DILI is broadly categorized into intrinsic and idiosyncratic types. Intrinsic hepatotoxins, such as APAP, cause dose-dependent injury, while idiosyncratic DILI involves complex immune and metabolic interactions that remain poorly understood. Mechanistic studies of intrinsic hepatotoxins have revealed oxidative stress and mitochondrial dysfunction as key contributors to injury. Historically, LPO and iron-catalyzed free radical generation were central to understanding DILI, but the focus shifted toward apoptosis in the late 20th century.
The discovery of ferroptosis—a regulated form of cell death characterized by glutathione depletion, inactivation of glutathione peroxidase 4 (GPx4), and iron-dependent propagation of LPO—has renewed interest in non-apoptotic cell death mechanisms. Although initially studied in cancer, ferroptosis is increasingly implicated in various diseases, including liver injury. However, the liver's robust antioxidant defenses, including glutathione (GSH) and vitamin E, question the widespread applicability of ferroptosis as a primary mode of cell death in DILI.
Mechanisms of Ferroptosis
Ferroptosis was first identified in RAS-mutant cancer cells exposed to erastin and related compounds, which inhibit the cystine/glutamate antiporter (system Xc-) and impair GSH synthesis. The resulting glutathione depletion disrupts GPx4 activity, preventing detoxification of lipid hydroperoxides and promoting LPO. This cascade leads to cellular dysfunction and death. The unique features of ferroptosis—iron dependency, ROS generation, and LPO propagation—distinguish it from apoptosis and necrosis.
In hepatocytes, ferroptosis faces physiological constraints due to the liver's multilayered antioxidant systems. These include ferritin-mediated iron sequestration, GPx4 activity, and high levels of vitamin E in membranes. The review emphasizes the need to critically evaluate whether ferroptosis contributes to hepatocyte death under normal conditions or whether it only becomes relevant in scenarios of severe antioxidant depletion or acute iron overload.
Reevaluating Lipid Peroxidation and Oxidative Stress
The relationship between LPO and APAP hepatotoxicity has been contentious. Early studies linked APAP overdose to severe LPO, particularly in animals fed vitamin E-deficient diets. However, subsequent research demonstrated minimal LPO under normal conditions, even in severe liver injury models. Instead, mitochondrial dysfunction emerged as a central driver of hepatotoxicity. The reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), formed during APAP metabolism, depletes GSH and forms protein adducts, particularly in mitochondria. This initiates oxidative stress, activates c-Jun N-terminal kinase (JNK), and disrupts mitochondrial integrity.
Peroxynitrite, a potent oxidant formed by the reaction of mitochondrial superoxide and nitric oxide, plays a key role in oxidative damage. Studies show that peroxynitrite mediates protein nitration, DNA damage, and mitochondrial dysfunction, rather than LPO, as the primary cytotoxic mechanism. These findings challenge the hypothesis that ferroptosis, characterized by extensive LPO, is the dominant mode of cell death in APAP-induced hepatotoxicity under normal conditions.
The Role of Ferroptosis Inhibitors
The authors discuss the use of ferroptosis inhibitors, such as ferrostatin-1, in assessing the relevance of ferroptosis in liver injury. Ferrostatin-1 and its analogs have shown inconsistent efficacy in APAP models, particularly under conditions of limited LPO. More potent inhibitors, like UAMC-3203, exhibit off-target effects, such as downregulating JNK signaling, complicating their interpretation. The review emphasizes that a significant increase in LPO (10- to 50-fold above baseline) is necessary for ferroptosis to play a biologically relevant role in liver injury.
Broader Implications and Other Hepatotoxins
Beyond APAP, several drugs and natural products have been investigated for their potential to induce ferroptosis. Methotrexate, an anti-cancer and anti-inflammatory drug, has been linked to ferritinophagy-mediated LPO and ferroptosis. However, LPO levels remain modest, and injury is mild. Similarly, rifampicin and isoniazid, used to treat tuberculosis, show ferroptosis-like features, such as GPx4 reduction and ACSL4 upregulation, but definitive causal evidence is lacking. Herbal compounds like Epimedium koreanum and toosendanin have also been associated with ferroptosis, though their effects are moderate and often correlative.
Discussion
The authors highlight an exponential increase in publications attributing DILI to ferroptosis. However, many studies rely on indirect markers, such as LPO or GPx4 depletion, without establishing causality. The liver's defense systems, including GSH, ferritin, and vitamin E, generally prevent excessive LPO, limiting ferroptosis under normal conditions. Scenarios like vitamin E deficiency or acute iron overload may enhance susceptibility to LPO and ferroptosis, but such conditions are rare in clinical practice.
Conclusions
Ferroptosis represents a compelling yet context-dependent mechanism in DILI. While it may contribute to liver injury under specific conditions of antioxidant compromise or iron overload, it is unlikely to play a dominant role in APAP-induced hepatotoxicity under normal conditions. The review underscores the need for rigorous experimental validation and a nuanced understanding of cell death mechanisms in liver diseases. Future research should focus on quantifying LPO thresholds and identifying conditions under which ferroptosis becomes pathologically relevant.
Full text
https://www.xiahepublishing.com/2310-8819/JCTH-2024-00324
The study was recently published in the Journal of Clinical and Translational Hepatology .
The Journal of Clinical and Translational Hepatology (JCTH) is owned by the Second Affiliated Hospital of Chongqing Medical University and published by XIA & HE Publishing Inc. JCTH publishes high quality, peer reviewed studies in the translational and clinical human health sciences of liver diseases. JCTH has established high standards for publication of original research, which are characterized by a study's novelty, quality, and ethical conduct in the scientific process as well as in the communication of the research findings. Each issue includes articles by leading authorities on topics in hepatology that are germane to the most current challenges in the field. Special features include reports on the latest advances in drug development and technology that are relevant to liver diseases. Regular features of JCTH also include editorials, correspondences and invited commentaries on rapidly progressing areas in hepatology. All articles published by JCTH, both solicited and unsolicited, must pass our rigorous peer review process.
