Acetaminophen should be a critical reference hepatotoxin for evaluating human-relevant in vitro models

We read with great interest the recent Research Brief by Livoti et al. (2025) where the authors question the utility of acetaminophen (APAP) as a reference hepatotoxin to calibrate human in vitro systems. The authors argue that induction of cell death in cultured human or rodent hepatocytes requires APAP concentrations of ≥10 mM, which they claim is much higher than hepatotoxic plasma levels in patients (≤1 mM). Based on simulation experiments, a hepatotoxic concentration of 0.8 mM APAP should be achieved with a single overdose of 10 g (Livoti et al. 2025). In addition, in vivo mechanisms of toxicity involving CYP2E1-mediated formation of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) do not apply to high doses in cell culture (Livoti et al. 2025). Having performed numerous in vitro experiments with primary human and mouse hepatocytes, in vivo experiments with rats and mice, and assessed biomarkers in APAP overdose patients, we strongly disagree with the authors’ comments and conclusions.

First, in contrast to the simulation experiments presented by the authors, when 6 healthy volunteers ingested a mild overdose of APAP (80 mg/kg = 6–7 g per volunteer) in a cross-over study design where each person served as its own control, the peak plasma levels were 0.8 mM in the absence of any hepatotoxicity (Kang et al. 2020). Thus, 10 g would likely result in plasma levels of >1 mM but still unlikely to cause toxicity in adult humans. In fact, patients in real danger for hepatotoxicity and acute liver failure are generally patients who take 50–100 g or more as single overdose. These patients have measured plasma APAP levels of 5–10 mM as reported in a case report and citations of similar cases (Zell-Kanter et al. 2013). These levels are in the same range as hepatotoxic concentrations in primary human hepatocytes from male and female donors (Xie et al. 2014). It is important to emphasize that these were freshly isolated cells, which were treated with APAP within 6 h after the tissue was removed from the patient and therefore do not show declining cytochrome P450 levels. It is unclear how long the delay was from tissue harvesting to APAP exposure of the hepatocytes in the authors’ experiments. In addition, the toxicity of primary human hepatocytes (release of alanine aminotransferase [ALT]) starts at 24 h and therefore should be assessed at 48–72 h, especially at lower doses, to be in line with the time course of liver injury observed in patients (Xie et al. 2014). The authors evaluated the time course of ATP depletion and not ALT release only up to 24 h (Livoti et al. 2025). This time course is appropriate for mouse hepatocytes (peak of injury between 12 and 24 h) but less for human hepatocytes with peak of injury at 48 h (Xie et al. 2014). In addition, ATP depletion is not comparable with ALT release reflecting necrotic cell death in vivo (Jaeschke 1990). Another aspect that is widely ignored is the fact that cell culture medium contains amino acids including cysteine and cystine, which increase cellular glutathione (GSH) synthesis as soon as the hepatocytes are exposed to the medium (Bajt et al. 2004). Thus, compared to the in vivo situation, isolated cells are supplied with a surplus of GSH precursors, which makes them less susceptible to APAP than hepatocytes in vivo. However, this is a problem of experimental designs with in vitro systems and not an intrinsic deficiency of using APAP as a reference substance.

The authors’ second argument against the use of APAP in vitro is that the P450 inhibitor 1-aminobenzotriazole reduced oxidative drug metabolism but not cell injury, again assessed as ATP decline (Livoti et al. 2025). However, this is not our experience. APAP-induced cell death after 10 mM APAP was completely inhibited by the CYP2E1 inhibitor fomepizole (Akakpo et al. 2018). Furthermore, early treatment with N-acetylcysteine, i.e. when the newly synthesized GSH scavenges NAPQI, eliminates protein adducts and completely protects against 10 mM APAP (Bajt et al. 2004; Xie et al. 2014). Importantly, the signaling mechanisms of toxicity in human and mouse hepatocytes are virtually identical to what is observed in humans and mice in vivo including CYP2E1-dependent NAPQI formation, GSH depletion, protein adducts formation, especially on mitochondria, activation, and mitochondrial translocation of c-jun N-terminal kinase, mitochondrial oxidant stress and dysfunction, DNA fragmentation and necrotic cell death (Jaeschke and Ramachandran 2024).

Thus, APAP is one of the most studied intrinsic hepatotoxins and should be a mandatory reference compound for all in vitro systems with relevance to the human pathophysiology of drug-induced liver injury. In fact, if APAP toxicity cannot be reproduced in the in vitro system, there is a good chance that it will not reliably detect intrinsic hepatotoxicity of other drugs in humans.

Funding

National Institute of Diabetes and Digestive and Kidney Diseases (DK102142, DK125465).

Conflicts of interest. The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.