Acetaminophen-induced liver injury in rats and mice: Comparison of protein adducts, mitochondrial dysfunction, and oxidative stress in the mechanism of toxicity

Abstract

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in the West. In mice, APAP hepatotoxicity can be rapidly induced with a single dose. Because it is both clinically relevant and experimentally convenient, APAP intoxication has become a popular model of liver injury. Early data demonstrated that rats are resistant to APAP toxicity. As a result, mice are the preferred species for mechanistic studies. Furthermore, recent work has shown that the mechanisms of APAP toxicity in humans are similar to mice. Nevertheless, some investigators still use rats. New mechanistic information from the last forty years invites a reevaluation of the differences between these species. Comparison may provide interesting insights and confirm or exclude the rat as an option for APAP studies. To this end, we treated rats and mice with APAP and measured parameters of liver injury, APAP metabolism, oxidative stress, and activation of the c-Jun N-terminal kinase (JNK). Consistent with earlier data, we found that rats were highly resistant to APAP toxicity. Although overall APAP metabolism was similar in both species, mitochondrial protein adducts were significantly lower in rats. Accordingly, rats also had less oxidative stress. Finally, while mice showed extensive activation and mitochondrial translocation of JNK, this could not be detected in rat livers. These data support the hypothesis that mitochondrial dysfunction is critical for the development of necrosis after APAP treatment. Because mitochondrial damage also occurs in humans, rats are not a clinically relevant species for studies of APAP hepatotoxicity.

Introduction

When used as directed, acetaminophen (APAP) is a safe and effective analgesic and fever reducer. However, large doses of APAP can cause serious liver injury. In fact, APAP overdose is the primary cause of acute liver failure in many countries throughout the West (Bernal, 2003, Gow et al., 2004, Larson et al., 2005), responsible for more than 70,000 hospitalizations each year in the U.S. alone (Budnitz et al., 2011). Research on the mechanism of APAP-induced liver injury began four decades ago, following the first published report of this toxicity in humans (Davidson and Eastham, 1966). Though many important questions have yet to be answered, the mechanism of APAP toxicity has been well investigated in rodents (Jaeschke et al., 2011, Jaeschke et al., 2012) and progress is now being made in humans (Antoine et al., 2012, Antoniades et al., 2012, Davern et al., 2006, McGill et al., 2012) and with in vitro human models (McGill et al., 2011). This profusion of data likely makes APAP the best characterized hepatotoxicant.

Because APAP-induced liver injury is clinically relevant, well studied, and can be rapidly induced in vivo with a single dose, it has become a standard model in the pharmacology and toxicology literature. In particular, APAP overdose in rodents is frequently used to test the hepatoprotective potential of herbal therapeutics. While this can be a valid approach, a number of concerns have been raised (Jaeschke et al., 2010, Jaeschke et al., 2011). For example, one of the most common issues in the complementary and alternative medicine literature is the use of rats to evaluate protection against APAP injury. It has been known since the early 1970s that rats are resistant to the liver-damaging effects of APAP (Mitchell et al., 1973). Doses which far exceed the LD50 for mice cause only minimal necrosis in rat liver. The reason for this difference in susceptibility is not well understood. In mice, APAP hepatotoxicity begins with metabolism of the parent compound to the reactive electrophile N-acetyl-p-benzoquinone imine (NAPQI). NAPQI depletes glutathione (GSH) and binds to proteins, primarily to the amino acid cysteine (Cohen et al., 1997, Nelson, 1990). Differences in APAP metabolism and protein binding could account for the difference between mice and rats. However, while protein binding appears to be a necessary first step toward injury, it is not sufficient to directly cause cell death (Jaeschke et al., 2012). 3′-Hydroxyacetanilide (AMAP), a non-hepatotoxic isomer of APAP, also binds to proteins (Tirmenstein and Nelson, 1989). Moreover, toxicity develops only after the onset of oxidative stress and mitochondrial dysfunction, and preventing these phenomena protects against APAP (Cover et al., 2005, Kon et al., 2004, Ramachandran et al., 2011a, Ramachandran et al., 2011b). Moreover, activation and mitochondrial translocation of c-Jun N-terminal kinase (JNK) have repeatedly been shown to play a role in APAP toxicity in the liver (Gunawan et al., 2006, Hanawa et al., 2008, Latchoumycandane et al., 2007, Saito et al., 2010). Thus, mitochondrial dysfunction, oxidative stress, and/or JNK activation may also be different between the two species.

A better understanding of the differences between rats and mice will not only aid future researchers in selection of the best model for their experiments, it may provide important new mechanistic insights into APAP toxicity. Therefore, the objective of the present study was to investigate potential differences in the mechanism of APAP-induced liver injury between rats and mice with emphasis on protein adduct formation, oxidative stress, and JNK activation.