Biological Activity

Bioactivity / Mechanism

Acetaminophen inhibition of prostaglandin production depends on the cellular conditions. In J774.2 macrophages, it suppressed the diclofenac-induced PGE2 response at concentrations >0.06 mM, whereas the LPS-induced response was relatively insensitive at similar concentrations. This experiment does not establish a universal COX-2 selectivity ratio. [1]

In a cell-free cardiolipin-liposome system, acetaminophen inhibited cytochrome c-catalyzed lipid oxidation with an IC50 of 22.8 μM ± 7.6 μM (mean ± SEM; n = 6). This functional redox endpoint is distinct from COX inhibition. [2]

Literature-reported observations and conditions; not Adooq-tested protocols or general treatment recommendations.

Cellular Effect

Cell Line	Activity	Value	Description	Reference
HepG2	EC50	594 μM	Cytotoxicity against human HepG2 cells assessed as intracellular ATP level after 24 hrs by luciferase reporter gene assay	[PMID: 24482730]
RAW264.7	GI50	1002 μM	Cytotoxicity against mouse RAW264.7 cells assessed as growth inhibition after 48 hrs by trypan blue assay	10.1039/C3MD00251A
Sf9	IC50	200 μM	Inhibition of human recombinant COX1 expressed in Sf9 cell microsomes assessed as reduction in conversion of arachidonic acid to PGE2 incubated for 5 mins by HTRF assay	[PMID: 27046190]

In Vitro Studies

In HepG2 cells, acetaminophen produced a 50% decrease in total cellular ATP at 594 ± 147 μM (mean ± SEM). The original main-text table supports this cytotoxicity endpoint; the exposure duration and detailed ATP assay conditions were unavailable in the retrieved material. [3]

Induction-dependent inhibition of PGE2 production

Cell-based study · Murine J774.2 monocyte/macrophage cells; diclofenac-induced apoptotic cultures compared with LPS-induced cultures. [1]

Acetaminophen inhibited diclofenac-induced PGE2 production dose dependently at concentrations >0.06 mM; the LPS-induced response was not appreciably inhibited at similar concentrations. This is a cellular functional comparison, not a purified-enzyme COX-1/COX-2 selectivity measurement.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
PGE2 production after arachidonic-acid challenge in the two induction conditions	Qualitative concentration-response Inhibition at >0.06 mM in diclofenac-induced cells; no appreciable inhibition at similar concentrations in LPS-induced cells.	[1]

Literature-Reported Treatment Conditions

Treatment concentration: 0.002–4 mM reported for the LPS-induced-cell assay; the diclofenac-induced concentration series is shown in Figure 3 without a complete text-listed range.
Exposure duration: 30 min acetaminophen preincubation, followed by 15 min with arachidonic acid.
Method: Cells washed twice; acetaminophen added at 37°C, then 30 μM arachidonic acid. Released PGE2 measured by radioimmunoassay.
Co-treatment: Sequential induction: 0.5 mM diclofenac for 48 h, washed out before acetaminophen; comparator induction with LPS 1 μg/ml for 12 h. Not simultaneous acetaminophen/diclofenac treatment.
Reported limits: Acetaminophen vehicle and replicate count/uncertainty definition for Figure 3 were not reported in the retrieved assay description. Absolute PGE2 outputs are not compared because the figure uses different units for the two induction conditions.

Methods and source locations

Compound reported in the paper: Acetaminophen; Compound label in the paper: Acetaminophen

Evidence applies to the named compound in this experimental model, without supplier or batch validation.

Methods: Measurement of PGE2 from Nonapoptotic Cells; Testing of NSAID-Induced COX Activity; Results: Effect of Acetaminophen on LPS and Diclofenac-Induced COX Activity; Figure 3.

PGE2 production after arachidonic-acid challenge in the two induction conditions: PGE2 radioimmunoassay; original text and visual inspection of Figure 3. — Results: Effect of Acetaminophen on LPS and Diclofenac-Induced COX Activity; Figure 3 and legend.

Inhibition of cytochrome c-catalyzed cardiolipin oxidation

Biochemical assay · Cell-free liposomes containing tetralinoleoyl cardiolipin (50 μM) and POPC (200 μM), with cytochrome c (5 μM) and H2O2 (100 μM). [2]

Acetaminophen inhibited cardiolipin oxidation with an IC50 of 22.8 μM ± 7.6 μM (SEM; n = 6). This measures suppression of lipid oxidation in a reconstituted system.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Inhibition of cytochrome c-catalyzed oxidation of tetralinoleoyl cardiolipin	IC50 22.8 μM ± 7.6 μM (mean ± SEM; n = 6)	[2]

Literature-Reported Treatment Conditions

Treatment concentration: 0–500 μM
Exposure duration: 30 min at 37°C after H2O2 initiation.
Method: Acetaminophen and cytochrome c added to liposomes; reaction initiated with H2O2 and stopped with 2 U/ml catalase. Oxidized cardiolipin quantified by LC-MS selective reaction monitoring.
Vehicle: Reaction buffer: PBS (50 mM, pH 7.4) with 100 μM DTPA; acetaminophen stock solvent not specified.
Co-treatment: Cytochrome c and H2O2 are assay components; no second pharmacological treatment.
Reported limits: Acetaminophen stock solvent and the biological/technical unit represented by n = 6 were not specified in the adopted sections.

Methods and source locations

Compound reported in the paper: Acetaminophen; Compound label in the paper: ApAP

Evidence applies to the named compound in this experimental model, without supplier or batch validation.

Methods 2.3 and 2.5; Results 3.2; Figure 2B.

Inhibition of cytochrome c-catalyzed oxidation of tetralinoleoyl cardiolipin: LC-MS; oxidized cardiolipin normalized to total cardiolipin using tetramyristoyl cardiolipin internal standard. — Results 3.2; Methods 2.3 and 2.5; Figure 2B and legend.

ATP-based cytotoxicity in HepG2 cells

Cell-based study · Human HepG2 cells; acetaminophen comparator in an analogue-development study. [3]

Acetaminophen reduced total cellular ATP with an EC50 of 594 ± 147 μM (mean ± SEM). This is an ATP-based cytotoxicity endpoint, not a target-inhibition potency.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Concentration causing a 50% decrease in total cellular ATP in HepG2 cells	EC50 594 ± 147 μM (mean ± SEM)	[3]

Literature-Reported Treatment Conditions

Method: Total intracellular ATP measured in HepG2 cells.
Reported limits: Exposure duration, tested concentration range, vehicle, replicate count and detailed ATP assay procedure could not be verified because the original supporting-information methods were not retrieved. The catalog’s 24 h/luciferase description is not adopted.

Methods and source locations

Compound reported in the paper: Acetaminophen; Compound label in the paper: ApAP

Evidence applies to the named compound in this experimental model, without supplier or batch validation.

Biological evaluation paragraphs preceding Table 1; Table 1, ApAP row and footnote a.

Concentration causing a 50% decrease in total cellular ATP in HepG2 cells: ATP-based cytotoxicity assessment; precise detection procedure unavailable in retrieved main text. — Table 1, ApAP row and footnote a; preceding cytotoxicity-assessment paragraph.

In Vivo Studies

In mice, oral acetaminophen at 200 mg/kg produced acute antinociception in formalin, tail immersion and von Frey tests without reducing spontaneous locomotor activity. These responses were absent in FAAH- or TRPV1-deficient mice. Route mattered: 100 mg/kg intraperitoneally increased thermal withdrawal latency but did not reproduce the oral effects in formalin or von Frey testing. [4]

Acute mouse antinociception: route and genetic dependence

Animal study · Adult mice; C57BL/6 animals (20–30 g) for dose-route work and FAAH or TRPV1 knockout mice with wild-type littermates. Only males used for nociceptive tests. [4]

A single oral 200 mg/kg dose reduced formalin-evoked behavior and increased thermal and mechanical withdrawal thresholds without reducing spontaneous locomotor activity. At the same oral dose, antinociception was absent in FAAH- or TRPV1-deficient mice. A 100 mg/kg intraperitoneal dose increased tail-withdrawal latency but did not reproduce the oral effect in the formalin or von Frey tests.

Measured endpoints — not treatment concentrations
Endpoint	Reported result	Reference
Acute antinociceptive phenotype with locomotor control	Behavioral response	[4]

Conditions shared by the study groups

Frequency: Single administration
Treatment course: Testing initiated 20 min after administration; formalin phases scored 0–5 and 15–40 min after formalin.
Vehicle: Saline or water (Drugs section); exact nociceptive-arm assignment unspecified.

Study groups and reported outcomes
Group / dose	Comparator	Observed result	Reference
Oral acetaminophen, 200 mg/kg 200 mg/kg; administration volume 10 ml/kg	Vehicle for formalin/locomotion; pretreatment thresholds for tail immersion/von Frey.	Reduced both formalin phases; increased tail-withdrawal latency and mechanical threshold. Locomotor activity was not reduced at 200 mg/kg. n = 6; plotted data mean ± SEM.	[4]
Intraperitoneal acetaminophen, 100 mg/kg 100 mg/kg; administration volume 10 ml/kg	Vehicle for formalin/locomotion; pretreatment thresholds for tail immersion/von Frey.	No reduction in locomotion at this dose; increased tail-withdrawal latency, with no significant effect in formalin or von Frey tests. n = 6; plotted data mean ± SEM.	[4]
FAAH genotype comparison 200 mg/kg; administration volume 10 ml/kg	Genotype-matched vehicle; wild-type littermates treated with acetaminophen.	Antinociception in formalin, tail immersion and von Frey tests was present in wild-type littermates and absent in FAAH−/− mice. n = 6–8 per group; plotted data mean ± SEM.	[4]
TRPV1 genotype comparison 200 mg/kg; administration volume 10 ml/kg	Genotype-matched vehicle; wild-type littermates treated with acetaminophen.	Antinociception in formalin, tail immersion and von Frey tests was present in wild-type littermates and absent in TRPV1−/− mice. Figure 5 reports n = 5–8 per group; plotted data mean ± SEM.	[4]

Oral acetaminophen, 200 mg/kg: Model: Adult C57BL/6 mice; male nociceptive cohorts.; Route: Oral; Reported limits: Locomotor cohort may include either sex; only nociceptive cohorts explicitly restricted to males.

Intraperitoneal acetaminophen, 100 mg/kg: Model: Adult C57BL/6 mice; male nociceptive cohorts.; Route: Intraperitoneal; Reported limits: Higher intraperitoneal doses reduced locomotion in Figure 1A; this arm does not establish an equivalent oral/intraperitoneal exposure.

FAAH genotype comparison: Model: Male FAAH−/− mice and FAAH+/+ littermates.; Route: Oral; Reported limits: Exact n for each panel/group is not separately given in the figure legend.

TRPV1 genotype comparison: Model: Male TRPV1−/− mice and TRPV1+/+ littermates.; Route: Oral; Reported limits: The n = 5–8 range covers Figure 5; exact n for each selected panel/group is not separately specified.

Methods and source locations

Compound reported in the paper: Acetaminophen; Compound label in the paper: AcAP

Evidence applies to the named compound in this experimental model, without supplier or batch validation.

Results; Figures 1, 2 and 5A–C; Methods: Animals and administration of drugs, In vivo tests, Drugs.

Acute antinociceptive phenotype with locomotor control: Formalin: 2.5%, 25 μl intraplantar; paw biting/licking at 0–5 and 15–40 min. Tail immersion: 46°C, 15 s cutoff. Von Frey: 2 g cutoff; genotype results displayed as maximal possible effect (%). Locomotor counts collected for 15 min starting 20 min after acetaminophen. — Figures 1, 2 and 5A–C; Methods: In vivo tests.

Oral acetaminophen, 200 mg/kg · Figure 1A–D, oral panels; Methods: Animals and administration of drugs, In vivo tests, Drugs.

Intraperitoneal acetaminophen, 100 mg/kg · Figure 1A–D, intraperitoneal panels; Methods: Animals and administration of drugs, In vivo tests, Drugs.

FAAH genotype comparison · Figure 2A–C; Methods: Animals and administration of drugs, In vivo tests, Drugs.

TRPV1 genotype comparison · Figure 5A–C; Methods: Animals and administration of drugs, In vivo tests, Drugs.

Specifications

Manufacturer	Adooq Bioscience
Actions	Inhibitor
CAS Number	103-90-2
Applications	COX inhibitor
Formula	C₈H₉NO₂
Molecular Weight	151.16
Synonyms	Paracetamol
SMILES	CC(=O)NC1=CC=C(C=C1)O

Storage & Solubility

In Vitro Solubility

Reported Solubility

No reported solubility data are available for this product.

Preparing Stock Solutions

Stock preparation is unavailable for this product molecular weight.

Calculated stock concentrations do not establish solvent solubility. Refer to the reported solubility data for experimental guidance.

Storage

Store powder at -20°C, keep desiccated.
In powder form, the chemical is stable for 36 months.
In solution, store at -20°C and use within 1 month to prevent loss of potency. Aliquot to avoid multiple freeze/thaw cycles.

Research References

[1] D L Simmons, R M Botting, P M Robertson, et al. Induction of an acetaminophen-sensitive cyclooxygenase with reduced sensitivity to nonsteroid antiinflammatory drugs. Proceedings of the National Academy of Sciences of the United States of America (1999). PMID: 10077674 DOI: 10.1073/pnas.96.6.3275 Source material
[2] Huiyong Yin, Aurélia Vergeade, Qiong Shi, et al. Acetaminophen inhibits cytochrome c redox cycling induced lipid peroxidation. Biochemical and biophysical research communications (2012). PMID: 22634010 DOI: 10.1016/j.bbrc.2012.05.058 Source material
[3] Roman V Shchepin, Wei Liu, Huiyong Yin, et al. Rational Design of Novel Pyridinol-Fused Ring Acetaminophen Analogues. ACS medicinal chemistry letters (2013). PMID: 24482730 DOI: 10.1021/ml4000904 Source material
[4] Christophe Mallet, David A Barrière, Anna Ermund, et al. TRPV1 in brain is involved in acetaminophen-induced antinociception. PloS one (2010). PMID: 20862299 DOI: 10.1371/journal.pone.0012748 Source material

Publications

5 publications citing this product

L Wang, .et al. , Physiol Res, 2024, Nov 15;73(5):769-778 PMID: 39545791
Jingyeong Shin, .et al. , Chemical Engineering Journal, 2023, December
Takashi Azuma, .et al. , SEP PURIF TECHNOL, 2019, Apr; 212: 483-489
Azuma T, .et al. , Environ Sci Pollut Res Int, 2017, Aug;24(23):19021-19030 PMID: 28660504
Azuma T, .et al. , Sci Total Environ, 2016, Apr 1;548-549:189-197 PMID: 26802347

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Acetaminophen

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Acetaminophen 500 mg

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Biological Activity

Bioactivity / Mechanism

Cellular Effect

In Vitro Studies

Induction-dependent inhibition of PGE2 production

Literature-Reported Treatment Conditions

Inhibition of cytochrome c-catalyzed cardiolipin oxidation

Literature-Reported Treatment Conditions

ATP-based cytotoxicity in HepG2 cells

Literature-Reported Treatment Conditions

In Vivo Studies

Acute mouse antinociception: route and genetic dependence

Conditions shared by the study groups

Specifications

Storage & Solubility

In Vitro Solubility

Reported Solubility

Preparing Stock Solutions

Storage

Research References

Publications

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