The development and hepatotoxicity of acetaminophen: reviewing over a century of progress

Abstract

Acetaminophen (APAP) was first synthesized in the 1800s, and came on the market approximately 65 years ago. Since then, it has become one of the most used drugs in the world. However, it is also a major cause of acute liver failure. Early investigations of the mechanisms of toxicity revealed that cytochrome P450 enzymes catalyze formation of a reactive metabolite in the liver that depletes glutathione and covalently binds to proteins. That work led to the introduction of N-acetylcysteine (NAC) as an antidote for APAP overdose. Subsequent studies identified the reactive metabolite N-acetyl-p-benzoquinone imine, specific P450 enzymes involved, the mechanism of P450-mediated oxidation, and major adducted proteins. Significant gaps remain in our understanding of the mechanisms downstream of metabolism, but several events appear critical. These events include development of an initial oxidative stress, reactive nitrogen formation, altered calcium flux, JNK activation and mitochondrial translocation, inhibition of mitochondrial respiration, the mitochondrial permeability transition, and nuclear DNA fragmentation. Additional research is necessary to complete our knowledge of the toxicity, such as the source of the initial oxidative stress, and to greatly improve our understanding of liver regeneration after APAP overdose. A better understanding of these mechanisms may lead to additional treatment options. Even though NAC is an excellent antidote, its effectiveness is limited to the first 16 hours following overdose.

Keywords: APAP-protein adducts; Acetaminophen overdose; biomarkers; drug metabolism; drug-induced liver injury; liver injury; liver regeneration; mitochondria; oxidative stress; reactive nitrogen.

Figure 1.. Histopathology of APAP-induced liver injury.

A mouse was treated with 300 mg/kg APAP. The liver was collected 24 h later. Liver tissue was fixed in 10% phosphate-buffered formalin and embedded in paraffin wax. 5 μm sections were cut, mounted on glass slides, and stained with hematoxylin and eoson. The dotted line indicates an area of hepatocellular necrosis displaying eosinophilia and loss of basophilic nuclei. Black arrowheads: Pyknosis and karyorrhexis. White arrowheads: Karyolysis. Red arrowheads: Cell swelling.

Figure 2.. The Rumack-Matthew nomogram.

When measured between 4 and 24 hours after APAP overdose, values above the dashed line (Rumack-Matthew line) are likely to result in hepatotoxicity, values between the dashed line and the solid line (Treatment line) may or may not lead to hepatotoxicity, and values below the solid line are unlikely to cause hepatotoxicity. All patients with values above the Treatment line should receive NAC.

Figure 3.. Proposed mechanisms of NAPQI formation.

Initially, it was thought that formation of the reactive metabolite of APAP may precede through N-hydroxylation (A) or epoxidation (B), as shown. These mechanisms were proven to be incorrect. (C) It is now thought that APAP is directly oxidized by a two-electron oxidation, a previously unrecognized cytochrome P-450 mechanism. Once formed, NAPQI readily reacts with sulfhydryl groups, such as on GSH or cysteine residues in proteins.

Figure 4.. Anti-APAP immunoblot.

Cytosolic fractions from the livers of control (−) and 600 mg/kg APAP-treated (+) mice, isolated 2 hours after treatment. Western blotting was performed using an anti-APAP antibody. A dominant band is visible around 58 kD. Adapted from(Cohen and Khairallah 1997), with permission from the publisher.

Figure 5.. Measurement of covalent APAP-protein adducts in the liver.

Mice were treated with a hepatotoxic dose of acetaminophen (APAP). Livers and serum were collected at multiple time points after APAP overdose. (A) Immunohistochemistry for APAP. (B) Serum alanine aminotransferase (ALT) activity (left y-axis) and serum APAP-protein adducts (right y-axis) over time. Adapted from (Roberts D. W. et al. 1991) and (McGill et al. 2013) with permission.

Figure 6.. Mechanisms of APAP hepatotoxicity.

Acetaminophen (APAP) hepatotoxicity begins with formation of a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Left side: NAPQI depletes hepatic glutathione (GSH) and binds to proteins. The initial protein binding leads to oxidative stress through an unknown mechanism that may involve covalent modification and inhibition of electron transport chain (ETC) complex proteins leading to increased superoxide [O₂⁻]. At the same time, activation of neuronal nitric oxide synthase (nNOS)/NOS1 occurs, possibly through redox modification or covalent modification of the transient receptor potential melastatin 2 (TRPM2) which increases intracellular calcium (Ca²⁺). Nitric oxide (NO) from nNOS then reacts with O₂⁻ to form peroxynitrite (ONOO⁻), which then reacts with tyrosine and other residues on intracellular proteins. Right side: The initial reactive oxygen species (ROS) also activate apoptosis-signaling kinase 1 (ASK1), ultimately leading to activation of the c-Jun N-terminal kinases 1 and 2 (JNK). JNK then translocates to mitochondria, binding to the protein Sab in the outer mitochondrial membrane (OMM). This interaction causes release of the protein tyrosine phosphatase, nonreceptor type 6 (SHP) from Sab on intermembrane side of the OMM. SHP then translocates to the inner mitochondrial membrane and inhibits the ETC enhancer Src, resulting in reduced ETC activity and therefore increased ROS production. This forms a feed-forward loop in which ROS activate JNK, and JNK increases ROS. Eventually, the mitochondrial membrane permeability transition (MPT) occurs with loss of ATP production. The mitochondria swell and the membranes rupture, liberating the endonucleases apoptosis-inducing factor (AIF) and endonuclease G. The free nucleases can then fragment nuclear DNA. In parallel, expression and activity of the receptor-interacting protein (RIP) kinases 1 and 3 increase, along with Drp1-mediated mitochondrial fragmentation, through underexplored mechanisms. Ultimately, the hepatocyte dies by oncotic necrosis.