Pain 139 (2008) 190–200

www.elsevier.com/locate/pain

Endocannabinoid and serotonergic systems are needed

for acetaminophen-induced analgesia
Christophe Mallet a,b, Laurence Daulhac a,c, Jérôme Bonnefont a,b, Catherine Ledent d,
Monique Etienne a,b, Eric Chapuy a,b, Frédéric Libert a,b,e, Alain Eschalier a,b,e,*
a
INSERM E9904, U766, Facultés de Médecine et de Pharmacie, 28, Place Henri Dunant, B.P. 38, F-63001 Clermont-Ferrand Cedex 01, France
b
Clermont Université, Université d’Auvergne, Faculté de Médecine, Laboratoire de Pharmacologie Médicale, F-63001 Clermont-Ferrand, France
c
Clermont Université, Université d’Auvergne, Faculté de Pharmacie, Laboratoire de Pharmacologie, F-63001 Clermont-Ferrand, France
d
IRIBHM, Université Libre de Bruxelles, B-1070 Bruxelles, Belgium
e
CHU Clermont-Ferrand, Service de Pharmacologie, Hôpital G. Montpied, F-63003 Clermont-Ferrand, France

Received 16 October 2007; received in revised form 18 January 2008; accepted 24 March 2008

Abstract

Acetaminophen is the most used analgesic/antipyretic drug. Its unclear mechanism of action could rely on cyclooxygenase inhi-
bition, NO synthesis blockade or reinforcement of the serotonergic system. Here we show that in thermal, mechanical and chemical
pain tests, AM-251, a specific CB1 receptor antagonist, abolished the analgesic action of acetaminophen, which was also lost in CB1
receptor knockout mice. Moreover, acetaminophen was shown unable to bind to CB1 receptors demonstrating an indirect involve-
ment of these receptors in the analgesic effect of this compound. Accordingly with these results, we also demonstrated that the inhi-
bition of FAAH, an enzyme involved in the cerebral metabolism of acetaminophen into AM404, known to reinforce the activity of
the endocannabinoid system, suppressed the antinociceptive effect of acetaminophen. In addition, similarly to the interaction of
acetaminophen with bulbospinal serotonergic pathways and spinal serotonin receptors, we observed that the antinociceptive activity
of ACEA, a CB1 receptor agonist, was inhibited by lesion of bulbospinal serotonergic pathways and antagonists of spinal 5-HT
receptors. We therefore propose that acetaminophen-induced analgesia could involve the following sequence: (1) FAAH-dependent
metabolism of acetaminophen into AM404; (2) indirect involvement of CB1 receptors by this metabolite; (3) endocannabinoid-
dependent reinforcement of the serotonergic bulbospinal pathways, and (4) involvement of spinal pain-suppressing serotonergic
receptors.
Ó 2008 Published by Elsevier B.V. on behalf of International Association for the Study of Pain.

Keywords: Acetaminophen; Endocannabinoids; Fatty acid amide hydrolase; Nociception; Serotonin

1. Introduction ity of the descending serotonergic pathways on spinal

The mechanism of the antinociception elicited by
acetaminophen is not yet elucidated. One main hypo-
thesis relies on the reinforcement of the inhibitory activ-
*
Corresponding author. Address: INSERM E9904, U766, Facul-
tés de Médecine et de Pharmacie, 28, Place Henri Dunant, B.P. 38,
F-63001 Clermont-Ferrand Cedex 01, France. Tel.: +33 4 73 17 82 32;
fax: +33 4 73 27 71 62.
E-mail address: alain.eschalier@u-clermont1.fr (A. Eschalier).
nociceptive processing, which has been supported by dif-
ferent groups [5,43,48]. Indeed, the lesion of the bulbo-
spinal descending serotonergic pathways abolishes the
antinociceptive action of acetaminophen [48]. More-
over, our group demonstrated that different spinal 5-
HT receptor subtypes are involved in the antinociceptive
effect of acetaminophen in rats [6,39] and recently con-
firmed this serotonergic mechanism in humans [42].
However, those mechanisms or others do not explain
how the action of acetaminophen is initiated.

0304-3959/$34.00 Ó 2008 Published by Elsevier B.V. on behalf of International Association for the Study of Pain.
doi:10.1016/j.pain.2008.03.030
 C. Mallet et al. / Pain 139 (2008) 190–200
The discovery of an involvement of endocannabi-
noids on pain modulation opens new mechanistic per-
spectives [12,44]. Anandamide and 2-arachidonoyl-
glycerol, two endogenous ligands of CB1 and CB2 recep-
tors, mainly metabolized by the fatty acid amide hydro-
lase (FAAH), and the monoacylglycerol lipase,
respectively, induce antinociceptive effects [24,51]. Simi-
larly, activation of this system by exogenous ligands for
cannabinoid (particularly CB1) receptors induces antin-
ociception in various acute pain tests in rodents
[16,24,33] but also in several animal models of chronic
pain [17]. Moreover, the combination of D9-THC and
cannabidiol is proposed in the treatment of pain for
patients with multiple sclerosis [46]. Several studies
reported that cerebral injection of cannabinoids in the
periaqueductal gray (PAG) or the rostroventral medulla
(RVM) elicits antinociception, therefore suggesting the
modulation of descending pathways to inhibit pain pro-
cessing at the spinal level [30,32,33].
Interestingly, recent findings showed that acetamino-
phen could be metabolized in the brain into AM404, a
compound able to inhibit the reuptake of anandamide
[14] thanks to FAAH [23] and that the antinociceptive
activity of acetaminophen may rely on an interaction
with the endocannabinoid system [37], even if this last
result has to be cautiously interpreted. We therefore
hypothesized that the interaction of acetaminophen with
the endocannabinoid system could be on the basis of the
reinforcement of the serotonergic system.
In this line, we investigated the possible interaction
between acetaminophen-induced antinoception and the
cannabinoid system by performing three experimental
series: (i) we first studied the involvement of CB1
receptors on the acetaminophen-elicited antinocicep-
tion; (ii) we determined if this involvement was direct
or not; and (iii) we assessed the involvement of the
serotonergic descending bulbospinal pathways and
spinal 5-HT receptors in the antinociceptive effect of
arachidonyl-20 -chloroethylamide (ACEA), a CB1 recep-
tor agonist to compare it with that demonstrated for
acetaminophen.
Our results demonstrated that acetaminophen,
devoid of any direct effect on CB1 receptors, needed a
FAAH-dependent metabolism to exert its CB1-mediated
antinociceptive effect that could lead to the reinforce-
ment of the activity of the 5-HT bulbospinal pathways.
2. Methods
2.1. Animals
Adult male Sprague–Dawley rats weighing 175–200 g were
purchased from Charles River. Male CB1 null mutant (Cb1/)
mice [28] and wild-type mice (CD1 background) were gener-
ously supplied by Dr Ledent. Animal care and experiments
were carried out according to the guidelines of the Committee
191
for Research and Ethical Issues of IASP [54] and to our Insti-
tutional Ethic Committee for animal experiments. Animals
were housed under controlled environmental conditions (21–
22 °C; 55% humidity) and kept under a 12/12 h light/dark
cycle, with food and water ad libitum for a week prior to start-
ing the experiments in order to acclimatize.
2.2. Intrathecal injections
Intrathecal (i.t.) injections were performed under isoflurane
anesthesia (4% induction, 2% maintenance) according to Mes-
tre et al. [34]. The anesthetized rat was held in one hand by the
pelvic girdle and a 25-gauge  1-inch needle connected to a
25 ll Hamilton syringe was inserted into the subarachnoidal
space between lumbar vertebrae L5 and L6, until a tail flick
was elicited. The syringe was held in position for few seconds
after the injection of a volume of 10 ll/rat.
2.3. Lesion of the descending serotonergic pathways
5,7-Dihydroxytryptamine (5,7-DHT, 100 lg/rat), dissolved
in saline containing 0.2 mg/ml ascorbic acid, was administered
intrathecally 7 days before the experiment. Desipramine
(10 mg/kg) was i.p. injected 30 min before 5,7-DHT to prevent
the reuptake of 5,7-DHT by catecholaminergic neurons. On
the day of the experiment, animals were treated with the active
drug and submitted to the paw pressure test (before and 15, 30,
45, 60, 90 and 120 min after administration) before sacrificing
them and removing spinal cords in order to confirm 5,7-DHT
efficacy by determining 5-HT lumbar levels by HPLC. Briefly,
lumbar dorsal horn samples were homogenized in a saturated
KCl solution and centrifuged at 15,000g for 15 min. The
resulting supernatants were mixed with 400 ll of an internal
standard (n-methyl-serotonin 0.6 mg/l, diluted in a pH 11 gly-
cin buffer) and extracted with 2.5 ml of dichloromethane/n-
butanol (75/25). The organic layer was back-extracted with
300 ll of 0.1 M phosphate buffer (pH 4.4). One-hundred
microliters of this buffer was injected in the HPLC system with
electrochemical detector.
2.4. Behavioral pain tests
2.4.1. Formalin test
Rats and mice received 50 and 25 ll of 2.5% formalin
injected subcutaneously (s.c.) into the dorsal surface of the
hind paw, respectively. Drugs were administered at different
times before formalin (40 min in rats or 30 min in mice for
acetaminophen; 10 min for ACEA in rats). Biting and licking
of the injected paw was monitored by measuring the total
duration of the response in seconds during the two typical
phases of nociceptive behavior (phase I: 0–5 min; phase II:
20–40 min for rats or 15–40 min for mice).
2.4.2. Paw pressure test
Rats were submitted to the paw pressure test using an Ugo
Basile analgesimeter (Apelex, tip diameter of the probe: 1 mm,
weight: 30 g). Nociceptive thresholds, expressed in grams (g),
were measured by applying an increasing pressure to the right
hind paw of the animals until a squeak (vocalization threshold)
was obtained (cut-off: 750 g). Treatments were done after the
192 C. Mallet et al. / Pain 139 (2008) 190–200
measurement of two consecutive stable vocalization threshold
values and testing was performed 15, 30, 45, 60, 90 and
120 min after drug administration.
2.4.3. Tail immersion test
Tail of the rats was immersed in a hot water bath main-
tained at 46 °C. The time latency for tail withdrawal was then
determined and a cut-off time of 30 s was applied to avoid
injury. Treatments were done after the measurement of two
consecutive stable withdrawal latency values and testing was
performed 15, 30, 45, 60, 90 and 120 min after drug
administration.
2.5. Assessment of tetrad effect
The general behavioral tests performed to screen for typical
high dosage cannabinoid effects, namely, antinociception,
motor impairment, catalepsy and hypothermia [8] were
conducted.
2.5.1. Antinociception
Antinociception was assessed using the paw pressure test
described above.
2.5.2. Spontaneous locomotor activity
The system used for recording spontaneous activity of the
rats (VideoTrack; ViewPoint, Champagne au Mont d’Or,
France) comprised a digital video camera set above 4 dark
25  25  40 cm enclosures. A computer was used to analyze
digital pictures (25 frames/second). Animal activity was
assessed by the total number of pixels that changed color
between 2 successive pictures (from black to white and from
white to black, when an animal changed its position). Sponta-
neous locomotor activity was counted for 5 min at 15, 30, 60
and 120 min post-administration.
2.5.3. Catalepsy
Catalepsy was measured using the ‘‘ring test” described by
Pertwee [41] modified for the rat [9]. The apparatus consisted
of a plastic ring (12 cm diameter) fixed horizontally at a height
allowing the hindpaws of rats to just touch the bench. The rat
was placed across the ring and the time (s) during which the rat
remained motionless on the ring was recorded. The test was
conducted for 4 min at 15, 30, 60 and 120 min post-
administration.
2.5.4. Body temperature
Rats were placed individually into an environmental room
maintained at a constant temperature of 21 ± 0.3 °C. The ani-
mals were allowed to acclimate for 60 min before the first tem-
perature reading. Prior to drug administration, baseline
temperatures were taken every 30 min for 90 min using a
thermistor probe (Ellab, Denmark), which was lubricated
and inserted approximately 7 cm into the colon. A digital ther-
mometer (Ellab, Denmark) was used to record body tempera-
ture. Rats were unrestrained throughout the experiment, with
only the tail being held gently between two fingers. Following
the baseline interval, treatments were administered. Body tem-
perature was recorded 15, 30, 60 and 120 min post-
administration.
2.6. Experimental procedure
All experiments were performed blind by a single experi-
menter using a parallel group design. Treatments were ran-
domized and administered according to the method of equal
blocks in order to assess the effect of the different treatments
(blindly administered) at the same time interval to avoid
unverifiable and time-variable environmental influences. One
block includes a number of animals corresponding to the num-
ber of the different treatments administered in each experi-
ment; the number of blocks corresponds to the number of
animals per treatment; treatments in the different blocks were
randomized, the order of treatments being different from one
block to an other; all animals in a same block were tested in
the same short laps of time; different animals were used in each
experiments (n = 6–10 per treatment, according to the experi-
ments) which were performed in a quiet room. When the
dose–effect relationship was studied, the following groups were
injected with active drug (acetaminophen or ACEA, 3–4 doses
for each) or vehicle. When the influence of antagonists or
inhibitors was tested, four groups of different animals were
injected as follows: vehicle + vehicle; vehicle + active drug
(acetaminophen or ACEA); antagonist or inhibitor + vehicle;
antagonist or inhibitor + active drug. Acetaminophen (100–
600 mg/kg) was administered orally (p.o.) while ACEA (1–
10 mg/kg), URB597 (0.15 mg/kg) haloperidol (5 mg/kg), desi-
pramine (10 mg/kg) and AM-251 (3 mg/kg) were administered
intraperitoneally (i.p.) and PMSF (10 mg/kg) subcutaneously
(s.c.). WAY-100635 (40 lg/rat), tropisetron (0.5 lg/rat) and
5,7-DHT (100 lg/rat) were administered intrathecally.
2.7. CB1 receptor binding assay
Evaluation of the affinity of acetaminophen for the CB1
cannabinoid receptor in transfected CHO cells was performed
in a radioligand binding assay using [H3]CP 55940, according
to the protocol of Rinaldi-Carmona et al. [45].
2.8. Drugs
Acetaminophen was generously provided by Bristol-Myers-
Squibb (France). N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-
ethyl]-N-2-pyridinylcyclohexane-carboxamide maleate salt
(WAY-100,635), 5,7-dihydroxytryptamine creatinine sulfate
(5,7-DHT), desipramine, haloperidol and Phenylmethylsul-
fonyl Fluoride (PMSF) were purchased from Sigma (France).
N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-
methyl-1H-pyrazole-3-carboxamide (AM-251) and Arachido-
nyl-20 -chloroethylamide (ACEA) were obtained from Tocris
Cookson (UK), and tropisetron from Novartis (France). 30 -
carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) was
from Cayman Chemical (France). PMSF, URB597, AM-251
and ACEA were dissolved in DMSO, while the other drugs
were dissolved in sterile physiological serum (0.9% NaCl).
2.9. Statistical analysis
Data are presented as the means ± SEM. Data from forma-
lin test, hypothermia and locomotor activity assessment (at 2 h
and 60 min, respectively) were analyzed by a one-way ANOVA
 C. Mallet et al. / Pain 139 (2008) 190–200
followed by a Student–Newman–Keuls’ test, when the F-value
was significant. For paw pressure, tail immersion and tetrad
tests, a two-way ANOVA analysis was performed and, when
the F-value was significant, a Dunnett’s test was used to ana-
lyze the time-course of the effects. The level of statistical signif-
icance was set at p < 0.05.
3. Results
3.1. Acetaminophen/endocannabinoid system relationship
3.1.1. Evidence of a CB1 receptor involvement in the
antinociceptive effect of acetaminophen
Acetaminophen (300 mg/kg, p.o.) induced a signifi-
cant antinociceptive activity in the formalin, (30.2 ±
7.8% and 44.3 ± 10.2% inhibition in phase I and II,
respectively; Fig. 1a), paw pressure (maximal vocalization
threshold increase: 97.4 ± 14.1%; Fig. 1b) and tail immer-
sion (maximal latency time increase: 37 ± 6%; Fig. 1c)
tests. In each test, this effect was completely abolished
by AM-251 (3 mg/kg, i.p.), a selective CB1 receptor
antagonist.
To confirm these observations, we examined the
antinociceptive activity of acetaminophen in CB1 recep-
tor knockout mice. While acetaminophen reduced the
nociceptive behavior elicited by formalin in phase I
(29.1 ± 6.1%) and phase II (37.1 ± 10.1%) in wild-
type mice, it did not produce any significant effect when
administered in Cb1/ mice (Fig. 1d).
3.1.2. Tetrad effects of acetaminophen
Those results suggested the involvement of CB1
receptors in the antinociceptive effect of acetaminophen.
We therefore looked at the ability of acetaminophen to
induce cannabinoid tetrad effects. Acetaminophen dose
dependently induced three of these effects: we observed
a dose-dependent antinociceptive activity from 100 to
300 mg/kg (Fig. 2a), as well as a reduction in locomotor
activity and a hypothermia which were delayed in com-
parison with the antinociceptive effect and needed higher
doses (300–600 mg/kg; Fig. 2b, 2c). However, acetami-
nophen did not induce any catalepsy, even at 600 mg/
kg, while haloperidol, used as positive control, was
active in our conditions (Fig. 2d).
Locomotor depressive and hypothermic effects
induced by acetaminophen (300 mg/kg, p.o.) were not
mediated by CB1 receptors as a pre-treatment with
AM-251 (3 mg/kg, i.p.) failed to abolish these two effects
(Fig. 3a and b).
3.2. Interaction between acetaminophen and CB1
receptors
3.2.1. Acetaminophen does not bind to CB1 receptors
We observed no significant displacement of the bind-
ing of [H3]CP 55940 by acetaminophen at concentra-
193
tions as high as 1 mM (percentage of specific binding:
8.6 ± 1.2%; 6.9 ± 2.8% and 11.2 ± 3.0% for acetamino-
phen concentrations of 107, 105 and 103 M, respec-
tively) in transfected CHO cells expressing CB1
receptor. These results indicated that acetaminophen
activates CB1 receptors via an indirect pathway.
3.2.2. Acetaminophen-induced antinociception relies on
FAAH-dependent AM404 formation
We assessed the influence of FAAH inhibitors on the
antinociceptive activity of acetaminophen. PMSF
(10 mg/kg, s.c), used at a dose shown to be able to abol-
ish the FAAH-dependent metabolism of acetaminophen
[23], significantly inhibited the antinociceptive effect of
acetaminophen in both the paw pressure and the forma-
lin tests (Fig. 4a and b). In addition, URB597 (0.15 mg/
kg, i.p.), an irreversible brain-penetrating FAAH inhib-
itor, also reversed the antinociception elicited by acet-
aminophen (Fig. 4c and d).
3.3. CB1 receptor agonist, ACEA, elicits antinociception
via a serotonergic bulbospinal mechanism
In order to demonstrate that the involvement of CB1
receptors in the antinociceptive effect of acetaminophen
was in line with its previously described serotonergic
mechanism, we studied the involvement of both bulbo-
spinal serotonergic pathways and spinal 5-HT receptors
in the effect of a CB1 receptor agonist.
We first performed a dose–response effect of ACEA
(i.p.) in order to look at the antinociceptive activity of this
CB1 agonist in the paw pressure test and compare its
potency with acetaminophen. ACEA induced a dose-
dependent antinociceptive effect for a maximal duration
of 45 min for 10 mg/kg (Fig. 5a). ED50s were 125.9 and
2.8 mg/kg for acetaminophen and ACEA, respectively,
at the peak effect time. The effect of ACEA was inhibited
by AM-251, a CB1 receptor antagonist (Fig. 5b).
We then examined the action of ACEA (3 mg/kg)
after lesioning the serotonergic bulbospinal pathways
using 5,7-DHT. Such a treatment significantly reduced
the 5-HT levels in the dorsal horn of the spinal cord
by 95.5 ± 0.3% (2301 ± 114 vs 103 ± 7 ng/g of spinal
cord tissue for control and 5,7-DHT-treated rats,
respectively) and led to the suppression of the effect of
ACEA in the paw pressure test (Fig. 5c).
To further compare the serotonergic profile of acet-
aminophen and a CB1 receptor agonist, we investigated
whether ACEA-elicited antinociception was involving
the same spinal serotonergic receptors than those identi-
fied with acetaminophen. We previously showed that
intrathecal injection of the 5-HT1A receptor antagonist
WAY-100,635 and tropisetron, a 5-HT3/4 receptor
antagonist, reversed the antinociceptive effect of acet-
aminophen in the formalin test and the paw pressure
test, respectively [1,5,6,40]. We observed that the
194 C. Mallet et al. / Pain 139 (2008) 190–200

Phase I Phase II
135 225

Biting & licking time (s)

Biting & licking time (s)

120 200
105 175
90 * 150
75 125 **
60 100
45 75
30 50
15 25
0 0
Aceta
a (p.o.) - + - + Aceta
a (p.o.) - + - +
AM251 (i.p.)
(i .) - - + + AM251 (i.p.)
(i .) - - + +

600 **
Veh
h - V eh
** Veh
h - Aceta
Vocalization Threshold (g)

500 AM -251 - Veh

AM -251 - Ace
Aceta
**
400
*
300

200
0
0 15 30 45 60 75 90 105 120
Time (min)

11 Veh
h - V eh
** **
** Veh
h - Aceta
Time withdrawal latency (s)

10 AM-251 - Veh
AM-251 - Ace
Aceta
9

6
0
0 15 30 45 60 75 90 105 120
Time (min)

Phase I Phase II
160 400
Biting & licking time (s)
Biting & licking time (s)

140 350
120 * 300
100 250 *
80 200
60 150
40 100
20 50
0 0
Acet a (p.o.) - + - + Aceta (p.o.) - + - +

WT C b1 - / - WT C b1 - / -

Fig. 1. The CB1 receptors are involved in the antinociceptive effect of acetaminophen. The effect of acetaminophen (Aceta; 300 mg/kg, p.o.) was
assessed (a) in the formalin, (b) the paw pressure, and (c) the tail immersion test in rats after i.p. administration of AM-251 (3 mg/kg) or vehicle. In all
tests, AM-251 was administered 10 min before acetaminophen. (d) Formalin test was performed in wild-type (WT) or in Cb1/ mice after treatment
by acetaminophen (Aceta; 300 mg/kg, p.o.) or vehicle. Error bars represent SEM. *p < 0.05, **p < 0.01 as compared with the vehicle-treated group;
n = 6–8 per group.

antinociceptive activity of ACEA was blocked by WAY- by tropisetron (0.5 lg/rat, i.t.) in the paw pressure test
100,635 (40 lg/rat, i.t.) in the formalin test (Fig. 6a) and (Fig. 6b).
 C. Mallet et al. / Pain 139 (2008) 190–200 195

Antinociception Motor impairment

600 * Veh
Aceta 100 mg/kg
* Veh
14

Vocalization Threshold (g)

Aceta 200 mg/kg

Locomotor activity (*100 cm)

500 Aceta 100 mg/kg 12 Aceta 300 mg/kg
* Aceta 200 mg/kg Aceta 600 mg/kg
* * Aceta 300 mg/kg 10
400 * 8

6
300 *
4 *

200 2 *

0 0
0 15 30 45 60 75 90 105 120 0 15 30 45 60 75 90 105 120
Time post dose (min) Time post dose (min)

Hypothermia Catalepsy
38.8

38.4 300
Temperature (°C)

38.0 * *
250
*
37.6
200

Latency( s)
* Veh
37.2 Aceta 100 mg/kg
150 * Aceta 200 mg/kg
36.8 Veh * Aceta 300 mg/kg
Aceta 100 mg/kg *
36.4 100 Aceta 600 mg/kg
Aceta 200 mg/kg Haloperidol5 mg/kg
36.0 Aceta 300 mg/kg 50
Aceta 600 mg/kg
*
0 0
0 15 30 45 60 75 90 105 120 0 15 30 45 60 75 90 105 120
Time post dose (min) Time post dose (min)

Fig. 2. Dose–effect relationship of acetaminophen in the behavioral tetrad effect. Animals were tested after administration (at time 0) of
acetaminophen (aceta; 100, 200, 300 or 600 mg/kg, p.o.) or vehicle (a) in the paw pressure test, (b) by video tracking, (c) in the ring test and (d) for
hypothermia. Haloperidol (5 mg/kg, i.p.) was used as a positive control for catalepsy (c). Error bars represent SEM. *p < 0.05, as compared with the
vehicle-treated group; n = 6–8 per group.

4. Discussion for instance, from the inhibition of cyclooxygenases

[36,38], to the inhibition of NO synthesis [4], to the
From its initiating molecular mechanism to the reinforcement of the serotonergic system [5,42,43,48].
mediators/receptors responsible for antinociception, They all failed to fully explain the action of the drug,
the singular mechanism of action of acetaminophen probably because some gaps remain to be filled
remains elusive and is still a matter of debate more between all those different systems. Here, we report
than one century after the discovery of this com- events that could serve as a new basis to resolve this
pound. Several hypotheses have been emitted going, mystery.

Before treatments
38.8 After treatments

38.4 8
Locomotor activity (*100 cm)

38.0 * 7
*
Temperature (ºC)

6 * *
37.6 **
5
37.2 ***
4
36.8
3
36.4
2
36.0 1

0 0
Veh Veh AM-251 AM-251 Veh Veh AM-251 AM-251
Treatments: Treatments:
Veh Aceta Veh Aceta Veh Aceta Veh Aceta

Fig. 3. The CB1 receptors are not involved in the motor impairment and hypothermia due to acetaminophen. (a) Rectal temperature was measured
in rats before and after treatments with an i.p. injection of AM-251 (3 mg/kg) or vehicle and an oral administration of acetaminophen (300 mg/kg) or
vehicle. Data shown were obtained 2 h after administration of acetaminophen. (b) Motor activity was assessed in rats treated with AM-251 (3 mg/kg,
i.p.) or vehicle and acetaminophen (300 mg/kg, p.o.) or vehicle. Data shown were obtained 1 h after administration of acetaminophen. In all tests,
AM-251 was administered 10 min before acetaminophen. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the same
treated group, before treatments (a) or with the vehicle-treated group (b); n = 6–8 per group.
196 C. Mallet et al. / Pain 139 (2008) 190–200

Phase I Phase II
135 250

Biting & licking time (s)

Biting & licking time (s)

120 225
105 200
90 * 175
75 150 *
125
60 100
45 75
30 50
15 25
0 0
Aceta (p.o.) - + - + Aceta (p.o.) - + - +
PMSF (s.c.) - - + + PMSF (s.c.) - - + +

700
** Veh - Veh
Vocalization Threshold (g) Veh - Aceta
600 **
PMSF - Veh
PMSF - Aceta
500

400

300

200
-15 0 15 30 45 60 75 90 105 120
Time (min)

Phase I Phase II
135 250
Biting & licking time (s)

Biting & licking time (s)

120 225
105 200
* 175
90
75 150 **
125
60 100
45 75
30 50
15 25
0 0
Aceta (p.o.) - + - + Aceta (p.o.) - + - +
URB597 (i.p.) - - + + URB597 (i.p.) - - + +

700
**
Veh - Veh
Vocalization Threshold (g)

600 ** Veh - Aceta

URB597 - Veh
500 URB597 - Aceta
**
400

300

200
-15 0 15 30 45 60 75 90 105 120
Time (min)

Fig. 4. The FAAH inhibitors, PMSF and URB597, block the antinociceptive effect of acetaminophen. The effect of acetaminophen (Aceta; 300 mg/
kg, p.o.) was assessed in the formalin (a and c) and the paw pressure tests (b and d) after administration of PMSF (10 mg/kg, s.c.) or vehicle (a and b)
and URB597 (0.15 mg/kg, i.p.) or vehicle (c and d). In all tests, PMSF and URB597 were administered 20 and 10 min before acetaminophen,
respectively. Error bars represent SEM. *p < 0.05, **p < 0.01 as compared with the vehicle-treated group; n = 6–8 per group.

Our results propose that the endocannabinoid system acetaminophen and AM-251, a specific antagonist of
would be a major component of the activity of acetami- CB1 receptors, which inhibited the effect of acetamino-
nophen and could be an essential link between some of phen. If our results are in accordance with a recent
the different hypotheses. Firstly, we observed that the report [37], it is noteworthy that this hypothesis needed
antinociceptive activity of acetaminophen relied on the to be verified using different noxious stimuli. Indeed, in
endocannabinoid system in different behavioral tests in this previous study, the effect of the drug was assessed
rats and mice. We demonstrated the involvement of only using high doses of acetaminophen (up to
CB1 receptors using both knockout mice insensitive to 1000 mg/kg, p.o.) in the thermal hot plate nociceptive
 C. Mallet et al. / Pain 139 (2008) 190–200 197

600 * *
ify this last point. Secondly, we looked at the effect of
acetaminophen on the classical ‘‘tetrad” which has
Veh
allowed to establish a cannabimimetic behavioral profile
Vocalization Threshold (g)

500 ACEA 1 mg/kg

* ACEA 3 mg/kg for the naturally occurring D9-THC and anandamide
* ACEA 10 mg/kg
400 *
[8,52] or several cannabinoid receptor agonists [17].
Using orally administered doses of acetaminophen from
300 100 to 600 mg/kg, we observed three of the four tetrad
effects (antinociception, motor impairment, hypother-
200 mia), but failed to observe catalepsy. According to Mar-
0 tin et al. [31] and Fride and Mechoulam [18] who
0 15 30 45 60 75 90 105 120 consider that only an activity of compounds in all four
Time (min)
tests would characterize them as cannabimimetics, we
600
could suspect that acetaminophen is not a cannabimi-
Veh - Veh
*** *** Veh - ACEA metic compound. This conclusion is reinforced by the
lack of inhibition of its locomotor depressive and hypo-
Vocalization Threshold (g)

AM - 251 - Veh
500
AM - 251 - ACEA thermic effects by a CB1 receptor antagonist and is in
400
line with the finding that non-cannabinoid compounds
(amphetamine, scopolamine, morphine, desipramine,
300
pimozide, pentobarbital, ethanol, and diazepam) can
induce some of the tetrad effects showing the lack of
200 specificity of this procedure [53].
0
Whatever, the results obtained here in pain tests dem-
0 15 30 45 60 75 90 105 120 onstrated that intact CB1 receptors are needed for the
Time (min) antinociceptive effect of acetaminophen. The question
would now be how CB1 receptors are activated. Indeed,
600 Veh - Veh
** Veh - ACEA
we did not observe any binding of acetaminophen to
CB1 receptors with a high affinity confirming the results
VocalizationT hreshold (g)

5,7 - DHT - Veh

500
**
400
300
200
0
0 15 30
Time (min)
Fig. 5. Serotonergic bulbospinal pathways are needed for CB1 agonist-
induced antinociception. (a) Animals were tested after administration
of ACEA (1, 3, 10 mg/kg, i.p.) or vehicle in the paw pressure test. (b)
The effect of ACEA (3 mg/kg, i.p.) was then assessed in the paw
pressure tests 10 min after i.p. injection of AM-251 (3 mg/kg) or
vehicle. (c) Vocalization thresholds were determined in rats after i.p.
injection of ACEA (3 mg/kg) or vehicle in healthy rats or in rats with
lesioned serotonergic bulbospinal pathways by 5,7-DHT. Error bars
represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with
the vehicle-treated group; n = 6–8 per group.
test. Those doses can induce toxicity [26], as well as
motor impairment and hypothermia, observed in the
present work, which induce a false antinociception when
motor reflexes and thermal stimulus are used [29]. More-
over, Haller et al. [22] failed to show any inhibitory
effect of a CB1 antagonist using phenylbenzoquinone-
induced abdominal writhes which could suggest differ-
ences in CB1 involvement due to the stimulus used or
to the site where the noxious stimulus was applied
(somatic versus visceral). Further work is needed to ver-
5,7 - DHT - ACEA of Fowler et al. [15]. A recent study suggested that every-
**
thing could start from the liver where acetaminophen is
metabolized into p-aminophenol, which can be further
transformed into AM404 in the brain after a FAAH-
dependent conjugation with arachidonic acid [23].
AM404 is in turn able to reinforce the activity of the
endocannabinoid system through both the inhibition
45 60 75 90 105 120 of cellular reuptake [3,19] and the reduction of degrada-
tion (by inhibiting FAAH) [20] of anandamide. Several
studies also reported that AM404 could induce a can-
nabinoid-dependent antinociception [21,27]. In line with
those data, we studied the effect of FAAH inhibitors on
the antinociceptive activity of acetaminophen to assess
the involvement of this metabolic pathway. We want,
first, to mention that the inhibition of FAAH can reduce
the degradation of anandamide [20], which could
account for an antinociceptive effect [25,47]. In order
to avoid such an effect, we used low FAAH-inhibiting
doses of URB597 (0.15 mg/kg, i.p.) and PMSF
(10 mg/kg, s.c.) that did not induce any intrinsic antino-
ciception in our conditions, as previously shown by
Kathuria et al. [25] and Compton and Martin [7],
respectively. Using these inhibitors, in these conditions,
we showed, for the first time, that this enzyme is needed
for the effect of acetaminophen. Thus FAAH-dependent
formation of AM404 seems to be necessary for acetami-
nophen-induced antinociceptive effect and could
account for an indirect involvement of CB1 receptors
by the analgesic drug. However, further studies are
198 C. Mallet et al. / Pain 139 (2008) 190–200

Phase I Phase II
150 350

Biting & licking time (s)

Biting & licking time (s)

135 300
120
250 **
105 **
90 200
75
60 150
45 100
30
50
15
0 0
ACEA (i.p.) - + - + ACEA (i.p.) - + - +
WAY-100635 (i.t.) - - + + WAY-100635 (i.t.) - - + +

600 Veh - Veh

** **
Veh - ACEA

Vocalization Threshold (g)

Tropisetron - Veh
500
Tropisetron - ACEA

400 **

300

200

0
0 15 30 45 60 75 90 105 120
Time (min)

Fig. 6. Spinal 5-HT receptors are involved in CB1 agonist-induced antinociception in rats. The effect of ACEA (3 mg/kg, i.p.) was assessed (a) in the
formalin and (b) the paw pressure tests after i.t. injection of (a) WAY-100,635 (40 lg/rat) or vehicle and (b) tropisetron (0.5 lg/rat) or vehicle.
Intrathecal administrations were performed 5 min before acetaminophen or saline. Error bars represent SEM. **p < 0.01 as compared with the
vehicle-treated group; n = 6–8 per group.

needed to assess the antinociceptive effect and the mech- That would lead to suspect that acetaminophen would
anism of action of AM404. first reinforce the activity of the endocannabinoid sys-
Different studies reported the implication of the tem and then that of the serotonergic one. However,
endocannabinoid system in pain modulation either at the demonstration that AM404 can be synthesized in
spinal or supraspinal levels [35]. Notably, the stimula- the spinal cord after acetaminophen treatment and the
tion of CB1 receptors in PAG and RVM reduced GABA presence of spinal CB1 receptors [23] might also suggest
release from the presynaptic boutons of local interneu- an involvement of these receptors. Thus, further studies
rons which can reduce the GABAergic negative influ- will be needed to determine the site of the interaction of
ence on the inhibitory descending pathways [49,50]. acetaminophen (or its metabolite AM404) with the
Since the antinociceptive activity of acetaminophen endocannabinoid system, the actual modalities of this
has been shown to depend on spinal serotonergic recep- interaction and how this leads to the reinforcement of
tors [6,11,39,40] and that the source of spinal 5-HT the serotonergic system.
comes exclusively from supraspinal centers and mainly It also remains possible that AM404 mediates the
from RVM [35], we suspected that the participation of antinociceptive activity of acetaminophen through other
the endocannabinoid system in the effect of acetamino- mechanisms. For instance, AM404 could activate the
phen would occur through the reinforcement of the TRPV1 receptor, whose stimulation has been shown to
activity of the bulbospinal serotonergic pathways. elicit antinociception in the PAG [30] and to be active in
Accordingly, we demonstrated that the antinociceptive the tetrad tests [13]. In addition, AM404 has been shown
effect induced by a CB1 receptor agonist (ACEA) needed to inhibit COX activities in vitro, with a potency close to
intact descending bulbospinal serotonergic pathways. those of NSAIDs [23]. The involvement of a central COX
Furthermore, ACEA-dependent recruitment of the sero- inhibition during the acetaminophen-induced antinoci-
tonergic system involved similar spinal 5-HT receptors ceptive and/or antipyretic activities will have to be con-
to those involved in the antinociceptive action of acet- firmed in vivo. On the contrary, the fact that AM404 is
aminophen and 5-HT [1,2,5,6,40], i.e. the 5-HT1A recep- not found in blood after administration of acetamino-
tor in the formalin test and the 5-HT3/4 receptor in the phen [23] might explain why acetaminophen does not
paw pressure test. This similar 5-HT-dependent mecha- exert any significant peripheral anti-inflammatory effect.
nism and the inhibition of the effect of acetaminophen This could therefore further explain why acetaminophen
by inactivation of CB1 receptors suggest that the endo- does not exert any significant anti-inflammatory effect.
cannabinoid system is an important link between acet- Other studies stated that the antinociceptive activity of
aminophen and 5-HT to produce antinociception. acetaminophen may rely on a decrease in spinal NO
 C. Mallet et al. / Pain 139 (2008) 190–200
Acetaminophen
LIVER 1 References
p-aminophenol
p-aminopheno
FAAH
BRAIN 2 involved in the spinal antinociceptive effect of 5-HT in rats. Pain
AM404
CB1 receptor 3 [4] Bjorkman R, Hallman KM, Hedner J, Hedner T, Henning M.
PAG
RVM Serotonergic
descending 4 [5] Bonnefont J, Alloui A, Chapuy E, Clottes E, Eschalier A. Orally
pathways
5-HT receptors 5
SPINAL CORD
Analgesia
Fig. 7. Schematic representation of the proposed sequence of events
going from an oral treatment with acetaminophen to its antinocicep-
tive activity. (1) Acetaminophen is deacetyled in p-aminophenol in the
liver; (2), then metabolized in the brain by FAAH into AM404. (3)
AM404 reinforces the activity of the supraspinal CB1 receptors, (4)
which in turn reinforce the activity of the serotonergic descending
pathways. (5) Spinal release of serotonin (5-HT) stimulates either the
5-HT3/4 receptor in a mechanical pain test or the 5-HT1A receptor in a
chemical pain test to inhibit the transmission of noxious stimuli
towards higher centers and therefore produce antinociception. PAG:
periaqueductal gray; RVM: rostroventral medulla.
[e.g. 4], which could also be a consequence of the rein-
forcement of the endocannabinoid system [10].
In conclusion, our present data together with results
published by Högestätt et al. [23] suggest that the activity
of acetaminophen, orally administered, would require a
multi-step process (Fig. 7). Firstly, the drug would need
to be metabolized in two steps to form cerebral FAAH-
dependent AM404. This metabolite could reinforce the
activity of the endocannabinoid system through CB1
receptors, which would in turn reinforce the activity of
the bulbospinal serotonergic inhibitory pathways.
Finally, antinociception would occur at the spinal level
where activation of stimulus-dependent 5-HT receptors
would block the transmission of nociceptive influx.
Acknowledgements
This work was funded by ‘Association Nationale de
la Recherche Technique’ and Bristol-Myers-Squibb
199
(grant 796/2004 to C. Mallet) and l’Institut Paul Hamel.
The authors have no conflicting financial interests.
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