The antipyretic effect of paracetamol occurs
independent of transient receptor potential
ankyrin 1–mediated hypothermia and is
associated with prostaglandin inhibition in the
brain
Elahe Mirrasekhian, Johan L. Å. Nilsson, Kiseko Shionoya, Anders Blomgren, Peter M. Zygmunt, David Engblom, Edward D. Högestätt and Anders Blomqvist
The self-archived postprint version of this journal article is available at Linköping University Institutional Repository (DiVA):
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N.B.: When citing this work, cite the original publication.
Mirrasekhian, E., Nilsson, J. L. Å., Shionoya, K., Blomgren, A., Zygmunt, P. M., Engblom, D., Högestätt, E. D., Blomqvist, A., (2018), The antipyretic effect of paracetamol occurs independent of transient receptor potential ankyrin 1–mediated hypothermia and is associated with prostaglandin inhibition in the brain, The FASEB Journal. https://doi.org/10.1096/fj.201800272R
Original publication available at:
https://doi.org/10.1096/fj.201800272R
Copyright: Federation of American Society of Experimental Biology (FASEB) http://www.faseb.org/
The antipyretic effect of paracetamol occurs independently of transient receptor potential ankyrin 1 mediated hypothermia and is associated with prostaglandin inhibition in the brain
Elahe Mirrasekhian1,*, Johan L. Å. Nilsson2,*, Kiseko Shionoya1, Anders Blomgren2, Peter M. Zygmunt2, David Engblom1, Edward D. Högestätt2#, Anders Blomqvist1
1Department of Clinical and Experimental Medicine, Linköping University, S-581 85
Linköping, Sweden, and 2Division of Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, S-221 85 Lund, Sweden
*These authors contributed equally to this work
#Deceased
Running title: Paracetamol antipyresis is TRPA1 independent
19 pages, 4 figures, 1 supplementary figure
Correspondence to: Dr. Anders Blomqvist, Division of Neurobiology, Department of Clinical and Experimental Medicine, Linköping University, S-581 85 Linköping, Sweden. E-mail: anders.blomqvist@liu.se; Dr. Peter M. Zygmunt, Division of Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, S-221 85 Lund, Sweden. E-mail: peter.zygmunt@med.lu.se
Nonstandard Abbreviations
Cox cyclooxygenase
KO knock-out
LPS lipopolysaccharide NAC N-acetyl cysteine
NAPQI N-acetyl-p-benzoquinoneimine NSAID nonsteroidal anti-inflammatory drug PG prostaglandin
TRPA1 transient receptor potential ankyrin 1 TRPV1 transient receptor potential vanilloid 1
TX thromboxane
Abstract
The mode of action of paracetamol (acetaminophen), which is widely used for treating pain and fever, has remained obscure, but may involve several distinct mechanisms, including cyclooxygenase inhibition and transient receptor potential ankyrin 1 (TRPA1) channel activation, the latter being recently associated with paracetamol’s propensity to elicit hypothermia at higher doses. Here we examined if the antipyretic effect of paracetamol was due to TRPA1 activation or cyclooxygenase inhibition. Treatment of wild-type and TRPA1 knock-out mice rendered febrile by immune challenge with lipopolysaccharide (LPS) with a dose of paracetamol that did not produce hypothermia (150 mg/kg), but known to be
analgetic, abolished fever in both genotypes. Paracetamol completely suppressed the LPS induced elevation of prostaglandin E2 in the brain, and also reduced the levels of several
other prostanoids in brain as well as in blood. The hypothermia induced by paracetamol was abolished in mice treated with the electrophile scavenger N-acetyl cysteine. We conclude that paracetamol’s antipyretic effect in mice is dependent on inhibition of cyclooxygenase
activity, including the formation of pyrogenic prostaglandin E2, whereas
paracetamol-induced hypothermia likely is mediated by the activation of TRPA1 by electrophilic metabolites of paracetamol, similar to its analgesic effect in some experimental paradigms.
Introduction
Paracetamol (acetaminophen) is one of the most commonly used over-the-counter drugs to treat pain and fever. However, its mechanism of action has remained a matter of discussion. Similar to nonsteroidal anti-inflammatory drugs (NSAIDs), paracetamol has been shown to inhibit prostaglandin synthesis (1, 2), but has been suggested to do so not by interfering with the active site of prostaglandin synthesizing cyclooxygenases, as done by NSAIDs, but by reducing their active oxidized form to their resting form (3). Paracetamol mediated
cyclooxygenase inhibition would therefore be more effective under conditions of low
peroxide concentration, such as in the brain, but inactive under conditions of high peroxidase concentrations, such as in an inflammatory site. Such a mechanism would be consistent with the known tissue selectivity of acetaminophen and explain why it is antipyretic and anti-nociceptive while possessing only minor anti-inflammatory properties (3, 4).
During recent years, the idea that paracetamol exerts its action by cyclooxygenase inhibition has been challenged. It has been shown that the anti-nociceptive effect of paracetamol may be executed by the formation of paracetamol metabolites, such as the N-acylphenolamine derivative N-arachidonoylaminophenol (AM404) and N-acetyl-p-benzoquinoneimine (NAPQI) activating the TRP ion channels transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1), respectively (5-8). Paracetamol given at a high dose is known to induce hypothermia both in mice and humans (9), and it was recently demonstrated that this effect is dependent on TRPA1 but not on TRPV1 (10). In the same study it was also reported that the same high dose of paracetamol that produced hypothermia in wild-type (WT) mice normalized body temperature in TRPA1 knock-out (KO) mice in a model of yeast induced fever, suggesting that paracetamol
produces antipyretic and hypothermic effects through distinct and independent mechanisms (10).
Here we examined the antipyretic effect of paracetamol, using a well-established lipopolysaccharide (LPS)-induced fever paradigm (11-13). We titrated a dose of paracetamol that does not produce hypothermia when given to WT mice, and after having verified that paracetamol-induced hypothermia is TRPA1 mediated, we administered the sub-hypothermic dose to TRPA1 KO and WT mice that had been rendered febrile by peripheral injection of bacterial cell wall lipopolysaccharide. We report that paracetamol exerts an antipyretic effect in both genotypes, i.e. it is independent of TRPA1, and we show that the antipyretic effect is associated with cyclooxygenase inhibition and normalized prostaglandin E2 levels in the
brain. Furthermore, we show that the glutathione precursor N-acetyl cysteine protects WT mice from the hypothermic effect of paracetamol, indicating that the paracetamol-induced hypothermia, similar to what was shown for its analgesic effect in some experimental paradigms, is mediated by the activation of TRPA1 by paracetamol’s electrophilic metabolites, such as NAPQI (5).
Materials and Methods Animals
Adult TRPA1 KO and WT mice of both sexes were used, and experimental groups were balanced with respect to sex and age. The KO mice were originally provided by Dr. David Julius (14) (RRID: MGI:3696956) and backcrossed for over 10 generations onto C57BL/6 mice (Taconic, Denmark) in the laboratory of Högestätt and Zygmunt. Homozygous breeding was then used for 2-3 generations to create KO and WT mice for experiments. The mice were kept at a 12:12 h light/dark cycle (light on at 7.00 a.m.) with free access to food and water. All injections were given early during the light period. Between and after injections animals were left undisturbed to avoid eliciting activity related temperature responses. All
experiments were approved by the Linköping and Lund Animal Ethics Committees, and were in accordance with the European Union directive for the use of animals for scientific
purposes (2010/63).
Drugs
In the experiments monitoring body temperature, LPS from Escherichia coli (100 µg/kg; Sigma, St. Louis, MO, USA; 0111:B4), paracetamol (100-200 mg/kg; Sigma), and parecoxib (10 mg/kg, Dynastat®; Pfizer, Sollentuna, Sweden) were all diluted in saline and injected intraperitoneally (i.p.; ~ 0.1 ml). N-acetyl cysteine (1g/kg; Meda, Solna, Sweden) was diluted in saline and injected subcutaneously (s.c.; ~0.3 ml).
Telemetric temperature recordings
The mice were briefly anesthetized with 1 % isoflurane (Abbot Scandinavia, Solna, Sweden) and implanted i.p. with a transmitter that records core body temperature (Data Sciences International, New Brighton, MN, USA; model TA11TAF10). Buprenorphine (Temgesic; 100 µg/kg, in about 0.1 ml saline i.p.; RB Pharmaceuticals, Sough, Berkshire, UK) was given for post-operative analgesia. Immediately after the surgery, the mice were transferred to a room in which the ambient temperature was set to 29°C, providing near-thermoneutral conditions (13). The animals were allowed to recover for at least 1 week before any
recordings were made. This time period was selected to permit e.g. the replenishment of cytochrome P450 2E1 (CYP2E1), the liver enzyme which metabolizes paracetamol to its electrophilic metabolite NAPQI, which activates TRPA1 (5), and which is known to be inhibited by isoflurane and other fluorinated anesthetics (15). Prior to drug injection, the basal temperature of each mouse was recorded for at least 24 h to assure that they displayed normal body temperature with normal circadian variation.
Mass spectrometry analyses of prostanoids
LPS (100 µg/kg i.p.) or saline was administered 4 h prior to i.p. injection of acetaminophen (150 mg/kg) or vehicle [Ringer’s acetate solution, Fresenius Kabi AB, Uppsala, Sweden (Na+ 131 mM, Cl- 112 mM, Acetate 30 mM, K+ 4 mM, Ca2+ 2 mM, Mg 2+ 1 mM)]. Forty minutes after the acetaminophen injection, the animals were anesthetized with isoflurane and
decapitated. The brain was removed, and the diencephalon was promptly dissected, snap frozen in liquid nitrogen, and stored at -80°C. Blood was collected in test tubes containing 100 µl buffered citrate and ibuprofen (500 µM), frozen on dry ice, and stored at -80°C.
Blood and diencephalon were homogenized in 1 ml ice-cold 10 mM Tris buffer, to which had been added 1 mM EDTA, 300 µM ascorbic acid, 10 µM methyl arachidonyl fluorophosphonate (Cayman Chemical Co., Ann Arbor, MI, USA) and 100 µM ibuprofen to prevent in vitro prostanoid formation and degradation. Aliquots (200 µl) of homogenates (blood and diencephalon) were precipitated with 1 ml ice-cold acetone, containing 300 µM ascorbic acid and 10 nM PGE1-d4 (Cayman Chemical),as internal standard. After
centrifugation at 17960 g for 10 min at 4°C, all supernatants were vacuum evaporated. Each extraction residual was reconstituted in 200 µl of a mixture of 80 % methanol, 19.5 % H2O
and 0.5 % acetic acid. The protein content of the pellet was determined by Coomassie protein assay (Pierce, Illinois, USA), using bovine serum albumin as standard reference.
All samples were analyzed on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) coupled to a Sciex 5500 tandem mass spectrometer (Applied Biosystems/MDS Sciex, Darmstadt, Germany). PGE2 (Sigma-Aldrich, Schnelldorf Germany), PGF2α
(Sigma-Aldrich), 6-keto-PGF1α (a stable metabolite of prostaglandin I2; Cayman Chemical) and
TXB2 (a stable metabolite of thromboxane A2; Biomol GmbH, Hamburg, Germany), were
quantified by LC-MS/MS in negative mode. Sample aliquots of 5 µl were injected onto a Waters CSH C18 column [50 × 2 mm, 3 µm (Waters, Milford, USA)] held at 50°C. All
mobile phases were water-acetonitrile gradients, containing 0.1 % formic acid and using a flow rate of 500 µl/min. The acetonitrile content was initially 20 %, then increased linearly to
100 % over 3 min and kept at this level for 3 min 15 s, followed by an equilibration period at 20 % for 45 s. The electrospray interface was operated in the positive ion mode at 550°C and the ion spray voltage was set to ~ 4500 volts. The mass transitions (m/z) were as follows: 351.2/315.3 for PGE2, 357.2/321.2 for PGE1-d4,369.2/169.1 for TXB2, 353.4/309.3 for
PGF2α, and 369.4/163.1 for 6-keto-PGF1α. The declustering potentials and the collision energies were between -105 and -120 volts and between -17 and -36 volts, respectively. All samples were normalized to an internal standard (PGE1-d4). The detection limits were
calculated as the concentrations corresponding to three times the standard deviation of the blanks. When levels were below detection limit (diencephalon: PGE2, 493,26 pmol/g protein;
TXB2,162,88; 6-keto-PGF1α,264,30; PGF2α, 1874,68; blood: PGE2,99,33; TXB2,21,57),
numerical values of 1/4 of the detection limit were used in the calculations.
Statistical analyses
Analysis of temperature differences (nadir temperatures) between treatments of WT mice with different doses of paracetamol or of differences in WT mice in prostanoid
concentrations in blood and brain following saline + vehicle, LPS + vehicle or LPS +
paracetamol treatment was done with a one-way ANOVA, followed by Dunnett’s or Tukey’s multiple comparisons test, respectively. Analysis of treatment effects (paracetamol) on LPS induced temperature responses of WT and KO mice were done with a two-way ANOVA, with genotype and treatment as main factors, followed by Tukey’s multiple comparisons test. All statistical analyses were done in Graph Pad Prism (GraphPad Software, La Jolla, CA, USA).
Results
TRPA1 KO mice display normal circadian temperature variation
We monitored basal temperature of WT and TRPA1 KO mice during a 48-h period. Both WT and TRPA1 KO mice showed normal circadian temperature rhythm, and there was no
significant difference between the genotypes (Fig. 1). However, for both genotypes, female mice displayed a slightly higher average temperature than male mice (37.4°C vs 36.8°C for WT mice, and 37.3°C vs 36.8 for KO mice).
Paracetamol given at 200 mg/kg, but not at 150 mg/kg, produces TRPA1-dependent hypothermia
mg/kg body weight injected i.p. to WT mice resulted in pronounced hypothermia, whereas 100 and 150 mg/kg did not (1-way ANOVA: F3,14 = 9.292, P = 0.0012 at nadir temperature;
P = 0.0153 for 200 mg vs 150 mg paracetamol; Fig. 2A, A1). This observation is consistent
with that previously reported by Li et al. (16), who found that 160 mg/kg paracetamol produced hypothermia, whereas doses <160 mg/kg did not. It is also in line with the
observation by Gentry et al. (10) that 300 mg/kg, but not 100 mg/kg produced hypothermia. We then verified that the hypothermia induced by the higher dose of paracetamol (200 mg/kg) indeed was TRPA1 mediated. We found that whereas WT mice displayed
hypothermia, as in the previous experiment (Fig. 2A), the body temperature of TRPA1 KO remained normal [2-way ANOVA (genotype x treatment): F1,17 = 4.795, P = 0.0428; P =
0.0463 for paracetamol treatment of WT and KO mice; Fig. 2B, B1].
N-acetyl cysteine abolishes paracetamol induced hypothermia
Because the analgesic effect of paracetamol may be mediated by the binding to TRPA1 of its electrophilic metabolites, such as NAPQI (5), we examined if NAPQI also was involved in paracetamol induced hypothermia. We therefore treated WT mice with N-acetyl cysteine (NAC), which replenishes the glutathione needed for metabolizing NAPQI into an inactive conjugate (17). We found that mice treated with 1g/kg NAC, given s.c. 20 min prior to paracetamol (200 mg/kg), did not develop hypothermia, in contrast to mice treated with vehicle [2-way ANOVA (treatment): F1,20 = 10.41, P = 0.0042; P = 0.0229 for NAC vs
saline treatment of paracetamol treated mice; Fig. 2C, C1].
TRPA1 KO mice display LPS induced fever, similar to WT mice, and in both genotypes 150 mg/kg paracetamol is antipyretic
Having established that a dose of 150 mg/kg paracetamol did not produce hypothermia in non-immune challenged mice, we used this dose, known to produce analgesia (7, 18), to treat LPS induced fever. We injected WT and TRPA1 KO mice with LPS i.p. (100 µg/kg) during the beginning of the light period. This procedure yielded a body temperature elevation of about 1°C compared to that of mice that were injected with saline [2-way ANOVA (treatment): F1,51 = 21.23 and P < 0.0001; P = 0.0446 and P = 0.0002 for LPS vs NaCl
treatment of WT and KO mice, respectively; Fig. 3A, A1]. Four hours after the LPS/saline
injection, i.e. at a time point when the LPS treated mice had displayed a sustained fever for about 2 h (Fig. 3A), we injected the mice i.p. with paracetamol (150 mg/kg) or saline. In mice of both genotypes that were first injected with saline, the second injection, irrespective of whether it was paracetamol or saline, yielded a handling stress induced hyperthermia (as did
the first injection; Fig. 3A). Similarly, mice of both genotypes that first had been injected with LPS and as a second injection received saline showed a stress induced increase of their body temperature, but from an already elevated level (Fig. 3A). Importantly, mice first injected with LPS and which in a second injection was given paracetamol displayed a prominent but transient temperature fall, which for TRPA1 KO mice normalized their body temperature [2-way ANOVA (treatment): F1,36 = 54.47 and P < 0.0001; P < 0.0001 and P =
0.0008 for paracetamol vs NaCl treatment of WT and KO mice respectively], hence
demonstrating that TRPA1 is not necessary for the antipyretic effect of paracetamol (Fig. 3A, A2). In WT mice, however, paracetamol not only normalized the body temperature, but
produced an additional hypothermia [2-way ANOVA (genotype): F1,36 = 5.116 and P =
0.0298; P = 0.0285 for paracetamol treated WT and KO mice; Fig. 3A, A2]. The nadir of the
temperature fall appeared at approximately 1 h after the paracetamol injection and lasted for about 2 ½ h. These observations fit well with published data on the pharmacokinetics of paracetamol in mice. After intraperitoneal injection of 200 mg/kg, maximal plasma
concentration was reached already at 15 min and half-life was about 60 min (19). There was no statistically significant difference between male and female mice, with WT mice of both sexes displaying hypothermia after paracetamol, whereas neither male or female KO mice did so.
To determine if the different sensitivity to 150 mg/kg paracetamol between febrile WT and TRPA1 KO mice was dependent on paracetamol induced activation of TRPA1, we repeated the experiment with antipyretic treatment of LPS-induced fever, but this time with the cyclooxygenase-2 (Cox-2) inhibitor parecoxib, for which no interaction with TRPA1 should be expected. Injection of parecoxib 4 h after LPS resulted in a temperature depression that was almost identical in magnitude between TRPA1 KO and WT mice (Fig. 3B),
suggesting that the differences seen between genotypes in the response to paracetamol were TRPA1 dependent.
Based on these observations we hypothesized that the increased sensitivity of febrile mice to the hypothermic effect of paracetamol was due to reduced glutathione stores and hence reduced capacity to neutralize paracetamol’s electrophilic metabolites. We tested this hypothesis by treating febrile WT mice with the cysteine prodrug NAC, which replenishes the intracellular levels of the antioxidant glutathione, before they were injected with paracetamol. As shown in Fig. 3C, NAC strongly attenuated the paracetamol induced hypothermia.
Paracetamol inhibits LPS-induced prostanoid synthesis in the brain
Finally, we examined to what extent paracetamol inhibited prostanoid synthesis when given during LPS-induced fever. WT mice were injected with LPS followed by either paracetamol or vehicle 4 h later. A control group was given saline followed 4 h later by vehicle. Animals were killed 40 min after the last injection and brain and blood were collected for analysis. This time point is about midway between Tmax for paracetamol (19) and nadir of its
antipyretic effect (Fig. 3A, A2). In selecting this time point we reasoned that the most
prominent suppression of prostaglandin levels in the brain (including the levels of PGE2)
would be seen prior to nadir of the temperature response when temperature still was falling rapidly, because at nadir, PGE2 levels should be rising to be able to reverse the temperature
fall.
As shown in Fig. 4A, LPS treatment induced PGE2 in the brain, and this induction was
completely suppressed by paracetamol (1-way ANOVA: F2,15 = 36.31, P < 0.0001; P <
0.0001 for LPS vs vehicle and LPS vs LPS + paracetamol). Notably, PGE2 levels were below
detection limit in blood (not shown). Although there was no or only modest increase in the levels of other analyzed prostanoids (TXB2, PGF2α and 6-keto-PGF1α; Fig. 4B-E) in the LPS
treated mice, paracetamol decreased the levels of these prostanoids in the brain, pointing to a general suppression of prostanoid synthesis by paracetamol [TXB2 in diencephalon: 1-way
ANOVA: F2,15 = 6.616, P = 0.0087; P = 0.0068 for LPS vs LPS + paracetamol (Fig. 4B);
PGF2α in diencephalon: F2,15 = 4.127, P = 0.0373; P = 0.0349 for LPS vs LPS + paracetamol
(Fig. 4D); 6-keto-PGF1α in diencephalon: F2,15 = 21.05, P < 0.0001; P = 0.0092 for LPS vs
vehicle; P < 0.0001 for LPS vs LPS + paracetamol; and P = 0.02 for LPS + paracetamol vs vehicle (Fig. 4E)]. Paracetamol treatment also yielded very low TXB2 levels in blood (Fig.
4C); however, the differences vs the non-paracetamol treated groups were not statistically significant.
Discussion
The present findings show that a dose of paracetamol that does not elicit hypothermia when given to naïve mice, still has the capacity to normalize the body temperature of mice rendered febrile by peripheral injection of LPS. The abolishment of fever was seen both in WT mice and in TRPA1 KO mice, demonstrating that the antipyretic effect of paracetamol is not dependent on TRPA1. Furthermore, the antipyretic effect of paracetamol was temporally associated with a complete suppression of the increased PGE2 levels in the brain that was
production of PGE2 is critical for fever (20-22), the present findings, taken together, indicate
that paracetamol exerts its antipyretic effect by inhibiting prostaglandin synthesis, a
mechanism that hence is different from the mechanism by which paracetamol, at high doses, evokes hypothermia (10). The latter mechanism depends on TRPA1 and seems to involve paracetamol metabolites, such as NAPQI, because, as shown here, it is abolished by NAC, a substance that promotes inactivation of NAPQI. Hence, it is similar to the mechanism by which paracetamol is anti-nociceptive, which has been shown also to depend on TRPA1, at least in some experimental paradigms (5).
The present study also shows that LPS-induced fever is independent of TRPA1 as LPS evoked the same temperature elevation in both WT and TRPA1 KO mice. In contrast, it has been shown that pain and acute vascular reactions caused by LPS are primarily dependent on TRPA1 activation in nociceptive sensory neurons (23) and that LPS-evoked mechanical hypersensitivity is dependent on TRPA1 via the endogenous activator hydrogen sulfide (24-26). LPS-induced fever is attenuated by genetic deletion in brain endothelial cells of IL-6 receptors (12) and of prostaglandin synthesizing enzymes (27), implying a mechanism by which humorally released proinflammatory cytokines act on the brain, which is distinct from the mechanisms by which LPS elicits pain. These data are well in line with two distinct mode of actions by which paracetamol is antipyretic and anti-nociceptive, respectively.
We previously demonstrated that the antipyretic action of paracetamol was dependent on the gene dose of the prostaglandin synthesizing enzyme Cox-2. We showed that mice heterozygous for the Cox-2 gene, and hence displaying lower levels of Cox-2, were more sensitive to the fever reducing effect of paracetamol than WT mice (28). Specifically, we demonstrated that the largest paracetamol dose that had no effect on LPS induced fever in WT mice strongly attenuated fever in Cox-2 heterozygous mice, whereas no difference was seen between mice homozygous and heterozygous, respectively, for the terminal PGE2
isomerase microsomal prostaglandin E synthase-1 (mPGES-1), implying that paracetamol indeed exerted its action by inhibiting Cox-2.
Although Flower and Vane already in 1972 suggested that the antipyretic activity of paracetamol could be explained by its inhibition of prostaglandin synthase in the brain (2), surprisingly few studies have demonstrated that paracetamol blocks pyrogen-induced
elevation of brain PGE2. Feldberg et al. (29) demonstrated in cats that elevation of PGE1-like
activity in the cerebrospinal fluid (and fever), evoked by intracerebral injection of bacterial pyrogen, was abolished when animals were given an intraperitoneal injection of paracetamol, and Li et al. (16) demonstrated in mice that paracetamol, albeit at a dose that yields
hypothermia, normalized PGE2 levels in animals that had been immune-challenged with
intravenous injection of LPS. However, studies have shown that paracetamol reduces brain PGE2 levels also in non-immune challenged mice (7, 30, 31), suggesting that it may act both
on constitutive and inducible PGE2-production. Although the ability of paracetamol to reduce
constitutive levels of PGE2 was not directly tested in the present study, it should be noted that
the average PGE2 level after paracetamol treatment of mice rendered febrile by LPS injection
was in fact lower than that seen in control mice that had been given two consecutive
injections of saline and vehicle only (Fig. 4A); however, the difference was not statistically significant. Furthermore, the present data showing that paracetamol not only blocked induced PGE2, implying an effect on Cox-2, but also reduced the levels of other prostaglandins and
TXB2 (Fig. 4B-E), indicate that paracetamol inhibits both Cox-1 and Cox-2. This idea is
consistent with the hypothesis that paracetamol interacts with the peroxidase site of the cyclooxygenase enzymes (32), present in both isoforms. This effect would preferentially be seen in tissues with low peroxide concentration, such as the brain.
It should also be noted that there was no induction of PGE2 in blood, with PGE2 levels
being under the detection limit in all examined treatment groups. Blood-borne PGE2, through
diffusion or transport into the brain, has been suggested to be involved in immune-induced fever (33, 34). However, it is unlikely to play such a role at the time point (280 min post injection) examined in the present study.
Interestingly, there seems to be a critical dose between 150 mg/kg and 200 mg/kg for paracetamol-induced hypothermia in mice. Li et al. (16) defined the dose to 160 mg/kg, and in the present study 200 mg/kg yielded hypothermia, whereas 150 mg did not, and Gentry et al. (10) reported hypothermia (in WT but not in TRPA1 mice) when 300 mg/kg was given s.c., whereas 100 mg/kg (given intrathecally) had no effect on body temperature. We found that WT mice treated with 150 mg/kg paracetamol when having been rendered febrile by a previous injection of LPS, displayed hypothermia, whereas TRPA1 KO mice did not (Fig. 3A, A2). Since the same treatment with a selective Cox-2 inhibitor instead of paracetamol
resulted in a normalized temperature in both TRPA1 KO and WT mice without eliciting hypothermia (Fig. 3B), the findings indicated that paracetamol at the given dose indeed interacted with TRPA1. TRPA1 is a unique chemosensor strictly controlled by the redox status (35, 36). The reason for the increased sensitivity of the febrile mice to the hypothermic effect of paracetamol is therefore probably explained by reduced glutathione stores as a consequence of the oxidative stress associated with inflammation and fever (37) and hence reduced capacity to neutralize the TRPA1-binding of paracetamol’s electrophilic metabolites.
This idea was supported by the finding that the cysteine prodrug NAC, which replenishes the intracellular levels of the antioxidant glutathione and itself is a scavenger of tissue damaging thiol reactive agents such as NAPQI (17), strongly attenuated the hypothermia elicited by 150 mg/kg paracetamol in the febrile mice (Fig. 3C).
In conclusion, we show that paracetamol exerts an antipyretic effect that is TRPA1-independent and that this effect is associated with inhibition of the synthesis of PGE2 (as well
as of other prostanoids) in the brain. Taken together with previous findings, these data support evidence that paracetamol exerts its antipyretic effect by inhibiting cyclooxygenase enzymes. However, paracetamol also activates TRPA1 through its electrophilic metabolite NAPQI. This activation elicits hypothermia when paracetamol is given at high doses, but may also contribute to the antipyretic effect of lower paracetamol doses, especially during febrile conditions.
Acknowledgements
This study was supported by the Swedish Medical Research Council (#20725 to DE, #07879 to AB and 2014-3801 to PMZ and EDH), the European Research Council (ERC-starting grant to DE), the Knut and Alice Wallenberg foundation (DE), the Swedish Brain Foundation (DE and AB), the Swedish Cancer Foundation (#213/692 to AB), the County Council of Östergötland (DE and AB), the Medical Faculty of Lund University (ALF) (EDH and PMZ) and AFA insurance (EDH and PMZ). We thank Anna Eskilsson for help with the
illustrations.
Author contributions
E.D.H., and P.M.Z. conceived the study. A. Blomqvist, D.E., E.D.H. and J.N. designed and directed research; E.M. and K.S. conducted the temperature measurements; J.N. conducted the biochemical experiments; A. Blomgren performed the mass spectrometry analyses; A. Blomqvist, D.E., E.D.H., E.M. and J.N. analyzed data; A. Blomqvist drafted the manuscript. All authors discussed the results and revised or commented on the manuscript.
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Legends to figures
Fig. 1. Diurnal body temperature variations in wild-type (WT) and TRPA1 knock-out (KO)
mice. Solid lines represent mean, and dotted lines SEM. Dark periods are indicated by horizontal bars on the abscissa. n = 6.
Fig. 2. Temperature responses to paracetamol in wild-type (WT) and TRPA1 knock-out (KO)
mice, and effect of N-acetyl cysteine. A. Paracetamol at a dose of 200 mg/kg (injected at time zero; arrow) elicits hypothermia, whereas a dose of 150 mg/kg or less does not. Note that the initial temperature peak is due to the handling stress associated with the intraperitoneal injection. Solid lines represent mean, and dotted lines SEM. A1 shows nadir temperatures
(means ± SEM) at 80 min after the paracetamol/saline injection (vertical line in A). * indicates P < 0.05. n = 4-6. B. TRPA1 KO mice, in contrast to WT mice, do not display hypothermia after treatment with 200 mg/kg paracetamol (injected at time zero; arrow). Solid lines represent mean, and dotted lines SEM. B1 shows nadir temperatures (means ± SEM) at
80 min after the paracetamol/saline injection (vertical line in B). * and ** indicate P < 0.05 and P < 0.01, respectively. n = 5-6. C. N-acetyl cysteine (NAC) blocks paracetamol induced hypothermia. NAC (1g/kg body weight) or vehicle was injected s.c. 40 min prior to the paracetamol injection (200 mg/kg, at time zero). C1 shows values at 106 min (vertical line in
C) after paracetamol/saline injection. * indicates P < 0.05. n = 7 for paracetamol treated animals and n = 5 for vehicle treated animals. While the initial temperatures differed between groups, there were no differences in the mean temperature of the different groups during 24 h preceding the experiments (not shown).
Fig. 3. Antipyretic effect of paracetamol. A. Intraperitoneal injection of LPS (100 µg/kg at
time zero; left arrow) results in fever in both wild-type (WT) and TRPA1 knock-out (KO) mice. 150 mg/kg paracetamol given during the LPS induced fever (at 240 min; right arrow) normalizes body temperature in TRPA1 KO mice, but results in hypothermia in WT mice. Solid and dashed lines represent mean, and dotted lines SEM. Note that half of the WT and KO mice (dashed blue and magenta lines) at 240 min were injected with either paracetamol (solid dark blue and magenta lines) or saline (solid light blue and pink lines). n = 20 and 10 before and after 240 min, respectively. Solid green line represent WT and KO mice injected with saline + saline and saline + paracetamol. n = 15. The temperature curves for the
individual groups are shown in Supplementary Fig. 1. A1 shows the febrile response to LPS
of WT and KO mice at 180 min after LPS injection (left vertical line in A). * and ** indicate
mice. Note that saline treated groups include for each genotype mice that at 240 min after LPS injection were treated with either paracetamol or saline, and the color of these
temperature traces as shown in Supplementary Fig. 1 is represented by the pattern color and fill color, respectively, of each bar. A2 shows nadir temperatures (means ± SEM) 70 min after
treatment with 150 mg/kg paracetamol (right vertical line) that was given at 240 min post LPS injection. Horizontal dashed line indicate nadir temperature for WT and KO mice treated with saline + saline (i.e. the ”normal” temperature in this experimental setting). * and *** indicate P < 0.05 and P < 0.001, respectively. n = 10. B. Parecoxib, 10 mg/kg, given 240 min after LPS injection (at time zero) normalizes LPS induced fever in both WT and TRPA1 KO mice. Solid lines represent mean, and dotted lines SEM. n = 8. C. N-acetyl cysteine (1g/kg s.c.) given 30 min prior to 150 mg/kg paracetamol in an experimental set-up similar to that shown in A (100 mg/kg LPS injected i.p. 4 h prior to paracetamol) attenuates the paracetamol induced hypothermia of WT mice. *** indicates P < 0.001. n = 8 (male mice).
Fig. 4. Concentrations of prostanoids in brain and blood of mice immune challenged with
LPS (100 µg/kg), with or without treatment with paracetamol (150 mg/kg). A. PGE2 levels in
the diencephalon in mice treated with saline and vehicle, LPS and vehicle, or LPS and paracetamol. PGE2 levels were also analyzed in blood, but was below detection level (not
shown). B. TXB2 levels in the diencephalon. C. TXB2 levels in blood. D. PGF2α levels in the
diencephalon. E. 6-keto-PGF1α levels in the diencephalon. *, **, *** indicate P < 0.05, 0.01 and 0.001, respectively. n = 6.
12 18 24 30 36 42 48 35 36 37 38 temperature ( oC) 6 0 time (h) Figure 1
-60 60 120 180 240 300 360 33.5 34.0 34.5 35.0 35.5 36.0 36.5 37.0 37.5 38.0 time (min) temperature ( oC) paracetamol 150 mg/kg saline paracetamol 200 mg/kg 0 34 saline 100 mg 150 mg 200 mg 35 36 37 paracetamol temperature ( oC) -60 60 120 180 240 300 360 34.5 35.0 35.5 36.0 36.5 37.0 37.5 38.0 38.5 time (min) WT paracetamol 200 mg/kg KO paracetamol 200 mg/kg WT saline KO saline B temperature ( oC) WT KO 35 36 37 38
paracet saline paracet saline
B1 0 ** * temperature ( oC) B1 A1 Figure 2 par ac etamol/saline par ac etamol/saline -60 60 120 180 240 300 360 420 35.0 35.5 36.0 36.5 37.0 37.5 time (min) NAC + paracetamol 200 mg/kg saline + paracetamol 200 mg/kg NAC + saline saline + saline 0 temperature ( oC) C C1 par ac etamol/saline NA C/saline NAC
paracet paracetNaCl salineNAC salineNaCl 35.0 35.5 36.0 36.5 37.0 37.5 temperature ( oC) * C1
35.5 36.0 36.5 37.0 37.5 A1 WT KO LPS saline LPS saline * ** WT KO 34 35 36 37 38 paracet
saline saline paracet
A2 *** *** temperature ( oC) temperature ( oC) Figure 3 -60 60 120 180 240 300 360 420 480 540 35.0 35.5 36.0 36.5 37.0 37.5 38.0 38.5 39.0 time (min) WT KO 0 B temperature ( oC) par ec oxib LPS C * -60 0 60 120 180 240 300 360 420 480 540 34.0 34.5 35.0 35.5 36.0 36.5 37.0 37.5 38.0 time (min) temperature (°C) WT LPS + paracetamol 150 mg/kg KO LPS + paracetamol 150 mg/kg saline/paracetamol controls KO LPS WT LPS KO LPS + saline WT LPS + saline LPS/saline par ac etamol/saline A1 A2 -60 0 60 120 180 240 300 360 420 480 33.5 34.0 34.5 35.0 35.5 36.0 36.5 37.0 37.5 time (min) vehicle NAC ∗∗∗ LPS NA C/saline par ac etamol temperature ( oC)
pmol/g protein saline + LPS + LPS + paracetamol 0 1000 2000 3000 4000 0 500 1000 1500 pmol/g protein 0 100 200 300 saline + LPS + LPS + paracetamol saline + LPS + LPS + paracetamol TXB2 blood 0 2000 4000 6000 PGF2a diencephalon
pmol/g protein pmol/g protein
saline + LPS + LPS + paracetamol 0 500 1000 1500 2000 2500 pmol/g protein 6-keto-PGF1a diencephalon saline + LPS + LPS + paracetamol C D E *** *** ** *** * ** * vehicle
vehicle vehicle vehicle
vehicle vehicle vehicle vehicle
vehicle vehicle
-60 60 120 180 240 300 360 420 480 540 34.5 35.0 35.5 36.0 36.5 37.0 37.5 time (min) WT LPS + saline KO LPS + saline WT saline + paracetamol 150 mg/kg KO saline + paracetamol 150 mg/kg WT saline + saline KO saline + saline 0 temperature ( oC)
Supplementary Fig. 1. Temperature traces for each group of mice in the experiment reported in Fig. 3A. The upper 4 traces (deep blue and light blue for WT mice, and magenta and pink for KO mice) seen between about 120 and 240 min represent mice that were given LPS, and the lower 4 traces (dark green and green for WT mice, and brown and yellow for KO mice) represent mice that instead were given saline. At about 300 min, the upper two traces (light blue and pink) represent LPS treated mice WT or KO, respectively, that were given saline at 240 min, whereas the deep blue and magenta traces represent LPS treated WT and KO mice that were given paracetamol at 240 min. Dark green, green, brown and yellow traces represent WT and KO animals that first were injected with saline (instead of LPS) and at 240 min given either paracetamol (dark green and brown) or saline (green and yellow). n = 10 for LPS treated mice and n = 3-4 for mice given saline instead of LPS.
