Abstract
Toxic injury occurs in the liver more often than any other organ. This can be attributed to the fact that virtually all ingested substances that are absorbed are first presented to the liver and that the liver is responsible for the metabolism and elimination of many substances. Drug-induced liver injury has become a serious health problem in contemporary society. Moreover, liver toxicity is a significant impediment to development of new pharmaceuticals. A classification of liver injury is presented as a means to better understand the spectrum of known mechanisms of liver injury and to assist in discovering novel pathways of toxic liver injury.
Keywords
The liver is subjected to toxic injury more often than any other organ. This is not surprising, since the portal vein blood that drains the absorptive surface of the intestinal tract flows directly to liver. Thus, the liver is exposed to all ingested substances that are absorbed into the portal blood. Advances in food safety and workplace chemical exposures have reduced liver injury through these sources. Drug-induced hepatotoxicity has now become a significant cause of acute liver failure, accounting for 50% of cases. Toxic injury of the liver is the most frequent impediment to development of candidate drugs by pharmaceutical companies and is a major cause of withdrawal of approved drugs from the market. Some chemicals, termed predictable hepatoxins, are generally well known and well-characterized hepatotoxins, while others, termed idiosyncratic hepatotoxins, cause liver injury only on rare occasions. However, individuals afflicted with serious idiosyncratic drug reactions are at risk of death without liver transplantation. Some idiosyncratic responses can be attributed to unusual inheritance of or lack of genes encoding enzymes involved in drug metabolism. The mechanisms of idiosyncratic drug reactions probably involve those discussed below as well as novel pathways that remain to be discovered. As a result there has been continuing interest in understanding the pathogenesis of drug-induced liver injury.
Liver injury can be categorized by a number of systems. Some systems are based on the histologic lesion produced (i.e., inflammation, necrosis, cholestasis), others the type of injury (i.e., cytotoxic, cholestatic, mixed), but the advent of a newer understanding that gives a view of injury at the molecular level has given rise to a more focused scheme for understanding and categorizing liver injury. A recent review article (Lee, 2003) has outlined 6 basic mechanisms or sites of action for chemical-induced liver injury (Table 1) and serves as the basis for this presentation. It is important to keep in mind that many of these mechanisms can be in play at the same time in an affected liver and the bright line that separates distinct processes such as necrosis and apoptosis is blurred more often than pathologists would like. For example, recent data has demonstrated that acetaminophen, a classic example of a chemical that produces dose-related centrilobular necrosis following metabolism by the cytochrome p450 system, also produces apoptosis in human hepatocytes (Kass et al., 2003).
Disruption of Calcium Homeostasis and Cell Membrane Injury
Cytosolic free calcium is maintained at a very low concentration compared to the extracellular levels in the healthy cell. Most intracellular calcium is sequestered within the endoplasmic reticulum or mitochondria. The integrity of membranes surrounding and within the cell as well as the balance of intracellular ions are maintained by a variety of energy consuming processes including the Ca2+ and Mg2+-ATPases. Bioactivation of certain drugs by the cytochrome p450 system (discussed below) can engender reactive intermediates that can bind to various cellular proteins leading to widespread cellular dysfunction. Drug-induced damage to cellular proteins that are involved in ion balance can lead to an influx of calcium that disrupts, among other processes, normal actin filament assembly and disturbs ATP production. The resulting dispersal of the cytoskeleton leads to blebbing of the cell membranes and, if cell membrane disruption is of sufficient magnitude, irreversible cell injury and cell lysis can occur. Similar effects can result from toxins such as phalloidin, an active component of the mushroom, Amanita phalloides (Watanabe and Phillips, 1986).
Canalicular and Cholestatic Injury
Cholestasis can be produced by chemicals that damage the structure and function of the bile canaliculi. More than 30 drugs have been identified that can lead to cholestasis (Lewis, 2000). A key component of bile secretion involves a series of ATP-dependant export pumps such as the canalicular bile salt transporter, that moves bile salts and other transporters that export other bile constituents from the hepatocyte cytoplasm to the lumen of the canaliculus. Some drugs bind these canalicular transporter molecules and lead to the arrest of bile formation or movement within the lumen of the canalicular system (Trauner et al., 1998). In rare circumstances affected individuals (~1%) develop progressive destruction of cholangiocytes leading to “vanishing bile duct syndrome” (Lewis, 2000). Additional investigation is needed to elucidate the mechanism of destruction of cells of the biliary tree. Secondary injury can result as bile salts have a detergent action that can damage cell membranes and injure biliary epithelium or hepatocytes in areas of cholestasis.
Another mechanism leading to cholestasis involves disruption of actin filaments situated around the bile canaliculi preventing the normal pulsatile contractions that move bile through the canalicular system to the bile ducts. Drugs that bind to actin filaments such as phalloidin and those that affect calcium homeostasis and cellular energy production can generate this type of injury.
Metabolic Bioactivation by Cytochrome P450 Enzymes
The most frequent mechanism of hepatocellular injury involves production of injurious metabolites by the cytochrome P450 system. This family of enzymes is located in the smooth endoplasmic reticulum of hepatocytes primarily, although they are also found in many other cells of the body. A major role of cytochrome P450 enzymes is to metabolize lipid soluble chemicals into water-soluble compounds for excretion from the body in bile or urine. In the first step of this 2-step biotransformation process, termed phase 1, chemicals are bioactivated to a high energy reactive intermediate molecule, in preparation for the second step, phase 2, which involves formation of covalent bonds with polar molecules such as glucuronic acid. Conjugation forms a water-soluble metabolite that can be excreted. However, in some circumstances, such as overdosage, the high-energy reactive metabolites can form adducts that are covalent bonds with other cellular constituents such as proteins and nucleic acids. In acute toxicity, adducts can form with essential cellular enzymes leading to cell injury or death. The site of toxic cellular injury within the hepatic acinus reflects the site of bioactivation of the chemical. For instance, carbon tetrachloride is metabolized by the cytochrome P450 system to CCl3 •, a free radical that induces cell membrane injury. Lesions induced by carbon tetrachloride are most severe in the periacinar (centrilobular) areas, because this is the area where the smooth endoplasmic reticulum is most abundant, and, therefore, where the active form of the chemical is present in greatest concentration. Consequently, the centrilobular region of the hepatic lobule is by far the most common site of acute toxic injury. Acetaminophen toxicity is another and more commonly encountered example of this mechanism of liver injury (Zhang et al., 2002).
Adducts can also form between bioactivated compounds and nucleic acids. Adducts formed with DNA are more likely to lead to long-term consequences such as neoplasia, but adducts formed with RNA can interfere with protein synthesis and lead to acute hepatic toxicity.
Stimulation of Autoimmunity
In addition to direct damage to cellular proteins and nucleic acids, adduct formation can lead to immune-mediated liver injury. This can occur when adducts form between drug metabolites and cellular proteins or nucleic acids and generate neoantigens. These neoantigens may be formed on the cell surface by interactions of chemicals with certain cell membrane receptors or they may be processed, transported to the cell surface and presented as antigens. This process has been demonstrated with several drugs that form adducts with the cytochrome P450 isoforms that are involved in their metabolism, including tienilic acid and dihydralazine. Depending on the MHC context of antigen presentation either cellular and humoral immunity can be involved (Lewis, 2000). Injury can occur through direct cellular cytotoxicity and antibody dependant cellular cytotoxicity. Hepatic injury may be significantly exacerbated by recruitment of inflammatory cells such as neutrophils and activation of sinusoidal lining cells, particularly Kupffer cells (Jaeschke, 2000).
Stimulation of Apoptosis
Apoptosis is a form of cell death characterized by organized nuclear and cellular fragmentation (Rust and Gores, 2000). In the course of apoptotic death intact cell organelles and cell membranes are fragmented into small membrane-bound bodies. Cellular DNA is cleaved by endonucleases to 120–180 base pair fragments. Classically apoptosis can be triggered through two basic mechanisms in the hepatocyte; interactions between death ligands (Fas-ligand, TNF) and death receptors (Fas and TNFR-1) that trigger caspase 8 activation or damage to mitochondrial inner membranes releasing cytochrome c that binds to Apaf-1 activating it, leading to downstream activation of caspase 9. These pathways are not completely separate as a protein named Bid that is activated by caspase 8 can cause mitochondria to release cytochrome c. Certain chemicals may be able to trigger apoptosis by direct stimulation of pro-apoptotic pathways in the hepatocytes. Alternatively, apoptosis can be stimulated by several other pathways including immune-mediated events such as those discussed above that lead to the release of tumor necrosis factor alpha or activate Fas pathways. Chemicals that damage mitochondria can also stimulate apoptosis through the release of cytochrome c. Cholestasis can also stimulate apoptosis through the action of pro-apoptotic bile acids such as glycodeoxycholic acid (GCDC) (Bissel et al., 2001; Jaeschke et al., 2002). Hepatocytes from Fas-deficient mice, but not TNF-R1-deficient mice are resistant to GCDC-mediated apoptosis and Fas-ligand deficient mice are also sensitive to GCDC-induced apoptosis (Faubion et al., 1999). Similarly bile duct ligation in Fas-deficient mice produced less apoptosis, caspase 8 activation and liver injury than in wild-type mice. Thus hydrophobic bile acids appear to function through a non-Fas receptor mechanism. A proposed mechanism involves hydrophobic bile acid-induced, microtubule-dependent, increased transport of Fas-containing cytoplasmic vesicles to the cell membrane where spontaneous oligomerization and death domain activation may occur (Soderman et al., 2000). This type of toxicant-induced transport of death receptors represents a novel form of hepatocyte injury.
Additional pathways may involve triggering of apoptosis by protein kinase C activation and mitochondrial injury. In addition, bioactivation by the cytochrome P450 system can produce reactive molecules that engender oxidative stress which can then be a stimulus to induce synthesis of Fas ligand and increase the susceptibility of hepatocytes to apoptosis.
Mitochondrial Injury
Chemicals that damage mitochondrial structure, enzymes or DNA synthesis can disrupt β-oxidation of lipids and oxidative energy production within the hepatocytes (Fromenty and Pessayre, 1995; Pessayre et al., 1999; Bissel et al., 2001). Prolonged interruption of β-oxidation leads to microvesicular steatosis within hepatocytes. Mild insult leads to macrovesicular steatosis. In severe cases microvesicular steatosis, hepatic failure and death can result. Some drugs may inhibit β-oxidation (asprin, valproic acid, tetracyclines) and others may disrupt oxidative phosphorylation alone or in addition (bile acids, amiodarone) depleting the hepatocytes of energy. Certain antiviral dideoxynucleoside analogues (i.e., FIAU) can disrupt mitochondrial DNA synthesis through inhibition of DNA polymerase gamma, leading to depletion of mitochondrial DNA and mitochondria leading to hepatocyte death.
Other Cells
It should be remembered that the liver is more than just hepatocytes and that liver injury can occur when other cell types are damaged. The biliary epithelium, Kupffer cells, endothelial cells, and the hepatic stellate cells can all suffer chemical-induced damage and contribute to liver injury and fibrosis (Bissel et al., 2001).