A Cytochrome P450–Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes

Abstract

Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAPinduced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

Cryopreserved Human Intestinal Mucosal Epithelium: A Novel In Vitro Experimental System for the Evaluation of Enteric Drug Metabolism, Cytochrome P450 Induction, and Enterotoxicity

“…Our results with CHIM are consistent with clinical findings showing that naproxen has higher enterotoxicity than acetaminophen (Lewis et al, 2002). Our results therefore suggest that CHIM can be useful in evaluation of the enterotoxic potential of orally administered drugs, especially for drugs that may be activated or detoxified by enteric metabolism, such as acetaminophen (Laine et al, 2009;Jaeschke and McGill, 2015;Jiang et al, 2015;Miyakawa et al, 2015) and naproxen (Miners et al, 1996;Rodrigues et al, 1996;Tracy et al, 1997). In our laboratory, we have initiated a study on the role of drug-metabolizing enzyme activities on the cytotoxicity of acetaminophen and naproxen on CHIM as an experimental approach to evaluate the usefulness of this novel experimental system in the definition of key pathways for toxic metabolite formation and/or detoxification.…”

Lactobacillus fermentum Postbiotic-induced Autophagy as Potential Approach for Treatment of Acetaminophen Hepatotoxicity

“…APAP exerts its toxic effects by two mechanisms: by CYP450-dependent NAPQI generation and by formation of PAP, as the result of APAP deacetylation (Miyakawa et al, 2015). CYP450-dependent pathway is activated immediately after APAP exposure and it lasts for the first several hours of exposure.…”

“…However, acute or cumulative overdose can cause hepatic necrosis and liver failure (Larson et al, 2005). The mechanisms of APAP-induced liver injury described to this moment include generation of reactive metabolite, N -acetyl- p -benzoquinone imine (NAPQI) and p -aminophenol (PAP) (Miyakawa et al, 2015). Given an important role of autophagy in elimination of damaged organelles, including mitochondria, it has been shown that activation of autophagy could serve as a cellular adaptive mechanism to counteract APAP-induced hepatotoxicity (Igusa et al, 2012; Ni et al, 2012).…”

Multiple microRNAs function as self-protective modules in acetaminophen-induced hepatotoxicity in humans

“…As described by Miyakawa et al (2015), the cell culture medium was collected and the remaining cells were lysed with an equal volume medium containing 1% Triton X-100. To eliminate unbroken cells or cell debris, the medium and cell lysate were centrifuged at 700 g for 5 min.…”