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Research Article
Cannabidiol Rescues Acute Hepatic Toxicity and
Seizure Induced by Cocaine
Luciano Rezende Vilela,1,2 Lindisley Ferreira Gomides,1
Bruna Araújo David,1Maísa Mota Antunes,1Ariane Barros Diniz,1
Fabrício de Araújo Moreira,2and Gustavo Batista Menezes1
1Laborat´
orio de Imunobiofotˆ
onica, Departamento de Morfologia, Instituto de Ciˆ
encias Biol´
ogicas,
Universidade Federal de Minas Gerais, Avenida Presidente Antˆ
onio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil
2Departamento de Farmacologia, Instituto de Ciˆ
encias Biol´
ogicas, Universidade Federal de Minas Gerais,
Avenida Presidente Antˆ
onio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil
Correspondence should be addressed to Fabr´
ıcio de Ara´
ujo Moreira; farmoreira@yahoo.com.br and
Gustavo Batista Menezes; menezesgb@gmail.com
Received January ; Accepted April
Academic Editor: Donna-Marie McCaerty
Copyright © Luciano Rezende Vilela et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Cocaine is a commonly abused illicit drug that causes signicant morbidity and mortality. e most severe and common
complications are seizures, ischemic strokes, myocardial infarction, and acute liver injury. Here, we demonstrated that acute
cocaine intoxication promoted seizure along with acute liver damage in mice, with intense inammatory inltrate. Considering the
protective role of the endocannabinoid system against cell toxicity, we hypothesized that treatment with an anandamide hydrolysis
inhibitor, URB, or with a phytocannabinoid, cannabidiol (CBD), protects against cocaine toxicity. URB (. mg/kg) abolished
cocaine-induced seizure, yet it did not protect against acute liver injury. Using confocal liver intravital microscopy, we observed that
CBD ( mg/kg) reduced acute liver inammation and damage induced by cocaine and prevented associated seizure. Additionally,
we showed that previous liver damage induced by another hepatotoxic drug (acetaminophen) increased seizure and lethality
induced by cocaine intoxication, linking hepatotoxicity to seizure dynamics. ese ndings suggest that activation of cannabinoid
system may have protective actions on both liver and brain induced by cocaine, minimizing inammatory injury promoted by
cocaine, supporting its further clinical application in the treatment of cocaine abuse.
1. Introduction
Drug abuse and addiction constitute a public health problem
of great importance with a high prevalence worldwide.
AccordingtotheUnitedNationsOceonDrugsandCrime
(UNODC), cocaine, a psychostimulant and psychotomimetic
drug, is among the most abused drugs in the world. e
estimated number of cocaine users globally ranges between
and million (.–.% of the population aged between
and years) []. Besides its toxicity for the cardiovascular
central nervous systems, cocaine causes liver injury in human
and animal models [,]. is drug may lead to severe
acute hepatotoxicity due to hepatocellular necrosis, which
can be life threatening. Indeed, as diminished liver function
contributes to various adverse health eects, hepatotoxicity
has been linked to the mortality in cocaine abusers [].
Oxidative stress (OS) plays a key role in cocaine-induced
hepatotoxicity and is well reported in both humans and ani-
mal models []. Furthermore, several lines of evidence indi-
cate that excessive OS with increased free radical generation,
impairment of mitochondrial respiration, and intense inam-
matory reaction plays a crucial role in cocaine-induced hepa-
totoxicity. OS is associated with the dysregulation of signaling
pathways modulating cell death and survival, which leads to
necrosis and apoptosis of hepatocytes []. Although previous
studies have yielded mechanistic information on cocaine-
induced hepatotoxicity, the current understanding on this
toxic event remains insucient []. Cocaine users have
Hindawi Publishing Corporation
Mediators of Inflammation
Volume 2015, Article ID 523418, 12 pages
http://dx.doi.org/10.1155/2015/523418
Mediators of Inammation
altered levels of proinammatory cytokines and chemokines
in the plasma, indicating that peripheral immune system
adaptations may contribute to the eects of cocaine [,].
It is well described that the prevention of OS generation
might have therapeutic advantages in several diseases [,,
]; therefore, the investigation of putative new compounds
with antioxidant properties might be promising. e herb
Cannabis sativa (“marijuana,” “hemp”) has been known
for centuries due to its abuse-related eects and possible
therapeutic uses, including the treatment of inammation,
diabetes, cancer, aective or neurodegenerative diseases, and
epilepsy-related disorders. Among more than chemical
compounds already characterized from this herb, the two
most investigated are Δ9-tetrahydrocannabinol (Δ9-THC)
and cannabidiol (CBD) [–]. Δ9-THC, which accounts
formostofthetypicalcannabisactions,mayhavethera-
peutic ecacy against several diseases, although its use is
limitedbyitspsychoactiveproperties.us,analternative
for developing cannabis-based medicines could be focusing
on CBD, the major nonpsychotomimetic phytocannabinoid.
is compound acts through multiple pharmacological tar-
gets, including facilitation of the endocannabinoid system,
activation of transient receptor potential vanilloid type-
(TRPV) channel, the peroxisome proliferator-activated
receptor 𝛾(PPAR𝛾), GPR, -hydroxytryptamine receptor
subtype A (-HT1A), the adenosine membrane transporter
phospholipase A2, lipoxygenase (LOX) and cyclooxygenase-
(COX-) enzymes, and Ca2+ homeostasis []. is rich
pharmacology, associated with the fact that it does not
share the psychotomimetic, amnestic, and sedative eects
of Δ9-THC, results in a wide range of potential thera-
peutic interest, including the treatment of epilepsies and
related disorders [,]. Accordingly, a recent clinical
study observed that CBD-enriched cannabis extracts signi-
cantly reduced seizure frequency in children with treatment-
resistant epilepsy, indicating that it could be used as a strategy
when other pharmacological treatments fail [].
Apart from searching for the therapeutic application
of phytocannabinoids, other potential strategy focuses on
the endocannabinoid system. is intercellular message
system comprises the CB1and CB2cannabinoid receptor,
the ligands arachidonoyl ethanolamide (anandamide) and
-arachidonoyl glycerol, their hydrolyzing enzymes (fatty
acid amide hydrolase, FAAH, and monoacyl glycerol lipase,
resp.), and their synthesizing machinery [,]. Among its
numerous biological functions, the endocannabinoid system
has been implicated in protection against deleterious stimuli
in various tissues, including the brain []andtheliver[,
]. Previous studies also demonstrated that CBD possesses
ecient antioxidant and anti-inammatory activities against
ischemia/reperfusion liver injury in rats [,]. Moreover,
CBD restored liver/brain function in a model of hepatic
encephalopathy associated with fulminant hepatic failure
induced in mice revealing that its eects may result from a
combination of itsactions in the liver and brain []. Here, to
investigate if CBD exerts protective by CBD exerts protective
eects in a model of seizures and liver damage resulting from
cocaine intoxication. We have compared the eects of these
phytocannabinoids with the FAAH inhibitor URB, which
facilitate endocannabinoid signaling by selectively increasing
anandamidelevels.Finally,weinvestigatedifprevioushepatic
lesion induced by other compounds (acetaminophen) [–
] would modulate SNC-related cocaine eects.
2. Materials and Methods
2.1. Mice. Male Swiss mice weighing – g from the animal
facility (Centro de Bioterismo, CEBIO) of UFMG were kept
on a h:h dark/light cycle at 22 ± 1∘Cwithfreeaccess
to food and water throughout the experiment. Each animal
was used only once. All experiments were conducted in
accordance with the Ethical Committee for Animal Exper-
imentation (CETEA) of the Federal University of Minas
Gerais (Universidade Federal de Minas Gerais (UFMG)) and
procedures for animal care were previously approved by this
organization under Protocol /.
2.2. Drugs. Cannabidiol ( mg/kg; THC-Pharm, Frankfurt,
Germany, and STI-Pharm, Brentwood, UK) was dissolved in
physiological saline containing tween- at %. URB
(. mg/kg; Tocris) was dissolved in ethanol/cremophor/
saline : : . Cocaine ( mg/kg; Merck, Darmstadt, Ger-
many) was dissolved in physiological saline. e solutions
were prepared immediately before use and injected via
intraperitoneal route in a volume of mL/kg. All doses
were chosen based on previously published studies [–
]. Cannabidiol dose was chosen based on dose-response
curves previously established in our group [–]. Although
most of these studies have focused on the anxiolytic and
antipsychotic eects of this compound, the dose range for its
antiseizure and antiepileptic eects is in the sane range [].
us, we tested anticonvulsant eect of cannabidiol (, ,
and mg/kg) minutes before cocaine administration. At
dose of mg/kg, behavioral parameters of seizure (latency
and duration seizure) are signicantly inhibited when com-
pared to doses of and mg/kg (data not shown).
2.3. Apparatus and Cocaine-Induced Seizures. e animals
were injected intraperitoneally (i.p.) with cocaine ( mg/kg)
and immediately placed in individual chambers ( cm in
diameter with a cm high Plexiglas wall) and observed
during min for the onset of behavior seizures and the
occurrence of death. Seizure was dened as the occurrence
of tail clonus with myoclonic jerks and wild jumping or
convulsions with loss of righting reex. Latency and duration
for myoclonic seizure were also measured in the subset of
mice that progressed to this state.
2.4. Model of Acetaminophen-Induced Liver Injury and Inam-
mation. Acetaminophen (paracetamol; APAP) was orally
administered ( or mg/kg; Sigma) in male Swiss
mice weighing – g from the animal facility (Centro de
Bioterismo, CEBIO) of UFMG aer h of fasting [–].
Control mice received warm sterile saline as vehicle. Aer
h, mice were anesthetized with a mixture of ketamine
( mg/kg) and xylazine ( mg/kg) and killed for serum and
Mediators of Inammation
liver harvesting. In a separate set of experiments, the survival
percentageofAPAP-challengedmicewasevaluatedduring
h. Neutrophil inltration into the liver was measured by
the myeloperoxidase (MPO) activity assay.
2.5. Alanine Aminotransferase Assay (ALT). e alanine
aminotransferase enzyme is present in the cytoplasm of hep-
atocytes and is highly specic for the liver. e measurement
of serum alanine aminotransferase (ALT) is a gold-standard
marker of liver damage. To determine the activity of ALT,
blood samples were centrifuged and the serum was collected
and dosed using a kinetic kit (Bioclin, Brazil) [,].
2.6. Liver Histological Analysis. Livers were collected h
aer induction of ALF and xed by overnight immersion in
% buered formalin. Paran-embedded specimens were
prepared and . 𝜇m sections were stained with hematoxylin
and eosin (H&E) according to a standard protocol. Liver
pathology was assessed by an investigator who was blinded
to the experimental treatment groups in an Olympus BX
microscope (Center Valley, PA, USA) [,].
2.7. Indocyanine Green (Cardiogreen; ICG). e ICG stan-
dard curve was drawn according to the manufacturer’s
instructions. Indocyanine green was completely dissolved
in distilled water and prepared to a nal concentration of
. mg/mL. According to the experimental design, each ani-
mal was given a caudal intravenous injection of indocyanine
green at mg/kg of body weight. One hour aer the ICG
injection, a laparotomy was performed and the aorta was
cut, followed by isolation of plasma with centrifugation at
rpm for min. Plasma (. mL) was extracted and
diluted with saline. Absorbance value of the plasma sam-
ple was determined by spectrophotometry determining the
absorbance of the plasma sample at nm, which was
compared with the value for normal plasma. e standard
curve was used to calculate the serum concentration of ICG.
2.8. Liver Confocal Intravital Microscopy. Liver microcircula-
tion was imaged as described previously [,]. Briey, mice
were anesthetized with a mixture of ketamine ( mg/kg)
and xylazine ( mg/kg). e liver was gently pulled out
through a laparotomy incision and positioned over a Plexiglas
stage for imaging under a confocal inverted microscope
(Nikon Eclipse Ti and C confocal head). For imaging liver
microcirculation and necrosis, mice received endovenously
𝜇g of phycoerythrin-labeled anti-PECAM- (stains liver
sinusoidal endothelial cells; eBiosciences, USA) and 𝜇Lof
the stock solution of Sytox Green (stains extracellular DNA
deposits and necrosis; Life Technologies, USA). e liver
surface was imaged in a custom made stage that holds the
liver in at position. Fluorophores were excited with
andnmlaserslineusingaxobjective.Allimageswere
generated by Volocity soware (.; PerkinElmer, USA).
2.9. Statistical Analysis. Experimental data analysis was per-
formed with one-way analysis of variance (ANOVA) (Tukey’s
post hoc test). 𝑃values <. were considered statistically
signicant. All experiments included 𝑛=6.Dataare
presented as mean ±SEM. Fisher’s exact probability test was
additionally used for specic comparisons between seizure
parameters and hepatic functions. Graphs and statistical
analysis were performed using Prism (GraphPad soware,
USA).
3. Results
3.1. Cocaine Administration Caused Acute Liver Injury and
Inammation. To investigate the hepatotoxic eects of acute
cocaine intake, mice received a single administration of
cocaine ( mg/Kg; i.p.) and livers were imaged under
confocal intravital microscopy. As shown in Figure (a),
control mice had a well-perfused liver microvasculature, as
evidenced by sinusoidal staining by anti-CD (PECAM-
, in red). Also, intravenous administration of a DNA-
binding probe (Sytox green) revealed almost no extracellular
DNA under basal conditions. We have previously shown
that hepatocytes released DNA into the liver, leading to a
widespread hepatic DNA accumulation, which was directly
correlated with injury severity and progression. In line
with this, mice overdosed with cocaine had massive liver
injury, evidenced by lack of perfusion (dark spots on red
channel), which were consistently lled by extracellular DNA,
linking cocaine intake to acute liver injury. Elevated serum
transaminases levels (ALT) conrmed acute hepatotoxicity
induced by cocaine (Figure (b)). To evaluate if such injury
was ultimately impairing liver function, we injected a known
amount of indocyanine green (cardiogreen) [], which is
rapidly cleared from the circulation under control conditions
by the liver. Interestingly, cocaine-overdosed mice presented
a signicant retention of cardiogreen in the serum, suggesting
that liver metabolic function was also impaired (Figure (c)).
In fact, liver histopathology showed that cocaine also caused
liver leukocyte inltration together with an extensive necrosis
(Figures (a) and (b)). Together, these data demonstrated
that acute cocaine intake led to liver injury and inammation.
3.2. Cocaine-Induced Seizure Is Directly Correlated with Liver
Injur y. In order to elucidate the relationship between liver
injury and seizures, we rst studied the dynamics of seizure
due to cocaine overdose. Following a single cocaine injection,
seizure was observed in all animals, manifested as rapid
myoclonic convulsions of forelimbs and tail clonus preceded
by locomotor depression (Figure (a)), as demonstrated by
reduced seizure latency and long lasting seizure (Figure (b)).
Interestingly, we observed a direct correlation between liver
injury and function with seizure duration, suggesting that
reducedlivermetabolismmightpredisposetomoresevere
seizureduetococaineintake(Figure (c)).isisparticularly
relevant in the clinics because it might suggest that patients
with previous liver damage are more susceptible to convulsive
seizure during cocaine overdose. In this direction, we next
investigated if a previous liver injury caused by another
hepatotoxic drug worsens seizure prole induced by cocaine.
For this, we established a model of hepatic injury due to
acetaminophen (APAP; mg/Kg) overdose. As shown in
Mediators of Inammation
PECAM-1
(sinusoids)
DNA (necrosis) Merge
Vehicle
Cocaine
(a)
0
1000
2000
3000
ALT (U/L)
Veh i c l e Cocaine
∗
(b)
Veh i c l e Cocaine
0
10
20
30
40
50 ∗
Cardiogreen (𝜇g/mL)
(c)
F : Cocaine intake causes severe liver injury and malfunction. (a) Liver confocal intravital microscopy showing that while control mice
have a well perfused liver vasculature (evidenced by PE-anti-CD antibody staining, in red) and absence of necrosis (in green), cocaine-
treated mice had several dark areas in the red channel, which are suggestive of malperfusion caused by necrosis (elicited by DNA staining
using Sytox Green). (b) Serum liver transaminase levels and (c) retention of cardiogreen conrmed necrosis. ∗Statistical signicance in
comparison to controls (𝑃 < 0.05). A x objective was used during imaging. One-way ANOVA with Tukey’s post hoc test. Data are expressed
as mean ±S.E.M. (𝑛=6mice/treatment).
Figure (a), APAP caused extensive liver injury following
hs of overdose, and overt DNA deposition (in green)
wasobservedinnonperfusedareas(blackareasinthered
channel). Elevated levels of serum ALT (Figure (b))and
histopathology examination conrmed acute liver injury
(Figure (a)), which occurred concomitantly to hepatic neu-
trophil inltration (Figure (b)). APAP-treated mice that
werechallengedwithasingledoseofcocainehadsig-
nicantly higher lethality in comparison to cocaine alone
(Figure (a)). However, mice treated with higher doses of
APAP had lower seizure incidence (Figure (b))andduration
(Figure (c)), with higher latency (Figure (d)), suggesting
that metabolic brain disorders induced by acute liver failure
can interfere in seizure dynamics, reinforcing the direct
correlation of liver metabolic status with brain function.
3.3. Pretreatment with Cannabidiol (CBD) Reduces Cocaine-
Induced Liver Injur y and Seizure. It is well accepted that
cannabinoids may have anti-inammatory activities and had
been proposed to treat neurological disorders. In this context,
the use of nonpsychotropic molecules is preferable in com-
parison to the herb itself, which also oers the opportunity
to dosage adjustments. Taking into account the relationship
between hepatic inammation and brain disorders, we next
investigated the participation of the cannabinoid system in
our model. For this, we pretreated mice with cannabidiol
(CBD; mg/Kg), a major nonpsychotomimetic phytocan-
nabinoid. In fact, pretreatment with CBD completely rescued
liver injury induced by cocaine, as assessed by reduced
liver DNA deposition (Figure (a)), and reduced serum
ALT (Figure (b))andcardiogreen(Figure (c))levels,
Mediators of Inammation
Cocaine
Veh i c l e
(a)
0
10
20
30
40
50
*
Necrosis area (%)
Veh i c l e C o cain e
∗
0
1
2
3
Histological score
Veh i c l e C o cain e
∗
0
50
100
150
200
Veh i c l e Cocaine
Neutrophils (×104)
∗
(b)
F : Histopathological assessment of cocaine-induced liver injury. (a) Liver histology (H&E) showing that while control mice have a
healthy liver architecture and absence of necrosis, cocaine-treated mice had several necrotic areas with leukocyte inltration (green arrow). (b)
Digital quantication of necrotic areas using ImageJ, histopathological score, and neutrophil inltration (assessed by MPO levels) conrmed
severe liver inammation and necrosis induced by cocaine. ∗Statistical signicance in comparison to controls (𝑃 < 0.05). A x objective was
used during imaging. One-way ANOVA with Tukey’s post hoc test. Data are expressed as mean ±S.E.M(𝑛=6mice/treatment).
which prevented hepatic dysfunction due to cocaine over-
dose. Histopathology analysis conrmed that CDB-treated
mice had no detectable injury due to cocaine injection
(Figure (a)), and lower liver inammation (estimated by
neutrophil inltration) was also observed (Figure (b)). In
line with this, CBD treatment also reduced cocaine-induced
seizure duration and higher latency (Figure (c)). Taking into
account the potent eects of CBD, we next investigated if the
inhibition of degradation of endogenous endocannabinoids
might mimic the benecial eects of the administration
of the exogenous compound (CBD). URB mimicked
the anticonvulsant eects of CBD, and URB-treated mice
had less severe seizure induced by cocaine (Figure (c)).
However, pretreatment with URB caused no detectable eects
of cocaine-induced liver injury, suggesting that despite its
protective central eects on seizure dynamics, exogenous
agonists and higher doses are necessary for reaching the
hepatoprotective eects of cannabinoid system activation.
4. Discussion
In the present study we demonstrated that CBD inhibited
cocaine-induced seizure and liver injury in mice, alleviating
hepatic inammatory process, and reduced cocaine lethality.
e FAAH inhibitor, URB, inhibited seizure but did
not interfere with hepatic inammatory process. We also
observed that previous hepatic dysfunction caused by APAP
increased cocaine-induced seizure and lethality.
Drugabuseandaddictionconstituteapublichealthprob-
lem of great importance with a high prevalence worldwide.
AccordingtotheUnitedNationsOceonDrugsandCrime
(UNODC, ), estimated number of cocaine users globally
ranges between and million (.–.% of the population
aged between and years) []. Acute cocaine abuse may
induce several neurological impairments, including seizures
and fulminant hepatic failure. Since there are no eective
treatments available, it is associated with severe intoxications
Mediators of Inammation
(a)
0
25
50
75
100
*
Seizure (%)
Veh i c l e Cocaine
0
200
400
600
800
∗
Total experimental time
Seizure latency (s)
Veh i c l e C o cain e
0
10
20
30
40
50
Seizure duration (s)
∗
Veh i c l e C o cain e
(b)
0 20406080
0
500
1000
1500
2000
2500
3000
3500
4000
Seizure duration
ALT (U/L)
r2= 0.86
0204060
30
35
40
45
50
Seizure duration
Cardiogreen (𝜇g/mL)
r2= 0.74
(c)
F : High doses of cocaine promote acute behavioral seizure. (a) Representative picture of seizure characterized by rapid myoclonic
convulsions of forelimbs and tail clonus preceded by locomotor depression. (b) Acute overdose induced seizure in all animals represented by
latency reduced and duration increased compared to group vehicle. (c) Positive correlation between seizure duration and acute hepatotoxicity
induced by cocaine (𝑟2= 0.86,𝑃 < 0.02for ALT; 𝑟2= 0.74,𝑃 < 0.05for cardiogreen). ∗Signicantly dierent from vehicle group (∗𝑃< 0.001).
One-way ANOVA with Tukey’s post hoc test. Data are expressed as mean ±S.E.M (𝑛=6mice/treatment).
and deaths [,]. us, our results showed that cannabidiol
displays neuroprotective and hepatoprotective eects against
cocaine toxicity and therefore might be used as future strategy
in clinical emergency. Although our data provide support for
the ecacy of CBD in this condition, careful pharmacologic
studies are needed to further delineate specic mechanisms
of CBD against cocaine toxicity.
e mechanisms involved in cocaine intoxication and
seizure induction have remained unclear. Its psychotomi-
metic and psychostimulant eects are mediated by blockade
of dopamine, serotonin, and norepinephrine reuptake. e
increased stimulation of dopamine D receptors may also
contribute to cocaine-induced seizures []. Clinical [–]
and experimental [,–] observations demonstrate that
the acute administration of high doses of this drug promotes
convulsive seizures and liver abnormalities or fulminant
hepatic failure. Less attention, however, has been directed
towards possible hepatotoxic eects []. is drug induces
increase in ALT activity and pervasive centrilobular foci of
necrosis crowded with pale and swollen hepatocytes [].
Several case reports have described hepatic injury from
cocaine use, including cases in which cocaine-induced liver
damage resulted in mortality [].
Here, we described for the rst time that previous treat-
ment with phytocannabinoid CBD abolished cocaine in-
duced acute seizure in mice, indicating anticonvulsant eect,
in parallel with anti-inammatory eect and protection
against liver injury. CBD is the major nonpsychoactive com-
ponent of cannabis that exerts multiple pharmacological
actions in the central nervous system and in the periphery,
Mediators of Inammation
PECAM-1
(sinusoids)
DNA (necrosis) Merge
Vehicle
APAP
(a)
0
1000
2000
3000
ALT (U/L)
Vehicle APAP
∗
(b)
F : Acetaminophen model of acute liver injury and failure. (a) Liver confocal intravital microscopy showing that while control mice
have a well perfused liver vasculature (evidenced by PE-anti-CD antibody staining, in red) and absence of necrosis (in green), APAP-
treated mice ( mg/Kg) had several dark areas in the red channel, which are suggestive of malperfusion caused by necrosis (elicited by
DNA staining using Sytox Green). (b) Serum liver transaminase levels conrmed necrosis. ∗Statistical signicance in comparison to controls
(𝑃 < 0.05). A x objective was used during imaging. One-way ANOVA with Tukey’s post hoc test. Data are expressed as mean ±S.E.M(𝑛=6
mice/treatment).
APAP
Veh i c l e
(a)
0
1
2
3∗
Histological score
Veh i c l e A PAP
0
100
200
300
400
∗
Neutrophils (×104)
Veh i c l e A PAP
0
20
40
60
80
∗
Necrosis area (%)
Veh i c l e A PAP
(b)
F : Histopathological assessment of acetaminophen-induced liver injury (APAP). (a) Liver histology (H&E) showing that while control
mice have a healthy liver architecture and absence of necrosis, APAP-treated mice had several necrotic areas with leukocyte inltration (green
arrow). (b) Digital quantication of necrotic areas using ImageJ, histopathological score, and neutrophil inltration (assessed by MPO levels)
conrmed severe liver inammation and necrosis induced by cocaine. ∗Statistical signicance in comparison to controls (𝑃 < 0.05). One-way
ANOVA with Tukey’s post hoc test. Data are expressed as mean ±S.E.M(𝑛=6mice/treatment).
Mediators of Inammation
Veh APAP 600 APAP 800
0
10
20
30
40
50
60
70
80
90
100
5/9
1/12
3/4
∗
∗
Lethality (%)
P = 0.04
P = 0.02
Cocaine (75 mg/kg)
(a)
Veh APAP 600 APAP 800
∗∗∗
0
10
20
30
40
50
60
70
80
90
100
Seizures (%)
P < 0.0001
Cocaine (75 mg/kg)
(b)
Veh APAP 600 APAP 800
0
10
20
30
40
50
∗∗∗
Seizure duration (s)
Cocaine (75 mg/kg)
(c)
Veh APAP 600 APAP 800
0
100
200
300
400
500
600 ∗∗
∗
Latency (s)
Cocaine (75 mg/kg)
(d)
F : Previous hepatic injury ( hours) with APAP interferes in seizure dynamics. (a) Administration of APAP at doses of
and mg/kg hours before cocaine administration increased lethality when compared to group vehicle/cocaine. (b) APAP at dose of
mg/kg reduced percentage of seizures and its duration (c). (d) Previous administration of APAP at dose of mg/kg decreased seizure
latency.APAP at dose of mg/kg enhanced seizure latency. ∗Signicantly dierent from vehicle group (∗𝑃< 0.05). ∗∗Signicantly dierent
from vehicle group (∗∗𝑃< 0.001). ∗∗∗Signicantly dierent from vehicle group (∗∗∗𝑃< 0.0001). One-way ANOVA with Tukey’s post hoc
test. Data are expressed as mean ±S.E.M(𝑛=6mice/treatment).
besides being well-tolerated and exhibiting a broad spec-
trum of therapeutic properties []. us, it is currently
attracting considerable interest as a potential medicine due
to its anti-inammatory, neuroprotective, antipsychotic, anx-
iolytic, antiepileptic, and anticancer eects [,,].
OneofthemainactionsofCBDisitsanticonvulsant
eect. In many experimental models, it exerted antiseizure
activities against PTZ-induced generalized seizures in rats
[], in the pilocarpine model temporal lobe seizures and the
penicillin model of partial seizures []aswellasinthePTZ-
and electroshock-induced seizures in rats []. us, consid-
eringtheecacyofCBDandthefactthatitdoesnotshare
thepsychotomimetic,amnestic,andsedativeeectsofΔ9-
THC, it is an interesting potential treatment for epilepsies. A
recently clinical study observed that CBD-enriched cannabis
extracts signicantly reduced seizure frequency in children
with treatment-resistant epilepsy, indicating that it could be
used as a strategy when other pharmacological treatments fail
[].
In addition, the mechanisms of action of this compound
have remained elusive and should be further investigated.
CBD is a compound with a wide plethora of pharmacolog-
ical actions, including anti-inammatory and antioxidative
eects, which may act through diverse mechanisms, facilitat-
ing the endocannabinoid system by blocker FAAH enzyme,
binding to TRPV channels, serotonin type-A receptors,
AA adenosine receptors, and peroxisome proliferator-
activated receptors (i.e., PPAR-𝛾)amongothers[,,].
In most in vivo models of inammation, CBD attenuates
inammatory cell migration/inltration (e.g., neutrophils)
[]. CBD suppresses T cell responses, decreases the release
of bioactive tumour necrosis factor (TNF𝛼), and reduces
prostaglandin E (PGE) levels, cyclooxygenase (COX) activ-
ity, and production of nitric oxide (NO). e suppressive
eects of CBD on cellular immune responses and on the
production of proinammatory mediators may indicate its
usefulness in several inammatory diseases []. Addition-
ally,CBDadministrationwasabletodecreaseleukocyte
migration into the lungs, myeloperoxidase activity in the lung
tissue, protein concentration and production of proinam-
matory cytokines (TNF𝛼and IL-), and chemokines (MCP-
and MIP-) in the bronchoalveolar lavage supernatant
showing a potent anti-inammatory eect in mice submitted
to LPS-induced acute lung injury []. In other context,
CBD modied the deleterious eects of inammation in
a viral model of multiple sclerosis through decreasing the
transmigration of blood leukocytes by downregulating the
expression of vascular cell adhesion molecule- (VCAM-),
chemokines (CCL and CCL), and the proinammatory
cytokine IL-𝛽[].
Mediators of Inammation
DNA (necrosis) Merge
PECAM-1
(sinusoids)
Cocaine
CBD ×cocaine
(a)
0
1000
2000
3000
ALT (U/L)
∗
Cocaine
Control Veh C B D URB
∗∗
(b)
**
Cocaine
Control Veh CBD URB
0
10
20
30
40
50
Cardiogreen (𝜇g/mL)
∗
∗∗
(c)
F : Cannabidiol (CBD; mg/Kg) prevented cocaine-induced liver injury. (a) Liver confocal intravital microscopy showing that while
CBD-treated mice have a well perfused liver vasculature (evidenced by PE-anti-CD antibody staining, in red) and absence of necrosis (in
green), cocaine-treated mice ( mg/Kg) had several dark areas in the red channel, which are suggestive of malperfusion caused by necrosis
(elicited by DNA staining using Sytox Green). (b) Reduced serum liver transaminase levels and (c) clearance of cardiogreen conrmed
inhibition of cocaine-induced liver injury by CBD. However, inhibition of anandamide hydrolysis by URB had no detectable eect. ∗Statistical
signicance in comparison to controls (𝑃 < 0.05)and ∗∗in comparison to vehicle-treated group. A x objective was used during imaging.
One-way ANOVA with Tukey’s post hoc test. Data are expressed as mean ±S.E.M(𝑛=6mice/treatment).
In respect to eects of CBD on liver, recent data showed
that CBD protected against liver toxicity induced by a single
dose of cadmium chloride []. Similarly, CBD attenuated
the deterioration in the measured biochemical parameters
and damages mediated by ischemia/reperfusion liver injury
besides reducing the inammatory response in tissue liver
[]. Previous studies already revealed that CBD pretreat-
ment signicantly protected against liver ischemia for min
followed by reperfusion for hs [].
Finally, our results on peripheric and central eects of
CBD against toxic eects of cocaine are in agreement with
previous results showing that this compound restored both
liver and brain function in a model of hepatic encephalopathy
associated with fulminant hepatic failure induced in mice by
thioacetamide []. Pretreatment with CBD was able to revert
neurological and cognitive deterioration induced by thioac-
etamideinparallelwithincreasesofliverfunction,conrmed
by improve of ammonia, bilirubin, aspartate transaminase
(AST), and alanine transaminase (ALT) levels. ese results
showed that CBD eects might result from a combination of
its actions in the liver and brain.
In conclusion, the present study demonstrates that co-
caine-induced seizures occur along with severe hepatic tox-
icity. e phytocannabinoid, CBD, but not the anandamide
Mediators of Inammation
Cocaine
Veh i c l e CBD ×cocaine
(a)
0
20
40
60
Cocaine
Necrosis area (%)
Veh CBD URBControl
∗
∗∗ 0
1
2
3
Cocaine
Histological score
Veh CBD URBControl
∗∗
∗
0
50
100
150
200
Cocaine
Neutrophils (×104)
Veh CBD URBControl
∗∗
∗
(b)
Veh CBD URB
0
100
200
300
400
500
600
Cocaine
Seizure latency (s)
∗∗ ∗∗
Veh CBD URB
0
10
20
30
40
50
Cocaine
Seizure duration (s)
∗∗ ∗∗
(c)
F : Cannabidiol (CBD; mg/Kg) prevented liver inammation and seizure due to cocaine intake. (a) Liver histology showing that
while CBD-treated mice have a normal liver architecture, cocaine-treated mice ( mg/Kg) had extensive areas which were suggestive of
malperfusion caused by necrosis. (b) Reduced necrotic area, histological score, and neutrophil inltration conrmed the anti-inammatory
and protective eects of CBD duringcocaine-induced hepatotoxicity. However, in hibition of anandamide hydrolysis by URB had no detectable
eect on liver parameters. (c) Despite lack of eect on liver injury, both CBD and URB inhibited cocaine-induced seizure, increasing latency
and reducing seizure duration. ∗Statistical signicance in comparison to controls (𝑃 < 0.05)and ∗∗in comparison to vehicle-treated group.
A x objective was used during imaging. One-way ANOVA with Tukey’s post hoc test. Data are expressed as mean ±S.E.M(𝑛=6
mice/treatment).
hydrolysis inhibitor, URB, protects against both eects
and warrants further investigation as a potential treatment for
both brain and liver consequences of cocaine intoxication.
Conflict of Interests
e authors declare that none of the authors have conict of
interests.
Authors’ Contribution
Luciano Rezende Vilela and Lindisley Ferreira Gomides
contributed equally to this work.
Acknowledgments
is work was supported by CNPq, CAPES, and Fapemig.
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