NAPQI

NAP Neutrophil-activating peptide NAPQI N-acetyl-p-benzoquinone imine... 

As an example, acetaminophen (APAP) in overdose has been used by several groups to identify hepatotoxicity biomarkers in mice. APAP-induced hepatotoxicity is characterized by hepatic centrilobular necrosis and hepatitis. APAP biotransformation by Phase I enzymes leads to the formation of the reactive metabolite N-acetyl-p-benzoquinone (NAPQI), which can deplete glutathione and form adducts with hepatic proteins (see Section 15.2). Protein adduction primes the hepatocytes for cytokines released by activated macrophages (Kupffer cells) and/or destructive insults by reactive nitrogen species. Although necrosis is recognized as the mode of cell death in APAP overdose, the precise mechanisms are still being elucidated [152]. 

GSH may also be coupled to electrophilic reaction intermediates nonenzymatically or by GSH transferase (GST)-catalyzed reactions. Many different types of substrates will undergo GSH conjugation, including epoxides, halogenated compounds, aromatic nitro compounds, and many others. In these reactions, GSH can interact with an electrophilic carbon or heteroatom (O, N, and S) [35]. One such substrate is a reactive metabolite of acetaminophen (APAP), N-acetyl-p-benzoquinonimine (NAPQI), which will readily form a GSH conjugate (Scheme 3.2). Other examples of Phase II bioactivation reactions that lead to toxic endpoints are shown in Table 3.1. 

APAP, although a safe drug in therapeutic doses, can lead to severe and potentially lethal liver and kidney injury in cases of overdose. Liver injury involves a characteristic centrilobular hepatic necrosis. The centrilobular region is rich in metabolic enzymes, such as the CYP family of isozymes. CYP2E1 is the predominant P450 isozyme in catalyzing the oxidation of APAP to a reactive intermediate, N-acetyl-p-benzoquinonimine (NAPQI), which possesses an electrophilic carbon that will covalently bind to cellular proteins [35], as shown in Scheme 3.2. 

However, the adverse effects of APAP bioactivation are not observed until high doses are administered, where there is sufficient depletion of natural reserves of antioxidants, for example, reduced glutathione (GSH). Depletion of GSH exacerbates arylation of cellular proteins by NAPQI and amplifies oxidative stress from ROS, eventually leading to a drop in cellular ATP levels and cell death. Hence it is not the advent of covalent binding of reactive intermediates that is solely responsible for APAP toxicity, but rather a combination of events in which protein binding plays an important role. 

Figure 7.19 Proposed metabolic activation of paracetamol to a toxic, reactive intermediate /V-acetyl-p-benzoquinone imine (NAPQI). This can react with glutathione (GSH) to form a conjugate or with tissue proteins. Alternatively, NAPQI can be reduced back to paracetamol by glutathione, forming oxidized glutathione (GSSG).

However, the reactive metabolite will cause other changes as well as binding to protein. Thus, NAPQI will react both chemically and enzymatically with GSH to form a conjugate and will also oxidize it to GSSG and in turn be reduced back to paracetamol. This cyclical process may explain the occurrence of extensive depletion of GSH. NADPH will also reduce NAPQI and in turn be oxidized to NADP, although reduction via GSH is probably preferential. NADPH oxidation may also result from GSSG reduction via GSH peroxidase (Fig. 7.18). 

The depletion of GSH and NADPH will allow the oxidation of protein sulfydryl groups, which may be an important step in the toxicity. Thus, GSH and protein sulfydryl groups, such as those on Ca2+-transporting proteins, are important for the maintenance of intracellular calcium homeostasis. Thus, paracetamol and NAPQI cause an increase in cytosolic calcium, and paracetamol inhibits the Na+/K+ ATPase pump in isolated hepatocytes. 

It promotes the synthesis of GSH used in the conjugation of the reactive metabolite NAPQI. 

Paracetamol is a widely used analgesic, which causes liver necrosis and sometimes renal failure after overdoses in many species. The half-life is increased after overdoses because of impaired conjugation of the drug. Toxicity is due to metabolic activation and is increased in patients or animals exposed to microsomal enzyme inducers. The reactive metabolite (NAPQI) reacts with GSH, but depletes it after an excessive dose and then binds to liver protein. Cellular target proteins for the reactive metabolite of paracetamol have been detected, some of which are enzymes that are inhibited. Therefore, a number of events occur during which ATP is depleted, Ca levels are deranged, and massive chemical stress switches on the stress response. 

Figure 8.5 illustrates the precolumn oxidation of a phenacetin solution with LC-EC-Array detection (discussed in more detail in the following section). A peak, which eluted at 3.8 min, shows a characteristic voltammetric profile (i.e., reduction followed by oxidation) of a quinone species. Based on EC-Array and MS data (not shown), this peak has been identified as NAPQI, the expected reactive intermediate. This peak was not evident in a microsomal incubate of phenacetin analyzed using the same conditions (not shown). A possible explanation for this is that... 

FIGURE 15-3 Acetaminophen metabolism. In the liver, acetaminophen is metabolized to a toxic intermediate N-acetyl-p-benzoquinoneimine (NAPQI). NAPQI is quickly detoxified by conjugation with glutathione (GSH), forming mercapturic acid, which is eliminated via the urine. High doses of acetaminophen or liver dysfunction can result in accumulation of NAPQI and subsequent toxicity to liver proteins. 

Figure 3.7 Products from the reaction of glutathione (GSH) with NAPQI...

Human CYP2E1 is one of the most efficient P450s to catalyze the oxidation of acetaminophen to NAPQI (157-159). Ethanol and isoniazid cause a time-dependent inhibition and induction of acetaminophen oxidation to NAPQI in humans (160,161) that can decrease risk for hepatotoxicity over the interval of concurrent administration and increase risk for hepatotoxicity a few hours after removal of ethanol or isoniazid. The latter induction phase of CYP2E1 may, in part, be responsible for cases of acetaminophen hepatotoxicity associated with the use of ethanol (162-165) or isoniazid (166-168). However, the induction is modest (2- to 3-fold) therefore, other susceptibility factors, genetic and others such as decreased glutathione stores and nutritional status, are likely to play an important role in some individuals (169-174). 

The mechanism of acetaminophen toxicity has been studied extensively in experimental animals. Oxidation of acetaminophen in the liver via cytochrome P450 results in the formation of a cytotoxic electrophile, N-acetyl-p-benzoquinoneimine (NAPQI), that binds to hepatic protein. In the kidney, the formation of a one-electron oxidation product, namely N-acetyl-benzosemiquinoneimine radical, occurs via prostaglandin H synthase. This free radical binds to renal proteins and damages the renal medulla. 

Figure 18.8. Structures of acetaminophen and N-accty l-/ -bcnzoquinone imine (NAPQI) metabolites and GSH defense.

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