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. 2025 Feb 27;21(2):229–240. doi: 10.1007/s13181-025-01065-w

Acute Acetaminophen Hepatotoxicity And Platelet Dysfunction

Michael L Ekaney 1, Trenton A Pritt 1, Neha Attal 1, Christine M Murphy 2, Iain H McKillop 1,✉
PMCID: PMC11933539  PMID: 40014260

Abstract

Introduction

Acetaminophen (APAP) overdose remains a common cause of liver injury, primarily due to its toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). This study sought to investigate APAP-induced platelet aggregation in vitro, and the implication of CYP2E1 in the metabolism of APAP and hepatic cell toxicity.

Methods

Co-cultures of platelets and hepatic cells that do not (HepG2) and do express CYP2E1 (HepG2E47) were exposed to APAP (0-20 mM), NAPQI (0-250 µM), APAP in the absence/presence of inhibitors of glutathione (50 μM buthionine sulphoximine (BSO)), or APAP in the absence/presence of inhibitors CYP2E1 (chlormethiazole (CMZ, 100 µM), or 4-methylpyrazole (4-MP, 5 mM)). Platelet aggregation, cell viability and reactive oxygen species (ROS) were analyzed. Changes in platelet aggregation was determined in platelets directly exposed to APAP/NAPQI.

Results

Exposure to APAP decreased platelet aggregation under co-culture conditions but not in platelet-only cultures. Conversely, NAPQI exposure decreased platelet aggregation in both co-culture and platelet-only conditions. Both APAP and NAPQI reduced cell viability in HepG2 and HepG2E47 cells, with BSO enhancing APAP toxicity, while 4-MP mitigated it. Acetaminophen exposure led to ROS production in HepG2E47 cells, with no effect of CMZ and 4-MP.

Conclusions

Acetaminophen exposure impacts platelet aggregation in co-cultures of platelets and HepG2/HepG2E47 cells with increased ROS production in HepG2E47 cells and 4-MP preventing APAP-induced cytotoxicity in HepG2E47 cells. While APAP had no direct effect on platelets, NAPQI exposure acted to decrease platelet aggregation. These findings enhance our understanding of the mechanisms of APAP-induced hepatotoxicity and the potential role of APAP-induced hepatocellular toxicity in platelet aggregation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13181-025-01065-w.

Keywords: Acetaminophen, Platelet aggregation, Reactive oxygen species, Cell viability

Introduction

Acetaminophen (APAP) is one of the most widely available over the counter (OTC) analgesics used in the United States [1]. The widespread availability of APAP has contributed to a high incidence of both accidental and intentional APAP overdose [2] that imposes a substantial clinical burden, accounting for approximately 56,000 emergency department visits, 2,600 hospitalizations, and 500 deaths per year in the United States [3, 4]. Most APAP-associated toxicity relates to liver injury resulting from hepatic metabolism [5]. Following ingestion APAP is extensively metabolized via glucuronidation and sulfation to form water-soluble metabolites that are excreted in urine [6]. Approximately 10% of APAP undergoes hepatic metabolism via the cytochrome P450 enzyme system, primarily via CYP2E1 [6]. The role of CYP2E1-APAP metabolism becomes more prominent at higher APAP doses (or under conditions in which other metabolic pathways are saturated) leading to the formation of the highly reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) [7, 8] (Suppl. Figure 1A).

Within the liver, NAPQI is usually detoxified by the endogenous antioxidant glutathione (GSH) [9]. In APAP overdose, excess NAPQI production leads to rapid GSH depletion, hepatic NAPQI accumulation, and formation of NAPQI-protein adducts. Additionally, GSH depletion leads to increased reactive oxygen species (ROS) production and oxidative stress [10–12] (Suppl. Figure 1A). The formation of NAPQI-adducts and elevated oxidative stress causes hepatocyte cell damage and progressively worsening liver injury, the degree of liver damage being dependent on the amount of APAP ingested and the metabolic pathways involved [13]. Current diagnostic markers of liver damage, such as aspartate and alanine transaminase (AST/ALT) levels, may not be detectable until significant liver damage has occurred. Typically, AST/ALT values increase within 24 h following APAP overdose, with maximal hepatoxicity detected between 72–96 h [14]. This delay in assessing APAP-induced liver damage has led to exploration of alternative approaches to detect earlier predictors of liver toxicity.

Previous studies report platelet activation occurs in response to organ damage in the brain [15], heart [16], and liver [17]. Ease of access to platelets has led to interest in detecting changes in platelet aggregation as a means to monitor extent of organ damage. Increasing evidence reports drug induced liver injury can induce changes in platelet activation [18], and that these changes can occur within a relatively short time frame following drug ingestion (4–12 h) [19]. Recent reports using intact and permeabilized platelets exposed to APAP report a concentration-dependent effect on mitochondrial respiration [20], and that platelet aggregation is driven by mitochondrial function [21]. These findings have led to evaluating bioenergetic function in platelets as a potential “peripheral signature” of mitochondrial dysfunction in metabolically active tissue [22]. This led us to hypothesize that hepatic APAP-metabolism to NAPQI, and the subsequent effects of NAPQI on cell damage and GSH levels/oxidative stress, could lead to measurable changes in platelet aggregation.

The potential mechanism[s] by which APAP/APAP metabolism impacts platelet function remain poorly defined. Since APAP toxicity leads to hepatocyte necrosis, complex local and systemic responses can make mechanistic studies using (circulating) platelet function difficult to interpret. The human hepatic HepG2 cell line is widely used to study hepatic toxicology due to stable growth characteristics and hepatocyte-like properties [23], but does not express CYP2E1 [24]. In 1998 Chen and Cederbaum stably transfected the HepG2 cell line with the human CYP2E1 gene [25] and the resultant CYP2E1-expressing cell line (HepG2E47) has been widely used to study ethanol and drug metabolism [26, 27]. Chlormethiazole (CMZ; a sedative/hypnotic used to treat alcohol withdrawal) and fomepizole (4-methylpyrazole 4-MP, used to treat ethyl glycol poisoning) inhibit CYP2E1 activity, albeit via different mechanisms; 4-MP is a competitive inhibitor at the substrate binding site [28], whereas CMZ is a non-competitive inhibitor binding to a site other than the substrate binding site to reduce catalytic efficiency (i.e. CMZ can inhibit CYP2E1 activity when a substrate is bound to the enzyme) [29] (Suppl. Figure 1B). In addition to pharmacological agents that target CYP2E1, buthionine sulphoximine (BSO, a synthetic amino acid) can be employed to inhibit γ-glutamylcysteine synthetase and deplete intracellular GSH levels [30].

The goals of this study were to address the role of APAP and CYP2E1-dependent APAP metabolism in hepatic cells and the subsequent effects on platelet aggregation using in vitro model systems and pharmacological agents.

Methods

Assurances

The study was approved by our Institutional Review Board and written, informed consent was obtained from platelet donors.

Research Strategy

This study integrates experimental approaches to investigate the effects of APAP and APAP metabolism on platelet aggregation and toxicity in hepatic cells. Platelet samples were prepared using apheresis, adjusted to a standardized concentration, and analyzed for aggregation in response to ADP, AA, and thrombin. Human hepatic cells that do not (HepG2) and do express CYP2E1 (HepG2E47) were cultured under defined conditions, with HepG2E47 cells being employed to model CYP2E1-mediated APAP metabolism. Pharmacological agents (BSO, CMZ, and 4-MP) were employed to alter GSH levels and CYP2E1 activity. The effect of APAP and its metabolite NAPQI on cell viability and oxidative stress were evaluated using fluorescent and spectrophotometric assays. Co-culture experiments were used to explored the impact of hepatic metabolism on platelet aggregation and provide insights into APAP-induced cellular and platelet dysfunction.

Experiment 1. Analysis of Platelet Aggregation in Hepatic Cell-Platelet Co-Cultures

HepG2 and HepG2E47 cells were seeded in high serum media (HSM) in the lower chamber of a [0.4 µm pore size] transwell plate (Corning Life Sciences, Glendale, AZ) prior to replacement with low serum media (LSM) (24 h). Culture medium was then replaced with fresh LSM containing APAP (20 mM) or NAPQI (100 µM). In parallel, HepG2 and HepG2E47 cells were exposed to BSO (50 µM), CMZ (100 µM), or 4-MP (5 µM) for 2 h prior to APAP addition (20 mM). Platelets in plasma (300,000 platelets/µl, 0.5 ml total volume) were added to the upper transwell chamber and co-cultured for 24 h. An aliquot of platelets was removed from the upper chamber and platelet aggregation in response to ADP (final concentration 10 μM), AA (0.5 mM final concentration), or thrombin (1U/ml final concentration) was measured [31]. Controls consisted of platelet poor plasma (PPP; 100% light transmission = 100% aggregation) and platelet rich plasma (PRP) (0% light transmission = 0% aggregation) [31].

Experiment 2. Direct Effect of APAP or NAPQI on Platelets

APAP (0–20 mM) or NAPQI (0–250 µM) were added to freshly isolated platelets (300,000 platelets/µl, 200 µl volume) and platelet aggregation in response to ADP, AA, or thrombin measured [31].

Experiment 3. Acetaminophen Toxicity in Hepatic Cells

HepG2 and HepG2E47 cells were cultured in HSM prior to replacement with LSM (24 h). Culture medium was replaced with fresh LSM, or LSM containing APAP (20 mM) or NAPQI (100 mM) for 24 h. In parallel, HepG2 and HepG2E47 cells were exposed to BSO (50 µM), CMZ (100 µM) or 4-MP (5 µM) for 2 h prior APAP addition (20 mM). A cell viability assay (Cell Counting Kit-8, Sigma Aldrich, St Louis, MO) was used to determine viability as per the manufacturer’s instructions.

Experiment 4. Live Cell Viability/Cytotoxicity Via Imaging

HepG2 and HepG2E47 cells were cultured in HSM prior to replacement with LSM (24 h). Culture medium was replaced with fresh LSM, or LSM containing APAP (20 mM) or NAPQI (100 mM) for 24 h. In parallel, HepG2 and HepG2E47 cells were exposed to BSO (50 µM), CMZ (100 µM) or 4-MP (5 µM) for 2 h prior APAP addition (20 mM). A fluorescent cell viability assay optimized for fluorescein isothiocyanate (FITC; excitation/emission λ = 488 nm/515 nm) was used to quantify live cells (Live/Dead Cell Imaging kit, Invitrogen, Waltham, MA) as per the manufacturer’s instructions.

Experiment 5. Measurement of Oxidative Stress

HepG2 and HepG2E47 cells were cultured in HSM prior to replacement with LSM (24 h). Culture medium was replaced with fresh LSM, or LSM containing APAP (20 mM) or NAPQI (100 mM) for 24 h. In parallel, HepG2 and HepG2E47 cells were exposed to BSO (50 µM), CMZ (100 µM) or 4-MP (5 µM) for 2 h prior APAP addition (20 mM). Cells were incubated with CellROX* Deep Red Reagent (Invitrogen) (30 min, 370C) as per the manufacturer’s protocol. Levels of ROS were measured by spectrophotometry (absorption/emission λ = 644/665 nm) and values reported as fold change versus control.

Overarching Methodology

Platelet Isolation and Preparation

Platelet samples were obtained using an apheresis technique into collection bags. The initial platelet count was determined using a Z2 Coulter counter (Beckman Coulter, Brea, CA) and adjusted to 300,000cells/µl using PPP.

Cells and Cell Culture Conditions

Human HepG2 cells were purchased from ATCC (Manassas, VA). The HepG2 cell line overexpressing CYP2E1 (HepG2E47 cells) was established using a transfection and limited dilution screening technique [25], and were a gift from Dr. Cederbaum (Icahn School of Medicine, NY). Hepatic cells were cultured in a humidified chamber (370C, 5% CO2) in high glucose Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Waltham, MA) supplemented with 10% (v/v) fetal bovine serum (FBS) [32].

CYP2E1 Expression in HepG2 and HepG2E47 Cells

HepG2 and HepG2E47 cells were plated in HSM for 24 h prior to replacement with LSM (24 h). Culture medium was removed and cell lysates prepared in radioimmunoprecipitation assay (RIPA) buffer (Sigma Aldrich). CYP2E1 protein levels were detected by Western blot using an anti-CYP2E1 antibody (Invitrogen, Waltham, MA) as previously reported [32].

Pharmacological Agents

Acetaminophen, NAPQI, and all other pharmacological agents used were purchased from Sigma Aldrich. Buthionine sulfoximine (50 µM; BSO) was used to deplete intracellular GSH [33]. Chlormethiazole (CMZ, 100 µM, non-competitive CYP2E1 inhibitor) and 4-methylpyrazole (5 mM, competitive CYP2E1 inhibitor) were employed to study CYP2E1-dependent APAP metabolism. To address potential differences in mechanisms of platelet activation, aggregation was investigated in the absence or presence of ADP (purinergic P2 agonist), arachidonic acid (AA; thromboxane A2 agonist), or thrombin (protease-activated receptor agonist).

Quantification of APAP

Acetaminophen was quantified using a spectrophotometric method as previously described by Chiou et al., in which the phenolic hydroxyl group of APAP reduces cuprous ions present in bicinchoninic acid (BCA) to form a chromophore detectable by spectrophotometry (562 nm absorption) [34]. HepG2 and HepG2E47 cells were seeded in HSM prior to replacement with LSM (24 h). Cell culture media was replaced with LSM containing APAP (20 mM) for 24 h prior to culture medium collection and centrifugation (10 min, 300xg, 370C). Concentration of APAP was determined spectrophotometrically in culture medium prior to (T0) and 24 h after (T24) drug addition against a standard curve.

Statistical Analysis

All measurements were performed in triplicate for all assays, and a minimum of three independent experiments performed for each condition. Statistical analysis was performed using GraphPad Prism v9.0 (La Jolla, CA). Data was tested for normality using the Shapiro Wilk test. Within group analyses comparing control group versus experimental groups were analyzed using a one-way ANOVA and the Tukey post hoc test for pairwise comparisons. Fluorescence intensity was determined using Image J (NIH, Bethesda, MD). Platelet aggregation was determined by dividing the absorbance values of the experimental group by the absorbance values of the control group × 100. Densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD). Unless otherwise stated, data are presented as means ± SEM. A P-value < 0.05 was considered significant.

Results

Cytochrome P450 2E1 Expression and APAP Metabolism

Western blot analysis of CYP2E1 expression confirmed the absence CYP2E1 protein in HepG2 cells and presence of CYP2E1 protein in HepG2E47 cells (Suppl. Figure 2A). Following APAP exposure (24 h) the concentration of APAP measured in HepG2 cell culture supernatant was unchanged compared to 0 h (Suppl. Figure 2B). Conversely, APAP in culture medium from HepG2E47 cells at T24 was significantly lower compared to T0 (Suppl. Figure 2B, *P < 0.05, T24 versus T0, N = 3).

Effect of Hepatic Cell Exposure to APAP on Platelet Aggregation in a Co-Culture System

ADP-Stimulated Aggregation

ADP-stimulated platelet aggregation remained unchanged following platelet co-culture with HepG2 cells exposed to 20 mM APAP, whereas HepG2 exposure to NAPQI led to decreased co-cultured platelet aggregation (Fig. 1A, *P < 0.05 NAPQI [85.2 ± 0.5%] versus Control [100.0 ± 0.0%], N = 3). Addition of BSO alone failed to alter co-cultured platelet aggregation while incubation with BSO prior to APAP exposure inhibited co-cultured platelet aggregation (Fig. 1A, #P < 0.05 BSO + APAP [84.5 ± 0.7%] versus BSO [97.0 ± 0.6%], N = 3). Addition of CMZ or 4-MP to HepG2 cell culture medium failed to affect co-cultured platelet aggregation with or without subsequent APAP exposure (Fig. 1A). Under identical conditions, both APAP and NAPQI significantly inhibited co-cultured platelet aggregation following addition to HepG2E47 culture medium (Fig. 1B, *P < 0.05 APAP [84.0 ± 1.6%] versus. Control [100.0 ± 0.0%]; NAPQI [70.0 ± 0.7%] versus Control [100.0 ± 0.0%], N = 3). Addition of BSO alone to HepG2E47 culture medium inhibited co-cultured platelet aggregation, and co-cultured platelet aggregation was further decreased when BSO was included prior to APAP (Fig. 1B, *P < 0.05 BSO [87.0 ± 0.9%] versus Control [100.0 ± 0.0%]; BSO + APAP [73.0 ± 1.1%] versus Control [100 ± 0.0%]; and BSO + APAP [73.0 ± 1.1%] versus. BSO [87.0 ± 0.9%], N = 3). Addition of either CMZ or 4-MP to HepG2E47 cell culture medium failed to affect co-cultured platelet aggregation. Pre-exposure to either CMZ or 4-MP did not restore co-cultured platelet aggregation to control levels following subsequent APAP exposure (Fig. 1B *P < 0.05, CMZ + APAP [93.6 ± 0.4%] versus Control or CMZ [98.0 ± 0.6%]; 4-MP + APAP [92.9 ± 0.3%] versus Control or 4-MP [98.0 ± 0.6%], N = 3).

Fig. 1.

Fig. 1

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Adenosine diphosphate (ADP) stimulated platelet aggregation following co-culture of platelets with A) HepG2 cells, and B) HepG2E47 cells in the absence (-) or presence (+) of acetaminophen (APAP, 20 mM), N-acetyl-p-benzoquinone imine (NAPQI, 100 µM), or APAP ± buthionine sulfoximine (BSO, 50 µM), chlormethiazole (CMZ, 100 µM) or 4-methylpyrazole (4-MP, 5 mM). *P < 0.05 versus control (C), N = 3; #P < 0.05, N = 3, APAP + BSO versus BSO. Arachidonic Acid (AA) stimulated platelet aggregation following co-culture of platelets with C) HepG2 cells and D) HepG2E47 cells ± APAP (20 mM), NAPQI (100 µM), or APAP ± BSO (50 µM), CMZ (100 µM), or 4-MP (5 mM), *P < 0.05 versus control (C), N = 3; #P < 0.05, N = 3, APAP + BSO versus BSO, #P < 0.05, N = 3, APAP + CMZ versus CMZ and APAP + 4-MP versus 4MP. Thrombin stimulated platelet aggregation following co-culture of platelets with E) HepG2 cells, and F) HepG2E47 cells ± APAP (20 mM), NAPQI (100 µM), or APAP ± BSO (50 µM), CMZ (100 µM), or 4-MP (5 mM), *P < 0.05 versus control (C), N = 3; #P < 0.05, N = 3, APAP + BSO versus BSO, APAP + CMZ versus CMZ and APAP + 4MP versus 4MP.

AA-Stimulated Aggregation

Under identical experimental conditions exposure of HepG2 cells to APAP or NAPQI resulted in decreased AA-stimulated co-cultured platelet aggregation (Fig. 1C, *P < 0.05 APAP [93.8 ± 0.4%] versus Control [100.0 ± 0.0%]; NAPQI [85.0 ± 1.0%] versus Control [100.0 ± 0.0%], N = 3). Addition of BSO alone to HepG2 cells failed to alter co-cultured platelet aggregation, while incubation with BSO prior to APAP exposure inhibited co-cultured platelet aggregation (Fig. 1C, *P < 0.05 BSO + APAP [89.0 ± 0.6%] versus BSO [98.0 ± 0.6%], N = 3). Addition of CMZ or 4-MP to HepG2 cell culture medium failed to affect co-cultured platelet aggregation with or without subsequent APAP exposure (Fig. 1C). Using HepG2E47 cells both APAP and NAPQI significantly inhibited co-cultured platelet aggregation (Fig. 1D, *P < 0.05 APAP [85.0 ± 0.6%] versus Control [100.0 ± 0.0%], *P < 0.05 NAPQI [80.0 ± 0.7%] versus Control [100.0 ± 0.0%], N = 3). Addition of BSO alone to HepG2E47culture medium inhibited co-cultured platelet aggregation. However, co-cultured platelet aggregation was not further decreased when BSO was included prior to APAP (Fig. 1D, *P < 0.05 BSO [95.0 ± 0.6%] vs Control [100.0 ± 0.0%], BSO + APAP [86.0 ± 0.6%] versus Control or BSO alone [95.0 ± 0.6%], N = 3). Addition of either CMZ or 4-MP to HepG2E47 cell culture medium failed to affect co-cultured platelet aggregation. Pre-exposure to either CMZ or 4-MP did not restore platelet aggregation in co-cultures to control levels following subsequent APAP exposure (Fig. 1D, #P < 0.05 CMZ + APAP [85.0 ± 0.6%] versus Control or CMZ [97.0 ± 0.9%]; 4-MP + APAP [91.5 ± 0.5%] vs. Control or 4-MP [98.0 ± 0.6%] N = 3).

Thrombin-Stimulated Aggregation

Exposure of HepG2 cells to either APAP or NAPQI resulted in a decline in thrombin-stimulated co-cultured platelet aggregation (Fig. 1E, *P < 0.05 APAP [89.9 ± 0.4%] versus Control [100.0 ± 0.0%]; NAPQI [80.0 ± 0.7%] versus Control [100.0 ± 0.0%], N = 3). Addition of BSO alone failed to alter co-cultured platelet aggregation, while incubation with BSO prior to APAP exposure inhibited co-cultured platelet aggregation (Fig. 1E, #P < 0.05 BSO + APAP [86.0 ± 0.6%] versus Control or BSO [98.0 ± 0.6%], N = 3). Addition of either CMZ or 4-MP to HepG2E47 culture medium failed to affect co-cultured platelet aggregation. Pre-exposure to either CMZ or 4-MP did not restore co-cultured platelet aggregation to control levels following subsequent APAP exposure (Fig. 1E, #P < 0.05 CMZ + APAP [91.1 ± 0.5%] versus Control or CMZ [98.0 ± 0.6%]; 4-MP + APAP [98.0 ± 0.6%] versus Control or 4-MP [93.0 ± 0.3%], N = 3). Using HepG2E47 cells under the same conditions, both APAP and NAPQI inhibited co-cultured platelet aggregation (Fig. 1F, *P < 0.05 APAP [89.9 ± 0.4%] versus Control [100.0 ± 0.0%]; NAPQI [80.0 ± 0.7%] versus Control [100.0 ± 0.0%], N = 3). Addition of BSO alone to HepG2E47 culture medium inhibited co-cultured platelet aggregation (Fig. 1F, *P < 0.05 BSO [85.0 ± 1.0%] versus Control [100.0 ± 0.0%], N = 3). Co-cultured platelet aggregation was not further decreased when BSO was included prior to APAP exposure. Addition of either CMZ or 4-MP to HepG2E47 cell culture medium did not affect co-cultured platelet aggregation. Pre-exposure to either CMZ or 4-MP did not restore co-cultured platelet aggregation to control levels following subsequent APAP exposure (Fig. 1F, #P < 0.05, CMZ + APAP [91.1 ± 0.5%] versus Control or CMZ [98.0 ± 0.6%]; 4-MP + APAP [93.0 ± 0.3] versus Control or 4-MP [98.0 ± 0.6%], N = 3).

Direct Effect of APAP or NAPQI on Platelet Aggregation

Exposure of platelets to APAP (0–20 mM) failed to alter platelet aggregation in response to ADP, AA, or thrombin (Fig. 2A-C). In contrast, direct exposure of platelets to NAPQI (0–250 μM) significantly inhibited platelet aggregation in response to ADP (Fig. 2A, *P < 0.05, 100 μM NAPQI [59.0 ± 1.1%] and 250 μM NAPQI [42.0 ± 1.1%] versus 0 μM NAPQI [100 ± 0.00%], #P < 0.05, 250 μM NAPQI versus 100 μM NAPQI, N = 3), AA (Fig. 2B, *P < 0.05, 100 μM NAPQI [51.0 ± 1.4%] and 250 μM NAPQI [35.0 ± 1.3%] versus 0 μM NAPQI [100.0 ± 0.0%], #P < 0.05, 250 μM NAPQI versus 100 μM NAPQI, N = 3, or thrombin (Fig. 2C, *P < 0.05, 100 μM NAPQI [56.0 ± 1.3%] and 250 μM NAPQI [41.0 ± 1.5%] versus 0 μM NAPQI [100.0 ± 0.0%], #P < 0.05, 250 μM NAPQI versus 100 μM NAPQI, N = 3).

Fig. 2.

Fig. 2

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A Adenosine diphosphate (ADP), B Arachidonic acid (AA), and C) Thrombin stimulated platelet aggregation following exposure to acetaminophen (APAP; 10 mM or 20 mM) or N-acetyl-p-benzoquinone imine (NAPQI; 100 µM or 250 mM), *P < 0.05 versus control (C), N = 3; #P < 0.05, N = 3, 250 µM NAPQI versus 100 µM NAPQI.

Effect of APAP on Cell Viability

Exposure of HepG2 cells to APAP led to decreased cell viability compared to control, whereas exposure to NAPQI did not affect viability (Fig. 3A, *P < 0.05, APAP [72.0 ± 0.8%] versus Control [100.0 ± 0.0%], N = 3). Addition of BSO decreased HepG2 cell viability, while CMZ or 4-MP did not affect HepG2 cell viability (Fig. 3A). However, pre-treatment of HepG2 cells with BSO, CMZ or 4-MP followed by APAP led to significantly reduced viability (Fig. 3A, #P < 0.05, BSO + APAP [62.0 ± 0.6%] versus Control and BSO [88.0 ± 0.8%], CMZ + APAP [70.0 ± 0.6%] versus Control or CMZ [98.0 ± 0.6%]; 4-MP + APAP [71.0 ± 0.6%] versus Control or 4-MP [98.0 ± 0.6%], N = 3).

Fig. 3.

Fig. 3

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Cell viability of A) HepG2 cells and B) HepG2E47 cells assessed using a CCK-8 kit in the absence (-) or presence (+) of acetaminophen (APAP; [20 mM]), N-acetyl-p-benzoquinone imine (NAPQI; [100 µM]), or APAP ± buthionine sulfoximine (BSO; [50 µM]), chlormethiazole (CMZ, 100 µM) or 4-methylpyrazole (4-MP, 5 mM), *P < 0.05 versus control (C), N = 3; #P < 0.05, N = 3, APAP + BSO versus BSO, APAP + CMZ versus CMZ and APAP + 4-MP versus 4-MP.

Exposure of HepG2E47 cells to APAP led to decreased cell viability compared to control, whereas exposure to NAPQI did not affect viability (Fig. 3B, *P < 0.05, APAP [52.0 ± 0.8%] versus Control 100.0 ± 0.0%], N = 3). Addition of BSO reduced HepG2E47 cell viability, and cell viability was further reduced by exposure to APAP following pre-exposure to BSO (Fig. 3B, *P < 0.05 BSO [52.0 ± 0.0%] versus Control [100.0 ± 0.0%], #P < 0.05 BSO + APAP [34.0 ± 1.5%] versus BSO [52.0 ± 0.8%], N = 3). Addition of CMZ and 4-MP alone failed to affect HepG2E47 cell viability (Fig. 3B). However, pre-treatment of HepG2E47 cells with CMZ or 4-MP prior APAP led to reduced cell viability (Fig. 3B, #P < 0.05 CMZ + APAP [53.0 ± 0.8%] versus Control and CMZ alone [98.0 ± 0.6%]; 4-MP + APAP [44.0 ± 0.9%] versus Control or 4-MP alone [98.0 ± 0.6%], N = 3).

Effect of APAP on Cell Viability Using Fluorescent Imaging

Using a fluorescent cell imaging approach both APAP or NAPQI led to decreased live cell number in HepG2 cells (Fig. 4A & B, *P < 0.05 APAP [13.0 ± 0.6] or NAPQI [17.0 ± 0.6] versus Control [28.0 ± 0.6], N = 3). Addition of BSO decreased live cell fluorescence in HepG2 cells versus control, effects that were magnified by pre-exposure to BSO, CMZ or 4-MP followed by APAP (Fig. 4A & B, *P < 0.05 BSO [24.0 ± 0.3] versus Control [28.00 ± 0.63]; #P < 0.05, BSO + APAP [11.0 ± 0.6] versus BSO [24.0 ± 0.3]; CMZ + APAP [14.0 ± 1.2] versus CMZ [24.8 ± 0.4]; 4-MP + APAP [15.0 ± 0.6] versus 4-MP [24.0 ± 0.6]).

Fig. 4.

Fig. 4

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Representative images of cell viability of A) HepG2 cells assessed using a fluorescein isothiocyanate Live/Dead Cell Imaging kit in the absence (-) or presence (+) of acetaminophen (APAP; [20 mM]), N-acetyl-p-benzoquinone imine (NAPQI; [100 µM]), or APAP ± buthionine sulfoximine (BSO; [50 µM]), chlormethiazole (CMZ; [100 µM]), or 4-methylpyrazole (4-MP; [5 mM]) and quantification of cell viability of B) HepG2 cells assessed using the Live/Dead Cell Imaging kit. Representative images of cell viability of C) HepG2E47 cells and quantification of cell viability of D) HepG2 cells assessed using the Live/Dead Cell Imaging kit. Cells were cultured ± APAP (20 mM), NAPQI (100 µM), or APAP ± BSO (50 µM), CMZ (100 µM), or 4-MP (5 mM), *P < 0.05 versus control (C), N = 3; #P < 0.05, N = 3, APAP + BSO versus BSO, APAP + CMZ versus CMZ, and APAP + 4-MP versus 4-MP.

Exposure of HepG2E47 cells to APAP led to decreased live cell number versus control. Conversely, NAPQI failed to decrease cell number (Fig. 4C & D, *P < 0.05, APAP [16.0 ± 0.5] versus Control [21.0 ± 0.5], N = 3). Addition of BSO decreased live cell number in HepG2E47 cells versus control, effects that were magnified by pre-exposure to BSO followed by APAP (Fig. 4C & D, *P < 0.05, BSO [15.0 ± 0.5] versus Control [21.0 ± 0.5]; #P < 0.05, BSO + APAP [11.0 ± 0.5] versus BSO [15.0 ± 0.5], N = 3). Addition of either CMZ or 4-MP did not alter live cell number in HepG2E47 cells versus control (Fig. 4C & D). However, while pre-exposure of HepG2E47 cells to CMZ led to a decrease in live cell number following APAP, pre-exposure to 4-MP abrogated the effect of APAP such that live cell number was equivalent to control (Fig. 4C & D).

Effect of APAP on Hepatic Cell Oxidative Stress

Analysis of ROS levels in HepG2 cells revealed moderate, though significant, increases in ROS following APAP (Fig. 5A, *P < 0.05 APAP [1.1 ± 0.0] versus Control [0.9 ± 0.0], N = 3). Pre-exposure to BSO followed by APAP did not affect oxidative stress compared to BSO alone (Fig. 5A). Exposure to CMZ or 4-MP did not affect oxidative stress compared to control, and this was not different in HepG2 cells pre-exposed to CMZ or 4-MP followed by APAP (Fig. 5A). Exposure of HepG2E47 to APAP led to a marked increase in oxidative stress, whereas NAPQI exposure failed to alter oxidative stress compared to control (Fig. 5B, *P < 0.05, APAP [2.1 ± 0.0] versus Control [1.0 ± 0.0], N = 3). Addition of BSO, CMZ or 4-MP failed to affect ROS in HepG2E47 cells (Fig. 5B). However, pre-treatment of HepG2E47 cells with BSO, CMZ or 4-MP followed by APAP led to significantly elevated ROS (Fig. 5B, #P < 0.05, BSO + APAP [1.5 ± 0.0] versus Control and BSO [1.1 ± 0.0], #P < 0.05, CMZ + APAP [2.2 ± 0.0] versus CMZ [0.9 ± 0.0]; 4-MP + APAP [1.9 ± 0.0] versus Control or 4-MP [1.0 ± 0.0], N = 3).

Fig. 5.

Fig. 5

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Reactive oxygen species (ROS) measured by fluorescence intensity (fold change) in A) HepG2 cells, and B) HepG2E47 cells in the absence (-) or presence (+) of acetaminophen (APAP; [20 mM]), N-acetyl-p-benzoquinone imine (NAPQI; [100 µM]), or APAP ± buthionine sulfoximine (BSO; [50 µM]), chlormethiazole (CMZ, 100 µM) or 4-methylpyrazole (4-MP, 5 mM). *P < 0.05 versus control (C), N = 3, #P < 0.05, N = 3, APAP + BSO versus BSO, APAP + CMZ versus CMZ and APAP + 4-MP versus 4-MP.

Discussion

Increasing evidence suggests changes in circulating platelet aggregation may be reflective of oxidative stress and cellular damage [35, 36]. This may be relevant in the pathogenesis of APAP toxicity and liver damage where changes in platelet aggregation can contribute to the development of microthrombi and enhanced hepatic damage [37]. In our studies, direct exposure of platelets to APAP, or co-culture of platelets with HepG2 cells exposed to APAP, did not affect platelet aggregation in response to ADP. Conversely, co-culture of platelets with hepatic cells that express CYP2E1 (HepG2E47) exposed to APAP, or direct exposure to NAPQI, led to inhibition of platelet aggregation. However, exposure of HepG2 cells to APAP inhibited co-cultured platelet aggregation in response to AA and thrombin, and while inhibition of CYP2E1 in HepG2E47 cells (CMZ or 4-MP) blunted the effect on aggregation of co-cultured platelets in response to ADP, similar effects were not observed in response to AA or thrombin. Although the reason for this observation requires experimental investigation, it would seem likely that these discrepancies relate to different mechanisms by which AA and thrombin activate platelets (cyclooxygenase-1 and protease-activator receptor pathway respectively) [38], mechanisms that may be less susceptible to the oxidative modifications or signaling disruptions caused by NAPQI. Conversely, the ADP-mediated pathway (P2Y receptor signaling) may be more sensitive to the effects of reactive metabolites produced by CYP2E1-dependent APAP metabolism [38].

In an attempt to address these discrepancies, we analyzed liver cell viability and oxidative stress. Measurement of APAP toxicity in HepG2 and HepG2E47 cells was performed using a cell viability assay (CCK-8) and a FITC-staining assay. Both approaches demonstrated increased APAP-dependent toxicity in HepG2E47 cells, supporting a role for CYP2E1-dependent APAP metabolism in mediating cell damage [39]. However, we detected notable variances between the assays that may reflect differences in sensitivity. The CCK-8 assay measures metabolic activity in cells, and thus may detect earlier cellular responses to APAP. Conversely, the FITC assay captures total live cell population, and may be more reflective of later stages of cell toxicity. This may explain the contradictory data obtained following direct addition of NAPQI to cells whereby the CCK-8 assay failed to detect a toxic response, yet a pronounced response to NAPQI was detected in HepG2 cells using the FITC/fluorescent detection approach. However, we failed to detect a similar response to NAPQI in HepG2E47 cells with the FITC approach.

Possible explanations of these differences may relate to differences in ROS production between cell types [40, 41]. Glutathione synthesis is vital for maintaining redox balance and detoxifying reactive intermediates [42]. In our studies, a role for GSH in mediating APAP toxicity is supported using BSO, which promoted APAP cell toxicity. Of note, other investigators have focused on GSH as a target to slow or reverse liver disease progression For example, S-adenosylmethionine (SAMe, a precursor for GSH synthesis) has been widely studied for its protective effects in a range of liver diseases [42–44]. Alternatively, exogenous antioxidants such as N-acetylcysteine (NAC) have been used as both a precursor for GSH synthesis, and for its ability to scavenge ROS, including the use of NAC to mitigate APAP-induced hepatotoxicity [45]. To further focus on the role of CYP2E1 in APAP toxicity, cells were exposed to CMZ [46] or 4-MP [47] followed by APAP. As expected, neither CMZ or 4-MP impacted APAP toxicity in HepG2 cells. Conversely, CMZ was more protective of APAP-induced cell toxicity in HepG2E47 cells using the CCK-8 assay, while 4-MP was more protective using the FITC approach. These data further support the need for care being taken when selecting assays of cell viability based on time of drug exposure. It is also worth noting that in healthy individuals CYP2E1 is normally expressed at low levels in the liver. However, following sustained, high levels of ethanol ingestion, CYP2E1 is induced in hepatocytes and stabilization of CYP2E1 occurs such that significant hepatic ethanol metabolism occurs via CYP2E1 [48]. This may have clinical significance in considering APAP toxicity in alcohol-dependent patients [48].

Analysis of oxidative stress in the cell lines revealed that pathways other than APAP-metabolism to NAPQI may contribute to cell toxicity and platelet aggregation. Using HepG2 cells, an effect of APAP, NAPQI, BSO, and BSO + APAP on ROS was detected, but was relatively low compared to control. In contrast, HepG2E47 cells revealed a greater than twofold increase in ROS following exposure to APAP, and a 1.5-fold increase in ROS following exposure to APAP in the presence of BSO. However, inhibition of CYP2E1 using CMZ or 4-MP led to equally dramatic increases in ROS following APAP exposure. These data may indicate inhibition of CYP2E1-APAP metabolism leads to metabolism via alternative pathway(s) [49]. Of note, previous studies report HepG2E47 cells have higher GSH, catalase, cytosolic and microsomal glutathione transferase, and heme oxygenase-1 expression than HepG2 cells, suggesting differences in basal antioxidant capacity may also need to be considered [50].

Given the dose-dependent decline in platelet aggregation following NAPQI exposure, the possibility is raised that measuring platelet aggregation via light transmission aggregometry could serve as an early indicator of hepatic APAP toxicity. The use of antioxidants may also prove beneficial in mitigating changes in platelet aggregation caused by APAP toxicity by maintaining the cellular redox balance. Thus, while NAC plays a central role as the standard therapy for APAP toxicity, our data suggest exploring other antioxidants as adjuncts to NAC to target the effects of ROS in platelets may also have merit. Determining the threshold for platelet damage and correlation with APAP toxicity is essential to assess damage. By correlating platelet dysfunction with liver damage, a better understanding of the extent of APAP-induced liver injury could be possible. However, it is unlikely that patients who have ingested potentially lethal doses of APAP have similar underlying liver health prior to ingestion, and those patients with underlying liver disease may have platelet dysfunction prior to APAP ingestion. Similarly, careful studies are required to fully understand the dose–effect relationship between APAP intake, hepatic damage, and consequences on changes in platelet aggregation at different time points.

In considering our data several important limitations should be noted. The use of a closed cell system to assess APAP and NAPQI toxicity in HepG2/HepG2E47 cells and platelet aggregation lacks the complexity of the in vivo environment. In the clinical setting, hepatic cell interaction with immune cells, extracellular matrix, and blood flow can lead to an oversimplified understanding of drug effects and toxicity using in vitro models. Similarly, while platelet aggregation is impacted by exogenous NAPQI platelets lack CYP2E1, meaning any NAPQI exposure results from hepatic cell production, and it is likely that NAPQI produced by hepatocytes undergoes interactions within these cells before reaching platelets. Of further note, the HepG2/HepG2E47 cells used were originally derived from a human hepatoma. Although these cells retain some hepatocyte-like functions they are cancerous cells that do not fully maintain the metabolic activities of hepatocytes. Finally, while genetically modified cells and pharmacological agents provide valuable mechanistic data, their use in vitro cannot fully replicate the complex pharmacokinetics and pharmacodynamics that exist in vivo, in which drug distribution, metabolism, and excretion involve multiple tissues and organs.

Conclusions

Direct exposure of platelets to APAP does not affect aggregation function, whereas exposure to NAPQI decreases aggregation in a concentration-dependent manner. However, while APAP and NAPQI impair platelet aggregation in co-cultures of hepatic cells expressing CYP2E1 (HepG2E47) and platelets, these effects are not abrogated by exposure to CMZ or 4-MP prior to APAP exposure. When analyzing the effects of APAP exposure in hepatic cells it was demonstrated that APAP decreased cell viability in both HepG2 and HepG2E47 cells, and increased ROS production in HepG2E47 cells, with 4-MP showing limited restoration of cell viability. These results highlight distinct toxic effects of APAP and NAPQI in hepatic cells and suggest APAP metabolism to NAPQI, and associated changes in ROS production/oxidative stress can influence platelet aggregation in vitro.

Supplementary Information

Below is the link to the electronic supplementary material.

13181_2025_1065_MOESM1_ESM.pdf (695.3KB, pdf)

Supplementary file1 Schematic representation of A) acetaminophen (APAP) metabolism and B) identical schematic including sites of action of the pharmacological agents used. Following therapeutic dose intake, the majority of APAP undergoes conjugation to form glucuronide and sulfate conjugates, which are excreted in urine. In addition, ~ 10% of APAP is metabolized via cytochrome P4502E1 (CYP2E1) to produce the reactive metabolite N-acetyl-p-benzoquinone imine NAPQI, which is neutralized by glutathione (GSH) to form a non-toxic conjugate. In the setting of APAP overdose, GSH depletion results in NAPQI accumulation and increased ROS production which bind to macromolecules forming protein adducts and cell damage. Pharmacological agents were employed to deplete GSH levels (Buthionine sulfoximine; BSO) or inhibit CYP2E1 via competitive inhibition (4-methyl pyrazole; 4-MP) or non-competitive inhibition (Chlormethiazole; CMZ) of the CYP2E1 substrate binding site (SBS). In addition, Adenosine diphosphate (ADP), Arachidonic acid (AA), and thrombin were used as activators of platelet aggregation targeting P2Y1/P2Y2, TXA2 and PAR1/PAR2 respectively. Validation of CYP2E1 expression in A) HepG2E47 cells via Western Blot Analysis in HepG2E47 cells and non-expression in HepG2 cells. Quantification of metabolized acetaminophen (APAP) by B) HepG2E47 cells and no metabolism by HepG2 cells, *P<0.05 Time point 0h (T0) versus Time Point 24 hours (T24), N=3. (PDF 695 KB)

Funding

Department of Surgery, Atrium Health’s Carolinas Medical Center.

Declarations

Conflicts of interest

None of the authors have any conflicts of interest to declare.

Footnotes

Publisher's Note

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

13181_2025_1065_MOESM1_ESM.pdf (695.3KB, pdf)

Supplementary file1 Schematic representation of A) acetaminophen (APAP) metabolism and B) identical schematic including sites of action of the pharmacological agents used. Following therapeutic dose intake, the majority of APAP undergoes conjugation to form glucuronide and sulfate conjugates, which are excreted in urine. In addition, ~ 10% of APAP is metabolized via cytochrome P4502E1 (CYP2E1) to produce the reactive metabolite N-acetyl-p-benzoquinone imine NAPQI, which is neutralized by glutathione (GSH) to form a non-toxic conjugate. In the setting of APAP overdose, GSH depletion results in NAPQI accumulation and increased ROS production which bind to macromolecules forming protein adducts and cell damage. Pharmacological agents were employed to deplete GSH levels (Buthionine sulfoximine; BSO) or inhibit CYP2E1 via competitive inhibition (4-methyl pyrazole; 4-MP) or non-competitive inhibition (Chlormethiazole; CMZ) of the CYP2E1 substrate binding site (SBS). In addition, Adenosine diphosphate (ADP), Arachidonic acid (AA), and thrombin were used as activators of platelet aggregation targeting P2Y1/P2Y2, TXA2 and PAR1/PAR2 respectively. Validation of CYP2E1 expression in A) HepG2E47 cells via Western Blot Analysis in HepG2E47 cells and non-expression in HepG2 cells. Quantification of metabolized acetaminophen (APAP) by B) HepG2E47 cells and no metabolism by HepG2 cells, *P<0.05 Time point 0h (T0) versus Time Point 24 hours (T24), N=3. (PDF 695 KB)

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