of adolescent rats. Here, we showed that the oxidative stress
induced by acute or chronic EtOH binging (notably at a very high
dose (6 g/kg)) was prevented by pre-treatment with a single dose
of 0.3 mg/kg URB597, an inhibitor of FAAH.
EtOH enhances ROS production and modulates the antioxidant
system in the brain [35]. Moreover, EtOH is well known to cause
severe toxicity and degeneration in the central nervous system [13].

As brain tissue has a moderate capacity to produce antioxidant

substances [36], the consumption of EtOH at high doses may produce excessive amount of free radicals, which in turn may damage
in neurons in different cerebral regions [37,38]. More specically,
consuming large amounts of EtOH (i.e., binging) induces oxidative stress and neuronal death [10]. Our results agree with those of
other studies showing increased oxidative stress in models of acute
and chronic EtOH binging [39,40]. The induction of oxidative stress
in the PFC caused by administration of a single high dose of EtOH
for three consecutive days (acute binging) most likely occurred due
to the direct effect of EtOH on free radical production [41].
Oxidative stress was also primarily observed in the PFC of adolescent rats after chronic (four weeks) EtOH binging, although only
at the higher dose of EtOH (6 g/kg). Physiological changes in the
brain may have occurred in response to successive EtOH exposure,
and such changes may explain the absence of oxidative stress at
the lower dose of EtOH (3 g/kg). This effect suggests that the oxidative stress observed after repetitive exposure to the higher dose of
EtOH may involve different mechanisms than those suggested to
mediate the effects of acute EtOH binging. The sustained generation of toxic metabolites during EtOH metabolism may contribute
to the persistent production of ROS during EtOH degradation. This
increase in ROS levels leads to protein oxidation, lipid peroxidation,
and DNA damage, which can stimulate apoptosis induction pathways [42], contributing to neurotoxicity and neurodegeneration in
individuals with alcoholism [43]. Another aspect to be considered is

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R. Pelico et al. / Neuroscience Letters 624 (2016) 1722

Fig. 2. Effect of URB597 on oxidative stress induced by chronic alcohol binging in the prefrontal cortex of adolescent rats. A, representative longitudinal PFC tissue sections
(14 m thick) showing the characteristic red DHE uorescence produced by the tissue when producing reactive oxygen species, comparing URB597-pre-treated (b) with
vehicle-pretreated rats (a). Magnication: 10. B, Average DHE-uorescence intensity values after chronic binging with or without URB597 pre-treatment. The values are
presented as the means SEM (n = 6 per group). *p < 0.05 and **p < 0.001 (two-way ANOVA). (For interpretation of the references to colour in this gure legend, the reader is
referred to the web version of this article.)

the excitotoxic damage caused by alcohol [44]. One study showed

an excessive glutamate release in a rodent binge drinking model
[45]. Furthermore, enhanced inammatory responses may occur
in response to EtOH binging. Under such conditions, the synthesis and release of pro-inammatory cytokines and the activation of
COX-2 exacerbate the increase in free radical productions [4648].
These potential mechanisms deserve further investigation in the
current model.
Our results demonstrated that the FAAH inhibitor URB597 prevented the induction of oxidative stress caused by acute and chronic
EtOH binging in the PFC of young rats. The dose of URB597 used
(0.3 mg/kg) in this study, which maximally blocks FAAH activity
and substantially increases the anandamide levels [49], does not
evoke classical cannabinoid-like effects (catalepsy, hypothermia or
hyperphagia) and, more importantly, does not have any effect on
rat models of abuse liability [50]. Inhibiting FAAH increases the
concentrations of the endocannabinoid anandamide in the brain
[51,52]. Increases in endocannabinoid concentrations in the central
nervous system have consistently been shown to have neuroprotective effects by inhibiting oxidative stress [53]. This effect is
mediated by CB1 receptors [54], but CB2 [55] and non-CB receptors
[56] are also involved. Additionally, URB597 may exert a neuroprotective effect by reducing ROS accumulation [57]. Therefore,
amplifying endocannabinoid signaling by inhibiting FAAH may be
a valuable strategy for decreasing brain oxidative stress resulting

from alcohol ingestion, ultimately potentially preventing the neurotoxic effects of EtOH without inducing the cannabimimetic side
effects [58].
Classic cannabinoids are potent antioxidants against ROS during ischemic metabolism or in different chronic brain pathologies in
which oxidative stress is a critical event in pathogenesis [59]. Endocannabinoids, such as anandamide and 2-arachidonoylglycerol
(2-AG), are both synthesized and released on demand in response
to different stimuli [60,61] and are involved in a variety of physiological, pharmacological and pathological processes [62], including
a series of synaptic plasticity events [63]. Therefore, as previously
suggested, any event that may increase endocannabinoid activity in
the brain, for example, through the selective inhibition of FAAH and
subsequent increases in anandamide concentrations, represents an
important target for the treatment of neuropsychiatric disorders
[6466] and, as suggested by our current results, for harm reduction
strategies for preventing the neurotoxicity of alcohol abuse.
This study has limitations that must be considered. The DHE uorescence assay is a very simple method that does not examine the
detailed potential antioxidant mechanisms associated with endocannabinoid activity. However, this assay efciently demonstrated
that the oxidative stress induced by EtOH in the PFC was reduced
when anandamide metabolism was inhibited. Its important to
highlight that although the preferred substrate of FAAH is anandamide [67], this enzyme also inhibits other N-acylethanolamines

R. Pelico et al. / Neuroscience Letters 624 (2016) 1722

(PEA and OEA). Therefore, further studies are necessary to elucidate whether these compounds also exert antioxidant effects
on oxidative stress caused by alcohol binging. Another pending research topic is the mechanism underlying the antioxidative
effects induced by FAAH inhibition. URB597 increases anandamide
(PEA and OEA) availability, and anandamide also interact with
PPAR family receptors, which also perform neuroprotective functions such as anti-excitotoxic, anti-inammatory and antioxidant
activities [68]. Further understanding of which pathways (including anti-inammatory pathways) of the endocannabinoid system
are involved in the protection of the brain against the oxidative
damage caused by EtOH as well as other drugs of abuse is needed
to establish the endocannabinoid mechanism as a novel potential
treatment target for the prevention of the harmful effects of drugs
of abuse, especially in young individuals.
In summary, our results demonstrated that acute and chronic
EtOH binging induces oxidative stress in the PFC of adolescent rats
and that this oxidative stress was prevented through the inhibition
of FAAH, an enzyme that metabolize the main endocannabinoid,
anandamide, by the URB597.

[14]

[15]

[16]

[17]

[18]

[19]

[20]

Conict of interest
The authors have no conicts of interest to declare.

[21]

Acknowledgements

[22]

This work was supported by grants from CNPq, CAPES, and

FAPES (Programa Primeiros Projetos (PPP) 53630408/2011).

[23]

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