image: Acetaminophen-induced liver injury remains a significant global public health concern due to its potential to cause acute liver failure and mortality in severe cases. The hepatotoxic effects of APAP are primarily mediated by its bioactivation to the reactive metabolite NAPQI via CYP2E1. Excessive accumulation of NAPQI depletes hepatic GSH stores, leading to oxidative stress, mitochondrial dysfunction, and covalent modification of cellular macromolecules. Multiple regulated and unregulated cell death pathways contribute to APAP-induced hepatotoxicity, including necrosis, apoptosis, necroptosis, and pyroptosis. Among these, sterile inflammation plays a pivotal role in amplifying liver injury. Inflammasome complexes, particularly NLRP3, have emerged as key regulators of this inflammatory response. Activation of the NLRP3 inflammasome leads to the cleavage of procaspase-1 into its active form, which in turn processes proinflammatory cytokines such as IL-1β and IL-18 and drives pyroptotic cell death via Gasdermin D activation. Despite the availability of NAC as the primary therapeutic agent for APAP overdose, treatment options remain limited, particularly when administered beyond the early therapeutic window. Consequently, current research has focused on identifying novel therapeutic strategies targeting the inflammatory and cell death pathways involved in APAP-induced hepatotoxicity. These include direct inhibition of inflammasome components, caspase-1 blockade, modulation of immune responses using anti-inflammatory agents, and targeted manipulation of gut microbiota and bile acid metabolism, which are increasingly recognized for their roles in liver homeostasis and injury response. Pharmacological inhibitors such as NLRP3 antagonists and caspase-1 inhibitors show promise in preclinical models but require further clinical validation. Emerging strategies targeting the gut-liver axis, such as microbiome modulation and bile acid receptor agonists, also offer new therapeutic avenues for APAP-induced liver injury. However, a deeper understanding of the molecular mechanisms leading to APAP-induced liver injury is crucial for developing effective, timely, and targeted therapies to reduce the global burden of APAP toxicity.
Credit: Latchoumycandane Calivarathan
Acetaminophen (APAP) is one of the most widely used analgesic and antipyretic agents and is generally safe at therapeutic doses. However, overdose remains a major cause of acute liver failure worldwide, progressing through stages ranging from preclinical toxic effects to hepatic injury, failure, and potential recovery. The hepatotoxicity of APAP is attributed to its metabolic activation in the liver, where overdose saturates detoxification pathways, leading to oxidative stress, mitochondrial dysfunction, and inflammation. Despite the availability of N-acetylcysteine (NAC) as the standard antidote, delayed treatment or severe overdoses often result in high morbidity and mortality, underscoring the need for new therapeutic strategies
Mechanistic Pathways of APAP Hepatotoxicity
Under normal dosing, most APAP is detoxified by conjugation pathways, while a small fraction undergoes cytochrome P450 (mainly CYP2E1) metabolism, producing the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). In overdose, glutathione stores are depleted, allowing NAPQI to bind covalently to proteins, impairing mitochondrial enzymes and disrupting ATP production. These events trigger oxidative stress, DNA damage, and regulated cell death pathways, including necrosis, apoptosis, necroptosis, and pyroptosis .
Role of Inflammasome Activation
A central feature of APAP-induced injury is sterile inflammation mediated by inflammasomes. Among these, the NLRP3 inflammasome has been most extensively studied. Activated by mitochondrial dysfunction, ROS production, and damage-associated molecular patterns (DAMPs), NLRP3 promotes caspase-1 activation, leading to maturation of pro-inflammatory cytokines IL-1β and IL-18. These cytokines amplify immune cell recruitment and liver inflammation, further aggravating injury. Inflammasome activation also drives pyroptosis through Gasdermin D cleavage, linking toxic injury to an amplified inflammatory cascade .
Therapeutic Approaches and Emerging Strategies
While NAC remains the primary therapy, its effectiveness declines if given late. Research is now focused on targeting inflammasome signaling and downstream pathways. Promising strategies include:
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NLRP3 inhibitors (e.g., MCC950, natural compounds such as salidroside, aloperine, and kaempferol) that reduce inflammasome activation.
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Caspase-1 inhibitors (VX-765, pralnacasan) that block cytokine processing and pyroptosis.
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Anti-inflammatory agents such as corticosteroids, NSAIDs, and cyclosporin A (sometimes in combination with NAC).
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Gut-liver axis modulation, including antibiotics, probiotics, and fecal microbiota transplantation, which alter microbiome composition to attenuate inflammatory responses and enhance regeneration.
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Bile acid receptor agonists and sequestrants, targeting metabolic and inflammatory cross-talk in the liver .
These approaches show strong preclinical potential, but translation into clinical therapies remains limited due to species differences and lack of robust clinical trial data.
Limitations and Future Directions
Most findings derive from animal and in vitro studies, which may not fully replicate human responses. Vulnerable populations such as children, pregnant women, and the elderly are underexplored. Future studies should bridge preclinical insights with clinical applications, focusing on personalized interventions and validating inflammasome-targeted therapies in humans .
Conclusions
APAP-induced liver injury remains a pressing clinical problem. Beyond oxidative stress and mitochondrial dysfunction, inflammasomes—particularly NLRP3—serve as central mediators linking toxic injury to sterile inflammation. Targeting inflammasome signaling and related pathways represents a promising frontier for novel therapies. However, a deeper mechanistic understanding and translational studies are essential to reduce the global burden of APAP hepatotoxicity .
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The study was recently published in the Gene Expression.
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Journal
Gene Expression
Article Title
Inflammasome Activation as a Key Driver of Acetaminophen-induced Hepatotoxicity: Mechanisms and Emerging Therapeutics
Article Publication Date
21-Jul-2025