Models of drug-induced liver injury for evaluation of phytotherapeutics and other natural products

Abstract

Extracts from medicinal plants, many of which have been used for centuries, are increasingly tested in models of hepatotoxicity. One of the most popular models to evaluate the hepatoprotective potential of natural products is acetaminophen (APAP)-induced liver injury, although other hepatotoxicity models such as carbon tetrachloride, thioacetamide, ethanol and endotoxin are occasionally used. APAP overdose is a clinically relevant model of drug-induced liver injury. Critical mechanisms and signaling pathways, which trigger necrotic cell death and sterile inflammation, are discussed. Although there is increasing understanding of the pathophysiology of APAP-induced liver injury, the mechanism is complex and prone to misinterpretation, especially when unknown chemicals such as plant extracts are tested. This review discusses the fundamental aspects that need to be considered when using this model, such as selection of the animal species or in vitro system, timing and dose-responses of signaling events, metabolic activation and protein adduct formation, the role of lipid peroxidation and apoptotic versus necrotic cell death, and the impact of the ensuing sterile inflammatory response. The goal is to enable researchers to select the appropriate model and experimental conditions for testing of natural products that will yield clinically relevant results and allow valid interpretations of the pharmacological mechanisms.

Figure 1

Mechanisms of acetaminophen hepatotoxicity. Acetaminophen (APAP) is converted to the electrophile NAPQI through phase I drug metabolism. NAPQI can be detoxified by glutathione (GSH) or bind to proteins. Binding to mitochondrial proteins causes mitochondrial dysfunction and oxidative stress, which activates apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK). Translocation of JNK into mitochondria enhances the oxidative stress. The mitochondrial membrane permeability transition (MPT) occurs and the mitochondrial membrane potential is lost resulting in depletion of cellular ATP levels. The release of mitochondrial endonucleases (AIF and EndoG) leads to nuclear DNA fragmentation. The result of this sequence is cell necrosis.

Figure 2

Timeline of acetaminophen hepatotoxicity. NAPQI formation begins immediately after acetaminophen (APAP) treatment. In mice, glutathione (GSH) levels drop precipitously in the first 0.5 h. Protein binding begins almost immediately after dosing and continues for 3-4 h (depending on the dose). Cell death begins during this time and continues until about 12 h post-treatment. A sterile inflammatory response occurs in the 6 – 24 h period. Finally, injury resolution, cell proliferation and liver regeneration begin at about 12 h and continue until about 72 h post-dosing.

Figure 3

A) Liver injury (plasma alanine aminotransferase activity) and B) malondialdehyde (MDA) levels were assessed in untreated controls and in different models of drug-induced liver injury. Data are shown for a model of allyl alcohol-induced liver injury (0.1 mmol/kg AA) in mice fed a regular diet (Vit E+/+) or a vitamin E–deficient diet for 4 weeks (data adapted from Knight et al., 2003 and Jaeschke et al., 1987). Other data are from models of acetaminophen (APAP)-induced liver injury in rats (adapted from Ajit et al., 2007) and mice (adapted from Wu et al., 2010) fed a regular diet. The comparison clearly indicates the high susceptibility to lipid peroxidation (LPO) of animals on a vitamin E-deficient diet. In contrast, APAP causes similar injury in mice on a regular diet but very little LPO indicating that LPO is not the mechanism of cell death. The comparison between rats and mice documents the low susceptibility of rats to APAP-induced LPO or liver injury.

Figure 4

Caspase-3 fluorescent activity assay and western blotting. C57BL/6 mice were treated intraperitoneally with saline vehicle (6 h), 300 mg/kg APAP (0.5, 6 or 24 h), or 700 mg/kg galactosamine (GalN)-100 μg/kg endotoxin (ET) (6 h). (A) Caspase-3 activity was determined by the Ac-DEVD-AFC fluorometric assay (Enzo Life Sciences, Plymouth Meeting, PA) using total liver homogenate and expressed as the change in relative fluorescence units (RFU) per minute per mg protein as previously described (Bajt et al., 2000). (B) Western blotting was also performed using total liver homogenate with an antibody which recognizes full length (pro-form) and cleaved (active) caspase-3 (Cell Signaling Technology, Beverly, MA) as previously described (Bajt et al., 2000).