The suggestion of dose dependence in some cases of drug-induced liver injury indicates that a host-dependent idiosyncrasy in the metabolism or excretion of these drugs may be responsible for hepatotoxicity. Although several xenobiotics are transformed by the cytochrome P450 system (CYPs) into stable metabolites, many others are oxidised into unstable, chemically reactive intermediates. These reactive intermediates attack hepatic constituents such as unsaturated lipids, proteins or DNA and can lead to liver cell death (Pessayre, 1995). The abundance of CYPs in the liver explains the major role of these metabolites in drug-induced hepatotoxicity. Furthermore, the centrilobular location of most CYPs accounts for the pericentral location of these lesions. When small amounts of reactive metabolites are formed, glutathione serves as a decoy target, sparing critical hepatic macromolecules. However, when large amounts of the reactive metabolite are formed, the formation of glutathione conjugates exceeds the capacity of the liver to synthesise glutathione. The resulting depletion of glutathione together with direct covalent binding of the metabolite to protein thiols has serious consequences. The oxidation of protein thiol groups results in the formation of disulphur bonds between different molecules of actin, resulting in destruction of the microfilamentous network beneath the plasma membrane (Mirabelli et al., 1988). Depletion of protein thiol groups also decreases the activity of calcium translocases resulting in increases in intracellular Ca2+ which further damages the cytoskeleton (Bellomo and Orrenius, 1985). These and other effects of oxidative stress lead to the swelling and disruption of intracellular organelles ultimately resulting in hepatocyte necrosis.
Although it was initially thought that the toxicity of reactive metabolites only caused cell necrosis, this idea has been challenged in recent years (Pessayre et al., 1999). It is now clear that the extensive formation of reactive metabolites can cause apoptosis, necrosis or both (Fau et al., 1997; Shi et al., 1998). Several compounds, such as acetaminophen and cocaine, transformed into reactive metabolites have been shown to cause DNA fragmentation of hepatocytes indicative of apoptosis (Shen et al., 1992; Cascales et al., 1994). The cellular mechanisms causing metabolite-induced apoptosis have been studied with germander, a medicinal plant used in weight control diets, the widespread use of which led to an epidemic of hepatitis in France (Larrey et al., 1992b). Germander contains furano diterpenoids, which are activated by CYP 3A into electrophilic metabolites (Lekehal et al., 1996). Extensive formation of glutathione conjugates results in glutathione depletion which, in combination with covalent binding of the metabolites, results in protein thiol oxidation (Lekehal et al., 1996). Oxidation of protein thiols inactivates plasma membrane calcium translocases and increases the permeability of the mitochondrial inner membrane (the mitochondrial membrane permeability transit or MMPT) which, through the release of cytochrome C, leads to the activation of caspases (Fagian et al., 1990). Caspases are cysteine proteases that cut proteins after an aspartate residue and are the major executioners of apoptosis (Thornberry and Lazebnik, 1998). Caspase activation in conjunction with increased intra-cellular calcium activates calcium-dependent endonucleases, which cut the DNA between nucleosomes, eventually resulting in apoptosis (Fau et al, 1997). Germander-induced apoptotic hepatocyte death is prevented by troleandomycin, which inhibits its metabolic activation by CYP 3A4 or by preventing depletion of glutathione with cysteine (Fau et al., 1997)
Factors Influencing Direct Toxicity Due to Reactive Metabolites
Hepatotoxicity from the reactive metabolites of drugs is a significant problem with drugs where the formation of reactive metabolites is low enough to ensure the absence of hepatotoxicity in most recipients (and therefore allowing the marketing of the drug), but is high enough to lead to "idiosyncratic" toxicity in some "susceptible" subjects. The reason for susceptibility could be either genetically determined or acquired.
The amount of reactive metabolite formed depends on a particular isoenzyme the hepatic level of which may vary between individuals. Genetic polymorphisms of drug metabolising enzymes may contribute to an individual's risk to an ADR. Polymorphism in debrisoquine oxidation (CYP 2D6) leads to accumulation of perhexiline resulting in liver injury in poor metabolisers (Morgan et al., 1984) and increases the formation of reactive metabolites leading to chlorpromazine hepatotox-icity in extensive metabolisers (Watson et al., 1988). Polymorphism in mephenytoin hydroxyla-tion (CYP 2C19) may predispose poor metaboli-sers to atrium (phenobarbital, febarbamate and difebarbamate) induced hepatotoxicity (Horsmans et al., 1994).
Individual susceptibility to hepatotoxicity due to reactive metabolites may also be related to physiological, nutritional or therapeutic modifica tions in drug metabolism. For example, fasting leads to glycogen depletion and decreased glucur-onidation, depletion of glutathione and induction of CYP2E1 leading to an increased risk of paracetamol-induced liver injury (Price et al., 1987; Whitcomb and Block, 1994). Acquired factors enhancing the rate of biotransformation of a drug to its reactive metabolites through the induction of cytochrome P450 isoenzymes play an important role in increasing the direct toxicity. Alcohol is a potent inducer of CYP2E1 and to a lesser extent CYP3A4. Subjects who consume alcohol regularly may therefore have increased bioactivation of paracetamol (which is metabolised by CYP2E1 and 3A4), resulting in hepatotoxicity at conventional "therapeutic" doses (Zimmerman and Maddrey, 1995). In individuals with heavy alcohol intake this is compounded by reduced glutathione synthesis and low glutathione stores due to inhibition of glutathione synthatase and ethanol-related oxidative stress, respectively. Isoniazid also increases the toxicity of paracetamol by inducing CYP2E1, while rifampicin, another microsomal enzyme inducer, increases the risk of hepatotoxicity due to isoniazid (Pessayre et al., 1977; Moulding et al., 1991). Anticonvulsants (phenytoin, carbamazepine, and phenobarbital) induce CYP3A4 and can also enhance the toxic effects of paracetamol (Bray et al., 1992). As an alternative mechanism of drug interaction leading to an increased risk of paracetamol-induced liver injury, zidovudine competes for glucuronidation of the toxic metabolite, thus reducing its excretion (Shriner and Goetz, 1992). Drug accumulation can result from metabolic inhibition caused by another drug. For instance, troleandomycin increases the risk of cholestasis with oral contraceptives by inhibiting the CYP3A responsible for estrogen oxidation (Miguet et al., 1980).
The presence of underlying liver disease may predispose to dose-dependent drug toxicity, especially if the margin between therapeutic and toxic concentrations is small (Schenker et al., 1999). It is generally believed that pre-existing liver disease would neither induce nor worsen idiosyncratic hepatotoxicity, although this issue has not been studied adequately. However, a recent study demonstrated a higher incidence of hepatotoxicity as well as more severe liver injury secondary to antituberculosis agents in hepatitis B virus (HBV) carriers when compared with non-carriers and with HBV carriers who did not receive antituberculosis therapy (Wong et al., 2000).
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