Acetaminophen (APAP) is a COX inhibitor that inhibits COX-1 and COX-2 (IC50=113.7/25.8 μM). Acetaminophen has antipyretic and analgesic activity as well as weak anti-inflammatory activity.

COX-2:5.8 μM

方法：非黑色素瘤和黑色素瘤细胞系用 Acetaminophen (100 µM) 处理 48 h，使用 MTT Assay 检测细胞活力。
结果：Acetaminophen 在黑色素瘤细胞系 SK-MEL-5、MeWo、B16-F0 和 B16-F10 中显示出相当大的毒性，分别导致 40±3%、45±7%、66±8% 和 60±5% 的细胞毒性。在非黑色素瘤细胞系 PC-3、BJ、Saos-2 和 SW-620 细胞中，Acetaminophen 在 100μM 时显示出可忽略不计的毒性。[1]
方法：神经母细胞瘤细胞 SH-SY5Y 用 Acetaminophen (2 mM) 处理 24-48 h，使用 Western Blot 方法检测靶点蛋白表达水平。
结果：Acetaminophen 以时间依赖的方式诱导 cytochrome c 从线粒体释放，在处理 48 h 后达到最大水平。此外，胞质和线粒体级分的免疫印迹分析表明，Acetaminophen 能够在处理后 24 h 诱导 Bax 积累到线粒体中。[2]

方法：为检测体内肝毒性，将 Acetaminophen 腹腔注射给小鼠 (300 mg/kg) 和大鼠 (1 g/kg)。
结果：小鼠肝脏大面积坏死，但在大鼠样本中几乎没有损伤。大鼠对 Acetaminophen 诱导的肝损伤具有高度抵抗力。[3]
方法：为检测体内肝毒性，将 Acetaminophen 急性 (300 mg/kg，灌胃给药)、慢性 (100 mg/kg，饮食给药，每天一次，持续六周) 或亚急性 (250 mg/kg，灌胃给药，每天三次，持续三天) 给药给老年和虚弱小鼠。
结果：尽管某些途径发生了变化，预计会影响对 Acetaminophen 毒性的易感性，但小鼠的 Acetaminophen 肝毒性并没有随着年龄的增长或虚弱而总体增加。[4]

Effect of inhibition of Acetaminophen on COX-1 and COX-2 activity in human whole blood: For COX-1 assay, aliquots of human whole blood drawn from healthy volunteers without anticoagulant are transferred to glass tubes containing Acetaminophen or DMSO, serum is separated by centrifugation after clotting, and serum TxB2 levels are determined. For COX-2 assay, aliquots of heparinized whole blood are incubated with LPS (10 μg/mL) and aspirin (10 μg/mL), plus Acetaminophen or DMSO for 24 hours at 37 °C, plasma is separated by centrifugation, and PGE2 levels are determined subsequently. The degree of COX-1 or COX-2 inhibition is calculated as the percentage change of plasma eicosanoid (TxB2 for COX-1 and PGE2 for COX-2).Concentration response curves are fitted by a sigmoidal regression with variable slope for both enzymatic assays, and the 50% inhibitory concentration (IC50) values are derived by using of PRISM Version 3.0.

Cells are exposed to Acetaminophen for 48 hours. Cell viability is determined by the trypan blue exclusion method. Intracellular GSH is measured by recording the disulfide, GS-TNB and 5-thio-nitrobenzoic acid (TNB), the yellow colored compound formed by the reaction between GSH with DTNB.(Only for Reference)