Effects of acetaminophen on constitutive and inducible prostanoid biosynthesis in human blood cells

Abstract

1. Acetaminophen, an analgesic and antipyretic drug with weak antiinflammatory properties, has been suggested to act as a tissue-selective inhibitor of prostaglandin H synthases (PGHSs) (e.g. COX-1 and COX-2) through its reducing activity, that is influenced by the different cellular levels of peroxides. 2. We have studied the effects of acetaminophen on inducible and constitutive prostanoid biosynthesis in monocytes and platelets in vitro. To discriminate between the inhibitory effect of the drug on PGHS-isozymes vs PGE-synthases (PGESs), parallel measurements of PGE(2) and thromboxane (TX) B(2) were carried out. Since antioxidant enzymes and cofactors, present in plasma, may affect acetaminophen-dependent inhibition of prostanoids, comparative experiments in whole blood vs isolated monocytes were performed. 3. Acetaminophen inhibited LPS-induced whole blood PGE(2) and TXB(2) production, in a concentration-dependent fashion [IC(50) microM (95% confidence intervals): 44 (27-70) and 94 (79-112), respectively]. Therapeutic plasma concentrations (100 and 300 microM) of the drug more profoundly reduced PGE(2) than TXB(2) (71 +/- 3 vs 54 +/- 4 and 95 +/- 0.8 vs 78 +/- 2%, respectively, mean +/- s.e.mean, n = 6, P < 0.01). 4. Differently, in isolated monocytes stimulated with LPS, both PGE(2) and TXB(2) production was maximally reduced by only 60%. 5 At 100 and 300 microM, the drug caused a similar and incomplete inhibition of platelet PGE(2) and TXB(2) production during whole blood clotting (45 +/- 4 vs 54 +/- 4 and 75 +/- 2 vs 75 +/- 1%, respectively, mean +/- s.e.mean, n = 4). 6 In conclusion, therapeutic concentrations of acetaminophen caused an incomplete inhibition of platelet COX-1 and monocyte COX-2 but in the presence of plasma, the drug almost completely suppressed inducible PGE(2) biosynthesis through its inhibitory effects on both COX-2 and inducible PGES.

Figure 1

Time-course of PGHS-isozyme and mPGES expression and prostanoid biosynthesis in LPS-stimulated monocytes. Isolated human monocytes (2–3×10⁶ ml⁻¹) were incubated for 0, 4 and 24 h at 37°C with LPS (10 μg ml⁻¹) and PGE₂ and TXB₂ levels were measured in the medium by RIA while COX-1, COX-2 and mPGES levels were evaluated in monocyte lysates by Western blot.

Figure 2

Effects of acetaminophen on prostanoid biosynthesis and PGHS-isozyme expression in LPS-stimulated monocytes. Increasing concentrations (0.1 to 300 μM) of acetaminophen were incubated with 2–3×10⁶ monocytes per millilitre at 37°C for 24 h in the presence of LPS (10 μg ml⁻¹). PGE₂ and TXB₂ released into the medium were assayed by RIA while COX-1 and COX-2 levels were evaluated in monocyte lysates by Western blot.

Figure 3

Effects of acetaminophen on prostanoid biosynthesis in whole blood stimulated with LPS or with endogenous thrombin. Increasing concentrations of acetaminophen (0.1–300 μM) were incubated with 1-ml heparinized whole blood samples, drawn from healthy volunteers pretreated with 300 mg of aspirin 48 h before sampling, in the presence of LPS (10 μg ml⁻¹) for 24 h, and plasma PGE₂ and TXB₂ levels were assayed by RIA as an index of inducible monocyte prostanoid biosynthesis. The same concentrations of the drug were also incubated with 1-ml whole blood samples (drawn from healthy subjects when they had not taken any NSAID during the 2 weeks preceding the study), allowed to clot for 1 h, and serum TXB₂ and PGE₂ levels were measured as a reflection of constitutive platelet prostanoid biosynthesis in response to endogenously formed thrombin. Results are depicted as percentage inhibition (mean±s.e.mean) from 4–6 separate experiments.

Figure 4

Effects of therapeutic concentrations of acetaminophen on inducible and constitutive PGE₂ and TXB₂ production in whole blood. One hundred and 300 μM of acetaminophen were incubated with 1-ml heparinized whole blood samples, drawn from healthy volunteers pretreated with 300 mg of aspirin 48 h before sampling, in the presence of LPS (10 μg ml⁻¹) for 24 h, and plasma PGE₂ and TXB₂ levels were assayed by RIA as an index of inducible monocyte prostanoid biosynthesis. The same concentrations of the drug were also incubated with 1-ml whole blood samples (drawn from healthy subjects when they had not taken any NSAID during the 2 weeks preceding the study), allowed to clot for 1 h, and serum TXB₂ and PGE₂ levels were measured as a reflection of constitutive platelet prostanoid biosynthesis. Results are depicted as percentage inhibition (mean±s.e.mean) from 4–6 separate experiments.