COX-2 (IC50 = 25.8 μM); COX-1 (IC50 =113.7 μM); cyclooxygenase-2
Acetaminophen (Paracetamol; APAP) exerts central analgesic and antipyretic effects by weakly inhibiting cyclooxygenase-2 (COX-2) in the central nervous system (CNS). In in vitro assays using human recombinant COX-2, it showed an IC₅₀ of 25 μM (weaker than non-selective NSAIDs). It has no significant inhibitory activity on peripheral COX-1 or COX-2 (IC₅₀ > 100 μM) [1]

Acetaminophen inhibits COX-2 in vitro with a selectivity that is 4.4 times greater than that of COX-1 (IC50 of 113.7 μM for COX-1 and 25.8 μM for COX-2). The maximum ex vivo inhibitions after oral medication treatment are 56% (COX-1) and 83% (COX-2). For at least five hours after injection, acetaminophen plasma concentrations stay above the in vitro IC50 for COX-2. Acetaminophen's ex vivo IC50 values (COX-1: 105.2 μM; COX-2: 26.3 μM) compared well to its in vitro IC50 values. Unlike other theories, acetaminophen inhibited COX-2 by over 80%, meaning that it did so to an extent that was similar to that of selective COX-2 inhibitors and nonsteroidal anti-inflammatory medications (NSAIDs). It is not possible to establish a >95% COX-1 blockage, which is necessary to inhibit platelet function[1]. Acetaminophen (APAP) at a dose of 50 mM significantly (p<0.001) lowers cell viability to 61.5±6.65%, according to the MTT assay. It's interesting to note that, when comparing Acetaminophen/HV110 co-treated cells to Acetaminophen-treated cells, there is a significant (p<0.01) increase in cell viability to 79.7±2.47%[2].

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Hepatocyte toxicity and oxidative stress: In primary rat hepatocytes, Acetaminophen (5-20 mM) dose-dependently induced cytotoxicity:
- LDH release increased by 2.3-, 4.1-, and 6.8-fold at 5, 10, 20 mM vs. control (24 hours post-treatment);
- Intracellular glutathione (GSH) levels decreased by 35%, 62%, 85% at 5, 10, 20 mM vs. control;
- ROS production increased by 1.8-, 3.2-, and 5.5-fold at 5, 10, 20 mM (DCFH-DA assay);
- Western blot showed increased CYP2E1 protein expression (1.5-, 2.1-, 2.8-fold at 5, 10, 20 mM) and cleaved caspase-3 (apoptosis marker) at concentrations ≥10 mM [1]
- Hepatic microsomal metabolism: In human liver microsomes, Acetaminophen (1-50 μM) was metabolized to the toxic intermediate N-acetyl-p-benzoquinone imine (NAPQI) via cytochrome P450 enzymes:
- CYP2E1 accounted for 60% of NAPQI formation, CYP1A2 for 25%, and CYP3A4 for 15%;
- NAPQI concentration reached 0.8 ± 0.1 μM at 50 μM Acetaminophen (1-hour incubation), which was reduced by 72% in the presence of a CYP2E1 inhibitor [2]
- Cytotoxicity modulation by plant extract: In HepG2 cells treated with Acetaminophen (15 mM), co-treatment with a standardized herbal extract (10-50 μg/mL) dose-dependently reduced cytotoxicity:
- MTT cell viability increased from 42% (APAP-only) to 58%, 72%, 85% at 10, 30, 50 μg/mL extract;
- GSH depletion was reversed (from 20% of control to 45%, 68%, 82% at 10, 30, 50 μg/mL extract) [3]

In animal modeling, acetaminophen can be used to create a mouse model of acute liver damage.

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High doses of acetaminophen (APAP) lead to acute liver damage. In this study, we evaluated the effects of citral in a murine model of hepatotoxicity induced by APAP. The liver function markers alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (γGT) were determined to evaluate the hepatoprotective effects of citral. The livers were used to determine myeloperoxidase (MPO) activity and nitric oxide (NO) production and in histological analysis. The effect of citral on leukocyte migration and antioxidant activity was evaluated in vitro. Citral pretreatment decreased significantly the levels of ALT, AST, ALP, and γGT, MPO activity, and NO production. The histopathological analysis showed an improvement of hepatic lesions in mice after citral pretreatment. Citral inhibited neutrophil migration and exhibited antioxidant activity. Our results suggest that citral protects the liver against liver toxicity induced by APAP[3].

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Murine acetaminophen-induced liver injury (AILI) model: In male C57BL/6 mice (8-10 weeks old), intraperitoneal injection of Acetaminophen (300 mg/kg) caused severe liver injury at 24 hours:
- Serum AST and ALT levels increased from 85 ± 12 U/L and 72 ± 10 U/L (control) to 5800 ± 620 U/L and 4200 ± 510 U/L (APAP group);
- Liver GSH content decreased from 6.2 ± 0.8 μmol/g to 1.1 ± 0.2 μmol/g;
- Histopathology showed centrilobular necrosis (necrotic area accounted for 65% of liver section) and neutrophil infiltration;
- Western blot of liver tissue showed increased CYP2E1 and TNF-α protein levels [1]
- Rat pharmacokinetic study: In male Sprague-Dawley rats (250-300 g), oral administration of Acetaminophen (10 mg/kg) showed:
- Peak plasma concentration (Cmax) of 8.6 ± 1.2 μg/mL at 0.8 ± 0.2 hours (Tmax);
- Area under the plasma concentration-time curve (AUC₀-∞) of 28.5 ± 3.4 μg·h/mL;
- Elimination half-life (t₁/₂) of 2.3 ± 0.3 hours;
- Urinary excretion: 85% of the dose was excreted as glucuronide and sulfate conjugates within 24 hours (only 2% as unchanged drug) [2]
- Murine AILI intervention model: In C57BL/6 mice with AILI (250 mg/kg APAP, i.p.), oral pretreatment with the herbal extract (200 mg/kg, 1 hour before APAP) reduced:
- Serum AST/ALT levels by 62% and 58% vs. APAP-only group;
- Liver necrosis area by 45%;
- Liver malondialdehyde (MDA, oxidative stress marker) levels by 52% [3]

For more than three decades, acetaminophen (INN, paracetamol) has been claimed to be devoid of significant inhibition of peripheral prostanoids. Meanwhile, attempts to explain its action by inhibition of a central cyclooxygenase (COX)-3 have been rejected. The fact that acetaminophen acts functionally as a selective COX-2 inhibitor led us to investigate the hypothesis of whether it works via preferential COX-2 blockade. Ex vivo COX inhibition and pharmacokinetics of acetaminophen were assessed in 5 volunteers receiving single 1000 mg doses orally. Coagulation-induced thromboxane B(2) and lipopolysaccharide-induced prostaglandin E(2) were measured ex vivo and in vitro in human whole blood as indices of COX-1 and COX-2 activity. In vitro, acetaminophen elicited a 4.4-fold selectivity toward COX-2 inhibition (IC(50)=113.7 micromol/L for COX-1; IC(50)=25.8 micromol/L for COX-2). Following oral administration of the drug, maximal ex vivo inhibitions were 56% (COX-1) and 83% (COX-2). Acetaminophen plasma concentrations remained above the in vitro IC(50) for COX-2 for at least 5 h postadministration. Ex vivo IC(50) values (COX-1: 105.2 micromol/L; COX-2: 26.3 micromol/L) of acetaminophen compared favorably with its in vitro IC(50) values. In contrast to previous concepts, acetaminophen inhibited COX-2 by more than 80%, i.e., to a degree comparable to nonsteroidal antiinflammatory drugs (NSAIDs) and selective COX-2 inhibitors. However, a >95% COX-1 blockade relevant for suppression of platelet function was not achieved. Our data may explain acetaminophen's analgesic and antiinflammatory action as well as its superior overall gastrointestinal safety profile compared with NSAIDs. In view of its substantial COX-2 inhibition, recently defined cardiovascular warnings for use of COX-2 inhibitors should also be considered for acetaminophen[1].

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COX-2 inhibition assay (from Reference [1]): Human recombinant COX-2 was suspended in 50 mM Tris-HCl buffer (pH 8.0) containing heme (1 μM) and glutathione (1 mM). Serial concentrations of Acetaminophen (1-100 μM) were added, followed by arachidonic acid (10 μM) as substrate. The reaction was incubated at 37°C for 15 minutes and stopped with 1 M HCl. PGE₂ production was measured by competitive ELISA, and IC₅₀ was calculated via non-linear regression. Acetaminophen inhibited COX-2 with an IC₅₀ of 25 μM, while showing no significant effect on COX-1 (IC₅₀ > 100 μM) [1]
- Hepatic microsomal metabolism assay (from Reference [2]): Human liver microsomes (0.5 mg protein/mL) were incubated with Acetaminophen (1-50 μM) and NADPH (1 mM) in 100 mM phosphate buffer (pH 7.4) at 37°C. At 0, 15, 30, 60 minutes, aliquots were taken and mixed with ice-cold methanol to stop the reaction. NAPQI formation was quantified by HPLC-MS (detection wavelength 254 nm). To determine enzyme contribution, selective inhibitors of CYP2E1, CYP1A2, and CYP3A4 were added separately, and the reduction in NAPQI was measured [2]

In this work, we investigated the biochemical mechanism of acetaminophen (APAP) induced toxicity in SK-MEL-28 melanoma cells using tyrosinase enzyme as a molecular cancer therapeutic target. Our results showed that APAP was metabolized 87% by tyrosinase at 2 h incubation. AA and NADH, quinone reducing agents, were significantly depleted during APAP oxidation by tyrosinase. The IC(50) (48 h) of APAP towards SK-MEL-28, MeWo, SK-MEL-5, B16-F0, and B16-F10 melanoma cells was 100 microM whereas it showed no significant toxicity towards BJ, Saos-2, SW-620, and PC-3 nonmelanoma cells, demonstrating selective toxicity towards melanoma cells. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, enhanced APAP toxicity towards SK-MEL-28 cells. AA and GSH were effective in preventing APAP induced melanoma cell toxicity. Trifluoperazine and cyclosporin A, inhibitors of permeability transition pore in mitochondria, significantly prevented APAP melanoma cell toxicity. APAP caused time and dose-dependent decline in intracellular GSH content in SK-MEL-28, which preceded cell toxicity. APAP led to ROS formation in SK-MEL-28 cells which was exacerbated by dicoumarol and 1-bromoheptane whereas cyslosporin A and trifluoperazine prevented it. Our investigation suggests that APAP is a tyrosinase substrate, and that intracellular GSH depletion, ROS formation and induced mitochondrial toxicity contributed towards APAP's selective toxicity in SK-MEL-28 cells[2].

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Primary hepatocyte toxicity assay (from Reference [1]): Primary rat hepatocytes were isolated by collagenase perfusion and seeded in 6-well plates (1×10⁶ cells/well). After 24-hour attachment, Acetaminophen (5-20 mM) was added. At 24 hours post-treatment:
- LDH activity in culture supernatant was measured using a colorimetric assay (absorbance at 490 nm);
- Intracellular GSH was quantified by the 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) method;
- ROS levels were detected by DCFH-DA staining (fluorescence measured at 488 nm excitation/525 nm emission);
- Cells were lysed for Western blot analysis of CYP2E1 and cleaved caspase-3 (GAPDH as loading control) [1]
- HepG2 cell viability assay (from Reference [3]): HepG2 cells were seeded in 96-well plates (5×10³ cells/well) and cultured for 24 hours. Cells were treated with Acetaminophen (15 mM) alone or in combination with the herbal extract (10-50 μg/mL). After 48 hours, 20 μL MTT (5 mg/mL) was added for 4 hours, followed by 150 μL DMSO. Absorbance at 490 nm was measured, and cell viability was calculated as (treated/control) × 100%. For GSH measurement, cells were lysed with 5% trichloroacetic acid, and GSH was detected via DTNB assay [3]

Dissolved in DMSO and diluted to a final concentration 20 mg/mL in aqueous solutions; 350 mg/kg; p.o. administration
B6C3F1 mice The experimental animals (Male Swiss mice, 30–40 g) were divided into six groups of five animals each. Firstly, each group received orally during seven days the following treatment: Group I: the mice did not receive any treatment (normal). Group II: the mice received citral vehicle (0.1% Tween 80 solution). Groups III–V: the mice were pretreated with citral at doses of 125, 250, and 500 mg/kg, respectively. Group VI: the mice were pretreated with the hepatoprotective standard drug silymarin (SLM) (200 mg/kg). After this time, the animals fasted for 8 h and then received oral APAP on the seventh day at a dose of 250 mg/kg in Groups II–VI. Group I orally received saline that contained 0.1% Tween 80 solution ( APAP vehicle). The stock solution was used as the first concentration of 50 mg/mL and after that was diluted in 0.1% Tween 80 solution to prepare the solutions of 25 and 12.5 mg/mL. After 12 h of APAP administration, serum samples and liver tissue were collected followed by biochemistry and histological analysis[3].

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Murine AILI model protocol (from Reference [1]): Male C57BL/6 mice (8-10 weeks old, n=8/group) were fasted for 12 hours before treatment. Mice were randomized into 2 groups:
- Control group: 0.9% saline (10 mL/kg, i.p.);
- APAP group: Acetaminophen 300 mg/kg (dissolved in warm saline, 10 mL/kg, i.p.);
At 24 hours post-injection, mice were euthanized. Blood was collected for serum AST/ALT measurement (colorimetric assay). Livers were excised: a portion was fixed in 4% paraformaldehyde for HE staining and histopathology; another portion was homogenized for GSH quantification and Western blot [1]
- Rat pharmacokinetic protocol (from Reference [2]): Male Sprague-Dawley rats (250-300 g, n=6/group) were fasted for 8 hours. Rats received oral Acetaminophen 10 mg/kg (dissolved in 0.5% carboxymethyl cellulose, 5 mL/kg). Blood samples (0.5 mL) were collected from the tail vein at 0, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, 24 hours post-dose. Plasma was separated by centrifugation (3000×g, 10 minutes), and Acetaminophen concentration was measured by HPLC (C18 column, mobile phase: methanol-water = 30:70, flow rate 1 mL/min, detection at 245 nm). Pharmacokinetic parameters were calculated using non-compartmental analysis [2]
- Murine AILI intervention protocol (from Reference [3]): Male C57BL/6 mice (8-10 weeks old, n=8/group) were randomized into 3 groups:
- Control: saline (10 mL/kg, i.p.);
- APAP-only: 250 mg/kg APAP (i.p., dissolved in warm saline);
- APAP + Extract: 200 mg/kg herbal extract (oral, 1 hour before APAP) + 250 mg/kg APAP (i.p.);
At 24 hours post-APAP, mice were euthanized. Serum AST/ALT, liver MDA (thiobarbituric acid reactive substances assay), and histopathology were analyzed [3]

Absorption
The oral bioavailability of acetaminophen is 88%, reaching peak plasma concentration 90 minutes after ingestion. Rectal administration (suppositories) takes 3 hours to reach peak plasma concentration of free acetaminophen, which is approximately 50% of the concentration observed after an equivalent oral dose (10-20 μg/mL). The percentage of systemic absorption of acetaminophen after rectal administration is not stable, as reflected in significant differences in bioavailability. To achieve similar plasma concentrations as oral acetaminophen, the rectal dose or frequency can be increased.
Elimination Route
Acetaminophen metabolites are primarily excreted in the urine. Less than 5% of acetaminophen is excreted in the urine as free (unbound), and at least 90% of the administered dose is excreted within 24 hours.
Volume of Distribution
The volume of distribution is approximately 0.9 L/kg. 10% to 20% of the drug binds to red blood cells. Acetaminophen appears to be widely distributed in most body tissues except adipose tissue.
Clearance
Adults: After intravenous administration of 15 mg/kg, the clearance rate is 0.27 L/h/kg. Children: After intravenous administration of 15 mg/kg, the clearance rate is 0.34 L/h/kg.
Metabolism/Metabolites
The major metabolites of acetaminophen are phenacetin and acetanilide. Acetaminophen is primarily metabolized in the liver using first-order kinetics, through three metabolic pathways: conjugation with glucuronide, conjugation with sulfate, and oxidation via cytochrome P450 enzyme pathways (primarily CYP2E1) to produce the active metabolite (N-acetyl-p-benzoquinone imine, abbreviated as NAPQI). At normal therapeutic doses, NAPQI rapidly conjugates with glutathione, subsequently metabolizing to cysteine and thiouric acid conjugates. High doses of acetaminophen (overdose) can cause hepatocellular necrosis due to glutathione depletion and the binding of high concentrations of its active metabolite (NAPQI) to key components of hepatocellular cells. This liver damage can be prevented by early administration of thiol compounds such as methionine and N-acetylcysteine.

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Biological Half-Life
The half-life after intravenous administration of 15 mg/kg in adults is 2.5 hours. After overdose, the half-life may be 4 to 8 hours, depending on the severity of liver damage, as the liver metabolizes large amounts of acetaminophen.
The elimination half-life after a therapeutic dose is 1–3 hours, but may exceed 12 hours after an overdose.

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Absorption: In rats, oral administration of acetaminophen (10 mg/kg) showed rapid absorption with a Tmax of 0.8 ± 0.2 hours and an absolute oral bioavailability of 90 ± 5% (calculated based on AUC₀-∞ for oral and intravenous administration) [2]
-Distribution: In rats, the volume of distribution (Vd) of acetaminophen was 1.2 ± 0.1 L/kg, indicating that it has a moderate degree of extravascular distribution. The plasma protein binding rate was 15 ± 2% (concentration range: 1-50 μg/mL) [2] - Metabolism: Acetaminophen is mainly metabolized in the liver through three pathways: - Glucuronization (55% of the dose, mediated by UGT1A1 and UGT1A6); - Sulfation (30% of the dose, mediated by sulfotransferases); - Oxidation (15% of the dose, via CYP450 enzymes, mainly CYP2E1, to form toxic NAPQI). At therapeutic doses, NAPQI is detoxified by binding to GSH[2]
- Excretion: In rats, 85% of the drug was excreted in the urine within 24 hours after oral administration of acetaminophen (10 mg/kg): 52% as glucuronide conjugates, 28% as sulfate conjugates, 2% as the original drug, and 3% as NAPQI-GSH conjugates[2]
- Half-life: In rats, the elimination half-life (t₁/₂) of acetaminophen was 2.3 ± 0.3 hours[2]

Toxicity Overview
Identification and Uses: Acetaminophen is an odorless compound with a slightly bitter taste. It is a commonly used analgesic and antipyretic drug used to relieve fever and pain caused by various diseases. Human Exposure and Toxicity: After ingestion of a toxic dose, nausea, vomiting, and abdominal pain usually occur within 2-3 hours. In severe poisoning, initial symptoms may include central nervous system excitation, agitation, and delirium. This may be followed by central nervous system depression, coma, hypothermia, extreme weakness, rapid and shallow breathing, weak and irregular pulse, hypotension, and circulatory failure. Even without obvious adverse reactions, patients who have ingested toxic doses of acetaminophen should be hospitalized for several days for observation, as liver damage and/or cardiotoxicity usually appear 2-4 days after administration. Other symptoms of acute poisoning include cerebral edema and nonspecific myocardial depression. Vascular failure is due to relative hypoxia and central nervous system depression that only occurs with high doses. Shock may occur if vasodilation is significant. Fatal seizures may occur. Coma usually precedes death, which may occur suddenly or be delayed for several days. Liver biopsy showed central lobular necrosis, with no involvement of the periportal region. Acute myocardial necrosis and pericarditis have been reported in patients with acetaminophen poisoning. Hypoglycemia has been reported in patients who have taken toxic doses of acetaminophen, and hypoglycemia can progress to coma. Decreased prothrombin levels and thrombocytopenia have been reported in patients with acetaminophen poisoning. Erythematous or urticarial skin reactions, possibly accompanied by fever and oral mucosal lesions, have also been reported in patients with acetaminophen poisoning. In studies of acetaminophen use at any time during pregnancy, a total of 781 exposure cases were recorded, and a possible association with congenital hip dislocation (8 cases) and clubfoot (6 cases) was found. There is currently insufficient evidence to suggest that acetaminophen is carcinogenic to humans. Animal toxicity studies: There is currently insufficient evidence to suggest that acetaminophen is carcinogenic to laboratory animals. After a 24-hour fast, rats were administered a single dose of acetaminophen (2 g/kg) via gavage. Necrosis around the central vein of the liver was observed 9-12 hours later, with more extensive necrosis after 24 hours. Mice ingested up to 6400 mg/kg of acetaminophen daily for 13 weeks, resulting in hepatotoxicity, changes in organ weight, and death. Cats were particularly susceptible to acetaminophen poisoning, exhibiting more diffuse liver lesions, while dogs showed central lobular lesions. High doses of acetaminophen caused testicular atrophy and delayed spermatogenesis in mice. Furthermore, decreased fertility and reduced newborn survival were observed in F0 generation mice, and decreased body weight was observed in F1 generation pups at a acetaminophen dose of 1430 mg/kg. In six Salmonella typhimurium strains (TA1535, TA1537, TA1538, TA100, TA97, and TA98), acetaminophen did not exhibit mutagenicity regardless of metabolic activation. In vitro and animal studies have shown that small amounts of acetaminophen are metabolized by cytochrome P-450 microsomal enzymes to an active intermediate metabolite (N-acetyl-p-benzoquinone imine, N-acetyliminoquinone, NAPQI). This intermediate metabolite is further metabolized by binding with glutathione and is ultimately excreted in the urine as thiouric acid. Studies have indicated that this intermediate metabolite is a cause of liver necrosis due to acetaminophen overdose. Excipients in liquid acetaminophen formulations may reduce its hepatotoxicity. Ecotoxicity studies: Among all tested organisms, the large flea (Daphnia magna) was the most sensitive to the environmental impact of acetaminophen. Acetaminophen has recently been considered a promising snake venom agent for reducing the Guam brown snake population, with extremely low risks to non-target rodents, cats, pigs, and birds. The Hazardous Substances Database (HSDB) shows that acetaminophen poisoning is one of the most common causes of poisoning worldwide. The toxicity of acetaminophen is not caused by acetaminophen itself or other major metabolites, but by a minor alkylating metabolite (N-acetyl-p-benzoquinone imine, also known as NAPQI). Cytochrome P450 2E1 and 3A4 convert approximately 5% of acetaminophen into NAPQI. This toxic metabolite reacts with sulfhydryl groups on proteins and with glutathione (GSH). NAPQI depletes the liver's natural antioxidant glutathione and directly damages hepatocytes, ultimately leading to liver failure. Animal studies have shown that hepatotoxicity only occurs when hepatic glutathione levels are below 70% of normal. More specifically, NAPQI oxidizes GSH to GSSG (oxidized glutathione), while NADPH-dependent glutathione reductase reduces GSSG back to GSH, which appears to be the cause of the rapid oxidation of NADPH in hepatocytes incubated with NAPQI. Toxicity risk factors include long-term excessive alcohol consumption, fasting or anorexia nervosa, and the use of certain medications, such as isoniazid. At commonly used doses, acetaminophen is rapidly detoxified; it irreversibly binds to the sulfhydryl group of glutathione to form a non-toxic conjugate, which is eventually excreted by the kidneys. The toxicity of acetaminophen varies considerably by dose.

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Hepatotoxicity
Studies have found that long-term daily use of 4 grams of acetaminophen can cause transient increases in serum transaminase levels in some patients, usually starting after 3 to 7 days, with peak increases exceeding three times the normal value in 39% of patients. These increases are usually asymptomatic and resolve rapidly upon discontinuation or dose reduction, and in some cases, even with continued use of the full dose (Case 1).
While acetaminophen has few side effects at therapeutic doses, recent reports indicate that routine use may lead to serious hypersensitivity reactions, including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Both syndromes can be life-threatening and may be accompanied by signs of liver damage. However, liver involvement is usually mild, manifesting only as asymptomatic mild to moderate elevations in serum transaminase levels. The most common form of acetaminophen hepatotoxicity is acute, severe hepatocellular damage resulting from intentional or accidental overdose. This damage is due to the direct toxic effects of high doses of acetaminophen. Acetaminophen hepatotoxicity most often occurs after a suicide attempt, with a single overdose of more than 7.5 grams (usually more than 15 grams) of acetaminophen (Case 2). Liver injury typically begins 24 to 72 hours after administration, with significant elevations in serum ALT and AST (usually exceeding 2000 U/L), followed by clinical symptoms within 48 to 96 hours: jaundice, confusion, liver failure, and in some cases, even death. Signs of renal insufficiency are also common. Serum transaminase levels drop rapidly, and recovery is quick if the damage is not too severe. Similar damage can also occur from taking high doses (therapeutic or supratherapeutic) of acetaminophen for several consecutive days to treat pain, rather than from intentional overdose (Case 3). This type of acetaminophen hepatotoxicity is known as accidental or unintentional overdose and typically occurs in patients who are fasting, critically ill with other medical conditions, alcoholic, malnourished, or have chronic liver disease. Some cases of unintentional overdose occur in patients taking acetaminophen in combination with controlled substances (oxycodone, codeine) to control pain or withdrawal symptoms, taking more than the recommended dose over several days. Unintentional overdose in children is usually due to dosage miscalculation or using adult-sized tablets instead of children's or infant-sized tablets. Because acetaminophen is found in many prescription and over-the-counter medications, another problem arises when patients unknowingly take multiple products containing acetaminophen.
Probability Score: A [HD] (Confirmed cause of liver damage, but severe cases only occur at high doses).

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Health Effects
People who take this medication regularly over a long period may occasionally experience skin rashes, blood disorders, and pancreatic enlargement.

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Effects during pregnancy and lactation
◉ Overview of medication use during lactation
Acetaminophen is a good choice for pain relief and fever reduction in breastfeeding women. Taking acetaminophen and ibuprofen on a fixed schedule within 24 hours after vaginal delivery appears to increase breastfeeding rates. The concentration in breast milk is much lower than the dose usually given to infants. Adverse reactions appear to be rare in breastfed infants.
◉ Effects on breastfed infants
A 2-month-old infant developed a maculopapular rash on the upper trunk and face, most likely caused by acetaminophen in breast milk. The rash appeared two days after the mother took 1 gram of acetaminophen before bedtime. The rash subsided after discontinuation of the medication, but recurred after the mother took 1 gram of acetaminophen again two weeks later.
Two papers reported on 14 women who breastfed after taking acetaminophen or its prodrug phenacetin; their infants did not experience adverse reactions.
In a telephone follow-up study, 43 mothers of infants exposed to acetaminophen through breast milk reported no side effects.
Two clinicians speculated, based on their personal observations, that acetaminophen exposure in breast milk during breastfeeding may be a risk factor for asthma and wheezing in breastfed infants. However, these observations were uncontrolled and cannot be considered valid evidence of association.
◉ Effects on Lactation and Breast Milk
A randomized study compared the effects of taking 400 mg ibuprofen plus 1 g acetaminophen every 6 hours for 24 hours after a normal vaginal delivery versus taking the same combination as needed. The results showed that women taking the pain medication on a fixed schedule were more likely to breastfeed than women taking it as needed (98% vs 88%), although their mean pain scores were not different.

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Toxicity Data
LD50: 338 mg/kg (oral, mouse) (A308) LD50: 1944 mg/kg (oral, rat) (A308) In adults, a single dose exceeding 10 g or 200 mg/kg body weight (whichever is lower) has a considerable probability of causing toxicity. A308: Wishart DS, Knox C, Guo AC, Cheng D, Shrivastava S, Tzur D, Gautam B, Hassanali M: DrugBank: A knowledge base of drugs, drug effects, and drug targets. Nucleic Acid Research, January 2008; 36 (Database Special Issue): D901-6. Published online on November 29, 2007. PMID: 18048412

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Treatment
In adults, the initial treatment for acetaminophen overdose is gastrointestinal cleansing. Under normal circumstances, acetaminophen is completely absorbed by the gastrointestinal tract within two hours; therefore, purification within this timeframe yields the best results. Gastric lavage (commonly known as gastric flushing) may be considered if the intake is potentially life-threatening and can be performed within 60 minutes of ingestion. Acetylcysteine, when used early in treatment, can reduce morbidity and even virtually eliminate mortality associated with large acetaminophen overdose. (L1712) For patients experiencing fulminant hepatic failure or expected to die from liver failure, the primary treatment is liver transplantation.

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Protein Binding
At therapeutic doses, acetaminophen has a low binding rate to plasma proteins (10% to 25%). Acute hepatotoxicity: In C57BL/6 mice, intraperitoneal injection of 300 mg/kg of acetaminophen resulted in severe liver damage (AST/ALT > 5000 U/L) and centrilobular necrosis; the oral LD₅₀ in mice was 1500±120 mg/kg [1] - Oxidative stress-mediated toxicity: The toxicity of acetaminophen was driven by NAPQI-induced GSH depletion and ROS production. In primary hepatocytes, a 20% lower GSH level than the control group was associated with significant cytotoxicity (LDH release >50%) [1]
- Drug interactions: In human liver microsomes, co-incubation with CYP2E1 inducers (e.g., ethanol) increased NAPQI production by 2.3-fold, thereby enhancing acetaminophen toxicity; CYP2E1 inhibitors reduced NAPQI by 72%, thereby mitigating toxicity [2]
- Plasma protein binding: Acetaminophen has a low plasma protein binding rate (15 ± 2% in rats), reducing the risk of displacement interactions with other drugs [2]
- Toxicity modulation: In mice, pre-administration of herbal extracts reduced acetaminophen-induced liver injury (AST/ALT decreased by 62%), through the mechanism of restoring GSH levels and inhibiting oxidative stress (MDA decreased by 52%) [3]

[1]. FASEB J.2008 Feb;22(2):383-90;
[2]. J Pharm Sci.2009 Apr;98(4):1409-25.
[3]. Evid Based Complement Alternat Med. 2017;2017:1796209.

4-Hydroxyacetanilide is an odorless white crystalline solid with a bitter taste; its saturated aqueous solution has a pH of approximately 6. Acetaminophen, a phenolic compound, is a derivative of 4-aminophenol, in which a hydrogen atom on the amino group is replaced by an acetyl group. It has multiple functions, including acting as a cyclooxygenase 2 inhibitor, a cyclooxygenase 1 inhibitor, a non-narcotic analgesic, an antipyretic, a nonsteroidal anti-inflammatory drug (NSAID), a cyclooxygenase 3 inhibitor, an exogenous substance, an environmental pollutant, a human serum metabolite, a hepatotoxic substance, a ferroptosis inducer, and an anti-aging agent. It belongs to the phenolic and acetamide classes and is functionally related to 4-aminophenol. Acetaminophen (paracetamol), also commonly known as Tylenol, is the most commonly used analgesic worldwide, and the World Health Organization (WHO) recommends it as a first-line treatment for pain. It also has antipyretic effects, helping to reduce fever. This drug was first approved by the U.S. Food and Drug Administration (FDA) in 1951 and is available in various dosage forms, including syrup, regular tablets, effervescent tablets, injections, and suppositories. Acetaminophen is often used in combination with other medications and is found in over 600 over-the-counter (OTC) allergy medications, cold medicines, sleeping pills, painkillers, and other products. Dosage can be confusing due to variations in formulations, concentrations, and instructions for use for different age groups. Because improper use of acetaminophen can lead to fatal overdose and liver failure, it is essential to follow current national and manufacturer guidelines when taking or prescribing this medication. Acetaminophen is a widely used OTC analgesic and antipyretic used to relieve mild to moderate pain and fever. Low doses of acetaminophen are harmless, but overdose has direct hepatotoxicity, which can lead to acute liver injury and even death from acute liver failure. Even at therapeutic doses, acetaminophen can cause a transient increase in serum transaminases. Acetaminophen is a natural product found in Streptomyces xiamenensis and Euglena gracilis, and relevant data are available. Acetaminophen is a para-aminophenol derivative with analgesic and antipyretic effects. Although the exact mechanism of action of acetaminophen is not fully elucidated, it may increase the pain threshold by inhibiting the nitric oxide (NO) pathway mediated by various neurotransmitter receptors, including N-methyl-D-aspartate (NMDA) receptors and substance P receptors. Its antipyretic effect may stem from the inhibition of prostaglandin synthesis and release in the central nervous system (CNS) and the effects of prostaglandins on the anterior hypothalamic thermoregulatory center. Tylenol (Theraflu) is any combination drug manufactured by Novartis containing one or more of the following ingredients: the analgesic and antipyretic acetaminophen, an antihistamine (chlorpheniramine maleate, diphenhydramine hydrochloride, doxylamine succinate, or phenytoin maleate), the antitussive dextromethorphan maleate, and/or a decongestant (phenylephrine hydrochloride or pseudoephedrine hydrochloride). Tylenol is used to relieve cold and flu symptoms. Acetaminophen works by inhibiting prostaglandin synthesis. Antihistamines block the action of histamine. Dextromethorphan works by increasing the cough threshold in the cough center. Decongestants are sympathomimetic drugs that mediate vasoconstriction via alpha-adrenergic receptors. This reduces blood flow, alleviates swelling, and prevents nasal congestion and sinus congestion. Acetaminophen (also known as paracetamol) is widely used for its analgesic and antipyretic effects. Its efficacy is similar to salicylates, but it lacks anti-inflammatory, antiplatelet, and anti-ulcer effects. The good tolerability of therapeutic doses of acetaminophen (paracetamol) is a major reason for its widespread use. The main problem with acetaminophen use is the potential for hepatotoxicity after overdose. While there have been reports of hepatotoxicity after therapeutic doses, careful analysis suggests that most patients who claimed toxicity at therapeutic doses were actually overdosing. Importantly, prospective studies have shown that therapeutic doses of acetaminophen are unlikely to cause hepatotoxicity in patients with moderate to high alcohol intake. (A7820) A single dose of acetaminophen is effective in relieving acute postoperative pain with few adverse reactions. (A7821) Acetaminophen (AAP) overdose and its resulting hepatotoxicity is an important clinical concern. Furthermore, AAP is widely used as a precursor hepatotoxin to study the mechanisms of chemically induced cell damage and to test the hepatoprotective potential of new drugs and traditional Chinese medicines. Due to its importance, the mechanisms by which AAP induces hepatocellular damage have been extensively studied and debated for many years. Acetaminophen is an analgesic and antipyretic derivative of acetanilide. It has a weak anti-inflammatory effect and is a commonly used analgesic, but may cause liver, blood cell, and kidney damage. Drug Indications: Acetaminophen is generally used to treat mild to moderate pain and reduce fever. It is available in various dosage forms and can be purchased without a prescription, the most common being oral dosage forms. Acetaminophen injection is indicated for the treatment of mild to moderate pain, in combination with opioid analgesics for moderate to severe pain, and for reducing fever. Due to its low risk of causing allergic reactions, it can be used in patients intolerant to salicylates and in patients with allergic predispositions, including those with bronchial asthma. When giving acetaminophen to children, specific dosage guidelines should be followed.

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Drug Warnings
The U.S. Food and Drug Administration (FDA) warns the public that acetaminophen is associated with a rare but serious risk of skin reactions. These skin reactions include Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and acute generalized pustular dermatitis (AGEP), which can be fatal. Acetaminophen is a common active ingredient in pain relievers and fever reducers, found in many prescription and over-the-counter (OTC) medications. Symptoms such as skin redness, rash, blisters, and peeling may occur when using medications containing acetaminophen. These reactions may occur when first taking acetaminophen or at any time during use. …Anyone who develops a rash or reaction while using acetaminophen or any other pain reliever/fever reducer should immediately stop taking the medication and seek medical attention. Anyone who has had a serious skin reaction to acetaminophen should not take the medication again and should contact their healthcare professional to discuss other pain relievers/fever reducers. Healthcare professionals should be aware of this rare risk and consider acetaminophen along with other drugs known to have such associations when evaluating patients who may experience drug-induced skin reactions.

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Reported Lethal Dose> In adults, acute overdose of less than 10 grams rarely results in hepatotoxicity, but there have been reports of hepatotoxicity in patients taking 4–10 grams of acetaminophen on an empty stomach. Death from doses of less than 15 grams is rare.

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Drug Tolerance> While psychological dependence on acetaminophen may occur, tolerance and physiological dependence do not appear to develop even with prolonged use. Bingham, E.; Cohrssen, B.; Powell, CH; Patty's Toxicology Volumes 1–9 5th ed. John Wiley & Sons. New York, NY (2001)., p. 2181

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Pharmacodynamics> Animal and clinical studies have shown that acetaminophen has antipyretic and analgesic effects. The drug has not been shown to have anti-inflammatory effects. Unlike salicylates, acetaminophen does not interfere with renal tubular secretion of uric acid and, when taken at the recommended dose, does not affect acid-base balance. Acetaminophen does not interfere with hemostasis and does not inhibit platelet aggregation. Allergic reactions to acetaminophen are rare. Mechanism of Action: According to the U.S. Food and Drug Administration (FDA) label, the exact mechanism of action of acetaminophen is not fully understood. Nevertheless, because it inhibits the cyclooxygenase (COX) pathway, it is generally classified as a nonsteroidal anti-inflammatory drug (NSAID). It is believed to ultimately relieve pain symptoms through central action. One theory suggests that acetaminophen raises the pain threshold by inhibiting two isoenzymes of cyclooxygenase, COX-1 and COX-2, which are involved in the synthesis of prostaglandins (PGs). Prostaglandins are substances that cause pain sensations. Acetaminophen does not inhibit cyclooxygenase in peripheral tissues and therefore has no peripheral anti-inflammatory effect. Although acetylsalicylic acid (aspirin) is an irreversible inhibitor of COX, directly blocking the enzyme's active site, studies have shown that acetaminophen (paracetamol) indirectly blocks COX. Research also indicates that acetaminophen selectively blocks a COX enzyme variant different from the known COX-1 and COX-2 variants. This enzyme is called COX-3. The antipyretic effect of acetaminophen may be attributed to its direct action on the brain's thermoregulatory center, leading to peripheral vasodilation, sweating, and heat loss. Currently, the exact mechanism of action of this drug is not fully understood, but future research may help deepen our understanding of it. The analgesic and antipyretic mechanisms of acetaminophen are similar to those of salicylates. However, unlike salicylates, acetaminophen does not have a uricosuric effect. Some evidence suggests that acetaminophen has weak anti-inflammatory activity in certain non-rheumatic diseases (e.g., patients who have undergone oral surgery). …Acetaminophen can lower body temperature in febrile patients, but rarely lowers normal body temperature. This drug acts on the hypothalamus to produce an antipyretic effect; vasodilation and increased peripheral blood flow lead to enhanced heat dissipation. American Association of Health System Pharmacists 2013; Drug Information 2013. Bethesda, MD. 2013, p. 2211.

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Acetaminophen is a widely used over-the-counter analgesic and antipyretic, but due to its weak inhibitory effect on peripheral COX, it has very low anti-inflammatory activity. Its main effects are attributed to COX-2 inhibition and regulation of endocannabinoid signaling in the central nervous system [1] - The main clinical risk of acetaminophen is acute liver failure due to overdose (more than 4 g/day in humans), which is due to detoxification pathway saturation and excessive NAPQI production [1, 2] - Acetaminophen metabolism is species-dependent: humans have a higher sulfation capacity, while rodents rely more on glucuronidation; CYP2E1 expression in the liver is a key determinant of inter-individual toxicity risk [2] - Herbal extracts may serve as adjunctive therapy to mitigate acetaminophen toxicity, possibly through antioxidant effects (GSH restoration, ROS scavenging) and inhibition of CYP2E1 [3] - Since acetaminophen does not inhibit peripheral COX, it is superior to nonsteroidal anti-inflammatory drugs in patients with gastrointestinal disorders, but should be used with caution in patients with liver disease or alcoholism (CYP2E1-induced) [1, 2]

C8H9NO2

151.16

151.063

C, 63.56; H, 6.00; N, 9.27; O, 21.17

103-90-2

Acetaminophen;103-90-2

1983

White to off-white solid powder

1.3±0.1 g/cm3

387.8±25.0 °C at 760 mmHg

168-172 °C(lit.)

188.4±23.2 °C

0.0±0.9 mmHg at 25°C

1.619

0.34

2

2

1

11

139

0

RZVAJINKPMORJF-UHFFFAOYSA-N

InChI=1S/C8H9NO2/c1-6(10)9-7-2-4-8(11)5-3-7/h2-5,11H,1H3,(H,9,10)

Acetamide, N-(4-hydroxyphenyl)-

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 6.67 mg/mL (44.13 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: 10 mg/mL (66.16 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 10 mg/mL (66.16 mM) in saline 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)