Canabis Sativa 1 [608418]

ORIGINAL ARTICLE
Study of the effect of Cannabis sativa on liver and brain damage
caused by thioacetamide
Omar M. E. Abdel-Salam &Marwa El-Sayed El-Shamarka &
Nermeen Shaffee &Alaa El-Din M. Gaafar
Received: 18 September 2012 /Accepted: 23 October 2012 /Published online: 8 November 2012
#Springer-Verlag London 2012
Abstract Cannabis sativa preparations are the most widely
used illicit drugs worldwide. The present study aimed to exam-
ine the effect of C.sativa extract on liver injury caused by
thioacetamide in the rat. Thioacetamide was administered at
50 mg/kg twice weekly via subcutaneous route (s.c.) for
2 weeks. Starting from the first dose of thioacetamide, rats weretreated with either C.sativa at doses of 10 or 20 mg/kg
(expressed as Δ
9-tetrahydrocannabinol), silymarin (25 mg/kg)
or saline, once daily s.c., for 2 weeks. Reduced glutathione(GSH), lipid peroxidation (mal ondialdehyde; MDA) and nitric
oxide concentrations were measured in the liver and brain.
Alanine aminotransferase (ALT), aspartate aminotransferase(AST), alkaline phosphatase (ALP), paraoxonase 1 activities
(PON1) and total proteins were determined in serum. Hepatic
injury was also determined via histological examination of liversections. The administration of only cannabis to saline-treated
rats had no significant effect on serum liver enzymes or on the
hepatic levels of GSH, MDA or nitric oxide. Serum PON1decreased by 21.9 % by 20 mg/kg cannabis. The level of MDA
a n dn i t r i co x i d ei nb r a i nd e c r e a s e db yo n l yc a n n a b i sa d m i n i s –
tration. In thioacetamide-treated rats, the administration of can-nabis extract (10 or 20 mg/kg) did not alter the level of MDA,
GSH or nitric oxide in hepatic tissue. Serum ALT or AST were
not significantly altered, but ALP increased significantly by38.9 % after treatment with 20 mg/kg cannabis. Serum PON1
activity which showed marked decrease in thioacetamide-
treated rats, increased by 18.9 and 151 % after C.sativa
treatment. Serum proteins increased after the administration of
cannabis (by 20.4 and 21.3 %, respectively). In brain tissue,
both MDA and nitric oxide were significantly decreased by
cannabis. Meanwhile, treatment with silymarin resulted in sig-
nificant decrease in MDA and increased GSH in the liver tissue.
Serum AST, ALT and ALP were significantly decreased, whilePON1 activity was increased af ter silymarin. In brain, MDA
decreased by 27.9 % after silymarin. Cannabis alone caused
histological liver damage and fibrosis and neuronal degenera-tion. The liver tissue damage and brain degeneration caused by
thioacetamide were enhanced by cannabis but almost prevented
by silymarin treatment. It is concluded that the administrationofC.sativa exacerbates the thioacetamide-induced liver and
brain injury.
Keywords Cannabis sativa extract .Silymarin .
Thioacetamide .Liver .Brain .Rat
Introduction
Cannabis sativa preparations are the most widely used illicit
drugs worldwide. They are usually used because of their
mood elevating properties, where a subjective feeling of thehigh and euphoria is perceived (Huestis 2002 ). The chem-
istry of C.sativa is complex, and the plant has been shown
to contain over 600 different constituents. Cannabinoids area group of C21 terpenophenolic compounds uniquely pro-
duced by C.sativa plant (Mechoulam and Gaoni 1967 ). One
cannabinoid that is delta-9-tetrahydrocannabinol ( Δ
9-THC)
is well known for being responsible for the psychotropic
activity of cannabis when herbal cannabis is smoked or
eaten. The pharmacology of other cannabinoids is startingto be delineated, and an anti-inflammatory, analgesic andO. M. E. Abdel-Salam ( *):M. E.-S. El-Shamarka
Department of Toxicology and Narcotics,National Research Centre,Tahrir St., Dokki,
Cairo, Egypt
e-mail: omasalam@hotmail.com
N. Shaffee
Department of Pathology, National Research Centre,
Cairo, Egypt
A. E.-D. M. Gaafar
Department of PhotoChemistry, National Research Centre,Cairo, EgyptComp Clin Pathol (2014) 23:495 –507
DOI 10.1007/s00580-012-1641-0

anticancer effects (Vara et al. 2011 ) have been ascribed to
individual cannabinoids. To date, two G protein-coupled
cannabinoid receptor subtypes —CB1 and CB2 —have been
cloned, with CB1 receptors being mainly expressed in the
central nervous system and CB2 receptors being primarily
localized in cells involved in immune and inflammatoryresponses. These receptors are activated by a family of
endogenous arachidonic acid derivatives, the endocannabi-
noids, and by Δ
9-THC, the principal active ingredient of the
plant C.sativa (Pertwee and Ross 2002 ).
Medical uses of cannabis and cannabinoids are on the rise.
Pain, insomnia and anxiety were the most common conditionsfor which evaluating physicians recommended medical mar-
ijuana (Reinarman et al. 2011 ). Herbal cannabis is also being
used by patients with Parkinson ’s disease (Venderová et al.
2004 ). Nabiximols (Sativex), an oromucosal spray of whole
plant extract containing equal proportions of Δ
9-tetrahydro-
cannabinol and cannabidiol, is being evaluated for decreasingspasticity in patients with multiple sclerosis (Sastre-Garriga et
al.2011 ). Smoking cannabis has also been utilized to lessen
neuropathic pain in patients with human immunodeficiencyvirus (HIV) (Ellis et al. 2009 )a n df o rp o s t – t r a u m a t i co rp o s t –
surgical neuropathic pain (Ware et al. 2010b ). Nabilone is a
synthetic THC for oral administration which is used as an
antiemetic in cancer patients receiving chemotherapy and as
an analgesic in pain conditions (Ware et al. 2010a ).
In recent years, the effect of C.sativa and different canna-
binoids on the development of liver injury has received con-
siderable interest. Smoking cannabis itself has beenimplicated both in accelerating liver fibrosis and steatosis
severity in untreated patients infected with hepatitis C virus
(Hézode et al. 2005 ,2008 ;I s h i d ae ta l . 2008 ) and in improv-
ing the tolerability to antiviral therapy with interferon- αplus
ribavirin (Sylvestre et al. 2006 ). Studies have also shown that
short-term administration of C.sativa enhances acute hepatic
damage caused by CCl
4or acetaminophen in rats (Abdel-
Salam et al. 2011 ). Cannabinoid receptors are up-regulated
in liver cirrhosis (Floreani et al. 2010 ). Moreover, research
into cannabinoid receptors suggested distinct roles for both
CB1 and CB2 receptors in modulating liver injury where
cannabinoid CB1 receptors enhance liver fibrogenesis andsteatogenesis (Yang et al. 2007 ; Trebicka et al. 2011 ) and
CB2 receptors display hepatic protective and antifibrogenic
effects (Julien et al. 2005 ;B á t k a ie ta l . 2007 ; Muñoz-Luque et
al.2008 ; Trebicka et al. 2011 ). Further research is thus re-
quired to delineate the effects of cannabis use in liver disease.
Given that the herbal cannabis is the most widely used
illicit drug and is abused by different age and patient pop-
ulations including patients with chronic liver disease, the
present study was therefore designed to examine the effectof daily cannabis administration on the development of liver
damage induced by thioacetamide in the rat. The effect of
cannabis was also studied on oxidative stress in the brainand liver in rats treated with thioacetamide. The administra-
tion of thioacetamide has been widely used to cause acute
liver failure and encephalopathy and also for inducing he-patic fibrosis when applied for prolonged time at lower
doses (Sanz et al. 2002 ; Sun et al. 2000 ; Túnez et al.
2005 ; Uskukovic-Markovic et al. 2007 ).
Materials and methods
Animals
Adult Sprague –Dawley rats of either sex (120 –130 g) were
used throughout the experiments and fed with standard labo-
ratory chow and water ad libitum. Animal procedures wereperformed in accordance with the Ethics Committee of the
National Research Centre and followed the recommendations
of the National Institutes of Health Guide for Care and Use ofLaboratory Animals (publication no. 85-23, revised 1985).
Equal groups of six rats each were used in all experiments.
Drugs and chemicals
Thioacetamide (Sigma, USA) and silymarin (SEICO Co,
Cairo, ARE) were used in the experiments. C.sativa L.
plant was supplied by the Ministry of Justice, Egypt.
Preparation of Cannabis extract
Dry extract of C. sativa
C.sativa extract was prepared from the dried flowering tops
and leaves of the plant. The method of extraction followed that
described by Turner and Mahlberg ( 1984 ) with modification.
In brief, 10 g of dried cannabis was ground with a mortar andpestle. Decarboxylation of the plant material was achieved by
placing the sample in a glass test tube (30 mL) and covering it
with aluminum foil. The test tube was placed in boiling waterbath (100 °C) for 2 h. Ten millilitres of analytical grade
chloroform was added and allowed to react for 1 h. The dried
cannabis was extracted three times, and fractions were com-bined, filtered over filter paper and collected in a 100-mL
volumetric flask. The filtrate was evaporated under a gentle
stream of nitrogen (on ice and protected from light) and storedat 4 °C and protected from light in an aluminum-covered
container, which provided the dry extract as residue.
Preparation diluted extract for injection
The residue (dry extract) was suspended in 2 mL of 96 %
ethanol, and the total volume in the volumetric flask in-
creased to 100 mL by adding distilled water. The extract was
injected via subcutaneous route (s.c.) at doses of 10 and496 Comp Clin Pathol (2014) 23:495 –507

20 mg/kg (expressed as delta-9-THC). The injection volume
was 0.2 ml/rat. Tetrahydrocannabinol (THC) content was
quantified using gas chromatography –mass spectrometry
(GC-MS). The THC content of the extract was 10 %.
Study designRats were randomly assigned into seven groups, each of six
animals. Group 1 (normal control) received s.c. saline0.2 mL/rat. Groups 2 and 3 received C.sativa only at doses
of 10 or 20 mg/kg daily. Groups 4 –7 were s.c. injected with
thioacetamide at the dose of 50 mg/kg twice weekly for2 weeks. Groups 4 and 5 were administered C.sativa extract
daily at doses of 10 or 20 mg/kg, group 6 received silymarin
at the dose of 25 mg/kg daily, while group 7 was adminis-tered saline and served as positive control. Rats had free
access to food and drinking water during the study. Rats
were killed 2 weeks after drug or vehicle administration bydecapitation under ether anaesthesia.
Biochemical assessmentBlood samples were obtained from the retro-orbital vein
plexus under ether anaesthesia. Rats were then killed by
decapitation under ether anaesthesia, livers and brains were
excised, washed with ice-cold saline solution (0.9 % NaCl)and parts of it were preserved in formalin 10 % for further
histopathological examination. Other parts were weighed
a n ds t o r e da t −80 °C for the biochemical analyses. The
tissue was homogenized with 0.1 M phosphate buffer saline
at pH7.4, to give a final concentration of 10 % w/vfor the
biochemical assays.
Determination of serum liver enzymes
The activities of aspartate aminotransferase (AST) and ala-
nine aminotransferase (ALT) enzymes, indicators of liver
damage, were measured in serum according to Reitman-Frankel colorimetric transaminase procedure (Crowley
1967 ), whereas colorimetric determination of alkaline phos-
phatase (ALP) activity was done according to the method ofBelfield and Goldberg ( 1971 ), using commercially available
kits (BioMérieux, France).
Determination of lipid peroxidation
Lipid peroxidation was assayed by measuring the level of
malondialdehyde (MDA) in the tissue homogenates. Malon-
dialdehyde was determined by measuring thiobarbituric re-
active species using the method of Ruiz-Larrea et al. ( 1994 ),
in which the thiobarbituric acid reactive substances react
with thiobarbituric acid to produce a red coloured complex
having peak absorbance at 532 nm.Determination of reduced glutathione
Reduced glutathione (GSH) was determined in tissue by
Ellman's method ( 1959 ). The procedure is based on the
reduction of Ellman ’s reagent by –SH groups of GSH to
form 2-nitro- s-mercaptobenzoic acid, the nitromercaptoben-
zoic acid anion has an intense yellow colour which can be
determined spectrophotometrically.
Determination of nitric oxide
Nitric oxide measured as nitrite was determined by using
Griess reagent, according to the method of Moshage et al.
(1995 ), where nitrite, stable end product of nitric oxide radical,
is mostly used as an indicator for the production of nitric oxide.
Determination of paraoxonase activity
Arylesterase activity of paraoxonase was measured spectro-
photometrically in supernatants using phenylacetate as a
substrate (Higashino et al. 1972 ; Watson et al. 1995 ).
Histopathological studiesThe brain and liver from all animals were dissected imme-
diately after death. The sections were fixed in 10 % neutral-buffered formal saline for 72 h at least. All the specimens
were washed in tap water for half an hour and then dehy-
drated in ascending grades of alcohol, cleared in xylene andembedded in paraffin. Serial sections of 6 μm thick were cut
and stained with haematoxylin and eosin (H&E) (Drury and
Walligton 1980 ) for histopathological investigation, and
with Van Geison ’
s stain for detection of fibrous tissue.
Images were captured and processed using Adobe Photo-
shop version 8.0.
Statistical analysis
Data are expressed as mean±SE. Statistical analysis of the
data was done using one-way ANOV A followed by Duncan
test for multiple group comparisons. Probability levels ofp<0.05 were considered statistically significant.
Results
Serum markers of hepatocellular damageWhen given to saline-treated rats, C.sativa at doses of 10 or
20 mg/kg had no significant effect on serum AST or ALT andALP levels (Fig. 1a–c). Rats treated with thioacetamide
exhibited significant elevations in the serum ALT, AST and
ALP levels by 68.6, 48.8 and 27.1 %, respectively, comparedComp Clin Pathol (2014) 23:495 –507 497

with the saline control group ( p<0.01). The administration of
C.sativa at 10 or 20 mg/kg did not significantly alter serum
ALT or AST. However, ALP activity increased by 38.9 % ( P<
0.05) by 20 mg/kg cannabis. In contrast, silymarin given to
thioacetamide-treated rats resulted in 33.7, 29.3 and 40.4 %
reduction in ALT, AST and ALP, respectively when comparedwith the thioacetamide control group.
Serum total proteinsSerum proteins were not affected by cannabis only treat-
ment. Thioacetamide-treated rats exhibited significant de-crease in serum proteins by 22.3 % compared with the
saline-treated group ( P<0.05). Serum proteins increased
by 20.4 and 21.3 % ( P<0.05 and P<0.05) after treatment
with C.sativa at doses of 10 or 20 mg/kg, respectively, as
compared to the thioacetamide control group (Fig. 2).
Serum paraoxonase activity
When given to saline-treated rats, cannabis at 20 mg/kg
resulted in 21.9 % decrease in serum PON1 activity
(P>0.05). A marked and significant decrease in serum
PON1 activity by 75.8 % ( P<0.05) was observed after the
administration of thioacetamide. Serum paraoxonase activi-
ty increased by 18.9 % ( P>0.05) and 151 % ( P<0.05) after
treatment with C.sativa at 10 or 20 mg/kg, respectively,
compared with the thioacetamide control group. Serum par-
aoxonase activity increased by 54.3 % after treatment withsilymarin ( P<0.05 vs thioacetamide control group) (Fig. 3).Saline
+ Cannabis 10 mg/kg
+Cannabis 20 mg/kg
Thioacetamide control+ Cannabis 10 mg/kg
+ Cannabis 20 mg/kg
+Silymarin 25 mg/kg010203040506070
**
*
+**Serum ALT (U/l)
Saline
+ Cannabis 10mg/kg
+ Cannabis 20mg/kg
Thioacetamidecontrol
+ Cannabis 10mg/kg
+ Cannabis 20mg/kg
+ Silymarin 25mg/kg050100150200250**
+*
*
*Serum AST (U/l)
Saline
+Cannabis10 mg/kg
+ Cannabis20 mg/kg
Thioacetamide control+Cannabis10 mg/kg
+ Cannabis20 mg/kg
+Silymarin25 mg/kg0100200300
+*
+*
*
**+Serum ALP(U/l)*a
b
c
Fig. 1 a –cEffect of daily C.sativa extract or silymarin on serum ALT,
AST and ALP activities. The effect of cannabis was studied in rats
given s.c. saline or thioacetamide (50 mg/kg) twice weekly for 2 weeksand compared with that of silymarin. Asterisks indicate significant
difference from the saline control group and between different groups
as indicated on the graph. The plus (+) sign indicates significantdifference from the thioacetamide control groupSaline
+ Cannabis 10mg/kg
+ Cannabis20mg/kg
Thioacetamidecontrol
+ Cannabis 10mg/kg
+ Cannabis 20mg/kg
+Silymarin 25mg/kg0.02.55.07.510.0
*+
*+Serum total protein (mg/dl)
Fig. 2 Effect of C.sativa extract or silymarin on serum total proteins
in saline and thioacetamide-treated rats. Asterisks indicate significant
difference from the saline control group and between different groupsas indicated on the graph. The plus (+)sign indicates significant
difference from the thioacetamide control group498 Comp Clin Pathol (2014) 23:495 –507

Serum glucose
Serum glucose showed significant increase in thioacetamide-
treated rats but decreased after cannabis (10 mg/kg) treatment
(Fig. 4)
Hepatic markers of oxidative stress
The administration of only C.sativa at 10 or 20 mg/kg did
not significantly alter MDA, nitric oxide or GSH in the liver
of saline-treated rats. The level of MDA increased by19.1 % ( P<0.05) while GSH decreased by 18.5 % ( P<
0.05) in rats treated with thioacetamide. The administration
ofC.sativa at 10 or 20 mg/kg to thioacetamide-treated rats
did not significantly alter hepatic MDA, while nitric oxide
decreased by 16.8 % by 20 mg/kg cannabis. Meanwhile,
treatment with silymarin resulted in significant decrease in
MDA by 24.3 %. Silymarin treatment also almost normal-
ized GSH registering 17.4 % increase compared with thethioacetamide control group (Fig. 5a–c).
Brain markers of oxidative stressIn saline-treated rats, the level of MDA was significantly
decreased by 19.1 % ( p<0.05) and 20.0 % ( p<0.05) by C.
sativa at 10 and 20 mg/kg, respectively. Reduced glutathi-
one was not significantly altered, but nitric oxide showed a
29.1 % decrease by 20 mg/kg cannabis compared with thesaline-treated group. The administration of thioacetamide
had no significant effect on MDA, GSH or nitric oxide in
the brain. In thioacetamide-treated rats, however, MDAdecreased by 26.9 and 21.9 % after treatment with 10 and
20 mg /kg cannabis, respectively. GSH showed slight yet
significant decrease after cannabis or silymarin treatment. Inaddition, the level of nitric oxide decreased by 30.2 % after
cannabis treatment at 20 mg/kg. Meanwhile, silymarin treat-
ment was associated with a 27.9 % decrease in brain MDAcompared with the thioacetamide control group (Fig. 6a–c).
Histopathological resultsLiverCannabis displayed a damaging effect on the liver tissue in
the form of dilatation of blood sinusoids, increased numberof Kupffer cells, slight fibrosis and cellular infiltration at
the periphery of the lobules when administered at the dose
of 10 mg/kg (Fig. 7a). The higher dose of 20 mg/kg
caused marked fibrosis with massive cellular infiltration
around blood vessels and local areas of degenerated cells
(Fig. 7b).
The administration of thioacetamide alone caused vac-
uolar degeneration and acidophilic cytoplasm in many
hepatocytes (Fig. 7e). Cannabis given to thioacetamide-Saline
+C a nnabis 10mg/kg
+C a nnabis 20mg/kg
Thioacetamidecontrol
+C a nnabis 10mg/kg
+C a nnabis20mg/kg
+ Silymarin 25mg/kg050100150
**
* *Serum glucose (mg/dl)## #
Fig. 4 Effect of C.sativa extract or silymarin on serum glucose in
saline and thioacetamide-treated rats. Asterisks indicate significant
difference from the saline control group and between different groupsas indicated on the graph. The number sign indicates significant dif-
ference from the silymarin groupSaline
+Cannabis10mg/kg
+ Cannabis 20mg/kg
Thioacetamidecontrol
+ Cannabis 10mg/kg
+ Cannabis 20mg/kg
+ Silymarin 25mg/kg051015
* **+** *
##
##Serum PON1 (kU/l)
Fig. 3 Effect of C.sativa extract or silymarin on serum paraoxonase
(PON1) activity in saline and thioacetamide-treated rats. Asterisks
indicate significant difference from the saline control group and be-tween different groups as indicated on the graph. The plus (+)sign
indicates significant difference from the thioacetamide control group.
The number sign indicates significant difference from the cannabis
only treatment groupsComp Clin Pathol (2014) 23:495 –507 499

Saline
+ Cannabis 10mg/kg
+Cannabis20mg/kg
Thioacetamidecontrol
+C a nnabis 10mg/kg
+C a nnabis 20mg/kg
+S i lymarin 25mg/kg050100150200250300350*
+Liver MDA (nmol/g tissue)
Saline
+ Cannabis 10mg/kg
+ Cannabis 20mg/kg
Thioacetamidecontrol
+ Cannabis 10mg/kg
+ Cannabis 20mg/kg
+ Silymarin 25mg/kg01020*
+
**
**Liver GSH ( µmol/g tissue)
Saline
+ Cannabis10mg/kg
+ Cannabis 20mg/kg
Thioacetamidecontrol
+ Cannabis10mg/kg
+ Cannabis 20mg/kg
+ Silymarin 25mg/kg01234567
+*Liver nitric oxide ( µmol/g tissue)a
b
c
Fig. 5 a –cEffect of C.sativa extract or silymarin on liver MDA,
reduced GSH or nitric oxide in saline and thioacetamide-treated rats.
Asterisks indicate significant difference from the saline control group
and between different groups as indicated on the graph. The plus (+)
sign indicates significant difference from the silymarin groupSaline
+Cannabis 10mg/kg
+ Cannabis20mg/kg
Thioacetamidecontrol
+ Cannabis 10mg/kg
+ Cannabis 20mg/kg
+Silymarin 25mg/kg050100150200250
+ *+ *** *+Brain MDA (nmol/g tissue)
Saline
+ Cannabis 10mg/kg
+Cannabis 20mg/kg
Thioacetamidecontrol
+Cannabis10mg/kg
+ Cannabis 20mg/kg
+S i lymarin 25mg/kg0123
+ +* *Brain GSH ( µmol/g tissue)
Saline
+C a nnabis10mg/kg
+C a nnabis 20mg/kg
Thioacetamidecontrol
+C a nnabis 10mg/kg
+C a nnabis 20mg/kg
+S i lymarin 25mg/kg0510152025
+*
***Brain nitric oxide ( µmol/g tissue)a
b
c
Fig. 6 a –cEffect of C.sativa extract or silymarin on brain MDA,
reduced GSH or nitric oxide in saline and thioacetamide-treated rats.Asterisks indicate significant difference from the saline control group
and between different groups as indicated on the graph. The plus(+)sign
indicates significant difference from the thioacetamide control group500 Comp Clin Pathol (2014) 23:495 –507

treated rats enhanced the effect of the latter, where sec-
tions from rats given 10 mg/kg cannabis+thioacetamideshowed fibrosis with cellular infiltration around blood
vessels, vacuolar degeneration, pyknotic nuclei and aci-
dophilic cytoplasm in many hepatocytes (Fig. 7c). Can-
nabis at the dose of 20 mg/kg along with thioacetamide
caused marked dilatation and congestion of central vein
and severe dilatation of the portal vein at the expense ofhepatocytes with massive fibrosis and cellular infiltration
distorting the normal architecture of the tissue (Fig. 7d). In
contrast, the administration of silymarin to thioacetamide-
treated rats resulted in slight fibrosis. Cellular infiltration
was also noticed around the central vein (Fig. 7f).
Van Geison ’s stain confirmed the above results as positive-
ly stained areas increased with the increasing cannabis dose
and on combining both cannabis and thioacetamide (Fig. 8).
Fig. 7 H&E stained sections of liver tissue from rat treated with: a
cannabis alone at a dose of 10 mg/kg showing dilatation of blood
sinusoids, increased number of Kupffer cells, slight fibrosis and cellu-
lar infiltration at the periphery of the lobules; the right lower corner of
the figure shows mild vacuolar degeneration in some hepatocytes; b
cannabis alone at a dose of 20 mg/kg showing marked fibrosis with
massive cellular infiltration around blood vessels; local areas of degen-
erated cells ( arrow ) are also observed; cthioacetamide+10 mg/kg
cannabis showing fibrosis with cellular infiltration around blood ves-
sels; vacuolar degeneration, pyknotic nuclei and acidophilic cytoplasm
are noticed over a wide area; dthioacetamide+cannabis 20 mg/kgshowing marked dilatation and congestion of central vein. Severe
dilatation of the portal vein at the expense of hepatocytes with massive
fibrosis and cellular infiltration distorting the normal architecture of the
tissue are observed; eonly thioacetamide showing degenerative signs
and acidophilic cytoplasm which are observed in many hepatocytes.The right lower corner of the figure shows many hepatocytes that are
suffering from marked vacuolar degeneration; fthioacetamide+sily-
marin showing restoration of the normal structure except for somehepatocytes that are still suffering from pyknotic nuclei and acidophilic
cytoplasm. Slight fibrosis with cellular infiltration is also noticed
around the central veinComp Clin Pathol (2014) 23:495 –507 501

Brain
The examination of brain sections from rats treated with
only cannabis showed brain tissue damage in the form
signs of degeneration (darkening and shrinking of nuclei)
in neurones. This effect was dose dependent as thenumber of neurones showing these signs of degeneration
was higher in rats treated with 20 mg/kg cannabis com-
pared with the lower dose of 10 mg/kg (Fig. 9c, b ).
Sections from only thioacetamide-treated rats showed
some neurones with dark small nuclei (Fig. 9e). Mean-
while, cannabis enhanced the damaging effect of thioa-
cetamide at both doses used (Fig. 9c, d ). In contrast to
the effects of cannabis, silymarin ameliorated the effectof thioacetamide on the brain tissue (Fig. 9f).Discussion
Since whole cannabis extracts are used to treat a variety of
medical conditions, the present study examined the effect of a
C.sativa extract on liver damage induced by thioacetamide in
the rat. Overall, the findings of the present study suggest thatthe administration of herbal cannabis increased liver injury
caused by thioacetamide. The release of the hepatocellular
enzymes alanine aminotransferase (ALT) and aspartate ami-
notransferase (AST) into the circulation, both being markers
of liver cell damage, was not altered by the extract. Mean-while, there was an increased alkaline phosphatase (ALP)
activity in serum, suggesting increased cholestasis by canna-
bis. In contrast to the effects of C.sativa on the activity of liver
enzymes, serum ALT and AST were almost normalized by
Fig. 8 Van Geison ’s stained
sections of liver tissue from rat
treated with: aonly cannabis at
a dose of 10 mg/kg showingslight increase in fibrous tissue
component around the main
blood vessels; bonly cannabis
at a dose of 20 mg/kg showing
marked increase in fibrous
tissue around blood vessels ifcompared with the previoussection; cthioacetamine+
cannabis 10 mg/kg showing
noticeable increase in fibroustissue around blood vessels,which is greater than that in the
first group (treated with
cannabis only); dthioacetamide
+cannabis at a dose of 20 mg/kg showing severe fibrosis
around the dilated and
congested blood vesselsdistorting the liver tissue
architecture; eonly
thioacetamide showing mildincrease in fibrous tissue aroundthe blood vessels; f
thioacetamide and silymarin
showing noticeable reduction infibrous tissue component ifcompared with the previous
section502 Comp Clin Pathol (2014) 23:495 –507

silymarin treatment which also markedly decreased serum
ALP activity, suggesting a protective effect for silymarin on
liver damage caused by thioacetamide. Indeed, silymarin hasbeen proved effective in reducing or alleviating hepatic injury
caused by several hepatotoxins e.g., ethanol, CCl
4or that
caused by ischaemia or irradiation (Flora et al. 1998 ;S a l l e r
et al. 2001 ). The histopathological studies confirmed the bio-
chemical findings and indicated cellular degeneration and the
development of fibrosis after the administration only cannabisand the exacerbation of thioacetamide liver damage and fibro-
sis on co-administration of cannabis. This contrasted with
restoration of the normal hepatic architecture by silymarin inthioacetamide-treated rats.
Increased oxidative stress due to excessive free radical
production or decreased antioxidant defence mechanismshas an important role in the pathogenesis of liver injury
due to viral, toxic, metabolic or cholestatic diseases (Poli
2000 ). It followed that decreasing oxidative stress by agents
such as ursodeoxycholic acid and silymarin is a well-
established strategy in the management of chronic liver
disease (Medina and Moreno-Otero 2005 ;S i n g a le ta l .
2011 ; Wang et al. 2012 ). Therefore, the present study also
assessed the effect of C.sativa on oxidative stress in the
liver and serum. Oxidative stress is largely thought to beinvolved in thioacetamide-induced liver damage. This is due
to the superoxide anion and hydrogen peroxide which is
generated during the biotransformation of thioacetamide bythe microsomal flavine-adenine dinucleotide-containing
monooxygenase and cytochrome P450 systems (Chieli and
Malvaldi 1984 ; Lee et al. 2003 ). The present study indicated
Fig. 9 H&E stained sections of
brain tissue from rat treated
with: acannabis alone at
10 mg/kg, showing somepyramidal cells with darkened
nuclei; bcannabis alone at
20 mg/kg, showing increasednumber of pyramidal cells with
darkened nuclei; c
thioacetamide and cannabis10 mg/kg showing someneurones that suffer from
ballooning and degeneration in
the left part of the figure, othersappeared with dark shrunkennuclei in the right part of the
figure; dthioacetamide and
cannabis 20 mg/kg showingincreased number of neuroneswith dark nuclei if compared
with the previous sections; e
only thioacetamide, showingsome neurones with dark small
nuclei; fthioacetamide and
silymarin, showing only a fewneurones with dark nuclei whilethe others appear normal in size
and shapeComp Clin Pathol (2014) 23:495 –507 503

a rise in liver MDA by thioacetamide treatment. Malondial-
dehyde is a marker of lipid peroxidation which suggests
increased free radical production and consequent attack ofmembrane lipids (Gutteridge 1995 ). Reduced glutathione,
an important intracellular antioxidant, is decreased in hepat-
ic tissue by thioacetamide, suggesting consumption by in-creased free radicals (Wang and Ballatori 1998 ). Under
these circumstances, cannabis failed to alter MDA or
GSH. In contrast, both hepatic MDA and GSH were nor-malized by silymarin treatment, thereby suggesting an im-
portant contribution of the antioxidant properties of
silymarin to its hepatic protective effects.
Paraoxonase is an esterase and lactonase which is in-
volved in protection against xenobiotic toxicity (Furlong
2008 ). Human PON1 is synthesized in the liver and secreted
into the blood, where it is mainly associated with high-
density lipoproteins, having a role in preventing the oxida-
tive modifications of low-density lipoprotein and henceprotecting against the development of atherosclerosis
(Mackness et al. 2006 ; Primo-Parmo et al. 1996 ; Furlong
et al. 2010 ). Decreased serum PON1 activity has been
observed in a number of disease processes in which in-
creased oxidative stress was evident, e.g. Alzheimer disease,
mixed dementia (Wehr et al. 2009 ), coronary heart disease
(Kotur-Stevuljevic et al. 2008 ) and bronchial asthma
(Cakmak et al. 2009 ). Baseline and stimulated PON1 activ-
ities were decreased in chronic hepatitis and in liver cirrho-
sis and correlated with serum total proteins, albumin and
bilirubin in patients but not in controls (Ferre et al. 2002 ).
Serum paraoxonase 1 activity decreased significantly in
non-alcoholic steatohepatitis compared to the control group
(Bașkol et al. 2005 ). Hence, measurement of serum PON1
activity has been proposed as a potential test for the evalu-
ation of liver function (Camps et al. 2009 ). An important
observation in the present study was the marked decrease inPON1 activity by thioacetamid e treatment. Interestingly,
PON1 activity increased in thioacetamide-treated rats ad-
ministered 20 mg/kg of cannabis as well as silymarin. Incontrast to the effect of thioacetamide in the liver, it failed to
alter brain MDA, GSH or nitric oxide at the doses employed
in the present study. In accordance with our earlier observa-tions in mice (Abdel-Salam et al. 2011 ), the administration
of only cannabis decreased both MDA and nitric oxide in
brain of saline-treated rats. These effects of cannabis werealso evident in rats given thioacetamide. Cannabinoids are
highly fat soluble and effectively penetrate the blood brain
barrier, and the brain tissue appears to be especially suscep-tible to the effects of herbal cannabis. In addition to its
cannabinoid content, other constituents of the plant cannabis
are flavonoids: flavocannabis ide, flavosativaside, glyco-
sides of vitexin and isovitexin and essential oil, composed
of olivetol, cannabene (a sesquiterpene), etc; alkaloids: can-
nabisativine, muscarine and trigonelline stilbene derivatives,e.g. 3,4 ′-dihydroxybibenzyl; and others: choline and calci-
um carbonate (ElShohly 2002 ). In animal models, cannabis
displayed acute neuroprotective effects (Sagredo et al. 2011 )
and improved parkinsonian symptoms (Venderová et al.
2004 ), possibly due to antioxidant properties of the flavo-
noids it contains. Cannabis, however, have long-term con-sequences on cognitive functions (Solowij et al. 2002 ;
Harvey et al. 2007 ). The inhibitory effect of cannabis on
nitric oxide could be one mechanism underlying the cogni-tive and memory impairing effects of long-term cannabis
u s e .I n t e r e s t i n g l y ,M D Ad e c r e a s e da l s ob ys i l y m a r i ni n
thioacetamide-treated rats.
Cannabis is abused by different age groups and patient
populations including patients with chronic liver disease and
even those on liver transplantation, and therefore, its effect onthe liver is of marked medical importance. Studies reported
the presence of hepatomegaly, splenomegaly, slightly elevated
serum AST, ALT and ALP among chronic users of onlymarijuana (Borini et al. 2004 ). Researchers also observed
increased ALT, gamma glutamyltransferase activity, total bile
acids, bilirubin, as well as decreased GSH and increased lipidperoxidation in sera of marijuana smokers with no history of
liver disease (Toson 2011 ). Smoking cannabis is also likely to
increase fibrosis progression rate and steatosis severity in
patients with chronic hepatitis C virus (Hézode et al. 2005 ,
2008 ; Ishida et al. 2008 ). Cannabis preparations exert their
effects by interacting with two G protein-coupled cannabinoid
receptor subtypes —CB1 and CB2. CB1 receptors are highly
up-regulated in human cirrhotic liver mainly in hepatic fibro-genic cells (Julien et al. 2005 ). These receptors also respond to
endogenous ligands, the endocannabinoids including the
arachidonic acid derivatives 2-arachidonoylglycerol andanandamide. It has been suggested that CB1 receptors en-
hance liver fibrogenesis and steatogenesis (Yang et al. 2007 ;
Trebicka et al. 2011 ) while CB2 receptors mediate hepatic
protective and antifibrogenic effects (Julien et al. 2005 ;B á t k a i
et al. 2007 ; Muñoz-Luque et al. 2008 ; Trebicka et al. 2011 ).
Blocking hepatic CB1 receptor with CB1 antagonists de-creased the wound healing response to acute liver injury,
inhibited the progression to fibrosis and lowered hepatic
TGF-β1 and reduced the activation of fibrogenic cells in the
liver (Teixeira-Clerc et al. 2006 ), decreased liver injury and
neutrophil infiltration, TNF- α, improved oxidative injury and
haemodynamic alterations (Caraceni et al. 2009 ), stimulated β
oxidation activity, and improved carbohydrate and lipid me-
tabolism (Jourdan et al. 2012 ). Hepatic CB2 receptor activa-
tion with specific agonists decreased inflammatory cellinfiltration and number of activated stellate cells, hepatic
pro-inflammatory cytokines, TNF- α, neutrophil infiltration,
oxidative stress, decreased fibrosis (Bátkai et al. 2007 ;
Muñoz-Luque et al. 2008 ; Horváth et al. 2012 ),
decreased hepatocyte steatosis, increased hepatic expression
of macrophage heme oxygenase-1 (Louvet et al. 2011 ).504 Comp Clin Pathol (2014) 23:495 –507

CB2−/−mice, on the other hand, developed increased tissue
damage and proinflammatory phenotype, hepatic stellate cell
activation and collagen production in response to a variety ofliver insults (Bátkai et al. 2007 ; Trebicka et al. 2011 ).
Some of the effects of cannabinoids are also mediated
through modulation of hepatic microcirculation and blood flow.Thus, the CB1 agonist anandamide induced marked vasodila-
tation in isolated vascular preparations (White et al. 2001 ;
Zygmunt et al. 1999 ). Anandamide effects appear to depend
on the activation of vanilloid rece ptors since they were blocked
by the vanilloid receptor antagonist capsazepine (Zygmunt et
al.1999 ). The endocannabinoid virodhamine caused endothe-
lium dependent relaxation of the rat isolated small mesenteric
artery (Hoi and Hiley 2006 ). Other endogenous CB1 and CB2
receptor agonists, e.g. 2-arachidonylglycerol and palmitoylethanolamide, were not vasodilators. The endocannabinoid
system appears to mediate the ext reme mesenteric vasodilation,
portal hypertension and systemic hypotension present in ad-vanced liver cirrhosis (Járai and Kúnos 2002 ). Ros et al. ( 2002 )
administered a cannabinoid CB1 r eceptor antagonist to cirrhot-
ic rats with ascites and found si gnificantly increased arterial
pressure and peripheral resista nce in cirrhotic but not control
rats. The authors suggested that monocytes from cirrhotic rats
are activated to produce anandamide, which in turn contributes
to arterial hypotension in experi mental cirrhosis. Meanwhile,
increased hepatic production of vasoconstrictor eicosanoidsmight be involved in the effect of endocannabinoids on hepatic
microcirculation in cirrhotics (increased intrahepatic resis-
tance). Thus, CB1 receptor blockade in cirrhotic ratsdecreased portal venous pressure and intrahepatic resistance
and superior mesenteric blood flow (Yang et al. 2007 ).
The present study used C.sativa extract consisting mainly
ofΔ
9-THC. The latter is a CB1 and CB2 receptor partial
agonist (Pertwee 2008 ). Cannabidiol, a non-psychotropic
cannabinoid in C.sativa , induced apoptosis in activated
hepatic stellate cells by inducing endoplasmic reticulum
stress response and activation of a pro-apoptotic pathway
(Lim et al. 2011 ). Cannabidiol displayed high potency as an
antagonist of CB1/CB2 receptor agonists in CB1- and
CB2-expressing cells or tissues, inhibiting evoked immune
cell migration (Pertwee 2008 ). Clearly, the effect of C.sativa
will depend on the relative amounts of Δ9-THC and other
cannabinoids or in other words on the degree of relative
stimulation of either CB1 or CB2 receptors by the extractconstituents. Not only this, but other cannabinoid and non-
cannabinoid compounds in herbal cannabis or its extracts
might modulate the pharmacological actions of Δ
9-THC
(Russo and McPartland 2003 ). In some liver disease, where
immunity has a role in the pathogenesis of liver injury, the
situation might be more complex, since cannabinoids canmodulate immunity by acting on CB2-receptors. Schwabe
and Siegmund ( 2005 ) suggested that the immune-
modulatory effects of cannabis ingredients are likely to affectdisease progression and fibrogenesis in chronic hepatitis C
by suppressing antiviral immunity.
In summary, the findings of the present study indicated
that the administration of C.sativa extract itself did not alter
serum alanine aminotransferase, aspartate aminotransferase
or alkaline phosphatase. The herb also displayed no effecton the oxidative status of the liver in terms of tissue levels of
MDA, GSH or nitric oxide. C.sativa administered to
thioacetamide-treated rats, however, worsened hepatic inju-ry and brain degeneration due to toxic agent.
Acknowledgments For the supply of cannabis samples, the authors
are indebted to Ministry of Justice of the Arab Republic of Egypt.
Conflicts of interest The authors declare that there are no conflicts
of interest.
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