Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity

Abstract

Hepatotoxicity is a serious problem during drug development and for the use of many established drugs. For example, acetaminophen overdose is currently the most frequent cause of acute liver failure in the United States and Great Britain. Evaluation of the mechanisms of drug-induced liver injury indicates that mitochondria are critical targets for drug toxicity, either directly or indirectly through the formation of reactive metabolites. The consequence of these modifications is generally a mitochondrial oxidant stress and peroxynitrite formation, which leads to structural alterations of proteins and mitochondrial DNA and, eventually, to the opening of mitochondrial membrane permeability transition (MPT) pores. MPT pore formation results in a collapse of mitochondrial membrane potential and cessation of adenosine triphosphate synthesis. In addition, the release of intermembrane proteins, such as apoptosis-inducing factor and endonuclease G, and their translocation to the nucleus, leads to nuclear DNA fragmentation. Together, these events trigger necrotic cell death. Alternatively, the release of cytochrome c and other proapoptotic factors from mitochondria can promote caspase activation and apoptotic cell death. Drug toxicity can also induce an inflammatory response with the formation of reactive oxygen species by Kupffer cells and neutrophils. If not properly detoxified, these extracellularly generated oxidants can diffuse into hepatocytes and trigger mitochondrial dysfunction and oxidant stress, which then induces MPT and necrotic cell death. This review addresses the formation of oxidants and the defense mechanisms available for cells and applies this knowledge to better understand mechanisms of drug hepatotoxicity, especially acetaminophen-induced liver injury.

Figure 1. Cellular mechanisms of oxidant generation and scavenging

Superoxide produced mainly from the mitochondrial electron transport chain can either react with nitric oxide to produce peroxynitrite or be converted to hydrogen peroxide by superoxide dismutases. The hydrogen peroxide thus formed can initiate lipid peroxidation by conversion to the hydroxyl radical or generate hypochlorous acid through the action of myeloperoxidase. Scavenging of hydrogen peroxide can occur through action of catalase, glutathione peroxidase or peroxyredoxin, which uses thioredoxin for its action. Glutathione helps as an anti-oxidant by functioning with glutathione peroxidase or directly scavenging species such as peroxynitrite and hypochlorous acid, while vitamin E interrupt the free radical chain reaction leading to lipid peroxidation.

Figure 2. Acetaminophen-induced mitochondrial oxidant stress and its influence on cellular signaling

Metabolism of APAP results in generation of the reactive intermediate NAPQI, which forms protein adducts and induces mitochondrial oxidative stress. The increased generation of superoxide, especially from the mitochondrial electron transport chain and its reaction with nitric oxide results in production of peroxynitrite. The superoxide can be scavenged by superoxide dismutase 2 (SOD2) and converted to hydrogen peroxide, though the generation of peroxynitrite can interfere in this process by nitration of SOD2. Mitochondrial oxidative stress and hydrogen peroxide can also activate the MAP kinase JNK by multiple pathways, resulting in its phosphorylation and translocation to the mitochondria. This then amplifies the mitochondrial oxidant stress, which subsequently leads to activation of the mitochondrial permeability transition, translocation of mitochondrial proteins such as apoptosis inducing factor (AIF) and endonuclease G to the nucleus. This then results in DNA fragmentation and finally oncotic necrosis.

Figure 3. Extracellular generation of ROS generation by inflammatory cells

Though both Kupffer cells as well as neutrophils can generate free radicals in the vicinity of hepatocytes, the location of generation influences the effect of oxidant generation. Radicals generated in the sinusoids by Kupffer cells need to diffuse through the vascular space as well as the space of Disse before reaching hepatocytes, and hence can be scavenged by GSH released from hepatocytes. The formation of reactive oxygen by extravasated neutrophils in close proximity to hepatocytes results in the diffusion of the oxidants into the target cell. The oxidant stress derived from neutrophils or Kupffer cells results mitochondrial dysfunction, mitochondrial oxidant stress and ultimately cell death.