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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 10, 2005 as doi:10.1096/fj.04-2437fje. |
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* Cardiothoracic Pharmacology, UCCM, Royal Brompton Hospital, NHLI, Imperial College London; and
The William Harvey Research Institute, Barts and the London, Queen Mary’s School of Medicine and Dentistry, Charterhouse Square, London, UK
1Correspondence: Cardiothoracic Pharmacology, UCCM, Royal Brompton Hospital, NHLI, Imperial College London, Dovehouse St., London SW3 6LY, UK. E-mail: j.a.mitchell{at}imperial.ac.uk
SPECIFIC AIMS
Acetaminophen is commonly used for safe and effective treatment of pain and fever. It works by preferentially lowering cyclo-oxygenase products in the central nervous system, where oxidant stress is strictly limited. The precise mechanism of action for acetaminophen on cyclo-oxygenase activity is debated. Two theories prevail: 1)acetaminophen selectively inhibits a distinct form of cyclo-oxygenase, cyclo-oxygenase-3; and 2) acetaminophen has no affinity for the active site of cyclo-oxygenase, but instead blocks activity by reducing the active oxidized form of cyclo-oxygenase to an inactive form.
We have used an in vitro model of cyclo-oxygenase-2 activity (A549 cells stimulated with IL-1ß) to compare mechanisms of action of acetaminophen with other nonsteroidal anti-inflammatory drugs (NSAIDs), including selective inhibitors of cyclo-oxygenase-2.
PRINCIPAL FINDINGS
1. Acetaminophen inhibits cyclo-oxygenase-2 activity in intact cells but not in broken cell preparations
When incubated with IL-1ß, acetaminophen caused a concentration-related inhibition in the accumulation of PGE2 in the medium over 24 h (Fig. 1
). When acetaminophen was added to cells in which cyclo-oxygenase-2 had been preinduced (24 h after IL-1ß), the A23187-induced burst of PGE2 was also inhibited in a concentration-dependent manner by acetaminophen (Fig. 1)
. When comparing the two experimental systems, acetaminophen was found to be more potent as an inhibitor of PGE2 production when added together with the inducing agent than when added to inhibit the acute formation (Fig. 1)
. Acetaminophen had no effect on the expression of cyclo-oxygenase-2 protein when incubated with the cells for 24 h (Fig. 1)
. In contrast, acetaminophen at concentrations (10–3 M), which were effective intact cells, had no inhibitory effect on cyclo-oxygenase-2 activity in broken cell preparations. In broken cells, acetaminophen caused a paradoxical increase in enzyme activity.
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2. The inhibitory effects of acetaminophen are fully reversed by increasing intracellular levels of lipid peroxide using t-butylOOH
The cell permeable organic hydroperoxide, t-butylOOH, had no affect on cyclo-oxygenase activity and did not affect the ability of acetaminophen to inhibit PGE2 production when given together with IL-1ß for 24 h (probably due to a short half life of the compound). However, the inhibitory effects of acetaminophen added after cyclo-oxygenase-2 induction, 1 h prior to stimulation with A23187, were complete blocked by cotreatment with t-butylOOH (5x10–5 M or 2x10–4 M).
3. The inhibitory effects of naproxen, ibuprofen, and rofecoxib, but not diclofenac or indomethacin, on cyclo-oxygenase-2 activity are significantly reduced by t-butylOOH
In addition to acetaminophen, the following NSAIDs induced concentration-dependent inhibition of cyclo-oxygenase-2 activity preinduced in A549 cells, naproxen, ibuprofen, rofecoxib, diclofenac, and indomethacin. Cotreatment of cells with t-butylOOH (2x10–4 M) did not influence the inhibitory effects of either diclofenac or indomethacin. However, t-butylOOH (2x10–4 M) significantly reduced the inhibitory effect of naproxen, ibuprofen, and rofecoxib (Fig. 2
).
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CONCLUSIONS AND SIGNIFICANCE
Acetaminophen is an efficacious antipyretic and analgesic, but is ineffective as an inhibitor of inflammation. These properties of acetaminophen suggest its therapeutic actions are somewhat restricted to central and peripheral nervous tissue. Selective inhibitors of cyclo-oxygenase-2 share the antipyretic and analgesic properties of acetaminophen, which suggests that the therapeutic target for acetaminophen is cyclo-oxygenase-2. In support of this, we have recently shown that under physiological conditions, cyclo-oxygenase-2 is constitutively expressed in the brain, while being absent in other organs and tissues. It has been known for some time that under some conditions, cyclo-oxygenase activity is influenced by local peroxide concentrations and that acetaminophen may inhibit cyclo-oxygenase activity indirectly by influencing the oxidation state of the enzyme. Here we show that in human intact cells expressing cyclo-oxygenase-2, the inhibitory effect of acetaminophen as well as some NSAIDs including rofecoxib, are acutely sensitive to hydroperoxide levels.
We present data here illustrating the complex nature of the interactions between acetaminophen and cyclo-oxygenase in different experimental conditions. In intact cells, when added together with the inducing agent, acetaminophen is a very potent inhibitor of cyclo-oxygenase-2-derived PGE2, with an IC50 
10-fold lower than therapeutic concentrations in humans. In this system, the effects of acetaminophen are on cyclo-oxygenase activity and not on enzyme induction because protein levels were unchanged by the drug. Acetaminophen is an effective, but less potent, inhibitor of cyclo-oxygeanse-2 activity in cells acutely stimulated to form prostanoids by the addition of calcium ionophore. Under these conditions, the inhibitory effects of acetaminophen were completely reversed by supplementation of intracellular levels of organic hydroperoxides. It seems logical to conclude that the reason for such dramatic differences in potency of acetaminophen in intact cells between the two protocols is intracellular levels of hydroperoxides. The activation of cells by addition of A23187 will certainly increase the local concentration of a range of intracellular mediators, including hydroperoxides.
In contrast to data obtained in intact cells, acetaminophen even at very high concentrations (10–3 M, 10-fold higher than the therapeutic range) was unable to inhibit cyclo-oxygenase-2 activity in broken cell preparations. Under these conditions, acetaminophen actually increased cyclo-oxygenase activity. This appears nonsensical and in complete contradiction to the data we obtained using intact cells. The stimulation of activity we note could be mediated by two possible mechanisms. First, acetaminophen may protect cyclo-oxygenase from the inactivating properties of hydrogen peroxide, which may be present in our homogenates. Under these conditions, H2O2 inactivation may occur via peroxidase-dependent destruction or via reduction of the oxidized, intermediated enzyme form, which undergoes irreversible inactivation. Second, acetaminophen may increase the rate of cyclo-oxygenase activity by donating electrons. In support of this theory, we noted that the stimulatory effect of acetaminophen on cyclo-oxygenase activity was abolished when the electron donating cofactor epinephrine was present. Alternatively, the reciprocal oxidation of acetaminophen by cyclo-oxygenase may result in the formation of a secondary radical that may enhance enzymatic activity. This phenomenon does not occur in intact cells or in vivo. Thus, the findings relating to acetaminophen and cyclo-oxygenase in homogenates or purified form remain important for our understanding of the biochemistry of the enzyme but have little relevance to its therapeutic mechanisms.
How is acetaminophen inhibiting cyclo-oxygenase-2 activity in intact cells? Two main theories prevail. Acetaminophen can reduce the oxidized from of cyclo-oxygenase to the inactive "resting" form. Our data showing that the inhibitory effects of acetaminophen are completely reversed by increasing hydroperoxide levels are in complete agreement with those published by Boutaud and co-workers and support this hypothesis. Elevated levels of peroxide antagonize the reductant function of acetaminophen and return the enzyme to its higher oxidation state. Thus, cells or tissues with high levels of peroxide such as activated platelets or inflammatory sites, are resistant to the action of acetaminophen, whereas the drug blocks prostanoid formation in endothelial cells at low micromolar concentrations. A second theory suggests that acetaminophen inhibits a structurally distinct form of cyclo-oxygenase (cyclo-oxygenase-3). Cyclo-oxygenase-3 is a product of the cyclo-oxygenase-1 gene identical to cyclo-oxygenase-1 but with the retention of intron 1 in its mRNA. The difference at the protein level between cyclo-oxygenase-3 and cyclo-oxygenase-1 is the insertion of 30–34 aa (depending on the mammalian species) into the hydrophobic signal peptide. In cyclo-oxygenase-3 this signal peptide is not cleaved and the protein is glycosylated, and displays cyclo-oxygenase activity. The initial report of cyclo-oxygenase-3 showed that in comparative assays, using canine cyclo-oxygenase-3, murine cyclo-oxygenase-1, and murine cyclo-oxygenase-2 expressed in transfected insect cells, cyclo-oxygenase-3 was selectively inhibited by acetaminophen. However, cyclo-oxygenase-3 is unlikely to be the target of acetaminophen in human tissues. In humans, the retention of intron 1 in cyclo-oxygenase-1 would shift the cyclo-oxygenase-3 sequence out of frame with respect to the open reading frame of cyclo-oxygenase-1, resulting in a completely different protein with no conceivable cyclo-oxygenase activity.
In addition to acetaminophen, the inhibitory effects of naproxen, ibuprofen, and rofecoxib were significantly blunted by increasing intracellular hydroperoxide levels with t-butylOOH. The inhibitory effects of diclofenac or indomethacin were, however, essentially unaffected by t-butylOOH. These findings show that inhibitors of cyclo-oxygenase might be reclassified with regard to their ability to interfere with oxidation state of the enzyme and/or essential radicals in the reaction. This is particularly relevant when our findings with rofecoxib are considered. Although this drug was developed to fit selectively into the active site of cyclo-oxygenase-2, it may well share some of the properties of acetaminophen. These data suggest that some inhibitory effects of rofecoxib and other NSAIDs are independent of the active site of the enzyme.
In summary, we present data that support the hypothesis that acetaminophen, as well as some NSAIDs, inhibit human cyclo-oxygenase-2 activity by reducing the enzyme to its inactive resting state. We suggest that our data illustrates an as yet unexplored therapeutic window for the "cyclo-oxygenase inhibitor." Compounds that combined active site selectivity with actions on enzyme oxidation state could display particular tissue selectivity.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2437fje;
This article has been cited by other articles:
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  S. Schildknecht, A. Daiber, S. Ghisla, R. A. Cohen, and M. M. Bachschmid Acetaminophen inhibits prostanoid synthesis by scavenging the PGHS-activator peroxynitrite FASEB J, January 1, 2008; 22(1): 215 - 224. [Abstract] [Full Text] [PDF]
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J. A. Mitchell, R. Lucas, I. Vojnovic, K. Hasan, J. R. Pepper, and T. D. Warner Stronger inhibition by nonsteroid anti-inflammatory drugs of cyclooxygenase-1 in endothelial cells than platelets offers an explanation for increased risk of thrombotic events FASEB J, December 1, 2006; 20(14): 2468 - 2475. [Abstract] [Full Text] [PDF]
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) or on the acute production of PGE2 from A23187-stimulated A549 cells (activity, 
). All data values represent mean ± SE, n = 6. B) Effect of vehicle (lane 1), vehicle plus acetaminophen 10–4 M (lane 2), IL-1ß (lane 3) or IL-1ß plus acetaminophen 10–4 M (lane 4) incubated for 24 h on COX-2 expression in A549 cells. Representative of 3 experiments.
